Functional ionizable phospholipids

Abstract

Provided herein is ionizable phospholipid and its related compositions and methods.In some embodiments, provided herein is ionizable phospholipid can be formulated into the composition that contains nucleic acid and one or more helper excipients.In some embodiments, these compositions can also be used to treat disease or disorder with therapeutic nucleic acid.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 068,944, filed on 21 August 2020, entitled “Functional Ionizable Phospholipids,” which is incorporated herein by reference.

[0002] Self-declaration of government support This invention was made with government support under authorization number EB025192, granted by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by referencing electronically provided sequence listings An electronic version of the sequence listing is submitted accompanying this specification, and its entire contents constitute part of this specification by reference. The electronic file is 2 kilobytes in size and is titled 106546-697678_UTSD 3759_SequenceListing_ST25.txt.

[0004] This disclosure relates to functional ionizable synthetic phospholipids, pharmaceutical compositions comprising the disclosed ionizable synthetic phospholipids, and methods for, among many applications, gene editing, selective protein expression, mRNA delivery, and / or delivery of pharmaceutical active ingredients in a target. This disclosure also relates to pharmaceutical compositions comprising cargo-carried lipid nanoparticles (LNPs). [Background technology]

[0005] Genome editing technology possesses many desirable therapeutic properties that offer unique opportunities to design highly precise medical therapies for treating human diseases. However, overcoming biological barriers remains a major challenge in the effective delivery of these therapeutic agents to desired cell and tissue targets. Currently, lipid nanoparticles (LNPs) are the most commonly used vehicle for delivering gene-editing therapies across cell membranes. While the most effective LNPs for gene delivery rely on ionizable amines as a key physiological parameter, many LNPs still have insufficient endosomal escape capacity, rendering significant amounts of gene therapy cargo nonfunctional. Therefore, improved LNPs for delivering gene-editing therapies are needed in this field. [Overview of the project]

[0006] A brief description is provided below to illustrate the nature of the subject matter disclosed herein. Certain aspects of the disclosure are described below, but this summary is not intended to limit the scope of the disclosure.

[0007] This disclosure is at least in part based on the identification of ionizable synthetic phospholipids for use in nanoparticles and lipid nanoparticles (LNPs). In some embodiments, the ionizable synthetic phospholipid is of formula (I): [ka] (wherein R1 is selected from the group consisting of C2-C20 unsubstituted alkyl, C2-C20 substituted alkyl, C2-C20 unsubstituted alkenyl, C2-C20 substituted alkenyl, C2-C20 unsubstituted alkynyl, C2-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, and C4-C20 substituted cycloalkyl; R2 and R3 are H, C1-C20 unsubstituted alkyl, C1-C20 substituted alkyl, C1-C20 unsubstituted alkenyl, C1-C20 substituted alkenyl, C1-C20 unsubstituted alkynyl, C1-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, and C4-C2 R4, R5, R6, and R7 are independently selected from the group consisting of 0-substituted cycloalkyls; R8 is selected from the group consisting of H, C1-C8 unsubstituted alkyls, C1-C8 substituted alkyls, C1-C8 unsubstituted alkenyls, C1-C8 substituted alkenyls, C1-C8 unsubstituted alkynyls, and C1-C8 substituted alkynyls; n may include an integer from 1 to 4. In some embodiments, the ionizable synthetic phospholipids of this specification may comprise formula (I), wherein R1 is selected from the group consisting of C2-C16 unsubstituted alkyls, C2-C16 substituted alkyls, or C4-C12 substituted cycloalkyls; R2 and R3 are independently selected from the group consisting of H, C1-C16 unsubstituted alkyls, C1-C16 substituted alkyls, or C4-C16 substituted cycloalkyls; R4, R5, R6, and R7 are independently selected from the group consisting of H, C1-C4 unsubstituted alkyls, or C1-C4 substituted alkyls; R8 is selected from C3-C18 unsubstituted alkyls; and n is an integer from 1 to 3. In some embodiments, the ionizable synthetic phospholipids of this specification may comprise formula (I), wherein R1 is a C2-C15 unsubstituted alkyl group; R2 and R3 are independently selected from the group consisting of H, C1-C16 substituted alkyl groups, or C4-C16 substituted cycloalkyl groups; R4, R5, R6, and R7 are independently selected from the group consisting of H, methyl, or ethyl groups; R8 is selected from a C4-C16 unsubstituted alkyl group; and n is an integer between 1 and 2.

[0008] In some embodiments, the ionizable synthetic phospholipids of this specification are of formula (II): [ka] (wherein R1 and R2 are independently selected from the group consisting of H, C1-C8 substituted alkyls, or C1-C8 unsubstituted alkyls; R3, R4, R5, and R6 are independently selected from the group consisting of H, C1-C8 unsubstituted alkyls, or C1-C8 substituted alkyls; R7 is selected from the group consisting of C3-C21 unsubstituted alkyls or C3-C21 substituted alkyls; n is an integer from 1 to 4) In some embodiments, the ionizable synthetic phospholipids of this specification may include formula (II), where R1 and R2 are independently selected from the group consisting of H, C1-C6 substituted alkyls, or C1-C6 unsubstituted alkyls; R3, R4, R5, and R6 are independently selected from the group consisting of H, C1-C4 unsubstituted alkyls, or C1-C4 substituted alkyls; R7 is selected from the group consisting of C3-C18 unsubstituted alkyls or C3-C18 substituted alkyls; and n is an integer from 1 to 3. In some embodiments, the ionizable synthetic phospholipids of this specification may comprise formula (II), where R1 and R2 are independently selected from the group consisting of H, C1-C4 substituted alkyls, or C1-C4 unsubstituted alkyls; R3, R4, R5, and R6 are independently selected from the group consisting of H, methyl, or ethyl; R7 is selected from the group consisting of C3-C15 unsubstituted alkyls or C3-C15 substituted alkyls; and n is an integer between 1 and 2.

[0009] In some embodiments, the ionizable synthetic phospholipids of this specification are such that R1 and R2 are independently selected from the group consisting of H, C1-C8 substituted alkyl, C1-C8 unsubstituted alkyl, C2-C8 unsubstituted alkenyl, C2-C8 substituted alkenyl, C2-C8 unsubstituted alkynyl, or C2-C8 substituted alkynyl; and R3, R4, R5, and R6 are H, C1-C8 substituted alkyl, C1-C8 unsubstituted alkyl, or C1-C8 unsubstituted The formula (II) may include R7, which is independently selected from the group consisting of alkenyls, C2-C8 substituted alkenyls, C2-C8 unsubstituted alkynyls, or C2-C8 substituted alkynyls; R7 is selected from the group consisting of C3-C21 unsubstituted alkyls, C3-C21 substituted alkyls, C3-C21 unsubstituted alkenyls, C3-C21 substituted alkenyls, C3-C21 unsubstituted alkynyls, or C3-C21 substituted alkynyls; and n is an integer from 1 to 4. In some embodiments, the ionizable synthetic phospholipids of this specification may include formula (II), where R1 and R2 are independently selected from the group consisting of H, C1-C6 substituted alkyl, C1-C6 unsubstituted alkyl, C2-C6 unsubstituted alkenyl, C2-C6 substituted alkenyl, C2-C6 unsubstituted alkynyl, or C2-C6 substituted alkynyl; R3, R4, R5, and R6 are independently selected from the group consisting of H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C2-C4 unsubstituted alkenyl, C2-C4 substituted alkenyl, C2-C4 unsubstituted alkynyl, or C2-C4 substituted alkynyl; R7 is selected from the group consisting of C3-C18 unsubstituted alkyl or C3-C18 substituted alkyl; and n is an integer from 1 to 3. In some embodiments, the ionizable synthetic phospholipids of this specification may comprise formula (II), where R1 and R2 are independently selected from the group consisting of H, C1-C4 substituted alkyls, or C1-C4 unsubstituted alkyls; R3, R4, R5, and R6 are independently selected from the group consisting of H, methyl, or ethyl; R7 is selected from the group consisting of C3-C15 unsubstituted alkyls or C3-C15 substituted alkyls; and n is an integer between 1 and 2.

[0010] In some embodiments, the ionizable synthetic phospholipids of this specification are of formula (III): [ka] (wherein R1 is selected from the group consisting of C2-C20 unsubstituted alkyl, C2-C20 substituted alkyl, C2-C20 unsubstituted alkenyl, C2-C20 substituted alkenyl, C2-C20 unsubstituted alkynyl, C2-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, or C4-C20 substituted cycloalkyl; R2 and R3 are H, C1-C20 unsubstituted alkyl, C1-C20 substituted alkyl, C1-C20 unsubstituted alkenyl, C1-C20 substituted alkenyl, C1-C20 unsubstituted alkynyl, C1-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, or C4-C20 substituted alkynyl, C1-C20 unsubstituted cycloalkyl, or C4-C20 substituted alkynyl, C1-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, or C4-C20 substituted alkynyl, C11-C20 substituted alkynyl, C4-C20 substituted cycloalkyl, or C4-C20 substituted alkynyl, C1-C20 substituted alkynyl, C1-C20 substituted alkynyl, C1-C20 substituted alkynyl, C1-C20 substituted alkynyl, C1-C20 substituted alkynyl, C R4 and R5 are independently selected from the group consisting of substituted cycloalkyls; R4 and R5 are independently selected from the group consisting of H, C1-C8 unsubstituted alkyls, C1-C8 substituted alkyls, C1-C8 unsubstituted alkenyls, C1-C8 substituted alkenyls, C1-C8 unsubstituted alkynyls, or C1-C8 substituted alkynyls; R6 is selected from the group consisting of C3-C21 unsubstituted alkyls, C3-C21 substituted alkyls, C3-C21 unsubstituted alkenyls, C3-C21 substituted alkenyls, C3-C21 unsubstituted alkynyls, or C3-C21 substituted alkynyls; n is an integer from 1 to 4; m is an integer from 1 to 4).In some embodiments, the ionizable synthetic phospholipids herein have R1 selected from the group consisting of C2-C16 unsubstituted alkyl, C2-C16 substituted alkyl, C2-C16 unsubstituted alkenyl, C2-C16 substituted alkenyl, C2-C16 unsubstituted alkynyl, C2-C16 substituted alkynyl, C4-C16 unsubstituted cycloalkyl, or C4-C16 substituted cycloalkyl; R2 and R3 independently selected from the group consisting of H, C1-C16 unsubstituted alkyl, C1-C16 substituted alkyl, C1-C16 unsubstituted alkenyl, C1-C16 substituted alkenyl, C1-C16 unsubstituted alkynyl, C1-C16 substituted alkynyl, C4-C16 unsubstituted cycloalkyl, or C4-C16 substituted cycloalkyl; R4 and R5 independently selected from the group consisting of H, C1-C6 unsubstituted alkyl, C1-C6 substituted alkyl, C1-C6 unsubstituted alkenyl, C1-C6 substituted alkenyl, C1-C6 unsubstituted alkynyl, or C1-C6 substituted alkynyl; R6 selected from the group consisting of C3-C18 unsubstituted alkyl, C3-C18 substituted alkyl, C3-C18 unsubstituted alkenyl, C3-C18 substituted alkenyl, C3-C18 unsubstituted alkynyl, or C3-C18 substituted alkynyl; n is an integer from 1 to 3; m is an integer from 1 to 3, and may include formula (III). In some embodiments, the ionizable synthetic phospholipids herein have R1 as C2-C15 unsubstituted alkyl; R2 and R3 independently selected from the group consisting of H, C1-C16 substituted alkyl, or C4-C16 substituted cycloalkyl; R4 and R5 independently selected from the group consisting of H, methyl, or ethyl; R6 selected from C4-C16 unsubstituted alkyl; n is an integer from 1 to 2; m is an integer from 1 to 2, and may include formula (III).

[0011] In some embodiments, the ionizable synthetic phospholipids herein may include at least one phosphate group and at least one zwitterion, and at least one zwitterion includes a pH-switchable zwitterion and / or an irreversible zwitterion. In some embodiments, the ionizable synthetic phospholipids herein may further include at least one tertiary amine.

[0012] In some embodiments, the ionizable synthetic phospholipids herein may further include a hydrophobic domain. In some embodiments, the ionizable synthetic phospholipids herein may further include one or more hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of one hydrophobic tail. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of two hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of three hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of four hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of five hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of six hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of seven hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of eight hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of nine hydrophobic tails. In some embodiments, the ionizable synthetic phospholipid comprising one or more hydrophobic tails herein may consist of ten hydrophobic tails.

[0013] In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl tail with an alkyl chain length of 8 to 16 carbon atoms. In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl tail with an alkyl chain length of 8 to 10 carbon atoms. In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl chain length of 9 to 12 carbon atoms. In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl chain length of 13 to 16 carbon atoms. In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl chain length of 8 to 16 carbon atoms. In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl tail having an alkyl chain length of 8 to 10 carbon atoms. In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl tail having an alkyl chain length of 9 to 12 carbon atoms. In some embodiments, an ionizable synthetic phospholipid comprising one or more hydrophobic tails according to this specification may comprise one or more hydrophobic tails having an alkyl tail having an alkyl chain length of 13 to 16 carbon atoms.

[0014] In some embodiments, the disclosure provides pharmaceutical compositions. In some embodiments, the pharmaceutical compositions herein may comprise one of the ionizable synthetic phospholipids disclosed herein.

[0015] In some embodiments, the compositions of this specification (e.g., pharmaceutical compositions, nanoparticles, LNPs) may further comprise at least one helper lipid. In some embodiments, the compositions of this specification may further comprise at least one helper lipid selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-methyldioctadecylamine (MDOA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecylammonium bromide salt (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), or any combination thereof. In some embodiments, the compositions of this specification may further comprise at least one helper lipid selected from the group consisting of zwitterionic helper lipids, ionizable cationic helper lipids, permanent cationic helper lipids, or any combination thereof. In some embodiments, the compositions herein may include a zwitterionic helper lipid that may contain 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the compositions herein may further include at least one ionizable cationic helper lipid comprising at least one lipid selected from the group consisting of N-methyldioctadecylamine (MDOA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and any combination thereof. In some embodiments, the compositions herein may further include at least one permanent cationic helper lipid comprising at least one lipid selected from the group consisting of dimethyldioctadecylammonium bromide salt (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and any combination thereof.

[0016] In some embodiments, the compositions herein may further comprise cholesterol and / or cholesterol derivatives. In some embodiments, the compositions herein may further comprise 1,2-dimyristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000). In some embodiments, the compositions herein may further comprise one or more multi-tail ionizable phospholipids in a 55:30:45 molar ratio, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. In some embodiments, the compositions herein may further comprise one or more multi-tail ionizable phospholipids in a 25:30:30 molar ratio, N-methyldioctadecylamine (MDOA), and cholesterol. In some embodiments, the composition of this specification may further comprise one or more multi-tail ionizable phospholipids in a 25:30:30 molar ratio, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and cholesterol. In some embodiments, the composition of this specification may further comprise one or more multi-tail ionizable phospholipids in a 25:30:30 molar ratio, 5A2-SC8, and cholesterol. In some embodiments, the composition of this specification may further comprise one or more multi-tail ionizable phospholipids in a 60:30:40 molar ratio, dimethyldioctadecylammonium bromide salt (DDAB), and cholesterol. In some embodiments, the composition of this specification may further comprise one or more multi-tail ionizable phospholipids in a 60:30:40 molar ratio, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol. In some embodiments, the compositions of this specification may further comprise 1,2-dimyristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000).

[0017] In some embodiments, the compositions herein may further comprise at least one cargo. In some embodiments, the compositions herein may further comprise at least one cargo, the cargo being mRNA. In some embodiments, the compositions herein may further comprise at least one cargo, the cargo being selected from the group consisting of pharmaceutical active ingredients, nucleic acids, mRNA, sgRNA, CRISPR / Cas9 DNA sequences, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), siRNA, miRNA, tRNA, ssDNA, base editors, peptides, proteins, CRISPR / Cas ribonucleoprotein (RNP) complexes, and any combination thereof.

[0018] In some embodiments, the compositions herein may be formulated for parenteral administration. In some embodiments, the compositions herein may be formulated for intravenous administration. In some embodiments, the compositions herein may be formulated for topical administration.

[0019] In some embodiments, the pharmaceutical compositions disclosed herein may include cargo-carrying lipid nanoparticles (LNPs) the LNPs comprising any ionizable phospholipid disclosed herein, or one or more multi-tailed ionizable phospholipids disclosed herein, wherein one or more multi-tailed ionizable phospholipids comprise a pH-switchable zwitterion and three hydrophobic tails.

[0020] In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more multi-tailed ionizable phospholipids comprising one tertiary amine, one phosphate group, and three alkyl tails. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more multi-tailed ionizable phospholipids in which the three hydrophobic tails or three alkyl tails may have an alkyl chain length of 8 to 16 carbon atoms. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more multi-tailed ionizable phospholipids in which the three hydrophobic tails or three alkyl tails may have an alkyl chain length of 8 to 10 carbon atoms. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more multi-tailed ionizable phospholipids in which the three hydrophobic tails or three alkyl tails may have an alkyl chain length of 9 to 12 carbon atoms. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more multi-tailed ionizable phospholipids in which three hydrophobic tails or three alkyl tails may have an alkyl chain length of 13 to 16 carbon atoms.

[0021] In some embodiments, the pharmaceutical composition disclosed herein may contain a cargo, the cargo being mRNA, and the pharmaceutical composition provides selective protein expression in the liver. In some embodiments, the pharmaceutical composition disclosed herein may contain a cargo, the cargo being mRNA, and the pharmaceutical composition provides selective protein expression in the spleen. In some embodiments, the pharmaceutical composition disclosed herein may contain a cargo, the cargo being mRNA, and the pharmaceutical composition provides selective protein expression in the lungs. In some embodiments, the pharmaceutical composition disclosed herein may contain a cargo, the cargo being mRNA, and the pharmaceutical composition provides selective protein expression in the skin. In some embodiments, the pharmaceutical composition disclosed herein may contain a cargo, the cargo being mRNA. In some embodiments, the pharmaceutical composition disclosed herein may contain a cargo, the cargo being located within the core of an LNP.

[0022] In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more multi-tailed ionizable phospholipids, where one or more multi-tailed ionizable phospholipids form a nanoparticle structure that substantially encapsulates the cargo.

[0023] In some embodiments, the pharmaceutical compositions disclosed herein may be used for gene delivery in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for gene editing in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for drug delivery in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for mRNA delivery in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for CRISPR / Cas9 gene editing in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for zinc finger nuclease (ZFN) gene editing in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for base editor gene editing in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for transcription activator-like effector nuclease (TALEN) gene editing in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for tissue-specific mRNA delivery in a subject. In some embodiments, the pharmaceutical compositions disclosed herein may be used for tissue-specific CRISPR / Cas9 gene editing in a subject.

[0024] In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one cargo, which may be selected from the group consisting of pharmaceutical active ingredients, nucleic acids, mRNA, sgRNA, CRISPR / Cas9 DNA sequences, zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), siRNA, miRNA, tRNA, ssDNA, base editors, peptides, proteins, cirRNA, CRISPR / Cas ribonucleoprotein (RNP) complexes, and any combination thereof.

[0025] In some embodiments, the pharmaceutical composition disclosed herein may comprise at least one nanoparticle. In some embodiments, the pharmaceutical composition disclosed herein may comprise at least one LNP. In some embodiments, the pharmaceutical composition disclosed herein may comprise at least one LNP, wherein the at least one LNP further comprises at least one helper lipid.

[0026] In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, the at least one LNP being a multicomponent LNP for organ-selective delivery, and the multicomponent LNP comprising a multitail ionizable phospholipid and one or more helper lipids. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, the cargo being mRNA, and the one or more helper lipids may enable selective protein expression in the skin, spleen, liver, and / or lungs.

[0027] In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, the one or more helper lipids being selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-methyldioctadecylamine (MDOA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and any combination thereof. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, the one or more helper lipids being selected from the group consisting of zwitterionic helper lipids, ionizable cationic helper lipids, and permanent cationic helper lipids. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the zwitterionic helper lipid may comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the ionizable cationic helper lipid may comprise at least one lipid selected from the group consisting of N-methyldioctadecylamine (MDOA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 5A2-SC8, and any combination thereof. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the permanent cationic helper lipid may comprise at least one lipid selected from the group consisting of dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and any combination thereof.In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may further comprise cholesterol or a cholesterol derivative. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may further comprise 1,2-dimiristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000). In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may comprise one or more multi-tail ionizable phospholipids in a 55:30:45 molar ratio, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may comprise one or more multi-tailed ionizable phospholipids in a 25:30:30 molar ratio, N-methyldioctadecylamine (MDOA), and cholesterol. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may comprise one or more multi-tailed ionizable phospholipids in a 25:30:30 molar ratio, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and cholesterol. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may comprise one or more multi-tailed ionizable phospholipids in a 25:30:30 molar ratio, 5A2-SC8, and cholesterol. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may comprise one or more multi-tail ionizable phospholipids in a 60:30:40 molar ratio, dimethyl dioctadecylammonium bromide (DDAB), and cholesterol.In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may comprise one or more multi-tail ionizable phospholipids in a 60:30:40 molar ratio, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may further comprise 1,2-dimyristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000).

[0028] In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, wherein the LNP may induce cargo release from endosomes in target cells.

[0029] In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, and the pharmaceutical compositions may be formulated for parenteral administration. In some embodiments, the pharmaceutical compositions disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, and the pharmaceutical compositions may be formulated for parenteral administration, and the parenteral administration may comprise at least one selected from the group consisting of subcutaneous, intradermal, intraperitoneal, intrathecal, and intramuscular administration.

[0030] In some embodiments, the pharmaceutical composition disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, and the pharmaceutical composition may be formulated for intravenous administration. In some embodiments, the pharmaceutical composition disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, and the pharmaceutical composition may be formulated for oral administration. In some embodiments, the pharmaceutical composition disclosed herein may comprise at least one LNP, a cargo, and one or more helper lipids, and the pharmaceutical composition may be formulated for topical administration.

[0031] In some embodiments, this disclosure provides a method for delivering a pharmaceutical active ingredient to a target. In some embodiments, the method of delivering a pharmaceutical active ingredient to a target may include the step of administering a therapeutically effective amount of any pharmaceutical composition to a target, including a cargo as disclosed herein, where the cargo is the pharmaceutical active ingredient.

[0032] In some embodiments, the Disclosure provides a method for delivering mRNA or mRNA / sgRNA for gene editing to a target. In some embodiments, the method herein for delivering mRNA or mRNA / sgRNA for gene editing to a target may include the step of administering a therapeutically effective amount of any pharmaceutical composition to a target, the cargo of which is mRNA.

[0033] In some embodiments, the disclosure provides a method for inducing selective protein expression in a target liver. In some embodiments, the method for inducing selective protein expression in a target liver may include the step of administering a therapeutically effective amount of the pharmaceutical composition herein to a target, wherein the cargo may be mRNA and the three hydrophobic tails or three alkyl tails may have an alkyl chain length of 9 to 12 carbon atoms. In some embodiments, the disclosure provides a method for inducing selective protein expression in a target spleen. In some embodiments, the method for inducing selective protein expression in a target spleen may include the step of administering a therapeutically effective amount of the pharmaceutical composition herein to a target, wherein the cargo may be mRNA and the three hydrophobic tails or three alkyl tails may have an alkyl chain length of 13 to 16 carbon atoms. In some embodiments, the disclosure provides a method for inducing selective protein expression in a target spleen, liver, skin and / or lung. In some embodiments, the method for inducing selective protein expression in a target spleen, liver, skin and / or lung may include the step of administering a therapeutically effective amount of the pharmaceutical composition herein to a target, wherein the cargo is mRNA.

[0034] In some embodiments, the Disclosure provides a method for gene delivery in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Disclosure provides a method for gene editing in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Disclosure provides a method for drug delivery in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Disclosure provides a method for RNA delivery in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Disclosure provides a method for CRISPR / Cas9 gene editing in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Disclosure provides a method for zinc finger nuclease (ZFN) gene editing in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Disclosure provides a method for base editor gene editing in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the present disclosure provides a method for editing a transcription activator-like effector nuclease (TALEN) gene in a subject, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition disclosed herein to the subject.

[0035] In some embodiments, the Disclosure provides a method for tissue-specific mRNA delivery in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject. In some embodiments, the Disclosure provides a method for tissue-specific CRISPR / Cas9 gene editing in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to the subject.

[0036] In some embodiments, the methods herein may include a step of administering any composition disclosed herein by parenteral administration. In some embodiments, the methods herein may include a step of administering any composition disclosed herein by intravenous administration. In some embodiments, the methods herein may include a step of administering any composition disclosed herein by topical administration.

[0037] In some embodiments, kits are provided herein. The kits disclosed herein may be used in conjunction with any of the compositions disclosed herein and / or to carry out any of the methods disclosed herein.

[0038] Additional embodiments and features are partially shown in the following description and may become apparent to those skilled in the art upon examination of the specification or may be learned through the practice of the disclosure. A further understanding of the nature and merits of the disclosure may be achieved by referring to the remainder of the specification and the drawings which form part of the disclosure.

[0039] A patent or application file must contain at least one drawing made in color. A copy of this patent or patent application publication containing the color drawing(s) must be provided by the Patent Office upon request and payment of the required fees.

[0040] This specification is better understood with reference to the following drawings and data graphs, which are presented as various embodiments of the disclosure and should not be construed as a complete enumeration of the scope of the disclosure. [Brief explanation of the drawing]

[0041] [Figure 1A]This figure shows a combinatorial library of iPhos lipids that have led to the elucidation of the physical mechanisms of action for chemically synthesized, tested, and enhanced endosomal escape. Figure 1A shows that effective iPhos lipids consisted of one ionizable amine, one phosphate group, and three hydrophobic alkyl tails; and that upon entry into acidic endosomes / lysosomes, protonation of the tertiary amine induced a zwitterionic head group that could be readily inserted into the membrane. [Figure 1B] This figure shows a combinatorial library of iPhos lipids that have led to the elucidation of the physical mechanisms of chemically synthesized, tested, and enhanced endosomal escape. Figure 1B shows that most biological membrane phospholipids possess zwitterions and adopt a lamellar phase; and that when iPhos lipids are mixed and inserted into the endosomal membrane, the conical shape formed by small ion-pair heads and multiple hydrophobic tails enables hexagonal phase transition. [Figure 1C] This figure shows a combinatorial library of iPhos lipids that have led to the elucidation of the physical mechanisms of chemically synthesized, tested, and enhanced endosomal escape. Figure 1C shows the synthesis route of iPhos. Alkylated dioxaphosphorane oxide molecules (Pm) were conjugated to amines (nA) to obtain iPhos(nAxPm) (where "x" in "nAxPm" indicates the number of modified Pm molecules on one amine molecule). [Figure 1D] This figure shows a combinatorial library of iPhos lipids that have led to the elucidation of the physical mechanisms of action for chemically synthesized, tested, and enhanced endosomal escape. Figure 1D shows 28 amine nA and 13 alkylated dioxaphosphoranoxide Pm molecules used in iPhos synthesis. [Figure 2A]This figure shows that the iPhos lipid, which has one pH-switchable zwitterion and three tails, was most effective for luciferase mRNA delivery in vitro. Figure 2A shows a heatmap of luciferase expression in IGROV1 cells after treatment with iPLNP (50 ng mRNA, n=3). RLU > 10000 was counted for hit rate calculation. [Figure 2B] This figure shows that iPhos lipids with one pH-switchable zwitterion and three tails were most effective for luciferase mRNA delivery in vitro. Figure 2B shows representative chemical structures of iPhos with different numbers of zwitterions and tails in an acidic endosomal environment. [Figure 2C] Figure C shows that iPhos lipids with one pH-switchable zwitterion and three tails were most effective for luciferase mRNA delivery in vitro. Figure C shows the relative hit rates of iPhos with a single zwitterion and iPhos with multiple zwitterions. [Figure 2D] This figure shows that iPhos lipids with one pH-switchable zwitterion and three tails were most effective for luciferase mRNA delivery in vitro. Figure 2D shows the relative hit rates of iPhos (1A1P4~18A1P16) with a single zwitterion and different numbers of tails. [Figure 2E] This figure shows that iPhos lipids with one pH-switchable zwitterion and three tails were most effective for luciferase mRNA delivery in vitro. Figure 2E shows that among the efficient iPhos (7A1P4-13A1P16), the tail length of the starting amine influenced the final in vitro efficacy. [Figure 3A] This figure shows that a model membrane study of endosomal escape demonstrated a mechanism of iPhos lipid-mediated RNA delivery correlated with chemical structure. Figure 3A shows hemolysis of 17A and 10A1P10 at pH 5.5, as well as the possibility that zwitterions may significantly aid in endosomal escape; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. [Figure 3B] This figure shows that a model membrane test of endosomal escape demonstrated a mechanism of iPhos lipid-mediated RNA delivery correlated with chemical structure. Figure 3B shows hemolysis of 9A1P9 and 10A1P10 at different pH levels; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. [Figure 3C] This figure shows that a model membrane test of endosomal escape demonstrated a mechanism of iPhos lipid-mediated RNA delivery correlated with chemical structure. Figure 3C shows hemolysis of iPLNP at different pH levels; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. [Figure 3D] This figure shows that a model membrane assay for endosomal escape demonstrated a mechanism of iPhos lipid-mediated RNA delivery correlated with chemical structure. Figure 3D shows lipid fusion and membrane rupture of 10A1P10 and iPLNP determined by FRET assay at pH 5.5; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. [Figure 3E] This figure shows that a model membrane test of endosomal escape demonstrated a mechanism of iPhos lipid-mediated RNA delivery correlated with chemical structure. Figure 3E shows iPLNP dissociation by FRET characterization after mixing with an anionic endosome mimetic at pH 5.5 for 10 minutes; and that a single zwitterion showed higher lipid fusion and iPLNP dissociation efficacy than multiple lamination ions; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. [Figure 3F] This figure shows that a model membrane study of endosomal escape demonstrated a mechanism of iPhos lipid-mediated RNA delivery correlated with chemical structure. Figure 3F shows 10A1P10 iPLNP dissociation at different time intervals at pH 5.5, with data shown as mean ± SD; n=3. [Figure 3G]This figure demonstrates that a model membrane study of endosomal escape demonstrated a mechanism of iPhos lipid-mediated RNA delivery correlated with chemical structure. Figure 3G shows 10A1P10 iPLNP dissociation at different time intervals at pH 5.5; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. Data in Figures 3B-3G are presented as mean ± sd (n=3 biologically independent samples). [Figure 4A] This figure shows a structure-activity study revealing that the chemical structure and alkyl length of iPhos lipids controlled in vivo efficacy and organ selectivity. Figure 4A shows the in vivo evaluation of 51 iPhos lipids at a low Fluc mRNA dose (0.1 mg / kg); bioluminescence images of various organs were recorded 6 hours after IV injection in C57BL / 6 mice; H: heart, Lu: lung, Li: liver, K: kidney, S: spleen. [Figure 4B] This figure shows a structure-activity study revealing that the chemical structure and alkyl length of iPhos lipids controlled in vivo efficacy and organ selectivity. Figure 4B shows that among effective 10A1P4-12A1P16 iPhos, the hydrophobic chain length on the amine side dramatically affected in vivo mRNA delivery efficacy. [Figure 4C] This figure shows a structure-activity study revealing that the chemical structure and alkyl length of iPhos lipids controlled in vivo efficacy and organ selectivity. Figure 4C shows mRNA expression in the liver by iPhos with different alkyl chain lengths on the phosphate side, and that carbon lengths of 9–12 were the most efficient. [Figure 4D] This figure shows a structure-activity study revealing that the chemical structure and alkyl length of iPhos lipids controlled in vivo efficacy and organ selectivity. Figure 4D shows mRNA expression in the spleen by iPhos with different alkyl chain lengths on the phosphate side, and that alkyl chain lengths of 13–16 were the most efficient. [Figure 5A]This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs that have different helper lipids to enable organ-selective RNA delivery. Figure 5A shows the proposed structure of iPhos 9A1P9 in an acidic endosomal environment. [Figure 5B] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5B shows images of recorded luciferase expression in the liver (Fluc mRNA, 0.25 mg / kg) demonstrating that iPhos 9A1P9 performed better than standard DOPE and DSPC in terms of mRNA delivery; H: heart, Lu: lung, Li: liver, K: kidney, S: spleen. [Figure 5C] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5C shows quantification of luciferase expression in recorded liver (Fluc mRNA, 0.25 mg / kg) demonstrating that iPhos 9A1P9 performed better than standard DOPE and DSPC in terms of mRNA delivery; data are shown as mean ± SD; n=3; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. [Figure 5D] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5D shows in vivo evaluations demonstrating that iPLNPs containing zwitterionic helper lipids mediated mRNA expression in the spleen, and that 9A1P9 with the helper lipid DOPE was efficient in the spleen (Fluc mRNA, 0.25 mg / kg). [Figure 5E]This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5E shows in vivo quantification demonstrating that iPLNPs containing zwitterionic helper lipids and 9A1P9 iPLNPs with helper lipid DOPE were efficient in the spleen, mediated by mRNA expression in the spleen (Fluc mRNA, 0.25 mg / kg). [Figure 5F] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied with iPLNPs having different helper lipids to enable organ-selective RNA delivery. Figure 5F shows the organ selectivity of Fluc mRNA expression by iPLNPs containing ionizable cationic helper lipids that resulted in mRNA translation in the liver, and by 9A1P9 iPLNPs having different ionizable cationic helper lipids that were assayed (0.25 mg / kg for Fluc mRNA, MDOA, and DODAP; 0.05 mg / kg for 5A2-SC8). [Figure 5G] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied with iPLNPs having different helper lipids to enable organ-selective RNA delivery. Figure 5G shows the quantification of Fluc mRNA expression by iPLNPs containing ionizable cationic helper lipids that resulted in mRNA translation in the liver, and by 9A1P9 iPLNPs having different ionizable cationic helper lipids that were assayed (0.25 mg / kg for Fluc mRNA, MDOA, and DODAP; 0.05 mg / kg for 5A2-SC8). [Figure 5H]This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5H shows organ images of fluc mRNA expression by iPLNPs containing permanent cationic helper lipids that induced mRNA translation in the lung, and by 9A1P9 iPLNPs using the permanent cationic helper lipids DDAB and DOTAP evaluated (fluc mRNA, 0.25 mg / kg). [Figure 5I] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5I shows iPLNPs containing permanent cationic helper lipids that induced mRNA translation in the lung, and quantification of Fluc mRNA expression by 9A1P9 iPLNPs using the permanent cationic helper lipids DDAB and DOTAP to be evaluated (Fluc mRNA, 0.25 mg / kg). [Figure 5J] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs, which have different helper lipids to enable organ-selective RNA delivery. Figure 5J shows a schematic diagram of a Cre-LoxP mouse model in which iPLNPs enabled selective Cre mRNA delivery in the liver or lung, and in which tdTomato expression was expressed by translating Cre recombinase mRNA to Cre protein, thereby removing the stoppage of mediated tdTomato expression in the liver and lung, respectively (Cre mRNA, 0.25 mg / kg). [Figure 5K]This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5K shows a 9A1P9-5A2-SC8 iPLNP that enabled selective Cre mRNA delivery in the liver. [Figure 5L] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5L shows a 9A1P9-DDAB iPLNP that enabled selective Cre mRNA delivery in the lung. [Figure 5M] This figure shows that iPhos is a platform technology that offers advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5M shows images of 9A1P9-5A2-SC8 iPLNP, which showed much higher mRNA delivery efficacy than the positive control DLin-MC3-DMA LNP, and recorded luciferase expression in the liver (Fluc mRNA, 0.05 mg / kg). [Figure 5N] This figure shows that iPhos is a platform technology that has advantages over conventionally used phospholipids, as it can be applied to iPLNPs with different helper lipids to enable organ-selective RNA delivery. Figure 5N shows 9A1P9-5A2-SC8 iPLNP, which showed much higher mRNA delivery efficacy than the positive control DLin-MC3-DMA LNP, and quantification of luciferase expression in the liver recorded (Fluc mRNA, 0.05 mg / kg); data are shown as mean ± SD; n=3; statistical significance: ***, P<0.001; **, P<0.01; *, P<0.05. [Figure 6A]This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6A shows a schematic diagram of co-delivery of Cas9 mRNA and sgTom1 with the stop cassette removed and the tdTomato protein activated. [Figure 6B] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lungs by co-delivering Cas9 mRNA and sgRNA. Figure 6B shows the 9A1P9-5A2-SC8 iPLNP that enabled specific gene editing in the liver. [Figure 6C] This figure shows iPLNPs that selectively achieve CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6C shows that tdTomato-positive cells were observed in the liver after IV administration of 9A1P9-5A2-SC8 iPLNPs containing Cas9 mRNA and sgTom1 to Ai9 mice. Scale bar, 50 μm. [Figure 6D] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6D shows the 9A1P9-DDAB iPLNP that enabled specific gene editing in the lung. [Figure 6E] This figure shows iPLNPs that selectively achieve CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6E shows a confocal fluorescence image of tdTomato-positive cells in the lung after administration of 9A1P9-DDAB iPLNP. Scale bar, 50 μm. [Figure 6F]This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6F shows the T7E1 assay for organ-selective gene editing in C57BL / 6 mice administered intravenously with 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs containing Cas9 mRNA and sgPTEN, respectively, enabling CRISPR / Cas9 gene editing in the liver and lung. For all CRISPR / Cas9 gene editing assays, a Cas9 mRNA / sgRNA weight ratio of 4:1 and a total RNA dose of 0.75 mg / kg were used. [Figure 6G] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6G shows 9A1P9-5A2-SC8 iPLNP (liver-specific, Fluc mRNA, 0.05 mg / kg) prepared by controlled microfluidic mixing, which resulted in reduced iPLNP size and conservation of efficacy and organ selectivity. [Figure 6H] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6H shows that the 9A1P9-5A2-SC8 iPLNP exhibited a small size and fully maintained precise organ selectivity. [Figure 6I] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lungs by co-delivering Cas9 mRNA and sgRNA. Figure 6I shows whole-body imaging performed 6 hours after each injection, demonstrating that 9A1P9-5A2-SC8 iPLNP (Fluc mRNA, 0.05 mg / kg) allowed for repeated administration without loss of efficacy. [Figure 6J]This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lungs by co-delivering Cas9 mRNA and sgRNA. Figure 6J shows the quantification of luciferase expression performed 6 hours after each injection, demonstrating that 9A1P9-5A2-SC8 iPLNP (Fluc mRNA, 0.05 mg / kg) allowed for repeated administration without loss of efficacy. [Figure 6K] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6K shows liver marker measurements (BUN) demonstrating that 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs were well-tolerated in vivo. [Figure 6L] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6L shows liver marker measurements (CREA) demonstrating that 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs were well-tolerated in vivo. [Figure 6M] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6M shows liver marker measurements (ATL) demonstrating that 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs were well-tolerated in vivo. [Figure 6N] This figure shows iPLNPs that selectively achieved CRISPR / Cas9 gene editing in the liver and lung by co-delivering Cas9 mRNA and sgRNA. Figure 6N shows liver marker measurements (AST) demonstrating the high tolerability of 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs in vivo. Data are presented as mean ± sd, and statistical significance was analyzed by two-sided unpaired t-tests. ****, P<0.0001; ***, P<0.001; **, P<0.01; *, P<0.05; NS, P>0.05. All data are from n=3 biologically independent mice. [Figure 7A] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 90.8% (P4; FIG. 7A). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7B] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 96.8% (P5; FIG. 7B). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7C] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 96.8% (P6; FIG. 7C). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7D] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 96.0% (P7; FIG. 7D). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7E]This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 93.0% (P8; FIG. 7E). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7F] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4-P16. The conversion yield to the corresponding alcohol product was 87.0% (P9; FIG. 7F). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7G] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4-P16. The conversion yield to the corresponding alcohol product was 93.0% (P10; FIG. 7G). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7H] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 95.3% (P11; FIG. 7H). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7I] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4-P16. The conversion yield to the corresponding alcohol product was 93.8% (P12; FIG. 7I). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7J] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 87.8% (P14; FIG. 7J). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7K] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4-P16. The conversion yield to the corresponding alcohol product was 95.3% (P13; FIG. 7K). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7L] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4-P16. The conversion yield to the corresponding alcohol product was 92.3% (P15; FIG. 7L). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 7M] This figure shows the ¹H NMR spectra (CDCl3) of alkylated dioxaphosphorane oxide molecules P4–P16. The conversion yield to the corresponding alcohol product was 88.5% (P16; FIG. 7M). The conversion yield was calculated from the integrals of peak a (4H, -OCH2CH2O- of the Pm molecule) and peak e (3H, -CH2CH2CH3 of both Pm and the corresponding alcohol molecule). [Figure 8] This figure shows the synthesis of iPhos using different amine starting materials. Amines with one primary, secondary, or tertiary amine group introduced a single zwitterion. Amines with several amine groups introduced multiple zwitterions. [Figure 9]This figure shows the 1H NMR spectra of P10 (DMSO-d6) and selected iPhos (CDCl3). The P10 spectrum was recorded after stirring in DMSO-d6 at 70°C and remained largely unchanged. For iPhos, the reaction was carried out in DMSO-d6 at 70°C for 3 days, and the peak a (4.4 ppm) where the methylene group disappeared after the reaction indicated that almost all of the P10 was consumed by the amine group. Furthermore, the less active P16 was also able to react completely with the amine. [Figure 10] This figure shows the cell viability of IGROV1 cells treated with iPLNP containing fluc mRNA. The majority of iPhos showed negligible cytotoxicity. [Figure 11] According to one embodiment of this disclosure, 9A1P9 in CDCl3; 1H NMR (CDCl3, ppm) δ 0.87 (m, 9H, -CH2CH2CH2CH3), 1.25 (m, 32H, -OCH2CH2(CH2)6CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH2CH3)2), 1.55-1.84 (m, 6H, -OCH2CH2(CH2)6CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH3)2), 2.80 (m, 4H, -N(CH2CH2CH2CH2CH2CH2CH2CH3)2), 3.87 (m, 2H, -NCH2CH2O-), 3.96-4.28 (m, 4H, -NCH2CH2O- and This figure shows the 1H NMR spectra of -OCH2CH2(CH2)6CH3).13C NMR (CDCl3, ppm) δ 14.08, 14.10, 22.61, 22.66, 26.00, 27.00, 27.03, 29.16, 29.22, 29.25, 29.32, 29.45, 29.49, 29.62, 31.73, 31.77, 47.72. 31P NMR (CDCl3, ppm) δ -0.91, 0.94. Calculated mass m / z 491.4, experimentally measured [M+H]+(LC-MS) m / z 492.4. [Figure 12]According to one embodiment of this disclosure, 10A1P10 in CDCl3; 1H NMR (CDCl3, ppm) δ 0.87 (t, 9H, -CH2CH2CH2CH3), 1.25 (m, 42H, -OCH2CH2(CH2)7CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3)2), 1.57-1.85 (m, 6H, -OCH2CH2(CH2)7CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3)2), 2.81 (m, 4H, -N(CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3)2), 3.87 (m, 2H, -NCH2CH2O-), 3.94-4.30 (m, 4H, This figure shows the 1H NMR spectra of -NCH2CH2O- and -OCH2CH2(CH2)7CH3). 3C NMR (CDCl3, ppm) δ values: 14.10, 22.68, 26.00, 27.01, 27.05, 29.28, 29.32, 29.35, 29.46, 29.51, 29.57, 29.62, 31.86, 31.89, 47.61. 3OP NMR (CDCl3, ppm) δ values: -1.08, 0.86. Calculated mass m / z 561.5, measured [M+H]+(LC-MS) m / z 563.6. [Figure 13]This figure shows the ¹H NMR spectrum of 9A1P15 in CDCl3 according to one embodiment of the present disclosure; a small amount of DMSO remained in the product by column flash chromatography, but this would not have affected the effect of 9A1P15; ¹H NMR (CDCl3, ppm) δ 0.86 (m, 9H, -CH2CH2CH2CH3), 1.24 (m, 44H, -OCH2CH2(CH2)12CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH3)2), 1.56-1.86 (m, 6H, -OCH2CH2(CH2)12CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH3)2), 2.81 (m, 4H, -N(CH2CH2CH2CH2CH2CH2CH2CH3)2), 3.86 (m, 2H, -NCH2CH2O-), 3.94-4.30 (m, 4H, -NCH2CH2O- and -OCH2CH2(CH2)12CH3).13C NMR (CDCl3, ppm) δ 14.07, 14.11, 22.61, 22.68, 25.87, 26.96, 27.01, 29.16, 29.19, 29.23, 29.35, 29.47, 29.65, 29.70, 31.72, 31.76, 47.49. 31P NMR (CDCl3, ppm) δ -0.95, 0.80; Calculated mass m / z575.5, measured [M+H]+(LC-MS) m / z576.6. [Figure 14]According to one embodiment of the present disclosure, 10A1P16 in CDCl3; 1H NMR (CDCl3, ppm) δ 0.87 (t, 9H, -CH2CH2CH2CH3), 1.25 (m, 54H, -OCH2CH2(CH2)13CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3)2), 1.54-1.78 (m, 6H, -OCH2CH2(CH2)13CH3 and -N(CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3)2), 2.76 (m, 4H, -N(CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH3)2), 3.85 (m, 2H, -NCH2CH2O-), 3.95-4.28 (m, 4H, This figure shows the 1H NMR spectra of -NCH2CH2O- and -OCH2CH2(CH2)13CH3). 13C NMR (CDCl3, ppm) δ values ​​14.11, 22.68, 25.91, 27.10, 27.20, 29.31, 29.34, 29.56, 29.59, 29.64, 29.66, 29.72, 31.88, 31.91, 47.78. 31P NMR (CDCl3, ppm) δ values ​​-1.00, 0.76. Calculated mass m / z 645.6, experimentally measured [M+H]+(LC-MS) m / z 646.6. [Figure 15A-15C] This figure shows the particle size and polydispersity index (PDI) (Figure 15A), zeta potential (Figure 15B), and mRNA binding efficacy (Figure 15C) of selected iPLNPs. When 9A1P9, 10A1P10, 9A1P15, and 10A1P16 were purified, their iPLNPs were approximately 150 nm in size. [Figures 16A-16B] Figure 16A shows the pKas of the selected iPLNPs, 9A1P9, 9A1P15, 10A1P10, and 10A1P16, and Figure 16B shows 16A1P10 and 25A3P9. [Figure 17A-17C]This figure shows how different tail lengths on the amine side of iPhos affected in vivo mRNA expression efficacy. Figure 17A shows bioluminescence imaging of various organs 6 hours after injection. C57BL / 6 mice were intravenously injected with 0.1 mg / kg FLuc mRNA using iPLNP, and luminescence was quantified 6 hours after injection. Figure 17B shows the structures of 7A1P11~11A1P11. Figure 17C shows the effect of alkyl chain length on the amine side on in vivo mRNA delivery efficacy. Both too short (4-6 carbon) and too long (over 10 carbon) carbon lengths reduced efficacy in the liver. Carbon lengths of 8 and 10 tended to show high FLuc mRNA expression. [Figure 18A] This figure shows that different tail lengths on the phosphate side of iPhos influenced organ-selective mRNA expression. Figure 18A shows bioluminescence imaging of various organs 6 hours after injection. C57BL / 6 mice were intravenously injected with 0.1 mg / kg FLuc mRNA using iPLNP. [Figure 18B] This figure shows that different tail lengths on the phosphate side of iPhos affected organ-selective mRNA expression. Figure 18B shows the structures of 10A1P4 to 10A1P16. [Figure 18C] This figure shows how different tail lengths on the phosphate side of iPhos affected organ-selective mRNA expression. Figure 18C shows the effect of alkyl chain length on organ selectivity. Short carbon lengths (4-8) did not show efficacy in vivo. Carbon lengths of 9 and 10 tended to mediate high Fluc mRNA expression, mainly in the liver. Interestingly, longer carbon lengths (13-16) shifted the majority of Fluc mRNA expression to the spleen. [Figures 19A-19C] This figure shows the particle size (Figure 19A), zeta potential (Figure 19B), and pKa (Figure 19C) of 10A1P4~10A1P16 iPLNPs. The 10A1P4~10A1P16 iPLNPs were generally negatively charged and had a size of approximately 200 nm. These nanoparticles exhibited pKa values ​​of 6.0~6.5. [Figure 20A]This figure shows the in vivo evaluation of 9A1P15 and 10A1P16 iPLNPs. Figure 20A shows the structures of 9A1P15 and 10A1P16 in an acidic endosomal environment. [Figure 20B] This figure shows the in vivo evaluation of 9A1P15 and 10A1P16 iPLNPs. Figure 20B shows the luminescence quantified in C57BL / 6 mice IV-injected with iPLNPs at 0.25 mg / kg FLuc mRNA and 6 hours after injection. [Figure 20C] This figure shows the in vivo evaluation of 9A1P15 and 10A1P16 iPLNPs. Figure 20C shows the quantification of luciferase expression in various organs, recorded by mean radiance. [Figure 21A] This figure shows that the 9A1P9 / mRNA weight ratio affected in vivo efficacy. Figure 21A shows the structure of 9A1P9 in an acidic endosomal environment. [Figure 21B] This figure shows that the 9A1P9 / mRNA weight ratio affected in vivo efficacy. Figure 21B shows the evaluation of 9A1P9 / mRNA with weight ratios of 9:1 (molar ratio 11622:1) and 18:1 (molar ratio 23244:1). The iPhos:MDOA:chol:DMG-PEG2000 molar ratio was fixed at 25:30:30:1. C57BL / 6 mice were intravenously injected with 0.25 mg / kg FLuc mRNA using iPLNP, and luminescence was quantified 6 hours after injection. [Figure 21C] This figure shows that the 9A1P9 / mRNA weight ratio affected in vivo efficacy. Figure 21C shows the quantification of luciferase expression in the liver, recorded by mean radiance (n=3). [Figure 22] This figure shows the structure of the helper lipids. Ionizable cationic lipids included MDOA, DODAP, and 5A2-SC8; permanent cationic lipids included DDAB and DOTAP. The zwitterionic lipid DOPE was used as a helper lipid. [Figure 23A]This figure shows the characterization of 9A1P9 iPLNPs with different helper lipids. The particle size and PDI of 9A1P9 iPLNPs with different helper lipids were evaluated. For all formulations, the ratios were: 9A1P9:MDOA (DODAP or 5A2-SC8):chol:DMG-PEG2000 (molar ratio) = 25:30:30:1; 9A1P9:DDAB (or DOTAP):chol:DMG-PEG2000 (molar ratio) = 60:30:40:0.4; 9A1P9:DOPE:chol:DMG-PEG2000 (molar ratio) = 55:30:45:0.2; 9A1P9:mRNA (w / w) = 18:1. [Figure 23B] This figure shows the characterization of 9A1P9 iPLNPs with different helper lipids. The zeta potential of 9A1P9 iPLNPs with different helper lipids was evaluated. For all formulations, the ratios were: 9A1P9:MDOA (DODAP or 5A2-SC8):chol:DMG-PEG2000 (molar ratio) = 25:30:30:1; 9A1P9:DDAB (or DOTAP):chol:DMG-PEG2000 (molar ratio) = 60:30:40:0.4; 9A1P9:DOPE:chol:DMG-PEG2000 (molar ratio) = 55:30:45:0.2; 9A1P9:mRNA (w / w) = 18:1. [Figure 23C] This figure shows the characterization of 9A1P9 iPLNPs with different helper lipids. The mRNA binding efficacy of 9A1P9 iPLNPs with different helper lipids was evaluated. For all formulations, the ratios were: 9A1P9:MDOA (DODAP or 5A2-SC8):chol:DMG-PEG2000 (molar ratio) = 25:30:30:1; 9A1P9:DDAB (or DOTAP):chol:DMG-PEG2000 (molar ratio) = 60:30:40:0.4; 9A1P9:DOPE:chol:DMG-PEG2000 (molar ratio) = 55:30:45:0.2; 9A1P9:mRNA (w / w) = 18:1. [Figure 23D]This figure shows the characterization of 9A1P9 iPLNPs with different helper lipids. The pKa of 9A1P9 iPLNPs with different helper lipids was evaluated. For all formulations, the ratios were: 9A1P9:MDOA (DODAP or 5A2-SC8):chol:DMG-PEG2000 (molar ratio) = 25:30:30:1; 9A1P9:DDAB (or DOTAP):chol:DMG-PEG2000 (molar ratio) = 60:30:40:0.4; 9A1P9:DOPE:chol:DMG-PEG2000 (molar ratio) = 55:30:45:0.2; 9A1P9:mRNA (w / w) = 18:1. [Figure 23E] This figure shows the characterization of 9A1P9 iPLNPs with different helper lipids. The in vitro mRNA delivery efficacy (25ng mRNA) of 9A1P9 iPLNPs with different helper lipids in IGROV-1 cells was evaluated. For all formulations, the ratios were: 9A1P9:MDOA (DODAP or 5A2-SC8):chol:DMG-PEG2000 (molar ratio) = 25:30:30:1; 9A1P9:DDAB (or DOTAP):chol:DMG-PEG2000 (molar ratio) = 60:30:40:0.4; 9A1P9:DOPE:chol:DMG-PEG2000 (molar ratio) = 55:30:45:0.2; 9A1P9:mRNA (w / w) = 18:1. [Figures 24A-24C] This figure shows the organ distribution of Cy5-mRNA. C57BL / 6 mice were intravenously injected with PBS (Figure 24A) at a dose of 0.25 mg / kg of Cy5-mRNA, or with iPLNP 9A1P9-5A2-SC8 (Figure 24B) or 9A1P9-DDAB (Figure 24C), and imaged 6 hours later. For the iPLNP preparations, a 9A1P9:5A2-SC8:chol:DMG-PEG2000 molar ratio of 25:30:30:1 and a 9A1P9:DDAB:chol:DMG-PEG2000 molar ratio of 60:30:40:0.4 were used; 9A1P9 / mRNA (w / w) was fixed with 18 / 1. [Figures 25A-25B]These figures show 9A1P9-5A2-SC8 (Figure 25A) and 9A1P9-DDAB (Figure 25B) iPLNPs, which demonstrated high mRNA delivery efficacy in the liver and lungs, respectively. 9A1P9:5A2-SC8:chol:DMG-PEG2000 (molar ratio) of 25:30:30:1 (Fluc mRNA, 0.05 mg / kg) and 9A1P9:DDAB:chol:DMG-PEG2000 (molar ratio) of 60:30:40:0.4 (Fluc mRNA, 0.25 mg / kg) were used. The 9A1P9:mRNA weight ratio was fixed at 18:1. C57BL / 6 mice were intravenously injected with iPLNPs and imaged 6 hours later. [Figures 26A-26C] This figure shows the kinetic analysis of Fluc protein expression induced by different organ-selective iPLNPs. 9A1P9-5A2-SC8 (liver-specific, 0.05 mg / kg FlucmRNA; Figure 26A), 9A1P9-DDAB (lung-specific, 0.25 mg / kg Fluc mRNA; Figure 26B), and 10A1P16-MDOA (spleen-specific, 0.25 mg / kg Fluc mRNA; Figure 26C) iPLNPs were administered intravenously to C57BL / 6 mice. Organs were imaged at 3, 6, 12, and 24 hours. Data are presented as mean ± sd (n=3 biologically independent mice). [Figures 27A-27C] This figure shows that 9A1P9-5A2-SC8 iPLNP achieved approximately 91% tdTomato editing in hepatocytes. Figure 27A shows 9A1P9-5A2-SC8 iPLNP (Cre mRNA, 0.25 mg / kg) administered intravenously to Ai9 mice. After 48 hours, hepatocytes were isolated and analyzed by flow cytometry. Figure 27B shows DLin-MC3-DMA LNP (Cre mRNA, 0.25 mg / kg) administered intravenously to Ai9 mice. After 48 hours, hepatocytes were isolated and analyzed by flow cytometry. Figure 27C shows the combined results of 9A1P9-5A2-SC8 iPLNP and DLin-MC3-DMA LNP. All data are from n=3 biologically independent mice. [Figure 28]This figure shows the percentage of tdTomato-positive lung cells. A FACS gating strategy was used to analyze tdTomato expression in lung cells. Briefly, Ghost Red 780 was used to distinguish between live and dead cells. Epithelial cells were defined using EpCam+, immune cells using CD45+ and CD31-, and endothelial cells using CD45- and CD31+. Gating for tdTomato+ in cell type was performed based on PBS-injected control Ai9 mice. Ai9 mice were IV-injected with 9A1P9-DDAB iPLNP (Cre mRNA, 0.25 mg / kg), and tdTomato+ of a given cell type was detected by flow cytometry after 48 hours. Data are presented as mean ± sd (n=3 biologically independent mice). [Figure 29] This figure shows the percentage of tdTomato-positive splenocytes. A FACS gating strategy was used to analyze tdTomato expression in splenocytes. Briefly, Ghost Red 780 was used to distinguish between live and dead cells. CD45+ was used to distinguish immune cells, then CD3+ and CD11b- were used for T cells, CD3- and CD11b+ for macrophage cells, and CD19+ and CD11b- for B cells. Gating for tdTomato+ in cell type was performed based on PBS-injected control mice. Ai9 mice were IV-injected with 10A1P16-MDOA iPLNP (Cre mRNA, 0.5 mg / kg), and tdTomato+ of a given cell type was detected by flow cytometry after 48 hours. Data are presented as mean ± sd (n=3 biologically independent mice). [Figures 30A-30B]This figure shows how iPLNP size can be reduced using the NanoAssemblr microfluidic mixing system. When 10A1P16-MDOA iPLNPs were prepared using the NanoAssemblr microfluidic mixing system, they exhibited a small size of less than 100 nm. High in vivo mRNA delivery efficacy and precise organ selectivity were fully retained even after reducing the iPLNP diameter (Figure 30A). 10A1P16-MDOA iPLNPs (Fluc mRNA, 0.25 mg / kg) mediated mRNA translation in the spleen (Figure 30B). (n=3 biologically independent mice). [Figure 31A-31D] This figure demonstrates the high in vivo tolerability of mRNA-carrying 10A1P16-MDOA iPLNP by measuring liver markers (BUN (Figure 31A); CREA (Figure 31B); ATL (Figure 31C); and AST (Figure 31D)). 10A1P16-MDOA (spleen-specific) iPLNP was administered intravenously (IV) to C57BL / 6 mice (0.25 mg / kg). Lipopolysaccharide (LPS, 5 mg / kg, IP) was used as a positive control, and PBS (IV) was used as a negative control. Renal function (BUN and CREA) and hepatic function (ALT and AST) were evaluated 24 hours after injection. LPS-treated mice showed severe renal and hepatic injury. There were no significant differences between the 10A1P16-MDOA iPLNP group and the PBS group. Data are presented as mean ± sd (n=3 biologically independent mice). Statistical significance was analyzed by a two-sided, unpaired t-test. ****, P<0.0001;***, P<0.001;**, P<0.01;*, P<0.05. [Figures 32A-32B]This figure shows the enablement of iPLNPs for efficient delivery of pDNA and siRNA. Figure 32A shows 9A1P9-5A2-SC8 iPLNPs and 9A1P9-DDAB iPLNPs used to deliver pCMV-Luc pDNA. 12.5 ng and 25 ng of pDNA were applied per well. Results were normalized to untreated cells. Figure 32B shows 9A1P9-5A2-SC8 iPLNPs and 9A1P9-DDAB iPLNPs that efficiently delivered siLuc(siRNA). 12.5 ng and 25 ng of siRNA were used per well. Data are presented as mean ± sd (n=3 biologically independent samples). [Figures 33A-33B] This figure shows the iPLNP-enabled efficient delivery of mRNA to subcutaneous tissue. Figure 33A shows that tdTomato-positive cells were observed via IVIS 44 hours after subcutaneous injection of PBS containing Cre recombinase (CRE) mRNA, 9A1-P9, 9A1-P15, and 10A1-P16 into Ai9 mice. Figure 33B shows the quantification of tdTomato expression in the subcutaneous tissue (e.g., skin) of Ai9 mice 44 hours after subcutaneous injection of PBS containing Cre recombinase (CRE) mRNA, 9A1-P9, 9A1-P15, and 10A1-P16. [Modes for carrying out the invention]

[0042] The following detailed description refers to the accompanying drawings illustrating various embodiments of the Disclosure. The drawings and description are intended to describe in sufficient detail to enable a person skilled in the art to implement the Disclosure. Other components may be utilized and modified without departing from the scope of the Disclosure. Therefore, the following description should not be construed as restrictive. The scope of the Disclosure is defined solely by the attached claims, together with the entire scope of the equivalent works for which the claims are granted.

[0043] This disclosure is at least in part based on the identification of ionizable synthetic phospholipids for use in lipid nanoparticles (LNPs). Accordingly, this disclosure provides ionizable synthetic phospholipid compositions and methods for preparing them, LNP compositions comprising the ionizable synthetic phospholipids disclosed herein and methods for preparing them, methods for using the compositions disclosed herein, and kits used for carrying out the methods disclosed herein.

[0044] I. Ionizable synthetic phospholipids This disclosure provides a novel class of ionizable synthetic phospholipids. In some embodiments, the ionizable synthetic phospholipids provided herein are of formula (I): [ka] (wherein R1 is selected from the group consisting of C2-C20 unsubstituted alkyl, C2-C20 substituted alkyl, C2-C20 unsubstituted alkenyl, C2-C20 substituted alkenyl, C2-C20 unsubstituted alkynyl, C2-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, and C4-C20 substituted cycloalkyl; R2 and R3 are H, C1-C20 unsubstituted alkyl, C1-C20 substituted alkyl, C1-C20 unsubstituted alkenyl, C1-C20 substituted alkenyl, C1-C20 unsubstituted alkynyl, C1-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, and C4 R4, R5, R6, and R7 are independently selected from the group consisting of ~C20 substituted cycloalkyls; R8 is selected from the group consisting of H, C1~C8 unsubstituted alkyls, C1~C8 substituted alkyls, C1~C8 unsubstituted alkenyls, C1~C8 substituted alkenyls, C1~C8 unsubstituted alkynyls, and C1~C8 substituted alkynyls; n is an integer from 1 to 4. It may have.

[0045] In at least some examples, R1 is selected from the group consisting of C2-C16 unsubstituted alkyls, C2-C16 substituted alkyls, or C4-C12 substituted cycloalkyls; R2 and R3 are independently selected from the group consisting of H, C1-C16 unsubstituted alkyls, C1-C16 substituted alkyls, or C4-C16 substituted cycloalkyls; R4, R5, R6, and R7 are independently selected from the group consisting of H, C1-C4 unsubstituted alkyls, or C1-C4 substituted alkyls; R8 is selected from C3-C18 unsubstituted alkyls; and n is an integer between 1 and 3.

[0046] In other examples, R1 is a C2-C15 unsubstituted alkyl group; R2 and R3 are independently selected from the group consisting of H, C1-C16 substituted alkyl groups, or C4-C16 substituted cycloalkyl groups; R4, R5, R6, and R7 are independently selected from the group consisting of H, methyl, or ethyl groups; R8 is selected from a C4-C16 unsubstituted alkyl group; and n is an integer between 1 and 2.

[0047] In some embodiments, the ionizable synthetic phospholipids provided herein are of formula (II): [ka] (In the formula, R1 and R2 are independently selected from the group consisting of H, C1-C8 substituted alkyls, or C1-C8 unsubstituted alkyls; R3, R4, R5, and R6 are independently selected from the group consisting of H, C1-C8 unsubstituted alkyls, or C1-C8 substituted alkyls; R7 is selected from the group consisting of C3-C21 unsubstituted alkyls or C3-C21 substituted alkyls; n is an integer from 1 to 4.) It may have.

[0048] In some examples, R1 and R2 are independently selected from the group consisting of H, C1-C6 substituted alkyls, or C1-C6 unsubstituted alkyls; R3, R4, R5, and R6 are independently selected from the group consisting of H, C1-C4 unsubstituted alkyls, or C1-C4 substituted alkyls; R7 is selected from the group consisting of C3-C18 unsubstituted alkyls or C3-C18 substituted alkyls; and n is an integer from 1 to 3.

[0049] In some examples, R1 and R2 are independently selected from the group consisting of H, C1-C4 substituted alkyl, or C1-C4 unsubstituted alkyl; R3, R4, R5, and R6 are independently selected from the group consisting of H, methyl, or ethyl; R7 is selected from the group consisting of C3-C15 unsubstituted alkyl or C3-C15 substituted alkyl; and n is an integer between 1 and 2.

[0050] In other examples, R1 and R2 are independently selected from the group consisting of H, C1-C8 substituted alkyl, C1-C8 unsubstituted alkyl, C2-C8 unsubstituted alkenyl, C2-C8 substituted alkenyl, C2-C8 unsubstituted alkynyl, or C2-C8 substituted alkynyl; R3, R4, R5, and R6 are independently selected from the group consisting of H, C1-C8 substituted alkyl, C1-C8 unsubstituted alkyl, C1-C8 unsubstituted alkenyl, C2-C8 substituted alkenyl, C2-C8 unsubstituted alkynyl, or C2-C8 substituted alkynyl; R7 is selected from the group consisting of C3-C21 unsubstituted alkyl, C3-C21 substituted alkyl, C3-C21 unsubstituted alkenyl, C3-C21 substituted alkenyl, C3-C21 unsubstituted alkynyl, or C3-C21 substituted alkynyl; and n is an integer from 1 to 4.

[0051] Furthermore, in other examples, R1 and R2 are independently selected from the group consisting of H, C1-C6 substituted alkyl, C1-C6 unsubstituted alkyl, C2-C6 unsubstituted alkenyl, C2-C6 substituted alkenyl, C2-C6 unsubstituted alkynyl, or C2-C6 substituted alkynyl; R3, R4, R5, and R6 are independently selected from the group consisting of H, C1-C4 unsubstituted alkyl, C1-C4 substituted alkyl, C2-C4 unsubstituted alkenyl, C2-C4 substituted alkenyl, C2-C4 unsubstituted alkynyl, or C2-C4 substituted alkynyl; R7 is selected from the group consisting of C3-C18 unsubstituted alkyl or C3-C18 substituted alkyl; and n is an integer from 1 to 3.

[0052] In yet another example, R1 and R2 are independently selected from the group consisting of H, C1-C4 substituted alkyl, or C1-C4 unsubstituted alkyl; R3, R4, R5, and R6 are independently selected from the group consisting of H, methyl, or ethyl; R7 is selected from the group consisting of C3-C15 unsubstituted alkyl or C3-C15 substituted alkyl; and n is an integer between 1 and 2.

[0053] In some embodiments, the ionizable synthetic phospholipids provided herein are of formula (III): [ka] (wherein R1 is selected from the group consisting of C2-C20 unsubstituted alkyl, C2-C20 substituted alkyl, C2-C20 unsubstituted alkenyl, C2-C20 substituted alkenyl, C2-C20 unsubstituted alkynyl, C2-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, or C4-C20 substituted cycloalkyl; R2 and R3 are H, C1-C20 unsubstituted alkyl, C1-C20 substituted alkyl, C1-C20 unsubstituted alkenyl, C1-C20 substituted alkenyl, C1-C20 unsubstituted alkynyl, C1-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, or C4-C R4 and R5 are independently selected from the group consisting of 20-substituted cycloalkyls; R6 is selected from the group consisting of H, C1-C8 unsubstituted alkyls, C1-C8 substituted alkyls, C1-C8 unsubstituted alkenyls, C1-C8 substituted alkenyls, C1-C8 unsubstituted alkynyls, or C1-C8 substituted alkynyls; R6 is selected from the group consisting of C3-C21 unsubstituted alkyls, C3-C21 substituted alkyls, C3-C21 unsubstituted alkenyls, C3-C21 substituted alkenyls, C3-C21 unsubstituted alkynyls, or C3-C21 substituted alkynyls; n is an integer from 1 to 4; m is an integer from 1 to 4. It may have.

[0054] In some examples, R1 is selected from the group consisting of C2-C16 unsubstituted alkyl, C2-C16 substituted alkyl, C2-C16 unsubstituted alkenyl, C2-C16 substituted alkenyl, C2-C16 unsubstituted alkynyl, C2-C16 substituted alkynyl, C4-C16 unsubstituted cycloalkyl, or C4-C16 substituted cycloalkyl; R2 and R3 are H, C1-C16 unsubstituted alkyl, C1-C16 substituted alkyl, C1-C16 unsubstituted alkenyl, C1-C16 substituted alkenyl, C1-C16 unsubstituted alkynyl, C1-C16 substituted alkynyl, C4-C16 unsubstituted cycloalkyl, or C4- R4 and R5 are independently selected from the group consisting of C16-substituted cycloalkyls; R4 and R5 are independently selected from the group consisting of H, C1-C6 unsubstituted alkyls, C1-C6 substituted alkyls, C1-C6 unsubstituted alkenyls, C1-C6 substituted alkenyls, C1-C6 unsubstituted alkynyls, or C1-C6 substituted alkynyls; R6 is selected from the group consisting of C3-C18 unsubstituted alkyls, C3-C18 substituted alkyls, C3-C18 unsubstituted alkenyls, C3-C18 substituted alkenyls, C3-C18 unsubstituted alkynyls, or C3-C18 substituted alkynyls; n is an integer from 1 to 3; and m is an integer from 1 to 3.

[0055] In other examples, R1 is a C2-C15 unsubstituted alkyl group; R2 and R3 are independently selected from the group consisting of H, C1-C16 substituted alkyl groups, or C4-C16 substituted cycloalkyl groups; R4 and R5 are independently selected from the group consisting of H, methyl, or ethyl groups; R6 is selected from a C4-C16 unsubstituted alkyl group; n is an integer between 1 and 2; and m is an integer between 1 and 2.

[0056] In some embodiments, the ionizable synthetic phospholipids provided herein are of formula (IV): nAxPm Formula (IV) (In the formula, "Pm" refers to an alkylated dioxaphosphorane oxide molecule, "nA" refers to an amine, and "x" in "nAxPm" refers to the number of modified Pm molecules on one amine molecule.) It may have. In some embodiments, the ionizable synthetic phospholipids of this specification have Pm [ka] Formula (IV) may have one or more selected from. In some embodiments, the ionizable synthetic phospholipids of this specification may have nA [ka] It may have an expression (IV) which may include one or more selected from.

[0057] In some embodiments, the ionizable synthetic phospholipids provided herein having nAxPm formula (IV) are 1AxP4, 1AxP5, 1AxP6, 1AxP7, 1AxP8, 1AxP9, 1AxP10, 1AxP11, 1AxP12, 1AxP13, 1AxP14, 1AxP15, 1AxP16, 2AxP4, 2AxP5, 2AxP6, 2AxP7, 2AxP8, 2AxP9, 2AxP10, 2AxP11, 2AxP12, 2AxP13, 2AxP14, 2AxP15, 2AxP16, 3AxP4, 3AxP5, 3AxP6, 3AxP7, 3AxP8, 3AxP9, 3AxP 10, 3AxP11, 3AxP12, 3AxP13, 3AxP14, 3AxP15, 3AxP16, 4AxP4, 4AxP5, 4AxP 6, 4AxP7, 4AxP8, 4AxP9, 4AxP10, 4AxP11, 4AxP12, 4AxP13, 4AxP14, 4AxP15 , 4AxP16, 5AxP4, 5AxP5, 5AxP6, 5AxP7, 5AxP8, 5AxP9, 5AxP10, 5AxP11, 5Ax P12, 5AxP13, 5AxP14, 5AxP15, 5AxP16, 6AxP4, 6AxP5, 6AxP6, 6AxP7, 6AxP8, 6AxP9, 6AxP10, 6AxP11, 6AxP12, 6AxP13, 6AxP14, 6AxP15, 6AxP16, 7AxP4, 7AxP5, 7AxP6, 7AxP7, 7AxP8, 7AxP9, 7AxP10, 7AxP11, 7AxP12, 7AxP13, 7Ax P14, 7AxP15, 7AxP16, 8AxP4, 8AxP5, 8AxP6, 8AxP7, 8AxP8, 8AxP9, 8AxP10, 8AxP11, 8AxP12, 8AxP13, 8AxP14, 8AxP15, 8AxP16, 9AxP4, 9AxP5, 9AxP6, 9A xP7, 9AxP8, 9AxP9, 9AxP10, 9AxP11, 9AxP12, 9AxP13, 9AxP14, 9AxP15, 9Ax P16, 10AxP4, 10AxP5, 10AxP6, 10AxP7, 10AxP8, 10AxP9, 10AxP10, 10AxP11 , 10AxP12, 10AxP13, 10AxP14, 10AxP15, 10AxP16, 11AxP4, 11AxP5, 11AxP6 , 11AxP7, 11AxP8, 11AxP9, 11AxP10, 11AxP11, 11AxP12, 11AxP13, 11AxP14,11AxP15、11AxP16、12AxP4、12AxP5、12AxP6、12AxP7、12AxP8、12AxP9、12AxP10、12AxP11、12AxP12、12AxP13、12AxP14、12AxP15、12AxP16、13AxP4、13AxP5、13AxP6、13AxP7、13AxP8、13AxP9、13AxP10、13AxP11、13AxP12、13AxP13、13AxP14、13AxP15、13AxP16、14AxP4、14AxP5、14AxP6、14AxP7、14AxP8、14AxP9、14AxP10、14AxP11、14AxP12、14AxP13、14AxP14、14AxP15、14AxP16、15AxP4、15AxP5、15AxP6、15AxP7、15AxP8、15AxP9、15AxP10、15AxP11、15AxP12、15AxP13、15AxP14、15AxP15、15AxP16、16AxP4、16AxP5、16AxP6、16AxP7、16AxP8、16AxP9、16AxP10、16AxP11、16AxP12、16AxP13、16AxP14、16AxP15、16AxP16、17AxP4、17AxP5、17AxP6、17AxP7、17AxP8、17AxP9、17AxP10、17AxP11、17AxP12、17AxP13、17AxP14、17AxP15、17AxP16、18AxP4、18AxP5、18AxP6、18AxP7、18AxP8、18AxP9、18AxP10、18AxP11、18AxP12、18AxP13、18AxP14、18AxP15、18AxP16、19AxP4、19AxP5、19AxP6、19AxP7、19AxP8、19AxP9、19AxP10、19AxP11、19AxP12、19AxP13、19AxP14、19AxP15、19AxP16、20AxP4、20AxP5、20AxP6、20AxP7、20AxP8、20AxP9、20AxP10、20AxP11、20AxP12、20AxP13、20AxP14、20AxP15、20AxP16、21AxP4、21AxP5、21AxP6、21AxP7、21AxP8、21AxP9、21AxP10、21AxP11、21AxP12、21AxP13、21AxP14、21AxP15、21AxP16、22AxP4, 22AxP5, 22AxP6, 22AxP7, 22AxP8, 22AxP9, 22AxP10, 22AxP11, 22AxP12, 22AxP13, 22AxP14, 22AxP15, 2 2AxP16, 23AxP4, 23AxP5, 23AxP6, 23AxP7, 23AxP8, 23AxP9, 23AxP10, 23AxP11, 23AxP12, 23AxP13, 23AxP14, 23A xP15, 23AxP16, 24AxP4, 24AxP5, 24AxP6, 24AxP7, 24AxP8, 24AxP9, 24AxP10, 24AxP11, 24AxP12, 24AxP13, 24Ax P14, 24AxP15, 24AxP16, 25AxP4, 25AxP5, 25AxP6, 25AxP7, 25AxP8, 25AxP9, 25AxP10, 25AxP11, 25AxP12, 25AxP1 3, 25AxP14, 25AxP15, 25AxP16, 26AxP4, 26AxP5, 26AxP6, 26AxP7, 26AxP8, 26AxP9, 26AxP10, 26AxP11, 26AxP12 , 26AxP13, 26AxP14, 26AxP15, 26AxP16, 27AxP4, 27AxP5, 27AxP6, 27AxP7, 27AxP8, 27AxP9, 27AxP10, 27AxP11, 2 This may include 7AxP12, 27AxP13, 27AxP14, 27AxP15, 27AxP16, 28AxP4, 28AxP5, 28AxP6, 28AxP7, 28AxP8, 28AxP9, 28AxP10, 28AxP11, 28AxP12, 28AxP13, 28AxP14, 28AxP15, and 28AxP16 (wherein "nAxPm", "x" refers to the number of modified Pm molecules on a single amine molecule).

[0058] In some embodiments, the ionizable synthetic phospholipids provided herein having nAxPm formula (IV) are 7A1P4, 7A1P5, 7A1P6, 7A1P7, 7A1P8, 7A1P9, 7A1P10, 7A1P11, 7A1P12, 7A1P13, 7A1P14, 7A1P15, 7A1P16, 8A1P4, 8A1P5, 8A1P6, 8A1P7, 8A1P8, 8A1P9, 8A1P10, 8A1P11, 8A1P12 , 8A1P13, 8A1P14, 8A1P15, 8A1P16, 9A1P4, 9A1P5, 9A1P6, 9A1P7, 9A1P8, 9A1P9, 9A1P10, 9A1P11, 9A1P12, 9 A1P13, 9A1P14, 9A1P15, 9A1P16, 10A1P4, 10A1P5, 10A1P6, 10A1P7, 10A1P8, 10A1P9, 10A1P10, 10A1P11, 10A 1P12, 10A1P13, 10A1P14, 10A1P15, 10A1P16, 11A1P4, 11A1P5, 11A1P6, 11A1P7, 11A1P8, 11A1P9, 11A1P10, 11A1P11, 11A1P12, 11A1P13, 11A1P14, 11A1P15, 11A1P16, 12A1P4, 12A1P5, 12A1P6, 12A1P7, 12A1P8, 12A1P 9, 12A1P10, 12A1P11, 12A1P12, 12A1P13, 12A1P14, 12A1P15, 12A1P16, 13A1P4, 13A1P5, 13A1P6, 13A1P7, 13A1P8, 13A1P9, 13A1P10, 13A1P11, 13A1P12, 13A1P13, 13A1P14, 13A1P15, 13A1P16, or any combination thereof. In some embodiments, the ionizable synthetic phospholipids provided herein having nAxPm formula (IV) may include 9A1P9, 9A1P15, 10A1P10, 10A1P16, or any combination thereof.

[0059] In some embodiments, the ionizable synthetic phospholipids of this specification are pH switchable. As used herein, “pH switchable” refers to lipids whose conformation changes upon protonation within a defined range of pH values. In some embodiments, the ionizable synthetic phospholipids of this specification are pH switchable at cytoplasmic pH (e.g., about 7.0 to about 7.5). In some embodiments, the ionizable synthetic phospholipids of this specification are pH switchable at endosomal and / or lysosomal lumen pH (e.g., about 6.5 to about 4.5). In some embodiments, the ionizable synthetic phospholipids of this specification are pH switchable in the range of about 4.0 to about 8.0 (e.g., about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0). In some embodiments, the ionizable synthetic phospholipids of this specification are irreversible (i.e., not pH switchable).

[0060] In some embodiments, the ionizable synthetic phospholipids of this specification may comprise at least one phosphate group and at least one zwitterion. A zwitterion, also known as an intramolecular salt or dipole ion, is a neutral species as a whole in which two or more atoms possess opposite formal charges. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one zwitterion. In some embodiments, the ionizable synthetic phospholipids of this specification may have multiple zwitterions (e.g., two or more zwitterions). In some embodiments, the ionizable synthetic phospholipids of this specification may have about 1 to about 10 zwitterions (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one irreversible zwitterion. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one pH-switchable zwitterion. In some embodiments, the ionizable synthetic phospholipids of this specification may have multiple pH-switchable zwitterions (e.g., two or more pH-switchable zwitterions). In some embodiments, the ionizable synthetic phospholipids of this specification may have about 1 to about 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) pH-switchable zwitterions.

[0061] In some embodiments, the ionizable synthetic phospholipids of this specification may comprise at least one phosphate group, at least one zwitterion, and at least one hydrophobic domain. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one tail. In some embodiments, the ionizable synthetic phospholipids of this specification may be multi-tailed (i.e., may have two or more tails). In some embodiments, the disclosed multi-tailed ionizable phospholipids may have endosomal membrane destabilization.

[0062] In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one hydrophobic tail. In some embodiments, the ionizable synthetic phospholipids of this specification may have two or more hydrophobic tails. In some embodiments, the ionizable synthetic phospholipids of this specification may have about 1 to about 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) hydrophobic tails. In some embodiments, the disclosure provides pH-switchable, multi-tailed ionizable phospholipids.

[0063] In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one hydrophobic tail, wherein the hydrophobic tail is an alkyl tail. In some embodiments, the ionizable synthetic phospholipids of this specification may have two or more alkyl tails. In some embodiments, the ionizable synthetic phospholipids of this specification may have about 1 to about 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) alkyl tails. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one alkyl tail containing an alkyl chain length of about 5 to about 20 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbons. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one alkyl tail containing an alkyl chain length of about 8 to about 16 carbons. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one alkyl tail having an alkyl chain length of about 8 to about 10 carbons. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one alkyl tail having an alkyl chain length of about 9 to about 12 carbons. In some embodiments, the ionizable synthetic phospholipids of this specification may have at least one alkyl tail having an alkyl chain length of about 13 to about 16 carbons. In some embodiments, the ionizable synthetic phospholipids of this specification may have multiple alkyl tails in which all alkyl tails are of the same length. In some embodiments, the ionizable synthetic phospholipids of this specification may have multiple alkyl tails in which all alkyl tails are of different lengths.

[0064] In some embodiments, the ionizable synthetic phospholipids of this specification may comprise at least one phosphate group, at least one zwitterion, at least one hydrophobic domain, and at least one tertiary amine. As used herein, a tertiary amine refers to an amine in which the nitrogen atom is directly bonded to any three carbon atoms of a hybrid that cannot be carbonyl group carbons. In some embodiments, in the ionizable synthetic phospholipids of this specification comprising at least one tertiary amine, the tertiary amine is not protonated at physiological pH (e.g., about 7.0 to about 7.5). In some embodiments, the lack of protonation of the tertiary amine in the ionizable synthetic phospholipids of this specification may result in the ionizable synthetic phospholipid being negatively charged. In some embodiments, the lack of protonation of the tertiary amine in the ionizable synthetic phospholipids of this specification may make it difficult for the ionizable synthetic phospholipid to fuse to the cell membrane. In some embodiments, in the ionizable synthetic phospholipids of this specification comprising at least one tertiary amine, the tertiary amine may be protonated at endosomal pH (e.g., about 6.5 to about 4.5). In some embodiments, protonation of a tertiary amine in the ionizable synthetic phospholipids of this specification may form at least one zwitterionic head.

[0065] In some embodiments, the ionizable synthetic phospholipids of this specification may comprise one tertiary amine, one phosphate group, and three hydrophobic tails. In some embodiments, the ionizable synthetic phospholipids of this specification may comprise one tertiary amine, one phosphate group, and three hydrophobic tails, the hydrophobic tails may comprise an alkyl chain length of about 10 to about 12 carbon atoms.

[0066] This disclosure provides methods for preparing ionizable synthetic phospholipids as disclosed herein. Those skilled in the art will recognize that standard techniques known in chemical synthesis are suitable for use herein. In some embodiments, the methods for preparing ionizable synthetic phospholipids herein may involve synthesis via orthogonal reactions of amines (e.g., 1A-28A) and alkylated dioxaphosphorone oxide molecules (e.g., P4-P16). In some embodiments, in the methods for preparing ionizable synthetic phospholipids herein, each alkylated dioxaphosphorone oxide molecule (e.g., P4-P16) may introduce at least one phosphate group and at least one hydrophobic alkyl chain into the ionizable synthetic phospholipid. In some embodiments, in the methods for preparing ionizable synthetic phospholipids herein, primary, secondary, and / or tertiary amines may consume about 1 equivalent of alkylated dioxaphosphorone oxide molecules (e.g., P4-P16) herein. In some embodiments, an amine of this specification having a single primary, secondary, or tertiary amine (e.g., 1A-18A) is reacted with about 1.1 equivalents of alkylated dioxaphosphorone oxide molecules (e.g., P4-P16) to obtain an ionizable synthetic phospholipid having nA1Pm according to formula IV of this specification. In some embodiments, an amine of this specification having multiple amine groups (e.g., 19A-28A) is reacted with about 2.2 equivalents of alkylated dioxaphosphorone oxide molecules (e.g., P4-P16) to obtain an ionizable synthetic phospholipid having nA2Pm according to formula IV of this specification. In some embodiments, an amine of this specification having multiple amine groups (e.g., 19A-28A) is reacted with about 3.3 equivalents of alkylated dioxaphosphorone oxide molecules (e.g., P4-P16) to obtain an ionizable synthetic phospholipid having nA3Pm according to formula IV of this specification. In some embodiments, an amine of this specification having multiple amine groups (e.g., 19A-28A) can be reacted with about 4.4 equivalents of alkylated dioxaphosphorane oxide molecules (e.g., P4-P16) to obtain an ionizable synthetic phospholipid having nA4Pm according to formula IV of this specification.In some embodiments, an amine of this specification having multiple amine groups (e.g., 19A-28A) can be reacted with about 5.5 equivalents of alkylated dioxaphosphorane oxide molecules (e.g., P4-P16) to obtain an ionizable synthetic phospholipid having nA5Pm according to formula IV of this specification.

[0067] In some embodiments, the method for preparing the ionizable synthetic phospholipids described herein may be carried out in a highly polar organic solvent. Non-limiting examples of highly polar organic solvents for use herein include water (H2O), methanol (CH3OH), dimethyl sulfoxide (DMSO; C2H6OS), dimethylformamide (C3H7NO), and acetonitrile (C2H3N). In some embodiments, the method for preparing the ionizable synthetic phospholipids described herein may be carried out in DMSO.

[0068] In some embodiments, the method for preparing the ionizable synthetic phospholipids described herein involves concentrations of approximately 0.1 g / mL to approximately 1.0 g / mL (for example, approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g / mL). -1 The starting materials may include (e.g., amines (e.g., 1A-28A) and alkylated dioxaphosphorane oxide molecules (e.g., P4-P16)). In some embodiments, the method for preparing ionizable synthetic phospholipids according to this specification may include about 0.3 g / mL of the starting materials (e.g., amines (e.g., 1A-28A) and alkylated dioxaphosphorane oxide molecules (e.g., P4-P16)).

[0069] In some embodiments, the method for preparing ionizable synthetic phospholipids according to this specification may include the step of stirring the starting materials (e.g., amines (e.g., 1A-28A) and alkylated dioxaphosphorane oxide molecules (e.g., P4-P16)) in a highly polar organic solvent for about 1 to about 5 days (e.g., about 1, 2, 3, 4, and 5 days). In some embodiments, the method for preparing ionizable synthetic phospholipids according to this specification may include the step of stirring the starting materials (e.g., amines (e.g., 1A-28A) and alkylated dioxaphosphorane oxide molecules (e.g., P4-P16)) in a highly polar organic solvent for about 3 days.

[0070] In some embodiments, the method for preparing ionizable synthetic phospholipids according to this specification may include a step of stirring the starting materials (e.g., amines (e.g., 1A-28A) and alkylated dioxaphosphorane oxide molecules (e.g., P4-P16)) in a highly polar organic solvent at about 60°C to about 80°C (e.g., about 60, 65, 70, 75, 80°C). In some embodiments, the method for preparing ionizable synthetic phospholipids according to this specification may include a step of stirring the starting materials (e.g., amines (e.g., 1A-28A) and alkylated dioxaphosphorane oxide molecules (e.g., P4-P16)) in a highly polar organic solvent at about 70°C.

[0071] In some embodiments, the method for preparing ionizable synthetic phospholipids as described herein may include purification. Non-limiting examples of purification methods suitable for use herein include column chromatography, vacuum drying, freeze-drying, and column fractionation.

[0072] II. Nanoparticles and lipid nanoparticles This disclosure provides a novel class of ionizable synthetic phospholipids for use in nanoparticles and / or lipid nanoparticles (LNPs). The term “nanoparticle” refers to a structure comprising a lipophilic core surrounded by a hydrophilic phase enclosing the core. In some embodiments, one or more nanoparticles can be formed using the ionizable synthetic phospholipids herein. Ionic interactions arising from the different lipophilic and hydrophilic components of the nanoparticles herein may result in independent and / or observable physical properties. In some embodiments, the nanoparticles herein may have an average size of about 1.0 μm or less (e.g., about 1000 nm, 750 nm, 500 nm, 250 nm, 150 nm, 100 nm, 75 nm, 50 nm, 25 nm, 10 nm, 7.5 nm, 5 nm, 2.5 nm, 1.0 nm). “Average size” is understood as the average diameter of a population of nanoparticles including the lipophilic and hydrophilic phases. The average size of the nanoparticles herein is known to those skilled in the art and can be measured, for example, by standard methods described in the following experimental section. In some embodiments, the nanoparticles herein may have an average particle size of 1.0 μm or less, or between 1.0 nm and 1000 nm, or between 100 nm and 350 nm. Those skilled in the art will recognize that the average size of the nanoparticles may be affected by the amount of lipid component (e.g., a large amount results in equal or larger sizes), the amount of surfactant (e.g., a large or high molecular weight results in equal or smaller sizes), and / or parameters of the preparation method, such as, but not limited to, the rate and type of stirring, the temperature of both phases, and the duration of the mixing period.

[0073] In some embodiments, the nanoparticles herein may have a surface charge whose magnitude can vary from about -50 mV to about +80 mV. The surface charge of the nanoparticles herein may be measured by standard methods known to those skilled in the art. In some embodiments, the surface charge of the nanoparticles herein may be measured by the Z potential.

[0074] In some embodiments, the nanoparticles of this specification can penetrate one or more cells upon contact with one or more cells. In some embodiments, the nanoparticles of this specification can deliver one or more bioactive molecules, small molecules, and / or gene-editing therapies to one or more cells upon contact with one or more cells. In some embodiments, the nanoparticles of this specification can penetrate one or more tissues upon contact with one or more tissues. In some embodiments, the nanoparticles of this specification can deliver one or more bioactive molecules, small molecules, and / or gene-editing therapies to one or more tissues upon contact with one or more tissues. In some embodiments, the nanoparticles of this specification can penetrate one or more organs upon contact with one or more organs. In some embodiments, the nanoparticles of this specification can deliver one or more bioactive molecules, small molecules, and / or gene-editing therapies to one or more cells upon contact with one or more organs. In some embodiments, the nanoparticles of this specification can penetrate specific cell types, tissue types, organs, or any combination thereof. In some embodiments, the nanoparticles of this specification can penetrate skin cells, lung cells, liver cells, spleen cells, or any combination thereof. In some embodiments, the nanoparticles of this specification may penetrate skin tissue, lung tissue, liver tissue, spleen tissue, or any combination thereof.

[0075] In some embodiments, one or more LNPs can be formed using the ionizable synthetic phospholipids of this specification. LNPs are spherical vesicles made of ionizable lipids that can be positively charged at low pH (enabling RNA complexation) and neutral at physiological pH (reducing potential toxic effects compared to positively charged lipids such as liposomes). LNPs are taken up by cells via endocytosis, and the ionizability of the lipids at low pH (presumably) can enable endosomal escape and release of the cargo into the cytoplasm.

[0076] In some embodiments, LNPs can be formed using any of the ionizable synthetic phospholipids disclosed herein. In some embodiments, the LNPs herein may comprise one or more multi-tail ionizable lipids. In some embodiments, each of the one or more multi-tail ionizable phospholipids may comprise one tertiary amine, one phosphate group, and two or more hydrophobic tails. In some embodiments, each of the one or more multi-tail ionizable phospholipids may comprise at least one tertiary amine, at least one phosphate group, and two or more hydrophobic tails, the hydrophobic tails may comprise an alkyl chain length of about 5 to about 20 carbons (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, each of one or more multi-tailed ionizable phospholipids may comprise at least one tertiary amine, at least one phosphate group, and two or more hydrophobic tails, the hydrophobic tails may comprise an alkyl chain length of about 10 to about 12 carbon atoms. In some embodiments, each of one or more multi-tailed ionizable phospholipids may comprise at least one tertiary amine, at least one phosphate group, and three hydrophobic tails, the hydrophobic tails may comprise an alkyl chain length of about 10 to about 12 carbon atoms.

[0077] In some embodiments, specification of LNPが、1AxP4、1AxP5、1AxP6、1AxP7、1AxP8、1AxP9、1AxP10、1AxP11、1AxP12、1AxP13、1AxP14、1AxP15、1AxP16、2AxP4、2AxP5、2AxP6、2 AxP7、2AxP8、2AxP9、2AxP10、2AxP11、2AxP12、2AxP13、2AxP14、2AxP15、2Ax P16、3AxP4、3AxP5、3AxP6、3AxP7、3AxP8、3AxP9、3AxP10、3AxP11、3AxP12、3 AxP13、3AxP14、3AxP15、3AxP16、4AxP4、4AxP5、4AxP6、4AxP7、4AxP8、4AxP9 、4AxP10、4AxP11、4AxP12、4AxP13、4AxP14、4AxP15、4AxP16、5AxP4、5AxP5、 5AxP6、5AxP7、5AxP8、5AxP9、5AxP10、5AxP11、5AxP12、5AxP13、5AxP14、5Ax P15、5AxP16、6AxP4、6AxP5、6AxP6、6AxP7、6AxP8、6AxP9、6AxP10、6AxP11、6A xP12、6AxP13、6AxP14、6AxP15、6AxP16、7AxP4、7AxP5、7AxP6、7AxP7、7AxP8 、7AxP9、7AxP10、7AxP11、7AxP12、7AxP13、7AxP14、7AxP15、7AxP16、8AxP4、 8AxP5、8AxP6、8AxP7、8AxP8、8AxP9、8AxP10、8AxP11、8AxP12、8AxP13、8AxP 14、8AxP15、8AxP16、9AxP4、9AxP5、9AxP6、9AxP7、9AxP8、9AxP9、9AxP10、9Ax P11、9AxP12、9AxP13、9AxP14、9AxP15、9AxP16、10AxP4、10AxP5、10AxP6、10 AxP7、10AxP8、10AxP9、10AxP10、10AxP11、10AxP12、10AxP13、10AxP14、10A xP15、10AxP16、11AxP4、11AxP5、11AxP6、11AxP7、11AxP8、11AxP9、11AxP10 、11AxP11、11AxP12、11AxP13、11AxP14、11AxP15、11AxP16、12AxP4、12AxP5、12AxP6、12AxP7、12AxP8、12AxP9、12AxP10、12AxP11、12AxP12、12AxP13、12AxP14、12AxP15、12AxP16、13AxP4、13AxP5、13AxP6、13AxP7、13AxP8、13AxP9、13AxP10、13AxP11、13AxP12、13AxP13、13AxP14、13AxP15、13AxP16、14AxP4、14AxP5、14AxP6、14AxP7、14AxP8、14AxP9、14AxP10、14AxP11、14AxP12、14AxP13、14AxP14、14AxP15、14AxP16、15AxP4、15AxP5、15AxP6、15AxP7、15AxP8、15AxP9、15AxP10、15AxP11、15AxP12、15AxP13、15AxP14、15AxP15、15AxP16、16AxP4、16AxP5、16AxP6、16AxP7、16AxP8、16AxP9、16AxP10、16AxP11、16AxP12、16AxP13、16AxP14、16AxP15、16AxP16、17AxP4、17AxP5、17AxP6、17AxP7、17AxP8、17AxP9、17AxP10、17AxP11、17AxP12、17AxP13、17AxP14、17AxP15、17AxP16、18AxP4、18AxP5、18AxP6、18AxP7、18AxP8、18AxP9、18AxP10、18AxP11、18AxP12、18AxP13、18AxP14、18AxP15、18AxP16、19AxP4、19AxP5、19AxP6、19AxP7、19AxP8、19AxP9、19AxP10、19AxP11、19AxP12、19AxP13、19AxP14、19AxP15、19AxP16、20AxP4、20AxP5、20AxP6、20AxP7、20AxP8、20AxP9、20AxP10、20AxP11、20AxP12、20AxP13、20AxP14、20AxP15、20AxP16、21AxP4、21AxP5、21AxP6、21AxP7、21AxP8、21AxP9、21AxP10、21AxP11、21AxP12、21AxP13、21AxP14、21AxP15、21AxP16、22AxP4、22AxP5、22AxP6、22AxP7、22AxP8、22AxP9, 22AxP10, 22AxP11, 22AxP12, 22AxP13, 22AxP14, 22AxP15, 22AxP16, 23AxP4, 23AxP5, 23AxP6, 23A xP7, 23AxP8, 23AxP9, 23AxP10, 23AxP11, 23AxP12, 23AxP13, 23AxP14, 23AxP15, 23AxP16, 24AxP4, 24AxP5, 24AxP6, 24AxP7, 24AxP8, 24AxP9, 24AxP10, 24AxP11, 24AxP12, 24AxP13, 24AxP14, 24AxP15, 24AxP16, 25Ax P4, 25AxP5, 25AxP6, 25AxP7, 25AxP8, 25AxP9, 25AxP10, 25AxP11, 25AxP12, 25AxP13, 25AxP14, 25AxP15, 25 AxP16, 26AxP4, 26AxP5, 26AxP6, 26AxP7, 26AxP8, 26AxP9, 26AxP10, 26AxP11, 26AxP12, 26AxP13, 26AxP14, 26AxP15, 26AxP16, 27AxP4, 27AxP5, 27AxP6, 27AxP7, 27AxP8, 27AxP9, 27AxP10, 27AxP11, 27AxP12, 27AxP1 3, may include one or more ionizable synthetic phospholipids provided herein having nAxPm formula (IV) selected from 27AxP14, 27AxP15, 27AxP16, 28AxP4, 28AxP5, 28AxP6, 28AxP7, 28AxP8, 28AxP9, 28AxP10, 28AxP11, 28AxP12, 28AxP13, 28AxP14, 28AxP15, 28AxP16 (wherein "x" in "nAxPm" refers to the number of modified Pm molecules on one amine molecule).

[0078] In some embodiments, the LNP of this specification is 7A1P4, 7A1P5, 7A1P6, 7A1P7, 7A1P8, 7A1P9, 7A1P10, 7A1P11, 7A1P12, 7A1P13, 7A1P14, 7A1P15, 7A1P16, 8A1P4, 8A1P5, 8A1P6, 8A1P7, 8A1P8, 8A1P9, 8A1P10, 8A1P11, 8A1P12, 8A1P13, 8A1P 14, 8A1P15, 8A1P16, 9A1P4, 9A1P5, 9A1P6, 9A1P7, 9A1P8, 9A1P9, 9A1P10, 9A1P11, 9A1P12, 9A1P13, 9A1P1 4, 9A1P15, 9A1P16, 10A1P4, 10A1P5, 10A1P6, 10A1P7, 10A1P8, 10A1P9, 10A1P10, 10A1P11, 10A1P12, 10A1P 13, 10A1P14, 10A1P15, 10A1P16, 11A1P4, 11A1P5, 11A1P6, 11A1P7, 11A1P8, 11A1P9, 11A1P10, 11A1P11, 1 1A1P12, 11A1P13, 11A1P14, 11A1P15, 11A1P16, 12A1P4, 12A1P5, 12A1P6, 12A1P7, 12A1P8, 12A1P9, 12A1P1 This may include one or more ionizable synthetic phospholipids provided herein having nAxPm formula (IV) selected from 0, 12A1P11, 12A1P12, 12A1P13, 12A1P14, 12A1P15, 12A1P16, 13A1P4, 13A1P5, 13A1P6, 13A1P7, 13A1P8, 13A1P9, 13A1P10, 13A1P11, 13A1P12, 13A1P13, 13A1P14, 13A1P15, 13A1P16, or any combination thereof. In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein having nAxPm formula (IV) selected from 9A1P9, 9A1P15, 10A1P10, 10A1P16, or any combination thereof.

[0079] LNPs typically contain helper lipids that promote cell binding, cholesterol that fills the gaps between lipids, and polyethylene glycol (PEG) that reduces opsonization by serum proteins and reticuloendothelial clearance.

[0080] In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein and at least one helper lipid. In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein and at least one helper lipid selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-methyldioctadecylamine (MDOA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecylammonium bromide salt (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and any combination thereof. In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein and at least one zwitterionic helper lipid (e.g., DOPE), an ionizable cationic helper lipid (e.g., MDOA, DODAP), a permanent cationic helper lipid (e.g., DDAB, DOTAP), or any combination thereof.

[0081] In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein and at least one cholesterol and / or cholesterol derivative. As used herein, “cholesterol derivative” refers to any compound that is essentially derived from a cholesterol structure, including its addition, substitution, and / or deletion. The term cholesterol derivative as used herein may also include steroid hormones and bile acids, as is commonly recognized in the art. Non-limiting examples of cholesterol derivatives suitable for use herein include dihydrocholesterol, ent-cholesterol, epi-cholesterol, desmosterol, cholestanol, cholestanone, cholestenone, sitosterol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, 3β-[N-(N'N'-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol Examples include olol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholesta-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, citcalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, thymosterol, diosgenin, fucosterol, fecosterol, or fecosterol, or salts or esters thereof.

[0082] In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein and at least one PEG or PEG-modified lipid. As used herein, PEG-modified lipid, or "PEG lipid," refers to a lipid modified with polyethylene glycol (PEG). Such species may instead be called PEGylated lipids. Non-limiting examples of PEG-modified lipids suitable for use herein include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol, and mixtures thereof. For example, but not limited to these, PEG-modified lipids for use in this specification include PEG-c-DOMG(R-3-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine poly(ethylene glycol)); PEG-DMG(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol poly(ethylene glycol)); PEG-DLPE(1,2-dilauroyl-sn-glycero-3-phosphorylglycerol sodium salt-poly(ethylene glycol) Possible examples include: PEG-DMPE (dimethyl-2-(dimethylphosphino)ethylphorphine-poly(ethylene glycol)); PEG-DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine-poly(ethylene glycol)); PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)); PEG-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxypoly(ethylene glycol))).

[0083] In some embodiments, the PEG-modified lipids for use herein may include a PEG moiety having a size of about 1,000 daltons to about 20,000 daltons. In some embodiments, the PEG-modified lipids for use herein may include a PEG moiety having a size of about 1,000 daltons, about 2,000 daltons, about 5,000 daltons, about 10,000 daltons, about 15,000 daltons, or about 20,000 daltons. In some embodiments, the LNPs herein may include one or more ionizable synthetic phospholipids provided herein and at least one PEG or PEG lipid, the PEG moiety may have a size of about 2,000 daltons.Examples of PEG lipids useful for the preparation of LNPs described herein include, but are not limited to, 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350](mPEG 350 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550](mPEG 550 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750](mPEG 750 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000](mPEG 1000 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](mPEG 2000 Examples include PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000](mPEG 3000 PE); 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](mPEG 5000 PE); N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)750](mPEG 750 ceramide); N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)2000](mPEG 2000 ceramide); and N-acyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)5000](mPEG 5000 ceramide). In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein and 1,2-dimiristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000).

[0084] The relative amounts of ionizable lipids, helper lipids, cholesterol, and PEG constituting the LNP can substantially affect the efficacy of the lipid nanoparticles. In some embodiments, the LNPs herein may comprise one or more ionizable synthetic phospholipids provided herein in a molar ratio of 55:30:45, 25:30:30, or 60:30:40, at least one helper lipid, and at least one cholesterol and / or cholesterol derivative. Those skilled in the art will recognize that the molar ratio of one or more ionizable synthetic phospholipids provided herein, at least one helper lipid, and at least one cholesterol and / or cholesterol derivative can be optimized as needed, particularly for a given application and / or route of administration of the LNPs herein. In some embodiments, the LNPs herein may comprise one or more ionizable synthetic phospholipids provided herein in a molar ratio of 55:30:45, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol. In some embodiments, the LNP of this specification may comprise one or more ionizable synthetic phospholipids provided herein in a 25:30:30 molar ratio, N-methyldioctadecylamine (MDOA), and cholesterol. In some embodiments, the LNP of this specification may comprise one or more ionizable synthetic phospholipids provided herein in a 25:30:30 molar ratio, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and cholesterol. In some embodiments, the LNP of this specification may comprise one or more ionizable synthetic phospholipids provided herein in a 25:30:30 molar ratio, 5A2-SC8, and cholesterol. In some embodiments, the LNP of this specification may comprise one or more ionizable synthetic phospholipids provided herein in a 60:30:40 molar ratio, dimethyldioctadecylammonium bromide salt (DDAB), and cholesterol.In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein in a 60:30:40 molar ratio, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol.

[0085] In some embodiments, the LNPs of this specification may comprise one or more ionizable synthetic phospholipids provided herein for targeting one or more cell types. In some embodiments, an LNP comprising ionizable synthetic phospholipids provided herein can selectively target the LNP to one or more cell types. In some embodiments, an LNP comprising ionizable synthetic phospholipids provided herein can cause the LNP to unload cargo at one or more selective cell types. In some embodiments, the LNPs of this specification can selectively target one or more cell types. In some embodiments, the LNPs of this specification can selectively target skin cells, spleen cells, liver cells, lung cells, or a combination thereof. In some embodiments, the LNPs of this specification can selectively target one or more tissue types. In some embodiments, the LNPs of this specification can selectively target skin, spleen, liver, lung, or a combination thereof. In some embodiments, an LNP of this specification comprising ionizable synthetic phospholipid 9A1P9 and helper lipid 5A2-SC8 can selectively target the liver. In some embodiments, LNPs according to this specification, comprising ionizable synthetic phospholipid 9A1P9 and helper lipid DDAB, can selectively target the lung. In some embodiments, LNPs according to this specification, comprising ionizable synthetic phospholipid 10A1P16 and helper lipid MDOA, can selectively target the spleen.

[0086] LNP size can influence the behavior of lipid nanoparticles in vivo. For example, in some descriptions where diameter is a relevant measurement such as spherical and other shaped vesicles with a measurable diameter, the terms “size” and “diameter” are used interchangeably. The sizes of LNPs disclosed herein may be determined by dynamic light scattering (DLS) and / or nanoparticle tracking analysis (NTA).

[0087] In some embodiments, the LNPs of this specification may have a diameter or size of about 20 nm to about 1000 nm. In some embodiments, the LNPs of this specification may have a size of about 20 nm to about 200 nm. In some embodiments, the LNPs of this specification may have a size of about 20 nm to about 190 nm or about 25 nm to about 190 nm. In some embodiments, the LNPs of this specification may have a size of about 30 nm to about 180 nm. In some embodiments, the LNPs of this specification may have a size of about 35 nm to about 170 nm. In some embodiments, the LNPs of this specification may have a size of about 40 nm to about 160 nm. In some embodiments, the LNPs of this specification may have a size of about 50 nm to about 150 nm, about 60 nm to about 140 nm, about 70 nm to about 130 nm, about 80 nm to about 120 nm, or about 90 nm to about 110 nm. In some embodiments, the LNPs of this specification may have a size or diameter of approximately 20 nm, approximately 25 nm, approximately 30 nm, approximately 35 nm, approximately 40 nm, approximately 45 nm, approximately 50 nm, approximately 55 nm, approximately 60 nm, approximately 65 nm, approximately 70 nm, approximately 75 nm, approximately 80 nm, approximately 85 nm, approximately 90 nm, approximately 95 nm, approximately 100 nm, approximately 105 nm, approximately 110 nm, approximately 115 nm, approximately 120 nm, approximately 125 nm, approximately 130 nm, approximately 135 nm, approximately 140 nm, approximately 145 nm, approximately 150 nm, approximately 155 nm, approximately 160 nm, approximately 165 nm, approximately 170 nm, approximately 175 nm, approximately 180 nm, approximately 185 nm, approximately 190 nm, approximately 195 nm, or approximately 200 nm.

[0088] In some embodiments, the average LNP size of an LNP composition or a group of LNPs may range from approximately 20 nm to approximately 1000 nm in diameter (for example, approximately 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000). In some embodiments, the LNP size of an LNP composition or a group of LNPs may be homogeneous, with a size diameter ranging from approximately 20 nm to approximately 1000 nm (for example, approximately 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000). In some embodiments, the LNP size of an LNP composition or a plurality of LNPs may be heterogeneous, with an average size diameter ranging from about 20 nm to about 1000 nm (e.g., about 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000). In some embodiments, the LNP size of the LNP composition or of the LNPs may be heterogeneous, with about 50% to about 99% of the LNPs having an average diameter of about 20 nm to about 1000 nm (e.g., about 20, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000).

[0089] In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified may have a negative charge. In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified may have a negative charge outside the cell (e.g., in serum). In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified may have a negative surface zeta potential. In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified may have a negative surface zeta potential in the range of about -20 mV to about -0.5 mV. In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified may have a negative surface zeta potential of about -20 mV, about -15 mV, about -10 mV, about -5 mV, about -2.5 mV, about -1.0 mV, or about -0.5 mV.

[0090] In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified herein may be chargeless (e.g., net neutral charge). In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified herein may be chargeless inside the cell (e.g., in the cell lumen). In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified herein may have a negative charge outside the cell and be chargeless after crossing the cell membrane and entering the cell lumen. In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified herein may have a negative surface zeta potential outside the cell and be chargeless after crossing the cell membrane and entering the cell lumen. In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified herein may have a negative surface zeta potential in the range of about -20mV to about -0.5mV outside the cell and be chargeless after crossing the cell membrane and entering the cell lumen (e.g., ~0mV).

[0091] In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified herein may have a pKa suitable for in vivo use. In some embodiments, LNPs comprising one or more ionizable synthetic phospholipids as specified herein may have a pKa in the range of about 5.5 to about 7.5 (e.g., about 5.5, 6.0, 6.5, 7.0, 7.5).

[0092] Various methods can be used to prepare the LNPs described herein. Such methods are either publicly known in the art or disclosed herein, including, for example, the method described in Lichtenberg and Barenholz in Methods of Biochemical Analysis, Volume 33, 337-462 (1988). See also Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); US Pat. Nos. 4,235,871, 4,501,728, 4,837,028; Liposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; and Hope, et al., Chem. Phys. Lip. 40:89 (1986).

[0093] Any of the LNPs described herein can be used as a vehicle for carrying biomolecules (cargo) to facilitate the delivery of biomolecules to a target. In addition to other excellent properties of the LNPs disclosed herein, the LNPs can protect the cargo they carry from degradation, such as enzymatic digestion. Thus, cargo-carrying LNPs are also provided herein that can be used to deliver cargo carried to a target for diagnostic and / or therapeutic purposes. In some embodiments, the cargo may be a therapeutic agent. In some embodiments, the cargo may be a gene editing agent.

[0094] In some embodiments, this disclosure provides cargo-loaded LNPs or therapeutic-loaded LNPs. The terms “cargo-loaded LNP,” “therapeutic-loaded LNP,” or “therapeutic-loaded LNP” mean the loading of one or more cargoes, including therapeutic agents, diagnostic agents, and drugs for use in gene editing. As used herein, the terms “loaded” or “loaded” as used with respect to “cargo-loaded LNP,” “therapeutic-loaded LNP,” or “therapeutic-loaded LNP” mean an LNP having one or more cargoes (which may be biomolecules, such as therapeutic agents, diagnostic agents, and drugs for use in gene editing) that are (1) encapsulated inside the LNP; (2) associated with or partially embedded in the lipid membrane of the LNP (i.e., partially protruding into the interior of the LNP); (3) associated with or bound to the outer portion of the lipid membrane and associated components (i.e., partially or completely protruding outside the LNP); or (4) completely located within the lipid membrane of the LNP (i.e., completely contained within the lipid membrane).

[0095] The term "cargo loading" refers to the process of loading, adding, or incorporating an exogenous cargo or treatment into an LNP such that one or more cargo loading or treatment-loading vesicles resulting from (1) to (4) above are achieved. Thus, in some embodiments, the cargo is encapsulated inside the LNP. In some embodiments, the cargo associates with or partially embeds in the lipid membrane of the LNP (i.e., partially protrudes into the interior of the vesicle). In some embodiments, the cargo associates with or binds to the outer portion of the lipid membrane (i.e., partially protrudes outside the LNP). In some embodiments, the cargo is fully located within the lipid membrane of the vesicle (i.e., fully contained within the lipid membrane). As used herein, the term "cargo" means any biomolecule or drug that can be loaded into or by an LNP, including, for example, biologics (e.g., peptides, proteins, antibodies, aptamers, nucleic acids, oligos), small molecules, therapeutic agents, and / or diagnostic agents.

[0096] In some embodiments, one or more cargoes may be present inside or on the inner surface of the LNP. In some embodiments, one or more cargoes present inside or on the inner surface of the LNP may associate with the LNP, for example, through chemical interactions, electromagnetic interactions, hydrophobic interactions, electrostatic interactions, van der Waals interactions, linkages, and bonds (hydrogen bonds, ionic bonds, covalent bonds, etc.). In some embodiments, the LNPs of this specification may encapsulate one or more cargoes.

[0097] In some embodiments, the LNPs of this specification may carry a single cargo, for example, a single therapeutic agent. In some embodiments, the LNPs of this specification may carry two or more different cargoes. In some embodiments, the LNPs of this specification may carry two or more molecules or copies of a single cargo or two or more different cargoes. In some embodiments, the LNPs of this specification may carry three or more molecules or copies of a single cargo or two or more different cargoes. In some embodiments, the LNPs of this specification may carry two to five molecules or copies of a single cargo or two or more different cargoes. In some embodiments, the LNPs and / or their pharmaceutical compositions according to this specification may carry 1 to 4,000, 10 to 4,000, 50 to 3,500, 100 to 3,000, 200 to 2,500, 300 to 1,500, 500 to 1,200, 750 to 1,000, 1 to 2,000, 1 to 1,000, 1 to 500, 10 to 400, 50 to 300, 1 to 250, 1 to 100, 2 to 50, 2 to 25, 2 to 15, 2 to 10, 3 to 50, 3 to 25, 3 to 25, 3 to 10, 4 to 50, 4 to 25, 4 to 15, 4 to 10, 5 to 50, 5 to 25, 5 to 15, or 5 to 10 molecules or copies, or any increment therein, in a single cargo or two (or more) different cargoes.

[0098] Methods for loading cargo onto LNPs are known in the art, and these methods can be used to load cargo onto the LNPs of this disclosure. Non-limiting examples of methods for loading cargo onto LNPs suitable for use herein include pH gradient, metal ion gradient, transmembrane gradient, surface loading, and fusion loading. The cargo of the cargo-loaded LNPs described herein may be of any type. In some embodiments, the LNP cargo as defined herein may be selected from the group consisting of pharmaceutical active ingredients, nucleic acids, ncRNA, siRNA, miRNA, tRNA, mRNA, shRNA, sgRNA, CRISPR / Cas9 DNA sequences, CRISPR / Cas12 DNA sequences, CRISPR / Cas13 sequences, RNA-acting adenosine deaminase (ADAR) sequences, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), base editors, single-stranded DNA (ssDNA), plasmid DNA (pDNA), circular RNA (circRNA), antisense oligonucleotides (AOs), small molecule drugs, proteins, and any combination thereof.

[0099] In some embodiments, the cargo of the cargo-loaded LNPs described herein may be a biomolecule. As used herein, the term “biomolecule” is used interchangeably with the term “biological therapeutic agent.” In some embodiments, the cargo of the cargo-loaded LNPs described herein may be a small molecule. In some embodiments, the cargo of the cargo-loaded LNPs described herein may be a pharmaceutical active ingredient.

[0100] In some embodiments, the LNPs herein may comprise a cargo (e.g., a biomolecule) which is a protein, peptide, aptamer, antibody, antibody fragment, or any combination thereof. In some embodiments, the LNPs herein may comprise a cargo (e.g., a biomolecule) which is a nucleic acid. In some embodiments, the nucleic acid cargo may be, for example, an oligonucleotide therapeutic agent, e.g., a single-stranded or double-stranded oligonucleotide therapeutic agent. In some examples, the oligonucleotide therapeutic agent may be single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense, e.g., antisense RNA, miRNA, morpholino oligonucleotide, peptide nucleic acid (PNA) or ssDNA (including, but not limited to, LNA, BNA, 2'-O-Me-RNA, 2'-MEO-RNA, 2'-F-RNA, having native and modified nucleotides), or analogs or conjugates thereof. In some embodiments, the cargo of the cargo-loaded LNPs herein may be mRNA.

[0101] In some embodiments, the LNPs described herein may be used to deliver the CRISPR-Cas system. “CRISPR / Cas” system or “CRISPR / Cas-mediated gene editing” refers to a type II CRISPR / Cas system (e.g., CRISPR / Cas9), a type V CRISPR / Cas system (e.g., CRISPR / Cas12), and / or a type VI CRISPR / Cas system (e.g., CRISPR / Cas13) modified for genome editing / engineering. This typically consists of a “guide” RNA (gRNA) and a nonspecific CRISPR-associated endonuclease (e.g., Cas9, Cas12, Cas13, or any variant thereof). “Guide RNA (gRNA)” is used herein interchangeably with “short guide RNA (sgRNA)” or “single-stranded guide RNA (sgRNA).” sgRNA is a short synthetic RNA composed of a “scaffold” sequence required for Cas binding and approximately 20 user-defined nucleotide “spacer” or “targeting” sequences that define the genomic target to be modified. The genomic target of Cas can be altered by changing the targeting sequence present in the sgRNA. In some embodiments, the LNPs herein may comprise a cargo having one or more components of the CRISPR-Cas system. In some embodiments, the cargo having one or more components of the CRISPR-Cas system may comprise mRNA, sgRNA, a CRISPR / Cas DNA sequence, a CRISPR / Cas ribonucleoprotein (RNP) complex, and any combination thereof.

[0102] Mai. Pharmaceutical composition This disclosure also provides a pharmaceutical composition comprising one or more of the LNPs described herein, capable of encapsulating one or more of the cargoes described herein, and a pharmaceutically acceptable carrier or excipient. The carrier of the pharmaceutical composition must be compatible with the active ingredient of the composition and preferably “acceptable” in the sense that it can stabilize the active ingredient and is not harmful to the subject being treated. The pharmaceutically acceptable excipient (carrier) includes buffers well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000), Lippincott Williams and Wilkins, Ed. KE Hoover, whose disclosure is incorporated herein by reference.

[0103] In some embodiments, the pharmaceutical compositions herein may contain pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosage and concentration used and include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin, ze It may contain proteins such as latin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalol, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0104] Depending on the route of administration and the form of the pharmaceutical product, different carriers and / or excipients may be used herein. Excipients for use herein include, but are not limited to, anti-tack agents, binders, coating disintegrants, fillers, flavoring agents (e.g., sweeteners) and colorants, lubricants, preservatives, and adsorbents. The carriers and / or excipients described herein may also include vehicles and / or diluents, where “vehicle” usually refers to any of the various media that act as a solvent or carrier; “diluent” refers to a diluent supplied to dilute the active ingredients of the composition; suitable diluents include any substance that can reduce the viscosity of the pharmaceutical preparation.

[0105] The type and amount of carrier and / or excipient are selected based on the function of the chosen pharmaceutical form; suitable pharmaceutical forms include liquid systems such as solutions, infusions, and suspensions; semi-solid systems such as colloids, gels, pastes, or creams; and solid systems such as powders, granules, tablets, capsules, pellets, microparticles, minitablets, microcapsules, micropellets, and suppositories. Each of these systems can be appropriately formulated for normal release, delayed release, or accelerated release using techniques well known in the art.

[0106] Pharmaceutical compositions comprising LNP as described herein can be prepared according to standard techniques as well as techniques described herein. In some examples, pharmaceutical compositions are formulated for parenteral administration, including intracapillary administration, intravenous administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intrathecal administration, and intramuscular administration. In some examples, pharmaceutical compositions herein may be administered intravenously by bolus injection or infusion. Formulations suitable for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985), whose disclosure is by reference to this specification.

[0107] In some embodiments, the pharmaceutical compositions herein may be formulated for injection, such as intravenous infusion. Sterile injectable compositions, such as sterile injectable aqueous or oily suspensions, may be formulated according to techniques known in the art, using appropriate dispersing or wetting agents (e.g., Tween 80) or suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile non-volatile oils are conventionally used as solvents or suspension media (e.g., synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectable preparations, as are naturally pharmaceutically acceptable oils, such as olive oil or castor oil, particularly their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants; other commonly used surfactants such as Tweens or Spans, or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceuticals.

[0108] In some embodiments, the pharmaceutical compositions described herein may be in the form of tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral, or rectal administration, or by inhalation or inhalation. To prepare solid compositions such as tablets, the LNPs disclosed herein may be mixed with a pharmaceutical carrier, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogeneous mixture of the compound of the present invention or a non-toxic, pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. The solid preformulation composition is then subdivided into the above-mentioned unit dosage forms containing 0.1 to about 500 mg of the active ingredient of the present invention. A dosage form can be obtained that provides the benefit of extended action by coating or otherwise compounding tablets or pills of novel compositions. For example, a tablet or pill may contain an internally administered component and an externally administered component, the latter in the form of an envelope over the former. The two components may be separated by an enteric coating that helps withstand disintegration in the stomach and allows the internal component to move intact into the duodenum, delaying its release. Various materials can be used for such an enteric coating or coating, and such materials include several polymer acids and mixtures of polymer acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0109] In some embodiments, the pharmaceutical compositions described herein may be emulsions. Suitable emulsions may be prepared using commercially available fat emulsions such as Intralipid®, Liposyn®, Infonutrol®, Lipofundin®, and Lipipysan®. The LNPs herein may be added to a premixed emulsion composition or added to an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and mixed with phospholipids (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) and water to form an emulsion. It will be recognized that the tonicity of the emulsion can be adjusted by adding other components, such as glycerol or glucose. Suitable emulsions typically contain up to 20% oil, for example, between 5 and 20%. Fat emulsions may contain fat droplets between 0.1 and 1.0 μm, particularly between 0.1 and 0.5 μm, and may have a pH in the range of 5.5 to 8.0. The emulsion composition may be prepared by mixing LNP with Intralipid® or its components (soybean oil, egg phospholipid, glycerol, and water). In some embodiments, the pharmaceutical composition described herein may be an emulsion for topical administration (for example, for treating the skin).

[0110] IV.How to use In certain embodiments, the Disclosure also provides a method for introducing one or more cargoes (e.g., nucleic acid molecules, pharmacoactive ingredients) into cells, comprising the step of contacting the cells with a composition disclosed herein. In some embodiments, the Method herein may include the step of delivering one or more cargoes herein (e.g., nucleic acid molecules, pharmacoactive ingredients) into cells, comprising contacting the cells or cell layers with the LNP disclosed herein. In some embodiments of the Method herein, the LNP can deliver one or more heterologous molecules to cells. According to these embodiments, the LNP herein can deliver one or more therapeutic heterologous molecules to cells. In some examples, the one or more therapeutic heterologous molecules delivered to cells using the Method herein may be therapeutic proteins, therapeutic DNA, and / or therapeutic RNA. In some embodiments, the therapeutic protein may be a monoclonal antibody or a fusion protein. In some embodiments, the therapeutic DNA and / or RNA may be antisense oligonucleotides, siRNA, shRNA, mRNA, DNA oligonucleotides, etc. In some embodiments, the LNP herein can deliver one or more therapeutic mRNAs to cells.

[0111] In some embodiments, the disclosure also provides a method for introducing a cargo (e.g., a nucleic acid molecule, a pharmaceutical active ingredient) into cells, comprising the step of contacting the cells with LNPs and / or pharmaceutical compositions disclosed herein. In some embodiments, the method herein may include the step of delivering the cargo (e.g., a nucleic acid molecule, a pharmaceutical active ingredient) to a specific cell type. In some embodiments, the method herein may include the step of delivering the cargo (e.g., a nucleic acid molecule, a pharmaceutical active ingredient) to a specific cell type selected from liver cells, lung cells, spleen cells, and / or skin cells. In some embodiments, the method herein may include the step of delivering the cargo (e.g., a nucleic acid molecule, a pharmaceutical active ingredient) to liver cells, comprising contacting the liver cells with LNPs disclosed herein. In some embodiments, the method herein may include the step of delivering the cargo (e.g., a nucleic acid molecule, a pharmaceutical active ingredient) to lung cells, comprising contacting the lung cells with LNPs disclosed herein. In some embodiments, the methods herein may include a step of delivering cargo (e.g., nucleic acid molecules, active pharmaceutical ingredients) to spleen cells, comprising contacting spleen cells with LNPs disclosed herein. In some embodiments, the methods herein may include a step of delivering cargo (e.g., nucleic acid molecules, active pharmaceutical ingredients) to skin cells, comprising contacting skin cells with LNPs disclosed herein. In certain embodiments, the disclosure also provides a method for introducing cargo (e.g., nucleic acid molecules, active pharmaceutical ingredients) into lung tissue, liver tissue, spleen tissue, skin tissue, or any combination thereof, comprising the step of contacting cells with LNPs and / or compositions disclosed herein.

[0112] Any of the LNPs and / or pharmaceutical compositions herein can be used to deliver therapeutic agents, diagnostic agents, or gene editing systems to a desired target site. In some embodiments, the LNPs and / or pharmaceutical compositions herein can be used to deliver therapeutic agents, diagnostic agents, or gene editing systems to the lungs, liver, skin, and / or spleen.

[0113] In some embodiments, any of the LNPs and / or pharmaceutical compositions described herein can be used to deliver a therapeutic agent, diagnostic agent, or gene editing system to treat and / or prevent a disease, condition, or disorder of interest. To carry out this use, an effective amount of a pharmaceutical composition containing the LNPs described herein can be administered to a subject in need of treatment (e.g., a mammalian subject, a human subject) via a suitable route, such as one of the routes described herein. Similarly, to carry out this use, an effective amount of a pharmaceutical composition containing any of the LNPs described herein, which encapsulates a therapeutic agent, diagnostic agent, or gene editing system, can be administered to a subject in need of treatment (e.g., a human subject) via a suitable route, such as one of the routes described herein. As used herein, “effective amount” refers to the amount of each activator required to produce a therapeutic effect on a subject, either alone or in combination with one or more other activators. The effective amount varies depending on the route of administration, the use of excipients, and the simultaneous use of other activators, as will be recognized by those skilled in the art. Such amounts naturally depend on the specific condition being treated, the severity of the condition, individual patient parameters including age, health status, size, sex, and weight, the duration of treatment, the nature of any concurrent therapies, the specific route of administration, and similar factors within the scope of the healthcare professional's knowledge and expertise. These factors are well known to those skilled in the art and can be addressed through routine experimentation. It is generally preferable to use the maximum dose of the individual component or its combination, i.e., the safest dose based on sound medical judgment. However, it will be understood by those skilled in the art that patients may request lower doses or tolerable doses for medical, psychological, or virtually any other reason.

[0114] In some embodiments, ionizable synthetic phospholipids, LNPS, pharmaceutical compositions, and methods described herein can be used to treat lung diseases or lung disorders. Non-limiting examples of lung diseases and / or lung disorders suitable for treatment via the methods herein include chronic obstructive pulmonary disease (COPD), asthma, acute tracheobronchitis, pneumonia, tuberculosis, lung cancer, influenza infection, SARS-CoV-2 infection, surfactant protein deficiency, cystic fibrosis, and alpha-1 antitrypsin (AAT) deficiency.

[0115] In some embodiments, ionizable synthetic phospholipids, LNPs, pharmaceutical compositions, and methods described herein can be used to treat liver diseases or liver disorders. Non-limiting examples of liver diseases and / or liver disorders suitable for treatment via the methods herein include phenylketonuria (PKU), ornithine transcarbamylase (OTC) deficiency, arginase-1 deficiency, alpha-1 antitrypsin deficiency, tyrosinemia type I (HT1), mucopolysaccharidosis, hemophilia, hypercholesterolemia, cirrhosis, liver cancer, non-alcoholic fatty liver disease (NAFLD), hepatocellular carcinoma (HCC), and non-alcoholic steatohepatitis (NASH).

[0116] In some embodiments, ionizable synthetic phospholipids, LNPs, pharmaceutical compositions, and methods described herein can be used to treat splenic diseases or disorders. Non-limiting examples of splenic diseases and / or disorders suitable for treatment via the methods herein include hereditary spherocytosis, Gaucher disease, sickle cell anemia, and beta-thalassemia.

[0117] In some embodiments, ionizable synthetic phospholipids, LNPs, pharmaceutical compositions, and methods described herein can be used to treat skin diseases or disorders. Non-limiting examples of skin diseases and / or disorders suitable for treatment via the methods herein include epidermolysis bullosa (EB), congenital onychomycosis, melanoma, ichthyosis, Hailey-Hailey disease, Sjögren-Larson syndrome (SLS), xeroderma pigmentosum (XP), wound healing, and Netherton syndrome.

[0118] V. Kit This disclosure also provides kits for use in delivering therapeutic agents, diagnostic agents, or gene editing systems to target sites (e.g., cells or tissues) or for treating / preventing target diseases, disorders, and / or conditions requiring such delivery. Such kits may include one or more containers containing any of the pharmaceutical compositions described herein.

[0119] In some embodiments, the kit may include instructions for use in accordance with any of the methods described herein. The included instructions may include instructions for administering a pharmaceutical composition to deliver a encapsulated therapeutic agent, diagnostic agent, or gene editing system, or to treat a subject in accordance with any of the methods described herein. Instructions for use of a pharmaceutical composition described herein, including LNP, generally include information about the dosage, administration schedule, and route of administration for the intended treatment.

[0120] The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. Instructions supplied with the kit of the present invention are typically written on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions may be provided for carrying out any of the methods described herein.

[0121] The kits described herein are in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). Packaging is also intended for use in combination with specific devices such as inhalers, nasal infusion devices (e.g., atomizers), or infusion devices such as minipumps. In some embodiments, suitable packaging may be an auto-injector. An auto-injector is a single-use, disposable, spring-loaded syringe. The kit may have a sterile access port (e.g., the container may be a vial with a stopper that can be punctured by an intravenous infusion bag or a subcutaneous needle). The container may also have a sterile access port (e.g., the container may be a vial with a stopper that can be punctured by an intravenous infusion bag or a subcutaneous needle).

[0122] The kits described herein may optionally provide additional components such as buffers and explanatory information. Typically, a kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, this disclosure provides a manufactured product comprising the components of the kit described above.

[0123] General technology Unless otherwise specified, the implementation of this invention will utilize conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, within the scope of the skills possessed by those skilled in the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds.This is fully explained in the following publications: *Current Protocols in Immunology* (JE Coligan et al., eds., 1991); *Short Protocols in Molecular Biology* (Wiley and Sons, 1999); *Immunobiology* (CA Janeway and P. Travers, 1997); *Antibodies* (P. Finch, 1997); *Antibodies: a practical approach* (D. Catty., ed., IRL Press, 1988-1989); *Monoclonal antibodies: a practical approach* (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); *Using antibodies: a laboratory manual* (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); *The Antibodies* (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0124] Without further detail, those skilled in the art will likely be able to make the most of the present invention based on the above description. Therefore, the following specific embodiments should be construed as merely illustrative and not limiting the remainder of the disclosure in any way. All publications cited herein constitute part of this specification by reference for the purposes or subject matter referred to herein. [Examples]

[0125] The following embodiments are included to demonstrate preferred embodiments of the present disclosure. Those skilled in the art will recognize that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that function well in the implementation of the present disclosure and thus constitute a preferred mode of implementation. However, those skilled in the art will recognize that, in light of the present disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure, and similar or comparable results can still be obtained.

[0126] [Example 1] The iPhos library was rationally designed with a membrane that destabilizes the superior endosomal escape mechanism. In accordance with this disclosure, an ionizable synthetic phospholipid ("iPhos lipid" or "iPhos") containing an ionizable amine, a phosphate group, and three hydrophobic tails was rationally designed. The small zwitterion composed of the amine and phosphate group was expected to be reversible at different pH levels. At physiological pH (approximately 7.4), the tertiary amine group was not protonated, making it difficult for the negatively charged iPhos to fuse to the membrane. In contrast, upon entry into acidic endosomes, the tertiary amine was protonated to form a zwitterionic head (Figure 1A). Substrate range testing revealed that the three hydrophobic tails were more readily mediating the membrane phase transition than the two chains. Thus, the synthetic iPhos lipid could be fully inserted into the natural phospholipid membrane, with the preferred small ion pair fused with the large tail to adopt a core shape, forming a hexagonal H II Because it promotes phase formation, its mechanism of action differs from that of classical gene delivery carriers (Figure 1B).

[0127] To overcome previous limitations in the synthetic pathway, the inventors focused on ring-opening reactions that could yield diverse products with chemical complexity satisfying the above design guidelines. Combinatorial reactions of amines (nA) and alkylated dioxaphosphorane oxide molecules (Pm) yielded 572 iPhos lipids (called nAxPm) (Figure 1C) (where "x" indicates the number of modified Pm molecules on one amine molecule). Pm molecules were synthesized via esterification of 2-chloro-2-oxo-1,3,2-dioxaphosphorane (COP) from corresponding alcohols with different alkyl chain lengths (Figures 7A-7M). Primary, secondary, and tertiary amine groups could all induce Pm ring-opening to introduce different zwitterions (Figure 8). To control the number of hydrophobic tails and zwitterions, amines with different alkyl chains and amine group numbers were used (Figures 1D and 9). Through this strategy, in addition to the number of groups, zwitterionic species (pH-switchable and irreversible) became available, greatly expanding the structure and diversity of phospholipids, making the chemical design of iPhos unique.

[0128] [Example 2] In vitro screening showed that the superior iPhos possesses a pH-switchable zwitterion and three tails. In accordance with this disclosure, ovarian cancer cells IGROV-1 were transfected using iPhos lipid nanoparticles (iPLNPs) to evaluate the potential for mRNA delivery. iPLNPs were formulated by mixing iPhos, a helper lipid, chol, and 1,2-dimiristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000)(25:30:30:1 mol / mol) using the ethanol dilution method. Initial screening demonstrated iPhos function using N-methyldioctadecylamine (MDOA), a structurally simple lipid, as the initial helper lipid. Therefore, iPLNPs can be considered a different concept from traditional LNPs, where the emphasis of the module is instead placed on the zwitterionic (functionally active iPhos) lipid, and all other lipids become helper lipids. All initial iPhos lipids with varying numbers and species of zwitterionic and tailed ions showed low toxicity (Figure 10). In vitro screening heatmaps concluded that iPhos with a single zwitterion (1A1P4–18A1P16) showed higher mRNA efficacy than those with multiple zwitterions (19A2P4–28A5P16) (Figures 2A–2C). This may be related to how multiple zwitterions construct a larger head, making membrane phase transitions more difficult. To further investigate the SAR of iPhos with a single zwitterion, two-tailed materials (1A1P4–6A1P16) showed much lower efficacy because the small tail body was unable to assemble a conical shape with the native membrane phospholipids. iPhos 14A1P4–18A1P16 possessed a permanent zwitterion and lacked structural flexibility in endosomal transit. Encouragingly, iPhos(7A1P4~13A1P16), composed of one tertiary amine, one phosphate group, and three hydrophobic tails, showed the highest expected mRNA delivery efficacy with a hit rate of approximately 60% (Figure 2D). The small zwitterionic head and large tail body fused to the membrane and lamellae, resulting in hexagonal H IIThis promoted the phase transition. Among these iPhos lipids, the amine tail length was particularly important, with hit rates for 10-12 chain lengths reaching up to 92% (Figure 2E). These findings contrasted with previously reported ionizable aminolipid and lipidoid libraries, where efficacy generally correlates with the polyamine core and a greater number of alkyl chains. 24~26 These results indicated that iPhos lipids may act through a different mechanism than ionizable aminolipids. Next, some selected top iPhos lipids (9A1P9, 9A1P15, 10A1P10, and 10A1P16) were purified (Figures 11-14), and the resulting iPLNPs exhibited a particle size suitable for endocytosis (approximately 150 nm), a slightly negative surface zeta potential for serum protein resistance (approximately -5 mV), and a pKa (6.0-6.5) suitable for in vivo assays (Figures 15A-15C and 16A-16B). These capabilities suggest great potential for in vivo applications of synthetic iPhos lipids.

[0129] [Example 3] Model membrane studies revealed the mechanism of iPhos-mediated endosomal rupture correlated with chemical structure. In accordance with this disclosure, the membrane-breaking activity of iPhos lipids and iPLNPs was first evaluated using a hemolysis model. 23、27 We investigated superior iPhos lipids (9A1P9 and 10A1P10) containing a pH-switchable zwitterionic head and three tails. 10A1P10 showed dramatically higher hemolysis than 17A, which has a single tertiary amine, confirming the zwitterionic superiority in membrane fusion and rupture (Figure 3A). In addition, 9A1P9, 10A1P10, and related iPLNPs showed higher membrane rupture activity in the acidic endosomal compartment compared to the neutral physiological environment (Figures 3B-3C).

[0130] Subsequently, iPhos lipid membrane fusion and iPLNP dissociation were evaluated using a fluorescence resonance energy transfer (FRET) assay. When two DOPE conjugate FRET probes, 7-nitrobenzo-2-oxa-1,3-diazole (NBD-PE) and lysamine rhodamine B (Rho-PE), were combined into a single endosome-mimicking liposome, FRET to rhodamine resulted in attenuation of NBD fluorescence. Once lipid fusion occurred, the resulting larger distance between the two probes led to an increase in the NBD signal. 28 As shown in Figure 3D, 10A1P10 iPLNPs exhibited higher lipid fusion than 25A3P9 iPLNPs, demonstrating that a small, single zwitterionic head exhibits a stronger tendency to insert into and disrupt the endosomal membrane compared to multiple zwitterions. Further testing of iPLNPs using FRET probes revealed that 10A1P10 iPLNPs, once mixed with endosomal-mimicking liposomes, were more readily disintegrated and released mRNA than 25A3P9 iPLNPs (Figures 3E-3G). These results demonstrate that, apart from the large tail body, the pH-switchable zwitterionic head of the iPhos lipid is essential for endosomal escape.

[0131] [Example 4] In vivo SARs of iPhos demonstrated that chemical structure and alkyl length control efficacy and organ selectivity. According to this disclosure, due to additional barriers to in vivo delivery, not all known carriers with in vitro activity have been transferred to animal models. 29、30 Furthermore, comparing siRNA / miRNA (18-22 bp) with long-chain RNA (1000-6000 nt), weaker electrostatic association was required to enable mRNA release after intracellular translocation. 12 Therefore, the chemistry of iPhos lipids has unique advantages over cationic lipids and can play an important role in mRNA delivery systems.

[0132] Fifty-one efficient iPhos lipids were selected from in vitro screening, and in vivo delivery was evaluated at a low mRNA dose of 0.1 mg / kg (Figure 4A). iPhos lipids containing multiple zwitterions were unable to deliver mRNA in vivo. Further SAR establishment revealed that iPhos lipids with one tertiary amine, one phosphate group, and three alkyl tails were the most effective. Interestingly, alkyl chain length had a tremendous impact on efficacy and organ selectivity. The amine chain length determined efficacy, with 8-10 carbon lengths mediating high in vivo mRNA expression (Figures 4B and 17). Surprisingly, alkyl length, in addition to the phosphate group, was found to influence mRNA transfection in selective organs (Figures 4C-4D). Short chains (9-12 carbons) indicated mRNA translation in the liver, while long chains (13-16 carbons) transferred protein expression to the spleen. In vivo evaluation of 10A1P4–10A1P16 clearly supported this inference (Figure 18). Subsequently, the nanoparticle size, zeta potential, and pKa of these iPLNPs were evaluated, and no significant differences were observed (Figure 19). Next, iPhos-based iPLNPs were evaluated at high doses (mRNA, 0.25 mg / kg) (Figures 20 and 21). Organ selectivity was achieved, with 9A1P9 iPLNP showing primary mRNA expression in the liver, while 9A1P15 and 10A1P16 iPLNPs mediated mRNA translation in the spleen. SAR provided guidelines for developing other effective vector materials with organ selectivity and specificity.

[0133] [Example 5] Synthetic iPhos lipids demonstrated broad compatibility with various helper lipids, mediated tissue-selective gene delivery and editing. According to the present disclosure, when used in iPLNPs containing a single helper lipid MDOA, the lipid 9A1P9 with the highest performance was first identified. To confirm that iPhos 9A1P9 is the most important and the active component of iPLNPs, a series of experiments were conducted. First, it was found that 9A1P9 showed 40 - 965 times higher in vivo efficacy compared to DOPE and DSPC, the currently most widely used phospholipids (Figures 5A - 5C). Second, when various other established lipids were evaluated as helper lipids in our 9A1P9 iPLNP mRNA delivery system, it showed broad applicability. Zwitterionic lipids (DOPE), ionizable cationic lipids (MDOA, DODAP, and 5A2 - SC8 26 ), and permanently cationic lipids (DDAB and DOTAP) were investigated as helper lipids (Figure 22). The molar ratios of the compositions were determined by an orthogonal design methodology 12、31 and are shown in Table 1. All formulated iPLNPs showed appropriate diameter, zeta potential, mRNA binding, pKa, and high in vitro mRNA delivery efficacy (Figure 23).

[0134]

Table 1

[0135] 9A1P9 coupled with various helper lipids achieved organ selectivity. 9A1P9 iPLNPs with zwitterionic, ionizable cationic, and permanently cationic helper lipids enabled selective mRNA expression in the spleen, liver, and lung, respectively (Figures 5D - 5I). When two extremely effective formulations were further tested, the in vivo biodistribution results revealed that 9A1P9 - 5A2 - SC8 and 9A1P9 - DDAB iPLNPs mediated high accumulation in the liver and lung, respectively (Figures 24A - 24C). Since iPhos lipids already enhance effective formulations in a modular manner, the combination of 9A1P9 - 5A2 - SC8 and 9A1P9 - DDAB had, respectively, in the liver (about 10 8 photons / second / cm 2 / sr, 0.05 mg / kg) and in the lung (about 10 8photons / second / cm 2 It was determined that iPLNP specifically exhibited ultra-high levels of mRNA expression at 0.25 mg / kg (0.25 mg / sr) (Figures 5F-5I and 25). High efficacy and organ selectivity were still maintained when Cre recombinase mRNA (Cre mRNA) was delivered (Figures 5J-5L). Compared with the "gold standard" DLin-MC3-DMA (used in FDA-approved Onpattro) LNP, 9A1P9-5A2-SC8 iPLNP still showed 13-fold higher mRNA delivery efficacy in vivo (Figures 5M-5N). Thus, iPLNP differs from conventional cationic lipid LNPs in achieving both high efficacy and controllable organ selectivity. Kinetic analysis revealed that protein expression occurred rapidly, peaking approximately 6 hours after injection (Figures 26A-26C).

[0136] Next, this model was used to quantify transfection of specific cell types in the liver, lung, and spleen organs. Following Cre mRNA delivery, liver-selective 9A1P9-5A2-SC8 iPLNP mediated mRNA delivery to approximately 91% of all hepatocytes (Figures 27A-27C). Lung-selective 9A1P9-DDAB iPLNP transfected approximately 34% of all endothelial cells, approximately 20% of all epithelial cells, and approximately 13% of immune cells (Figure 28). Spleen-selective 10A1P16-MDOA iPLNP transfected approximately 30% of all macrophages and 6% of all B cells (Figure 29). The iPLNPs proposed here represent one of the most effective mRNA delivery systems and hold great potential for organ-selective CRISPR / Cas9 gene editing.

[0137] [Example 6] 9A1P9 iPLNP achieved liver or lung-selective CRISPR / Cas9 gene editing. According to this disclosure, while LNPs have been used to deliver mRNA, there are still very few reports of successful in vivo Cas9 mRNA / sgRNA delivery for CRISPR / Cas gene editing, and even fewer reports of accurate delivery to specific organs.1 The iPLNP system demonstrated high mRNA delivery efficacy and organ selectivity, so it was then used to co-deliver Cas9 mRNA and sgRNA for gene editing. 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs, containing Cas9 mRNA and Tom1 sgRNA (sgTom1) in a 4:1 weight ratio, were administered intravenously (IV) to Ai9 mice at a total RNA dose of 0.75 mg / kg, with the stop cassette removed, which would activate the tdTomato protein (Figure 6A). Exovivo organ imaging specifically observed fluorescent tdTomato protein in the liver after administration of 9A1P9-5A2-SC8 iPLNP (Figure 6B). Sectioned organ analysis by confocal fluorescence microscopy showed tdTomato-positive cells in liver tissue (Figure 6C). Similarly, 9A1P9-DDAB iPLNP induced specific gene editing in the lungs (Figures 6D-6E). Subsequently, PTEN sgRNA (sgPTEN) was co-delivered to C57BL / 6 mice with Cas9 mRNA for gene editing, targeting endogenous genes (Cas9 mRNA / sgPTEN weight ratio, 4:1; total RNA dose, 0.75 mg / kg). The T7E1 assay demonstrated efficient targeted gene editing in the liver and lungs using 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs, respectively (Figure 6F). CRISPR / Cas9 gene editing in specific organs has remained a long-standing challenge in research and clinical translation. This study demonstrates highly efficient and organ-selective gene editing, expanding the application of iPLNPs to a wider range of genetic disorders.

[0138] Considering the potential preclinical activity, lead iPLNPs were fabricated on a higher scale using controlled microfluidic mixing. Precise control of mixing rate and volume ratio enabled the preparation of smaller 9A1P9-5A2-SC8 iPLNPs (77.2 nm, liver-specific), 9A1P9-DDAB iPLNPs (108.1 nm, lung-specific), and 10A1P16-MDOA iPLNPs (96.1 nm, spleen-specific). Importantly, high in vivo mRNA delivery efficacy and precise organ selectivity were fully retained even after reducing iPLNP diameter (Figures 6G-6H and 30A-30B). Furthermore, the iPLNPs allowed for repeated administration, and high efficacy was maintained after each repeated injection (Figures 6I-6J). Analysis of liver enzymes and tissue section histology showed that these iPLNPs exhibited negligible in vivo toxicity at the tested doses (Figures 6K–6N and 31A–31D). These results highlight the potential of the iPLNP system for future applications.

[0139] [Example 7] iPLNP achieved nucleic acid delivery after subcutaneous injection. iPLNPs were formulated using the ethanol dilution method. The molar ratio of lipid components for each iPLNP was 25:30:30:1, iPhos lipid:cholesterol:DODAP:PEG-DMG. The weight ratio of iPhos lipid to Cre recombinase (CRE) mRNA was fixed at 18:1. 5 μg of CRE mRNA iPLNP was subcutaneously injected into Ai9 mice. 44 hours after subcutaneous injection of CRE mRNA iPLNP, the mice were imaged for the tdTomato signal using IVIS. 9A1-P9, 9A1-P15, and 10A1-P16 iPLNPs were able to deliver CRE mRNA to cells to achieve gene editing, editing the DNA and turning on the expression of the red fluorescent reporter tdTomato protein (Figures 33A-33B).

[0140] Discussion of Examples 1-7 The CRISPR / Cas9 gene editing system is attracting increasing attention due to its tremendous potential for treating genetic diseases. While cells use phospholipids to construct membranes and mediate transport, almost all effective lipid nanoparticles for gene delivery rely on ionizable amines as a crucial physicochemical parameter that mediates endosomal escape via charge acquisition. In carrier development, synthetic zwitterionic lipids have been largely unexplored despite their potential to readily enable endosomal membrane fusion and leakage due to their homology with biological membranes. Although zwitterions have been reported to be beneficial for nanoparticle stability, RNA encapsulation, cellular uptake, and pharmacokinetics, current phospholipids are limited by a lack of flexibility in their chemical structure.

[0141] Therefore, this disclosure aims to develop novel phospholipids using chemical synthesis, revealing highly attractive candidates for insertion into biological membranes for efficient cargo escape from endosomes. Meanwhile, the structure and function of the phospholipids were well-tuned to specifically enable acidic endosomal rupture and prevent hemolytic effects in physiological environments. The rational design of the iPhos lipid involved a pH-switchable zwitterionic head and a three-tailed subgroup. This unique structure facilitates insertion into naturally occurring membrane phospholipids and induction of phase transitions to release RNA from endosomes. SAR revealed that the iPhos chain length can control in vivo mRNA delivery efficacy and organ selectivity. Furthermore, evaluation of a variety of existing zwitterionic, ionizable cationic, and permanent cationic helper lipids using our iPLNP system showed that they selectively mediated mRNA translation in the spleen, liver, and lung. Ultimately, we achieved organ-selective CRISPR / Cas9 gene editing, a long-standing challenge, by co-delivering mRNA and sgRNA using the superior 9A1P9-5A2-SC8 and 9A1P9-DDAB iPLNPs. Furthermore, these iPLNPs demonstrated broad applicability for delivering plasmid DNA and other nucleic acids, including siRNA (Figures 32A-32B). These profiles offer significant potential for ionizable synthetic phospholipids to treat a variety of genetic disorders with minimal side effects.

[0142] Materials and methods used in Examples 1-7 Materials - Chemicals and reagents for synthesis. 2-Chloro-2-oxo-1,3,2-dioxaphosphoran (COP) and triethylamine (TEA) were purchased from Fisher Scientific. Amines, alcohols, cholesterol (chol), and N-methyldioctadecylamine (MDOA) were purchased from Sigma-Aldrich. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazole-4-yl) (ammonium salt) (NBD-PE), 1,2-Dioleoyl- sn-glycero-3-phosphoethanolamine-N-(lysamine rhodamine B sulfonyl)(ammonium salt)(N-Rh-PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3-trimethylammonium-propane(chloride salt)(DOTAP), and dimethyldioctadecylammonium(bromide salt)(DDAB) were purchased from Avanti Lipids. 1,2-dimyristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000) was obtained from NOF America. The ionizable cationic lipid 5S2-SC8 was prepared according to the inventors' previous literature report. DLin-MC3-DMA was purchased from MedKoo Biosciences and used according to the formulation details reported in the literature. The organic solvent was purchased from Sigma-Aldrich.

[0143] Materials - Reagents for biological assays. Dulbecco's modified phosphate-buffered saline (PBS), RPMI-1640 medium, fetal bovine serum (FBS), and trypsin-EDTA (0.25%) were purchased from Sigma-Aldrich. Firefly luciferase messenger RNA (mRNA), Cre mRNA, and Cas9 mRNA were purchased from TriLink Biotechnologies. The Quant-iT RiboGreen RNA assay kit was purchased from Life Technologies. The ONE-Glo+Tox luciferase assay kit was purchased from Promega. Firefly D-luciferin, sodium salt monohydrate was purchased from Gold Biotechnology.

[0144] Synthesis of alkylated dioxaphosphorane oxide molecules P4-P16. P4-P16 were synthesized via esterification of 2-chloro-2-oxo-1,3,2-dioxaphosphoran (COP) with corresponding alcohols having different alkyl chain lengths. For example, to prepare P4, 1-butanol (30 mmol) and triethylamine (TEA, 30 mmol) were dissolved in 25 mL of anhydrous tetrahydrofuran (THF). Then, a solution of COP (30 mmol) in 10 mL of THF was added dropwise to the mixture at -15°C. The reaction was then continued at 25°C for 12 hours. The mixture was filtered to remove triethylamine hydrochloride, and the filtrate was concentrated by rotary evaporation to obtain P4. P5-P10 molecules were synthesized using their respective alcohols according to the general protocol described above. For the synthesis of P11-P16, COP was added to the corresponding alcohol at 0°C, with the other procedures remaining the same. All P4-P16 synthesis yielded yields of over 90%.

[0145] General synthesis of ionizable phospholipid (iPhos) libraries. iPhos(nAxPm) were synthesized via orthogonal reactions of amines (1A-28A) and alkylated dioxaphosphorane oxide molecules (Pm, m=4-16). "x" indicates the number of modified Pm molecules on a single amine molecule, and each Pm molecule could introduce one phosphate group and one hydrophobic alkyl chain into the iPhos. Each primary, secondary, or tertiary amine was designed to consume one equivalent of alkylated dioxaphosphorane oxide molecule Pm. For amine nA(n=1-18) with a single primary, secondary, or tertiary amine, 1.1 equivalents of Pm were reacted with the amine to obtain nA1Pm. For nA(n=19-28) with multiple amine groups, each amine group was designed to introduce at most one zwitterion. In short, amines were reacted with 2.2 equivalents, 3.3 equivalents, 4.4 equivalents, and 5.5 equivalents of Pm to obtain nA2Pm, nA3Pm, nA4Pm, and nA5Pm iPhos, respectively. All reactions were carried out in anhydrous dimethyl sulfoxide (DMSO) at a starting material concentration of 0.3 g / mL. The mixtures were stirred at 70°C for 3 days, and then the DMSO was removed by vacuum drying.

[0146] Crude iPhos were used for initial mRNA delivery (in vitro and in vivo screening) experiments. Selected top iPhos (e.g., 9A1P9, 10A1P10, 9A1P15, and 10A1P16) were purified by column flash chromatography and used for additional characterization (including size, zeta potential, mRNA binding, pKa, hemolysis, FRET testing, etc.) and in vivo evaluation. The products were eluted and fractionated using a solvent gradient from 3% chloroform in methanol to 10% chloroform in methanol. The final iPhos were concentrated by rotational evaporation and dried under vacuum for 24 hours.

[0147] In vitro iPhos nanoparticle (iPLNP) formulation and characterization. iPLNPs were prepared by ethanol dilution. mRNA was diluted in citrate / sodium citrate buffer (10 mM, pH 4.4). A lipid mixture containing synthetic iPhos, MDOA, cholesterol, and DMG-PEG2000 was prepared in ethanol. The two solutions were rapidly mixed by pipette in a 3:1 aqueous:ethanol volume ratio. After 15 minutes of incubation, the nanoparticles were 3-fold diluted in 1×PBS buffer for in vitro mRNA delivery.

[0148] For particle size and Ribogreen mRNA binding measurement, nanoparticles were diluted 5-fold with 1×PBS buffer. Zeta potentials were recorded for nanoparticles diluted 10-fold with 1×PBS buffer. A Zetasizer Nano ZS (Malvern) equipped with a He-Ne laser (λ=632nm) was used for particle size and zeta potential measurements. Particle size was measured by dynamic light scattering (DLS), and zeta potentials were determined by electrophoretic light scattering.

[0149] pKa determination was performed using the 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay. The pKa of each iPLNP was determined by the TNS assay. iPLNPs composed of synthetic iPhos / MDOA / chol / DMG-PEG2000 (25 / 30 / 30 / 1 mol%) were formulated in PBS at a concentration of 0.6 mM total lipids. TNS was prepared as a 100 μM stock solution in milliQ water. Nanoparticles were diluted to 6 μM total lipids in a 96-well plate with a buffer containing 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 10 mM 4-morpholine ethanesulfonic acid (MES), 10 mM ammonium acetate, and 130 mM NaCl (pH ranged from 2.5 to 11), with a volume of 100 μL per well. The TNS stock solution was added to each well to obtain a final concentration of 5 μM. Subsequently, plates were read using excitation and emission wavelengths of 321 nm and 445 nm, respectively. Sigmoid fit analysis was applied to the fluorescence data, and the pKa was measured as the pH that yielded the maximum half of the fluorescence intensity.

[0150] Hemolysis assay. Mouse erythrocytes (RBCs) were isolated from freshly collected whole blood by centrifugation at 10000 × g for 5 minutes, and the RBCs were then washed five times with PBS buffer (pH 7.4). The RBCs were then suspended in PBS at pH 7.4 and 5.5, respectively. iPLNP was formulated using the in vitro iPLNP formulation method outlined above. The lipids were dissolved in chloroform, evaporated by rotation, and then vacuum-dried for 2 hours to obtain a thin lipid film. PBS (pH 7.4) was then added, and the film was sonicated for 20 minutes to obtain a particle suspension. The RBC suspension was added to a 96-well plate, and the calculated iPLNP or lipids were added to the wells. After incubation at 37°C for 1 hour, the RBC solution was centrifuged at 10000 × g for 5 minutes, and the supernatant containing hemoglobin was collected. Hemoglobin content was assessed at a wavelength of 540 nm using a microplate reader. The RBC suspension incubated in PBS was set as the negative control, and the RBC suspension incubated in Triton-X solution (1 wt%) was set as the positive control.

[0151] Evaluation of lipid mixing and fusion characteristics by fluorescence resonance energy transfer (FRET) assay. Lipid mixing and fusion with endosome-mimicking anionic liposomes were determined by FRET assay. NBD-PE and N-Ph-PE, DOPE conjugate FRET probes, were formulated into the same endosome-mimicking nanoparticles to induce FRET-induced attenuation of NBD fluorescence to rhodamine. Once lipid fusion occurred, the NBD signal would increase as the distance between the two probes increased. Endosome-mimicking anionic liposomes were prepared by mixing DOPS:DOPC:DOPE:NBD-PE:N-Ph-PE (molar ratio 25:25:48:1:1) in chloroform, followed by rotational evaporation and vacuum drying for 2 hours to obtain a thin lipid film. The dried film was then resuspended in PBS (pH 7.4) by sonication for 20 minutes to fix the total lipid concentration at 1 mM. iPLNPs were formulated at an iPhos concentration of 1 mM using the in vitro iPLNP formulation method outlined above. iPhos 10A1P10 was dissolved in chloroform and rotated to evaporate to obtain a thin lipid membrane. Then, PBS (pH 7.4) was added and sonication was performed for 20 minutes to obtain a particle suspension (10 mM). Since 25A3P10, which has multiple zwitterions, could not form a particle suspension after sonication, only lipid fusion of 25A3P10 iPLNPs was evaluated. PBS (pH 5.5) was added to a black 96-well plate (100 μL / well), and 1 μL of endosome-mimicking anionic liposomes (1 mM) was added to each well. Then, 10 μL of iPLNP or 1 μL of lipid suspension was added to the wells. After incubation at 37°C for 5 minutes, fluorescence measurements (F) were performed using a microplate reader at Ex / Em = 465 / 520 nm. Only endosome-mimicking anionic liposomes in PBS were used as a negative control (F min ) was set as the positive control (F max ) was set as. Lipid fusion (%) was set to (FF min ) / (F max -F min ) * I calculated it as 100%.

[0152] iPLNP dissociation by FRET assay. iPLNP dissociation was measured by mixing iPLNP with endosome-mimicking anionic liposomes. DOPE conjugate FRET probes NBD-PE and N-Rh-PE were formulated into the same iPLNP. iPLNP was prepared using an iPhos:MDOA:chol:DMG-PEG2000:NBD-PE:N-Rh-PE lipid mixture (molar ratio 25:30:30:1:0.86:0.86) at a final total lipid concentration of 1 mM. Other procedures were the same as the in vitro iPLNP formulation method outlined above. Endosome-mimicking anionic liposomes were prepared by mixing DOPS:DOPC:DOPE (molar ratio 25:25:50) in chloroform, followed by rotary evaporation and further vacuum drying for 2 hours to obtain a thin lipid film. Subsequently, the dried film was resuspended in PBS (pH 7.4) by sonication for 20 minutes, and the total lipid concentration was fixed at 10 mM. PBS (pH 5.5) was added to a black 96-well plate (100 μL / well), and 1 μL of iPLNP was added to each well. Then, 1 μL of endosome-mimicking anionic liposomes was added to the wells. After incubation at 37°C for 10 minutes (or other mentioned time intervals), fluorescence measurements (F) were performed using a microplate reader at Ex / Em = 465 / 520 nm. iPLNP in PBS (containing NBD-PE and N-Rh-PE internally) was used as a negative control (F). min ) was set as the positive control. iPLNP (containing NBD-PE and N-Rh-PE inside) incubated with Triton-X solution (2 wt%) was used as the positive control (F max ) was set as iPLNP dissociation (%) (FF min ) / (F max -F min ) * I calculated it as 100%.

[0153] Cell culture. Human ovarian adenocarcinoma cells (IGROV1) were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin (P / S). The cells were cultured at 37°C and 5% CO2 in a humidified atmosphere.

[0154] In vitro screening of iPhos for mRNA delivery. IGROV1 cells were placed in a white opaque 96-well plate at a rate of 1 × 10⁶ 4 Cells were seeded at a density of 100 μL of RPMI-1640 medium supplied with 10% FBS and 1% P / S. After 24 hours, nanoparticles containing Fluc mRNA were prepared in a 96-well plate using the in vitro iPLNP formulation method outlined above by rapidly mixing the aqueous and ethanol phases (v / v=3:1) using a multichannel pipette. iPLNPs were prepared with a synthetic iPhos:mRNA molar ratio of 11622:1 and a lipid mixture synthetic iPhos:MDOA:chol:DMG-PEG2000 molar ratio of 25:30:30:1. With the synthetic iPhos:mRNA molar ratio fixed at 11622:1, 10A1P4-12A1P16:mRNA showed an average weight ratio of 10±2.5. This ensured that each iPLNP contained the same molar amount of lipid mixture. Unless otherwise noted, these ratios were also used for other characterization and in vivo evaluations. 50 ng of mRNA / well was used. The previous medium was then replaced with 150 μL of fresh cell culture medium, and the formulated iPLNP was added to the cells. After a further 24-hour incubation, luciferase expression and cell viability were evaluated using the ONE-Glo+Tox luciferase assay kit. All transfection assays were performed in triplicate, and the mean + standard deviation is reported.

[0155] In vivo iPLNP formulation and characterization. mRNA was diluted in citrate / sodium citrate buffer (10 mM, pH 3.2). A lipid mixture containing synthetic iPhos, MDOA, cholesterol, and DMG-PEG2000 was prepared in ethanol. The two phases were rapidly mixed by pipette in a 3:1 aqueous:ethanol volume ratio. After 15 minutes of incubation, the iPLNPs were dialyzed against 1×PBS in a Pur-A-Lyzer midi dialysis chamber (Sigma-Aldrich) for in vivo use.

[0156] Animal experimentation. All experiments were approved by the Animal Experimentation Committee of the University of Texas Southwestern Medical Center and did not conflict with local, state, and federal regulations where applicable. Female C57BL / 6 mice were purchased from the UT Southwestern Animal Husbandry Core. B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J mice (also known as Ai9 or Ai9(RCL-tdT) mice) were obtained from The Jackson Laboratory (007909) and reared to maintain homozygous expression of a Cre reporter allele with a LoxP-adjacent STOP cassette that inhibits transcription of the red fluorescent tdTomato protein driven by the CAG promoter. After Cre-mediated recombination, Ai9 mice express tdTomato fluorescence. Ai9 mice are congenic on the C57BL / 6J genetic background.

[0157] In vivo luciferase mRNA delivery. For iPhos in vivo screening, nanoparticles containing Fluc mRNA were prepared as in the in vivo iPLNP formulation method described above. Unless otherwise noted, formulation ratios followed those of the in vitro screening. Briefly, iPLNPs were prepared with a synthetic iPhos:Fluc mRNA molar ratio of 11622:1 and a lipid mixture synthetic iPhos:MDOA:chol:DMG-PEG 2000 molar ratio of 25:30:30:1. The nanoparticles were then administered intravenously (IV) to female C57BL / 6 mice (6-8 weeks old). After 6 hours, luciferase expression was evaluated by bioluminescence imaging of live animals. Briefly, mice were anesthetized under isoflurane and intraperitoneally injected with 100 μL of D-luciferin (GoldBio, 30 mg / mL in PBS) substrate. Luciferase activity was imaged using the IVIS Lumina system (Perkin Elmer) 5 minutes after anesthesia. Organs were then isolated and imaged using the same method. Images were processed using Living Image analysis software (Perkin Elmer).

[0158] iPhos 9A1P9 was compared with commercially available phospholipids DOPE and DSPC. C57BL / 6 mice were intravenously injected with 0.25 mg / kg Fluc mRNA via nanoparticles, and luminescence was quantified 6 hours after injection. A 25:30:30:1 molar ratio of 9A1P9:MDOA:chol:DMG-PEG 2000 and an 18:1 weight ratio of 9A1P9 / mRNA were used. For comparison with commercially available phospholipids, equimolar amounts of DOPE or DSPC were used for substitution with 9A1P9. Other procedures were performed as described above.

[0159] For 9A1P9 iPLNPs with different helper lipids, we used 9A1P9:DOPE:chol:DMG-PEG2000 (molar ratio) of 55:30:45:0.2, 9A1P9:MDOA (DODAP or 5A2-SC8):chol:DMG-PEG2000 (molar ratio) of 25:30:30:1, and 9A1P9:DDAB (or DOTAP):chol:DMG-PEG2000 (molar ratio) of 60:30:40:0.4. For all formulations, the 9A1P9:mRNA weight ratio was fixed at 18:1. Other procedures were carried out as described above.

[0160] In vivo Cre mRNA delivery. Nanoparticles containing Cre mRNA were prepared as described above in the in vivo iPLNP formulation method. The nanoparticles were then administered to Ai9 mice via IV injection. After 48 hours, the mice were sacrificed, organs were isolated, and imaged using the IVIS Spectrum in vivo imaging system (Perkin Elmer).

[0161] In vivo biodistribution. Nanoparticles containing Cy5-labeled Fluc mRNA (Cy5-mRNA, 0.25 mg / kg) were prepared as described above in the in vivo iPLNP formulation method. iPLNP was administered via IV injection to female C57BL / 6 mice (6-8 weeks old). After 6 hours, the mice were sacrificed, organs were isolated, and imaged using the IVIS Spectrum in vivo imaging system (Perkin Elmer).

[0162] In vivo co-delivery of Cas9 mRNA and sgTom1 for gene editing. Nanoparticles containing Cas9 mRNA and modified sgTom1 (mRNA / sgRNA weight ratio 4:1, total RNA dose 0.75 mg / kg) were prepared as in the in vivo iPLNP formulation method described above. 9A1P9:5A2-SC8:chol:DMG-PEG2000 (molar ratio) of 25:30:30:1 and 9A1P9:DDAB:chol:DMG-PEG2000 (molar ratio) of 60:30:40:0.4 were used for 9A1P9-5A2-SC8 iPLNP and 9A1P9-DDAB iPLNP, respectively. The 9A1P9 / RNA weight ratio was fixed at 18:1. Subsequently, the iPLNPs were administered to Ai9 mice via IV injection. The PBS group was used as a negative control. Ten days later, the mice were sacrificed, organs were isolated, and imaged using the IVIS Spectrum in vivo imaging system (Perkin Elmer). The tissue was then embedded in an optimal cutting temperature (OCT) compound and cut into 10 μm sections. These sections were fixed with 4% paraformaldehyde for 20 minutes in RT and washed three times with PBS. Subsequently, one drop of ProLong Gold Mountant containing DAPI was applied, and coverslips were placed over these slides. These slides were then imaged using confocal microscopy (Zeiss LSM 700).

[0163] In vivo co-delivery of Cas9 mRNA and sgPTEN for gene editing in C57BL / 6 mice. PTEN was selected to investigate endogenous gene editing in vivo. iPLNPs containing Cas9 mRNA and modified sgPTEN (mRNA / sgRNA weight ratio 4:1, total RNA dose 0.75 mg / kg) were prepared as described above in the in vivo iPLNP formulation method. 9A1P9:5A2-SC8:chol:DMG-PEG2000 (molar ratio) of 25:30:30:1 and 9A1P9:DDAB:chol:DMG-PEG2000 (molar ratio) of 60:30:40:0.4 were used for 9A1P9-5A2-SC8 iPLNPs and 9A1P9-DDAB iPLNPs, respectively. The 9A1P9 / RNA weight ratio was fixed at 18:1. Subsequently, iPLNP was administered to wild-type C57BL / 6 mice (6-8 weeks old) via IV injection. After 10 days, tissue was collected and genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (ThermoFisher). After obtaining the PTEN PCR product, the gene editing efficacy was confirmed by performing the T7E1 assay (NEB) according to a standard protocol. Furthermore, the gene editing efficacy of PTEN was evaluated by Image J based on the formula indel (%) = 100 × (1 - (1 - cleavage fraction)^0.5) (where cleavage fraction = (fragment 1 + fragment 2) / (fragment 1 + fragment 2 + parent fragment)). The sgRNA sequences and PCR primers used herein are provided in Tables 2 and 3, respectively.

[0164] [Table 2]

[0165] [Table 3]

[0166] Statistical analysis. Statistical analysis was performed using GraphPad Prism version 7 (GraphPad Software). Two groups were compared using a two-tailed, unpaired Student's t-test, and multiple replicate groups were compared using one-way ANOVA. P-value < 0.05. * ), P<0.01( ** ) and P<0.001( *** ) was considered statistically significant.

[0167] References used in Examples 1-7 1. Wang, HX et al. CRISPR / Cas9-based genome editing for disease modeling and therapy: Challenges and opportunities for nonviral delivery. Chem. Rev. 117, 9874-9906 (2017). 2. Hajj, KA & Whitehead, KA Tools for translation: Non-viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2, 17056 (2017). 3. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012). 4. Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013). 5. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013). 6. Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179 (2017). 7. Liu, S. et al. Highly branched poly(β-amino ester) delivery of minicircle DNA for transfection of neurodegenerative disease related cells. Nat. Commun. 10, 3307 (2019). 8. Zhou, J. et al. Biodegradable poly(amine-co-ester) terpolymers for targeted gene delivery. Nat. Mater. 11, 82-90 (2012). 9. Liu, S. et al. Bioreducible zinc(II)-coordinative polyethylenimine with low molecular weight for robust gene delivery of primary and stem cells. J. Am. Chem. Soc. 139, 5102-5109 (2017). 10. Kanasty, R., Dorkin, J.R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967-977 (2013). 11. Semple, S.C. et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 28, 172-176 (2010). 12. Cheng, Q. et al. Dendrimer-based lipid nanoparticles deliver therapeutic FAH mRNA to normalize liver function and extend survival in a mouse model of hepatorenal tyrosinemia type I. Adv. Mater. 30, e1805308 (2018). 13. van Meer, G., Voelker, D.R. & Feigenson, G.W. Membrane lipids: Where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112-124 (2008). 14. Sahay, G., Alakhova, D.Y. & Kabanov, A.V. Endocytosis of nanomedicines. J. Controlled Release 145, 182-195 (2010). 15. Gilleron, J. et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 31, 638-646 (2013). 16. Sahay, G. et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 31, 653-658 (2013). 17. Wittrup, A. et al. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 33, 870-876 (2015). 18. Miller, J.B. & Siegwart, D.J. Design of synthetic materials for intracellular delivery of RNAs: From siRNA-mediated gene silencing to CRISPR / Cas gene editing. Nano Res. 11, 5310-5337 (2018). 19. Truong, B. et al. Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency. Proc. Natl. Acad. Sci. U.S.A. 116, 21150-21159 (2019). 20. Mout, R., Ray, M., Lee, Y.W., Scaletti, F. & Rotello, V.M. In vivo delivery of CRISPR / Cas9 for therapeutic gene editing: Progress and challenges. Bioconjugate Chem. 28, 880-884 (2017). 21. Luther, D.C., Lee, Y.W., Nagaraj, H., Scaletti, F. & Rotello, V.M. Delivery approaches for CRISPR / Cas9 therapeutics in vivo: Advances and challenges. Expert Opin. Drug Deliv. 15, 905-913 (2018). 22. Miller, J.B. et al. Non-viral CRISPR / Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew. Chem. Int. Ed. 56, 1059-1063 (2017). 23. Alabi, C.A. et al. Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery. Proc. Natl. Acad. Sci. U.S.A. 110, 12881-12886 (2013). 24. Akinc, A. et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 26, 561-569 (2008). 25. Love, K. et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl. Acad. Sci. U.S.A. 107, 1864-1869 (2010). 26. Zhou, K. et al. Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl. Acad. Sci. U.S.A. 113, 520-525 (2016). 27. Wei, T. et al. Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance. Proc. Natl. Acad. Sci. USA 112, 2978-2983 (2015). 28. Zhang, Y. et al. The development of an in vitro assay to screen lipid based nanoparticles for siRNA delivery. J. Controlled Release 174, 7-14 (2014). 29. Cheng, Y., Yumul, RC & Pun, SH Virus-inspired polymer for efficient in vitro and in vivo gene delivery. Angew. Chem. Int. Ed. 55, 12013-12017 (2016). 30. Zhou, D. et al. The transition from linear to highly branched poly(beta-amino ester)s: Branching matters for gene delivery. Science Adv. 2, e1600102 (2016). 31. Li, B. et al. An orthogonal array optimization of lipid-like nanoparticles for mRNA delivery in vivo. Nano Lett. 15, 8099-8107 (2015). The claims at the time of filing were as follows: [Claim 1] Equation (I): [ka] (In the formula, R1 is selected from the group consisting of C2-C20 unsubstituted alkyl, C2-C20 substituted alkyl, C2-C20 unsubstituted alkenyl, C2-C20 substituted alkenyl, C2-C20 unsubstituted alkynyl, C2-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, and C4-C20 substituted cycloalkyl; R2 and R3 are independently selected from the group consisting of H, C1-C20 unsubstituted alkyl, C1-C20 substituted alkyl, C1-C20 unsubstituted alkenyl, C1-C20 substituted alkenyl, C1-C20 unsubstituted alkynyl, C1-C20 substituted alkynyl, C4-C20 unsubstituted cycloalkyl, and C4-C20 substituted cycloalkyl; R4, R5, R6, and R7 are independently selected from the group consisting of H, C1-C8 unsubstituted alkyl, C1-C8 substituted alkyl, C1-C8 unsubstituted alkenyl, C1-C8 substituted alkenyl, C1-C8 unsubstituted alkynyl, and C1-C8 substituted alkynyl; R8 is selected from the group consisting of C3-C21 unsubstituted alkyl, C3-C21 substituted alkyl, C3-C21 unsubstituted alkenyl, C3-C21 substituted alkenyl, C3-C21 unsubstituted alkynyl, and C3-C21 substituted alkynyl; n is an integer between 1 and 4. Ionizable synthetic phospholipids containing these lipids. [Claim 2] R1 is selected from the group consisting of C2-C16 unsubstituted alkyl groups, C2-C16 substituted alkyl groups, or C4-C12 substituted cycloalkyl groups; R2 and R3 are independently selected from the group consisting of H, C1-C16 unsubstituted alkyl, C1-C16 substituted alkyl, or C4-C16 substituted cycloalkyl; R4, R5, R6, and R7 are independently selected from the group consisting of H, C1-C4 unsubstituted alkyl, or C1-C4 substituted alkyl; R8 is selected from C3-C18 unsubstituted alkyl groups; n is an integer between 1 and 3. The ionizable synthetic phospholipid according to claim 1. [Claim 3] R1 is C2 - C15 unsubstituted alkyl; R2 and R3 are independently selected from the group consisting of H, C1 - C16 substituted alkyl, or C4 - C16 substituted cycloalkyl; R4, R5, R6, and R7 are independently selected from the group consisting of H, methyl, or ethyl; R8 is selected from C4 - C16 unsubstituted alkyl; n is an integer from 1 to 2, The ionizable synthetic phospholipid according to claim 1. [Claim 4] Formula (II):

Chemical formula

Claims

1. Equation (I): 【Chemistry 1】 (In the formula, R １ These are C2-C10 unsubstituted alkyl groups; R ２ is H, or a C4-C12 unsubstituted alkyl group; R ３ These are C4-C12 unsubstituted alkyl groups; R ４ , R ５ , R ６ , and R ７ Each of these is independently H; R ８ These are C3-C15 unsubstituted alkyl groups; n is an integer of 1. Ionizable synthetic phospholipids.

2. R １ is selected from the group consisting of C2, C4, C6, C8, C9, or C10 unsubstituted alkyl; R ３ However, it is a C4, C6, C8, C10, C11, or C12 unsubstituted alkyl; R 2, R ４ , R ５ , R ６ , and R ７ However, each is independently H; R ８ is selected from C3-C18 unsubstituted alkyl groups; n is an integer of 1. The ionizable synthetic phospholipid according to claim 1.

3. R １ is a C6 unsubstituted alkyl group; R ３ However, it is a C8 unsubstituted alkyl; R ２ , R ４ , R ５ , R ６ , and R ７ However, each is independently H; R ８ is a C8 unsubstituted alkyl group; n is an integer of 1. The ionizable synthetic phospholipid according to claim 1.

4. Formula (III): 【Chemistry 2】 (In the formula, R １ These are C2-C10 unsubstituted alkyl groups; R3 is an unsubstituted alkyl group from C4 to C12; R 2, R ４ and R ５ Each of these is independently H; R ６ These are C3-C15 unsubstituted alkyl groups; n is an integer of 1; (m is an integer between 1 and 4) Ionizable phospholipids.

5. R １ However, it is a C2, C4, C6, C8, C9, or C10 unsubstituted alkyl; R ３ However, it is a C4, C6, C8, C10, C11, or C12 unsubstituted alkyl; R 2, R ４ and R ５ However, each is independently H; R6 is a C3-C15 unsubstituted alkyl group; n is an integer of 1; m is an integer between 1 and 3. The ionizable phospholipid according to claim 4.

6. A pharmaceutical composition comprising the ionizable synthetic phospholipid described in claim 1.

7. The composition according to claim 6, further comprising a helper lipid, wherein the helper lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-methyldioctadecylamine (MDOA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), dimethyldioctadecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and any combination thereof.

8. The composition according to claim 6, further comprising cholesterol or a cholesterol derivative.

9. The composition according to claim 6, further comprising 1,2-dimyristoyl-rac-glycero-3-methoxy(poly(ethylene glycol-2000))(DMG-PEG2000).

10. The composition according to claim 6, further comprising one or more multi-tailed ionizable phospholipids in a molar ratio of 55:30:45, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol, wherein the one or more multi-tailed ionizable phospholipids comprise a tertiary amine, a phosphate group, and three hydrophobic tails.

11. The composition according to claim 6, further comprising one or more multi-tailed ionizable phospholipids in a molar ratio of 25:30:30, N-methyldioctadecylamine (MDOA), and cholesterol, wherein the one or more multi-tailed ionizable phospholipids comprise a tertiary amine, a phosphate group, and three hydrophobic tails.

12. The composition according to claim 6, further comprising one or more multi-tailed ionizable phospholipids in a molar ratio of 25:30:30, 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), and cholesterol, wherein the one or more multi-tailed ionizable phospholipids comprise a tertiary amine, a phosphate group, and three hydrophobic tails.

13. The composition according to claim 6, further comprising one or more multi-tailed ionizable phospholipids in a molar ratio of 25:30:30, 5A2-SC8, and cholesterol, wherein the one or more multi-tailed ionizable phospholipids comprise a tertiary amine, a phosphate group, and three hydrophobic tails.

14. The composition according to claim 6, further comprising one or more multi-tailed ionizable phospholipids in a molar ratio of 60:30:40, dimethyldioctadecylammonium bromide (DDAB), and cholesterol, wherein the one or more multi-tailed ionizable phospholipids comprise a tertiary amine, a phosphate group, and three hydrophobic tails.

15. The composition according to claim 6, further comprising one or more multi-tailed ionizable phospholipids in a molar ratio of 60:30:40, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol, wherein the one or more multi-tailed ionizable phospholipids comprise a tertiary amine, a phosphate group, and three hydrophobic tails.

16. The composition according to claim 6, further comprising cargo.

17. The composition according to claim 16, wherein the cargo is selected from the group consisting of a pharmaceutical active ingredient, nucleic acid, mRNA, sgRNA, CRISPR / Cas9 DNA sequence, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), siRNA, miRNA, tRNA, ssDNA, base editor, peptide, protein, CRISPR / Cas ribonucleoprotein (RNP) complex, and any combination thereof.

18. The composition according to claim 6, which is formulated for parenteral administration, intravenous administration, oral administration, topical administration, or any combination thereof.

19. A pharmaceutical composition comprising lipid nanoparticles (LNPs) carrying cargo, The aforementioned LNP is The ionizable phospholipid described in claim 1; or One or more multi-tailed ionizable phospholipids A pharmaceutical composition comprising, wherein the one or more multi-tailed ionizable phospholipids comprises a tertiary amine, a phosphate group, and three hydrophobic tails.

20. The pharmaceutical composition according to claim 6, wherein the cargo is located within the core of the LNP.

21. The pharmaceutical composition according to claim 6, wherein the one or more multi-tailed ionizable phospholipids form a nanoparticle structure that substantially encapsulates the cargo, and the one or more multi-tailed ionizable phospholipids comprise a tertiary amine, a phosphate group, and three hydrophobic tails.

22. The pharmaceutical composition according to claim 6, wherein the cargo is selected from the group consisting of a pharmaceutical active ingredient, nucleic acid, mRNA, sgRNA, CRISPR / Cas9 DNA sequence, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), siRNA, miRNA, tRNA, ssDNA, base editor, peptide, protein, cirRNA, CRISPR / Cas ribonucleoprotein (RNP) complex, and any combination thereof.

23. A pharmaceutical composition for use in the delivery of a pharmaceutical active ingredient, comprising a therapeutically effective amount of the pharmaceutical composition according to claim 6, wherein the cargo is the pharmaceutical active ingredient.

24. A pharmaceutical composition for use in in vivo delivery of mRNA or mRNA / sgRNA for gene editing, comprising a therapeutically effective amount of the pharmaceutical composition according to claim 6, wherein the cargo is a pharmaceutical active ingredient.

25. A pharmaceutical composition for use in inducing selective protein expression in the spleen, liver, and / or lungs of a target, comprising a therapeutically effective amount of the pharmaceutical composition according to claim 6, wherein the cargo is mRNA.

26. A pharmaceutical composition for use in gene delivery, gene editing, drug delivery, mRNA delivery, CRISPR / Cas9 gene editing, zinc finger nuclease (ZFN) gene editing, base editor gene editing, and transcription activator-like effector nuclease (TALEN) gene editing, comprising the pharmaceutical composition described in claim 6.

27. A pharmaceutical composition for use in tissue-specific cargo delivery to a target, comprising a therapeutically effective amount of the pharmaceutical composition according to claim 6.

28. The pharmaceutical composition according to claim 27, wherein the cargo comprises mRNA, a CRISPR / CasDNA sequence, or any combination thereof.

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