Lipids for delivery of nucleic acids to eukaryotic cells

Abstract

Ionizable lipids useful for delivering macromolecules, such as nucleic acids, into eukaryotic cells are provided. The lipids may be used alone, in combination with other lipids, and / or in combination with other transfection enhancing agents to make transfection complexes.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and cell and gene therapy, and more specifically relates to novel compounds and methods for introducing payloads (such as nucleic acids, ribonucleoproteins, etc.) into eukaryotic cells.

[0002] This application contains a sequence list, which is hereby incorporated in its entirety by reference. The sequence list .xml file (identified as TP386504WO1) is 667,141 bytes in size and was created on September 18, 2024. The sequence list submitted electronically with this document does not exceed the scope of the specification and does not contain any new information. Background Technology

[0003] Transfection is the process of introducing nucleic acids into eukaryotic cells through non-viral methods. Transfection methods allow the introduction of negatively charged molecules (such as the phosphate backbone of DNA and RNA) into cells with negatively charged membranes. Chemicals such as calcium phosphate and DEAE-glucan, or cationic lipid-based reagents, coat DNA, neutralizing the charge or even making the molecule positively charged overall. DNA-transfection reagent complexes readily cross cell membranes, especially for lipids with "fusion-enhancing" components that strengthen fusion with the cellular lipid bilayer.

[0004] With recent advances in nucleic acid-based therapeutics and the continued demand for low-toxicity transfection reagents, there is a persistent need for novel ionizable lipids that can be used in vitro and in vivo. Summary of the Invention

[0005] This document discloses compounds, compositions, and methods for improving the efficiency of introducing macromolecules (such as nucleic acids) or small molecules (e.g., therapeutic agents) into cells. Compounds, compositions containing these compounds, and methods for delivering payloads (e.g., nucleic acids or small molecules) to cells using these novel compounds and compositions are provided. These compounds can be used alone for transfection, or they can be combined with additional reagents in transfection compositions. For example, the novel compounds can be combined with one or more ionizable lipids and / or neutral lipids, one or more cell surface ligands, one or more fusion enhancers, and one or more nuclear localizers and one or more amphiphilic peptides, and any combination thereof. The resulting compositions can be complexed with one or more macromolecules (e.g., nucleic acids (such as DNA or RNA), proteins, ribonucleoproteins, etc.) and used for delivering these macromolecules into cells.

[0006] Therefore, in one embodiment, this disclosure provides a compound having formula I:

[0007] ,

[0008] Or its pharmaceutically acceptable salt, wherein:

[0009] A1 is —(CH2) x — or —(CH2) y —A4—(CH2) z —;

[0010] A4 Choose the group consisting of the following items: —(CH2) x —, —(CO)O—, —O(CO)—,

[0011] —SS—、 , , , and ;

[0012] Q1 is N or Q2 is N or ;

[0013] R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups, or —(CH2) 1-7 COOR;

[0014] R3 is —COR—(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or or C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0015] C4-C 30Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0016] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0017] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0018] C3-C6 cycloalkyl groups, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; or

[0019] C3-C6 cycloalkenyl groups, optionally replaced by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0020] C1-C 30Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7- or aryl groups, or

[0021] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups; or

[0022] Only if R1 and R2 are independently -(CH2) 1-7 R3 is H only when COOR is used;

[0023] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0024] R5 and R6 are independently selected from H and CH3;

[0025] R7 is H or an optionally substituted C1-C6 straight-chain or branched alkyl group, or an optionally substituted monounsaturated or polyunsaturated C1-C6 straight-chain or branched alkenyl group. ,or ;

[0026] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, —(CH2) 0-3 Het;

[0027] A2 and A3 independently choose from the following groups: H, , , , , , , , , , , and ;

[0028] R8 is —COR or ;

[0029] Where R9 is H, or ;

[0030] Where R 10 Choose the group consisting of the following items: H;

[0031] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cycloalkyl substitution;

[0032] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups;

[0033] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces;

[0034] C3-C6 cycloalkyl groups, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; and

[0035] C3-C6 cycloalkenyl groups, optionally replaced by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces; where R 11 Choose from the following groups: -NH2, -NHR, -N(R)2, , and ;

[0036] Where AA represents any natural or non-natural amino acid side chain; and

[0037] a is an integer from 1 to 6;

[0038] b is an integer from 0 to 6;

[0039] x is an integer from 1 to 9;

[0040] y is an integer from 1 to 4;

[0041] z is an integer from 1 to 4;

[0042] n is an integer from 1 to 5;

[0043] n1, n2, n3, n4, and n5 are independently integers from 1 to 5.

[0044] n6 is an integer from 0 to 7.

[0045] n7 and n8 are independent integers from 0 to 5;

[0046] n9 is an integer from 1 to 5;

[0047] m1 is an integer from 1 to 5;

[0048] m2 is an integer from 0 to 5;

[0049] m3 is an integer from 1 to 7;

[0050] p is an integer from 1 to 50; and Het is an optionally substituted 5-7 member monocyclic basic heterocycle or an optionally substituted 8-11 member bicyclic basic heterocycle.

[0051] In another embodiment, this disclosure provides a compound having the structure of formula Ia:

[0052]

[0053] Or its pharmaceutically acceptable salt, wherein:

[0054] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, and optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or —(CH2) 1- 7COOR;

[0055] A2 and A3 independently choose from the following groups: , , , , , , and ;

[0056] R3 is —COR, —(CH2) n COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or or

[0057] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0058] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0059] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0060] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0061] C3-C6 cycloalkyl groups, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; or

[0062] C3-C6 cycloalkenyl groups, optionally replaced by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0063] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7- or aryl groups, or

[0064] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups; or

[0065] Only if R1 and R2 are independently -(CH2) 1-7 R3 is H only when COOR is used;

[0066] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0067] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het; and

[0068] Where R 10 Choose the group consisting of the following items: H;

[0069] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cycloalkyl substitution;

[0070] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups;

[0071] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces;

[0072] Where AA represents any natural or non-natural amino acid side chain;

[0073] x is an integer from 1 to 9;

[0074] n is an integer from 1 to 5;

[0075] n5 is an integer from 1 to 5;

[0076] n6 is an integer from 0 to 7;

[0077] n7 and n8 are independent integers from 0 to 5;

[0078] n9 is an integer from 1 to 5;

[0079] m3 is an integer from 1 to 3;

[0080] p is an integer from 1 to 50; and

[0081] Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

[0082] In other embodiments, this disclosure provides compositions and methods for introducing nucleic acids, proteins, or peptides into eukaryotic cells by contacting the cells with a compound of formula I or a composition thereof, thereby introducing the nucleic acids, proteins, or peptides into the cells.

[0083] In other embodiments, this disclosure provides a composition comprising:

[0084] (i) One or more compounds according to formula I, and

[0085] (ii) one or more of structural lipids, ionizable lipids, and stabilizers; and (iii) optional payload.

[0086] In other embodiments, this disclosure provides a kit comprising a compound of formula I, and one or more cationic lipids, and / or one or more neutral lipids, and / or one or more cell surface ligands, and / or one or more fusion agents, and / or one or more nuclear-localized peptides or proteins and / or one or more amphiphilic peptides.

[0087] In other embodiments, this disclosure provides a kit comprising:

[0088] (i) One or more compounds according to formula I, and

[0089] (ii) one or more of structural lipids, ionizable lipids, and stabilizers; and (iii) optional payload. Attached Figure Description

[0090] Figure 1 is a graph depicting the particle size (d.nm) and polydispersity index of lipid-mRNA formulations.

[0091] Figure 2 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse liver after intravenous administration of the lipid-mRNA formulation.

[0092] Figure 3 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse liver to luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse spleen after intravenous administration of the lipid-mRNA formulation.

[0093] Figure 4 depicts the luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver of mice after intravenous administration of the lipid-mRNA formulation.

[0094] Figure 5 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse liver to luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse spleen after intravenous administration of the lipid-mRNA formulation.

[0095] Figure 6 is a graph depicting the transfection efficiency two days after transfection.

[0096] Figure 7 is a graph depicting GFP expression levels two days after transfection.

[0097] Figure 8 is a diagram depicting live T cells two days after transfection.

[0098] Figure 9 is a graph depicting the particle size (d.nm) of the lipid-mRNA formulation.

[0099] Figure 10 is a graph depicting the polydispersity index (PDI) of lipid-mRNA formulations.

[0100] Figure 11 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse liver after intravenous administration of a lipid-mRNA formulation.

[0101] Figure 12 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse liver to luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse spleen after intravenous administration of the lipid-mRNA formulation.

[0102] Figure 13 is a graph depicting the particle size (d.nm) and polydispersity index of the lipid-mRNA formulation.

[0103] Figure 14 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse liver after intravenous administration of the lipid-mRNA formulation.

[0104] Figure 15 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse liver to luciferase activity (in bioluminescence flux, photons / second (p / s)) in mouse spleen after intravenous administration of the lipid-mRNA formulation.

[0105] Figure 16 depicts GFP expression 48 hours after transfection with Compound 8, prepared in water at a 1:4 ratio with DOPE. Adding peptide SEQ ID NO. 350 to the lipids improved transfection efficiency, as evidenced by increased GFP expression. The addition of a second peptide (SEQ ID NO. 47) further increased GFP expression compared to using only 15-24.

[0106] Figure 17 is a graph depicting the fluorescence of Prestoblue, where the lipid-only formulation showed the lowest toxicity, while the formulation containing one or two peptides showed similar toxicity characteristics to the Expi293 transfection reagent.

[0107] Figure 18 is a graph depicting the particle size (d.nm) of the lipid-mRNA formulation.

[0108] Figure 19 is a diagram depicting the PDI of the lipid-mRNA formulation.

[0109] Figure 20 depicts the luciferase activity (in bioluminescence flux, photons / second (p / s)) in the spleen of mice after intravenous administration of the lipid-mRNA formulation.

[0110] Figure 21 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in the lungs of mice after intravenous administration of the lipid-mRNA formulation. Detailed Implementation

[0111] This disclosure relates to ionizable lipids and lipid compositions comprising the lipids provided herein (e.g., lipid complexes and lipid nanoparticle compositions). This disclosure also provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells, specifically to mammalian organs, generating target peptides in mammalian cells, and treating diseases or ailments in mammals in need of such treatment. For example, a method for generating a target peptide in cells involves contacting a nanoparticle composition comprising mRNA with mammalian cells, thereby translating the mRNA to generate the target peptide. A method for delivering therapeutic and / or prophylactic agents to mammalian cells or organs may involve administering a nanoparticle composition comprising the therapeutic and / or prophylactic agent to a subject, wherein such administration involves contacting the cell or organ with the composition, thereby delivering the therapeutic and / or prophylactic agent to the cell or organ.

[0112] Ionizable molecules are provided for improving methods of delivering macromolecules to eukaryotic cells. These compositions and methods are effective in a variety of cell types and provide high transfection efficiency. Specifically, molecules based on Formula I cores have been found to be useful for the efficient delivery of macromolecules into cells. These molecules can be advantageously used with one or more neutral lipids and additional components (such as fusion-promoting or fusion-enhancing molecules, additional cationic / ionizable lipids, cell surface ligands, cell adhesion molecules, nuclear localizers, and endosomal release agents) and payloads (e.g., complexes formed with macromolecules or pharmaceutical formulations or nutrients).

[0113] This complex is easy to prepare using a simple method, can be used in a variety of cell types, and is stable, therefore it is suitable for... in vitro , In vitro and in vivo Applications include the delivery of therapeutic nucleic acids (such as siRNA therapeutic agents, mRNA vaccine formulations, etc.), liposomes, drug formulations delivered to cells, nutrients, etc., for example, in cosmetic, nutritional health or therapeutic applications.

[0114] General definition

[0115] To understand this subject matter and the appended patent claims, the following definitions are included. The abbreviations used herein have their conventional meanings in the fields of chemistry and biology.

[0116] While various embodiments and aspects of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous modifications, variations, and substitutions will now be apparent to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of this invention.

[0117] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions thereof cited in this application (including, but not limited to, patents, patent applications, articles, books, manuals, and monographs) are expressly incorporated herein by reference in their entirety for any purpose.

[0118] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. See, for example,Singleton et al., *Discussionary of Microbiology and Molecular Biology*, 2nd edition, J. Wiley & Sons (New York, NY 1994); Sambrook et al., *Molecula Cloning*, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0119] The term "about," when referring to a range of values, critical values, or specific values, indicates that the listed values ​​may deviate from the actual values ​​by a maximum of 25%. Since many of the values ​​used herein were determined experimentally, those skilled in the art will understand that such determinations may and often will vary across different experiments. This inherent deviation should not be construed as an undue limitation on the values ​​used herein. The term "about" is used to cover deviations of ±25%, ±20%, 10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly indicates otherwise, all values ​​provided herein are modified by the term "about."

[0120] In the specification and claims herein, phrases such as “at least one of…” or “one or more of…” may appear after a list of elements or features. The term “and / or” may also appear in a list of two or more elements or features. Unless otherwise implied or explicitly contradicted by the context in which it is used, such phrases are intended to mean any element or feature listed alone, or any element or feature combined with other listed elements or features. For example, the phrases “at least one of A and B”; “one or more of A and B”; and “A and / or B” are each intended to mean “A alone, B alone, or A together with B”. Similar interpretations apply to lists comprising three or more items. For example, the phrases “at least one of A, B, and C”; “one or more of A, B, and C”; and “A, B, and / or C” are each intended to mean “A alone, B alone, C alone, A together with B, A together with C, B together with C, or A together with B and C together.” Furthermore, the use of the term “based on” in the foregoing and claims is intended to mean “at least partially based on,” meaning that features or elements not listed are also permitted.

[0121] It should be understood that when a parameter range is provided, all integers and their decimal places within that range are also considered to be provided by the present invention. For example, "0.2-5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc., up to 5.0 mg (and inclusive).

[0122] This document generally uses standard nomenclature to describe compounds. For compounds with asymmetric centers, all stereoisomers of the compound and mixtures thereof are covered unless otherwise specified. Non-limiting examples of stereoisomers include enantiomers, diastereomers, and E or Z isomers. When the compound is present in various tautomeric forms, the document aims to include all tautomeric forms. Include variables (e.g., X, L1, L2, L3, Y) are used herein. wait Compounds are described using general formulas. Unless otherwise specified, each variable in such formulas is defined independently of any other variable, and any variable that appears more than once in the formula is defined independently each time it appears. If a part is described as being selected “independently” from a set, then each part is chosen independently of the others. Therefore, each part may be the same as or different from one or more other parts.

[0123] The number of carbon atoms in the hydrocarbon group can be represented by the prefix "C". x -C yThe symbol “” indicates that x is the minimum number of carbon atoms in the moiety and y is the maximum number of carbon atoms. Therefore, for example, “C1-C6 alkyl” refers to an alkyl substituent containing 1 to 6 carbon atoms. Further exemplifying, C3-C6 cycloalkyl refers to a saturated hydrocarbon ring containing 3 to 6 carbon atoms. The prefix attached to a multicomponent substituent applies only to the first component immediately following that prefix. For clarity, the term “carbocycloalkyl” encompasses two components: the carbocyclo group and the alkyl group. Therefore, for example, C3-C6 carbocycloalkyl C1-C6 alkyl refers to a C3-C6 carbocyclo group attached to the parent molecule moiety via a C1-C6 alkyl group.

[0124] Unless otherwise specified, when a linking element connects two other elements in the depicted chemical structure, the leftmost component described by the linking element binds to the left-hand element in the depicted structure, and the rightmost component described by the linking element binds to the right-hand element in the depicted structure. For illustration, if the chemical structure is -L... S -ML S ''- and M is -N(R B If S(O)-, then the chemical structure is -L S -N(R B )S(O)-L S ''-.

[0125] If the linking elements in the depicted structure are bonds, then the elements to the left of the linking element are directly connected to the elements to the right of the linking element via covalent bonds. For example, if the chemical structure is depicted as -LS-M-LS' and M is chosen as the bond, then the chemical structure will be -LS-LS''-. If two or more adjacent linking elements in the depicted structure are bonds, then the elements to the left of these linking elements are directly connected to the elements to the right of these linking elements via covalent bonds. For example, if the chemical structure is described as -L S -ML S ''-M'-L S ''- and M and L S If ' is chosen as the bond, then the chemical structure will be -L S -M'-L S Similarly, if the chemical structure is described as -L S -ML S ''-M'-L S ''- and M, L S If ' and M' are bonds, then the chemical structure will be -L. S -L S ''-。 When using a chemical formula to describe a part, the dash indicates that the part has a free valence.

[0126] If a portion is described as “optionally substituted,” then the portion can be substituted or unsubstituted. If a portion is described as being optionally substituted by up to a specific number of non-hydrogen radicals, then the portion can be unsubstituted, or substituted by up to that specific number of non-hydrogen radicals or by up to the maximum number of substituted positions on the portion, whichever is less. Thus, for example, if a portion is described as a heterocycle optionally substituted by up to three non-hydrogen radicals, then any heterocycle having fewer than three substituted positions will be optionally substituted by up to only as many non-hydrogen radicals as the heterocycle has substituted positions. For example, a tetrazolium group (which has only one substituted position) will be optionally substituted by up to one non-hydrogen radical. Similarly, if an amino nitrogen is described as being optionally substituted by up to two non-hydrogen radicals, then a primary amino nitrogen will be optionally substituted by up to two non-hydrogen radicals, while a secondary amino nitrogen will be optionally substituted by up to only one non-hydrogen radical.

[0127] When a portion is substituted with an oxo or thio group, it means that the portion contains a carbon atom covalently bonded to at least two hydrogen atoms (e.g., CH2), and the two hydrogen radicals are substituted with an oxo or thio group to form C=O or C=S, respectively.

[0128] The terms “alkyl” or “alkyl group” or “alkylene group” refer to a fully saturated (i.e., without double or triple bonds) straight or branched hydrocarbon chain. Alkyl groups can have 1 to 20 carbon atoms (wherever they appear in this document, numerical ranges such as “1 to 20” refer to every integer within a given range); For example "1 to 20 carbon atoms" means that alkyl groups can be composed of 1, 2, or 3 carbon atoms. wait The alkyl group consists of a maximum of 20 carbon atoms, but this definition also covers the use of the term "alkyl" when no numerical range is specified, and may be optionally substituted. The alkyl group may also be a medium-sized alkyl group having 1 to 9 carbon atoms. The alkyl group may also be a lower alkyl group having 1 to 4 carbon atoms. The alkyl group of a compound may be represented as "C". 1-4 Alkyl", C 1-20 Alkyl", C 4-30 Alkyl or similar name. Mark with "C". 4-30 "Alkyl" means a straight-chain or branched saturated hydrocarbon with optional substitutions of 4 to 30 carbon atoms.

[0129] Unless otherwise stated, alkyl groups as used herein refer to unsubstituted and substituted alkyl groups. For example, alkyl groups in the above-described straight-chain or branched hydrocarbon chains may be substituted with hydroxyl groups. In another embodiment, the methylene group is connected via a disulfide bridge (—S—S—).

[0130] Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, n-hexyl, lauryl, palmitoyl, stearyl, etc.

[0131] The term "alkenyl," alone or in combination with any other term, refers to a straight-chain or branched monounsaturated or polyunsaturated aliphatic hydrocarbon radical containing a specified number of carbon atoms, or an unspecified number, in one embodiment, 2 to 30 carbon atoms (i.e., (C1...). 2-30 (alkenyl), in one embodiment, is 4 to 30 carbon atoms (i.e., (C) 4-30 ()alkenyl), and in another embodiment, 2 to 6 carbon atoms (i.e., (C) 2-6 (Alkenyl). As used herein, the term "alkenyl" or "alkenyl group" means a optionally substituted straight-chain or branched hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one double bond. (Note "C") 2-14 "Alkenyl" refers to a straight-chain or branched hydrocarbon with optional substitutions, comprising 2 to 14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, a cis-alkenyl group may include one or more double bonds. A C5 alkenyl group comprising two double bonds may be an linoleyl group.

[0132] Unless otherwise stated, alkenyl groups as used herein refer to unsubstituted and substituted alkenyl groups. For example, alkenyl groups in the aforementioned straight-chain or branched hydrocarbon chains may be substituted with hydroxyl groups. In another embodiment, the alkenyl groups are linked via disulfide bridges (—S—S—).

[0133] Examples of alkenyl radicals include, but are not limited to, vinyl, E-propenyl and Z-propenyl, isopropenyl, E-butenyl and Z-butenyl, E-isobutenyl and Z-isobutenyl, E-pentenyl and Z-pentenyl, E-hexenyl and Z-hexenyl, E,E-hexadienyl, E,Z-hexadienyl, Z,E-hexadienyl and Z,Z-hexadienyl, oleoyl, palmitoleoyl, etc.

[0134] The term "alkynyl," alone or in combination with any other term, refers to a straight-chain or branched hydrocarbon radical having one or more triple bonds, containing a specified number of carbon atoms, or an unspecified number, in one embodiment, from 2 to 20 carbon atoms. As used herein, the term "alkynyl" or "alkynyl group" means a optionally substituted straight-chain or branched hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond. The designation "C2-14 alkynyl" refers to an optionally substituted straight-chain or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may contain one, two, three, four, or more carbon-carbon triple bonds. For example, a C18 alkynyl group may contain one or more carbon-carbon triple bonds. Unless otherwise stated, the alkynyl groups referred to herein are both unsubstituted and substituted alkynyl groups.

[0135] Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, propynyl, butynyl, and pentyynyl.

[0136] The term "basic heterocycle" refers to a stable, optionally substituted 5-7 member monocyclic heterocycle or optionally substituted 8-11 member bicyclic heterocycle, which, if monocyclic, may optionally be benzene-fused and optionally substituted at one or more carbon atoms with halogen, alkyl, alkoxy, oxo, etc., and / or substituted at a secondary nitrogen atom (i.e., -NH-) with alkyl, arylalkoxycarbonyl, alkanoyl, phenyl, or phenylalkyl, or optionally substituted at a tertiary nitrogen atom (i.e., +N-) with an oxonium group and attached via a carbon atom.

[0137] The terms "heterocyclic," "heterocyclic," or "heterocyclic group" refer to a saturated (e.g., "heterocyclic alkyl"), partially unsaturated (e.g., "heterocyclic alkenyl" or "heterocyclic alkynyl"), or fully unsaturated (e.g., "heteroaryl") ring system, wherein at least one ring atom is a heteroatom (i.e., nitrogen, oxygen, or sulfur), and the remaining ring atoms are independently selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. A heterocycle can be, but is not limited to, a single ring, two fused rings, a bridged ring, or a spirocyclic ring. A heterocyclic group can be attached to a parent molecule moiety via any substituted carbon or nitrogen atom in the group. In the case where the heterocyclic group is a divalent portion connecting two other elements in the depicted chemical structure, the heterocyclic group can be attached to the two other elements via any two substituted ring atoms. Similarly, in the case where the heterocyclic group is a trivalent portion connecting three other elements in the depicted chemical structure, the heterocyclic group can be attached to the three other elements respectively via any three substituted ring atoms.

[0138] In the compounds of this invention, "Het" represents a heterocycle containing 4 to 12 carbon atoms, wherein at least one nitrogen atom is present in the ring. The heterocyclic group can be, but is not limited to, a monocyclic group containing a single ring. Non-limiting examples of monocyclic groups include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrroliyl, isopyrroliyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isomizolyl, imidazolinyl, imidazolinyl, pyrazolyl, pyrazololinyl, pyrazolyl, triazolyl, tetraazolyl, dithiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolinyl, isothiazolyl, thiazolinyl, isothiazolyl, thiazolinyl, isothiazolyl, thiazodiazolyl, oxazolyl (including 1,2,3-oxazolyl) Diazolyl, 1,2,4-oxadiazolyl (also known as "azoximyl"), 1,2,5-oxadiazolyl (also known as "furazanyl") and 1,3,4-oxadiazolyl), oxatriazolyl (including 1,2,3,4-oxatriazolyl and 1,2,3,5-oxatriazolyl), dioxazolyl (including 1,2,3-dioxazolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl and 1,3,4-dioxazolyl), pyridyl, piperidine alkyl, diazinyl (including pyridazinyl (also known as "1,2-diazinyl"), pyrimidinyl (also known as "1,3-diazinyl") and pyrazinyl (also known as "1,4-diazinyl")), piperazinyl, triazinyl (including s-triazinyl (also known as "1,3,5-triazinyl"), as-triazinyl (also known as "1,2,4-triazinyl") and v-triazinyl (also known as "1,2,3-triazinyl")), oxazinyl (including 1,2,3-oxazinyl, 1, 3,2-oxazinyl, 1,3,6-oxazinyl (also known as "pentazolatriyl", 1,2,6-oxazinyl and 1,4-oxazinyl)), isoxazinyl (including o-isooxazinyl and p-isooxazinyl), oxazolidinyl, isoxazolidinyl, oxazinyl (including 1,2,5-oxazinyl or 1,2,6-oxazinyl), oxadiazinyl (including 1,4,2-oxadiazinyl and 1,3,5,2-oxadiazinyl), morpholinyl, aziridine, and diaziridine.

[0139] Heterocyclic groups can also be, but are not limited to, bicyclic groups containing two fused rings, such as, for example, naphridinyl (including [1,8]naphridinyl and [1,6]naphridinyl), thiazopyrimidinyl, thienopyrimidinyl, pyrimidinyl, pyridinylpyrimidinyl, pyrazolyl, indolazidyl, pyridinylindenyl, pyranopyrroleyl, 4H-quinazinyl, purine, pyridinylpyridinyl (including pyridinyl[3,4-b]-pyridinyl, pyridinyl[3,2-b]-pyridinyl and pyridinyl[4,3-b]-pyridinyl), pyridinylpyrimidine and pteridinyl. Other non-limiting examples of fused-ring heterocycles include benzofused heterocyclic groups such as indolyl, isoindolyl, indoleninyl (also known as "pseudoindolyl"), isoindazole (also known as "benzopyrazolyl" or indazole), benzozizyl (including quinolinyl (also known as "1-benzozizyl") and isoquinolinyl (also known as "2-benzozizyl")), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including cenylyl (also known as...)) The terms are “1,2-benzodiazinyl” and quinazolinyl (also known as “1,3-benzodiazinyl”), benzothiazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, benzothiadiazolyl, benzimidazolyl, benzotriazolyl, benzooxazinyl (including 1,3,2-benzooxazinyl, 1,4,2-benzooxazinyl, 2,3,1-benzooxazinyl and 3,1,4-benzooxazinyl), benzoisooxazinyl (including 1,2-benzoisooxazinyl and 1,4-benzoisooxazinyl) and tetrahydroisoquinolinyl.

[0140] The heterocyclic group can also be, but is not limited to, spirocyclic systems, such as, for example, 1,4-dioxa-8-azaspiro[4.5]decyl. The heterocyclic group may contain one or more sulfur atoms as ring members; and in some cases, the sulfur atoms are oxidized to SO or SO2. The nitrogen heteroatom in the heterocyclic group may be quaternized or not quaternized, and may be oxidized or not oxidized to N-oxides. Furthermore, the nitrogen heteroatom may or may not be N-protected.

[0141] Heterocyclic or carbocyclic rings may be further substituted. Unless otherwise specified, the term "substituted" means substitution by independently replacing one, two, three, or more hydrogen atoms with a substituent, including but not limited to -F, -Cl, -Br, -I, hydroxyl, protected hydroxyl, -NO2, -N3, -CN, -NH2, protected amino, oxo, thiooxo, -NH-C2-C8-alkenyl, -NH-C2-C8-ynyl, -NH-C3-C 12 -cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-Cl-C 12-alkyl, O-C2-C8-alkenyl, -O-C3-C 12 -cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, -C(O)-C1-C 12 -alkyl, -C(O)-C2-C8-alkenyl, -C(O)-C2-C8-ynyl, -C(O)-C3-C 12 -cycloalkyl, -C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl, -CONH2, -CONH-C1-C 12 -alkyl, -CONH-C2-C8-alkenyl, -CONH-C2-C8-ynyl, -CONH-C3-C 12 -cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-C1-C 12 -alkyl, -OCO2-C2-C8-alkenyl, -OCO2-C2-C8-ynyl, OCO2-C3-C 12 -cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2-heterocycloalkyl, -OCONH2, -OCONH-C1-C 12 -alkyl, -OCONH-C2-C8-alkenyl, -OCONH-C2-C8-ynyl, -OCONH-C3-C 12 -cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl, -OCONH-heterocycloalkyl, -NHC(O)-C1-C 12 -alkyl, -NHC(O)-C2-C8-alkenyl, -NHC(O)-C2-C8-ynyl, -NHC(O)-C3-C 12 -cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, -NHC(O)-heterocycloalkyl, -NHCO2-C1-C 12 -alkyl, -NHCO2-C2-C8-alkenyl, -NHCO2-C2-C8-alkynyl, -NHCO2-C3-C 12 -cycloalkyl, -NHCO2-aryl, -NHCO2-heteroaryl, -NHCO2-heterocycloalkyl, -NHC(O)NH2, -NHC(O)NH-C1-C 12 -alkyl, -NHC(O)NH-C2-C8-alkenyl, -NHC(O)NH-C2-C8-ynyl, -NHC(O)NH-C3-C 12 -cycloalkyl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, -NHC(O)NH-heterocycloalkyl, -NHC(S)NH2, -NHC(S)NH-C1-C 12-alkyl, -NHC(S)NH-C2-C8-alkenyl, -NHC(S)NH-C2-C8-ynyl, -NHC(S)NH-C3-C 12 -cycloalkyl, -NHC(S)NH-aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH2, -NHC(NH)NH-C1-C 12 -alkyl, -NHC(NH)NH-C2-C8-alkenyl, NHC(NH)NH-C2-C8-alkynyl, -NHC(NH)NH-C3-C 12 -cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl, -NHC(NH)NH-heterocycloalkyl, -NHC(NH)-C1-C 12 -alkyl, -NHC(NH)-C2-C8-alkenyl, -NHC(NH)-C2-C8-ynyl, -NHC(NH)-C3-C 12 -cycloalkyl, -NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl, -C(NH)NH-C1-C 12 -alkyl, -C(NH)NH-C2-C8-alkenyl, -C(NH)NH-C2-C8-ynyl, -C(NH)NH-C3-C 12 -cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NH-heterocycloalkyl, -S(O)-C1-C 12 -alkyl, -S(O)-C2-C8-alkenyl, -S(O)-C2-C8-ynyl, -S(O)-C3-C 12 -cycloalkyl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heterocycloalkyl, -SO2NH2, -SO2NH-C1-C 12 -alkyl, -SO2NH-C2-C8-alkenyl, -SO2NH-C2-C8-alkynyl, -SO2NH-C3-C 12 -cycloalkyl, -SO2NH-aryl, -SO2NH-heteroaryl, -SO2NH-heterocycloalkyl, -NHSO2-C1-C 12 -alkyl, -NHSO2-C2-C8-alkenyl, -NHSO2-C2-C8-alkynyl, -NHSO2-C3-C 12 -cycloalkyl, -NHSO2-aryl, -NHSO2-heteroaryl, -NHSO2-heterocycloalkyl, -CH2NH2, -CH2SO2CH3, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -C3-C 12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-Cl-C 12 -alkyl, -S-C2-C8-alkenyl, -S-C2-C8-ynyl, -S-C3-C 12 -cycloalkyl, -S-aryl, -heteroaryl, -S-heterocycloalkyl, or methylthiomethyl. It should be understood that aryl, heteroaryl, alkyl, etc., can be further substituted.

[0142] The term "N-protecting group" or "N-protected" refers to those groups that protect amino groups from unwanted reactions. Commonly used N-protecting groups are described in Greene and Wuts. Protecting Groups in Chemical Synthesis (3rd edition, John Wiley & Sons, NY (1999)). Non-limiting examples of N-protecting groups include acyl groups such as formyl, acetyl, propionyl, neopentyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, or 4-nitrobenzoyl; sulfonyl groups such as benzenesulfonyl or p-toluenesulfonyl; sulfenyl groups such as phenylsulfenyl (phenyl-S-) or triphenylmethylsulfenyl (triphenylmethyl-S-); sulfinyl groups such as p-methylphenylsulfinyl (p-methylphenyl-S(O)-) or tert-butylsulfinyl (t-Bu-S(O)-); and carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl Carbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, dimethyl-3,5-dimethoxybenzyloxycarbonyl, diphenylmethoxycarbonyl, tert-butyloxycarbonyl The N-protecting groups include: alkyl groups such as benzyl, p-methoxycarbonyl, triphenylmethyl, or benzyloxycarbonyl; p-methoxyphenyl; and silyl groups such as trimethylsilyl. Preferred N-protecting groups include formyl, acetyl, benzoyl, neopentyl, tert-butylacetyl, phenylsulfonyl, benzyl, tert-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0143] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0144] As used herein, the term "natural amino acid side chain" refers to the substituents on the side chain of a naturally occurring amino acid. Naturally occurring amino acids have substituents attached to the α-carbon. Naturally occurring amino acids include arginine, lysine, aspartic acid, glutamic acid, glutamine, asparagine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan, alanine, isoleucine, leucine, phenylalanine, valine, proline, and glycine.

[0145] As used in this article, "non-natural amino acid side chain" refers to the substituents on the side chain of amino acids that do not exist naturally. Non-natural amino acids include β-amino acids (β... 3 and β 2 (This includes) homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, pyrrolidone, glycine derivatives, cyclic substituted phenylalanine and tyrosine derivatives, linear core amino acids, and N-methyl amino acids. Exemplary non-natural amino acids are available from Sigma-Aldrich and are listed under “Non-natural Amino Acids and Derivatives”. See also Travis S. Young and Peter G. Schultz, “Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon”, J. Biol. Chem. 2010 285: 11039-11044, which is incorporated herein by reference in its entirety.

[0146] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, wherein, in embodiments, the polymer may bind to a portion not composed of amino acids. These terms also apply to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. “Fusion protein” refers to a chimeric protein encoding two or more separate protein sequences recombinantly expressed or chemically synthesized as a single part.

[0147] The term "alkoxy" refers to an alkyl, alkenyl, or alkynyl ether group, wherein the terms "alkyl," "alkenyl," or "alkynyl" are as defined above. Examples of suitable alkyl ether groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, oleoyloxy, palmitoyloxy, palmitoleoyloxy, etc.

[0148] The term "aryl," alone or in combination with any other term, refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing a specified number of carbon atoms, in one embodiment 6 to 15 carbon atoms (i.e., (C... 6-15 aryl), and in another embodiment, 6 to 10 carbon atoms (i.e., (C) 6-10 The aryl radical is optionally substituted with one or more substituents selected from alkyl, alkoxy (e.g., methoxy), nitro, halogen (e.g., chlorine), amino, carboxylic acid ester, and hydroxyl groups. Examples of aryl radicals include, but are not limited to, phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, chamomilecyclol, fluorenyl, anthracene, etc.

[0149] The term "arylalkyl" or "arylalkyl" refers to an aryl group that is a substituent linked by an alkylene group, such as "C". 7-14 Aryl groups, including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C12). 1-4 Alkylene groups). In addition, the terms "aralkyl" or "arylalkyl" used alone or in combination refer to alkyl groups as defined above, in which one hydrogen atom is replaced by phenyl, benzyl, 2-phenylethyl, etc.

[0150] The term "aranekoxycarbonyl" used alone or in combination refers to a free radical of the formula -C(O)-O-araneyl, where the term "araneyl" has the meaning given above. An example of an aranekoxycarbonyl free radical is the benzyloxycarbonyl.

[0151] The term “aryloxy” used alone or in combination refers to a free radical of the formula aryl-O-, wherein the term “aryl” has the meaning given above.

[0152] The term "alkanoyl" used alone or in combination refers to an acyl radical derived from an alkane carboxylic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, etc.

[0153] The term "aryloxyalkanoyl" refers to an acyl radical of the formula aryl-O-alkanoyl, where aryl and alkanoyl have the meanings given above.

[0154] The term "araneacyl" refers to an acyl radical derived from an aryl-substituted alkyl carboxylic acid, such as phenylacetyl, 3-phenylpropionyl (hydrogenated cinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-phenylbutyryl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl, etc.

[0155] The term "aromatic acyl" refers to an acyl radical derived from an aromatic carboxylic acid. Examples of such radicals include aromatic carboxylic acids, optionally substituted benzoic or naphtholic acids, such as benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4-(benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6-(benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3-(benzyloxyformamido)-2-naphthoyl, etc.

[0156] The term "aminocarbonyl" used alone or in combination refers to an amino-substituted carbonyl group (carbamoyl) derived from an amino-substituted carboxylic acid, wherein the amino group can be a primary amino group with successive substituents, a secondary amino group with successive substituents, or a tertiary amino group with successive substituents, and the substituents are selected from hydrogen, alkyl radicals, aryl radicals, aralkyl radicals, cycloalkyl radicals, cycloalkylalkyl radicals, etc.

[0157] The term "aminoalkyl acyl" refers to an acyl radical derived from an amino-substituted alkane carboxylic acid, wherein the amino group can be a primary, secondary, or tertiary amino group containing a substituent selected from hydrogen, cycloalkyl radicals, cycloalkylalkyl radicals, etc. Examples include N,N-dimethylaminoacetyl and N-benzylaminoacetyl.

[0158] The term "carbon ring" refers to a non-aromatic, stable 3- to 8-membered carbon ring, which can be saturated, monounsaturated, or polyunsaturated. The carbon ring can be attached to any carbon atom within a ring to produce a stable structure. In one embodiment, the carbon ring has 5 to 7 carbon atoms.

[0159] The term "cycloalkyl" used alone or in combination refers to an alkyl radical containing about 3 to about 8 carbon atoms and being cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0160] The term "cycloalkylalkyl" means an alkyl radical as defined above that is substituted with a cycloalkyl radical containing about 3 to about 8 (about 3 to about 6 in one embodiment) carbon atoms.

[0161] The term "cycloalkylcarbonyl" refers to an acyl group derived from a monocyclic or bridged cycloalkyl carboxylic acid, such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, etc., or an acyl group derived from a benzofused monocyclic cycloalkyl carboxylic acid optionally substituted with, for example, an alkylamino group, such as 1,2,3,4-tetrahydro-2-naphthoyl or 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.

[0162] The term "cycloalkylalkoxycarbonyl" refers to the acyl group of a cycloalkylalkoxycarboxylic acid derived from the formula cycloalkylalkyl-O-COOH, wherein the cycloalkylalkyl group has the meaning given above.

[0163] The term "alkylation reaction" may include the alkylation reaction center of the target compound according to SN known in the art. 1 or SN 2 The mechanism involves nucleophilic attack on the electron-deficient regions of the alkylating agent (see, for example, Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. 293-871 (p. 4)). (John Wiley & Sons 1992). Those skilled in the art will understand that in some embodiments, the alkylating agent further comprises one or more leaving groups (LG). The term "leaving group" (X in Scheme IV) and its grammatical equivalents herein refer to an atom or molecule that is detached from a molecule (e.g., an organic molecule). In some embodiments, the residual portion may be an alkyl group covalently bonded to the target compound. Thus, in various exemplary embodiments, the leaving group may be a charged or uncharged atom or group that detaches from an atom or molecule considered to be the substrate residue or main portion in a particular reaction. The ability of the leaving group to detach from the alkylating agent may be a function of the leaving group's own capability. Thus, the leaving group can affect the inherent reactivity of the alkylating agent in the alkylation reaction. In some embodiments, the lower the pKa value of the leaving group's conjugate acid, the better, because in some embodiments, the leaving group may more easily stabilize the negative charge that may grow during the alkylation reaction. Thus, in some embodiments, the leaving group may be an electronegative atom or molecule. Examples of leaving groups include, but are not limited to: acetate (AcO) - p-Nitrobenzoate (PNBO) - ), sulfonate (e.g., mesylate: MsO) - ), p-Tosylate (TsO) - ), p-bromobenzenesulfonate (BsO) - ), p-nitrobenzenesulfonate (NsO) - ), fluoromethanesulfonate, difluoromethanesulfonate, trifluoromethanesulfonate (Triflate: TfO) - ) and ethanesulfonate ions), haloesters and halide ions (e.g., I) - ,Br - Cl - ).

[0164] The term "solid-bonded arylalkyl chloride" includes arylalkyl chlorides covalently linked to polystyrene-based resins. Those skilled in the art will understand that polystyrene-based resins can have various reactive functional groups, such as chlorine as a leaving group, or they can have other leaving groups as described above. Since Merrifield's pioneering work on polystyrene (2% divinylbenzene crosslinked) as a solid-phase support for peptide synthesis (Merrifield, RB (1963), J. Am. Chem. Soc., 85, 2149-2153), the properties of solid-phase supports have been extensively improved to meet the specific needs of new organic chemistry.

[0165] The composition may also include a salt of one or more compounds (e.g., compounds of Formula I). ​​The salt may be a pharmaceutically acceptable salt. As used herein, “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound wherein the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts with a basic residue, such as amines; alkali metal or organic salts with an acidic residue, such as carboxylic acids. Representative acid addition salts include: acetates, adipates, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, hydrogen sulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, diglucuronides, dodecyl sulfates, ethanesulfonates, fumarates, glucohepanoates, glycerophosphates, hemisulfates, heptarates, hexanoates, hydrobromide salts, hydrochlorides, hydroiodates, 2-hydroxyethanesulfonates, lacturonates, lactic acid salts. Salts, including laurates, lauryl sulfates, malates, maleates, malonates, mandelates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, dihydroxynaphthalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pentanoates, propionates, pyruvates, salicylates, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, and valerates, are disclosed. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, ethanolamine, trimethylamine, diethylamine, triethylamine, ethylamine, and isopropylamine. Pharmaceutically acceptable salts disclosed herein include conventional non-toxic salts formed from parent compounds, such as salts formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts disclosed herein can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic moiety. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof; typically, non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.For a list of suitable salts, see: Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418; Pharmaceutical Salts: Properties, Selection, and Use, PH Stahl and CG Wermuth (eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0166] The term "surface ligand" or "cell surface ligand" refers to a chemical compound or structure that binds to a surface receptor on a cell. As used herein, the term "cell surface receptor" refers to a specific chemical group on the cell surface to which the ligand can attach. Cell surface receptors can be cell-specific, meaning they are found primarily in one type of cell rather than another (e.g., LDL and desialyl glycoprotein receptors are specific to hepatocytes). Receptors facilitate the internalization of ligands and attachment molecules. Cell surface receptors include, but are not limited to, folic acid receptors, biotin receptors, lipoic acid receptors, low-density lipoprotein receptors, desialyl glycoprotein receptors, insulin-like growth factor type II / cation-independent mannose-6-phosphate receptors, calcitonin gene-related peptide receptors, insulin-like growth factor I receptors, nicotinic acetylcholine receptors, hepatocyte growth factor receptors, endothelin receptors, bile acid receptors, bone morphogenetic protein receptors, chondroitin receptors, or glycosylphosphatidylinositol (GPI)-anchored proteins (e.g., β-adrenergic receptors, T cell activating protein, Thy-1 protein, GPI-anchored 5' nucleotidases). These are non-limiting examples.

[0167] A receptor is a molecule that binds specifically to a ligand and has a relatively high affinity. It is typically a protein or glycoprotein, but can also be coated with glycolipids, lipopolysaccharides, glycosaminoglycans, or polysaccharides. For the purposes of this disclosure, epitopes to which antibodies or fragments thereof bind are interpreted as receptors because antigen:antibody complexes undergo endocytosis. Furthermore, surface ligands include any substance capable of entering the cell via endocytosis (e.g., endocytosis, pit-mediated endocytosis, pinocytosis).

[0168] As used herein, the term "ligand" refers to a chemical compound or structure that binds to a receptor. This includes, but is not limited to, ligands such as desialylated serum mucoprotein, desialylated glycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, and RGD-containing cell adhesion peptides.

[0169] Those skilled in the art will readily recognize that the ligand chosen will depend on which receptor it binds to. Because different cell types have different receptors, this provides a method for targeting nucleic acids to a specific cell type, depending on which cell surface ligand is used. Therefore, the preferred cell surface ligand may depend on the cell type being targeted.

[0170] As used herein, the terms “nuclear localizer,” “nuclear localization signal,” or “nuclear ligand” refer to a ligand, such as a peptide, that will localize a reagent covalently or non-covalently linked to it to the cell nucleus, typically by binding to a nuclear receptor. As used herein, the term “nuclear receptor” refers to a chemical group on the nuclear membrane that will bind a specific ligand and facilitate the transport of the ligand and its associated linker across the nuclear membrane. Nuclear receptors can be, but are not limited to, those that bind nuclear localization sequences. Non-limiting examples of nucleoli include GYSTPPKKKRKVEDP (SEQ ID NO:1), GYSTPPKTRRRP (SEQ ID NO:2), GYSTPGRKKR (SEQ ID NO:3), GYSTPRRNRRRRW (SEQ ID NO:4), PDEVKRKKKPPTSYG (SEQ ID NO:5), PRRRTKPPTSYG (SEQ ID NO:6), RKKRGPTSYG (SEQ ID NO:7), WRRRRNRRPTSYG (SEQ ID NO:8), and GYGPPKKKRKVEAPYKA(K). 8-40 K (SEQ ID NO:9) can be used to transport nucleic acids to the cell nucleus.

[0171] As used herein, the term "lysing agent" refers to a molecule, compound, protein, or peptide capable of breaking down endosome membranes and releasing DNA transporters into the cytoplasm of the cell. This term includes, but is not limited to, viruses, synthetic compounds, lysing peptides, or derivatives thereof. The term "lysing peptide" refers to a chemical group that permeates the membrane, causing a loss of its structural organization and integrity. Due to the presence of lysing agents, membranes undergo lysis, fusion, or both. Examples of lysing agents / endosome release agents include chloroquine, polyamines, and polyamides. Suitable reagents are described, for example, by Pei and Buyanova. Bioconjugate Chem , 30:273-283 (2009) and Juliano, Nucleic Acid Therapeutics, 28:166-177 (2018)

[0172] As used herein, the term "polycationic nucleic acid binding moiety" refers to a moiety containing multiple positive charges at physiological pH, which allows the moiety to bind negatively charged nucleic acids. Polycationic nucleic acid binding moieties can be linked to, for example, cell surface ligands, fusion agents, and / or nuclear-targeting peptides. This linkage can be covalent. Suitable polycationic nucleic acid binding moieties include polyamines (such as PEI, spermine, spermidine, carboxysemine) and polybasic peptides containing, for example, multiple lysine, ornithine, histidine, or arginine residues.

[0173] The term "nucleic acid," when not applied to specific types of molecules such as unmodified DNA or RNA, refers to any type of nucleic acid currently known or that may be prepared or identified in the future, provided that when mixed with any lipid of Formula I, the nucleic acid carries a sufficient negative charge to form lipid aggregates, liposomes, or liposome-like complexes. As used herein, nucleic acid refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, as well as mixtures and polymers thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, have binding properties similar to a reference nucleic acid, and are metabolized in a manner similar to a reference nucleotide. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNAs), 5-methylcytosine, pseudouridine, N... 1 5-Methyl-pseudouridine, 5-methoxyuridine, etc.

[0174] In some embodiments, the payload described herein may include mRNA modified with one or more nucleotides selected from the group consisting of: pseudouridine (abbreviated as the Greek letters "psi" or "ψ"), 5-methylcytosine (m... 5 C), 5-methyluridine (m) 5 U), 2′-O-methyluridine (Um or m) 2′-O U), 2-thiouridine (s) 2 U) and N 6 -Methyladenosine (m 6 A) Replace at least a portion of the corresponding unmodified canonical nucleosides (e.g., replace substantially all of the corresponding unmodified canonical nucleosides A, C, G, or T).

[0175] In some embodiments, the payload described herein may include a ribonucleoprotein complex (e.g., Cas9 / guide RNA) for efficient delivery into cells. As used herein, “ribonucleoprotein complex” or “ribonucleoprotein particle” refers to a complex or particle comprising a nucleoprotein and ribonucleic acid. As used herein, “guide RNA” or “gRNA” refers to a ribonucleotide sequence capable of binding a nucleoprotein to form a ribonucleoprotein complex. In embodiments, the guide RNA comprises one or more RNA molecules. As used herein, “nucleoprotein” refers to a protein capable of binding nucleic acids (e.g., RNA, DNA). When a nucleoprotein binds to ribonucleic acid, it is referred to as a “ribonucleoprotein”. The interaction between the ribonucleoprotein and ribonucleic acid can be direct, such as through covalent bonds, or indirect, such as through non-covalent bonds (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interactions, etc.). Non-limiting examples of ribonucleoproteins include ribosomes, telomeres, RNAseP, hnRNP, CRISPR-associated protein 9 (Cas9), and small nucleus RNPs (snRNP). Ribonucleoproteins can be enzymes. In an embodiment, the ribonucleoprotein is an endonuclease. Therefore, in an embodiment, the ribonucleoprotein complex comprises an endonuclease and ribonucleic acid. In an embodiment, the endonuclease is CRISPR-associated protein 9.

[0176] Nucleic acids can be in the form of antisense molecules, such as "gap-mers" containing an RNA-DNA-RNA structure with activated RNAseH. Nucleic acids can be, for example, DNA or RNA, or RNA-DNA hybrids, and can be oligonucleotides, plasmids, portions of plasmid DNA, pre-condensed DNA, polymerase chain reaction (PCR) products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups, or other forms of nucleic acid molecules. Nucleic acids can be double-stranded RNA molecules used to suppress gene expression through RNA interference. Nucleic acids can be short interfering double-stranded RNA molecules (siRNA). Nucleic acid molecules can also be Stealth molecules. TM RNAi molecules (Invitrogen Corporation / LifeTechnologies Corporation, Carlsbad, CA).

[0177] As used herein, “RNA” refers to ribonucleic acid, which may be naturally occurring or non-natural. For example, RNA may include one or more modified and / or non-naturally occurring components such as bases, nucleosides, nucleotides, or linkers. RNA may include cap structures, chain-terminating nucleosides, stem-loops, polyA sequences, and / or polyadenylation signals. RNA may have a nucleotide sequence encoding a target polypeptide. For example, RNA may be messenger RNA (mRNA). Translation of mRNA encoding a specific polypeptide (e.g., in vivo translation of mRNA within mammalian cells) can produce the encoded polypeptide. RNA may be selected from the non-restrictive group including: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-guide RNA (sgRNA), self-replicating RNA (srRNA), self-amplifying RNA, stRNA, Cas9 mRNA, or combinations thereof.

[0178] The term "amphiphilic peptide" refers to a peptide whose secondary structure places hydrophobic and hydrophilic amino acid residues on different sides of the peptide. Peptides typically employ a helical secondary structure. In some cases, amphiphilic peptides can also function as fusion agents. Examples of amphiphilic peptides suitable for use in the compositions described herein include, but are not limited to, peptides comprising sequences selected from the group consisting of: FEAALAEALAEALA (SEQ ID No.: 10), Ac-LARLLPRLLARL-NHCH3 (SEQ ID No.: 11), GLLEELLELLEELWEELLEG (SEQ ID No.: 12), GWEGLIEGIEGGWEGLIEG (SEQ ID No.: 13), GLFEALAEFIEGGWEGLIEG (SEQ ID No.: 14), GLFEALLELLESLWELLLEA (SEQ ID No.: 15), GGYCLEKWMIVASELKCFGNTA (SEQ ID No.: 16), GGYCLTRWMLIEAELKCFGNTAV (SEQ ID No.: 17), and WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID No.: 18). The amphiphilic peptide may, for example, be optionally covalently linked to a polycationic nucleic acid binding moiety.

[0179] Exosomes, exosome lipids

[0180] The term "exosome" refers to a small membrane vesicle secreted by most cells, containing a cellular payload of proteins, lipids, genetic material, and other biomolecules transported to other cells at different locations in the tissue. Exosomes can be considered liposome particles. Exosomes, or mixtures of lipids obtained therefrom, can be used in combination with other transfection agents or helper lipid mixtures. Exosomes are also known as microvesicles, epididymal bodies, spermatozoa, exosome-like vesicles, microparticles, prostate bodies, dendritic bodies, tumor bodies, archaeosomes, and cancer bodies. Exosomes suitable for use in the compositions and methods described herein also include synthetic exosomes. Non-limiting examples of synthetic exosomes suitable for use in the embodiments described herein are described, for example, by: Li, YJ., Wu, JY., Liu, J. et al. Artificial exosomes for translational nanomedicine. J Nanobiotechnol U.S. Patent No. 19,242 (2021), U.S. Patent No. 11,938,219, U.S. Patent Application No. US2023 / 0181466, etc., are each incorporated herein by reference in their entirety.

[0181] Examples of lipid components isolated from exosomes include, but are not limited to: lysophosphatidylcholine (non-limiting examples: C-18, C-16, C-14 and mixtures thereof), lysophosphatidylcholine (non-limiting examples: C-18, C-16 and C-14), sphingomyelin, ceramide (non-limiting examples: C-8 to C-24), disaturated phosphatidylcholine (non-limiting examples: DSPC, DPPC, DMPC and other Cn (n= 8 – 25)), mixtures of diunsaturated phosphatidylcholine (non-limiting examples: DOPC, DP(db)PC), phosphatidylserine (PS), phosphatidylinositol (PI), disaturated phosphatidylethanolamine (non-limiting examples: DSPE, DPPE, DMPE), mixtures of diunsaturated phosphatidylethanolamine (non-limiting examples: DOPE, DP(db)PE), phosphatidylglycerol (PG) (non-limiting example: C-18). It contains C-22), cholesterol, and diglycerides, such as cardiolipin.

[0182] It is also anticipated that mixtures of any combination of the ionizable lipids, neutral lipids, exosomes, and lipid mixtures isolated from exosomes listed above are also possible.

[0183] The lipid compositions described herein can also be combined with one or more exosomes, or biological materials derived from or purified from exosomes (e.g., lipids, proteins, nucleic acids, etc.).

[0184] Structural lipids

[0185] The lipid component of the lipid nanoparticle composition may include one or more structural lipids. The structural lipids may be selected from, but are not limited to, the group consisting of: sterols, such as cholesterol, codostrum, sitosterol, ergosterol, campesterol, stigmasterol, brassicosterol, tomatine, tomatine glycosides, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes sterols (e.g., cholesterol) and corticosteroids (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0186] stabilizer

[0187] The lipid compositions described herein may also include stabilizers, such as stabilized lipids. Stabilized lipids may be neutral lipids, or they may be charged. Stabilized lipids that may be advantageously used in the formulations described herein include, but are not limited to, polyethylene glycol (PEG) modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amine. Such lipids are also referred to as PEGylated lipids. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. Other stabilized lipids that may be used in the compositions disclosed herein include, for example, polyethylene glycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether copolymers, triblock polyoxyethylene alkyl ether copolymers, and amphiphilic branched polymers. In the implementation scheme, the stabilizer may be polyoxyethylene (20) oleoyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate (“Myrj52”), poly(propylene glycol) 11-block-poly(ethylene glycol) 16-block-poly(propylene glycol) 11, poly(propylene glycol) 12-block-poly(ethylene glycol) 28-block-poly(propylene glycol) 12, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxacyclopentan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-olefin ester), Myrj52 (polyoxyethylene (40) stearate), Brij TM S10 (polyoxyethylene (10) stearyl ether), Brij TM L4 = Polyoxyethylene (4) lauryl ether; BRIJ TM S20 = Polyoxyethylene (20) stearyl ether; Brij TMS35 = Polyoxyethylene (23) lauryl ether; TPGS 1000 = D-α-tocopherol polyethylene glycol 1000 succinate; and equal proportions of Tween 20 / polysorbate 80 / tridecyl-D-maltodextrin and combinations thereof. In some compositions, the stabilizer content is about 0.1-5 mol% of the lipid composition. For example, in some compositions, the stabilizer content is about 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, or any value between these values, of the lipid component of the composition described herein. In other examples, the stabilizer content is about 0.5 mol% to 5 mol% of the lipid component of the composition described herein. In other examples, the stabilizer content is about 0.5 mol% to 4 mol% of the lipid component of the composition described herein. In other examples, the stabilizer content is approximately 0.5 mol% to 3 mol% of the lipid component in the composition described herein. In other examples, the stabilizer content is approximately 0.5 mol% to 2 mol% of the lipid component in the composition described herein. In other examples, the stabilizer content is approximately 0.5 mol% to 1 mol% of the lipid component in the composition described herein. In other examples, the stabilizer content is approximately 1 mol% to 5 mol% of the lipid component in the composition described herein. In other examples, the stabilizer content is approximately 1 mol% to 4 mol% of the lipid component in the composition described herein. In other examples, the stabilizer is present in an amount of approximately 1 mol% to approximately 3 mol% of the lipid component in the composition. In other examples, the stabilizer content is approximately 1 mol% to 2 mol% of the lipid component in the composition described herein.

[0188] Payload complexation :

[0189] This disclosure provides compositions for delivering payloads (including, but not limited to, nucleic acids) to cells. The nucleic acids may be complexed with the exterior of lipid complexes (e.g., liposomes, lipid nanoparticles) described herein. In some embodiments, the composition has about 20% to about 50% of the nucleic acid complexed to the exterior of the lipid complex. In other embodiments, the composition has about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the nucleic acid complexed to the exterior of the lipid complex. The external complexation of the nucleic acids can be measured using methods known in the art, such as Blakney. et al. The method disclosed in (2019)Gene Therapy 26:363-372.

[0190] The compound shown in Formula I :

[0191] Compounds based on the core structure shown in Formula I have been found to be useful for the efficient delivery of macromolecules into eukaryotic cells. These compositions and methods are effective in a variety of cell types and provide high transfection efficiency. These compounds can be advantageously used with one or more neutral lipids and other components, such as fusion molecules or fusion-enhancing molecules, other cationic lipids, cell surface ligands, cell adhesion molecules, amphiphilic peptides, and nuclear localizers, to form complexes with macromolecules. These complexes can be easily prepared using simple methods and are applicable to a variety of cell types.

[0192] In one embodiment, this disclosure provides a compound of formula I:

[0193] ,

[0194] Or its pharmaceutically acceptable salt, wherein:

[0195] A1 is —(CH2) x — or —(CH2) y —A4—(CH2) z —;

[0196] A4 Choose the group consisting of the following items: —(CH2) x —, —(CO)O—, —O(CO)—,

[0197] —SS—、 , , , and ;

[0198] Q1 is N or Q2 is N or ;

[0199] R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups, or —(CH2) 1-7 COOR;

[0200] R3 is —COR, —(CH2) n COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or or

[0201] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0202] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0203] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0204] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0205] C3-C6 cycloalkyl groups, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; or

[0206] C3-C6 cycloalkenyl groups, optionally replaced by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0207] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7- or aryl groups, or

[0208] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups; or

[0209] Only if R1 and R2 are independently -(CH2) 1-7 R3 is H only when COOR is used;

[0210] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0211] R5 and R6 are independently selected from H and CH3;

[0212] R7 is H or an optionally substituted C1-C6 straight-chain or branched alkyl group, or an optionally substituted monounsaturated or polyunsaturated C1-C6 straight-chain or branched alkenyl group. ,or ;

[0213] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, —(CH2) 0-3 Het;

[0214] A2 and A3 independently choose from the following groups: H, , , , , , , , ; , , ;

[0215] R8 is —COR, or ;

[0216] Where R9 is H, or ;

[0217] Where R 10 Choose the group consisting of the following items: H;

[0218] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cycloalkyl substitution;

[0219] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups;

[0220] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces;

[0221] C3-C6 cycloalkylamines, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; and

[0222] C3-C6 cycloalkenylamines, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces;

[0223] Where R 11 Choose from the following groups: -NH2, -NHR, -N(R)2, , and ;

[0224] Where AA represents any natural or non-natural amino acid side chain; and

[0225] a is an integer from 1 to 6;

[0226] b is an integer from 0 to 6;

[0227] x is an integer from 1 to 9;

[0228] y is an integer from 1 to 4;

[0229] z is an integer from 1 to 4;

[0230] n is an integer from 1 to 5;

[0231] n1, n2, n3, n4, and n5 are independently integers from 1 to 5.

[0232] n6 is an integer from 0 to 7.

[0233] n7 and n8 are independent integers from 0 to 5;

[0234] n9 is an integer from 1 to 5;

[0235] m1 is an integer from 1 to 5;

[0236] m2 is an integer from 0 to 5;

[0237] m3 is an integer from 1 to 7;

[0238] p is an integer from 1 to 50; and Het is an optionally substituted 5-7 member monocyclic basic heterocycle or an optionally substituted 8-11 member bicyclic basic heterocycle.

[0239] In some embodiments of the compound shown in Formula I,

[0240] A1 is —(CH2) x — or —(CH2) y —A4—(CH2) z —;

[0241] A4 Choose the group consisting of the following items: —(CH2) x —, —(CO)O—, —O(CO)—,

[0242] —SS—、 , , , and ;

[0243] Q1 is N; Q2 is N;

[0244] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups;

[0245] R3 is —COR, —(CH2) n COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or ;

[0246] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, and optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; where AA represents any natural or non-natural amino acid side chain;

[0247] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het;

[0248] A2 and A3 independently choose from the following groups: H, , , , , , , , , and ;

[0249] Where R 10 Choose the group consisting of the following items: H;

[0250] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cycloalkyl substitution;

[0251] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups;

[0252] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces;

[0253] Where AA represents any natural or non-natural amino acid side chain;

[0254] a is an integer from 1 to 6;

[0255] b is an integer from 0 to 6;

[0256] x is an integer from 1 to 9;

[0257] y is an integer from 1 to 4;

[0258] z is an integer from 1 to 4;

[0259] n is an integer from 1 to 5;

[0260] n1, n2, n3, n4, and n5 are independent integers from 1 to 5;

[0261] n6 is an integer from 0 to 7;

[0262] n7 and n8 are independent integers from 0 to 5;

[0263] n9 is an integer from 1 to 5;

[0264] m1 is an integer from 1 to 5;

[0265] m2 is an integer from 0 to 5;

[0266] m3 is an integer from 0 to 5;

[0267] p is an integer from 1 to 50; and

[0268] Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

[0269] In another embodiment, this disclosure provides a compound having the structure of formula Ia:

[0270]

[0271] Or its pharmaceutically acceptable salt, wherein:

[0272] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, and optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or —(CH2) 1- 7COOR;

[0273] A2 and A3 independently choose from the following groups: , , , , , , and ;

[0274] R3 is —COR, —(CH2) n COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or or

[0275] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0276] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0277] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or

[0278] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0279] C3-C6 cycloalkyl groups, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; or

[0280] C3-C6 cycloalkenyl groups, optionally replaced by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or

[0281] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7- or aryl groups, or

[0282] C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups; or

[0283] Only if R1 and R2 are independently -(CH2) 1-7 R3 is H only when COOR is used;

[0284] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0285] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het;

[0286] Where R 10 Choose the group consisting of the following items: H;

[0287] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) nAryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cycloalkyl substitution;

[0288] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups;

[0289] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces;

[0290] Where AA represents any natural or non-natural amino acid side chain; and

[0291] x is an integer from 1 to 9;

[0292] n is an integer from 1 to 5;

[0293] n5 is an integer from 1 to 5;

[0294] n6 is an integer from 0 to 7;

[0295] n7 and n8 are independent integers from 0 to 5;

[0296] n9 is an integer from 1 to 5;

[0297] m3 is an integer from 1 to 3;

[0298] p is an integer from 1 to 50; and Het is an optionally substituted 5-7 member monocyclic basic heterocycle or an optionally substituted 8-11 member bicyclic basic heterocycle.

[0299] In some embodiments of the compound shown in formula Ia, R3 is —COR or —(CH2). n COOR.

[0300] In some embodiments of the compound shown in Formula Ia, R is selected from the group consisting of: oleyl, oleoyl, linoleyl, linoleoyl, palmyl, palmoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0301] In some embodiments of the compound shown in Formula Ia, n5 is 1, or n7 is 1, or n9 is 1, and these values ​​are independent of each other.

[0302] In some embodiments of the compound shown in formula Ia, x is 4.

[0303] In another embodiment, this disclosure provides a compound having the structural formula Ib:

[0304]

[0305] Or its pharmaceutically acceptable salt, wherein:

[0306] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0307] R3 is —COR, —(CH2) n COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or ;

[0308] A2 and A3 independently choose from the following groups: , , , as well as ;

[0309] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0310] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het;

[0311] Where R 10 Choose the group consisting of the following items: H;

[0312] C1-C 30A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cycloalkyl substitution;

[0313] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups;

[0314] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces;

[0315] C3-C6 cycloalkylamines, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; and

[0316] C3-C6 cycloalkenylamines, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces;

[0317] Where AA represents any natural or non-natural amino acid side chain; and

[0318] y is an integer from 1 to 4;

[0319] z is an integer from 1 to 4;

[0320] n is an integer from 1 to 5;

[0321] n5 is an integer from 1 to 5;

[0322] m2 is an integer from 1 to 5;

[0323] p is an integer from 1 to 50; and

[0324] Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

[0325] In some embodiments of the compound shown in formula Ib, R3 is —COR or —(CH2). n COOR.

[0326] In some embodiments of the compound shown in Formula Ib, R is selected from the group consisting of: oleyl, oleoyl, linoleyl, linoleoyl, palmyl, palmoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0327] In some embodiments of the compound shown in Formula Ib, n5 is 1 or m2 is 2 or n9 is 1, and these values ​​are independent of each other.

[0328] In some embodiments of the compound of formula Ib, y and z are independently integers from 1 to 2.

[0329] In another embodiment, this disclosure provides a compound having the structural formula Ic:

[0330]

[0331] Or its pharmaceutically acceptable salt, wherein:

[0332] R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0333] R3 is —COR, —(CH2) n COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or , or optionally replace C1-C 30 Straight-chain or branched alkyl groups, or optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups;

[0334] A4. Choose the group consisting of the following items: —(CO)O—, —O(CO)—, —SS—. , , and ;

[0335] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0336] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het;

[0337] Where AA represents any natural or non-natural amino acid side chain; and

[0338] y is an integer from 1 to 4;

[0339] z is an integer from 1 to 4;

[0340] n is an integer from 1 to 5;

[0341] n1, n2, n3, and n4 are independent integers from 1 to 5;

[0342] m1 is an integer from 1 to 5;

[0343] p is an integer from 1 to 50; and

[0344] Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

[0345] In some embodiments of the compound shown in formula Ic, R3 is —COR or —(CH2). n COOR.

[0346] In some embodiments of the compound shown in Formula Ic, R is selected from the group consisting of: oleyl, oleoyl, linoleyl, linoleoyl, palmyl, palmoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0347] In some embodiments of the compound of formula Ic, n1, n2, n3 and n4 are independently integers from 1 to 2.

[0348] In some embodiments of the compound of formula Ic, y and z are independently integers from 1 to 2.

[0349] In some embodiments of the compound shown in formula Ic, m1 is an integer from 1 to 2.

[0350] In another embodiment, this disclosure provides a compound having the structural formula Id:

[0351]

[0352] Or its pharmaceutically acceptable salt, wherein:

[0353] A2 and A3 independently choose from the following groups: H, , , , as well as ;

[0354] R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0355] R3 is —COR, —(CH2) n COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or , or optionally replace C1-C 30 Straight-chain or branched alkyl groups, or optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups;

[0356] R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0357] R5 and R6 are independently selected from H and CH3;

[0358] R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het;

[0359] A4. Choose the group consisting of the following items: —(CO)O—, —O(CO)—, —SS—. , , , as well as ;

[0360] Where R 10 Choose the group consisting of the following items: H;

[0361] C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cycloalkyl substitution;

[0362] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups;

[0363] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces;

[0364] C3-C6 cycloalkylamines, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; and

[0365] C3-C6 cycloalkenylamines, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) nSR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces;

[0366] Where AA represents any natural or non-natural amino acid side chain; and

[0367] a is an integer from 1 to 2;

[0368] b is an integer from 0 to 3;

[0369] y and z are independent integers from 1 to 4;

[0370] z is an integer from 1 to 4;

[0371] m1 and m2 are independent integers from 1 to 5;

[0372] n1, n2, n3, n4, and n5 are independent integers from 1 to 5;

[0373] p is an integer from 1 to 50; and

[0374] Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

[0375] In another embodiment, this disclosure provides a compound having the structural formula Ik:

[0376]

[0377] Or its pharmaceutically acceptable salt, wherein:

[0378] A1 is —(CH2) x — or —(CH2) y —S—S—(CH2) z —;

[0379] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0380] Where R 10 Choose from the following groups: H; or

[0381] C1-C30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl; or

[0382] C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups; or

[0383] C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces; and

[0384] a is an integer from 1 to 6;

[0385] b is an integer from 1 to 6;

[0386] x is an integer from 1 to 9;

[0387] y is an integer from 1 to 4;

[0388] z is an integer from 1 to 4;

[0389] n5 is an integer from 1 to 5; and

[0390] Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

[0391] In some embodiments of the compound represented by formula Ik, A1 is...

[0392] —(CH2)4— or —(CH2)2—S—S—(CH2)2—.

[0393] In some embodiments of the compound shown by formula Ik, n5 is 1.

[0394] In some embodiments of the compound of formula Ik, a and b are independently integers from 1 to 3.

[0395] In some embodiments of the compound shown in formula Ik, R 10 Functional groups selected from, but not limited to, the following: H,

[0396]

[0397] ,

[0398]

[0399] .

[0400] In another embodiment, this disclosure provides a compound having the structural formula In:

[0401]

[0402] Or its pharmaceutically acceptable salt, wherein:

[0403] A1 is —(CH2) y —S—S—(CH2) z —;

[0404] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0405] Where AA represents any natural or non-natural amino acid side chain; and

[0406] a is an integer from 1 to 6;

[0407] b is an integer from 1 to 6;

[0408] y is an integer from 1 to 4;

[0409] z is an integer from 1 to 4; and

[0410] n5 is an integer from 1 to 5.

[0411] In some embodiments of the compound represented by formula In, n5 is 1.

[0412] In some embodiments of the In compound, a and b are independently integers from 1 to 3.

[0413] In some embodiments of the compound represented by formula In, AA is a functional group selected from, but not limited to, the following:

[0414] .

[0415] In another embodiment, this disclosure provides a compound having the structural formula Iy:

[0416]

[0417] Or its pharmaceutically acceptable salt, wherein:

[0418] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0419] y is an integer from 1 to 4; and

[0420] z is an integer from 1 to 4;

[0421] In some embodiments of the compound of formula Iy, y and z are independently integers from 1 to 3.

[0422] In another embodiment, this disclosure provides a compound having the structural formula Iz:

[0423]

[0424] Or its pharmaceutically acceptable salt, wherein:

[0425] R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups;

[0426] R3 is an optional substituted C1-C 30 Straight-chain or branched alkyl groups, or optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups;

[0427] y is an integer from 1 to 4; and

[0428] z is an integer from 1 to 4;

[0429] In some embodiments of the compound of formula Iz, y and z are independently integers from 1 to 3.

[0430] In some embodiments of the compound shown in Formula Id, R3 is —COR or —(CH2). n COOR.

[0431] In some embodiments of the compound shown in Formula Id, R is selected from the group consisting of: oleyl, oleoyl, linoleyl, linoleoyl, palmyl, palmoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0432] In some embodiments of the compound shown in Formula Id, a is 1 and b is an integer from 0 to 1.

[0433] In some embodiments of the compound of formula Id, y and z are independently integers from 1 to 2.

[0434] In some embodiments of the compound represented by formula Id, A2 is... ;

[0435] A3 is H;

[0436] A4 is ;

[0437] R1 and R5 are CH3;

[0438] R2 and R6 are H;

[0439] R3 is —COR, —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or ;

[0440] R7 is H;

[0441] b is 0; and its pharmaceutically acceptable salts.

[0442] In some embodiments of the compound of formula Id, A2 and A3 are independently selected from the group consisting of: and ;

[0443] A4 is ;

[0444] R5 and R6 are H;

[0445] R3 is either —COR or —(CH2) n COOR, —(CO)NHR—, —(CO)N(R)2—, or ;

[0446] R7 is H;

[0447] a and b are both 1; and

[0448] Pharmaceutically acceptable salt.

[0449] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (Ig), (Ik), (In), (Io), (II-o-1), or (II-o-2), R1 and R2 are —(CH2). 13 CH3.

[0450] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (Ig), (Ik), (In), (Io), (II-o-1), or (II-o-2), A1 is —(CH2)4— or —(CH2)—A4—(CH2.)—.

[0451] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (In), (Io), (Iv), or (Iz), AA is glycine, histidine, serine, tryptophan, arginine, aspartic acid, or pyrrolidone, tyrosine. Preferably, the AA is histidine.

[0452] In some embodiments of the compounds shown in formula (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain containing an RGD or RYD tripeptide unit.

[0453] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain containing a histidine or RGD or RYD tripeptide unit, wherein the RGD or RYD tripeptide unit is preceded by a spacer group, such as a glycine or serine spacer group (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0454] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain comprising repeating RGD or RYD tripeptide units, each repeating RGD or RYD tripeptide unit being preceded by a spacer group, such as a glycine or serine spacer group (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0455] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain comprising repeating GLF or WYG tripeptide units, said repeating GLF or WYG tripeptide units being preceded by a spacer group, such as a glycine or serine spacer group (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0456] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain comprising repeating poly-Arg or poly-His units, said repeating poly-Arg or poly-His units being preceded by a spacer group, such as a glycine or serine spacer group (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0457] In some embodiments of the compounds shown in formula (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain selected from any peptide chain listed in Table 1.

[0458] In some embodiments of the compounds shown in formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (Io), (Ir), or (Iw), R 7 Neurotransmitter-based functional groups selected from, but not limited to, the following: , , , , , as well as .

[0459] In some embodiments of formula (I), (Ia), (Ib), (Ic), (Id), (Ig), or (II-o-2), R is selected from the group consisting of: oleyl, oleoyl, linoleyl, linoleoyl, palm oil, palmoyl, palmyl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0460] In some embodiments of formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (Iw), or (II-o-2), the 5-7 member monocyclic basic heterocycle is selected from the group consisting of:

[0461] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0462] Some implementation schemes include compounds selected from the group consisting of:

[0463] ,

[0464] And its pharmaceutically acceptable salts.

[0465] Some implementation schemes include compounds selected from the group consisting of:

[0466] ,

[0467] And its pharmaceutically acceptable salts.

[0468] Some implementation schemes include compounds selected from the group consisting of:

[0469] ,

[0470] And its pharmaceutically acceptable salts.

[0471] Some implementation schemes include compounds selected from the group consisting of:

[0472] ,

[0473] And its pharmaceutically acceptable salts.

[0474] Some implementation schemes include compounds selected from the group consisting of:

[0475]

[0476] ,

[0477] And its pharmaceutically acceptable salts.

[0478] Some implementation schemes include compounds selected from the group consisting of:

[0479] ,

[0480] And its pharmaceutically acceptable salts.

[0481] Some implementation schemes include compounds selected from the group consisting of:

[0482]

[0483] in:

[0484] ,

[0485] And its pharmaceutically acceptable salts.

[0486] Some implementation schemes include compounds selected from the group consisting of:

[0487]

[0488] in:

[0489] n6 is either 0 or 1;

[0490] R 10 For H;

[0491]

[0492] ;

[0493]

[0494] And its pharmaceutically acceptable salts.

[0495] Some implementation schemes include compounds selected from the group consisting of:

[0496]

[0497] in:

[0498] ,

[0499] And its pharmaceutically acceptable salts.

[0500] Some implementation schemes include compounds selected from the group consisting of:

[0501]

[0502] in:

[0503] n6 is either 0 or 1;

[0504] R 10 For H;

[0505]

[0506] ,

[0507] And its pharmaceutically acceptable salts.

[0508] Some implementation schemes include compounds selected from the group consisting of:

[0509]

[0510] in:

[0511] R is

[0512] , , , and its pharmaceutically acceptable salts.

[0513] This document provides compositions comprising: (i) one or more compounds of Formula I; (ii) one or more of structural lipids, ionizable lipids, and stabilizers; and (iii) optional payload.

[0514] Compositions comprising: (i) the compound of Formula I; (ii) one or more structural lipids; (iii) one or more stabilizers; and (iv) optional payload.

[0515] Compositions comprising: (i) a compound according to Formula I; (ii) one or more structural lipids; (iii) one or more stabilizers; (iv) one or more transfection enhancers; and (v) optional payload.

[0516] Compositions comprising: (i) one or more compounds of formula I; and (ii) a payload.

[0517] Compositions comprising one or more compounds of Formula I are also provided, in an amount of 10 to 80 mol% (excluding any payload), or as a percentage of the total lipids contained in the composition.

[0518] Compositions comprising one or more compounds of Formula I are also provided, wherein the content of the structural lipids is 14-50 mol% of the composition (excluding any payload) or mol% of the total lipids contained in the composition.

[0519] Compositions comprising one or more compounds of Formula I are also provided, wherein the stabilizer is present in an amount of 0.1-10 mol% of the composition (excluding any payload) or in mol% of the total lipids contained in the composition.

[0520] Compositions comprising one or more compounds of Formula I are also provided, the compositions further comprising exosomes or biological material derived from or purified from exosomes.

[0521] Compositions comprising one or more compounds of Formula I are also provided, the compositions further comprising a polymer.

[0522] Compositions comprising one or more compounds of Formula I are also provided, wherein the polymer is selected from the group consisting of: dense star dendritic polymers, PAMAM dendritic polymers, NH3 core dendritic polymers, ethylenediamine core dendritic polymers, fifth-generation or higher dendritic polymers, dendritic polymers having substituted groups, dendritic polymers containing one or more amino acids, grafted dendritic polymers, activated dendritic polymers, polyethyleneimine (PEI), polyethyleneimine conjugates, polylysine, polyarginine, polyornithine, histones, and any combination thereof.

[0523] For example, the composition may include one or more compounds of Formula I and linear or branched PEI.

[0524] This document provides compositions comprising one or more compounds of Formula I and a stabilizer selected from the group consisting of surfactants, neutral lipids, polymer-coupled lipids, polyethylene glycols, phospholipids, and any combination thereof.

[0525] For example, compositions comprising one or more of Formula I compositions and a stabilizer for PEG-modified lipids are provided.

[0526] This document provides compositions comprising one or more compounds of Formula I and one or more transfection enhancers, such as polycationic nucleic acid binding moieties, or transfection enhancers selected from the group consisting of endosome releasers, cell surface ligands, nuclear localizers, cell-penetrating peptides, fusion peptides, amphiphilic peptides, and any combination thereof.

[0527] This document provides compositions comprising one or more compounds of Formula I and a payload.

[0528] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the payload comprises nucleic acid.

[0529] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the compound contains a charge N and the nucleic acid molecule contains a charge P, wherein, upon contact with a cell, the N / P ratio of the composition formed by the compound and the nucleic acid is about 1 to 20.

[0530] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the payload comprises a nucleic acid, wherein the nucleic acid is RNA.

[0531] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, the composition further comprising a nucleic acid, wherein the nucleic acid is RNA, and the RNA is mRNA, siRNA, shRNA, self-replicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a Dicer substrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.

[0532] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the payload comprises a nucleic acid, wherein the nucleic acid is DNA.

[0533] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the payload further comprises one or more peptides, and optionally comprises nucleic acids.

[0534] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the payload comprises a nucleic acid, wherein the nucleic acid is RNA, and the RNA is mRNA.

[0535] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the payload comprises a nucleic acid, wherein the nucleic acid is RNA, and wherein the RNA encodes an immunogen.

[0536] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, wherein the payload comprises a nucleic acid, wherein the nucleic acid is RNA, and the RNA encodes a cancer antigen.

[0537] In another embodiment, this disclosure provides compositions comprising one or more compounds of formula I, wherein the structural lipid is selected from the group consisting of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, tomatine, ursolic acid, α-tocopherol, hopane compounds, phytosterols, steroids, and any combination thereof.

[0538] In another embodiment, this disclosure provides a composition of a compound of formula I with one or more stabilizers, wherein the stabilizer comprises one or more phospholipids selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-eicosanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0Diether) 1,2-Oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME16.0) The formulations include PE, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In some embodiments, these formulations may also contain one or more lipids derived from the viral capsid, for example, those derived from enveloped viruses.

[0539] In another embodiment, this disclosure provides a composition comprising one or more compounds of Formula I, said composition further comprising a transfection enhancer selected from the group consisting of: endosome releasing agents, cell surface ligands, nuclear localizers, cell-penetrating peptides, fusion peptides, and any combination thereof.

[0540] In another aspect, this disclosure provides compositions of compounds of Formula I with at least one or more neutral lipids, wherein the one or more neutral lipids are selected from DOPE, DPhPE, cholesterol, sterols, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatoamine, ursolic acid, α-tocopherol, DOPC, Lyso-PE (1-acyl-2-hydroxy- sn1-glycerol-3-phosphate ethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycerol-3-phosphate choline), and 3-alkoxy-2-hydroxy-1-acetamidopropane, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), and dimyristoylphosphatidylethanolamine (DM). PE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (transDOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphate choline (DUPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate ethanolamine, or combinations thereof.

[0541] On the other hand, this disclosure provides compositions of a compound of formula I with at least one or more cationic lipids and / or at least one or more neutral lipids, wherein the cationic lipids are selected from GeneIn TM LipofectAmine TM 2000, Lipofect Amine TM Lipofectin ® DMRIE-C, CellFectin® (Invitrogen)、Oligofectamine ® (Invitrogen)、LipofectAce ® (Invitrogen)、Fugene ® (Roche, Basel, Switzerland)、Fugene ® HD(Roche)、Transfectam ® (Tranfectam, Promega, Madison, WI)、Tfx-10 ® (Promega)、Tfx-20 ® (Promega)、Tfx-50 ® (Promega)、Transfectin TM (BioRad, Hercules,CA)、SilentFect TM (Bio-Rad)、Effectene ® (Qiagen, Valencia, CA)、DC-chol(AvantiPolar Lipids)、GenePorter ® (Gene Therapy Systems, San Diego, CA)、DharmaFect 1 ® (Dharmacon, Lafayette, CO)、DharmaFect 2 ® (Dharmacon)、DharmaFect 3 ® (Dharmacon)、DharmaFect 4 ® (Dharmacon)、Escort TM III(Sigma, St. Louis, MO)、Escort TMIV (Sigma), DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxy-propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-(3-amino-2- Hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)-propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)-propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine), 3,5-(N,N-di-lysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine), L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide disalt L-Lysine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine, L-Lysine-bis-(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl]piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4,-bis[(3-amino-2-hydroxyethyl ... [(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitoylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-myristoylamino]-butane-2,3-diol, 1,4-bis[(3-oleyl-amino)propyl]-piperazine, L-arginine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, bis[(3-(3-aminopropyl)-myristoylamino)2-hydroxypropyl]piperazine, L-arginine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-serine-bis-(O,O'-oleoyl-β-hydroxyethyl)-amide dihydrochloride, 1,4-Bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesterol-3β-carboxy-amide vinyltrimethyl-ammonium iodide, 1,4-bis[(3-myristoylamino)propyl]piperazine, 1-dimethylamino-3-trimethyl-amino-DL-2-propyl-cholesterol carboxylate iodide, cholesterol-3β-carboxyamide vinyl -amine, cholesterol-3β-oxysuccinamide-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylaminonium-DL-2-propyl-cholesterol-3β-oxysuccinate iodide, 2-[(2-trimethylaminonium)-ethylmethyl-amino]ethyl-cholesterol-3β-oxysuccinate iodide, 3β[N-(N',N'-dimethylamino-ethane)-carbamoyl]cholesterol and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitoylamino)propyl]piperazine, L-guanyloylglycyl-N-(1-heptadecyloctadecyl)glycamide, N, 2 N 5 -bis(3-aminopropyl)-L-guanyloylglycyl-N-(1-heptadecyloctadecyl)glycamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-octadecyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-octadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleenylamino)2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-octadecyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethyl-ethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-octadecyl-L-glutamine]-L-glutamic acid, N 2 -[N2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-diolenyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-diolenyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristoylamino)-2-hydroxy-propyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-diolenyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-diolenoyl-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-olenoylamino)propyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-glutamine, N 2 -[N 2 N 5 -bis(amino-propyl)-L-ornithine]-NN-dipalmitoyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)-carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dipalmitoyl-L-glutamine]-L-glutamic acid, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-guanyl]-N,N-dimyristic-L-glutamine, N 2 -[N 2 N 5 [-bis(amino-propyl)-L-ornithine]-NN-dimyristoyl-L-α-glutamine, N2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristoyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoyl-amino)-2-hydroxypropyl]-piperazine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-guanylo-NN-dimyristoyl-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristoylamino)propyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dilauroyl-L-glutamine, N 2 -[N 2 N 5 [-bis(amino-propyl)-L-ornithine]-NN-dilauroyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauroyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)-carbonyl]-N 2 N 5-Bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-guaninyl-NN-dilauroyl-L-glutamine]-L-glutamic acid, 3-[N',N"-bis(2-tert-butoxycarbonyl-amino-ethyl)guanidinyl]-N,N-octadecano-9-enyl-propionamide, 3-[N',N"-bis(2-tert-butoxy-carbonylamino-ethyl)guanidinyl]-N,N-dipalmitoyl-propionamide, 3-[N',N"-bis(2-tert-butyl-oxycarbonylamino-ethyl)guanidinyl]-N,N-dimyristoyl-propionamide, 1,4-bis[(3-(3-amino-propyl)palmitoylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxy-propionyl] [(3-(3-amino-2-hydroxypropyl)-myristoylamino)-propyl]piperazine, N,N-(2-hydroxy-3-amino-propyl)-N-2-hydroxypropyl-3-N,N-diolenoylaminopropane, N,N-(2-hydroxy-3-amino-propyl)-N-2-hydroxypropyl-3-N,N-dipalmitoylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristoylaminopropane, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristoylamino)-propyl]piperazine, [(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-olenoyloxyethyl)]methylammonium bromide [(3-aminopropyl)-bis-(2-palmitoyloxyethyl)]methylammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-decanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-aminopropyl)-›4-(3-aminopropylamino)-4-tetradecyl-carbamoyl-butylcarbamate cholesterol ester, (3-aminopropyl)-›4-(3-aminopropylamino)-4-tetradecyl-carbamoyl-butylcarbamate cholesterol ester, (3-aminopropyl)-›4-(3-aminopropylamino)- (3-Aminopropyl)-4-(3-Aminopropylamino)-4-(2-Dimethylamino-ethylcarbamoyl)-butylcarbamoyl cholesterol ester, Spermine-5-carboxyglycine (N'-stearoyl-N'-oleyl)amide tetratrifluoroacetate, Spermine-5-carboxyglycine (N'-stearoyl-N'-transoleyl)amide tetratrifluoroacetate, Guanidylbutylaminocarboxycholesterol acetate, Spermine-5-carboxy-β-alanine cholesterol ester tetratrifluoroacetate, 2,6-Diaminohexanoyl β-alanine cholesterol ester ditrifluoroacetate, 2,4-Diaminobutyryl β-alanine cholesterol ester ditrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesterol ester trifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxylic acid cholesterol ester, stearoyl carnitine ester, palmitoyl carnitine ester, myristoyl carnitine ester, stearoylstearoyl carnitine ester chloride salt, L-stearoylstearoyl carnitine ester, stearoyloleoyl carnitine ester chloride, palmitoyl palmitoyl carnitine ester chloride, myristoyl myristoyl carnitine ester chloride, L-myristoyl myristoyl carnitine ester... Alkali ester chloride, 1,4-bis[(3-(3-amino-2-hydroxy-propyl)-palmitoyl-amino)-propyl]-piperazine, N-(3-aminopropyl)-N,N'-bis-(dodecyloxyethyl)-piperazine bromide, N-(3-amino-propyl)-N,N'-bis-(oleyloxyethyl)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(palmitoyloxyethyl)-piperazine bromide, N-(3-aminopropyl)-N, N'-bis-(myristoyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleenyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmitoyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmitoyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl) )-N'-methyl-N,N'-(bis-2-myristoyloxyethyl)-piperazine bromide, 1,4-bis[(3-(3-amino-propyl)-oleenylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-amino-propyl)-myristoylamino)-2-hydroxy-propyl]piperazine or 1,4-bis[(3-(3-amino-propyl)-palmitoylamino)-2-hydroxy-propyl]piperazine, 2,3-dioleenoxy-1,4-N,N, ’ -dimethyl-N,N ’ -Di(2-hydroxy-3-aminopropyl)-diamino-butane, 2,3-dipalmitoyloxy-1,4-N,N ’ -dimethyl-N,N ’ -Di(2-hydroxy-3-amino-propyl)-diamino-butane, 2,3-dimyristoleenoxy-1,4-N,N ’ -dimethyl-N,N ’ -Di(2-hydroxy-3-aminopropyl)-diamino-butane, 2,3-dioleenooxy-1,4-N,NN,N ’ -dimethyl-N,N ’ -Di(3-aminopropyl)-diaminobutane, 2,3-dipalmitoylenoxy-1,4-N,N ’ -dimethyl-N,N ’-Di(3-aminopropyl)-diaminobutane, 2,3-dimyristoleenoyl-1,4-N,N ’ -dimethyl-N,N ’ -Di(3-aminopropyl)-diaminobutane, 2,3-dioleenooxy-1,4-N,N ’ -dimethyl-N,N ’ -Di(5-carboxamido-spermine)-diamino-butane, 2,3-dipalmitoyloxy-1,4-N,N ’ -dimethyl-N,N ’ -Di(5-carboxamidospermine)-diaminobutane, 2,3-dimyristoleenoyl-1,4-N,N ’ -dimethyl-N,N ’ -Di(5-carboxamidospermine)-diaminobutane, 2,3-dioleenooxy-1,4-N,N ’ -dimethyl-N,N ’ -Di(lysyl)-diaminobutane, 2,3-dipalmitoyl-1,4-N,N ’ -dimethyl-N,N ’ -Di(lysyl)-diamino-butane, 2,3-dimyristoleenoyl-1,4-N,N ’ -dimethyl-N,N ’ -Di(lysyl)-diaminobutane, 2,3-dioleenooxy-1,4-N,N ’ -dimethyl-N,N ’ -Di(histyl)-diaminobutane, 2,3-dipalmitoyl-1,4-N,N ’ -dimethyl-N,N ’ -Di(histyl)-diaminobutane, 2,3-dimyristoleenoxy-1,4-N,N ’ -dimethyl-N,N ’ -Di(histyl)-diaminobutane, 2,3-dioleenooxy-N,N ’ -Dimethyl-1,4-diaminobutane, 2,3-dipalmitoyloxy-N,N ’ -Dimethyl-1,4-diaminobutane, 2,3-dimyristyl-oleenoyl-N,N ’ -Dimethyl-1,4-diaminobutane; PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, polyethyleneimine (PEI), and polyethyleneimine conjugates.

[0542] On the other hand, this disclosure provides compositions of compounds of Formula I with at least one or more cationic lipids and / or at least one or more neutral lipids, wherein the one or more neutral lipids are selected from DOPE, DPhPE, cholesterol, sterols, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatoamine, ursolic acid, α-tocopherol, DOPC, Lyso-PE (1-acyl-2-hydroxy- sn 1-glycerol-3-phosphate ethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycerol-3-phosphate choline), and 3-alkoxy-2-hydroxy-1-acetamidopropane, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidyl-3-phosphate Acylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (trans-DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphate choline (DUPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate ethanolamine, or combinations thereof.

[0543] In another embodiment, this disclosure provides compositions of the compound of Formula I with at least one or more cationic lipids and / or at least one or more neutral lipids and / or at least one or more PEG-modified lipids, wherein the PEG-modified lipids are selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-ceramide conjugate, PEG-modified 1,2-diacoxypropyl-3-amine, or any combination thereof.

[0544] In another embodiment, this disclosure provides compositions of compounds of Formula I, wherein the one or more PEG-modified lipids are selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and any combination thereof.

[0545] In another embodiment, this disclosure provides a composition of the compound of Formula I with at least one or more cationic lipids and / or at least one or more neutral lipids, wherein the alkoxy group is selected from oleyl, palmyl, palmitoleoyl, myristoleoyl, myristyl, and lauryl.

[0546] In another embodiment, this disclosure provides a composition of the compound of Formula I with one or more polyamine transfection agents.

[0547] In another embodiment, this disclosure provides a composition of the compound of Formula I with one or more fusion agents.

[0548] In another embodiment, this disclosure provides a composition of the compound of Formula I with one or more amphiphilic peptides.

[0549] In another aspect, this disclosure provides compositions of compounds of Formula I with one or more amphiphilic peptides, wherein the one or more amphiphilic peptides act as fusion agents.

[0550] In another embodiment, this disclosure provides a method for delivering a payload to a cell, the method comprising: (i) providing a composition comprising a compound of formula I; (ii) providing a cell; and (iii) contacting the cell with the composition. In some embodiments, the payload comprises a nucleic acid encoding a therapeutic protein, such as an antibody, growth factor, cytokine, enzyme, etc.

[0551] In another embodiment, this disclosure provides a method for introducing nucleic acids, proteins, or peptides into eukaryotic cells, the method comprising contacting the cells with a composition of any of the disclosed compositions, thereby introducing the nucleic acids, proteins, or peptides into the cells.

[0552] In another embodiment, this disclosure provides a method for introducing nucleic acids, proteins, or peptides into eukaryotic cells, wherein the cells are human cells, the method comprising contacting the cells with a composition of any of the disclosed compositions to introduce the nucleic acids, proteins, or peptides into the cells.

[0553] In another embodiment, this disclosure provides a method for introducing nucleic acids, proteins, or peptides into eukaryotic cells, wherein the cells are mammalian cells, the method comprising contacting the cells with a composition of any of the disclosed compositions to introduce the nucleic acids, proteins, or peptides into the cells.

[0554] In another embodiment, this disclosure provides a method of delivering a composition to a subject, the method comprising administering the composition to the subject according to a compound of Formula I.

[0555] In another embodiment, this disclosure provides a method for delivering a payload to a cell, wherein contact with the cell is in vitro It was carried out.

[0556] In another embodiment, this disclosure provides a method for delivering a payload to a cell, wherein contact with the cell is in vivo It was carried out.

[0557] In another embodiment, this disclosure provides a method for delivering a payload to a cell, wherein contact with the cell is In vitro It was carried out.

[0558] In another embodiment, this disclosure provides a method of administering any of the disclosed compositions to a subject, wherein the administration is systemic.

[0559] In another embodiment, this disclosure provides a method of administering any of the disclosed compositions to a subject, wherein the administration is selected from the group consisting of: subcutaneous administration, intramuscular administration, intranasal administration, intratumoral administration, administration to the brain, administration to the spinal cord, administration to the eye, administration to the subject's lymph nodes, and any combination thereof.

[0560] In another embodiment, this disclosure provides a kit comprising a compound of formula I, and one or more structural lipids, ionizable lipids, and stabilizers.

[0561] In another embodiment, this disclosure provides a kit comprising a compound of formula I, and one or more structural lipids, and / or one or more stabilizers, and / or optional payloads.

[0562] In another embodiment, this disclosure provides a kit comprising a compound of Formula I, and one or more structural lipids, and / or one or more stabilizers, and / or one or more fusion agents, and / or optional payloads.

[0563] In another embodiment, this disclosure provides a method for inhibiting protein expression in cells, the method comprising contacting the cells with an RNAi molecule and a compound of Formula I as described herein or any of the disclosed compositions.

[0564] Those skilled in the art will recognize that, although the compounds of the present invention are shown herein in their electrically neutral (unprotonated) form for convenience, these compounds may also be present in partially or fully protonated forms in solutions of appropriate pH, and unless otherwise expressly stated, the present invention covers compounds in all protonated, unprotonated, ionized and non-ionized forms without limitation.

[0565] Preparation method :

[0566] The compounds disclosed herein can be synthesized by the methods described below or by modifications thereof. Modifications to these methods include, among others, temperatures, solvents, reagents, etc., known to those skilled in the art. Typically, in any process of preparing the compounds disclosed herein, it may be necessary and / or required to protect any sensitive or reactive groups on the relevant molecules. This can be achieved by conventional protecting groups, such as those described in *Protective Groups in Organic Chemistry* (JFW McOmie, ed., Plenum Press, 1973) and *Protecting Groups in Organic Synthesis* by PGMGreen and TW Wutts (3rd edition, Wiley, New York (1999)), both of which are incorporated herein by reference in their entirety. Protecting groups can be removed at a subsequent convenient stage using methods known in the art. Synthetic chemical transformations that can be used to synthesize suitable compounds are known in the art and include, for example, those described in *Comprehensive Organic Transformations* by R. Larock (VCH Publishers, 1989) or *Encyclopedia of Reagents for Organic Synthesis* edited by L. Paquette (John Wiley and Sons, 1995), both of which are incorporated herein by reference in their entirety. The routes shown and illustrated herein are merely illustrative and are neither intended nor intended to limit the scope of the claims in any way. Those skilled in the art will be able to identify modifications to the disclosed synthetic methods and to devise alternative routes based on the disclosure herein; all such modifications and alternative routes are within the scope of the claims.

[0567] In the following schemes, the protecting group of the oxygen atom is selected based on its compatibility with the necessary synthetic steps, as well as the compatibility of the introduction and deprotection steps with the overall synthetic scheme (Protecting Groups in Organic Synthesis, PGM Green and TW Wutts, 3rd edition, Wiley, New York (1999)).

[0568] If the compounds of this technique contain one or more chiral centers, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or d(l) stereoisomers, or as mixtures rich in stereoisomers. Unless otherwise stated, all such stereoisomers (and their enriched mixtures) are included within the scope of this technique. Pure stereoisomers (or enriched mixtures) can be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, etc.

[0569] The starting materials for the following reactions are typically known compounds, or can be prepared using known procedures or obvious modifications thereof. For example, many starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka-Chemce, or Sigma (St. Louis, Missouri, USA). Other starting materials can be prepared by procedures described in standard reference texts, such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March's Advanced Organic Chemistry (John Wiley, and Sons, 5th ed., 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0570] The following example schemes are for reference only and collectively represent exemplary methods for preparing the compounds described herein. Furthermore, other methods for preparing the compounds described herein will be readily apparent to those skilled in the art based on the following reaction schemes and examples. Unless otherwise stated, all variables are as defined above.

[0571] In one embodiment, as shown in Scheme I, the method involves treating intermediate Ie with a BOC-anhydride in the presence of a base to obtain a bis-BOC derivative Ih, followed by treatment with an acyl chloride II-h to obtain intermediate If. This is then subjected to BOC-removal in the presence of an acid to obtain a compound of formula In.

[0572] Option I

[0573]

[0574] In another embodiment, as shown in Scheme II, the bis-BOC derivative Ih is treated with methanesulfonyl chloride in the presence of a base to produce the bis-methanesulfonate intermediate Ij. The intermediate Ij is then treated with an amine represented by formula II-k in the presence of a base, followed by BOC-removal in the presence of an acid to produce the compound shown in formula Ik.

[0575] Option II

[0576]

[0577] In one embodiment, for example, compounds 8-12 and 38-92 are synthesized according to the reaction conditions shown in Scheme II.

[0578]

[0579] .

[0580]

[0581] .

[0582] In another embodiment, as shown in Scheme III, the bis-BOC derivative Ih is treated with a BOC-protected amino acid (II-m) in the presence of a suitable base and coupling agent to obtain derivative Im, which is then subjected to BOC-removal in the presence of an acid to produce a compound of formula In.

[0583] Option III

[0584]

[0585] In one embodiment, for example, compounds 13-21 are synthesized according to the reaction conditions shown in Scheme III.

[0586]

[0587] In another embodiment, as shown in Scheme IV, the bis-BOC derivative Ih is subjected to alkylation of the hydroxyl group with the alkylating agent R3-X (II-j; X is a leaving group), followed by BOC-removal in the presence of an acid to produce the compound shown in Formula Io.

[0588] Option IV

[0589]

[0590] In one embodiment, for example, compounds 22-23 are synthesized according to the reaction conditions shown in Scheme IV.

[0591]

[0592]

[0593] In another embodiment, as shown in Scheme V, isoindoline-1,3-dione is condensed with 3-chloro-2-(chloromethyl)prop-1-ene to obtain the adduct 2-(2-(chloromethyl)allyl)isoindoline-1,3-dione, which is then treated with diamine Ix to obtain intermediate Ip. Intermediate Ip is then treated with hydrazine in ethanol, followed by treatment with (BOC)₂O to obtain derivative Iq. Derivative Iq is then treated with amine II-k, followed by BOC-removal in the presence of an acid to produce a compound of formula Ir.

[0594] Option V

[0595]

[0596] In one embodiment, for example, compound 24 is synthesized according to the reaction conditions shown in scheme V, wherein the reaction conditions are as follows: N 1 , N 4 -Di(tetradecyl)butane-1,4-diamine is coupled as amine Ix with 2-(2-(chloromethyl)allyl)isoindoline-1,3-dione, and 2-aminoethane-1-ol is coupled as amine Ik with the corresponding intermediate Iq.

[0597]

[0598] In another embodiment, as shown in Scheme VI, a solid-bound aralkyl chloride II-a is reacted with an aminothiol II-b to produce a solid-bound amine II-c. Amine II-c is then reductively amination in the presence of an aldehyde II-n to produce intermediate II-d, which is subsequently treated with 2-(oxepane-2-ylmethyl)isoindoline-1,3-dione in the presence of a base and heated in a suitable solvent to produce adduct II-e. This adduct II-e is then released from the solid phase and subsequently oxidized to produce disulfide Is. Disulfide Is is then deprotected with hydrazine in the presence of ethanol to produce a compound of formula Ix-1.

[0599] Solution VI

[0600]

[0601] In another embodiment, as shown in Scheme VII, disulfide II-f is reductively amination with aldehyde II-n to produce intermediate II-g, which is then treated with 2-(oxepane-2-ylmethyl)isoindoline-1,3-dione in the presence of a base and heated in a suitable solvent to produce adduct It. Adduct It is then treated with hydrazine in ethanol, followed by treatment with (BOC)₂O to obtain derivative Iu.

[0602] In another embodiment, as shown in Scheme VII, the bis-BOC derivative Iu is treated with a BOC-protected amino acid (II-m) in the presence of a suitable base and coupling agent, and the resulting product is then subjected to BOC-removal in the presence of an acid to produce the compound shown in Formula Iv.

[0603] In another embodiment, as shown in Scheme VII, the bis-BOC derivative Iu is treated with methanesulfonyl chloride in the presence of a base to produce a bis-methanesulfonate intermediate, which is then treated with an amine represented by formula II-k in the presence of a base, followed by BOC-removal in the presence of an acid to produce a compound shown in formula Iw.

[0604] Option VII

[0605]

[0606]

[0607] In one embodiment, for example, compounds 25-37 are synthesized according to the reaction conditions shown in Scheme VII, wherein 2,2'-dithiodi(ethane-1-amine) is used as the diamine II-f and tetradecanoal is used as the aldehyde II-n to produce the corresponding intermediate II-g. The corresponding intermediate II-g is converted to produce intermediate Iu. This intermediate is then further converted to compound 25-37 using the reaction shown in Scheme VII.

[0608]

[0609]

[0610]

[0611]

[0612]

[0613] The following example schemes are provided for reference and represent preferred methods for preparing the compounds illustrated herein. These methods are not limiting, and other routes may obviously be used to prepare these compounds. Such methods specifically include solid-phase based chemical methods, including combinatorial chemistry. Given the existing literature and the content of this disclosure, those skilled in the art are fully capable of preparing these compounds by those methods. The compound numbers used in the synthetic schemes shown below are only for these specific schemes and should not be construed as or confused with the same numbers used in other sections of this application.

[0614] Examples of compounds shown in Formula I can be synthesized using methods well known in the art, such as compound 1 shown in scheme IA below.

[0615] Solution IA

[0616]

[0617]

[0618] As shown in scheme IA, DHDMS was treated with BOC-anhydride in the presence of a base to obtain a bis-BOC derivative (1A). This intermediate (1A) was treated with oleyl chloride to obtain intermediate 1B. This intermediate was then treated with TFA to obtain a bis-oleyl derivative 1. Other analogs have been synthesized using various acyl chlorides using a similar method.

[0619] Similarly, compounds 2-4 were also synthesized using the method described above.

[0620]

[0621]

[0622]

[0623] Scheme VIII

[0624]

[0625] In another embodiment, as shown in scheme VIII, diamine Ix is treated with epoxide II-o in a polar solvent in the presence of a base and heated to produce the compound shown in formula II-o-1. Compound II-o-1 is treated with acyl chloride II-h, followed by acid treatment to form a salt of the compound shown in formula II-o-2.

[0626] Compounds 5-7 were synthesized using the scheme shown in scheme VIIIA below.

[0627] Scheme VIIIA

[0628]

[0629]

[0630] As shown in scheme VIIIA, make N 1 , N 4 1,4-Diamine (7A) was reacted with 2-ethyloxetine in 2,2,2-trifluoroethanol at 80 °C in the presence of DIPEA to obtain compound 7. In the next step, compound 7 was treated with oleyl chloride to obtain intermediate 6A. This intermediate was then treated with HCl to obtain bis-oleyl derivative 6 as a hydrochloride. Similarly, compound 5 was also synthesized using the method described above by treating compound 7 with linoleyl chloride.

[0631]

[0632] Compound 8-12 was synthesized using the scheme shown in Scheme IIA below.

[0633] Option IIA

[0634]

[0635]

[0636] As shown in Scheme IIA, the bis-BOC derivative was treated with methanesulfonyl chloride in the presence of DIPEA to produce bis-methanesulfonate intermediate 8A. Intermediate 8A was treated with 2-aminoethanol in the presence of a base to produce derivative 8B, which was then treated with TFA to produce compound 8. Other analogs were synthesized using a similar method by substituting various amines for the methanesulfonate group in intermediate 8A.

[0637] Similarly, compounds 9-12, 39-41, 43, 46 and 80 were synthesized using 4-aminobut-1-ol (for compound 9), 3-aminopropane-1,2-diol (for compound 10), 4-(aminomethyl)phenol (for compound 11), 2-aminoethane-1-thiol (for compound 12), 3-aminopropane-1-ol (for compound 39), 5-aminopentane-1-ol (for compound 40), 6-aminohexane-1-ol (for compound 41), 2-amino-3-phenylpropane-1-ol (for compound 43), 2-amino-2-methylpropane-1-ol (for compound 46), 2-amino-2-methylpropane-1,3-diol (for compound 48) and 1-aminopropane-2-ol (for compound 80) in accordance with the method described above.

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644] Solution IX

[0645]

[0646]

[0647] In another embodiment, as shown in Scheme IX, diamine II-u is reacted under reductive amination conditions using aldehyde II-q and NaBH(OAc)3 to produce intermediate II-r. Those skilled in the art will understand that many other reductive amination conditions and reagents are available within the scope of this disclosure for this reaction. The resulting product II-r is then treated with 2-(oxecyclopropane-2-ylmethyl)isoindoline-1,3-dione in the presence of a base, followed by treatment with hydrazine in ethanol to produce product II-s. Intermediate II-s is then treated with (BOC)2O, followed by treatment with 2,2-dimethoxypropane in the presence of a Lewis acid (e.g., BF3·Et2O) to obtain derivative II-s-1. Intermediate II-s-1 is then subjected to an indium-catalyzed transesterification reaction with alcohol II-p, followed by removal of the acetal group and BOC under acid-catalyzed conditions to produce the compound shown in formula II-t. Those skilled in the art will understand that many other transesterification conditions, as well as conditions for the removal of acetal groups and BOC, can be used to carry out these reactions within the scope of this disclosure.

[0648] In one embodiment, for example, the compound shown below was synthesized according to the reaction conditions shown in Scheme IX.

[0649]

[0650]

[0651]

[0652] Lipid nanoparticle formulation :

[0653] The lipid of formula (I) can be bound to a nucleic acid and / or protein payload to produce a lipid nanoparticle formulation. For example, the lipid of formula (I) can be bound to a payload selected from one or more of the following: a siRNA, a miRNA, an mRNA, a shRNA, an autoamplifying RNA, an oRNA (or a non-naturally occurring circular RNA), an antisense oligonucleotide (ASO), a gRNA, a ribonucleoprotein (e.g., a CRISPR complex), a dsDNA, a plasmid DNA, etc.

[0654] In the case of nucleic acid delivery, the amount of nucleic acid (e.g., mRNA, self-amplified RNA, etc.) in the lipid nanoparticle formulation may depend on the size, sequence, and other properties of the nucleic acid. The amount of nucleic acid in the lipid nanoparticle formulation may also depend on the size, composition, desired target, and other properties of the lipid nanoparticle formulation. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. In some embodiments, the weight ratio of the lipid component to the nucleic acid (e.g., mRNA) in the nanoparticle composition may be from about 5:1 to about 50:1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the weight ratio of the lipid component to the nucleic acid (e.g., mRNA) may be from about 10:1 to about 40:1. For example, the amount of nucleic acid in a nanoparticle composition can be determined using absorption spectroscopy ( ). For example It is measured by ultraviolet-visible (UV-vis) spectroscopy.

[0655] The lipid nanoparticle formulation may contain nucleic acids at concentrations from about 0.1 mg / ml to 2 mg / ml, such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml, or greater than 2.0 mg / ml.

[0656] Preferably, the one or more nucleic acids ( For example,The mRNA, lipids, and their amounts are selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of the number of nitrogen atoms in one or more lipids to the number of phosphate groups in the nucleic acid (e.g., mRNA). Generally, a lower N:P ratio is preferred. One or more mRNAs, lipids, and their amounts can be selected to provide an N:P ratio of about 2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In some embodiments, the N:P ratio may be about 2:1 to about 5:1. In a preferred embodiment, the N:P ratio may be about 4:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

[0657] Other lipid components :

[0658] Advantageously, in addition to the lipids shown in formula (I), lipid nanoparticle formulations also contain one or more auxiliary lipids, most preferably neutral auxiliary lipids, but those skilled in the art will recognize that other lipids, including cationic / ionizable lipids, may also be used. However, some formulations only include a combination of lipids of formula (I) and nucleic acids.

[0659] The ionizable lipids described herein refer to lipids having at least one protonable or deprotonable group. In some embodiments, the ionizable lipids may be positively charged at or below a physiological pH (e.g., pH 7.4) and neutral at a second pH (e.g., at or above a physiological pH). For example, when incorporated into lipid nanoparticles, the pKa of the protonable group of the ionizable lipids described herein may be in the range of about 4 to about 11, for example, about 4 to about 7. For example Between approximately 5 and 7, such as between approximately 5.5 and 6.9.

[0660] Therefore, in addition to lipids of formula (I), lipid nanoparticle formulations may also include neutral lipids, such as phospholipids. Exemplary phospholipids that may be used in the lipid nanoparticle formulations described herein include, but are not limited to, phospholipids used in the compositions disclosed herein, including but not limited to DOPE, DPhPE, DOPC, Lyso-PE (1-acyl-2-hydroxy- sn4-(N-maleimide methyl)-cyclohexane-1-carboxylic acid (CMA), Lyso-PC (1-acyl-3-hydroxy-sn-glycerol-3-phosphocholine), distearate phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimide methyl)-cyclohexane-1-carboxylic acid The phospholipids used in the compositions described herein may be, for example, from about 5 mol% to about 20 mol% of the total lipid nanoparticle formulation. Advantageously, the phospholipid content ranges from about 1 mol% to 40 mol%, for example, from about 1 mol% to about 25 mol%. Preferably, the phospholipid content in the lipid nanoparticle formulation disclosed herein is at least about 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 12 mol%, 14 mol%, 16 mol%, 18 mol%, or 20 mol%, or any amount between any of the above values, of the total lipid nanoparticle formulation.

[0661] The lipid nanoparticle formulations described herein may also advantageously include other neutral lipids, such as sterols or lipids containing a sterol moiety (“sterol derivatives”). As defined herein, “sterol” is a subgroup of steroids composed of steroid alcohols. Exemplary sterols and lipids containing a sterol moiety used in the lipid nanoparticle formulations described herein include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicosterol, tomatine, tomatine, ursolic acid, α-tocopherol, humulanes, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. Some lipid nanoparticle formulations described herein include sterols or sterol derivatives. The content of sterols or sterol derivatives in the entire lipid nanoparticle formulation is approximately 5-60 mol% of the total. Advantageously, the content of sterols or sterol derivatives is approximately 15-50 mol%, for example, 25-40 mol%. Preferably, the amount of sterols (such as cholesterol) or sterol derivatives in the lipid compositions disclosed herein is at least about 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, or 60 mol% of the total lipid formulation. Some of the lipid nanoparticle formulations described in this article do not include sterols or sterol derivatives.

[0662] The lipid nanoparticle formulations described herein may also include stabilizers such as surfactants, neutral lipids, polymerically conjugated lipids, polyethylene glycol, phospholipids, and any combinations thereof. Examples of nonionic stabilizers include: polysorbates (Tweens), Brij... TM S20 (polyoxyethylene (20) stearyl ether), Brij TM 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij TM S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether) and Myrj TM52 (polyoxyethylene (40) stearate). Other exemplary stabilizers used in the embodiments described herein include TPGS 1000 (D-α-tocopherol polyethylene glycol 1000 succinate); or a mixture of Tween 20 / polysorbate 80 / tridecyl-D-maltodextrin (referred to as Lipid H in Table 15) in equal proportions. Furthermore, other stabilizing lipids that may be advantageously used in the formulations described herein include, but are not limited to, polyethylene glycol (PEG) modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amine. Such lipids are also referred to as PEGylated lipids. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. Other stable lipids that can be used in the compositions disclosed herein include, for example, polyethylene glycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether copolymers, triblock polyoxyethylene alkyl ether copolymers, and amphiphilic branched polymers. In the implementation scheme, the stabilizer may be polyoxyethylene (20) oleoyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate (“Myrj52”), poly(propylene glycol) 11-block-poly(ethylene glycol) 16-block-poly(propylene glycol) 11, poly(propylene glycol) 12-block-poly(ethylene glycol) 28-block-poly(propylene glycol) 12, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxacyclopentan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-olefin ester), Myrj52 (polyoxyethylene (40) stearate), Brij TM S10 (polyoxyethylene (10) stearyl ether), Brij TM L4 = polyoxyethylene (4) lauryl ether; BRIJ TM S20 = Polyoxyethylene (20) stearyl ether; Brij TMS35 = Polyoxyethylene (23) lauryl ether; TPGS 1000 = D-α-tocopherol polyethylene glycol 1000 succinate; and equal proportions of Tween 20 / polysorbate 80 / tridecyl-D-maltodextrin and combinations thereof. In some compositions, the stabilizer content is approximately 0.1-5 mol% of the lipid nanoparticle formulation. For example, in some compositions, the stabilizer content is approximately 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, or any value between these values.

[0663] In addition to the lipids shown in formula (I), lipid nanoparticle formulations may also contain one or more cationic / ionizable lipids. In some embodiments, at least one auxiliary lipid may be selected from, for example, the group consisting of: DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmit ...di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di-di- N-1-(2,3-dioleoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine), 3,5-(N,N-dilysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl- β-hydroxyethylamine), L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, L-lysine-bis(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl]piperazine, L-lysine-bis(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine, L-ornithine-bis(O,O'-palmitoyl- β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-amino-2-hydroxypropyl)-oleoylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-palmitoylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-myristoylamino]-butane-2,3-diol, 1,4-bis[(3-oleoylamino)propyl]piperazine, L-arginine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, bis[(3-(3-aminopropyl)-myristoylamino)-2-hydroxypropyl]piperazine, L-arginine-bis(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-serine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochlorideO'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, glycine-bis(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesterol-3β-carboxy-amidoethyltrimethylammonium iodide, 1,4-bis[(3-myristoylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol carboxylate iodide, choline Sterol-3β-carboxamide ethylamine, cholesterol-3β-oxosuccinamide ethyltrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol-3β-oxosuccinate iodide, 2-[(2-trimethylammonium)-ethylmethylamino]ethyl-cholesterol-3β-oxosuccinate iodide, 3β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol, 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitoylamino)propyl]piperazine, L-ornithine glycyl-N-(1-heptadecyloctadecyl)glycamide, N, 2 N 5 -Bis(3-aminopropyl)-L-guanyloylglycyl-N-(1-heptadecyloctadecyl)glycamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-distearatel-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-distearatel-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-distearyl-L-α-asparagine, N-[N 2 -[N 2 N 5 bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-guanylo-N,N-distearatel-L-glutamine]-L-glutamic acid, N 2 -[N 2 N 5-bis(3-aminopropyl)-L-ornithine]-N,N-dioleoyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-dioleoyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristoylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-dioleoyl-L-α-asparagine, N-[N 2 -[N 2 N 5 bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-N,N-dioleoyl-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleoylamino)propyl]piperazine, N 2 -[N 2 N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-N,N-dipalmitoyl-L-glutamine]-L-glutamic acid, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-guanyl]-N,N-dimyristic-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-guanyl]-N,N-dimyristic-L-α-glutamine, N 2 -[N 2 N 5-bis(aminopropyl)-L-ornithine]-N,N-dimyristoyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-guanylo-N,N-dimyristoyl-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristoylamino)propyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dilauroyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-dilauroyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-N,N-dilauroyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-guanyl-N,N-dilauroyl-L-glutamine]-L-glutamic acid, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidinyl]-N,N-di(octadecyl-9-enyl)propionamide, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidinyl]-N,N-dipalmitoylpropionamide, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidinyl]-N,N-dimyristoylpropionamide, 1,4-bis[(3-(3-aminopropyl)palmitoylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino)propyl] Piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dioleoylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitoylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristoylaminopropane, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristoylamino)propyl]piperazine, [(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleoyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmitoyloxyethyl)]methylammonium bromide Methylammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-decanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-amino-propyl)-›4-(3-amino-propylamino)-4-tetradecylcarbamoyl-butylcarbamoyl cholesterol ester, (3-amino-propyl)-›4-(3-amino-propylamino)-4-carbamoyl-butylcarbamoyl cholesterol ester, (3-amino-propyl)- ›4-(3-aminopropylamino)-4-(2-dimethylamino-ethylcarbamoyl)-butylcarbamate cholesterol ester, spermine-5-carboxyglycine (N'-stearoyl-N'-oleyl)amide tetratrifluoroacetate, spermine-5-carboxyglycine (N'-stearoyl-N'-transoleyl)amide tetratrifluoroacetate, guanidine-butylaminocarboxycholesterol acetate, spermine-5-carboxy-β-alanine cholesterol ester tetratrifluoroacetate, 2,6-diaminohexanoyl β-alanine cholesterol ester ditrifluoroacetate, 2,4-diaminobutyroyl β-alanine cholesterol ester ditrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesterol ester tritrifluoroacetate, [N,[N-bis(2-hydroxyethyl)-2-aminoethyl]carbamoyl cholesterol ester, stearoyl carnitine ester, palmitoyl carnitine ester, myristoyl carnitine ester, stearoylstearoyl carnitine ester chloride, L-stearoylstearoyl carnitine ester, stearoyloleoyl carnitine ester chloride, palmitoylpalmitoyl carnitine ester chloride, myristoylmyristoyl carnitine ester chloride, L-myristoylmyristoyl carnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoylamino)propyl]piperazine, N-(3- N-(3-aminopropyl)-N,N'-bis(dodecyloxyethyl)-piperazine ammonium bromide, N-(3-aminopropyl)-N,N'-bis(oleoyloxyethyl)-piperazine ammonium bromide, N-(3-aminopropyl)-N,N'-bis(palmitoyloxyethyl)-piperazine ammonium bromide, N-(3-aminopropyl)-N,N'-bis(myristoyloxyethyl)-piperazine ammonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazine ammonium bromide, N-(3-aminopropyl)- N'-Methyl-N,N'-(bis-2-oleoyloxyethyl)-piperazine ammonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmitoyloxyethyl)-piperazine ammonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristoyloxyethyl)-piperazine ammonium bromide, 1,4-bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristoylamino)-2- [Hydroxypropyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, KL22, KL25, 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecyl-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) Or MC3), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA) (2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,[12Z)-Octadeca-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLinDMA (2S)).

[0664] Preferably, the lipid represented by formula (I), or a combination of the lipid represented by formula (I) and one or more cationic / ionizable lipids, comprises approximately 5-80 mol% (excluding the payload) in the lipid nanoparticle formulation, for example, approximately 5-80 mol% of the lipid component of the lipid nanoparticle formulation. For example, in some lipid nanoparticle formulations, the content of the compound represented by formula (I), or a combination of the compound represented by formula (I) and one or more other cationic / ionizable lipids, is less than 50 mol%. In other lipid nanoparticle formulations, the content of the compound represented by formula (I), or a combination of the compound represented by formula (I) and one or more other cationic / ionizable lipids, is greater than 50 mol.

[0665] Therefore, some lipid nanoparticle formulations comprise lipids of formula (I), or combinations of lipids of formula (I) with one or more cationic / ionizable lipids, wherein the content of cationic / ionizable lipids is 15-80 mol%, the content of sterols is 20-60 mol%, the content of stabilizers is 0.5-5 mol%, and the content of phospholipids is 1-40 mol% (all based on the total mol% of the lipid nanoparticle formulation). An exemplary lipid nanoparticle formulation may comprise about 20-60 mol% of lipids of formula (I) or combinations of lipids of formula (I) with one or more other cationic / ionizable lipids, about 5-25 mol% of phospholipids, about 25-55 mol% of sterols or sterol derivatives; and about 0.5-15 mol% of stabilizers. Another exemplary lipid nanoparticle formulation comprises about 50 mol% of the lipid of formula (I) or a combination of the lipid of formula (I) and one or more cationic / ionizable lipids, about 1.5 mol% of a stabilizer, about 38.5 mol% of a sterol or sterol derivative, and about 10 mol% of phospholipids. Another exemplary lipid nanoparticle formulation comprises about 55% of the lipid of formula (I) or a combination of the lipid of formula (I) and one or more cationic / ionizable lipids, about 2.5 mol% of a stabilizer, about 32.5 mol% of a sterol or sterol derivative, and about 10 mol% of phospholipids.

[0666] Polyamine components :

[0667] Other formulations may also contain one or more polyamine transfection agents, such as dense star dendrimers, PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, 5th generation or higher dendrimers, dendrimers with substituted groups, dendrimers including one or more amino acids, grafted dendrimers, activated dendrimers, polyethyleneimine (PEI) and / or polyethyleneimine conjugates.

[0668] Transfection enhancer :

[0669] Other formulations may contain transfection enhancers, such as fusion agents (e.g., endosome releasers), cell surface ligands, and / or nuclear localizers (e.g., nuclear receptor ligand peptides). Examples of transfection enhancers include, but are not limited to, reovirus-related fusion peptides (see WO07 / 130073, which is incorporated herein by reference in its entirety), insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell-targeting antibodies, lactoferrin, fibronectin, adenovirus pentagonal basement membrane, Knob, hexagonal protein, vesicular stomatitis virus glycoprotein, Semliki Forest virus core protein, influenza hemagglutinin, hepatitis B core protein, HIV Tat protein, herpes simplex virus VP22 protein, histones, arginine-rich cell permeability proteins, high-mobility group proteins, invasion proteins, internalization proteins, endotoxins, diphtheria toxin, Shigella toxin, melitrix venom peptides, Xenopus antimicrobial peptides, bacitracin, cephalosporin, defensins, proteoglycans, etc. Limulin, thionine, indoleglycoside, bacitracinolone, Drosophila extract, bee antimicrobial peptide, cardilidine, bactericidal permeability-increasing protein, nisin, bufotoxin and fragments thereof. Other cell-penetrating peptides used in the compositions described herein include those provided in Table 1 below:

[0670] Table 1

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702] The lipid nanoparticle compositions described herein can also be combined with one or more exosomes, or biomaterials derived from or purified from exosomes (e.g., lipids, proteins, nucleic acids, etc.).

[0703] Exemplary compositions may include, for example: lipids of formula (I) and one or more exosomes; lipids of formula (I), one or more exosomes, and one or more neutral lipids; lipids of formula (I), one or more exosomes, one or more neutral lipids, and one or more stabilizers; lipids of formula (I), one or more exosomes, one or more neutral lipids, optionally one or more stabilizers, and optionally one or more cell-penetrating peptides.

[0704] Other exemplary compositions include, for example: lipids of formula (I), and one or more biomaterials derived from or purified from exosomes; lipids of formula (I), one or more biomaterials derived from or purified from exosomes, and one or more neutral lipids; lipids of formula (I), one or more biomaterials derived from or purified from exosomes, one or more neutral lipids, and one or more stabilizers; lipids of formula (I), one or more biomaterials derived from or purified from exosomes, one or more neutral lipids, optionally one or more stabilizers, and optionally one or more cell-penetrating peptides.

[0705] Preparation and application of the compound of formula I for transfection :

[0706] The liposomes described above can be prepared by various methods for transfection. One of the simplest preparation methods is reverse evaporation (as described in U.S. Patent No. 9,259,475, which is incorporated herein by reference in its entirety). Other available preparation methods include sonication and microfluidics. Advantageously, the lipids can be prepared into lipid nanoparticles (e.g., Roces) using microfluidic mixing techniques. et al., Pharmaceutics (As described in , 12:1095 (2020)). Suitable microfluidic mixing devices are available, for example, from Precision Nanosystems (Vancouver, BC). Typically, microfluidic mixing combines two fluid streams, one containing nucleic acids and the other containing lipids and other components as shown in formula (I), such as peptides, ligands and other lipid components described below.

[0707] For the lipid nanoparticle composition containing RNA, a stock solution was prepared by diluting a 0.1 mg / ml deionized aqueous solution of RNA with 50 mM sodium citrate buffer at a pH between 3 and 4. The nanoparticle composition can be treated by dialysis to remove ethanol and achieve buffer exchange. Dialysis against phosphate-buffered saline (PBS, pH 7.4) can be performed using the desired molecular weight cutoff (e.g., 10 kD). The resulting nanoparticle suspension can be filtered through a 0.2 μm sterile filter (Sarstedt, Numbrecht, Germany) into a glass vial and sealed.

[0708] Methods for determining the particle size in nanoparticle formulations are well known in the art. For example, the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) can be used to determine the particle size, polydispersity index (PDI), and zeta potential of nanoparticle compositions. Ultraviolet-visible spectrophotometry can be used to determine the concentration of the payload (e.g., nucleic acids such as mRNA) in a nanoparticle composition. A certain amount of the composition is diluted in a suitable solvent, and the absorption spectrum of the solution is recorded on a spectrophotometer, for example, between 230 nm and 330 nm. The concentration of the therapeutic and / or preventative agent in the nanoparticle composition can be calculated based on the extinction coefficient of the therapeutic and / or preventative agent used in the composition, and the difference between the absorbance at a wavelength (e.g., 260 nm) and a baseline value at a wavelength (e.g., 330 nm).

[0709] For nanoparticle compositions containing RNA, QUANT-IT can be used. TM RIBOGREEN ® The RNA detection kit (Invitrogen Corporation, Carlsbad, CA) was used to evaluate the encapsulation efficiency of the nanoparticle composition for RNA, following the manufacturer's instructions. The fluorescence intensity generated after adding the RIBOGREEN reagent was measured using a fluorescence plate reader at, for example, an excitation wavelength of approximately 480 nm and an emission wavelength of approximately 520 nm. The fluorescence value of the reagent blank was subtracted from the fluorescence value of each sample, and the percentage of free RNA was determined by dividing the fluorescence intensity of the intact sample (without Triton X-100) by the fluorescence value of the damaged sample (caused by the addition of Triton X-100).

[0710] The lipids shown in formula (I) can be prepared using one or more auxiliary lipids (most advantageously neutral auxiliary lipids), but those skilled in the art will understand that other lipids, including the cationic lipids described above, can also be used. Other neutral lipids include, for example (but not limited to), DPhPE, cholesterol, DOPC, Lyso-PE (1-acyl-2-hydroxy- sn 1-glycerol-3-phosphate ethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycerol-3-phosphate choline) and / or 3-alkoxy-2-hydroxy-1-acetamidopropane can be used in place of DOPE or in combination with it.

[0711] The lipids represented by formula (I) can be prepared from one or more ionizable / cationic lipids and / or one or more neutral lipids. Exemplary ionizable / cationic lipids that can be used in the compositions provided herein include, but are not limited to, GeneIn. TM LipofectAmine TM 2000, Lipofect Amine TM , Lipofectin®, DMRIE-C, CellFectin® (Invitrogen), Oligofectamine® (Invitrogen), LipofectAce® (Invitrogen), Fugene® (Roche, Basel, Switzerland), Fugene® HD (Roche), Transfectam® (Tranfectam, Promega, Madison, WI), Tfx-10® (Promega), Tfx-20® (Promega), Tfx-50® (Promega), Transfectin TM (BioRad, Hercules, CA), SilentFect TM DharmaFect 4®(Dharmacon)、Escort TMIII(Sigma, St. Louis, MO)、Escort TMIV (Sigma), DOTMA, DOTAP, DMRIE, DC-cholesterol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-(3-amino) -2-hydroxy-propoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethyl-amine, 3,5-(N,N-di-lysyl)-diamino-benzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethyl-amine), L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl) L-Lysine-bis-(O,O'-palmitoyl-β-hydroxyethyl)-amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine, L-Lysine-bis-(O,O'-myristoyl-β-hydroxyethyl)-amide dihydrochloride, L-ornithine-bis-(O,O'-myristoyl-β-hydroxyethyl)-amide dihydrochloride, L-ornithine-bis-(O,O'-oleoyl-β-hydroxyethyl)-amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]-piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)-amide dihydrochloride, 1,4,-Bis[(3-amino-2-hydroxypropyl)-oleoylamino]-butane-2,3-diol, 1,4,-Bis[(3-amino-2-hydroxypropyl)-palmitoylamino]-butane-2,3-diol, 1,4,-Bis[(3-amino-2-hydroxypropyl)-myristoyl-amino]-butane-2,3-diol, 1,4-bis[(3-oleoylamino)propyl]piperazine, L-arginine-bis-(O,O'-oleoyl-β-hydroxyethyl)-amide dihydrochloride, bis[(3-(3-aminopropyl)-myristoylamino)2-hydroxypropyl]piperazine, L-arginine-bis-(O,O'-palmitoyl-β-hydroxyethyl)-amide dihydrochloride, L-serine-bis-(O,O'-oloyl-β-hydroxyethyl)-amide dihydrochloride,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesterol-3β-carboxy-amidinyl ethylidene-trimethylammonium iodide, 1,4-bis[(3-myristoyl-amino)propyl]-piperazine, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol carboxylic acid ester iodide Cholesteryl-3β-carboxy-amidinyl ethylamine, cholesterol-3β-oxysuccinamide-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesteryl-3β-oxysuccinate iodide, 2-[(2-trimethylammonium)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinate iodide, 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitoylamino)propyl]piperazine, L-ornithine glycyl-N-(1-heptadecyl-octadecyl)glycamide, N, 2 N 5 -bis(3-aminopropyl)-L-guanyloylglycyl-N-(1-heptadecyl-octadecyl)glycamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-octadecyl-L-glutamine,N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-octadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleoamino)2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-octadecyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5-bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithine-NN-octadecyl-L-glutamine]-L-glutamic acid, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dioleoyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-diole-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristoylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-diole-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)-carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dioleo-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleoamino)propyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-glutamine,N 2 -[N 2 N 5 -bis(amino-propyl)-L-ornithine]-NN-dipalmitoyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dipalmitoyl-L-glutamine]-L-glutamic acid, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-guanyl]-N,N-dimyristic-L-glutamine, N 2-[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristoyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(amino-propyl)-L-ornithine]-NN-dimyristoyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoyl-amino)-2-hydroxypropyl]piperazine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-guanylo-NN-dimyristoyl-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristoylamino)propyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dilauroyl-L-glutamine, N 2 -[N 2 N 5 [-bis(amino-propyl)-L-ornithine]-NN-dilauroyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauroyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-guaninyl-NN-dilauroyl-L-glutamine]-L-glutamic acid, 3-[N',N"-bis(2-tert-butoxycarbonyl-amino-ethyl)guanidinyl]-N,N-octadecano-9-enyl-propionamide, 3-[N',N"-bis(2-tert-butoxy-carbonylaminoethyl)guanidinyl]-N,N-dipalmitoyl-propionamide, 3-[N',N"-bis(2-tert-butoxy-carbonylaminoethyl)guanidinyl]-N,N-dimyristoyl-propionamide, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleoylamino) [(3-(3-amino-2-hydroxypropyl)-N-2-hydroxypropyl-3-N,N-dioleoyl-aminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitoyl-aminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristoyl-aminopropane, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristoylamino)-propyl]-piperazine, [(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleoyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleoyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-( 2-Palmyloxyethyl)]methylammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-aminopropyl)-›4-(3-aminopropylamino)-4-tetradecylcarbamoyl-butylcarbamate cholesterol ester, (3-aminopropyl)-›4-(3-aminopropyl-amino-4- ...-aminopropyl-4-carbamoyl-butylcarbamate cholesterol ester, (3-aminopropyl-aminopropyl-4-carbamoyl-butylcarbamate cholesterol ester, (3-aminopropyl-aminopropyl-4-carbamoyl-butylcarbamate cholesterol ester, (3-aminopropyl-4-carbamoyl-2-hydroxy-3-dimethylaminopropyl- (-propyl)-›4-(3-amino-propyl-amino)-4-(2-dimethylamino-ethylcarbamoyl)-butylcarbamate cholesterol ester, spermine-5-carboxyglycine (N'-stearoyl-N'-oleyl)amide tetratrifluoroacetate, spermine-5-carboxyglycine (N'-stearoyl-N'-transoleyl)amide tetratrifluoroacetate, guanidine-butylaminocarboxylic acid cholesterol acetate, spermine-5-carboxy-β-alanine cholesterol ester tetratrifluoroacetate, 2,6-diaminohexanoyl β-alanine cholesterol ester ditrifluoroacetate, 2,4-diamino-butyryl β-alanine cholesterol ester ditrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesterol ester tritrifluoroacetate, [N,[N-bis(2-hydroxy-ethyl)-2-aminoethyl]aminocarboxylic acid cholesterol ester, stearoylcarnitine ester, palmitoylcarnitine ester, myristoylcarnitine ester, stearoylstearoylcarnitine ester chloride salt, L-stearoylstearoylcarnitine ester, stearoyloleoylcarnitine ester chloride, palmitoylpalmitoylcarnitine ester chloride, myristoylmyristoylcarnitine ester chloride, L-myristoylmyristoylcarnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoylamino)propyl]piperazine, N-(3-amino-propyl)-N,N'-bis-(dodecyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N,N'-bis-(oleyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl) -N,N'-bis-(palmitoyloxy-ethyl)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(myristoyloxy-ethyl)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxy-ethyl)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleyloxy-ethyl)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmitoyloxy-ethyl)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristoyloxy-ethyl)-piperazine bromide, 1,4-bis[(3-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristoyloxy-ethyl)-piperazine bromide, [(3-(3-aminopropyl)-myristoylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristoylamino)-2-hydroxy-propyl]piperazine or 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxy-propyl]piperazine, 2,3-dioleoyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dimyristoyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-Dioleoyl-oxy-1,4-N,N'-dimethyl-N,N'-di(3-aminopropyl)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(3-aminopropyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(3-aminopropyl)-diaminobutane, 2,3-dioleoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamide spermine)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamide spermine)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N'-Dimethyl-N,N'-Di(5-carboxamide spermine)-diaminobutane, 2,3-dioleoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dioleoyloxy-1,4-N,N'-dimethyl-N,N'-dimethyl-N N'-Di(histyl)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(histyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(histyl)-diaminobutane, 2,3-dioleoyloxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dipalmitoyloxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dimyristoleoyloxy-N,N, ’ -Dimethyl-1,4-diaminobutane; PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, polyethyleneimine, and polyethyleneimine couplings.

[0712] Other formulations may contain transfection enhancers, such as fusion agents, cell surface ligands, and / or nuclear localizers (e.g., nuclear receptor ligand peptides). Examples of transfection enhancers that can be used in the embodiments described herein include, but are not limited to, reovirus-related fusion peptides, insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell-targeting antibodies, lactoferrin, fibronectin, adenovirus pentavalent base, Knob, hexavalent protein, vesicular stomatitis virus glycoprotein, Semliki forest virus core protein, influenza hemagglutinin, hepatitis B core protein, HIV Tat protein, herpes simplex virus VP22 protein, histones, arginine-rich cell permeability proteins, high-mobility group proteins, invasion proteins, internalization proteins, endotoxins, diphtheria toxin, Shigella toxin, melione, Xenopus antimicrobial peptides, bacitracin, cephalosporin, defensins, proteoglycans, horseshoe crab, thionine, indoleglycosides, bacitracin, Drosophila melione, bee antimicrobial peptides, cardilidine, bactericidal permeability-increasing proteins, nisin, bufotoxin and fragments thereof.

[0713] The compositions described herein may advantageously include, for example, one or more peptide sequences that enhance transfection efficiency, such as linkers, spacers, or nuclear targeting sequences. The peptides described herein may be included alone in the compositions described herein (i.e., not covalently linked to other molecules), or may be covalently linked to one or more molecules in the compositions described herein (e.g., covalently linked to ionizable lipids or other lipids described herein, covalently linked to other transfection enhancers described herein, or covalently linked to the payload described herein, etc.). In some embodiments, the covalent linking can be via an indirector. The terms “spacer” or “linker” as used herein (which are used interchangeably herein) refer to the chemical structure that links two molecules together. In some embodiments, the spacer is attached to each molecule at a different portion of the spacer molecule. In other embodiments, the spacer is a hydrophilic portion and comprises about 6 to 30 carbon atoms. In other embodiments, the spacer comprises a polyether, such as -CH2-O-(CH2-CH2-O-)iCH2-. In other embodiments, the spacer comprises a hydrophilic polymer, such as [(gly)i(ser)j]k (sequence identifier: 585). In these formulas, i ranges from 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20. In some embodiments, the spacer is a peptide of the sequence APYKAWK (sequence identifier: 505). In other embodiments, the spacer is a sequence that is degraded in vivo by peptidase.

[0714] method :

[0715] The compositions described herein can be used to introduce payloads into cells using methods known in the art, wherein the components of the transfection complex are mixed in different orders before being added to the cell culture. Typically, a liposome component of the lipid (with or without accessory lipids) is prepared first, and then mixed with the payload (e.g., a nucleic acid such as DNA or RNA) to prepare the transfection complex. The complex is then added to the cell culture, and transfection is monitored using well-known methods. Other components, such as cell surface ligands, fusion agents, and nuclear localizers, may be added to the nucleic acid before mixing it with the liposome; alternatively, these components may be added to the liposome before the nucleic acid is added.

[0716] Cells that can be transfected using these methods include, but are not limited to, virtually any eukaryotic cell, including primary cells, cultured cells, passaged cell cultures or cell lines, and cells in cultured tissues. Suitable cells include human cell lines and animal cell lines. The cells may be fibroblasts. Cells may be attached cells or cells in suspension (suspension cells). In some illustrative respects, the cells are suspension CHO-S cells and suspension 293-F cells. Other cell types that can be used include, but are not limited to, 293, 293-S, CHO, Cos, 3T3, HeLa, primary fibroblasts, A549, Be2C, SW480, CHOK1, Griptite 293, HepG2, Jurkat, LNCaP, MCF-7, NIH-3T3, PC12, C6, Caco-2, COS-7, HL60, HT-1080, IMR-90, K-562, SK-BR3, PHP1, HUVEC, MJ90, NHFF, NDFF, and primary neurons.

[0717] These formulations are used in a method of protein production, the method comprising contacting cells with a lipid-nucleic acid complex as described above, wherein the nucleic acid encodes the protein. The cells are incubated to produce the protein and the protein is collected. Cells suitable for protein production have been described above. Furthermore, any composition containing the lipids shown in Formula I can be used for cell transfection. Such compositions are discussed further herein and include, but are not limited to, compositions containing the lipids shown in Formula I, auxiliary lipids, and optional transfection enhancers such as fusion peptides or proteins.

[0718] Lipids prepared into lipid nanoparticle (LNP) formulations were screened, and the RNA payload of the complex was used to... in vivo Functional testing was performed. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the protein encoded by the payload. The lipids prepared in this manner were used for transfection. Transfection was performed using a formulation described below containing compound 1, which is a lipid of formula (I). These results are shown in Figures 1 through 5.

[0719] Reagent test kit :

[0720] The components of the transfection composition described above may be provided in the form of a kit. The kit contains the lipids shown in Formula (I), as well as other components such as neutral lipids, cationic lipids, cell surface ligands, fusion agents, amphiphilic peptides, and / or nuclear localizers. Kit components may be individual or may be premixed in any manner. For example, the lipids shown in Formula I may be mixed with one or more neutral lipids. Other components may also be present in the same container or in one or more separate containers. Kits typically include containers, such as vials and / or tubes, which are packaged together, for example, in a cardboard box. Kits may be shipped from the supplier to the consumer. For example, in one example, this document provides a kit comprising vials containing the liposome formulation described above, along with optional transfection agents and transfection-enhancing peptides. The kit may also include, for example, a separate container containing a transfection enhancer, such as a transfection-enhancing peptide, such as PlusReagent. TM (Invitrogen Corp., Carlsbad, CA). The kit may also contain cells, cell culture medium, and reporter nucleic acid sequences, such as plasmids expressing reporter genes, in separate containers. In some examples, the culture medium may be serum-reduced medium and / or protein expression medium.

[0721] In one embodiment, the kit comprises ionizable lipids (such as lipids represented by Formula I) and individual portions or mixtures of peptides, proteins or fragments thereof, or modified peptides, proteins or fragments thereof. In another embodiment, the kit comprises a multicationic polymer and individual portions or mixtures of peptides, proteins or fragments thereof, or modified peptides, proteins or fragments thereof. The cationic lipid transfection kit may optionally contain neutral lipids and other transfection enhancers or other additives, and the relative amounts of components in the kit may be adjusted to facilitate the preparation of the transfection composition. Kit components may include suitable media or solvents for other kit components.

[0722] Payloads that can be delivered by the methods of this invention include nucleic acids, proteins, ribonucleoproteins, etc., including DNA and RNA of any size (including RNAi / siRNA) from any source, containing natural or non-natural bases, and including payloads that encode and are capable of expressing therapeutic or other useful proteins in cells, payloads that inhibit undesirable expression of nucleic acids in cells, payloads that inhibit undesirable enzyme activity or activate desired enzymes, payloads that catalyze reactions (ribozymes), and payloads that play a role in diagnostic analysis (e.g., diagnostic nucleic acids). Therapeutic nucleic acids include those that encode or are capable of expressing therapeutically useful proteins, peptides, or polypeptides in cells, those that inhibit undesirable nucleic acid expression in cells, and those that inhibit undesirable enzyme activity in cells or activate desired enzymes. In some embodiments, the payload includes an RNA molecule. These compositions can be used to deliver RNA payloads, such as mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, or combinations thereof. In some embodiments, the RNA molecule comprises multiple RNA molecules, for example, multiple mRNAs. In view of the disclosure herein, the compositions and methods provided herein can also be readily applied to the introduction of bioactive macromolecules other than nucleic acids (including polyamines, polyamino acids, polypeptides, and proteins) into eukaryotic cells. Other useful materials, such as therapeutic agents, diagnostic materials, and research reagents, can be bound to peptides and modified peptides and introduced into eukaryotic cells by the methods of the present invention.

[0723] The compositions described herein can be delivered to cells via in vivo administration. For in vivo For administration, the pharmaceutical composition is preferably administered parenterally (e.g., intra-articular, intravenous, intraperitoneal, subcutaneous, intrathecal, intradermal, intratracheal, intraosseous, intramuscular, or intratumoral). In certain embodiments, the pharmaceutical composition is administered via intravenous, intrathecal, or intraperitoneal bolus injection. Other routes of administration include local (skin, eyes, mucous membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal administration.

[0724] Typical applications include using established methods to deliver siRNA into cells for use in... in vitro and in vivo Knockdown or silencing of specific cellular targets. Furthermore, its applications include delivering DNA or mRNA sequences encoding polypeptides with therapeutic value. In this way, hereditary diseases can be treated by supplementing missing or insufficient gene products. The method of the present invention can be used in… In vitro, in vitro or in vivo Implementation. For example, the compositions described in this invention can also be used to deliver payloads using methods known to those skilled in the art. in vivoCells. In another example, the composition described in this invention can be used to deliver a payload to... In vitro The patient's cell samples were then returned to the patient.

[0725] for in vivo For administration, the pharmaceutical composition is preferably administered parenterally (e.g., intra-articular, intravenous, intraperitoneal, subcutaneous, intrathecal, intradermal, intratracheal, intraosseous, intramuscular, or intratumoral). In certain embodiments, the compositions described herein are administered intravenously, intrathecally, or intraperitoneally by bolus injection. Other routes of administration include local (skin, eyes, mucous membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.

[0726] for In vitro The preferred application involves applying the composition described herein to a biological sample that has been removed from an organism, followed by washing the cells and reinjecting them into the organism. The organism can be a mammal, particularly a mammal (e.g., a primate), such as a human. This process is used for cell reprogramming, gene repair, immunotherapy, and the like.

[0727] In one embodiment, the present invention provides a method for regulating the expression of a target polynucleotide or polypeptide. These methods typically involve contacting cells with a composition of the present invention associated with a payload (e.g., a nucleic acid) capable of regulating the expression of the target polynucleotide or polypeptide. As used herein, the term "regulation" refers to altering the expression of a target polynucleotide or polypeptide. Regulation can mean increasing or enhancing, or decreasing or reducing.

[0728] In related implementations, this document provides a method for treating a disease or condition characterized by overexpression of a polypeptide in a subject, comprising providing the subject with the composition described herein, wherein the composition comprises a payload selected from siRNA, microRNA, antisense oligonucleotide, and plasmids capable of expressing siRNA, microRNA, or antisense oligonucleotide, wherein the siRNA, microRNA, or antisense RNA comprises a polynucleotide or its complement that specifically binds to the polypeptide.

[0729] In related implementations, this document provides methods for treating diseases or conditions characterized by insufficient peptide expression in a subject. These methods may include providing a subject with the compositions described herein, wherein the compositions comprise a payload selected from mRNA, self-amplifying RNA (SAM), self-replicating DNA, or plasmids, and contain a nucleic acid therapeutic agent that specifically encodes or expresses an insufficiently expressed peptide or its complement.

[0730] In one embodiment, the compounds, compositions, methods, and uses described herein are for delivering a bioactive agent to hepatocytes (e.g., hepatocytes). In one embodiment, the compounds, compositions, methods, and uses of the present invention are for delivering a bioactive agent to a tumor or tumor cells (e.g., primary tumors or metastatic cancer cells). In another embodiment, the compounds, compositions, methods, and uses are for delivering a bioactive agent to skin fat, muscle, and lymph nodes (subcutaneous administration).

[0731] To deliver the bioactive agent to the liver or hepatocytes, in one embodiment, the composition of the present invention is brought into contact with the patient's liver or hepatocytes via parenteral administration (e.g., intravenous, intramuscular, subcutaneous) or local administration (e.g., direct injection, portal vein injection, catheter insertion, stent placement) to facilitate delivery. To deliver the bioactive agent to the kidneys or renal cells, in one embodiment, the composition of the present invention is brought into contact with the patient's kidneys or renal cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous) or local administration (e.g., direct injection, catheter insertion, stent placement) to facilitate delivery. To deliver the bioactive agent to a tumor or tumor cells, in one embodiment, the composition of the present invention is brought into contact with the patient's tumor or tumor cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous) or local administration (e.g., direct injection, catheter insertion, stent placement) to facilitate delivery.

[0732] To deliver a payload to the central nervous system or central nervous system cells, in one embodiment, the composition described herein may be brought into contact with the patient's central nervous system or central nervous system cells (e.g., brain cells and / or spinal cord cells) via parenteral administration (e.g., intravenous, intramuscular, subcutaneous) or local administration (e.g., direct injection, catheter insertion, stent placement, osmotic pump administration (e.g., intrathecal or intraventricular)) to facilitate delivery. To deliver a payload to the peripheral nervous system (PNS) or PNS cells, the composition described herein may be brought into contact with the patient's PNS or PNS cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous) or local administration (e.g., direct injection) to facilitate delivery. To deliver a payload to the lungs or lung cells, the composition described herein may be brought into contact with the patient's lungs or lung cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous) or local administration (e.g., direct administration to lung tissue and cells) to facilitate delivery. In some embodiments, the composition comprises lipid molecules functionalized with neurotransmitter-based functional groups, thereby enabling payload delivery to the brain across the blood-brain barrier (BBB).

[0733] To deliver the payload to the vascular system or vascular cells, the compositions described herein may be brought into contact with the patient's vascular system or vascular cells via parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration) or local administration (e.g., clamping, catheter insertion, stent placement) to facilitate delivery.

[0734] To deliver a payload to the skin or skin cells (e.g., dermal cells and / or hair follicle cells), the compositions described herein may be brought into contact with the patient's skin or skin cells (e.g., dermal cells and / or hair follicle cells) via parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration) or topical administration (e.g., direct application to the skin, iontophoresis) to facilitate delivery. To deliver a payload to the eye or ocular cells (e.g., macula, fovea, cornea, retina), in one embodiment, the compositions of the invention are brought into contact with the patient's eye or ocular cells (e.g., macula, fovea, cornea, retina) via parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration) or topical administration (e.g., direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eye drops, implantation) to facilitate delivery. To deliver a payload to the ear or ear cells (e.g., inner ear, middle ear, and / or outer ear cells), the composition described herein can be brought into contact with the patient's ear or ear cells (e.g., inner ear, middle ear, and / or outer ear cells) in a manner known in the art, such as by parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration) or local administration (e.g., direct injection) to facilitate delivery. To deliver a payload (e.g., RNA encoding an immunogen) to immune system cells (e.g., antigen-presenting cells, including specialized antigen-presenting cells), the composition described herein can be delivered intramuscularly, after which immune cells can infiltrate the delivery site and process the delivered RNA and / or process antigens encoded by non-immune cells (e.g., muscle cells). Such immune cells may include macrophages (e.g., bone marrow-derived macrophages), dendritic cells (e.g., bone marrow-derived plasmacytoid dendritic cells and / or bone marrow-derived myeloid dendritic cells), monocytes (e.g., human peripheral blood mononuclear cells), etc. (e.g., see Geall, Andy et al., WO2012 / 006372).

[0735] Immunization. For immunization purposes, the compositions described herein can be prepared as injections, pulmonary or nasal aerosols, or packaged in a delivery device (e.g., syringe, nebulizer, sprayer, inhaler, skin patch, etc.). Such delivery devices can be used to administer the pharmaceutical composition to a subject (e.g., a human) for immunization.

[0736] For immunization purposes, in some embodiments, the embodiments provided herein include delivering one or more RNAs encoding an immunogen. This immunogen can induce an immune response recognizing the immunogen, thereby providing immunity against pathogens, allergens, or tumor antigens. Immunization is preferably performed against diseases and / or infections caused by pathogens.

[0737] The embodiments described herein will be more readily understood by referring to the following examples. These examples are for illustrative purposes only and are not intended to limit the invention.

[0738] The following examples are provided to illustrate and further explain certain preferred embodiments and aspects of the invention, but should not be construed as limiting the scope of the invention.

[0739] Example

[0740] Unless otherwise defined, scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terms and techniques related to cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are well-known and commonly used in the art. Recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection) are performed using standard techniques. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The terms, laboratory procedures, and techniques related to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well-known and commonly used in the art. Chemical synthesis, chemical analysis, drug preparation, formulation, administration, and patient treatment are performed using standard techniques.

[0741] Preparation of the compound shown in Formula I :

[0742] The compounds disclosed herein can be synthesized by the methods described below or by modifications thereof. Modifications to the methods include, among others, temperatures, solvents, reagents, etc., known to those skilled in the art. Typically, in any process preparing the compounds disclosed herein, it may be necessary and / or required to protect any sensitive or reactive groups on the relevant molecules. This can be achieved through conventional protecting groups, such as those on… Protecting Groups in Organic Chemistry Groups in Organic Chemistry) (JFW McOmie, ed., Plenum Press, 1973) and TW Green, PGM Wutts Protecting Groups in Organic Synthesis SynthesisThose described in (3rd edition, Wiley, New York (1999)), both of which are incorporated herein by reference in their entirety. Protecting groups may be removed at a subsequent convenient stage using methods known in the art. Synthetic chemical transformations that can be used to synthesize suitable compounds are known in the art and include, for example, those described in R. Larock's work. Comprehensive Organic Transformation Comprehensive Organic Transformations (VCH Publishers, 1989) or edited by L. Paquette Encyclopedia of Reagents for Organic Synthesis The methods described in (John Wiley and Sons, 1995), both of which are incorporated herein by reference in their entirety. The routes shown and illustrated herein are merely illustrative and are neither intended nor should be construed as limiting the scope of the claims in any way. Those skilled in the art will be able to identify improvements to the disclosed synthetic methods and to devise alternative routes based on the disclosure herein; all such improvements and alternative routes are within the scope of the claims.

[0743] In the following scheme, the protecting group of the oxygen atom is selected based on its compatibility with the necessary synthetic steps, as well as the compatibility of the introduction and deprotection steps with the overall synthetic scheme (Protecting Groups in Organic Synthesis, TW Green and PGM Wutts, 3rd edition, Wiley, New York (1999)).

[0744] If the compounds of this technique contain one or more chiral centers, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or d(l) stereoisomers, or as mixtures rich in stereoisomers. Unless otherwise stated, all such stereoisomers (and their enriched mixtures) are included within the scope of this technique. Pure stereoisomers (or enriched mixtures) can be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, etc.

[0745] The starting materials for the following reactions are typically known compounds, or can be prepared using known procedures or obvious modifications thereof. For example, many starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka-Chemce, or Sigma (St. Louis, Missouri, USA). Other starting materials can be prepared by procedures described in standard reference texts, such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March's Advanced Organic Chemistry (John Wiley, and Sons, 5th ed., 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0746] Example

[0747] General Program

[0748] It will be apparent to those skilled in the art that methods for preparing precursors and functional groups associated with the compounds described in the claims herein are generally described in the literature. Variations known to those skilled in the art in these reactions may also be used, but will not be elaborated upon here. Based on the relevant literature and this disclosure, those skilled in the art are fully capable of preparing any of the described compounds.

[0749] It should be recognized that those skilled in the art of organic chemistry can easily perform these operations without further instruction; that is, these operations are entirely within the scope of their expertise and practice. These reactions include the reduction of carbonyl compounds to the corresponding alcohols, oxidation reactions, acylation reactions, aromatic substitution reactions (including electrophilic and nucleophilic substitution), etherification reactions, esterification reactions, and saponification reactions. These operations are discussed in standard textbooks, such as March's *Advanced Organic Chemistry* (Wiley) and Carey and Sundberg's *Advanced Organic Chemistry* (incorporated herein by reference in its entirety). Unless otherwise stated, all intermediate compounds of this invention are used directly without further purification.

[0750] It will be readily understood by those skilled in the art that certain reactions are best carried out with other functional groups in the molecule masked or protected, thereby avoiding any unwanted side reactions and / or increasing the reaction yield. Typically, those skilled in the art utilize protecting groups to increase yields or avoid unwanted reactions. These reactions are well documented in the literature and are entirely within the capabilities of those skilled in the art. Many examples of these operations can be found, for example, in *Protecting Groups in Organic Synthesis* by TW Greene and PGM Watts (4th edition, John Wiley & Sons, 2007), which is hereby incorporated in its entirety.

[0751] The following example schemes are provided for reference and represent preferred methods for preparing the compounds illustrated herein. These methods are not limiting, and other routes may obviously be used to prepare these compounds. Such methods specifically include solid-phase based chemical methods, including combinatorial chemistry. Given the existing literature and the content of this disclosure, those skilled in the art are fully capable of preparing these compounds by those methods. The compound numbers used in the synthetic schemes shown below are only for these specific schemes and should not be construed as or confused with the same numbers used in other sections of this application.

[0752] The trademarks used herein are merely illustrative and reflect the explanatory material used in this invention. Those skilled in the art will recognize that variations in batches, manufacturing processes, etc., are normal. Therefore, these examples and the trademarks used are not limiting and are not intended to restrict the scope of the invention, but merely to illustrate how those skilled in the art may choose to implement one or more embodiments of the invention.

[0753] The following abbreviations have the stated meanings:

[0754]

[0755] The following example schemes are for reference only and collectively represent exemplary methods for preparing the compounds described herein. Furthermore, other methods for preparing the compounds described herein will be readily apparent to those skilled in the art based on the following reaction schemes and examples. Unless otherwise stated, all variables are as defined above.

[0756] Example 1

[0757] (Butane-1,4-diylbis(tetradecylazadiyl))bis(3-aminopropane-1,2-diyl) 9E , 9'E )- Synthesis of bis(octadec-9-enoic acid) ester (compound 1 shown in Scheme I)

[0758] Under a nitrogen atmosphere, a solution of DHDMS (1.7 g, 2.7 mmol) in ethyl acetate (90 mL) was stirred at room temperature. Then, a solution of di-tert-butoxycarbonate (DiBoc) in ethyl acetate (5 mL) (1.7 g, 7.8 mmol, 2.9 equivalents) was added to the solution, and the mixture was stirred at room temperature for 16 hours under a nitrogen atmosphere. The resulting solution was diluted with ethyl acetate (100 mL), washed with brine (2 x 100 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give a yellow oil. Then, the oil was purified by silica gel rapid column chromatography using methanol / dichloromethane as solvent to obtain di-tert-butyl((butane-1,4-diylbis-(tetradecylazane-diyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate (1A) (1.1 g, yield 48%).

[0759] To a DMF (50 mL) solution of ((butane-1,4-diylbis(tetradecylazadiyl))-bis(2-hydroxypropane-3,1-diyl))-dicarbamate (1A) (0.89 g, 1.1 mmol), diisopropylethylamine (DIPEA) (1.67 g / 2.25 mL, 12.9 mmol, 12.0 equivalent) was added, and the mixture was stirred at room temperature for 10 min under a nitrogen atmosphere. Oleoyl chloride (2.8 g, 9.3 mmol, 8.7 equivalent) was added to the resulting solution, and the mixture was stirred at room temperature for 10 min. The solution was then heated and stirred at 60 °C for 16 h. The reaction mixture was concentrated under reduced pressure to give a yellow oil. The resulting oil was then diluted with DCM (100 mL) and washed with water (3 x 50 mL). The combined aqueous layers were then back-extracted with DCM (2 x 50 mL), and the combined organic layers were dried over Na2SO4, filtered, and concentrated to give a yellow oil. The crude oil was then purified by silica gel rapid column chromatography with (10-100%) DCM / hexane and (0-100%) MeOH to give a BOC-protected diester intermediate (1B) (1.1 g, 73% yield) (m / z MH+ = 1356.21).

[0760] A DCM (10 mL) solution of the Boc-protected diester (1B) (1.1 g, 0.785 mmol) was cooled to 0°C in an ice bath and stirred under a nitrogen atmosphere. TFA (1 mL) was added, and the resulting solution was stirred for 5 hours while being heated to room temperature. After the reaction was complete, the solution was concentrated under reduced pressure to obtain a maroon oil. The oil was dissolved in DCM (1 mL) and MeOH (4 mL), and the solution was loaded onto a C18 reversed-phase column (Innoval ODS-2, 1.0 x 25 cm) and purified using an (85-100%) water / MeOH gradient to obtain the tetrafluoroacetate of compound 1 (71.6 mg) (m / z MH). + 1156.11).

[0761] Compounds 2-4 were synthesized using the same steps as described above. Intermediate 1A was treated with linoleic acid chloride, followed by TFA treatment, to give trifluoroacetate of compound 2 (1.0 g) (M + H). + = 1100.05614 m / Z (free base)).

[0762] Intermediate 1A was treated with myristoyl chloride, followed by TFA treatment, to give trifluoroacetate of compound 3 (323 mg) (M + H). += 1048.02888 m / z (free base)).

[0763] Intermediate 1A was treated with palmitoleyl chloride, followed by TFA treatment, to give trifluoroacetate of compound 4 (264 mg) (M + H). + = 1152.08786 m / z (free base)).

[0764] Example 2

[0765] synthesis N 1 , N 4 -double(2-((( E )-Octadeca-9-enoyl)oxy)butyl)- N 1 , N 4 -Ditetradecylbutane-1, 4-Diammonium hydrochloride (compound 6 in scheme VIIIA)

[0766] Add to a heavy-walled, sealable, round-bottomed container N 1 , N 4 A solution of bis(tetradecylbutane)-1,4-diamine (2.1 g, 4.3 mmol) in 2,2,2-trifluoroethanol (50 mL) was added. DIPEA (3.62 mL, 20.8 mmol, 4.8 equivalents) and 1,2-butene oxide (0.9 mL, 10.4 mmol, 2.4 equivalents) were added, and the container was sealed and heated to 80 °C with stirring for 20 hours. The solution was concentrated under reduced pressure to give a maroon oil. The oil was dissolved in DCM (5 mL) and purified by a (0–10%) MeOH / DCM gradient on a normal-phase silica gel column to give 1,1'-(butane-1,4-diylbis(tetradecylazadiyl))bis(but-2-ol, i.e., compound 7) (1.34 g, 50% yield) as a white solid. M + H + =625.62639.

[0767] DIPEA (1.8 mL, 9.9 mmol, 12.0 equivalents) was added to a DMF (24 mL) solution of 1,1'-(butane-1,4-diylbis(tetradecylazadiyl))bis(but-2-ol) (0.5 g, 0.8 mmol), and the mixture was stirred at room temperature for 30 min under a nitrogen atmosphere. Oleyl chloride (1.9 g, 6.5 mmol, 7.8 equivalents) was added to the resulting solution, and the mixture was stirred at room temperature for 10 min. The resulting solution was then heated to 60 °C and stirred for 16 h. The reaction solution was concentrated under reduced pressure to give a yellow oil. The resulting oil was diluted with water (100 mL) and then extracted with EtOAc (6 x 50 mL). The combined organic layers were then washed with brine, dried over Na₂SO₄, filtered, and concentrated to give a yellow oil. The resulting oil was purified on a normal-phase column by (0-100%) EtOAc / DCM to give (butane-1,4-diylbis(tetradecylazadiyl))bis(butane-1,2-diyl)bis(octadecane-2,4,6,8,10,12,14,16-octatylacetate), compound 6A (210 mg, yield 22%). M + H + =1154.087.

[0768] To a round-bottom flask containing a 3 mL solution of dichloromethane (DCM) of (butane-1,4-diylbis(tetradecylazadiyl))bis(butane-1,2-diyl)bis(octadecane-2,4,6,8,10,12,14,16-octatyryne ester) (188 mg, 163 μmole), a 4.0 M solution of dioxane (100 μL / 14.6 mg, 400 μmoles, 2.45 equivalents) was added, and the mixture was stirred at room temperature for 1 hour under nitrogen protection. The resulting solution was concentrated under reduced pressure to obtain... N 1 , N 4 -double(2-((( E )-Octadecano-9-enoyl)oxy)butyl)- N 1 , N 4 -Tetradecylbutane-1,4-diamine (compound 6), a white oil (186.4 mg, 94% yield). M + H + = 1154.15064.

[0769] Compound 5 was synthesized using the same steps as described above. The intermediate 1,1'-(butane-1,4-diylbis(tetradecylazadiyl))bis(but-2-ol) was treated with linoleyl chloride, followed by treatment with HCl, to give the hydrochloride salt. N 1 , N 4 -bis(2-(((9Z,12Z)-octadecyl-9,12-dienoyl)oxy)butyl)- N 1 , N 4 -Ditetradecylbutane-1,4-diammonium, namely compound 5 (139.5 mg, yield 53.6%). M + H + = 1150.11951.

[0770] Example 3

[0771] synthesis N 1 ,N 1' -(butane-1,4-diyl)bis( N 2 -(2-hydroxyethyl)- N 1 -Tetradecylpropane-1,2,3-triammonium) Trifluoroacetate (compound 8 in Scheme IIA)

[0772] DCM (20 mL) was added to a 250 mL round-bottom flask containing di-tert-butyl((butane-1,4-diylbis(tetradecylazadiyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate 1A (1.2 g, 1.5 mmol), and the solution was stirred at room temperature until the compound dissolved. Then, at 0... o Add TEA (1.4 mL, 10 mmol, 6.8 equivalents) at C, and incubate the resulting solution at 0°C. o The mixture was stirred at C for 40 minutes, and a solution of methanesulfonyl chloride (787 mg, 6.9 mmol, 4.7 equivalents) in DCM (30 mL) was added. After stirring for 16 hours, the reaction was allowed to warm to room temperature. After the reaction was complete, the solution was diluted with DCM (200 mL) and washed with water (3 x 100 mL). The combined aqueous layers were back-extracted with DCM (4 x 100 mL). The combined organic layers were dried over Na2SO4, filtered, and the solution was concentrated under reduced pressure to give 2,2,21,21-tetramethyl-4,19-dioxo-9,14-ditetradecyl-3,20-dioxa-5,9,14,18-tetraazacosicosodeane-7,16-dimethyldimethanesulfonate 8A, which was an oil and was used directly in the next step without further purification. Yield 1.27 g, 87%, LC-MS (ESI) m / z 1027.82 [M + 2Na + H] + ].

[0773] To a 100 mL round-bottom flask containing a 13 mL solution of 2,2,21,21-tetramethyl-4,19-dioxo-9,14-ditetradecyl-3,20-dioxa-5,9,14,18-tetraazacosicosode-7,16-dimethyldimethanesulfonate 8A (620 mg, 0.63 mmol) in ethanol, add a 1 mL solution of 2-aminoethane-1-ol (0.4 g, 6.5 mmol, 10.3 equivalents). Potassium carbonate (0.4 g, 2.9 mmol, 4.6 equivalents) is added to the solution. The resulting solution is heated to 65 °C and stirred overnight. After the reaction is complete, the solution is concentrated to dryness under reduced pressure to give a white oil. The resulting oil is diluted with 20 mL of EtOAc and washed with 3 x 10 mL of water. The combined aqueous layers were back-extracted with EtOAc (4 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give di-tert-butyl((butane-1,4-diylbis(tetradecylazinediyl))bis(2-((2-hydroxyethyl)amino)propane-3,1-diyl))dicarbamate 8B oil, which was used directly for the next step without purification. 556 mg, yield 96%, LC-MS (ESI) m / z 913.79 [M + H + ].

[0774] Trifluoroacetic acid (5 mL) was added to a round-bottom flask containing a 15 mL solution of di-tert-butyl((butane-1,4-diylbis(tetradecylazinediyl))bis(2-((2-hydroxyethyl)amino)propane-3,1-diyl))dicarbamate 8B (556 mg, 0.6 mmol). The resulting solution was stirred at room temperature for 5 hours. The reaction solution was concentrated under reduced pressure to obtain a yellow oil. The oil was then dissolved in MeOH (2 mL) and water (4 mL). The resulting solution was then loaded onto a reverse-phase column and purified with (50–100%) MeOH / water to obtain... N 1 ,N 1' -(butane-1,4-diyl)bis( N 2 -(2-hydroxyethyl)- N 1 -Tetradecylpropane-1,2,3-triammonium) trifluoroacetate (compound 8). 126 mg, yield 16%, LC-MS (ESI); m / z 713.70 [M + H + ].

[0775] Compounds 9-12, 39-41, 43, 46, and 80 were synthesized using the same steps as described above. Intermediate 8A was treated with 4-aminobutyric acid followed by TFA treatment to obtain... N 1 , N 1' -(butane-1,4-diyl)bis( N 2 -(2-hydroxyethyl)- N 1 -Tetradecylpropane-1,2,3-triammonium) trifluoroacetate, namely compound 9. (138 mg, yield 16%, LC-MS (ESI) m / z 769.75 [M + H]) + .

[0776] Intermediate 8A was treated with 3-aminopropane-1,2-diol, followed by TFA treatment, to obtain... N 1 , N 1' -(butane-1,4-diyl)bis( N 2 -(2,3-Dihydroxypropyl)- N 1 -Tetradecylpropane-1,2,3-triammonium) trifluoroacetate, i.e., compound 10. LC-MS (ESI) m / z 773.70 [M + H] + .

[0777] Intermediate 8A was treated with 4-(aminomethyl)phenol, followed by TFA treatment, to obtain N 1 , N 1' -(butane-1,4-diyl)bis( N 2 -(4-hydroxybenzyl)- N 1 -Tetradecylpropane-1,2,3-triammonium) trifluoroacetate, i.e., compound 11. LC-MS (ESI) m / z 837.71 [M + H] + .

[0778] Intermediate 8A was treated with 2-aminoethane-1-thiol, followed by TFA treatment, to obtain... N 1 , N 1' -(butane-1,4-diyl)bis( N 2 -(2-Mercaptoethyl)- N1 -Tetradecylpropane-1,2,3-triammonium)trifluoroacetate, i.e., compound 12. LC-MS (ESI) m / z 745.64 [M + H] + .

[0779] Intermediate 8A was treated with 3-aminopropane-1-ol, followed by TFA treatment, to yield 14-((3-ammonium-2-((3-hydroxypropyl)amino)propyl)(4-((3-ammonium-2-((3-hydroxypropyl)ammonium)propyl)(tetradecyl)ammonium)butyl)ammonium)tetradecane-1-cationic trifluoroacetate, i.e., compound 39. LC-MS (ESI) m / z 741.72 [M + H] + .

[0780] Intermediate 8A was treated with 5-aminopentan-1-ol, followed by TFA treatment, to yield 14-((3-ammonium-2-((5-hydroxypentyl)amino)propyl)(4-((3-ammonium-2-((5-hydroxypentyl)ammonium)propyl)(tetradecyl)ammonium)butyl)ammonium)tetradecane-1-cationic trifluoroacetate, i.e., compound 40. LC-MS (ESI) m / z 797.78 [M + H] + .

[0781] Intermediate 8A was treated with 6-aminohexane-1-ol, followed by TFA treatment, to obtain... N 1 , N 1' -(butane-1,4-diyl)bis(N2-(6-hydroxyhexyl)- N 1 -Tetradecylpropane-1,2,3-triammonium) trifluoroacetate, i.e., compound 41. LC-MS (ESI) m / z 825.82 [M + H] + .

[0782] Intermediate 8A was treated with 2-amino-3-phenylpropan-1-ol, followed by TFA treatment, to yield 14-((3-ammonium-2-((1-hydroxy-3-phenylpropan-2-yl)amino)propyl)(4-((3-ammonium-2-((1-hydroxy-3-phenylpropan-2-yl)ammonium)propyl)(tetradecyl)ammonium)butyl)ammonium)tetradecane-1-cationic trifluoroacetate, i.e., compound 43. LC-MS (ESI) m / z 893.77 [M + H] + .

[0783] Intermediate 8A was treated with 2-amino-2-methylpropane-1-ol, followed by TFA treatment, to yield 14-((3-ammonium-2-((1-hydroxy-2-methylpropane-2-yl)amino)propyl)(4-((3-ammonium-2-((1-hydroxy-2-methylpropane-2-yl)ammonium)propyl)(tetradecyl)ammonium)tetradecane-1-cationic trifluoroacetate, i.e., compound 46. LC-MS (ESI) m / z 769.75.72 [M + H] + .

[0784] Intermediate 8A was treated with 2-amino-2-methylpropane-1,3-diol, followed by TFA treatment, to obtain trifluoroacetic acid 14-((3-ammonium-2-((1,3-dihydroxy-2-methylpropane-2-yl)amino)propyl)(4-((3-ammonium-2-((1,3-dihydroxy-2-methylpropane-2-yl)ammonium)propyl)(tetradecyl)ammonium)tetradecane-1-dimethylamine, i.e., compound 48. LC-MS (ESI) m / z 801.73 [M + H] + .

[0785] Intermediate 8A was treated with 1-aminopropane-2-ol, followed by TFA treatment, to yield 14-((3-ammonium-2-((2-hydroxypropyl)amino)propyl)(4-((3-ammonium-2-((2-hydroxypropyl)ammonium)propyl)(tetradecyl)ammonium)butyl)ammonium)tetradecane-1-cationic trifluoroacetate, i.e., compound 80. LC-MS (ESI) m / z 741.72 [M + H] + .

[0786] Example 4

[0787] Utilizing the RNA payload of the complex, through in vivo Functional testing was used to screen and evaluate lipid nanoparticle (LNP) formulations. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the protein encoded by the payload. Compositions comprising compounds 1-9 and helper lipids were prepared and complexed with mRNA. As shown in Table 2, the studied formulations varied in the molar ratio of compound 1 to helper lipids.

[0788] Table 2: Exemplary LNP Formulations

[0789]

[0790] All LNP formulations contained lipid compounds, DOPE, cholesterol, and DMG-PEG. Some formulations contained a peptide (sequence identifier: 47). All lipids were weighed and dissolved in ethanol at the desired molar ratio. The lipid mixture was complexed with firefly luciferase (fLuc) mRNA into the LNPs using a microfluidic device. The LNPs were dialyzed in phosphate-buffered saline (LNP1–LNP5, LNP13–LNP60) or TRIS buffer (LNP6–LNP10), and particle size and homogeneity were measured using dynamic light scattering. The LNPs were injected into mice the following day.

[0791] Female BALB / c mice aged 6 to 10 weeks were purchased from The Jackson Laboratory and acclimatized for 7 days prior to the study. LNPs equivalent to 10 µgfLuc mRNA were injected into the mice via intravenous tail vein injection. Four hours post-injection, the mice were anesthetized with isoflurane, and imaging was performed 10 minutes after intraperitoneal injection of 100 µL of Rediject D-luciferin (Perkin Elmer). Bioluminescence imaging was performed using the IVIS Lumina III imaging system (Perkin Elmer). in vivo (Whole body) and In vitro Organ quantification was performed, and the Living Image software was used for analysis.

[0792] All LNP formulations containing compound 1 had particle sizes >100 nm and polydispersity indices <0.3 (Figure 1). Following intravenous administration of the LNP formulations containing compound 1, mRNA delivery and luciferase expression were detected in the liver (Figure 2) and spleen (Figure 3) of injected mice. Compared to the spleen, LNPs containing compound 1 were expressed at higher levels in the liver, indicating that LNPs containing compound 1 are a highly efficient mRNA delivery system for liver delivery.

[0793] Example 5

[0794] LNP formulations were screened, and the RNA payloads of the complexes were evaluated using in vivo functional assays. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the proteins encoded by the payloads. Compositions comprising compounds 1-9 and helper lipids were prepared and complexed with mRNA. As shown in Table 3, the formulations studied varied in the molar ratio of compounds 1-9 to helper lipids.

[0795] Table 3: Exemplary LNP Formulations

[0796]

[0797]

[0798] All LNP formulations contained lipid compounds (DOPE or DSPC, or both), cholesterol, and DMG-PEG or C16-PEG. Some formulations contained a peptide (sequence identifier: 47). All lipids were weighed and dissolved in ethanol at the desired molar ratio. The lipid mixture was complexed with firefly luciferase (fLuc) mRNA into the LNPs using a microfluidic device. The LNPs were dialyzed in phosphate buffer, and particle size and uniformity were measured using dynamic light scattering. The LNPs were injected into mice the following day.

[0799] Female BALB / c mice aged 6 to 10 weeks were purchased from The Jackson Laboratory and acclimatized for 7 days prior to the study. LNPs equivalent to 10 µg fLuc mRNA were injected intravenously via the tail vein into the mice at a total volume of 200 µl. Four hours post-injection, the mice were anesthetized with isoflurane, and imaging was performed 10 minutes after intraperitoneal injection of 100 µL LDiject D-fluorescein (Perkin Elmer). Imaging was performed using the IVIS Lumina III imaging system (Perkin Elmer). in vivo (Whole body) and In vitro Quantitative bioluminescence imaging of organs was performed and analyzed using Living Image software.

[0800] All LNP formulations containing compounds 1–9 had particle sizes >400 nm and polydispersity indices <0.3 (Figs. 9, 10, 13, 18, 19). Intravenous administration of LNP formulations containing compounds 1–9 induced mRNA delivery and luciferase expression in the liver (Figs. 4, 11, 14), spleen (Figs. 5, 12, 15, 20), and lungs (Fig. 21) of injected mice. Compared to the spleen, LNPs containing compounds 1–4 were expressed at higher levels in the liver, indicating that LNPs containing compounds 1–4 are a highly efficient mRNA delivery system for liver delivery. Compared to the liver, LNPs containing compounds 5–9 were expressed at higher levels in the spleen, indicating that LNPs containing compounds 5–9 are a highly efficient mRNA delivery system for liver delivery.

[0801] Example 6

[0802] Human primary T cell transfection protocol:

[0803] Compound 1 was dissolved in ethanol to a final concentration of 25 mg / mL. Compound 1 was mixed with DOPE at a molar ratio of 1:1 and 1:2 at a concentration of 25 mg / mL to prepare formulations. 4 mL of the prepared lipid was added to 90 mL of diluted 5-methoxyuridine (5 molU)-modified enhanced green fluorescent protein (eGFP) mRNA solution (1 mg mRNA dissolved in 100 mM pH ~5.2 sodium acetate buffer), and vortexed three times to prepare LNP. After incubation at room temperature for 10 minutes, an appropriate amount of mRNA was added to activated human primary T cells. Two days after transfection, transfection efficiency was assessed by flow cytometry. EP: Electroporation was used as a control / reference, with 100 ng of 5 molU-modified eGFP mRNA mixed with 10 µL of 2 x 10⁻⁶ ppm of DOPE. 5 The T cells were mixed and electroporated under the conditions of 1400 V, 20 ms and 1 pulse.

[0804] Table 4. Exemplary lipid formulations:

[0805]

[0806] Table 5. Exemplary lipid complex formulations:

[0807]

[0808] Compared to LNP11 and LNP12, the lipid formulations LP24 and LP25 showed higher transfection efficiency, which appears to indicate that a high proportion of DOPE and compound 1 played a role in the transfection of human primary T cells, as shown in Figures 6 and 7. Furthermore, as shown in Figure 8, formulations LP24 and LP25, as well as the lipid nanoparticles LNP11 and LNP12, all exhibited good cell viability compared to the electroporated samples.

[0809] Example 7

[0810] Compound 8: Preparation and Screening

[0811] Compound 8 was dissolved in chloroform to obtain a stock solution with a concentration of 25 mg / mL. Similarly, DOPE and cholesterol were dissolved in chloroform as cofactor lipids to a concentration of 25 mg / mL. The cationic lipids (compound 8) were mixed with DOPE or cholesterol in different molar ratios (2:1, 1:1, 1:2, 1:4, and 1:8) in glass vials. The chloroform was removed from these vials, and the lipids were dried using a rotary evaporator. The vials were placed in a vacuum desiccator overnight to completely remove the chloroform. The following day, the required amount of 95% ethanol was added to each vial to achieve a final lipid concentration of 2 mg / mL.

[0812] Liposome preparation:

[0813] For formulations in the aqueous phase, a reverse-phase evaporation method was used. Lipids dissolved in chloroform were added to a vial along with an appropriate amount of water required to achieve a final lipid concentration of 2 mg / mL, followed by removal of the chloroform using a rotary evaporator. Lipids dissolved in water or 95% ethanol (used alone or in combination with peptides such as 15-24 (0.5 mg / mL), 09-04 (0.025 mg / mL), or 15-24 + 09-04 (0.5 + 0.025 mg / mL)) were tested on VPC 1.0 cells (suspension HEK 293 cells), and transfection efficiency was assessed using the antibody fragment-GFP plasmid (Dulaglutide-T2A GFP).

[0814] suspension cell transfection

[0815] VPC 1.0 cells (virus-producing cells derived from the HEK293F cell line) were cultured in ExpiFectamine 293 (Expi293 expression medium) and passaged every 3-4 days to maintain a cell density below 6 x 10⁻⁶. 6 Cells / mL. For routine maintenance, passage cells to 0.3 x 10⁻⁶ cells / mL. 6 Up to 0.5 x 10 6 Cells were stored at a density of 10 cells / mL. The day before transfection, cells were stocked at a density of 3 x 10⁻⁶ cells / mL. 6 Cells were passaged at a density of 10 cells / mL; on the day of transfection, the cells were redissolved to 3 x 10⁻⁶ cells / mL. 6Cells / mL were aliquoted into 96-well deep-well plates. Approximately 800 µL of cells were aliquoted into 2 mL of 96-well deep-well plates (60 wells only) and incubated on a shaker at 900 rpm until ready for transfection. 100 µL of Opti-MEM medium containing DNA (0.8 µg / mL) was aliquoted into 96-well plates to prepare the transfection complex. The transfection reagent was added to the wells containing DNA, mixed by repeated pipetting, and then incubated for 20 minutes to form the lipid complex / lipid nanoparticles. After 20 minutes, 100 µL of the DNA-transfection reagent complex was added to the cells, and transfection efficiency and toxicity were measured at 24 and 48 hours post-transfection.

[0816] Transfection efficiency and toxicity

[0817] Potency was determined at 24 and 48 hours post-transfection using a microplate reader-based assay and compared with existing catalog products, such as 2.5 µL of ExpiFectamine 293 (Expi293) reagent. GFP fluorescence intensity in cells was also measured using a microplate reader. See Figure 16. 10 µL of cells were diluted in 40 µL of culture medium, and the GFP fluorescence intensity was measured (485 / 515). Then, PrestoBlue (PB) HS reagent (50 µL of PB diluted 5-fold with culture medium) was added. The contents of the well plate were mixed using an orbital oscillator for 2 minutes, and then incubated at 37°C for 1 hour. The fluorescence intensity was then measured again (560 / 590). Higher PrestoBlue fluorescence intensity indicates stronger cell viability. See Figure 17.

[0818] Other implementation plans :

[0819] Although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0820] The patents and scientific literature mentioned in this article establish the knowledge available to those skilled in the art. All references, For exampleU.S. patents, U.S. patent applications, PCT patent applications designating the U.S., published foreign patents, and patent applications cited herein are incorporated herein by reference in their entirety. GenBank and NCBI filings cited herein by accession number are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and not intended to be restrictive.

[0821] While the invention has been specifically shown and described with reference to its preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the invention as covered by the appended claims.

[0822] Although the present disclosure has been described with reference to the above embodiments, it should be understood that modifications and variations are covered within the spirit and scope of this disclosure. Therefore, this disclosure is limited only by the following claims.

Claims

1. A compound having universal structure I, or a pharmaceutically acceptable salt thereof: I in: A1 is —(CH2) x — or —(CH2) y —A4—(CH2) z —; A4 Choose the group consisting of the following items: —(CH2) x —, —(CO)O—, —O(CO)—, —SS—, , , , and ; Q1 is N or Q2 is N or ; R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups; R3 is —COR, —(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or or C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or C3-C6 cycloalkyl groups, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; or C3-C6 cycloalkenyl groups, optionally replaced by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7- or aryl groups, or C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups; or Only if R1 and R2 are independently -(CH2) 1-7 R3 is H only when COOR is applied; R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R5 and R6 are independently selected from H and CH3; R7 is H or an optionally substituted C1-C6 straight-chain or branched alkyl group, or an optionally substituted monounsaturated or polyunsaturated C1-C6 straight-chain or branched alkenyl group. ; R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, —(CH2) 0-3 Het; A2 and A3 independently choose from the following groups: H, , , , , , , , , , , and ; R8 is —COR or or ; Where R9 is H or or ; Where R 10 Choose the group consisting of the following items: H; C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cyclic alkyl substitution; C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups; C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces; C3-C6 cycloalkylamines, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; and C3-C6 cycloalkenylamines, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces; Where R 11 Choose from the following groups: -NH2, -NHR, -N(R)2, , and ; Where AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 0 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, n4, and n5 are independently integers from 1 to 5. n6 is an integer from 0 to 7. n7 and n8 are independent integers from 0 to 5; n9 is an integer from 1 to 5; m1 is an integer from 1 to 5; m2 is an integer from 0 to 5; m3 is an integer from 1 to 7; p is an integer from 1 to 50; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

2. The compound according to claim 1, wherein: A1 is —(CH2) x — or —(CH2) y —A4—(CH2) z —; A4 Choose the group consisting of the following items: —(CH2) x —, —(CO)O—, —O(CO)—, —SS—、 , , , and ; Q1 is N; Q2 is N; R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups; R3 is —COR, —(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or ; R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups -(CH2) 0-3 It; A2 and A3 independently choose from the following groups: H, , , , , , , , , and ; Where R 10 Choose the group consisting of the following items: H; C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cyclic alkyl substitution; C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups; C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl-(CH2) n Het replaces; Where AA represents any natural or non-natural amino acid side chain; a is an integer from 1 to 6; b is an integer from 0 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, n4, and n5 are independent integers from 1 to 5; n6 is an integer from 0 to 7; n7 and n8 are independent integers from 0 to 5; n9 is an integer from 1 to 5; m1 is an integer from 1 to 5; m2 is an integer from 0 to 5; m3 is an integer from 0 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

3. The compound according to claim 1, having the structure of formula Ia: Or its pharmaceutically acceptable salt, wherein: R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; A2 and A3 independently choose from the following groups: , , , , , , and ; R3 is —COR, —(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or or C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are substituted by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7- or aryl groups, or C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl, or C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces, or C3-C6 cycloalkyl groups, wherein the ring carbon atoms are replaced by -O-, -S-, -SS-, or -NR7-; or C3-C6 cycloalkenyl groups, optionally replaced by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces; R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het; and Where R 10 Choose the group consisting of the following items: H; C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optional cyclic alkyl substitution; C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups; C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces; Where AA represents any natural or non-natural amino acid side chain; x is an integer from 1 to 9; n is an integer from 1 to 5; n5 is an integer from 1 to 5; n6 is an integer from 0 to 7; n7 and n8 are independent integers from 0 to 5; n9 is an integer from 1 to 5; m3 is an integer from 1 to 3; p is an integer from 1 to 50; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

4. The compound according to claim 3, wherein n5 is 1, or n7 is 1, or n9 is 1, independently of each other.

5. The compound according to claim 3, wherein n6 is 1, 2, or 3.

6. The compound according to claim 3, wherein n8 is 0, 1, or 2.

7. The compound according to claim 3, wherein x is 4.

8. The compound according to claim 3, wherein R3 is or .

9. The compound according to claim 3, wherein R3 is an optionally substituted C1-C 30 Straight-chain or branched alkyl groups, or optionally substituted C4-C groups 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups.

10. The compound according to claim 1, having the structure of formula Ib: Or its pharmaceutically acceptable salt, wherein: R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R3 is —COR, —(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or ; A2 and A3 independently choose from the following groups: , , , and ; R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het; and Where AA represents any natural or non-natural amino acid side chain; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n5 is an integer from 1 to 5; m2 is an integer from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

11. The compound according to claim 10, wherein n5 is 1, or m2 is 2, or n9 is 1, independently of each other.

12. The compound according to claim 10, wherein y and z are independently integers from 1 to 2.

13. The compound according to claim 1, having the structure of formula Ic: Or its pharmaceutically acceptable salt, wherein: R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R3 is —COR, —(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or ; A4. Choose the group consisting of the following items: —(CO)O—, —O(CO)—, —SS—. , , and ; R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het; and Where AA represents any natural or non-natural amino acid side chain; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, and n4 are independent integers from 1 to 5; m1 is an integer from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

14. The compound according to claim 13, wherein n1, n2, n3 and n4 are independently integers from 1 to 2.

15. The compound according to claim 13, wherein y and z are independently integers from 1 to 2.

16. The compound according to claim 13, wherein m1 is an integer from 1 to 2.

17. The compound according to claim 1, having the structure of formula Id: Or its pharmaceutically acceptable salt, wherein: A2 and A3 independently choose from the following groups: H, , and ; R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R3 is —COR, —(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or ; R4 is H or an optionally substituted C1-C 20 Straight-chain or branched alkyl groups, optionally substituted C1-bC 20 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R5 and R6 are independently selected from H and CH3; R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het; A4. Choose the group consisting of the following items: —(CO)O—, —O(CO)—, —SS—. , , , and ;and Where AA represents any natural or non-natural amino acid side chain; a is an integer from 1 to 2; b is an integer from 0 to 3; y and z are independent integers from 1 to 4; z is an integer from 1 to 4; m1 and m2 are independent integers from 1 to 5; n1, n2, n3, n4, and n5 are independent integers from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

18. The compound according to claim 17, wherein a is 1 and b is an integer from 0 to 1.

19. The compound according to claim 17, wherein y and z are independently integers from 1 to 2.

20. The compound according to claim 17, wherein when A2 is ; A3 is H; A4 is ; R1 and R5 are CH3; R2 and R6 are H; R3 is —COR, —(CH2) n COOR or ; R7 is H; b is 0; and its pharmaceutically acceptable salts.

21. The compound according to claim 17, wherein A2 and A3 are independently selected from the group consisting of: and ; A4 is ; R5 and R6 are H; R3 is —COR, —(CH2) n COOR or ; R7 is H; a and b are both 1; and Its pharmaceutically acceptable salt.

22. The compound according to claim 1, having the structure of formula Ia: Or its pharmaceutically acceptable salt, wherein: A1 is —(CH2) x — or —(CH2) y —A4—(CH2) z —; A4 Choose the group consisting of the following items: —(CH2) x —, —(CO)O—, —O(CO)—, and —SS—; R1 and R2 are independently chosen from the following groups: H, arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C groups. 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups; R is selected from the group consisting of the following: optional substitutions of C4-C 30 Straight-chain or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, -(CH2) 0-3 Het; and a is an integer from 1 to 6; b is an integer from 1 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n5 is an integer from 1 to 5; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

23. The compound according to claim 22, wherein A1 is —(CH2)4— or —(CH2)2—S—S—(CH2)2—.

24. The compound according to claim 22, wherein n5 is 1, 2, or 3.

25. The compound according to claim 22, wherein x is 2, 3, or 4.

26. The compound according to claim 22, wherein y and z are independently integers from 1 to 3.

27. The compound according to claim 1, having the structure of formula Ik: Or its pharmaceutically acceptable salt, wherein: A1 is —(CH2) x — or —(CH2) y —S—S—(CH2) z —; R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; Where R 10 Choose from the following groups: H; or C1-C 30 A straight-chain or branched alkyl group, optionally surrounded by -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted cycloalkyl; or C1-C 30 Straight-chain or branched alkyl groups, wherein the carbon atoms of the chain are replaced by -O-, -S-, -SS-, -NR7-, or aryl groups; or C3-C6 cycloalkyl groups, optionally prefixed with -OR7, -N(R7)2, -SR7, or -(CH2). n OR7、-(CH2) n SR7, -(CH2) n N(R7)2、-(CH2) n Aryl, -(CH2) n Arylalkyl, -(CH2) n Het replaces; and a is an integer from 1 to 6; b is an integer from 1 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n5 is an integer from 1 to 5; and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocycle.

28. The compound according to claim 27, wherein A1 is —(CH2)4— or —(CH2)2—S—S—(CH2)2—.

29. The compound according to claim 27, wherein n5 is 1.

30. The compound according to claim 27, wherein a and b are independently integers from 1 to 3.

31. The compound according to claim 27, wherein R 10 Choose the group consisting of the following items: H, 。 32. The compound according to claim 1, having the structure of formula In: Or its pharmaceutically acceptable salt, wherein: A1 is —(CH2) y —S—S—(CH2) z —; R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; Where AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 1 to 6; y is an integer from 1 to 4; z is an integer from 1 to 4; and n5 is an integer from 1 to 5.

33. The compound according to claim 32, wherein n5 is 1.

34. The compound according to claim 32, wherein a and b are independently integers from 1 to 3.

35. The compound according to claim 32, wherein AA is selected from the group consisting of: 。 36. The compound according to claim 1, having the structure of Iy: Or its pharmaceutically acceptable salt, wherein: R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; y is an integer from 1 to 4; and z is an integer from 1 to 4.

37. The compound according to claim 36, wherein y and z are independently integers from 1 to 3.

38. The compound according to claim 1, having the structure of formula Iz: Or its pharmaceutically acceptable salt, wherein: R1 and R2 are independently chosen from the following groups: arbitrarily substituted C1-C 30 Straight-chain or branched alkyl groups and optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups; R3 is —COR, —(CH2) n COR or —(CH2) n COOR or —(CO)NHR— or —(CO)N(R)2— or , or optionally replace C1-C 30 Straight-chain or branched alkyl groups, or optionally substituted C4-C groups 30 Monounsaturated or polyunsaturated straight-chain or branched alkenyl groups, or optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched alkynyl groups; y is an integer from 1 to 4; and z is an integer from 1 to 4.

39. The compound according to claim 38, wherein y and z are independently integers from 1 to 3.

40. The compound according to any one of claims 3, 10, 13, 17 and 38, wherein R3 is —COR or —(CH2). n COOR.

41. The compound according to any one of claims 1 to 40, wherein R1 and R2 are —(CH2). 13 CH3.

42. The compound according to any one of claims 1 to 35, wherein A1 is —(CH2)4— or —(CH2)—A4—(CH2)—.

43. The compound according to any one of claims 1 to 26 and 38 to 39, wherein R is selected from the group consisting of: oleoyl, oleyl, linoleoyl, linoleoyl, palmitoyl, palmitoyl, palmitoyl, palmitoyl, myristoyl, myristoyl, lauroyl and lauroyl groups.

44. The compound according to any one of claims 1 to 39, wherein R is —(CH2). 0-3 Het, where Het is an optionally substituted 5- to 7-membered monocyclic basic heterocyclic compound.

45. The compound according to any one of claims 1 to 39, wherein the optionally substituted 5- to 7-membered monocyclic basic heterocycle is selected from the group consisting of: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

46. ​​A compound having a structure selected from the group consisting of: in: ， And its pharmaceutically acceptable salts.

47. A compound having a structure selected from the group consisting of: ， And its pharmaceutically acceptable salts.

48. A compound having a structure selected from the group consisting of: ， And its pharmaceutically acceptable salts.

49. A compound having a structure selected from the group consisting of: ， And its pharmaceutically acceptable salts.

50. A compound having a structure selected from the group consisting of: ， And its pharmaceutically acceptable salts.

51. A compound having a structure selected from the group consisting of: in: ， And its pharmaceutically acceptable salts.

52. A compound having a structure selected from the group consisting of: in: R 10 For: H, 、 、 ， And its pharmaceutically acceptable salts.

53. A compound having a structure selected from the group consisting of: in: ， And its pharmaceutically acceptable salts.

54. A compound having a structure selected from the group consisting of: in: n6 is either 0 or 1; R 10 For: H, 、 、 ， And its pharmaceutically acceptable salts.

55. A compound having a structure selected from the group consisting of: in: R is 、 、 , and its pharmaceutically acceptable salts.

56. A composition comprising: (i) one or more compounds according to claims 1 to 55, and (ii) one or more of structural lipids, ionizable lipids, and stabilizers; and (iii) Optional payload.

57. The composition of claim 56, wherein the composition comprises: (i) the compound according to any one of claims 1 to 55; (ii) One or more structural lipids; (iii) one or more stabilizers; and (iv) Optional payload.

58. The composition of claim 56, wherein the composition comprises: (i) the compound according to any one of claims 1 to 55; (ii) One or more structural lipids; (iii) One or more stabilizers; (iv) one or more transfection enhancers; and (v) Optional payload.

59. A composition comprising: (i) one or more compounds according to any one of claims 1 to 55; and (ii) Payload.

60. The composition according to any one of claims 56 to 59, wherein one or more of the compounds according to claims 1 to 55 are present in the composition at 10 mol% to 80 mol%, and any effective load is not included if present.

61. The composition according to any one of claims 56 to 59, wherein the structural lipid is present at 14 mol% to 50 mol% of the composition, and any effective load is not included if present.

62. The composition according to any one of claims 56 to 59, wherein the stabilizer is present in the composition at 0.1 mol% to 10 mol%, and any effective load is not included if present.

63. The composition according to any one of claims 56 to 59, wherein the composition further comprises exosomes or biological material derived from or purified from exosomes.

64. The composition according to any one of claims 56 to 63, wherein the composition further comprises a polymer.

65. The composition of claim 64, wherein the polymer is selected from the group consisting of: dense star-shaped dendritic polymers, PAMAM dendritic polymers, NH3 core dendritic polymers, ethylenediamine core dendritic polymers, fifth-generation or higher dendritic polymers, dendritic polymers having substituent groups, dendritic polymers containing one or more amino acids, grafted dendritic polymers, activated dendritic polymers, polyethyleneimine, polyethyleneimine conjugates, polylysine, polyarginine, polyornithine, histones, and any combination thereof.

66. The composition of claim 64, wherein the polymer is linear or branched PEI.

67. The composition according to any one of claims 56 to 58 and 60 to 66, wherein the stabilizer is selected from the group consisting of surfactants, neutral lipids, polymer-coupled lipids, polyethylene glycol, phospholipids, and any combination thereof.

68. The composition according to any one of claims 56 to 58 and 60 to 67, wherein the stabilizer is a PEG-modified lipid.

69. The composition according to any one of claims 56 to 58 and 60 to 67, wherein the one or more transfection enhancers comprise a polycationic nucleic acid binding moiety.

70. The composition of claim 69, wherein the transfection enhancer is selected from the group consisting of: endosome release agents, cell surface ligands, nuclear localizers, cell-penetrating peptides, fusion-promoting peptides, and any combination thereof.

71. The composition of claim 58, wherein the one or more transfection enhancers comprise an amphiphilic peptide.

72. The composition according to any one of claims 56 to 58 and 60 to 71, wherein the composition further comprises a payload.

73. The composition according to claim 59 or 72, wherein the payload comprises nucleic acid.

74. The composition of claim 73, wherein the compound of claims 1 to 55 comprises a charge N and the nucleic acid molecule comprises a charge P, and wherein the combination of the compound of claims 1 to 55 and the nucleic acid contacting the cell comprises an N / P ratio of about 1 to 20.

75. The composition according to claim 73 or 74, wherein the nucleic acid is RNA.

76. The composition according to claim 75, wherein the RNA is mRNA, siRNA, shRNA, self-replicating RNA (srRNA), o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), or any combination thereof.

77. The composition according to claim 73, wherein the nucleic acid is DNA.

78. The composition of claim 72, wherein the payload further comprises one or more peptides and optionally nucleic acids.

79. The composition of claim 78, wherein the peptide is covalently linked to a nucleic acid.

80. The composition according to claim 76, wherein the RNA is mRNA.

81. The composition according to claim 80, wherein the composition comprises two or more different mRNAs.

82. The composition according to claim 81, wherein the RNA encodes an immunogen.

83. The composition of claim 82, wherein the RNA encodes a cancer antigen.

84. The composition according to any one of claims 56 to 58, wherein the structural lipid is selected from the group consisting of: cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, campesterol, lycopene, tomato saponin, ursolic acid, α-tocopherol, hopanes, phytosterols, steroids, and any combination thereof.

85. The composition according to any one of claims 56 to 58, wherein the stabilizer comprises one or more phospholipids selected from the group consisting of: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-di(undecanoyl)-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether PC), 1-oleoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG) and sphingomyelin.

86. The composition according to any one of claims 56 to 58, wherein the ionizable lipid comprises one or more cationic lipids selected from the group consisting of: GeneIn™, LipofectAmine™ 2000, LipofectAmine™, Lipofectin®, DMRIE-C, CellFectin® (Invitrogen), Oligofectamine® (Invitrogen), LipofectAce® (Invitrogen), Fugene® (Roche, Basel, Switzerland), Fugene® HD (Roche), Transfectam® (Tranfectam, Promega, Madison, WI), Tfx-10® (Promega), Tfx-20® (Promega), Tfx-50® (Promega), Transfectin™ (BioRad, Hercules, CA), SilentFect™ (Bio-Rad), Effectene® (Qiagen, Valencia, CA), DC-chol (Avanti PolarLipids), GenePorter® (Gene Therapy Systems, San Francisco). Diego, CA), DharmaFect 1® (Dharmacon, Lafayette, CO), DharmaFect 2® (Dharmacon), DharmaFect 3® (Dharmacon), DharmaFect 4® (Dharmacon), Escort™ III (Sigma, St. Louis, MO), Escort™ IV (Sigma), DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl) -2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-Diamine, N-1-dimethyl-N-1-(2,3-dipalmitoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, L-spermine-5-carboxy-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine, 3,5-(N,N-dilysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine), L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, L-lysine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)-piperazine] L-Lysine-bis-(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleoylamino)-2-hydroxypropyl]piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4,-bis[(3-amino-2-hydroxypropyl)-oleoylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitoylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitoylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitoylamino]-butane-2,3-diol, [(3-aminopropyl)-myristylamino]-butane-2,3-diol, 1,4-bis[(3-oleylamino)propyl]-piperazine, L-arginine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, bis[(3-(3-aminopropyl)-myristylamino)2-hydroxypropyl]piperazine, L-arginine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-serine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis-(O,O'-palmitoyl-β-hydroxyethyl]piperazine L-Histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesterol-3β-carboxy-amide vinyltrimethylammonium iodide, 1,4-bis[(3-myristyl-amino)-propyl]-piperazine, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol carboxylate iodide, cholesterol-3β-carboxyamide vinylamine, cholesterol-3β-oxysuccinamide-vinyl-trimethylammonium iodide, 1-dimethylamino-3-trimethylammonium-DL-2-propyl-cholesterol-3β-oxysuccinate iodide, 2-[(2-trimethylammonium)-ethylmethyl-amino]ethyl-cholesterol-3β-oxysuccinate iodide, 3β[N-(N',N'-Dimethylamino-ethane)-carbamoyl]cholesterol and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitoylamino)propyl]piperazine, L-ornithine glycyl-N-(1-heptadecyloctadecyl)glycamide, N, 2 N 5 -bis(3-aminopropyl)-L-guanylo-glycyl-N-(1-heptadecyloctadecyl)-glycamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkyl-amino)-2-hydroxypropyl]-piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-octadecyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-octadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleoamino)2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-octadecyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-octadecyl-L-glutamine]-L-glutamic acid, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dioleoyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-diole-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-diole-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-diole-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleyl-amino)-propyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)-carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dipalmitoyl-L-glutamine]-L-glutamic acid, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dimyristyl-L-glutamine,N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoylamino)-2-hydroxypropyl]-piperazine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 N 5 -bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithoyl-NN-dimyristyl-L-glutamine]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristyl-amino)-propyl]piperazine, N 2 -[N 2 N 5 [-bis(3-aminopropyl)-L-ornithine]-N,N-dilauroyl-L-glutamine, N 2 -[N 2 N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauroyl-L-α-glutamine, N 2 -[N 2 N 5 -bis-(aminopropyl)-L-ornithine]-NN-dilauroyl-L-α-asparagine, N-[N 2 -[N 2 N 5 -bis[(1,1-dimethylethoxy)-carbonyl]-N 2 N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-guaninyl-NN-dilauroyl-L-glutamine]-L-glutamic acid, 3-[N',N”-bis(2-tert-butoxycarbonyl-amino-ethyl)guanidinyl]-N,N-octadec-9-enylpropionamide, 3-[N',N”-bis(2-tert-butoxy-carbonyl-amino-ethyl)guanidinyl]-N,N-dipalmitoylpropionamide, 3-[N',N”-bis(2-tert-butyl-oxycarbonyl-amino-ethyl)guanidinyl]-N,N-dimyristylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmitoyl-amino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleyl- [(3-(3-amino-2-hydroxypropyl)-myristylamino)-propyl]piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dioleoylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitoylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristylaminopropane, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-propyl]piperazine, [(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleoyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmitoylamino ... Acyloxyethyl)]methylammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-aminopropyl)-›4-(3-aminopropylamino)-4-tetradecyl-carbamoyl-butylcarbamate cholesterol ester, (3-aminopropyl)-›4-(3-aminopropylamino-4 ...2-dimethylamino-3-dimethylamino-3-dimethylamino- (-propyl)-›4-(3-amino-propylamino)-4-(2-dimethylamino-ethylcarbamoyl)-butylcarbamate cholesterol ester, spermine-5-carboxyglycine (N'-stearoyl-N'-oleyl)amide tetratrifluoroacetate, spermine-5-carboxyglycine (N'-stearoyl-N'-transoleyl)amide tetratrifluoroacetate, guanidinobutylamine carboxycholesterol acetate, spermine-5-carboxy-β-alanine cholesterol ester tetratrifluoroacetate, 2,6-diaminohexanoyl β-alanine cholesterol ester ditrifluoroacetate, 2,4-diaminobutyroyl β-alanine cholesterol ester ditrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesterol ester tritrifluoroacetate, [N,[N-bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxylic acid cholesterol ester, stearoylcarnitine ester, palmitoylcarnitine ester, myristoylcarnitine ester, stearoylstearoylcarnitine ester chloride, L-stearoylstearoylcarnitine ester, stearoyloleoylcarnitine ester chloride, palmitoylpalmitoylcarnitine ester chloride, myristoylmyristoylcarnitine ester chloride, L-myristoylmyristoylcarnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoyl-amino)propyl]-piperazine, N-(3-aminopropyl)-N,N'-bis-(dodecyloxyethyl)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(oleyloxyethyl)-piperazine bromide, N-(3-aminopropyl)-N, N'-bis-(palmitoyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N,N'-bis-(myristyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmitoyloxyethyl)-piperazineonium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristyloxyethyl)-piperazineonium bromide, 1,4-bis[(3-(3-aminopropyl)-oleylamino) [-2-hydroxypropyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxypropyl]piperazine or 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dimyristoyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dioleyloxy-1,4- N,N'-dimethyl-N,N'-di(3-amino-propyl)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(3-amino-propyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(3-amino-propyl)-diaminobutane, 2,3-dioleoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamide-spermine)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamide-spermine)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N'-dimethyl-N,N'-Di(5-carboxamide spermine)-diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dipalmitoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(histyl)-diaminobutane Butane, 2,3-dipalmitoyl-oxy-1,4-N,N'-dimethyl-N,N'-di(histyl)-diaminobutane, 2,3-dimyristoleoyl-oxy-1,4-N,N'-dimethyl-N,N'-di(histyl)-diaminobutane, 2,3-dioleoyl-oxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dipalmitoyl-oxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dimyristoleoyl-oxy-N,N'-dimethyl-1,4-diaminobutane; PAMAM dendrimers, NH3-core dendrimers, ethylenediamine-core dendrimers, polyethyleneimine, and polyethyleneimine conjugates.

87. The composition of claim 68, wherein the one or more PEG-modified lipids are selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-ceramide conjugate, PEG-modified dialkylamine, PEG-modified 1,2-diacoxypropyl-3-amine, and any combination thereof.

88. The composition of claim 68, wherein the one or more PEG-modified lipids are selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and any combination thereof.

89. The composition according to any one of claims 56 to 58, wherein the composition further comprises a transfection enhancer selected from the group consisting of endosome releasing agents, cell surface ligands, nuclear localizers, cell-penetrating peptides, fusion-promoting peptides, and any combination thereof.

90. A method for delivering a payload to a cell, the method comprising: (i) Providing a composition according to any one of claims 56 to 89; (ii) Provide cells; as well as (iii) Contact the cells with the composition.

91. A method for delivering a composition to a subject, the method comprising: The composition according to any one of claims 56 to 89 is administered to the subject.

92. The method of claim 90, wherein the contact with the cells is... in vitro It was carried out.

93. The method of claim 90, wherein contact with the cells is In vitro It was carried out.

94. The method of claim 90, wherein the contact with the cells is... in vivo It was carried out.

95. The method of claim 90, wherein the cell is a eukaryotic cell.

96. The method of claim 95, wherein the eukaryotic cell is a mammalian cell.

97. The method of claim 91, wherein the application is systemic.

98. The method of claim 91, wherein the administration is selected from the group consisting of: subcutaneous administration, intramuscular administration, intranasal administration, intratumoral administration, brain administration, spinal cord administration, ocular administration, administration to the lymph nodes of the subject, and any combination thereof.

99. A kit comprising: (i) one or more compounds according to claims 1 to 55, and (ii) One or more of structural lipids, ionizable lipids, and stabilizers.

100. The kit according to claim 99, wherein the kit comprises: (i) the compound according to any one of claims 1 to 55; (ii) One or more structural lipids; (iii) one or more stabilizers; and (iv) Optional payload.

101. The kit according to claim 99, wherein the kit comprises: (i) the compound according to any one of claims 1 to 55; (ii) One or more structural lipids; (iii) One or more stabilizers; (iv) one or more fusion agents; and (v) Optional payload.

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