Lipids for delivery of nucleic acids to eukaryotic cells

Abstract

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

Description

LIPIDS FOR DELIVERY OF NUCLEIC ACIDS TO EUKARYOTIC CELLS FIELD

[0001] The present invention is in the field of molecular biology and cell and gene therapy, and more particularly relates to new compounds and methods for the introduction of payloads such as nucleic acids, ribonucleoproteins, etc. into eukaryotic cells. BACKGROUND

[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. phosphate backbones of DNA and RNA) into cells having a negatively charged membrane. Chemicals such as calcium phosphate and DEAE-dextran, or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule. The DNA-transfection reagent complex easily crosses the cell membrane, especially for lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer of the cell.

[0003] With the recent advances in nucleic-acid based therapeutics, and continued need for transfection reagents with low toxicity, there is a continued need for novel ionizable lipids that can be used in vitro and in vivo. SUMMARY

[0004] Disclosed herein are compounds, compositions and methods that improve the efficiency of introducing macromolecules, such as nucleic acids, or small molecules (e.g, therapeutics), into cells. Compounds are provided, together with compositions containing these compounds and methods for using these new compounds and compositions for delivery of payloads, (e.g. a nucleic acid or small molecule), to cells. The compounds may be used alone for transfection, or they may be used in combination with additional reagents in transfection compositions. For example, the new compounds may be combined with one or more ionizable lipids and / or neutral lipids, with one or more cell surface ligands, with one or more fusion enhancing agents, and with one or more nuclear localization agents and one or more amphipathic peptides and any combinations thereof. The resulting compositions may be complexed with one or more macromolecules (e.g, nucleic acids, such as DNA or RNA, proteins, ribonucleoproteins, and the like) and used to deliver these macromolecules into cells.

[0005] Thus, in one embodiment, the disclosure provides a compound having Formula I:or pharmaceutically acceptable salts thereof, wherein: A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A4 is selected from the group consisting of —(CH2)x—, —(CO)O—, —O(CO)—,R1 and R2 are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, optionally substituted C4- C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group, or —(CH2)1-7COOR; O)NHR— or —(CO)N(R)2— chain orbranched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl, or C3-C6 cycloalkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet, or C3-C6 cycloalkyl groups wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; or C3-C6 cycloalkenyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, or C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S-NR7-, -NR7-S-NR7- or aryl groups, or C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S- NR7-, or -NR7-S-NR7- groups; or R3 is H only when R1 and R2 are independently of each other —(CH2)1-7COOR; R4is H or optionally substituted C1-C20straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R5 and R6 are independently selected from H and CH3; R7 is H or optionally substituted C1-C6 straight-chain or branched-chain alkyl, optionally substituted monounsaturated or polyunsaturated C1-C6straight-chain or branched- chain alkenylR is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, —(CH2)0-3Het; A2and A3are independently selected from the group consisting of H,wherein R9 is H,wherein R10 is selected from the group consisting of H; C1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30 straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6 cycloalkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet;C3-C6cycloalkyl groups wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; and C3-C6 cycloalkenyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet; wherein R11is selected from the group consisting of -NH2, -NHR, -N(R)2,wherein AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 0 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, n4, n5 are independently an integer from 1 to 5 n6is an integer from 0 to 7 n7 and n8 are independently an integer from 0 to 5; n9is an integer from 1 to 5; m1 is an integer from 1 to 5; m2is an integer from 0 to 5; m3 is an integer from 1 to 7; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0006] In another embodiment, the disclosure provides a compound having the structure of Formula I-a:or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group, or —(CH2)1-7COOR; A2and A3are independently selected from the group consisting ofC1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl or C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl, or C3-C6 cycloalkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet, or C3-C6 cycloalkyl groups wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; or C3-C6 cycloalkenyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, or C1-C30 straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S-NR7-, -NR7-S-NR7- or aryl groups, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S- NR7-, or -NR7-S-NR7- groups; or R3 is H only when R1 and R2 are independently of each other —(CH2)1-7COOR; R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30 straight-chain or branched-chain alkyl, optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; and wherein R10is selected from the group consisting of H; C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6 cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; wherein AA represents any natural or non-natural amino acid side chain; x is an integer from 1 to 9; n is an integer from 1 to 5; n5is an integer from 1 to 5; n6 is an integer from 0 to 7; n7and n8are independently an integer from 0 to 5;n9is an integer from 1 to 5; m3is an integer from 1 to 3; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0007] In other embodiments, the disclosure provides compositions and methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell by contacting the cell with a compound of Formula I or a composition thereof, thereby introducing the nucleic acid, protein, or peptide into the cell.

[0008] In other embodiments, the disclosure provides a composition comprising: (i) one or more compounds according to a compound of Formula I, and (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizing agent; and (iii) optionally, 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 localization peptides or proteins and / or one or more amphipathic peptide.

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

[0011] Figure 1 is a graph depicting size (d.nm) and polydispersity index of the lipid-mRNA formulations.

[0012] Figure 2 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.

[0013] Figure 3 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver by luciferase activity (in bioluminescence flux, photons / second (p / s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.

[0014] Figure 4 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.

[0015] Figure 5 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver by luciferase activity (in bioluminescence flux, photons / second (p / s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.

[0016] Figure 6 is a graph depicting transfection efficiency two days post- transfection.

[0017] Figure 7 is a graph depicting GFP expression level two days post- transfection.

[0018] Figure 8 is a graph depicting viable T cells two days post-transfection.

[0019] Figure 9 is a graph is a graph depicting size (d.nm) of the lipid-mRNA formulations..

[0020] Figure 10 is a graph depicting the polydispersity index (PDI) of the lipid-mRNA formulations..

[0021] Figure 11 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.

[0022] Figure 12 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver by luciferase activity (in bioluminescence flux, photons / second (p / s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.

[0023] Figure 13 is a graph depicting size (d.nm) and polydispersity index of the lipid-mRNA formulations.

[0024] Figure 14 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver of mice following intravenous administration of lipid- mRNA formulations.

[0025] Figure 15 is a graph depicting the ratio of luciferase activity (in bioluminescence flux, photons / second (p / s)) in the liver by luciferase activity (in bioluminescence flux, photons / second (p / s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations.

[0026] Figure 16 is a graph showing GFP expression 48h post transfection using Compound 8 formulated with DOPE at a ratio of 1:4 in water. Adding peptide SEQ IDNO. 350 to the lipid increases the transfection efficiency as seen with increased GFP expression. Addition of a second peptide, SEQ ID NO.47 further increases the GFP expression compared to just 15-24.

[0027] Figure 17 is a graph showing Prestoblue fluorescence of lipid only formulation shows least toxicity while the formulations with one or two peptides show similar toxicity profiles as that of the Expi293 transfection reagent.

[0028] Figure 18 is a graph depicting size (d.nm) of the lipid-mRNA formulations.

[0029] Figure 19 is a graph depicting PDI of the lipid-mRNA formulations..

[0030] Figure 20 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in the spleen of mice following intravenous administration of lipid- mRNA formulations.

[0031] Figure 21 is a graph depicting luciferase activity (in bioluminescence flux, photons / second (p / s)) in the lung of mice following intravenous administration of lipid- mRNA formulations. DETAILED DESCRIPTION

[0032] The disclosure relates to ionizable lipids and lipid compositions (e.g., lipoplexes and lipid nanoparticle compositions) including the lipids provided herein. The disclosure also provides methods of delivering a therapeutic and / or prophylactic to a mammalian cell, specifically delivering a therapeutic and / or prophylactic to a mammalian organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof. For example, a method of producing a polypeptide of interest in a cell involves contacting a nanoparticle composition comprising an mRNA with a mammalian cell, whereby the mRNA may be translated to produce the polypeptide of interest. A method of delivering a therapeutic and / or prophylactic to a mammalian cell or organ may involve administration of a nanoparticle composition including the therapeutic and / or prophylactic to a subject, in which the administration involves contacting the cell or organ with the composition, whereby the therapeutic and / or prophylactic is delivered to the cell or organ.

[0033] Ionizable molecules are provided that are useful for improved methods of delivering macromolecules into eukaryotic cells. The compositions and methods are effective in a wide variety of cells and provide a high efficiency of transfection. Specifically, it has been found that molecules based on a core of Formula I are useful for efficient deliveryof macromolecules into cells. These molecules advantageously can be used with one or more neutral lipids and additional components such as fusogenic or fusion-enhancing molecules, additional cationic / ionizable lipids, cell surface ligands, cell adhesion molecules, nuclear localization agents, and endosomal release agents, together with the payload (e.g, in a complex with the macromolecule or pharmaceutical agent, or nutrient, or the like).

[0034] The complex is easily prepared by straightforward methods and can be used on a wide variety of cells, are stable, and therefore are suitable for use in in vitro, ex vivo and in vivo applications, for example, delivery of therapeutic nucleic acids (e.g., siRNA therapeutics, mRNA vaccine preparations, and the like), osomes), delivery of pharmaceutical agents, nutrients and the like to cells, e.g, in for cosmetic, nutraceutical, or therapeutic applications. General Definitions

[0035] The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. The abbreviations used herein have their conventional meanings within the chemical and biological arts.

[0036] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0037] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0038] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); 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 can be used in the practice of this invention. The following definitions are provided tofacilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0039] The term “about” when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 25% from the listed value. As many of the numerical values used herein are experimentally determined, it should be understood by those skilled in the art that such determinations can, and often times will, vary among different experiments. The values used herein should not be considered unduly limiting by virtue of this inherent variation. The term “about” is used to encompass variations of ±25% or less, variations of ±20% or less, variations of 10% or less, variations of ±5% or less, variations of ±1% or less, variations of ±0.5% or less, or variations of ±0.1% or less from the specified value. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0040] In the descriptions herein and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and / or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and / or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and / or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

[0041] It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.

[0042] Compounds are generally described herein using standard nomenclature. For a recited compound having asymmetric center(s), all of the stereoisomers of the compound and mixtures thereof are encompassed unless otherwise specified. Non-limiting examples of stereoisomers include enantiomers, diastereomers, and E or Z isomers. Where a recited compound exists in various tautomeric forms, the compound is intended to encompass all tautomeric forms. Certain compounds are described herein using general formulas that include variables (e.g., X, L1, L2, L3, Y, etc.). Unless otherwise specified, each variable within such a formula is defined independently of any other variable, and any variable that occurs more than one time in a formula is defined independently at each occurrence. If moieties are described as being "independently" selected from a group, each moiety is selected independently from the other. Each moiety therefore can be identical to or different from the other moiety or moieties.

[0043] The number of carbon atoms in a hydrocarbyl moiety can be indicated by the prefix "Cx-Cy," where x is the minimum and y is the maximum number of carbon atoms in the moiety. Thus, for example, "C1-C6 alkyl" refers to an alkyl substituent containing from 1 to 6 carbon atoms. Illustrating further, C3-C6 cycloalkyl means a saturated hydrocarbyl ring containing from 3 to 6 carbon ring atoms. A prefix attached to a multiple-component substituent only applies to the first component that immediately follows the prefix. To illustrate, the term "carbocyclylalkyl" contains two components: carbocyclyl and alkyl. Thus, for example, C3-C6carbocyclyl C1-C6alkyl refers to a C3-C6carbocyclyl appended to the parent molecular moiety through a C1-C6 alkyl group.

[0044] Unless otherwise specified, when a linking element links two other elements in a depicted chemical structure, the leftmost-described component of the linking element is bound to the left element in the depicted structure, and the rightmost-described component of the linking element is bound to the right element in the depicted structure. To illustrate, if the chemical structure is -LS-M-LS''- and M is -N(RB)S(O)-, then the chemical structure is -LS-N(RB)S(O)-LS''-.

[0045] If a linking element in a depicted structure is a bond, then the element left to the linking element is joined directly to the element right to the linking element via a covalent bond. For example, if a chemical structure is depicted as -LS-M-LS' and M is selected as bond, then the chemical structure will be -LS-LS''-. If two or more adjacent linking elements in a depicted structure are bonds, then the element left to these linking elements is joined directly to the element right to these linking elements via a covalent bond. For instance, if a chemical structure is depicted as -LS-M-LS''-M'-LS''-, and M and LS' are selected as bonds, then the chemical structure will be -LS-M'-LS''-. Likewise, if a chemical structure is depicted as -LS-M-LS''-M'-LS''-, and M, LS' and M' are bonds, then the chemical structure will be -LS-LS''. When a chemical formula is used to describe a moiety, the dash(es) indicates the portion of the moiety that has the free valence(s).

[0046] If a moiety is described as being "optionally substituted", the moiety may be either substituted or unsubstituted. If a moiety is described as being optionally substituted with up to a particular number of non-hydrogen radicals that moiety may be either unsubstituted or substituted by up to that particular number of non-hydrogen radicals or by up to the maximum number of substitutable positions on the moiety, whichever is less. Thus, for example, if a moiety is described as a heterocycle optionally substituted with up to three non- hydrogen radicals, then any heterocycle with less than three substitutable positions will be optionally substituted by up to only as many non-hydrogen radicals as the heterocycle has substitutable positions. For example, tetrazolyl (which has only one substitutable position) will be optionally substituted with up to one non-hydrogen radical. Similarly, if an amino nitrogen is described as being optionally substituted with up to two non-hydrogen radicals, then a primary amino nitrogen will be optionally substituted with up to two non-hydrogen radicals, whereas a secondary amino nitrogen will be optionally substituted with up to only one non-hydrogen radical.

[0047] Where a moiety is substituted with oxo or thioxo, it means that the moiety contains a carbon atom covalently bonded to at least two hydrogens (e.g., CH2), and the two hydrogen radicals are substituted with oxo or thioxo to form C=O or C=S, respectively.

[0048] The term “alkyl” or “alkyl group” or “alkylene group” refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated) which is optionally substituted. The alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 4 carbon atoms. The alkyl group of the compounds may be designated as “C1-4 alkyl”, “C1-20 alkyl”, “C4-30 alkyl” or similar designations. The notation “C4-30alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 4-30 carbon atoms.

[0049] Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups. For example, the alkyl groups in the straightor branched hydrocarbon chains described above, may be substituted with hydroxyl groups. In another embodiment, the methylene groups are linked via disulfide bridges (—S—S—).

[0050] Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, n-hexyl, lauryl, palmityl, stearyl and the like.

[0051] The term “alkenyl”, alone or in combination with any other term, refers to a straight-chain or branched-chain monounsaturated or polyunsaturated aliphatic hydrocarbon radical containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2-30 carbon atoms (i.e. (C2-30)alkenyl), in one embodiment from 4-30 carbon atoms (i.e. (C4-30)alkenyl), and in another embodiment from 2-6 carbon atoms (i.e. (C2-6)alkenyl). As used herein, the term “alkenyl” or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation “C2-14 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, Cis alkenyl may include one or more double bonds. A C5 alkenyl group including two double bonds may be a linoleyl group.

[0052] Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups. For example, the alkenyl groups in the straight or branched hydrocarbon chains described above, may be substituted with hydroxyl groups. In another embodiment, the alkenyl groups are linked via disulfide bridges (—S—S—).

[0053] 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, palmitoleoyl and the like.

[0054] The term “alkynyl,” alone or in combination with any other term, refers to a straight-chain or branched-chain hydrocarbon radical having one or more triple bonds containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2 to about 20 carbon atoms. As used herein, the term “alkynyl” or “alkynyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least onecarbon-carbon triple bond, which is optionally substituted. The notation “C2-14 alkynyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. For example, C18 alkynyl may include one or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

[0055] Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl and the like.

[0056] The term “basic heterocycle” refers to a stable optionally substituted 5-7 membered monocyclic heterocyclic ring or an optionally substituted 8-11 membered bicyclic heterocyclic ring which is either saturated or partially unsaturated, and which may be optionally benzofused if monocyclic and which is optionally substituted on one or more carbon atoms by halogen, alkyl, alkoxy, oxo, and the like, and / or on a secondary nitrogen atom (i.e., —NH—) by alkyl, aralkoxycarbonyl, alkanoyl, phenyl or phenylalkyl or on a tertiary nitrogen atom (i.e., +N—) by oxido and which is attached via a carbon atom.

[0057] The term "heterocycle" or "heterocyclo" or "heterocyclyl" refers to a saturated (e.g., "heterocycloalkyl"), partially unsaturated (e.g., "heterocycloalkenyl" or "heterocycloalkynyl") or completely unsaturated (e.g., "heteroaryl") ring system where at least one of the ring atoms is a heteroatom (i.e., nitrogen, oxygen or sulfur), with the remaining ring atoms being independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur. A heterocycle may be, without limitation, a single ring, two fused rings, or bridged or spiro rings. A heterocycle group can be linked to the parent molecular moiety via any substitutable carbon or nitrogen atom(s) in the group. Where a heterocycle group is a divalent moiety that links two other elements in a depicted chemical structure, the heterocycle group can be attached to the two other elements through any two substitutable ring atoms. Likewise, where a heterocycle group is a trivalent moiety that links three other elements in a depicted chemical structure, the heterocycle group can be attached to the three other elements through any three substitutable ring atoms, respectively.

[0058] In the instant compounds, “Het” indicates a heterocycle containing 4- 12 carbon atom, where at least one nitrogen atom is present in the ring(s). A heterocyclyl may be, without limitation, a monocycle which contains a single ring. Non-limiting examples of monocycles include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiolyl, oxathiolyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiodiazolyl, oxathiazolyl, oxadiazolyl (including 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl (also known as "azoximyl"), 1,2,5-oxadiazolyl (also known as "furazanyl"), and 1,3,4-oxadiazolyl), oxatriazolyl (including 1,2,3,4-oxatriazolyl and 1,2,3,5-oxatriazolyl), dioxazolyl (including 1,2,3-dioxazolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl, and 1,3,4-dioxazolyl), pyridinyl, piperidinyl, diazinyl (including pyridazinyl (also known as "1,2-diazinyl"), pyrimidinyl (also known as "1,3-diazinyl"), and pyrazinyl (also known as "1,4-diazinyl")), piperazinyl, triazinyl (including s-triazinyl (also known as "1,3,5-triazinyl"), as-triazinyl (also known 1,2,4- triazinyl), and v-triazinyl (also known as "1,2,3-triazinyl), oxazinyl (including 1,2,3-oxazinyl, 1,3,2-oxazinyl, 1,3,6-oxazinyl (also known as "pentoxazolyl"), 1,2,6-oxazinyl, and 1,4- oxazinyl), isoxazinyl (including o-isoxazinyl and p-isoxazinyl), oxazolidinyl, isoxazolidinyl, oxathiazinyl (including 1,2,5-oxathiazinyl or 1,2,6-oxathiazinyl), oxadiazinyl (including 1,4,2-oxadiazinyl and 1,3,5,2-oxadiazinyl), morpholinyl, azepinyl, and diazepinyl.

[0059] A heterocyclyl may also be, without limitation, a bicycle containing two fused rings, such as, for example, naphthyridinyl (including [1,8]naphthyridinyl, and [1,6]naphthyridinyl), thiazolpyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, pyridopyrimidinyl, pyrazolopyrimidinyl, indolizinyl, pyrindinyl, pyranopyrrolyl, 4H- quinolizinyl, purinyl, pyridopyridinyl (including pyrido[3,4-b]-pyridinyl, pyrido[3,2-b]- pyridinyl, and pyrido[4,3-b]-pyridinyl), pyridopyrimidine, and pteridinyl. Other non-limiting examples of fused-ring heterocycles include benzo-fused heterocyclyls, such as indolyl, isoindolyl, indoleninyl (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, benzodiazinyl (including cinnolinyl (also known as "1,2- benzodiazinyl") and quinazolinyl (also known as "1,3-benzodiazinyl")), 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 tetrahydroisoquinolinyl.

[0060] A heterocyclyl may also be, without limitation, a spiro ring system, such as, for example, 1,4-dioxa-8-azaspiro[4.5]decanyl. A heterocyclyl may comprise one or more sulfur atoms as ring members; and in some cases, the sulfur atom(s) is oxidized to SO or SO2. The nitrogen heteroatom(s) in a heterocyclyl may or may not be quaternized and mayor may not be oxidized to N-oxide. In addition, the nitrogen heteroatom(s) may or may not be N-protected.

[0061] A heterocycle or carbocycle may be further substituted. Unless specified, the term "substituted" refers to substitution by independent replacement of one, two, or three or more of the hydrogen atoms with substituents including, but not limited to, -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-C12-cycloalkyl, -NH-aryl, -NH-heteroaryl, - NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-C1-C12-alkyl, -O- C2-C8-alkenyl, alkynyl, -O-C3-C12-cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, - C(O)-C1-C12-alkyl, -C(O)-C2-C8-alkenyl, -C(O)-C2-C8-alkynyl, -C(O)-C3-C12-cycloalkyl, - C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl, -CONH2, -CONH-C1-C12-alkyl, - CONH-C2-C8-alkenyl, -CONH-C2-C8-alkynyl, -CONH-C3-C12-cycloalkyl, -CONH-aryl, - CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-C1-C12-alkyl, -OCO2-C2-C8-alkenyl, - OCO2-C2-C8-alkynyl, -OCO2-C3-C12-cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2- heterocycloalkyl, -OCONH2, -OCONH-C1-C12-alkyl, -OCONH-C2-C8-alkenyl, -OCONH- C2-C8-alkynyl, -OCONH-C3-C12-cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl, -OCONH- heterocycloalkyl, -NHC(O)-C1-C12-alkyl, -NHC(O)-C2-C8-alkenyl, -NHC(O)-C2-C8-alkynyl, -NHC(O)-C3-C12-cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, -NHC(O)- heterocycloalkyl, -NHCO2-C1-C12-alkyl, -NHCO2-C2-C8-alkenyl, -NHCO2-C2-C8-alkynyl, - NHCO2-C3-C12-cycloalkyl, -NHCO2-aryl, -NHCO2-heteroaryl, -NHCO2-heterocycloalkyl, - NHC(O)NH2, -NHC(O)NH-C1-C12-alkyl, -NHC(O)NH-C2-C8-alkenyl, -NHC(O)NH-C2-C8- alkynyl, -NHC(O)NH-C3-C12-cycloalkyl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, - NHC(O)NH-heterocycloalkyl, -NHC(S)NH2, -NHC(S)NH-C1-C12-alkyl, -NHC(S)NH-C2- C8-alkenyl, -NHC(S)NH-C2-C8-alkynyl, -NHC(S)NH-C3-C12-cycloalkyl, -NHC(S)NH-aryl, - NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH2, -NHC(NH)NH-C1- C12-alkyl, -NHC(NH)NH-C2-C8-alkenyl, NHC(NH)NH-C2-C8-alkynyl, -NHC(NH)NH-C3- C12-cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl, -NHC(NH)NH- heterocycloalkyl, -NHC(NH)-C1-C12-alkyl, -NHC(NH)-C2-C8-alkenyl, -NHC(NH)-C2-C8- alkynyl, -NHC(NH)-C3-C12-cycloalkyl, -NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)- heterocycloalkyl, -C(NH)NH-C1-C12-alkyl, -C(NH)NH-C2-C8-alkenyl, -C(NH)NH-C2-C8- alkynyl, -C(NH)NH-C3-C12-cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NH- heterocycloalkyl, -S(O)-C1-C12-alkyl, -S(O)-C2-C8-alkenyl, -S(O)-C2-C8-alkynyl, -S(O)-C3- C12-cycloalkyl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heterocycloalkyl, -SO2NH2, -SO2NH-C1- C12-alkyl, -SO2NH-C2-C8-alkenyl, -SO2NH-C2-C8-alkynyl, -SO2NH-C3-C12-cycloalkyl, -SO2NH-aryl, -SO2NH-heteroaryl, -SO2NH-heterocycloalkyl, -NHSO2-C1-C12-alkyl, - NHSO2-C2-C8-alkenyl, -NHSO2-C2-C8-alkynyl, -NHSO2-C3-C12-cycloalkyl, -NHSO2-aryl, - NHSO2-heteroaryl, -NHSO2-heterocycloalkyl, -CH2NH2, -CH2SO2CH3, -aryl, -arylalkyl, - heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -C3-C12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-C1-C12-alkyl, -S-C2-C8-alkenyl, -S- C2-C8-alkynyl, -S-C3-C12-cycloalkyl, -S-aryl, -heteroaryl, -S-heterocycloalkyl, or methylthiomethyl. It is understood that the aryls, heteroaryls, alkyls, and the like can be further substituted.

[0062] The term "N-protecting group" or "N-protected" refers to those groups capable of protecting an amino group against undesirable reactions. Commonly used N- protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3rded., 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; sulfenyl groups such as phenylsulfenyl (phenyl-S-) or triphenylmethylsulfenyl (trityl-S-); sulfinyl groups such as p-methylphenylsulfinyl (p- methylphenyl-S(O)-) or t-butylsulfinyl (t-Bu-S(O)-); carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p- nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4- dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4- dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5- dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1- methylethoxy carbonyl, dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxy carbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloro-ethoxy-carbonyl, phenoxy carbonyl, 4- nitro-phenoxy carbonyl, 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).

[0063] The term "halogen" means fluorine, chlorine, bromine, or iodine.

[0064] As used herein, a “natural amino acid side chain” refers to the side- chain substituent of a naturally occurring amino acid. Naturally occurring amino acids have a substituent attached to the α–carbon. Naturally occurring amino acids include Arginine, Lysine, Aspartic acid, Glutamic acid, Glutamine, Asparagine, Histidine, Serine, Threonine, Tyrosine, Cysteine, Methionine, Tryptophan, Alanine, Isoleucine, Leucine, Phenylalanine, Valine, Proline, and Glycine.

[0065] As used herein, a “non-natural amino acid side chain” refers to the side- chain substituent of a non-naturally occurring amino acid. Non-natural amino acids include β-amino acids (β3and β2), Homo-amino acids, Proline and Pyruvic acid derivatives, 3- substituted Alanine derivatives, Pyrrolysine, 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 “unnatural amino acids & derivatives.” See also, Travis S. Young and Peter G. Schultz, “Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon,” J. Biol. Chem. 2010285: 11039-11044, which is incorporated by reference in its entirety.

[0066] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed or chemically synthesized as a single moiety.

[0067] The term “alkoxy” refers to an alkyl, alkenyl or alkynyl ether radical, wherein the terms “alkyl”, “alkenyl” or “alkynyl” are defined above. Examples of suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, oleyloxy, palmityloxy, palmitoleoyloxy and the like.

[0068] The term “aryl,” alone or in combination with any other term, refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, in one embodiment from 6-15 carbon atoms (i.e. (C6-15)aryl), and in another embodiment from 6-10 carbon atoms (i.e. (C6-10)aryl), optionally substituted with one or more substituents selected from alkyl, alkoxy, (for example methoxy), nitro, halogen,(for example chloro), amino, carboxylate and hydroxy. Examples of aryl radicals include, but are not limited to phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl and the like.

[0069] The term “aralkyl” or “arylalkyl” is an aryl group connected, as a substituent, via an alkylene group, such “C7-14 aralkyl” and the like, including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-4 alkylene group). Additionally, the term “aralkyl” or “arylalkyl”, alone or in combination, means an alkyl radical as defined above in which one hydrogen atom is phenyl, benzyl, 2-phenylethyl and the like.

[0070] The term “aralkoxycarbonyl”, alone or in combination, means a radical of the formula -C(O)-O-aralkyl in which the term “aralkyl” has the significance given above. An example of an aralkoxycarbonyl radical is benzyloxycarbonyl.

[0071] The term “aryloxy”, alone or in combination, means a radical of the formula aryl-O- in which the term “aryl” has the significance given above.

[0072] The term “alkanoyl”, alone or in combination, means an acyl radical derived from an alkanecarboxylic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, and the like.

[0073] The term “aryloxyalkanoyl” means an acyl radical of the formula aryl- O-alkanoyl wherein aryl and alkanoyl have the significance given above.

[0074] The term “aralkanoyl” means an acyl radical derived from an aryl- substituted alkanecarboxylic acid such as phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4- aminohydrocinnamoyl, 4-phenylbutyryl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4- aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl, and the like.

[0075] The term “aroyl” means an acyl radical derived from an aromatic carboxylic acid. Examples of such radicals include aromatic carboxylic acids, an optionally substituted benzoic 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, 3- (benzyloxyformamido)-2-naphthoyl, and the like.

[0076] The term “aminocarbonyl” alone or in combination, means an amino- substituted carbonyl (carbamoyl) group derived from an amino-substituted carboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group continuingsubstituents selected from hydrogen, alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl radicals and the like.

[0077] The term “aminoalkanoyl” means an acyl radical derived from an amino substituted alkanecarboxylic acid wherein the amino group can be a primary, secondary or tertiary amino group containing substituents selected from hydrogen, cycloalkyl, cycloalkylalkyl radicals and the like, examples of which include N,N- dimethylaminoacetyl and N-benzylaminoacetyl.

[0078] The term “carbocycle” refers to a non-aromatic stable 3- to 8- membered carbon ring which may be saturated, mono-unsaturated or poly-unsaturated. The carbocycle may be attached at any endocyclic carbon atom which results in a stable structure. Carbocycles in one embodiment have 5-7 carbons.

[0079] The term “cycloalkyl”, alone or in combination, means an alkyl radical which contains from about 3 to about 8 carbon atoms and is cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

[0080] The term “cycloalkylalkyl” means an alkyl radical as defined above which is substituted by a cycloalkyl radical containing from about 3 to about 8, in one embodiment from about 3 to about 6, carbon atoms.

[0081] The term “cycloalkylcarbonyl” means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, and the like, or from a benz-fused monocyclic cycloalkanecarboxylic acid which is optionally substituted by, for example, alkanoylamino, such as 1,2,3,4-tetrahydro-2-naphthoyl, 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.

[0082] The term “cycloalkylalkoxycarbonyl” means an acyl group derived from a cycloalkylalkoxycarboxylic acid of the formula cycloalkylalkyl-O-COOH wherein cycloalkylalkyl has the significance given above.

[0083] The term “alkylation reaction” may comprise a nucleophilic attack of an alkylation reactive center of a target compound to an electron deficient region of an alkylating agent according to an SN1or SN2mechanism, as known in the art (see, e.g., Jerry March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure.293-871 (4thed. John Wiley & Sons 1992)). A person of ordinary skill in the art will appreciate that in some embodiments, an alkylating agent further comprises one or more leaving groups (LGs). By “leaving group” (X in Scheme IV) and grammatical equivalents herein are meant an atom or molecule that detaches from a molecule, e.g., an organic molecule. In some embodiments, the residual part can be the alkyl group which becomes covalently bonded toa target compound. Accordingly, in various exemplary embodiments, a leaving group can be an atom or group, charged or uncharged, that becomes detached from an atom or molecule in what is considered the residual or main part of the substrate in a specified reaction. The ability of a leaving group to leave the alkylating agent can be a function of the leaving group's ability. Thus, a leaving group can affect the intrinsic reactivity of the alkylating agent in an alkylation reaction. In some embodiments, the lower the pKa of the conjugate acid of the leaving group, the better the leaving group, because, in some embodiments, the leaving group can more easily stabilize the developing negative charge that can occur in an alkylation reaction. Therefore, in some embodiments, a leaving group can be an electronegative atom or molecule. Examples of leaving groups include, but are not limited to, acetate (AcO−), p-nitrobenzoate (PNBO−), sulfonates (e.g., methanesulfonate (Mesylate: MsO−), p-toluenesulfonate (tosylate: TsO−), p-bromobenzenesulfonate (Brosylate: BsO−), p- nitrobenzenesulfonate (Nosylate: NsO−), fluoromethanesulfonate, difluoromethanesulfonate, trifluoromethanesulfonate (Triflate: TfO−) and ethanesulfonate), halide esters, and halogen ions (e.g., I−, Br−, Cl−).

[0084] The term “solid-phase bound arylalkyl chloride” comprises an arylalkyl chloride covalently linked to a polystyrene based resin. A person of skill in the art will appreciate that the polystyrene based resin can have various reactive functionalities, for example a chloride as a leaving group or it can have other leaving groups as discussed above. Since the pioneering work of Merrifield (Merrifield, R. B. (1963), J. Am. Chem. Soc., 85,2149- 2153) on polystyrene (2% divinylbenzene cross-linked) as solid support for peptide synthesis, several improvements on the nature of the solid support were brought about to meet special needs of new organic chemistry.

[0085] Compositions may also include salts of one or more compounds, e.g., compounds of Formula I. Salts may be pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, ethanolamine, trimethylamine, diethylamine, triethylamine, ethylamine, isopropylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1- 19 (1977), each of which is incorporated herein by reference in its entirety..

[0086] The term “surface ligand” or “cell surface ligand” refers to a chemical compound or structure which will bind to a surface receptor of a cell. The term “cell surface receptor” as used herein refers to a specific chemical grouping on the surface of a cell to which the ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes). The receptor facilitates the internalization of the ligand and attached molecules. A cell surface receptor includes but is not limited to a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II / cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilageinduction factor receptor or glycosylphosphatidylinositol (GPI)-anchored proteins (e.g., β- andrenargic receptor, T-cell activating protein, Thy-1 protein, GPI-anchored 5’ nucleotidase). These are nonlimiting examples.

[0087] A receptor is a molecule to which a ligand binds specifically and with relatively high affinity. It is usually a protein or a glycoprotein, but may also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan or a glycocalyx. For purposes of this disclosure, epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen:antibody complex undergoes endocytosis. Furthermore, surface ligand includes anything which is capable of entering the cell through cytosis (e.g. endocytosis, potocytosis, pinocytosis).

[0088] As used herein, the term “ligand” refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, RGD-containing cell adhesion peptides and the like.

[0089] One skilled in the art will readily recognize that the ligand chosen will depend on which receptor is being bound. Since different types of cells have different receptors, this provides a method of targeting nucleic acid to specific cell types, depending on which cell surface ligand is used. Thus, the preferred cell surface ligand may depend on the targeted cell type.

[0090] The term “nuclear localization agent,” “nuclear localization signal,” or “nuclear ligand” as used herein refers to a ligand, such as a peptide, which will cause an agent covalently or non-covalently linked to it to localize at the cell nucleus, typically by binding a nuclear receptor. The term “nuclear receptor” as used herein refers to a chemical grouping on the nuclear membrane which will bind a specific ligand and help transport the ligand, and accompanying linked moieties, through the nuclear membrane. Nuclear receptors can be but are not limited to those receptors which bind nuclear localization sequences. Nonlimiting 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)8-40K (SEQ ID NO:9), may be used to transport nucleic acid to the nucleus.

[0091] The term “lysis agent” as used herein refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane and freeing the DNA transporter into the cytoplasm of the cell. This term includes but is not limited to viruses, synthetic compounds, lytic peptides, or derivatives thereof. The term “lytic peptide” refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both. Examples of lysis agents / endosomal release agents include choroquine, polyamines and polyamidoamines. Suitable agents are described in, for example, Pei and Buyanova, Bioconjugate Chem, 30:273-283 (2009) and Juliano, Nucleic Acid Therapeutics, 28:166-177 (2018).

[0092] The term “polycationic nucleic acid binding moiety” as used herein refers to a moiety containing multiple positive charges at physiological pH that allow the moiety to bind a negatively charged nucleic acid. A polycationic nucleic acid binding moiety may be linked to, for example, a cell surface ligand, a fusion agent, and / or a nuclear localization peptide. The linkage may be covalent. Suitable polycationic nucleic acid binding moieties include polyamines such as PEI, spermine, spermidine, carboxyspermine and polybasic peptides containing, for example, multiple lysine, ornithine, histidine, or arginine residues.

[0093] The term “nucleic acid,” when not applied to a specific type of molecule such as unmodified DNA or RNA, refers to any type of nucleic acid that presently is known or that may be prepared or identified in the future, provided that the nucleic acid is sufficiently negatively charged to form a lipid aggregate, liposome, or liposome-like complex when admixed with any lipid of Formula I. Nucleic acid, as used herein, refers to deoxyribonucleotides or ribonucleotides and mixtures and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as a reference nucleic acid, and which are metabolized in a manner similar to a reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), 5 methyl cytosine, pseudouridine, N1-methyl-pseudouridine, 5 methoxyuridine, and the like.

[0094] In some embodiments, the payloads described herein can include mRNAs modified with one or more nucleotides selected from the group consisting of:pseudouridine (abbreviated by the Greek letter “psi” or “ψ”), 5-methylcytosine (m5C), 5- methyluridine (m5U), 2′-O-methyluridine (Um or m2′-OU), 2-thiouridine (s2U), and N6- methyladenosine (m6A)) in place of at least a portion of the corresponding unmodified canonical nucleoside (e.g., in place of substantially all of the corresponding unmodified A, C, G, or T canonical nucleoside.

[0095] In some embodiments, the payloads described herein can include ribonucleoprotein complexes (e.g., Cas9 / guide RNA) which are delivered into a cell at high efficiencies. A “ribonucleoprotein complex,” or “ribonucleoprotein particle” as provided herein refers to a complex or particle including a nucleoprotein and a ribonucleic acid. A “guide RNA” or “gRNA” as provided herein refers to a ribonucleotide sequence capable of binding a nucleoprotein, thereby forming ribonucleoprotein complex. In embodiments, the guide RNA includes one or more RNA molecules. A “nucleoprotein” as provided herein refers to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid, it is referred to as “ribonucleoprotein.” The interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). Non-limiting examples of ribonucleoproteins include ribosomes, telomerase, RNAseP, hnRNP, CRISPR associated protein 9 (Cas9) and small nuclear RNPs (snRNPs). The ribonucleoprotein may be an enzyme. In embodiments, the ribonucleoprotein is an endonuclease. Thus, in embodiments, the ribonucleoprotein complex includes an endonuclease and a ribonucleic acid. In embodiments, the endonuclease is a CRISPR associated protein 9.

[0096] Nucleic acids may be in the form of an antisense molecule, for example a “gap-mer” containing an RNA-DNA-RNA structure that activates RNAseH. The nucleic acid can be, for example, DNA or RNA, or RNA-DNA hybrid, and can be an oligonucleotide, plasmid, parts of a plasmid DNA, pre-condensed DNA, product of a polymerase chain reaction (PCR), vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups or other form of nucleic acid molecule. The nucleic acid may be a double-stranded RNA molecule of the type used for inhibiting gene expression by RNA interference. The nucleic acid may be a short interfering double stranded RNA molecule (siRNA). The nucleic acid molecule can also be a StealthTMRNAi molecule (Invitrogen Corporation / Life Technologies Corporation, Carlsbad, CA).

[0097] As used herein, an “RNA” refers to a ribonucleic acid that may be naturally or non-naturally occurring. For example, an RNA may include modified and / or non- naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. RNAs may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetrical 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.

[0098] The term “amphipathic peptide” refers to a peptide whose secondary structure places hydrophobic and hydrophilic amino acid residues on different faces of the peptide. The peptides often adopt a helical secondary structure. In some circumstances an amphipathic peptide may also function as a fusion agent. Examples of amphipathic peptides suitable for use in the compositions described herein include, but are not limited to, peptides comprising a sequence 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 amphipathic peptide may optionally be linked to a polycationic nucleic acid binding moiety, for example via a covalent linkage. Exosomes, exosome lipids

[0099] The term “exosome” refers to the small membrane vesicles secreted by most cells that contain cell specific payloads of proteins, lipids and, genetic material and other biomolecules that are transported to other cells in different location of the tissue. Exosomes can be considered liposomal particles. Exosomes or lipid mixtures obtained therefrom, can be used in combination with other transfection agents or helper lipid mixtures. Exosomes are also referred to as microvesicles, epididimosomes, argosomes, exosome-like vesicles,microparticles, promininosomes, prostasomes, dexosomes, texosomes, archeosomes 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 described, e.g., in Li, YJ., Wu, JY., Liu, J. et al. Artificial exosomes for translational nanomedicine. J Nanobiotechnol 19, 242 (2021), US Patent No. 11938219, US Patent Application Publication No. US2023 / 0181466, and the like, each of which is herein incorporated by reference in its entirety.

[0100] Examples of lipid constituents isolated from exosomes include, but are not limited to, Lyso-PC (non-limiting examples of which C-18, C-16, C-14 and mixture), Lyso- bisphospahtidic acid (non-limiting example of which is C-18, C-16 and C-14), sphingomyelin, ceramides (non-limiting examples C-8- C-24), disaturated PC (non-limiting examples DSPC, DPPC, DMPC and others where Cn (n= 8 – 25)), diunsaturated PC-MIX (non-limiting examples of which are DOPC, DP(db)PC) phosphatidyl serine (PS), phosphatidyl inositol (PI)), disaturated PE non-limiting example, DSPE, DPPE, DMPE), di-unsaturated PE-MIX (non- limiting example DOPE DP(db)PE), phosphatidyl glycerol (PG) (non-limiting examples of which are C-18 – C-22), cholesterol, and diglycerides, such as cardiolipin.

[0101] Also contemplated are any mixtures of combination of the above listed ionizable lipids, neutral lipids, exosomes, and lipid mixtures isolated from exosomes.

[0102] The lipid compositions provided herein can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, or the like) derived or purified from exosomes. Structural Lipids

[0103] The lipid component of a lipid nanoparticle composition may include one or more structural lipids. Structural lipids can be selected from the group consisting of, but are not limited to a sterol, e.g., cholesterol, fecosterol, 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 a sterol, (e.g., cholesterol) and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. Stabilizing Agents

[0104] The lipid compositions provided herein can also include a stabilizing agent, such as a stabilizing lipid. Stabilizing lipids can be neutral lipids, or they can be charged.Stabilizing lipids that can advantageously be 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-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. Other stabilizing lipids useful in the compositions disclosed herein include, e.g., polyglycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether co-polymers, triblock polyoxyethylene alkyl ethers co-polymers, and amphiphilic branched polymers. In embodiments, stabilizing agent can be In polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate ("Myrj52"), poly(propylene glycol)11-block-poly(ethylene glycol)16-block-poly(propylene glycol)11, poly(propylene glycol)12-block-poly(ethylene glycol)28-block-poly(propylene glycol)12, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan- 2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (Polyoxyethylene (40) stearate), Brij™ S10 (Polyoxyethylene (10) stearyl ether), BRIJ™ L4 = Polyoxyethylene (4) lauryl ether; BRIJ™ S20= Polyoxyethylene (20) stearyl ether; BRIJ™ S35= Polyoxyethylene (23) lauryl ether; TPGS 1000 =D-α-Tocopherol polyethylene glycol 1000 succinate; Tween 20 / Polysorbate 80 / Tridecyl-D-maltoside in equal ratios, and combinations thereof. In certain compositions, the stabilizing agent is present at about 0.1 - 5 mol% of the lipid composition. For example, in some compositions, the stabilizing agent is present at 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 stabilizing agent is present at about 0.5 mol% to about 5 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 0.5 mol% to about 4 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 0.5 mol% to about 3 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 0.5 mol% to about 2 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 0.5 mol% to about 1 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 1 mol% to about 5 mol% of the lipid component of the compositions provided herein. In other examples, the stabilizing agent is present at about 1 mol% to about 4 mol% of the lipid component of the compositions provided herein. In other examples, thestabilizing agent is present at about 1 mol% to about 3 mol% of the lipid component of the compositions. In other examples, the stabilizing agent is present at about 1 mol% to about 2 mol% of the lipid component of the compositions provided herein. Complexation of Payloads:

[0105] The disclosure herein provided compositions for the delivery of payloads, including but not limited to nucleic acids, to cells. Nucleic acids can be complexed to the exterior of the lipid complexes provided herein (e.g., liposomes, lipid nanoparticles). In some embodiments, the compositions have from about 20% to about 50% of the nucleic acid complexed to the exterior of the lipid complex. In other embodiments, the compositions have about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% of the nucleic acid complexed to the exterior of the lipid complex. Exterior complexation of a nucleic acid can be measured by methods know in the art, such as in Blakney et al. (2019) Gene Therapy 26:363-372. Compounds of Formula I:

[0106] It has been found that compounds based on a core structure of Formula I are useful for the efficient delivery of macromolecules into eukaryotic cells. The compositions and methods are effective in a wide variety of cells and provide a high efficiency of transfection. These compounds advantageously can be used with one or more neutral lipids and additional components such as fusogenic or fusion-enhancing molecules, additional cationic lipids, cell surface ligands, cell adhesion molecules, amphipathic peptides and nuclear localization agents, in a complex with the macromolecule. The complex is easily prepared by straightforward methods and can be used on a wide variety of cells.

[0107] In one embodiment, the disclosure provides a compound having Formula I:or pharmaceutically acceptable salts thereof, wherein: A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A4is selected from the group consisting of —(CH2)x—, —(CO)O—, —O(CO)—,R1 and R2 are independently of one another selected from the group consisting of H, optionally substituted C1-C30straight-chain or branched-chain alkyl, optionally substituted C4- C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group, or —(CH2)1-7COOR; R3 is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—,C1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl, orC3-C6cycloalkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet, or C3-C6 cycloalkyl groups wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; or C3-C6 cycloalkenyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, or C1-C30 straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S-NR7-, -NR7-S-NR7- or aryl groups, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S- NR7-, or -NR7-S-NR7- groups; or R3 is H only when R1 and R2 are independently of each other —(CH2)1-7COOR; R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R5 and R6 are independently selected from H and CH3; R7is H or optionally substituted C1-C6straight-chain or branched-chain alkyl, optionally substituted monounsaturated or polyunsaturated C1-C6 straight-chain or branched- chain alkenyl,R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, —(CH2)0-3Het; A2 and A3 are independently selected from the group consisting of H,wherein R9 is H,wherein R10is selected from the group consisting of H; C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; C3-C6cycloalkyl amines wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; and C3-C6 cycloalkenyl amines, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet;wherein R11is selected from the group consisting of -NH2, -NHR, -N(R)2,wherein AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 0 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, n4, n5 are independently an integer from 1 to 5 n6 is an integer from 0 to 7 n7 and n8 are independently an integer from 0 to 5; n9 is an integer from 1 to 5; m1 is an integer from 1 to 5; m2 is an integer from 0 to 5; m3is an integer from 1 to 7; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0108] In some embodiments of compounds of Formula I, A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A4is selected from the group consisting of —(CH2)x—,—(CO)O—, —O(CO)—,Q1 is N; Q2 is N;R1and R2are independently of one another selected from the group consisting of optionally substituted C1-C30straight-chain or branched-chain alkyl, optionally substituted C4- C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group; R3is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—,R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; wherein AA represents any natural or non-natural amino acid side chain; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; A2 and A3 are independently selected from the group consisting of H,wherein R10 is selected from the group consisting of H; C1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl;C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6 cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet;; wherein AA represents any natural or non-natural amino acid side chain; a is an integer from 1 to 6; b is an integer from 0 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, n4, n5 are independently an integer from 1 to 5; n6 is an integer from 0 to 7; n7 and n8 are independently an integer from 0 to 5; n9 is an integer from 1 to 5; m1 is an integer from 1 to 5; m2is an integer from 0 to 5; m3 is an integer from 0 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0109] In another embodiment, the disclosure provides a compound having the structure of Formula I-a:or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group, or —(CH2)1-7COOR;A2and A3are independently selected from the group consisting ofC1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl or C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl, or C3-C6cycloalkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet, or C3-C6cycloalkyl groups wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; or C3-C6cycloalkenyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, orC1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S-NR7-, -NR7-S-NR7- or aryl groups, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S- NR7-, or -NR7-S-NR7- groups; or R3is H only when R1and R2are independently of each other —(CH2)1-7COOR; R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; wherein R10 is selected from the group consisting of H; C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; wherein AA represents any natural or non-natural amino acid side chain; and x is an integer from 1 to 9; n is an integer from 1 to 5; n5 is an integer from 1 to 5; n6is an integer from 0 to 7; n7 and n8 are independently an integer from 0 to 5; n9 is an integer from 1 to 5; m3is an integer from 1 to 3; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0110] I some embodiments of the compounds of Formula I-a, R3is —COR or —(CH2)nCOOR.

[0111] In some embodiments of the compounds of Formula I-a, R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.

[0112] In some embodiments of the compounds of Formula I-a, independently of each other, n5 is 1 or n7 is 1 or n9 is 1.

[0113] In some embodiments of the compounds of Formula I-a, x is 4.

[0114] In another embodiment, the disclosure provides a compound having the structure of Formula I-b:or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3 is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—,A2and A3are independently selected from the group consisting ofR4is H or optionally substituted C1-C20straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30 straight-chain or branched-chain alkyl, optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; wherein R10 is selected from the group consisting of H; C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30 straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6 cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; C3-C6 cycloalkyl amines wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; and C3-C6 cycloalkenyl amines, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet; wherein AA represents any natural or non-natural amino acid side chain; and y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n5is an integer from 1 to 5; m2 is an integer from 1 to 5; p is an integer from 1 to 50; andHet is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0115] In some embodiments of the compounds of Formula I-b, R3 is —COR or —(CH2)nCOOR.

[0116] In some embodiments of the compounds of Formula I-b, R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.

[0117] In some embodiments of the compounds of Formula I-b, independently of each other, n5 is 1 or m2 is 2 or n9 is 1.

[0118] In some embodiments of the compounds of Formula I-b, y and z are independently an integer from 1 to 2.

[0119] In another embodiment, the disclosure provides a compound having the structure of Formula I-c:or a pharmaceutically acceptable salt thereof, wherein: R1and R2are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3 is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—.ituted C1-C30straight-chain or branched-chain alkyl, or optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group or optionally substituted C4- C30monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl group;A4is selected from the group consisting of —(CO)O—, —O(CO)—, —S-S—,R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; wherein AA represents any natural or non-natural amino acid side chain; and y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, and n4are independently an integer from 1 to 5; m1 is an integer from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0120] In some embodiments of the compounds of Formula I-c, R3 is —COR or —(CH2)nCOOR.

[0121] In some embodiments of the compounds of Formula I-c, R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.

[0122] In some embodiments of the compounds of Formula I-c, n1, n2, n3, and n4are independently an integer from 1 to 2.

[0123] In some embodiments of the compounds of Formula I-c, y and z are independently an integer from 1 to 2.

[0124] In some embodiments of the compounds of Formula I-c, m1 is an integer from 1 to 2.

[0125] In another embodiment, the disclosure provides a compound having the structure of Formula I-d:or a pharmaceutically acceptable salt thereof, wherein: A2 and A3 are independently selected from the group consisting of H,R1and R2are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3is —COR, —(CH2)nCOR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—,straight-chain or branched-chain alkyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group or optionally substituted C4- C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl group; R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R5and R6are independently selected from H and CH3; R is selected from the group consisting of optionally substituted C4-C30 straight-chain or branched-chain alkyl, optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het;A4is selected from the group consisting of —(CO)O—, —O(CO)—, —S-S—,wherein R10 is selected from the group consisting of H; C1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; C3-C6cycloalkyl amines wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; and C3-C6cycloalkenyl amines, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet; wherein AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 2; b is an integer from 0 to 3; y and z are independently an integer from 1 to 4; z is an integer from 1 to 4; m1and m2are independently an integer from 1 to 5; n1, n2, n3, n4, and n5 are independently an integer from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0126] In another embodiment, the disclosure provides a compound having the structure of Formula I-k:or a pharmaceutically acceptable salt thereof, wherein: A1is —(CH2)x—, or —(CH2)y—S—S—(CH2)z—; R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; wherein R10 is selected from the group consisting of H; or C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; or C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; or C3-C6cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; and a is an integer from 1 to 6; b is an integer from 1 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n5 is an integer from 1 to 5; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

[0127] In some embodiments of the compounds of Formula I-k, A1 is —(CH2)4— or —(CH2)2—S—S—(CH2)2—.

[0128] In some embodiments of the compounds of Formula I-k, n5 is 1.

[0129] In some embodiments of the compounds of Formula I-k, a and b are independently an integer from 1 to 3.

[0130] In some embodiments of the compounds of Formula I-k, R10is a functional group selected from but not limited to H,HO

[0131] In another embodiment, the disclosure provides a compound having the structure of Formula I-n:or a pharmaceutically acceptable salt thereof, wherein: A1 is —(CH2)y—S—S—(CH2)z—; R1and R2are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; wherein AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 1 to 6; y is an integer from 1 to 4; z is an integer from 1 to 4; and n5is an integer from 1 to 5.

[0132] In some embodiments of the compounds of Formula I-n; n5 is 1.

[0133] In some embodiments of the compounds of Formula I-n; a and b are independently an integer from 1 to 3.

[0134] In some embodiments of the compounds of Formula I-n, AA is a functional group selected from but not limited to:

[0135] In another embodiment, the disclosure provides a compound having the structure of Formula I-y:or a pharmaceutically acceptable salt thereof, wherein: R1and R2are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; y is an integer from 1 to 4; and z is an integer from 1 to 4;

[0136] In some embodiments of the compounds of Formula I-y; y and z are independently an integer from 1 to 3.

[0137] In another embodiment, the disclosure provides a compound having the structure of Formula I-z:or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3 is optionally substituted C1-C30 straight-chain or branched-chain alkyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group or optionally substituted C4-C30 monounsaturated or polyunsaturated straight- chain or branched-chain alkynyl group; y is an integer from 1 to 4; and z is an integer from 1 to 4;

[0138] In some embodiments of the compounds of Formula I-z; y and z are independently an integer from 1 to 3.

[0139] In some embodiments of the compounds of Formula I-d, R3is —COR or —(CH2)nCOOR.

[0140] In some embodiments of the compounds of Formula I-d, R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl group.

[0141] In some embodiments of the compounds of Formula I-d, a is 1 and b is an integer from 0-1.

[0142] In some embodiments of the compounds of Formula I-d, y and z are independently an integer from 1 to 2.

[0143] In some embodiments of the compounds of Formula I-d, when A2isA3is H; A4isR1 and R5 are CH3; R2and R6are H; R3 is —COR, —(CH2)nCOOR, —(CO)NHR—, —(CO)N(R)2—,R7is H; b is 0; and a pharmaceutically acceptable salt thereof.

[0144] In some embodiments of the compounds of Formula I-d, A2 and A3 are independently selected from the group consisting ofR7is H; a and b are 1; and pharmaceutically acceptable salt thereof.

[0145] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-g), (I-k), (I-n), (I-o), (II-o-1), or (II-o-2), R1 and R2 are —(CH2)13CH3.

[0146] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-g), (I-k), (I-n), (I-o), (II-o-1), or (II-o-2), A1 is —(CH2)4— or —(CH2)—A4—(CH2)—.

[0147] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-n), (I-o), (I-v), or (I-z), AA is glycine, histidine, serine, tryptophan, arginine, aspartic acid, or pyrrolysine, tyrosine. Preferably, the AA is histidine.

[0148] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k), (I-n), (I-o), (I-r), (I-v), (I-w), or (I-z), R9 is a peptide chain that includes RGD or RYD tripeptide units.

[0149] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k), (I-n), (I-o), (I-r), (I-v), (I-w), or (I-z), R9 is a peptide chain that includes histidine or an RGD or RYD tripeptide unit preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).

[0150] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k), (I-n), (I-o), (I-r), (I-v), (I-w), or (I-z), R9 is a peptide chain that includes repeat RGD or RYD tripeptide units preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).

[0151] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k), (I-n), (I-o), (I-r), (I-v), (I-w), or (I-z), R9 is a peptide chain that includes repeat GLF or WYG tripeptide units preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).

[0152] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k), (I-n), (I-o), (I-r), (I-v), (I-w), or (I-z), R9is a peptide chain that includes repeat Poly-Arg or Poly-His units preceded by spacer such as a glycine or serine spacer (e.g., one or multiple glycine residues, one or more serine residues, or combination of glycine and serine residues).

[0153] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k), (I-n), (I-o), (I-r), (I-v), (I-w), or (I-z), R9is a peptide chain selected from any of the peptide chains listed in Table 1.

[0154] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k), (I-o), (I-r), or (I-w), R7is a neurotransmitter-based functional group selected from but not limited

[0155] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-g), or (II-o-2). R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl groups.

[0156] In some embodiments of Formulas (I), (I-a), (I-b), (I-c), (I-d), (I-k) (I- w), or (II-o-2), the 5-7 membered monocyclic basic heterocycle is selected from the group consisting of

[0157] Some embodiments include a compound selected from the group consisting of:and pharmaceutically acceptable salts thereof.

[0158] Some embodiments include a compound selected from the group consisting of:and pharmaceutically acceptable salts thereof.

[0159] Some embodiments include a compound selected from the group consisting of:and pharmaceutically acceptable salts thereof.

[0160] Some embodiments include a compound selected from the group consisting of:and pharmaceutically acceptable salts thereof.

[0161] Some embodiments include a compound selected from the group consisting of:and pharmaceutically acceptable salts thereof.

[0162] Some embodiments include a compound selected from the group consisting of:and pharmaceutically acceptable salts thereof.

[0163] Some embodiments include a compound selected from the group consisting of:wherein:and pharmaceutically acceptable salts thereof.

[0164] Some embodiments include a compound selected from the group consisting of: wn6 is 0 or 1; R10 is H;and pharmaceutically acceptable salts thereof.

[0165] Some embodiments include a compound selected from the group consisting of:wherein:and pharmaceutically acceptable salts thereof.

[0166] Some embodiments include a compound selected from the group consisting of:pharmaceutically acceptable salts thereof.

[0167] Some embodiments include a compound selected from the group consisting of:acceptable salts thereof.

[0168] Provided herein are compositions comprising (i) one or more compounds according to Formula I (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizing agent; and (iii) optionally, a payload.

[0169] Also provided are compositions comprising (i) a compound according to Formula I (ii) one or more structural lipids (iii) one or more of stabilizing agents; and (iv) optionally, a payload.

[0170] Also provided are compositions comprising (i) a compound according to Formula I (ii) one or more structural lipids (iii) one or more of stabilizing agents; (iv) one or more transfection enhancing agents, and (v) optionally, a payload.

[0171] Also provided are compositions comprising (i) one or more compounds according to Formula I and (ii) a payload.

[0172] Also provided are compositions comprising one or more compounds according to Formula I at 10 to 80 mol% (excluding any payload), or mol% of the total lipid present in the composition.

[0173] Also provided are compositions comprising one or more compounds according to Formula I and wherein a structural lipid is present at 14-50 mol% (excluding any payload) of the composition, or mol% of the total lipid present in the composition.

[0174] Also provided are compositions comprising one or more compounds according to Formula I and wherein a stabilizing agent is present at 0.1-10 mol% (excluding any payload) of the composition, or mol% of the total lipid present in the composition.

[0175] Also provided are compositions comprising one or more compounds according to Formula I and further comprising an exosome or a biological material derived or purified from an exosome.

[0176] Also provided are compositions comprising one or more compounds according to Formula I and further comprising a polymer.

[0177] Also provided are compositions comprising one or more compounds according to Formula I and wherein the polymer is selected from the group consisting of: a dense star dendrimer, a PAMAM dendrimer, an NH3 core dendrimer, an ethylenediamine core dendrimer, a dendrimer of generation 5 or higher, a dendrimer with a substituted group, a dendrimer comprising one or more amino acids, a grafted dendrimer, an activated dendrimer, polyethylenimine (PEI), polyethylenimine conjugates, polylysine, polyarginine, polyornithine, histone, and any combination thereof.

[0178] For example, compositions can include one or more compounds according to Formula I and a linear or branched PEI.

[0179] Provided herein are compositions comprising one or more compounds according to Formula I, and a stabilizing agent selected from the group consisting of: a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof.

[0180] For example, provided are compositions that include one or more compositions of Formula I, and a stabilizing agent that is a PEG-modified lipid.

[0181] Provided herein are compositions including one or more compounds according to Formula I and one or more transfection enhancing agents, such as a polycationic nucleic acid binding moiety, or a transfection enhancing agent selected from the group consisting of: an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell-penetrating peptide, a fusogenic peptide, an amphipathic peptide, and any combination thereof.

[0182] Provided herein are compositions comprising one or more compounds according to Formula I and a payload.

[0183] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid.

[0184] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the compound comprises a charge N and the nucleic acid molecule comprises a charge P and wherein the combination of the compound and the nucleic acid contacting the cell comprises an N / P ratio from about 1 to 20.

[0185] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid and wherein the nucleic acid is an RNA.

[0186] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and further comprises a nucleic acid wherein the nucleic acid is an RNA and wherein the RNA is mRNA, siRNA, shRNA, self-replicating RNA (srRNA), an o-RNA, self-amplifying RNA, stRNA, trRNA, crRNA, sgRNA, RNAi molecule, an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), or any combination thereof.

[0187] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid and wherein the nucleic acid is an DNA.

[0188] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload further comprises one or more peptides, and optionally a nucleic acid.

[0189] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid wherein the nucleic acid is an RNA and further wherein the RNA is mRNA.

[0190] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid wherein the nucleic acid is an RNA, and the RNA encodes an immunogen.

[0191] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the payload comprises a nucleic acid wherein the nucleic acid is an RNA, and the RNA encodes a cancer antigen.

[0192] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I and wherein the structural lipidis 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.

[0193] In another embodiment, the disclosure provides compositions of a compound of Formula I and one or more of stabilizing agent, wherein the stabilizing agent comprises one or more phospholipids selected from the group consisting of: 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-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-dioleoyl-sn-glycero-3-phosphoethanola mine (DOPE), 1,2- diphytanoyl-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, 1,2-dioleoyl-sn-glycero-3- phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In some embodiments, the formulations may also include one or more lipids derived from viral capsids, e.g, from enveloped viruses.

[0194] In another embodiment, the disclosure provides compositions comprising one or more compounds according to Formula I further comprising a transfection enhancing agent selected from the group consisting of an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell-penetrating peptide, a fusogenic peptide, and any combination thereof.

[0195] In another aspect, the disclosure provides compositions of a compound of Formula I and at least one or more neutral lipids, wherein the one or more neutral lipids is 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-phospho-ethanolamine), 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), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanol amine (SOPE), and 1,2-dioleoyl-sn-glycero-3- phophoethanolamine (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-diphytanoyl-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.

[0196] In another aspect, the disclosure provides compositions of a compound of Formula I, and at least one or more cationic lipids, and / or at least one or more neutral lipids, wherein the cationic lipid is selected from GeneInTM, LipofectAmineTM2000, LipofectAmineTM, Lipofectin®, DMRIE-C, CellFectin®(Invitrogen), Oligofectamine®(Invitrogen), LipofectAce®(Invitrogen), Fugene®(Roche, Basel, Switzerland), Fugene®HD (Roche), Transfectam®(Tranfectam, Promega, Madison, WI), Tfx-10®(Promega), Tfx-20®(Promega), Tfx-50®(Promega), TransfectinTM(BioRad, Hercules, CA), SilentFectTM(Bio- Rad), Effectene®(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), EscortTMIII (Sigma, St. Louis, MO), EscortTMIV (Sigma), DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N- 1-(2,3-diaoleoyloxypropyl)-2-hydroxy-propane-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-diaoleoyl- oxypropyl)-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-hyd roxyethylamine, 3,5-(N,N-di-lysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl- dimethylaminopropyl-β-hydroxyethylamine), L-Lysine-bis(O,O’-oleoyl-β-hydroxy- ethyl)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-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-oleyl-amino)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, Sarcosine-bis-(O,O’- palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-Histidine-bis-(O,O’-palmitoyl-β- hydroxyethyl)amide dihydrochloride, cholesteryl-3β-carboxyl-amidoethylenetrimethyl- ammonium iodide, 1,4-bis[(3-myristylamino)propyl]-piperazine, 1-dimethylamino-3- trimethyl-ammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3β- carboxyamidoethylene-amine, cholesteryl-3β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysucc inate iodide, 2-[(2-trimethylammonio)-ethylmethyl-amino]ethyl-cholesteryl-3β-oxysuccinate iodide, 3β[N-(N’, N’-dimethylamino-ethane)-carbamoyl]cholesterol, and 3β-[N- (polyethyleneimine)-carbamoyl] cholesterol,1,4-bis[(3-palmitylamino)propyl]piperazine, L- Ornithylglycyl-N-(1-heptadecyloctadecyl)-glycinamide, N2,N5-Bis(3-aminopropyl)-L- ornithylglycyl-N-(1-heptadecyloctadecyl)-glycin-amide, 1,4-bis[(3-(3-amino-2- hydroxypropyl)-alkylamino)-2-hydroxypropyl]-piperazine, N2-[N2,N5-Bis(3-aminopropyl)- L-ornithyl]-N,N-dioctadecyl-L-glutamine,N2-[N2,N5-Bis(amino-propyl)-L-ornithyl]-N-N- dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxy-propyl)-oleylamino)2- hydroxypropyl]piperazine, N2-[N2,N5-Bis(amino-propyl)-L-ornithyl]-N-N-dioctadecyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethyl-ethoxy)carbonyl]-N2,N5-bis[3-[(1,1- dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioctadecyl-L-glutaminyl]-L- glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-diolyl-L-glutamine, N2- [N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dioleyl-L-α-glutamine,4-bis[(3-(3-amino-2- hydoxypropyl)-myristylamino)-2-hydroxy-propyl]piperazine, N2-[N2,N5-Bis(aminopropyl)- L-ornithyl]-N-N-dioleyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]- N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioleyl-L- glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N2- [N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-dipalmityl-L-glutamine,N2-[N2,N5-Bis(amino- propyl)-L-ornithyl]-N-N-dipalmityl-L-α-glutamine, N2-[N2,N5-Bis(aminopropyl)-L- ornithyl]-N-N-dipalmityl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)- carbonyl]-N2,N5-bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N- dipalmityl-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N- dimyristyl-L-glutamine, N2-[N2,N5-Bis(amino-propyl)-L-ornithyl]-N-N-dimyristyl-L-α- glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dimyristyl-L-α-asparagine, 1,4- bis[(3-(3-amino-2-hydroxypropyl)-palmityl-amino)-2-hydroxypropyl]-piperazine, N-[N2- [N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1- dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dimyristyl-L-glutaminyl]-L- glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine, N2-[N2,N5- Bis(3-amino-propyl)-L-ornithyl]-N,N-dilaureyl-L-glutamine, N2-[N2,N5-Bis(amino-propyl)- L-ornithyl]-N-N-dilaureyl-L-α-glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N- dilaureyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)-carbonyl]-N2,N5-bis[3- [(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dilaureyl-L-glutaminyl]-L- glutamic acid, 3-[N’,N”-bis(2-tertbutyloxycarbonyl-amino-ethyl)guanidino]-N,N-dioctadec- 9-enyl-propionamide, 3-[N’,N”-bis(2-tertbutyloxy-carbonylaminoethyl)guanidino]-N,N- dipalmityl-propionamide, 3-[N’,N”-bis(2-tertbutyl-oxycarbonylaminoethyl)guanidino]-N,N- dimyristyl-propionamide, 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-hydroxy-propyl-3-N,N-dipalmitylaminopropane, N,N-(2-hydroxy-3- aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristylaminopropane, 1,4-bis[(3-(3-amino-2- hydoxypropyl)-myristylamino)-propyl]-piperazine, [(3-aminopropyl)-bis-(2- tetradecyloxyethyl)]methyl ammonium bromide, [(3-aminopropyl)-bis-(2- oleyloxyethyl)]methyl ammonium bromide, [(3-aminopropyl)-bis-(2-palmityloxyethyl)]methyl ammonium bromide, Oleoyl-2-hydroxy-3-N,N-dimethyamino propane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethy- amino propane, 1,2-dipalmitoyl-1-N,N-dimethylamino-propane, myristoyl-2-hydroxy-3- N,N-dimethyamino propane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-Amino- propyl)-›4-(3-amino-propylamino)-4-tetradecyl-carbamoyl-butylcarbamic acid cholestryl ester, (3-Amino-propyl)-›4-(3-amino-propylamino-4-carbamoylbutylcarbamic acid cholestryl ester, (3-Amino-propyl)-›4-(3-amino-propylamino)-4-(2-dimethylamino-ethylcarbamoy l)- butylcarbamic acid cholesteryl ester, Spermine-5-carboxyglycine (N’-stearyl-N’-oleyl) amide tetratrifluoro-acetic acid salt, Spermine-5-carboxyglycine (N’-stearyl-N’-elaidyl) amide tetratri-fluoroacetic acid salt, Agmatinyl carboxycholesterol acetic acid salt, Spermine-5- carboxy-β-alanine cholesteryl ester tetratrifluoroacetic acid salt, 2,6-Diaminohexanoeyl β- alanine cholesteryl ester bistrifluoroacetic acid salt, 2,4-Diaminobutyroyl β-alanine cholesteryl ester bistrifluoroacetic acid salt, N,N-Bis (3-aminopropyl)-3-aminopropionyl β- alanine cholesteryl ester tristrifluoroacetic acid salt, [N,N-Bis(2-hydroxyethyl)-2- aminoethyl]aminocarboxy cholesteryl 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-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-(palmityl- oxyethyl)-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-oleyl-oxyethyl)- 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)-diamino-butane, 2,3- dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(2-hydroxy-3-amino-propyl)-diamino-butane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(2-hydroxy-3-aminopropyl)-diamino- butane, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(3-amino-propyl)-diaminobutane, 2,3- dipalm-itoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(3-amino-propyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(3-amino-propyl)-diaminobutane, 2,3- dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(5-carboxamido-spermine)-diamino-butane, 2,3- dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(5-carboxamidospermine)-diamino-butane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(5-caqrboxamidospermine)- diaminobutane, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(lysyl)-diaminobutane, 2,3- dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(lysyl)-diamino-butane, 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-dipalmitoleoyl-oxy-1,4-N,N’-dimethyl- N,N’-di(histidyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’- di(histidyl)-diaminobutane, 2,3-dioleyloxy-N,N’-dimethyl-1,4 -diaminobutane, 2,3- dipalmitoleoyloxy-N,N’-dimethyl-1,4-diaminobutane, 2,3-dimyrist-oleoyloxy-N,N’- dimethyl-1,4-diaminobutane; PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, polyethylene-imine (PEI), and polyethylenimine conjugates.

[0197] In another aspect, the disclosure provides compositions of a compound of Formula I, and at least one or more cationic lipids, and / or at least one or more neutral lipids, wherein the one or more neutral lipids is 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-phospho- ethanolamine), 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), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl- phosphatidyethanol amine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phophoethanolamine (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-diphytanoyl-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.

[0198] In another embodiment, the disclosure provides compositions of a compound of Formula I, and at least one or more cationic lipids, and / or at least one or more neutral lipids, and / or at least one or more PEG-modified lipids, wherein the PEG-modified lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, a PEG-ceramide conjugate, a PEG-modified 1,2-diacyloxypropan-3-amine, or any combinations thereof.

[0199] In another embodiment, the disclosure provides compositions of a compound of Formula I, and wherein the one or more PEG-modified lipids is selected from the group consisting of: PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and any combination thereof.

[0200] In another embodiment, the disclosure provides compositions of a compound of Formula I, and at least one or more cationic lipids, and / or at least one or more neutral lipids, wherein the alkoxy is selected form oleyl, palmityl, palmitoleoyl, myristoleyl, myristyl, and lauryl.

[0201] In another embodiment, the disclosure provides compositions of a compound of Formula I, and one or more polyamine transfection agents.

[0202] In another embodiment, the disclosure provides compositions of a compound of Formula I, and one or more fusion agents.

[0203] In another embodiment, the disclosure provides compositions of a compound of Formula I, and one or more amphipathic peptides.

[0204] In another aspect, the disclosure provides compositions of a compound of Formula I, and one or more amphipathic peptides, wherein the one or more amphipathic peptide functions as a fusion agent.

[0205] In another embodiment, the disclosure provides method of delivering a payload to a cell, comprising: (i) providing a composition comprising a compound of Formula I; (ii) providing a cell; and (iii) contacting the cell with the composition. In some embodiments, the payload includes a nucleic acid that encodes a therapeutic protein, such as an antibody, growth factor, cytokine, enzyme, or the like.

[0206] In another embodiment, the disclosure provides methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell, comprising contacting the cell with a composition of any of the disclosed compositions, thereby introducing the nucleic acid, protein, or peptide into the cell.

[0207] In another embodiment, the disclosure provides methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell, wherein the cell is a human cell, comprising contacting the cell with a composition of any of the disclosed compositions, thereby introducing the nucleic acid, protein, or peptide into the cell.

[0208] In another embodiment, the disclosure provides methods for introducing a nucleic acid, protein, or peptide into a eukaryotic cell, wherein the cell is a mammalian cell, comprising contacting the cell with a composition of any of the disclosed compositions, thereby introducing the nucleic acid, protein, or peptide into the cell.

[0209] In another embodiment, the disclosure provides method for delivering a composition to a subject, comprising administering the composition according to a compound of Formula I to the subject.

[0210] In another embodiment, the disclosure provides methods of delivering a payload to a cell, wherein contacting the cell is in vitro.

[0211] In another embodiment, the disclosure provides methods of delivering a payload to a cell, wherein contacting the cell is in vivo.

[0212] In another embodiment, the disclosure provides methods of delivering a payload to a cell, wherein contacting the cell is ex vivo.

[0213] In another embodiment, the disclosure provides methods for administering any of the disclosed compositions, to subject, wherein the administration is systemic.

[0214] In another embodiment, the disclosure provides methods for administering any of the disclosed compositions, to subject, wherein the administration is selected from the group consisting of subcutaneous administration, intramuscular administration, intranasal administration, intra-tumoral administration, administration to the brain, administration to the spinal cord, administration to the eye, administration to the lymph node of a subject, and any combination thereof.

[0215] In another embodiment, the disclosure provides kits including a compound of Formula I, and one or more structural lipids, ionizable lipid, and a stabilizing agent.

[0216] In another embodiment, the disclosure provides kits including a compound of Formula I, and one or more structural lipids, and / or one or more stabilizing agents, and / or optionally a payload.

[0217] In another embodiment, the disclosure provides kits including a compound of Formula I, and one or more structural lipids, and / or one or more stabilizing agents, and / or one or more fusion agents, and / or optionally a payload.

[0218] In another embodiment, the disclosure provides methods for inhibiting expression of a protein in a cell, comprising contacting the cell with an RNAi molecule and a compound of Formula I, as described herein, or the composition of any one of the disclosed compositions.

[0219] The skilled artisan will recognize that, although the compounds of the invention are shown here for convenience in their neutral (unprotonated) forms, these compounds may also exist in a partially or fully protonated form in solutions of appropriate pH, and that the present invention encompasses the compounds in all their protonated, unprotonated, ionized and non-ionized forms without limitation, unless specifically indicated otherwise. Methods of Preparation:

[0220] The compounds disclosed herein may be synthesized by methods described below, or by modification of these methods. Ways of modifying the methodology include, among others, temperature, solvent, reagents etc., known to those skilled in the art. In general, during any of the processes for preparation of the compounds disclosed herein, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry (ed. J.F.W. McOmie, Plenum Press, 1973); and P.G.M. Green, T.W. Wutts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999), which are both hereby incorporated herein by reference in their entirety. The protecting groups may be removed at a convenient subsequent stage using methods known from the art. Synthetic chemistry transformations useful in synthesizing applicable compounds are known in the art and include e.g. 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, which are both hereby incorporated herein by reference in their entirety. The routes shown and described herein are illustrative only and are not intended, nor are they to be construed, to limit the scope of the claims in any manner whatsoever. Those skilled in the art will be able to recognizemodifications of the disclosed syntheses and to devise alternate routes based on the disclosures herein; all such modifications and alternate routes are within the scope of the claims.

[0221] In the following schemes, protecting groups for oxygen atoms are selected for their compatibility with the requisite synthetic steps as well as compatibility of the introduction and deprotection steps with the overall synthetic schemes (P.G.M. Green, T.W. Wutts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999)).

[0222] If the compounds of the present technology contain one or more chiral centers, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or d(l) stereoisomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the present technology, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.

[0223] 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), Emka-Chemce or Sigma (St. Louis, Missouri, USA). Others may be prepared by procedures, or obvious modifications thereof, 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).

[0224] The following example schemes are provided for the guidance of the reader, and collectively represent an example method for making the compounds encompassed herein. Furthermore, other methods for preparing compounds described herein will be readily apparent to the person of ordinary skill in the art in light of the following reaction schemes and examples. Unless otherwise indicated, all variables are as defined above.

[0225] In one embodiment, as shown in Scheme I, the method involves treatment of intermediate I-e with BOC-anhydride in presence of a base to obtain the bis-BOC derivative I-h which is subsequently treated with an acid chloride II-h to obtain theintermediate I-f. This is then subjected to BOC-removal in presence of an acid to obtain compounds of formula I-n. SCHEME I

[0226] In another embodiment, as shown in Scheme II, the bis-BOC derivative I-h is treated with mesyl chloride in presence of a base to yield the bis-mesylate intermediate I-j. The intermediate I-j is then treated with amines represented by formula II-k in presence of a base followed by BOC-removal in presence of an acid to yield the compounds of formula I-k. SCHEME II

[0227] In one embodiment, for example, compounds 8-12, 38-92 are synthesized following reactions conditions as shown in Scheme II.

[0228] In another embodiment, as shown in Scheme III, the bis-BOC derivative I-h is treated with a BOC-protected amino acid (II-m) in presence of an appropriate base and coupling reagent to obtain the derivative I-m which is then subjected to BOC- removal in presence of an acid to yield the compounds of formula I-n. SCHEME III

[0229] In one embodiment, for example, compounds 13-21 are synthesized following reactions conditions as shown in Scheme III.

[0230] In another embodiment, as shown in Scheme IV, the bis-BOC derivative I-h is subjected to alkylation reaction of the hydroxyl group with an alkylating agent R3-X (II-j; X is a leaving group) followed by BOC-removal in presence of an acid to yield the compounds of formula I-o.SCHEME IV

[0231] In one embodiment, for example, compounds 22-23 are synthesized following reactions conditions as shown in Scheme IV.

[0232] In another embodiment, as shown in Scheme V, isoindoline-1,3-dione is condensed with 3-chloro-2-(chloromethyl)prop-1-ene to obtain the adduct 2-(2- (chloromethyl)allyl)isoindoline-1,3-dione which is subsequently treated with the diamine I-x to obtain the intermediate I-p. The intermediate I-p is then subjected to treatment with hydrazine in ethanol followed by treatment with (BOC)2O to obtain the derivative I-q. The derivative I-q is then treated with amine II-k followed by BOC-removal in presence of an acid to yield the compounds of formula I-r. SCHEME V

[0233] In one embodiment, for example, compound 24 is synthesized following reactions conditions as shown in Scheme V using N1,N4-ditetradecylbutane-1,4- diamine as the amine I-x to couple with 2-(2-(chloromethyl)allyl)isoindoline-1,3-dione and 2-aminoethan-1-ol as the amine I-k to couple with the corresponding intermediate I-q.

[0234] In another embodiment, as shown in Scheme VI, solid-phase bound arylalkyl chloride II-a is reacted with amino-thiol II-b to yield the solid-phase bound amine II-c. The amine II-c is then subjected to reductive amination in presence of aldehyde II-n to yield 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 an appropriate solvent to yield the adduct II-e. This adduct II-e is then subjected to release from the solid-phase followed by oxidation to yield the disulfide I-s. The disulfide I-s is then subjected to deprotection using hydrazine in presence of ethanol to yield the compounds of formula I-x-1. SCHEME VI

[0235] In another embodiment, as shown in Scheme VII, disulfide II-f is subjected to reductive amination with aldehyde II-n to yield intermediate II-g which is subjected to treatment with 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione in the presence of a base and heated in an appropriate solvent to yield the adduct I-t. The adduct I-t is thensubjected to treatment with hydrazine in ethanol followed by treatment with (BOC)2O to obtain the derivative I-u.

[0236] In another embodiment, as shown in Scheme VII, the bis-BOC derivative I-u is treated with a BOC-protected amino acid (II-m) in presence of an appropriate base and coupling reagent followed by subjecting the resulting product to BOC-removal in presence of an acid to yield the compounds of formula I-v.

[0237] In another embodiment, as shown in Scheme VII, the bis-BOC derivative I-u is treated with mesyl chloride in presence of a base to yield the bis-mesylate intermediate which is then treated with amines represented by formula II-k in presence of a base followed by BOC-removal in presence of an acid to yield the compounds of formula I- w. SCHEME VII

[0238] In one embodiment, for example, compounds 25-37 are synthesized following reactions conditions as shown in Scheme VII using 2,2'-disulfanediylbis(ethan-1- amine) as the diamine II-f and tetradecanal as the aldehyde II-n to yield the corresponding intermediate II-g. The corresponding intermediate II-g was elaborated to yield the intermediate I-u. Then this intermediate was further converted to compounds 25-37 using reactions shown in Scheme VII.

[0239] The following example schemes are provided for the guidance of the reader, and represent preferred methods for making the compounds exemplified herein. These methods are not limiting, and it will be apparent that other routes may be employed to prepare these compounds. Such methods specifically include solid-phase based chemistries, including combinatorial chemistry. The skilled artisan is thoroughly equipped to prepare these compounds by those methods given the literature and this disclosure. The compound numberings used in the synthetic schemes depicted below are meant for those specific schemes only, and should not be construed as or confused with same numberings in other sections of the application.

[0240] The examples of compounds of Formula I may be synthesized using methods that are well known in the art, as shown for Compound 1 shown below, in Scheme IA. SCHEME IAAs shown in Scheme IA, the DHDMS is treated with BOC-anhydride in presence of a base to obtain the bis-BOC derivative (1A). This intermediate (1A) was treated with oleoyl chloride to obtain the intermediate 1B. This was subsequently treated with TFA to obtain the bis-oleyl derivative 1. Using similar procedures, additional analogs have been synthesized using various acid chlorides.

[0241] Similarly Compounds 2-4 were also synthesized using procedures as described above.SCHEME VIII

[0242] In another embodiment, as shown in Scheme VIII, diamine I-x is treated with an epoxide II-o in the presence of a base in a polar solvent and heated to yield the compounds of formula II-o-1. The compound II-o-1 was treated with an acid chloride II-h followed by treatment with an acid to form the salts of the compounds of formula II-o-2.

[0243] Compounds 5-7 were synthesized using scheme as shown below in Scheme VIIIA. SCHEME VIIIA

[0244] As shown in Scheme VIIIA, N1,N4-ditetradecylbutane-1,4-diamine (7A) is treated with 2-ethyloxirane in 2,2,2-trifluoroethanol in presence of DIPEA at 80 °C to obtain the compound 7. In the next step, compound 7 was treated with oleoyl chloride to obtain the intermediate 6A. This was subsequently treated with HCl to obtain the bis-oleoyl derivative 6 as a hydrochloride salt. Similarly compound 5 was also synthesized using procedures as described above by treating compound 7 with linoleoyl chloride.

[0245] Compounds 8-12 were synthesized using schemes as shown below in Scheme IIA.SCHEME IIA

[0246] As shown in Scheme IIA, the bis-BOC derivative was treated with mesyl chloride in presence of DIPEA to yield the bis-mesylate intermediate 8A. The intermediate 8A was the treated with 2-aminoethanol in presence of a base to yield the derivative 8B which was treated with TFA to yield the compound 8. Using similar procedures, additional analogs have been synthesized by displacing the mesylate group in intermediate 8A using various amines.

[0247] Similarly Compounds 9-12, 39-41, 43, 46, and 80 were also synthesized following procedures as described above 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-aminopropan-1-ol (for compound 39), 5- aminopentan-1-ol (for compound 40), 6-aminohexan-1-ol (for compound 41), 2-amino-3- phenylpropan-1-ol (for compound 43), 2-amino-2-methylpropan-1-ol (for compound 46), 2-amino-2-methylpropane-1,3-diol (for compound 48), and 1-aminopropan-2-ol (for compound 80).SCHEME IX

[0248] In another embodiment, as shown in Scheme IX, diamine II-u is subjected to reductive amination conditions using the aldehyde II-q and NaBH(OAc)3to yield the intermediate II-r. The skilled artisan will appreciate that there are many other reductive amination conditions and reagents which are within the scope of this disclosure to carry out the reaction. The resulting 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 yield the product II-s. The intermediate II-s is then treated with (BOC)2O followed by treatment with 2,2-dimethoxypropane in the presence of a Lewis acid (for example BF3.Et2O) to obtain the derivative II-s-1. Intermediate II-s-1 is then subjected to indium catalyzed transesterification with alcohol II-p followed by acetonide-removal and BOC-removal under acid-catalyzed conditions to yield the compounds of formula II-t. The skilled artisan will appreciate that there are many other transesterification conditions and acetonide-removal andBOC-removal conditions which are within the scope of this disclosure to carry out these reactions.

[0249] In one embodiment, for example, compounds shown below are synthesized following reactions conditions as shown in Scheme IX.Lipid Nanoparticle Formulations:

[0250] The lipids of formula (I) can be combined with a nucleic acid and / or protein payload in a to produce a lipid nanoparticle formulation. For example, the lipids of formula (I) can be combined with a payload selected from one or more of the following: an siRNA, an miRNA, an mRNA, a shRNA, a self-amplifying RNA, an oRNA (or non-naturally occurring circular RNA), an anti-sense oligonucleotide (ASO), a gRNA, a ribonucleoprotein (e.g., a CRISPR complex), a dsDNA, a plasmid DNA, or the like.

[0251] In the instance of delivery of a nucleic acid, the amount of nucleic acid (e.g., mRNA, self-amplifying RNA or the like) in 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 lipid nanoparticle formulation. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. In some embodiments, the wt / wt ratio of the lipid component to a nucleic acid, such as an mRNA, in a nanoparticle composition may be from about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the wt / wt ratio of the lipid component to a nucleic acid, such as an mRNA may be from about 10:1 to about 40:1. The amount of nucleic acid in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible (UV-vis) spectroscopy).

[0252] The lipid nanoparticle formulations can comprise a nucleic acid in a concentration from approximately 0.1 mg / ml to 2 mg / ml such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml or greater than 2.0 mg / ml.

[0253] Preferably, the one or more nucleic acids (e.g. mRNAs), lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in a nucleic acid (e.g., an mRNA). In general, a lower N:P ratio is preferred. The one or more mRNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In certain embodiments, the N:P ratio may be from 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 from 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. Additional lipid components:

[0254] Advantageously, in addition to the lipids of formula (I), the lipid nanoparticle formulations include one or more co-lipids, most advantageously neutral co- lipids, although the skilled artisan will recognize that other lipids, including cationic / ionizable lipids, may be used. Some formulations, however, include just the lipids of formula (I), in combination with a nucleic acid.

[0255] Ionizable lipids described herein refer to lipids that have at least one protonatable or deprotonatable group. In some embodiments, ionizable lipids can be positively charged at a pH at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, e.g., at or above physiological pH. For example, the ionizable lipids provided herein can have a pKa of the protonatable group in the range of about 4 to about 11, e.g., about 4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and 6.9, when incorporated into lipid nanoparticles.

[0256] Accordingly, in addition to the lipids of Formula (I), the lipid nanoparticle formulations can include neutral lipids such as phospholipids. Exemplary 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 include, but are not limited to, 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), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanol amine (SOPE), and 1,2-dioleoyl-sn-glycero-3- phophoethanolamine (trans DOPE), or any combination thereof. Phospholipids useful in the compositions provided herein can be present, for example at about 5 mol% to about 20 mol% of the lipid nanoparticle formulation. Advantageously, phopsolipids are present at a ragne from about 1 mol % to about 40 mol%, e.g., from 1 mol% to about 25 mol %. Preferably, the amount of the phospholipid 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 nanoparticle formulations.

[0257] Other neutral lipids that can be advantageously included in the lipid nanoparticle formulations provided herein include sterols, or lipids containing sterol moieties (“sterol derivatives”). As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. Exemplary sterols and lipids containing sterol moieties useful in the lipid nanoparticle formulations provided herein include, but are not limited to holesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. Some lipid nanoparticle formulations provided herein include a sterol or sterol derivative. The sterols or sterol derivatives can be present at about 5-60 mol% of the overall lipid nanoparticle formulation. Advantageously, the sterol or sterol derivatives are present from about 15-50 mol%, e.g., 25-40 mol %. Preferably, the amount of the sterol (such as cholesterol) or sterol derivative in the lipid composition 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 include a sterol or sterol derivative.

[0258] The lipid nanoparticle formulations provided herein can also include a stabilizing agent, such as a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof Examples of non-ionic stabilizing agentsinclude: Polysorbates (Tweens), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™35 (Polyoxyethylene lauryl ether, Polyethyleneglycol lauryl ether), Brij™S10 (Polyethylene glycol octadecyl ether, Polyoxyethylene (10) stearyl ether), and Myrj™52 (polyoxyethylene (40) stearate). Other exemplary stabilizing agents useful in the embodiments provided herein include TPGS 1000 (D-α-Tocopherol polyethylene glycol 1000 succinate); or Tween 20 / Polysorbate 80 / Tridecyl-D-maltoside in equal ratios (called Lipid H in Table 15). Yet other stabilizing lipids that can advantageously be 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-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG- DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. Other stabilizing lipids useful in the compositions disclosed herein include, e.g., polyglycol lipids, yoxyethylene alkyl ethers, diblock polyoxyethylene ether co-polymers, triblock polyoxyethylene alkyl ethers co- polymers, and amphiphilic branched polymers. In embodiments, stabilizing agent can be In polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate ("Myrj52"), poly(propylene glycol)11-block-poly(ethylene glycol)16-block- poly(propylene glycol)11, poly(propylene glycol)12-block-poly(ethylene glycol)28-block- poly(propylene glycol)12, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4- bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (Polyoxyethylene (40) stearate), Brij™ S10 (Polyoxyethylene (10) stearyl ether), BRIJ™ L4 = Polyoxyethylene (4) lauryl ether; BRIJ™ S20= Polyoxyethylene (20) stearyl ether; BRIJ™ S35= Polyoxyethylene (23) lauryl ether; TPGS 1000 =D-α-Tocopherol polyethylene glycol 1000 succinate; Tween 20 / Polysorbate 80 / Tridecyl-D-maltoside in equal ratios, and combinations thereof. In certain compositions, the stabilizing agent is present at about 0.1 - 5 mol% of the lipid nanoparticle formulation. For example, in some compositions, the stabilizing agent is present at 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 nanoparticle formulation.

[0259] Lipid nanoparticle formulations can include one or more cationic / ionizable lipids, in addition to the lipid of Formula (I). For example, some lipid nanoparticle formulations include a cationic / ionizable lipid 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, 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-di- lysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylami nopropyl-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-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, Sarcosine-bis-(O,O'-palmitoyl-β- hydroxyethyl)amide dihydrochloride, L-Histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesteryl-3β-carboxyl-amidoethylenetrimethylammonium iodide, 1,4- bis[(3-myristylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonio-DL-2- propyl-cholesteryl carboxylate iodide, cholesteryl-3β-carboxyamidoethyleneamine, cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium 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-Ornithylglycyl-N-(1- heptadecyloctadecyl)glycinamide, N2,N5-Bis(3-aminopropyl)-L-ornithylglycyl-N- (1-heptadecyloctadecyl)glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2- hydroxypropyl]piperazine N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L- glutamine,N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dioctadecyl-L-α-glutamine, 1,4- bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N2-[N2,N5- Bis(aminopropyl)-L-ornithyl]-N-N-dioctadecyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1- dimethylethoxy)carbonyl]- N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L- ornithyl-N-N-dioctadecyl-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L- ornithyl]-N,N-diolyl-L-glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dioleyl-L- α-glutamine,4-bis[(3-(3-amino-2-hydoxypropyl)-myristylamino)-2- hydroxypropyl]piperazine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dioleyl-L-α- asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]- N2,N5-bis[3-[(1,1- dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N2-[N2,N5-Bis(3- aminopropyl)-L-ornithyl]-N,N-dipalmityl-L-glutamine,N2-[N2,N5-Bis(aminopropyl)-L- ornithyl]-N-N-dipalmityl-L-α-glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N- dipalmityl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]- N2,N5-bis[3- [(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dipalmityl-L-glutaminyl]-L- glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-dimyristyl-L-glutamine, N2- [N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dimyristyl-L-α-glutamine, N2-[N2,N5- Bis(aminopropyl)-L-ornithyl]-N-N-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- ornithyl-N-N-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)- myristylamino)propyl]piperazine, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N- dilaureyl-L-glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dilaureyl-L-α- glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dilaureyl-L-α-asparagine, N-[N2- [N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]- N2,N5-bis[3-[(1,1- dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dilaureyl-L-glutaminyl]-L-glutamic acid, 3-[N',N"-bis(2-tertbutyloxycarbonylaminoethyl)guanidino]-N,N-dioctadec-9- enylpropionamide, 3-[N',N"-bis(2-tertbutyloxycarbonylaminoethyl)guanidino]-N,N- dipalmitylpropionamide, 3-[N',N"-bis(2-tertbutyloxycarbonylaminoethyl)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-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- hydoxypropyl)-myristylamino)propyl]piperazine, [(3-aminopropyl)-bis-(2- tetradecyloxyethyl)]methyl ammonium bromide, [(3-aminopropyl)-bis-(2- oleyloxyethyl)]methyl ammonium bromide, [(3-aminopropyl)-bis-(2- palmityloxyethyl)]methyl ammonium bromide, Oleoyl-2-hydroxy-3-N,N-dimethyamino propane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N- dimethyamino propane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2- hydroxy-3-N,N-dimethyamino propane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3- Amino-propyl)-›4-(3-amino-propylamino)-4-tetradecylcarbamoyl-butylcarbamic acid cholesteryl ester, (3-Amino-propyl)-›4-(3-amino-propylamino-4-carbamoylbutylcarbamic acid cholesteryl ester, (3-Amino-propyl)-›4-(3-amino-propylamino)-4-(2-dimethylamino- ethylcarbamoy l)-butylcarbamic acid cholesteryl ester, Spermine-5-carboxyglycine (N'- stearyl-N'-oleyl) amide tetratrifluoroacetic acid salt, Spermine-5-carboxyglycine (N'-stearyl- N'-elaidyl) amide tetratrifluoroacetic acid salt, Agmatinyl carboxycholesterol acetic acid salt, Spermine-5-carboxy-β-alanine cholesteryl ester tetratrifluoroacetic acid salt, 2,6- Diaminohexanoeyl β-alanine cholesteryl ester bistrifluoroacetic acid salt, 2,4- Diaminobutyroyl β-alanine cholesteryl ester bistrifluoroacetic acid salt, N,N-Bis (3- aminopropyl)-3-aminopropionyl β-alanine cholesteryl ester tristrifluoroacetic acid salt., [N,N- Bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxy cholesteryl 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-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, and 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]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-yl 4-(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.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- -octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z- ,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), and (2S)- 2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-- octadeca-9,12-dien- 1-yloxy]prop an-1-amine (Octyl-CLinDMA (2S)).

[0260] Preferably, the lipid of Formula (I), or the combination of the lipid of Formula (I) with one or more cationic / ionizable lipids, is present at about 5-80 mol% of the lipid nanoparticle formulation (excluding payload), e.g., about 5-80 mol% of the lipid component of the lipid nanoparticle formualtion. For example, some lipid nanoparticle formulations include less than 50 mol% Formula (I), or combination of Formula (I) and one or more additional cationic / ionizable lipids. Other lipid nanoparticle formulations include more than 50 mol% Formula (I), or combination of Formula (I) and one or more additional cationic / ionizable lipids.

[0261] Accordingly, some lipid nanoparticle formulations include a lipid of Formula (I), or a combination of Formula (I) and one or more cationic / ionizable lipids at 15- 80 mol%, a sterol at 20-60 mol%, a stabilizing agent at 0.5-5 mol%, and a phospholipid at 1- 40 mol% of the lipid nanoparticle formulation. An exemplary lipid nanoparticle formulation can include about 20-60 mol % Formula (I) lipid or combination of Formula (I) lipid and one or more additional cationic / ionizable lipids, about 5-25 mol % phospholipid, about 25-55 mol% sterol or sterol derivative; and about 0.5-15 mol% stabilizing agent. Another exemplary lipid nanoparticle formulation includes a about 50 mol % lipid of Formula (I) or combination of lipid of Formula (I) and one or more cationic / ionizable lipids, about 1.5 mol% stabilizing agent, about 38.5 mol% sterol or sterol derivative, and about 10 mol% phospholipid. Another exemplary lipid nanoparticle formulation comprises about 55% lipid of Formula (I) or combination of lipid of Formula (I) and one or more cationic / ionizable lipids, about 2.5 mol % stabilizing agent, about 32.5 mol % sterol or sterol derivative, and about 10 mol % phospholipid. Polyamine Components:

[0262] Other formulations may also include one or more polyamine transfection agents, such as dense star dendrimers, PAMAM dendrimers, NH3core dendrimers, ethylenediamine core dendrimers, dendrimers of generation 5 or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethylenimine (PEI), and / or polyethylenimine conjugates. Transfection Enhancing Agents:

[0263] Still other formulations can include transfection enhancing agents such as a fusion agent (such as an endosomal release agent), a cell surface ligand and / or a nuclear localization agent such as a nuclear receptor ligand peptide, Examples of transfection enhancing agents include, but are not limited to, reovirus-related fusogenic peptides (see WO07 / 130073, which is hereby incorporated by reference in its entirety), insulin, a transferrin, epidermal growth factor, fibroblast growth factor, a cell targeting antibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexon protein, a vesicular stomatitis virus glycoprotein, a Semliki Forest Virus core protein, a influenza hemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpes simplex virus VP22 protein, a histone protein, a arginine rich cell permeability protein, a high mobility group protein, and invasin protein, and internalin protein, an endotoxin, a diphtheria toxin, a shigella toxin, a melittin, a magainin, a gramicidin, a cecrophin, a defensin, a protegrin, a tachyplesin, a thionin, a indolicidin, a bactenecin, a drosomycin, an apidaecin, a cathelicidin, a bactericidal- permeability-increasing protein, a nisin, a buforin, and fragments thereof. Other cell penetrating peptides useful in the compositions provided herein include those provided in Table 1 below: Table 1

[0264] The lipid nanoparticle compositions provided herein can also be combined with one or more exosomes, or biological materials (e.g., lipids, proteins, nucleic acids, or the like) derived or purified from exosomes.

[0265] Exemplary compositions can 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 stabilizing agents; a lipid of Formula (I), and one or more exosomes, and one or more neutral lipids, optionally one or more stabilizing agents, and optionally one or more cell penetrating peptides.

[0266] Other exemplary compositions include, for example; a lipid of Formula (I), and one or more biological materials derived or purified from exosomes; a lipid of Formula (I), and one or more biological materials derived or purified from exosomes, and one or more neutral lipids; a lipid of Formula (I), and one or more biological materials derived or purifiedfrom exosomes, one or more neutral lipids, and or more stabilizing agents; a 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 stabilizing agents, and optionally one or more cell penetrating peptides. Formulation and Use of Compounds of Formula I for Transfection:

[0267] The lipids described above may be formulated by various methods to be used in transfection. One of the simplest methods for formulation is reverse evaporation, as described in U.S. Pat. No. 9,259,475, which is hereby incorporated by reference in its entirety. Other methods for formulation that can be used are sonication and microfluidization. Advantageously, the lipids are formulated as lipid nanoparticles using microfluidic mixing as described, for example, in Roces et al., Pharmaceutics, 12:1095 (2020). Suitable microfluidic mixing devices are commercially available from, for example, Precision Nanosystems (Vancouver, BC). Typically, microfluidic mixing combines two fluid streams, one containing the nucleic acid(s) and one containing the lipid of Formula (I) and other components, such as the peptide, ligand and other lipid components as described below.

[0268] For lipid nanoparticle compositions including an RNA, solutions of the RNA at concentrations of 0.1 mg / ml in deionized water are diluted in 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution. Nanoparticle compositions can be processed by dialysis to remove ethanol and achieve buffer exchange. Formulations may 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 filters (Sarstedt, Numbrecht, Germany) into glass vials and sealed.

[0269] Methods of determining particle size in nanoparticles formulations are well-known in the art. For example, a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) can be used to determine the particle size, the polydispersity index (PDI) and the zeta potential of the nanoparticle compositions. UV-visible spectroscopy can be used to determine the concentration of payload such as nucleic acid (e.g., mRNA) in nanoparticle compositions. A quantity of the composition is diluted in a suitable solvent and the absorbance spectrum of the solution is recorded, for example, between 230 nm and 330 nm on a spectrophotometer. The concentration of therapeutic and / or prophylactic in the nanoparticle composition can be calculated based on the extinction coefficient of the therapeutic and / or prophylactic used in the composition and on the difference between the absorbance at a wavelength of, for example, 260 nm and the baseline value at a wavelength of, for example, 330 nm.

[0270] For nanoparticle compositions including an RNA, a QUANT-IT™ RIBOGREEN®RNA assay (Invitrogen Corporation, Carlsbad, CA) can be used to evaluate the encapsulation of an RNA by the nanoparticle composition using methods provided by the manufacturer. The fluorescence intensity generated after addition of the RIBOGREEN reagent can be measured using a fluorescence plate reader at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence values of the reagent blank are subtracted from that of each of the samples 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 disrupted sample (caused by the addition of Triton X-100).

[0271] The lipids of Formula (I) can be formulated with one or more co-lipids, most advantageously neutral co-lipids, although the skilled artisan will recognize that other lipids, including cationic lipids described above, may be used. Other neutral lipids such as, but not limited to, 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-acetamido-propane can be used instead of, or in combination with DOPE in the formulation.

[0272] The lipids of Formula (I) can be formulated with one or more ionizable / cationic lipids and / or one or more neutral lipids. Exemplary ionizable / cationic lipids useful in the compositions provided herein include, but are not limited to GeneInTM, LipofectAmine™2000, LipofectAmine™, Lipofectin®, DMRIE-C, CellFectin®(Invitrogen), Oligofectamine® (Invitrogen), LipofectAce® (Invitrogen), Fugene® (Roche, Basel, Switzerland), Fugene® HD (Roche), Transfectam® (Tranfectam, Promega, Madison, WI), Tfx-10® (Promega), Tfx-20® (Promega), Tfx-50® (Promega), Transfectin™(BioRad, Hercules, CA), SilentFect™(Bio-Rad), Effectene® (Qiagen, Valencia, CA), DC-chol (Avanti 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-oxy-propyl)-2- hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diapalmityl-oxypropyl)-2-hydroxy- propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diaoleoyloxypropyl)-2-(3-amino-2-hydroxy- propyloxy)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-diapalmityloxy-propyl)- 2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, L-spermine-5-carboxyl-3-(DL-1,2- dipalmitoyl-dimethylaminopropyl-β-hydroxyethyl-amine, 3,5-(N,N-di-lysyl)-diamino- benzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylami nopropyl-β-hydroxyethylamine), L- Lysine-bis(O,O’-oleoyl-β-hydroxyethyl)amide dihydro-chloride, L-Lysine-bis-(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]-piperazine, L-Ornithine-bis-(O,O’-palmitoyl- β-hydroxyethyl)-amide dihydro-chloride, 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-hydroxy-propyl]piperazine, L- Arginine-bis-(O,O’-palmitoyl-β-hydroxy-ethyl)-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-hydroxy-ethyl)amide dihydrochloride, Sarcosine-bis-(O,O’-palmitoyl-β-hydroxy-ethyl)amide dihydro-chloride, L- Histidine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydro-chloride, cholesteryl-3β- carboxyl-amidoethylene-trimethylammonium iodide, 1,4-bis[(3-myristyl-amino)propyl]- piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3β-carboxy-amidoethyleneamine, cholesteryl-3β-oxysuccinamido- ethylenetrimethyl-ammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl- cholesteryl-3β-oxysucc inate iodide, 2-[(2-trimethylammonio)-ethylmethylamino] ethyl- cholesteryl-3β-oxysuccinate iodide, 3β[N-(N’,N’-dimethyl-aminoethane)- carbamoyl]cholesterol, and 3β-[N-(polyethylene-imine)-carbamoyl] cholesterol,1,4-bis[(3- palmitylamino)propyl]piperazine, L-Ornithylglycyl-N-(1-heptadecyl-octadecyl)glycinamide, N2,N5-Bis(3-aminopropyl)-L-ornithylglycyl-N-(1-hepta-decyl-octadecyl)glycinamide, 1,4- bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxy-propyl]piperazine N2-[N2,N5- Bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L-glutamine,N2-[N2,N5-Bis(aminopropyl)- L-ornithyl]-N-N-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)- oleylamino)2-hydroxypropyl]piperazine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N- dioctadecyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioctadecyl-L-glutaminyl]-L- glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-diolyl-L-glutamine, N2- [N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dioleyl-L-α-glutamine,4-bis[(3-(3-amino-2- hydoxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N2-[N2,N5-Bis(amino-propyl)- L-ornithyl]-N-N-dioleyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)-carbonyl]- N2,N5-bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioleyl-L- glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N2- [N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-dipalmityl-L-glutamine,N2-[N2,N5-Bis(amino- propyl)-L-ornithyl]-N-N-dipalmityl-L-α-glutamine, N2-[N2,N5-Bis(aminopropyl)-L- ornithyl]-N-N-dipalmityl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]- N2,N5-bis[3-[(1,1-dimethyl-ethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dipalmityl-L- glutaminyl]-L-glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-dimyristyl-L- glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dimyristyl-L-α-glutamine, N2- [N2,N5-Bis(amino-propyl)-L-ornithyl]-N-N-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino- 2-hydroxypropyl)-palmityl-amino)-2-hydroxypropyl]piperazine, N-[N2-[N2,N5-Bis[(1,1- dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L- ornithyl-N-N-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)- myristylamino)propyl]piperazine, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-dilaureyl- L-glutamine, N2-[N2,N5-Bis(amino-propyl)-L-ornithyl]-N-N-dilaureyl-L-α-glutamine, N2- [N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dilaureyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1- dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L- ornithyl-N-N-dilaureyl-L-glutaminyl]-L-glutamic acid, 3-[N’,N”-bis(2-tertbutyloxycarbonyl- amino-ethyl)guanidino]-N,N-dioctadec-9-enyl-propionamide, 3-[N’,N”-bis(2-tertbutyloxy- carbonylaminoethyl)guanidino]-N,N-dipalmityl-propionamide, 3-[N’,N”-bis(2-tertbutyloxy- carbonylaminoethyl)guanidino]-N,N-dimyristyl-propionamide, 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-diolyl- aminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxy-propyl-3-N,N-dipalmityl- aminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristyl- aminopropane, 1,4-bis[(3-(3-amino-2-hydoxypropyl)-myristylamino)-propyl]-piperazine, [(3-aminopropyl)-bis-(2-tetradecyloxyethyl)]methyl ammonium bromide, [(3-aminopropyl)- bis-(2-oleyloxyethyl)]methyl ammonium bromide, [(3-aminopropyl)-bis-(2- palmityloxyethyl)]methyl ammonium bromide, Oleoyl-2-hydroxy-3-N,N-dimethyamino propane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethy-aminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2-hydroxy-3-N,N- dimethyamino propane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-Amino-propyl)- ›4-(3-aminopropylamino)-4-tetradecylcarbamoyl-butylcarbamic acid cholestryl ester, (3- Amino-propyl)-›4-(3-amino-propyl-amino-4-carbamoylbutylcarbamic acid cholestryl ester, (3-Amino-propyl)-›4-(3-amino-propyl-amino)-4-(2-dimethylamino-ethylcarbamoy l)- butylcarbamic acid cholesteryl ester, Spermine-5-carboxyglycine (N’-stearyl-N’-oleyl) amide tetratrifluoroacetic acid salt, Spermine-5-carboxy-glycine (N’-stearyl-N’-elaidyl) amide tetratrifluoroacetic acid salt, Agmatinyl carboxycholesterol acetic acid salt, Spermine-5- carboxy-β-alanine cholesteryl ester tetratrifluoroacetic acid salt, 2,6-Diaminohexanoeyl β- alanine cholesteryl ester bistrifluoroacetic acid salt, 2,4-Diamino-butyroyl β-alanine cholesteryl ester bistrifluoroacetic acid salt, N,N-Bis (3-aminopropyl)-3-aminopropionyl β- alanine cholesteryl ester tristrifluoroacetic acid salt, [N,N-Bis(2-hydroxy-ethyl)-2- aminoethyl]aminocarboxy cholesteryl 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- amino-propyl)-N,N’-bis-(dodecyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N’- bis-(oleyloxyethyl)-piperazinium bromide, N-(3-aminopropyl)-N,N’-bis-(palmityloxy- ethyl)-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-amino-propyl)-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-hydroxy-propyl]piperazine, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(2- hydroxy-3-aminopropyl)-diaminobutane, 2,3-dipalmit-oleoyloxy-1,4-N,N’-dimethyl-N,N’- di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dimyrist-oleoyloxy-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-aminopropyl)-diaminobutane, 2,3-dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’- di(3-aminopropyl)-diaminobutane, 2,3-dimyrist-oleoyloxy-1,4-N,N’-dimethyl-N,N’-di(3- aminopropyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(5- carboxamidospermine)-diaminobutane, 2,3-dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(5-carboxamidospermine)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(5- caqrboxamidospermine)-diaminobutane, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(lysyl)- diaminobutane, 2,3-dipalmitoleoyloxy-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-dipalmitoleoyloxy-1,4-N,N’-dimethyl- N,N’-di(histidyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’- di(histidyl)-diamino-butane, 2,3-dioleyloxy-N,N’-dimethyl-1,4 -diaminobutane, 2,3- dipalmitoleoyloxy-N,N’-dimethyl-1,4-diaminobutane, 2,3-dimyristoleoyloxy-N,N’-dimethyl- 1,4-diaminobutane; PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, polyethylene-imine, and polyethylenimine conjugates.

[0273] Still other formulations can include transfection enhancing agents such as a fusion agent, a cell surface ligand and / or a nuclear localization agent such as a nuclear receptor ligand peptide, Examples of transfection enhancing agents useful in the embodiments provided herein include, but are not limited to, reovirus-related fusogenic peptides, insulin, a transferrin, epidermal growth factor, fibroblast growth factor, a cell targeting antibody, a lactoferrin, a fibronectin, an adenovirus penton base, Knob, a hexon protein, a vesicular stomatitis virus glycoprotein, a Semliki Forest Virus core protein, a influenza hemagglutinin, a hepatitis B core protein, an HIV Tat protein, a herpes simplex virus VP22 protein, a histone protein, a arginine rich cell permeability protein, a high mobility group protein, and invasin protein, and internalin protein, an endotoxin, a diptheria toxin, a shigella toxin, a melittin, a magainin, a gramicidin, a cecrophin, a defensin, a protegrin, a tachyplesin, a thionin, a indolicidin, a bactenecin, a drosomycin, an apidaecin, a cathelicidin, a bacteriacidal- permability-increasing protein, a nisin, a buforin, and fragments thereof.

[0274] The compositions provided herein can advantageously include, e.g., one or more peptide sequences that enhance transfection efficiency, such as linkers, spacers, or nuclear targeting sequences. The peptides provided herein can be included in the compositions herein independently (i.e., not covalently attached to another molecule) or alternatively, can be covalently linked to one or more molecules of the compositions provided herein (e.g., covalently linked to an ionizable or other lipid as described herein, covalently linked to another transfection enhancer as described herein, or covalently linked to a payload as described herein, or the like). In some embodiments, the covalent linkage can be via a spacer. The term "spacer," or "linker," which are used interchangeably herein, as used herein refers to a chemical structure that links two molecules to each other. In some embodiments, the spacer binds each molecule on a different part of the spacer molecule. In other embodiments, the spacer is a hydrophilic moietyand comprises about 6 to 30 carbon atoms. In other embodiments, the spacer comprises 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 formulae i ranges from 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20. In some embodiments, the spacer is a peptide of sequence APYKAWK (SEQ ID NO:505). In other embodiments, the spacer is a sequence that is degraded in vivo by a peptidase. Methods:

[0275] Use of these compositions provided herein in introducing payloads into cells can be carried out by methods that are known in the art where the components of a transfection complex are mixed in differing orders prior to addition to a cell culture. Typically, a liposomal preparation of the lipid, with or without co-lipid is prepared, and is then mixed with the payload(e.g., a nucleic acid such as DNA or RNA) to form a transfection complex. The complex is then added to a cell culture and transfection is monitored using well known methods. Additional components such as cell surface ligands, fusion agents, nuclear localization agents and the like may be added to the nucleic acid prior to admixture with the liposome, or may be added to the liposome prior to addition of nucleic acid.

[0276] Cells which can be transfected according to these methods include, but are not limited to, virtually any eukaryotic cell including primary cells, cells in culture, a passaged cell culture or a cell line, and cells in cultured tissue. Suitable cells include human cell lines and animal cell lines. The cell may be a fibroblast. The cells can be attached cells or cells in suspension (suspension cells). In certain illustrative aspects, the cells are suspension CHO-S cells and suspension 293-F cells. Other cells that may be used include, without limitation, 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.

[0277] The formulations are used in a method for producing a protein which includes contacting a cell with a lipid-nucleic acid complex as described above, where the nucleic acid encodes the protein. The cells are incubated to produce the protein and the protein is collected. Cells which can be used for protein production are described above. In addition, any composition which includes a lipid of Formula I can be used for transfection of cells. Such compositions are further discussed herein, and include, but are not limited to compositions comprising lipids of Formula I, a co-lipid and an optional transfection enhancing agent such as a fusogenic peptide or protein.

[0278] The lipids formulated into Lipid nanoparticle (LNP) formulations, were screened, and assessed by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the payload-encoded protein. The lipids formulated in this manner were used in transfection. The transfection of was carried out using the formulations described below, which contain Compound 1, which is a lipid of Formula (I). These results are shown in Figures 1-5. Reagent Kits:

[0279] Components of the transfection compositions described above can be provided in a reagent kit. The kits contain the lipid of Formula (I), together with additional components, such as a neutral lipid, a cationic lipid, cell surface ligands, fusion agents, amphipathic peptide and / or nuclear localization agents and the like. The kit components may be separate or may be premixed in any manner. For example, the lipid of Formula I may be admixed with one or more neutral lipid. Additional components may also be present in the same container or may be present in one or more separate containers. The kits typically include vessels, such as vials and / or tubes, which are packaged together, for example in a cardboard box. The kits can be shipped from a supplier to a customer. For example, in one example provided herein is a kit that includes a vial that includes a liposomal formulation as described above and, optionally, a transfection agent and a transfection enhancing peptide. The kit can also include, for example, a separate vessel that includes a transfection enhancing agent, such as a transfection enhancing peptide, for example Plus ReagentTM(Invitrogen Corp., Carlsbad, CA). The kit can also include in separate containers, cells, cell culture medium, and a reporter nucleic acid sequence, such as a plasmid that expresses a reporter gene. In certain examples, the culture medium can be reduced-serum medium and / or protein expression medium.

[0280] In one embodiment, a kit comprises individual portions of, or a mixture of, ionizable lipid, such as a lipid of Formula I, and peptide, protein, or fragment thereof or modified peptide, protein or fragment thereof. In another embodiment, a kit comprises individual portions of, or a mixture of, polycationic polymers and peptide, protein or fragments thereof or modified peptide, protein or fragments thereof. Cationic lipid transfection kits can optionally include neutral lipid as well as other transfection-enhancing agents or other additives, and the relative amounts of components in the kit may be adjusted to facilitate preparation of transfection compositions. Kit components can include appropriate medium or solvents for other kit components.

[0281] Payloads that can be delivered by the methods of this invention include nucleic acids, proteins, ribonucloeproteins, and the like, including DNA and RNA (including RNAi / siRNA) of any size from any source comprising natural bases or non- natural bases, and include those encoding and capable of expressing therapeutic or otherwise useful proteins in cells, those which inhibit undesired expression of nucleic acids in cells, those which inhibit undesired enzymatic activity or activate desired enzymes, those which catalyze reactions (ribozymes), and those which function in diagnostic assays (e.g., diagnostic nucleic acids). Therapeutic nucleic acids include those nucleic acids that encode or can express therapeutically useful proteins, peptides or polypeptides in cells, those which inhibit undesired expression of nucleic acids in cells, and those which inhibit undesired enzymatic activity or activate desired enzymes in cells. In certain embodiments, the payload comprises an RNA molecule. The compositions can be used to deliver RNA payloads such as mRNA, siRNA, shRNA, miRNA, self-replicating RNA (srRNA), self-amplifying RNA, stRNA, sgRNA, or combinations thereof. In some embodiments, the RNA molecule comprises more than one RNA molecule, e.g., more than one mRNA. The compositions and methods provided herein can also be readily adapted in view of the disclosure herein to introduce biologically active macromolecules other than nucleic acids including, among others, polyamines, polyamine acids, polypeptides and proteins into eukaryotic cells. Other materials useful, for example as therapeutic agents, diagnostic materials, research reagents, which can be bound to the peptides and modified peptides and introduced into eukaryotic cells by the methods of this invention.

[0282] The compositions provided herein can be delivered to cells via in vivo administration. For in vivo administration, the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally). In particular embodiments, the pharmaceutical compositions are administered intravenously, intrathecally, or intraperitoneally by a bolus injection. Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal administration.

[0283] Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets in vitro and in vivo. Alternatively, applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides. In this manner, therapy is provided for genetic diseases by supplying deficient or absent gene products. Methods of the present invention maybe practiced in vitro, ex vivo, or in vivo. For example, the compositions of the present invention can also be used for delivery of payloads to cells in vivo, using methods which are known to those of skill in the art. In another example, the compositions of the invention can be used for delivery of a payload to a sample of patient cells that are ex vivo, then are returned to the patient.

[0284] For in vivo administration, the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally). In particular embodiments, the compositions provided herein are administered intravenously, intrathecally, or intraperitoneally by a bolus injection. Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal.

[0285] For ex vivo applications, the compositions provided herein are preferably administered to biological samples that have been removed from the organism, then the cells are washed and restored to the organism. The organism may be a mammal, and in particular may be a mammal (e.g., a primate), such as a human. This process is used for cell reprogramming, genetic restoration, immunotherapy, for example.

[0286] In one embodiment, the present invention provides a method of modulating the expression of a target polynucleotide or polypeptide. These methods generally include contacting a cell with a composition of the present invention that is associated with a payload (e.g., a nucleic acid) that is capable of modulating the expression of a target polynucleotide or polypeptide. As used herein, the term "modulating" refers to altering the expression of a target polynucleotide or polypeptide. Modulating can mean increasing or enhancing, or it can mean decreasing or reducing.

[0287] In related embodiments, provided herein are methods of treating a disease or disorder characterized by overexpression of a polypeptide in a subject, including providing to the subject a composition provided herein, wherein the composition includes a payload that is a therapeutic agent selected from an siRNA, a microRNA, an antisense oligonucleotide, and a plasmid capable of expressing an siRNA, a microRNA, or an antisense oligonucleotide, and wherein the siRNA, microRNA, or antisense RNA includes a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a complement thereof.

[0288] In related embodiments, provided herein are methods of treating a disease or disorder characterized by under-expression of a polypeptide in a subject. Thesemethods can include providing to the subject a composition as provided herein, wherein the composition includes a payload that is a therapeutic agent selected from an mRNA, a self- amplifying RNA (SAM), a self-replicating DNA, or a plasmid, comprises a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof.

[0289] In one embodiment, the compounds, compositions, and methods and uses of the described herein are for delivering a biologically active agent to liver cells (e.g. hepatocytes). In one embodiment, the compounds, compositions, and methods and uses of the invention are for delivering a biologically active agent to a tumor or to tumor cells (e.g. a primary tumor or metastatic cancer cells). In another embodiment, the compounds, compositions, and methods and uses are for delivering a biologically active agent to the skin adipose, muscle and lymph nodes (subcutaneous dosing).

[0290] For delivery of a biologically active agent to the liver or liver cells, in one embodiment a composition of the invention is contacted with the liver or liver cells of the via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, portal vein injection, catheterization, stenting), to facilitate delivery. For delivery of a biologically active agent to the kidney or kidney cells, in one embodiment a composition of the invention is contacted with the kidney or kidney cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, catheterization, stenting), to facilitate delivery. For delivery of a biologically active agent to a tumor or tumor cells, in one embodiment, a composition of the invention is contacted with the tumor or tumor cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, catheterization, stenting), to facilitate delivery.

[0291] For delivery of a payload to the CNS or CNS cells), in one embodiment compositions described herein can be contacted with the CNS or CNS cells (e.g. brain cells and / or spinal cord cells) of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, catheterization, stenting, osmotic pump administration (e.g. intrathecal or ventricular)), to facilitate delivery. For delivery of a payload to the Peripheral Nervous System (PNS) or PNS cells, compositions described herein can be contacted with the PNS or PNS cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection), to facilitate delivery. For delivery of a payloadto a lung or lung cells, compositions provided herein can be contacted with the lung or lung cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. pulmonary administration directly to lung tissues and cells), to facilitate delivery. In some embodiments, the compositions include lipid molecules functionalized with neurotransmitter-based functional groups allowing for the delivery of the payload to the brain via the blood-brain barrier (BBB).

[0292] For delivery of a payload to the vasculature or vascular cells, compositions provided herein can be contacted with the vasculature or vascular cells of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. clamping, catheterization, stenting), to facilitate delivery.

[0293] For delivery of a payload to the skin or skin cells (e.g. dermis cells and / or follicular cells), compositions described herein can be contacted with the skin or skin cells (e.g. dermis cells and / or follicular cells) of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct dermal application, iontophoresis), to facilitate delivery. For delivery of a payload to an eye or ocular cells (e.g. macula, fovea, cornea, retina), in one embodiment a composition of the invention is contacted with the eye or ocular cells (e.g. macula, fovea, cornea, retina) of the patient via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection, intraocular injection, periocular injection, subretinal, iontophoresis, use of eyedrops, implants), to facilitate delivery. For delivery of a payload to an ear or cells of the ear (e.g. cells of the inner ear, middle ear and / or outer ear), compositions provided herein can be contacted with the ear or cells of the ear (e.g. cells of the inner ear, middle ear and / or outer ear) of the patient as is generally known in the art, such as via parenteral administration (e.g. intravenous, intramuscular, subcutaneous administration) or local administration (e.g. direct injection), to facilitate delivery. For delivery of a payload (e.g. RNA encoding an immunogen) to cells of the immune system (e.g. antigen-presenting cells, including professional antigen presenting cells), compositions provided herein can be delivered intramuscularly, after which immune cells can infiltrate the delivery site and process delivered RNA and / or process encoded antigen produced by non- immune cells, such as muscle cells. Such immune cells can include macrophages (e.g. bone marrow derived macrophages), dendritic cells (e.g. bone marrow derived plasmacytoid dendritic cells and / or bone marrow derived myeloid dendritic cells), monocytes (e.g. human peripheral blood monocytes), etc. (for example, see WO2012 / 006372 by Geall, Andy et al.).

[0294] Immunization. For immunization purposes, compositions provided herein can be prepared as an injectable, a pulmonary or nasal aerosol, or in a delivery device (e.g. syringe, nebulizer, sprayer, inhaler, dermal patch, etc.). This delivery device can be used to administer a pharmaceutical composition to a subject, e.g. to a human, for immunization.

[0295] For immunization purposes, in some embodiments, the embodiments provided herein include delivering one or more RNAs that encode(s) an immunogen. The immunogen can elicit an immune response which recognizes the immunogen, to provide immunity against a pathogen, or against an allergen, or against a tumor antigen. Immunizing against disease and / or infection caused by a pathogen is preferred.

[0296] The embodiments described herein, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

[0297] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. EXAMPLES

[0298] Unless otherwise defined, scientific and technical terms used in connection with the disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those 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). Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.Preparation of the Compounds of Formula I:

[0299] The compounds disclosed herein may be synthesized by methods described below, or by modification of these methods. Ways of modifying the methodology include, among others, temperature, solvent, reagents etc., known to those skilled in the art. In general, during any of the processes for preparation of the compounds disclosed herein, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry (ed. J.F.W. McOmie, Plenum Press, 1973); and T.W. Green, P.G.M. Wuts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999), which are both hereby incorporated herein by reference in their entirety. The protecting groups may be removed at a convenient subsequent stage using methods known from the art. Synthetic chemistry transformations useful in synthesizing applicable compounds are known in the art and include e.g. 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, which are both hereby incorporated herein by reference in their entirety. The routes shown and described herein are illustrative only and are not intended, nor are they to be construed, to limit the scope of the claims in any manner whatsoever. Those skilled in the art will be able to recognize modifications of the disclosed syntheses and to devise alternate routes based on the disclosures herein; all such modifications and alternate routes are within the scope of the claims.

[0300] In the following schemes, protecting groups for oxygen atoms are selected for their compatibility with the requisite synthetic steps as well as compatibility of the introduction and deprotection steps with the overall synthetic schemes (T.W. Green, P.G.M. Wuts, Protecting Groups in Organic Synthesis (3rd ed.) Wiley, New York (1999)).

[0301] If the compounds of the present technology contain one or more chiral centers, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or d(l) stereoisomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of the present technology, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.

[0302] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. Forexample, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California , USA), Emka-Chemce or Sigma (St. Louis, Missouri, USA). Others may be prepared by procedures, or obvious modifications thereof, 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). EXAMPLES General procedures

[0303] It will be apparent to the skilled artisan that methods for preparing precursors and functionality related to the compounds claimed herein are generally described in the literature. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art, but are not mentioned in greater detail. The skilled artisan given the literature and this disclosure is well equipped to prepare any of the compounds.

[0304] It is recognized that the skilled artisan in the art of organic chemistry can readily carry out manipulations without further direction, that is, it is well within the scope and practice of the skilled artisan to carry out these manipulations. These include reduction of carbonyl compounds to their corresponding alcohols, oxidations, acylations, aromatic substitutions, both electrophilic and nucleophilic, etherifications, esterification and saponification and the like. These manipulations are discussed in standard texts such as March Advanced Organic Chemistry (Wiley), Carey and Sundberg, Advanced Organic Chemistry (incorporated herein by reference in their entirety) and the like. All the intermediate compounds of the present invention were used without further purification unless otherwise specified.

[0305] The skilled artisan will readily appreciate that certain reactions are best carried out when other functionality is masked or protected in the molecule, thus avoiding any undesirable side reactions and / or increasing the yield of the reaction. Often the skilled artisan utilizes protecting groups to accomplish such increased yields or to avoid the undesired reactions. These reactions are found in the literature and are also well within the scope of the skilled artisan. Examples of many of these manipulations can be found for example in T. W.Greene and P.G.M. Wuts Protecting Groups in Organic Synthesis, 4th Ed., John Wiley & Sons (2007), incorporated herein by reference in its entirety.

[0306] The following example schemes are provided for the guidance of the reader, and represent preferred methods for making the compounds exemplified herein. These methods are not limiting, and it will be apparent that other routes may be employed to prepare these compounds. Such methods specifically include solid phase based chemistries, including combinatorial chemistry. The skilled artisan is thoroughly equipped to prepare these compounds by those methods given the literature and this disclosure. The compound numberings used in the synthetic schemes depicted below are meant for those specific schemes only, and should not be construed as or confused with same numberings in other sections of the application.

[0307] Trademarks used herein are examples only and reflect illustrative materials used at the time of the invention. The skilled artisan will recognize that variations in lot, manufacturing processes, and the like, are expected. Hence the examples, and the trademarks used in them are non-limiting, and they are not intended to be limiting, but are merely an illustration of how a skilled artisan may choose to perform one or more of the embodiments of the invention.

[0308] The following abbreviations have the indicated meanings: (BOC)2O = di-tert-butyl dicarbonate DCM = dichloromethane DIEA = N,N-Diisopropylethylamine DIPEA = N,N-Diisopropylethylamine DMF = N,N-dimethylformamide DMP = Dess Martin Periodinane DNs = dinitrosulfonyl ESBL = extended-spectrum β-lactamase EtOAc = ethyl acetate EA = ethyl acetate FCC = Flash Column Chromatography HATU = 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate MeCN = acetonitrileNMR = nuclear magnetic resonance PE = Petroleum Ether Prep = preparatory Py = pyridine RT or rt = room temperature Sat. = saturated aqueous TBDMSCl = tert-butyldimethylsilyl chloride TBS = tert-butyldimethylsilyl TFA = trifluoroacetic acid TEA = triethylamine THF = tetrahydrofuran TLC = thin layer chromatography

[0309] The following example schemes are provided for the guidance of the reader, and collectively represent an example method for making the compounds provided herein. Furthermore, other methods for preparing compounds described herein will be readily apparent to the person of ordinary skill in the art in light of the following reaction schemes and examples. Unless otherwise indicated, all variables are as defined above. Example 1 Synthesis of (butane-1,4-diylbis(tetradecylazanediyl))bis(3-aminopropane-1,2-diyl) (9E,9'E)-bis(octadec-9-enoate) (Compound 1 in Scheme I)

[0310] To a solution of DHDMS (1.7 g, 2.7 mmol), triethylamine (0.79 g / 1.1 mL, 7.8 mmol, 2.9 eq.) in ethyl acetate (90 mL), stirred at room temperature, under nitrogen atmosphere, di-tertio-butyloxycarbonate (DiBoc) (1.7 g, 7.8 mmol, 2.9 eq.) in ethyl acetate (5 mL) was added to the solution and stirred at room temperature under nitrogen for 16 h. The resulting solution was diluted with ethyl acetate (100 mL), washed with brine (2 x 100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to produce a yellow oil. The oil was then purified by silica-gel flash column chromatography with MeOH / DCM as solvents to produce di-tert-butyl ((butane-1,4-di-ylbis-(tetradecylazane-di-yl)) bis(2- hydroxypropane-3,1-di-yl)) dicarbamate (1A) (1.1g, 48% yield).

[0311] To a solution of di-tert-butyl ((butane-1,4- diylbis(tetradecylazanediyl))-bis(2-hydroxypropane-3,1-diyl))-dicarbamate (1A) (0.89 g, 1.1 mmol) in DMF (50 mL), was added diisopropylethylamine (DIPEA) (1.67 g / 2.25 mL, 12.9mmol, 12.0 eq.) and stirred at room temperature under nitrogen atmosphere for 10 min. To the resulting solution, oleoyl chloride (2.8 g, 9.3 mmol, 8.7 eq.) was added and allowed to stir at room temperature for 10 minutes after which the solution was heated and stirred for 16 h at 60 °C. The reaction mixture was then concentrated under reduced pressure to produce a yellow oil. The resulting oil was then diluted with DCM (100 mL) and washed with water (3 x 50 mL). The combined aqueous layers were then back extracted with DCM (2 x 50 mL), and the combined organic layers were dried over Na2SO4, filtered, and concentrated to produce a yellow oil. The crude oil was then purified using silica-gel flash column chromatography with (10-100%) DCM / Hexanes and (0-100% MeOH) to produce the BOC-protected diester intermediate (1B) (1.1g, 73% yield) (m / z MH+ = 1356.21).

[0312] A solution of Boc-protected diester (1B) (1.1g, 0.785 mmol.) in DCM (10 mL) was cooled to 0 °C using an ice-bath and stirred under nitrogen atmosphere. TFA (1 mL) was added, and the resulting solution was stirred for 5 h, while letting the solution to warm up to room temperature. Upon completion the solution was concentrated under reduced pressure to produce a maroon oil. The oil was dissolved in DCM (1 mL) and MeOH (4 mL) and the solution was loaded onto a C-18 reverse phase column (Innoval ODS-2, 1.0 x 25cm) and purified by using (85-100%) Water / MeOH gradient which resulted in the tetra- trifluoroacetate salt of compound 1 (71.6 mg) (m / z MH+1156.11).

[0313] Compounds 2-4 were synthesized using same procedures as above. Intermediate 1A was treated with linoleoyl chloride followed by TFA treatment to yield the trifluoroacetate salt of compound 2 (1.0 g) (M + H+= 1100.05614 m / Z free base)).

[0314] Intermediate 1A was treated with myristoyl chloride followed by TFA treatment to yield the trifluoroacetate salt of compound 3 (323 mg) (M + H+= 1048.02888 m / z (free base)).

[0315] Intermediate 1A was treated with palmitoleoyl chloride followed by TFA treatment to yield the trifluoroacetate salt of compound 4 (264 mg) (M + H+= 1152.08786 m / z free base)). Example 2 Synthesis of N1,N4-bis(2-(((E)-octadec-9-enoyl)oxy)butyl)-N1,N4-ditetradecylbutane- 1,4-diaminium hydrochloride salt (Compound 6 in Scheme VIIIA)

[0316] A heavy walled sealable round bottom vessel was charged with N1,N4- ditetradecylbutane-1,4-diamine (2.1 g, 4.3 mmol) in 2,2,2-trifluoroethanol (50 ml). DIPEA (3.62 mL, 20.8 mmol, 4.8 equiv.) and 1,2-butylene oxide (0.9 mL, 10.4 mmol, 2.4 equiv.) wereadded and the vessel was sealed, heated to 80 °C and stirred 20 h. The solution was concentrated under reduced pressure to produce a maroon oil. The oil was dissolved in DCM (5 ml) and purified on a normal phase silica-gel column with (0-10%) MeOH / DCM gradient to produce 1,1'-(butane-1,4-diylbis(tetradecylazanediyl))bis(butan-2-ol, Compound 7) (1.34 g, 50% yield) as a white solid. M + H+= 625.62639.

[0317] To a solution of 1,1'-(butane-1,4- diylbis(tetradecylazanediyl))bis(butan-2-ol) (0.5 g, 0.8mmol) in DMF (24 mL), was added DIPEA (1.8 mL, 9.9 mmol, 12.0 equiv.) and stirred at rt under nitrogen for 30 min. To the resulting solution, oleoyl Chloride (1.9 g, 6.5 mmol, 7.8 equiv.) was added to the solution and allowed to stir at rt for 10 minutes. The resulting solution was then heated to 60ۥ°C, and stirred for 16 h. The reaction solution was then concentrated down under reduced pressure to produce a yellow oil. The resulting oil was then diluted with Water (100 mL) and extracted with EtOAc (6 x 50 mL). The combined organic layers were then washed with brine, dried over Na2SO4, filtered, and concentrated to produce a yellow oil. The resulting oil was then purified on a normal phase column with (0-100%) EtOAc / DCM to produce (butane-1,4- diylbis(tetradecylazanediyl))bis(butane-1,2-diyl) bis(octadeca-2,4,6,8,10,12,14,16- octaynoate, Compound 6A) (210 mg, 22% yield). M + H+=1154.087.

[0318] To a round bottom flask charged with (butane-1,4- diylbis(tetradecylazanediyl))bis(butane-1,2-diyl) bis(octadeca-2,4,6,8,10,12,14,16- octaynoate) (188mg, 163 µmole), in DCM (3 mL), 4.0 M HCl in dioxane (100 uL / 14.6 mg, 400 µmoles, 2.45 equiv.). was added and stirred at rt under Nitrogen gas for 1 h. The resulting solution was concentrated under reduced pressure to produce N1,N4-bis(2-(((E)-octadec-9- enoyl)oxy)butyl)-N1,N4-ditetradecylbutane-1,4-diaminium (Compound 6) as a white oil (186.4 mg, 94% yield). M + H+= 1154.15064.

[0319] Compound 5 was synthesized using same procedures as above. Intermediate 1,1'-(butane-1,4-diylbis(tetradecylazanediyl))bis(butan-2-ol was treated with linoleoyl chloride followed by HCl treatment to yield the hydrochloride salt of N1,N4-bis(2- (((9Z,12Z)-octadeca-9,12-dienoyl)oxy)butyl)-N1,N4-ditetradecylbutane-1,4-diaminium, Compound 5. (139.5 mg, 53.6% yield). M + H+= 1150.11951. Example 3 Synthesis of N1,N1'-(bune-1,4-diyl)bis(N2-(2-hydroxyethyl)-N1-tetradecylpropane- 1,2,3-triaminium) trifluoroacetate salt (Compound 8 in Scheme IIA)

[0320] To a 250 mL round bottom flask charged with di-tert-butyl ((butane- 1,4-diylbis(tetradecylazanediyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate 1A (1.2 g, 1.5 mmol), DCM (20 mL) was added and the solution was stirred at rt until the compound was dissolved and then at 0oC, added TEA (1.4 mL, 10 mmol, 6.8 equiv) and stirred the resulting solution for 40 minutes at 0oC, and added methanesulfonyl chloride (787 mg, 6.9 mmol, 4.7 equiv.) in DCM (30 mL). Stirred for 16 h and allowed the reaction to warm to rt. Upon completion, diluted the solution with DCM (200 ml) and washed with water (3 x 100ml). The combined aqueous layers were back extracted with DCM (4 x 100ml). The combined organic layers were dried over Na2SO4, filtered and concentrated the solution under reduced pressure to produce 2,2,21,21-tetramethyl-4,19-dioxo-9,14-ditetradecyl-3,20-dioxa-5,9,14,18- tetraazadocosane-7,16-diyl dimethanesulfonate 8A as an oil which was moved on as is without further purification. 1.27 g produced, 87% yield, LC-MS (ESI) m / z 1027.82 [M + 2Na + H+].

[0321] To a 100 mL round bottom flask charged with 2,2,21,21-tetramethyl- 4,19-dioxo-9,14-ditetradecyl-3,20-dioxa-5,9,14,18-tetraazadocosane-7,16-diyl dimethanesulfonate 8A (620 mg, 0.63 mmol) in ethanol (13 mL) was added 2-aminoethan-1-ol (0.4 g, 6.5 mmol, 10.3 equiv.) in ethanol (1 mL). Potassium carbonate (0.4 g, 2.9 mmol, 4.6 equiv.) was added to the solution. The resulting solution was heated to 65 G°C and stirred overnight and was concentrated to dryness under reduced pressure upon completion of the reaction to produce a white oil. The resulting oil was diluted in EtOAc (20 mL) and washed with water (3 x 10 mL). The combined aqueous layers were back extracted with EtOAc (4 x 10 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to produce di-tert-butyl ((butane-1,4-diylbis(tetradecylazanediyl))bis(2-((2- hydroxyethyl)amino)propane-3,1-diyl))dicarbamate 8B as an oil, which was used for the next step without purification. 556 mg, 96% yield, LC-MS (ESI) m / z 913.79 [M + H+].

[0322] To a round bottom flask charged with di-tert-butyl ((butane-1,4- diylbis(tetradecylazanediyl))bis(2-((2-hydroxyethyl)amino)propane-3,1-diyl))dicarbamate 8B (556 mg, 0.6mmol.) in DCM (15 ml) was added trifluoroacetic acid (5 ml). The resulting solution was stirred at rt for 5 h. The reaction solution was concentrated under reduced pressure to produce a yellow oil. The oil was then dissolved in MeOH (2 mL) and water (4 mL). The solution was then loaded onto a reverse phase column and purified with (50-100%) MeOH / Water to produce N1,N1'-(butane-1,4-diyl)bis(N2-(2-hydroxyethyl)-N1- tetradecylpropane-1,2,3-triaminium) trifluoroacetate (Compound 8). 126 mg, 16% yield, LC- MS (ESI); m / z 713.70 [M + H+].

[0323] Compounds 9-12, 39-41, 43, 46, and 80 were synthesized using same procedures as above. Intermediate 8A was treated with 4-aminobutan-1-ol followed by TFA treatment to yield N1,N1'-(butane-1,4-diyl)bis(N2-(2-hydroxyethyl)-N1-tetradecylpropane-1,2,3- triaminium) trifluoroacetate, Compound 9. (138 mg, 16% yield, LC-MS (ESI) m / z 769.75 [M + H]+.

[0324] Intermediate 8A was treated with 3-aminopropane-1,2-diol followed by TFA treatment to yield N1,N1'-(butane-1,4-diyl)bis(N2-(2,3-dihydroxypropyl)-N1- tetradecylpropane-1,2,3-triaminium) trifluoroacetate, Compound 10. LC-MS (ESI) m / z 773.70 [M + H]+.

[0325] Intermediate 8A was treated with 4-(aminomethyl)phenol followed by TFA treatment to yield N1,N1'-(butane-1,4-diyl)bis(N2-(4-hydroxybenzyl)-N1- tetradecylpropane-1,2,3-triaminium) trifluoroacetate, Compound 11. LC-MS (ESI) m / z 837.71 [M + H]+.

[0326] Intermediate 8A was treated with 2-aminoethane-1-thiol followed by TFA treatment to yield N1,N1'-(butane-1,4-diyl)bis(N2-(2-mercaptoethyl)-N1-tetradecylpropane- 1,2,3-triaminium) trifluoroacetate, Compound 12. LC-MS (ESI) m / z 745.64 [M + H]+.

[0327] Intermediate 8A was treated with 3-aminopropan-1-ol followed by TFA treatment to yield 14-((3-ammonio-2-((3-hydroxypropyl)amino)propyl)(4-((3-ammonio-2-((3- hydroxypropyl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecan-1-ylium trifluoroacetate, Compound 39. LC-MS (ESI) m / z 741.72 [M + H]+.

[0328] Intermediate 8A was treated with 5-aminopentan-1-ol followed by TFA treatment to yield 14-((3-ammonio-2-((5-hydroxypentyl)amino)propyl)(4-((3-ammonio-2-((5- hydroxypentyl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecan-1- yliumtrifluoroacetate, Compound 40. LC-MS (ESI) m / z 797.78 [M + H]+.

[0329] Intermediate 8A was treated with 6-aminohexan-1-ol followed by TFA treatment to yield N1,N1'-(butane-1,4-diyl)bis(N2-(6-hydroxyhexyl)-N1-tetradecylpropane- 1,2,3-triaminium)trifluoroacetate, Compound 41. LC-MS (ESI) m / z 825.82 [M + H]+.

[0330] Intermediate 8A was treated with 2-amino-3-phenylpropan-1-ol followed by TFA treatment to yield 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)butyl)ammonio)tetradecan-1-ylium trifluoroacetate, Compound 43. LC-MS (ESI) m / z 893.77 [M + H]+.

[0331] Intermediate 8A was treated with 2-amino-2-methylpropan-1-ol followed by TFA treatment to yield 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)tetradecan-1-ylium trifluoroacetate, Compound 46. LC-MS (ESI) m / z 769.75.72 [M + H]+

[0332] Intermediate 8A was treated with 2-amino-2-methylpropane-1,3-diol followed by TFA treatment to yield 14-((3-ammonio-2-((1,3-dihydroxy-2-methylpropan-2- yl)amino)propyl)(4-((3-ammonio-2-((1,3-dihydroxy-2-methylpropan-2- yl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecan-1-ylium trifluoroacetate, Compound 48. LC-MS (ESI) m / z 801.73 [M + H]+

[0333] Intermediate 8A was treated with 1-aminopropan-2-ol followed by TFA treatment to yield 14-((3-ammonio-2-((2-hydroxypropyl)amino)propyl)(4-((3-ammonio-2-((2- hydroxypropyl)ammonio)propyl)(tetradecyl)ammonio)butyl)ammonio)tetradecan-1-ylium trifluoroacetate, Compound 80. LC-MS (ESI) m / z 741.72 [M + H]+. Example 4

[0334] Lipid Nanoparticle (LNP) formulations were screened and assessed by invivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the payload- encoded protein. Compositions including Compound 1-9 and helper lipids were made and complexed with mRNA. As shown in Table 2, the formulations examined varied in molar ratios of Compound 1 and helper lipids. Table 2: Exemplary LNP formulations

[0335] All the LNP formulations contained the lipid compound, DOPE, Cholesterol, and DMG-PEG. Some formulations included the peptide SEQ ID NO: 47. All the lipids were weighed and solubilized in ethanol at the desired molar ratio. This lipid mixand firefly luciferase (fLuc) mRNA were complexed into 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 LNPs were injected in mice the next day.

[0336] Female BALB / c mice aged 6-10 weeks old were purchased from the Jackson Laboratory and were acclimatized for 7 days before the study. Mice were injected with LNPs equivalent to 10 µg fLuc mRNA using intravenous tail vein injection in a total volume of 200 µl. At 4 h post-injection, mice were anesthetized with isofluorane anesthesia and imaged 10 min after intraperitoneal injection of 100 µL Rediject D-Luciferin (Perkin Elmer). Bioluminescence imaging was quantified invivo (whole body) and exvivo (organ) using an IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.

[0337] All the LNP formulations with Compound 1 had particle size >100nm and polydispersity index < 0.3 (Figure 1). Intravenous administration of the LNP formulations with Compound 1 resulted in mRNA delivery and luciferase expression in the liver (Figure 2) and in the spleen (Figure 3) of the injected mice. LNPs with Compound 1 show higher liver expression compared to spleen indicating that LNPs with Compound 1 are efficient mRNA delivery systems for liver delivery. Example 5

[0338] LNP formulations were screened and assessed by in vivo functional testing using the RNA payload of the complex. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of the payload-encoded protein. Compositions including Compound 1-9 and helper lipids were made and complexed with mRNA. As shown in Table 3, the formulations examined varied in molar ratios of Compounds 1-9 and helper lipids. Table 3: Exemplary LNP formulations

[0339] All the LNP formulations contained the lipid compound, either DOPE or DSPC or both, Cholesterol, and either DMG-PEG or C16-PEG. Some formulations included the peptide SEQ ID NO: 47. All the lipids were weighed and solubilized in ethanol at the desired molar ratio. This lipid mix and firefly luciferase (fLuc) mRNA were complexed into LNPs using a microfluidic device. The LNPs were dialyzed in phosphate buffer and particle size and homogeneity were measured using dynamic light scattering. The LNPs were injected in mice the next day.

[0340] Female BALB / c mice aged 6-10 weeks old were purchased from the Jackson Laboratory and were acclimatized for 7 days before the study. Mice were injected with LNPs equivalent to 10 µg fLuc mRNA using intravenous tail vein injection in a total volume of 200µl. At 4 h post-injection, mice were anesthetized with isofluorane anesthesia and imaged 10 min after intraperitoneal injection of 100 µL Rediject D-Luciferin (Perkin Elmer). Bioluminescence imaging was quantified in vivo (whole body) and ex vivo (organ) using an IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Imagesoftware.

[0341] All the LNP formulations with Compound 1-9 had particle size >400nm and polydispersity index < 0.3 (Figure 9, 10, 13, 18, 19). Intravenous administration of the LNP formulations with Compound 1-9 resulted in mRNA delivery and luciferase expression in the liver (Figure 4, 11, 14) and in the spleen (Figure 5, 12, 15, 20) and in the lung (Figure 21) of the injected mice. LNPs with Compound 1-4 show higher liver expression compared to spleen indicating that LNPs with Compound 1-4 are efficient mRNA delivery systems for liver delivery. LNPs with Compound 5-9 show higher spleen expression compared to liver indicating that LNPs with Compound 5-9 are efficient mRNA delivery systems for liver delivery. Example 6 Human Primary T Cell Transfection Protocol:

[0342] Compound 1 was dissolved in ethanol to obtain a final concentration at 25 mg / mL. Formulation was prepared by mixing Compound 1 with DOPE, 25 mg / mL, at molar ratio 1:1 and 1:2. LNP was prepared by adding 4 mL formulated lipids to 90 mL diluted 5-methoxyuridine (5moU) modified enhanced green fluorescent protein (eGFP) mRNA, 1 mg mRNA in 100 mM sodium acetate pH ~5.2 buffer, and vortexed three times. After 10 minutes incubation at room temperature, appropriate amount of mRNA was added to activated human primary T cells. Transfection efficiency was evaluated by flow cytometry two days post- transfection. EP: Electroporation as control / reference, 100 ng 5moU modified eGFP mRNA mixed with 2 x 105T cells in 10 µL, electroporation conditions, 1400V, 20ms and 1 pulse. Table 4. Exemplary lipid formulations:Table 5. Exemplary lipid complex formulations:

[0343] Lipid formulations LP24 and LP25 showed higher transfection efficiency compared to LNP11 and LNP12 which seemed to indicate that a high ratio of DOPE and Compound 1 played a role in primary human T cells transfection as shown in Figures 6 and 7. Furthermore, formulations LP24, LP25, and lipid nanoparticles LNP11 and LNP12 showed good cell viability compared to the electroporation sample as seen in Figure 8. Example 7 Compound 8 Formulation and Screening:

[0344] Compound 8 was dissolved in chloroform to make a stock of 25 mg / mL. Similarly, DOPE and cholesterol as co-lipids were dissolved in chloroform at 25 mg / mL. Cationic lipid, Compound 8 with DOPE or Cholesterol were mixed at different molar ratios (2:1, 1:1, 1:2, 1:4, and 1:8) in a glass vial. Chloroform from these vials was removed and the lipids dried using a rotary evaporator. The vials were left overnight in a vacuum desiccator to completely remove the chloroform. The following day, desired amount of 95% ethanol was added to each of the vials to get a final lipid concentration of 2 mg / mL. Liposome preparation:

[0345] For formulations in water, reverse phase evaporation method was used. Lipids in chloroform were added to a vial along with the desired volume of water needed for a final lipid concentration of 2 mg / mL followed by removal of chloroform using a rotary evaporator. Lipids in water or 95% ethanol by themselves or in combination with peptides like 15-24 (0.5 mg / mL), 09-04 (0.025 mg / mL), or 15-24 + 09-04 (0.5 + 0.025 mg / mL) weretested on VPC 1.0 cells (suspension HEK 293 cells) for their transfection efficiency using an antibody fragment-GFP plasmid (Dulaglutide-T2A GFP). Transfection of suspension cells

[0346] VPC 1.0 cells (Viral Production Cells derived from HEK293F cell lines) were maintained in ExpiFectamine 293 (Expi293 expression media by splitting every 3-4 days to maintain a cell density less than 6 x 106cells / mL. The cells are split to a density of 0.3 x 106to 0.5 x 106cells / mL for regular maintenance. A day before transfection, the cells are split at 3 x 106cells / mL and the day of transfection, cells are re-diluted to 3 x 106cells / mL and aliquoted into a 96-deep well plate. About 800 µL of cells were aliquoted into 2-mL 96- deep well plates (inner 60 wells only) and incubated on a shaker set at 900 rpm until ready for transfection. Transfection complexes were made in 100 µL of Opti-MEM containing DNA (0.8 µg / mL) aliquoted in a 96-well plate. Transfection reagent was added to the DNA containing wells and mixed by pipetting up and down followed by incubation for 20 min for the lipoplex / lipid nanoparticle to form. After 20 min, 100 µL of the DNA-transfection reagent complex was added to the cells and transfection efficiency and toxicity determined at 24 and 48h post-transfection. Transfection efficiency and toxicity

[0347] Plate reader-based assays were used at 24 and 48h post transfection to determine the efficacy in comparison with existing catalog products like the ExpiFectamine 293 (Expi293) reagent (2.5 µL). GFP fluorescence of the cells was also measured using a plate reader. See Figure 16. 10 µL cells were diluted in media (40 µL) and GFP fluorescence measured (485 / 515), followed by addition of PrestoBlue (PB) HS reagent (50 µL of 5X diluted PB in media). The contents of the plate were mixed using an orbital shaker for 2 min followed by 1 h incubation at 37°C before measuring fluorescence (560 / 590). Higher PrestoBlue fluorescence indicates greater viability. See Figure 17. OTHER EMBODIMENTS:

[0348] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0349] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All references, e.g., U.S. patents, U.S. patent application publications, PCT patent applications designating the U.S., publishedforeign patents and patent applications cited herein are incorporated herein by reference in their entireties. GenBank and NCBI submissions indicated by accession number 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 case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0350] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

[0351] Although the disclosure has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the disclosure. Accordingly, the disclosure is limited only by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A compound having the general structure I, or pharmaceutically acceptable salts thereof:wherein: A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A4 is selected from the group consisting of —(CH2)x—, —(CO)O—, —O(CO)—, —R1and R2are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, optionally substituted C4- C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group;R3is —COR, —(CH2)nCOR or —(CH2)nCOOR or —(CO)NHR— or —(CO)N(R)2—C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl, or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl or C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl, or C3-C6 cycloalkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet, or C3-C6 cycloalkyl groups wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; or C3-C6cycloalkenyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, or C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S-NR7-, -NR7-S-NR7- or aryl groups, or C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S- NR7-, or -NR7-S-NR7- groups; or R3 is H only when R1 and R2 are independently of each other —(CH2)1-7COOR; R4is H or optionally substituted C1-C20straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R5 and R6 are independently selected from H and CH3;R7is H or optionally substituted C1-C6straight-chain or branched-chain alkyl, optionally substituted monounsaturated or polyunsaturated C1-C6straight-chain or branched- chain alkenyl or; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, —(CH2)0-3Het; A2 and A3 are independently selected from the group consisting of H,wherein R9 is H orwherein R10is selected from the group consisting of H;C1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; C3-C6cycloalkyl amines wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; and C3-C6cycloalkenyl amines, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet; wherein R11 is selected from the group consisting of -NH2, -NHR, -N(R)2,wherein AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 0 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, n4, n5 are independently an integer from 1 to 5 n6is an integer from 0 to 7 n7and n8are independently an integer from 0 to 5; n9is an integer from 1 to 5; m1is an integer from 1 to 5; m2 is an integer from 0 to 5; m3is an integer from 1 to 7; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

2. The compound of claim 1, wherein: A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A4is selected from the group consisting of —(CH2)x—, —(CO)O—, —O(CO)—,Q1 is N; Q2 is N; R1and R2are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, optionally substituted C4- C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group; R3 is —COR, —(CH2)nCOR, or —(CH2)nCOOR or —(CO)NHR— or —R4is H or optionally substituted C1-C20straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30 straight-chain or branched-chain alkyl, optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het;A2and A3are independently selected from the group consisting of H,wherein R10is selected from the group consisting of H; C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30 straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6 cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; wherein AA represents any natural or non-natural amino acid side chain; a is an integer from 1 to 6; b is an integer from 0 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, n4, n5 are independently an integer from 1 to 5; n6is an integer from 0 to 7; n7 and n8 are independently an integer from 0 to 5; n9is an integer from 1 to 5; m1 is an integer from 1 to 5; m2is an integer from 0 to 5;m3is an integer from 0 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

3. The compound of claim 1 having the structure of formula I-a:or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; A2 and A3 are independently selected from the group consisting ofC1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl, or C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -S-NR7-S-, -NR7-S-S-NR7-, -NR7-S-NR7- or aryl groups, or C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, - (CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet, optionally substituted cycloalkyl or C3-C6cycloalkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet, or C3-C6 cycloalkyl groups wherein the ring carbons are replaced with -O-, -S-, -S-S-, - NR7-; or C3-C6 cycloalkenyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, -(CH2)nHet; R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; and wherein R10 is selected from the group consisting of H; C1-C30straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; C1-C30 straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; C3-C6cycloalkyl alkyl groups, optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; wherein AA represents any natural or non-natural amino acid side chain; x is an integer from 1 to 9; n is an integer from 1 to 5; n5 is an integer from 1 to 5; n6is an integer from 0 to 7;n7and n8are independently an integer from 0 to 5; n9is an integer from 1 to 5; m3 is an integer from 1 to 3; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

4. The compound of claim 3, wherein independently of each other, n5is 1 or n7 is 1 or n9 is 1.

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

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

7. The compound of claim 3, wherein x is 4.

8. The compound of claim 3, wherein R3 is9. The compound of claim 3, wherein R3is optionally substituted C1-C30straight-chain or branched-chain alkyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group.

10. The compound of Claim 1 having the structure of formula I-b:or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3is —COR, —(CH2)nCOR, or —(CH2)nCOOR or —(CO)NHR— orR4is H or optionally substituted C1-C20straight-chain or branched-chain alkyl, optionally substituted C1-C20 monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; and wherein AA represents any natural or non-natural amino acid side chain; y is an integer from 1 to 4;z is an integer from 1 to 4; n is an integer from 1 to 5; n5 is an integer from 1 to 5; m2is an integer from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

11. The compound of claim 10, wherein independently of each other, n5 is 1 or m2is 2 or n9is 1.

12. The compound of claim 10, wherein y and z are independently an integer from 1 to 2.

13. The compound of Claim 1 having the structure of formula I-c:or a pharmaceutically acceptable salt thereof, wherein: R1and R2are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3 is —COR, —(CH2)nCOR, or —(CH2)nCOOR or —(CO)NHR— or —A4is selected from the group consisting of —(CO)O—, —O(CO)—, —S-S—,R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-C20monounsaturated or polyunsaturated straight-chain or branched- chain alkenyl; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; and wherein AA represents any natural or non-natural amino acid side chain; y is an integer from 1 to 4; z is an integer from 1 to 4; n is an integer from 1 to 5; n1, n2, n3, and n4are independently an integer from 1 to 5; m1 is an integer from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

14. The compound of claim 13, wherein n1, n2, n3, and n4are independently an integer from 1 to 2.

15. The compound of claim 13, wherein y and z are independently an integer from 1 to 2.

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

17. The compound of Claim 1 having the structure of formula I-d:or a pharmaceutically acceptable salt thereof, wherein: A2and A3are independently selected from the group consisting of H,R1 and R2 are independently of one another selected from the group consisting of H, optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3is —COR, —(CH2)nCOR, or —(CH2)nCOOR or —(CO)NHR— or —R4 is H or optionally substituted C1-C20 straight-chain or branched-chain alkyl, optionally substituted C1-bC20monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl; R5and R6are independently selected from H and CH3; R is selected from the group consisting of optionally substituted C4-C30 straight-chain or branched-chain alkyl, optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; A4is selected from the group consisting of —(CO)O—, —O(CO)—, —S-S—,wherein AA represents any natural or non-natural amino acid side chain; a is an integer from 1 to 2; b is an integer from 0 to 3; y and z are independently an integer from 1 to 4;z is an integer from 1 to 4; m1and m2are independently an integer from 1 to 5; n1, n2, n3, n4, and n5 are independently an integer from 1 to 5; p is an integer from 1 to 50; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

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

19. The compound of claim 17, wherein y and z are independently an integer from 1 to 2.

20. The compound of claim 17, wherein when A2is ;R7 is H; b is 0; and a pharmaceutically acceptable salt thereof.

21. The compound of claim 17, wherein A2and A3are independently selected from the group consisting ofR7is H; a and b are 1; and a pharmaceutically acceptable salt thereof.

22. The compound of claim 1 having the structure of formula I-a:or a pharmaceutically acceptable salt thereof, wherein: A1 is —(CH2)x—, or —(CH2)y—A4—(CH2)z—; A4 is selected from the group consisting of —(CH2)x—, —(CO)O—, —O(CO)—, and —S-S—;R1and R2are independently of one another selected from the group consisting of H, optionally substituted C1-C30straight-chain or branched-chain alkyl, optionally substituted C4- C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, or optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched- chain alkynyl group; R is selected from the group consisting of optionally substituted C4-C30straight-chain or branched-chain alkyl, optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl, -(CH2)0-3Het; and a is an integer from 1 to 6; b is an integer from 1 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n5 is an integer from 1 to 5; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

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

24. The compound of claim 22, wherein n5is 1 or 2 or 3.

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

26. The compound of claim 22, wherein y and z are independently an integer from 1 to 3.

27. The compound of claim 1 having the structure of formula I-k:or a pharmaceutically acceptable salt thereof, wherein:A1is —(CH2)x—, or —(CH2)y—S—S—(CH2)z—; R1and R2are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; wherein R10is selected from the group consisting of H; or C1-C30 straight-chain or branched-chain alkyl groups optionally substituted with -OR7, -N(R7)2, -SR7, -(CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl, - (CH2)nHet, optionally substituted cycloalkyl; or C1-C30straight-chain or branched-chain alkyl groups wherein the chain carbons are replaced with -O-, -S-, -S-S-, -NR7-, aryl groups; or C3-C6 cycloalkyl alkyl groups , optionally substituted with -OR7, -N(R7)2, -SR7, - (CH2)nOR7, -(CH2)nSR7, -(CH2)nN(R7)2, -(CH2)nAryl, -(CH2)nArylalkyl -(CH2)nHet; and a is an integer from 1 to 6; b is an integer from 1 to 6; x is an integer from 1 to 9; y is an integer from 1 to 4; z is an integer from 1 to 4; n5is an integer from 1 to 5; and Het is an optionally substituted 5-7 membered monocyclic basic heterocycle, or an optionally substituted 8-11 membered bicyclic basic heterocyclyl.

28. The compound of claim 27, wherein A1is —(CH2)4— or —(CH2)2—S—S—(CH2)2—.

29. The compound of claim 27, wherein n5 is 1.

30. The compound of claim 27, wherein a and b are independently an integer from 1 to 3.

31. The compound of claim 27, wherein R10is selected from the group consisting of: H,32. The compound of claim 1 having the structure of formula I-n:or a pharmaceutically acceptable salt thereof, wherein: A1 is —(CH2)y—S—S—(CH2)z—; R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; wherein AA represents any natural or non-natural amino acid side chain; and a is an integer from 1 to 6; b is an integer from 1 to 6; y is an integer from 1 to 4; z is an integer from 1 to 4; and n5 is an integer from 1 to 5.

33. The compound of claim 32, wherein n5 is 1.

34. The compound of claim 32, wherein a and b are independently an integer from 1 to 3.

35. The compound of claim 32, wherein AA is selected from the group consisting of:

36. The compound of claim 1 having the structure of formula I-y:or a pharmaceutically acceptable salt thereof, wherein: R1 and R2 are independently of one another selected from the group consisting of optionally substituted C1-C30straight-chain or branched-chain alkyl, and optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; y is an integer from 1 to 4; and z is an integer from 1 to 4; 37. The compound of claim 36, wherein y and z are independently an integer from 1 to 3.

38. The compound of claim 1 having the structure of formula I-z:or a pharmaceutically acceptable salt thereof, wherein: R1and R2are independently of one another selected from the group consisting of optionally substituted C1-C30 straight-chain or branched-chain alkyl, and optionally substituted C4-C30monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group; R3is —COR —(CH2)nCOR or —(CH2)nCOOR or —(CO)NHR— or —(CO)N(R)2—C30 straight-chain or branched-chain alkyl, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkenyl group, or optionally substituted C4-C30 monounsaturated or polyunsaturated straight-chain or branched-chain alkynyl group; y is an integer from 1 to 4; and z is an integer from 1 to 4; 39. The compound of claim 38, wherein y and z are independently an integer from 1 to 3.

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

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

42. The compound of any one of claims 1-35, wherein A1is —(CH2)4— or —(CH2)—A4—(CH2)—.

43. The compound of any one of claims 1-26, and 38-39, wherein R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, palmitoleyl, palmitoleoyl, palmityl, palmitoyl, myristyl, myristoyl, lauryl and lauroyl groups.

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

45. The compound of any one of claims 1-39, wherein the optionally substituted 5-7 membered monocyclic basic heterocycle is selected from the group consisting of46. A compound having the structure selected from the group consisting ofand pharmaceutically acceptable salts thereof.

47. A compound having the structure selected from the group consisting ofand pharmaceutically acceptable salts thereof.

48. A compound having the structure selected from the group consisting ofand pharmaceutically acceptable salts thereof.

49. A compound having the structure selected from the group consisting ofand pharmaceutically acceptable salts thereof.

50. A compound having the structure selected from the group consisting ofand pharmaceutically acceptable salts thereof.

51. A compound having the structure selected from the group consisting ofand pharmaceutically acceptable salts thereof.

52. A compound having the structure selected from the group consisting ofwherein: R10is: H,and pharmaceutically acceptable salts thereof.

53. A compound having the structure selected from the group consisting ofwherein:, and pharmaceutically acceptable salts thereof.

54. A compound having the structure selected from the group consisting ofwherein: n6is 0 or 1; R10 is: H,pharmaceutically acceptable salts thereof.

55. A compound having the structure selected from the group consisting ofwherein: R is56. A composition comprising: (i) one or more compounds according to claims 1-55, and (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizing agent; and (iii) optionally, a payload.

57. The composition according to claim 56, comprising: (i) a compound according to any one of claims 1-55; (ii) one or more structural lipids, (iii) one or more stabilizing agents; and (iv) optionally, a payload.

58. The composition according to claim 56, comprising: (i) a compound according to any one of claims 1-55; (ii) one or more structural lipids, (iii) one or more stabilizing agents; (iv) one or more transfection enhancing agents; and(v) optionally, a payload.

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

60. The composition according to any one of claims 56 to 59, wherein the one or more compounds according to claims 1-55 is present at 10 to 80 mol% of the composition, excluding any payload, if present.

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

62. The composition according to any one of claims 56 to 59, wherein the stabilizing agent is present at 0.1-10 mol% of the composition, excluding any payload, if present.

63. The composition of any one of claims 56 to 59, further comprising an exosome or a biological material derived or purified from an exosome.

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

65. The composition of claim 64, wherein the polymer is selected from the group consisting of: a dense star dendrimer, a PAMAM dendrimer, an NH3core dendrimer, an ethylenediamine core dendrimer, a dendrimer of generation 5 or higher, a dendrimer with a substituted group, a dendrimer comprising one or more amino acids, a grafted dendrimer, an activated dendrimer, polyethylenimine, polyethylenimine conjugates, polylysine, polyarginine, polyornithine, histone, and any combination thereof.

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

67. The composition of any one of claims 56 to 58 and 60 to 66, wherein the stabilizing agent is selected from the group consisting of: a surfactant, a neutral lipid, a polymer-conjugated lipid, polyethylene glycol, a phospholipid, and any combination thereof.

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

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

70. The composition according to claim 69, wherein the transfection enhancing agent selected from the group consisting of: an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell-penetrating peptide, a fusogenic peptide, and any combination thereof.

71. The composition of claim 58, wherein the one or more transfection enhancing agents comprises an amphipathic peptide.

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

73. The composition of claim 59 or 72, wherein the payload comprises a nucleic acid.

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

75. The composition of claim 73 or 74, wherein the nucleic acid is an RNA.

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

77. The composition of claim 73, wherein the nucleic acid is a DNA.

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

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

80. The composition of claim 76, wherein the RNA is an mRNA.

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

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

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

84. The composition according to any one of claims 56 to 58, wherein the structural lipid is selected from the group consisting of: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha- tocopherol, hopanoids, phytosterols, steroids, and any combination thereof.

85. The composition according to any one of claims 56 to 58, wherein the stabilizing agent comprises one or more phospholipids selected from the group consisting of: 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-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-dioleoyl-sn- glycero-3-phosphoethanola mine (DOPE), 1,2-diphytanoyl-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, 1,2-dioleoyl-sn-glycero-3-phospho- rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

86. The composition according to any one of claims 56 to 58, wherein the ionizable lipid comprises one or more cationic lipid selected from GeneInTM, LipofectAmineTM 2000, LipofectAmineTM, Lipofectin®, DMRIE-C, CellFectin® (Invitrogen), Oligofectamine® (Invitrogen), LipofectAce® (Invitrogen), Fugene® (Roche, Basel, Switzerland), Fugene® HD (Roche), Transfectam® (Tranfectam, Promega, Madison, WI), Tfx-10® (Promega), Tfx-20® (Promega), Tfx-50® (Promega), TransfectinTM (BioRad, Hercules, CA), SilentFectTM (Bio-Rad), Effectene® (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), EscortTM III (Sigma, St. Louis, MO), EscortTM 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- diapalmityloxy-propyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-diaoleoyl- oxypropyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3- diamyristyloxy-propyl)-2-(3-amino-2-hydroxypropyloxy)propane-1,3-diamine, N-1- dimethyl-N-1-(2,3-diapalm-ityloxypropyl)-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-di-lysyl)-diamino-benzoyl-glycyl-3-(DL-1,2-dipalmitoyl- dimethylaminopropyl-β-hydroxyethylamine), L-Lysine-bis(O,O’-oleoyl-β-hydroxy- ethyl)amidedihydrochloride, L-Lysine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amidedihydrochloride, 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-hydroxy-propyl]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-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)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-hydroxy-ethyl)amide dihydrochloride, Sarcosine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L- Histidine-bis-(O,O’-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesteryl-3β- carboxyl-amidoethylenetrimethylammonium iodide, 1,4-bis[(3-myristyl-amino)-propyl]- piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3β-carboxyamidoethyleneamine, cholesteryl-3β-oxysuccinamido- ethylene-trimethylammonium 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’-dimethylamino-ethane)- carbamoyl]cholesterol, and 3β-[N-(polyethyleneimine)-carbamoyl] cholesterol,1,4-bis[(3- palmitylamino)propyl]piperazine, L-Ornithylglycyl-N-(1-heptadecyloctadecyl)-glycinamide, N2,N5-Bis(3-aminopropyl)-L-ornithyl-glycyl-N-(1-heptadecyloctadecyl)-glycinamide, 1,4- bis[(3-(3-amino-2-hydroxypropyl)-alkyl-amino)-2-hydroxypropyl]-piperazine, N2-[N2,N5- Bis(3-aminopropyl)-L-ornithyl]-N,N-diocta-decyl-L-glutamine,N2-[N2,N5- Bis(aminopropyl)-L-ornithyl]-N-N-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2- hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N2-[N2,N5-Bis(amino-propyl)-L- ornithyl]-N-N-dioctadecyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethyl- ethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N- N-dioctadecyl-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]- N,N-diolyl-L-glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dioleyl-L-α- glutamine,4-bis[(3-(3-amino-2-hydoxypropyl)-myristylamino)-2-hydroxy-propyl]piperazine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dioleyl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1- dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleyl-amino)-propyl]piperazine, N2-[N2,N5-Bis(3- aminopropyl)-L-ornithyl]-N,N-dipalmityl-L-glutamine,N2-[N2,N5-Bis(amino-propyl)-L- ornithyl]-N-N-dipalmityl-L-α-glutamine, N2-[N2,N5-Bis(amino-propyl)-L-ornithyl]-N-N- dipalmityl-L-α-asparagine, N-[N2-[N2,N5-Bis[(1,1-dimethylethoxy)-carbonyl]-N2,N5- bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dipalmityl-L- glutaminyl]-L-glutamic acid, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N-dimyristyl-L- glutamine, N2-[N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dimyristyl-L-α-glutamine, N2- [N2,N5-Bis(aminopropyl)-L-ornithyl]-N-N-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino- 2-hydroxy-propyl)-palmitylamino)-2-hydroxypropyl]-piperazine, N-[N2-[N2,N5-Bis[(1,1- dimethyl-ethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L- ornithyl-N-N-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)- myristyl-amino)-propyl]piperazine, N2-[N2,N5-Bis(3-aminopropyl)-L-ornithyl]-N,N- dilaureyl-L-glutamine, N2-[N2,N5-Bis(amino-propyl)-L-ornithyl]-N-N-dilaureyl-L-α- glutamine, N2-[N2,N5–Bis-(amino-propyl)-L-ornithyl]-N-N-dilaureyl-L-α-asparagine, N- [N2-[N2,N5-Bis[(1,1-dimethylethoxy)-carbonyl]-N2,N5-bis[3-[(1,1- dimethylethoxy)carbonyl]aminopropyl]-L-ornithyl-N-N-dilaureyl-L-glutaminyl]-L-glutamic acid, 3-[N’,N”-bis(2-tertbutyloxycarbonyl-amino-ethyl)guanidino]-N,N-dioctadec-9- enylpropionamide, 3-[N’,N”-bis(2-tertbutyloxy-carbonylamino-ethyl)guanidino]-N,N- dipalmitylpropionamide, 3-[N’,N”-bis(2-tertbutyl-oxycarbonyl-aminoethyl)guanidino]-N,N- dimyristylpropionamide, 1,4-bis[(3-(3-amino-propyl)-palmityl-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- hydoxypropyl)-myristylamino)-propyl]piperazine, [(3-aminopropyl)-bis-(2-tetradecyl- oxyethyl)]methyl ammonium bromide, [(3-aminopropyl)-bis-(2-oleyloxyethyl)]methyl ammonium bromide, [(3-aminopropyl)-bis-(2-palmityloxyethyl)]methyl ammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethyamino propane, 2-didecanoyl-1-N,N- dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethyamino propane, 1,2- dipalmitoyl-1-N,N-dimethylamino-propane, myristoyl-2-hydroxy-3-N,N-dimethyamino propane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-amino-propyl)-›4-(3-amino- propylamino)-4-tetradecyl-carbamoyl-butylcarbamic acid cholestryl ester, (3-Amino-propyl)-›4-(3-amino-propylamino-4-carbamoylbutylcarbamic acid cholestryl ester, (3-Amino- propyl)-›4-(3-amino-propylamino)-4-(2-dimethylamino-ethylcarbamoy-l)-butylcarbamic acid cholesteryl ester, Spermine-5-carboxyglycine (N’-stearyl-N’-oleyl) amide tetratrifluoro- acetic acid salt, Spermine-5-carboxyglycine (N’-stearyl-N’-elaidyl) amide tetratri- fluoroacetic acid salt, Agmatinyl carboxycholesterol acetic acid salt, Spermine-5-carboxy-β- alanine cholesteryl ester tetratrifluoroacetic acid salt, 2,6-Diaminohexanoeyl β-alanine cholesteryl ester bistrifluoroacetic acid salt, 2,4-Diaminobutyroyl β-alanine cholesteryl ester bistrifluoroacetic acid salt, N,N-Bis (3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl ester tristrifluoroacetic acid salt, [N,N-Bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxy cholesteryl 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)-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-dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’- di(2-hydroxy-3-amino-propyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl- N,N’-di(2-hydroxy-3-aminopropyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N’-dimethyl- N,N’-di(3-amino-propyl)-diaminobutane, 2,3-dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’- di(3-amino-propyl)-diamino-butane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(3- amino-propyl)-diaminobutane, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(5-carboxamido- spermine)-diaminobutane, 2,3-dipalm-itoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(5- carboxamidospermine)-diaminobutane, 2,3-dimyrist-oleoyloxy-1,4-N,N’-dimethyl-N,N’- di(5-caqrboxamidospermine)-diaminobutane, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’- di(lysyl)-diaminobutane, 2,3-dipalmitoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(lysyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N’-dimethyl-N,N’-di(lysyl)-diamino-butane, 2,3-dioleyloxy-1,4-N,N’-dimethyl-N,N’-di(histidyl)-diaminobutane, 2,3-dipalmitoleoyl-oxy- 1,4-N,N’-dimethyl-N,N’-di(histidyl)-diaminobutane, 2,3-dimyristoleoyloxy-1,4-N,N’- dimethyl-N,N’-di(histidyl)-diaminobutane, 2,3-dioleyloxy-N,N’-dimethyl-1,4 - diaminobutane, 2,3-dipalmitoleoyloxy-N,N’-dimethyl-1,4-diaminobutane, 2,3-dimyrist- oleoyloxy-N,N’-dimethyl-1,4-diaminobutane; PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, polyethylenimine, and polyethylenimine conjugates.

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

88. The composition of claim 68, wherein the one or more PEG-modified lipids is 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 enhancing agent selected from the group consisting of an endosomal release agent, a cell surface ligand, a nuclear localization agent, a cell- penetrating peptide, a fusogenic peptide, and any combination thereof.

90. A method of delivering a payload to a cell, comprising: (i) providing a composition according to any one of claims 56 to 89; (ii) providing a cell; and (iii) contacting the cell with the composition.

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

92. The method of claim 90, wherein the contacting the cell is in vitro.

93. The method of claim 90, wherein the contacting the cell is ex vivo.

94. The method of claim 90, wherein the contacting the cell is in vivo.

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

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

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

98. The method of claim 91, wherein the administration is selected from the group consisting of: subcutaneous administration, intramuscular administration, intranasal administration, intra-tumoral administration, administration to the brain, administration to the spinal cord, administration to the eye, administration to the lymph node of a subject, and any combination thereof.

99. A kit comprising: (i) one or more compounds according to claims 1-55, and (ii) one or more of a structural lipid, an ionizable lipid, and a stabilizing agent.

100. The kit according to claim 99, comprising: (i) a compound according to any one of claims 1-55; (ii) one or more structural lipids, (iii) one or more stabilizing agents; and (iv) optionally, a payload. 101 The kit according to claim 99, comprising: (i) a compound according to any one of claims 1-55; (ii) one or more structural lipids, (iii) one or more stabilizing agents; (iv) one or more fusion agent; and (v) optionally, a payload.

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