Conical shaped diacyl glyceroethylphosphocholines as cationic lipids and uses thereof

Abstract

A compound having the structure (I) is provided. The compound is useful as a therapeutic agent in the form of a lipoplex, or in a form of a polyplex, in a form of a lipid nanoparticle, or in a form of a liposome, or in a form of a micelle, or in a form of an emulsion including a drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide.

Description

[0001] CONICAL SHAPED DIACYL GLYCEROETHYLPHOSPHOCHOLINES AS CATIONIC LIPIDS AND USES THEREOF

[0002] CROSS REFERENCE TO PRIOR APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 663,528 filed

[0004] June 24, 2024, the content of which is incorporated by reference herein in its entirety for all purposes.

[0005] FIELD OF THE INVENTION

[0006] The invention relates to cationic lipids. These lipids may be utilized as part of lipid nanoparticle compositions, cationic emulsions, liposomes, lipoplexes, micelles or polyplexes. These vehicles may be useful for delivering pharmaceuticals to humans or animals or for agricultural uses, for example.

[0007] BACKGROUND

[0008] Lipid compounds are used to prepare delivery systems for drugs, oligonucleotides, antibodies, enzymes, proteins, small molecules, or nucleotides. The delivery systems may encompass lipid nanoparticles, lipoplex, or in a form of a polyplex, in a form of a lipid nanoparticle, or in a form of a liposome, or in a form of a micelle. The delivery system together with the drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide thus form a therapeutic agent. It is desirable for such therapeutic agents to be non-toxic, while at the same time effectively delivering the drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide in order to achieve the desired therapeutic effect. In case of oligonucleotides and nucleotides, for example, the objective is to induce a cell to express a desired protein, without causing harm to the target cell or other cells in the organism.

[0009] Other lipids that may be used in such delivery systems are those referred to as ALC-0315 lipids (e.g. [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate)), which are described in WO 2018 / 08081480 Al. These lipids suffer from higher than desirable cell toxicity and lower encapsulation efficiency of the therapeutic agents, as well as a reduced protein expression from targeted cells, if used to deliver a nucleotide or oligonucleotide to the cells. Accordingly, there remains a need for alternative lipids that simultaneously provide low cell toxicity and high encapsulation efficiency of the drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide. SUMMARY

[0010] The present disclosure is directed to lipid compounds that are low toxicity and according to an embodiment may provide increased protein expression in the target cells when used as part of a composition to deliver nucleic acids to a cell. The lipid compounds may also provide reduced toxicity and improved encapsulation efficiency. The individual lipids were designed based on the inventors’ understanding that the spatial geometry related to the fatty acid and headgroups are related to encapsulation efficiency, transfection, and expression. In particular, the fixed ion of these lipids may provide improved properties compared to ionizable groups. When tested, these lipids showed high cell viability and good protein expression in vitro and in vivo, compared to marketed products in this field. These may be used in lipid nanoparticles (LNPs) and lipoplexes for nucleic acid delivery. Additionally, cationic lipids can be used for specific applications in liposomes and other lipid vesicle morphologies and may be used for delivery of pharmaceuticals to humans and animals and also for agricultural applications. According to some embodiments, the inclusion of the lipids disclosed herein may be used in a human or veterinary therapeutic agent, such as a vaccine, drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide. The inclusion of the lipids disclosed herein may also be used for agriculture applications, for delivery of sensitive compounds before release. For example, liposomes, lipid nanoparticles, micelles, lipoplexes polyplexes, or emulsions may be used to slow, delay, or meter delivery of seed enhancement or pest control agents, for example.

[0011] It has been found that in lipid nanoparticle (LNP) formulations, cationic ionizable lipids may complex with negatively charged DNA or RNA. Aside from playing a major role in the LNP formulation itself, cationic lipids also serve a major role in the biological administration delivery of DNA or RNA to target cells.

[0012] According to a particular embodiment, the lipid compounds may be used to form lipid nanoparticles that are the therapeutic agent that delivers a drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to another embodiment, the lipid compounds may be used to form liposomes that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to another embodiment, the lipid compounds may be used to form micelles that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to other embodiments, the lipid compounds may be used to form lipoplexes that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. According to other embodiments, the lipid compounds may be used to form polyplexes that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide to a person in need thereof. According to other embodiments, the lipid compounds may be used to form emulsions that deliver the drug, vaccine, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, to a person in need thereof. The inclusion of the lipids disclosed herein may also be used for agriculture applications, for delivery of sensitive compounds before release. For example, liposomes, lipid nanoparticles, micelles, lipoplexes polyplexes, or emulsions may be used to slow, delay, or meter delivery of seed enhancement or pest control agents, for example. According to an embodiment, the compounds have the structure (I) or stereoisomers thereof: where: Y is -N+(R)3X-, each R is independently selected from methyl, ethyl, or C3-C6 linear or branched alkyl, and X is a halide or triflate anion; a, b, r, p are independently selected integers from 1-8; n is an integer from 1-3; m and q are independently selected integers from 0-8; A and B are each independently selected from -O-C(=O)-, -C(=O)-O-, -NH-C(=O)-, and -C(=O)-NH-; L and M are each independently selected from -CH2-, -O-C(=O)-, -C(=O)-O-, -NH-C(=O)-, and -C(=O)-NH-; and F and G are independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the provisos: i) at least one of F and G comprises at least one branch, and ii) F and G are not both H. A therapeutic agent comprising a drug or oligonucleotide in a lipid nanoparticle, a liposome or a micelle including a compound having the structure (I) or stereoisomers thereof or combinations of two of more thereof is provided. A method of treatment, comprising administering the therapeutic agent to a patient in need thereof is also provided. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows mass spectroscopy results for Compound (I), Figure 2 shows mass spectroscopy results for Compound (III), Figure 3 shows LNP average particle size as detailed in Example 8b, Figure 4 shows LNP Zeta potential as detailed in Example 8b, Figure 5 shows LNP Ribogreen Assay encapsulation efficiency and RNA yield data as detailed in Example 8b, Figure 6 shows HepG2 cell transfection as detailed in Example 8c, Figure 7 shows C2C12 cell transfection as detailed in Example 8c, Figure 8 shows differentiation of THP-1 cells in dendritic cell transfection, Figure 9 shows data for differentiation of THP-1 Cells in macrophage cell transfection, Figure 10 shows date for IL1ß production in dendritic cells and monocytes, Figure 11 shows date for IL1ß production in macrophages, Figure 12 shows IL6 production in monocytes, dendritic cells and macrophages, and Figure 13 shows data for TNFα production in monocytes, dendritic cells and macrophages. DETAILED DESCRIPTION Definitions As used herein, the terms “prevention”, “prevent”, “preventing”, “suppression”, “suppress” and “suppressing” as used herein refer to a course of action (such as administering a compound or pharmaceutical composition) initiated prior to the onset of a symptom, aspect, or characteristics of a disease or condition so as to prevent or reduce such symptom, aspect, or characteristics. Such preventing and suppressing need not be absolute to be useful. As used herein, the terms “treatment”, “treat” and “treating” as used herein refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a symptom, aspect, or characteristics of a disease or condition so as to eliminate or reduce such symptom, aspect, or characteristics. Such treating need not be absolute to be useful. As used herein, the term “in need of treatment” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure. As used herein, the term “in need of prevention” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a disease or condition that is preventable by a method or compound of the disclosure. As used herein, the terms “individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female. As used herein, the term “therapeutically effective amount” as used herein refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease or condition. Such effect need not be absolute to be beneficial. As used herein, the term “alkyl”, whether used alone or as part of a substituent or linking group, includes straight hydrocarbon groups comprising from one to 100 carbon atoms. Thus the phrase includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following which are provided by way of example: —CH(CH3)2, —CH(CH3)(CH2CH3), —CH(CH2CH3)2, —C(CH3), —C(CH2CH3)3, —CH2CH(CH3)2, —CH2CH(CH3)(CH2CH3), —CH2CH(CH2CH3)2, — CHC(CH3)3, —CH2C(CH2CH3)3, —CH(CH3)CH(CH3)(CH2CH3), —CH2CH2CH(CH3)2, — CH2CH2CH(CH3)(CH2CH3)—, CH2CH2CH(CH2CH3)2, —CH2CH2C(CH3)3, — CH2CH2C(CH2CH3)3, —CH(CH3)CH2CH(CH3)2, —CH(CH3)CH(CH3)CH(CH3)CH(CH3)2, — CH(CH2CH3)CH(CH3)CH(CH3)(CH2CH3) and others. The phrase also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted with straight and branched chain alkyl groups as defined above. The phrase also includes polycyclic alkyl groups such as, but not limited to, adamantyl, norbornyl, and bicyclo[2.2.2]octyl and such rings substituted with straight and branched chain alkyl groups as defined above. As used herein, the term “alkylene”, whether used alone or as part of a substituent group, includes any group obtained by removing a hydrogen atom from an alkyl group; an alkylene group forms two bonds with other groups. As used herein, the term “alkenyl”, whether used alone or as part of a substituent group, includes an alkyl group having at least one double bond between any two adjacent carbon atoms. As used herein the term “alkynyl”, whether used alone or as part of a substituent group, includes an alkyl group having at least one triple bond between any two adjacent carbon atoms. As used herein, the terms “unsubstituted alkyl,” “unsubstituted alkenyl” and “unsubstituted alkenyl” refer to alkyl, alkenyl and alkynyl groups that do not contain heteroatoms. The phrases “substituted alkyl,” “substituted alkenyl,” and “substituted alkynyl” refer to alkyl, alkenyl, and alkynyl groups as defined above in which one or more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen or non-carbon atoms such as, but not limited to, a halogen atom in halides such as F, Cl, Br, and I; and oxygen atom in groups such as carbonyl, carboxyl, hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, enamines imines, oximes, hydrazones, and nitriles; a silicon atom in groups such as in trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. Other alkyl groups include those in which one or more bonds to a carbon or hydrogen atom is replaced by a bond to an oxygen atom such that the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxy group. Still other alkyl groups include alkyl groups that have an amine, alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine, heterocyclylamine, (alkyl)(heterocyclyl)-mine, (aryl)(heterocyclyl)amine, or diheterocyclylamine group. As used herein the term “alkynyl”, whether used alone or as part of a substituent group, includes an alkyl group having at least one triple bond between any two adjacent carbon atoms. Compounds According to an embodiment, a compound having a structure (I) or stereoisomers thereof or a combination of any two or more thereof is provided.

[0013] In this structure (I) ,Y is -N+(R)3X-, each R is independently selected from methyl, ethyl, or C3-C6 linear or branched alkyl, and X is a halide or triflate anion; a, b, r, p are independently selected integers from 1-8; n is an integer from 1-3; m and q are independently selected integers from 0-8; A and B are each independently selected from -O-C(=O)-, -C(=O)-O-, -NH-C(=O)-, and -C(=O)-NH-; L and M are each independently selected from -CH2-, -O-C(=O)-, -C(=O)-O-, - NH-C(=O)-, and -C(=O)-NH-; and F and G are independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the provisos: i) at least one of F and G comprises at least one branch, and ii) F and G are not both H. This structure covers any chirality of the asymmetric carbons or mixtures thereof. According to some embodiments, the at least one branch of at least one of F and G may be via an ester group. According to some embodiments, the at least one branch of at least one of F and G may be via an amide group. According to some embodiments, at least one of F or G may be substituted with at least one ester group. According to some embodiments, at least one of F or G may be substituted with at least one alkenyl group. According to some embodiments, at least one of F or G may be substituted with at least one amide group. According to some embodiments, at least one of F or G may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more alkenyl groups, with the provisos that F and G are not both H and at least one of F and G is branched. According to some embodiments, F and G may be independently selected from H and aliphatic alkyl C4-C75 groups optionally substituted with one or more alkenyl groups, with the provisos that F and G are not both H and at least one of F and G is branched. According to some embodiments, F and G may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more alkenyl groups, with the provisos that F and G are not both H and at least one of F and G is branched. According to some embodiments, F and G may be independently selected from H and aliphatic alkyl C4-C50 groups optionally substituted with one or more alkenyl groups, with the provisos that F and G are not both H and at least one of F and G is branched. According to some embodiments, F and G may be independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more alkenyl groups, with the provisos that F and G are not both H and at least one of F and G is branched. According to some embodiments, F and G may be independently selected from H and aliphatic alkyl C4-C24 groups optionally substituted with one or more alkenyl groups, with the provisos that F and G are not both H and at least one of F and G is branched.

[0014] According to some embodiments, a may be 1, 2, 3, 4, 5, 6, 7, or 8. According to some embodiments, a may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, a may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, a may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, a may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, a may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, a may be an integer selected from 6 to 8.

[0015] According to some embodiments, b may be 1, 2, 3, 4, 5, 6, 7, or 8. According to some embodiments, b may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, b may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, b may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, b may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, b may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, b may be an integer selected from 6 to 8.

[0016] According to some embodiments, r may be 1, 2, 3, 4, 5, 6, 7, or 8. According to some embodiments, r may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, r may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, r may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, r may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, r may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, r may be an integer selected from 6 to 8.

[0017] According to some embodiments, p may be 1, 2, 3, 4, 5, 6, 7, or 8. According to some embodiments, p may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, p may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, p may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, p may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, p may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, p may be an integer selected from 6 to 8.

[0018] According to some embodiments, n may be 1, 2, or 3.

[0019] According to some embodiments, m may be an integer selected from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. According to some embodiments, m may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, m may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, m may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, m may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, m may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, m may be an integer selected from 6 to 8. According to some embodiments, m may be 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0020] According to some embodiments, q may be an integer selected from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, or from 0 to 2. According to some embodiments, q may be an integer selected from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 3. According to some embodiments, q may be an integer selected from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4. According to some embodiments, q may be an integer selected from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5. According to some embodiments, q may be an integer selected from 4 to 8, from 4 to 7, from 4 to 6. According to some embodiments, q may be an integer selected from 5 to 8, from 5 to 7. According to some embodiments, q may be an integer selected from 6 to 8. According to some embodiments, q may be 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0021] According to some embodiments, A may be -O-C(=O)- or -C(=O)-O-. According to an embodiment, B may be at least of -O-C(=O)- or -C(=O)-O-. According to some embodiments, A may be of-NH-C(=O)- or C(=O)-NH-. According to some embodiments, B may be -NH-C(=O)-, or C(=O)-NH-.

[0022] According to some embodiments, L may be -O-C(=O)- or -C(=O)-O-. According to some embodiments, M may be -O-C(=O)- or -C(=O)-O-. According to some embodiments, L may be -NH-C(=O)- or C(=O)-NH-. According to some embodiments, M may be -NH-C(=O)-, and C(=O)-NH-. According to some embodiments, L may be -CH2-. According to some embodiments, M may be -CH2-.

[0023] According to an embodiment, the compound comprises at least one of the structures (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), or stereoisomers thereof or a combination of one or more thereof:

[0024] In any of the above structures the Cl- anion may be replaced with Br-, F-, or I- or a triflate ion (-OSO2CF3.) Therapeutic Agent The immune system of higher organisms has been characterized as distinguishing foreign agents (or “non-self”) agents from familiar or “self” components, such that foreign agents elicit immune responses while “self” components are ignored or tolerated. Immune responses have traditionally been characterized as either humoral responses, in which antibodies specific for antigens are produced by differentiated B lymphocytes known as plasma cells, or cell mediated responses, in which various types of T lymphocytes act to eliminate antigens by a number of mechanisms. For example, CD4+ helper T cells that are capable of recognizing specific antigens may respond by releasing soluble mediators such as cytokines to recruit additional cells of the immune system to participate in an immune response. Also, CD8+ cytotoxic T cells that are also capable of specific antigen recognition may respond by binding to and destroying or damaging an antigen-bearing cell or particle. It is known in the immunological arts to provide certain vaccines according to a variety of formulations, usually for the purpose of inducing a desired immune response in a host. Several strategies for eliciting specific immune responses through the administration of a vaccine to a host include immunization with heat-killed or with live, attenuated infectious pathogens such as viruses, bacteria or certain eukaryotic pathogens; immunization with a non- virulent infective agent capable of directing the expression of genetic material encoding the antigen(s) to which an immune response is desired; and immunization with subunit vaccines that contain isolated immunogens (such as proteins) from a particular pathogen in order to induce immunity against the pathogen. (See, e.g., Liu, 1998 Nature Medicine 4(5 suppl.):515.) For certain antigens there may be one or more types of desirable immunity for which none of these approaches has been particularly effective, including the development of vaccines that are effective in protecting a host immunologically against human immunodeficiency viruses or other infectious pathogens, cancer, autoimmune disease, or other clinical conditions. In other embodiments there is provided a vaccine composition comprising (a) an antigen; the compound having structures (I) – (IX) or stereoisomers thereof or a mixture of two or more thereof; and a toll-like receptor (TLR) agonist, wherein in certain further embodiments the TLR agonist may be selected from lipopolysaccharide, peptidoglycan, polyI:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF) and at least one hepatitis C antigen. In another embodiment there is provided a vaccine composition comprising: an antigen; the compound having structures (I) – (IX) or stereoisomers thereof or a combination of any two or more thereof; and at least one co-adjuvant that is selected from saponins and saponin mimetics. In another embodiment there is provided a vaccine composition comprising an antigen; the compound having structures (I) – (IX) or stereoisomers thereof or a combination of any two or more thereof; and a carrier that comprises at least one of an oil and ISCOMATRIX™. In another embodiment there is provided a vaccine composition comprising an antigen; the compound having structures (I) – (IX) or stereoisomers thereof or a combination of any two or more thereof; and one or more of: (i) at least one co-adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinoline immune response modifier, and (iv) at least one double stem loop immune modifier (dSLIM). In certain further embodiments (i) the co- adjuvant, when present, may be selected from alum, a plant alkaloid and a detergent, wherein the plant alkaloid may be selected from tomatine and the detergent may be selected from saponin, Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist, when present, may be selected from lipopolysaccharide, peptidoglycan, polyI:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF) and at least one hepatitis C antigen, and (iii) the imidazoquinoline immune response modifier, when present, may be selected from resiquimod (R848), imiquimod and gardiquimod. In another embodiment there is provided a vaccine composition comprising: an antigen; the compound having structures (I) – (IX) or stereoisomers thereof or a combination of any two or more thereof; and at least one of a co-adjuvant and a pharmaceutically acceptable carrier, wherein: the co-adjuvant is selected from a cytokine, a detergent, and a block copolymer or biodegradable polymer, and the pharmaceutically acceptable carrier comprises a carrier that is selected from calcium phosphate, an oil-in-water emulsion, a water-in-oil emulsion, a liposome, a novosome, a non-ionic surfactant vesicle (e.g., niosome) and a microparticle. In a particular embodiment, where a liposome or similar carrier is used, a compound having structures (I) – (IX) or stereoisomers thereof or a combination of any two or more thereof is in the laminar structure of the liposome or is encapsulated. In another particular embodiment, where a microparticle is used, the microparticle is one that is based on or comprises polymer fat lipids. In certain further embodiments the cytokine is selected from GM-CSF, IL-2, IL-7, IL-12, TNF-α and IFN-gamma, the block copolymer or biodegradable polymer may be selected from Pluronic L121, CRL1005, PLGA, PLA, PLG, and polyI:C, and the detergent may be selected from the group consisting of saponin, Polysorbate 80, Span 85 and Stearyl tyrosine. In other embodiments there is provided a vaccine composition comprising: at least one recombinant expression construct which comprises a promoter operably linked to a nucleic acid sequence encoding an antigen and the compound having structures (I) – (IX) or stereoisomers thereof or a combination of any two or more thereof. In one embodiment the recombinant expression construct may be present in a viral vector, which in certain further embodiments is present in a virus that is selected from an adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus, a poxvirus, and a retrovirus. According to certain of any of the above-described embodiments that include a TLR agonist, the TLR agonist may be capable of delivering a biological signal by interacting with at least one TLR that may be selected from TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9. In certain further embodiments the TLR agonist may be selected from lipopolysaccharide, peptidoglycan, polyI:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LeIF) and at least one hepatitis C antigen. In a particular embodiment, where a TLR-7 and / or TLR-8 agonist is used, the TLR-7 and / or TLR-8 agonist may be entrapped within a vesicle. According to certain of any of the above-described embodiments, the antigen may be derived from at least one infectious pathogen that may be selected from a bacterium, a virus, and a fungus. In certain further embodiments the bacterium may be an Actinobacterium, and in certain still further embodiments the Actinobacterium is a Mycobacterium. In certain other related embodiments, the Mycobacterium may be selected from M. tuberculosis and M. leprae. In certain other related embodiments, the bacterium may be selected from Salmonella, Neisseria, Borrelia, Chlamydia and Bordetella. In certain other related embodiments, the virus may be selected from a herpes simplex virus, a human immunodeficiency virus (HIV), a feline immunodeficiency virus (FIV), cytomegalovirus, Varicella Zoster Virus, hepatitis virus, Epstein Barr Virus (EBV), respiratory syncytial virus, human papilloma virus (HPV) and a cytomegalovirus. According to certain of any of the above-described embodiments, the antigen may be derived from a human immunodeficiency virus, which in certain further embodiments is selected from HIV-1 and HIV- 2. In certain other related embodiments, the fungus may be selected from Aspergillus, Blastomyces, Coccidioides and Pneumocystis. In certain other related embodiments, the fungus may be a yeast, which in certain further embodiments may be a Candida, wherein in certain still further embodiments the Candida may be selected from C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. tropicalis and C. parapsilosis.

[0025] According to certain of any of the above-described embodiments, the antigen may be derived from a parasite, which in certain further embodiments may be a protozoan, which in certain further embodiments may be a Plasmodium, which in certain still further embodiments may be selected from P. falciparum, P. vivax, P. malariae and P. ovale. In certain other embodiments the parasite may be selected from Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosoma mansonii, Schistosoma haematobium, Schistosoma japoni cum, Schistosoma mekongi, Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coli, Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereria bancrofti, Giardia, Leishmania, Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale, Brugia malayi, Onchocerca volvulus, Dracanculus medinensis, Trichinella spiralis, Strongyloides stercoralis, Opisthorchis sinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciola gigantica, Taenia saginata and Taenia solium.

[0026] According to certain of any of the above-described embodiments, the antigen may be derived from at least one cancer cell. In certain further embodiments the cancer cell may originate in a primary solid tumor, and in certain other embodiments the cancer cell may originate in a cancer that is a metastatic or secondary solid tumor, and in certain other embodiments the cancer cell originates in a cancer that may be a circulating tumor or an ascites tumor. In certain related embodiments the cancer cell may originate in a cancer that may be selected from cervical cancer, ovarian cancer, breast cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, pseudomyxoma peritonei, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma and Wilms' tumor. In certain other related embodiments, the cancer cell originates in a cancer that is selected from testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oliodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease.

[0027] According to certain of any of the above-described embodiments, the antigen may be derived from, or may be immunologically cross-reactive with, at least one epitope, biomolecule, cell or tissue that is associated with an autoimmune disease. In certain further embodiments the epitope, biomolecule, cell or tissue that is associated with an autoimmune disease may be selected from snRNP when the autoimmune disease is systemic lupus erythematosus, at least one of thyroglobulin, thyrotropin receptor and a thyroid epithelial cell when the autoimmune disease is Graves' disease, a platelet when the autoimmune disease is thrombocytopenic purpura, at least one of pemphigus antigen, desmoglein-3, desmoplakin, envoplakin and bullous pemphigoid antigen 1 when the autoimmune disease is pemphigus, myelin basic protein when the autoimmune disease is multiple sclerosis, a pancreatic islet beta cell when the autoimmune disease is type 1 diabetes, and an acetylcholine receptor when the autoimmune disease is myasthenia gravis.

[0028] In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response, comprising a compound having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier or excipient. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising an antigen and a compound having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier or excipient. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising an antigen; a compound having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof; a toll-like receptor (TLR) agonist; and a pharmaceutically acceptable carrier or excipient. In a further embodiment the TLR agonist is selected from lipopolysaccharide, peptidoglycan, polyLC, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LelF) and at least one hepatitis C antigen. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising: an antigen; a compound having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof; at least one adjuvant; and a pharmaceutically acceptable carrier or excipient. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising antigen; a compound having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier that comprises at least one of an oil and ISCOMATRIX™. In another embodiment there is provided a pharmaceutical composition for inducing or enhancing an immune response comprising: (a) an antigen; a compound having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof; (c) one or more of: (i) at least one adjuvant, (ii) at least one TLR agonist, (iii) at least one imidazoquinoline immune response modifier, and (iv) at least one double stem loop immune modifier (dSLIM); and (d) a pharmaceutically acceptable carrier or excipient. In certain further embodiments (i) the adjuvant, when present, may be selected from alum, a plant alkaloid and a detergent, wherein the plant alkaloid may be tomatine and the detergent may be selected from Polysorbate 80, Span 85 and Stearyl tyrosine, (ii) the TLR agonist, when present, may be selected from lipopolysaccharide, peptidoglycan, polyI:C, CpG, 3M003, flagellin, Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a (LelF) and at least one hepatitis C antigen, and (iii) the imidazoquinoline immune response modifier, when present, is selected from resiquimod (R848), imiquimod and gardiquimod.

[0029] In another embodiment there is provided a pharmaceutical composition comprising: at least one recombinant expression construct which comprises a promoter operably linked to a nucleic acid sequence encoding an antigen; a compound having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof; and a pharmaceutically acceptable carrier or excipient. In certain further embodiments the recombinant expression construct may be present in a viral vector, which in certain further embodiments is present in a virus that is selected from an adenovirus, an adeno-associated virus, a herpesvirus, a lentivirus, a poxvirus, and a retrovirus. According to some embodiments, RNA or DNA may be delivered in lipid nanoparticles (LNP). The present lipid compounds having structures (I) - (IX) or stereoisomers thereof or mixture of any two or more thereof are part of the lipids in the formulation of LNP. The outer layer of LNP could contain targeting groups projecting out of the LNP.

[0030] According to an embodiment, a therapeutic agent comprising a drug or oligonucleotide conjugated with and / or covalently bound to the compound having structures (I) - (X) or stereoisomers thereof or a combination of any two or more thereof is provided.

[0031] According to some embodiments, the compound having structures (I) - (IX) or stereoisomers thereof or a combination of any two or more thereof may be in the form of a liposome.

[0032] According to some embodiments, the compound having structures (I) - (IX) or stereoisomers thereof or a combination of any two or more thereof may be in the form of a micelle.

[0033] According to some embodiments, the compound having structures (I) - (IX) or stereoisomers thereof or a combination of any two or more thereof may be in the form of a polyplex.

[0034] According to some embodiments, the compound having structures (I) - (IX) or stereoisomers thereof or a combination of any two or more thereof may be in the form of a lipoplex.

[0035] According to some embodiments, the compound having structures (I) - (IX) or stereoisomers thereof or a combination of any two or more thereof may be in the form of an emulsion.

[0036] As in known in the art, these compounds of structures (I) - (IX) or stereoisomers thereof or mixtures of any two or more thereof may be used in conjunction with other lipids to form a LNP that encapsulates a therapeutic agent. Non-limiting examples of other lipids that may be used in conjunction with compounds having structures (I) - (IX) or stereoisomers thereof or mixtures of any two or more thereof are other ionizable cationic lipids, permanently charged cationic lipids, polyethylene glycol (PEG) lipids, and / or cholesterol. The lipids of structures (I) – (IX) or stereoisomers thereof or mixtures of any two or more thereof as described herein may be used to provide adjuvant systems. Such systems may include three or four or more components used together including at least one of the lipids disclosed herein. These adjuvants may be provided for example in the form of liposomes, emulsions, lipid nanoparticles, lipoplexes, or polyplexes. Non-limiting examples of additional components that may be used with the present lipids of structures (I) – (IX) or stereoisomers thereof or mixtures of any two or more thereof are squalene (and / or derivative thereof such as squalane), such as the sustainably sourced squalene (Croda). Other such adjuvant lipids that may be used in combination with the lipids of structures (I) – (X) or stereoisomers thereof thereof disclosed herein to provide pharmaceutical compositions include the family of the PHAD® adjuvants (Croda) which are synthetic structural analogs of monophosphoryl Lipid A (MPLA). Non- limiting examples include the following. PHAD®-504 a synthetic structural analog of detoxified MPLA derived from E. coli lipopolysaccharide (LPS) is suitable optional adjuvant. It is structurally similar to PHAD®, differing only in the length of a single fatty acid chain.3D- PHAD® which is a homogeneous synthetic equivalent for the 3-deacylated MPLA derived from bacterial LPS is also a suitable adjuvant that may be used in combination with the lipids disclosed herein. The MPLA structural analog, 3D(6-acyl)-PHAD® is also suitable to be using in conjunction with the lipids of structures (I) – (X) or stereoisomers thereof thereof disclosed here. Other adjuvants that may be used to prepare liposomes in combination with the present lipids include sterols. Suitable sterols may include but are not limited to s-sitosterol, beta- sitosterol, campesterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. Sterols, such as cholesterol may also be used to prepare lipid nanoparticles. The sterol works as a stability enhancer by filling gaps in the lipid layer and assists in the transfection of the pharmaceutical ingredient, such as RNA. Structural analogs of cholesterol, such as β-sitosterol, can also be used to create LNPs with a comparable particle size (diameter ~100 nm) and encapsulation efficiency (~95% using 200 ng mRNA) as cholesterol. β-sitosterol-substituted LNPs may improve transfection efficiency when compared to cholesterol containing LNPs. Neutral lipids may also be used with the present lipids of structures (I) – (IX) or stereoisomers thereof or mixtures thereof. A neutral lipid, for example phosphatidylcholine, which may be non-crystalline at room temperature. Eggyolk phosphatidylcholine, dioleoyl phosphatidylcholine, dioleoyl ethanolamine and dilauryl phosphatidylcholine are non-limiting examples. Delivery of various drugs, vaccines, or oligonucleotides, antibodies, enzymes, proteins, small molecules, or nucleotides via these LNPs that include compounds of structures (I) – (IX) or stereoisomers thereof are especially attractive, since the present compounds are in a form of a cation. Thus, these compounds can be useful to deliver gene therapy to a patient in need thereof. Suitable drugs are antibodies, enzymes, and especially drugs that are used to treat diseases. Other suitable pharmaceuticals are anti-tumor antigens that may be useful for immunotherapeutically treating cancers. Suitable oligonucleotides are ABE (adenine base editor), miRNA inhibitors, antisense oligonucleotides, ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids, antagomir, antimir, mimic, supermir, and aptamers, allele-specific oligonucleotide, antisense oligonucleotide, ribonucleoproteins (RNP), saRNA, siRNA, dsRN, mRNA, siRNA, sgRNA, gRNA, ss-siRNA, cDNA, and cRNA where the c = circular. Vaccines and adjuvants for vaccines may be delivered by the use of the lipids of structures (I) – (IX) or mixtures of any two or more thereof disclosed herein. For example, optional inclusion of adjuvants (particular for vaccines) may include immunologically active saponin fractions having adjuvant activity, typically derived from the bark of the South American tree Quillaja Saponaria Molina, including for example QS21 as a preferred material which may also be derived more sustainably from plant tissue culture such as QS21 (Croda). The lipids disclosed herein may also be used with aluminum hydrogels available as Alhydrogel™ (Croda). QS21 may be used in combination with Alhydrogel as a useful adjuvant together with the lipid disclosed herein. According to some embodiments, CRISPR (clustered regularly interspaced short palindromic repeats) base editing therapy can be delivered using the present structures of formulas (I)-(IX) or stereoisomers thereof or mixture of any two or more thereof. The therapeutic agents may be formulated into a composition for administration to a patient in need thereof. A formulated composition can assume a variety of states. In some examples, the composition may be at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the formulation is in an aqueous phase, e.g., in a solution that includes water.

[0037] The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a lipid nanoparticle. Generally, the composition is formulated in a manner that is compatible with the intended method of administration.

[0038] In some embodiments, the therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle, and / or in the form of a liposome including a compound having the structure of formulas (I)-(IX) or stereoisomers thereof or a combination of any two or more thereof may be prepared by at least one of the following methods: microfluidics, impinging jet mixing, lipid film hydration, high shear mixing, high pressure homogenization, high pressure extrusion, spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

[0039] The therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle including a compound having a structure (I) - (IX) or stereoisomers thereof or mixtures of any two or more thereof may be formulated into pharmaceutical compositions suitable for administration. The therapeutic agent comprising a drug or oligonucleotide and the compound having a structure (I) - (IX) or stereoisomers thereof or mixtures of any two or more thereof may also be in the form of a lipid nanoparticle, or may be in the form of a liposome. As is known in the art, liposomes have one or more rings of lipid bilayer surrounding an aqueous pocket, but lipid nanoparticles usually only have a single phospholipid outer layer that encapsulates the interior, which may be non-aqueous.

[0040] The therapeutic agent, e.g. drug and / or oligonucleotide preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include multivesicular liposomes (MVLs), unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material comprising a compound having structure (I) - (IX) or stereoisomers thereof or mixtures of any two or more thereof or a combination of any two or more thereof and an aqueous interior. A liposome containing an oligonucleotide or a drug can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can include the present compound having structure (I) - (IX) or stereoisomers thereof or mixtures of any two or more thereof. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS (3-((3-cholamidopropyl) dimethylammonio)-!- propanesulfonate), octylglucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide or a drug preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the oligonucleotide or drug and condense around the oligonucleotide or a drug to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of an oligonucleotide or a drug.

[0041] The interior aqueous or non-aqueous portion contains the therapeutic agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the therapeutic agent composition, although in some examples, it may. Liposomes, micelles and lipid nanoparticles are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal or lipid or micelle membrane is structurally similar to biological membranes, when liposomes, lipid nanoparticles or micelles are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the lipid nanoparticle and cell progresses, the internal contents that include the therapeutic agent composition are delivered into the cell.

[0042] "Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0043] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the oligonucleotide or drug composition, an alkali metal C8 to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.

[0044] The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing will provide smaller size micelles.

[0045] In one method a first micellar composition is prepared which contains the oligonucleotide or drug composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the oligonucleotide or drug composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0046] Phenol and / or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after fol mation of the mixed micellar composition.

[0047] For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray. Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1, 1, 1,2 tetrafluoroethane) may be used.

[0048] The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Bio phys. Acta 858: 161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired. Lipid nanoparticles may include four types of lipids. These are phospholipids (helper lipids), ionizable cationic lipids, cholesterol, and polyethylene glycol (PEG) lipids. The present compounds (I) – (IX) or stereoisomers thereof or mixtures of any two or more thereof may be used as the ionizable cationic lipids. The purpose of the compounds having structure (I) – (IX) or stereoisomers thereof or mixtures of any two or more thereof is thus to improve the stability of the nanoparticle. Lipid nanoparticles may be prepared by mixing an ethanolic lipid mixture including the present compounds (I) – (IX) or mixtures of any two or more thereof with an acidic aqueous solution containing oligonucleotides and / or a drug. A 1:3 ratio of ethanolic lipid mixture to aqueous buffer is generally used. The general method is to combine the lipid elements with the ethanol, the therapeutic agent (oligonucleotides or drug) in aqueous, and rapidly mix them to form the lipid nanoparticles. Such compositions include the therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle including a compound having the structure (I) – (IX) or stereoisomers thereof or mixture of any two or more thereof or as a liposome, or as a micelle and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The compounds of structures (I)-(IX) or stereoisomers thereof or mixture of any two or more thereof, may be used to form polyplexes. The polyplexes may be formed by the combination of cationic polymers (e.g. poly-L-Lysine (PLL), poly-L-ornithine (POL), polyethyleneimine (PEI), polyamidoamine (PAM) or combinations thereof) together with the lipids of structures (I)-(IX) or stereoisomers thereof or mixture of any two or more thereof, helper lipids and a nucleic acid (e.g. DNA, RNA, etc.) These may be mixed together and will self-assemble via electrostatic interactions into the polyplexes. Non-limiting methods of combining include microfluidics, sonication, and extrusion for example.

[0049] The compounds of structures (I)-(IX) or stereoisomers thereof or mixture of any two or more thereof, may be used to form lipoplexes. The lipoplexes may be formed by combining the present cationic lipids of structures (I)-(IX) or stereoisomers thereof or mixture of any two or more thereof, optionally with neutral and / or amphoteric lipids and a therapeutic agent, such as anionic charged polynucleic acids (e.g. DNA, RNA, etc.). Typically lipoplexes are formed as suspensions in water, and so the lipid and therapeutic agent may suitably be provided in an aqueous environment; each may be provided as an aqueous solution and subsequently the two aqueous solutions mixed together, alternatively the lipid may be provided as an aqueous solution and the therapeutic agent subsequently added to this or conversely the therapeutic agent may be provided as an aqueous solution and the lipid subsequently added. Desirably, the lipid may be provided as a liposome or lipid nanoparticle prior to subsequent introduction of the therapeutic agent. Lipoplexes utilizing cationic lipids are known to be readily formed with negatively charged DNA or RNA where spontaneous self-assembly occurs from simple mixing to provide the nano-sized lipid based lipoplex materials. Like polyplexes, lipoplexes may be formed by mixing together the components which may self-assemble via electrostatic interaction into lipoplexes. Non-limiting methods of combining include microfluidics, sonication, and extrusion for example.

[0050] The compounds of structures (I)-(IX) or stereoisomers thereof or mixture of any two or more thereof, may be formed into emulsions to deliver therapeutic agent by combining the compounds of structures (I)-(IX) or mixture of any two or more thereof, with the therapeutic agent and an optional emulsifier. Such emulsions may be prepared using standard techniques. Suitable emulsions may be oil-in-water (o / w) or water-in-oil (w / o) emulsions. The water-in-oil emulsions may also be processed into a water-in-oil-in-water emulsion (also called "double emulsions"), where the internal and external aqueous phases are separated by an oil phase.

[0051] In general, the aqueous phase, the oil phase comprising the compounds of structures (I)- (IX) or stereoisomers thereof or salts thereof or mixture of any two or more thereof, optionally any emulsifiers and / or surfactants and therapeutic agent(s) are brought together and emulsified until a stable emulsion having the desired viscosity is obtained (the desired viscosity is application-dependent. Suitable viscosities would be known to the relevant skilled person). Compounds of structures (I)-(IX) or stereoisomers thereof or salts thereof or mixture of any two or more thereof may be present in the oil phase. Water-in-oil (w / o) emulsions are two phase systems including an oil phase (continuous phase) comprising the compounds of structures (I)- (IX) or stereoisomers thereof or salts thereof or mixture of any two or more thereof and an aqueous emulsions phase (discontinuous phase). The aqueous phase may be dispersed as small droplets in the oil phase, and the emulsion may contain one or more surfactants and / or emulsifiers. Water-in-oil emulsions are generally used in pharmaceutical formulations as a vehicle for delivery of therapeutic agents, especially in case of water-insoluble or water-sensitive active agents. In vaccinations, water-in-oil emulsions are commonly used as an adjuvant to stimulate the immune response against target antigens derived from one or more infectious agents. These water-in-oil emulsion vaccines may be delivered to a patient in need thereof via injection.

[0052] When emulsions are prepared, energy must be expended to form an interface between the oil phase and aqueous phase. Therefore, emulsification equipment suitable to prepare emulsions includes a wide variety of agitators, homogenizers, colloid mills, jet mixers and ultrasonic devices for example. Production-size agitators can be propeller shaped or paddle shaped stirring systems, with rotation speed usually up to 2,000 rpm, and are considered as low shear mixing procedures. Another type of production-site agitator is the colloid mill. The principle of operation of the colloid mill is the passage of the mixed phases of an emulsion formula between a stator and a high-speed rotor revolving at speeds of 2,000 to 18,000 rpm that is considered as a high shear mixing procedure.

[0053] Optionally, once the emulsion has been prepared a drying step may be utilized to provide a dried powder product comprising the compounds of structures (I)-(IX) or stereoisomers thereof or salts thereof or mixture of any two or more thereof and therapeutic agent. In this case the therapeutic agent may be encapsulated in the compounds of structures (I)-(IX) or stereoisomers thereof or salts thereof or mixture of any two or more thereof and optionally other lipids as well. Such a drying step may advantageously utilize a spray drier, although other drying methods are known in the art. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated.

[0054] Methods of Treatment

[0055] According to an embodiment, a method of treatment is provided. The method comprising administering the therapeutic agent to a patient in need thereof. Suitable administration to a subject in need thereof includes a variety of routes. Non-limiting examples include: parenteral, intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, or ocular.

[0056] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0057] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.

[0058] Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0059] Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the therapeutic agent comprising a drug or oligonucleotide in the form of a lipid nanoparticle, liposome or micelle including a compound having the structure (I) - (IX) or mixture of any two or more thereof can be combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring agents can be added. According to some embodiments, compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. According to some embodiments, the administration of the pharmaceutical composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, intratumor, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic. According to some embodiments the administration of the pharmaceutical composition is intramuscular. EXAMPLES Materials Anhydrous chloroform (CHCl3), tetrahydrofuran (THF), dichloromethane (DCM), glutaric anhydride (2), triethylamine (TEA), and ethyl triflate (EtOTf) were purchased from Sigma-Aldrich. Dicyclohexylcarbodimide (DCC), dimethylaminopyridine (DMAP), and Compound 4a, or cadmium-bound GPC are Avanti raw materials.18:1 Lyso PC (Compound 4b) was synthesized at Avanti as catalog #845875. Example 1: Synthetic Scheme for Compounds (II) and (III): Example 1a: Synthetic Scheme for Compound 3 (above): Alcohol 1 (Example 1) (20g, 78 mmol, 1 eq) was dissolved in THF (110 mL). Glutaric anhydride (Compound 2, 10g, 88 mmol, 1.12 eq), DMAP (1.071 g, 8.76 mmol, 0.112 eq), and TEA (20 mL, 140 mmol, 1.8 eq) were also added. The combined materials were refluxed for 3 hours. The reaction was monitored by thin layer chromatography in 9:1 chloroform-methanol until the alcohol was consumed. After cooling to room temperature, the reaction was acidified with 160 mL of 1M hydrochloric acid and extracted with 160 mL of diethyl ether three times. It was then washed with water twice and then brine once. The organic layer was dried with sodium sulfate, and the solvent was removed via rotary evaporation. The resultant crude was purified by ethyl acetate-hexane gradient, eluting in 8:2 to 7:2 hexane-ethyl acetate, to give 21.1 g (65%) of Compound 3. Rf= 0.4 (7:3 hexane-ethyl acetate).1H NMR (400 MHz, CDCl3): δ 0.88 (6 H, m), 1.26 ppm (24H, m), 1.50 (4H, m), 1.96 (2H, m), 2.05 (ethyl acetate), 2.30-2.50 (4H, m), 2.59 (water), 4.04 (unknown), 4.13 (ethyl acetate), 4.88 (1H, CH, m). MS (ESI+) calculated for C22H41O4- [M-Cl]-: 369.30, found 369.302. Example 1b: Synthetic Scheme for Compound 5a (above): To Compound 3 (13.46 g, 36.3 mmol, 2 eq) dissolved in 250 mL of anhydrous amylene- stabilized chloroform, was added DCC (18.73g, 91 mmol, 5 eq) in three additions. After 15 minutes of activation, DMAP (5.32 g, 2.40 mmol, 2.40 eq) and 4a (8g, 18.16 mmol, 1 eq) were added. The reaction was then sealed under nitrogen and stirred overnight at room temperature. Thin layer chromatography in 65:25:4 chloroform-methanol-ammonium hydroxide and visualization in iodide showed mostly PC at Rf~ 0.4 with < 5% of Lyso PC as a bottom spot of Rf~ 0.3. The material was then purified by normal phase silica chromatography. After rotary evaporation, approximately 12.2 g of compound 5a (70%) was obtained as a neat oil. Rf= 0.4 (65:25:4 chloroform-methanol-water).1H NMR (400 MHz, CDCl3): δ 0.88 (12 H, m), 1.28 ppm (48H, m), 1.50 (8H, m), 1.90 (4H, m), 2.35 (8H, m), 2.59 (water), 3.34 (9H, s), 3.76 (2H, m), 3.95 (2H, m), 4.15 (1H, m), 4.31 (2H, m), 4.39 (1H, m), 4.85 (2H, m), 5.21 (1H, m). MS (ESI+) calculated for C52H101NO12P+[M+H]+: 962.706, found 962.707. Example 1c: Synthetic scheme for Compound 5b (above): To Compound 3 (8.95 g, 24.2 mmol, 1.8 eq) dissolved in 150 mL of anhydrous amylene- stabilized chloroform, was added DCC (8.31 g, 40.3 mmol, 3 eq) in three additions. After 15 minutes of activation, DMAP (4.10 g, 33.5 mmol, 2.5 eq) and 4b (7g, 13.4 mmol, 1 eq) were added. The reaction was then sealed under nitrogen and stirred overnight at room temperature. Thin layer chromatography in 65:25:4 chloroform-methanol-ammonium hydroxide and visualization in iodide showed mostly PC at Rf~ 0.4 with < 5% of Lyso PC as a bottom spot of Rf~ 0.3. The material was then purified by normal phase silica chromatography. The product eluted between 73:23:3 and 65:25:4 chloroform-methanol-water. After rotary evaporation, approximately 10 g of compound 5 (82%) was obtained as a neat oil. Rf= 0.4 (65:25:4 chloroform-methanol-water).1H NMR (400 MHz, CDCl3): δ 0.87 (9 H, m), 1.26 ppm (44H, m), 1.50 (4H, m), 1.57 (2H, m), 1.89 (2H, m), 1.99 (4H, m), 2.33 (6H, m), 2.58 (water), 3.34 (9H, s), 3.83 (2H, m), 3.97 (2H, m), 4.12 (1H, m), 4.32 (2H, m), 4.39 (1H, m), 4.83 (1H, m), 5.23 (1H, m), 5.34 (2H, m). MS (ESI+) calculated for C52H101NO12P+[M+H]+: 874.653, found 874.653. Example 1d: Synthesis of Compound (II): Compound 5a (7.0 g, 7.98 mmol, 1 eq) as a stock solution in chloroform was cycled with toluene under rotary evaporation and then dissolved in 200 mL of dichloromethane. Subsequently EtOTf (1.42 g, 7.98 mmol, 1 eq) and sodium bicarbonate (6.70 g, 80 mmol, 10 eq) were added. The reaction was stirred vigorously under nitrogen for 30 minutes. Thin layer chromatography with 65:25:4 chloroform-methanol-water, and iodide visualization showed an absence of the starting PC at Rf~ 0.4 and a higher spot at Rf~ 0.5. The crude material was then dissolved in chloroform and purified on silica gel Pure product fractions were partitioned with 0.1 M sodium chloride (4 g) and washed with fresh methanol-water, backwashing if needed to recover product from the aqueous phase. After drying via rotary evaporation, 4.78 g of Compound (II) (63 %) was obtained. Rf= 0.5 (65:25:4 chloroform-methanol-water).1H NMR (400 MHz, 8:2 CDCl3-CD3OD): δ 0.88 (12 H, m), 1.19 (isopropyl alcohol), 1.27 ppm (48H, m), 3.29 (3H, m), 1.52 (8H, m), 1.96 (4H, m), 2.42 (8H, m), 3.29 (9H, s), 3.36 (methanol), 3.66 (ethanol), 3.85 (2H, m), 4.02 (water + isopropyl alcohol), 4.22 (5H, m), 4,35 (1H, m) 4.52 (2H, m), 4.86 (2H, m), 5.29 (1H, m). MS (ESI+) calculated for C54H105NO12P+[M-Cl]+: 990.7, found 991.5.

[0060] Example 1e: Synthesis of Compound (III): Compound 5b (7.0 g, 8.00 mmol, 1 eq) as a stock solution in chloroform was cycled with toluene under rotary evaporation and then dissolved in 200 mL of dichloromethane. Subsequently EtOTf (1.43 g, 8.00 mmol, 1 eq) and sodium bicarbonate (6.72 g, 80 mmol, 10 eq) were added. The reaction was stirred vigorously under nitrogen for 30 minutes. Thin layer chromatography with 65:25:4 chloroform-methanol-water, and iodide visualization showed an absence of the starting PC at Rf~ 0.4 and a higher spot at Rf~ 0.5. The material was then dried via rotary evaporation. The crude material was then dissolved in chloroform and purified on silica gel. After drying via rotary evaporation, 6.2 g of Compound 6b (83 %) was obtained. Rf= 0.5 (65:25:4 chloroform-methanol-water).1H NMR (400 MHz, 8:2 CDCl3-CD3OD): δ 0.88 (9 H, m), 1.19 (isopropyl alcohol), 1.27 ppm (47H, m), 1.52, (4H, m), 1.62 (2H, m), 2.01 (6H, m), 2.34 (4H, m), 2.44 (2H, m), 3.28 (9H, s), 3.36 (methanol), 3.66 (ethanol), 3.83 (2H, m), 4.02 (water + isopropyl alcohol), 4.21 (5H, m), 4,35 (1H, m) 4.52 (2H, m), 4.86 (1H, m), 5.27 (1H, m), 5.35 (2H, m). MS (ESI+) calculated for C54H105NO12P+[M-Cl]+: 902.684, found 902.681.

[0061] Example 2: Synthetic Scheme for Compounds (IV) and (V): Example 2a: Synthesis of Compound 8 (above): Alcohol 7 (30g, 25.31 mmol, 1 eq) was refluxed in toluene in toluene (100 mL) with glutaric anhydride (Compound 2, 15g, 125.31 mmol, 1.05 eq). The reaction was monitored by thin layer chromatography in 9:1 chloroform-methanol until the alcohol was consumed. After cooling to room temperature, the solvent was removed. The resultant crude was purified by ethyl acetate-hexane gradient, eluting in 8:2 to 7:2 hexane-ethyl acetate, to give 21.1 g (75%) of Compound 8 with 11g of material available for a reprocess column. Rf= 0.4 (7:3 hexane-ethyl acetate).1H NMR (400 MHz, CDCl3): δ 0.88 (6 H, t), 1.26 ppm (24H, m), 1.61 (1H, m), 1.96 (2H, t), 2.44 (4H, m), 4.00 (m, 2H). MS (ESI+) calculated for C22H41O4- [M-Cl]-: 369.30, found 369.302. Example 2b: Synthesis of Compound 9a (above): To Compound 7 (13.46 g, 2.96 mmol, 2 eq) dissolved in 100 mL of anhydrous amylene- stabilized chloroform, was added DCC (2.41 g, 12 mmol, 3 eq). Then DMAP (0.48 g, 2.40 mmol, 1.0 eq) and RGPC (Compound 4a, 4g, 7.78 mmol, 1.0 eq) were added. The reaction was then sealed under nitrogen and stirred overnight at room temperature. Thin layer chromatography in 65:25:4 chloroform-methanol-ammonium hydroxide and visualization in iodide showed mostly PC at Rf~ 0.4 with < 5% of Lyso PC as a bottom spot of Rf~ 0.3. The material was then purified by normal phase silica chromatography. After rotary evaporation, approximately 3.24 g of 8 (89%) was obtained as a neat oil. Rf= 0.4 (65:25:4 chloroform-methanol-water).1H NMR (400 MHz, CDCl3): δ 0.88 (12 H, t), 1.28 ppm (48H, m), 1.63 (2H, m), 1.91 (4H, t), 2.39 (8H, m), 3.22 (9H, s), 3.36 (methanol), 3.61 (2H, m), 3.97-4.01 (6H, t), 4.02 (water), 4.14 (1H, q), 4.25 (2H, t), 4.43 (1H, m), 5.23 (1H, m). MS (ESI+) calculated for C52H101NO12P+[M+H]+: 934.297, found 934.675. Example 2c: Synthesis of Compound 9b (above): To Compound 8 (2.82 g, 7.68 mmol, 2 eq) dissolved in 100 mL of anhydrous amylene- stabilized chloroform, was added DCC (4.75 g, 23.00 mmol, 3 eq), then DMAP (0.47 g, 4.00 mmol, 1 eq) and Compound 4 (2.00 g, 3.84 mmol, 1 eq). The reaction was then sealed under nitrogen and stirred overnight at room temperature. Thin layer chromatography in 65:25:4 chloroform-methanol-ammonium hydroxide and visualization in iodide showed mostly PC at Rf~ 0.4 with < 5% of Lyso PC as a bottom spot of Rf~ 0.3. The material was then purified by normal phase silica chromatography. After rotary evaporation, approximately 2.96 g of compound 9b (87%) was obtained as a neat oil. Rf= 0.4 (65:25:4 chloroform-methanol-water). 1H NMR (400 MHz, 8:2 CDCl3-CD3OD): δ 0.88 (9 H, t), 1.28 ppm (44H, m), 1.60 (3H, m), 1.91 (2H, m), 2.01 (4H, m), 2.31 (2H, m), 2.41 (4H, m), 3.22 (9H, s), 3.36 (methanol), 3.58 (2H, m), 3.70 (methanol), 3.97-4.02 (4H, m), 4.02 (water), 4.16 (1H, m), 4.22 (2H, m), 4.43 (1H, m), 5.24 (1H, m), 5.35 (2H, m). MS (ESI+) calculated for C54H105NO12P+[M-Cl]+: 860.6375, found 860.6389.MS (ESI+) calculated for C47H91NO10P+[M+H]+: 860.6375, found 860.6389. Example 2d: Synthesis of Compound (IV): Compound 9a (3.24 g, 3.47 mmol, 1 eq) as a stock solution in chloroform was cycled with toluene under rotary evaporation and then dissolved in 50 mL of dichloromethane. Subsequently ethyl triflate (0.43 mL,3.29 mmol, 0.95 eq) was added. The reaction was stirred vigorously for 30 minutes. Thin layer chromatography with 65:25:4 chloroform-methanol-water, and iodide visualization showed minimal to no starting PC at Rf~ 0.4 and a higher spot at Rf~ 0.5. The crude material was then dissolved in chloroform and purified on silica gel. After drying via rotary evaporation, 2.37 g of Compound 10a (68 %) was obtained. Rf= 0.5 (65:25:4 chloroform-methanol-water).1H NMR (400 MHz, CDCl3): δ 0.88 (12 H, m), 1.19 (isopropyl alcohol), 1.27 ppm (48H, m), 1.38 (3H, m), 1.60 (2H, s), 1.86 (water), 1.93 (4H, m), 2.42 (8H, m), 3.47 (9H, s), 3.95 (4H, m), 4.13-4.36 (8H, m), 4.56 (2H, m), 5.26 (1H, m). Example 2e: Synthesis of Compound (V): Compound 9b (2.96 g, 3.44 mmol, 1 eq) as a stock solution in chloroform was cycled with toluene under rotary evaporation and then dissolved in 50 mL of dichloromethane. Subsequently ethyl triflate (0.42 mL, 3.27 mmol, 0.95 eq) was added. The reaction was stirred vigorously under nitrogen for 30 minutes. Thin layer chromatography with 65:25:4 chloroform- methanol-water, and iodide visualization showed an absence of the starting PC at Rf~ 0.4 and a higher spot at Rf~ 0.5. The crude material was then dissolved in chloroform and purified on silica gel. After drying via rotary evaporation, 1.6 g of Compound 10b (50%) was obtained. Rf= 0.5 (65:25:4 chloroform-methanol-water).1H NMR (400 MHz, CDCl3): δ 0.88 (9 H, m), 1.20- 1.40 ppm (47H, m), 1.60 (3H, m), 1.70 (water), 1.90-2.00 (6H, m) 2.30-2.45 (6H, m), 3.45 (9H, m), 4.00 (2H, m), 4.20 (6H, m), 4.23 (2H, m), 4.54 (2H, m), 5.26 (1H, m), 5.33 (2H, m). Example 3: Preparation of Lipid Nanoparticle: 40mg of quaternary conical ethyl phosphate cationic lipid (EPC) of formula (II), 14 mg cholesterol, 7.6 mg DSPC, and 3.5 mg DMG-PEG2000 were mixed together in ethanol at 50 mg / mL. This was combined with RNAi under controlled conditions with microfluidics to produce lipid nanoparticles and then dialyze against PBS to remove ethanol. Resulting particles had a particle size of 80-120 nm, and RNA encapsulation efficiency of 96%. Example 4: Preparation of Lipid nanoparticle (LNP):

[0062] 32mg tertiary conical ethyl phosphate cationic lipid (EPC) of formula (III), 13 mg cholesterol, 6.7 mg DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine), and 3.2 mg DMG (Dimethylglyoxime)-PEG2000 were dissolved in ethanol at 50 mg / mL. This solution was combined with dsRNA in aqueous under controlled conditions using microfluidics. Dialysis was used to remove ethanol and unencapsulated RNA. Final product was 70-100 nm with 94% encapsulation efficiency of RNA.

[0063] Example 5: Preparation of Lipoplex:

[0064] A lipid film composed of 40 mg quaternary conical ethyl phosphate cationic lipid (EPC) of formula (II) and 10 mg 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) was prepared and hydrated with an aqueous solution containing DNA then sonicated to form a lipoplex.

[0065] Example 6: Preparation of Lipoplex:

[0066] Tertiary conical ethyl phosphate cationic lipid (EPC) of formula (III) was hydrated with an aqueous solution containing DNA at a 1 : 1 ratio lipid:DNA. The lipoplex was sonicated and extruded to reduce size.

[0067] Example 7: Preparation and testing of Lipid nanoparticles (Prophetic):

[0068] Lipid nanoparticles are prepared by combining 1.5mol% DMG-PEG (Dimethylglyoxime)-PEG2000 50 mol% compound of formula (II) and / or formula (III), 38.5 mol% cholesterol and 10 mol% DSPC (1,2-Distearoyl-sn-glycero-3-phosphorylcholine). These are combined with mRNA in a citrate buffer and formed into lipid nanoparticles using microfluidics. The nanoparticles are isolated with dialysis using phosphate buffered saline (7.4 pH). The N / P ratio (amine groups / phosphate groups) is 6. The size of the LNP ranges from 60-80 d nm.

[0069] Example 8: Preparation and testing of Lipid nanoparticles: Lipid nanoparticles were prepared using microfluidics. These prepared lipid nanoparticles contained the following lipids: “APL-719” a lipid with the structure of formula (II) in accordance with the present invention prepared as described above , or alternatively, “ALC-0315”, a commercially available comparative example lipid having the structure shown below, which is utilized in the commercially available Comirnaty ® Covid-19 mRNA vaccine (ex. Pfizer Inc) and this commercial use means that ALC-0315 is considered to be a good benchmark material for comparative testing. Example 8a: Preparation of LNPs using microfluidic mixing technique: Lipid nanoparticles (LNPs) were prepared using a microfluidic mixing technique. Either lipid APL-719 or lipid ALC-0315 were dissolved in 99% ethanol (EtOH) to provide final lipid stocks with a concentration of between 5 mM and 50 mM depending on the lipid and the test to be performed. These lipid stocks were then pipetted into a desirable ratio to produce a variety of LNPs, and in particular to provide “Comirnaty like” benchmark LNPs having similar formulation properties to the commercially available Comirnaty ® Covid-19 mRNA vaccine (test formulation ratio of 50:38.5:10:1.5 of lipid to be tested:cholesterol:phospholipid (DSPC):PEG-lipid(DMG-PEG)) in Eppendorf tubes, and total concentration adjusted with EtOH. RNA encoding firefly luciferase (ex. TriLink L-7202) was diluted in 25 mM sodium acetate (pH 4.0) for formulations with lipid ALC-0315, and 25 mM Tris (hydroxymethyl) aminomethane (TRIS, pH 7.6) for formulations with lipid APL-719; this formulation difference is due to the fact that ALC-0315 is an ionizable lipid. Subsequently, the aqueous and organic phases where then mixed in a 3:1 flow rate ratio with a total flow rate of 4 mL / min using a 100 XT Sunny microfluidic mixing chip (ex. Unchained labs) integrated into an Automated Nanoparticle (ANP) System (ex. ParticleWorks). Following mixing, the buffer was exchanged into phosphate buffered saline (PBS, pH 7.4) using continuous diafiltration. Briefly, LNP samples (1-2 mL) in 25% EtOH was transferred into Amicon ® Ultra (ex.Merck KGaA) spin filters (regenerated nitrocellulose membrane, 4 mL volume, 100 kDa MWCO) and the volume adjusted to 4.5 mL using PBS. The filters were then centrifuged at 500g until the volume had decreased to 500 µL (typically 5-20 minutes depending on the formulation). Then, the volume was adjusted to 4.5 mL again using PBS. This procedure was repeated four times. Finally, the 500µL LNP sample (exact volume was determined with a pipette) was transferred to Eppendorf tubes and stored short-term at 4°C. When necessary, for long-term storage, 49% sucrose (percentage by weight, w / w) was added to the formulation to a final concentration of 10% sucrose (w / w) and the samples kept at -80 °C. Example 8b: Characterization of LNPs: To determine the size of the “Comirnaty like” LNPs prepared as above, 1 µL of the LNP sample was diluted in 80 µL PBS (approx. concentration 100 ng / mL RNA) and transferred to single-use microcuvettes (ex. Brand GMBH) and the size determined using a Malvern Zetasizer Nano-ZS. Size is reported as the Z-Average value and is an average of three independent measurements on the same sample. Results are shown in Figure 3. To determine the zeta potential of the LNPs, 10 µL of the LNP sample was diluted in either 800 µL 5 mM TRIS (pH 7.8) or 5 mM MES (pH 5.8). The sample to be tested was transferred to a folded capillary cell (Malvern DTS1060) using a 1 mL syringe, and the zeta potential determined using M3-PALS using a Malvern Zetasizer Nano-ZS. Zeta potential is reported as an average of three independent measurements on the same sample. Results are shown in Figure 4. RNA concentration of the “Comirnaty like” benchmark LNPs prepared as above and encapsulation efficiency was determined using the Ribogreen Assay, a sensitive fluorescent method used for quantifying RNA in solution. It utilizes the Quant-iT RiboGreen RNA Reagent, which can detect as little as 1 ng / mL of RNA and provides a linear detection range of 1–200 ng of RNA. Briefly, the LNPs to be tested were diluted x50 in TE buffer (pH 7.5) in Eppendorf tubes. These samples were then further diluted x10 in 0.5% Tween-20 (in TE buffer) for a final dilution of x500. Samples in 0.5% Tween-20 were incubated at 60 °C for 30 minutes on a thermoshaker to ensure disassembly of the LNPs, and allowed to cool to room temperature before proceeding. Eight-step standards with RNA concentrations ranging from 0 to 100 ng / mL was prepared in both TE buffer and 0.5% Tween-20 using the same FLuc RNA stock as used for preparing the particles. One-hundred µL of each sample was transferred to black polystyrene 96- well plates, and 100 µL Quant-iT Ribogreen dye (ex. Invitrogen) pre-diluted 1000x in TE buffer was added to each well. Plate was gently tapped on the side to ensure mixing and allowed to incubate in the dark at room temperature for 5 minutes. Finally, the Ribogreen fluorescence was read on a SpectraMax i3x Microplate Reader (ex. Molecular Devices) by exciting at 488 nm and reading emission at 535 nm. Total RNA concentration was calculated by comparing fluorescence intensity to the standard in 0.5% Tween-20, and concentration of dye-accessible (non- encapsulated) RNA by comparing fluorescence intensity to the standard in TE buffer: encapsulated RNA concentration was determined as the difference between these values, and encapsulation efficiency calculated as the percent encapsulated RNA made up of the total RNA. RNA yield was calculated as the total RNA mass in the final sample compared to the input RNA in the microfluidic mixing procedure. Results are shown in Figure 5. Example 8c: Cell culture tests: It is desirable for RNA vaccine developers to have access to a range of LNPs providing different immune profiles in use and suitable for different purposes. Selection and optimization of LNPs for successful RNA vaccination products is a developing area and as such, there is not a “one size fits all” approach to selection of LNP constituents for use in RNA delivery. The lipids of the present invention are believed to have utility in LNPs for the purpose of RNA (amongst other materials) delivery and cell transfection and the following cell culture tests demonstrate this utility, although the person skilled in the art of vaccine formulation would be able to take this information and seek to optimize vaccine formulations based on desirable immune profile and intended purpose, for example, other lipids as described above may be used in conjunction with lipids of the present invention as “helper” lipids which could further increase cell uptake of the LNP encapsulated RNA vs. use of the lipid of the present invention alone. In the following tests LNPs are formulated as detailed above but in this case the LNP formulation has a ratio as follows: Lipid to be tested :cholesterol:other “helper” lipid:PEG lipid 50:38.5:10:1.5 molar ratio Where the other “helper” lipid is DSPC the overall LNP formulation is akin to a “Comirnaty like” LNP prepared as above in Example 8a, but alternatively the other “helper” lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), however, in either case the RNA concentration is kept at around 25-35 µg / mL. Furthermore, in some cases LNPs samples for testing were prepared in a method which varied to that described in Example 8a, in that the spin filtration step was replaced by a dialysis step – this change in preparation method appears to have an effect on cell transfection, but the effect can be positive or negative depending on the overall formulation or cell type and it appears that method of manufacture of LNP could affect effectiveness of transfection in actual use. In the Figures 6, 7 and 9 the method of production is identified by reference to “Dialysis” or “Spin Filtration”, save for this difference the LNPs which are prepared for testing are prepared as detailed in Example 8a above. Cell types tested include, i) HepG2: Hepatocyte cell line (an easy to transfect, positive control), ii) C2C12: Murine skeletal muscle cell line (bystander cell model), iii) Monocytes: undifferentiated THP-1 cells (human monocytes from a leukemia patient), iv) Macrophages: THP-1 cells differentiated by incubating 48h in PMA (40 ng / mL) followed by 24h rest, and v) Dendritic cells: THP-1 cells differentiated by incubating one week with cytokines IL4 (100 ng / mL) and GM-CSF (100 ng / mL). The tests are described in more detail below, but this cell type selection was chosen to show utility of the lipids of the present invention for transfection over a wide range of cell types, although it should be appreciated that optimisation of any suggested formulation has not been undertaken and is for illustrative purposes only. Cell cultures: HepG2 cells (human hepatocytes) were acquired from ATCC (HB-8065). Cells were grown in a medium consisting of MEMα w. GlutaMax (Gibco) supplied with 10% FBS (Gibco) and Pen / Strep (BioWest) in cell-culture treated T75 flasks (VWR) incubated at 37 °C and 5% CO2. Cells were split when they reached a confluency of 75% using TrypLe and discarded when reaching passage number 20. C2C12 (murine skeletal muscle) cells were acquired from ATCC (CRL-1772). Cells were grown in a medium consisting of high-glucose DMEM w. GlutaMax (Gibco) supplied with 10% FBS (Gibco) and Pen / Strep (BioWest) in cell-culture treated T75 flasks (VWR) incubated at 37 oC and 5% CO2. Cells were split when they reached a confluency of 60% using TrypLe, and discarded when reaching passage number 25. THP-1 (human monocyte) cells were acquired from ATCC (TIB-202). Cells were grown in a medium consisting of RPMI 1640 w. L-Glutamine and 25 mM HEPES (VWR) supplied with 10% FBS (Gibco), 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and Pen / Strep (BioWest) in non-treated T25 flasks (VWR) incubated at 37 °C and 5% CO2. Cells were kept in a concentration between 3 x 105 and 1 x 106 cells / mL. When subculturing cells, 10% conditioned medium was always used. Cells were discarded before reaching 20 passages. For differentiation of THP-1 cells into dendritic cells, the medium was supplemented with 100 ng / mL recombinant human GM-CSF (ProSci) and 100 ng / mL recombinant human IL4 (ProSci). The cells were kept in this medium for a total of six days (with medium replaced on day three), before being used for experiments. For differentiation of THP-1 cells into macrophages, the medium was supplemented with 40 ng / mL PMA (Calbiochem) and the cells kept in this medium in 48 hours. Then, medium was replaced with fresh growth medium without PMA, and the cells incubated for further 24h before being used for experiments. As the cells became adherent during differentiation, the differentiation process was always carried out in the same 96 well plate as in where the experiment was performed. Treatment of cell cultures with LNPs: THP-1 cells differentiated into macrophages were seeded in flat-bottom cell culture- treated 96 well plates three days before initiation of the experiment at a concentration of 10.000 cells / well. HepG2 and C2C12 cells were seeded in similar plates on the day before the experiment at a concentration of 20.000 cells / well. THP-1 cells differentiation into DCs as well as non-differentiated THP-1 cells were seeded on the day of the experiment into U-bottom non- treated 96 well plates at a concentration of 20.000 cells / well. LNPs were pre-diluted in PBS to the desired concentration, typically 7 µg / mL. Then, 10 µL of the diluted LNPs were added to the cells (100 µL) thereby achieving 70 ng RNA per well (or 3.5 pg RNA per cell).10 µL PBS were added to control wells for comparison. Triplicate wells were made of each sample. Plates were then allowed to incubate overnight (18h) at 37 °C and 5% CO2. Cell culture sample testing: 1. Toxicity determination Toxicity was determined by quantifying LDH release into the cell medium using the CyQyant kit (Invitrogen). Following incubation with LNPs, 20 µL cell medium from each well was transferred to black flat-bottom 96-well plates with optical bottom. Plates with non-adherent cells were centrifuged 1000g for 3 minutes to ensure only medium was collected. Then, 30 µL MQ water was added to each well containing cell medium, followed by 50 µL Reaction Mixture supplied with the kit (containing a tetrazolium salt that can be converted to a red formazan product by the LDH enzyme released by dying cells). After 30 minutes incubation at room temperature in dark, the reaction was stopped using the buffer supplied with the kit, and the absorbance read at 490 nm (background subtraction at 680 nm) on a SpectraMax i3x Microplate Reader (Molecular Devices). To determine maximum LDH release (corresponding to 100% cell death), control wells were treated with the lysis buffer supplied with the kit. To ensure equal volume in all wells, no PBS was added to these control wells upon treating with LNPs the day before. Cytotoxicity was reported in percent, with 0% defined as the LDH release in wells treated with PBS and 100% defined as LDH release in lysed cells. 2. Transfection quantification Transfection was determined from the FLuc luminescence using the lyophilized Firefly Luciferase Assay (ex. Millipore). Following sampling of cell medium for determination of toxicity, the remaining medium in each well was carefully removed with a pipette. Then, 50 µL lysis buffer supplied with the kit was added to each well and the plate incubated at room temperature for 15 minutes. Thereafter, 20 µL sample was transferred from each well into a white-bottom 96-well plate. One hundred µL Luciferin Working Solution was then added to each well, and luminescence at 560 nm read immediately on a SpectraMax i3x Microplate Reader (Molecular Devices) with 1000 ms integration time. To normalize the luminescence to the number of cells in each well, 10 µL of the lysed cells were transferred to a black flat-bottom 96- well plates with optical bottom. Following addition of 40 µL cell medium to each well, the cell number was semi-quantified by measuring the total LDH content in each well using the CyQuant kit as described above. Figure 6 shows normalized transfection of HepG2 cells for a sample of “Comirnaty-like” LNPs (containing lipid ALC0315:Cholesterol:DSPC:lipid ALC019 at a molar ratio of 46.3 : 42.7 : 9.4 : 1.6), compared to samples prepared as detailed above for comparative lipid ALC- 0315 and lipid APL-719 in accordance with the present invention both in combination with either other “helper” lipid DSPC or DOPE and lipid APL-719 in accordance with the present invention (lipid to be tested:Cholesterol:other “helper” lipid:PEG lipid at 50:38.5:10:1.5 molar ratio). In this case it can be clearly seen that ALC-0315 transfection efficiency is much reduced for particles prepared by dialysis, compared to particles prepared by spin filtration and also that APL-719 containing LNPs are capable of HepG2 transfection as campared to the PBS control, but this is at a lower rate than seen for ALC-0315 in some cases, highlighting the need for selection and optimisation of vaccine formulations for real life use. Figure 7 shows normalized transfection of C2C12 cells for a sample of “Comirnaty-like” LNP (containing lipid ALC0315:Cholesterol:DSPC:lipid ALC019 at a molar ratio of 46.3 : 42.7 : 9.4 : 1.6), compared to samples prepared as detailed above for comparative lipid ALC- 0315 and lipid APL-719 in accordance with the present invention both in combination with either other “helper” lipid DSPC or DOPE and lipid APL-719 in accordance with the present invention (lipid to be tested:Cholesterol:other “helper” lipid:PEG lipid at 50:38.5:10:1.5 molar ratio). In this case it can be clearly seen that in the C2C12 cells, ALC-0315 transfection efficiency is much reduced for LNPs prepared by dialysis, compared to those prepared by spin filtration. Again, APL-719 containing LNPs are capable of C2C12 cell transfection as compared to the PBS control, but this is at lower rate than observed for ALC-0315 in this cell type. Figure 8 shows data for differentiation of THP-1 cells in dendritic cell transfection; all LNPs prepared for use in this test were prepared using a dialysis method. In this test improvement in transfection can be observed when using APL-719 versus ALC-0315 when the other “helper” lipid is DSPC. Again, transfection is observed for all LNPs versus the PBS control. Figure 9 shows data for differentiation of THP-1 cells in macrophages. Here it can be seen that in macrophages APL-719 prepared by spin filtration method, and when DOPE is the other “helper” lipid, the rate of transaction is best. 3. Cytokine release Transfection of THP-1 cells was performed as described above; however, the cell number was adjusted to 27.000 cells / well and the growth medium volume increased to 270 µL. RNA mass per well was 100 ng per well (corresponding to 3.7 pg / cell). As positive controls, cells were treated with 2 µg / mL LPS (Lipopolysaccharide from E. coli ex. Enzo Life Sciences) or 10 µg / mL CAFTM01(liposomal adjuvant ex. Croda, under license from SSI); LPS is known to illicit a strong immune response and CAFTM01is utilized in many earlier studies looking at vaccine adjuvency. Final volume following addition of adjuvants was 290 µL. Following the overnight incubation, 250 µL cell medium was collected from each well. Medium from two wells was pooled, thereby achieving a 500 µL sample. Six wells were treated with the same formulation, thereby resulting in triplicate samples of medium. For determination of medium concentration of cytokines IL1β, IL6, and TNFα, ELISA kits from Invitrogen was used;100 µL medium was used. ELISAs were performed according to manufacturer’s instructions. n.b. The sample identified in Figures 10 to 13 as “APL-143 Sitosterol” is not further disclosed herein, and in these Figures reference to Comirnaty is a reference to the “Comirnaty-like” material as described above. Figures 10 to 13 relate to cytokine release test data. Figure 10 shows date for IL1ß production Important pro-inflammatory cytokine, stimulating both Th1 and Th17 polarized responses. No response in dendritic cells (DCs) towards any of the immunostimulants (not even positive control) . In monocytes, the only LNP giving an immune response significantly above the PBS background is APL-719. In macrophages, Figure 11, it can be noted that the PBS background is very high indicating overstimulation in maturation phase. Figure 12 shows IL6 production in monocytes, dendritic cells (DCs) and macrophages. IL6 production is an important cytokine, stimulating IgG production, often associated with Th2 polarization. It can be noted that there is a very high signal observed for the PBS control for macrophages. The “Comirnaty-like” LNP leads to very high production in macrophages and to some degree in DCs. Aside from LPS, APL-719 seems overall as strongest inducer of IL6. Figure 13 shows data for TNFα production an important pro-inflammatory cytokine, activating innate immune cells, driving Thl responses. It can be seen that APL-719 containing LNPs stimulate a response to a similar extent as CAF01 in monocytes and DCs.

[0070] The foregoing description of various forms of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Numerous modifications or variations are possible in light of the above teachings. The forms discussed were chosen and described to provide the best illustration of the principles of the invention and its practical application to thereby enable one of ordinary skill in the art to utilize the invention in various forms and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention as determined by the appended claims when interpreted in accordance with the breadth to which they are fairly, legally, and equitably entitled.

Claims

What is claimed is:

1. A compound having structure (I) or stereoisomers thereof or mixtures thereof:where: Y is -N+(R)3X-, each R is independently selected from methyl, ethyl, or C3-C6 linear or branched alkyl, and X is a halide or triflate anion; a, b, r, p are independently selected integers from 1-8; n is an integer from 1-3; m and q are independently selected integers from 0-8; A and B are each independently selected from -O-C(=O)-, -C(=O)-O-, -NH-C(=O)-, and -C(=O)-NH-; L and M are each independently selected from -CH2-, -O-C(=O)-, -C(=O)-O-, -NH-C(=O)-, and -C(=O)-NH-; and F and G are independently selected from H and aliphatic alkyl C4-C100 groups optionally substituted with one or more of alkenyl, alkynyl, hydroxyl, amide, ester, and / or ether groups, with the provisos: i) at least one of F and G comprises at least one branch, and ii) F and G are not both H.

2. The compound of claim 1, wherein the at least one branch of at least one of F and G is via an ester group.

3. The compound of claim 1 or claim 2, wherein the at least one branch of at least one of F and G is via an amide group.

4. The compound of any of claims 1-3, wherein at least one of F or G is substituted with at least one ester group.

5. The compound of any of claims 1-4, wherein at least one of F or G is substituted with at least one alkenyl group.

6. The compound of any of claims 1-5, wherein at least one of F or G is substituted with at least one amide group.

7. The compound of any of claims 1-6, wherein A is -O-C(=O)- or -C(=O)-O8. The compound of any of claims 1-7, wherein B is -O-C(=O)- or -C(=O)-O-.

9. The compound of any of claims 1-8, wherein A is -NH-C(=O)- or C(=O)-NH-.

10. The compound of any of claims 1-9, wherein B is -NH-C(=O)-, or C(=O)-NH-11. The compound of any of claims 1-10, wherein L is -O-C(=O)- or -C(=O)-O-.

12. The compound of any of claims 1-11, wherein M is -O-C(=O)- or -C(=O)-O13. The compound of any of claims 1-12, wherein L is -NH-C(=O)- or C(=O)-NH-.

14. The compound of any of claims 1-13, wherein M is -NH-C(=O)-, and C(=O)-NH- 15. The compound of any of claims 1-14, wherein L is -CH2-.

16. The compound of any of claims 1-15, wherein M is -CH2-.

17. The compound of claim 1, wherein the compound comprises at least one of the structures (II), (III), (IV), (V), (VI), (VII), (VIII), (IX),or stereoisomers thereof, or a combination of one or more thereof:

18. A therapeutic agent comprising a drug, oligonucleotide, antibody, enzyme, protein, small molecule, or nucleotide, in a form of a cationic emulsion, or in a form of a lipoplex, or in a form of a polyplex, or in a form of a lipid nanoparticle, or in a form of a liposome, or in a form of a micelle, or in a form of an emulsion comprising a compound having a structure of any of claims 1-17 or a mixture of any one of more thereof.

19. A pharmaceutical composition comprising the therapeutic agent of claim 18 and a pharmaceutically acceptable carrier.

20. A method of treatment, comprising administering the therapeutic agent of claim 18 or the pharmaceutical composition of claim 19 to a patient in need thereof.

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