Compounds for use in the preparation of lipid nanoparticles encapsulating active agents, nanoparticle compositions comprising the compounds, and methods related thereto

Abstract

Provided are compounds comprising a structure represented by general formula (1) or its ionized form for the preparation of lipid nanoparticles encapsulating therapeutic agents, preventative agents, and / or biological agents: wherein R5 and each R6 are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R3 and each R 7 Independently, each A is an optionally substituted alkylene group, optionally substituted alkenyl group, or optionally substituted ynylene group; n is 0 to 100; l and m are each independently 0 or 1; each A is independently selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, optionally substituted ynylene groups, epoxide ring-opening products, and / or their derivatives; and B is R1R2N- or R23O-, wherein R1 and R2 are each independently H or hydrophobic tails, provided that R 1 and R 2 Both are not H; and R23 is an optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

Description

Technical Field

[0001] This disclosure broadly relates to compounds for preparing lipid nanoparticles encapsulating surfactants and methods for preparing said compounds. This disclosure also relates to nanoparticle compositions comprising said compounds, and related methods and uses.

[0002] background

[0003] Lipid nanoparticles (LNPs) are widely used for delivering therapeutics, prophylactic agents, and / or biological agents (e.g., polynucleotides such as mRNA). However, safe, stable, and efficient delivery systems remain a challenge. In particular, adverse health effects and cytotoxicity associated with the use of lipid nanoparticles for delivery have been reported.

[0004] Currently, the US Food and Drug Administration (FDA) has approved only two mRNA Covid-19 vaccines for human use: Moderna and Pfizer-BioNtech. Both vaccines utilize SARS-CoV-2 mRNA as the antigen and lipids as the carrier. LNPs consist of four components: ionizable lipids, PEG-lipid conjugates, cofactor lipids, and cholesterol. The lipids assemble with mRNA or siRNA to form nanoparticles for effective in vivo delivery. Another lipid nanoparticle-based siRNA drug, Patisiran, has been approved for the treatment of hATTR amyloidosis. These lipids contain a single tertiary amine group that ionizes at low pH values ​​(e.g., pH 4-5), allowing positively charged lipids to aggregate RNA into RNA-encapsulated nanoparticles (mRNA LNPs) upon mixing for mRNA delivery.

[0005] However, currently available formulations have several drawbacks and shortcomings and are far from ideal. First, the ionizable lipids used in Moderna and Pfizer-Biotec mRNA vaccine formulations are extremely expensive. Furthermore, in a physiological environment (pH 7.4), the positive charge on the ionized tertiary amine groups is neutralized, causing the lipids to revert to their original form, which may lead to the dissociation of mRNA from LNPs.

[0006] In view of the above, there is a need to address or at least improve the aforementioned problems. In particular, there is a need to provide cost-effective, substantially safe and stable, and / or effective delivery of compound and / or nanoparticle compositions for therapeutic, preventative, and / or biological agents.

[0007] Overview

[0008] In one aspect, compounds comprising the structure represented by general formula (1) or ionized forms thereof are provided for the preparation of lipid nanoparticles encapsulating therapeutic agents, preventative agents, and / or biological agents:

[0009]

[0010] in

[0011] R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0012] R 3 and each R 7 Independently, it is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0013] n is between 0 and 100;

[0014] l and m are each independently 0 or 1;

[0015] Each A is independently selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, optionally substituted alkynyl groups, epoxy ring-opening products and / or their derivatives; and

[0016] B is R 1 R 2 N- or R 23 O-, where R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; and R is one of them. 23 It can be an alkyl group, an alkenyl group, or an alkynyl group that are optionally substituted.

[0017] In one embodiment, the compound comprises three or more tertiary amines.

[0018] In one implementation, R 1 and R 2 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

[0019] In one implementation, A is represented by general formula (2):

[0020]

[0021] Where R 8 R 8’ R 9 and R 9’ Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0022] In one implementation, R 8R 8’ and R 9’ For H and R 9 C y H 2y+1 , where y is from 1 to 8.

[0023] In one embodiment, the compound has a molecular weight of 500 g / mol to 50,000 g / mol.

[0024] In one embodiment, the compound comprises a structure selected from one or more of the following:

[0025]

[0026] PL (n = 15);

[0027]

[0028] LP1 (n = 5);

[0029]

[0030] LP2 (n = 8);

[0031]

[0032] LP3 (n = 10);

[0033]

[0034] LP4 (n = 15);

[0035]

[0036] LPL1 (n = 1);

[0037]

[0038] LPL2 (n = 15);

[0039]

[0040] LPL3 (n = 5);

[0041] LPL4 (n = 8);

[0042] LPL5 (n = 10); and

[0043]

[0044] LPL6 (n = 15).

[0045] In another aspect, a method is provided for preparing compounds represented by the general formula (1) disclosed herein, the method comprising:

[0046] (a) Reacting a lipid-b-poly(Lys) compound represented by general formula (1b') with an epoxy-containing compound represented by general formula (3) to obtain a compound represented by general formula (1):

[0047]

[0048] in

[0049] R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0050] R 3 And each R is independently an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0051] n is between 1 and 100;

[0052] Each R 11 To R 13 Independently selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, or optionally substituted alkynyl groups;

[0053] R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; and

[0054] R 8 and R 9 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0055] In one embodiment, the method further includes, prior to step (a):

[0056] (ai) React the compound represented by general formula (4) with a protected amino acid containing an N-carboxylic anhydride (NCA) represented by general formula (5) to obtain a protected lipid-b-poly(Lys) compound represented by general formula (6) via ring-opening polymerization (ROP):

[0057]

[0058] in

[0059] R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0060] R 3 and each R 7 Independently, it is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0061] n is between 1 and 100;

[0062] R 11 To R 12 Each is independently selected from H, fatty alcohol, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl;

[0063] R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H;

[0064] R 10 H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0065] PG 1 For the protection of the base; and

[0066] (a-ii) Deprotect the protected lipid-b-poly(Lys) compound represented by general formula (6) obtained in step (ai) to obtain the compound represented by general formula (1b'):

[0067]

[0068] in

[0069] R 13 It is selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, or optionally substituted alkynyl groups.

[0070] In one implementation, the method further includes, prior to step (ai):

[0071] (aii) Reaction of the protected amino acid represented by general formula (7) with a carbonylating agent to obtain the protected amino acid containing NCA represented by general formula (5):

[0072]

[0073] in

[0074] R 6 and R 16 Each is independently H, an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, or an alkynyl group that is optionally substituted;

[0075] R 7 It is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0076] R 11 Selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, or optionally substituted alkynyl groups; and

[0077] PG 1 To protect the base.

[0078] In one implementation, the method further includes, prior to step (ai):

[0079] (ai-ii) Reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (9) in the presence of one or more coupling agents and a base to obtain a carbamate compound represented by general formula (10).

[0080]

[0081] in

[0082] R 5 R 14 and R 15 Each is independently H, an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, or an alkynyl group that is optionally substituted;

[0083] R 3 Independently, it is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0084] n is between 1 and 100;

[0085] R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; and

[0086] PG 2 For the protection of the base; and

[0087] (ai-iii) Deprotect the carbamate compound represented by general formula (10) obtained from step (ai-ii) to obtain the compound represented by general formula (4).

[0088]

[0089] in

[0090] R 10 H represents an alkyl group, an alkenyl group, or an alkynyl group, which may be optionally substituted.

[0091] In another aspect, a method is provided for preparing compounds represented by the general formula (1) disclosed herein, the method comprising:

[0092] (b) Reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (11) in the presence of one or more coupling agents and a base to obtain a dicarboxylate compound represented by general formula (12).

[0093]

[0094] in

[0095] R 7 It is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0096] R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H;

[0097] R 14 and R 17 To R 19 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; and

[0098] PG 4 and PG 5 Each serves as an independent protective base;

[0099] (c) Deprotect the dicarbamate compound of general formula (12) obtained in step (b) to obtain the amide compound of general formula (13).

[0100]

[0101] in

[0102] R 20 To R 21 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; and

[0103] (d) Reacting the amide compound represented by general formula (13) with the epoxy represented by general formula (3) to obtain the compound represented by general formula (1):

[0104]

[0105] R 8 To R 9 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0106] In another aspect, a nanoparticle composition for delivering therapeutic agents, preventative agents, and / or biological agents is provided, the nanoparticle composition comprising:

[0107] The compounds disclosed herein; and

[0108] Therapeutic agents, preventative agents, and / or biological agents encapsulated in the compounds disclosed herein.

[0109] In one embodiment, the nanoparticle composition comprises nanoparticles having an N / P ratio of 1:1 to 50:1.

[0110] In one embodiment, the nanoparticle composition comprises nanoparticles having an average particle size of 20 nm to 650 nm.

[0111] In one embodiment, the composition further comprises:

[0112] (i) Auxiliary lipids;

[0113] (ii) cholesterol or its derivatives; and

[0114] (iii) Polyethylene glycol (PEG) lipid conjugates or amphiphilic lipids.

[0115] In one embodiment, the compound represented by the general formula (1), the auxiliary lipid, cholesterol or a derivative thereof, and the PEG-lipid conjugate or amphiphilic lipid are mixed in a weight ratio of 30 to 50:5 to 50:5 to 60:1 to 5.

[0116] In one embodiment, the auxiliary lipid is selected from 1,2-distearyl-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), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. 1,2-Diundecanoyl-sn-glycero-choline (DPPC), 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-cholesterolylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16) LysoPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphatecholine, 1,2-Diarachidonicoyl-sn-glycero-3-phosphatecholine, 1,2-bis(docohexanoyl-sn-glycero-3-phosphatecholine), 1,2-diphydanoyl-sn-glycero-3-phosphateethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycero-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycero-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof.

[0117] In one embodiment, the cholesterol or its derivatives are selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, alfalfa sterol, and combinations thereof.

[0118] In one embodiment, the polyethylene glycol (PEG)-lipid conjugate or amphiphilic lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(PEG)-2000]-N,N-bistetradecylacetamide (ALC-0159), R-3-[(ω-methoxy-poly(ethylene glycol)2000)aminomethyl... [Acyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-methoxy](sodium salt)), PEG-DPPC, PEG-DSPE lipids and combinations thereof.

[0119] In another aspect, the nanoparticle compositions disclosed herein are provided for use in medicine.

[0120] In another aspect, nanoparticle compositions disclosed herein are provided for the treatment or prevention of diseases, disorders, or conditions in subjects in need.

[0121] In another aspect, the use of the nanoparticle compositions disclosed herein in the preparation of medicaments for the treatment or prevention of diseases, disorders or conditions in subjects in need is provided.

[0122] In another aspect, methods are provided for treating or preventing diseases, disorders, or conditions in a subject in need, the methods comprising administering to the subject a therapeutically effective amount of the nanoparticle composition disclosed herein.

[0123] In one embodiment, an immune response in the subject is induced by administering the nanoparticle composition to the subject.

[0124] In one implementation, the disease, disorder, or symptom is mediated by a coronavirus.

[0125] In one implementation, the coronavirus is the SARS-CoV-2 coronavirus.

[0126] definition

[0127] As used herein, the term "particle" broadly refers to a discrete entity or discrete body. Particles described herein may include organic particles, inorganic particles, composite particles, or biological particles. The particles used herein may also be macroscopic particles formed from aggregates of multiple subparticles or fragments of small objects. The particles of this disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidal particles. When used to refer to particles, the term "size" broadly refers to the maximum size of the particle. For example, when used in the context of nanoparticles, the term "size" may refer to the diameter of the nanoparticle, but is not limited thereto. In various embodiments, when the particle is substantially spherical, the term "size" may refer to the diameter of the particle; or when the particle is substantially non-spherical, the term "size" may refer to the maximum length of the particle.

[0128] As used herein, the term "nano" should be broadly interpreted to include dimensions at the nanoscale, i.e., less than about 1000 nm, about 1 nm to less than about 1000 nm, about 1 nm to about 900 nm, about 1 nm to about 800 nm, about 1 nm to about 700 nm, about 1 nm to about 600 nm, about 1 nm to about 500 nm, about 1 nm to about 400 nm, about 1 nm to about 300 nm, about 1 nm to about 200 nm, or about 1 nm to about 100 nm. Therefore, as used herein, the terms "nanostructure," "nanoparticle," "nanomaterial," etc., may include structures having at least one dimension not exceeding the range described herein. As used herein, the terms “nanostructure,” “nanoparticle,” “nanomaterial,” etc., may include structures having at least one size not greater than about 200 nm, not greater than about 150 nm, not greater than about 100 nm, not greater than about 90 nm, not greater than about 80 nm, not greater than about 70 nm, not greater than about 60 nm, not greater than about 50 nm, not greater than about 40 nm, not greater than about 30 nm, not greater than about 20 nm, or not greater than about 10 nm.

[0129] The term "micrometer" as used herein should be interpreted broadly to include particles of about 1 micrometer to about 1000 micrometers, about 1 micrometer to less than about 1000 micrometers, about 1 micrometer to about 900 micrometers, about 1 micrometer to about 800 micrometers, about 1 micrometer to about 700 micrometers, about 1 micrometer to about 600 micrometers, about 1 micrometer to about 500 micrometers, about 1 micrometer to about 400 micrometers, about 1 micrometer to about 300 micrometers, about 1 micrometer to about 200 micrometers, about 1 micrometer to about 100 micrometers, or about 1 micrometer to about 5 micrometers. In various embodiments, particles of about 5 micrometers or smaller may be used for intranasal spray delivery.

[0130] As used herein, the terms “treatment,” “treat,” and “therapy,” and their synonyms, refer to therapeutic treatments and preventative or preventative measures aimed at preventing or alleviating a medical condition, including but not limited to diseases, symptoms, and disorders. A medical condition also includes the body’s response to a disease or disorder such as inflammation. Those who require such treatment include those who already have a medical condition, those who are prone to developing a medical condition, and those who wish to prevent a medical condition.

[0131] As used herein, the term "therapeuticly effective amount" for a compound is intended to refer to an amount sufficient to prevent or at least alleviate (mitigate) a medical condition such as an infectious disease or a respiratory illness (e.g., a coronavirus caused by the SARS-CoV-2 virus or influenza caused by an influenza virus). The dosage and administration of the compounds, compositions, and formulations of this disclosure can be determined by those skilled in the art of clinical pharmacology or pharmacokinetics. The effective amount of the active agent of this disclosure used therapeutically will depend, for example, on the therapeutic purpose, route of administration, and the patient's condition. Therefore, it may be necessary for the therapist to adjust the dosage and change the route of administration as needed to obtain the best therapeutic effect.

[0132] The term “subject” is intended to refer broadly to any animal, such as a mammal, and includes humans. Exemplary subjects include, but are not limited to, humans and non-human primates. As used herein, the term “subject” also includes patients and non-patients. The term “patient” refers to an individual who has or may have a disease such as an infectious disease (e.g., a coronavirus caused by the SARS-CoV-2 virus), while “non-patient” refers to an individual who does not have or may not have a medical condition. “Non-patient” includes healthy individuals, non-ill individuals, and / or individuals without a medical condition. As used herein, the term “mammal” includes vertebrates such as humans or large veterinary mammals (e.g., horses, cattle, deer, sheep, llamas, goats, pigs).

[0133] In the following definitions of various substituents, it is stated that "the group may be a terminal group or a bridging group." This is intended to indicate that the use of the term is intended to cover both cases where the group is a terminal group / part and cases where the group is a linking group between two other parts of the molecule. Using the term "alkyl" with one carbon atom as an example, it should be understood that when present as a terminal group, the term "alkyl" with one carbon atom can mean -CH3, and when present as a bridging group, the term "alkyl" with one carbon atom can mean -CH2-, etc.

[0134] The term "bond" refers to the connection between atoms in a compound or molecule. A bond can be a single bond, a double bond, or a triple bond.

[0135] The term "alkyl" as a group or part of a group refers to a straight-chain or branched aliphatic hydrocarbon group having 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Examples of suitable straight-chain and branched alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hexyl, pentyl, 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, etc. The groups may be terminal groups or bridging groups.

[0136] The term "alkenyl," used as a group or part of a group, refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight-chain or branched, having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group may contain multiple double bonds, and the orientation of each double bond is independently E or α. Exemplary alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 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, etc. The groups may be terminal groups or bridging groups.

[0137] The term "alkynyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be straight-chain or branched, having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group may contain 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-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-hepynyl, 2-hepynyl, 6-hepynyl, 1-octyynyl, 2-octyynyl, 7-octyynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 9-decynyl, etc. The group may be a terminal group or a bridging group.

[0138] As used herein, the term "cyclic" broadly refers to a structure in which one or more series of atoms are linked to form at least one ring. This term includes, but is not limited to, saturated and unsaturated 5-membered rings and saturated and unsaturated 6-membered rings. Examples of groups having cyclic structures include, but are not limited to, cyclopentane, cyclopentene, cyclohexane, cyclohexene, benzene, etc. The term "cyclic" as used herein includes "heterocyclic".

[0139] As used herein, the term "heterocycle" broadly refers to a structure in which two or more atoms of different kinds are linked to form at least one ring. For example, a heterocycle may be formed from a carbon atom and at least one other atom (i.e., a heteroatom) selected from oxygen (O), nitrogen (N) or (NR) and sulfur (S), wherein R is independently hydrogen or an organic group. The term also includes, but is not limited to, saturated and unsaturated 5-membered rings and saturated and unsaturated 6-membered rings. Examples of groups having heterocyclic structures include, but are not limited to, furan, thiophene, 1H-pyrrole, 2H-pyrrole, 1-pyrrololine, 2-pyrroleline, 3-pyrroleline, 1-pyrazoline, 2-pyrazoline, 3-pyrazoline, 2-imidazoline, 3-imidazoline, 4-imidazoline, pyrazole, imidazoline, 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-oxothiopentane, 1,3-oxothiopentane, pyrazolidine, imidazoline, pyridine, 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-pyranone, 4-pyranone, 1,4-dioxin, 2H-thiaran, 4H-thiaran, tetrahydropyran, thiazide, piperidine, 1,4-dioxane, 1,2-dithiaran, 1,3-dithiaran, 1,4-dithiaran, 1,3,5-trithiaran, piperazine, morpholine, thiomorpholine, etc.

[0140] The term "amine group" is intended to refer broadly to groups containing -NR2, where R is independently hydrogen or an organic group. These groups can be terminal groups or bridging groups.

[0141] The term "amide group" is intended to refer broadly to groups containing -C(=O)NR2, where R is independently hydrogen or an organic group. The group may be a terminal group or a bridging group.

[0142] The term "aryl" as a group or part of a group means (i) an optionally substituted monocyclic or fused polycyclic aromatic carbon ring (a ring structure having all ring atoms as carbon), preferably having 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, anthracene, phenanthryl, fluorenyl, indene or indanyl, etc.

[0143] The term "heteroaryl" as a group or part of a group refers to a group containing an aromatic ring (preferably a 5- or 6-membered aromatic ring) in which one or more carbon atoms (e.g., 1 to 6 carbon atoms) are replaced by heteroatoms. Suitable heteroatoms may 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, benzoisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xanthan, phenoxazine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazolium, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthidine, quinoxaline, cyclophosphine, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isoxazole, furazine, phenothiazine, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5- or 8-quinoline, 1-, 3-, 4- or 5-isoquinoline, 1-, 2- or 3-indolyl and 2- or 3-thiophene, etc. The group can be a terminal group or a bridging group.

[0144] The term "halogen" refers to chlorine, fluorine, bromine, or iodine. The term "halide" refers to chloride, fluoride, bromide, or iodide.

[0145] As used herein, the term "epoxy ring-opening product" broadly refers to one or more products obtained from the ring-opening reaction of an epoxy ring. The term "epoxy" or "epoxy ring" may include the terms "epoxide," "cyclic ether," "three-membered cyclic ether," etc., and / or may be used interchangeably with the terms "epoxide," "cyclic ether," "three-membered cyclic ether," etc. Examples of such epoxy ring-opening products include, but are not limited to, compounds containing one or more hydroxyl (-OH) groups. Such epoxy ring-opening products may be compounds containing primary alcohols, secondary alcohols, tertiary alcohols, or diols.

[0146] The term "optionally substituted" when used to describe a chemical structure or part refers to one or more hydrogen atoms in said chemical structure or part being optionally substituted by a chemical moiety or functional group, such as alcohol, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, tert-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), carbamoyl (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., -CCl3, -CF3, ... -C(CF3)3), heteroalkyl, isocyanate, isothiocyanate, nitrile, nitro, phosphate diester, sulfide, sulfonamide (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl), sulfoxide, thiol (e.g., mercapto, thioether) or urea (-NHCONH-alkyl-).

[0147] Unless otherwise stated, the terms “coupled” or “connected” as used in this specification are intended to cover a direct connection or a connection between the two through one or more intermediate means.

[0148] As used herein, the term "associated with" refers to a broad relationship between two elements. This relationship includes, but is not limited to, physical, chemical, or biological relationships. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other, or element A may contain element B, or vice versa.

[0149] The term “and / or”, such as “X and / or Y”, is understood to mean “X and Y” or “X or Y”, and should be considered to provide explicit support for both meanings or either meaning.

[0150] Furthermore, in the description herein, the word “substantially” is always understood to include, but is not limited to, “completely” or “entirely”. Additionally, terms such as “comprising” and “including” are intended to be non-restrictive descriptive language whenever used, as they broadly include elements / components described after these terms, in addition to other components not explicitly stated. For example, when “comprising” is used, a reference to “one” feature is also intended to refer to “at least one” of that feature. In the appropriate context, terms such as “consisting of” can be considered a subset of terms such as “comprising”. Therefore, in the embodiments disclosed herein that use terms such as “consisting of”, it should be understood that these embodiments provide teachings on corresponding embodiments using terms such as “consisting of”. Furthermore, terms such as “about” and “approximately”, whenever used, generally mean reasonable variations, such as a variation of + / -5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, or a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

[0151] Furthermore, in the description herein, certain values ​​may be disclosed within a range. Values ​​showing the endpoints of a range are intended to illustrate a preferred range. Whenever a range is described, it is intended to encompass and teach all possible subranges as well as individual numerical values ​​within that range. That is, the endpoints of a range should not be interpreted as inflexible limitations. For example, the description of a range of 1% to 5% is intended to have specifically disclosed subranges of 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., and individually, values ​​within that range such as 1%, 2%, 3%, 4%, and 5%. The above specific disclosures are intended to apply to any depth / width of the range.

[0152] Additionally, when describing some embodiments, this disclosure may have disclosed methods and / or processes in a specific order of steps. However, unless otherwise required, it will be appreciated that the methods or processes should not be limited to the specific order of steps disclosed. Other orders of steps are also possible. The specific order of steps disclosed herein should not be construed as an undue limitation. Unless otherwise required, the methods and / or processes disclosed herein should not be limited to steps performed in the order written. The order of steps may vary and still remain within the scope of this disclosure.

[0153] Furthermore, it should be understood that while this disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may be omitted in other alternative embodiments, and this disclosure provides support for such omissions and these associated alternative embodiments.

[0154] It will also be recognized that, in cases where priority is claimed in an earlier application, the entire contents of the earlier application are also considered to form part of this disclosure and can be used to support the implementation thereof.

[0155] Description of the implementation plan

[0156] The following discloses compounds for preparing lipid nanoparticles encapsulating active agents, methods for preparing said compounds, exemplary non-limiting embodiments of nanoparticle compositions comprising said compounds and related methods / uses.

[0157] compound

[0158] Compounds for preparing lipid nanoparticles are provided. In various embodiments, the compounds comprise one or more ionizable and / or capable of ionization amine groups. The amine groups may 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 ionization and / or present in ionized form at, for example, 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 encapsulation / loading agents, delivery media / systems, and / or transfection media / systems. In various embodiments, the compounds are designed / configured to allow loading / encapsulation of one or more types of molecules or payloads. In various embodiments, the compounds are also designed / configured to allow the loaded / encapsulated agent to be released from the compound and / or subsequently delivered to a desired target (e.g., cells, cytosols, tissues, or organs). Molecules / loads to be loaded / encapsulated onto / in / within the compound may include, but are not limited to, therapeutic agents, prophylactic agents, biological agents, etc. In various embodiments, the molecule / loador to be loaded / encapsulated comprises nucleic acids. For example, the molecule / loador to be loaded / encapsulated may be a nucleic acid selected from ribonucleic acid (RNA), microRNA (miRNA), messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), plasmid deoxyribonucleic acid (pDNA), oligonucleotides such as antisense oligonucleotides or allele-specific oligonucleotides (ASO), or combinations thereof. In various embodiments, the molecule / loador to be loaded / encapsulated comprises a therapeutic agent. For example, the molecule / loador to be loaded / encapsulated may be a therapeutic agent selected from negatively charged therapeutic agents, drug molecules, vaccines (e.g., dengue vaccines, Covid-19 vaccines, etc.), or combinations thereof. Advantageously, the compound is 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, cells, cytosol, tissue, or organ).

[0159] Therefore, in various embodiments, supports, nanocarriers or delivery systems / media comprising the compound or its ionized form are also provided.

[0160] Advantageously, the compound is designed / configured to be ionizable in a low pH range, for example, in the pH ranges of about 3.0 to about 6.5, about 3.1 to about 6.4, about 3.2 to about 6.3, about 3.3 to about 6.2, about 3.4 to about 6.1, about 3.5 to about 6.0, about 3.6 to about 5.9, about 3.7 to about 5.8, about 3.8 to about 5.7, about 3.9 to about 5.6, about 4 to about 5.5, about 4.1 to about 5.4, about 4.2 to about 5.3, about 4.3 to about 5.2, about 4.4 to about 5.1, about 4.5 to about 5.0, about 4.6 to about 4.9, about 4.7 to about 4.8, or about 4.75, depending on the type or nature of the amine group. In various embodiments, the compound is designed / configured to be in an ionized form, for example, the compound can be ionized to become positively charged.

[0161] In various embodiments, the compound comprises a structure represented by general formula (1) or its ionized form:

[0162]

[0163] in

[0164] R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0165] R 3 and each R 7 Independently, it is an optionally substituted alkylene group (e.g., -CH2-), an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0166] n is approximately 0 to approximately 100, approximately 0 to approximately 50, approximately 0 to approximately 40, approximately 0 to approximately 30, approximately 0 to approximately 20, approximately 1 to approximately 19, approximately 2 to approximately 18, approximately 3 to approximately 17, approximately 4 to approximately 16, approximately 5 to approximately 15, approximately 6 to approximately 14, approximately 7 to approximately 13, approximately 8 to approximately 12, approximately 9 to approximately 11, or approximately 10;

[0167] l and m are each independently 0 or 1;

[0168] Each A is independently selected from H, fatty alcohols, optionally substituted alkyl groups (e.g., alkyl alcohols), optionally substituted alkenyl groups (e.g., alkenyl alcohols), optionally substituted alkynyl groups (e.g., alkynyl alcohols), epoxy ring-opening products and / or their derivatives; and

[0169] B is R1 R 2 N- or R 23 O-, where R 1 and R 2 Each is independently H, a hydrophobic tail / chain / group, or contains at least a straight-chain aliphatic, branched aliphatic, and / or cyclic hydrocarbon, provided that R is R. 1 and R 2 The two are not both H; and R is one of them. 23 The radical can be an alkyl group, an alkenyl group, or an alkynyl group that are optionally substituted. The compound is suitable for preparing lipid nanoparticles.

[0170] In various embodiments, the compound represented by general formula (1) comprises one or more hydrophobic tails / chains / groups. For example, one or two hydrophobic tails / chains / groups may be present in general formula (1). In various embodiments, R 1 and R 2 Only one of them can be H. For example, in various implementations, when R 1 When it is H, R 2 Not H. For example, in various implementations, when R 2 When it is H, R 1 Not H, or vice versa. In various implementation schemes, R 1 and R 2 Only one of them is a hydrophobic tail / chain / group. For example, when R 1 When it is H, R 2 It is a hydrophobic tail / chain / group, or vice versa. In various embodiments, R 1 and R 2 Both are hydrophobic tails / chains / groups.

[0171] In various implementation schemes, R 1 and R 2 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

[0172] In various implementation schemes, R 1 and R 2 Each of the hydrophobic tail chains / groups independently comprises an optionally substituted alkyl group. The alkyl group may have at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 carbon atoms. For example, R 1 and R 2 Each independently is C x H2x+1 , where x≥3, x≥4, x≥5, x≥6, x≥7, x≥8, x≥9, x≥10, x≥11, x≥12, x≥13, x≥14, x≥15, x≥16, x≥17, x≥18, x≥19, x≥20, x≥21, x≥22, x≥23, ​​x≥24 or x≥25.

[0173] In various implementation schemes, R 5 and each R 6 Independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. For example, R 5 and / or R 6 Optional from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hexyl, pentyl, 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, etc., or combinations thereof.

[0174] In various implementation schemes, R 3 and each R 7 Independently selected from optionally substituted alkylene, optionally substituted alkenyl, or optionally substituted ynylene. For example, R 3 and / or R 7Optional components include methylene, ethylene, n-propylene, 2-propylene, isopropylene, n-butylene, isobutylene, sec-butylene, tert-butylene, hexylene, pentylene, 1,2-dimethylpropylene, 1,1-dimethylpropylene, pentylene, isopentylene, hexylene, 4-methylpentylene, 1-methylpentylene, 2-methylpentylene, 3-methylpentylene, 2,2-dimethylbutylene, 3,3-dimethylbutylene, 1,2-dimethylbutylene, 1,3-dimethylbutylene, and 1,2,2-trimethylpropylene. 1,1,2-Trimethylpropylidene, 2-Ethylpentane, 3-Ethylpentane, Heptane, 1-Methylhexane, 2,2-Dimethylpentane, 3,3-Dimethylpentane, 4,4-Dimethylpentane, 1,2-Dimethylpentane, 1,3-Dimethylpentane, 1,4-Dimethylpentane, 1,2,3-Trimethylbutylene, 1,1,2-Trimethylbutylene, 1,1,3-Trimethylbutylene, 5-Methylheptane, 1-Methylheptane, Octylene, Nonylene, Decylene, etc., or combinations thereof.

[0175] In various implementation schemes, A is represented by general formula (2):

[0176]

[0177] Where R 8 R 8’ R 9 and R 9’ Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0178] In various implementation schemes, R 8 R 8’ R 9 and / or R 9’ Each can be selected from methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hexyl, pentyl, 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, etc., or combinations thereof.

[0179] In various implementation schemes, R 8 R 8’ and / or R 9’ For H. In various implementation schemes, R 9 C y H 2y+1 Where y is 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various implementation schemes, R 9 For hexyl (i.e. -C6H) 13 ).

[0180] In various implementation schemes, n is an integer ≥ 1. In various implementation schemes, n≥1, n≥2, n≥3, n≥4, n≥5, n≥6, n≥7, n≥8, n≥9, n≥10, n≥11, n≥12, n≥13, n≥14, n≥15, n≥16, n≥17, n≥18, n≥19, n≥20, n≥21, n≥22, n≥23, n≥24, n≥25, n≥26, n≥27, n≥28, n≥29, n≥30, n≥31, n≥32, n≥33, n≥34, n≥35, n≥36, n≥37, n≥38, n≥39, n≥40, n≥41, n≥42, n≥43, n≥44, n≥45, n≥46, n≥47, n≥48, n≥49, n≥50 n≥51, n≥52, n≥53, n≥54, n≥55, n≥56, n≥57, n≥58, n≥59, n≥60, n≥61, n≥62, n≥63, n≥64, n≥65, n≥66, n≥67, n≥68, n≥69, n≥70, n≥71, n≥72, n≥73, n≥74, n≥75, n≥76, n≥77, n≥78, n≥79, n≥80, n≥81, n≥82, n≥83, n≥84, n≥85, n≥86, n≥87, n≥88, n≥89, n≥90, n≥91, n≥92, n≥93, n≥94, n≥95, n≥96, n≥97, n≥98 n ≥ 99 or n ≥ 100. In various embodiments, n is about 1 to about 100, about 10 to about 90, about 20 to about 80, about 30 to about 70, about 40 to about 60 or about 50.

[0181] In various implementations, each A can be the same as or different from each other. In various implementations, all A are the same. In various implementations where n≥2, A can be the same as or different from each other. For example, when n=20 and there are a total of twenty A, each of the twenty A in the structure represented by general formula (1) can be the same as or different from each other.

[0182] In various embodiments, the compound comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more tertiary amine groups (e.g., ionizable tertiary amine groups). In various embodiments, the tertiary amine (3°) groups are ionizable. In various embodiments, unlike the ionizable lipids containing only a single tertiary amine group used in Moderna and Pfizer-Biotec (ALC-0315) mRNA vaccines and partecillan formulations, the lipids of this disclosure contain multiple tertiary amine groups (e.g., up to 15) and peptides. In various embodiments, advantageously, the presence of multiple tertiary amine groups enhances RNA binding, thereby providing stability during the preparation of LNPs. In various embodiments, advantageously, the presence of multiple tertiary amine groups facilitates the release of RNA into endosomes in the cytosol for efficient RNA transfection. In various embodiments, the tertiary amine is chemically conjugated to an amino acid backbone (e.g., lysine), a polypeptide backbone (e.g., polylysine), or a derivative thereof. In various embodiments, the compound is an ionizable lipid comprising a long-tailed lipid and multiple tertiary amine groups (having 1, 5, 8, 10, or 15 tertiary amine groups) chemically linked by a fully biodegradable polypeptide backbone (polylysine). Advantageously, the use of polypeptides in embodiments of the lipids disclosed herein allows for biodegradability, mitigating potential toxicity from long-term accumulation and cellular / systemic inflammation caused by non-biodegradable ionizable lipids.

[0183] In various embodiments, the polypeptide backbone or its derivatives are biodegradable.

[0184] In various embodiments, -NA2 is a tertiary (3°) amine group (e.g., an ionizable tertiary amine group). In various embodiments, the compound is in an ionized form, wherein -NA2 has been ionized to become a positively charged group. For example, a tertiary amine group can be ionized to become a quaternary ammonium cation. In various embodiments, -NA2 is ionized / protonated in a pH range of about 3.0 to about 6.5 to become a positively charged group / ion / cation. In various embodiments, -NA2 is protonated to become -NA2H. + .

[0185] In various embodiments, the compound comprises a lipid compound. The term "compound" may be used interchangeably with the terms "lipid," "lipid compound," "ionizable lipid," "ionizable lipid compound," "cationic lipid compound," "ionizable cationic lipid compound," etc. In various embodiments, the compound is amphiphilic and comprises both hydrophilic and hydrophobic moieties. In various embodiments, the lipid moieties of the compound are hydrophobic, while groups such as amines and / or hydroxyl groups in the compound are hydrophilic. In various embodiments, the compound comprises one or more hydrophilic moieties at an amine group (e.g., ionizable NA2). In various embodiments, the compound comprises a hydrophilic moiety at a B (e.g., R) group ... 1 and / or R 2 The compound contains a hydrophobic portion / tail / chain / group. Advantageously, in various embodiments, the presence of the hydrophobic portion / tail / chain / group in the compound helps to confer improved cell uptake and / or transfection, thereby resulting in higher and / or better transfection efficiency.

[0186] Advantageously, in various embodiments, the presence of multiple tertiary amine groups enhances RNA binding, thereby providing stability during the preparation of lipid nanoparticles (LNPs).

[0187] It should be understood that, in various embodiments, the tertiary amine group ionizes and becomes positively charged at low pH values ​​(e.g., pH 3.0 to 6.5). Advantageously, the positively charged lipids can condense RNA into RNA-encapsulated LNPs after mixing.

[0188] It should be understood that, in various embodiments, the positive charge on the tertiary amine group is neutralized to produce the original tertiary amine form in the lipids in a physiological environment (pH 7.4). Advantageously, this neutralization process can reduce the toxicity of LNP.

[0189] It should be understood that, in various implementations, the presence of multiple tertiary amine groups can increase proton uptake in the endosome, which can promote the endosome release of RNA LNPs for efficient RNA transfection.

[0190] In various embodiments, the compound represented by general formula (1) comprises a structure selected from one or more of the following:

[0191] General formula (1a) or (1a'),

[0192]

[0193]

[0194] in

[0195] R 5 and each R 6Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0196] R 3 and each R 7 Independently, it is an optionally substituted alkylene group (e.g., -CH2-), an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0197] Each A is independently -CHR 8 -CHR 9 OH represents a derivative of an epoxy ring-opening product, where R 8 and R 9 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0198] B is R 1 R 2 N-, where R 1 and R 2 Each is independently H, a hydrophobic tail / chain / group, or contains a straight-chain aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon, provided that R is present. 1 and R 2 The two are not both H;

[0199] General formula (1b) or (1b'),

[0200]

[0201] in

[0202] R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0203] R 3 and each R 7 Independently, it is an optionally substituted alkylene group (e.g., -CH2-), an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0204] n is approximately 1 to approximately 100, approximately 1 to approximately 50, approximately 1 to approximately 40, approximately 1 to approximately 30, approximately 1 to approximately 20, approximately 1 to approximately 19, approximately 2 to approximately 18, approximately 3 to approximately 17, approximately 4 to approximately 16, approximately 5 to approximately 15, approximately 6 to approximately 14, approximately 7 to approximately 13, approximately 8 to approximately 12, approximately 9 to approximately 11 or 10;

[0205] Each A is composed of R 11 To R 13 It means that R 11 To R 13Each is independently selected from H, fatty alcohols, optionally substituted alkyl groups (e.g., alkyl alcohols), optionally substituted alkenyl groups (e.g., alkenyl alcohols), or optionally substituted alkynyl groups (e.g., alkynyl alcohols); and

[0206] B is R 1 R 2 N-, where R 1 and R 2 Each is independently H, a hydrophobic tail / chain / group, or contains a straight-chain aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon, provided that R is present. 1 and R 2 The two are not both H;

[0207] General formula (1c) or (1c'),

[0208]

[0209] in

[0210] R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0211] R 3 and each R 7 Independently, it is an optionally substituted alkylene group (e.g., -CH2-), an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0212] Each A is independently -CHR 8 -CHR 9 OH represents a derivative of an epoxy ring-opening product, where R 8 and R 9 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0213] B is R 1 R 2 N-, where R 1 and R 2 Each is independently H, a hydrophobic tail / chain / group, or contains a straight-chain aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon, provided that R is present. 1 and R 2 The two are not both H; or

[0214] General formula (1d) or (1d')

[0215]

[0216] in

[0217] R5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl;

[0218] R 3 and each R 7 Independently, it is an optionally substituted alkylene group (e.g., -CH2-), an optionally substituted alkenyl group, or an optionally substituted ynylene group;

[0219] Each A is independently -CHR 8 -CHR 9 OH represents a derivative of an epoxy ring-opening product, where R 8 and R 9 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0220] B is R 23 O-, where R 23 It can be an alkyl group, an alkenyl group, or an alkynyl group that are optionally substituted.

[0221] In various embodiments, in general formula (1), when m=1, n=1 to 100, l=0, A is H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl, and B is R 1 R 2 When N-, the compound represented by the general formula (1) is a lipid-b-poly(Lys)(LP). For example, the compound may be represented by the general formula (1b) or (1b').

[0222] In various embodiments, in general formula (1), when m=0, n=0, l=1, A is a fatty alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e.g., alkenyl alcohol), optionally substituted alkynyl (e.g., alkynyl alcohol), epoxy ring-opening product and / or its derivative, and B is R 1 R 2 When N-, the compound represented by the general formula (1) is a lipid-Lys-lipid derivative (LPL1) with a degree of polymerization (DP) of 1. For example, the compound may be represented by the general formula (1c) or (1c').

[0223] In various embodiments, in general formula (1), when m=1, n=1 to 100, l=1, A is a fatty alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e.g., alkenyl alcohol), optionally substituted alkynyl (e.g., alkynyl alcohol), epoxy ring-opening product and / or its derivative, and B is R 1 R 2When N-, the compound represented by the general formula (1) is a lipid-b-ionizable poly(Lys)-lipid (LPL). For example, the compound may be represented by the general formula (1a) or (1a').

[0224] In various embodiments, in general formula (1), when m=0, n=1 to 100, l=1, A is a fatty alcohol, optionally substituted alkyl (e.g., alkyl alcohol), optionally substituted alkenyl (e.g., alkenyl alcohol), optionally substituted alkynyl (e.g., alkynyl alcohol), epoxy ring-opening product and / or its derivative, and B is R 23 When O-, the compound represented by the general formula (1) is an ionizable poly(Lys)-lipid (PL). For example, the compound may be represented by the general formula (1d) or (1d').

[0225] In various implementation schemes, l and m are not both 0 in general formula (1).

[0226] In various embodiments, the compound comprises one or more structures selected from the following:

[0227]

[0228] PL (n = 15);

[0229]

[0230] LP1 (n = 5);

[0231]

[0232] LP2 (n = 8);

[0233]

[0234] LP3 (n = 10);

[0235]

[0236] LP4 (n = 15);

[0237]

[0238] LPL1 (n = 1);

[0239]

[0240] LPL2 (n = 15);

[0241]

[0242] LPL3 (n = 5);

[0243]

[0244] LPL4 (n = 8);

[0245]

[0246] LPL5 (n = 10); and

[0247]

[0248] LPL6 (n = 15).

[0249] In various embodiments, the molecular weight of the compound is from about 500 g / mol to about 50,000 g / mol, from 1,000 g / mol to about 49,000 g / mol, from about 2,000 g / mol to about 48,000 g / mol, from about 3,000 g / mol to about 47,000 g / mol, from about 4,000 g / mol to about 46,000 g / mol, from about 5,000 g / mol to about 45,000 g / mol, and from about 600 g / mol. 0 g / mol to about 44,000 g / mol, about 7,000 g / mol to about 43,000 g / mol, about 8,000 g / mol to about 42,000 g / mol, about 9,000 g / mol to about 41,000 g / mol, about 10,000 g / mol to about 40,000 g / mol, about 11,000 g / mol to about 39,000 g / mol, about 12,000 g / mol to about 38,000 g / mol, about 13,000 g / mol to about 37,000 g / mol, about 14,000 g / mol to about 36,000 g / mol, about 15,000 g / mol to about 35,000 g / mol, about 16,000 g / mol to about 34,000 g / mol, about 17,000 g / mol to about 33,000 g / mol, about 18,000 g / mol to about 32,000 g / mol, about 19, From about 31,000 g / mol, from about 20,000 g / mol to about 30,000 g / mol, from about 21,000 g / mol to about 29,000 g / mol, from about 22,000 g / mol to about 28,000 g / mol, from about 23,000 g / mol to about 27,000 g / mol, from about 24,000 g / mol to about 26,000 g / mol or about 25,000 g / mol.

[0250] Methods for preparing compounds

[0251] A method for preparing compounds represented by general formula (1) disclosed herein is provided.

[0252] In various embodiments, the method includes the step of preparing a compound represented by general formula (1a) or (1a'), wherein the compound represented by general formula (1a) or (1a') is a compound in which m=1, n=1 to 100, l=1, and A (corresponding to -CHR) 8 -CHR 9 OH) is a derivative of the ring-opening product of epoxy, and B is R 1 R 2 The method comprises the general formula (1) for N- (i.e., lipid-b-ionizable poly(Lys)-lipid (LPL)), the method being:

[0253]

[0254]

[0255] (a) Reacting a lipid-b-poly(Lys) compound represented by general formula (1b') with an epoxy (e.g., 1,2-epoxyalkane) represented by general formula (3) to obtain a compound represented by general formula (1a) or (1a'):

[0256]

[0257] in

[0258] R 1 To R 3 R 5 To R 9 and n contain one or more features and / or share one or more properties, said one or more features and one or more properties being similar to those described above; and

[0259] R 11 To R 13 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0260] In various implementations, the method further includes, prior to step (a):

[0261] (ai) To make one or more N-carboxylic anhydride (NCA) monomers represented by general formula (5) (e.g., protected amino acids containing NCA, such as N 6 -benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA)) reacts / polymerizes with an amide compound represented by general formula (4) (e.g., 2-amino-N,N-bistetradecylacetamide) to obtain a protected lipid-b-poly(Lys) compound represented by general formula (6):

[0262]

[0263] in

[0264] R 1 To R 3 R 5 To R 7 R 11 R 12 And n contains one or more features and / or shares one or more properties, which are similar to those described above;

[0265] R 10 H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and

[0266] PG 1 Protecting groups include, for example, N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), or 9-fluorenylmethoxycarbonyl (Fmoc).

[0267] (a-ii) Deprotect the protected lipid-b-poly(Lys) compound represented by general formula (6) obtained in step (ai) to obtain the compound represented by general formula (1b'):

[0268]

[0269] in

[0270] R 1 To R 3 R 5 To R 7 R 11 To R 13 n and PG 1 It includes one or more features and / or shares one or more properties, which are similar to those described above;

[0271] In various embodiments, the polymerization / reaction step (ai) comprises ring-opening polymerization (ROP) of the NCA ring / group in the compound represented by general formula (5). It should be understood that in step (ai), one or more NCA monomers (i.e., represented by general formula (5)) may be sequentially added to an initiator (e.g., an amide compound represented by general formula (4)) to form a block copolymer polypeptide.

[0272] In various embodiments, the deprotection of the protected lipid-b-poly(Lys) compound represented by general formula (6) is carried out in the presence of one or more acids. In various embodiments, the deprotection / deprotection step (a-ii) comprises subjecting the protected lipid-b-poly(Lys) compound to an acetic acid (AcOH) solution of one or more acidic compounds such as trifluoroacetic acid (TFA) and hydrobromic acid (HBr).

[0273] In various embodiments, the method includes the step of preparing a compound represented by general formula (1b) or (1b'), wherein the compound represented by general formula (1b) or (1b') is a compound in which m=1, n=1 to 100, l=0, and A (corresponding to R) 11 R 12 R 13 H is an optionally substituted alkyl group, or an optionally substituted alkenyl group or an optionally substituted alkynyl group, and B is R. 1 R 2 The general formula (1) for N- (i.e., lipid-b-poly(Lys)(LP)) is used, and the method includes steps (ai) and (a-ii) as described above.

[0274]

[0275]

[0276] In various implementations, the method further includes, prior to step (ai):

[0277] (aii) makes the protected amino acid (e.g., N) represented by general formula (7) 6 -Cbz-lysine) reacts with a carbonylating agent (e.g., triphosgene) to yield a protected amino acid (e.g., N-Cbz-lysine) containing NCA, represented by general formula (5). 6 -benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA):

[0278]

[0279] in

[0280] R 6 R 7 R 11 and PG 1 It contains one or more features and / or shares one or more properties, said one or more features and one or more properties being similar to those described above; and

[0281] R 16 H represents an alkyl group, an alkenyl group, or an alkynyl group, which may be optionally substituted.

[0282] In various implementation schemes, PG 1 It is N-carboxybenzyl or benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), or 9-fluorenylmethoxycarbonyl (Fmoc).

[0283] In various implementations, the method further includes, prior to step (ai):

[0284] (ai-ii) Reaction of an amine compound represented by general formula (8) (e.g., bistetradecylamine) with a protected amino acid compound represented by general formula (9) (e.g., Boc-glycine) in the presence of one or more coupling agents (e.g., 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HBTU)) and a base (e.g., N,N-diisopropylethylamine (DIPEA)) to obtain a carbamate compound represented by general formula (10) (e.g., (2-(bistetradecylamino)-2-oxoethyl)carbamate tert-butyl ester):

[0285]

[0286] in

[0287] R 1 To R 3 and R 5 It includes one or more features and / or shares one or more properties, which are similar to those described above;

[0288] R 14 and R 15 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; and

[0289] PG 2 Protecting groups, such as tert-butoxycarbonyl (Boc), N-carboxybenzyl or benzyloxycarbonyl (Cbz) or 9-fluorenylmethoxycarbonyl (Fmoc); and

[0290] (ai-iii) Deprotect the urethane compound of general formula (10) obtained by step (ai-ii) to obtain an amide compound of general formula (4) (e.g., 2-amino-N,N-bistetradecylacetamide):

[0291]

[0292] in

[0293] R 1 To R 3 R 5 R 10and PG 2 It includes one or more features and / or shares one or more properties, which are similar to those described above.

[0294] In various embodiments, the coupling agent in steps (ai-ii) may be HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate, hexafluorophosphate N,N-diisopropylethylamine (DIPEA) azabenzotriazol tetramethylureon (HATU) or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0295] In various embodiments, the base in steps (ai-ii) may be N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), hydroxybenzotriazole (HOBt), or 4-dimethylaminopyridine (DMAP).

[0296] In various embodiments, the deprotection of the carbamate compound represented by general formula (10) is carried out in the presence of one or more acids. In various embodiments, the deprotection / deprotection steps (ai-iii) include subjecting the carbamate compound to one or more acidic compounds such as trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, formic acid, or acetic acid.

[0297] In various embodiments, the method includes the step of preparing a compound represented by general formula (1c) or (1c'), wherein the compound represented by general formula (1c) or (1c') is a compound in which m=0, n=0, l=1, and A (corresponding to -CHR) 8 -CHR 9 OH) is defined as, for example, a derivative of an epoxy ring-opening product, and B is R. 1 R 2 The general formula (1) for N- (i.e., lipid-Lys-lipid (LPL1) with DP of 1), the method comprising:

[0298]

[0299] (b) Mixing an amine compound represented by general formula (8) (e.g., bistetradecylamine) with a protected amino acid compound represented by general formula (11) (e.g., N-acetylglucosamine). 2 N 6-bis(tert-butoxycarbonyl)lysine) is reacted in the presence of one or more coupling agents (e.g., 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate (HBTU)) and a base (e.g., N,N-diisopropylethylamine (DIPEA)) to give a dicarboxylate compound of general formula (12) (e.g., (6-(bistetradecylamino)-6-oxohexane-1,5-diyl)dicarboxylate ditert-butyl ester):

[0300]

[0301] in

[0302] R 1 To R 2 R 7 and R 14 It includes one or more features and / or shares one or more properties, which are similar to those described above;

[0303] R 17 To R 19 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; and

[0304] PG 4 and PG 5 Each can be an independent protecting group, such as tert-butoxycarbonyl (Boc), N-carboxybenzyl or benzyloxycarbonyl (Cbz) or 9-fluorenylmethoxycarbonyl (Fmoc);

[0305] (c) Deprotect the dicarbamate compound of general formula (12) obtained in step (b) to obtain an amide compound of general formula (13) (e.g., 2,6-diamino-N,N-bistetradecylhexamide):

[0306]

[0307] in

[0308] R 1 To R 2 R 7 R 18 To R 19 PG 4 and PG 5 It contains one or more features and / or shares one or more properties, said one or more features and one or more properties being similar to those described above; and

[0309] R 20 and R21 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; and

[0310] (d) Reacting an amide compound represented by general formula (13) with an epoxy (e.g., a 1,2-epoxyalkane) represented by general formula (3) to obtain a compound represented by general formula (1c) or (1c'):

[0311]

[0312]

[0313] in

[0314] R 1 To R 3 R 5 To R 9 and R 18 To R 21 It contains one or more features and / or shares one or more properties, said one or more features and one or more properties being similar to those described above; and

[0315] R 20 and R 21 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0316] In various embodiments, the coupling agent in step (b) may be HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium hexafluorophosphate, hexafluorophosphate N,N-diisopropylethylamine (DIPEA) azabenzotriazol tetramethylureon (HATU) or a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0317] In various embodiments, the base in step (b) may be N,N-diisopropylethylamine (DIPEA), triethylamine (TEA), hydroxybenzotriazole (HOBt), or 4-dimethylaminopyridine (DMAP).

[0318] In various embodiments, the deprotection of the dicarbamate compound represented by general formula (12) is carried out in the presence of one or more acids. In various embodiments, the deprotection / deprotection step (c) includes subjecting the carbamate compound to one or more acidic compounds such as trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, formic acid, or acetic acid.

[0319] In various embodiments, the method includes the step of preparing a compound represented by the general formula (1d) or (1d'), wherein the compound represented by the general formula (1d) or (1d') is a compound in which m=0, n=1 to 100, l=1, and A (corresponding to -CHR) 8 -CHR 9 OH) is defined as, for example, a derivative of an epoxy ring-opening product, and B is R. 23 The general formula (1) for O- (i.e., poly(Lys)-lipid derivative (PL)) is used, and the method includes:

[0320]

[0321]

[0322] (e) Reacting the poly(Lys) derivative represented by general formula (14) with an epoxy (e.g., 1,2-epoxyalkane) represented by general formula (3) to obtain a compound represented by general formula (1d) or (1d'):

[0323]

[0324] in

[0325] R 6 To R 9 and R 11 It contains one or more features and / or shares one or more properties, said one or more features and one or more properties being similar to those described above; and

[0326] R 22 To R 24 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0327] In various implementations, the method further includes, prior to step (e):

[0328] (ei) make a protected amino acid containing an amine group represented by general formula (15) (e.g., H-Lys(Boc)-Ome) and a protected amino acid containing an N-carboxylic anhydride (NCA) represented by general formula (5) (e.g., N-Lys(Boc)-Ome) 6 -benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA) is subjected to ring-opening polymerization (ROP) followed by deprotection of the resulting product to give the poly-(Lys) derivative represented by general formula (14):

[0329]

[0330] in

[0331] R6 R 7 R 11 R 22 To R 24 and PG 1 It contains one or more features and / or shares one or more properties, said one or more features and one or more properties being similar to those described above; and

[0332] PG 6 Protecting groups include, for example, tert-butoxycarbonyl (Boc), N-carboxybenzyl or benzyloxycarbonyl (Cbz) or 9-fluorenylmethoxycarbonyl (Fmoc).

[0333] In various embodiments, the deprotection of the ring-opening polymerization product in step (ei) is carried out in the presence of one or more acids. In various embodiments, the deprotection / deprotection step (a-ii) includes subjecting the resulting product to one or more acidic compounds such as trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, formic acid, or acetic acid.

[0334] In various embodiments, the method includes optionally ionizing one or more -NA2 groups to become positively charged groups, such as -NA2H. + Group.

[0335] In various embodiments, the reaction / polymerization / deprotection steps (a), (ai), (a-ii), (aii), (ai-ii), (ai-iii), (b), (c), (d), (e) and / or (ei) include one or more of the following steps: suspension, dispersion, mixing, stirring and / or dissolution.

[0336] In various embodiments, the reaction / polymerization / deprotection steps (a), (ai), (a-ii), (aii), (ai-ii), (ai-iii), (b), (c), (d), (e), and / or (ei) are carried out in the presence of an organic solvent. The organic solvent may be, for example, hexane, diethyl ether, methanol, ethanol, dichloromethane (DCM), tetrahydrofuran (THF), acetonitrile, chloroform, or ethyl acetate. In various embodiments, the organic solvent may be provided in a dry or anhydrous form.

[0337] In various embodiments, the reaction / polymerization / deprotection steps (a), (ai), (a-ii), (aii), (ai-ii), (ai-iii), (b), (c), (d), (e), and / or (ei) are carried out under vacuum or in an inert atmosphere. For example, the suspension, dispersion, mixing, stirring, and / or dissolution steps may be carried out in the presence of an inert gas such as argon or nitrogen.

[0338] In various embodiments, the reaction / polymerization / deprotection steps (a), (ai), (a-ii), (aii), (ai-ii), (ai-iii), (b), (c), (d), (e) and / or (ei) are carried out over a duration of about 1 hour to about 200 hours, about 10 hours to about 190 hours, about 20 hours to about 180 hours, about 30 hours to about 170 hours, about 40 hours to about 160 hours, about 50 hours to about 150 hours, about 60 hours to about 140 hours, about 70 hours to about 130 hours, about 80 hours to about 120 hours, about 90 hours to about 110 hours, or about 100 hours.

[0339] In various embodiments, the reaction / polymerization / deprotection steps (a), (ai), (a-ii), (aii), (ai-ii), (ai-iii), (b), (c), (d), (e) and / or (ei) are optionally carried out at temperatures of about -10°C to about 100°C, about -5°C to about 95°C, about 0°C to about 90°C, about 5°C to about 85°C, about 10°C to about 80°C, about 15°C to about 75°C, about 20°C to about 70°C, about 25°C to about 65°C, about 30°C to about 60°C, about 35°C to about 55°C, about 40°C to about 50°C, or about 45°C.

[0340] In various implementations, the method further includes:

[0341] (fi) The step of separating the compound represented by general formula (1a) or (1a') after step (a);

[0342] (f-ii) The step of separating the compound represented by general formula (1b) or (1b') after step (a-ii);

[0343] (f-iii) The step of separating the protected amino acid containing NCA represented by general formula (5) after step (aii);

[0344] (f-iv) The step of separating the amide compound represented by general formula (4) after step (ai-iii);

[0345] (fv) The step of separating the compound represented by general formula (1c) or (1c') in step (d);

[0346] (f-vi) The step of separating the compound represented by general formula (1d) or (1d') after step (e); and

[0347] (f-vii) Step (ei) is the step of separating the poly(Lys) derivative represented by general formula (14).

[0348] In various embodiments, the separation step includes one or more of the following steps: redissolution, purification, centrifugation, washing, precipitation, recrystallization, and / or dialysis.

[0349] In various implementations, the dialysis medium includes a solution miscible with water, such as ethanol, methanol, or deionized water.

[0350] In various embodiments, the washing medium includes an aqueous medium / solution, such as a salt solution, water, or acid. The salt solution may be a chloride salt solution, such as a saturated sodium chloride solution (salt water). The acid may be citric acid.

[0351] In various embodiments, the salt solution comprises a highly concentrated / saturated salt solution.

[0352] In various embodiments, the purification, centrifugation, recrystallization and / or washing steps are repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times or at least eight times with a washing medium.

[0353] In various embodiments, the method further includes one or more of the following post-reaction steps: optionally drying at low temperature (e.g., freeze-drying), drying under vacuum, or drying in an inert atmosphere.

[0354] In various embodiments, the drying step is carried out in the presence of a desiccant such as anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium sulfate, and anhydrous calcium chloride, or combinations thereof.

[0355] In various embodiments, the currently disclosed lipids can be synthesized in just 5 steps via an amino-epoxy reaction, while the synthesis of ALC-0315 requires 6 to 7 steps, which significantly reduces the manufacturing cost of the lipids.

[0356] Nanoparticle Composition

[0357] Advantageously, in various embodiments, the ionizable nature of the compound represented by general formula (1) (due to the presence of ionizable amine groups) allows for the aggregation and encapsulation / loading of molecules / loads into embodiments of the compound, thereby forming nanoparticles in the composition. In various embodiments, embodiments of the compound are capable of forming nanoparticles in the composition. In various embodiments, in the presence of a composition comprising molecules / loads (e.g., therapeutic agents, preventative agents, and / or biological agents), one or more amine groups in the compound (e.g., ionized / protonated amine groups in lipid compounds) aggregate and encapsulate / load molecules / loads into the compound to form nanoparticles (e.g., lipid nanoparticles (LNPs)) in the composition.

[0358] The term "nanoparticle" may include the terms "lipid nanoparticles," "encapsulated lipid nanoparticles," "loaded lipid nanoparticles," "LNP," etc., and / or may be used interchangeably with the terms "lipid nanoparticles," "encapsulated lipid nanoparticles," "loaded lipid nanoparticles," "LNP," etc.

[0359] A nanoparticle composition is provided, comprising:

[0360] (i) the compounds disclosed herein (e.g., those represented by general formula (1)); and

[0361] (ii) therapeutic agents and / or preventive agents and / or biological agents encapsulated / loaded / coupled / linked / bound in / on the compounds disclosed herein (e.g., represented by general formula (1)).

[0362] In various embodiments, the compounds disclosed herein (e.g., represented by general formula (1)) are capable of ionization (e.g., protonation) in a low pH range of 3.0 to 6.5, such that the composition encapsulates therapeutic agents and / or preventative agents and / or biological agents coupled / bonded / linked / bound to the composition / nanoparticles. In various embodiments, the compounds represented by general formula (1) are capable of ionization (e.g., protonation) in a low pH range, such that the composition encapsulates therapeutic agents and / or preventative agents and / or biological agents coupled / bonded / linked / bound to the composition / nanoparticles. The therapeutic agents and / or preventative agents and / or biological agents may be coupled / bonded / linked / bound to the composition / nanoparticles via electrostatic interactions and / or other physical interactions. In various embodiments, the therapeutic agents and / or preventative agents and / or biological agents are electrostatically and / or physically coupled / bonded / linked / bound to the composition / nanoparticles.

[0363] Advantageously, the composition is suitable for encapsulating, delivering and / or transfecting one or more therapeutic, prophylactic and / or biological agents, for example, to a desired target (e.g., a subject, cell, cytosol, tissue or organ).

[0364] In various embodiments, the composition further comprises:

[0365] (i) Neutral / helper lipids;

[0366] (ii) cholesterol or its derivatives; and

[0367] (iii) Polyethylene glycol (PEG) lipid conjugates, or other amphiphilic lipids.

[0368] The term “polyethylene glycol (PEG) modified lipids” may include the terms “polyethylene glycolated lipids” and “lipids modified with PEG” and / or may be used interchangeably with the terms “polyethylene glycolated lipids” and “lipids modified with PEG”.

[0369] In various embodiments, the compounds disclosed herein (e.g., represented by general formula (1)), neutral / auxiliary lipids, cholesterol or derivatives thereof, and PEG-lipid conjugates are mixed / dissolved in an organic solvent. In various embodiments, any organic solvent can be used as a medium effectively containing the components (e.g., reactants / substrates) of the reaction mixture disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the mixture. In various embodiments, the organic solvent includes ethanol, methanol, isopropanol, acetonitrile, ethyl acetate, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and combinations thereof.

[0370] In various embodiments, the compounds disclosed herein (e.g., represented by general formula (1)), neutral / auxiliary lipids, cholesterol or derivatives thereof, and PEG-lipid conjugates are mixed in a molar ratio of about 30 to 50: about 5 to 50: about 5 to 60: about 1 to 5.

[0371] In various embodiments, the compounds disclosed herein (e.g., represented by general formula (1)), neutral / auxiliary lipids, cholesterol or derivatives thereof, and PEG-lipid conjugates are mixed in a molar ratio of 46:9.4:42:1.6.

[0372] In various embodiments, the compounds disclosed herein (e.g., represented by general formula (1)), neutral / auxiliary lipids, cholesterol or derivatives thereof, and PEG-lipid conjugates are mixed in a molar ratio of 46:42:9.4:1.6.

[0373] In various embodiments, the compounds disclosed herein (e.g., represented by general formula (1)), neutral / auxiliary lipids, cholesterol or derivatives thereof, and PEG-lipid conjugates are mixed in a molar ratio of 30:12.3:55.7:2.

[0374] In various embodiments, the compounds disclosed herein (e.g., represented by general formula (1)), neutral / auxiliary lipids, cholesterol or derivatives thereof, and PEG-lipid conjugates are mixed in a molar ratio of 46.3:9.4:42.7:1.6.

[0375] In various embodiments, the neutral / auxiliary lipid comprises phospholipids such as unsaturated lipids. Examples of phospholipids include, but are not limited to, 1,2-distearate-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-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), and 1-palmitoyl-2-oleoyl-sn 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 hemolytic PC), 1,2-dilinanoyl-sn-glycero-3-phosphocholine, 1,2-disarachidonicyl-sn-glycero-3-phosphocholine, 1,2-bis(docohexanoyl)-sn-glycero-3-phosphocholine, 1,2-di-phytanoyl-sn-glycero-3-phosphoethanolamine (ME) 16.0 PE), 1,2-distearyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycero-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycero-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphate racemic (1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

[0376] In various embodiments, the cholesterol or its derivatives are selected from cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, alfalfa sterol, and combinations thereof.

[0377] In various embodiments, the polyethylene glycol (PEG)-lipid conjugate or amphiphilic lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(PEG)-2000]-N,N-bistetradecylacetamide (ALC-0159), R-3-[(ω-methoxy-poly(ethylene glycol)2000)aminomethyl... [Acyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DOMG), 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-methoxy](sodium salt)), PEG-DPPC, PEG-DSPE lipids and combinations thereof.

[0378] In various embodiments, the therapeutic agent, preventative agent, and / or biological agent is provided in an aqueous buffer solution. The aqueous buffer solution may be sodium acetate.

[0379] In various embodiments, the nanoparticle composition comprises nanoparticles formed from a compound represented by general formula (1) or its ionized form.

[0380] Nanoparticles

[0381] Nanoparticles (e.g., lipid nanoparticles) are provided, comprising:

[0382] (i) the compounds disclosed herein (e.g., those represented by general formula (1)); and

[0383] (ii) Therapeutic agents and / or preventive agents and / or biological agents that are encapsulated / loaded / coupled / bonded / linked / integrated in / to a compound disclosed herein (e.g., represented by general formula (1)).

[0384] In various embodiments, the nanoparticles have an N:P or N / P ratio (i.e., the molar ratio of ionizable (within the physiological pH range) nitrogen atoms in the nanoparticles / compound to phosphate groups in the therapeutic and / or preventative and / or biological agents (e.g., nucleic acids)) of about 1:1 to about 50:1. The N:P or N / P ratio of the nanoparticles may be about 1:1 to about 50:1, about 2:1, about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, or about 45:1.

[0385] It should be understood that, in various embodiments, the optimal N / P ratio depends on the type of therapeutic and / or prophylactic and / or biological agent (e.g., nucleic acids such as mRNA, siRNA, miRNA, pDNA, DNA, and oligonucleotides). For example, the optimal N / P ratio may differ for siRNA, mRNA, DNA, and oligonucleotides. The optimal N / P ratio may also differ for siRNA, pDNA, and oligonucleotides. In various embodiments, it should be understood that shorter nucleic acid therapeutics (e.g., siRNA) or prophylactics (e.g., mRNA) require more (i.e., larger amounts / concentrations / volumes) of ionizable lipids to encapsulate them into lipid nanoparticles. Therefore, in various embodiments, N / P ratios up to about 20:1 are used for encapsulating and delivering nucleic acid therapeutics (e.g., shorter nucleic acid therapeutic siRNA).

[0386] In various embodiments, the encapsulation / loading / binding efficiency / capacity of the therapeutic agent and / or prophylactic agent and / or biological agent in the composition / nanoparticles is at least about 1.0%, at least about 5.0%, at least about 10.0%, at least about 15.0%, at least about 20.0%, at least about 25.0%, at least about 30.0%, at least about 35.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, or at least about 99.0%. In various embodiments, the composition / nanoparticles can facilitate the successful encapsulation of siRNA and mRNA into lipid nanoparticles, resulting in high transfection efficiency of both siRNA and mRNA.

[0387] In various embodiments, the nucleic acid transfection efficiency of the therapeutic and / or prophylactic and / or biological agents in the composition / nanoparticles is greater than or equivalent to that of those using other ionizable lipids (e.g., ALC-0315). Advantageously, in various embodiments, the amount of amino groups in the lipids of this disclosure required for effective siRNA transfection is half the amount of amino groups in ALC-0315 LNP (Pfizer-Biotech mRNA vaccine formulation), indicating the lower cost and better biocompatibility of the LNPs of this disclosure.

[0388] In various embodiments, the nanoparticles have nucleic acid transfection efficiencies that are lower, not lower, or higher than those of corresponding nanoparticles using ALC-0315 ionizable lipids under similar conditions. For example, the mRNA transfection efficiency can be 0 times, at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least about 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, or at least 50 times higher than that of corresponding nanoparticles using ALC-0315 ionizable lipids under similar conditions.

[0389] It should be understood that, in various implementation schemes, the nanoparticles can still induce therapeutic and / or vaccination effects even when their transfection efficiency is lower than that of the ionizable lipids of ALC-0315.

[0390] In various embodiments, the average particle size (or diameter) of the nanoparticles is about 20 nm to about 650 nm, about 30 nm to about 640 nm, about 40 nm to about 630 nm, about 50 nm to about 620 nm, about 60 nm to about 610 nm, about 70 nm to about 600 nm, about 80 nm to about 590 nm, about 90 nm to about 580 nm, about 100 nm to about 570 nm, about 110 nm to about 560 nm, about 120 nm to about 550 nm, about 130 nm to about 540 nm, about 140 nm to about 530 nm, about 150 nm to about 520 nm, about 160 nm to about 510 nm, about 170 nm to about 500 nm, about 180 nm to about 490 nm, about 190 nm to about 480 nm, about 200 nm to about 470 nm, and about 210 nm to about 460 nm. The nanometer diameters are approximately 220 nm to approximately 450 nm, approximately 230 nm to approximately 440 nm, approximately 240 nm to approximately 430 nm, approximately 250 nm to approximately 420 nm, approximately 260 nm to approximately 410 nm, approximately 270 nm to approximately 400 nm, approximately 280 nm to approximately 390 nm, approximately 290 nm to approximately 380 nm, approximately 300 nm to approximately 370 nm, approximately 310 nm to approximately 360 nm, approximately 320 nm to approximately 350 nm, approximately 330 nm to approximately 340 nm, or approximately 335 nm. In various embodiments, the siRNA LNP may comprise nanoparticles with an average particle size of 70 nm and a neutral charge, while the mRNA LNP may comprise nanoparticles with an average particle size of 100 nm and a neutral charge.

[0391] In various embodiments, the polydispersity index (PDI) of the composition comprising nanoparticles is about 0.020 to about 0.700, about 0.030 to about 0.690, about 0.040 to about 0.680, about 0.050 to about 0.670, about 0.060 to about 0.660, about 0.070 to about 0.650, about 0.080 to about 0.640, about 0.090 to about 0.630, about 0.100 to about 0.620, about 0.110 to about 0.610, about 0.120 to about 0.600, about 0.130 to about 0.590, about 0.140 to about 0.580 nm, about 0.150 to about 0.570 nm, about 0.160 to about 0.560 nm, about 0.170 to about 0.550 nm. Approximately 0.180 to approximately 0.540 nm, approximately 0.190 to approximately 0.530 nm, approximately 0.200 to approximately 0.520 nm, approximately 0.210 to approximately 0.510 nm, approximately 0.220 to approximately 0.500 nm, approximately 0.230 to approximately 0.490 nm, approximately 0.240 to approximately 0.480 nm, approximately 0.250 to approximately 0.470 nm, approximately 0.260 to approximately 0.460 nm, approximately 0.270 to approximately 0.450 nm, approximately 0.280 to approximately 0.440 nm, approximately 0.290 to approximately 0.430 nm, approximately 0.300 to approximately 0.420 nm, approximately 0.310 to approximately 0.410 nm, approximately 0.320 to approximately 0.400 nm, approximately 0.330 to approximately 0.390 nm. The nanoparticles are about 0.340 to about 0.380 nm, about 0.350 to about 0.370 nm, or about 0.360 nm. In various embodiments, the nanoparticles have a narrow particle size distribution (PDI < 0.3), and / or the nanoparticle composition is relatively / substantially uniformly distributed and has a size acceptable for in vivo application.

[0392] In various embodiments, the nanoparticles exhibit a zeta potential of approximately -15.0 mV to approximately +20.0 mV in saline solution (e.g., phosphate-buffered saline (PBS)) or in a physiological environment, approximately -14.0 mV to approximately +19.0 mV, approximately -13.0 mV to approximately +18.0 mV, approximately -12.0 mV to approximately +17.0 mV, approximately -11.0 mV to approximately +16.0 mV, approximately -10.0 mV to approximately +15.0 mV, approximately -9.0 mV to approximately +14.0 mV, approximately -8.0 mV to approximately +13.0 mV, approximately -7.0 mV to approximately +12.0 mV, approximately -6.0 mV to approximately +11.0 mV, approximately -5.0 mV to approximately +10.0 mV, and approximately -4.0 mV to approximately +9.0 mV. Approximately -3.0 mV to approximately +8.0 mV, approximately -2.0 mV to approximately +7.0 mV, approximately -1.0 mV to approximately +6.0 mV, approximately 0 mV to approximately +5.0 mV, approximately +1.0 mV to approximately +4.0 mV, or approximately +2.0 mV to approximately +3.0 mV. Advantageously, in various embodiments, the nanoparticles have a substantially neutral surface charge, making the nanoparticles suitable / desirable for in vivo applications.

[0393] In various embodiments, the composition / compound / nanoparticle is biocompatible, meaning it is partially compatible with a biological system and, when used in humans or animals, substantially or significantly does not induce adverse physiological reactions, such as toxic reactions (e.g., cytotoxicity), immune reactions, damage, etc. In various embodiments, the composition / compound / nanoparticle is substantially free of substances that would induce adverse physiological reactions. Advantageously, the nanoparticles (e.g., lipid nanoparticles) are capable of effectively binding therapeutic agents, prophylactic agents, and / or biological agents (e.g., RNA) and / or providing high transfection efficiency without causing / inducing substantial or any cytotoxicity.

[0394] Methods for preparing nanoparticles

[0395] A method for preparing the nanoparticles disclosed herein is provided, the method comprising:

[0396] (g) Preparing an aqueous composition comprising a therapeutic agent and / or a preventive agent and / or a biological agent;

[0397] (h) The aqueous composition is mixed with the composition as described above to obtain nanoparticles.

[0398] In various embodiments, step (g) includes mixing the therapeutic agent and / or prophylactic agent and / or biological agent in an aqueous buffer solution. The aqueous buffer solution may be a sodium acetate buffer solution, a citrate buffer solution, a phosphate buffer solution, a glycine buffer solution, or a combination thereof.

[0399] In various embodiments, the mixing step (g) is measured at approximately 2.00 to approximately 6.00, approximately 2.10 to approximately 5.90, approximately 2.20 to approximately 5.80, approximately 2.30 to approximately 5.70, approximately 2.40 to approximately 5.60, approximately 2.50 to approximately 5.50, approximately 2.60 to approximately 5.40, approximately 2.70 to approximately 5.30, approximately 2.80 to approximately 5.20, approximately 2.90 to approximately 5.10, approximately 3.00 to approximately 5.00, approximately 3.10 to approximately 4.90, approximately 3.20 to approximately 4.80, approximately 3.30 to approximately 4.70, approximately 3.40 to approximately 4.60, approximately 3.50 to approximately 4.50, approximately 3.60 to approximately 4.40, approximately 3.70 to approximately 4.30, and approximately 3.80 to approximately 4.20. The experiment was conducted at a pH of approximately 3.90 to approximately 4.10 or approximately 4.00.

[0400] In various embodiments, step (h) includes mixing the aqueous composition with the composition of any one of the preceding AS at a volume ratio of about 6:1 to about 1:1, about 5:1 to about 1:1, about 4:1 to about 1:1, about 3:1 to about 1:1 or about 2:1 to about 1:1.

[0401] In various embodiments, the step (h) of mixing the aqueous composition with the composition includes using a microfluidic device or pulsed eddy current. For example, a microfluidic device can be used to perform microfluidic mixing. The micromixing can be performed by passive mixing using a passive micromixer, such as a T-shaped or Y-shaped microfluidic mixer, parallel lamination, sequential, focused enhancement mixer, or droplet micromixer. Micromixing can also be performed by active mixing using external forces such as pressure fields, electrokinetics, dielectrophoresis, electrowetting, magnetohydrodynamics, or ultrasound. Advantageously, since microfluidic mixing involves mixing two compositions (i.e., an 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 ionizable lipids and therapeutic agents, preventative agents, and / or biological agents) is modulated, thereby producing nanoparticles with smaller particle size and / or narrow particle size distribution or uniformity (e.g., smaller PDI).

[0402] In various embodiments, the yield of the compound represented by general formula (1) is about 1% to about 100%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, about 40% to about 60% or about 50%.

[0403] In various embodiments, the method further includes removing an organic phase (e.g., ethanol). For example, removing the organic phase may include dialysis of the nanoparticles to remove any residual organic solvents present. Advantageously, removing the organic phase by dialysis can improve the encapsulation efficiency of therapeutic and / or preventative and / or biological agents.

[0404] In various embodiments, carriers, nanocarriers, or delivery systems / media comprising the compositions / compounds / nanoparticles disclosed herein are also provided.

[0405] In various embodiments, vaccine compositions comprising the compositions / compounds / nanoparticles disclosed herein are also provided.

[0406] In various embodiments, carriers, nanocarriers, delivery systems / media, compounds or ionized forms thereof, nanoparticle compositions, nanoparticles (or lipid nanoparticles) disclosed herein are also provided for use in medicine (e.g., for the treatment or prevention of one or more diseases, disorders or conditions mentioned herein).

[0407] In various embodiments, carriers, nanocarriers, delivery systems / media, compounds or ionized forms thereof, nanoparticle compositions, nanoparticles (or lipid nanoparticles) disclosed herein for treating or preventing diseases, disorders, or conditions are also provided. The use of said carriers, nanocarriers, delivery systems / media, compounds or ionized forms thereof, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) in the manufacture of medicaments for treating or preventing diseases, disorders, or conditions and / or in methods of treating or preventing diseases, disorders, or conditions, said methods comprising dispensing said carriers, nanocarriers, delivery systems / media, compounds or ionized forms thereof, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) into the manufacture of medicaments for treating or preventing diseases, disorders, or conditions, and in ..., or in methods comprising dispensing said carriers, nanocarriers, delivery systems / media, compounds or ionized forms thereof, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) into the manufacture of medicaments, The procedure involves administering (e.g., in a therapeutically effective dose) granules to a subject in need (e.g., a vertebrate, such as a human, or a large veterinary mammal, such as a horse, cattle, deer, sheep, llama, goat, pig). The disease, disorder, or symptom may be selected from infectious / contagious diseases, viral infections (i.e., diseases caused by viruses), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), respiratory diseases, etc., or combinations thereof. In various embodiments, the disease, disorder, or symptom is mediated by an influenza virus (e.g., influenza A, B, C, and / or D viruses). For example, the disease may be influenza A, B, C, or D, such as H1N1, H3N2. In various embodiments, the disease, disorder, or symptom is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronavirus, such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, disorder, or symptom may be SARS-CoV-2 coronavirus disease.

[0408] In various embodiments, the carriers, nanocarriers, delivery systems / media, compounds or their ionized forms, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) disclosed herein are also provided for encapsulating and / or delivering therapeutic agents, prophylactic agents, and / or biological agents to subjects, cells, cytosols, tissues, or organs (e.g., mammalian cells, cytosols, tissues, or organs), and the use of said carriers, nanocarriers, delivery systems / media, compounds or their ionized forms, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) in the preparation of medicaments for encapsulating and / or delivering therapeutic agents, prophylactic agents, and / or biological agents to subjects, cells, cells, cytosols, tissues, or organs, for delivery to subjects, cells, cytosols, tissues, or organs, and the use of said carriers, nanocarriers, delivery systems / media, compounds or their ionized forms, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) in the preparation of medicaments for encapsulating and / or delivering therapeutic agents, prophylactic agents, and / or biological agents to subjects, cells, cytosols, tissues, or organs, for delivery to subjects, cells, cytosols, tissues, or organs, and the use of said carriers, nanocarriers, delivery systems / media, compounds or their ionized forms, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) in the preparation of medicaments for encapsulating and / or delivering therapeutic agents, prophylactic agents, and / or biological agents ... Methods for delivering prophylactic and / or biological agents to a cytosol, tissue, or organ (e.g., mammalian cells, cytosol, tissue, or organ), and / or for delivering therapeutic, prophylactic, and / or biological agents to a subject, cell, cytosol, tissue, or organ (e.g., mammalian cells, cytosol, tissue, or organ), the methods comprising the steps of administering (e.g., in a therapeutically effective amount) the carrier, nanocarrier, delivery system / mediator, compound or ionized form thereof, nanoparticle composition, nanoparticle (or lipid nanoparticle) to the subject (e.g., a vertebrate such as a human or a large veterinary mammal (e.g., a horse, cattle, deer, sheep, llama, goat, pig).

[0409] In various embodiments, the carriers, nanocarriers, delivery systems / mediators, compounds or ionized forms thereof, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) disclosed herein are also provided for inducing an immune response in a subject (e.g., a vertebrate, such as a human or a large veterinary mammal (e.g., a horse, cattle, deer, sheep, llama, goat, pig)); the use of said carriers, nanocarriers, delivery systems / mediators, compounds or ionized forms thereof, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) in the manufacture of a medicament for inducing an immune response in a subject; and / or methods for inducing an immune response in a subject, said methods comprising the step of administering (e.g., in a therapeutically effective amount) said carriers, nanocarriers, delivery systems / mediators, compounds or ionized forms thereof, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) to a subject in need. In various embodiments, an immune response in a subject will be induced by administering said compound or ionized form thereof, nanoparticle composition, and nanoparticle (or lipid nanoparticle) to the subject. In various implementations, an immune response is induced in the subject to protect them from various diseases, disorders, or conditions, such as infectious / contagious diseases, viral infections (i.e., diseases caused by viruses), bacterial infections (i.e., diseases caused by bacteria), fungal infections (i.e., diseases caused by fungi), respiratory diseases, etc., or combinations thereof as mentioned herein. The carrier, nanocarrier, delivery system / medium, compound, or ionized form thereof, nanoparticle composition, or nanoparticles may be delivered to the subject in the form of a vaccine or as a component of a vaccine.

[0410] In various implementations, 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 could be SARS-CoV-2 coronavirus disease.

[0411] In various embodiments, carriers, nanocarriers, delivery systems / media, compounds or their ionized forms, nanoparticle compositions, and nanoparticles prepared by embodiments of the methods disclosed herein comprise one or more of the following features or properties: broad applicability (e.g., usability for encapsulating, delivering and / or transfecting a wide range of therapeutic, preventative and / or biological agents), nano-sized, substantially neutral surface charge, high encapsulation efficiency, high transfection efficiency, high stability, low toxicity (e.g., low cytotoxicity), and low production / synthesis costs, thus making them suitable for in vivo applications requiring efficient cellular uptake and / or gene transfection. Brief description of the attached diagram

[0413] Figure 1 The N synthesized according to various embodiments disclosed herein is shown. 6-benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA) 1 1H NMR spectra, where NMR analysis was performed in deuterated dimethyl sulfoxide (DMSO-d6) as a solvent.

[0414] Figure 2 The N synthesized according to various embodiments disclosed herein is shown. 6 -benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA) 13 C10 NMR spectra, where NMR analysis was performed in deuterated dimethyl sulfoxide (DMSO-d6) as a solvent.

[0415] Figure 3 The 2-amino- synthesized according to various embodiments disclosed herein are shown. N,N -Ditetradecylacetamide 1 1H NMR spectrum, in which NMR analysis was performed in deuterated chloroform (CDCl3) as a solvent.

[0416] Figure 4 This document illustrates lipid-block-poly(lysine) (lipid-b-poly(Lys)) (LP) with a degree of polymerization (DP) of 5 synthesized according to various embodiments disclosed herein. 1 1H NMR spectroscopy, wherein the NMR analysis is performed in heavy water (D2O) as a solvent.

[0417] Figure 5 This document illustrates lipid-block-poly(lysine) (lipid-b-poly(Lys)) (LP) with a degree of polymerization (DP) of 8 synthesized according to various embodiments disclosed herein. 1 1H NMR spectroscopy, wherein the NMR analysis is performed in heavy water (D2O) as a solvent.

[0418] Figure 6 This document illustrates lipid-block-poly(lysine) (lipid-b-poly(Lys)) (LP) with a degree of polymerization (DP) of 10 synthesized according to various embodiments disclosed herein. 1 1H NMR spectroscopy, wherein the NMR analysis is performed in heavy water (D2O) as a solvent.

[0419] Figure 7 This document illustrates lipid-block-poly(lysine) (lipid-b-poly(Lys)) (LP) synthesized according to various embodiments disclosed herein, with a degree of polymerization (DP) of 15. 1 1H NMR spectra, where NMR analysis was performed in deuterated dimethyl sulfoxide (DMSO-d6) as a solvent.

[0420] Figure 8This document illustrates lipid-block-poly(lysine) (lipid-b-poly(Lys)) (LP) synthesized according to various embodiments disclosed herein, with a degree of polymerization (DP) of 15. 1 1H NMR spectra, where NMR analysis was performed in deuterated dimethyl sulfoxide (DMSO-d6) as a solvent.

[0421] Figure 8 This document illustrates lipid-block-ionizable poly(lysine)-lipid (lipid-b-ionizable poly(Lys)-lipid) (LPL3) with a degree of polymerization (DP) of 5 synthesized according to various embodiments disclosed herein. 1 1H NMR spectrum, in which NMR analysis was performed in deuterated chloroform (CDCl3) as a solvent.

[0422] Figure 9 This document illustrates a lipid-block-ionizable poly(lysine)-lipid (lipid-b-ionizable poly(Lys)-lipid) (LPL4) with a degree of polymerization (DP) of 8 synthesized according to various embodiments disclosed herein. 1 1H NMR spectrum, in which NMR analysis was performed in deuterated chloroform (CDCl3) as a solvent.

[0423] Figure 10 This document illustrates a lipid-block-ionizable poly(lysine)-lipid (lipid-b-ionizable poly(Lys)-lipid) (LPL5) with a degree of polymerization (DP) of 10 synthesized according to various embodiments disclosed herein. 1 1H NMR spectrum, in which NMR analysis was performed in deuterated chloroform (CDCl3) as a solvent.

[0424] Figure 11 This document illustrates a lipid-block-ionizable poly(lysine)-lipid (lipid-b-ionizable poly(Lys)-lipid) (LPL6) with a degree of polymerization (DP) of 15 synthesized according to various embodiments disclosed herein. 1 1H NMR spectrum, in which NMR analysis was performed in deuterated chloroform (CDCl3) as a solvent.

[0425] Figure 12 This illustrates a lipid-block-ionizable poly(lysine)-lipid (lipid-b-ionizable poly(Lys)-lipid) (LPL2) with a degree of polymerization (DP) of 15, derived from 1,2-epoxypropane and synthesized according to various embodiments disclosed herein. 1 1H NMR spectra, where NMR analysis was performed in deuterated dimethyl sulfoxide (DMSO-d6) as a solvent.

[0426] Figure 13The following are examples of lipid-lysine-lipid (lipid-Lys-lipid) (LPL1) synthesized according to various embodiments disclosed herein. 1 1H NMR spectrum, in which NMR analysis was performed in deuterated chloroform (CDCl3) as a solvent.

[0427] Figure 14 The following are examples of ionizable poly(lysine)-lipids (ionizable poly(Lys)-lipids) synthesized according to various embodiments disclosed herein. 1 1H NMR spectrum, in which NMR analysis was performed in deuterated chloroform (CDCl3) as a solvent.

[0428] Figure 15 The following are examples of preparations made according to various embodiments disclosed herein, each with a concentration of 3 ( Figure 15 A) and 6 ( Figure 15 B) N / P ratio size distribution of LDL4-loaded GFP-siRNA-containing lipid nanoparticles (siRNA-LNP), compared with ALC-0315 ( Figure 15 C) Compare the size distribution of LNPs.

[0429] Figure 16 The efficiency of GFP knockdown in HepG2 cells after incubation for 72 hours with various siRNA-LNP formulations synthesized according to the various embodiments disclosed herein is shown. Figure 16 A) and vitality ( Figure 16 B). Lipofectamine 3000, a commercially available transfection agent, was used as a positive control.

[0430] Figure 17 The efficiency of GFP knockdown in HepG2 cells after incubation for 72 hours with various siRNA-LNP formulations having different lipid tail lengths at N / P ratios of 3 and 6 is demonstrated. These formulations contain LPL3, LPL4, LPL5, or LPL6 synthesized according to various embodiments disclosed herein.

[0431] Figure 18 The efficiency of GFP knockdown in HepG2 cells after incubation for 72 hours with various siRNA-LNP formulations having different lipid tail lengths at N / P ratios of 3 and 6 is demonstrated. These formulations contain LPL2 or LPL6 synthesized according to various embodiments disclosed herein.

[0432] Figure 19 The efficiency of GFP knockdown in HepG2 cells after incubation for 72 hours with various siRNA-LNP formulations at N / P ratios of 3 and 6 is demonstrated. These formulations contain PL or LPL6 synthesized according to various embodiments disclosed herein.

[0433] Figure 20The results show the GFP knockdown efficiency of HepG2 cells after incubation with siRNA-LNP containing LDL4 at N / P ratios of 3 and 6 for 72 hours, compared to ALC-0315LNP prepared according to the various embodiments disclosed herein. Figure 20 A) and vitality ( Figure 20 B). Cell viability of treated wells is expressed as a percentage relative to negative control wells that did not receive any treatment.

[0434] Figure 21 Fluorescence microscopy and bright-field images of HepG2 cells expressing eGFP transfected with siRNA-LNP containing LDL4 at N / P ratios of 3 and 6, respectively, are shown, compared to ALC-0315LNP prepared according to the various embodiments disclosed herein. Images of untreated HepG2 cells expressing eGFP are included as controls. The top row shows the fluorescence images, while the bottom row shows the bright-field images.

[0435] Figure 22 The transfection efficiency of HEK-293T cells, indicated by relative luciferase expression, is shown after 48 hours of transfection or incubation with LNP formulations loaded with various FLuc mRNAs prepared according to the various embodiments disclosed herein. Figure 22 (A) and Viability 22 (B)). Commercial transfection agent Lipofectamine 3000 was used as a positive control, and untreated HEK-293T cells were used as a negative control. Statistical significance was calculated using one-way ANOVA (analysis of variance), and data are presented as mean ± SD (ns: no significant difference). P < 0.05, P < 0.01, P < 0.001, P < 0.0001).

[0436] Example

[0437] Exemplary embodiments of this disclosure will be better understood and readily conceived by those skilled in the art through the following examples, tables, and, where applicable, the accompanying drawings. It should be understood that other modifications relating to structural, biological, and / or chemical changes may be made without departing from the scope of the invention. The exemplary embodiments are not necessarily mutually exclusive, as some may be combined with one or more embodiments to form new exemplary embodiments. The exemplary embodiments should not be construed as limiting the scope of this disclosure.

[0438] Those skilled in the art will understand that other changes and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of this disclosure as broadly described. For example, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included, etc., across different exemplary embodiments in the description herein. Therefore, the embodiments herein are to be considered illustrative rather than restrictive in all respects.

[0439] Example 1: Materials and Methods

[0440] 1.1. Materials

[0441] All chemicals were purchased from Sigma-Aldrich, Alfa Chemistry, GL Biochem (Shanghai), and Tokyo Chemical Industry (Singapore) and used as is unless otherwise specified. Solvents purchased from VWR or JT Baker were of HPLC or analytical grade and used as is. Lipids ALC-0315, 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and ALC-0159 used to prepare LNP as controls were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Fetal bovine serum (FBS) was purchased from Corning (USA), and 0.9% w / v saline (pH 7.4) was purchased from Braun (Singapore). AlamarBlue cell viability reagent was purchased from ThermoFisher Scientific, Singapore. Phosphate-buffered saline (PBS, 10×) was purchased from 1st BASE and diluted to 1×PBS before use. pmaxGFP siRNA VSC-1001 was purchased from Lonza as a lyophilized powder and prepared according to the manufacturer's instructions. Lipofectamine RNAi Max and Lipofectamine 3000 were purchased from Invitrogen and Thermofisher.

[0442] 1.2. N 6 Synthesis of β-benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA)

[0443] The synthesis of Lys-NCA is shown in Scheme 1. The general synthesis method for Lys-NCA: N... 6-Benzyloxycarbonyl-lysine (7.0 g, 25 mmol) was suspended in 100 mL of dry tetrahydrofuran (THF), and then triphosgene (3.2 g) was added under N2. The mixture was stirred at 70 °C under N2 for 3 h. After cooling the reaction mixture to room temperature, the crude product was precipitated by pouring the mixture into ice-cold hexane (1000 mL), and collected by filtration. The resulting crude product was purified by recrystallization three times with a THF / hexane mixture. The yield of Lys-NCA was 78%. The structure of Lys-NCA is given by... 1 H NMR and 13 C NMR spectroscopy ( Figure 1 and 2 This has been confirmed.

[0444]

[0445] Option 1. N 6 Synthesis of β-benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA)

[0446] 1.3. 2-Amino- N,N - Lipid synthesis of bis(tetradecylacetamide)

[0447] 2-Amino- N,N The synthesis of the lipid from bis(tetradecyl)acetamide is shown in Scheme 2. First, tert-butyl (2-(bis(tetradecylamino)-2-oxoethyl)carbamate was synthesized. Boc-glycine (876 mg, 5.0 mmol) and bis(tetradecylamino) (2.05 g, 5.0 mmol) were dissolved in dry dichloromethane (DCM) (100 mL). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureonium hexafluorophosphate (HBTU) (2.38 g, 6.25 mmol) and... N -Ethyl- N -(1-Methylethyl)-2-propylamine (DIPEA) (1.62 g, 12.5 mmol). The reaction mixture was stirred at room temperature under N2 for 24 h. The mixture was diluted with 100 mL of DCM and washed with 7% citric acid, brine, and H2O. The resulting organic layer was collected and dried over anhydrous Na2SO4. The DCM was evaporated under vacuum to produce the crude product. The crude product was purified by rapid silica gel column chromatography (hexane:ethyl ether 8:2, v / v) to give the final product as a yellow oil. The yield of the compound was 87%. Then, 2-amino- N,N- Ditetradecylacetamide. (2-(ditetradecylamino)-2-oxoethyl)carbamate tert-butyl ester was dissolved in 15 mL of anhydrous DCM, and 12 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred under N2 atmosphere for 2 h. The solvent was evaporated under vacuum to give the crude product. The resulting crude product was dissolved in 40 mL of DCM, and then 40 mL of NaHCO3 aqueous solution (10%) was added. The mixture was stirred under N2 atmosphere for 12 h. The organic layer was collected and dried over anhydrous Na2SO4. DCM was evaporated under vacuum. The resulting product was dried under vacuum to give a white powder. The yield of the compound was 92%. The structure of the product is determined by... 1 H NMR spectrum ( Figure 3 This has been confirmed.

[0448]

[0449] Option 2. 2-Amino- N,N - Lipid synthesis of bis(tetradecylacetamide).

[0450] 1.4. Synthesis of lipid-block-ionizable polylysine (lipid-b-poly(Lys)) (LP) and lipid-block-ionizable polylysine lipid (lipid-b-poly(Lys)-lipid) (LPL)

[0451] The synthesis of lipid-block-ionizable polylysine and lipid-block-ionizable polylysine lipids is shown in Scheme 3. First, lipid-b-poly(N... 6 -benzyloxycarbonyl-L-Lys). Lipid-b-poly(N 6 A general synthetic method for 2-amino-N,N-bistetradecylacetamide (46.6 mg, 0.1 mmol) and Lys-NCA was dissolved in 40 mL of anhydrous DMF in a glove box. The mixture was stirred in a glove box at room temperature for 24 h. The crude product was precipitated by centrifugation by pouring the mixture solution into ice-cold diethyl ether (300 mL). The crude product was purified by dissolving in MeOH and precipitating by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum. Lipid-b-poly(N... 6 The number of polymeric units of (-benzyloxycarbonyl-Lys) can be adjusted by changing the amount of Lys-NCA.

[0452]

[0453] Scheme 3. Synthesis of lipid-block-ionizable polylysine (lipid-b-poly(Lys)) (LP) and lipid-block-ionizable polylysine lipid (lipid-b-poly(Lys)-lipid) (LPL).

[0454] Then lipid-β-polymerization (N) was performed.6 Deprotection of (-benzyloxycarbonyl-Lys) to generate lipid-β-poly(Lys). A general synthetic method for lipid-β-peptides: 300 mg of lipid-β-poly(N...) 6 (-benzyloxycarbonyl-Lys) was dissolved in 6 mL of TFA, and 2.0 mL of acetic acid solution of 33% HBr was added. The mixture was stirred in an ice bath for 2 h. The solvent was removed under vacuum. The resulting crude product was suspended in 15 mL of methanol, and then precipitated by pouring the mixture solution into ice-cold diethyl ether (100 mL), and collected by centrifugation. The crude product was purified by suspending it in methanol and precipitating by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum. The crude product was purified by dialysis with deionized (DI) water. The product was obtained by freeze-drying under vacuum. The yield of lipid-b-poly(Lys) was 65%. The structure of lipid-b-poly(Lys) was determined by... 1 H NMR ( Figure 4-7 This has been confirmed.

[0455] The lipid-b-ionizable polypeptide lipid (LPL) was finally synthesized. 150 mg of lipid-b-poly(Lys) was dissolved in 6.0 mL of anhydrous ethanol, and 1.8 g of 1,2-epoxyalkane was added. The mixture was stirred at 90 °C for 48 h under a nitrogen atmosphere. The solvent was evaporated under vacuum to obtain the crude product. The crude product was purified by ethanol dialysis. The solvent was removed under vacuum. The yield of the compound was 92%. The structure of the lipid-b-ionizable polypeptide lipid was determined by… 1 H NMR ( Figure 8-12 This has been confirmed.

[0456] 1.5. Synthesis of lipid-ionizable lysine lipid derivatives (LPL1)

[0457] The synthesis of the lipid-ionizable Lys derivative is shown in Scheme 4. First, lipid-Lys(Boc) was synthesized. The general synthetic method for lipid-Lys(Boc) was as follows: Boc-Lys(Boc)-OH (1.73 g, 5.0 mmol) and ditetradecylamine (2.05 g, 5.0 mmol) were dissolved in dry dichloromethane (DCM) (100 mL). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureon hexafluorophosphate (HBTU) (2.38 g, 6.25 mmol) and N-ethyl-N-(1-methylethyl)-2-propylamine (DIPEA) (1.62 g, 12.5 mmol) were added to the solution. The reaction mixture was stirred at room temperature under N2 for 24 h. The mixture was diluted with 100 mL of DCM and washed with brine. The resulting organic layer was collected and dried over anhydrous Na2SO4. DCM was evaporated under vacuum to produce a crude product. The crude product was purified by rapid silica gel column chromatography (hexane:diethyl ether 8:2, v / v) to give the final product as a yellow oil. The yield of the compound was 85%.

[0458]

[0459] Scheme 4. Synthesis of lipid-Lys-lipid derivative (LPL1).

[0460] Then, lipid-Lys was synthesized. Lipid-Boc-Lys (Boc) was dissolved in 15 mL of anhydrous DCM, and 12 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred under N2 atmosphere for 2 h. The solvent was evaporated under vacuum to obtain the crude product. The obtained crude product was dissolved in 40 mL of DCM, and then 15 g of NaHCO3 was added. The mixture was stirred under N2 atmosphere for 12 h. The organic layer was collected and dried over anhydrous Na2SO4. DCM was evaporated under vacuum. The obtained crude product was purified by rapid silica gel column chromatography (DCM:MeOH 95:5, v / v). The yield of the compound was 80%.

[0461] The lipid-ionizable Lys derivative was finally synthesized. Lipid-Lys was dissolved in 8.0 mL of anhydrous ethanol, and 2.56 g of 1,2-epoxyoctane was added. The mixture was stirred at 90 °C for 48 h under a nitrogen atmosphere. The solvent was evaporated under vacuum to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (DCM:MeOH 95:5, v / v). The yield of the lipid-ionizable Lys derivative was 72%. The structure of the lipid-Lys-lipid derivative was determined by… 1 H NMR ( Figure 13 This has been confirmed.

[0462] 1.6. Synthesis of Electrolytically Ionizable Poly(Lys)-Lipid Derivatives (PL)

[0463] The synthesis of poly(Lys)-lipids (PL) is shown in Scheme 5. First, poly(N)-lipids were synthesized. 6 -benzyloxycarbonyl-Lys). Poly(N 6 A general synthetic method for H-Lys(Boc)-OMe (29.7 mg, 0.1 mmol) and Lys-NCA was dissolved in 40 mL of anhydrous DMF in a glove box. The mixture was stirred in a glove box at room temperature for 24 h. The crude product was precipitated by centrifugation by pouring the mixture solution into ice-cold diethyl ether (300 mL). The crude product was purified by dissolving it in MeOH and precipitating it by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum. Poly(N 6 The number of polymeric units of (-benzyloxycarbonyl-Lys) is adjusted by changing the amount of Lys-NCA. 6 The yield of (-benzyloxycarbonyl-Lys) was 82%.

[0464]

[0465] Scheme 5. Synthesis of poly(Lys)-lipid derivatives (PL).

[0466] Then perform aggregation (N) 6 Deprotection of (-benzyloxycarbonyl-Lys) yields poly(Lys). A general synthetic method for poly(Lys) involves adding 500 mg of poly(N...)... 6 (-benzyloxycarbonyl-Lys) was dissolved in 8 mL of TFA, and 2.7 mL of acetic acid solution of 33% HBr was added. The mixture was stirred in an ice bath for 2 h. The solvent was removed under vacuum. The resulting crude product was suspended in 30 mL of methanol, and then precipitated by centrifugation by pouring the mixture solution into ice-cold diethyl ether (300 mL). The crude product was purified by suspending it in methanol and precipitating by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum. The crude product was purified by dialysis with deionized water. The product was obtained by freeze-drying under vacuum. The yield of poly(Lys) was 85%.

[0467] The ionizable poly(Lys)-lipid derivative was finally synthesized. 300 mg of poly(Lys) was dissolved in 8.0 mL of anhydrous ethanol, and 1.28 g of 1,2-epoxyoctane was added. The mixture was stirred at 90 °C for 48 h under a nitrogen atmosphere. The solvent was evaporated under vacuum to obtain the crude product. The crude product was purified by ethanol dialysis. The solvent was removed under vacuum. The yield of the ionizable poly(Lys)-lipid derivative was 90%. The structure of the ionizable poly(Lys)-lipid derivative was determined by… 1 H NMR ( Figure 14 This has been confirmed.

[0468] Example 2: Preparation of siRNA-loaded lipid nanoparticle (siRNA LNP) formulation by manual mixing

[0469] siRNA LNPs were prepared by rapidly adding an ethanol solution containing specified amounts of ionizable lipids (LPL), DSPC, cholesterol, and PEG-lipids to a sodium acetate buffer solution (pH 4, 10 mM) containing GFP-siRNA (2.5 μg), followed by three pulsed vortex mixing cycles. The molar ratio of LPL:DSPC:cholesterol:PEG-lipids was typically 46:9.4:42:1.6. The prepared mixture was allowed to stand for 30 minutes, then diluted 1-fold with 0.9% w / v saline, filtered through a sterile filter (0.2 μm), and stored at 4°C until use. The N / P ratio was calculated using the amount of nitrogen per mole of polymer versus the amount of phosphate ester in the siRNA.

[0470] Example 3: Preparation of siRNA-loaded lipid nanoparticles (siRNA LNPs) formulations using microfluidic devices

[0471] siRNA LNPs were prepared using the Precision Nanosystems NanoAssemblr Ignite system. The microfluidic flow rate was set to 12 mL / min, and the volume ratio of aqueous to organic phase was 3:1. To prepare the aqueous phase, GFP-siRNA (75 μg) was diluted in 10 mM sodium acetate buffer at pH 4. In the organic phase, LPL, cholesterol, DSPC, and PEG-lipid (ALC-0159) were dissolved in ethanol at a molar ratio of (46:9.4:42:1.6). These molar ratios were kept constant at different N / P ratios, calculated based on the molar nitrogen content of each LPL and the molar p content of the siRNA. After preparation, the LNPs were diluted 15-fold in 0.9% saline solution at pH 7.4. Subsequently, prior to characterization, they were concentrated using centrifugal ultrafiltration with a Vivaspin® 10k MWCutoff centrifuge unit (Sartorius) to remove ethanol.

[0472] Example 4: Preparation of mRNA-loaded lipid nanoparticle (mRNA LNP) formulations by manual mixing

[0473] mRNA LNPs were prepared by dissolving ionizable lipids, DSPC, cholesterol, and PEG-lipid (ALC-0159) in ethanol at different molar ratios (30:12.3:55.7:2 and 46.3:9.4:42.7:1.6) to a final volume of 50 μL. The organic phase was then rapidly mixed with sodium acetate buffer (pH 4 or pH 5.25, 10 mM) containing 10 μg Fluc mRNA (TriLink BioTechnologies, San Diego, CA) at an N / P ratio of 3 or 6 to a final volume of 200 μL. The N / P ratio was calculated based on the molar nitrogen content of each ionizable lipid and the molar phosphate content of the mRNA. The prepared LNPs were stabilized at room temperature for 2 hours prior to characterization.

[0474] Example 5: Characterization of siRNA LNP and mRNA LNP (nanoparticle size, polydispersity (PDI), zeta potential and encapsulation efficiency)

[0475] Nanoparticle size, polydispersity index (PDI), and zeta potential were measured using a Zetasizer (Malvern, UK). siRNA and mRNA LNPs were diluted 40-fold and 20-fold, respectively, in 0.9% w / v saline, and the average of three technical replicates was reported for size / zeta potential measurements. The encapsulation efficiency of the formulated siRNA and mRNA LNPs was determined using the Quant-it™ RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA). Nanoparticles were diluted to ~1-5 ng μL with nuclease-free water (pH 7). -1 The RiboGreen RNA reagent was diluted 200-fold with Tris-EDTA buffer containing 5% Triton-X100 or Tris-EDTA (TE) buffer alone. Then, 90 μL of the buffer solution containing RiboGreen reagent was added to 10 μL of siRNA and mRNA LNPs, and the plates were incubated in black 96-well plates at 37°C for 30 min. Afterward, the fluorescence intensity of the wells was read using a microplate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The obtained values ​​were then used to calculate the encapsulation efficiency of siRNA and mRNA in the LNPs based on the following equation.

[0476]

[0477] Example 6: Transfection study of mRNA LNPs in HEK293T cells

[0478] The transfection efficiency of mRNA LNPs was characterized by delivering FLuc mRNA into HEK293T cells. HEK293T cell lines were cultured to the logarithmic growth phase in DMEM medium supplemented with 10% fetal bovine serum (FBS) (v / v) and 1% penicillin / streptomycin (v / v). In short, cells were cultured at 1 × 10⁶ cells / year. 4 Cells were seeded at a density of 100 μL / well in 96-well white clear plates and incubated at 37°C and 5% CO2 for 24 h. After overnight incubation, 100 μL of mRNA LNP was injected into each well, diluted to a final concentration of 100 ng / well with DMEM containing 10% FBS. The transfected cells were then incubated at 37°C and 5% CO2 for 48 h. Lipofectamine was prepared according to the manufacturer's protocol. After 48 h, 100 μL of One-Glo™ luciferase reagent (Promega) was added to each well and incubated in the dark for 3 min. The luminescence signal was immediately measured using a microplate reader (Tecan, Männedorf, Switzerland).

[0479] Example 7: Evaluation of the cytotoxicity of mRNA LNPs

[0480] The cytotoxicity of mRNA LNPs was assessed in HEK293T cells. HEK293T cells were cultured at a rate of 1 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 μL per well in 96-well black clear plates and incubated at 37°C and 5% CO2 for 24 h. After incubation, 100 μL of mRNA LNP was injected into each well, diluted to a final concentration of 100 ng / well with DMEM containing 10% FBS, and then incubated at 37°C and 5% CO2 for 48 h. After 48 h, the used medium was removed from the wells and replaced with 10% Alamar Blue reagent in DMEM medium. The plates were incubated at 37°C for 2 h, and then fluorescence intensity was measured using a microplate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 570 nm and an emission wavelength of 600 nm. Cell viability of the treated wells was then expressed as a percentage relative to the untreated negative control wells.

[0481] Example 8: Transfection study using siRNA LNP in HepG2 cells stably expressing eGFP

[0482] HepG2 eGFP-expressing cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS) (v / v) and 1% penicillin / streptomycin (v / v). Cells were incubated at 37°C with 5% CO2. Cells were trypsinized at 80–90% confluence and seeded at a density of 5,000 cells / well in 96-well black clear plates. Prior to transfection, they were incubated at 37°C with 5% CO2 for 24 h. On the day of transfection, siRNA LNP solution was mixed with DMEM containing 10% FBS to a final volume of 100 μL per well, with an siRNA dose of 100 ng / well. The plates were then incubated at 37°C with 5% CO2 for 72 h. Lipofectamine was prepared according to the manufacturer's protocol.

[0483] Example 9: Measurement of GFP knockdown efficiency of siRNA LNP in HepG2 cells and cell viability of HepG2 cells

[0484] Total mean GFP fluorescence was measured using a Tecan Spark Control microplate reader (Männedorf, Switzerland) at excitation and emission wavelengths of 485 nm and 535 nm, respectively. The knockdown percentage was calculated using the formula (100% - (fluorescence reading of treated cells / fluorescence reading of untreated cells)) with reference to untreated cells. After 72 h of incubation, the medium was removed from each well and replaced with 100 μL of fresh DMEM medium containing 10% Alamar Blue reagent. The plates were then incubated at 37 °C for 2 h. Fluorescence intensity was then measured using a microplate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 570 nm and an emission wavelength of 600 nm. Cell viability in the treated wells was then expressed as a percentage relative to the untreated negative control wells.

[0485] Example 10: Fluorescence Microscopy Imaging

[0486] Fluorescence images were captured using an Olympus BX53 fluorescence microscope equipped with a DP80 digital camera and a FITC filter set suitable for GFP imaging (excitation 488 nm, emission 507 nm). Images were collected using a 10x immersion objective. Image acquisition and analysis were performed using Olympus cellSens Dimension imaging software. Exposure time, gain, and offset settings were kept constant throughout the image acquisition process for all samples to ensure comparability.

[0487] Example 11: Statistical Analysis

[0488] Welch's t-test was used. Figure 17-19 Statistical analysis was performed using one-way ANOVA and Tukey's post-hoc test with the commercially available GraphPad Prism. Figure 22 Statistical analysis. Values ​​marked with an asterisk (ns, p > 0.05) showed significant differences. p < 0.05; p < 0.01; p < 0.001; (p < 0.0001).

[0489] Example 12: Results and Discussion

[0490] 12.1. Synthesis of lipid-β-ionizable peptides

[0491] As shown in Scheme 3, N-amino-N,N-bistetradecylacetamide lipids (Scheme 2) are used as initiators. 6 Ring-opening polymerization (ROP) of β-benzyloxycarbonyl-lysine-N-carboxylic anhydride (Lys-NCA) (Scheme 1), followed by lipid-poly(N... 6 The deprotection of β-benzyloxycarbonyl-Lys in TFA and HBr / CH3COOH first synthesized lipid-β-poly(Lys). The successful synthesis of Lys-NCA, 2-amino-N,N-bistetradecylacetamide, and lipid-poly(Lys) was achieved through... 1 H NMR and 13 C NMR spectrum ( Figure 1-3 (This is confirmed.) Figure 4 As shown, the peaks (-Ph) at δ 7.35 and 5.25 ppm are... H -and Ph-C H The disappearance of 2- indicates successful deprotection of the lipid-β-poly(Lys). The degree of polymerization (DP) of lysine in lipid-β-poly(Lys) was modulated by changing the amount of Lys-NCA. The DP of lysine in lipid-β-poly(Lys) was determined by the integral area (peak j of the peptide; peak a of the lipid). Figure 4-7 Lipids with DP values ​​of 5, 8, 10, and 15 (Lys) were further used to synthesize lipid-b-ionizable poly(Lys)-lipid derivatives (LPL). An ionizable poly(Lys)-lipid derivative with DP of 15, denoted as PL, was synthesized as a control ionizable lipid (LPL). Figure 14 A lipid-b-poly(Lys) denoted as LP was also prepared as a control lipid.

[0492] To facilitate RNA encapsulation and delivery, a series of lipid-β-electrolyzable poly(Lys) derivatives (LPLs) were synthesized via an amino-epoxy reaction. These ionizable lipids possess long, bi-tailed lipids and polypeptide backbones with alkyl side chains of varying lengths. The structure of LPLs is determined by... 1 Confirmed by H NMR spectroscopy. Figure 5-12 As shown, the peak j(NH2-C) of lipid-b-poly(Lys) at δ 2.9 ppm is... H The disappearance of 2-) and the peak of lipid-b-ionizable polymers (Lys)-lipids at δ 2.6 ppm j(-NC) H The presence of 2- indicates the successful addition of primary amines to 1,2-epoxyalkanes. The proton signal and integral ratio of the epoxyalkyl chain are related to the ionizable lipid... 1 The theoretical values ​​in the H NMR spectrum consistently reveal the integrity of the LPL. Figure 8-12 Simultaneously, lipid-Lys-lipids were also synthesized as ionizable lipids for RNA delivery. The successful synthesis of lipid-Lys-lipids was achieved by… 1 H NMR spectroscopy ( Figure 13 This has been confirmed. The characteristics of lipids—Lys-lipids, PL, LP and LPL are listed in Table 1.

[0493] Table 1. Characteristics of ionizable lipids.

[0494]

[0495] 12.2. Size, size distribution (PDI), zeta potential, and encapsulation efficiency of siRNA-loaded LNPs

[0496] The size, polydispersity index (PDI), zeta potential, and encapsulation efficiency of LNPs loaded with GFP-siRNA were assessed using Zetasizer (Malvern, UK). These results are shown in Tables 2-5 and Figure 15The siRNA LNPs formed from LPs containing 15 primary amines exhibit a high surface charge because the LPs are positively charged even at pH 7.4 (measured in saline). The remaining lipids with tertiary amine groups form LNPs with a neutral surface (ζ potential within ±10 mV) at pH 7.4, which is ideal for in vivo applications. Most formulated LDL nanoparticles are smaller than 100 nm in size. Although a range of size distributions was observed, this variability can be attributed to the use of a manual mixing formulation method. This method was chosen because of its speed and efficiency in screening different lipids and formulations, thus aiding in the selection of the optimal mixture for further studies. Encapsulation efficiency was lowest when the N / P ratio was 2. Microfluidic mixing was performed after determining the polymers and N / P ratios that performed optimally. The results were then directly compared to a clinically approved ionizable lipid (ALC-0315) used in Pfizer-Biotech's COVID-19 mRNA vaccine formulation. The best-performing candidate, LPL4, exhibited comparable size, PDI, ζ-potential, and siRNA encapsulation efficiency to ALC-0315 (Table 5). Both types of siRNA LNPs showed narrow size distributions. Figure 15 ).

[0497] Table 2. Characteristics of manually formulated siRNA-loaded LNPs with different lipid lengths.

[0498]

[0499] Table 3. Characteristics of manually prepared PL, LPL 2 and 6 LNPs loaded with siRNA.

[0500]

[0501] Table 4. Characteristics of manually formulated LPL LNPs loaded with siRNA with different degrees of polymerization.

[0502]

[0503] Table 5. Characteristics of LPL4 and ALC-0315 LNP loaded with siRNA prepared by microfluidic mixing.

[0504]

[0505] 12.3. In vitro knockdown efficiency of siRNA LNP in HepG2 eGFP cells

[0506] To evaluate the efficacy of LNP as an siRNA delivery medium, an siRNA encoding GFP knockdown was selected as an optical reporter gene. The average total fluorescence reading of GFP after siRNA delivery allowed for the quantification of the average knockdown of GFP protein expression. The knockdown efficiency after cell treatment with siRNA LNP was compared with that of the commercial transfection agent Lipofectamine 3000 as a positive control.

[0507] The optimal carbon length for achieving optimal siRNA transfection was determined through initial evaluation, which included testing the carbon lengths of various lipids attached to polylysine at two different N / P ratios. Figure 16-19 Of these, the C8 portion exhibited the highest performance (LPL6). vs LPLs with longer carbon chain lengths were synthesized, but they were insoluble in the solvents used in the formulations and therefore were not tested. No cytotoxicity was observed in any of the LPL formulations.

[0508] The degree of polymerization (DP) of LPL LNPs was investigated to obtain the most efficient lipids while maintaining a constant attached carbon chain length (from LPL3 to LPL6). Knockdown was less efficient at low and high DP, peaking at LPL4 and LPL5. At N / P3, siRNA LPL4 LNP mediated higher gene knockdown than siRNA LPL5 LNP. Figure 17 ).

[0509] At N / P 3, the knockdown of LPL2 LNP was significantly lower than that of LPL6, indicating the importance of having the C8 portion over the C3 portion while maintaining DP constant. Figure 18 Ditetradecylamine as an initiator is essential for efficient siRNA delivery of LPL LNPs because the knockdown mediated by siRNA PL LNPs is more potent than that induced by siRNA LPL6 LNPs, particularly at N / P 3. Figure 19 ). The siRNA LPL4 LNP and siRNA ALC-0315 LNP induction at N / P 3 resulted in comparable knockdown ( Figure 20 Furthermore, as shown in the fluorescence microscopy images, the GFP expression level of the LPL4 formulation was significantly reduced, with an efficiency comparable to that of the ALC-0315 formulation.

[0510] 12.4. mRNA-LNP characterization and in vitro transfection

[0511] In addition to siRNA delivery, the ability of lipids LPL1 and LPL3 to encapsulate and deliver mRNA into cells was further investigated. The N / P ratio and pH of the sodium acetate buffer used to dissolve mRNA were varied, while the molar ratio of ionizable lipids was fixed at 30% for the mRNA-loaded LPL1 and LPL3 LNP formulations. The size, polydispersity index (PDI), and zeta potential of the mRNA-loaded LNPs were similarly evaluated using a Zetasizer (Malvern, UK). Although all formulations had a near-neutral surface charge of ±10 mV, significant differences in size, PDI, and encapsulation efficiency were observed when the pH and N / P ratio of the sodium acetate buffer used were varied. For initial screening, the pH of the sodium acetate buffer used was kept the same as that used in the Pfizer formulation (pH 4). At pH 4, the LPL1 LNP formulations with N / P ratios of 6 and 12 both had sizes greater than 200 nm and exhibited a large size distribution (Table 6). Therefore, when compared with Pfizer formulations using ALC-0315, these formulations exhibited less than 20% poorer encapsulation efficiency (Table 6) and lower transfection efficiency, described by poorer luminescence intensity measured in HEK293T cells. Figure 22 A). Unlike LPL1 LNPs, LPL3 LNPs formulated with N / P 21 and 42 have a narrow size distribution (PDI < 0.3) and an acceptable size for in vivo applications. However, their encapsulation efficiency is less than 50% (Table 6), and their transfection efficiency is lower than ALC-0315 ( Figure 22 A) indicates that further optimization is needed.

[0512] The pH of the sodium acetate buffer used was then increased to pH 5.25. At pH 5.25, LPL1 LNPs prepared with N / P 12 did not show positive encapsulation efficiency when Ribogreen assays were performed (Table 6). This is likely a result of the tight binding between LPL1 lipids and FLuc mRNA, which prevents mRNA release for accurate measurement of encapsulation efficiency. The same hypothesis can also explain the low transfection efficiency observed in vitro when LPL1 LNPs prepared with N / P 12 at pH 5.25 were incubated with HEK293T cells for 48 h. Figure 22 A). However, at the same pH of 5.25, the LPL1 LNP formulated at a lower N / P ratio of 6 had a smaller size of 102 nm and a good PDI of 0.14. Furthermore, among all the LNPs tested, it had the highest encapsulation efficiency of 78.8% (Table 6) and a significantly higher transfection efficiency (5 times higher than ALC-0315). Figure 22A). Although LPL3LNP formulated with N / P 42 resulted in a much higher encapsulation efficiency than that formulated with N / P 21, the transfection efficiency using N / P 21 was significantly higher than that using N / P 42. Figure 22 A). Notably, LPL3 LNP with N / P 21 and pH 5.25 exhibited significantly higher transfection activity than ALC-0315. Figure 22 A).

[0513] Table 6. Characteristics of manually prepared LNPs loaded with mRNA.

[0514]

[0515] 12.5. Evaluation of the cytotoxicity of mRNA-loaded LNPs in HEK293T cells

[0516] The cytotoxicity of LNPs encapsulated in mRNA was assessed using a metabolic cell viability assay. HEK293T cells transfected with LPL 1 LNPs prepared at N / P 6 and 12, pH 4 and N / P 12, pH 5.25 showed significantly reduced viability compared to untreated cells. Figure 22 B). The observed low cell viability may be due to free lipid-induced cytotoxicity, as these formulations have low encapsulation efficiency (Table 6). No cytotoxicity was observed in any of the LPL3 LNP formulations and LPL1 LNPs prepared at N / P ratios of 6 and pH 5.25. Of all the LNP formulations tested, LPL1 and LPL3 LNPs prepared at N / P ratios of 6 and 21 at pH 5.25, respectively, showed higher transfection activity than ALC-0315 and were non-toxic to cells; making them suitable candidates for both in vitro and in vivo applications. Figure 22 B)

[0517] 12.6. Summary

[0518] A novel ionizable lipid based on lipid-β-ionizable peptides was developed, successfully encapsulating siRNA and mRNA into LNPs. Through extensive screening of different formulations and N / P ratios, several promising candidates exhibiting not only efficient siRNA transfection but also mRNA transfection were identified. These candidates were compared with ALC-0315 used in the Pfizer-BNT Covid19 mRNA vaccine formulation, showing a size of approximately 70–110 nm, neutral surface charge, high siRNA and mRNA encapsulation efficiency, and comparable GFP knockdown efficiency. Therefore, this study introduces a novel peptide-based LNP system for efficient siRNA and mRNA delivery, offering potential for future therapeutic applications in siRNA- and mRNA-based therapies. LNPs can also be used to deliver other nucleic acid therapeutic agents.

Claims

1. A compound comprising a structure represented by general formula (1) or an ionized form thereof, used for the preparation of lipid nanoparticles encapsulating therapeutic agents, preventative agents, and / or biological agents: in R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl; R 3 and each R 7 Independently, it is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group; n is between 0 and 100; l and m are each independently 0 or 1; Each A is independently selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, optionally substituted alkynyl groups, epoxy ring-opening products and / or their derivatives; and B is R 1 R 2 N- or R 23 O-, where R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; and R is one of them. 23 It can be an alkyl group, an alkenyl group, or an alkynyl group that are optionally substituted.

2. The compound of claim 1, wherein the compound comprises three or more tertiary amines.

3. The compound according to any one of the preceding claims, wherein R 1 and R 2 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and combinations thereof.

4. The compound according to any one of the preceding claims, wherein A is represented by general formula (2): Where R 8 R 8’ R 9 and R 9’ Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

5. The compound according to claim 4, wherein R 8 R 8’ and R 9’ For H and R 9 C y H 2y+1 , where y is from 1 to 8.

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

7. The compound according to any one of the preceding claims, wherein the compound comprises a structure selected from one or more of the following: PL (n = 15); LP1 (n = 5); LP2 (n = 8); LP3 (n = 10); LP4 (n = 15); LPL1 (n = 1); LPL2 (n = 15); LPL3 (n = 5); LPL4 (n = 8); LPL5 (n = 10); and LPL6 (n = 15).

8. A method for preparing a compound of general formula (1) according to any one of claims 1 to 7, the method comprising: (a) Reacting a lipid-b-poly(Lys) compound represented by general formula (1b') with an epoxy-containing compound represented by general formula (3) to obtain a compound represented by general formula (1): in R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl; R 3 And each R is independently an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group; n is between 1 and 100; Each R 11 To R 13 Independently selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, or optionally substituted alkynyl groups; R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; and R 8 and R 9 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

9. The method of claim 8, wherein the method further comprises, prior to step (a): (ai) React the compound represented by general formula (4) with a protected amino acid containing an N-carboxylic anhydride (NCA) represented by general formula (5) to obtain a protected lipid-b-poly(Lys) compound represented by general formula (6) via ring-opening polymerization (ROP): in R 5 and each R 6 Independently H, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted ynyl; R 3 and each R 7 Independently, it is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group; n is between 1 and 100; R 11 To R 12 Each is independently selected from H, fatty alcohol, optionally substituted alkyl, optionally substituted alkenyl or optionally substituted alkynyl; R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; R 10 H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and PG 1 For the protection of the base; and (a-ii) Deprotect the protected lipid-b-poly(Lys) compound represented by general formula (6) obtained in step (ai) to obtain the compound represented by general formula (1b'): in R 13 It is selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, or optionally substituted alkynyl groups.

10. The method according to claim 8 or claim 9, wherein the method further comprises, prior to step (ai),: (aii) Reaction of the protected amino acid represented by general formula (7) with a carbonylating agent to obtain the protected amino acid containing NCA represented by general formula (5): in R 6 and R 16 Each is independently H, an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, or an alkynyl group that is optionally substituted; R 7 It is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group; R 11 Selected from H, fatty alcohols, optionally substituted alkyl groups, optionally substituted alkenyl groups, or optionally substituted alkynyl groups; and PG 1 To protect the base.

11. The method according to any one of claims 8 to 10, wherein the method further comprises, prior to step (ai),: (ai-ii) Reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (9) in the presence of one or more coupling agents and a base to obtain a carbamate compound represented by general formula (10). in R 5 R 14 and R 15 Each is independently H, an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, or an alkynyl group that is optionally substituted; R 3 Independently, it is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group; n is between 1 and 100; R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; and PG 2 For the protection of the base; and (ai-iii) Deprotect the carbamate compound represented by general formula (10) obtained from step (ai-ii) to obtain the compound represented by general formula (4). in R 10 H represents an alkyl group, an alkenyl group, or an alkynyl group, which may be optionally substituted.

12. A method for preparing a compound of formula (1) according to any one of claims 1 to 7, the method comprising: (b) Reacting an amine compound represented by general formula (8) with a protected amino acid compound represented by general formula (11) in the presence of one or more coupling agents and a base to obtain a dicarboxylate compound represented by general formula (12). in R 7 It is an optionally substituted alkylene group, an optionally substituted alkenyl group, or an optionally substituted ynylene group; R 1 and R 2 Each can be independently either H or hydrophobic tails, provided that R is present. 1 and R 2 The two are not both H; R 14 and R 17 To R 19 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; and PG 4 and PG 5 Each serves as an independent protective base; (c) Deprotect the dicarbamate compound of general formula (12) obtained in step (b) to obtain the amide compound of general formula (13). in R 20 To R 21 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; and (d) Reacting the amide compound represented by general formula (13) with the epoxy represented by general formula (3) to obtain the compound represented by general formula (1): R 8 To R 9 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

13. A nanoparticle composition for delivering therapeutic agents, preventative agents, and / or biological agents, said nanoparticle composition comprising: The compound according to any one of claims 1 to 7; and Therapeutic agents, preventative agents, and / or biological agents encapsulated in the compounds according to any one of claims 1 to 7.

14. The nanoparticle composition of claim 13, wherein the nanoparticle composition comprises nanoparticles having an N / P ratio of 1:1 to 50:

1.

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

16. The nanoparticle composition according to any one of claims 13 to 15, wherein the composition further comprises: (i) Auxiliary lipids; (ii) cholesterol or its derivatives; and (iii) Polyethylene glycol (PEG) lipid conjugates or amphiphilic lipids.

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

18. The nanoparticle composition of claim 16, wherein the auxiliary lipid is selected from 1,2-distearyl-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 1,2-Diundecanoyl-sn-glycero-3-phosphocholine (DPPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:O diether PC), 1-Oleoyl-2-cholesterolylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16) Lyso PC), 1,2-Dilinoleoyl-sn-glycero-3-phosphatecholine, 1,2-Diarachidonicoyl-sn-glycero-3-phosphatecholine, 1,2-bis(docohexanoyl-sn-glycero-3-phosphatecholine), 1,2-diphydanoyl-sn-glycero-3-phosphateethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycero-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycero-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycero-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof.

19. The nanoparticle composition according to claim 16, wherein the cholesterol or its derivatives are selected from cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, alfalfa sterol, and combinations thereof.

20. The nanoparticle composition according to claim 16, wherein the polyethylene glycol (PEG)-lipid conjugate or amphiphilic lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(PEG)-2000]-N,N-bistetradecylacetamide (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-dimyristyloxypropylamine (PEG-S-DMG), PEG-DMPE (1,2-dimyristyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-methoxy](sodium salt)), PEG-DPPC, PEG-DSPE lipids and combinations thereof.

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

22. The nanoparticle composition according to any one of claims 13 to 20, for use in treating or preventing diseases, disorders or ailments of a subject in need.

23. Use of the nanoparticle composition according to any one of claims 13 to 20 in the preparation of a medicament for treating or preventing a disease, disorder or ailment of a subject in need.

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

25. The nanoparticle composition according to any one of claims 13 to 20, the use according to claim 23, or the method according to claim 24, wherein an immune response in the subject is induced by administering the nanoparticle composition to the subject.

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

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

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