Lipids for nucleic acid delivery to eukaryotic cells

Abstract

An ionizable lipid useful for delivering macromolecules such as nucleic acids to eukaryotic cells is provided. This lipid can be used alone, in combination with other lipids, and / or in combination with other transfection-enhancing reagents to prepare transfection complexes.

Description

[Technical Field]

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

[0002] Transfection is the process of introducing nucleic acids into eukaryotic cells by non-viral methods. Transfection methods allow the introduction of negatively charged molecules (e.g., the phosphate backbone of DNA and RNA) into cells with negatively charged membranes. Chemicals such as calcium phosphate and DEAE-dextran, or cationic lipid-based reagents, coat the DNA, neutralizing or even generating the overall positive charge on the molecule. DNA transfection reagent complexes readily cross the cell membrane, especially lipids with "fusing" components that enhance fusion with the cell's lipid bilayer.

[0003] With recent advances in nucleic acid-based therapies and the ongoing need for low-toxicity transfection reagents, there is a continuing need for novel ionizable lipids that can be used in vitro and in vivo. [Overview of the project]

[0004] Compounds, compositions, and methods for improving the efficiency of introducing macromolecules such as nucleic acids or small molecules (e.g., therapeutic agents) into cells are disclosed herein. The compounds are provided together with compositions containing these compounds, and methods for using these novel compounds and compositions to deliver a payload (e.g., nucleic acids or small molecules) to cells. The compounds may be used alone for transfection or in combination with additional reagents in the transfection composition. For example, the novel compounds may be complexed with one or more ionizable lipids and / or neutral lipids, one or more cell surface ligands, one or more fusion enhancers, one or more nuclear localizers, and one or more amphiphilic peptides, and any combination thereof, and may be used to deliver these macromolecules to cells.

[0005] Therefore, in one embodiment, the present disclosure relates to a compound having formula I: JPEG2026528786000002.jpg2590, or a pharmaceutically acceptable salt thereof, in the formula, A1 is -(CH2) x - or - (CH2) y -A4-(CH2) z -and, A4 is -(CH2) x -, -(CO)O-, -O(CO)-, -SS-, JPEG2026528786000003.jpg1420, JPEG2026528786000004.jpg1624, JPEG2026528786000005.jpg1621 JPEG2026528786000006.jpg2032, and Selected from the group consisting of JPEG2026528786000007.jpg2039, Q1 is N or The file is JPEG2026528786000008.jpg2521, and Q2 is N or is JPEG2026528786000009.jpg2521, R1 and R2 are, independently of each other, H, optionally substituted C1 - C 30 linear or branched alkyl, optionally substituted C4 - C 30 monounsaturated or polyunsaturated linear or branched alkenyl, or optionally substituted C4 - C 30 monounsaturated or polyunsaturated linear or branched alkynyl group, or -(CH2) 1~7 selected from the group consisting of COOR, R3 is -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n aryl, -(CH2) n arylalkyl, -(CH2) n Het, optionally substituted by optionally substituted cycloalkyl, -COR-(CH2) n COR or -(CH2) n COOR or -(CO)NHR- or -(CO)N(R)2-, is JPEG2026528786000010.jpg2230, or is JPEG2026528786000011.jpg3254, or C1 - C 30 linear or branched alkyl group, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n aryl, -(CH2) n arylalkyl, -(CH2) n Het, optionally substituted by optionally substituted cycloalkyl, C4 - C 30 monounsaturated or polyunsaturated linear or branched alkenyl group, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) nSR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C4~C 30 Monounsaturated or polyunsaturated linear or branched alkynyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl-(CH2) n C3-C6 cycloalkyl groups optionally substituted with Het, or C3-C6 cycloalkyl groups in which the ring carbon is replaced by -O-, -S-, -SS-, or -NR7-, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n A C3-C6 cycloalkenyl group optionally substituted with Het, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7-, or aryl groups, C1~C 30 Linear or branched alkyl groups, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups, C4~C 30 Whether it is a monounsaturated or polyunsaturated linear or branched alkenyl group, R3 is such that R1 and R2 are independent of each other -(CH2) 1~7 H is only true if COOR is true. R4 is replaced by H, or C1-C which are optionally replaced. 20 Linear or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R5 and R6 are independently selected from H and CH3. R7 is H, or optionally substituted C1-C6 linear or branched alkyl, or optionally substituted monounsaturated or polyunsaturated C1-C6 linear or branched alkenyl. JPEG2026528786000012.jpg2434, or The file is JPEG2026528786000013.jpg3356, R is optionally substituted into C4~C 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3 Selected from the group consisting of Het, A2 and A3 independently, H, JPEG2026528786000014.jpg1730, JPEG2026528786000015.jpg2431, JPEG2026528786000016.jpg2531 JPEG2026528786000017.jpg2030, JPEG2026528786000018.jpg1629, JPEG2026528786000019.jpg1628, JPEG2026528786000020.jpg1529, JPEG2026528786000021.jpg1530, JPEG2026528786000022.jpg1428, JPEG2026528786000023.jpg2133, JPEG2026528786000024.jpg2133, and Selected from the group consisting of JPEG2026528786000025.jpg2133, R8, -COR JPEG2026528786000026.jpg2329 or The file is JPEG2026528786000027.jpg3051, In the formula, R9 is H, JPEG2026528786000028.jpg2227 or The file is JPEG2026528786000029.jpg3051, In the formula, R 10 However, H, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C 30 Linear or branched alkyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl-(CH2) n A C3-C6 cycloalkyl group optionally substituted with Het, C3-C6 cycloalkyl groups in which the ring carbon is replaced by -O-, -S-, -SS-, -NR7-, and -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2)n Ariel, -(CH2) n Arylalkyl, -(CH2) n Selected from the group consisting of C3-C6 cycloalkenyl groups optionally substituted with Het, where R 11 However, -NH2, -NHR, -N(R)2, JPEG2026528786000030.jpg3044, JPEG2026528786000031.jpg2741, and Selected from the group consisting of JPEG2026528786000032.jpg2743, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 6. b is an integer between 0 and 6. x is an integer from 1 to 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n1, n2, n3, n4, and n5 are independent integers between 1 and 5. n6 is an integer between 0 and 7. n7 and n8 are independent integers between 0 and 5. n9 is an integer between 1 and 5. m1 is an integer between 1 and 5. m2 is an integer between 0 and 5. m3 is an integer between 1 and 7. The present invention provides a compound, or a pharmaceutically acceptable salt thereof, in which p is an integer between 1 and 50, and Het is an optionally substituted 5-7 member monocyclic basic heterocycle or an optionally substituted 8-11 member bicyclic basic heterocyclil.

[0006] In another embodiment, the present disclosure relates to a compound having the structure of formula Ia, JPEG2026528786000033.jpg2591 or a pharmaceutically acceptable salt thereof, wherein the formula, R1 and R2 are substituted independently and arbitrarily into C1-C 30Linear or branched alkyl groups, and optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, or -(CH2) 1~7 Selected from the group consisting of COOR, A2 and A3 are independent of each other. JPEG2026528786000034.jpg1530, JPEG2026528786000035.jpg2431, JPEG2026528786000036.jpg2131, JPEG2026528786000037.jpg1628, JPEG2026528786000038.jpg1531, JPEG2026528786000039.jpg2032, JPEG2026528786000040.jpg2234, and Selected from the group consisting of JPEG2026528786000041.jpg2234, R3 is -COR, -(CH2) n COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2- JPEG2026528786000042.jpg2231, or JPEG2026528786000043.jpg3051, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) nN(R7)2, -(CH2) n aryl, -(CH2) n arylalkyl, -(CH2) n Het, optionally substituted by optionally substituted cycloalkyl, C4 - C 30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n aryl, -(CH2) n arylalkyl, -(CH2) n Het, optionally substituted by optionally substituted cycloalkyl, C4 - C 30 monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl group, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n aryl, -(CH2) n arylalkyl-(CH2) n C3 - C6 cycloalkyl group optionally substituted by Het, or C3 - C6 cycloalkyl group in which the ring carbon is replaced by -O-, -S-, -S - S-, -NR7-, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n aryl, -(CH2) n arylalkyl, -(CH2) n C3 - C6 cycloalkenyl group optionally substituted by Het, or C1 - C in which the chain carbon is replaced by -O-, -S-, -S - S-, -S - NR7 - S-, -NR7 - S - S - NR7-, -NR7 - S - NR7-, or an aryl group 30Linear or branched alkyl groups, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups, C4~C 30 Whether it is a monounsaturated or polyunsaturated linear or branched alkenyl group, R3 is such that R1 and R2 are independent of each other -(CH2) 1~7 H is only true if COOR is true. R4 is replaced by H, or C1-C which are optionally replaced. 20 Linear or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is optionally substituted into C4~C 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3 Selected from the group consisting of Het, In the formula, R 10 However, H, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C 30 Linear or branched alkyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) nArylalkyl-(CH2) n Selected from the group consisting of C3-C6 cycloalkylalkyl groups that are optionally substituted with Het, In the formula, AA represents any natural or unnatural amino acid side chain, x is an integer from 1 to 9. n is an integer between 1 and 5. n5 is an integer between 1 and 5. n6 is an integer between 0 and 7. n7 and n8 are independent integers between 0 and 5. n9 is an integer between 1 and 5. m3 is an integer between 1 and 3. p is an integer between 1 and 50. The present invention provides compounds, or pharmaceutically acceptable salts thereof, in which Het is optionally substituted with a 5-7 member monocyclic basic heterocycle or an optionally substituted with an 8-11 member bicyclic basic heterocyclil.

[0007] In other embodiments, the Disclosure provides compositions and methods for introducing nucleic acids, proteins, or peptides into cells by contacting eukaryotic cells with a compound of formula I or a composition thereof.

[0008] In other embodiments, this disclosure is, (i) One or more compounds formed by the compound of formula I, (ii) one or more of structural lipids, ionizable lipids, and stabilizers, (iii) optionally, provide a composition comprising a payload.

[0009] In other embodiments, the 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.

[0010] In other embodiments, the present disclosure provides a kit comprising: (i) one or more compounds by a compound of Formula I; and (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizer; and (iii) optionally, a payload. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] [Figure 1] A graph showing the size (d.nm) and polydispersity index of a lipid-mRNA formulation. [Figure 2] A graph showing luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice after intravenous administration of a lipid-mRNA formulation. [Figure 3] A graph showing the ratio of luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice to luciferase activity (bioluminescence flux, photons / second (p / s)) in the spleen after intravenous administration of a lipid-mRNA formulation. [Figure 4] A graph showing luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice after intravenous administration of a lipid-mRNA formulation. [Figure 5] A graph showing the ratio of luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice to luciferase activity (bioluminescence flux, photons / second (p / s)) in the spleen after intravenous administration of a lipid-mRNA formulation. [Figure 6] A graph showing transfection efficiency two days after transfection. [Figure 7] A graph showing GFP expression levels two days after transfection. [Figure 8] A graph showing viable T cells two days after transfection. [Figure 9] A graph showing the size (d.nm) of a lipid-mRNA formulation. [Figure 10]A graph showing the polydispersity index (PDI) of the lipid-mRNA formulation. [Figure 11] A graph showing the luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice after intravenous administration of the lipid-mRNA formulation. [Figure 12] A graph showing the ratio of luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice to luciferase activity (bioluminescence flux, photons / second (p / s)) in the spleen after intravenous administration of the lipid-mRNA formulation. [Figure 13] A graph showing the size (d.nm) and polydispersity index of the lipid-mRNA formulation. [Figure 14] A graph showing the luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice after intravenous administration of the lipid-mRNA formulation. [Figure 15] A graph showing the ratio of luciferase activity (bioluminescence flux, photons / second (p / s)) in the liver of mice to luciferase activity (bioluminescence flux, photons / second (p / s)) in the spleen after intravenous administration of the lipid-mRNA formulation. [Figure 16] A graph showing GFP expression 48 hours after transfection using compound 8 formulated with DOPE at a ratio of 1:4 in water. By adding peptide, SEQ ID NO: 350 to the lipid, the transfection efficiency increases as seen by the increase in GFP expression. By adding a second peptide, SEQ ID NO: 47, the GFP expression further increases compared to only 15 - 24. [Figure 17] A graph showing that the Prestoblue fluorescence of the lipid-only formulation shows minimal toxicity, while the formulation with one or two peptides shows a toxicity profile similar to that of the Expi293 transfection reagent. [Figure 18] A graph showing the size (d.nm) of the lipid-mRNA formulation. [Figure 19] A graph showing the PDI of the lipid-mRNA formulation. [Figure 20]This graph shows the luciferase activity (bioluminescence flux, photons / second (p / s)) in the spleen of mice after intravenous administration of a lipid-mRNA preparation. [Figure 21] This graph shows the luciferase activity (bioluminescence flux, photons / second (p / s)) in the lungs of mice after intravenous administration of a lipid-mRNA preparation. [Modes for carrying out the invention]

[0012] This disclosure relates to ionizable lipids and lipid compositions (e.g., lipoplexes and lipid nanoparticle compositions) containing lipids provided herein. This disclosure also provides methods for delivering therapeutic and / or prophylactic agents to mammalian cells, methods for specifically delivering therapeutic and / or prophylactic agents to mammalian organs, methods for generating a polypeptide of interest within mammalian cells, and methods for doing so in mammals requiring treatment of a disease or disorder. For example, a method for generating a polypeptide of interest within cells comprises contacting a nanoparticle composition containing mRNA with mammalian cells, thereby allowing the mRNA to be translated to produce the polypeptide of interest. A method for delivering therapeutic and / or prophylactic agents to mammalian cells or organs may comprise administering a nanoparticle composition containing the therapeutic and / or prophylactic agent to a target, the administration comprising contacting the cell or organ with the composition, thereby delivering the therapeutic and / or prophylactic agent to the cell or organ.

[0013] An improved method for delivering macromolecules to eukaryotic cells is provided, comprising ionizable molecules useful for this purpose. The composition and method are effective for a variety of cells and offer high transfection efficiency. Specifically, molecules based on the core of Formula I have been found to be useful for the efficient delivery of macromolecules to cells. These molecules can be advantageously used together with a payload, one or more neutral lipids, and additional components, such as fusion-promoting or fusion-advancing molecules, additional cationic / ionizable lipids, cell surface ligands, cell adhesion molecules, nuclear localization agents, and endosomal release agents (for example, in complexes with macromolecules or pharmaceuticals, or nutrients).

[0014] This complex is easily prepared by a simple method, can be used on a wide variety of cells, is stable, and is therefore suitable for in vitro, ex vivo, and in vivo applications, such as the delivery of therapeutic nucleic acids (e.g., siRNA therapeutics, mRNA vaccine preparations, etc.), osomes, pharmaceuticals, nutrients, etc. to cells, for example, in cosmetics, nutritional supplements, or therapeutic applications.

[0015] general definition The following definitions are included for the purpose of understanding this subject matter and constructing the appended claims. Abbreviations used herein have their conventional meanings within the art of chemistry and biology.

[0016] While various embodiments and aspects of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided only as examples. Many variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in carrying out the present invention.

[0017] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein. However, all documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, manuals, and papers, are expressly incorporated herein in whole by reference for any purpose.

[0018] Unless otherwise defined, technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994) and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices, and materials similar or equivalent to those described herein may be used in the practice of the present invention. The following definitions are provided to facilitate the understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0019] When used in relation to numerical ranges, cutoffs, or specific values, the term “approximately” is used to indicate that the listed values ​​may vary by up to 25% from the listed values. Since many of the numerical values ​​used herein are determined experimentally, it should be understood by those skilled in the art that such determinations may vary between different experiments and will often fluctuate. The values ​​used herein should not be considered overly restrictive due to this inherent variability. The term “approximately” is used to encompass variations of ±25%, ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from a given value. “Approximately” may 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 described value. Unless otherwise evident from the context, all numerical values ​​provided herein are qualified by the term “approximately.”

[0020] In the description and claims herein, phrases such as “at least one” or “one or more of the following” may appear, followed by a connecting list of elements or features. The term “and / or” may also appear in lists of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such phrases are intended to mean any of the enumerated elements or features individually, or any of the enumerated elements or features in combination with any of the other enumerated 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 intended to mean “A only, B only, or A and B together,” respectively. A similar interpretation is also intended for lists containing 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 intended to mean "A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together," respectively. In addition, the use of the term "based on" in the foregoing and in the claims is intended to mean "at least partially based on" so that features or elements not enumerated are also permitted.

[0021] If a parameter range is provided, it is understood that all integers within that range and their tenths are also provided by the present invention. For example, "0.2 to 5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc., up to a maximum of 5.0 mg and including 5.0 mg.

[0022] Compounds are generally described herein using standard nomenclature. For listed compounds having a chiral center, all stereoisomers and mixtures thereof are included unless otherwise specified. Non-restrictive examples of stereoisomers include enantiomers, diastereomers, and E or Z isomers. Where listed compounds exist in various tautomeric forms, the compound is intended to encompass all tautomeric forms. A particular compound is described herein using a general formula containing variables (e.g., X, L1, L2, L3, Y, etc.). Unless otherwise specified, each variable in such a formula is defined independently of any other variable, and any variable appearing more than once in the formula is defined independently in each appearance. When a part is described as being selected "independently" from a group, each part is selected independently of one another. Thus, each part may be identical or different from one or more other parts.

[0023] The number of carbon atoms in the hydrocarbyl moiety is determined by the prefix "C". x ~C y This can be shown by the formula, where x is the minimum number of carbon atoms in the part and y is the maximum number. Therefore, for example, "C1-C6 alkyl" refers to an alkyl substituent containing 1 to 6 carbon atoms. To illustrate further, C3-C6 cycloalkyl means a saturated hydrocarbyl ring containing 3 to 6 carbon ring atoms. Prefixes attached to multicomponent substituents apply only to the first component immediately following the prefix. To illustrate, the term "carbocyclylalkyl" contains two components, namely a carbocyclyl and an alkyl. Therefore, for example, C3-C6 carbocyclyl C1-C6 alkyl refers to a C3-C6 carbocyclyl added to the parent molecule through a C1-C6 alkyl group.

[0024] Unless otherwise specified, when a linking element links two other elements in the depicted chemical structure, the leftmost component of the linking element is linked to the leftmost element in the depicted structure, and the rightmost component of the linking element is linked to the rightmost element in the depicted structure. For example, if the chemical structure is -L S-M-L S ”- and when M is -N(R B )S(O)-, the chemical structure is -L S -N(R B )S(O)-L S ”- is.

[0025] When the linking element in the described structure is a bond, the element on the left side of the linking element is directly bonded to the element on the right side of the linking element via a covalent bond. For example, when the chemical structure is -L S -M-L S ’ is described and M is selected as a bond, the chemical structure is -L S -L S ’’-. When two or more adjacent linking elements in the described structure are bonds, the elements on the left side of these linking elements are directly bonded to the elements on the right side of these linking elements via covalent bonds. For example, when the chemical structure is -L S -M-L S ’’-M’-L S ’’- is described and M and L S ’ are selected as bonds, the chemical structure is -L S -M’-L S ’’-. Similarly, when the chemical structure is -L S -M-L S ’’-M’-L S ’’- is described and M, L S ’, and M’ are bonds, the chemical structure is -L S -L S ’’-. When a chemical formula is used to describe a part, the dash indicates a part with a free valence.

[0026] When a part is described as being "optionally substituted," that part may be either substituted or unsubstituted. When a part is described as being optionally substituted by up to a certain number of non-hydrogen radicals, that part may be unsubstituted or substituted by up to that certain number of non-hydrogen radicals or up to a maximum number of substituted positions on that part, whichever is less. For example, when a part is described as a heterocycle optionally substituted by up to three non-hydrogen radicals, any heterocycle with fewer than three substituted positions may be optionally substituted by only the same number of non-hydrogen radicals as the heterocycle has substituted positions. For example, tetrazolyl (which has only one substituted position) may be optionally substituted by up to one non-hydrogen radical. Similarly, when an amino nitrogen is described as being optionally substituted by up to two non-hydrogen radicals, the primary amino nitrogen may be optionally substituted by up to two non-hydrogen radicals, while the secondary amino nitrogen may be optionally substituted by only one non-hydrogen radical.

[0027] When a portion is substituted with an oxo or thioxo, it means that the portion contains a carbon atom covalently bonded to at least two hydrogens (e.g., CH2), and the two hydrogen radicals are substituted with an oxo or thioxo to form C=O or C=S, respectively.

[0028] The terms “alkyl” or “alkyl group” or “alkylene group” refer to a straight or branched hydrocarbon chain that is fully saturated (i.e., does not contain double or triple bonds). Alkyl groups may have 1 to 20 carbon atoms (wherever they appear herein, numerical ranges such as “1 to 20” refer to each integer within a given range; for example, “1 to 20 carbon atoms” means that an alkyl group may consist of 20 or fewer carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., but this definition also covers appearances of the term “alkyl” where no numerical range is specified) and are optionally substituted. Alkyl groups may also be medium-sized alkyl groups having 1 to 9 carbon atoms. Alkyl groups may also be lower alkyl groups having 1 to 4 carbon atoms. The alkyl group of a compound is “C 1~4 Alkyl," "C 1~20 Alkyl," "C 4~30 It may be specified as "alkyl" or a similar designation. Notation "C 4~30 "Alkyl" refers to a linear or branched saturated hydrocarbon containing 4 to 30 carbon atoms, which are optionally substituted.

[0029] Unless otherwise specified, alkyl groups as used herein refer to both unsubstituted and substituted alkyl groups. For example, alkyl groups in the linear or branched hydrocarbon chains described above may be substituted with hydroxyl groups. In another embodiment, the methylene group is bonded via a disulfide bridge (-SS-).

[0030] Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, ter-butyl, pentyl, isoamyl, hexyl, lauryl, palmityl, and stearyl.

[0031] The term "alkenyl," either alone or in combination with any other term, refers to a specified number of carbon atoms, or, if no number is specified, in one embodiment, 2 to 30 carbon atoms (i.e., (C) 2~30(Alkenyl), in one embodiment 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 The term "alkenyl" refers to a linear or branched monounsaturated or polyunsaturated aliphatic hydrocarbon radical containing an alkenyl. As used herein, the terms "alkenyl" or "alkenyl group" mean a linear or branched hydrocarbon containing two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one optionally substituted double bond. 2~14 An "alkenyl" refers to an optionally substituted straight or branched hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may contain one, two, three, four, or more carbon-carbon double bonds. For example, a cis-alkenyl may contain one or more double bonds. A C5 alkenyl group containing two double bonds may be a linoleyl group.

[0032] Unless otherwise specified, the alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups. For example, the alkenyl groups in the linear or branched hydrocarbon chains described above may be substituted with hydroxyl groups. In another embodiment, the alkenyl groups are linked via disulfide bridges (-SS-).

[0033] Examples of alkenyl radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl, E,E-, E,Z-, Z,E-, and Z,Z-hexadienyl, oleoyl, palmitreol, etc.

[0034] The term "alkynyl," alone or in combination with any other term, refers to a linear or branched hydrocarbon radical having one or more triple bonds, comprising a specified number of carbon atoms, or, if no number is specified, in one embodiment, 2 to about 20 carbon atoms. As used herein, the terms "alkynyl" or "alkynyl group" mean a linear or branched hydrocarbon comprising two or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more carbon atoms) and at least one optionally substituted carbon-carbon triple bond. The notation "C2-14 alkynyl" means an optionally substituted linear or branched hydrocarbon comprising 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 may contain one or more carbon-carbon triple bonds. Unless otherwise specified, the alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

[0035] Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, and pentynyl.

[0036] The term "basic heterocycle" refers to a stable 5- to 7-membered monocyclic heterocycle or an 8- to 11-membered bicyclic heterocycle that is either saturated or partially unsaturated, and in the case of monocyclics, may be optionally benzo-condensed, and bonded on one or more carbon atoms by halogens, alkyls, alkoxys, oxos, etc., and / or on a secondary nitrogen atom (i.e., -NH-) by alkyls, aralkoxycarbonyls, alkanoyls, phenyls, or phenylalkyls, or on a tertiary nitrogen atom (i.e., +N-) by oxides, and via carbon atoms.

[0037] The terms “heterocyclic,” “heterocyclo,” or “heterocyclyl” refer to a saturated (e.g., “heterocycloalkyl”), partially unsaturated (e.g., “heterocycloalkenyl” or “heterocycloalkynyl”), or fully unsaturated (e.g., “heteroaryl”) ring system in which at least one of the ring atoms 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 heterocyclic group can be a monocyclic, a fused ring of two rings, or a bridged or spirocyclic ring. A heterocyclic group can be linked to the parent molecular part via any two substituteable carbon or nitrogen atoms in the group. If the heterocyclic group is a divalent moiety linking two other elements in the described chemical structure, the heterocyclic group can be linked to the two other elements via any two substituteable ring atoms. Similarly, if a heterocyclic group is a trivalent moiety that links three other elements in the described chemical structure, then each heterocyclic group can be bonded to the three other elements through any three substitutable ring atoms.

[0038] In this compound, "Het" indicates a heterocycle containing 4 to 12 carbon atoms with at least one nitrogen atom present in the ring. A heterocyclyl can be a monocycle containing a single ring, but is not limited to this.Non-restrictive examples of monocyclic compounds include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinil, pyrrolidinil, imidazolyl, isoimidazolyl, imidazolinil, imidazolidinil, pyrazolyl, pyrazolinil, pyrazolidinil, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinil, isothiazolinil, thiazolidinil, isothiazolidinil, thiodiazolyl, oxathiazolyl, oxadiazolyl (1, 2,3-Oxadiazolyl, 1,2,4-Oxadiazolyl (also known as "Azoximil"), 1,2,5-Oxadiazolyl (also known as "Furazanil") and 1,3,4-Oxadiazolyl, Oxatriazolyl (also known as 1,2,3,4-Oxatriazolyl and 1,2,3,5-Oxatriazolyl), Dioxazolyl (also known as 1,2,3-Dioxazolyl, 1,2,4-Dioxazolyl, 1,3,2-Dioxazolyl, and 1,3,4-Dioxazolyl), Pyridinyl, Piperidinyl, Diazinyl ( Pyridanidyl (also known as "1,2-diadinyl"), pyrimidinyl (also known as "1,3-diadinyl"), and pyrazinyl (also known as "1,4-diadinyl"), 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 (1,2,3-oxazinyl, 1,3,2-oxazinyl, Examples include 1,3,6-oxazinyl (also known as "pentoxazolyl"), 1,2,6-oxazinyl, and 1,4-oxazinyl), isoxazinyl (including o-isoxazinyl and p-isoxazinyl), oxazolidinyl, isoxazolidinyl, oxathiadinyl (including 1,2,5-oxathiadinyl or 1,2,6-oxathiadinyl), oxadiadinyl (including 1,4,2-oxadiadinyl and 1,3,5,2-oxadiadinyl), morpholinyl, azepinyl, and diazepinyl.

[0039] Heterocyclines can also be bicyclic compounds containing two fused rings, such as naphthilidinyl (including [1,8]naphthilidinyl and [1,6]naphthilidinyl), thiazolepyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, pyridopyrimidinyl, pyrazolopyrimidinyl, indolidinyl, pyrindinyl, pyranopyrrolyl, 4H-quinolidinyl, purinyl, pyridopyrimidinyl (including pyrido[3,4-b]-pyrimidinyl, pyrido[3,2-b]-pyrimidinyl, and pyrido[4,3-b]-pyrimidinyl), pyridopyrimidine, and pteridinyl. Other non-restrictive examples of fused heterocycles include indolyl, isoindolyl, indreninyl (also known as "pseudoindolyl"), isoindazolyl (also known as "benzpyrazolyl" or indazolyl), benzazinyl (including quinolinyl (also known as "1-benzazinyl") and isoquinolinyl (also known as "2-benzazinyl")), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiadinyl (sinnolinyl (also known as "1,2-benzodiadinyl") and quinazolinyl ("1,3-benzodiadinyl") Examples include benzothiazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, benzothiadiazolyl, benzimidazolyl, benzotriazolyl, benzoxazinyl (including 1,3,2-benzoxazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl, and 3,1,4-benzoxazinyl), benzisoxazinyl (including 1,2-benzisoxazinyl and 1,4-benzisoxazinyl), and benzo-condensed heterocyclines such as tetrahydroisoquinolinyl.

[0040] Heterocyclines can also be spirocyclic systems, such as, but are not limited to, 1,4-dioxa-8-azaspiro[4.5]decanyl. Heterocyclines may contain one or more sulfur atoms as ring members, and in some cases, the sulfur atoms are oxidized to SO or SO2. Nitrogen heteroatoms in heterocyclines may or may not be quaternized, and may or may not be oxidized to N-oxides. In addition, nitrogen heteroatoms may or may not be N-protected.

[0041] Heterocyclic or carbocyclic rings may be further substituted. Unless otherwise specified, the term “substituted” means that one, two, or three or more hydrogen atoms are replaced with -F, -Cl, -Br, -I, hydroxy, protected hydroxy, -NO2-N3, -CN, -NH2, protected amino, oxo, thioxo, -NH-C2~C8-alkenyl, -NH-C2~C8-alkynyl, -NH-C3~C 12 -Cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-C1~C 12 -alkyl, -O-C2~C8-alkenyl, alkynyl, -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-alkynyl, -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-alkynyl, -CONH-C3~C 12 -Cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-C1~C 12 -alkyl, -OCO2-C2~C8-alkenyl, -OCO2-C2~C8-alkynyl, -OCO2-C3~C 12-Cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2-heterocycloalkyl, -OCONH2-OCONH-C1-C 12 -alkyl, -OCONH-C2~C8-alkenyl, -OCONH-C2~C8-alkynyl, -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-alkynyl, -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-alkynyl, -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-alkynyl, -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-alkynyl, -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-alkynyl, -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-alkynyl, -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-C1~C 12-alkyl, -S-C2~C8-alkenyl, -S-C2~C8-alkynyl, -S-C3~C 12 This refers to substitution by independently replacing substituents, including but not limited to -cycloalkyl, -S-aryl, -heteroaryl, -S-heterocycloalkyl, or methylthiomethyl. It is understood that aryl, heteroaryl, alkyl, etc., may be further substituted.

[0042] The term "N-protecting group" or "N-protected" refers to a group that can protect an amino group from undesirable reactions. Commonly used N-protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3 rd(ed., John Wiley & Sons, NY (1999))) Non-limiting examples of N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-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; phenylsulfenyl (phenyl-S-) or triphenylmethylsulfenyl ( Sulfenyl groups such as trityl-S-; sulfinyl groups such as p-methylphenylsulfinyl (p-methylphenyl-S(O)-) or t-butylsulfinyl (t-Bu-S(O)-); benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl Cicarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, Examples include carbamate-forming groups such as methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, or phenylthiocarbonyl; alkyl groups such as benzyl, p-methoxybenzyl, triphenylmethyl, or benzyloxymethyl; p-methoxyphenyl; and silyl groups such as trimethylsilyl.Preferred N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

[0043] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0044] As used herein, “natural amino acid side chain” refers to a side chain substituent of a naturally occurring amino acid. Naturally occurring amino acids have substituents bonded to the α-carbon. Examples of 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.

[0045] As used herein, "unnatural amino acid side chain" refers to a side chain substituent of an unnatural amino acid. Examples of unnatural amino acids include β-amino acids (β 3 and β 2 Examples include homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, pyrrolicine, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids and N-methyl amino acids. Exemplary non-natural amino acids are available from Sigma-Aldrich, 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 (the whole is incorporated by reference).

[0046] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, and polymers may, in embodiments, be conjugated to portions that are not amino acids. The above terms apply to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring and non-naturally occurring amino acid polymers. “Fusion protein” refers to a chimeric protein that encodes two or more distinct protein sequences, expressed recombinantly as a single portion or chemically synthesized.

[0047] The term "alkoxy" refers to alkyl, alkenyl, or alkynyl ether radicals, and the terms "alkyl," "alkenyl," or "alkynyl" are defined above. Suitable examples of alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, oleyloxy, palmityloxy, and palmitoleoyloxy.

[0048] The term "aryl" alone or in combination with any other term refers to a specified number of carbon atoms, in one embodiment 6 to 15 carbon atoms (i.e., (C)) optionally substituted with one or more substituents selected from alkyl, alkoxy, (e.g., methoxy), nitro, halogen, (e.g., chloro), amino, carboxylate, and hydroxyl. 6~15 In another embodiment, aryl atoms, 6 to 10 carbon atoms (i.e., (C) 6~10 This refers to carbocyclic aromatic radicals (such as phenyl or naphthyl) that contain aryl radicals. Examples of aryl radicals include, but are not limited to, phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azlenyl, fluorenyl, and anthracenyl.

[0049] The term "aralkyl" or "arylalkyl" refers to an aryl group linked via an alkylene group as a substituent, such as "C 7~14 "Aralkyl" and similar compounds include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group may be a lower alkylene group (i.e., C 1~4 It is an alkylene group. Furthermore, the terms "aralkyl" or "arylalkyl" mean, alone or in combination, an alkyl radical as defined above, in which one hydrogen atom is phenyl, benzyl, 2-phenylethyl, etc.

[0050] The term "aralkoxycarbonyl," either alone or in combination with "aralkyl," refers to a radical of the formula -C(O)-O-aralkyl, where "aralkyl" has the meaning defined above. An example of an aralkoxycarbonyl radical is benzyloxycarbonyl.

[0051] The term "aryloxy," either alone or in combination with "aryl," means a radical of the formula aryl-O-, where the term "aryl" has the meaning defined above.

[0052] The term "alkanoyl," either alone or in combination, refers to an acyl radical derived from an alkanecarboxylic acid, such as acetyl, propionyl, butyryl, valeryl, and 4-methylvaleryl.

[0053] The term "aryloxyalkanoyl" refers to the acyl radical of the formula aryl-O-alkanoyl, where aryl and alkanoyl have the meanings set forth above.

[0054] The term "aralkanoyl" refers to acyl radicals derived from aryl-substituted alkanecarboxylic acids such as phenylacetyl, 3-phenylpropionyl(hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-phenylbutyryl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, and 4-methoxyhydrocinnamoyl.

[0055] The term "aloyl" refers to an acyl radical derived from an aromatic carboxylic acid. Examples of such radicals include aromatic carboxylic acids, optionally substituted benzoic acid, or naphthoic acid, 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, and 3-(benzyloxyformamide)-2-naphthoyl.

[0056] The term "aminocarbonyl" refers to an amino-substituted carbonyl (carbamoyl) group derived from an amino-substituted carboxylic acid, which, alone or in combination, may be a primary, secondary, or tertiary amino group, with the amino group followed by a substituent selected from hydrogen, alkyl, aryl, aralkyl, cycloalkyl, or cycloalkylalkyl radical.

[0057] The term "aminoalkanoyl" refers to an acyl radical derived from an amino-substituted alkanecarboxylic acid, where the amino group may be a primary, secondary, or tertiary amino group containing a substituent selected from hydrogen, cycloalkyl, or cycloalkylalkyl radical, such as N,N-dimethylaminoacetyl and N-benzylaminoacetyl.

[0058] The term "carbocyclic ring" refers to a stable, non-aromatic 3- to 8-membered carbon ring, which may be saturated, monounsaturated, or polyunsaturated. A carbocyclic ring can be bonded at any intra-ring carbon atom that results in a stable structure. In one embodiment, the carbocyclic ring has 5 to 7 carbon atoms.

[0059] The term "cycloalkyl" refers to alkyl radicals that, alone or in combination, contain approximately 3 to 8 carbon atoms and are cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0060] The term "cycloalkylalkyl" means the alkyl radical as defined above, which is substituted with a cycloalkyl radical containing approximately 3 to approximately 8 carbon atoms, and in one embodiment, approximately 3 to approximately 6 carbon atoms.

[0061] The term "cycloalkylcarbonyl" refers to an acyl group derived from a benz-condensed monocyclic cycloalkanecarboxylic acid that is optionally substituted with a monocyclic or crosslinked cycloalkanecarboxylic acid such as cyclopropanecarbonyl, cyclohexanecarbonyl, or adamantanecarbonyl, or with an alkanoylamino such as 1,2,3,4-tetrahydro-2-naphthoyl or 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.

[0062] The term "cycloalkylalkoxycarbonyl" refers to an acyl group derived from a cycloalkylalkoxycarboxylic acid of the formula cycloalkylalkyl-O-COOH, where cycloalkylalkyl has the meanings described above.

[0063] The term "alkylation reaction" is as is known in the art (for example, Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. 293-871 (4) thed. John Wiley & Sons 1992)), SN 1 or SN 2 The mechanism may include a nucleophilic attack of the alkylation reaction center of the target compound on the electron-deficient region of the alkylating agent. Those skilled in the art will understand that in some embodiments the alkylating agent further comprises one or more leaving groups (LGs). "Leaving group" (X in scheme IV) and its grammatical equivalent herein means an atom or molecule separated 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 separated from an atom or molecule in what is considered a residue or major part of the substrate in a particular reaction. The ability of the leaving group to leave the alkylating agent may be a function of the ability of the leaving group. Thus, the leaving group may affect the intrinsic reactivity of the alkylating agent in the alkylation reaction. In some embodiments, the lower the pKa of the conjugate acid of the leaving group, the better the leaving group is, because in some embodiments, the leaving group can more easily stabilize the negative charge that may develop in the alkylation reaction. Therefore, in some embodiments, the leaving group may be an electronegative atom or molecule. An example of a leaving group is acetate (AcO - ), p-nitrobenzoate (PNBO - ), sulfonates (for example, methanesulfonate (mesylate: MsO) - ), p-toluenesulfonate (tosylate: TsO - ), p-bromobenzenesulfonate (brosilate: BsO - ), p-nitrobenzenesulfonate (nosilate: NsO - ), fluoromethanesulfonate, difluoromethanesulfonate, trifluoromethanesulfonate (triflate: TfO - ) and ethanesulfonates), halogen esters, and halogen ions (for example, I - , Br - Cl - ) are some examples, but are not limited to these.

[0064] The term "solid-phase bonded arylalkyl chloride" includes arylalkyl chlorides covalently bonded to polystyrene resins. Those skilled in the art will understand that polystyrene resins can have various reactive functional groups, such as chlorides as leaving groups, or other leaving groups as described above. Since Merrifield's (Merrifield, RB (1963), J. Am. Chem. Soc., 85, 2149-2153) pioneering work on polystyrene (2% divinylbenzene crosslinked) as a solid support for peptide synthesis, several improvements in the properties of solid supports have been made to meet the specific needs of new organic chemistry.

[0065] The composition may also contain one or more compounds, for example, a salt of the compound of formula I. The salt may be a pharmaceutically acceptable salt. As used herein, “pharmaceutically acceptable salt” means a derivative of the disclosed compound, the parent compound being modified by converting an existing acidic or base moiety to its salt form (for example, by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Typical acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphosulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, and lactobionate. Examples include phosphate, lactate, laurate, lauryl sulfate, malate, maleate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, parsulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Typical alkali 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, isopropylamine, etc.).Examples of pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. These pharmaceutically acceptable salts can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acidic or basic forms of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of the two, generally in non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., 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) (the entire work is incorporated herein by reference).

[0066] The terms “surface ligand” or “cell surface ligand” refer to a chemical compound or structure that binds to a cell surface receptor. As used herein, the term “cell surface receptor” refers to a specific chemical group on the cell surface to which a ligand can bind. Cell surface receptors are specific to a particular cell, i.e., they may be predominantly found in one type of cell rather than another (for example, LDL and asialoglycoprotein receptors are specific to hepatocytes). Receptors facilitate the internalization of ligands and bound molecules. Examples of cell surface receptors include, but are not limited to, folate receptors, biotin receptors, lipoic acid receptors, low-density lipoprotein receptors, asialoglycoprotein 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, osteomorphonidate receptors, cartilage-inducing factor receptors, or glycosylphosphatidylinositol (GPI)-anchored proteins (e.g., β-andrenaline receptors, T cell activating proteins, Thy-1 protein, GPI-anchored 5' nucleotidases). These are non-limiting examples.

[0067] A "receptor" is a molecule to which a ligand binds specifically and with relatively high affinity. Receptors are usually proteins or glycoproteins, but can also be glycolipids, lipid polysaccharides, glycosaminoglycans, or glycocalyxes. For the purposes of this disclosure, an epitope to which an antibody or fragment thereof binds is interpreted as a receptor because the antigen:antibody complex undergoes endocytosis. Furthermore, surface ligands include anything that can enter a cell through cytosis (e.g., endocytosis, potocytosis, pinocytosis).

[0068] As used herein, the term “ligand” refers to a chemical compound or structure that binds to a receptor. These ligands include, but are not limited to, asialo-orosomucoids, asialo-glycoproteins, lipoic acid, biotin, apolipoprotein E sequences, insulin-like growth factor II, calcitonin gene-related peptides, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, and RGD-containing cell adhesion peptides.

[0069] Those skilled in the art will readily recognize that the choice of ligand depends on which receptor is bound. Since different cell types have different receptors, this provides one method for targeting nucleic acids to specific cell types, depending on which cell surface ligand is used. Therefore, the preferred cell surface ligand may depend on the targeted cell type.

[0070] The terms “nuclear localization agent,” “nuclear localization signal,” or “nuclear ligand,” as used herein, refer to ligands such as peptides that, by typically binding to a nuclear receptor, localize a drug covalently or noncovalently bound to it to the cell nucleus. The term “nuclear receptor,” as used herein, refers to a chemical group on the nuclear membrane that binds to a particular ligand and helps transport the ligand and its associated binding site across the nuclear membrane. Nuclear receptors may, but are not limited to, those receptors that bind to nuclear localization sequences. Non-exclusive examples of nuclear ligands 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) is one example, and it can be used to transport nucleic acids to the nucleus.

[0071] As used herein, the term “solubilant” refers to a molecule, compound, protein, or peptide capable of degrading the endosomal membrane and releasing DNA transporters into the cytoplasm of a cell. This term includes, but is not limited to, viruses, synthetic compounds, lytic peptides, or derivatives thereof. The term “cytolytic peptide” refers to a chemical group that penetrates the membrane in such a way that the membrane’s structural organization and integrity are lost. As a result of the presence of a solubilant, the membrane undergoes lysis, fusion, or both. Examples of solubilants / endosomal release agents include chloroquine, polyamines, and polyamidoamines. Preferred agents are described, for example, in Pei and Buyanova, Bioconjugate Chem, 30:273-283 (2009) and Juliano, Nucleic Acid Therapeutics, 28:166-177 (2018).

[0072] As used herein, the term “polycationic nucleic acid binding moiety” refers to the portion containing multiple positive charges that enables the binding of negatively charged nucleic acids at physiological pH. Polycationic nucleic acid binding moieties can be linked, for example, to cell surface ligands, fusion agents, and / or nuclear localization peptides. The binding may be covalent. Suitable polycationic nucleic acid binding moieties include polyamines such as PEI, spermine, spermidine, and carboxyspermine, and polybasic peptides containing, for example, multiple lysine, ornithine, histidine, or arginine residues.

[0073] The term "nucleic acid" refers to any type of nucleic acid currently known or that may be prepared or identified in the future, unless otherwise applied to a specific type of molecule such as unmodified DNA or RNA, provided that the nucleic acid has a sufficiently negative charge to form lipid aggregates, liposomes, or liposome-like complexes when mixed with any lipid of formula I. As used herein, nucleic acid refers to single-stranded or double-stranded deoxyribonucleotides or ribonucleotides, as well as mixtures and polymers thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified skeletal residues or bindings, synthetic, naturally occurring, and non-naturally occurring, that have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include phosphorothioates, phosphoramides, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA), 5-methylcytosine, pseudouridine, and N 1 Examples include, but are not limited to, methyl-pseudridine and 5-methoxyuridine.

[0074] In some embodiments, the payload described herein may include mRNA modified with one or more nucleotides selected from the group consisting of: pseudouridine (abbreviated by 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).

[0075] In some embodiments, the payload described herein may include a ribonucleoprotein complex (e.g., Cas9 / guide RNA) that is delivered to cells with high efficiency. “Ribonucleoprotein complex” or “ribonucleoprotein particle” as provided herein refers to a complex or particle comprising a nucleoprotein and ribonucleic acid. “Guide RNA” or “gRNA” as provided herein refers to a ribonucleotide sequence that can bind to a nucleoprotein and thereby form a ribonucleoprotein complex. In embodiments, the guide RNA comprises one or more RNA molecules. “Nucleoprotein” as provided herein refers to a protein that can bind to nucleic acids (e.g., RNA, DNA). When a nucleoprotein binds to ribonucleic acid, it is referred to as a “ribonucleoprotein.” Interactions between a ribonucleoprotein and ribonucleic acid may be direct, for example, by covalent bonding, or indirect, for example, by non-covalent bonding (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 (PI effect), hydrophobic interactions, etc.). Non-limiting examples of ribonucleoproteins include ribosomes, telomerases, RNAseP, hnRNP, CRISPR-related protein 9 (Cas9), and micronuclear RNP (snRNP). Ribonucleoproteins can be enzymes. In some embodiments, the ribonucleoprotein is an endonuclease. Therefore, in some embodiments, the ribonucleoprotein complex includes an endonuclease and ribonucleic acid. In some embodiments, the endonuclease is CRISPR-related protein 9.

[0076] Nucleic acids may be in the form of antisense molecules, such as “gapmers” containing an RNA-DNA-RNA structure that activates RNAseH. Nucleic acids may be, for example, DNA or RNA, or RNA-DNA hybrids, and may be oligonucleotides, plasmids, parts 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 may be double-stranded RNA molecules of the type used to inhibit gene expression by RNA interference. Nucleic acids may be small interfering double-stranded RNA molecules (siRNA). Nucleic acid molecules may also be Stealth® RNAi molecules (Invitrogen Corporation / Life Technologies Corporation, Carlsbad, CA).

[0077] As used herein, “RNA” refers to ribonucleic acid that may or may not be naturally occurring. For example, RNA may contain modifications such as one or more nucleic acid bases, nucleosides, nucleotides, or linkers, and / or non-natural components. RNA may contain cap structures, strand termination nucleosides, stem-loops, poly(A) sequences, and / or polyadenylation signals. RNA may have a nucleotide sequence that codes for a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of mRNA that codes for a particular polypeptide, for example, in vivo translation of mRNA in a mammalian cell, may produce the coded polypeptide. RNA may be selected from a non-limiting group consisting of 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.

[0078] The term "amphiphilic peptide" refers to a peptide having a secondary structure in which hydrophobic and hydrophilic amino acid residues are arranged on different faces of the peptide. Peptides often adopt a helical secondary structure. In some situations, 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 containing 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 optionally be linked to the polycationic nucleic acid binding site, for example, via a covalent bond.

[0079] exosomes, exosomal lipids The term “exosome” refers to a small membrane vesicle secreted by most cells that contains cell-specific payloads of proteins, lipids, genetic material, and other biomolecules that are transported to other cells at different locations in tissue. Exosomes can be considered liposome particles. Exosomes or lipid mixtures derived therefrom can be used in combination with other transfection agents or helper lipid mixtures. Exosomes are also referred to as microvesicles, epididymosomes, algosomes, exosome-like vesicles, microparticles, prominosomes, prostasomes, dexosomes, texosomes, archaeosomes, and oncosomes. Exosomes useful in the compositions and methods described herein also include synthetic exosomes. Non-limiting examples of synthetic exosomes useful in the embodiments described herein are, for example, described in Li, YJ., Wu, JY., Liu, J. et al. Artificial exosomes for translational nanomedicine. J Nanobiotechnol 19, 242 (2021), U.S. Patent No. 11938219, U.S. Patent Application Publication No. 2023 / 0181466, etc. (the entirety of which is incorporated herein by reference).

[0080] Examples of lipid components isolated from exosomes include lyso-PC (non-limiting examples include C-18, C-16, C-14, and mixtures), lyso-bisphosphatidic acid (non-limiting examples include C-18, C-16, and C-14), sphingomyelin, ceramide (non-limiting examples include C-8 to C-24), diunsaturated PC (non-limiting examples include DSPC, DPPC, DMPC, and others with Cn (n=8 to 25)), diunsaturated PC-MIX (non-limiting examples include DOPC, DP(db)PC), phosphatidylserine (PS), phosphatidylinositol (PI), diunsaturated PE (non-limiting examples include DSPE, DPPE, DMPE), and diunsaturated PE-MIX (non-limiting examples include DOPE). Examples include, but are not limited to, diglycerides such as DP(db)PE, phosphatidylglycerol (PG) (non-limited examples of which are C-18 to C-22), cholesterol, and cardiolipin.

[0081] Furthermore, any combination of the ionizable lipids, neutral lipids, exosomes, and lipid mixtures isolated from exosomes listed above is also intended.

[0082] The lipid compositions provided herein can also be combined with one or more exosomes, or biological substances derived from or purified from exosomes (e.g., lipids, proteins, nucleic acids, etc.).

[0083] structural lipids The lipid components of the lipid nanoparticle composition may include one or more structural lipids. The structural lipids can be selected from, but are not limited to, sterols, such as cholesterol, festerol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-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 (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0084] Stabilizer Lipid compositions provided herein may also contain stabilizers such as stabilized lipids. Stabilized lipids may be neutral lipids or may be charged. Examples of stabilized lipids that can be advantageously used in formulations provided 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-amines. 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 useful in compositions disclosed herein include, for example, polyglycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether copolymers, triblock polyoxyethylene alkyl ether copolymers, and amphiphilic branched polymers.In this embodiment, the stabilizers are 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, and polysorbate 80 (Tween It may also be 80 (IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadec-9-enoate), Myrj52 (polyoxyethylene(40) stearate), Brij(trademark)S10 (polyoxyethylene(10) stearyl ether), BRIJ(trademark)L4 = polyoxyethylene(4) lauryl ether, BRIJ(trademark)S20 = polyoxyethylene(20) stearyl ether, BRIJ(trademark)S35 = polyoxyethylene(23) lauryl ether, TPGS 1000 = D-α-tocopherol polyethylene glycol 1000 succinate, Tween 20 / polysorbate 80 / tridecyl-D-maltoside in equal proportions, and combinations thereof. In certain compositions, the stabilizer is present in about 0.1 to 5 mol% of the lipid composition. For example, in some compositions, the stabilizer is present in 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 in between, of the lipid component of the compositions provided herein. In other examples, the stabilizer is present in about 0.5 mol% to about 5 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizer is present in about 0.5 mol% to about 4 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizer is present in about 0.5 mol% to about 3 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizer is present in about 0.5 mol% to about 2 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizer is present in about 0.5 mol% to about 1 mol% of the lipid component of the compositions provided herein.In other examples, the stabilizer is present in about 1 mol% to about 5 mol% of the lipid component of the composition provided herein. In other examples, the stabilizer is present in about 1 mol% to about 4 mol% of the lipid component of the composition provided herein. In other examples, the stabilizer is present in about 1 mol% to about 3 mol% of the lipid component of the composition. In other examples, the stabilizer is present in about 1 mol% to about 2 mol% of the lipid component of the composition provided herein.

[0085] Payload integration : The disclosures herein provide compositions for delivering payloads (including, but not limited to, nucleic acids) to cells. Nucleic acids can be complexed externally to lipid complexes (e.g., liposomes, lipid nanoparticles) provided herein. In some embodiments, the compositions have about 20% to about 50% nucleic acids complexed externally to the lipid complexes. In other embodiments, the compositions have about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% nucleic acids complexed externally to the lipid complexes. External complexation of nucleic acids can be measured by methods known in the art, such as those described in Blakney et al. (2019) Gene Therapy 26:363-372.

[0086] Compound of formula I : Compounds based on the core structure of Formula I have been found to be useful for the efficient delivery of macromolecules to eukaryotic cells. The compositions and methods are effective for a variety of cells and provide high transfection efficiency. These compounds can be advantageously used in complex with macromolecules, along with one or more neutral lipids and additional components, such as fusion-promoting or fusion-advancing molecules, additional cationic lipids, cell surface ligands, cell adhesion molecules, amphiphilic peptides, and nuclear localization agents. The complexes can be easily prepared by simple methods and used for a variety of cells.

[0087] In one embodiment, the present disclosure relates to a compound having formula I, JPEG2026528786000044.jpg2591, or a pharmaceutically acceptable salt thereof, in the formula, A1 is -(CH2) x - or - (CH2) y -A4-(CH2) z And, A4 is -(CH2) x -, -(CO)O-, -O(CO)-, -SS-, JPEG2026528786000045.jpg1421, JPEG2026528786000046.jpg1725, JPEG2026528786000047.jpg1723 JPEG2026528786000048.jpg2134, and Selected from the group consisting of JPEG2026528786000049.jpg2141, Q1 is N or The file is JPEG2026528786000050.jpg2621, and Q2 is N or The file is JPEG2026528786000051.jpg2621, R1 and R2 are independently of each other, and C1~C are optionally substituted for H. 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched-chain alkenyls, or optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkynyl groups, or -(CH2) 1~7 Selected from the group consisting of COOR, R3 is -COR, -(CH2) n COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2- JPEG2026528786000052.jpg2230, or JPEG2026528786000053.jpg3051, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2)n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C4~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C4~C 30 Monounsaturated or polyunsaturated linear or branched alkynyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl -(CH2) n C3-C6 cycloalkyl groups optionally substituted with Het, or C3-C6 cycloalkyl groups in which the ring carbon is replaced by -O-, -S-, -SS-, or -NR7-, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n A C3-C6 cycloalkenyl group optionally substituted with Het, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7-, or aryl groups, C1~C 30 Linear or branched alkyl groups, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups, C4~C 30 Whether it is a monounsaturated or polyunsaturated linear or branched alkenyl group, R3 is such that R1 and R2 are independent of each other -(CH2) 1~7 H is only true if COOR is true. R4 is replaced by H, or C1-C which are optionally replaced. 20 Linear or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R5 and R6 are independently selected from H and CH3. R7 is H, or optionally substituted C1-C6 linear or branched alkyl, or optionally substituted monounsaturated or polyunsaturated C1-C6 linear or branched alkenyl. JPEG2026528786000054.jpg2231, or The file is JPEG2026528786000055.jpg3051, R is optionally substituted into C4~C 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3Selected from the group consisting of Het, A2 and A3 independently, H, JPEG2026528786000056.jpg1628, JPEG2026528786000057.jpg2330, JPEG2026528786000058.jpg2531, JPEG2026528786000059.jpg2030, JPEG2026528786000060.jpg1528, JPEG2026528786000061.jpg1526, JPEG2026528786000062.jpg1529, JPEG2026528786000063.jpg1528, JPEG2026528786000064.jpg1226, JPEG2026528786000065.jpg2032, JPEG2026528786000066.jpg2133, and Selected from the group consisting of JPEG2026528786000067.jpg2133, R8 is -COR, JPEG2026528786000068.jpg2330 or The file is JPEG2026528786000069.jpg3051, In the formula, R9 is H, JPEG2026528786000070.jpg2227 or The file is JPEG2026528786000071.jpg3051, In the formula, R 10 However, H, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) nHet, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C 30 Linear or branched alkyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl-(CH2) n Selected from the group consisting of C3-C6 cycloalkylalkyl groups that are optionally substituted with Het, C3-C6 cycloalkylamines in which the ring carbon is replaced by -O-, -S-, -SS-, -NR7-, and -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n A C3-C6 cycloalkenylamine optionally substituted with Het, In the formula, R 11 However, -NH2, -NHR, -N(R)2, JPEG2026528786000072.jpg2740, JPEG2026528786000073.jpg2640, and Selected from the group consisting of JPEG2026528786000074.jpg2641, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 6. b is an integer between 0 and 6. x is an integer from 1 to 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n1, n2, n3, n4, and n5 are independent integers between 1 and 5. n6 is an integer between 0 and 7. n7 and n8 are independent integers between 0 and 5. n9 is an integer between 1 and 5. m1 is an integer between 1 and 5. m2 is an integer between 0 and 5. m3 is an integer between 1 and 7. The present invention provides a compound, or a pharmaceutically acceptable salt thereof, in which p is an integer between 1 and 50, and Het is an optionally substituted 5-7 member monocyclic basic heterocycle or an optionally substituted 8-11 member bicyclic basic heterocyclil.

[0088] In some embodiments of the compound of formula I, A1 is -(CH2) x - or - (CH2) y -A4-(CH2) z -and, A4 is -(CH2) x -, -(CO)O-, -O(CO)-, -SS-, JPEG2026528786000075.jpg1421, JPEG2026528786000076.jpg1826, JPEG2026528786000077.jpg1824 JPEG2026528786000078.jpg2032, and Selected from the group consisting of JPEG2026528786000079.jpg2141, Q1 is N, and Q2 is N. R1 and R2 are independently and arbitrarily substituted with C1~C 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched-chain alkenyls, or optionally substituted C4-C 30Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkynyl groups, R3 is -COR, -(CH2) n COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2-, JPEG2026528786000080.jpg2332, or The file is JPEG2026528786000081.jpg3253, R4 is H, or optionally substituted with C1-C 20 Linear or branched alkyl groups, optionally substituted C1-C 20 A monounsaturated or polyunsaturated linear or branched alkenyl, where AA represents any natural or unnatural amino acid side chain. R is a C4~C that is optionally substituted. 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3 Selected from the group consisting of Het, A2 and A3 are independently H, JPEG2026528786000082.jpg1632, JPEG2026528786000083.jpg2734, JPEG2026528786000084.jpg2335, JPEG2026528786000085.jpg1731, JPEG2026528786000086.jpg1631, JPEG2026528786000087.jpg1631, JPEG2026528786000088.jpg1328, JPEG2026528786000089.jpg2235, JPEG2026528786000090.jpg2234, and Selected from the group consisting of JPEG2026528786000091.jpg2233, In the formula, R10 H, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C 30 Linear or branched alkyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n A C3-C6 cycloalkylalkyl group that is optionally substituted with Het. In the formula, AA represents any natural or unnatural amino acid side chain. a is an integer between 1 and 6. b is an integer between 0 and 6. x is an integer between 1 and 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n1, n2, n3, n4, and n5 are independent integers between 1 and 5. n6 is an integer between 0 and 7. n7 and n8 are independent integers between 0 and 5. n9 is an integer between 1 and 5. m1 is an integer between 1 and 5. m2 is an integer between 0 and 5. m3 is an integer between 0 and 5. p is an integer between 1 and 50. Het is a 5- to 7-membered monocyclic basic heterocycle or a 8- to 11-membered bicyclic basic heterocycline that has been selectively substituted.

[0089] In another embodiment, the present disclosure relates to a compound having the structure of formula Ia, JPEG2026528786000092.jpg2591 or a pharmaceutically acceptable salt thereof, in the formula, R1 and R2 are substituted independently and arbitrarily into C1-C 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, or -(CH2) 1~7 Selected from the group consisting of COOR, A2 and A3 are independent of each other. JPEG2026528786000093.jpg1733, JPEG2026528786000094.jpg2734, JPEG2026528786000095.jpg2234, JPEG2026528786000096.jpg2033, JPEG2026528786000097.jpg1634, JPEG2026528786000098.jpg2235, JPEG2026528786000099.jpg2234, and Selected from the group consisting of JPEG2026528786000100.jpg2234, R3 is -COR, -(CH2) n COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2- JPEG2026528786000101.jpg2433, or JPEG2026528786000102.jpg3051, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) nSR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C4~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C4~C 30 Monounsaturated or polyunsaturated linear or branched alkynyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl -(CH2) n C3-C6 cycloalkyl groups optionally substituted with Het, or C3-C6 cycloalkyl groups in which the ring carbon is replaced by -O-, -S-, -SS-, or -NR7-, or -OR7, -N(R7)2, -SR7, -(CH2)n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n A C3-C6 cycloalkenyl group optionally substituted with Het, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, -NR7-S-NR7-, or aryl groups, C1~C 30 Linear or branched alkyl groups, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -S-NR7-S-, -NR7-SS-NR7-, or -NR7-S-NR7- groups, C4~C 30 Whether it is a monounsaturated or polyunsaturated linear or branched alkenyl group, R3 is such that R1 and R2 are independent of each other -(CH2) 1~7 H is only true if COOR is true. R4 is replaced by H, or C1-C which are optionally replaced. 20 Linear or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is a C4~C that is optionally substituted. 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3 Selected from the group consisting of Het, In the formula, R 10 However, H, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) nHet, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C 30 Linear or branched alkyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl-(CH2) n Selected from the group consisting of C3-C6 cycloalkylalkyl groups that are optionally substituted with Het, In the formula, AA represents any natural or unnatural amino acid side chain, x is an integer from 1 to 9. n is an integer between 1 and 5. n5 is an integer between 1 and 5. n6 is an integer between 0 and 7. n7 and n8 are independent integers between 0 and 5. n9 is an integer between 1 and 5. m3 is an integer between 1 and 3. The present invention provides a compound, or a pharmaceutically acceptable salt thereof, in which p is an integer between 1 and 50, and Het is an optionally substituted 5-7 member monocyclic basic heterocycle or an optionally substituted 8-11 member bicyclic basic heterocyclil.

[0090] In some embodiments of the compound of formula Ia, R3 is -COR or -(CH2) n It is COOR.

[0091] In some embodiments of the compound of formula Ia, R is selected from the group consisting of oleyl, oleoil, linoleyl, linoleoil, palmitrail, palmitreoil, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0092] In some embodiments of the compound of formula Ia, independently of each other, n5 is 1, or n7 is 1, or n9 is 1.

[0093] In some embodiments of the compound of formula Ia, x is 4.

[0094] In another embodiment, the present disclosure relates to a compound having the structure of formula Ib, JPEG2026528786000103.jpg4198 or a pharmaceutically acceptable salt thereof, in the formula, R1 and R2 are substituted independently and arbitrarily into C1-C 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R3 is -COR, -(CH2) n COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2-, JPEG2026528786000104.jpg2231, or The file is JPEG2026528786000105.jpg3355, A2 and A3 are independent of each other. JPEG2026528786000106.jpg1735, JPEG2026528786000107.jpg1526, JPEG2026528786000108.jpg2235, JPEG2026528786000109.jpg2234, and Selected from the group consisting of JPEG2026528786000110.jpg2335, R4 is replaced by H, or C1-C which are optionally replaced. 20 Linear or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is optionally substituted into C4~C30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3 Selected from the group consisting of Het, In the formula, R 10 However, H, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C 30 Linear or branched alkyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl-(CH2) n Selected from the group consisting of C3-C6 cycloalkylalkyl groups that are optionally substituted with Het, C3-C6 cycloalkylamines in which the ring carbon is replaced by -O-, -S-, -SS-, -NR7-, and -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n A C3-C6 cycloalkenylamine optionally substituted with Het, In the formula, AA represents any natural or unnatural amino acid side chain, y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n5 is an integer between 1 and 5. m2 is an integer between 1 and 5. p is an integer between 1 and 50. The present invention provides compounds, or pharmaceutically acceptable salts thereof, in which Het is optionally substituted with a 5-7 member monocyclic basic heterocycle or an optionally substituted with an 8-11 member bicyclic basic heterocyclil.

[0095] In some embodiments of the compound of formula Ib, R3 is -COR or -(CH2) n It is COOR.

[0096] In some embodiments of the compound of formula Ib, R is selected from the group consisting of oleyl, oleoil, linoleyl, linoleoil, palmitrail, palmitreoil, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0097] In some embodiments of the compound of formula Ib, independently of each other, n5 is 1, m2 is 2, or n9 is 1.

[0098] In some embodiments of the compound of formula Ib, y and z are independently integers between 1 and 2.

[0099] In another embodiment, the present disclosure relates to a compound having the structure of formula Ic, JPEG2026528786000111.jpg25117 or a pharmaceutically acceptable salt thereof, in the formula, R1 and R2 are independently of each other, and C1~C are optionally substituted for H. 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R3 is -COR, -(CH2) n COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2- JPEG2026528786000112.jpg2332 or JPEG2026528786000113.jpg3253 or C1~C which are optionally replaced. 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched-chain alkenyls, or optionally substituted C4-C 30 A monounsaturated or polyunsaturated linear or branched alkynyl group. A4 is -(CO)O-, -O(CO)-, -SS-, JPEG2026528786000114.jpg1722, JPEG2026528786000115.jpg1722, JPEG2026528786000116.jpg2037, and Selected from the group consisting of JPEG2026528786000117.jpg2139, R4 is replaced by H, or C1-C which are optionally replaced. 20 Linear or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is optionally substituted into C4~C 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3 Selected from the group consisting of Het, In the formula, AA represents any natural or unnatural amino acid side chain, y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n1, n2, n3, and n4 are independent integers between 1 and 5. m1 is an integer between 1 and 5. p is an integer between 1 and 50. The present invention provides compounds, or pharmaceutically acceptable salts thereof, in which Het is optionally substituted with a 5-7 member monocyclic basic heterocycle or an optionally substituted with an 8-11 member bicyclic basic heterocyclil.

[0100] In some embodiments of the compound of formula Ic, R3 is -COR or -(CH2) n It is COOR.

[0101] In some embodiments of the compound of formula Ic, R is selected from the group consisting of oleyl, oleoil, linoleyl, linoleoil, palmitrail, palmitreoil, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0102] In some embodiments of the compound of formula Ic, n1, n2, n3, and n4 are independently integers between 1 and 2.

[0103] In some embodiments of the compound of formula Ic, y and z are independently integers between 1 and 2.

[0104] In some embodiments of the compound of formula Ic, m1 is an integer between 1 and 2.

[0105] In another embodiment, the present disclosure relates to a compound having the structure of formula Id, JPEG2026528786000118.jpg25113 or a pharmaceutically acceptable salt thereof, in the formula, A2 and A3 independently, H, JPEG2026528786000119.jpg1934, JPEG2026528786000120.jpg1429, JPEG2026528786000121.jpg1631, JPEG2026528786000122.jpg2235, and Selected from the group consisting of JPEG2026528786000123.jpg2335, R1 and R2 are independently of each other, and C1~C are optionally substituted for H. 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R3 is -COR-(CH2) n COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2-, JPEG2026528786000124.jpg2231 or JPEG2026528786000125.jpg3051, or C1~C which have been optionally replaced. 30 Linear or branched alkyl groups, or optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, or optionally substituted C4-C 30 A monounsaturated or polyunsaturated linear or branched alkynyl group. R4 is replaced by H, or C1-C which are optionally replaced. 20 Linear or branched alkyl groups, optionally substituted C1-C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R5 and R6 are independently selected from H and CH3. R is optionally substituted into C4~C 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH2) 0~3 Selected from the group consisting of Het, A4 is -(CO)O-, -O(CO)-, -SS-, JPEG2026528786000126.jpg1520, JPEG2026528786000127.jpg1722, JPEG2026528786000128.jpg2134, JPEG2026528786000129.jpg2037, and Selected from the group consisting of JPEG2026528786000130.jpg1426, In the formula, R 10 However, H, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C 30 Linear or branched alkyl groups, -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl-(CH2) n Selected from the group consisting of C3-C6 cycloalkylalkyl groups that are optionally substituted with Het, C3-C6 cycloalkylamines in which the ring carbon is replaced by -O-, -S-, -SS-, -NR7-, and -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n A C3-C6 cycloalkenylamine optionally substituted with Het, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 2. b is an integer between 0 and 3. y and z are independent integers between 1 and 4. z is an integer between 1 and 4. m1 and m2 are independent integers between 1 and 5. n1, n2, n3, n4, and n5 are independent integers between 1 and 5. p is an integer between 1 and 50. The present invention provides compounds, or pharmaceutically acceptable salts thereof, in which Het is optionally substituted with a 5-7 member monocyclic basic heterocycle or an optionally substituted with an 8-11 member bicyclic basic heterocyclil.

[0106] In another embodiment, the present disclosure relates to a compound having the structure of formula Ik, JPEG2026528786000131.jpg2096 or a pharmaceutically acceptable salt thereof, wherein the formula, A1 is -(CH2) x - or - (CH2) y -SS-(CH2) z -and, R1 and R2 are substituted independently and arbitrarily into C1-C 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, In the formula, R 10 However, H, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl, -(CH2) n Het, optionally substituted with cycloalkyl, C1-C 30 Linear or branched alkyl groups, or The chain carbon atoms are replaced by -O-, -S-, -SS-, -NR7-, and aryl groups, C1~C30 Linear or branched alkyl groups, or -OR7, -N(R7)2, -SR7, -(CH2) n OR7, -(CH2) n SR7, -(CH2) n N(R7)2, -(CH2) n Ariel, -(CH2) n Arylalkyl-(CH2) n Selected from the group consisting of C3-C6 cycloalkylalkyl groups that are optionally substituted with Het, a is an integer between 1 and 6. b is an integer between 1 and 6. x is an integer from 1 to 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n5 is an integer between 1 and 5. The present invention provides compounds, or pharmaceutically acceptable salts thereof, in which Het is optionally substituted with a 5-7 member monocyclic basic heterocycle or an optionally substituted with an 8-11 member bicyclic basic heterocyclil.

[0107] In some embodiments of the compound of formula Ik, A1 is It is -(CH2)4- or -(CH2)2-SS-(CH2)2-.

[0108] In some embodiments of the compound of formula Ik, n5 is 1.

[0109] In some embodiments of the compound of formula Ik, a and b are independently integers from 1 to 3.

[0110] In some embodiments, the compound of formula Ik is R 10 H, JPEG2026528786000132.jpg9098 JPEG2026528786000133.jpg88108, JPEG2026528786000134.jpg134110 The functional group is selected from JPEG2026528786000135.jpg93130 (but is not limited to these).

[0111] In another embodiment, the present disclosure relates to a compound having the structure of formula In, JPEG2026528786000136.jpg30103 or a pharmaceutically acceptable salt thereof, in the formula, A1 is -(CH2) y -SS-(CH2) z -and, R1 and R2 are substituted independently and arbitrarily into C1-C 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 6. b is an integer between 1 and 6. y is an integer between 1 and 4. z is an integer between 1 and 4. The present invention provides a compound, or a pharmaceutically acceptable salt thereof, in which n5 is an integer between 1 and 5.

[0112] In some embodiments of the compound of formula In, n5 is 1.

[0113] In some embodiments of the compound of formula In, a and b are independently integers from 1 to 3.

[0114] In some embodiments of compounds of formula In, AA is The functional group is selected from JPEG2026528786000137.jpg114116 (but is not limited to these).

[0115] In another embodiment, the present disclosure relates to a compound having the structure of formula Iy, JPEG2026528786000138.jpg2476 or a pharmaceutically acceptable salt thereof, in the formula, R1 and R2 are substituted independently and arbitrarily into C1-C 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, y is an integer between 1 and 4. The present invention provides compounds, or pharmaceutically acceptable salts thereof, in which z is an integer between 1 and 4.

[0116] In some embodiments of the compound of formula Iy, y and z are independently integers between 1 and 3.

[0117] In another embodiment, the present disclosure relates to a compound having the structure of formula Iz, JPEG2026528786000139.jpg2676 or a pharmaceutically acceptable salt thereof, in the formula, R1 and R2 are substituted independently and arbitrarily into C1-C 30 Linear or branched alkyl groups, and optionally substituted C4-C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R3 is optionally replaced by C1~C 30 Linear or branched alkyl groups, optionally substituted C4-C 30 Monounsaturated or polyunsaturated straight-chain or branched-chain alkenyls, or optionally substituted C4-C 30 A monounsaturated or polyunsaturated linear or branched alkynyl group. y is an integer between 1 and 4. The present invention provides compounds, or pharmaceutically acceptable salts thereof, in which z is an integer between 1 and 4.

[0118] In some embodiments of the compound of formula Iz, y and z are independently integers between 1 and 3.

[0119] In some embodiments of the compound of formula Id, R3 is -COR or -(CH2) n It is COOR.

[0120] In some embodiments of the compound of formula Id, R is selected from the group consisting of oleyl, oleoil, linoleyl, linoleoil, palmitrail, palmitreoil, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0121] In some embodiments of the compound of formula Id, a is 1 and b is an integer between 0 and 1.

[0122] In some embodiments of the compound of formula Id, y and z are independently integers between 1 and 2.

[0123] In some embodiments of the compound of formula Id, A2 is When the filename is JPEG2026528786000140.jpg1732, A3 is H, A4 is The file is JPEG2026528786000141.jpg2035, R1 and R5 are CH3. R2 and R6 are H, R3 is -COR, -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2-, JPEG2026528786000142.jpg2230, or The file is JPEG2026528786000143.jpg3356, R7 is H, b is 0, and its pharmaceutically acceptable salt.

[0124] In some embodiments of the compound of formula Id, A2 and A3 are independently JPEG2026528786000144.jpg1733 and Selected from the group consisting of JPEG2026528786000145.jpg1426, A4 is The file is JPEG2026528786000146.jpg1629, R5 and R6 are H, R3 is -COR or -(CH2) n COOR, -(CO)NHR-, -(CO)N(R)2-, JPEG2026528786000147.jpg2333, or The file is JPEG2026528786000148.jpg3051, R7 is H, a and b are 1, and The pharmaceutically acceptable salt.

[0125] In some embodiments of formulas (I), (Ia), (Ib), (Ic), (Id), (Ig), (Ik), (In), (Io), (II-o-1), or (II-o-2), R1 and R2 are -(CH2) 13 It is CH3.

[0126] In some embodiments of 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)-.

[0127] In some embodiments of formula (I), (Ia), (Ib), (Ic), (Id), (In), (Io), (Iv), or (Iz), AA is glycine, histidine, serine, tryptophan, arginine, aspartic acid, or pyrrolicin, tyrosine. Preferably, AA is histidine.

[0128] In some embodiments of 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.

[0129] In some embodiments of formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain comprising a histidine or RGD or RYD tripeptide unit preceded by a spacer such as a glycine or serine spacer (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0130] In some embodiments of 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 preceded by a spacer such as a glycine or serine spacer (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0131] In some embodiments of 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 preceded by a spacer such as a glycine or serine spacer (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0132] In some embodiments of formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain containing a poly-Arg or poly-His unit preceded by a spacer such as a glycine or serine spacer (e.g., one or more glycine residues, one or more serine residues, or a combination of glycine and serine residues).

[0133] In some embodiments of formula (I), (Ia), (Ib), (Ic), (Id), (Ik), (In), (Io), (Ir), (Iv), (Iw), or (Iz), R9 is a peptide chain selected from any of the peptide chains listed in Table 1.

[0134] In some embodiments of formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (Io), (Ir), or (Iw), R 7 teeth, JPEG2026528786000149.jpg2542, JPEG2026528786000150.jpg3040, JPEG2026528786000151.jpg3643, JPEG2026528786000152.jpg3245, JPEG2026528786000153.jpg3840, JPEG2026528786000154.jpg3054, and A neurotransmitter-based functional group selected from (but not limited to) JPEG2026528786000155.jpg2935.

[0135] In some embodiments of formulas (I), (Ia), (Ib), (Ic), (Id), (Ig), or (II-o-2), R is selected from the group consisting of oleyl, oleoil, linoleyl, linoleoil, palmitrail, palmitoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

[0136] In some embodiments of formulas (I), (Ia), (Ib), (Ic), (Id), (Ik), (Iw), or (II-o-2), the 5-7 member monocyclic basic heterocycle is JPEG2026528786000156.jpg2227 JPEG2026528786000157.jpg3030, JPEG2026528786000158.jpg3631, JPEG2026528786000159.jpg3026, JPEG2026528786000160.jpg2239, JPEG2026528786000161.jpg2123, JPEG2026528786000162.jpg2532, JPEG2026528786000163.jpg3033, JPEG2026528786000164.jpg2523, JPEG2026528786000165.jpg2226, JPEG2026528786000166.jpg3028, JPEG2026528786000167.jpg3028, JPEG2026528786000168.jpg2525, JPEG2026528786000169.jpg3028, JPEG2026528786000170.jpg3620, JPEG2026528786000171.jpg3018, JPEG2026528786000172.jpg2026, JPEG2026528786000173.jpg2026, JPEG2026528786000174.jpg3018, JPEG2026528786000175.jpg3618, JPEG2026528786000176.jpg2527, JPEG2026528786000177.jpg2025, JPEG2026528786000178.jpg3019, JPEG2026528786000179.jpg3021, JPEG2026528786000180.jpg2025, JPEG2026528786000181.jpg3021, JPEG2026528786000182.jpg3021, JPEG2026528786000183.jpg3019, JPEG2026528786000184.jpg3019, JPEG2026528786000185.jpg3017, JPEG2026528786000186.jpg1928, JPEG2026528786000187.jpg3019, JPEG2026528786000188.jpg1826, JPEG2026528786000189.jpg1826, JPEG2026528786000190.jpg2224, JPEG2026528786000191.jpg3019, JPEG2026528786000192.jpg2028, JPEG2026528786000193.jpg2527, and Selected from the group consisting of JPEG2026528786000194.jpg3019.

[0137] Some embodiments include, JPEG2026528786000195.jpg4696 This includes compounds selected from the group consisting of JPEG2026528786000196.jpg113101 and pharmaceutically acceptable salts thereof.

[0138] Some embodiments include, JPEG2026528786000197.jpg56124 This includes compounds selected from the group consisting of JPEG2026528786000198.jpg118105 and pharmaceutically acceptable salts thereof.

[0139] Some embodiments include, JPEG2026528786000199.jpg56111 This includes compounds selected from the group consisting of JPEG2026528786000200.jpg113100 and pharmaceutically acceptable salts thereof.

[0140] Some embodiments include, JPEG2026528786000201.jpg41140 This includes compounds selected from the group consisting of JPEG2026528786000202.jpg114101 and pharmaceutically acceptable salts thereof.

[0141] Some embodiments include, [ka] This includes compounds selected from the group consisting of JPEG2026528786000204.jpg118105 and pharmaceutically acceptable salts thereof.

[0142] Some embodiments include, [ka] This includes compounds selected from the group consisting of JPEG2026528786000206.jpg118105 and pharmaceutically acceptable salts thereof.

[0143] Some embodiments include, A compound selected from the group consisting of JPEG2026528786000207.jpg46117, During the ceremony, The compound, JPEG2026528786000208.jpg92115, and its pharmaceutically acceptable salts are included.

[0144] Some embodiments include, A compound selected from the group consisting of JPEG2026528786000209.jpg29128, During the ceremony, n6 is either 0 or 1. R 10 H, JPEG2026528786000210.jpg8390 JPEG2026528786000211.jpg85104; JPEG2026528786000212.jpg129106 The compound, JPEG2026528786000213.jpg87122, and its pharmaceutically acceptable salts are included.

[0145] Some embodiments include, A compound selected from the group consisting of JPEG2026528786000214.jpg51109, wherein in the formula, The compound, JPEG2026528786000215.jpg104104, and its pharmaceutically acceptable salts are included.

[0146] Some embodiments include, A compound selected from the group consisting of JPEG2026528786000216.jpg31110, wherein in the formula, n6 is either 0 or 1. R 10 H, JPEG2026528786000217.jpg8491 JPEG2026528786000218.jpg7896, JPEG2026528786000219.jpg122100 The compound, JPEG2026528786000220.jpg85119, and its pharmaceutically acceptable salts are included.

[0147] Some embodiments include, A compound selected from the group consisting of JPEG2026528786000221.jpg3073, wherein in the formula, R is JPEG2026528786000222.jpg7786, JPEG2026528786000223.jpg51101, The compound, JPEG2026528786000224.jpg9398, and its pharmaceutically acceptable salts are included.

[0148] This specification provides compositions comprising (i) one or more compounds described in Formula I, (ii) one or more structural lipids, ionizable lipids, and stabilizers, and (iii) optionally, a payload.

[0149] A composition is also provided comprising (i) a compound described in Formula I, (ii) one or more structural lipids, (iii) one or more stabilizers, and (iv) optionally, a payload.

[0150] Furthermore, compositions comprising (i) a compound described in Formula I, (ii) one or more structural lipids, (iii) one or more stabilizers, (iv) one or more transfection enhancers, and (v) optionally, a payload are also provided.

[0151] A composition comprising (i) one or more compounds described in Formula I, and (ii) a payload is also provided.

[0152] Compositions are also provided that contain 10 to 80 mol% (excluding any payload) or mol% of the total lipids present in the composition, one or more compounds described in Formula I.

[0153] Compositions are also provided that contain one or more compounds described in Formula I, wherein the structural lipids are present in 14 to 50 mol% of the composition (excluding any payload), or in mol% of the total lipids present in the composition.

[0154] Furthermore, compositions are also provided that contain one or more compounds described in Formula I, wherein the stabilizer is present in an amount of 0.1 to 10 mol% of the composition (excluding any payload) or in a mol% of the total lipids present in the composition.

[0155] A composition is also provided which comprises one or more compounds described in Formula I, and further comprises an exosome or a biological substance derived from or purified from an exosome.

[0156] Compositions are also provided that contain one or more compounds described in Formula I, and further contain a polymer.

[0157] Furthermore, compositions are also provided that contain one or more compounds described in Formula I, wherein the polymer is selected from the group consisting of high-density star dendrimers, PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, fifth-generation or higher dendrimers, substituted dendrimers, dendrimers containing one or more amino acids, graft dendrimers, activated dendrimers, polyethyleneimine (PEI), polyethyleneimine conjugates, polylysine, polyarginine, polyornithine, histones, and any combination thereof.

[0158] For example, the composition may include one or more compounds described in Formula I and a linear or branched PEI.

[0159] Compositions comprising one or more compounds described in Formula I and a stabilizer selected from the group consisting of: surfactants, neutral lipids, polymer conjugate lipids, polyethylene glycols, phospholipids, and any combination thereof are provided herein.

[0160] For example, a composition is provided that comprises one or more compositions of formula I and a stabilizer that is a PEG-modified lipid.

[0161] This specification provides compositions comprising one or more compounds described in 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 release agents, cell surface ligands, nuclear localizers, cell-permeable peptides, fusion peptides, amphiphilic peptides, and any combination thereof.

[0162] A composition comprising one or more compounds described in Formula I and a payload is provided herein.

[0163] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the payload comprises nucleic acids.

[0164] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the compound comprises charge N, the nucleic acid molecule comprises charge P, and the combination of the compound and the nucleic acid in contact with the cell comprises an N / P ratio of about 1 to 20.

[0165] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the payload comprises a nucleic acid, and the nucleic acid is RNA.

[0166] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, further comprising a nucleic acid, wherein the nucleic acid is RNA, and 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.

[0167] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the payload comprises a nucleic acid, and the nucleic acid is DNA.

[0168] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the payload further comprises one or more peptides and optionally a nucleic acid.

[0169] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the payload comprises a nucleic acid, the nucleic acid is RNA, and further, the RNA is mRNA.

[0170] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the payload comprises a nucleic acid, the nucleic acid is RNA, and the RNA encodes an immunogen.

[0171] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the payload comprises a nucleic acid, the nucleic acid is RNA, and the RNA encodes a cancer antigen.

[0172] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and any combination thereof.

[0173] In another embodiment, the present disclosure relates to a composition of a compound of formula I and one or more stabilizers, wherein the stabilizer is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 The present invention provides a composition comprising one or more phospholipids selected from the group consisting of PE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In some embodiments, the formulation may also comprise one or more lipids derived from a viral capsid, such as an enveloped virus.

[0174] In another embodiment, the present disclosure provides a composition comprising one or more compounds described in Formula I, and further comprising a transfection enhancer selected from the group consisting of endosomal release agents, cell surface ligands, nuclear localization agents, cell-permeable peptides, fusion peptides, and any combination thereof.

[0175] In another aspect, the present disclosure provides compositions of a compound of formula I and at least one neutral lipid, wherein the one or more neutral lipids include DOPE, DPhPE, cholesterol, sterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), and 3-alkoxy-2-hydroxy-1-acetamidopropane, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine (POP E) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleo Ilphosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Trans-DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,Selected from 2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, or a combination thereof.

[0176] In another aspect, the Disclosure provides a composition comprising a compound of formula I and at least one cationic lipid and / or at least one neutral lipid, wherein the cationic lipid is selected from: GeneIn(trademark), LipofectAmine(trademark)2000, LipofectAmine(trademark), Lipofectin(registered trademark), DMRIE-C, CellFectin(registered trademark)(Invitrogen), Oligofectamine(registered trademark)(Invitrogen), LipofectAce(registered trademark)(Invitrogen), Fugene(registered trademark)(Roche, Basel, Switzerland), Fugene(registered trademark)HD(Roche) ), Transfectam (registered trademark) (Tranfectam, Promega, Madison, WI), Tfx-10 (registered trademark) (Promega), Tfx-20 (registered trademark) (Promega), Tfx-50 (registered trademark) (Promega), Transfectin (trademark) (BioRad, Hercules, CA), SilentFect (trademark) (Bio-Rad), Effectene (registered trademark) (Qiagen, Valencia, CA), DC-chol (Avanti Polar Lipids), GenePorter (registered trademark) (Gene Therapy Systems, San Diego, CA), DharmaFect 1 (registered trademark) (Dharmacon, Lafayette, CO), DharmaFect 2 (registered trademark) (Dharmacon), DharmaFect 3 (registered trademark) (Dharmacon), DharmaFect 4(Registered Trademark)(Dharmacon), Escort(Trademark)III(Sigma, St. Louis, MO), Escort(Trademark)IV(Sigma), DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-Diapalmityloxypropyl)-2-Hydroxypropane-1,3-Diamine, N-1-Dimethyl-N-1-(2,3-Diaoleoyloxypropyl)-2-(3-Amino-2-Hydroxypropyloxy)propane-1,3-Diamine, N-1-Dimethyl-N-1-(2,3-Diamyristyloxypropyl)-2-(3-Amino-2-Hydroxypropyloxy)propane-1,3-Diamine, N-1-Dimethyl-N-1-(2,3-Diapalmityloxypropyl)-2-(3-Amino-2-Hydroxypropyl Xypropyloxy)propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyldimethylaminopropyl-β-hydroxyethylamine, 3,5-(N,N-dilysyl)-diaminobenzoylglycyl-3-(DL-1,2-dipalmitoyldimethylaminopropyl-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)-oleylamino)-2-hydroxypropyl]piperazine, L-ornithine Chin-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-myristylamino]-butane-2,3-diol, 1,4-bis[(3-oleylamino)propyl]-piperazine, L-arginine-bis-(O,O'-Oleoyl-β-hydroxyethyl)amido dihydrochloride, bis[(3-(3-aminopropyl)-myristylamino)2-hydroxypropyl]piperazine, L-arginine bis(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, L-serine bis(O,O'-oleoyl-β-hydroxyethyl)amido dihydrochloride, 1,4-bis[(3-(3-aminopropyl)palmitylamino)-2-hydroxypropyl]piperazine, glycin bis(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, sarcosin bis(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, L-histidine bis(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, iodide cholesteryl-3β-carboxylamide ethylenetrimethylammonium, 1,4-bis[(3-myristylamino)propyl [L]piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propylcholesterylcarboxylate iodide, cholesteryl-3β-carboxyamide ethyleneamine, cholesteryl-3β-oxysuccinate-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propylcholesteryl-3β-oxysuccinate iodide, 2-[(2-trimethylammonio)-ethylmethylamino]ethylcholesteryl-3β-oxysuccinate iodide, 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitylamino)propyl]piperazine, L-ornitylglycyl-N-(1-heptadecyloctadecyl)-glycinamide, N, 2 ,N 5 -Bis(3-aminopropyl)-L-ornitylglycyl-N-(1-heptadecyloctadecyl)-glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]-piperazine, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornityl]-N,N-dioctadecyl-L-glutamine, N 2-[N 2 ,N 5 -Bis(amino-propyl)-L-ornityl]-NN-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -Bis(amino-propyl)-L-ornityl]-NN-dioctadecyl-L-α-asparagine, N 2 -[N 2 ,N 5 -Bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornityl]-N,N-diolyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dioleyl-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-ornityl-NN-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N 2 -[N 2 ,N 5-Bis(3-aminopropyl)-L-ornityl]-N,N-dipalmytil-L-glutamine, N 2 -[N 2 ,N 5 -Bis(amino-propyl)-L-ornityl]-NN-dipalmytil-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dipalmytil-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-ornityl-NN-dipalmytil-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornityl]-N,N-dimyristyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(amino-propyl)-L-ornityl]-NN-dimyristyl-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmityl-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-ornityl-NN-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine, N 2 -[N 2 ,N 5-Bis(3-aminopropyl)-L-ornityl]-N,N-dilaureyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dilaureyl-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dilaureyl-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-ornityl-NN-dilaureyl-L-glutaminyl]-L-glutamic acid, 3-[N',N”-bis(2-tertbutyloxycarbonylaminoethyl)guanidino]-N,N-dioctadeca-9-enylpropionamide, 3-[N',N”-bis(2-tertbutyloxycarbonylaminoethyl)guanidino]-N,N-dipalmytilpropionamide, 3-[N',N”-bis(2-ter [t-butyloxy-carbonylaminoethyl)guanidino]-N,N-dimyristylpropionamide, 1,4-bis[(3-(3-amino-propyl)-palmitylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxy-propyl)-oleyl-amino)propyl]piperazine, N,N-(2-hydroxy-3-amino-propyl)-N-2-hydroxypropyl-3-N,N-diolylaminopropane, N,N-(2-hydroxy-3-amino-propyl)-N -2-Hydroxypropyl-3-N,N-Dipalmitylaminopropane, N,N-(2-Hydroxy-3-amino-propyl)-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-oleyloxyethyl)]methylammonium Umbromide, [(3-aminopropyl)-bis(2-palmityloxyethyl)]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-tetradecylcarbamoylbutylcarbamate cholesteryl ester, (3-aminopropyl)-4-(3-aminopropylamino-4-carbamoylbutylcarbamate cholesteryl ester, (3-aminopropyl)-4-(3-aminopropylamino)-4-(2-dimethylaminoethylcarbamoyl)-butylcarbamate cholesteryl ester, spermine-5-carboxyglycine (N'-stearyl-N'-oleyl)amide tetratrifluoroacetate, spermine-5-carboxyglycine (N'-stearyl-N'-elaidyl)amide tetratrifluoroacetate, agmatinyl carboxycholesterol acetate, spermine-5-carboxy-β-alanine cholesteryl ester tetratrifluoroacetate, 2,6-Diaminohexanoyl β-alanine cholesteryl ester bistrifluoroacetate, 2,4-Diaminobutyroyl β-alanine cholesteryl ester bistrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl ester tristrifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxycholesteryl ester, stearyl carnitine ester, palmityl carnitine ester, myristyl carnitine ester, stearyl stearoyl carnitine N-ester chloride salt, L-stearyl stearoyl carnitine ester, stearyl oleoyl carnitine ester chloride, palmityl palmitoyl carnitine ester chloride, myristyl myristoyl carnitine ester chloride, L-myristyl myristoyl carnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxy-propyl)-palmityl-amino)-propyl]-piperazine, N-(3-aminopropyl)-N,N'-bis(dodecyloxyethyl)-piperazinium bromide, N-(3-amino-propyl)-N,N'- Bis(oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(palmityloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(myristyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl- N,N'-(bis-2-palmityloxyethyl)-piperazinium bromide, N-(3-amino-propyl)-N'-methyl-N,N'-(bis-2-myristyloxyethyl)-piperazinium bromide, 1,4-bis[(3-(3-amino-propyl)-oleylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxy-propyl]piperazine, or 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]-piperazine, 2,3-Dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-Dipalmitreoyloxy-1,4-N,NN'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-Dimyristreoyloxy-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-aminopropyl) -Diaminobutane, 2,3-Dipalmitreoyloxy-1,4-N,N'-Dimethyl-N,N'-Di(3-amino-propyl)-Diaminobutane, 2,3-Dimyristreoyloxy-1,4-N,N'-Dimethyl-N,N'-Di(3-amino-propyl)-Diaminobutane, 2,3-Dioleyloxy-1,4-N,N'-Dimethyl-N,N'-Di(5-Carboxamide-Spermine)-Diaminobutane, 2,3-Dipalmitreoyloxy-1,4-N,N'-Dimethyl-N,N'-Di(5-Carboxamide-Spermine)-Di Amino-butane, 2,3-dimyristreoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamidespermine)diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dipalmitreoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dimyristreoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N' -dimethyl-N,N'-di(histidyl)-diaminobutane, 2,3-dipalmitreoyloxy-1,4-N,N'-dimethyl-N,N'-di(histidyl)-diaminobutane, 2,3-dimyristooleoyloxy-1,4-N,N'-dimethyl-N,N'-di(histidyl)-diaminobutane, 2,3-dioleyloxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dipalmitreoyloxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dimyristooleoyloxy-N,N'-dimethyl-1,4-Diaminobutane; PAMAM dendrimer, NH3 core dendrimer, ethylenediamine core dendrimer, polyethyleneimine (PEI), and polyethyleneimine conjugate.

[0177] In another embodiment, the Disclosure provides compositions of a compound of formula I and at least one cationic lipid and / or at least one neutral lipid, wherein the one or more neutral lipids are selected from: DOPE, DPhPE, cholesterol, sterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, DOPC, Lyso-PE(1-acyl-2) (hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), and 3-alkoxy-2-hydroxy-1-acetamidopropane, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphospha Tidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearo Il-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Trans-DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, or combinations thereof.

[0178] In another embodiment, the Disclosure provides a composition comprising a compound of formula I and at least one cationic lipid and / or at least one neutral lipid and / or at least one PEG-modified lipid, wherein the PEG-modified lipid is 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-diacyloxypropane-3-amine, or any combination thereof.

[0179] In another embodiment, the disclosure provides a composition of a compound of formula I, wherein 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.

[0180] In another embodiment, the disclosure provides a composition comprising a compound of formula I and at least one cationic lipid and / or at least one neutral lipid, wherein the alkoxy is selected from oleyl, palmityl, palmitreyl, myristreyl, myristyl, and lauryl.

[0181] In another embodiment, the present disclosure provides a composition of a compound of formula I and one or more polyamine transfection agents.

[0182] In another embodiment, the present disclosure provides a composition of a compound of formula I and one or more fusion agents.

[0183] In another embodiment, the disclosure provides a composition of a compound of formula I and one or more amphiphilic peptides.

[0184] In another embodiment, the present disclosure provides a composition comprising a compound of formula I and one or more amphiphilic peptides, wherein the one or more amphiphilic peptides function as a fusion agent.

[0185] In another embodiment, the present disclosure provides a method for delivering a payload to cells, comprising (i) providing a composition comprising a compound of formula I, (ii) providing cells, and (iii) contacting the cells with the composition. In some embodiments, the payload comprises nucleic acids encoding therapeutic proteins such as antibodies, growth factors, cytokines, and enzymes.

[0186] In another embodiment, the Disclosure provides a method for introducing nucleic acids, proteins, or peptides into eukaryotic cells, comprising introducing the nucleic acids, proteins, or peptides into the cells by contacting the cells with any of the compositions disclosed.

[0187] In another embodiment, the Disclosure provides a method for introducing nucleic acids, proteins, or peptides into eukaryotic cells, wherein the cells are human cells, and the method comprises introducing the nucleic acids, proteins, or peptides into the cells by contacting the cells with one of the compositions disclosed.

[0188] In another embodiment, the Disclosure provides a method for introducing nucleic acids, proteins, or peptides into eukaryotic cells, wherein the cells are mammalian cells, and the method comprises introducing the nucleic acids, proteins, or peptides into the cells by contacting the cells with any of the compositions disclosed.

[0189] In another embodiment, the present disclosure provides a method for delivering a composition to a target, comprising administering a composition comprising a compound of formula I to a target.

[0190] In another embodiment, the present disclosure provides a method for delivering a payload to a cell, wherein contact with the cell is in vitro.

[0191] In another embodiment, the present disclosure provides a method for delivering a payload to a cell, wherein contact with the cell is in vivo.

[0192] In another embodiment, the present disclosure provides a method for delivering a payload to a cell, wherein contact with the cell is ex vivo.

[0193] In another embodiment, the present disclosure provides a method for administering any of the disclosed compositions to a subject, wherein the administration is systemic.

[0194] In another embodiment, the present disclosure provides a method for administering any of the disclosed compositions to a target, wherein the administration is selected from the group consisting of subcutaneous, intramuscular, intranasal, intratumoral, brain, spinal cord, eye, lymph node of the target, and any combination thereof.

[0195] In another embodiment, the disclosure provides a kit comprising a compound of formula I, one or more structural lipids, an ionizable lipid, and a stabilizer.

[0196] In another embodiment, the disclosure provides a kit comprising a compound of formula I, and one or more structural lipids, and / or one or more stabilizers, and / or optionally, a payload.

[0197] In another embodiment, the 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 optionally, a payload.

[0198] In another embodiment, the Disclosure provides a method for inhibiting protein expression in cells, comprising contacting the cells with an RNAi molecule and a compound of formula I, as described herein, or one of the compositions disclosed herein.

[0199] Those skilled in the art will recognize that, although the compounds of the present invention are shown herein in their neutral (non-protonated) forms for convenience, these compounds may also exist in partially or fully protonated forms in solutions of appropriate pH, and the present invention encompasses all compounds without limitation in their protonated, non-protonated, ionized, and non-ionized forms unless otherwise specifically indicated.

[0200] Preparation method : The compounds disclosed herein may be synthesized by the methods described below, or by modifications thereof. Modifications to the methodologies include, among other things, temperatures, solvents, reagents, etc., known to those skilled in the art. Generally, during any of the processes for preparing the compounds disclosed herein, it may be necessary and / or desirable to protect the sensitive or reactive groups of any of the relevant molecules. This can be achieved by conventional protecting groups, such as those described in Protective Groups in Organic Chemistry (ed. JFW McOmie, Plenum Press, 1973) and PGM Green, TW Wutts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999) (both incorporated herein in their entirety by reference). Protecting groups may be removed at a convenient later stage using methods known from the art. Synthetic chemical transformations useful for the synthesis of applicable compounds are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers, 1989, or L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons, 1995 (both incorporated herein in their entirety by reference). The routes shown and described herein are illustrative only and are not intended to, and should not be construed to, limit the claims in any way. Those skilled in the art will recognize modifications of the disclosed synthesis and devise alternative routes based on the disclosure herein. All such modifications and alternative routes are within the scope of the claims.

[0201] In the following scheme, oxygen atom protecting groups are selected based on their compatibility with the required synthetic steps, as well as their compatibility with the introduction and deprotection steps and the overall synthetic scheme (PGM Green, TW Wutts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999)).

[0202] If a compound in this technology contains one or more chiral centers, such a compound can be prepared or isolated as a pure stereoisomer, i.e., individual enantiomers or d(l) stereoisomers, or a stereoisomer-enriched mixture. All such stereoisomers (and enriched mixtures) are included within the scope of this technology unless otherwise indicated. Pure stereoisomers (or enriched mixtures) can be prepared, for example, using optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated, for example, using chiral column chromatography, chiral decomposition agents, etc.

[0203] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), and Emka-Chemce or Sigma (St. Louis, Missouri, USA). Others 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 Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley, and Sons, 5th Edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or by obvious modifications thereof.

[0204] The following exemplary schemes are provided for the guidance of the leader and represent exemplary methods for preparing the compounds contained herein. Furthermore, other methods for preparing the compounds described herein will be readily apparent to those skilled in the art in light of the following reaction schemes and examples. Unless otherwise indicated, all variables are as defined above.

[0205] In one embodiment, as shown in Scheme I, the method involves treating intermediate Ie with BOC-anhydride in the presence of a base, and then treating it with acid chloride II-h to obtain the bis-BOC derivative Ih, which yields intermediate If. Subsequently, BOC removal is performed in the presence of an acid to obtain the compound of formula In. [ka]

[0206] In another embodiment, as shown in Scheme II, the bis-BOC derivative Ih is treated with mesyl chloride in the presence of a base to obtain the bis-mesylate intermediate Ij. The intermediate Ij is then treated with an amine represented by formula II-k in the presence of a base, followed by removal of BOC in the presence of an acid to obtain the compound of formula Ik. [ka]

[0207] In one embodiment, for example, compounds 8-12 and 38-92 are synthesized according to reaction conditions as shown in Scheme II. JPEG2026528786000227.jpg8590 JPEG2026528786000228.jpg85102. JPEG2026528786000229.jpg144115 JPEG2026528786000230.jpg98130

[0208] 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 reagent to obtain the derivative Im, and then the BOC is removed in the presence of an acid to obtain the compound of formula In. [ka]

[0209] In one embodiment, for example, compounds 13 to 21 are synthesized according to reaction conditions as shown in Scheme III. JPEG2026528786000232.jpg134114

[0210] In another embodiment, as shown in Scheme IV, the bis-BOC derivative Ih is subjected to an alkylation reaction of its hydroxyl group with an alkylating agent R3-X (II-j; X is a leaving group), followed by removal of BOC in the presence of an acid, to obtain the compound of formula Io. [ka]

[0211] In one embodiment, for example, compounds 22-23 are synthesized according to reaction conditions as shown in Scheme IV. JPEG2026528786000234.jpg69107 JPEG2026528786000235.jpg67111

[0212] In another embodiment, as shown in scheme V, isoindoline-1,3-dione is condensed with 3-chloro-2-(chloromethyl)propa-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 removal of BOC in the presence of an acid to obtain the compound of formula Ir. [ka]

[0213] In one embodiment, for example, compound 24 is N as amine Ix. 1 ,N 4-Ditetradecylbutane-1,4-diamine is used and synthesized according to the reaction conditions shown in Scheme V, by coupling with 2-(2-(chloromethyl)allyl)isoindoline-1,3-dione and 2-aminoethane-1-ol as amine Ik, and then coupling with the corresponding intermediate Iq. JPEG2026528786000237.jpg30102

[0214] In another embodiment, as shown in Scheme VI, solid-bonded arylalkyl chloride II-a is reacted with amino-thiol II-b to obtain solid-bonded amine II-c. Amine II-c is then reduced and aminated in the presence of aldehyde II-n to obtain intermediate II-d, which is then treated with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione in the presence of a base and heated in a suitable solvent to obtain adduct II-e. Adduct II-e is then released from the solid phase and subsequently oxidized to obtain disulfide Is. Disulfide Is is then deprotected using hydrazine in the presence of ethanol to obtain the compound of formula Ix-1. [ka]

[0215] In another embodiment, as shown in Scheme VII, disulfide II-f is subjected to reductive amination with aldehyde II-n to obtain intermediate II-g, which is treated with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione in the presence of a base and heated in a suitable solvent to obtain adduct It. Adduct It is then treated with hydrazine in ethanol, followed by treatment with (BOC)2O to obtain derivative Iu.

[0216] 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 reagent, and the resulting product is subsequently subjected to BOC removal in the presence of an acid to obtain the compound of formula Iv.

[0217] In another embodiment, as shown in scheme VII, the bis-BOC derivative Iu is treated with mesyl chloride in the presence of a base, then with an amine represented by formula II-k in the presence of a base, and subsequently the BOC is removed in the presence of an acid to obtain the compound of formula Iw. [ka]

[0218] In one embodiment, for example, compounds 25-37 were synthesized using 2,2'-disulfanediylbis(ethane-1-amine) as diamine II-f and tetradecanal as aldehyde II-n, according to the reaction conditions shown in Scheme VII, to obtain the corresponding intermediate II-g. The corresponding intermediate II-g was purified to obtain intermediate Iu. This intermediate was then further converted to compounds 25-37 using the reaction shown in Scheme VII. JPEG2026528786000240.jpg3298 JPEG2026528786000241.jpg75116 JPEG2026528786000242.jpg77129 JPEG2026528786000243.jpg140110 JPEG2026528786000244.jpg32110

[0219] The following exemplary schemes are provided for the guidance of the reader and represent preferred methods for preparing the compounds illustrated herein. These methods are not limiting, and it will be apparent that other routes may be used to prepare these compounds. Such methods specifically include solid-phase-based chemistry, including combinational chemistry. Those skilled in the art will be fully equipped to prepare these compounds by these methods given in the literature and this disclosure. The numbering of compounds used in the synthesis schemes shown below means only those specific schemes and should not be construed as or confused with the same numbering in other sections of this application.

[0220] Examples of compounds of formula I may be synthesized using methods well known in the art, as shown for compound 1 shown below in scheme IA. [ka] As shown in Scheme IA, DHDMS was treated with BOC anhydride in the presence of a base to obtain the bis-BOC derivative (1A). This intermediate (1A) was treated with oleoyl chloride to obtain intermediate 1B. This was then treated with TFA to obtain bisoleyl derivative 1. Using a similar procedure, additional analogues were synthesized using various acid chlorides.

[0221] Similarly, compounds 2-4 were synthesized using the procedure described above. JPEG2026528786000246.jpg56147 JPEG2026528786000247.jpg62121 JPEG2026528786000248.jpg62127 [ka]

[0222] In another embodiment, as shown in Scheme VIII, diamine Ix was treated with epoxide II-o in the presence of a base in a polar solvent and heated to obtain the compound of formula II-o-1. Compound II-o-1 was treated with acid chloride II-h, followed by treatment with an acid to form a salt of the compound of formula II-o-2.

[0223] Compounds 5-7 were synthesized using the scheme shown in Scheme VIIIA below. [ka]

[0224] As shown in scheme VIIIA, N 1 ,N 4-Ditetradecylbutane-1,4-diamine (7A) was treated with 2-ethyloxirane 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 oleoyl chloride to obtain intermediate 6A. This was then treated with HCl to obtain bis-oleoyl derivative 6 as a hydrochloride salt. Similarly, compound 5 was synthesized using the above procedure by treating compound 7 with oleoyl chloride. JPEG2026528786000251.jpg56140

[0225] Compounds 8-12 were synthesized using the scheme shown in Scheme IIA below. [ka]

[0226] As shown in Scheme IIA, the bis-BOC derivative was treated with mesyl chloride in the presence of DIPEA to obtain bis-mesylic acid intermediate 8A. Intermediate 8A was treated with 2-aminoethanol in the presence of a base to obtain derivative 8B, which was then treated with TFA to obtain compound 8. Using a similar procedure, additional analogues were synthesized by substituting the mesylic acid group in intermediate 8A with various amines.

[0227] Similarly, compounds 9-12, 39-41, 43, 46, and 80 were also synthesized using 4-aminobutan-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-aminopentan-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-1,3-ol (for compound 46), 2-amino-2-methylpropane-1,3-diol (for compound 48), and 1-aminopropane-2-ol (for compound 80), following the procedure described above. JPEG2026528786000253.jpg59142 JPEG2026528786000254.jpg56139 JPEG2026528786000255.jpg50139 JPEG2026528786000256.jpg68140 JPEG2026528786000257.jpg59149 JPEG2026528786000258.jpg59105 [ka]

[0228] In another embodiment, as shown in scheme IX, diamine II-u is subjected to reductive amination conditions using aldehyde II-q and NaBH(OAc)3 to obtain intermediate II-r. Those skilled in the art will understand that many other reductive amination conditions and reagents exist within the scope of this disclosure to carry out the reaction. The obtained product II-r is then treated with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione in the presence of a base, followed by treatment with hydrazine in ethanol to obtain 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 indium-catalyzed transesterified with alcohol II-p, followed by acetonide removal and BOC removal under acid-catalyzed conditions to obtain the compound of formula II-t. Those skilled in the art will understand that many other transesterification and acetonide removal and BOC removal conditions exist within the scope of this disclosure for carrying out these reactions.

[0229] In one embodiment, for example, the compounds shown below are synthesized according to reaction conditions as shown in Scheme IX. JPEG2026528786000260.jpg144108 JPEG2026528786000261.jpg51100 JPEG2026528786000262.jpg9397

[0230] Lipid nanoparticle formulations : Lipids of formula (I) can be combined with nucleic acid and / or protein payloads to produce lipid nanoparticle formulations. For example, lipids of formula (I) can be combined with payloads selected from one or more of the following: siRNA, miRNA, mRNA, shRNA, auto-amplified RNA, oRNA (or non-natural circular RNA), antisense oligonucleotides (ASOs), gRNA, ribonucleoproteins (e.g., CRISPR complexes), dsDNA, plasmid DNA, etc.

[0231] In the case of nucleic acid delivery, the amount of nucleic acid (e.g., mRNA, auto-amplified RNA, etc.) in a lipid nanoparticle formulation may depend on the size, sequence, and other characteristics of the nucleic acid. The amount of nucleic acid in a lipid nanoparticle formulation may also depend on the size, composition, desired target, and other characteristics of the lipid nanoparticle formulation. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. In some embodiments, the weight / weight ratio of the lipid component to the nucleic acid such as mRNA in the nanoparticle composition may be 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, etc. For example, the weight / weight ratio of lipid components to nucleic acids such as mRNA may be approximately 10:1 to approximately 40:1. The amount of nucleic acids in the nanoparticle composition may be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible (UV-vis) spectroscopy).

[0232] Lipid nanoparticle formulations may contain nucleic acids at concentrations of approximately 0.1 mg / ml to 2 mg / ml, for example, but not limited to these, 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.

[0233] Preferably, one or more nucleic acids (e.g., mRNA), lipids, and their amounts may be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the nucleic acid (e.g., mRNA). Generally, lower N:P ratios are preferred. One or more mRNAs, lipids, and their amounts may be selected to provide N:P ratios of 2:1 to about 8:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In certain embodiments, the N:P ratio may be about 2:1 to about 5:1. In preferred embodiments, 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.

[0234] Additional lipid components : Advantageously, in addition to the lipid of formula (I), the lipid nanoparticle formulation contains one or more copolymers, most advantageously neutral copolymers, although those skilled in the art will recognize that other lipids, including cationic / ionizable lipids, may be used. However, some formulations contain only the lipid of formula (I) in combination with nucleic acids.

[0235] 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 a pH below the physiological pH (e.g., pH 7.4) and neutral at a second pH, for example, above the physiological pH. For example, the ionizable lipids provided herein, when incorporated into lipid nanoparticles, have a pKa of the protonable group in the range of about 5 to 7, such as about 4 to about 11, for example, about 4 to about 7, for example, about 5.5 to 6.9.

[0236] Therefore, in addition to the lipid of formula (I), the lipid nanoparticle formulation may include neutral lipids such as phospholipids. Examples of phospholipids that can be used in the lipid nanoparticle formulations provided herein include, but are not limited to, phospholipids useful in the compositions disclosed herein, including, but are not limited to, the following: DOPE, DPhPE, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl Phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (transDOPE), or combinations thereof. Useful phospholipids in the compositions provided herein may be present, for example, in about 5 mol% to about 20 mol% of the lipid nanoparticle formulation. Advantageously, phospholipids are present in a range of about 1 mol% to about 40 mol%, for example, 1 mol% to about 25 mol%. Preferably, the amount of phospholipids in the lipid nanoparticle formulations 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 in between, of the overall lipid composition formulation.

[0237] Other neutral lipids that may be advantageously included in the lipid nanoparticle formulations provided herein include sterols or lipids containing a sterol moiety ("sterol derivatives"). Wherein use herein, "sterol" is a subgroup of steroids consisting of steroid alcohols. Exemplary sterols and lipids containing a sterol moiety that are useful in the lipid nanoparticle formulations provided herein include, but are not limited to, phoresterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. Some lipid nanoparticle formulations provided herein contain sterols or sterol derivatives. Sterols or sterol derivatives may be present in about 5 to 60 mol% of the overall lipid nanoparticle formulation. Advantageously, sterols or sterol derivatives are present in about 15 to 50 mol%, for example, 25 to 40 mol%. Preferably, the amount of sterols (such as cholesterol) or sterol derivatives in the lipid compositions disclosed herein is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% of the overall lipid formulation. Some lipid nanoparticle formulations provided herein do not contain sterols or sterol derivatives.

[0238] Lipid nanoparticle formulations provided herein may also include stabilizers, such as surfactants, neutral lipids, polymer-conjugated lipids, polyethylene glycol, phospholipids, and any combination thereof. Examples of nonionic stabilizers include polysorbate (Tweens), Brij™ S20 (polyoxyethylene (20) stearyl ether), BRIJ™ 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij™ S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether), and Myrj™ 52 (polyoxyethylene (40) stearate). Other exemplary stabilizers useful in the embodiments provided herein include TPGS 1000 (D-α-tocopherol polyethylene glycol 1000 succinate); or equal ratios of Tween 20 / polysorbate 80 / tridecyl-D-moltoside (referred to as lipid H in Table 15). Other stabilized lipids that can be advantageously used in the formulations provided 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-amines. 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 useful in the compositions disclosed herein include, for example, polyglycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether copolymers, triblock polyoxyethylene alkyl ether copolymers, and amphiphilic branched polymers.In this embodiment, the stabilizers are 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, and polysorbate 80 (Tween It may also be 80 (IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadec-9-enoate), Myrj52 (polyoxyethylene(40) stearate), Brij(trademark)S10 (polyoxyethylene(10) stearyl ether), BRIJ(trademark)L4 = polyoxyethylene(4) lauryl ether, BRIJ(trademark)S20 = polyoxyethylene(20) stearyl ether, BRIJ(trademark)S35 = polyoxyethylene(23) lauryl ether, TPGS 1000 = D-α-tocopherol polyethylene glycol 1000 succinate, Tween 20 / polysorbate 80 / tridecyl-D-maltoside in equal proportions, and combinations thereof. In certain compositions, the stabilizer is present in approximately 0.1–5 mol% of the lipid nanoparticle formulation. For example, in some compositions, the stabilizer is present in 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 in between.

[0239] Lipid nanoparticle formulations may contain one or more cationic / ionizable lipids in addition to the lipid of formula (I). For example, lipid nanoparticle formulations may contain cationic / ionizable lipids selected from 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-diaoleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine Cypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1 -(2,3-diapalmityloxypropyl)-2-(3-amino-2-hydroxypropyloxy)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 )amido dihydrochloride, L-lysine-bis(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine, L-lysine-bis-(O,O'-myristoyl-β-hydroxyethyl)amido dihydrochloride, L-ornithine-bis-(O,O'-myristoyl-β-hydroxyethyl)amido dihydrochloride, L-ornithine-bis-(O,O'-oleoyl-β-hydroxyethyl)amido 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-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-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)-palmitylamino)-2-hydroxypropyl]piperazine, glycine -Bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosin-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesteryl-3β-carboxyl-amide ethylenetrimethylammonium iodide, 1,4-bis[(3-myristylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl Lyl-3β-carboxyamide ethyleneamine, cholesteryl-3β-oxysuccinate ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinate iodide, 2-[(2-trimethylammonio)-ethylmethylamino]-ethyl-cholesteryl-3β-oxysuccinate iodide, 3β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol, and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-Bis[(3-palmitylamino)propyl]piperazine, L-ornitylglycyl-N-(1-heptadecyloctadecyl)glycinamide, N2,N5-bis(3-aminopropyl)-L-ornitylglycyl-N-(1-heptadecyloctadecyl)glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-dioctadecyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dioctadecyl-L-α-glutamine Glutamin, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dioctadecyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]L-ornityl-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-diolyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dioleyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN- [Dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-dipalmytil-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dipalmytil-L-α-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dipalmytil-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethyle [Toxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN-dipalmytil-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-dimyristyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dimyristyl-L-α-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dimyristyl-L-α-asparagine, 1,4 -Bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)-2-hydroxypropyl]piperazine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]L-ornityl-NN-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-dilaureyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dilaureyl-L-α-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dilaureyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN-dilaureyl-L-glutaminyl]-L-glutamic acid, 3-[N',N”-bis [(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dioctadeca-9-enylpropionamide, 3-[N',N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dipalmitylpropionamide, 3-[N',N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dimyristylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxy [Cipropyl)-oleylamino)propyl]piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-diolylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitylaminopropane, 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-oleyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmityloxyethyl)]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-tetradecylcarbamoylbutylcarbamate cholesteryl ester, (3-aminopropyl)-4-(3-aminopropylamino-4-carbamoylbutylcarbamate cholesteryl ester, (3-aminopropyl)-4-(3-aminopropylamino)-4-(2-dimethylaminoethylcarbamoyl)-butylcarbamate cholesteryl ester, spermine-5-carboxyglycine (N'-stearyl-N'-oleyl)amide tetratrifluoroacetate, spermine-5-carboxyglycine (N'-stearyl-N'-elaidyl)amide tetratrifluoroacetate, Agmatinyl carboxycholesterol acetate, spermine-5-carboxy-β-alanine cholesteryl ester tetratrifluoroacetate, 2,6-diaminohexanoyl β-alanine cholesteryl ester bistrifluoroacetate, 2,4-diaminobutyroyl β-alanine cholesteryl ester bistrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl ester tritrifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxycholesteryl ester Stearyl carnitine ester, palmityl carnitine ester, myristyl carnitine ester, stearyl stearoyl carnitine ester chloride salt, L-stearyl stearoyl carnitine ester, stearyl oleoyl carnitine ester chloride, palmityl palmitoyl carnitine ester chloride, myristyl myristoyl carnitine ester chloride, L-myristyl myristoyl carnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine, N-(3-A N-(3-aminopropyl)-N,N'-bis(dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(palmityloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(myristyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl Lu-N,N'-(bis-2-oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmityloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristyloxyethyl)-piperazinium bromide, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxypropyl]piperazine, and 1,4-Bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxypropyl]piperazine, KL22, KL25, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholesta-5-e N-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA),(2R)-2-({8-[(3.β.)-cholesta-5-ene-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadeca- 9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2R)), and (2S)-2-({8-[(3)-cholesta-5-ene-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-1-amine(octyl-CLinDMA(2S)).

[0240] Preferably, the lipid of formula (I), or a combination of the lipid of formula (I) and one or more cationic / ionizable lipids, is present in about 5 to 80 mol% of the lipid nanoparticle formulation (excluding the payload), for example, about 5 to 80 mol% of the lipid component of the lipid nanoparticle formulation. For example, some lipid nanoparticle formulations contain less than 50 mol% of formula (I), or a combination of formula (I) and one or more additional cationic / ionizable lipids. Other lipid nanoparticle formulations contain more than 50 mol% of formula (I), or a combination of formula (I) and one or more additional cationic / ionizable lipids.

[0241] Therefore, some lipid nanoparticle formulations contain 15–80 mol% of the lipid of formula (I) or a combination of formula (I) and one or more cationic / ionizable lipids, 20–60 mol% of sterols, 0.5–5 mol% of a stabilizer, and 1–40 mol% of a phospholipid. An example lipid nanoparticle formulation may contain about 20–60 mol% of the lipid of formula (I), or a combination of the lipid of formula (I) and one or more additional cationic / ionizable lipids, about 5–25 mol% of a phospholipid, about 25–55 mol% of a sterol or sterol derivative, and about 0.5–15 mol% of a stabilizer. 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 / ionic lipids, about 1.5 mol% of a stabilizer, about 38.5 mol% of a sterol or sterol derivative, and about 10 mol% of a phospholipid. 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 / ionic lipids, about 2.5 mol% of a stabilizer, about 32.5 mol% of a sterol or sterol derivative, and about 10 mol% of a phospholipid.

[0242] Polyamine components : Other formulations may also include one or more polyamine transfection agents such as high-density star dendrimers, PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, fifth-generation or higher dendrimers, substituted dendrimers, dendrimers containing one or more amino acids, grafted dendrimers, activated dendrimers, polyethyleneimine (PEI), and / or polyethyleneimine conjugates.

[0243] Transfection enhancer : Further formulations include transfection enhancers such as fusion agents (e.g., endosomal release agents), cell surface ligands, and / or nuclear localization agents such as nuclear receptor ligand peptides. Examples of transfection enhancers include retrovirus-related fusion peptides (see WO07 / 130073, the whole of which is incorporated herein by reference), insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell-targeted antibodies, lactoferrin, fibronectin, adenovirus penton base, Knob, hexone protein, vesicular stomatitis virus glycoprotein, semliki forest fever virus core protein, influenza hemagglutinin, hepatitis B core protein, and HIV. Examples of such proteins include, but are not limited to, Tat protein, herpes simplex virus VP22 protein, histone proteins, arginine-rich cell permeability proteins, high-mobility group proteins, and invesin proteins, as well as internarin proteins, endotoxins, diphtheria toxin, Shigella toxin, melittin, magainin, gramicidin, cecrophin, defensin, protegurin, tachypressin, thionine, indolicidin, bactenesin, drosomycin, apidaesin, cathelicidin, proteins that increase bacterial permeability, nisin, buforin, and fragments thereof. Other cell permeability peptides useful in the compositions provided herein are listed in Table 1 below.

[0244] Table 1 JPEG2026528786000263.jpg231153 JPEG2026528786000264.jpg223153 JPEG2026528786000265.jpg222153 JPEG2026528786000266.jpg227153 JPEG2026528786000267.jpg222153 JPEG2026528786000268.jpg222153 JPEG2026528786000269.jpg221153 JPEG2026528786000270.jpg222153 JPEG2026528786000271.jpg222153 JPEG2026528786000272.jpg222153 JPEG2026528786000273.jpg222153 JPEG2026528786000274.jpg222153 JPEG2026528786000275.jpg222153 JPEG2026528786000276.jpg222153 JPEG2026528786000277.jpg222153 JPEG2026528786000278.jpg236153 JPEG2026528786000279.jpg225153 JPEG2026528786000280.jpg229153 JPEG2026528786000281.jpg222153 JPEG2026528786000282.jpg235153 JPEG2026528786000283.jpg222153 JPEG2026528786000284.jpg216153 JPEG2026528786000285.jpg60153

[0245] The lipid nanoparticle compositions provided herein can also be combined with one or more exosomes, or biological substances derived from or purified from exosomes (e.g., lipids, proteins, nucleic acids, etc.).

[0246] Exemplary compositions may include, for example, a lipid of formula (I) and one or more exosomes, a lipid of formula (I) and one or more exosomes, and one or more neutral lipids, a lipid of formula (I) and one or more exosomes, one or more neutral lipids, and one or more stabilizers, a lipid of formula (I) and one or more exosomes, and one or more neutral lipids, optionally one or more stabilizers, and optionally one or more cell-permeable peptides.

[0247] Other exemplary compositions include, for example, the lipid of formula (I) and one or more biological materials derived or purified from exosomes; the lipid of formula (I) and one or more biological materials derived or purified from exosomes, and one or more neutral lipids; the lipid of formula (I) and one or more biological materials derived or purified from exosomes, one or more neutral lipids, and / or multiple stabilizers; the lipid of formula (I) and one or more biological materials derived or purified from exosomes, and one or more neutral lipids, optionally one or more stabilizers, and optionally one or more cell-permeable peptides.

[0248] Formulation and use of compound I for transfection : The lipids described above may be formulated by various methods used in transfection. One of the simplest methods for formulation is reverse-phase evaporation, as described in U.S. Patent No. 9,259,475 (which is incorporated herein by reference in its entirety). Other methods for formulation that can be used are sonication and microfluidication. Advantageously, the lipids are formulated as lipid nanoparticles using a microfluidic mixing, as described, for example, in Roces et al., Pharmaceutics, 12:1095 (2020). Suitable microfluidic mixing devices are commercially available, for example, from Precision Nanosystems (Vancouver, BC). Typically, a microfluidic mixing combines two fluid streams, one containing nucleic acids(s) and the other containing lipids of formula (I) and other components, such as the peptides, ligands and other lipid components described below.

[0249] For a lipid nanoparticle composition containing RNA, a solution of RNA at a concentration of 0.1 mg / ml in deionized water is diluted in 50 mM sodium citrate buffer at a pH of 3-4 to form a stock solution. The nanoparticle composition can be treated by dialysis to remove ethanol and achieve buffer exchange. The formulation may also be dialyzed against phosphate-buffered saline (PBS), pH 7.4, using a desired molecular weight cutoff, e.g., 10 kD. The resulting nanoparticle suspension may be filtered through a 0.2 μm sterile filter (Sarstedt, Numbrecht, Germany) into a glass vial and sealed.

[0250] Methods for determining the particle size in nanoparticle formulations are well known in the art. For example, the particle size, polydispersity index (PDI), and zeta potential of a nanoparticle composition can be determined using Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). The concentration of a payload, such as nucleic acid (e.g., mRNA), in a nanoparticle composition can be determined using UV-Vis spectroscopy. The amount of composition is diluted in a suitable solvent, and the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm using a spectrophotometer. The concentration of therapeutic and / or prophylactic agents in a nanoparticle composition can be calculated based on the extinction coefficient of the therapeutic and / or prophylactic agent used in the composition, as well as the difference between the absorbance at a wavelength of, for example, 260 nm and a baseline value at a wavelength of, for example, 330 nm.

[0251] For RNA-containing nanoparticle compositions, RNA encapsulation by the nanoparticle composition can be evaluated using the QUANT-IT® RIBOGREEN® RNA assay (Invitrogen Corporation, Carlsbad, CA) and the method provided by the manufacturer. The fluorescence intensity generated after the addition of the RIBOGREEN reagent can be measured using a fluorescence plate reader at an excitation wavelength of approximately 480 nm and an emission wavelength of approximately 520 nm, for example. The fluorescence value of the reagent blank is subtracted from the respective fluorescence values ​​of the sample, and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-100) by the fluorescence value of the destroyed sample (induced by addition of Triton X-100).

[0252] Those skilled in the art will recognize that other lipids, including the cationic lipids described above, may be used, but the lipid of formula (I) can be formulated with one or more copolymers, most advantageously neutral copolymers. Other neutral lipids, such as DPhPE, cholesterol, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), and / or 3-alkoxy-2-hydroxy-1-acetamidopropane, may be used in place of or in combination with DOPE in the formulation.

[0253] Lipids of formula (I) can be formulated together with one or more ionizable / cationic lipids and / or one or more neutral lipids. Examples of ionic / cationic lipids useful in the compositions provided herein include, but are not limited to, GeneIn® and LipofectAmine®. 2000, LipofectAmine (trademark), Lipofectin (registered trademark), DMRIE-C, CellFectin (registered trademark) (Invitrogen), Oligofectamine (registered trademark) (Invitrogen), LipofectAce (registered trademark) (Invitrogen), Fugene (registered trademark) (Roche, Basel, Switzerland), Fugene (registered trademark) HD ​​(Roche), Transfectam (registered trademark) (Tranfectam, Promega, Madison, WI), Tfx-10 (registered trademark) (Promega), Tfx-20 (registered trademark) (Promega), Tfx-50 (registered trademark) (Promega), Transfectin (trademark) (BioRad, Hercules, CA), SilentFect (trademark) (Bio-Rad), Effectene (registered trademark) (Qiagen, Valencia, CA), DC-chol (Avanti Polar Lipids), GenePorter® (Gene Therapy Systems, San 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-diaoleoyl-oxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-Diamyristyl-oxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diapalmityl-oxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-Dimeth Tyl-N-1-(2,3-diapalmityloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyldimethylaminopropyl-β-hydroxyethylamine, 3,5-(N,N-dilysyl)-diamino-benzoyl-glycyl-3-(DL-1,2-dipalmitoyldimethylaminopropyl-β-hydroxyethylamine), L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, L-lysine-bis Su(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxy-propyl]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-hydroxy-ethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl [Pill]-piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)-amido dihydrochloride, 1,4-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxy-propyl)-myristyl-amino]-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)-palmitylamino)-2-hydroxy-propyl]piperazine, glycine bis(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosin bis(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesteryl-3β-carboxyl-amide ethylene-trimethylammonium iodide, 1,4-bis[(3-myristylamino) [(N',N'-dimethylaminoamino)propyl]-piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propylcholesteryl carboxylate iodide, cholesteryl-3β-carboxyamide ethyleneamine, cholesteryl-3β-oxysuccinate-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propylcholesteryl-3β-oxysuccinate iodide, 2-[(2-trimethylammonio)-ethylmethylamino]ethylcholesteryl-3β-oxysuccinate iodide, 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitylamino)propyl]piperazine, L-ornitylglycyl-N-(1-heptadecyloctadecyl)glycinamide, N, 2 ,N 5 -Bis(3-aminopropyl)-L-ornitylglycyl-N-(1-heptadecyloctadecyl)glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornityl]-N,N-dioctadecyl-L-glutamine, N2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dioctadecyl-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-ornityl-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornityl]-N,N-diolyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dioleyl-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-ornityl-NN-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N 2 -[N 2 ,N 5-Bis(3-aminopropyl)-L-ornityl]-N,N-dipalmytil-L-glutamine, N 2 -[N 2 ,N 5 -Bis(amino-propyl)-L-ornityl]-NN-dipalmytil-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dipalmytil-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-ornitylyl-NN-dipalmytil-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -Bis(3-aminopropyl)-L-ornityl]-N,N-dimyristyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dimyristyl-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(amino-propyl)-L-ornityl]-NN-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmityl-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-ornityl-NN-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine, N 2 -[N 2 ,N 5-Bis(3-aminopropyl)-L-ornityl]-N,N-dilaureyl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dilaureyl-L-α-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornityl]-NN-dilaureyl-L-α-asparagine,N-[N 2 -[N 2 ,N 5 -Bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN-dilaureyl-L-glutaminyl]-L-glutamic acid, 3-[N',N”-bis( 2[-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dioctadeca-9-enylpropionamide, 3-[N',N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dipalmytilpropionamide, 3-[N',N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dimyristylpropionamide, 1,4-bis[(3-(3-aminopropyl) [Palmitylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)oleylamino)-propyl]piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-diolylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxy-propyl-3-N,N-dipalmitylaminopropane, N,N-(2-hydroxy-3- Minopropyl)-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-oleyloxyethyl)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmityloxyethyl)]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-tetradecylcarbamoylbutylcarbamate cholesteryl ester, (3-aminopropyl)-4-(3-aminopropylamino-4-carbamoylbutylcarbamate cholesteryl ester, (3-aminopropyl)-4-(3-aminopropylamino)-4-(2-dimethylaminoethylcarbamoyl)-butylcarbamate cholesteryl ester, spermine-, 5 -Carboxyglycine (N'-stearyl-N'-oleyl)amide tetratrifluoroacetate, spermine-5-carboxyglycine (N'-stearyl-N'-elaidyl)amide tetratrifluoroacetate, agmatinyl carboxycholesterol acetate, spermine-5-carboxy-β-alanine cholesteryl ester tetratrifluoroacetate, 2,6-diaminohexanoyl β-alanine cholesteryl ester bistrifluoroacetate, 2,4-diaminobutyroyl β-alanine cholesteryl ester bistrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl ester tritrifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxycholesteryl ester, stearylcarnitine ester, Palmitylcarnitine ester, myristylcarnitine ester, stearylstearoylcarnitine ester chloride salt, L-stearylstearoylcarnitine ester, stearyloleoylcarnitine ester chloride, palmitylpalmitoylcarnitine ester chloride, myristylmyristoylcarnitine ester chloride, L-myristylmyristoylcarnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)palmitylamino)propyl]piperazine, N-(3-amino-propyl)-N,N'-bis( Decyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(palmityloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis(myristyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-o 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl]piperazine, 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)-palmitylamino)-2-hydroxypropyl]piperazine, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dipalmitoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)diaminobutane, 2,3-dimyristooleoyloxy-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-aminopropyl)diaminobutane, 2,3-dipalmitoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(3-aminopropyl)-diaminobutane, 2,3-dimyristooleoyloxy-1,4-N,N'-dimethyl-N,N'-di(3-aminopropyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamidespermine)-diaminobutane Tan, 2,3-dipalmitreoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamidespermine)diaminobutane, 2,3-dimyrystreoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamidespermine)diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dipalmitreoyloxy-1,4-N,N'-dimethyl-N ,N'-di(lysyl)-diaminobutane, 2,3-dimyristreoyloxy-1,4-N,N'-dimethyl-N,N'-di(lysyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(histidyl)-diaminobutane, 2,3-dipalmitreoyloxy-1,4-N,N'-dimethyl-N,N'-di(histidyl)-diaminobutane, 2,3-dimyristreoyloxy-1,4-N,N'-dimethyl-N ,N'-di(histidyl)-diaminobutane, 2,3-dioleyloxy-N,NN'-dimethyl-1,4-diaminobutane, 2,3-dipalmitreoyloxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dimyristreoyloxy-N,N'-dimethyl-1,4-diaminobutane; PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, polyethyleneimines, and polyethyleneimine conjugates.

[0254] Other formulations may include transfection enhancers such as fusion agents, cell surface ligands, and / or nuclear localization agents such as nuclear receptor ligand peptides. Examples of transfection enhancers useful in embodiments provided herein include, but are not limited to, insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell-targeted antibodies, lactoferrin, fibronectin, adenovirus penton base, Knob, hexone protein, vesicular stomatitis virus glycoprotein, semuliki forest fever virus core protein, influenza hemagglutinin, hepatitis B core protein, HIV Tat protein, herpes simplex virus VP22 protein, histone proteins, arginine-rich cell permeability proteins, high-mobility group proteins, and invesin proteins, as well as internarin proteins, endotoxins, diphtheria toxin, Shigella toxin, melittin, magainin, gramicidin, cecrophin, defensin, protegurin, tachypressin, thionine, indolicidin, bactenesin, drosomycin, apidaesin, cathelicidin, proteins that increase bacterial permeability, nisin, buforin, and fragments thereof.

[0255] The compositions provided herein may, for example, advantageously include one or more peptide sequences that improve transfection efficiency, such as linkers, spacers, or nuclear target sequences. The peptides provided herein may be included independently in the compositions herein (i.e., not covalently bonded to another molecule) or, alternatively, may be covalently bonded to one or more molecules of the compositions provided herein (e.g., may be covalently bonded to an ionizable or other lipid described herein, may be covalently bonded to another transfection enhancer described herein, or may be covalently bonded to a payload described herein, etc.). In some embodiments, the covalent bond may be via a spacer. The terms “spacer” and “linker,” as used interchangeably herein, refer to a chemical structure that links two molecules together, as used herein. In some embodiments, the spacer is bonded to each molecule on different parts of the spacer molecule. In other embodiments, the spacer is a hydrophilic portion containing about 6 to 30 carbon atoms. In other embodiments, the spacer contains a polyether, for example, -CH2-0-(CH2-CH2-0-)iCH2-. In other embodiments, the spacer comprises a hydrophilic polymer, for example, [(gly)i(ser)j]k (SEQ ID NO: 585). In these formulas, i is in the range of 1 to 6, j is in the range of 1 to 6, and k is in the range of 3 to 20. In some embodiments, the spacer is a peptide of the sequence APYKAWK (SEQ ID NO: 505). In other embodiments, the spacer is a sequence that is degraded in vivo by a peptidase.

[0256] method : The use of these compositions provided herein for introducing a payload into cells can be carried out by methods known in the art, in which the components of the transfection complex are mixed in different orders before being added to the cell culture. Typically, a liposome preparation of lipids, with or without copolymers, is prepared and then mixed with the payload (e.g., nucleic acids such as DNA or RNA) to form a transfection complex. The complex is then added to the cell culture, and transfection is monitored using well-known methods. Additional components, such as cell surface ligands, fusion agents, and nuclear localizers, may be added to the nucleic acid before mixing with the liposomes, or to the liposomes before the addition of the nucleic acid.

[0257] Cells that can be transfected according to these methods include, but are not limited to, substantially any eukaryotic cells, including primary cells, cells in culture, passaged cell cultures or cell lines, and cells in cultured tissues. Preferred cells include human cell lines and animal cell lines. The cells may be fibroblasts. The cells may be adherent cells or cells in suspension (suspension cells). In certain exemplary embodiments, the cells are suspension CHO-S cells and suspension 293-F cells. Other cells that may 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.

[0258] The formulation is used in a method for producing a protein, comprising contacting cells with a lipid-nucleic acid complex as described above, wherein the nucleic acid encodes a protein. The cells are incubated to produce the protein, and the protein is collected. Cells that can be used for protein production are described above. Furthermore, any composition comprising the lipid of formula I can be used for cell transfection. Such compositions are further described herein and include, but are not limited to, compositions comprising the lipid of formula I, copolymers, and any transfection enhancers, such as fusion peptides or proteins.

[0259] Lipids formulated into lipid nanoparticle (LNP) formulations were screened and evaluated by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, in vivo distribution, and expression of the payload-encoding protein. Lipids formulated using this method were used for transfection. Transfection was performed using the formulation described below, which contains compound 1, a lipid of formula (I). These results are shown in Figures 1-5.

[0260] Reagent kit : The components of the transfection composition described above can be provided in a reagent kit. The kit comprises the lipid of formula (I) and additional components such as neutral lipids, cationic lipids, cell surface ligands, fusion agents, amphiphilic peptides and / or nuclear localizers. The kit components may be separated or pre-mixed in any way. For example, the lipid of formula I may be mixed with one or more neutral lipids. The additional components may also be present in the same container or in one or more separate containers. The kit typically includes containers such as vials and / or tubes, which are packaged together, for example, in a cardboard box. The kit can be shipped from the supplier to the customer. For example, one example provided herein is a kit comprising the liposome formulation described above and a vial optionally containing a transfection agent and a transfection-enhancing peptide. The kit may also include a separate container containing, for example, a transfection-enhancing agent, e.g., a transfection-enhancing peptide, e.g., Plus Reagent® (Invitrogen Corp., Carlsbad, CA). The kit may also include separate containers, cells, cell culture medium, and a reporter nucleic acid sequence such as a plasmid expressing the reporter gene. In certain examples, the culture medium may be reduced serum medium and / or protein expression medium.

[0261] In one embodiment, the kit comprises an ionizable lipid such as a lipid of formula I, and a peptide, protein or fragment thereof, or individual parts of a modified peptide, protein or fragment thereof, or a mixture thereof. In another embodiment, the kit comprises a polycationic polymer and a peptide, protein or fragment thereof, or individual parts of a modified peptide, protein or fragment thereof, or a mixture thereof. The cationic lipid transfection kit may optionally include a neutral lipid 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 a suitable medium or solvent for other kit components.

[0262] The payloads that can be delivered by the methods of the present invention include nucleic acids, proteins, ribonucleoproteins, etc., including DNA and RNA (including RNAi / siRNA) of any size from any source containing native or non-native bases, which can encode and express proteins that are therapeutically or otherwise useful in cells, which inhibit the undesirable expression of nucleic acids in cells, which inhibit undesirable enzyme activity or activate desired enzymes, which catalyze reactions (ribozymes), and which function in diagnostic assays (e.g., diagnostic nucleic acids). Examples of therapeutic nucleic acids include nucleic acids that encode or can express therapeutically useful proteins, peptides or polypeptides in cells, which inhibit the undesirable expression of nucleic acids in cells, and which inhibit undesirable enzyme activity or activate desired enzymes in cells. In certain embodiments, the payload includes an RNA molecule. The composition 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 includes two or more RNA molecules, for example, two or more mRNAs. The compositions and methods provided herein can also be readily adapted, from the perspective of the disclosure herein, to introduce biologically active macromolecules other than nucleic acids, including polyamines, polyamic acids, polypeptides, and proteins, into eukaryotic cells. For example, other materials useful 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.

[0263] The compositions provided herein can be delivered to cells by in vivo administration. For in vivo administration, the pharmaceutical compositions are preferably administered parenterally (e.g., intra-articular, intravenous, intraperitoneal, subcutaneous, intrathecal, intradermal, intratracheal, intraosseous, intramuscular, or intratumor). In certain embodiments, the pharmaceutical compositions are administered intravenously, intrathecally, or intraperitoneally by bolus injection. Other routes of administration include topical administration (skin, eye, mucous membrane), oral administration, pulmonary administration, intranasal administration, sublingual administration, rectal administration, and vaginal administration.

[0264] Typical applications include using well-known procedures to provide intracellular delivery of siRNA to knock down or silence specific cell targets in vitro and in vivo. Alternatively, applications include the delivery of DNA or mRNA sequences encoding therapeutically useful polypeptides. In this way, treatment for genetic diseases is provided by supplying a deficient or deficient gene product. The methods of the present invention may be carried out in vitro, ex vivo, or in vivo. For example, the compositions of the present invention can also be used to deliver a payload to cells in vivo using methods known to those skilled in the art. In another embodiment, the compositions of the present invention are ex vivo and can be used to deliver a payload to a sample of patient cells that are then returned to the patient.

[0265] For in vivo administration, the pharmaceutical composition is preferably administered parenterally (e.g., intra-articular, intravenous, intraperitoneal, subcutaneous, intrathecal, intradermal, intratracheal, intraosseous, intramuscular, or intratumor). In certain embodiments, the compositions provided herein are administered intravenously, intrathecally, or intraperitoneally by bolus injection. Other routes of administration include topical (skin, eye, mucous membrane), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.

[0266] For ex vivo applications, the compositions provided herein are preferably administered to a biological sample removed from an organism, which is then washed and restored to the organism. The organism may be a mammal, and in particular may be a primate such as a human. This process is used, for example, in cell reprogramming, gene repair, and immunotherapy.

[0267] In one embodiment, the present invention provides methods for regulating the expression of a target polynucleotide or polypeptide. These methods generally involve contacting cells with a composition of the present invention relating to a payload (e.g., nucleic acid) that can regulate the expression of a target polynucleotide or polypeptide. As used herein, the term “regulation” means altering the expression of a target polynucleotide or polypeptide. Regulation may mean increasing or enhancing, or decreasing or reducing.

[0268] In related embodiments, a method for treating a disease or disorder characterized by the overexpression of a polypeptide in a subject is provided herein, comprising providing a composition provided herein to a subject, wherein the composition comprises a payload which is a therapeutic agent selected from siRNA, microRNA, antisense oligonucleotide, and plasmids capable of expressing siRNA, microRNA, or antisense oligonucleotide, and the siRNA, microRNA, or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding a polynucleotide or its complement.

[0269] In related embodiments, the foregoing provides methods for treating diseases or disorders characterized by deficiency of polypeptide expression in a subject. These methods may include providing a composition provided herein to a subject, the composition comprising a payload which is a therapeutic agent selected from mRNA, self-amplified RNA (SAM), self-replicating DNA, or plasmid, and comprising a nucleic acid therapeutic agent which specifically encodes or expresses a low-expression polypeptide or its complement.

[0270] In one embodiment, the compounds, compositions, methods, and uses described herein are for delivering a biologically active agent to liver cells (e.g., hepatocytes). In another embodiment, the compounds, compositions, methods, and uses of the present invention are for delivering a biologically active agent to a tumor or tumor cells (e.g., a primary tumor or metastatic cancer cells). In yet another embodiment, the compounds, compositions, methods, and uses are for delivering a biologically active agent to skin fat, muscle, and lymph nodes (subcutaneous administration).

[0271] For the delivery of a bioactive drug to the liver or liver cells, in one embodiment, the composition of the present invention is brought into contact with the liver or liver cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, portal vein injection, catheter placement, stent placement) to facilitate delivery. For the delivery of a bioactive drug to the kidney or kidney cells, in one embodiment, the composition of the present invention is brought into contact with the patient's kidney or kidney cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., direct injection, catheter placement, stent placement) to facilitate delivery. For the delivery of a bioactive drug 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 administration) or local administration (e.g., direct injection, catheter placement, stent placement) to facilitate delivery.

[0272] For the delivery of a payload to the CNS or CNS cells, in one embodiment, the compositions described herein can be brought into contact with the patient's CNS or CNS cells (e.g., brain cells and / or spinal cord cells) via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, catheter placement, stent placement, osmotic pump administration (e.g., subarachnoid space or intravenous)) to facilitate delivery. For the delivery of a payload to the peripheral nervous system (PNS) or PNS cells, the compositions described herein can be brought into contact with the patient's PNS or PNS cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection) to facilitate delivery. For the delivery of a payload to the lungs or lung cells, the compositions provided herein can be brought into contact with the patient's lungs or lung cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct pulmonary administration to lung tissue and cells) to facilitate delivery. In some embodiments, the composition comprises lipid molecules functionalized with neurotransmitter-based functional groups that enable the delivery of the payload to the brain across the blood-brain barrier (BBB).

[0273] For the delivery of payloads to the vascular system or vascular cells, the compositions provided herein can come into contact with the patient's vascular system or vascular cells via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or local administration (e.g., clamp, catheter placement, stent placement) to facilitate delivery.

[0274] To deliver the payload to the skin or skin cells (e.g., dermal cells and / or follicular cells), the compositions described herein can be brought into contact with the patient's skin or skin cells (e.g., dermal cells and / or follicular cells) via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct skin application, ion electrophoresis) to facilitate delivery. To deliver the payload to the eye or eye cells (e.g., macula, fovea, cornea, retina), in one embodiment, the compositions of the present invention can be brought into contact with the patient's eye or eye cells (e.g., macula, fovea, cornea, retina) via parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection, intraocular injection, periorbital injection, subretinal injection, iontophoresis, use of eye drops, implantation) to facilitate delivery. To deliver a payload to the ear or ear cells (e.g., cells of the inner ear, middle ear, and / or outer ear), the compositions provided herein can be brought into contact with the patient's ear or ear cells (e.g., cells of the inner ear, middle ear, and / or outer ear) as is commonly known in the art, such as by parenteral administration (e.g., intravenous, intramuscular, subcutaneous administration) or topical administration (e.g., direct injection). To deliver a payload (e.g., RNA encoding an immunogen) to cells of the immune system (e.g., antigen-presenting cells, including professional antigen-presenting cells), the compositions provided herein can be delivered intramuscularly, after which the immune cells can infiltrate the delivery site, process the delivered RNA, and / or process the encoded antigen produced by non-immune cells such as 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), and monocytes (e.g., human peripheral blood monocytes) (see, for example, WO2012 / 006372 by Geall, Andy et al.).

[0275] Immunization. For immunization purposes, the compositions provided herein may be prepared as injectable, pulmonary or nasal aerosols, or in a delivery device (e.g., syringe, nebulizer, sprayer, inhaler, skin patch, etc.). Using this delivery device, the pharmaceutical compositions may be administered to a subject, e.g., a human, for immunization purposes.

[0276] For immunization purposes, in some embodiments, the embodiments provided herein include delivering one or more RNAs encoding immunogens. The immunogens can induce an immune response that recognizes them in order to provide immunity against pathogens, allergens, or tumor antigens. Immunization against diseases and / or infections caused by pathogens is preferred.

[0277] The embodiments described herein are provided for illustrative purposes only and are not intended to limit the invention; they will be more readily understood by referring to the following examples.

[0278] The following embodiments are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting its scope. [Examples]

[0279] Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. Generally, the terms and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques are carried out according to the manufacturer's specifications, or as generally achieved in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for the chemical synthesis, chemical analysis, drug preparation, formulation, and delivery of patients, and for treatment.

[0280] Preparation of the compound of formula I : The compounds disclosed herein may be synthesized by the methods described below, or by modifications thereof. Modifications to the methodologies include, among other things, temperatures, solvents, reagents, etc., known to those skilled in the art. Generally, during any of the processes for preparing the compounds disclosed herein, it may be necessary and / or desirable to protect the sensitive or reactive groups of any of the relevant molecules. This can be achieved by conventional protecting groups, such as those described in Protective Groups in Organic Chemistry (ed. JFW Mc Omie, Plenum Press, 1973) and TW Green, PGM Wuts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999) (both incorporated herein in their entirety by reference). Protecting groups may be removed at a convenient later stage using methods known from the art. Synthetic chemical transformations useful for the synthesis of the compound in question are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers, 1989, or L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons, 1995 (both incorporated herein in their entirety by reference). The routes shown and described herein are illustrative only and are not intended to, and should not be construed as, limiting the claims in any way. Those skilled in the art will recognize modifications of the disclosed synthesis and be able to devise alternative routes based on the disclosure herein. All such modifications and alternative routes are within the scope of the claims.

[0281] In the following scheme, oxygen atom protecting groups are selected based on their compatibility with the required synthetic steps, as well as their compatibility with the introduction and deprotection steps and the overall synthetic scheme (TW Green, PGM Wuts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999)).

[0282] If a compound in this technology contains one or more chiral centers, such a compound can be prepared or isolated as a pure stereoisomer, i.e., individual enantiomers or d(l) stereoisomers, or a stereoisomer-enriched mixture. All such stereoisomers (and enriched mixtures) are included within the scope of this technology unless otherwise indicated. Pure stereoisomers (or enriched mixtures) can be prepared, for example, using optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds can be separated, for example, using chiral column chromatography, chiral decomposition agents, etc.

[0283] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), and Emka-Chemce or Sigma (St. Louis, Missouri, USA). Others 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 Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley, and Sons, 5th Edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or by obvious modifications thereof.

[0284] Examples General procedure It will be apparent to those skilled in the art that methods for preparing precursors and functional groups related to the compounds claimed herein are generally described in the literature. These reactions may also utilize variants known to those skilled in the art but not described in more detail. Those skilled in the art, given the literature and this disclosure, are well equipped to prepare any of the compounds.

[0285] Those skilled in organic chemistry will recognize that the operations can be easily carried out without further instructions, i.e., that performing these operations is within the scope and practice of those skilled in the art. These include the reduction, oxidation, acylation, aromatic substitution, electrophilic and nucleophilic processes, etherification, esterification, and saponification of carbonyl compounds to their corresponding alcohols. These operations are described in standard texts such as March Advanced Organic Chemistry (Wiley), Carey and Sundberg, and Advanced Organic Chemistry (which is incorporated herein by reference in its entirety). Unless otherwise specified, all intermediate compounds of the present invention were used without further purification.

[0286] Those skilled in the art readily understand that certain reactions are best performed when other functional groups are masked or protected intramolecularly, thus avoiding any undesirable side effects and / or increasing the reaction yield. Often, those skilled in the art utilize protecting groups to achieve such yield increases or to avoid undesirable reactions. These reactions are found in the literature and are within the scope of those skilled in the art. Many examples of these operations can be found, for example, in TW Greene and PGM Wuts, Protecting Groups in Organic Synthesis, 4th Ed., John Wiley & Sons (2007) (the whole of which is incorporated herein by reference).

[0287] The following exemplary schemes are provided for the guidance of the reader and represent preferred methods for preparing the compounds illustrated herein. These methods are not limiting, and it will be apparent that other routes may be used to prepare these compounds. Such methods specifically include solid-phase-based chemistry, including combinational chemistry. Those skilled in the art will be fully equipped to prepare these compounds by these methods given in the literature and this disclosure. The numbering of compounds used in the synthesis schemes shown below means only those specific schemes and should not be construed as or confused with the same numbering in other sections of this application.

[0288] The trademarks used herein are merely examples and reflect exemplary materials used at the time of the invention. Those skilled in the art will recognize that variations in lot, manufacturing process, etc., are to be expected. Accordingly, the examples and the trademarks used therein are not limiting and are not intended to limit, but merely illustrate how those skilled in the art may choose to carry out one or more embodiments of the invention.

[0289] The following abbreviations have the meanings indicated. JPEG2026528786000286.jpg143153

[0290] The following exemplary schemes are provided for the guidance of the leader and represent exemplary methods for preparing the compounds provided herein. Furthermore, other methods for preparing the compounds described herein will be readily apparent to those skilled in the art in light of the following reaction schemes and examples. Unless otherwise indicated, all variables are as defined above.

[0291] Example 1 (Butane-1,4-diylbis(tetradecylazanediyl))bis(3-aminopropane-1,2-diyl)(9E,9'E)-bis(octadeca-9-enoate) (Synthesis of compound 1 in Scheme I) A solution of DHDMS (1.7 g, 2.7 mmol) and triethylamine (0.79 g / 1.1 mL, 7.8 mmol, 2.9 equivalents) in ethyl acetate (90 mL), stirred at room temperature under a nitrogen atmosphere, was mixed with di-tertio-butyloxycarbonate (DiBoc) (1.7 g, 7.8 mmol, 2.9 equivalents) in ethyl acetate (5 mL). The mixture was stirred at room temperature under nitrogen for 16 hours. The resulting solution was diluted with ethyl acetate (100 mL), washed with brine (2 × 100 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure to produce a yellow oily substance. Next, the oily substance was purified by silica gel flash column chromatography using MeOH / DCM as the solvent to produce di-tert-butyl((butane-1,4-diylbis-(tetradecylazane-diyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate (1A) (1.1 g, yield 48%).

[0292] To a solution of di-tert-butyl((butane-1,4-diylbis(tetradecylazandiyl))-bis(2-hydroxypropane-3,1-diyl))-dicarbamate (1A) (0.89 g, 1.1 mmol) in DMF (50 mL), diisopropylethylamine (DIPEA) (1.67 g / 2.25 mL, 12.9 mmol, 12.0 equivalents) was added and the mixture was stirred at room temperature under a nitrogen atmosphere for 10 minutes. To the resulting solution, oleoyl chloride (2.8 g, 9.3 mmol, 8.7 equivalents) was added and the mixture was stirred at room temperature for 10 minutes. The solution was then heated and stirred at 60°C for 16 hours. The reaction mixture was then concentrated under reduced pressure to produce a yellow oily substance. The resulting oily substance was then diluted with DCM (100 mL) and washed with water (3 × 50 mL). Next, the combined aqueous layer was back-extracted with DCM (2 × 50 mL), the combined organic layer was dried over Na2SO4, filtered, and concentrated to produce a yellow oily substance. The crude oil was then purified using silica gel flash column chromatography with (10-100%) DCM / hexane and (0-100% MeOH) to produce a BOC-protected diester intermediate (1B) (1.1 g, yield 73%) (m / z MH+ = 1356.21).

[0293] A solution of Boc-protected diester (1B) (1.1 g, 0.785 mmol) in DCM (10 mL) was cooled to 0°C using an ice bath and stirred under a nitrogen atmosphere. TFA (1 mL) was added, and the resulting solution was stirred for 5 hours while warming to room temperature. Upon completion, the solution was concentrated under reduced pressure to produce a chestnut-colored oil. The oil was dissolved in DCM (1 mL) and MeOH (4 mL), and the solution was packed into a C-18 reversed-phase column (Innoval ODS-2, 1.0 × 25 cm) and purified using an (85-100%) water / MeOH gradient to obtain tetra-trifluoroacetate of compound 1 (71.6 mg) (m / z MH). + 1156.11).

[0294] Compounds 2-4 were synthesized using the same procedure as above. Intermediate 1A was treated with linoleic acid chloride, followed by TFA treatment to obtain the trifluoroacetate salt of compound 2 (1.0 g) (M+H + = 1100.05614 m / z free base).

[0295] Intermediate 1A was treated with myristoyl chloride, followed by TFA treatment to obtain the trifluoroacetate salt of compound 3 (323 mg) (M+H + = 1048.02888 m / z (free base).

[0296] Intermediate 1A was treated with palmitreoyl chloride, followed by TFA treatment to obtain the trifluoroacetate salt of compound 4 (264 mg) (M+H + = 1152.08786 m / z free base).

[0297] Example 2 N 1 ,N 4 Bis(2-(((E)-octadeca-9-enoyl)oxybutyl)-N 1 ,N 4 -Ditetradecylbutane-1,4-diaminium hydrochloride (synthesis of compound 6 in scheme VIIIA) In a heavy-walled, airtight, round-bottomed container, add 2,2,2-trifluoroethanol (50 ml) N 1,N 4 -Ditetradecylbutane-1,4-diamine (2.1 g, 4.3 mmol) was packed into the container. DIPEA (3.62 mL, 20.8 mmol, 4.8 equivalents) and 1,2-butylene oxide (0.9 mL, 10.4 mmol, 2.4 equivalents) were added, the container was sealed, heated to 80°C, and stirred for 20 hours. The solution was concentrated under reduced pressure to produce a chestnut-colored oily substance. The oily substance was dissolved in DCM (5 ml) and purified on a normal-phase silica gel column with a (0-10%) MeOH / DCM gradient to produce 1,1'-(butane-1,4-diylbis(tetradecylazanediyl))bis(butane-2-ol, compound 7) (1.34 g, yield 50%) as a white solid. M+H + = 625.62639.

[0298] A solution of 1,1'-(butane-1,4-diylbis(tetradecylazandiyl))bis(butan-2-ol) (0.5 g, 0.8 mmol) in DMF (24 mL) was mixed with DIPEA (1.8 mL, 9.9 mmol, 12.0 equivalents). The mixture was stirred under nitrogen at room temperature for 30 minutes. To the resulting solution, oleoyl chloride (1.9 g, 6.5 mmol, 7.8 equivalents) was added and the mixture was stirred at room temperature for 10 minutes. The resulting solution was then heated to 60°C and stirred for 16 hours. The reaction solution was then concentrated under reduced pressure to produce a yellow oil. The resulting oil was then diluted with water (100 mL) and extracted with toluene (6 × 50 mL). The combined organic layers were then washed with brine, dried over Na₂SO₄, filtered, and concentrated to produce a yellow oil. Next, the resulting oily substance was purified on a normal phase column with (0-100%) ethyl acetate / dimethyl chloride to produce (butane-1,4-diylbis(tetradecylazandiyl))bis(butane-1,2-diyl)bis(octadeca-2,4,6,8,10,12,14,16-octinoate, compound 6A) (210 mg, yield 22%). M+H + = 1154.087.

[0299] A round-bottom flask filled with (butane-1,4-diylbis(tetradecylazandiyl))bis(butane-1,2-diyl)bis(octadeca-2,4,6,8,10,12,14,16-octinoate) (188 mg, 163 mM) in DCM (3 mL) was filled with 4.0 M HCl (100 μL / 14.6 mg, 400 mM, 2.45 equivalents) in dioxane and stirred at room temperature for 1 hour under nitrogen gas. The resulting solution was concentrated under reduced pressure and N 1 ,N 4 -Bis(2-(((E)-Octadeca-9-enoyl)oxybutyl)-N 1 ,N 4 -Ditetradecylbutane-1,4-diaminium (compound 6) was produced as a white oily substance (186.4 mg, yield 94%). M+H + = 1154.15064.

[0300] Compound 5 was synthesized using the same procedure as above. The intermediate 1,1'-(butane-1,4-diylbis(tetradecylazanediyl))bis(butane-2-ol) was treated with linoleic acid chloride, followed by HCl treatment, and then N 1 ,N 4 -Bis(2-((9Z,12Z)-Octadeca-9,12-dienoyl)oxy)butyl)-N 1 ,N 4 -Ditetradecylbutane-1,4-diaminium, the hydrochloride salt of compound 5 was obtained (139.5 mg, yield 53.6%). M+H + = 1150.11951.

[0301] Example 3 N 1 ,N 1 '-(butane-1,4-diyl)bis(N2-(2-hydroxyethyl)-N 1 Synthesis of tetradecylpropane-1,2,3-triaminium)trifluoroacetate (compound 8 in Scheme IIA) Di-tert-butyl((butane-1,4-diylbis(tetradecylazandiyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate 1A (1.2 g, 1.5 mmol) was packed into a 250 mL round-bottom flask, and 20 mL of DCM was added. The solution was stirred at room temperature until the compound dissolved, then 1.4 mL of TEA (10 mmol, 6.8 equivalents) was added at 0 °C, and the resulting solution was stirred at 0 °C for 40 minutes. Methanesulfonyl chloride (787 mg, 6.9 mmol, 4.7 equivalents) in 30 mL of DCM was added. The mixture was stirred for 16 hours, and the reaction was warmed to room temperature. Once complete, the solution was diluted with 200 mL of DCM and washed with 3 × 100 mL of water. The combined aqueous layer was back-extracted with 4 × 100 mL of DCM. The combined organic layers were dried over Na2SO4, filtered, and the solution was concentrated under reduced pressure to produce 2,2,21,21-tetramethyl-4,19-dioxo-9,14-ditetradecyl-3,20-dioxa-5,9,14,18-tetraazadocosan-7,16-diyldimethanesulfonate 8A as an oily substance without further purification. This was then transferred without further purification. 1.27 g was produced, yielding 87%, and LC-MS (ESI) m / z 1027.82[M+2Na+H + ].

[0302] A 100 mL round-bottom flask filled with 2,2,21,21-tetramethyl-4,19-dioxo-9,14-ditetradecyl-3,20-dioxa-5,9,14,18-tetraazadocosan-7,16-diyldimethanesulfonate 8A (620 mg, 0.63 mmol) in ethanol (1 mL) was used to add 2-aminoethane-1-ol (0.4 g, 6.5 mmol, 10.3 equivalents) in ethanol (1 mL). Potassium carbonate (0.4 g, 2.9 mmol, 4.6 equivalents) was added to the solution. The resulting solution was heated to 65 °C and stirred overnight. Once the reaction was complete, it was concentrated to dryness under reduced pressure to produce a white oily substance. The resulting oily substance was diluted with ELISA (20 mL) and washed with water (3 × 10 mL). The combined aqueous layer was back-extracted with ELISA (4 × 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to produce di-tert-butyl((butane-1,4-diylbis(tetradecylazandiyl))bis(2-((2-hydroxyethyl)amino)propane-3,1-diyl)dicarbamate 8B as an oily substance, which was used in the next step without purification. 556 mg, yield 96%, LC-MS (ESI) m / z 913.79[M+H + ].

[0303] Trifluoroacetic acid (5 ml) was added to a round-bottom flask packed with di-tert-butyl((butane-1,4-diylbis(tetradecylazandiyl))bis(2-((2-hydroxyethyl)amino)propane-3,1-diyl))dicarbamate 8B (556 mg, 0.6 mmol) in DCM (15 ml). The resulting solution was stirred at room temperature for 5 hours. The reaction solution was concentrated under reduced pressure to produce a yellow oily substance. The oily substance was then dissolved in MeOH (2 mL) and water (4 mL). The solution was then packed into a reverse-phase column and purified with (50-100%) MeOH / water, and N 1 ,N 1 '-(butane-1,4-diyl)bis(N 2 -(2-hydroxyethyl)-N 1-Tetradecylpropane-1,2,3-triaminium)trifluoroacetate (compound 8) was prepared. 126 mg, yield 16%, LC-MS (ESI); m / z 713.70[M+H + ].

[0304] Compounds 9-12, 39-41, 43, 46, and 80 were synthesized using the same procedure as above. Intermediate 8A was treated with 4-aminobutan-1-ol, followed by TFA treatment, and then N 1 ,N 1 '-(butane-1,4-diyl)bis(N 2 Compound 9, -(2-hydroxyethyl)-N1-tetradecylpropane-1,2,3-triaminium)trifluoroacetic acid, was obtained. (138 mg, yield 16%, LC-MS (ESI) m / z 769.75 [M+H]) + .

[0305] Intermediate 8A is treated with 3-aminopropane-1,2-diol, followed by TFA treatment, and then N 1 ,N 1 '-(butane-1,4-diyl)bis(N 2 -(2,3-dihydroxypropyl)-N 1 Compound 10 was obtained as -tetradecylpropane-1,2,3-triaminium)trifluoroacetic acid. LC-MS (ESI) m / z 773.70 [M + H] + .

[0306] Intermediate 8A is treated with 4-(aminomethyl)phenol, followed by TFA treatment, and then N 1 ,N 1 '-(butane-1,4-diyl)bis(N 2 -(4-hydroxybenzyl)-N 1 Compound 11 was obtained as -tetradecylpropane-1,2,3-triaminium)trifluoroacetic acid. LC-MS (ESI) m / z 837.71 [M + H] + .

[0307] Intermediate 8A is treated with 2-aminoethane-1-ol, followed by TFA treatment, and then N 1 ,N1 '-(butane-1,4-diyl)bis(N 2 -(2-mercaptoethyl)-N 1 Compound 12 was obtained as -tetradecylpropane-1,2,3-triaminium)trifluoroacetic acid. LC-MS (ESI) m / z 745.64 [M + H] + .

[0308] Intermediate 8A was treated with 3-aminopropan-1-ol, followed by TFA treatment to obtain compound 39, 14-((3-ammonio-2-((3-hydroxypropyl)amino)propyl)(4-((3-ammonio-2-((3-hydroxypropyl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecane-1-illiumtrifluoroacetic acid. LC-MS (ESI) m / z 741.72 [M + H] + .

[0309] Intermediate 8A was treated with 5-aminopentan-1-ol, followed by TFA treatment to obtain compound 40, 14-((3-ammonio-2-((5-hydroxypentyl)amino)propyl)(4-((3-ammonio-2-((5-hydroxypentyl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecane-1-illiumtrifluoroacetic acid. LC-MS (ESI) m / z 797.78 [M + H] + .

[0310] Intermediate 8A is treated with 6-aminohexane-1-ol, followed by TFA treatment, and then N 1 ,N 1 '-(butane-1,4-diyl)bis(N2-(6-hydroxyhexyl)-N 1 Compound 41, compound 41, was obtained using -tetradecylpropane-1,2,3-triaminium)trifluoroacetic acid. LC-MS (ESI) m / z 825.82 [M + H] + .

[0311] Intermediate 8A was treated with 2-amino-3-phenylpropan-1-ol, followed by TFA treatment to obtain compound 43, 14-((3-ammonio-2-((1-hydroxy-3-phenylpropan-2-yl)amino)propyl)(4-((3-ammonio-2-((1-hydroxy-3-phenylpropan-2-yl)ammonio)propyl)(tetradecyl)ammonio)ammonio)tetradecane-1-illiumtrifluoroacetic acid. LC-MS (ESI) m / z 893.77 [M + H] + .

[0312] Intermediate 8A was treated with 2-amino-2-methylpropan-1-ol, followed by TFA treatment to obtain compound 46, 14-((3-ammonio-2-((1-hydroxy-2-methylpropan-2-yl)amino)propyl)(4-((3-ammonio-2-((1-hydroxy-2-methylpropan-2-yl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecane-1-illium trifluoroacetic acid. LC-MS (ESI) m / z 769.75.72 [M + H] + .

[0313] Intermediate 8A was treated with 2-amino-2-methylpropane-1,3-diol, followed by TFA treatment to obtain compound 48, 14-((3-ammonio-2-((1,3-dihydroxy-2-methylpropane-2-yl)amino)propyl)(4-((3-ammonio-2-((1,3-dihydroxy-2-methylpropane-2-yl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecane-1-illium trifluoroacetate. LC-MS (ESI) m / z 801.73 [M + H] + .

[0314] Intermediate 8A was treated with 1-aminopropan-2-ol, followed by TFA treatment to obtain 14-((3-ammonio-2-((2-hydroxypropyl)amino)propyl)(4-((3-ammonio-2-((2-hydroxypropyl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecane-1-illiumtrifluoroacetic acid, compound 80. LC-MS (ESI) m / z 741.72 [M + H] + .

[0315] Example 4 Lipid nanoparticle (LNP) formulations were screened and evaluated by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, in vivo distribution, and expression of the payload-encoded protein. Compositions containing compounds 1-9 and helper lipids were prepared and complexed with mRNA. As shown in Table 2, the tested formulations differed in the molar ratio of compound 1 to helper lipids.

[0316] Table 2: Exemplary LNP preparations JPEG2026528786000287.jpg44160

[0317] All LNP formulations contained lipid compounds, DOPE, cholesterol, and DMG-PEG. Some formulations contained the peptide of SEQ ID NO: 47. All lipids were weighed and solubilized in ethanol at the desired molar ratio. This lipid mixture and firefly luciferase (fLuc) mRNA were complexed with LNPs using a microfluidic device. The LNPs were dialyzed in phosphate buffer (LNP1-LNP5, LNP13-LNP60) or TRIS buffer (LNP6-LNP10), and particle size and homogeneity were measured using dynamic light scattering. The following day, the LNPs were injected into mice.

[0318] Female BALB / c mice aged 6–10 weeks were purchased from the Jackson Laboratory and acclimatized for 7 days prior to the study. Mice were injected with LNP equivalent to 10 μg of fLuc mRNA via intravenous tail vein injection of 200 μl total volume. Four hours after injection, the mice were anesthetized with isofluorane, and imaging was performed 10 minutes after intraperitoneal injection of 100 μL of Rediject D-luciferin (Perkin Elmer). Bioluminescence images were quantified in vivo (whole body) and ex vivo (organ) using the IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.

[0319] All LNP formulations containing compound 1 had a particle size >100 nm and a polydispersity index <0.3 (Figure 1). Intravenous administration of LNP formulations containing compound 1 resulted in mRNA delivery and luciferase expression in the liver (Figure 2) and spleen (Figure 3) of injected mice. LNPs containing compound 1 showed higher hepatic expression compared to spleen expression, indicating that LNPs containing compound 1 are an efficient mRNA delivery system for hepatic delivery.

[0320] Example 5 LNP formulations were screened and evaluated by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, in vivo distribution, and expression of the payload-encoded protein. Compositions containing compounds 1-9 and helper lipids were prepared and complexed with mRNA. As shown in Table 3, the tested formulations differed in the molar ratio of compounds 1-9 to helper lipids.

[0321] Table 3: Exemplary LNP formulations JPEG2026528786000288.jpg179153 JPEG2026528786000289.jpg201153

[0322] All LNP formulations contained lipid compounds, either DOPE or DSPC, or both, cholesterol, and either DMG-PEG or C16-PEG. Some formulations contained peptide SEQ ID NO: 47. All lipids were weighed and solubilized in ethanol at the desired molar ratio. This lipid mixture and firefly luciferase (fLuc) mRNA were complexed with LNPs using a microfluidic device. The LNPs were dialyzed in phosphate buffer, and particle size and uniformity were measured using dynamic light scattering. The following day, the LNPs were injected into mice.

[0323] Female BALB / c mice aged 6–10 weeks were purchased from the Jackson Laboratory and acclimatized for 7 days prior to the study. Mice were injected with LNP equivalent to 10 μg of fLuC mRNA via intravenous tail vein injection of 200 μl total volume. Four hours after injection, the mice were anesthetized with isofluorane, and imaging was performed 10 minutes after intraperitoneal injection of 100 μL of Rediject D-luciferin (Perkin Elmer). Bioluminescence images were quantified in vivo (whole body) and ex vivo (organ) using the IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.

[0324] All LNP formulations containing compound 1 had a particle size >400 nm and a polydispersity index <0.3 (Figures 9, 10, 13, 18, 19). Intravenous administration of LNP formulations containing compounds 1-9 resulted in mRNA delivery and luciferase expression in the liver (Figures 4, 11, 14), spleen (Figures 5, 12, 15, 20), and lung (Figure 21) of injected mice. LNPs containing compounds 1-4 showed higher liver expression compared to the spleen, indicating that LNPs containing compounds 1-4 are efficient mRNA delivery systems for liver delivery. LNPs containing compounds 5-9 showed higher spleen expression compared to the liver, indicating that LNPs containing compounds 5-9 are efficient mRNA delivery systems for liver delivery.

[0325] Example 6 Human primary T cell transfection protocol: Compound 1 was dissolved in ethanol to obtain a final concentration of 25 mg / mL. Formulations were prepared by mixing Compound 1 with DOPE, 25 mg / mL, in molar ratios of 1:1 and 1:2. LNPs were prepared by adding 4 mL of formulated lipid to 90 mL of diluted 5-methoxyuridine (5 moU) modified green fluorescent protein (eGFP) mRNA and 1 mg of mRNA in 100 mM sodium acetate buffer at approximately pH 5.2, and vortexing three times. After incubation at room temperature for 10 minutes, an appropriate amount of mRNA was added to activated human primary T cells. Transfection efficiency was evaluated by flow cytometry 2 days after transfection. EP: Electroporation as control / reference, 100 ng of 5 moU modified eGFP mRNA, 10 μL, electroporation conditions, 1400 V, 20 ms, and 2 × 10⁶ in one pulse. 5 It was mixed with T cells.

[0326] Table 4. Exemplary lipid preparations: JPEG2026528786000290.jpg28153

[0327] Table 5. Exemplary lipid complex formulations: JPEG2026528786000291.jpg34153

[0328] Lipid formulations LP24 and LP25 showed higher transfection efficiency compared to LNP11 and LNP12, which appeared to indicate that a high proportion of DOPE and compound 1 played a role in primary human T cell transfection, as shown in Figures 6 and 7. Furthermore, formulations LP24, LP25, and lipid nanoparticles LNP11 and LNP12 showed better cell viability compared to electroporated samples, as seen in Figure 8.

[0329] Example 7 Formulation and screening of compound 8: Compound 8 was dissolved in chloroform to prepare a 25 mg / mL stock solution. Similarly, DOPE and cholesterol were dissolved in 25 mg / mL chloroform as copolymers. Compound 8 containing the cationic lipid, DOPE, or cholesterol was mixed in glass vials at different molar ratios (2:1, 1:1, 1:2, 1:4, and 1:8). Chloroform was removed from these vials, and the lipids were dried using a rotary evaporator. The vials were left overnight in a vacuum dryer to completely remove the chloroform. The following day, the desired amount of 95% ethanol was added to each vial to obtain a final lipid concentration of 2 mg / mL.

[0330] Liposome preparation: For formulation in water, reverse-phase evaporation was used. Lipids in chloroform were added to vials with the desired amount of water required to achieve a final lipid concentration of 2 mg / mL, and the chloroform was subsequently removed using a rotary evaporator. Lipids in water or 95% ethanol were tested for transfection efficiency on VPC1.0 cells (suspended HEK293 cells) using antibody fragment-GFP plasmids (Dulaglutide-T2A GFP), either 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).

[0331] Transfection of suspension cells VPC1.0 cells (virus-producing cells derived from the HEK293F cell line) divide every 3-4 days in ExpiFectamine293 (Expi293 expression medium, resulting in 6 × 10⁶ cells). 6 The cells were maintained by keeping the cell density below cells / mL. For regular maintenance, the cells were 0.3 × 10⁶. 6 ~0.5 × 10 6 The cells are divided into 3 × 10⁶ cells / mL densities. The day before transfection, the cells are divided into 3 × 10⁶ cells. 6 Divide the cells by the amount of cells / mL, and on the day of transfection, 3 × 10 cells 6The DNA was re-diluted to cells / mL and divided equally into 96-well plates. Approximately 800 μL of cells were divided equally into 2 mL 96-well plates (only the inner 60 wells) and incubated on a 900 rpm shaker set until ready for transfection. The transfection complex was prepared in 100 μL of Opti-MEM containing the DNA (0.8 μg / mL) divided equally in the 96-well plates. The transfection reagent was added to the DNA in the wells, mixed up and down with a pipette, and then incubated for 20 minutes to form lipoplex / lipid nanoparticles. After 20 minutes, 100 μL of the DNA transfection reagent complex was added to the cells, and transfection efficiency and toxicity were determined at 24 and 48 hours post-transfection.

[0332] Transfection efficiency and toxicity To determine efficacy compared with existing catalog products such as ExpiFectamine293 (Expi293) reagent (2.5 μL), a plate reader-based assay was used at 24 and 48 hours post-transfection. GFP fluorescence of cells was also measured using a plate reader. See Figure 16. 10 μL of cells were diluted in medium (40 μL), GFP fluorescence was measured (485 / 515), followed by the addition of PrestoBlue (PB)HS reagent (50 μL of 5-fold dilution of PB in medium). The plate contents were mixed for 2 minutes using an orbital shaker, followed by incubation at 37°C for 1 hour, and then fluorescence was measured (560 / 590). Higher PrestoBlue fluorescence indicates higher viability. See Figure 17.

[0333] Other Embodiments : The present invention is described in conjunction with embodiments for carrying out the invention, but the foregoing description is intended to illustrate, rather than limit, the scope of the invention as defined by the appended claims. Other embodiments, advantages, and modifications are within the scope of the following claims.

[0334] The patents and scientific literature referenced herein establish knowledge available to those skilled in the art. All references cited herein, including U.S. patents, published U.S. patent applications, PCT patent applications designating the United States, published foreign patents, and patent applications, are incorporated herein by reference in their entirety. Filings to GenBank and NCBI, indicated by accession numbers cited herein, are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference. In the event of any conflict, this specification, including definitions, shall prevail. In addition, materials, methods, and examples are illustrative only and not intended to be limiting.

[0335] While the present invention is specifically illustrated and described with reference to its preferred embodiments, those skilled in the art will understand that various modifications of form and detail can be made within the scope of the invention as encompassed by the appended claims without departing from it.

[0336] While this disclosure is described with reference to the embodiments described above, modifications and variations will be understood to be included within the spirit and scope of this disclosure. Accordingly, this disclosure is limited only by the following claims.

Claims

1. A compound having general structure I, or a pharmaceutically acceptable salt thereof, I During the ceremony, A 1 However, - (CH 2 ) x - or - (CH 2 ) y -A 4 - (CH 2 ) z -and, AND 4 が、-(CH 2 ) x -、-(CO)O-、-O(CO)-、-SS-、 、 、 , and Selected from the group consisting of, Q 1 However, N or Q 2 However, N or And, R 1 and R 2 However, H and C are substituted independently of each other. 1 ~C 30 Linear or branched alkyl groups, optionally substituted with carbon 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyls, or optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkynyl groups, R 3 However, -COR, -(CH 2 ) n COR, or - (CH 2 ) n COOR, or -(CO)NHR-, or -(CO)N(R) 2 - , or , or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 1 ~C 30 Linear or branched alkyl groups, or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, -OR 7 ,-N(R 7 ) 2 ,-SR 7 ,-(CH 2 ) n OR 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R 7 ) 2 ,-(CH 2 ) n aryl,-(CH 2 ) n arylalkyl,-(CH 2 ) n Het,an optionally substituted cycloalkyl optionally substituted with,C 4 to C 30 a monounsaturated or polyunsaturated straight or branched chain alkynyl group,or -OR 7 ,-N(R 7 ) 2 ,-SR 7 ,-(CH 2 ) n [[ID=X12]]OR 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R 7 ) 2 ,-(CH 2 ) n aryl,-(CH 2 ) n arylalkyl,-(CH 2 [[ID=X38]]) n a C 3 to C 6 cycloalkyl group optionally substituted with Het, or It should be noted that there may be some unclear or potentially incorrect notations in the original text. This translation is based on the best understanding of the content. If possible, it is recommended to clarify the original text for a more accurate translation. The ring carbons are -O-, -S-, -SS-, -NR 7 - is replaced by C 3 ~C 6 Cycloalkyl groups, or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 Cycloalkenyl group, or Chain carbon atoms are -O-, -S-, -SS-, -S-NR 7 -S-, -NR 7 -SS-NR 7 -, -NR 7 -S-NR 7 -, or replaced by an aryl group, C 1 ~C 30 Linear or branched alkyl groups, or Chain carbon atoms are -O-, -S-, -SS-, -S-NR 7 -S-, -NR 7 -SS-NR 7 -, or -NR 7 -S-NR 7 - Replaced by the base, C 4 ~C 30 Whether it is a monounsaturated or polyunsaturated linear or branched alkenyl group, R 3 However, R 1 and R 2 (CH 2 ) 1～7 H is only true if it is COOR. R 4 However, H, or C which is optionally substituted. 1 ~C 20 Linear or branched alkyl groups, optionally substituted with carbon 1 ~C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R 5 and R 6 However, independently, H and CH 3 Selected from, R 7 However, H, or C which is optionally substituted. 1 ~C 6 Linear or branched alkyl groups, optionally substituted monounsaturated or polyunsaturated carbon atoms. 1 ~C 6 Linear or branched alkenyls, or And, R is replaced by C in an arbitrary choice. 4 ~C 30 Linear or branched alkyl groups, optionally substituted with C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH 2 ) 0～3 Selected from the group consisting of Het, A 2 and A 3 However, independently, H, 、 、 、 、 、 、 、 、 、 , and Selected from the group consisting of, R 8 However, -COR or or And, In the formula, R 9 However, H or or And, In the formula, R 10 However, H, -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 1 ~C 30 Linear or branched alkyl groups, Chain carbon atoms are -O-, -S-, -SS-, -NR 7 -, replaced by an aryl group, C 1 ~C 30 Linear or branched alkyl groups, -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl-(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 Cycloalkylalkyl groups, The ring carbons are -O-, -S-, -SS-, -NR 7 - is replaced by C 3 ~C 6 Cycloalkylamines, and -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 Selected from the group consisting of cycloalkenylamines, In the formula, R 11 However, -NH 2 -NHR, -N(R) 2 , 、 , and Selected from the group consisting of, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 6. b is an integer between 0 and 6. x is an integer from 1 to 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n 1 , n 2 , n 3 , n 4 , n 5 However, they are independent integers from 1 to 5. n 6 However, these are integers from 0 to 7. n 7 and n 8 However, they are independent integers from 0 to 5. n 9 However, these are integers from 1 to 5. m 1 However, these are integers from 1 to 5. m 2 However, these are integers from 0 to 5. m 3 However, these are integers from 1 to 7. A compound, or a pharmaceutically acceptable salt thereof, in which p is an integer between 1 and 50, and Het is an optionally substituted 5- to 7-membered monocyclic basic heterocycle or an optionally substituted 8- to 11-membered bicyclic basic heterocyclil.

2. A 1 However, - (CH 2 ) x - or - (CH 2 ) y -A 4 - (CH 2 ) z -and, AND 4 が、-(CH 2 ) x -、-(CO)O-、-O(CO)、 -SS-, 、 、 , and Selected from the group consisting of, Q 1 However, N is Q 2 However, N is, R 1 and R 2 However, C is substituted independently and at will. 1 ~C 30 Linear or branched alkyl groups, optionally substituted with carbon 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyls, or optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkynyl groups, R 3 However, -COR, -(CH 2 ) n COR, or - (CH 2 ) n COOR, or -(CO)NHR-, or -(CO)N(R) 2 - or And, R 4 However, H, or C which is optionally substituted. 1 ~C 20 Linear or branched alkyl groups, optionally substituted with carbon 1 ~C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is replaced by C in an arbitrary choice. 4 ~C 30 Linear or branched alkyl groups, optionally substituted with C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyls, - (CH 2 ) 0～3 Selected from the group consisting of Het, A 2 and A 3 However, independently, H, 、 、 、 、 、 、 、 、 , and Selected from the group consisting of, In the formula, R 10 However, H, -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 1 ~C 30 Linear or branched alkyl groups, Chain carbon atoms are -O-, -S-, -SS-, -NR 7 -, replaced by an aryl group, C 1 ~C 30 Linear or branched alkyl groups, -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl-(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 Selected from the group consisting of cycloalkylalkyl groups, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 6. b is an integer between 0 and 6. x is an integer from 1 to 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n 1 , n 2 , n 3 , n 4 , n 5 However, they are independent integers from 1 to 5. n 6 However, these are integers from 0 to 7. n 7 and n 8 However, they are independent integers from 0 to 5. n 9 However, these are integers from 1 to 5. m 1 However, these are integers from 1 to 5. m 2 However, these are integers from 0 to 5. m 3 However, these are integers from 0 to 5. p is an integer between 1 and 50. The compound according to claim 1, wherein Het is an optionally substituted 5-7 member monocyclic basic heterocycle or an optionally substituted 8-11 member bicyclic basic heterocyclil.

3. A compound according to claim 1 having the structure of formula Ia, or a pharmaceutically acceptable salt thereof, in the formula, R 1 and R 2 However, C is substituted independently and at will. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, A 2 and A 3 However, they became independent, 、 、 、 、 、 、 , and Selected from the group consisting of, R 3 However, -COR, -(CH 2 ) n COR, or - (CH 2 ) n COOR, or -(CO)NHR-, or -(CO)N(R) 2 - or or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 1 ~C 30 Linear or branched alkyl groups, or Chain carbon atoms are -O-, -S-, -SS-, -S-NR 7 -S-, -NR 7 -SS-NR 7 -, -NR 7 -S-NR 7 -, or replaced by an aryl group, C 1 ~C 30 Linear or branched alkyl groups, or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl-(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 Cycloalkyl groups, or The ring carbons are -O-, -S-, -SS-, -NR 7 - is replaced by C 3 ~C 6 Cycloalkyl groups, or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 It is a cycloalkenyl group, R 4 However, H, or C which is optionally substituted. 1 ~C 20 Linear or branched alkyl groups, optionally substituted with carbon 1 ~C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is replaced by C in an arbitrary choice. 4 ~C 30 Linear or branched alkyl groups, optionally substituted with C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH 2 ) 0～3 Selected from the group consisting of Het, In the formula, R 10 However, H, -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 1 ~C 30 Linear or branched alkyl groups, Chain carbon atoms are -O-, -S-, -SS-, -NR 7 -, replaced by an aryl group, C 1 ~C 30 Linear or branched alkyl groups, -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl-(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 Selected from the group consisting of cycloalkylalkyl groups, In the formula, AA represents any natural or unnatural amino acid side chain, x is an integer from 1 to 9. n is an integer between 1 and 5. n 5 However, these are integers from 1 to 5. n 6 However, these are integers from 0 to 7. n 7 and n 8 However, they are independent integers from 0 to 5. n 9 However, these are integers from 1 to 5. m 3 However, these are integers from 1 to 3. p is an integer between 1 and 50. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Het is optionally substituted with a 5- to 7-membered monocyclic basic heterocycle or an optionally substituted with an 8- to 11-membered bicyclic basic heterocyclil.

4. n, independently of each other 5 is 1, or n 7 is 1, or n 9 The compound according to claim 3, wherein is 1.

5. n 6 The compound according to claim 3, wherein the compound is 1, 2, or 3.

6. n 8 The compound according to claim 3, wherein the coefficient is 0, 1, or 2.

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

8. R 3 but, or The compound according to claim 3.

9. R 3 However, C is optionally substituted. 1 ~C 30 Linear or branched alkyl groups, or optionally substituted C 4 ~C 30 The compound according to claim 3, wherein the compound is a monounsaturated or polyunsaturated linear or branched alkenyl group.

10. A compound according to claim 1 having the structure of formula Ib, or a pharmaceutically acceptable salt thereof, in the formula, R 1 and R 2 However, C is substituted independently and at will. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R 3 However, -COR, -(CH 2 ) n COR, or - (CH 2 ) n COOR, or -(CO)NHR-, or -(CO)N(R) 2 - or And, A 2 and A 3 However, they became independent, 、 、 、 , and Selected from the group consisting of, R 4 However, H, or C which is optionally substituted. 1 ~C 20 Linear or branched alkyl groups, optionally substituted with carbon 1 ~C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is replaced by C in an arbitrary choice. 4 ~C 30 Linear or branched alkyl groups, optionally substituted with C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH 2 ) 0～3 Selected from the group consisting of Het, In the formula, AA represents any natural or unnatural amino acid side chain, y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n 5 However, these are integers from 1 to 5. m 2 However, these are integers from 1 to 5. p is an integer between 1 and 50. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Het is optionally substituted with a 5- to 7-membered monocyclic basic heterocycle or an optionally substituted with an 8- to 11-membered bicyclic basic heterocyclil.

11. n, independently of each other 5 Is it 1, or m 2 is 2, or n 9 The compound according to claim 10, wherein is 1.

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

13. A compound according to claim 1 having the structure of formula Ic, or a pharmaceutically acceptable salt thereof, in the formula, R 1 and R 2 However, H and C are substituted independently of each other. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R 3 However, -COR, -(CH 2 ) n COR, or - (CH 2 ) n COOR, or -(CO)NHR-, or -(CO)N(R) 2 - or And, AND 4 It is -(CO)O-, -O(CO)-, -SS-, 、 、 , and Selected from the group consisting of, R 4 However, H, or C which is optionally substituted. 1 ~C 20 Linear or branched alkyl groups, optionally substituted with carbon 1 ~C 20 Monounsaturated or polyunsaturated linear or branched alkenyls R is replaced by C in an arbitrary choice. 4 ~C 30 Linear or branched alkyl groups, optionally substituted with C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH 2 ) 0～3 Selected from the group consisting of Het, In the formula, AA represents any natural or unnatural amino acid side chain, y is an integer between 1 and 4. z is an integer between 1 and 4. n is an integer between 1 and 5. n 1 , n 2 , n 3 , and n 4 However, they are independent integers from 1 to 5. m 1 However, these are integers from 1 to 5. p is an integer between 1 and 50. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Het is optionally substituted with a 5- to 7-membered monocyclic basic heterocycle or an optionally substituted with an 8- to 11-membered bicyclic basic heterocyclil.

14. n 1 , n 2 , n 3 , and n 4 The compound according to claim 13, wherein each of the elements is independently an integer between 1 and 2.

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

16. m 1 The compound according to claim 13, wherein the compound is an integer between 1 and 2.

17. A compound according to claim 1 having the structure of formula Id, or a pharmaceutically acceptable salt thereof, in the formula, A 2 and A 3 However, independently, H, 、 , and Selected from the group consisting of, R 1 and R 2 However, H and C are substituted independently of each other. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R 3 However, -COR, -(CH 2 ) n COR, or - (CH 2 ) n COOR, or -(CO)NHR-, or -(CO)N(R) 2 - or And, R 4 However, H, or C which is optionally substituted. 1 ~C 20 Linear or branched alkyl groups, optionally substituted with carbon 1 ~bC 20 Monounsaturated or polyunsaturated linear or branched alkenyls R 5 and R 6 However, independently, H and CH 3 Selected from, R is replaced by C in an arbitrary choice. 4 ~C 30 Linear or branched alkyl groups, optionally substituted with C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH 2 ) 0～3 Selected from the group consisting of Het, AND 4 It is -(CO)O-, -O(CO)-, -SS-, 、 、 、 , and Selected from the group consisting of, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 2. b is an integer between 0 and 3. y and z are independent integers between 1 and 4. z is an integer between 1 and 4. m 1 and m 2 However, they are independent integers from 1 to 5. n 1 , n 2 , n 3 , n 4 , and n 5 However, they are independent integers from 1 to 5. p is an integer between 1 and 50. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Het is optionally substituted with a 5- to 7-membered monocyclic basic heterocycle or an optionally substituted with an 8- to 11-membered bicyclic basic heterocyclil.

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

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

20. A 2 but, When that is the case, A 3 However, H is, A 4 but, And, R 1 and R 5 However, CH 3 And, R 2 and R 6 However, H is, R 3 However, -COR, -(CH 2 ) n COOR or And, R 7 However, H is, The compound according to claim 17, wherein b is 0, and a pharmaceutically acceptable salt thereof.

21. A 2 and A 3 However, they became independent, and Selected from the group consisting of, A 4 but, And, R 5 and R 6 However, H is, R 3 However, -COR, or -(CH 2 ) n COOR or And, R 7 However, H is, The compound according to claim 17, wherein a and b are 1, and The pharmaceutically acceptable salt.

22. A compound according to claim 1 having the structure of formula Ia, or a pharmaceutically acceptable salt thereof, in the formula, A 1 However, - (CH 2 ) x - or - (CH 2 ) y -A 4 - (CH 2 ) z -and, AND 4 が、-(CH 2 ) x -、-(CO)O-、-O(CO)-、 and -SS-, R 1 and R 2 However, H and C are substituted independently of each other. 1 ~C 30 Linear or branched alkyl groups, optionally substituted with carbon 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyls, or optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkynyl groups, R is replaced by C in an arbitrary choice. 4 ~C 30 Linear or branched alkyl groups, optionally substituted with C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl, -(CH 2 ) 0～3 Selected from the group consisting of Het, a is an integer between 1 and 6. b is an integer between 1 and 6. x is an integer from 1 to 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n 5 However, these are integers from 1 to 5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Het is optionally substituted with a 5- to 7-membered monocyclic basic heterocycle or an optionally substituted with an 8- to 11-membered bicyclic basic heterocyclil.

23. A 1 However, - (CH 2 ) 4 - or - (CH 2 ) 2 -SS-(CH 2 ) 2 - The compound according to claim 22.

24. n 5 The compound according to claim 22, wherein the compound 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. A compound according to claim 1 having the structure of formula Ik, or a pharmaceutically acceptable salt thereof, in the formula, A 1 However, - (CH 2 ) x - or - (CH 2 ) y -SS-(CH 2 ) z -and, R 1 and R 2 However, C is substituted independently and at will. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, In the formula, R 10 However, H, or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl, -(CH 2 ) n Het, optionally substituted with an optionally substituted cycloalkyl, C 1 ~C 30 Linear or branched alkyl groups, or Chain carbon atoms are -O-, -S-, -SS-, -NR 7 -, replaced by an aryl group, C 1 ~C 30 Linear or branched alkyl groups, or -OR 7 , -N(R 7 ) 2 , -SR 7 ,-(CH 2 ) n Ure 7 ,-(CH 2 ) n SR 7 ,-(CH 2 ) n N(R) 7 ) 2 ,-(CH 2 ) n Ariel, - (CH 2 ) n Arylalkyl-(CH 2 ) n C, which is optionally replaced with Het. 3 ~C 6 Selected from the group consisting of cycloalkylalkyl groups, a is an integer between 1 and 6. b is an integer between 1 and 1, x is an integer from 1 to 9. y is an integer between 1 and 4. z is an integer between 1 and 4. n 5 However, these are integers from 1 to 5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Het is optionally substituted with a 5- to 7-membered monocyclic basic heterocycle or an optionally substituted with an 8- to 11-membered bicyclic basic heterocyclil.

28. A 1 However, - (CH 2 ) 4 - or - (CH 2 ) 2 -SS-(CH 2 ) 2 - The compound according to claim 27.

29. n 5 The compound according to claim 27, wherein the compound is 1.

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

31. R 10 However, H, A compound according to claim 27, selected from the group consisting of the following.

32. A compound according to claim 1 having the structure of formula In, or a pharmaceutically acceptable salt thereof, in the formula, A 1 However, - (CH 2 ) y -SS-(CH 2 ) z -and, R 1 and R 2 However, C is substituted independently and at will. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, In the formula, AA represents any natural or unnatural amino acid side chain, a is an integer between 1 and 6. b is an integer between 1 and 6. y is an integer between 1 and 4. z is an integer between 1 and 4. n 5 The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the integer is between 1 and 5.

33. n 5 The compound according to claim 32, wherein the compound 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 the following:

36. A compound according to claim 1 having the structure of formula Iy, or a pharmaceutically acceptable salt thereof, in the formula, R 1 and R 2 However, C is substituted independently and at will. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, y is an integer between 1 and 4. z is an integer between 1 and 4.

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

38. A compound according to claim 1 having the structure of formula Iz, or a pharmaceutically acceptable salt thereof, in the formula, R 1 and R 2 However, C is substituted independently and at will. 1 ~C 30 Linear or branched alkyl groups, and optionally substituted C 4 ~C 30 Selected from the group consisting of monounsaturated or polyunsaturated linear or branched alkenyl groups, R 3 However, -COR- (CH 2 ) n COR or - (CH 2 ) n COOR or -(CO)NHR- or -(CO)N(R) 2 -, or , or C optionally replaced 1 ~C 30 Linear or branched alkyl groups, or optionally substituted C 4 ~C 30 Monounsaturated or polyunsaturated linear or branched alkenyl groups, or optionally substituted C 4 ~C 30 A monounsaturated or polyunsaturated linear or branched alkynyl group. y is an integer between 1 and 4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein 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. R 3 However, -COR or -(CH 2 ) n A compound according to any one of claims 3, 10, 13, 17, and 38, which is COOR.

41. R 1 and R 2 However, - (CH 2 ) 13 CH 3 The compound according to any one of claims 1 to 40.

42. A 1 However, - (CH 2 ) 4 - or - (CH 2 )-A 4 - (CH 2 ) - The compound according to any one of claims 1 to 35.

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 oleyl, oleoil, linoleyl, linoleoil, palmitrail, palmitreoil, palmityl, palmitoyl, myristyl, myristoyl, lauryl, and lauroyl groups.

44. R is - (CH 2 ) 0～3 The compound according to any one of claims 1 to 39, wherein the compound is Het, and Het is a 5- to 7-membered monocyclic basic heterocycle that is optionally substituted.

45. The arbitrarily substituted 5-7 member monocyclic basic heterocycles are 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and A compound according to any one of claims 1 to 39, selected from the group consisting of the following.

46. A compound having a structure selected from the group consisting of the following: During the ceremony, The compound, and its pharmaceutically acceptable salt.

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

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

49. A compound having a structure selected from the group consisting of the following: 【Chemistry 1】 【change】 and its pharmaceutically acceptable salts.

50. A compound having a structure selected from the group consisting of the following: 【Chemistry 2】 【change】 and its pharmaceutically acceptable salts.

51. A compound having a structure selected from the group consisting of the following: During the ceremony, The compound, and its pharmaceutically acceptable salt.

52. A compound having a structure selected from the group consisting of the following: During the ceremony, R 10 However, H, 、 、 The compound, and its pharmaceutically acceptable salt.

53. A compound having a structure selected from the group consisting of the following: During the ceremony, The compound, and its pharmaceutically acceptable salt.

54. A compound having a structure selected from the group consisting of the following: During the ceremony, n 6 However, it is either 0 or 1, R 10 However, H, 、 、 The compound, and its pharmaceutically acceptable salt.

55. A compound having a structure selected from the group consisting of the following: During the ceremony, R 、 、 The compound, and its pharmaceutically acceptable salt.

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

57. (i) A compound according to any one of claims 1 to 55, (ii) One or more structural lipids, (iii) One or more stabilizers, (iv) The composition according to claim 56, optionally comprising a payload.

58. (i) A 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, (v) The composition according to claim 56, optionally comprising a payload.

59. A composition, (i) one or more compounds according to any one of claims 1 to 55, (ii) A composition comprising a payload.

60. The composition according to any one of claims 56 to 59, wherein one or more compounds according to claims 1 to 55 are present in 10 to 80 mol% of the composition, except for any payload, if present.

61. The composition according to any one of claims 56 to 59, wherein the structural lipid is present in 14 to 50 mol% of the composition, except for any payload, if present.

62. The composition according to any one of claims 56 to 59, wherein the stabilizer is present in an amount of 0.1 to 10 mol% of the composition, except for any payload, if present.

63. The composition according to any one of claims 56 to 59, further comprising exosomes, or biomaterials derived from or purified from exosomes.

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

65. The aforementioned polymers include high-density star dendrimers, PAMAM dendrimers, and NH 3 The composition according to claim 64, selected from the group consisting of core dendrimers, ethylenediamine core dendrimers, fifth-generation or higher dendrimers, substituted dendrimers, dendrimers containing one or more amino acids, graft dendrimers, activated dendrimers, polyethyleneimines, polyethyleneimine conjugates, polylysine, polyarginine, polyornithine, histones, and any combination thereof.

66. The composition according to any one of claims 64, wherein the polymer is a 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 conjugate 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 one or more transfection enhancers include a polycationic nucleic acid binding moiety.

70. The composition according to claim 69, wherein the transfection enhancer is selected from the group consisting of an endosome release agent, a cell surface ligand, a nuclear localization agent, a cell permeable peptide, a fusion peptide, and any combination thereof.

71. The composition according to claim 58, wherein the one or more transfection enhancers include an amphiphilic peptide.

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

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

74. The composition according to claim 73, wherein the compound according to claims 1 to 55 contains charge N, the nucleic acid molecule contains charge P, and the combination of the compound according to claims 1 to 55 and the nucleic acid in contact with the cell contains 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 according to claim 72, wherein the payload further comprises one or more peptides and optionally nucleic acids.

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

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

81. The composition according to claim 80, comprising two or more different mRNAs.

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

83. The composition according to 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, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and any combination thereof.

85. The stabilizers include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-di-O-octadenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-difytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 The composition according to any one of claims 56 to 58, comprising one or more phospholipids selected from the group consisting of PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

86. The ionizable lipids include GeneIn (trademark), LipofectAmine (trademark) 2000, LipofectAmine (trademark), Lipofectin (registered trademark), DMRIE-C, CellFectin (registered trademark) (Invitrogen), Oligofectamine (registered trademark) (Invitrogen), LipofectAce (registered trademark) (Invitrogen), Fugene (registered trademark) (Roche, Basel, Switzerland), Fugene (registered trademark) HD ​​(Roche), Transfectam (registered trademark) (Tranfectam, Promega, Madison, WI), Tfx-10 (registered trademark) (Promega), Tfx-20 (registered trademark) (Promega), Tfx-50 (registered trademark) (Promega), Transfectin (trademark) (BioRad, Hercules, CA), SilentFect (trademark) (Bio-Rad), Effectene (registered trademark) (Qiagen, Valencia, CA), and DC-chol (Avanti Polar Lipids), GenePorter® (Gene Therapy Systems, San 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-diaoleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-Diaoleauroyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diamyristyloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diapalmityloxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyldimethyl Minopropyl-β-hydroxyethylamine, 3,5-(N,N-di-lysyl)-diamino-benzoyl-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'- Listyl-β-hydroxyethyl)amido dihydrochloride, L-ornithine-bis-(O,O'-myristoyl-β-hydroxyethyl)amido dihydrochloride, L-ornithine-bis-(O,O'-oleoyl-β-hydroxyethyl)amido dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, 1,4-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane -2,3-diol, 1,4-bis[(3-amino-2-hydroxy-propyl)-palmitylamino]-butane-2,3-diol, 1,4-bis[(3-amino-2-hydroxypropyl)-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-hydroxy-propyl]piperazine, L-arginine-bis-(O,O'-Palmitoyl-β-hydroxyethyl)amido dihydrochloride, L-serine-bis-(O,O'-oleoyl-β-hydroxyethyl)amido dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O'-palmitoyl-β-hydroxy-ethyl)amido dihydrochloride, sarcosine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, L-histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amido dihydrochloride, co Resteryl-3β-carboxyl-amide ethylenetrimethylammonium iodide, 1,4-bis[(3-myristyl-amino)propyl]-piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-carboxylate iodide, cholesteryl-3β-carboxyamide ethyleneamine, cholesteryl-3β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinate iodide 2-[(2-trimethylammonio)-ethylmethyl-amino]ethyl-cholesteryl-3β-oxysuccinate iodide, 3β[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, and 3β[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitylamino)propyl]piperazine, L-ornitylglycyl-N-(1-heptadecyloctadecyl)-glycinamide, N2,N5-bis(3-aminopropyl)-L-ornityl-glycyl-N-(1-heptadecyl Siloctadecyl)-glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkyl-amino)-2-hydroxypropyl]-piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-dioctadecyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N2-[N2,N5-bis(amino-propyl)-L-ornityl]-NN-dioctadecyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]L-ornityl-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-diolyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-o [Lunityl]-NN-Dioleyl-L-α-Glutamine, 4-Bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxy-propyl]piperazine, N2-[N2,N5-Bis(aminopropyl)-L-ornityl]-NN-Dioleyl-L-α-Asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN-Dioleyl-L-Glutaminyl]-L- Glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleyl-amino)-propyl]piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-dipalmytil-L-glutamine, N2-[N2,N5-bis(amino-propyl)-L-ornityl]-NN-dipalmytil-L-α-glutamine, N2-[N2,N5-bis(amino-propyl)-L-ornityl]-NN-dipalmytil-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)-cal [N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN-dipalmytil-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-bis(3-aminopropyl)-L-ornityl]-N,N-dimyristyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dimyristyl-L-α-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornityl]-NN-dimyristyl-L-α-asparagine, 1,4-Bis[(3-(3-amino-2-hydroxy-propyl)-palmitylamino)-2-hydroxypropyl]-piperazine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]L-ornityl-NN-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristyl-amino)-propyl]piperazine, N2-[N2,N5-bis(3-aminopropyl N2-[N2,N5-bis(amino-propyl)-L-ornityl]-N,N-dilaureyl-L-glutamine, N2-[N2,N5-bis(amino-propyl)-L-ornityl]-NN-dilaureyl-L-α-glutamine, N2-[N2,N5-bis(amino-propyl)-L-ornityl]-NN-dilaureyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)-carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornityl-NN-dilaureyl-L-glutamine [L]-L-glutamic acid, 3-[N',N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dioctadeca-9-enylpropionamide, 3-[N',N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dipalmytilpropionamide, 3-[N',N”-bis(2-tert-butyloxycarbonylaminoethyl)guanidino]-N,N-dimyristylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmytil -amino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleyl-amino)propyl]piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-diolylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitylaminopropane, 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-tetradecyl-oxyethyl)]methylammonium bromide,[(3-aminopropyl)-bis-(2-oleyloxyethyl)]methylammonium bromide,[(3-aminopropyl)-bis-(2-palmityloxyethyl)]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-amino-propylamino)-4-tetradecyl-carbamoyl-butylcarbamate cholesteryl ester, (3- (Aminopropyl)->4-(3-amino-propylamino-4-carbamoylbutylcarbamate cholesteryl ester, (3-aminopropyl)->4-(3-amino-propylamino)-4-(2-dimethylamino-ethylcarbamoyl)-butylcarbamate cholesteryl ester, spermine-5-carboxyglycine (N'-stearyl-N'-oleyl)amide tetratrifluoroacetate, spermine-5-carboxyglycine (N'-stearyl-N'-elaidyl)amide tetratrifluoroacetate Tratrifluoroacetate, agmatinylcarboxycholesterol acetate, spermine-5-carboxy-β-alanine cholesteryl ester tetratrifluoroacetate, 2,6-diaminohexanoyl β-alanine cholesteryl ester bistrifluoroacetate, 2,4-diaminobutyroyl β-alanine cholesteryl ester bistrifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl ester tritrifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxycholesteryl ester, stearyl carnitine ester, palmityl carnitine ester, myristyl carnitine ester, stearyl stearoyl carnitine ester chloride, L-stearyl stearoyl carnitine ester, stearyl oleoyl carnitine ester chloride, palmityl palmitoyl carnitine ester chloride, myristyl myristoyl carnitine ester salt Modified, L-myristylmyristoylcarnitine ester chloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmityl-amino)propyl]piperazine, N-(3-aminopropyl)-N,N'-bis-(dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis-(oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis-(palmityloxyethyl, )-piperazinium bromide, N-(3-aminopropyl)-N,N'-bis-(myristyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmityloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N'-methyl-N,N '-(bis-2-myristyloxyethyl)-piperazinium bromide, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxy-propyl]piperazine, or 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxypropyl]-piperazine, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3 -Dipalmitre oil oxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-amino-propyl)-diaminobutane, 2,3-dimyristre oil oxy-1,4-N,N'-dimethyl-N,N'-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dioleyl oxy-1,4-N,N'-dimethyl-N,N'-di(3-amino-propyl)-diaminobutane, 2,3-dipalmitre oil oxy-1,4-N,N'-dimethyl-N,N'-di(3-amino-propyl)-diaminobutane, 2,3-dimyrist Leoyloxy-1,4-N,N'-dimethyl-N,N'-di(3-amino-propyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamide-spermine)-diaminobutane, 2,3-dipalmitoleoyloxy-1,4-N,N'-dimethyl-N,N'-di(5-carboxamide-spermine)-diaminobutane, 2,3-dimyristo-oleoyloxy-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-dipalmitreoyloxy-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(histidyl)-diaminobutane, 2,3-dipalmitreoyloxy-1,4-N,N'-dimethyl-N,N'-di(histidyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N The composition according to any one of claims 56 to 58, comprising one or more cationic lipids selected from N'-dimethyl-N,N'-di(histidyl)-diaminobutane, 2,3-dioleyloxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dipalmitreoyloxy-N,N'-dimethyl-1,4-diaminobutane, 2,3-dimyristooleoyloxy-N,N'-dimethyl-1,4-diaminobutane; PAMAM dendrimer, NH3 core dendrimer, ethylenediamine core dendrimer, polyethyleneimine, and polyethyleneimine conjugate.

87. The composition according to 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-diacyloxypropane-3-amine, and any combination thereof.

88. The composition according to 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, further comprising a transfection enhancer selected from the group consisting of an endosome release agent, a cell surface ligand, a nuclear localization agent, a cell permeable peptide, a fusion peptide, and any combination thereof.

90. A method for delivering a payload to a cell, (i) To provide the composition according to any one of claims 56 to 89, (ii) providing cells, (iii) A method comprising bringing the cells into contact with the composition.

91. A method for delivering a composition to a target, comprising administering the composition according to any one of claims 56 to 89 to the target.

92. The method according to claim 90, wherein the contact of the cells is performed in vitro.

93. The method according to claim 90, wherein the contact of the cells is ex vivo.

94. The method according to claim 90, wherein the contact of the cells is performed in vivo.

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

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

97. The method according to claim 91, wherein the administration is systemic.

98. The method according to claim 91, wherein the administration is selected from the group consisting of subcutaneous administration, intramuscular administration, intranasal administration, intratumoral administration, administration to the brain of the target, administration to the spinal cord, administration to the eye, administration to the lymph node, and any combination thereof.

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

100. (i) A compound according to any one of claims 1 to 55, (ii) One or more structural lipids, (iii) One or more stabilizers, (iv) The kit according to claim 99, optionally comprising a payload.

101. (i) A 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, (v) The kit according to claim 99, optionally comprising a payload.

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