Compounds for preparing lipid nanoparticles encapsulating pharmaceutical agents, nanoparticle compositions comprising the compounds, and related methods

Abstract

Provided are compounds represented by general formula (1) for preparing lipid nanoparticles encapsulating therapeutic, prophylactic, and / or biological agents, wherein A R comprises units from a polyamino acid; R 1 and R 2 are each independently a hydrophobic group; R 3 , R 4 , and R 5 are each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R 7 is -H or -C(=O)R 8 , wherein R 8 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted alkoxy; m > 1; and n > 1.

Description

Technical Field

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

[0002] background

[0003] Lipid nanoparticles are widely used to deliver therapeutics, prophylactic agents, and / or biological agents (e.g., polynucleotides such as mRNA). However, a safe, stable, and efficient delivery system remains a challenge. Specifically, adverse health effects and cytotoxicity associated with delivery using lipid nanoparticles have been reported.

[0004] Currently, lipid nanoparticles have been successfully used to deliver Moderna's and Pfizer-BioNtech's mRNA Covid-19 vaccines, which have been approved by the US Food and Drug Administration (FDA) for human use. Both vaccines utilize SARS-CoV-2 mRNA as an antigen and lipids as a carrier. The lipids consist of three different types of lipids (ionizable lipids, PEG-lipid conjugates, and helper lipids) and cholesterol. The lipids assemble with the mRNA to form nanoparticles, which stimulate immune cells to produce a prophylactic response against the SARS-CoV-2 virus.

[0005] However, currently available formulations have several drawbacks and shortcomings and are far from ideal. First, these formulations clearly generate anti-lipid and anti-PEG antibodies (due to the use / presence of PEG-lipid conjugates), which can cause hypersensitivity and allergic reactions in some subjects. Polyethylene glycol (PEG) has been reported as a high-risk allergen often hidden in pharmaceuticals / foods. PEG can lead to the release of allergenic compounds through IgE binding to basophils, and individuals may develop anaphylactic symptoms due to the presence of PEG in the drug. PEG has also been identified as a cause of the accelerated blood clearance (ABC) phenomenon. Furthermore, the presence of anti-PEG antibodies in the body may reduce the plasma half-life of mRNA LNPs and its vaccination efficacy.

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

[0007] Overview

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

[0009]

[0010] in

[0011] A R Contains units derived from polyamino acids;

[0012] R 1 and R 2 Each is an independent hydrophobic group;

[0013] R 3 R 4 and R 5 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group;

[0014] R 7 It is -H or -C(=O)R 8 , where R 8 It is an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, an alkynyl group that is optionally substituted, or an alkoxy group that is optionally substituted;

[0015] m≥1; and

[0016] n≥1.

[0017] In one implementation, A R It comprises a structure represented by general formula (2), wherein A contains a hydrophilic organic group that is part of an amino acid represented by general formula (3):

[0018]

[0019] in

[0020] R 6 It is H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0021] In one embodiment, the polyamino acid is selected from polyserine, polyglutamic acid, and combinations thereof.

[0022] In one embodiment, A is selected from -CH2OH, -CH2CH2C(=O)OH, and combinations thereof.

[0023] In one implementation, A R Includes structures represented by general formulas (2A) and / or (2B):

[0024]

[0025] in

[0026] p + q = n; and

[0027] R 6’ =R 6’’ =R 6 .

[0028] In one embodiment, the compound represented by general formula (1) comprises a structure represented by general formula (1A) and / or (1B):

[0029]

[0030]

[0031] in

[0032] p + q = n; and

[0033] R 6’ =R 6’’ =R 6 .

[0034] In one implementation, n ≤ 60.

[0035] In one implementation scheme, in R 1 and R 2 Each of the hydrophobic groups independently comprises an optionally substituted alkyl group having at least 8 carbon atoms.

[0036] In one implementation scheme, in R 1 and R 2 Each of the hydrophobic groups independently comprises an optionally substituted alkyl group having no more than 18 carbon atoms.

[0037] In one implementation, R 3 R 4 R 5 and R 6 Both are H.

[0038] In one embodiment, the compound comprises a structure selected from one or more of the following:

[0039] ,

[0040] C14-pDLS1 (n=18)

[0041] ,

[0042] C14-pDLS2 (n=18)

[0043] ,

[0044] C14-pDLS3 (n=21)

[0045] ,

[0046] C14-pDLS4 (n=27)

[0047] ,

[0048] C14-pDLS5 (n=32)

[0049] ,

[0050] C14-pDLS6 (n=45)

[0051] ,

[0052] C18-pDLS (n=30)

[0053] ,and

[0054] C12-pDLS (n=32)

[0055]

[0056] C8-pDLS (n=34).

[0057] In another aspect, a method for preparing the compound claimed in any of the preceding claims is provided, the method comprising:

[0058] (i) Polymerizing one or more N-carboxylic anhydride (NCA) monomers represented by general formula (5) with a lipid initiator / molecule represented by general formula (6) to obtain a first intermediate compound represented by general formula (7):

[0059]

[0060]

[0061] in

[0062] A PG A represents A protected by one or more protecting groups, wherein A contains a hydrophilic organic group that is part of an amino acid represented by general formula (3);

[0063] R 11 and R12 Each is an independent hydrophobic group;

[0064] R 13 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;

[0065] m≥1; and

[0066] n≥1;

[0067] (ii) Optionally, the first intermediate compound represented by general formula (7) is reacted with an acylating agent to obtain the second intermediate compound represented by general formula (8):

[0068]

[0069] Where R 17 It is -H or -C(=O)R 18 , where R 18 It is an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, an alkynyl group that is optionally substituted, or an alkoxy group that is optionally substituted;

[0070] (iii) Deprotect the first intermediate compound represented by general formula (7) or the second intermediate compound represented by general formula (8) to obtain the compound represented by general formula (1).

[0071] In one implementation, the method further includes, prior to step (i):

[0072] (ai) Reaction of the protected amino acid represented by general formula (10) with a carbonylating agent to obtain an N-carboxylic anhydride (NCA) monomer represented by general formula (5):

[0073]

[0074] In one embodiment, the N-carboxylic anhydride (NCA) monomer represented by general formula (5) comprises a structure represented by general formula (5A) and / or (5B):

[0075]

[0076] In one embodiment, step (i) comprises mixing general formula (5A) with general formula (5B) in a molar ratio of 1:5 to 5:1.

[0077] In one aspect, a nanoparticle composition for delivering therapeutic agents, preventative agents, and / or biological agents is provided, the nanoparticle composition comprising:

[0078] The compounds disclosed herein; and

[0079] Therapeutic agents, preventative agents, and / or biological agents.

[0080] In one embodiment, the composition further comprises:

[0081] (a) Ionizable lipids;

[0082] (b) assisting lipids; and

[0083] (c) Cholesterol and / or its derivatives.

[0084] In one embodiment, the ionizable lipids, auxiliary lipids, cholesterol and / or their derivatives, and compounds represented by general formula (1) are mixed in a molar ratio of 5 – 65 : 4 – 20 : 10 – 60 : 0.1 – 20.

[0085] In one embodiment, the ionizable lipid is selected from ALC-0315, SM-102, Lipid 5, DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-O16B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-O17O, NT1-O14B, 306-O12B-3, 306-O12B, 113-O16B, 306Oi10, 30 6Oi9-cis2, BAMEA-O16B, Al-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, LipidC24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-three-tailed, C13-113-three-tailed, C13-112-four-tailed or C13-113-four-tailed, C12-200 and their combinations.

[0086] In one embodiment, the auxiliary lipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline. Ole-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof.

[0087] In one embodiment, the cholesterol and / or its derivatives are selected from cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, alfalfa sterol, and combinations thereof.

[0088] In one embodiment, the nanoparticle composition comprises nanoparticles having an N / P ratio of 1:1 to 50:1.

[0089] In one embodiment, the nanoparticle composition comprises nanoparticles having an average particle size of no more than 300 nm.

[0090] In one embodiment, the nanoparticle composition comprises nanoparticles having a zeta potential of -20 mV to +20 mV.

[0091] In another aspect, nanoparticle compositions disclosed herein are provided for use in medicine.

[0092] In another aspect, the nanoparticle compositions disclosed herein are provided for use in treating or preventing diseases, disorders, or conditions in subjects with such need.

[0093] 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 of such treatment is provided.

[0094] In another aspect, a method is provided for treating or preventing a disease, disorder, or condition in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition disclosed herein.

[0095] In one embodiment, the immune response in the subject is induced by administering the nanoparticle composition to them.

[0096] In one implementation, the disease, disorder, or symptom is mediated by a coronavirus.

[0097] In one implementation, the coronavirus is the SARS-CoV-2 coronavirus.

[0098] definition

[0099] As used herein, the term "particle" refers to a discrete entity or discrete object. The particles described herein may include organic particles, inorganic particles, composite particles, or biological particles. The particles described herein may also be large 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" refers to the maximum scale of the particle. For example, the term "size" may refer to the diameter of a nanoparticle when used in the context of nanoparticles, 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.

[0100] The term "nano" as used herein should be interpreted broadly to include 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, the terms "nanostructure," "nanoparticle," "nanomaterial," etc., as used herein may include structures having at least one dimension within the range not exceeding those described. As used herein, the terms “nanostructure,” “nanoparticle,” “nanomaterial,” etc., can include structures having at least one dimension not exceeding about 1,000 nm, not exceeding about 950 nm, not exceeding about 900 nm, not exceeding about 850 nm, not exceeding about 800 nm, not exceeding about 750 nm, not exceeding about 700 nm, not exceeding about 650 nm, not exceeding about 600 nm, not exceeding about 550 nm, not exceeding about 500 nm, not exceeding about 450 nm, not exceeding about 400 nm, not exceeding about 350 nm, not exceeding about 300 nm, not exceeding about 250 nm, not exceeding about 200 nm, not exceeding about 150 nm, not exceeding about 100 nm, not exceeding about 90 nm, not exceeding about 80 nm, not exceeding about 70 nm, not exceeding about 60 nm, not exceeding about 50 nm, not exceeding about 40 nm, not exceeding about 30 nm, not exceeding about 20 nm, or not exceeding about 10 nm.

[0101] The term “micrometer” as used herein should be interpreted broadly to include dimensions 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.

[0102] As used herein, “treatment,” “treat,” and “therapeutic method,” and their synonyms, refer both to therapeutic treatment and preventative or avoidant measures aimed at preventing or alleviating medical conditions, including but not limited to diseases, symptoms, and disorders. Medical conditions also include the body’s response to a disease or disorder (e.g., inflammation). Those who require such treatment include people who already have a medical condition, people who are susceptible to a medical condition, or people who need to prevent a medical condition.

[0103] The term "therapeuticly effective amount" as used herein is intended to mean an amount sufficient to prevent or at least slow down (alleviate) a medical condition, such as infectious diseases (e.g., dengue fever caused by the dengue virus), respiratory diseases (e.g., coronavirus caused by the SARS-CoV-2 virus or influenza caused by the influenza virus), cancer, autoimmune diseases, and cardiovascular diseases. 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 to be used in treatment will depend on, for example, the therapeutic target, the route of administration, and the patient's condition. Therefore, therapists may need to adjust the dosage and change the route of administration as needed to achieve the best therapeutic effect.

[0104] The term “subject” is intended to broadly refer to any animal, such as mammals, and includes humans. Exemplary subjects include, but are not limited to, humans and non-human primates. The term “subject” as used herein also includes patients and non-patients. The term “patient” means an individual who has or may have a medical condition such as an infectious disease (e.g., coronavirus caused by the SARS-CoV-2 virus, dengue fever caused by the dengue virus, etc.), while “non-patient” means an individual who does not have or may not have a medical condition. “Non-patient” includes healthy individuals, individuals who are not ill, and / or individuals who do not have a medical condition. The term “mammal” as used herein includes vertebrates such as humans or large veterinary mammals (e.g., horses, cattle, deer, sheep, llamas, goats, pigs).

[0105] 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.

[0106] In the following definitions of many substituents, it is stated that "the group may be a terminal group or a bridging group." This is intended to indicate that the term is used to cover both cases where the group is a terminal group / part and cases where the group is a linker between two other parts of the molecule. Taking the term "alkyl" having one carbon atom as an example, it should be understood that when present as a terminal group, the term "alkyl" having one carbon atom can refer to -CH3, and when present as a bridging group, the term "alkyl" having one carbon atom can refer to -CH2-, etc.

[0107] 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 can be terminal groups or bridging groups.

[0108] The term "alkenyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, and it can be a straight or branched chain 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 can contain multiple double bonds, and the orientation around each double bond is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-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-Decanyl, 2-Decanyl, 3-Decanyl, etc. The groups can be terminal groups or bridging groups.

[0109] 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 it can be a straight or branched chain 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, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-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 can be a terminal group or a bridging group.

[0110] As used herein, the term "cyclic" refers to a structure in which one or more atomic series are linked together 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. As used herein, the term "cyclic" includes "heterocyclic".

[0111] As used herein, the term "heterocycle" refers to a structure in which two or more different kinds of atoms are linked together to form at least one ring. For example, a heterocycle can be formed by a carbon atom and at least one other atom selected from oxygen (O), nitrogen (N), or (NR) and sulfur (S) (i.e., a heteroatom), where 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-pyrrolin, 2-pyrrolin, 3-pyrrolin, 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, and pyrrole. Alkane, 1,3-dioxacyclopentane, 1,2-oxathiacyclopentane, 1,3-oxathiacyclopentane, pyrazolidine, imidazoline, pyridine, pyrazine, pyrimidine, 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, thiocyclohexane, piperidine, 1,4-dioxacyclohexane, 1,2-dithiaran, 1,3-dithiaran, 1,4-dithiaran, 1,3,5-trithiacyclohexane, piperazine, morpholine, thiomorpholine, etc.

[0112] The term "amine" or similar term is intended to refer generally to a group containing -NR2, where R is independently hydrogen or an organic group. The group may be a terminal group or a bridging group.

[0113] The term "amide group" or similar term is intended to refer generally to a group containing -C(=O)NR2, where R is independently hydrogen or an organic group. The group can be a terminal group or a bridging group.

[0114] The term "aryl" as a group or part of a group means (i) a optionally substituted monocyclic or fused polycyclic aromatic carbon ring (the ring structure having ring atoms that are all 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.

[0115] 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) in the ring 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, naphtha[2,3-b]thiophene, furan, isoindazine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazolium, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthidine, quinoxaline, cyclophosphine, carbazole, phenantridine, 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-indole, and 2- or 3-thiophene, etc. The group can be a terminal group or a bridging group.

[0116] The term "halogen" refers to chlorine, fluorine, bromine, or iodine. The term "halogen" refers to chloride, fluoride, bromide, or iodide.

[0117] The term "optionally substituted" when used to describe a chemical structure or part indicates a chemical structure or part in which one or more of its hydrogen atoms are optionally substituted by a chemical part 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 (such as 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-) substitution.

[0118] Unless otherwise stated, the terms “coupled” or “connected” as used in this specification are intended to cover direct connection or connection via one or more intermediate means.

[0119] The term "related" as used in this article to refer to two elements indicates a broad relationship between them. This relationship includes, but is not limited to, physical, chemical, or biological relationships. For example, when element A is related to element B, elements A and B may be directly or indirectly connected to each other, or element A may contain element B, or vice versa.

[0120] As used herein, the term “adjacent” means, when referring to two elements, that one element is closely adjacent to another element, and may include, but is not limited to, elements in contact with each other, or may further include elements separated by one or more other elements placed between them.

[0121] The term “and / or”, for example, “X and / or Y”, is understood to mean “X and Y” or “X or Y”, and should be understood to provide explicit support for both meanings or either meaning.

[0122] Furthermore, throughout this specification, the word “substantially” is always understood to include, but is not limited to, “completely” or “thoroughly”. Additionally, terms such as “comprising” and “including” are always intended as non-restrictive descriptive language, as they broadly include elements / groups listed after such terms, in addition to other components not explicitly listed. For example, when “comprising” is used, a reference to “one” feature is also intended to refer to “at least one” of that feature. Terms such as “consisting” and “consist” can be considered, in appropriate context, as a subset of terms such as “comprising” and “including”. Therefore, in the disclosed embodiments using terms such as “comprising” and “including”, it should be understood that these embodiments provide instruction for corresponding embodiments using terms such as “consisting” and “consist”. Furthermore, terms such as “about” and “approximately” generally refer to reasonable deviations, such as ±5% of the disclosed value, or 4% of the disclosed value, or 3% of the disclosed value, or 2% of the disclosed value, or 1% of the disclosed value.

[0123] Furthermore, certain values ​​may be disclosed in the form of ranges in this specification. The values ​​showing the endpoints of a range are intended to indicate a preferred range. Whenever a range is described, it is intended to encompass and teach all possible subranges within that range, as well as individual numerical values. That is, the endpoints of a range should not be interpreted as rigid limitations. For example, a description of a range of 1% to 5% is intended to explicitly disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, and individual values ​​within that range, such as 1%, 2%, 3%, 4%, and 5%. The intent of the specific disclosures above is applicable to ranges of any depth / breadth.

[0124] Furthermore, in describing certain embodiments, this disclosure may have disclosed methods and / or processes in a specific order of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific order of steps disclosed. Other orders of steps may be 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 performing the steps in the order they are written. The order of steps may vary and remains within the scope of this disclosure.

[0125] 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 supports such omissions and these related alternative embodiments.

[0126] It should also be understood that if priority is claimed over an earlier application, the entire contents of the earlier application should also be considered as part of this disclosure and may serve as support for the implementation schemes disclosed herein.

[0127] Description of the implementation plan

[0128] The following discloses exemplary, non-limiting embodiments of compounds for preparing lipid nanoparticles for encapsulating pharmaceutical agents, methods for preparing said compounds, nanoparticle compositions comprising said compounds, and related methods / uses thereof.

[0129] compound

[0130] Compounds for preparing lipid nanoparticles are provided. In various embodiments, the compounds comprise one or more peptide units / blocks and / or derivatives thereof. For example, the compounds may comprise one or more oligopeptides, polypeptides / polyamino acids, and / or derivatives thereof. In various embodiments, the total number of peptide units / blocks and / or derivatives thereof in the compound (or the total length of oligopeptides, polypeptides / polyamino acids, and / or derivatives thereof) can be adjusted as needed. Advantageously, in various embodiments, the compounds are designed / constructed to allow the hydrophilic / hydrophobic balance of the compounds to be customized / adjusted by adjusting the number of peptide units / blocks and / or derivatives thereof (or the length of oligopeptides, polypeptides / polyamino acids, and / or derivatives thereof). In various embodiments, the compounds comprise hydrophobic tails / chains / groups. For example, the compounds may comprise one or more straight-chain aliphatic hydrocarbons, branched-chain aliphatic hydrocarbons, and / or cyclic hydrocarbons. In various embodiments, the total length of hydrophobic tails / chains / groups in the compound can be adjusted as needed. Advantageously, in various embodiments, the compound is also designed / constructed to allow the hydrophilicity / hydrophobicity balance of the compound to be customized / tuned by adjusting the length of the hydrophobic tail / chain / group. In various embodiments, the peptide unit / block and / or its derivatives are hydrophilic. Advantageously, the presence of the peptide unit / block and / or its derivatives increases the hydrophilicity of the compound and thus increases its solubility. Even more advantageously, the structure of the compound allows for its use in / formation into nanoparticles in a composition, which can be used as encapsulation / loading agents, delivery media / systems, and / or transfection media / systems. In various embodiments, the design of the compound helps prevent nonspecific protein uptake, particle aggregation, and control the size of the formed nanoparticles. In various embodiments, the configuration of the compound helps maintain the colloidal stability (of the nanoparticles) and facilitates molecular / cargo condensation and encapsulation / loading into the nanoparticle composition. In various embodiments, the compound is designed / constructed to allow loading / encapsulation of one or more types of molecules or cargoes. In various embodiments, the compound is also designed / constructed to allow the loaded / encapsulated agent to be released from the composition containing the compound and / or subsequently delivered to a desired target (e.g., cells, cytosol, tissue, or organ). The molecules / cargo to be loaded / encapsulated onto / inside / in the composition containing the compound may include, but are not limited to, therapeutic agents, prophylactic agents, biological agents, etc. In various embodiments, the molecules / cargo to be loaded / encapsulated comprise nucleic acids.For example, the molecule / cargo to be loaded / encapsulated may be a nucleic acid selected from ribonucleic acid (RNA), messenger ribonucleic acid (mRNA), microRNA (miRNA), 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 / cargo to be loaded / encapsulated comprises a therapeutic agent, for example, a negatively charged therapeutic agent. For example, the molecule / cargo to be loaded / encapsulated may be a therapeutic agent selected from drug molecules, vaccines (e.g., dengue fever vaccine, Covid-19 vaccine), or combinations thereof. Advantageously, the compound is suitable for formulation into nanoparticles 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). Advantageously, the compound is suitable for use in compositions / nanoparticle compositions for delivering one or more therapeutic agents, prophylactic agents, and / or biological agents.

[0131] Therefore, in various embodiments, supports, nanocarriers, or delivery systems / media comprising the compound are also provided.

[0132] In various embodiments, the compound comprises a structure represented by general formula (1):

[0133]

[0134] in

[0135] A R It contains units derived from polyamino acids / peptides / oligopeptides;

[0136] R 1 and R 2 Each is independently a hydrophobic tail / chain / group or contains at least a straight-chain aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon;

[0137] R 3 R 4 and R 5 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group;

[0138] R 7 It is -H or -C(=O)R 8 , where R 8 It is an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, an alkynyl group that is optionally substituted, or an alkoxy group that is optionally substituted;

[0139] m≥1; and

[0140] n≥1.

[0141] In the various implementation schemes where n≥2, A R They can be the same or different. For example, when n=20 and there are a total of 20 A's. R At that time, there are 20 A's in the structure represented by general formula (1). R Each of them can be the same as or different from each other.

[0142] In various embodiments, the compound comprises peptides / polyamino acids / oligopeptides and / or derivatives thereof, such as block peptides and / or derivatives thereof. In various embodiments, the peptide / polyamino acid / oligopeptide and / or derivative components / parts of the compound are composed of -[A R ] n -represent.

[0143] In various implementation schemes, A R A repeating unit containing polyamino acids, peptides, or oligopeptides. Repeating unit A of polyamino acids, peptides, or oligopeptides. R It can be an amino acid residue, which is the remainder / residue of amino acids when they react to form a peptide, while removing a water molecule (H2O). In various embodiments, A R Includes the structure represented by general formula (2):

[0144]

[0145] in

[0146] R 6 It is H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

[0147] In various embodiments, A comprises a hydrophilic and / or polar portion. In various embodiments, A comprises a side chain of an amino acid residue. In various embodiments, A comprises an organic group, which is a portion of an amino acid represented by general formula (3):

[0148]

[0149] In various embodiments, the amino acid comprises an α-amino acid. In various embodiments, the α-amino acid comprises an amino group (-NH2) and a carboxyl group (-COOH) attached to the same carbon atom.

[0150] In various embodiments, the amino acid comprises one or more hydrophilic groups. For example, the amino acid may comprise 1, 2, 3, 4, or 5 hydrophilic groups. The hydrophilic groups may be -OH, -C(=O)OH, and / or -NR. x R y , where R xand R y Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or combinations thereof. It should be understood that various hydrophilic and / or polar moieties can be used as A represented by general formula (2). R In implementation scheme A, the hydrophilic and / or polar portion can impart hydrophilicity as an alternative form of PEG.

[0151] In various embodiments, the amino acid is selected from serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, etc., and combinations thereof. In various embodiments, A is selected from -CH2OH, -CH2CH2C(=O)OH, or combinations thereof. In various embodiments, the polyamino acid or polypeptide / oligopeptide is selected from polyserine, polyglutamic acid, etc., and combinations thereof.

[0152] Advantageously, the presence of peptide units / blocks and / or their derivatives (or oligopeptides, polypeptides / polyamino acids and / or their derivatives) increases the hydrophilicity of the compound and thus increases its solubility. Advantageously, in various embodiments, the presence of peptide units / blocks and / or their derivatives (or oligopeptides, polypeptides / polyamino acids and / or their derivatives) in the compound eliminates the need for hydrophilic polyethylene glycol (PEG), which is otherwise essential in conventional PEG-lipid conjugates used in LNP formulations. In various embodiments, by eliminating the presence of polyethylene glycol (PEG) in the compound, embodiments of the compound and the lipid nanoparticles formed therefrom are not shielded by long PEG chains and / or avoid this possibility. In various embodiments, the compound is substantially free of polyethylene glycol (PEG). In various embodiments, the compound is substantially free of polyethylene glycol (PEG)-modified lipid conjugates, polyethylene glycol (PEG)-modified lipids, polyethylene glycol-treated lipids, PEG-conjugated lipids, PEG-lipid conjugates and / or lipids modified with PEG. Advantageously, in various embodiments, the structure of the compound is designed to allow it to be used as a substitute or replacement for conventional PEG-lipid conjugates (e.g., ALC-0159).

[0153] In various implementation schemes, A R That is, the structural / repetitive unit represented by general formula (2) can exist in the form of L-enantiomers (e.g., L-amino acids and their derivatives) and / or D-enantiomers (e.g., D-amino acids and their derivatives):

[0154]

[0155] In various implementation schemes, A RIt contains L-amino acids and / or their derivatives. In various embodiments, A R It contains D-amino acids and / or their derivatives. In various embodiments, A R The compound comprises a mixture of two enantiomers: an L-amino acid and / or its derivatives; and a D-amino acid and / or its derivatives. In various embodiments, the compound comprises an L-enantiomer and / or a D-enantiomer randomly distributed within / in the structural / repeating units of the compound.

[0156] In various embodiments, the compound comprises a mixture containing L-enantiomers and D-enantiomers (e.g., a racemic mixture of enantiomers). For example, the compound / polyamino acid / peptide / oligopeptide may contain repeating units or blocks of L-enantiomers and D-enantiomers (e.g., poly(D,L-amino acid) or poly(L,D-amino acid). In such embodiments, A R That is, the structural / repeating unit represented by general formula (2) contains the structure represented by general formula (2A) and / or (2B):

[0157]

[0158] in

[0159] p + q = n;

[0160] R 6’ =R 6’’ =R 6 ;and

[0161] A contains one or more features and / or shares one or more properties similar to those described above (e.g., as defined in general formula (3)).

[0162] In various embodiments, the compound represented by general formula (1) comprises a structure represented by general formula (1A) and / or (1B):

[0163]

[0164]

[0165] Where R 1 R 2 R 3 R 4 R 5 R 6’ R 6’’ R 7A, m, p, q contain one or more features and / or share one or more properties similar to those described above (e.g., as defined in general formulas (1) and (3)).

[0166] It should be understood that, in the various embodiments of the compounds disclosed herein, L-enantiomers and D-enantiomers are randomly distributed within / in the structural / repeating units of the compound. In various embodiments, the compound / polyamino acid / peptide / oligopeptide is a random copolymer or a block copolymer.

[0167] Advantageously, in various embodiments, the compound is designed / constructed to allow the hydrophilicity / hydrophobicity balance of the compound to be customized as follows: by adjusting R 1 and R 2 The length or hydrophobicity of n, and / or the number of peptide units / blocks and / or their derivatives (or the length of oligopeptides or polypeptides / polyamino acids and / or their derivatives), that is, by adjusting / changing / regulating the value of n and / or controlling the degree of polymerization. It should be understood that in various embodiments, the hydrophilicity of the compound can be increased by increasing the value of n and / or the degree of polymerization, and vice versa.

[0168] 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, or n≥60. In some implementations, n≤45, n≤44, n≤43, n≤42, n≤41, n≤40, n≤39, n≤38, n≤37, n≤36, n≤35, n≤34, n≤33, or n≤32. For example, 1 ≤ n ≤ 60, 5 ≤ n ≤ 60, 1 ≤ n ≤ 40, 1 ≤ n ≤ 32, 18 ≤ n ≤ 40, or 18 ≤ n ≤ 32. In various embodiments, n is about 1 to about 60, about 5 to about 60, about 1 to about 40, about 1 to about 32, about 18 to about 40, or about 18 to about 32. In some embodiments, n is no more than about 60, no more than about 50, no more than about 48, no more than about 45, no more than about 44, no more than about 43, no more than about 42, no more than about 41, no more than about 40, no more than about 39, no more than about 38, no more than about 37, no more than about 36, no more than about 35, no more than about 34, no more than about 33, no more than about 32, no more than about 31, or no more than about 30.

[0169] Advantageously, in various embodiments, the compound is designed / constructed to allow for adjustment of the length of the hydrophobic tail / chain / group (e.g., in R...). 1 and / or R 2 The hydrophilicity / hydrophobicity balance of the compound can be customized / adjusted by (the length of the hydrophobic tail / chain / group at R). It should be understood that, in various embodiments, this can be achieved by increasing the length of the hydrophobic tail / chain / group at R. 1 and / or R 2 The length of the hydrophobic tail / chain / group at the site can be used to increase the hydrophobicity of the compound, and vice versa.

[0170] In various implementation schemes, R 1 and R 2 Each is independently a hydrophobic tail / chain / group, or contains at least a straight-chain aliphatic hydrocarbon, a branched aliphatic hydrocarbon, and / or a cyclic hydrocarbon. In various embodiments, in R... 1 and R2 The hydrophobic tail / chain / group at each location independently comprises an optionally substituted alkyl group. In various embodiments, at R 1 and R 2 The hydrophobic tail / chain / group at each location independently contains unsaturated hydrocarbons such as optionally substituted alkenyl groups. For example, R 1 and R 2 It may contain one or more C=C double bonds. The alkyl or alkenyl group may have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 carbon atoms. For example, R 1 and R 2 Each can be C independently x H 2x+1 Or C x H 2x , where x is an integer ≥ 8, x ≥ 9, x ≥ 10, x ≥ 11, x ≥ 12, x ≥ 13, x ≥ 14, x ≥ 15, x ≥ 16, x ≥ 17, x ≥ 18, x ≥ 19, x ≥ 20, x ≥ 21, x ≥ 22, x ≥ 23, x ≥ 24, x ≥ 25, x ≥ 26, x ≥ 27, x ≥ 28, x ≥ 29, x ≥ 30, x ≥ 31, x ≥ 32, x ≥ 33, x ≥ 34, x ≥ 35, or x ≥ 36. In some implementations, in R 1 and R 2 The hydrophobic tail / chain / group at each location independently comprises an alkyl or alkenyl group having no more than 36 carbon atoms, no more than 35 carbon atoms, no more than 34 carbon atoms, no more than 33 carbon atoms, no more than 32 carbon atoms, no more than 31 carbon atoms, no more than 30 carbon atoms, no more than 29 carbon atoms, no more than 28 carbon atoms, no more than 27 carbon atoms, no more than 26 carbon atoms, no more than 25 carbon atoms, no more than 24 carbon atoms, no more than 23 carbon atoms, no more than 22 carbon atoms, no more than 21 carbon atoms, no more than 20 carbon atoms, no more than 19 carbon atoms, or no more than 18 carbon atoms. For example, R 1 and R 2 Each can be C independently x H 2x+1 Or C x H 2xWhere 1 ≤ x ≤ 36, 5 ≤ x ≤ 30, 5 ≤ x ≤ 20, or 8 ≤ x ≤ 18. Advantageously, in various embodiments, the presence of the hydrophobic portion / tail / chain / group in the compound allows the compound to be readily integrated into the lipid domains of lipid nanoparticles (LNPs), thereby presenting polyamino acid / peptide domains on the surface of the LNP to improve stability and cell targeting ability (e.g., immune cell targeting ability).

[0171] In various implementation schemes, m is an integer ≥ 1. In various implementation schemes, m≥1, m≥2, m≥3, m≥4, m≥5, m≥6, m≥7, m≥8, m≥9, m≥10, m≥11, m≥12, m≥13, m≥14, m≥15, m≥16, m≥17, m≥18, m≥19, m≥20, m≥21, m≥22, m≥23, m≥24, m≥25, m≥26, m≥27, m≥28, m≥29, m≥30, m≥31, m≥32, m≥33, m≥34, m≥35, m≥36, m≥37, m≥38, m≥39, m≥40, m≥41, m≥42, m≥43, m≥44, m≥45, m≥46, m≥47, m≥48, m≥49, or m≥50.

[0172] In various implementations, m=1. In such implementations, the compound represented by general formula (1) comprises a structure represented by general formula (1C):

[0173]

[0174] In various embodiments, the compound comprises a lipid compound. Therefore, in various embodiments, the term "compound" may include the terms "lipopeptide," "lipid-peptide," "peptide-lipid," "peptide-lipid," "lipid-block-peptide," etc., and / or may be used interchangeably therewith.

[0175] In various implementation schemes, R 3 R 4 R 5 and R 6 Each is independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl. In various embodiments, R 3 R 4 R 5 and R 6 Each is independently an alkyl group that has been optionally substituted, an alkenyl group that has been optionally substituted, or an alkynyl group that has been optionally substituted. For example, R 3 R 4 R 5 R 6 and R8 It 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. In various embodiments, R 3 and R 4 It is H. In various implementation schemes, R 5 It is H. In various implementation schemes, R 6 It's H.

[0176] In various implementation schemes, R 8 It is an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, an alkynyl group that is optionally substituted, or an alkoxy group that is optionally substituted. For example, R 8 It 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.

[0177] In various embodiments, the compound has concentrations of about 500 g / mol to about 10,000 g / mol, about 750 g / mol to about 9,750 g / mol, about 1,000 g / mol to about 9,500 g / mol, about 1,250 g / mol to about 9,250 g / mol, about 1,500 g / mol to about 9,000 g / mol, about 1,750 g / mol to about 8,750 g / mol, about 2,000 g / mol to about 8,500 g / mol, about 2,250 g / mol to about 8,250 g / mol, about 2,500 g / mol to about 8,000 g / mol, about 2,750 g / mol to about 7,750 g / mol, about 3,000 g / mol to about 7,500 g / mol, about 3,250 g / mol to about 7,250 g / mol, about 3,500 g / mol, about 3,500 g / mol, about 1,000 g / mol, about 1,250 g / mol to about 9,250 g / mol, about 1,500 g / mol, about 1,750 g / mol, about 1,0 ... Number average molecular weight (Mg) from about 7,000 g / mol to about 7,000 g / mol, from about 3,750 g / mol to about 6,750 g / mol, from about 4,000 g / mol to about 6,500 g / mol, from about 4,250 g / mol to about 6,250 g / mol, from about 4,500 g / mol to about 6,000 g / mol, from about 4,750 g / mol to about 5,750 g / mol, or from about 5,000 g / mol to about 5,500 g / mol, or from about 5,250 g / mol. n ).

[0178] In various embodiments where the amino acid comprises serine, A is -CH2OH and the compound is represented by general formula (4):

[0179]

[0180] In various embodiments, the compound represented by general formula (4) may comprise structures represented by general formula (4A) and / or (4B):

[0181]

[0182]

[0183] In various embodiments, lipid nanoparticles (LNPs) comprising a lipid composition comprising a lipid-polyserine represented by general formula (11) are provided:

[0184]

[0185] in

[0186] R 7It is H, C(=O)CH3 or C(=O)CH2CH3;

[0187] n is from 1 to 100 (e.g., 18, 21, 27, 32, 45); and

[0188] Nucleic acid (e.g., mRNA) encapsulated in the lipid composition,

[0189] The lipid composition therein does not contain PEG.

[0190] In various embodiments, the LNP comprises repeating units / segments of L- and D-enantiomers (i.e., lipid-block-poly(D,L-serine)) and is represented by general formula (12):

[0191]

[0192] in

[0193] R 7 It is H, C(=O)CH3 or C(=O)CH2CH3;

[0194] p + q is 1 to 100 (e.g., 18, 21, 27, 32, 45).

[0195] In various embodiments, the compound comprises one or more structures selected from the following:

[0196]

[0197] pDLS1 or C14-pDLS1 (n=18)

[0198]

[0199] pDLS2 or C14-pDLS2 (n=18)

[0200]

[0201] pDLS3 or C14-pDLS3 (n=21)

[0202]

[0203] pDLS4 or C14-pDLS4 (n=27)

[0204]

[0205] pDLS5 or C14-pDLS5 (n=32)

[0206]

[0207] pDLS6 or C14-pDLS6 (n=45)

[0208]

[0209] C18-pDLS (n=30)

[0210]

[0211] C12-pDLS (n=32)

[0212]

[0213] C8-pDLS (n=34).

[0214] In various embodiments, the polyserine in C14-pDLS1, C14-pDLS2, C14-pDLS3, C14-pDLS4, C14-pDLS5, C14-pDLS6, C18-pDLS, C12-pDLS, and C8-pDLS is poly(D,L-serine) and may be replaced by poly(L-serine), poly(D-serine), and / or poly(L,D-serine). In various embodiments, the polyserine in C14-pDLS1, C14-pDLS2, C14-pDLS3, C14-pDLS4, C14-pDLS5, C14-pDLS6, C18-pDLS, C12-pDLS, and C8-pDLS may also be replaced by polyglutamic acid. In various implementation schemes, one or more of C14-pDLS1, C14-pDLS2, C14-pDLS3, C14-pDLS4, C14-pDLS5, C14-pDLS6, C18-pDLS, C12-pDLS, and C8-pDLS are used. Groups can be Group substitution.

[0215] Methods for preparing compounds

[0216] A method for preparing compounds represented by general formula (1) as disclosed herein is provided, the method comprising:

[0217] (i) Polymerizing / reacting one or more N-carboxylic anhydride (NCA) monomers represented by general formula (5) with a lipid initiator represented by general formula (6) to obtain a first intermediate compound represented by general formula (7):

[0218]

[0219]

[0220] in

[0221] A PG A represents A functionalized / protected with one or more protecting groups, wherein A contains a hydrophilic organic group that is part of an amino acid represented by general formula (3);

[0222] R 11 and R 12 Each is an independent hydrophobic group;

[0223] R 13 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;

[0224] m≥1; and

[0225] n≥1;

[0226] (ii) Optionally, the first intermediate compound represented by general formula (7) is reacted with an acylating agent to obtain the second intermediate compound represented by general formula (8):

[0227]

[0228] in

[0229] R 17 It is -H or -C(=O)R 18 , where R 18 It is an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, an alkynyl group that is optionally substituted, or an alkoxy group that is optionally substituted;

[0230] (iii) Deprotect the first intermediate compound represented by general formula (7) and / or the second intermediate compound represented by general formula (8) to obtain the compound represented by general formula (1).

[0231] In various embodiments, the polymerization / reaction step (i) comprises ring-opening polymerization of the NCA ring / group in a compound represented by general formula (5). It should be understood that in step (i), one or more NCA monomers (i.e., represented by general formula (5)) may be simultaneously added to an initiator to polymerize and form a random copolymer polypeptide, or an initiator may be added sequentially to form a block copolymer polypeptide.

[0232] In various embodiments, the lipid initiator comprises a primary amine group.

[0233] In various implementation schemes, A PGThis represents A, which is functionalized / protected with one or more protecting groups. In various embodiments, the protecting group is benzyl, tert-butyl, or a combination thereof. For example, one or more hydroxyl groups (i.e., -OH) in A can be functionalized / protected with protecting groups and converted into a state where A... PG -OBn or -O t Bu. In various implementation schemes, A PG Contains benzyl (Bn) groups, tert-butyl ( t A protected / functionalized by groups such as Bu or combinations thereof. For example, A PG It can be -CH2O- t Bu、-CH2O-Bn、-CH(CH3)O- t Bu or -CH(CH3)O-Bn. In various implementations, A PG Contains -CH2O-Bn:

[0234]

[0235] In various implementation schemes, R 11 To R 17 Contains one or more features and / or shares one or more features with respect to R as described above. 1 To R 7 The similar performance described above. In various implementations, w, m, and n contain one or more features and / or share one or more similar performance characteristics to those described above.

[0236] In various embodiments, step (ii) comprises reacting a first intermediate compound represented by general formula (7) with an acylating agent to react R 17 Transform from –H to -C(=O)R 18 .

[0237] In various embodiments, the acylating agent (in step (ii)) is selected from acid anhydrides (e.g., acetic anhydride (Ac2O)), acyl halides (e.g., acyl halides), N-hydroxysuccinimide (NHS) esters, imine esters, and combinations thereof. In various embodiments, the acylating agent is represented by general formula (9):

[0238]

[0239] Where R 18 and R 18’ Each is independently an alkyl group that has been optionally substituted, an alkenyl group that has been optionally substituted, or an alkynyl group that has been optionally substituted. In various embodiments, R 18 and R 18’Each is independently 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. In various embodiments, R 18 and R 18’ Contains one or more features and / or shares one or more features with respect to R as described above. 8 Those described (e.g., those described in general formula (1)) have similar performance.

[0240] In various embodiments, the deprotection / deprotection step (iii) of the first intermediate compound represented by general formula (7) and / or the second intermediate compound represented by general formula (8) comprises deprotecting / removing one or more protecting groups (e.g., benzyl or tert-butyl) from the intermediate compound. In various embodiments, the deprotection / deprotection step (iii) comprises subjecting the first intermediate compound represented by general formula (7) and / or the second intermediate compound represented by general formula (8) to acidic conditions. For example, the deprotection step (iii) may be carried out in the presence of one or more acids (e.g., trifluoroacetic acid (TFA), hydrobromic acid (HBr), and acetic acid (AcOH)).

[0241] In various embodiments, the NCA monomer is derived from a protected amino acid. In various embodiments, the method further includes, prior to step (i):

[0242] (ai) Reaction of the protected amino acid represented by general formula (10) with a carbonylating agent to obtain the amino acid represented by general formula (5) - N -Carboxylic anhydride (NCA):

[0243]

[0244] In various embodiments, the reaction step (ai) comprises cyclizing the protected amino acid represented by general formula (10), for example, to obtain a cyclic product, namely the amino acid represented by general formula (5). N-Carboxylic anhydride (NCA). In various embodiments, the carbonylating agent used in step (ai) is selected from phosgene, diphosgene, triphosgene, etc., or combinations thereof. It should be understood that in various embodiments, the protected amino acid represented by general formula (10) contains -NH2 and -COOH to allow cyclization to occur in order to obtain / prepare NCA prior to ring-opening polymerization.

[0245] In various embodiments, the polymerization step (i) comprises suspending / dispersing / mixing / dissolving one or more N-carboxylic anhydride (NCA) monomers represented by general formula (5) in a molar ratio of about 5:1 to about 70:1, about 5:1 to about 65:1, about 5:1 to about 60:1, about 10:1 to about 55:1, about 15:1 to about 50:1, about 20:1 to about 45:1, about 25:1 to about 40:1, or about 30:1 to about 35:1 with a lipid initiator / molecule represented by general formula (6).

[0246] In various embodiments, the one or more N-carboxylic anhydride (NCA) monomers (i.e., represented by general formula (5)) comprise structures represented by general formula (5A) and / or (5B):

[0247]

[0248] In various implementation schemes, A PG It contains one or more features and / or shares one or more performance characteristics similar to those described above.

[0249] In various embodiments, the one or more N-carboxylic anhydride (NCA) monomers comprise an L-enantiomer (e.g., an L-protected amino acid-NCA) or may be present in the form of an L-enantiomer (e.g., an L-protected amino acid-NCA). In various embodiments, the one or more N-carboxylic anhydride (NCA) monomers comprise a D-enantiomer (e.g., a D-protected amino acid-NCA) or may be present in the form of a D-enantiomer (e.g., a D-protected amino acid-NCA). In various embodiments, the one or more N-carboxylic anhydride (NCA) monomers comprise a mixture of L-enantiomers and D-enantiomers (e.g., a racemic mixture). For example, the one or more N-carboxylic anhydride (NCA) monomers may comprise a mixture of L-protected amino acid-NCA and D-protected amino acid-NCA or may be present in the form of a mixture of L-protected amino acid-NCA and D-protected amino acid-NCA. In some embodiments, the NCA monomer comprises a racemic mixture, for example, o-benzyl-L-serine-N-carboxylic anhydride and o-benzyl-D-serine-N-carboxylic anhydride. In various embodiments, the lipid-peptide is formed from a mixture of L-serine-NCA and D-serine-NCA (e.g., a racemic mixture).

[0250] In various embodiments, in step (i), general formula (5A) is mixed with general formula (5B) in a molar ratio of about 1:5 to about 5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, about 1:2 to about 2:1, or about 1:1. Advantageously, the method is designed to allow the hydrophilicity / hydrophobicity (or the value of n and / or the degree of polymerization) of the compound to be adjusted / customized / adjusted by changing the amount of N-carboxylic anhydride (NCA) monomers (e.g., general formula (5A) and / or general formula (5B)).

[0251] In various embodiments, the protected amino acid comprises a structure represented by general formula (10A) and / or (10B):

[0252]

[0253] In various implementation schemes, A PG It contains one or more features and / or shares one or more performance characteristics similar to those described above.

[0254] In various embodiments, the protected amino acid represented by general formula (10) comprises an L-enantiomer (e.g., an L-protected amino acid) or may be present in the form of an L-enantiomer (e.g., an L-protected amino acid). In various embodiments, the protected amino acid represented by general formula (10) comprises a D-enantiomer (e.g., a D-protected amino acid) or may be present in the form of a D-enantiomer (e.g., a D-protected amino acid). In various embodiments, the protected amino acid represented by general formula (10) comprises a mixture of L-enantiomers and D-enantiomers (e.g., a racemic mixture). For example, the protected amino acid represented by general formula (10) may comprise a mixture of L-protected amino acids and D-protected amino acids or be present in the form of a mixture of L-protected amino acids and D-protected amino acids. In some embodiments, the protected amino acid comprises o-benzyl-L-serine and / or o-benzyl-D-serine.

[0255] In various embodiments, the first intermediate compound represented by general formula (7) comprises repeating units or blocks of L-enantiomers and / or D-enantiomers (e.g., poly(D-protected amino acid), poly(L-protected amino acid), poly(D,L-protected amino acid), or poly(L,D-protected amino acid)). For example, the first intermediate compound represented by general formula (7) may comprise structures represented by general formulas (7A) and / or (7B) or be present in the form of structures represented by general formulas (7A) and / or (7B):

[0256]

[0257] In various embodiments, the second intermediate compound represented by general formula (8) comprises repeating units or blocks of L-enantiomers and / or D-enantiomers (e.g., poly(D-protected amino acid), poly(L-protected amino acid), poly(D,L-protected amino acid), or poly(L,D-protected amino acid)). For example, the second intermediate compound represented by general formula (8) may comprise structures represented by general formulas (8A) and / or (8B) or be present in the form of structures represented by general formulas (8A) and / or (8B):

[0258]

[0259] It should be understood that in various embodiments of the first intermediate compound and / or the second intermediate compound disclosed herein, the L-enantiomer (e.g., ) and D-enantiomers (e.g., The compounds are randomly distributed within the structural / repeating units of the compound. Therefore, in various embodiments, the compounds / polyamino acids / peptides / oligopeptides designed / prepared according to the various embodiments disclosed herein are random copolymers or block copolymers. In various embodiments, the lipid-poly(protected amino acid) comprises repeating units / segments of L- and D-enantiomers, i.e., lipid-block-poly(D,L-protected amino acid). In various embodiments, the lipid-poly(o-benzyl-serine) comprises repeating units / segments of L- and D-enantiomers, i.e., lipid-block-poly(o-benzyl-D,L-serine).

[0260] In various embodiments, the polymerization / reaction / deprotection steps (i), (ii), (iii) and / or (ai) include one or more of the following steps: suspension, dispersion, mixing, stirring, dissolving, acoustic treatment and / or ultrasonic treatment.

[0261] In various embodiments, the polymerization / reaction / deprotection steps (i), (ii), (iii), and / or (ai) are carried out in the presence of an organic solvent. In various embodiments, any organic solvent that effectively serves as a medium for containing the components (e.g., reactants / substrates) of the reaction mixture can be used in the embodiments of the reaction mixtures disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the reaction mixture. The organic solvent can be an organic solvent such as dichloromethane (DCM), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), acetonitrile, ethyl acetate, dimethylformamide (DMF), etc., or combinations thereof. In various embodiments, the organic solvent can be provided in a dry or anhydrous form. For example, the polymerization / reaction step (i) may comprise suspending, dispersing, mixing, stirring, dissolving, acoustically treating, and / or sonicating one or more N-carboxylic anhydride (NCA) monomers represented by general formula (5) with a lipid initiator represented by general formula (6) in the presence of a dry / anhydrous organic solvent. For example, the reaction step (ai) may involve suspending, dispersing, mixing, stirring, dissolving, acoustically treating and / or sonicating the protected amino acid represented by general formula (10) in the presence of a dry / anhydrous organic solvent.

[0262] In various embodiments, the polymerization / reaction / deprotection steps (i), (ii), (iii), and / or (ai) are carried out under vacuum or in an inert atmosphere. For example, steps such as suspension, dispersion, mixing, stirring, dissolving, acoustic treatment, and / or ultrasonic treatment may be carried out in a glove box in the presence of an inert gas (such as argon or nitrogen) or in the absence of a reactive gas such as oxygen (e.g., dissolved oxygen).

[0263] In various embodiments, the polymerization / reaction / deprotection steps (i), (ii), (iii), and / or (ai) are carried out for a duration of about 1 hour to about 200 hours, about 1 hour to about 190 hours, about 10 hours to about 180 hours, about 20 hours to about 170 hours, about 30 hours to about 160 hours, about 40 hours to about 150 hours, about 50 hours to about 140 hours, about 60 hours to about 130 hours, about 70 hours to about 120 hours, about 80 hours to about 110 hours, about 90 hours to about 100 hours, or about 95 hours. The polymerization / reaction / deprotection steps (i), (ii), (iii), and / or (ai) may also be carried out for a duration of about 1 hour to about 72 hours, about 2 hours to about 60 hours, about 3 hours to about 48 hours, about 4 hours to about 36 hours, about 5 hours to about 24 hours, or about 6 hours to about 12 hours.

[0264] In various embodiments, the polymerization / reaction / deprotection steps (i), (ii), (iii), and / or (ai) are carried out at temperatures ranging from about 10.0°C to about 100.0°C, from about 20.0°C to about 90.0°C, from about 30.0°C to about 80.0°C, from about 40.0°C to about 70.0°C, from about 50.0°C to about 60.0°C, or about 55.0°C. In various embodiments, the polymerization / reaction / deprotection steps (i), (ii), (iii), and / or (ai) are carried out at room temperature, for example, from about 20°C to about 30°C, from about 21°C, from about 22°C, from about 23°C, from about 24°C, from about 25°C, from about 26°C, from about 27°C, from about 28°C, from about 29°C, or from about 30°C.

[0265] In various embodiments, the polymerization / reaction / deprotection steps (i), (ii), (iii), and / or (ai) are optionally carried out at temperatures of about -10°C to about 10°C, about -9°C to about 9°C, about -8°C to about 8°C, about -7°C to about 7°C, about -6°C to about 6°C, about -5°C to about 5°C, about -4°C to about 4°C, about -3°C to about 3°C, about -2°C to about 2°C, about -1°C to about 1°C, or 0°C, for example, to control reaction kinetics. For example, the reaction steps can be carried out in an ice bath.

[0266] In various embodiments, the deprotection step (iii) is performed at a temperature of about -20.0°C to about 50.0°C, about -10.0°C to about 40.0°C, about 0.0°C to about 30.0°C, about 10.0°C to about 20.0°C, or about 15.0°C. For example, the deprotection step can be performed in an ice bath.

[0267] In various implementations, the method further includes:

[0268] (bi) The step of separating the first intermediate compound represented by general formula (7) after step (i);

[0269] (b-ii) The step of separating the second intermediate compound represented by general formula (8) after step (ii);

[0270] (b-iii) The step of separating the compound represented by general formula (1) after step (iii); and / or

[0271] (b-iv) Step (ai) to separate the N-carboxylic anhydride (NCA) monomer represented by general formula (5).

[0272] In various embodiments, the separation step comprises one or more of the following steps: redissolving, purifying, centrifuging, quenching, washing, precipitating, filtering, and / or recrystallizing a first intermediate compound represented by general formula (7), a second intermediate compound represented by general formula (8), a compound represented by general formula (1), and / or an N-carboxylic anhydride (NCA) monomer represented by general formula (5). In various embodiments, the separation step is performed to remove byproducts from the first intermediate compound represented by general formula (7), the second intermediate compound represented by general formula (8), the compound represented by general formula (1), and / or the N-carboxylic anhydride (NCA) monomer represented by general formula (5).

[0273] In various embodiments, the steps of purification, centrifugation, quenching, recrystallization, and / or washing are performed 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, at least eight times, at least nine times, at least ten times, at least fifteen times, or at least twenty times using a washing medium. In various embodiments, the steps of purification, centrifugation, recrystallization, and / or washing are typically / frequently repeated at least three times.

[0274] In various embodiments, the washing medium comprises an aqueous medium / solution such as a salt solution, deionized water, an acid, or a combination thereof. The salt solution may be a bicarbonate such as sodium bicarbonate, or a hydrochloride such as saturated sodium chloride (salt water). The acid may be citric acid. In various embodiments, the salt solution comprises a highly concentrated / saturated salt solution.

[0275] In various embodiments, the method further comprises one or more of the following post-reaction steps: optionally drying at low temperature (e.g., freeze-drying), under vacuum and / or in an inert atmosphere.

[0276] In various implementation schemes, the drying step is performed in the presence of a desiccant such as anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium sulfate, and anhydrous calcium chloride, or combinations thereof.

[0277] In various embodiments, the yields of compounds represented by general formula (1) are about 1.0% to about 100.0%, about 5.0% to about 99.0%, about 10.0% to about 98.0%, about 20.0% to about 97.0%, about 30.0% to about 96.0%, about 40.0% to about 95.0%, about 50.0% to about 90.0%, about 55.0% to about 85.0%, about 60.0% to about 80.0%, about 65.0% to about 70.0%, or about 75.0%. The yields of compounds represented by general formula (1) can be about 50.0% to about 98.0%.

[0278] Advantageously, the implementation of the method is simple and easy to carry out, and has low production / manufacturing costs (i.e., cost-effective), due to the mild reaction conditions and the absence of harsh and / or cumbersome steps. Advantageously, the implementation of the method includes simple purification steps (e.g., easy separation from byproducts, etc.) and synthesizes products in high yields. Advantageously, the implementation of the method is scalable and / or has a considerably high degree of scalability.

[0279] Nanoparticle Composition

[0280] Advantageously, in various embodiments, the structural design of the compound represented by general formula (1) enables the compound to replace or substitute for conventional lipid-PEG conjugates (e.g., ALC-0159) in the formulation of nanoparticles in the composition. In various embodiments, the formulation of the compound enables it to be formulated into nanoparticles in the composition. Advantageously, in various embodiments, the design of the compound represented by general formula (1) helps prevent nonspecific protein uptake, particle aggregation, and control the size of the formed nanoparticles. In various embodiments, the formulation of the compound represented by general formula (1) helps maintain the colloidal stability (of the lipid nanoparticles), promotes the aggregation and encapsulation / loading of molecules / cargo in the nanoparticle composition, and thereby delivers nucleic acids (e.g., mRNA), thus enhancing vaccination or therapeutic efficiency.

[0281] The term “nanoparticle” may include the terms “lipid nanoparticles”, “encapsulated lipid nanoparticles”, “loaded lipid nanoparticles”, “LNP”, “cell-targeting lipid nanoparticles”, “immune cell-targeting lipid nanoparticles” and / or may be used interchangeably with them.

[0282] A nanoparticle composition is provided, comprising:

[0283] (i) the compounds represented by general formula (1) disclosed herein; and

[0284] (ii) Encapsulating / loading therapeutic agents, preventative agents and / or biological agents in the composition.

[0285] Advantageously, the composition is suitable for encapsulating, delivering, and / or transfecting one or more therapeutic agents, preventative agents, and / or biological agents to, for example, desired targets (such as subjects, cells, cytosols, tissues, or organs).

[0286] In various embodiments, the composition further comprises:

[0287] (a) Ionizable lipids;

[0288] (b) Neutral / auxiliary lipids; and

[0289] (c) Cholesterol and / or its derivatives.

[0290] In various embodiments, the composition is substantially free of polyethylene glycol (PEG). In various embodiments, the composition is substantially free of PEG-modified lipid conjugates, PEG-modified lipids, PEG-conjugated lipids, PEG-lipid conjugates, and / or PEG-modified lipids. In various embodiments, the compound is a substitute / alternative to PEG-lipid conjugates.

[0291] In various embodiments, the compound, ionizable lipid, neutral / auxiliary lipid, and cholesterol and / or its derivatives are mixed / dissolved in an organic solvent. In various embodiments, the formation of lipid nanoparticles involves the self-assembly of the lipid component and one or more types of molecules or cargoes. In various embodiments, any organic solvent that effectively serves as a medium for containing the lipid component can be used in the embodiments of the lipid materials disclosed herein. In various embodiments, the organic solvent is capable of substantially dissolving the components present in the mixture. The organic solvent may comprise methanol, ethanol, isopropanol, acetonitrile, ethyl acetate, tetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc., or combinations thereof.

[0292] In various embodiments, the ionizable lipids, neutral / co-lipids, cholesterol and / or their derivatives, and compounds represented by general formula (1) are in the following proportions: about 5-65: about 4-20: about 10-60: about 0.1-20, about 10-60: about 6-18: about 15-55: about 0.2-18, about 15-55: about 7-17: about 20-50: about 0.5-16, about 20-50: about 8-16: about 25-45: about 1-14, about 25-45: about 9-15: about 30-40: about 1-, about 30-40: about 10-14: about 32-38: about 4-12 or about 33-37: about 11- A mixture of 13: approximately 34-36: approximately 6-10 molar ratios.

[0293] In various embodiments, the ionizable lipids include, but are not limited to, ALC-0315, SM-102, Lipid 5, DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, CL1, BP Lipid 310, ATX-001, ATX-100, Lipid 2, 80-O16B, BP Lipid 309, and BP Lipid 307, 93-O17S, 93-O17O, NT1-O14B, 306-O12B-3, 306-O12B, 113-O16B, 306Oi10, 30 6Oi9-cis2, BAMEA-O16B, Al-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-tritailed, C13-113-tritailed, C13-112-tetratailed or C13-113-tetratailed, C12-200 and combinations thereof. It should be understood that any suitable ionizable lipid capable of effectively adjusting / modifying / changing its charge according to ambient pH can be used in embodiments of the compositions disclosed herein. Therefore, any kind / type of ionizable lipid can be used with embodiments of the compounds disclosed herein (e.g., lipid-poly(D,L-serine), but not limited to those listed above).

[0294] In various embodiments, the composition comprises about 5.0 mol% to about 65.0 mol%, about 10.0 mol% to about 60.0 mol%, about 15.0 mol% to about 55.0 mol%, about 20.0 mol% to about 50.0 mol%, about 25.0 mol% to about 45.0 mol%, about 30.0 mol% to about 40.0 mol%, or about 35.0 mol% of ionizable lipids. In various embodiments, the ionizable lipids are present in the composition at approximately 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, and 44 mol%. It exists in amounts of approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%, approximately 49 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, approximately 60 mol%, approximately 61 mol%, approximately 62 mol%, approximately 63 mol%, approximately 64 mol%, or approximately 65 mol%.

[0295] In various embodiments, the neutral / auxiliary lipid comprises phospholipids such as unsaturated lipids. Examples of phospholipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine. 1,2-Diundecanoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-Diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-Palmyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-Di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-Ooleoyl-2-cholestylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycerol-3-phosphocholine (C16) Lyso PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

[0296] In various embodiments, the composition comprises about 1.0 mol% to about 20.0 mol%, about 1.5 mol% to about 19.5 mol%, about 2.0 mol% to about 19.0 mol%, about 2.5 mol% to about 18.5 mol%, about 3.0 mol% to about 18.0 mol%, about 3.5 mol% to about 17.5 mol%, about 4.0 mol% to about 17.0 mol%, about 4.5 mol% to about 16.5 mol%, about 5.0 mol% to about 16.0 mol%, about 5.5 mol% to about 15.5 mol%, about 6.0 mol% to about 15.0 mol%, about 6.5 mol% to about 14.5 mol%, about 7.0 mol% to about 14.0 mol%, about 7.5 mol% to about 13.5 mol%, about 8.0 mol% to about 13.0 mol%, and about 8.5 mol% to about 12.5 mol%. Neutral / auxiliary lipids in amounts of about 9.0 mol% to about 12.0 mol%, about 9.5 mol% to about 11.5 mol%, about 10.0 mol% to about 11.0 mol%, or about 10.5 mol%. In various embodiments, the neutral / auxiliary lipids are present in amounts of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, or about 20 mol%.

[0297] In various embodiments, the cholesterol and / or its derivatives include, but are not limited to, cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, alfalfa sterol, etc., or combinations thereof.

[0298] In various embodiments, the composition comprises about 10.0 mol% to about 60.0 mol%, about 15.0 mol% to about 55.0 mol%, about 20.0 mol% to about 50.0 mol%, about 25.0 mol% to about 45.0 mol%, about 30.0 mol% to about 40.0 mol%, or about 35.0 mol% of cholesterol and / or its derivatives. In various embodiments, the cholesterol and / or its derivatives are present in the composition at approximately 10 mol%, approximately 11 mol%, approximately 12 mol%, approximately 13 mol%, approximately 14 mol%, approximately 15 mol%, approximately 16 mol%, approximately 17 mol%, approximately 18 mol%, approximately 19 mol%, approximately 20 mol%, approximately 21 mol%, approximately 22 mol%, approximately 23 mol%, approximately 24 mol%, approximately 25 mol%, approximately 26 mol%, approximately 27 mol%, approximately 28 mol%, approximately 29 mol%, approximately 30 mol%, approximately 31 mol%, approximately 32 mol%, approximately 33 mol%, approximately 34 mol%, approximately 35 mol%, approximately 36 mol%, approximately 37 mol%, approximately 38 mol%, approximately 39 mol%, approximately 40 mol%, approximately 41 mol%, approximately 42 mol%, approximately 43 mol%, approximately 44 mol%, approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%. It exists in amounts of approximately 49 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, or approximately 60 mol%.

[0299] In various embodiments, the composition comprises about 0.10 mol% to about 20.0 mol%, about 0.20 mol% to about 19.0 mol%, about 0.30 mol% to about 18.0 mol%, about 0.40 mol% to about 17.0 mol%, about 0.50 mol% to about 16.0 mol%, about 0.60 mol% to about 15.0 mol%, about 0.70 mol% to about 14.0 mol%, about 0.80 mol% to about 13.0 mol%, about 0.90 mol% to about 12.0 mol%, about 1.0 mol% to about 11.0 mol%, about 1.5 mol% to about 10.0 mol%, about 2.0 mol% to about 9.5 mol%, about 2.5 mol% to about 9.0 mol%, about 3.0 mol% to about 8.5 mol%, about 3.5 mol% to about 8.0 mol%, and about 4.0 mol%. Compounds represented by general formula (1) in the range of about 7.5 mol%, about 4.5 mol%, about 7.0 mol%, about 5.0 mol%, about 6.5 mol%, or about 5.5 mol% to about 6.0 mol%. In various embodiments, the compound represented by general formula (1) is the major component of the composition, and is present in the following proportions of the composition: about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1.0 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2.0 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3.0 mol%, about 3.1 mol%, about 3.2 mol%. It exists in amounts of approximately 3.3 mol%, approximately 3.4 mol%, approximately 3.5 mol%, approximately 3.6 mol%, approximately 3.7 mol%, approximately 3.8 mol%, approximately 3.9 mol%, approximately 4.0 mol%, approximately 4.1 mol%, approximately 4.2 mol%, approximately 4.3 mol%, approximately 4.4 mol%, approximately 4.5 mol%, approximately 4.6 mol%, approximately 4.7 mol%, approximately 4.8 mol%, approximately 4.9 mol%, approximately 5.0 mol%, approximately 10.0 mol%, approximately 15.0 mol%, or approximately 20.0 mol%.

[0300] 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.

[0301] In various embodiments, the nanoparticle composition comprises nanoparticles formed from a compound represented by general formula (1).

[0302] Nanoparticles

[0303] Nanoparticles (e.g., lipid nanoparticles) are provided, comprising:

[0304] (i) the compounds represented by general formula (1) disclosed herein; and

[0305] (ii) Encapsulating / loading / coupling / bonding / connecting / binding therapeutic agents and / or preventive agents and / or biological agents into / to the nanoparticles.

[0306] In various embodiments, the nanoparticles have an N:P or N / P ratio of about 1:1 to about 50:1 (i.e., the molar ratio of ionizable nitrogen atoms in ionizable lipids to phosphate groups in therapeutic agents, preventative agents, and / or biological agents (e.g., nucleic acids)). The nanoparticles may have an N:P or N / P ratio of about 1:1 to about 50:1, about 2:1, about 5:1, about 10:1 to about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, or about 45:1.

[0307] 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, and oligonucleotides). For example, the optimal N / P ratio for siRNA, pDNA, and oligonucleotides can be different. 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, an N / P ratio of up to about 20:1 is used to encapsulate and deliver nucleic acid therapeutics (e.g., shorter nucleic acid therapeutic siRNA).

[0308] In various embodiments, the encapsulation / loading / binding efficiency / capacity of the therapeutic agent, preventive agent, and / or biological agent in the composition / nanoparticles is at least about 10.0%, at least about 15.0%, at least about 20.0%, at least about 25.0%, at least about 30.0%, at least about 35.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, or at least about 99.9%.

[0309] In various embodiments, the encapsulation efficiency of the nanoparticles is slightly lower, comparable, but not lower than or higher than that of corresponding nanoparticles using ALC-0159 as a PEG-lipid conjugate under similar conditions. For example, the encapsulation efficiency may be at least about 50% of the encapsulation efficiency of corresponding nanoparticles using ALC-0159 as a PEG-lipid conjugate under similar conditions. In another embodiment, the encapsulation efficiency may be at least about 1% to at least about 50% higher than that of corresponding nanoparticles using ALC-0159 as a PEG-lipid conjugate under similar conditions.

[0310] In various embodiments, the cell / nucleic acid transfection efficiency (percentage of cells / nucleic acids transfected with the gene) of the composition / nanoparticles is at least about 1.0%, at least about 5.0%, at least about 10.0%, at least about 15.0%, at least about 20.0%, at least about 25.0%, at least about 30.0%, at least about 35.0%, at least about 40.0%, at least about 45.0%, at least about 50.0%, at least about 55.0%, at least about 60.0%, at least about 65.0%, at least about 70.0%, at least about 75.0%, at least about 80.0%, at least about 85.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, or at least about 99.9%. In various embodiments, the cell transfection efficiency is not required to reach 100%. For example, it should be understood that vaccine applications may not require / not require 100% cell transfection to mediate an immune response, which is different from the case of cancer treatment applications.

[0311] In various embodiments, the nucleic acid transfection efficiency of the therapeutic and / or preventive and / or biological agents in the composition / nanoparticles is greater than that of those using PEG-conjugated lipids (e.g., ALC-0159).

[0312] In various embodiments, the nucleic acid transfection efficiency of the nanoparticles is not less than or is more than that of the corresponding nanoparticles using ALC-0159 PEG-modified lipids under similar conditions. For example, the nucleic acid (e.g., mRNA) transfection efficiency may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least about 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 times that of the corresponding nanoparticles using ALC-0159 PEG-modified lipids under similar conditions.

[0313] In various embodiments, the cell transfection efficiency of the nanoparticles is comparable to, and no less than, or more than, that of corresponding nanoparticles using ALC-0159 as a PEG-lipid conjugate under similar conditions. For example, the cell transfection efficiency may be at least about 30% of the cell transfection efficiency of corresponding nanoparticles using ALC-0159 as a PEG-lipid conjugate under similar conditions. In another embodiment, the cell transfection efficiency may be at least about 1% to at least about 50% higher than the cell transfection efficiency of corresponding nanoparticles using ALC-0159 as a PEG-lipid conjugate under similar conditions.

[0314] In various implementations, transfection efficiencies in certain cell lines were lower than, but still on the same order of magnitude, those of corresponding nanoparticles using ALC-0159 as a PEG-lipid conjugate under similar conditions. It should be understood that such levels of gene transfection may still be suitable for gene therapy.

[0315] Advantageously, in various implementations, the average or mean particle size (or diameter) of the nanoparticles can be designed to be customizable / adjustable to suit the desired application.

[0316] In various embodiments, the nanoparticles have an average or mean particle size (or diameter) in the nanometer range. In various embodiments, the nanoparticles have an average or mean particle size (or diameter) not exceeding about 300 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm. For example, the nanoparticles have an average particle size (or diameter) of about 50.0 nm to about 300.0 nm, about 75.0 nm to about 250.0 nm, about 100.0 nm to about 200.0 nm, about 105.0 nm, about 110.0 nm, about 115.0 nm, about 120.0 nm, about 125.0 nm, about 130.0 nm, about 135.0 nm, about 140.0 nm, about 145.0 nm, about 150.0 nm, about 155.0 nm, about 160.0 nm, about 165.0 nm, about 170.0 nm, about 175.0 nm, about 180.0 nm, about 185.0 nm, about 190.0 nm, about 195.0 nm, about 200.0 nm, or about 205.0 nm.

[0317] In various embodiments, the composition comprising the nanoparticles has a polydispersity index (PDI) of not more than about 0.50, not more than about 0.40, not more than about 0.30, or not more than about 0.20. For example, the nanoparticles have a polydispersity index (PDI) of about 0.005 to about 0.50, about 0.01 to about 0.40, or about 0.05 to about 0.30. In various embodiments, the composition comprising the nanoparticles has a polydispersity index (PDI) of about 0.01 to about 0.50, about 0.0125 to about 0.45, about 0.015 to about 0.40, about 0.020 to about 0.35, about 0.025 to about 0.30, about 0.030 to about 0.25, about 0.035 to about 0.20, about 0.040 to about 0.15, about 0.045 to about 0.10, about 0.050 to about 0.095, about 0.055 to about 0.090, about 0.060 to about 0.085, about 0.065 to about 0.080, or about 0.070 to about 0.075. Advantageously, in various embodiments, the nanoparticles have a narrow particle size distribution (e.g., not exceeding about 0.2) and / or the nanoparticles or nanoparticle composition are relatively / substantially homogeneous.

[0318] In various embodiments, the nanoparticles exhibit voltages of approximately -20.0 mV to +20.0 mV, approximately -19.5 mV to +19.5 mV, approximately -19.0 mV to +19.0 mV, approximately -18.5 mV to +18.5 mV, approximately -18.0 mV to +18.0 mV, approximately -17.5 mV to +17.5 mV, approximately -17.0 mV to +17.0 mV, approximately -16.5 mV to +16.5 mV, approximately -16.0 mV to +16.0 mV, approximately -15.5 mV to +15.5 mV, approximately -15.0 mV to +15.0 mV, approximately -14.5 mV to +14.5 mV, and approximately -14.0 mV to +14.0 mV in saline (e.g., phosphate-buffered saline (PBS)) or in a physiological environment. mV, approximately -13.5 mV to approximately +13.5 mV, approximately -13.0 mV to approximately +13.0 mV, approximately -12.5 mV to approximately +12.5 mV, approximately -12.0 mV to approximately +12.0 mV, approximately -11.5 mV to approximately +11.5 mV, approximately -11.0 mV to approximately +11.0 mV, approximately -10.5 mV to approximately +10.5 mV, approximately -10.0 mV to approximately +10.0 mV, approximately -9.5 mV to approximately +9.5 mV, approximately -9.0 mV to approximately +9.0 mV, approximately -8.5 mV to approximately +8.5 mV, approximately -8.0 mV to approximately +8.0 mV, approximately -7.5 mV to approximately +7.5 mV, approximately -7.0 mV to approximately +7.0 mV, approximately -6.5 mV to approximately +6.5 mV Zeta potentials of approximately mV, about -6.0 mV to about +6.0 mV, about -5.5 mV to about +5.5 mV, about -5.0 mV to about +5.0 mV, about -4.5 mV to about +4.5 mV, about -4.0 mV to about +4.0 mV, about -3.5 mV to about +3.5 mV, about -3.0 mV to about +3.0 mV, about -2.5 mV to about +2.5 mV, about -2.0 mV to about +2.0 mV, about -1.5 mV to about +1.5 mV, about -1.0 mV to about +1.0 mV, about -0.5 mV to about +0.5 mV, or about +0.0 mV. Advantageously, in various embodiments, the nanoparticles have a substantially neutral surface charge, either negative or positive, making the nanoparticles suitable for / applicable to in vivo applications. In various embodiments, negatively charged nanoparticles can be used to target the spleen and lymph nodes. In various embodiments, positively charged nanoparticles can be used for localized delivery of goods through the mucus layer, such as nasal delivery. In various embodiments, negatively charged nanoparticles can be used to target the spleen or lymph nodes.

[0319] In various embodiments, the cell viability of the composition / nanoparticles is at least about 50.0%, at least about 60.0%, at least about 70.0%, at least about 80.0%, at least about 90.0%, at least about 95.0%, at least about 96.0%, at least about 97.0%, at least about 98.0%, at least about 99.0%, at least about 99.5%, or at least about 99.9%.

[0320] In various embodiments, the cell viability of the nanoparticles is comparable to, or no less than, or greater than, that of corresponding nanoparticles using ALC-0519 as a PEG-lipid conjugate under similar conditions. For example, the cell viability may be at least comparable to that of corresponding nanoparticles using ALC-0519 as a PEG-lipid conjugate under similar conditions. Therefore, in various embodiments, the nanoparticles contain / have high cell compatibility and / or negligible cytotoxicity.

[0321] In various embodiments, the composition / compound / nanoparticle is biocompatible, meaning it is compatible with a biological system or parts thereof and will not substantially or significantly cause adverse physiological responses such as toxic reactions / responses (e.g., cytotoxicity), immune reactions / responses, allergic reactions, damage, etc., when used in humans or animals. In various embodiments, the composition / compound / nanoparticle is substantially free of substances that would cause adverse physiological responses. It should be understood that the composition / compound / nanoparticle may trigger / cause an immune response (e.g., enhance vaccine efficacy), and in such embodiments, the composition / compound / nanoparticle is still considered biocompatible. 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.

[0322] Methods for preparing nanoparticles

[0323] A method for preparing the nanoparticles disclosed herein is provided, the method comprising:

[0324] (ci) to prepare an aqueous composition comprising a therapeutic agent and / or a preventive agent and / or a biological agent;

[0325] (c-ii) The aqueous composition obtained from (ci) is mixed with the composition disclosed herein to obtain nanoparticles.

[0326] In various embodiments, step (ci) comprises mixing the therapeutic agent and / or prophylactic agent and / or biological agent in an aqueous buffer solution. The aqueous buffer solution may be sodium acetate, citrate buffer, phosphate buffer, glycine buffer, or combinations thereof.

[0327] In various embodiments, the mixing step (ci) is performed at pH values ​​of about 2.0 to about 6.0, about 2.1 to about 5.9, about 2.2 to about 5.8, about 2.3 to about 5.7, about 2.4 to about 5.6, about 2.5 to about 5.5, about 2.6 to about 5.4, about 2.7 to about 5.3, about 2.8 to about 5.2, about 2.9 to about 5.1, about 3.0 to about 5.0, about 3.1 to about 4.9, about 3.2 to about 4.8, about 3.3 to about 4.7, about 3.4 to about 4.6, about 3.5 to about 4.5, about 3.6 to about 4.4, about 3.7 to about 4.3, about 3.8 to about 4.2, about 3.9 to about 4.1, or about 4.0.

[0328] In various embodiments, the compositions disclosed herein comprise an organic phase (e.g., ethanol). In various embodiments, the aqueous compositions comprise an aqueous phase. In various embodiments, step (c-ii) comprises mixing the aqueous composition with the organic composition disclosed herein at an aqueous phase:organic phase volume ratio of about 10:1 to about 1:1. For example, the aqueous phase and organic phase may be mixed at a volume ratio of about 10:1 to about 1:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, or about 2:1.

[0329] In various embodiments, the step (c-ii) of mixing the aqueous composition with the organic composition comprises injecting (e.g., direct injection) the organic composition into the aqueous composition. In various embodiments, the step (c-ii) of mixing the aqueous composition with the organic composition comprises inhaling (e.g., rapid inhalation) the organic composition into the aqueous composition.

[0330] In various embodiments, the step (c-ii) of mixing the aqueous composition with the composition includes micromixing, for example, microfluidic mixing using a microfluidic device. Micromixing can be achieved passively using passive micromixers such as T-shaped or Y-shaped microfluidic mixers, parallel stacked, sequential, focused-enhanced mixers, or microdroplet micromixers. Micromixing can also be achieved actively using external forces such as pressure fields, electrodynamics, dielectrophoresis, electrowetting, magnetohydrodynamics, or ultrasound. Advantageously, because microfluidic mixing involves mixing two compositions (i.e., the aqueous composition and the composition disclosed herein) in a controlled manner and / or at a specified / fixed / controlled / precise mixing ratio, the interactions between the two compositions (e.g., between ionizable lipids and therapeutic, preventative, and / or biological agents) can be tuned, resulting in nanoparticles with smaller particle sizes and / or narrow size distributions or homogeneity (e.g., smaller PDI).

[0331] In various embodiments, the method further includes removing an organic phase (e.g., ethanol). For example, removing the organic phase may include dialysis or filtration. Advantageously, removing the organic phase by dialysis or filtration can solidify the LNP and improve the encapsulation efficiency of therapeutic and / or preventative and / or biological agents.

[0332] In various embodiments, supports, nanocarriers, or delivery systems / media that include the compositions / compounds / nanoparticles disclosed herein are also provided.

[0333] In various embodiments, vaccine compositions comprising the compositions / compounds / nanoparticles disclosed herein are also provided.

[0334] In various embodiments, carriers, nanocarriers, delivery systems / media, compounds, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) disclosed herein are also provided for pharmaceutical uses (e.g., for the treatment or prevention of one or more of the diseases, disorders, or conditions mentioned herein).

[0335] In various embodiments, the carriers, nanocarriers, delivery systems / media, compounds, nanoparticle compositions, nanoparticles (or lipid nanoparticles) disclosed herein are also provided for the treatment or prevention of diseases, disorders, or conditions; the use of said carriers, nanocarriers, delivery systems / media, compounds, nanoparticle compositions, nanoparticles (or lipid nanoparticles) in the preparation of medicaments for the treatment or prevention of diseases, disorders, or conditions; and / or methods for treating or preventing diseases, disorders, or conditions, said methods comprising the step of administering (e.g., in a therapeutically effective amount) said carriers, nanocarriers, delivery systems / media, compounds, nanoparticle compositions, nanoparticles (or lipid nanoparticles) to a subject in need (e.g., a vertebrate such as a human or a large veterinary mammal (e.g., a horse, a cow, a deer, a sheep, a llama, a goat, a pig) to a subject in need (e.g., a vertebrate such as a human or a large veterinary mammal (e.g., a horse, a cow, a delivery system / media, a compound, a nanoparticle composition, a nanoparticle (or lipid nanoparticle)).

[0336] The disease, obstacle, or condition may be selected from infectious / contact-transmitted 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, cancer, cardiovascular diseases, skin diseases, or combinations thereof. In various embodiments, the disease, obstacle, or condition 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, obstacle, or condition is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronaviruses such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, obstacle, or condition may be SARS-CoV-2 coronavirus disease. In various embodiments, the disease, obstacle, or condition is mediated by a dengue virus (e.g., DEN-1, DEN-2, DEN-3, and / or DEN-4 viruses). For example, the disease may be dengue fever.

[0337] In various embodiments, carriers, nanocarriers, delivery systems / media, compounds, 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, 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 (e.g., mammalian cells, cytosols, tissues, or organs). A method of delivering a therapeutic agent, prophylactic agent, and / or biological agent to a subject, cell, cytosol, tissue, or organ (e.g., mammalian cell, cytosol, tissue, or organ), the method comprising the steps of administering (e.g., in a therapeutically effective amount) the carrier, nanocarrier, delivery system / mediator, compound, nanoparticle composition, nanoparticle (or lipid nanoparticle) to a subject in need (e.g., a vertebrate such as a human or a large veterinary mammal (e.g., a horse, cattle, deer, sheep, llama, goat, pig)).

[0338] In various embodiments, the carriers, nanocarriers, delivery systems / mediators, compounds, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) disclosed herein are also provided for inducing an immune response in subjects (e.g., vertebrates such as humans or large veterinary mammals (e.g., horses, cattle, deer, sheep, llamas, goats, pigs)); the use of said carriers, nanocarriers, delivery systems / mediators, compounds, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) in the preparation of a medicament for inducing an immune response in subjects; and / or methods for inducing an immune response in subjects, said methods comprising the step of administering (e.g., in a therapeutically effective amount) said carriers, nanocarriers, delivery systems / mediators, compounds, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) to a subject in need. In various embodiments, the immune response in said subject is induced by administering said compounds, nanoparticle compositions, and nanoparticles (or lipid nanoparticles) to the subject. In various embodiments, the subject is protected from various diseases, disorders, or conditions by inducing an immune response, such as infectious / contact-transmitted 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, and combinations thereof. The carrier, nanocarrier, delivery system / medium, compound, nanoparticle composition, or nanoparticle can be delivered to the subject in the form of a vaccine or as a component of a vaccine. In various embodiments, the methods for inducing an immune response are specifically targeted at coronaviruses such as SARS-CoV-2.

[0339] In various embodiments, the disease, impairment, or condition is mediated by an influenza virus (e.g., influenza A, B, C, and / or D viruses). For example, the disease could be influenza A, B, C, or D such as H1N1, H3N2. In various embodiments, the disease, impairment, or condition is mediated by a coronavirus (e.g., severe acute respiratory syndrome coronaviruses such as SARS-CoV-2 or SARS-CoV-1). For example, the disease, impairment, or condition could be SARS-CoV-2 coronavirus disease.

[0340] In various embodiments, the carriers, nanocarriers, delivery systems / media, compounds, nanoparticle compositions, and nanoparticles prepared from embodiments of the methods disclosed herein include one or more of the following features or properties: broad applicability (e.g., usability for encapsulating, delivering, and / or transfecting a wide range of therapeutics, prophylactic agents, and / or biological agents), nanoscale size, substantially neutral or negative surface charge, high encapsulation efficiency (e.g., > 80%), high transfection efficiency (e.g., > 80%), high stability, low toxicity (e.g., low cytotoxicity), and low production / synthesis cost, thus making them suitable for in vivo applications requiring efficient cellular uptake and / or gene transfection.

[0341] In various embodiments, this technology differs from existing techniques that use cationic polymers or peptides to directly concentrate mRNA into nanoparticles. Those skilled in the art will understand that such nanoparticles are extremely unstable in aqueous solutions, making them difficult to store, transport, and / or use in vivo. Furthermore, the nanoassemblies of such nanoparticles lack a hydrophilic shell, thus leading to instability in vivo and during storage.

[0342] In various embodiments, this technique differs from existing techniques for conjugating lipids to peptides, where the peptides are formed using small amine compounds as initiators. It should be understood that such synthetic routes are impractical, cumbersome, and the resulting products are difficult to purify, thus making it challenging to obtain pure products. Furthermore, not every peptide chain contains lipids in the products obtained from such synthetic routes.

[0343] In contrast, this technology involves using lipids as initiators to polymerize amino acid N-carboxylic anhydrides (NCA). That is, in various embodiments, lipids are used as initiators to link lipids to the final product. Advantageously, in various embodiments, each molecule of the final product will therefore contain lipids. In various embodiments, the lipid nanoparticles of this technology differ from those of prior art lipid nanoparticles at least in the length of the hydrocarbon chain and / or linker between the hydrophobic tail and the peptide.

[0344] In various implementations, this technology differs from existing technologies using lipid-block-hydrophilic polycarbonates. Those skilled in the art will understand that lipid-block-hydrophilic homopolymers result in the formation of large mRNA-loaded LNPs and are not suitable as alternatives to lipid-PEG conjugates.

[0345] In various embodiments, this technique includes capping a primary amine group in the peptide. Advantageously, in various embodiments, capping a primary amine group in the peptide plays a key role in providing high mRNA transfection efficiency (see, for example, pDLS2). Compared to(pDLS1). Unbound by theory, it is believed that the amine group interacts strongly with the mRNA, resulting in incomplete release of the mRNA from the LNP into the cell. Advantageously, in various embodiments, the capping of the primary amine group in the peptide allows embodiments of this technology (e.g., lipid nanoparticles of this technology) to directly replace / substitute ALC-0159.

[0346] Advantageously, in various embodiments, the hydrophilicity / hydrophobicity balance in the lipid-peptide or lipid-PEG conjugates of this technology is customizable / tunable / adjustable. Even more advantageously, compared to prior art, the lipid nanoparticles of this technology require shorter peptide (e.g., polyserine) chain lengths to achieve a sufficient / appropriate / suitable degree / level of hydrophilicity for the desired application. In various embodiments, the mRNA transfection efficiency is significantly lower with pDLS6, which has n=45 (similar to the length of PEG), than with pDLS2-5, which have shorter peptide chains. Without being bound by theory, it is believed that polyserine is more hydrophilic than PEG because each serine molecule contains two hydrophilic components (i.e., -NH- and -OH), while each PEG monomer contains only one hydrophilic component (i.e., -O-). Therefore, in various embodiments, a chain length of only 18-32 repeating units is required in polyserine (compared to 45-50 repeating units in PEG) to provide sufficient hydrophilicity for the lipid-peptide conjugate.

[0347] In various embodiments, when the repeat unit (or degree of polymerization - DP) is 45 or higher, the mRNA encapsulation efficiency and mRNA transfection efficiency are significantly lower than the corresponding values ​​for lipid-poly(D,L-serine) where the peptide DP is 18-32. As an example, this invention discloses the synthesis of three lipid-poly(D,L-serine) using lipids of different chain lengths (C8, C12, and C18, relative to C14). From the results (see, for example, Examples 3 and 4, Tables 14-15 and...),... Figures 24 to 25 As can be seen, the critical lipid length is C8. With lipids of this length (product: C8-pDLS), lipid-poly(D,L-serine) cannot form nanoparticles (micrometer size, with a wide size distribution, PDI: 0.62) with mRNA. Conversely, C12-pDLS, C14-pDLS, and C18-pDLS produced nanoparticles with a narrow size distribution (PDI < 0.12). Specifically, C18-pDLS produced the highest transfection efficiency in both HeLa and macrophage cell lines, followed by C14-pDLS.

[0348] In various embodiments, the lipid compositions of this technology involve directly replacing PEG-lipid conjugates with lipid-block-peptides in the lipid composition to achieve enhanced encapsulation efficiency and mRNA transfection efficiency. Such substitution and technical effects are not taught or anticipated from the prior art. Brief description of the attached diagram

[0350] Figure 1 The L-Ser-NCA (solvent, DMSO-d6) according to various embodiments disclosed herein is shown. 1 HNMR.

[0351] Figure 2 The L-Ser-NCA (solvent, DMSO-d6) according to various embodiments disclosed herein is shown. 13 CNMR.

[0352] Figure 3 The following are examples of D-Ser-NCA (solvent, DMSO-d6) according to various embodiments disclosed herein. 1 HNMR.

[0353] Figure 4 The following are examples of D-Ser-NCA (solvent, DMSO-d6) according to various embodiments disclosed herein. 13 CNMR.

[0354] Figure 5 The lipid (solvent, CDCl3) of 2-amino-N,N-bistetradecylacetamide according to various embodiments disclosed herein is shown. 1 H NMR.

[0355] Figure 6 The lipid-block-poly(o-benzyl-D,L-serine) embodiments disclosed herein are shown. 18 (solvent, DMSO-d6) 1 H NMR.

[0356] Figure 7 The lipid-block-poly(o-benzyl-D,L-serine) embodiments disclosed herein are shown. 21 (solvent, DMSO-d6) 1 H NMR.

[0357] Figure 8 The lipid-block-poly(o-benzyl-D,L-serine) embodiments disclosed herein are shown. 27 (solvent, DMSO-d6) 1 H NMR.

[0358] Figure 9 The lipid-block-poly(o-benzyl-D,L-serine) embodiments disclosed herein are shown. 32 (solvent, DMSO-d6) 1 H NMR.

[0359] Figure 10 The lipid-block-poly(o-benzyl-D,L-serine) embodiments disclosed herein are shown. 45 (solvent, DMSO-d6) 1 H NMR.

[0360] Figure 11 The lipid-block-poly(D,L-serine) embodiments disclosed herein are shown. 18 [pDLS2] (solvent, DMSO-d6) 1 H NMR.

[0361] Figure 12 The lipid-block-poly(D,L-serine) embodiments disclosed herein are shown. 21 [pDLS3] (solvent, DMSO-d6) 1 H NMR.

[0362] Figure 13 The lipid-block-poly(D,L-serine) embodiments disclosed herein are shown. 27 [pDLS4] (solvent, DMSO-d6) 1 H NMR.

[0363] Figure 14 The lipid-block-poly(D,L-serine) embodiments disclosed herein are shown. 32 [pDLS5] (solvent, DMSO-d6) 1 H NMR.

[0364] Figure 15 The lipid-block-poly(D,L-serine) embodiments disclosed herein are shown. 45 [pDLS6] (solvent, DMSO-d6) 1 H NMR.

[0365] Figure 16The viability and mRNA transfection efficiency of HELA cells transfected with mRNA LNP for 48 hours according to the various embodiments disclosed in this paper are shown. The Mann-Whitney test was used to compare the statistical significance of ALC-0159 with other pDLS formulations at a 1.0% molar ratio. p < 0.01).

[0366] Figure 17 The viability and mRNA transfection efficiency of HELA cells transfected with mRNA LNP for 48 hours according to the various embodiments disclosed herein were demonstrated. The Mann-Whitney test was used to compare the statistical significance of ALC-0159 with other pDLS formulations at a 1.2% molar ratio. p < 0.01).

[0367] Figure 18 The viability and mRNA transfection efficiency of HELA cells transfected with mRNA LNP for 48 hours according to the various embodiments disclosed in this paper are shown. The Mann-Whitney test was used to compare the statistical significance of ALC-0159 with other pDLS formulations at a 1.4% molar ratio. p < 0.01).

[0368] Figure 19 The viability and mRNA transfection efficiency of HELA cells transfected with mRNA LNP for 48 hours according to the various embodiments disclosed in this paper are shown. The Mann-Whitney test was used to compare the statistical significance of ALC-0159 with other pDLS formulations at a 1.6% molar ratio. p < 0.01).

[0369] Figure 20 C18-lipid-block-poly(D,L-serine) embodiments according to various embodiments disclosed herein are shown. 30 [C18-pDLS] (solvent, DMSO-d6) LNP 1 H NMR.

[0370] Figure 21 C14-lipid-block-poly(D,L-serine) embodiments according to various embodiments disclosed herein are shown. 30 [C14-pDLS or pDLS5] (solvent, DMSO-d6) LNP 1 H NMR.

[0371] Figure 22 C12-lipid-block-poly(D,L-serine) embodiments according to various embodiments disclosed herein are shown.32 [C12-pDLS] (solvent, DMSO-d6) 1 H NMR.

[0372] Figure 23 C8-lipid-block-poly(D,L-serine) embodiments according to various embodiments disclosed herein are shown. 34 [C8-pDLS] (solvent, DMSO-d6) 1 H NMR.

[0373] Figure 24 The viability and mRNA transfection efficiency of HeLa cells transfected with mRNA LNPs of different lipid tail lengths for 48 hours according to the various embodiments disclosed herein were demonstrated. The Mann-Whitney test was used to compare the statistical significance of ALC-0159 with other pDLS formulations at a 1.0% molar ratio. p < 0.01).

[0374] Figure 25 The viability and mRNA transfection efficiency of RAW264.7 cells transfected with mRNA LNPs of different lipid tail lengths for 48 hours according to the various embodiments disclosed herein were demonstrated. The Mann-Whitney test was used to compare the statistical significance of ALC-0159 with other pDLS formulations at a 1.0% molar ratio. p < 0.01).

[0375] Example

[0376] The embodiments of this disclosure will be better understood and readily grasped by those skilled in the art from the following examples, tables, and (if applicable) accompanying drawings. It should be understood that other modifications related to structural and / or chemical changes may be made without departing from the scope of the invention. The embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more embodiments to form new embodiments. The embodiments should not be construed as limiting the scope of this disclosure.

[0377] The following examples describe a series of lipid- b - Development of hydrophilic peptides as alternatives to PEG-lipid conjugates. Lipid- b- The peptide (pDLS) is formed by ring-opening polymerization of L-serine NCA and D-serine NCA, using a lipid containing a primary amine group as an initiator. The hydrophilicity / hydrophobicity of pDLS is modulated by varying the length of the peptide. Compared to LNPs formulated using ALC-0159, mRNA LNPs encapsulated using pDLS as an alternative to ALC-0159 exhibit nanoscale size, narrow size distribution (PDI < 0.2), and greater encapsulation efficiency. The transfection efficiency of mRNA LNPs formulated with pDLS having the optimal peptide length is greater than that of mRNA LNPs formulated with ALC-0159. The pDLS can serve as a viable alternative to ALC-0159 in mRNA delivery, potentially reducing the risk of allergic reactions caused by PEG-lipid conjugates, prolonging the plasma half-life of mRNA LNPs, and thus enhancing vaccination efficacy.

[0378] Advantageously, the lipid-block-peptide embodiments disclosed herein are intended to replace / substitute conventional PEG-lipid conjugates such as ALC-0159. This application demonstrates the application of a series of polyD,L-serine (pDLS) in varying molar amounts as alternatives to PEG-conjugated lipids in mRNA lipid nanoparticles (LNPs). The lipid-block-peptide-encapsulated mRNAs designed according to the various embodiments disclosed herein exhibit nanoscale (< 200 nm) and good size distribution (PDI < 0.2), and possess near-neutral zeta potentials. The lipid-block-peptide-encapsulated mRNA LNPs designed according to the various embodiments disclosed herein also demonstrate greater encapsulation and in vitro transfection efficiency with negligible cytotoxicity.

[0379] Example 1: Materials and Methods

[0380] 1.1. Material

[0381] Unless otherwise specified, all chemical reagents used in the synthesis of lipids were purchased from Sigma-Aldrich and used as is. 1,2-Distearayoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and ALC-0315 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Triton®-X100, Tris-EDTA, and VivoGlo luciferin (in vivo grade) were purchased from Promega (Madison, WI, USA). Alamar blue and Pierce Firefly Luciferase Glow assay kits were purchased from Invitrogen (Waltham, MA, USA). All other reagents used were analytical grade.

[0382] 1.2. Synthesis of o-benzyl-L-serine-N-carboxylic anhydride (L-Ser-NCA) (Scheme 1)

[0383] The synthetic strategy for L-Ser-NCA is shown in Scheme 1. The general synthetic method for L-Ser-NCA is as follows: o-benzyl-L-serine (6.0 g, 2.53 mmol) was suspended in 100 mL of dry tetrahydrofuran (THF), and then triphosgene (3.4 g) was added under N2. The mixture was stirred at 70 °C for 3 h under N2. After cooling the reaction mixture to room temperature, the crude product was precipitated by pouring the mixture into hexane (800 mL), and collected by filtration. The obtained crude product was purified by recrystallization three times with a THF / hexane mixture. The yield of L-Ser-NCA was 67%. 1 H NMR and 13 The structure of L-Ser-NCA was verified by C10 NMR spectroscopy. Figure 1 and Figure 2 ).

[0384] 1.3. Synthesis of o-benzyl-D-serine-N-carboxylic anhydride (D-Ser-NCA) (Scheme 1)

[0385] The synthetic strategy for D-Ser-NCA is shown in Scheme 1. The general synthetic method for D-Ser-NCA is as follows: o-benzyl-D-serine (6.0 g, 2.53 mmol) was suspended in 100 mL of dry tetrahydrofuran (THF), and then triphosgene (3.4 g) was added under N2. The mixture was stirred at 70 °C for 3 h under N2. After cooling the reaction mixture to room temperature, the crude product was precipitated by pouring the mixture into hexane (800 mL), and collected by filtration. The obtained crude product was purified by recrystallization three times with a THF / hexane mixture. The yield of D-Ser-NCA was 62%. 1H NMR and 13 The structure of D-Ser-NCA was verified by C10 NMR spectroscopy. Figure 3 and Figure 4 ).

[0386]

[0387] Option 1. Synthetic strategy for serine-N-carboxylic anhydride (Ser-NCA)

[0388] 1.4. Lipid synthesis of 2-amino-N,N-bistetradecylacetamide

[0389] First, tert-butyl (2-(bis(tetradecylamino)-2-oxoethyl)carbamate was synthesized. Boc-glycine (350.3 mg, 2.0 mmol) and bis(tetradecylamino) (819.6 mg, 2.0 mmol) were dissolved in dry dichloromethane (DCM) (40 mL). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylureonium hexafluorophosphate (HBTU) (910.5 mg, 2.4 mmol) and N-ethyl-N-(1-methylethyl)-2-propylamine (DIPEA) (646.1 mg, 5.0 mmol) were added to the solution. The reaction mixture was stirred at room temperature under N2 for 24 h. The mixture was diluted with 50 mL of DCM and washed with 7% citric acid, brine, and H2O. The resulting organic layer was collected and dried over anhydrous MgSO4. 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 85%.

[0390] Then, 2-amino-N,N-bistetradecylacetamide was synthesized. Tert-butyl (2-(bistetradecylamino)-2-oxoethyl)carbamate was dissolved in 5 mL of anhydrous DCM, and 4 mL of trifluoroacetic acid (TFA) was added. The mixture was stirred under N2 atmosphere for 2 h. The solvent was evaporated under vacuum to produce a crude product. The obtained crude product was dissolved in 20 mL of DCM, and then 20 mL of a 10% aqueous solution of NaHCO3 was added. The mixture was stirred under N2 atmosphere for 12 h. The organic layer was collected and dried over anhydrous MgSO4. DCM was evaporated under vacuum. The resulting product was dried under vacuum to give a white powder. The yield of the compound was 95%. 1 The structure of the product was verified by H NMR spectroscopy. Figure 5 ).

[0391] 1.5. Synthesis of lipid-block-poly(D,L-serine)

[0392] The synthetic strategy for lipid-block-poly(D, L-serine) [lipid-poly(D, L-Ser)] is as described in Scheme 2. First, lipid-block-poly(o-benzyl-D, L-serine) [lipid-poly(o-benzyl-D, L-Ser)] was synthesized. The general synthetic method for lipid-poly(o-benzyl-D, L-Ser) was as follows: In a glove box, 2-amino-N,N-bistetradecylacetamide (46.6 mg, 0.1 mmol), L-Ser-NCA (221 mg, 1.0 mmol), and D-Ser-NCA (221 mg, 1.0 mmol) were dissolved in 40 mL of anhydrous DCM. The mixture was stirred in a glove box at room temperature for 48 h. Then, 3.0 mL of acetic anhydride was added, and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture into ice-cold diethyl ether (300 mL) and collected by centrifugation. The crude product was purified by dissolution with DCM, followed by precipitation by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum. The number of polymeric units of the lipid-poly(o-benzyl-D, L-Ser) (protected pDLS) could be adjusted by varying the amounts of L-Ser-NCA and D-Ser-NCA. The yields of the protected peptides pDLS2, pDLS3, pDLS4, pDLS5, and pDLS6 were 58%, 63%, 70%, 72%, and 78%, respectively. 1 H NMR confirmed the typical structure of lipid-poly(o-benzyl-D, L-Ser). Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 and Figure 11 The reactant feed ratios used for the synthesis of lipid-block-poly(o-benzyl-D, L-serine) are listed in Table 1.

[0393] Table 1. Reactant feed ratios for lipid-block-poly(o-benzyl-D, L-serine).

[0394]

[0395] Then, the lipid-block-poly(o-benzyl-D, L-Ser) was deprotected to produce lipid-poly(D, L-Ser). The general synthetic method for lipid-poly(D, L-Ser) (pDLS) is as follows: 1.0 g of lipid-poly(o-benzyl-D, L-Ser) was dissolved in 10 mL of TFA, and 3 mL of 33% HBr in acetic acid 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 pouring the mixture solution into ice-cold diethyl ether (300 mL), 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 lipid-poly(D, L-Ser) was purified by dialysis with deionized water. The final lipid-poly(D, L-Ser) was obtained by freeze-drying under vacuum. The yields of deprotected lipid-peptides of pDLS2, pDLS3, pDLS4, pDLS5, and pDLS6 were 55%, 58%, 64%, 75%, and 87%, respectively. 1 H NMR confirmed the typical structure of lipid-poly(D, L-Ser). Figure 11 , Figure 12 , Figure 13 , Figure 14 and Figure 15 The characteristics of lipid-poly(D,L-serine) conjugates (pDLS) are listed in Table 2.

[0396]

[0397] Table 2. Characteristics of lipid-poly(D,L-serine) (pDLS)

[0398]

[0399] 1.6. Formulation of mRNA-loaded lipid nanoparticles (mRNA LNPs)

[0400] mRNA LNPs were prepared by manual mixing, and the different molar ratios used are shown in Table 3. The dose of mRNA used in each formulation was normalized to 10 μg. Furthermore, the ratio of ionizable lipids to mRNA was normalized using an N / P ratio of 6.

[0401] As an alternative to the lipid-PEG conjugate (ALC-0159), lipid-pDLS conjugates with different PDLS lengths were prepared. mRNA LNPs were prepared by mixing lipid-pDLS with other lipids at varying molar concentrations of lipid-pDLS to optimize the mRNA LNPs. The molar concentrations of the other lipids used were adjusted to compensate for the difference between the molar concentration of the pDLS compound and that of ALC-0159 (1.6%).

[0402] In the preparation of mRNA LNPs, two separate phases were manually mixed together. The organic phase consisted of an organic phase (a lipid mixture dissolved in ethanol to a final volume of 50 μL). The aqueous phase consisted of 140 μL of a 10 mM sodium acetate solution (pH 4) containing 1 mg / mL firefly luciferase mRNA (TriLink Biotechnologies) to a final volume of 150 μL. The organic phase was added to the aqueous phase and thoroughly mixed by rapid pipetting. The mRNA LNP solution was then incubated at room temperature for 30 minutes to allow the LNPs to encapsulate and self-assemble.

[0403] Table 3. Molar ratio (content) of lipids used in the formulation

[0404]

[0405] 1.7. Evaluate the encapsulation efficiency of mRNA in LNPs

[0406] To evaluate the encapsulation efficiency of different lipid mixtures for mRNA, Quant-it was used. TM The encapsulation efficiency of the prepared mRNA LNPs was determined using the RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA). The RiboGreen RNA reagent was diluted 200-fold with Tris-EDTA buffer containing 5% Triton-X100 or Tris-EDTA buffer alone. Then, 90 μL of the buffer solution containing RiboGreen reagent was added to 10 μL of the mRNA LNP sample, and the plate was incubated at 37°C for 20 min in a black 96-well plate. Subsequently, 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 encapsulation efficiency of the mRNA in the LNPs was then calculated using the obtained values ​​according to the following equation.

[0407]

[0408] in ConcTrit The mRNA concentration was obtained by adding mRNA LNPs to 5% Triton-X100 diluted in Tris-EDTA buffer. Conc TE The corresponding mRNA concentration was obtained by adding mRNA LNP to Tris-EDTA buffer that does not contain Triton-X100.

[0409] 1.8. Characterization of mRNA LNP

[0410] The size, polydispersity index (PDI), and zeta potential of mRNA LNPs were characterized using a Zetasizer (Malvern, UK). Size and PDI were obtained by dynamic light scattering (DLS), where 25 μL of mRNA LNP sample was diluted with saline solution to a final volume of 500 μL. The sample was measured three times at 25 °C, with 20 runs per run. The sample was also measured at 1.68 s per run. The surface zeta potential of the mRNA LNPs was measured by diluting 25 μL of sample with saline solution to a final volume of 1 mL. The sample was also measured three times at 25 °C, with 20 runs per run during the measurement.

[0411] 1.9. Cell culture and administration of mRNA LNP to HELA cells

[0412] To test the cytotoxicity and transfection efficiency of mRNA LNP, HELA cells were cultured and administered mRNA LNP. HELA cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS) (v / v) and 1% penicillin / streptomycin (v / v). Cells were incubated in a Thermo Fisher, Waltham, MA, USA at 37°C and 5% CO2. Cells were then seeded at 10,000 cells / well in white 96-well plates for mRNA transfection efficiency and in black 96-well plates for mRNA LNP cytotoxicity evaluation. The amount of mRNA LNP added to each well was normalized to a dose of 100 ng of mRNA per well. After administration, the 96-well plates were incubated for 48 hours, and cell viability and transfection efficiency were then assessed.

[0413] 1.10. In vitro viability of HELA cells after incubation with mRNA LNP

[0414] After 48 hours of incubation, the old medium containing mRNA LNPs was removed from the wells of the black 96-well plates. 100 μL of fresh DMEM medium containing 10% Alamar blue reagent was added to each well. The plates were then incubated at 37°C for 2 hours. 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.

[0415] 1.11. In vitro transfection efficiency of mRNA LNP in HELA cells after incubation with mRNA LNP

[0416] After 48 hours of incubation, the transfection efficiency of mRNA LNP in HELA cells was measured by removing the culture medium containing mRNA LNP and replacing it with a 100 μL cell lysis buffer / D-luciferin mixture. The mixture contained 50 μL of 2x cell lysis buffer diluted with phosphate-buffered saline (PBS) and 50 μL of 100x D-luciferin diluted with firefly assay buffer. Cells were incubated with the mixture at 37°C for 10 minutes to promote cell lysis and signal stabilization. Subsequently, the luminescence intensity was read using a microplate reader (Tecan, Männedorf, Switzerland) at an exposure time of 1000 ms.

[0417] Example 2: Results and Discussion

[0418] 2.1. Synthesis of pDLS

[0419] Using 2-amino-N,N-bistetradecylacetamide lipids as initiators, lipid-block-poly(D,L-serine) (pDLS) was synthesized via ring-opening polymerization (ROP) of o-benzyl-L-serine-N-carboxylic anhydride and o-benzyl-D-serine-N-carboxylic anhydride (Scheme 1), followed by deprotection of the lipid-block-poly(o-benzyl-D,L-serine) in TFA and HBr / CH3COOH (Scheme 2). 1 H and 13 C10 NMR spectroscopy confirmed the successful synthesis of D-Ser-NCA and L-Ser-NCA. Figure 1 , Figure 2 , Figure 3 and Figure 4 ), and also through 1 The successful synthesis of 2-amino-N,N-bistetradecylacetamide was confirmed by 1H NMR spectroscopy. Figure 5 ).pass 1 The structure of lipid-block poly(o-benzyl-D, L-serine)n was confirmed by 1H NMR spectroscopy. Figure 6 , Figure 7 , Figure 8 , Figure 9 and Figure 10 The ko(-Ph) from the peptide is clearly shown at δ 7.24 and 0.84 ppm, respectively. H -) and a(-C) from lipids H The peak at 3) indicates the successful synthesis of the lipid-poly(o-benzyl-D,L-serine) conjugate. It was also obtained through... 1 1H NMR spectroscopy was used to verify the deprotection of lipid-block-poly(o-benzyl-D,L-serine) to form lipid-block-poly(D,L-serine) (pDLS). Figure 11 , Figure 12 , Figure 13 , Figure 14 and Figure 15 As shown, the peaks ko (-Ph) at δ 7.24 and 4.41 ppm are respectively. H -) and e (Ph-C H The disappearance of 2- indicates successful deprotection of the lipid-block-poly(o-benzyl-D, L-serine). The degree of polymerization (DP) of serine in pDLS was modulated by varying the amounts of L-Ser-NCA and D-Ser-NCA. The DP of serine in the lipid-poly(D, L-serine) was determined by the integral area (peak g of peptide; peak a of lipid). Figure 11 , Figure 12 , Figure 13 , Figure 14 and Figure 15 Lipid-poly(D, L-serine) compounds with different DP values ​​(including 18, 21, 27, 32, and 45) were synthesized and designated as pDLS2, pDLS3, pDLS4, pDLS5, and pDLS6, respectively. Compared to pDLS2, the synthesis of pDLS1 did not involve the addition of acetic anhydride to shield the terminal amino groups.

[0420] 2.2. mRNA LNP size, size distribution (PDI), and zeta potential

[0421] The size, PDI, and zeta potential of the mRNA LNPs were determined using a Zetasizer (Malvern, UK) and are shown in Tables 4–7. All formulations produced small particle sizes of approximately 200 nm or less and narrow size distributions with PDI < 0.2. Furthermore, all formulations produced near-neutral zeta potentials (or neutral surface charges) at < ± 6 mV, which is desirable / ideal for in vivo application.

[0422] Table 4. Characteristics of hand-prepared mRNA LNPs at a pDLS molar ratio of 1.0%

[0423]

[0424] Table 5. Characteristics of hand-formulated mRNA LNPs at a pDLS molar ratio of 1.2%.

[0425]

[0426] Table 6. Characteristics of hand-formulated mRNA LNPs at a pDLS molar ratio of 1.4%

[0427]

[0428] Table 7. Characteristics of hand-formulated mRNA LNPs at a pDLS molar ratio of 1.6%

[0429]

[0430] 2.3. mRNA encapsulation efficiency in LNP

[0431] The encapsulation efficiency of mRNA LNPs was determined by the RiboGreen assay and is shown in Tables 8–11. All formulations containing pDLS showed higher encapsulation efficiency than the formulation using the lipid-PEG conjugate (ALC-0159) (17.5%). This indicates that pDLS provides a better alternative to encapsulating mRNA by manual mixing, thus reducing mRNA waste because a larger amount of mRNA can be encapsulated in the LNP if pDLS is used. Among the different pDLS compounds used, pDLS6 showed the lowest encapsulation efficiency. This is likely due to the larger polypeptide chain length (48 subunits) in pDLS6, resulting in a poorer hydrophilicity / hydrophobicity balance and consequently lower mRNA encapsulation efficiency.

[0432] Table 8. Encapsulation efficiency of mRNA LNP at a molar ratio of 1.0%

[0433]

[0434] Table 9. Encapsulation efficiency of mRNA LNP at a molar ratio of 1.2%

[0435]

[0436] Table 10. Encapsulation efficiency of mRNA LNP at a molar ratio of 1.4%

[0437]

[0438] Table 11. Encapsulation efficiency of mRNA LNP at a molar ratio of 1.6%

[0439]

[0440] 2.4. In vitro cell compatibility and transfection efficiency of mRNA LNP in HELA cells

[0441] To determine the cytotoxicity and transfection efficiency of the pDLS compound as an alternative to ALC-0159, HELA cells were treated with the formulated mRNA LNP. Results of cell viability and luminescence intensity assays showed... Figure 16 , Figure 17 , Figure 18 and Figure 19 All formulations showed negligible cytotoxicity. Furthermore, all pDLS formulations except pDLS1 showed significantly higher transfection efficiency than ALC-0159. This indicates that pDLS, as an alternative to ALC-0159, can achieve greater mRNA transfection efficiency in HELA cells containing mRNA LNPs. The lower mRNA transfection efficiency mediated by pDLS1 compared to pDLS2 is likely due to the presence of a primary amine group in the peptide of pDLS1, which may bind too strongly to the mRNA, preventing its release into the cytosol. Additionally, the higher mRNA transfection efficiency induced by pDLS2-pDLS5 compared to pDLS6, which contains a longer hydrophilic peptide (45 subunits), suggests a balance between hydrophobicity and hydrophilicity for optimal transfection efficiency.

[0442] Example 3: Materials and Methods

[0443] 3.1. Material

[0444] Unless otherwise specified, all chemical reagents used in the synthesis of lipids were purchased from Sigma-Aldrich and used as is. 1,2-Distearayoyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and ALC-0315 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Triton®-X100 and Tris-EDTA were purchased from Promega (Madison, WI, USA). The Alamar Blue and PierceFirefly Luciferase Glow assay kits were purchased from Invitrogen (Waltham, MA, USA). All other reagents used were analytical grade.

[0445] 3.2. Synthesis of C18-lipid-block-poly(D,L-serine)

[0446] The synthetic strategy for C18-lipid-block-poly(D, L-serine) [C18-lipid-poly(D, L-Ser)] is shown in Scheme 3. First, C18-lipid-block-poly(o-benzyl-D, L-serine) [C18-lipid-poly(o-benzyl-D, L-Ser)] was synthesized. The general synthetic method for C18-lipid-poly(o-benzyl-D, L-Ser) was as follows: In a glove box, 2-amino-N,N-octadecylacetamide (57.9 mg, 0.1 mmol), L-Ser-NCA (663 mg, 1.0 mmol), and D-Ser-NCA (663 mg, 1.0 mmol) were dissolved in 40 mL of anhydrous DCM. The mixture was stirred in a glove box at room temperature for 48 h. Then, 3.0 mL of acetic anhydride was added, and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture into 300 mL of ice-cold diethyl ether and collected by centrifugation. The obtained crude product was purified by dissolving in DCM and precipitating by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum.

[0447] Then, the protection of C18-lipid-block-poly(o-benzyl-D, L-Ser) was deprotected to produce C18-lipid-poly(D, L-Ser). The general synthetic method for C18-lipid-poly(D, L-Ser) (C18-pDLS) is as follows: 1.0 g of C18-lipid-poly(o-benzyl-D, L-Ser) was dissolved in 10 mL of TFA, and 3 mL of a solution of 33% HBr in acetic acid 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 pouring the mixture into 300 mL of ice-cold diethyl ether, 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 C18-lipid-poly(D, L-Ser) was purified by dialysis with deionized water. The final C18-lipo-poly(D,L-Ser) was obtained by freeze-drying under vacuum. The yield of the deprotected lipid-peptide of C18-pDLS was 85%. 1 H NMR confirmed the typical structure of C18-lipid-poly(D, L-Ser). Figure 20 The characteristics of the C18-lipid-poly(D,L-serine) conjugate (C18-pDLS) are listed in Table 12.

[0448]

[0449] 3.3. Synthesis of C14-lipid-block-poly(D,L-serine)

[0450] The synthetic strategy for C14-lipid-block-poly(D, L-serine) [C14-lipid-poly(D, L-Ser)] is shown in Scheme 4. First, C14-lipid-block-poly(o-benzyl-D, L-serine) [C14-lipid-poly(o-benzyl-D, L-Ser)] was synthesized. The general synthetic method for C14-lipid-poly(o-benzyl-D, L-Ser) was as follows: In a glove box, 2-amino-N,N-bistetradecylacetamide (46.6 mg, 0.1 mmol), L-Ser-NCA (663 mg, 1.0 mmol), and D-Ser-NCA (663 mg, 1.0 mmol) were dissolved in 40 mL of anhydrous DCM. The mixture was stirred in a glove box at room temperature for 48 h. Then, 3.0 mL of acetic anhydride was added, and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture into 300 mL of ice-cold diethyl ether and collected by centrifugation. The obtained crude product was purified by dissolving in DCM and precipitating by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum.

[0451] Then, the protection of C14-lipid-block-poly(o-benzyl-D, L-Ser) was deprotected to produce lipid-poly(D, L-Ser). The general synthetic method for C14-lipid-poly(D, L-Ser) (C14-pDLS) is as follows: 1.0 g of C14-lipid-poly(o-benzyl-D, L-Ser) was dissolved in 10 mL of TFA, and 3 mL of a solution of 33% HBr in acetic acid 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 pouring the mixture solution into 300 mL of ice-cold diethyl ether, 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 C14-lipid-poly(D, L-Ser) was purified by dialysis with deionized water. The final C18-lipo-poly(D,L-Ser) was obtained by freeze-drying under vacuum. The yield of the deprotected lipid-peptide of C14-pDLS was 82%. 1 H NMR confirmed the typical structure of C14-lipid-poly(D, L-Ser). Figure 21 The characteristics of C14-lipid-poly(D,L-serine) conjugate (C14-pDLS) are listed in Table 12.

[0452]

[0453] 3.4. Synthesis of C12-lipid-block-poly(D,L-serine)

[0454] The synthetic strategy for C12-lipid-block-poly(D, L-serine) [C12-lipid-poly(D, L-Ser)] is shown in Scheme 5. First, C12-lipid-block-poly(o-benzyl-D, L-serine) [C12-lipid-poly(o-benzyl-D, L-Ser)] was synthesized. The general synthetic method for C12-lipid-poly(o-benzyl-D, L-Ser) was as follows: In a glove box, 2-amino-N,N-docosahexadecylacetamide (41 mg, 0.1 mmol), L-Ser-NCA (663 mg, 1.0 mmol), and D-Ser-NCA (663 mg, 1.0 mmol) were dissolved in 40 mL of anhydrous DCM. The mixture was stirred in a glove box at room temperature for 48 h. Then, 3.0 mL of acetic anhydride was added, and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture into 300 mL of ice-cold diethyl ether and collected by centrifugation. The obtained crude product was purified by dissolving in DCM and precipitating by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum.

[0455] Then, the protection of C12-lipid-block-poly(o-benzyl-D, L-Ser) was deprotected to produce C12-lipid-poly(D, L-Ser). The general synthetic method for C12-lipid-poly(D, L-Ser) (C12-pDLS) was as follows: 1.0 g of C12-lipid-poly(o-benzyl-D, L-Ser) was dissolved in 10 mL of TFA, and 3 mL of a solution of 33% HBr in acetic acid 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 pouring the mixture into 300 mL of ice-cold diethyl ether, 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 C12-lipid-poly(D, L-Ser) was purified by dialysis with deionized water. The final C12-lipid-poly(D,L-Ser) was obtained by freeze-drying under vacuum. The yield of the deprotected lipid-peptide of C12-pDLS was 80%. 1 H NMR confirmed the typical structure of C12-lipid-poly(D, L-Ser). Figure 22 The characteristics of C12-lipid-poly(D,L-serine) conjugates (C12-pDLS) are listed in Table 12.

[0456]

[0457] 3.5. Synthesis of C8-lipid-block-poly(D,L-serine)

[0458] The synthetic strategy for C8-lipid-block-poly(D, L-serine) [C8-lipid-poly(D, L-Ser)] is shown in Scheme 6. First, C8-lipid-block-poly(o-benzyl-D, L-serine) [C8-lipid-poly(o-benzyl-D, L-Ser)] was synthesized. The general synthetic method for C8-lipid-poly(o-benzyl-D, L-Ser) was as follows: In a glove box, 2-amino-N,N-dioctylacetamide (29.8 mg, 0.1 mmol), L-Ser-NCA (663 mg, 1.0 mmol), and D-Ser-NCA (663 mg, 1.0 mmol) were dissolved in 40 mL of anhydrous DCM. The mixture was stirred in a glove box at room temperature for 48 h. Then, 3.0 mL of acetic anhydride was added, and the reaction was continued for 2 h. The crude product was precipitated by pouring the mixture into ice-cold diethyl ether (300 mL) and collected by centrifugation. The crude product was purified by dissolution with DCM, followed by precipitation by pouring the solution into ice-cold diethyl ether. The resulting product was dried under vacuum.

[0459] Then, the protection of C8-lipid-block-poly(o-benzyl-D, L-Ser) was deprotected to produce C8-lipid-poly(D, L-Ser). The general synthetic method for C8-lipid-poly(D, L-Ser) (C8-pDLS) is as follows: 1.0 g of C8-lipid-poly(o-benzyl-D, L-Ser) was dissolved in 10 mL of TFA, and 3 mL of a solution of 33% HBr in acetic acid 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 pouring the mixture into 300 mL of ice-cold diethyl ether, 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 C8-lipid-poly(D, L-Ser) was purified by dialysis with deionized water. The final C8-lipo-poly(D,L-Ser) was obtained by freeze-drying under vacuum. The yield of the deprotected lipid-peptide of C8-pDLS was 86%. 1 H NMR confirmed the typical structure of C8-lipid-poly(D, L-Ser). Figure 23 The characteristics of C8-lipid-poly(D,L-serine) conjugates (C8-pDLS) are listed in Table 12.

[0460]

[0461] Table 12. Characteristics of lipid-poly(D,L-serine) with alkyl chain length

[0462]

[0463] 3.6. Formulation of mRNA-loaded lipid nanoparticles (mRNA LNPs)

[0464] mRNA LNPs were prepared by manual mixing, and the different molar ratios used are shown in Table 13. The dose of mRNA used in each formulation was normalized to 10 μg. Furthermore, the ratio between ionizable lipids and mRNA was normalized using an N / P ratio of 6.

[0465] As an alternative to the lipid-PEG conjugate (ALC-0159), lipid-pDLS conjugates with different PDLS lengths were prepared. mRNA LNPs were prepared by mixing lipid-pDLS with other lipids at a molar concentration of 1.6%.

[0466] In the preparation of mRNA LNPs, two separate phases were manually mixed together. The organic phase consisted of an organic phase (a lipid mixture dissolved in ethanol to a final volume of 50 μL). The aqueous phase consisted of 140 μL of a 10 mM sodium acetate solution (pH 4) containing 1 mg / mL firefly luciferase mRNA (TriLink Biotechnologies) to a final volume of 150 μL. The organic phase was added to the aqueous phase and thoroughly mixed by rapid pipetting. The mRNA LNP solution was then incubated at room temperature for 30 minutes to allow the LNPs to encapsulate and self-assemble.

[0467] Table 13. Molar ratio (content) of lipids used in the formulation

[0468]

[0469] 3.7. Evaluate the encapsulation efficiency of mRNA in LNPs

[0470] To evaluate the encapsulation efficiency of different lipid mixtures for mRNA, Quant-it was used. TMThe encapsulation efficiency of the prepared mRNA LNPs was determined using the RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA). The RiboGreen RNA reagent was diluted 200-fold with Tris-EDTA buffer containing 5% Triton-X100 or Tris-EDTA buffer alone. Then, 90 μL of the buffer solution containing RiboGreen reagent was added to 10 μL of the mRNA LNP sample, and the plate was incubated at 37°C for 20 min in a black 96-well plate. Subsequently, 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 encapsulation efficiency of the mRNA in the LNPs was then calculated using the obtained values ​​according to the following equation.

[0471]

[0472] in Conc Trit The mRNA concentration was obtained by adding mRNA LNPs to 5% Triton-X100 diluted in Tris-EDTA buffer. Conc TE The corresponding mRNA concentration was obtained by adding mRNA LNP to Tris-EDTA buffer that does not contain Triton-X100.

[0473] 3.8. Characterization of mRNA LNP

[0474] The size, polydispersity index (PDI), and zeta potential of mRNA LNPs were characterized using a Zetasizer (Malvern, UK). Size and PDI were obtained by dynamic light scattering (DLS), where 25 μL of mRNA LNP sample was diluted with saline solution to a final volume of 500 μL. The sample was measured three times at 25 °C, with 20 runs per run. The sample was also measured at 1.68 s per run. The surface zeta potential of the mRNA LNPs was measured by diluting 25 μL of sample with saline solution to a final volume of 1 mL. The sample was also measured three times at 25 °C, with 20 runs per run during the measurement.

[0475] 3.9. Cell culture and administration of mRNA LNP to HeLa and RAW264.7 cells.

[0476] To test the cytotoxicity and transfection efficiency of mRNA LNPs, HeLa and RAW264.7 cells were cultured and administered mRNA LNPs. HeLa and RAW264.7 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 and 5% CO2 in a Thermo Fisher, Waltham, MA, USA. Cells were then seeded at 10,000 cells / well in white 96-well plates for mRNA transfection efficiency and in black 96-well plates for evaluating mRNA LNP cytotoxicity. The amount of mRNA LNP added to each well was normalized to a dose of 100 ng of mRNA per well. After administration, the 96-well plates were incubated for 48 hours, and cell viability and transfection efficiency were then assessed.

[0477] 3.10. In vitro viability of HeLa and RAW264.7 cells after incubation with mRNA LNP

[0478] After 48 hours of incubation, the old medium containing mRNA LNPs was removed from the wells of the black 96-well plates. 100 μL of fresh DMEM medium solution containing 10% Alamar blue reagent was added to each well. The plates were then incubated at 37°C for 2 hours. 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.

[0479] 3.11. In vitro transfection efficiency of mRNA LNP in HeLa and RAW264.7 cells after incubation with mRNA LNP

[0480] After 48 hours of incubation, the transfection efficiency of mRNA LNP in HeLa and RAW264.7 cells was measured by removing the culture medium containing mRNA LNP and replacing it with a 100 μL cell lysis buffer / D-luciferin mixture. The mixture contained 50 μL of 2x cell lysis buffer diluted with phosphate-buffered saline (PBS) and 50 μL of 100x D-luciferin diluted with firefly assay buffer. Cells were incubated with the mixture at 37°C for 10 minutes to promote cell lysis and signal stabilization. Subsequently, the luminescence intensity was read using a microplate reader (Tecan, Männedorf, Switzerland) at an exposure time of 1000 ms.

[0481] Example 4: Results and Discussion

[0482] 4.1. Synthesis of pDLS with different lipid alkyl chain tail lengths

[0483] Using lipids with different alkyl chain tail lengths as initiators, lipid-block-poly(D,L-serine) with different alkyl chain tail lengths were synthesized via ring-opening polymerization (ROP) of o-benzyl-L-serine-N-carboxylic anhydride and o-benzyl-D-serine-N-carboxylic anhydride, followed by deprotection of the lipid-block-poly(o-benzyl-D,L-serine) in TFA and HBr / CH3COOH (schemes 3, 4, 5, and 6). Lipid-poly(D,L-serine) with different alkyl chain tail lengths (including 18, 14, 12, and 8) were synthesized and designated as C18-pDLS, C14-pDLS, C12-pDLS, and C8-pDLS, respectively. 1 1H NMR spectroscopy confirmed the successful synthesis of C18-pDLS, C14-pDLS, C12-pDLS and C8-pDLS. Figure 20 , Figure 21 , Figure 22 and Figure 23 The characteristics of lipid-poly(D,L-serine) conjugates with different lipid alkyl chain tail lengths are listed in Table 12.

[0484] 4.2. mRNA LNP size, size distribution (PDI), and zeta potential

[0485] Using a Zetasizer (Malvern, UK), the size, PDI, and zeta potential of the mRNA LNPs were determined and are shown in Table 14. Most formulations produced small particle sizes of approximately 200 nm or less and narrow size distributions with a PDI < 0.2, except for the LNP formulation including C8-pDLS, which showed a size of 1166 nm and a PDI of 0.620. This indicates a lower limit for lipid tail length, beyond which it is no longer possible to formulate stable nanoparticles. Furthermore, all formulations produced near-neutral zeta potentials of <±4 mV, which is desirable / ideal for in vivo application.

[0486] Table 14. Characteristics of mRNA LNPs manually formulated at a pDLS molar ratio of 1.6% with different lipid tail lengths.

[0487]

[0488] 4.2. mRNA encapsulation efficiency in LNP

[0489] The encapsulation efficiency of mRNA LNPs was determined by the RiboGreen assay and is shown in Table 15. All formulations containing pDLS showed higher encapsulation efficiency than the formulation using the lipid-PEG conjugate (ALC-0159) (71.8%). This indicates that pDLS provides a better alternative to encapsulating mRNA by manual mixing, thus reducing mRNA waste, as a larger amount of mRNA can be encapsulated in the LNP if pDLS is used. Among the different pDLS compounds used, C18-pDLS showed the lowest encapsulation efficiency. This indicates that encapsulation efficiency begins to decline after the upper limit of lipid tail length. Combined with the results of the physicochemical properties of the tested LNPs, the results clarify the range of effective lipid tail lengths that can form stable lipid nanoparticles. This range may be greater than C8 and less than C18.

[0490] Table 15. Encapsulation efficiency of mRNA LNPs at a molar ratio of 1.6% with different lipid tail lengths.

[0491]

[0492] 4.4. In vitro cell compatibility and transfection efficiency of mRNA LNP in HeLa and RAW264.7 cells

[0493] To determine the cytotoxicity and transfection efficiency of the pDLS compound as an alternative to ALC-0159, HeLa and RAW264.7 cells were treated with the formulated mRNA LNP. Cell viability and luminescence intensity measurements showed... Figure 24 and Figure 25 All formulations showed negligible cytotoxicity. Furthermore, all pDLS formulations except C12-pDLS showed significantly higher transfection efficiencies than ALC-0159. The results also appear to highlight the trend that longer lipid tails lead to greater transfection efficiencies. C8-pDLS does not appear to follow this trend, possibly due to its larger size, which leads to nanoparticle aggregation to form microparticles that deliver a larger dose of mRNA after transfection. The results indicate that pDLS, as an alternative to ALC-0159, can achieve greater mRNA transfection efficiencies in HeLa and RAW264.7 cells with mRNA LNPs, with C18-pDLS achieving twice the transfection efficiency of ALC-0159 in HeLa cells and 20 times the transfection efficiency of ALC-0159 in RAW264.7 cells.

[0494] Example 5: Summary

[0495] mRNA LNPs formulated with pDLS compounds instead of ALC-0159 exhibited nanoscale size (< 200 nm), narrow size distribution (PDI < 0.2), and near-neutral zeta potential (< ± 10 mV). Furthermore, the pDLS formulation produced greater encapsulation efficiency compared to the ALC-0159 formulation, demonstrating significantly greater mRNA transfection efficiency and negligible cytotoxicity in both HeLa and RAW264.7 cells. These results suggest that pDLS can serve as a superior alternative to ALC-0159 in encapsulating mRNA to reduce mRNA waste and transfecting cells with mRNA to produce a greater amount of protein at similar doses. The results also indicate that a feasible range of lipid tail lengths exists, achieved by increasing the lipid tail length for greater transfection efficiency while keeping it below an upper limit to maintain acceptable encapsulation efficiency. In a clinical context, this may reduce the risk of immunogenic reactions induced by PEG-conjugated lipids ALC-0159. The commercial application of mRNA LNPs formulated using pDLS may lead to the development of mRNA vaccines that address the allergic reactions induced by currently available mRNA vaccines.

[0496] 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, which is broadly described herein. For example, features of different exemplary embodiments may be mixed, combined, interchanged, merged, adopted, modified, included, etc., among different exemplary embodiments. Therefore, the embodiments herein should be considered exemplary and non-limiting in all respects.

Claims

1. Compounds represented by general formula (1) for the preparation of lipid nanoparticles encapsulating therapeutic agents, preventative agents, and / or biological agents: in A R Contains units derived from polyamino acids; R 1 and R 2 Each is an independent hydrophobic group; R 3 R 4 and R 5 Each is independently H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group; R 7 It is -H or -C(=O)R 8 , where R 8 It is an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, an alkynyl group that is optionally substituted, or an alkoxy group that is optionally substituted; m≥1; and n≥1。 2. The compound of claim 1, wherein A R It comprises a structure represented by general formula (2), wherein A contains a hydrophilic organic group that is part of an amino acid represented by general formula (3): in R 6 It is H, an optionally substituted alkyl group, an optionally substituted alkenyl group, or an optionally substituted alkynyl group.

3. The compound according to any one of the preceding claims, wherein the polyamino acid is selected from polyserine, polyglutamic acid, and combinations thereof.

4. The compound according to any one of claims 2 to 3, wherein A is selected from -CH2OH, -CH2CH2C(=O)OH, and combinations thereof.

5. The compound according to any one of the preceding claims, wherein A R Includes structures represented by general formulas (2A) and / or (2B): in p + q = n; and R 6’ =R 6’’ =R 6 。 6. The compound according to any one of the preceding claims, wherein the compound represented by general formula (1) comprises a structure represented by general formula (1A) and / or (1B): in p + q = n; and R 6’ =R 6’’ =R 6 。 7. The compound according to any one of the preceding claims, wherein n ≤ 60.

8. The compound according to any one of the preceding claims, wherein in R 1 and R 2 Each of the hydrophobic groups independently comprises an optionally substituted alkyl group having at least 8 carbon atoms.

9. The compound according to any one of the preceding claims, wherein in R 1 and R 2 Each of the hydrophobic groups independently comprises an optionally substituted alkyl group having no more than 18 carbon atoms.

10. The compound according to any one of the preceding claims, wherein R 3 R 4 R 5 and R 6 Both are H.

11. The compound according to any one of the preceding claims, wherein the compound comprises a structure selected from one or more of the following: C14-pDLS1 (n=18) C14-pDLS2 (n=18) C14-pDLS3 (n=21) C14-pDLS4 (n=27) C14-pDLS5 (n=32) C14-pDLS6 (n=45) C18-pDLS (n=30) C12-pDLS (n=32) C8-pDLS (n=34).

12. A method for preparing the compound claimed in any one of the preceding claims, the method comprising: (i) Polymerizing one or more N-carboxylic anhydride (NCA) monomers represented by general formula (5) with a lipid initiator / molecule represented by general formula (6) to obtain a first intermediate compound represented by general formula (7): in A PG A represents A protected by one or more protecting groups, wherein A contains a hydrophilic organic group that is part of an amino acid represented by general formula (3); R 11 and R 12 Each is an independent hydrophobic group; R 13 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; m≥1; and n≥1； (ii) Optionally, the first intermediate compound represented by general formula (7) is reacted with an acylating agent to obtain the second intermediate compound represented by general formula (8): Where R 17 It is -H or -C(=O)R 18 , where R 18 It is an alkyl group that is optionally substituted, an alkenyl group that is optionally substituted, an alkynyl group that is optionally substituted, or an alkoxy group that is optionally substituted; (iii) Deprotect the first intermediate compound represented by general formula (7) or the second intermediate compound represented by general formula (8) to obtain the compound represented by general formula (1).

13. The method of claim 12, wherein the method further comprises, prior to step (i): (ai) Reaction of the protected amino acid represented by general formula (10) with a carbonylating agent to obtain the N-carboxylic anhydride (NCA) monomer represented by general formula (5): 。 14. The method of any one of claims 12 to 13, wherein the N-carboxylic anhydride (NCA) monomer represented by general formula (5) comprises a structure represented by general formula (5A) and / or (5B): 。 15. The method of claim 14, wherein step (i) comprises mixing general formula (5A) with general formula (5B) in a molar ratio of 1:5 to 5:

1.

16. A nanoparticle composition for delivering therapeutic agents, preventative agents, and / or biological agents, said nanoparticle composition comprising: The compound claimed in any one of claims 1 to 11; and Therapeutic agents, preventative agents, and / or biological agents.

17. The nanoparticle composition of claim 16, wherein the composition further comprises: (a) Ionizable lipids; (b) assisting lipids; and (c) Cholesterol and / or its derivatives.

18. The nanoparticle composition of claim 17, wherein the ionizable lipids, auxiliary lipids, cholesterol and / or their derivatives and compounds represented by general formula (1) are mixed in a molar ratio of 5-65 : 4-20 : 10-60 : 0.1-20.

19. The nanoparticle composition according to any one of claims 17 to 18, wherein the ionizable lipid is selected from ALC-0315, SM-102, Lipid 5, DLinDMA, D-Lin-MC2-DMA, DLin-MC3-DMA, D-Lin-MC4-DMA, Dlin-KC2-DMA, YSK05, AA3-Dlin, SSPalmM, SSPalmO-Phe, Lipid A9, L319, DODMA, C1, BPLipid 310, ATX-001, ATX-100, Lipid 2, 80-O16B, BP Lipid 309, BP Lipid 307, 93-O17S, 93-O17O, NT1-O14B, 306-O12B-3, 306-O12B, 113-O16B, 306Oi10, 30 6Oi9-cis2, BAMEA-O16B, Al-28, 113-O12B, 98N12-5, Ckk-E12, OF-02, C12-200, BP Lipid 311, BP Lipid 308, BP Lipid 314, BP Lipid 312, LP01, TCL053, Lipid C24, BP Lipid 315, Lipid 29, 9A1P9, C13-112-three-tailed, C13-113-three-tailed, C13-112-four-tailed or C13-113-four-tailed, C12-200 and their combinations.

20. The nanoparticle composition of any one of claims 17 to 19, wherein the auxiliary lipid is selected from 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2- Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholestylhemisuccinoyl-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16) Lyso PC), 1,2-dilinoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate choline, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0 PE), 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof.

21. The nanoparticle composition according to any one of claims 17 to 20, wherein the cholesterol and / or its derivatives are selected from cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, alfalfa sterol, and combinations thereof.

22. The nanoparticle composition according to any one of claims 16 to 21, wherein the nanoparticle composition comprises nanoparticles having an N / P ratio of 1:1 to 50:

1.

23. The nanoparticle composition of any one of claims 16 to 22, wherein the nanoparticle composition comprises nanoparticles having an average particle size of not more than 300 nm.

24. The nanoparticle composition according to any one of claims 16 to 23, wherein the nanoparticle composition comprises nanoparticles having a zeta potential of -20 mV to +20 mV.

25. A nanoparticle composition claimed in any one of claims 16 to 24 for pharmaceutical use.

26. A nanoparticle composition claimed in any one of claims 16 to 24 for use in treating or preventing diseases, disorders or conditions in subjects in need of such treatment.

27. Use of the nanoparticle composition claimed in any one of claims 16 to 24 in the preparation of a medicament for treating or preventing a disease, disorder, or ailment in a subject in need of such treatment.

28. A method for treating or preventing a disease, disorder, or ailment in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition claimed in any one of claims 16 to 24.

29. The nanoparticle composition of claim 26, the use of claim 27, or the method of claim 28, wherein an immune response in the subject is induced by administration of the nanoparticle composition.

30. The nanoparticle composition of claim 26, the use of claim 27, or the method of claim 28, wherein the disease, disorder, or symptom is mediated by a coronavirus.

31. The nanoparticle composition, use, or method of claim 30, wherein the coronavirus is SARS-CoV-2 coronavirus.

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