Lipids for nucleic acid delivery

Abstract

Novel lipids for delivery of macromolecules, such as nucleic acids, into eukaryotic cells and tissues are provided. The lipids may be used alone, in combination with other lipids, and / or in combination with other transfection enhancing agents to prepare transfection complexes and complexes for in vivo delivery of bioactive agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a 371st national phase application of international application number PCT / US2022 / 040256 filed on August 12, 2022, which claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 397,287 filed on August 11, 2022 and U.S. Provisional Application Serial No. 63 / 232,535 filed on August 12, 2021 under 35 U.S.C. §119(e), the contents of which are hereby expressly incorporated herein by reference in their entirety, as if fully set forth herein. Technical Field

[0003] The present disclosure generally relates to lipid compositions and methods for delivering macromolecules, such as nucleic acids, into eukaryotic cells and tissues. Background Art

[0004] Transfection agents (such as lipid aggregates comprising cationic lipid components) have been used to deliver large anionic molecules (such as nucleic acids) to certain types of cells. See Felgner et al., Nature 337: 387-388 (1989); Proc. Natl. Acad. Sci. USA 84: 7413 (1987). However, these agents are not universally effective in all cell types, and their effectiveness is different for different types of nucleic acids. In many cases, a single cationic lipid is ineffective or only partially effective for transfection. In addition, these methods do not work for all cell types, generally require relatively complex protocols and are inconvenient. In particular, these methods are not suitable for in vivo delivery of nucleic acids or delivery to specific cells or tissues, such as delivery of DNA or RNA vaccines. Therefore, it is clearly desirable to introduce new and improved methods for macromolecules and specifically nucleic acids into cells. Specifically, it is highly desirable to introduce nucleic acids into a wider variety of cells and specifically into improved methods in tissues. Summary of the invention

[0005] Provide new compound, composition and method, it improves the efficiency of macromolecule such as nucleic acid introduced into cell, for example, for nucleic acid and / or protein in vivo or in vitro delivery.Provide new lipid molecules, and compositions containing these lipids and methods for transfection using these new lipid molecules and compositions.Cationic lipid / ionizable lipid can be used for transfection alone, or advantageously, they can be used in transfection compositions with additional reagent combination.For example, cationic lipid can be combined with any combination of one or more neutral lipids, additional cationic lipid / ionizable lipid, one or more cell surface ligands, one or more fusion enhancers, one or more endosome release agents and / or one or more nuclear localization agents or aforementioned substances.The composition gained can be compounded with one or more macromolecules, such as ribose (for example, DNA, siRNA, mRNA, miRNA etc.), protein or nucleic acid and protein combination (for example, ribonucleoprotein complex), and for macromolecule delivery to eukaryotic cells, and for nucleic acid delivery to tissues particularly effective.

[0006] Provided are compounds having structure (I)

[0007]

[0008] in

[0009] L2 is selected from optionally -OR at up to 2 positions 9 Substituted C3-C 12 Alkylene, C3-C 12 Carbon ring, C3-C 12 Heterocyclic and -(CH2) m CHOR 9 (CH2) n CHOR 9 (CH2) p - the group consisting of, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8;

[0010] L1 and L3 are selected from chemical bonds, -CO q -, wherein q is 1 or 2, C2-C8 alkyl optionally interrupted by N, O or -C(O)O-, monounsaturated C4-C8 alkenyl, -CO optionally interrupted by N, O or -C(O)O- q C2-C8 alkyl and -CO q -monounsaturated C4-C8 alkenyl group, or L1X and L3Y are independently -CR 3 =C(R 4 )R 5 ;

[0011] Each L1 and L3 can be independently -OR'd at up to 2 positions 9 replace;

[0012] R 1 and R 2 Independently H, C8-C 20 Alkyl or monounsaturated C8-C 20 alkenyl, provided that when L1X and / or L3Y are independently -CR 3 =C(R 4 )R 5 When R 1 and R 2 Not H;

[0013] R 9 Selected from H, -OH, -M-C8-C 20 Alkyl and -M-C8-C 20 alkenyl, wherein M is selected from the group consisting of a chemical bond, -C(=O)-, -C(=O)N(R6)-, -N(R6)C(=O)-, -N(R6)C(=O)N(R7)-, -C(=O)O-, -OC(=O)-, -S-, -SS-, -C=S-, -C(=S)O-, -C(=O)S-, -SC(=O)- and -(R6)P(=O)-;

[0014] X and Y are independently selected from the group consisting of:

[0015] L4-Het, wherein L4 is C1-C4 alkylene or -(CH2) m CHOR 9 (CH2) n CHOR 9 (CH2) p -, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8, and Het is a C4-C 12 Heterocyclic rings,

[0016]

[0017] Provided that either X or Y, but not both, may be selected from -H, -C1-C 20 Alkyl, -NH2 and -NH-C1-C 20 The group composed of;

[0018] R 3 and R 4 Each independently selected from H, C1-C 20 Alkyl and C3-C6 cycloalkyl, HDMS, DHDMS, R 12 And by N* , R 3 and R 4 A group consisting of an optionally substituted 3 to 7 membered heterocyclic ring;

[0019] R 5 Selected from the group consisting of H, C1-C6 alkyl and C3-C6 cycloalkyl;

[0020] R 6 To R 8 Selected from the group consisting of H and C1-C4 alkyl;

[0021] Q is selected from C3-C4 alkyl optionally interrupted by -N(H)- or -N(C1-C4 alkyl)- 12 Alkyl, C4-C 12 the group consisting of alkenyl and cycloheteroalkyl;

[0022] R 10 is selected from the group consisting of H, C1-C4 alkyl or may be absent;

[0023] R 11 Selected from H, C1-C4 alkyl and -OR 9 The group composed of;

[0024] When present, R 12 selected from the group consisting of PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinyl pyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acids), wherein (i) the PEG or polymer is linear or branched; (ii) the PEG or polymer is polymerized from n subunits; (iii) n is a number average degree of polymerization between 5 and 200 units; and (iv) the compound of structure (I) has at most two R 10 Group.

[0025] In some embodiments, X and Y may be the same or different, and L1 and L3 may be the same or different. Advantageously, R 1 and R 2 Can be the same or different.

[0026] In some embodiments, X or Y or both are

[0027]

[0028] In other embodiments, X or Y or both are

[0029]

[0030] In other embodiments, L1 is -CO-, -C(=O)-, -OC(=O)-, or -C(=O)O, and X is L4Het, and in certain other embodiments, L4X is -CH=C(R 3 )R 4 .

[0031] R 3 To R 8 Independently may be H or -C1-C3 alkyl.

[0032] In some embodiments, L2 can be -CH2CH(OR 9 )CH(OR 9 )CH2-.

[0033] In certain embodiments, R 9 is H, and R 1 and R 2 is not H. In other embodiments, R 9 Not H, and R 1 and R 2 is H; for example, R 9 Can be -C 18 Alkyl or -CO-C 14 -C 18 Alkyl, and R 1 and R 2 It's H.

[0034] Also provided are compositions containing a compound as described above and at least one additional lipid, wherein the lipid can be a neutral lipid or a cationic lipid / ionizable lipid. The cationic lipid can be selected, for example, from the group consisting of DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, dihydroxy dimyristyl spermine (DHDMS), hydroxy dimyristyl spermidine (HDMS), N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2 ,3-dipalmityloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmityloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, L-spermine-5-carboxy-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine, 3,5-(N, N-dilysyl)-diaminobenzoyl-glycyl-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine), L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, L-lysine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine, L-lysine-bis-(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl]piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4,-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-myristylamino]-butane-2,3-diol, 1,4,-bis[(3-oleylamino)propyl]piperazine, L-arginine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, bis[(3-(3-aminopropyl)-myristylamino)2-hydroxypropyl]piperazine, L-arginine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-serine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesteryl-3β-carboxy-amidoethylenetrimethylammonium iodide, 1,4-bis[( 3-Myristylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol carboxylate iodide, cholesteryl-3β-carboxamidoethyleneamine, cholesteryl-3β-oxysuccinamideethylenetrimethylamine iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinic acid iodide, 2-[(2-trimethylammonio)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinic acid iodide, 3β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitylamino)propyl]piperazine, L-ornithineglycyl-N-(1-heptadecylooctadecyl)glycinamide, N, 2 ,N 5 -bis(3-aminopropyl)-L-ornithineglycyl-N-(1-heptadecanyloctadecyl)glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dioctadecyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine,

[0035] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioctadecyl-L-α-asparagine, N-[N 2 -[N2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-diol-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine,

[0036] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioleyl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-asparagine,

[0037] N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dipalmitoyl-L-glutamine]-L-glutamic acid, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dimyristyl-L-glutamine,

[0038] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)-2-hydroxypropyl]piperazine, N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dilauryl-L-glutamine, N 2 -[N 2 ,N 5 -Bis(aminopropyl)-L-ornithine]-NN-dilauryl-L-α-glutamine,

[0039] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauryl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dilauryl-L-glutaminyl]-L-glutamic acid, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-dioctadecyl-9-enylpropionamide, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-bispalmitylpropionamide, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-bispalmitylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)- [(3-aminopropyl)-bis-(2-tetradecylethoxy)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleylethoxy)]methylammonium bromide,

[0040] [(3-aminopropyl)-bis-(2-palmitylethoxy)]methylammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane,

[0041] Myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-amino-propyl)-4-(3-amino-propylamino)-4-tetradecylcarbamoyl-butylcarbamoyl cholesterol ester, (3-amino-propyl)-4-(3-amino-propylamino)-4-carbamoyl-butylcarbamoyl cholesterol ester, (3-amino-propyl)-4-(3-amino-propylamino)-4-(2-dimethylamino-ethylcarbamoyl)-butylcarbamoyl cholesterol ester, spermine-5-carboxyglycine (N'-stearyl-N'-oleyl) amide tetrakis trifluoroacetate, spermine-5-carboxyglycine (N'-stearyl-N'-oleyl) amide Amine tetrakis trifluoroacetate, agmatine carboxy cholesterol acetate, spermine-5-carboxy-β-alanine cholesterol ester tetrakis trifluoroacetate, 2,6-diaminohexenyl β-alanine cholesterol ester bis trifluoroacetate, 2,4-diaminobutyryl β-alanine cholesterol ester bis trifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesterol ester tris trifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl]aminocarboxy cholesterol ester, stearyl carnitine ester, palmitoyl carnitine ester, myristyl carnitine ester, stearyl stearoyl carnitine ester hydrochloride, L-stearyl stearoyl carnitine ester, stearyl oleoyl carnitine ester hydrochloride, palmityl palmitoyl carnitine ester hydrochloride, myristyl myristyl Carnitine ester hydrochloride, L-myristyl myristoyl carnitine ester hydrochloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine, N-(3-aminopropyl)-N,N'-bis-(dodecylethoxy)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(oleylethoxy)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(palmitylethoxy)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(myristylethoxy)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(myristylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecylethoxy)-piperazine bromide, N-(3-aminopropyl)- N'-Methyl-N,N'-(bis-2-oleylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmitylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristylethoxy)-piperazine bromide, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxy-propyl]piperazine and 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazine, KL22, KL25, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]dioxolane (DLin-K-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA or MC3), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z ,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA(2S)). ,

[0042] The neutral lipid may, for example, be selected from the group consisting of sterols or sterol derivatives, phospholipids, or a combination thereof.

[0043] The composition may also contain a phospholipid selected from the group consisting of, for example, DOPE, DPhPE, cholesterol, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine). The composition may also contain at least one additional neutral lipid, which may be, for example, a phospholipid selected from the group consisting of DOPE, DPhPE, cholesterol, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine) and Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine).

[0044] The composition may also contain a cell targeting peptide, a nuclear localization peptide, a fusogenic agent and / or a peptide endosomal releasing agent. Each of these peptides may optionally contain a polycationic nucleic acid binding moiety.

[0045] The composition may further contain a stabilizer or a non-peptide endosomal releasing agent.

[0046] The composition may contain a nucleic acid, which may be, for example, an RNA molecule. In some embodiments, the nucleic acid may be an mRNA molecule.

[0047] Also provided is a method for introducing a nucleic acid into a eukaryotic cell, wherein the cell is contacted with the above-mentioned composition and the nucleic acid. The cell can be, for example, an animal cell or a human cell. The contact between the composition and the nucleic acid and the cell can occur in vivo, in vitro or in vitro.

[0048] Also provided are methods for delivering a lipid composition to spleen tissue and / or lung tissue of a subject, wherein the composition as described above is administered to the subject.

[0049] Also provided is a method for delivering a bioactive agent to the spleen tissue and / or lung tissue of a subject, the method comprising: (i) mixing the bioactive agent with the composition as described above to form a bioactive agent-lipid complex bioactive agent; and (ii) administering an effective amount of the bioactive agent-lipid complex to the subject, thereby delivering the bioactive agent-lipid complex to the spleen tissue and / or lung tissue of the subject.

[0050] Through the following specific embodiments, other purposes, features and advantages of the present invention will become apparent. However, it should be understood that the specific embodiments and specific examples, although indicating preferred embodiments of the present invention, are given by way of illustration only, because according to the specific embodiments, various changes and modifications within the essence and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figures 1A to 1C Figure 3 depicts HeLa cells transfected with lipid-DNA formulations ( Figure 1A )、HDFa cells( Figure 1B ) and A549 cells ( Figure 1C ) is a graph of GFP fluorescence intensity (in relative fluorescence units) in .

[0052] FIG. 2A to FIG. 2C Figure 3 is a graph depicting MCF7 cells transfected with lipid-DNA formulations ( Figure 2A )、HEK293( Figure 2B ) and CHO-K1 cells ( Figure 2C ) is a graph of GFP fluorescence intensity (in relative fluorescence units) in .

[0053] Figure 3 is a graph depicting GFP fluorescence intensity (in relative fluorescence units) in HEK-293 cells after transfection using lipid-DNA formulations.

[0054] Figure 4is a graph depicting GFP fluorescence intensity (in relative fluorescence units) in HeLa cells after transfection using lipid-DNA formulations.

[0055] Figure 5 is a graph depicting GFP fluorescence intensity (in relative fluorescence units) in MCF7 cells after transfection using lipid-DNA formulations.

[0056] Figure 6 is a graph depicting GFP fluorescence intensity (in relative fluorescence units) in HDFa cells after transfection using lipid-DNA formulations.

[0057] Figure 7 is a graph depicting GFP fluorescence intensity (in relative fluorescence units) in A549 cells after transfection using lipid-DNA formulations.

[0058] Figure 8 is a graph depicting GFP fluorescence intensity (in relative fluorescence units) in HEK293 cells after transfection using lipid-DNA formulations.

[0059] Fig. 9 is a graph depicting luciferase activity (measured in bioluminescent flux, photons / second (p / s)) in the lungs of mice following intravenous administration of lipid-mRNA formulations.

[0060] Fig.10 is a graph depicting luciferase activity (measured in bioluminescent flux, photons / second (p / s)) in the spleen of mice following intravenous administration of lipid-mRNA formulations. DETAILED DESCRIPTION

[0061] Lipid molecules are provided, which can be used for improved methods of delivering macromolecules to eukaryotic cells, and are particularly effective for the delivery of various cells, tissues and organs, and provide efficient transfection. These lipid molecules are positively charged at specific pH (e.g., physiological pH), and are advantageously used for preparing complexes with one or more neutral lipids and additional components (such as fusion molecules or fusion enhancing molecules, additional cationic lipids / ionizable lipids, cell surface ligands, cell adhesion molecules, nuclear localization agents and endosome release agents) and macromolecules. Such complexes are easily prepared and stable, and are therefore suitable for in vitro, ex vivo and in vivo applications, for example, delivery of therapeutic nucleic acids (e.g., siRNA therapeutic agents, mRNA vaccine preparations, etc.), cell therapy applications (e.g., delivery of gene editing reagents), etc. For example, as shown herein, the lipid composition comprising new cationic lipids / ionizable lipids and nucleic acid payloads is used in vivo so that nucleic acid payloads are effectively delivered to lung tissue and spleen tissue.

[0062] Surprisingly, it is also found that common cationic lipids / ionizable lipids, particularly the nucleic acid transfection efficiency of new cationic lipids / ionizable lipids described herein can be significantly improved by reducing the net positive charge on lipids by any free primary amine functional groups and secondary amine functional groups on partially acylated or alkylated lipids in many cases. Unexpectedly, the reduction of this charge has been shown to greatly increase the ability of transfection complexes containing modified lipids to effectively transfect cells. Therefore, for lipids with N primary or secondary amines, acylation or alkylation up to N-1 amine groups can be performed. Those skilled in the art will recognize that in most cases, the distribution of acyl groups in lipid preparations with different amino groups will be statistical, because unless acylation is performed as a part of a more sophisticated synthesis scheme, regiospecific acylation may be impossible. Therefore, initially (in non-acylated amines) and during the reaction, the distribution of acyl groups is not only affected by the stoichiometric influence of acylating agents relative to lipids, but also by the reactivity of amine groups, because the acylation activity at free amines is potentially affected by the acylation at another amine in other places in the molecule.

[0063] The enhancement of transfection is particularly pronounced for lipids containing 4 or more reactive amines in addition to possible tertiary or quaternary amines, but is not necessarily limited to these lipids. This observation is surprising in view of the bias in the art that cationic lipids are expected to have relatively high charges on them to enhance binding of negatively charged nucleic acids.

[0064] As used herein, the term "ionizable lipid" refers to a lipid having one or more functional groups that can be reversibly ionized (protonated or deprotonated) depending on the pH of the medium containing the lipid. The functional group can be basic, such as an amino functional group, or can be acidic, such as a carboxylic acid moiety. Those skilled in the art will appreciate that other ionizable functional groups can also be used. The ionizable lipid can contain both a basic moiety and an acidic moiety. Advantageously, the ionizable lipid carries an overall positive charge at physiological pH.

[0065] Cationic lipids / ionizable lipids

[0066] Provided are compounds having structure (I)

[0067]

[0068] in

[0069] L2 is selected from optionally -OR at up to 2 positions 9 Substituted C3-C 12 Alkylene, C3-C 12 Carbon ring, C3-C 12 Heterocyclic and -(CH2) mCHOR 9 (CH2) n CHOR 9 (CH2) p - the group consisting of, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8;

[0070] L1 and L3 are selected from chemical bonds, -CO q -, wherein q is 1 or 2, C3-C8 alkyl optionally interrupted by N, O or -C(O)O-, monounsaturated C4-C8 alkenyl, -CO optionally interrupted by N, O or -C(O)O- q C2-C8 alkyl and -CO q -monounsaturated C4-C8 alkenyl group, or L1X and L3Y are independently -CR 3 =C(R 4 )R 5 ;

[0071] Each L1 and L3 can be independently -OR'd at up to 2 positions 9 replace;

[0072] R 1 and R 2 Independently H, C8-C 20 Alkyl or monounsaturated C8-C 20 alkenyl, provided that when L1X and / or L3Y are independently -CR 3 =C(R 4 )R 5 When R 1 and R 2 Not H;

[0073] R 9 Selected from H, -OH, -M-C8-C 20 Alkyl and -M-C8-C 20 alkenyl, wherein M is selected from the group consisting of a chemical bond, -C(=O)-, -C(=O)N(R6)-, -N(R6)C(=O)-, -N(R6)C(=O)N(R7)-, -C(=O)O-, -OC(=O)-, -S-, -SS-, -C=S-, -C(=S)O-, -C(=O)S-, -SC(=O)- and -(R6)P(=O)-;

[0074] X and Y are independently selected from the group consisting of:

[0075] L4-Het, wherein L4 is C1-C4 alkylene or -(CH2) m CHOR 9 (CH2) nCHOR 9 (CH2) p -, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8, and Het is a C4-C 12 Heterocyclic rings,

[0076]

[0077] Provided that either X or Y, but not both, may be selected from -H, -C1-C 20 Alkyl, -NH2 and -NH-C1-C 20 The group composed of;

[0078] R 3 and R 4 Each independently selected from H, C1-C 20 Alkyl and C3-C6 cycloalkyl, HDMS, DHDMS, R 12 And by N * , R 3 and R 4 A group consisting of an optionally substituted 3 to 7 membered heterocyclic ring;

[0079] R 5 Selected from the group consisting of H, C1-C6 alkyl and C3-C6 cycloalkyl;

[0080] R 6 To R 8 Selected from the group consisting of H and C1-C4 alkyl;

[0081] Q is selected from C3-C4 alkyl optionally interrupted by -N(H)- or -N(C1-C4 alkyl)- 12 Alkyl, C4-C 12 the group consisting of alkenyl and cycloheteroalkyl;

[0082] R 10 is selected from the group consisting of H, C1-C4 alkyl or may be absent;

[0083] R 11 Selected from H, C1-C4 alkyl and -OR 9 The group composed of;

[0084] When present, R 12selected from the group consisting of PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinyl pyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acids), wherein (i) the PEG or polymer is linear or branched; (ii) the PEG or polymer is polymerized from n subunits; (iii) n is a number average degree of polymerization between 5 and 200 units; and (iv) the compound of structure (I) has at most two R 10 Group.

[0085] In certain embodiments, the compound of structure (I), wherein X or Y is each independently selected from the group consisting of:

[0086]

[0087] as well as

[0088]

[0089] s = 0, 1, or 2

[0090] t=2, 3, 4 or 5.

[0091] In certain embodiments, the cationic lipid / ionizable lipid provided herein is a compound of structure (I) or other compound selected from the group comprising the compounds in Table 1.

[0092] Table 1

[0093]

[0094]

[0095]

[0096]

[0097] In some embodiments of compounds having structure (I), X and Y may be the same or different, and L1 and L3 may be the same or different. Advantageously, R 1 and R 2 Can be the same or different.

[0098] In some embodiments, X or Y or both are

[0099]

[0100] In other embodiments, X or Y or both are

[0101]

[0102] In other embodiments, L1 is -CO-, -C(=O)-, -OC(=O)-, or -C(=O)O, and X is L4Het, and in certain other embodiments, L4X is -CH=C(R 3 )R 4 .

[0103] R 3 To R 8 Independently may be H or -C1-C3 alkyl.

[0104] In some embodiments, L2 can be -CH2CH(OR 9 )CH(OR 9 )CH2-.

[0105] In certain embodiments, R 9 is H, and R 1 and R 2 is not H. In other embodiments, R 9 Not H, and R 1 and R 2 is H; for example, R 9 Can be -C 18 Alkyl or -CO-C 14 -C 18 Alkyl, and R 1 and R 2 It's H.

[0106] R 1 and R 2 independently can be H, C8-C 20 Alkyl or monounsaturated C8-C 20 Alkenyl.

[0107] R 3 To R 8 can be independently selected from H, -C1-C6 alkyl- and C3-C6 cycloalkyl. In a specific example, R 3 To R 8 are independently H or C1-C3 alkyl.

[0108] R 9 Can be H, -CO-, C8-C 20 Alkyl, monounsaturated -CO-, C8-C 20 Alkenyl, C8-C 20 Alkyl or monounsaturated C8-C 20 Specific examples include: 9 It is C 14 -C 18 Alkyl or monounsaturated alkenyl; wherein R9 is H, and R 1 and R 2 Not H; where R 9 Not H, and R 1 and R 2 is H; and where R 9 It is C 14 -C 18 Alkyl or -CO, -C 14 -C 18 Alkyl, and R 1 and R 2 is H. Advantageously, when present, R 9 The double bond in is a cis double bond.

[0109] In these molecules, X and Y may be the same or different, L1 and L3 independently may be the same or different, and R 1 and R 2 Can be the same or different.

[0110] In a specific embodiment, L2 can be C4-C 12 In other embodiments, L2 is advantageously -(CH2) m CHOR 9 (CH2) n CHOR 9 (CH2) p -, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8. In a specific example, L2 is -CH2CH(OR 9 )CH(OR 9 )CH2-.

[0111] In other embodiments, the molecule may contain a heterocyclic moiety connected via an amide bond or a carbamate bond, wherein L1 is -CO-, -C(=O)-, -OC(=O)-, or -C(=O)O-, and X is L4Het, wherein Het is a heterocyclic ring as defined below. In yet other embodiments, when R 1 and / or R 2 Instead of H, the molecule may contain an enamine moiety where L1X is -CR 5 =C(R 3 )R 4 In such enamine moieties, R3 and R5 may together form a C3-C7 carbocyclic ring.

[0112] The molecule of structure (I) has a substituent R 1 , R 2 Each or all of L2, L3, X, and Y may be independently symmetrical or asymmetrical; that is, each R 1can be the same or different, each R 2 can be the same or different, L1 and L3 can be the same or different, and X and Y can be the same or different. In addition, the structure of L2 does not have to be symmetrical.

[0113] definition

[0114] Compounds are generally described herein using standard nomenclature. For compounds with asymmetric centers, unless otherwise specified, all stereoisomers of the compound and their mixtures are encompassed. Non-limiting examples of stereoisomers include enantiomers, diastereomers, and E isomers or Z isomers. When the compound exists in various tautomeric forms, the compound is intended to include all tautomeric forms. General formulas including variables (e.g., X, L1, L2, L3, Y, etc.) are used herein to describe certain compounds. Unless otherwise specified, each variable in such a formula is defined independently of any other variable, and any variable that appears more than once in the formula is defined independently at each occurrence. If a part is described as being "independently" selected from a group, each part is selected independently of one another. Therefore, each part may be the same or different from another part.

[0115] The number of carbon atoms in the hydrocarbon portion may be indicated by the prefix "C x -C y ” means, where x is the minimum number of carbon atoms in the moiety and y is the maximum number of carbon atoms. Thus, for example, “C1-C6 alkyl” refers to an alkyl substituent containing from 1 to 6 carbon atoms. By further example, C3-C6 cycloalkyl means a saturated hydrocarbon ring containing from 3 to 6 carbon ring atoms. A prefix attached to a multi-component substituent applies only to the first component immediately following the prefix. For illustration, the term “carbocyclylalkyl” contains two components: a carbocyclyl group and an alkyl group. Thus, for example, C3-C6 carbocyclylC1-C6 alkyl refers to a C3-C6 carbocyclyl group attached to the parent molecular moiety through a C1-C6 alkyl group.

[0116] Unless otherwise indicated, when a linking element connects two other elements in a depicted chemical structure, the component depicted on the far left of the linking element is bound to the left element in the depicted structure, and the component depicted on the far right of the linking element is bound to the right element in the depicted structure. S -ML S ”- and M is-N(R B )S(O)-, the chemical structure is -L S -N(R B )S(O)-L S ”-.

[0117] If a connecting element in a depicted structure is a bond, then the element to the left of the connecting element is directly connected to the element to the right of the connecting element via a covalent bond. For example, if a chemical structure is depicted as -L S -ML S ' and M is chosen as a bond, the chemical structure will be "-L S -L S -". If two or more adjacent connecting elements in a depicted structure are bonds, then the elements to the left of those connecting elements are directly connected to the elements to the right of those connecting elements via a covalent bond. For example, if a chemical structure is depicted as "-L S -ML S -M'-L S '-" and M and L S ' is selected as a bond, the chemical structure will be "-L S -M'-L S- Similarly, if the chemical structure is depicted as -L S -ML S ”-M'-L S ”-, and M, L S ' and M' are bonds, then the chemical structure will be -L S -L S ”-. When a chemical formula is used to describe a moiety, a dash indicates the portion of the moiety that has a free valence.

[0118] If a part is described as "optionally substituted", the part can be substituted or unsubstituted. If a part is described as optionally substituted by a non-hydrogen radical of at most a specific number, the part can be unsubstituted, or substituted by a non-hydrogen radical of at most the specific number or by a maximum number of substitutable positions on the part, whichever is smaller. Thus, for example, if a part is described as a heterocycle optionally substituted by at most three non-hydrogen radicals, any heterocycle with less than three substitutable positions will be optionally substituted by at most only as many non-hydrogen radicals as the heterocycle has substitutable positions. For example, tetrazolyl (which has only one substitutable position) will be optionally substituted by at most one non-hydrogen radical. Similarly, if amino nitrogen is described as optionally substituted by at most two non-hydrogen radicals, primary amino nitrogen will be optionally substituted by at most two non-hydrogen radicals, and secondary amino nitrogen will be optionally substituted by at most only one non-hydrogen radical.

[0119] When a moiety is substituted with oxo or thio, it means that the moiety contains a carbon atom (eg, CH2) covalently bonded to at least two hydrogens, and the two hydrogen radicals are substituted with oxo or thio to form C=O or C=S, respectively.

[0120] The term "alkenyl" means a straight or branched hydrocarbon chain containing one or more double bonds. Relative to the group substituted on the double bond carbon, each carbon-carbon double bond can have an E (cis) geometry or a Z (trans) geometry in the alkenyl portion. Examples of alkenyl radicals include, but are not limited to, vinyl, E-propenyl and Z-propenyl, isopropenyl, E-butenyl and Z-butenyl, E-isobutenyl and Z-isobutenyl, E-pentenyl and Z-pentenyl, E-hexenyl and Z-hexenyl, E, E-hexadienyl, E, Z-hexadienyl, Z, E-hexadienyl and Z, Z-hexadienyl, etc.

[0121] The term "alkenylene" refers to a divalent unsaturated hydrocarbon chain which may be linear or branched and has at least one carbon-carbon double bond. Non-limiting examples of alkenylene include -C(H)=C(H)-, -C(H)=C(H)-CH2-, -C(H)=C(H)-CH2-CH2-, -CH2-C(H)=C(H)-CH2-, -C(H)=C(H)-CH2-, -C(H)=C(H)-CH-(CH3)-, and -CH2-C(H)=C(H)-CH-(CH2CH3)-.

[0122] The term "alkyl" means a straight or branched saturated hydrocarbon chain. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl.

[0123] The term "alkylene" refers to a divalent saturated hydrocarbon chain, which may be linear or branched. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH(CH3)CH2-.

[0124] The term "alkynyl" means a straight or branched hydrocarbon chain containing one or more triple bonds. Non-limiting examples of alkynyl include ethynyl, 1-propynyl, 2-propynyl, 3-propynyl, decynyl, 1-butynyl, 2-butynyl, and 3-butynyl.

[0125] The term "alkynyl", alone or in combination with any other terms, refers to a straight or branched chain hydrocarbon radical having one or more triple bonds, containing the specified number of carbon atoms, or where no number is specified, in one embodiment from 2 to about 10 carbon atoms. Examples of alkynyl radicals include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, pentynyl, and the like.

[0126] The term "alkoxy" refers to an alkyl ether radical, wherein the term "alkyl" is as defined above. Examples of suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and the like.

[0127] The term "aryl", alone or in combination with any other term, refers to a carbocyclic aromatic radical (such as phenyl or naphthyl) containing the specified number of carbon atoms, in one embodiment 6 to 15 carbon atoms (i.e. (C 6-15 ) aryl), and in another embodiment 6 to 10 carbon atoms (i.e. (C 6-10 ) aryl), optionally substituted with one or more substituents selected from alkyl, alkoxy (e.g., methoxy), nitro, halogen (e.g., chlorine), amino, carboxylate and hydroxy. Examples of aryl radicals include, but are not limited to, phenyl, p-tolyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl, etc.

[0128] The term "aralkyl", alone or in combination, means an alkyl radical as defined above wherein one of the hydrogen atoms is phenyl, benzyl, 2-phenylethyl, and the like.

[0129] The term "aralkyloxycarbonyl", used alone or in combination, means a radical of the formula -C(O)-O-aralkyl, wherein the term "aralkyl" has the meaning given above. An example of an aralkyloxycarbonyl radical is benzyloxycarbonyl.

[0130] The term "aryloxy", used alone or in combination, means a radical of the formula aryl-O-, the term "aryl" having the significance given above.

[0131] The term "alkynylene" refers to a divalent unsaturated hydrocarbon group that may be linear or branched and has at least one carbon-carbon triple bond. By way of example, representative alkynylene groups include -C≡C-, -C≡C-CH2-, -C≡C-CH2-CH2-, -CH2-C≡C-CH2-, -C≡C-CH(CH3)-, and -CH2-C≡C-CH(CH2CH3)-.

[0132] The term "alkanoyl," used alone or in combination, means an acyl radical derived from an alkanoic acid, examples of which include acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, and the like.

[0133] The term "aryloxyalkanoyl" is intended to indicate an acyl radical of the formula aryl-O-alkanoyl in which aryl and alkanoyl have the meanings given above.

[0134] The term "aralkanoyl" means an acyl radical derived from an aryl-substituted alkanecarboxylic acid, such as phenylacetyl, 3-phenylpropionyl(hydrocinnamoyl), 4-phenylbutyroyl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-phenylbutyroyl, (1-naphthyl)acetyl, 4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl, and the like.

[0135] The term "aroyl" means an acyl radical derived from an aromatic carboxylic acid. Examples of such radicals include aromatic carboxylic acids, optionally substituted benzoic acid or naphthoic acid, such as benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl, 4-(benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2-naphthoyl, 6-(benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl, 3-hydroxy-2-naphthoyl, 3-(benzyloxycarbonyl)-2-naphthoyl, etc.

[0136] The term "aminocarbonyl", used alone or in combination, means an amino-substituted carbonyl (carbamoyl) derived from an amino-substituted carboxylic acid, wherein the amino group can be a primary amino group continuous substituent, a secondary amino group continuous substituent, or a tertiary amino group continuous substituent selected from hydrogen radicals, alkyl radicals, aryl radicals, aralkyl radicals, cycloalkyl radicals, cycloalkylalkyl radicals, and the like.

[0137] The term "aminoalkanoyl" means an acyl radical derived from an amino-substituted alkanecarboxylic acid, wherein the amino group may be a primary, secondary or tertiary amino group containing a substituent selected from the group consisting of a hydrogen radical, a cycloalkyl radical, a cycloalkylalkyl radical, and the like, examples of which include N,N-dimethylaminoacetyl and N-benzylaminoacetyl.

[0138] The term "carbocycle" or "carbocyclic" or "carbocyclyl" refers to a saturated (e.g., "cycloalkyl"), partially saturated (e.g., "cycloalkenyl" or "cycloalkynyl"), or fully unsaturated (e.g., "aryl") 3- to 8-membered carbocyclic ring system containing zero heteroatom ring atoms. "Ring atoms" or "ring members" are atoms that are bound together to form a ring. A carbocyclyl can be, but is not limited to, a single ring, two fused rings, or a bridged ring or a spirocycle. Substituted carbocyclyls can have cis or trans geometry. Representative examples of carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclopentadienyl, cyclohexadienyl, adamantyl, decahydro-naphthyl, octahydro-indenyl, cyclohexenyl, phenyl, naphthyl, indanyl, 1,2,3,4-tetrahydro-naphthyl, indenyl, isoindenyl, decahydronaphthyl and norpinyl.Carbocyclyl groups can be attached to the parent molecular moiety by any replaceable carbon ring atom. In the case where the carbocyclyl group is a divalent part connecting two other elements in the depicted chemical structure, the carbocyclyl group can be attached to the two other elements by any two replaceable ring atoms. Similarly, in the case where the carbocyclyl group is a trivalent part connecting three other elements in the depicted chemical structure, the carbocyclyl group can be attached to the three other elements by any three replaceable ring atoms respectively. Carbocycle can be attached to any bridge ring carbon atom producing a stable structure. In one embodiment, the carbocycle has 5 to 7 carbons.

[0139] The term "cycloalkyl" refers to a saturated carbocyclyl group containing zero heteroatom ring members. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decalinyl, and norpinanyl.

[0140] The term "cycloalkyl", used alone or in combination, means an alkyl radical containing about 3 to about 8 carbon atoms and being cyclic. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0141] The term "cycloalkylalkyl" means an alkyl radical as defined above substituted with a cycloalkyl radical containing from about 3 to about 8, and in one embodiment from about 3 to about 6, carbon atoms.

[0142] The term "cycloalkylcarbonyl" means an acyl group derived from a monocyclic or bridged cycloalkanecarboxylic acid, such as cyclopropanecarbonyl, cyclohexanecarbonyl, adamantanecarbonyl, etc., or an acyl group derived from a benzo-fused monocyclic cycloalkanecarboxylic acid optionally substituted with, for example, alkanoylamino, such as 1,2,3,4-tetrahydro-2-naphthoyl, 2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl.

[0143] The term "cycloalkylalkoxycarbonyl" means an acyl radical derived from a cycloalkylalkoxycarboxylic acid of formula cycloalkylalkyl-O-COOH wherein cycloalkylalkyl has the significance given above.

[0144] The term "carbocyclylalkyl" refers to a carbocyclyl group attached to the parent molecular moiety through an alkylene group. For example, C3-C6carbocyclylC1-C6alkyl refers to a C3-C6carbocyclyl group attached to the parent molecular moiety through a C1-C6alkylene group.

[0145] The term "cycloalkenyl" refers to a non-aromatic, partially unsaturated carbocyclyl moiety having zero heteroatom ring members. Representative examples of cycloalkenyl groups include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, and octahydronaphthyl.

[0146] The prefix "halo" means that the substituent to which the prefix is ​​attached is substituted by one or more independently selected halogen radicals. For example, "C1-C6 haloalkyl" means a C1-C6 alkyl substituent in which one or more hydrogen atoms are substituted by independently selected halogen radicals. Non-limiting examples of C1-C6 haloalkyl include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl and 1,1,1-trifluoroethyl. It should be appreciated that if a substituent is substituted by more than one halogen radical, those halogen radicals may be the same or different (unless otherwise stated).

[0147] The term "heterocycle" or "heterocyclic" or "heterocyclyl" refers to a saturated (e.g., "heterocycloalkyl"), partially unsaturated (e.g., "heterocycloalkenyl" or "heterocycloalkynyl") or fully unsaturated (e.g., "heteroaryl") ring system, wherein at least one of the ring atoms is a heteroatom (i.e., nitrogen, oxygen or sulfur), and the remaining ring atoms are independently selected from the group consisting of carbon, nitrogen, oxygen and sulfur. The heterocycle can be, but is not limited to, a single ring, two fused rings, or a bridged ring or a spirocycle. The heterocyclic group can be connected to the parent molecular part via any substitutable carbon atom or nitrogen atom in the group. In the case where the heterocyclic group is a divalent part connecting two other elements in the depicted chemical structure, the heterocyclic group can be attached to the two other elements by any two substitutable ring atoms. Similarly, in the case where the heterocyclic group is a trivalent part connecting three other elements in the depicted chemical structure, the heterocyclic group can be attached to the three other elements by any three substitutable ring atoms, respectively.

[0148] In the compounds of the present invention, "Het" represents a heterocycle containing 4 to 12 carbon atoms, wherein at least one nitrogen atom is present in the ring. The heterocyclic radical can be, but is not limited to, a monocyclic ring containing a single ring. Non-limiting examples of monocyclic rings include furanyl, dihydrofuranyl, tetrahydrofuranyl, pyrrolyl, isopyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, isoimidazolyl, imidazolinyl, imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl, tetrazolyl, dithiol, oxathiol, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, thiadiazolyl, oxathiazolyl, oxadiazolyl (including 1,2, 3-oxadiazolyl, 1,2,4-oxadiazolyl (also known as "oxadiazolyl"), 1,2,5-oxadiazolyl (also known as "furoxyl") and 1,3,4-oxadiazolyl), oxatriazolyl (including 1,2,3,4-oxatriazolyl and 1,2,3,5-oxatriazolyl), dioxazolyl (including 1,2,3-dioxazolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl and 1,3,4-dioxazolyl), pyridinyl, piperidinyl, dioxazolyl azinyl (including pyridazinyl (also known as "1,2-diazinyl"), pyrimidinyl (also known as "1,3-diazinyl") and pyrazinyl (also known as "1,4-diazinyl")), piperazinyl, triazinyl (including s-triazinyl (also known as "1,3,5-triazinyl), as-triazinyl (also known as "1,2,4-triazinyl) and v-triazinyl (also known as "1,2,3-triazinyl), oxazinyl (including 1,2,3-oxazinyl, 1,3,2- The invention also includes oxazinyl, 1,3,6-oxazinyl (also known as "pentyloxazolone", 1,2,6-oxazinyl and 1,4-oxazinyl), isoalloxazinyl (including o-isoalloxazinyl and p-isoalloxazinyl), oxazolidinyl, isoxazolidinyl, oxathiazinyl (including 1,2,5-oxathiazinyl or 1,2,6-oxathiazinyl), oxadiazinyl (including 1,4,2-oxadiazinyl and 1,3,5,2-oxadiazinyl), morpholinyl, azepinyl and diazepinyl.

[0149] The heterocyclic group can also be, but is not limited to, a bicyclic ring containing two fused rings, such as, for example, naphthyridinyl (including [1,8] naphthyridinyl and [1,6] naphthyridinyl), thiazolylpyrimidinyl, thienopyrimidinyl, pyrimidopyrimidinyl, pyridopyrimidinyl, pyrazolopyrimidinyl, indolizinyl, pyridoindenyl, pyranopyrrolyl, 4H-quinolizinyl, purinyl, pyridopyridinyl (including pyrido[3,4-b]-pyridinyl, pyrido[3,2-b]-pyridinyl and pyrido[4,3-b]-pyridinyl), pyridopyrimidine and pteridinyl. Other non-limiting examples of fused-ring heterocycles include benzo-fused heterocyclic groups such as indolyl, isoindolyl, indoleninyl (also known as "pseudoindolyl"), isoindazolyl (also known as "benzopyrazolyl" or indazolyl), benzopyridinyl (including quinolinyl (also known as "1-benzopyridinyl") and isoquinolinyl (also known as "2-benzopyridinyl")), benzimidazolyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including quinolinyl, phthalazinyl, quinoxalinyl, benzodiazinyl (including quinolinyl, phthalazinyl, quinoxalinyl, benzodiazinyl), benzophenone, ... quinazolinyl (also known as "1,2-benzodiazinyl" and quinazolinyl (also known as "1,3-benzodiazinyl")), benzothiazolyl, 4,5,6,7-tetrahydrobenzo[d]thiazolyl, benzothiadiazolyl, benzimidazolyl, benzotriazolyl, benzoxazinyl (including 1,3,2-benzoxazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl and 3,1,4-benzoxazinyl), benzisoxazinyl (including 1,2-benzoisoxazinyl and 1,4-benzoisoxazinyl) and tetrahydroisoquinolinyl.

[0150] The heterocyclic group may also be, but is not limited to, a spirocyclic system, such as, for example, 1,4-dioxa-8-azaspiro[4.5]decyl. The heterocyclic group may contain one or more sulfur atoms as ring members; and in some cases, the sulfur atom is oxidized to SO or SO2. The nitrogen heteroatom in the heterocyclic group may be quaternized or not, and may be oxidized or not to N-oxide. In addition, the nitrogen heteroatom may or may not be N-protected.

[0151] The heterocyclic or carbocyclic ring may be further substituted. Unless otherwise indicated, the term "substituted" refers to substitution by independently replacing one, two, three or more hydrogen atoms with substituents, including but not limited to -F, -Cl, -Br, -I, hydroxy, protected hydroxy, -NO2, -N3, -CN, -NH2, protected amino, oxo, thio, -NH-C2-C8-alkenyl, -NH-C2-C8-alkynyl, -NH-C3-C 12 -cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-C1-C 12 -alkyl, -O-C2-C8-alkenyl, alkynyl, -O-C3-C12 -cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, -C(O)-C1-C 12 -alkyl, -C(O)-C2-C8-alkenyl, -C(O)-C2-C8-alkynyl, -C(O)-C3-C 12 -cycloalkyl, -C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl, -CONH2, -CONH-C1-C 12 -alkyl, -CONH-C2-C8-alkenyl, -CONH-C2-C8-alkynyl, -CONH-C3-C 12 -cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-C1-C 12 -alkyl, -OCO2-C2-C8-alkenyl, -OCO2-C2-C8-alkynyl, -OCO2-C3-C 12 -cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2-heterocycloalkyl, -OCONH2, -OCONH-C1-C 12 -alkyl, -OCONH-C2-C8-alkenyl, -OCONH-C2-C8-alkynyl, -OCONH-C3-C 12 -cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl, -OCONH-heterocycloalkyl, -NHC(O)-C1-C 12 -alkyl, -NHC(O)-C2-C8-alkenyl, -NHC(O)-C2-C8-alkynyl, -NHC(O)-C3-C 12 -cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, -NHC(O)-heterocycloalkyl, -NHCO2-C1-C 12 -alkyl, -NHCO2-C2-C8-alkenyl, -NHCO2-C2-C8-alkynyl, -NHCO2-C3-C 12 -cycloalkyl, -NHCO2aryl, -NHCO2-heteroaryl, -NHCO2-heterocycloalkyl, -NHC(O)NH2, -NHC(O)NH-C1-C 12 -alkyl, -NHC(O)NH-C2-C8-alkenyl, -NHC(O)NH-C2-C8-alkynyl, -NHC(O)NH-C3-C 12 -aryl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, -NHC(O)NH-heterocycloalkyl, -NHC(S)NH2, -NHC(S)NH-C1-C 12-alkyl, -NHC(S)NH-C2-C8-alkenyl, -NHC(S)NH-C2-C8-alkynyl, -NHC(S)NH-C3-C 12 -cycloalkyl, -NHC(S)NH-aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH2, -NHC(NH)NH-C1-C 12 -alkyl, -NHC(NH)NH-C2-C8-alkenyl, NHC(NH)NH-C2-C8-alkynyl, -NHC(NH)NH-C3-C 12 -cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl, -NHC(NH)NH-heterocycloalkyl, -NHC(NH)-C1-C 12 -alkyl, -NHC(NH)-C2-C8-alkenyl, -NHC(NH)-C2-C8-alkynyl, -NHC(NH)-C3-C 12 -cycloalkyl, -NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl, -C(NH)NH-C1-C 12 -alkyl, -C(NH)NH-C2-C8-alkenyl, -C(NH)NH-C2-C8-alkynyl, -C(NH)NH-C3-C 12 -cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NH-heterocycloalkyl, -S(O)-C1-C 12 -alkyl, -S(O)-C2-C8-alkenyl, -S(O)-C2-C8-alkynyl, -S(O)-C3-C 12 -cycloalkyl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heterocycloalkyl, -SO2NH2, -SO2NH-C1-C 12 -alkyl, -SO2NH-C2-C8-alkenyl, -SO2NH-C2-C8-alkynyl, -SO2NH-C3-C 12 -cycloalkyl, -SO2NH-aryl, -SO2NH-heteroaryl, -SO2NH-heterocycloalkyl, -NHSO2-C1-C 12 -alkyl, -NHSO2-C2-C8-alkenyl, -NHSO2-C2-C8-alkynyl, -NHSO2-C3-C 12 -cycloalkyl, -NHSO2-aryl, -NHSO2-heteroaryl, -NHSO2-heterocycloalkyl, -CH2NH2, -CH2SO2CH3, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -C3-C 12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-C1-C 12 -alkyl, -S-C2-C8-alkenyl, -S-C2-C8-alkynyl, -S-C3-C 12 -cycloalkyl, -S-aryl, -heteroaryl, -S-heterocycloalkyl or methylthiomethyl. It should be understood that aryl, heteroaryl, alkyl, etc. may be further substituted.

[0152] The term "N-protecting group" or "N-protected" refers to those groups that are capable of protecting an amino group from undesired reactions. Commonly used N-protecting groups are described in Greene and Wuts, Protecting Groups in Chemical Synthesis (3rd ed., John Wiley & Sons, NY (1999). Non-limiting examples of N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, tert-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl or 4-nitrobenzoyl; sulfonyl groups such as benzenesulfonyl or p-toluenesulfonyl; sulfinyl groups, Such as phenylsulfinyl (phenyl-S-), triphenylmethylsulfinyl (trityl-S-); sulfinyl groups such as p-methylphenylsulfinyl (p-methylphenyl-S(O)-), tert-butylsulfinyl (t-Bu-S(O)-); carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, Oxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, dimethyl-3,5-dimethoxybenzyloxycarbonyl, diphenylmethyloxycarbonyl, tert-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloro-ethoxy- The N-protecting groups include phenyl, phenyl, tert-butyl ...

[0153] The term "halogen" means fluorine, chlorine, bromine or iodine.

[0154] The term "ectosome" refers to a small membrane vesicle secreted by a large portion of a cell that contains a cell-specific payload of proteins, lipids, and genetic materials and other biomolecules that are transported to other cells at different locations in the tissue. The ectosome can be considered a liposomal particle. The ectosome or lipid mixture obtained therefrom can be used in combination with other transfection agents or auxiliary lipid mixtures. The ectosome is also known as a microvesicle, epididymal body, acrosome, ectosome-like vesicle, microparticle, prosome, prostate body, exosome, tumor cell extracellular body, archaeal cell body and cancer body. In one example of an exosome, the lipid components are Lyso-PC (non-limiting examples of which are C-18, C-16, C-14 and mixtures), Lyso-bisphosphatidic acid (non-limiting examples of which are C-18, C-16 and C-14), sphingomyelin, ceramide (non-limiting examples of which are C-8-C-24), disaturated PC (non-limiting examples of which are DSPC, DPPC, DMPC and other examples where Cn (n=8-25)), diunsaturated PC-MIX (non-limiting examples of which are DOPC, DP(db)PC), phosphatidylserine (PS), phosphatidylinositol (PI), disaturated PE (non-limiting examples of which are DSPE, DPPE, DMPE), diunsaturated PE-MIX (non-limiting examples of which are DOPE, DP(db)PE), phosphatidylglycerol (PG) (non-limiting examples of which are C-18-C-22, cholesterol, diglycerides such as cardiolipin).

[0155] The term "surface ligand" or "cell surface ligand" refers to a chemical compound or structure that will bind to a surface receptor of a cell. As used herein, the term "cell surface receptor" refers to a specific grouping of chemicals on the surface of a cell to which a ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one type of cell but not in another type of cell (e.g., LDL and asialoglycoprotein receptors are specific for hepatocytes). Receptors promote internalization of ligands and attachment molecules. Cell surface receptors include, but are not limited to, folate receptors, biotin receptors, lipoic acid receptors, low-density lipoprotein receptors, asialoglycoprotein receptors, insulin-like growth factor type II / cation-independent mannose-6-phosphate receptors, calcitonin gene-related peptide receptors, insulin-like growth factor I receptors, nicotinic acetylcholine receptors, hepatocyte growth factor receptors, endothelin receptors, bile acid receptors, bone morphogenetic protein receptors, cartilage inducing factor receptors, or glycosylphosphatidylinositol (GPI) anchored proteins (e.g., β-adrenergic receptors, T cell activation protein, Thy-1 protein, GPI anchored 5' nucleotidase). These are non-limiting examples.

[0156] A receptor is a molecule to which a ligand specifically binds and has a relatively high affinity. It is typically a protein or glycoprotein, but may also be a glycolipid, lipopolysaccharide, glycosaminoglycan, or polysaccharide coating. For the purposes of this disclosure, the epitope to which an antibody or fragment thereof binds is interpreted as a receptor because the antigen: antibody complex is endocytosed. In addition, surface ligands include any substance that can enter a cell by cellular engulfment (e.g., endocytosis, uptake, pinocytosis).

[0157] As used herein, the term "ligand" refers to a compound or structure that will bind to a receptor. This includes, but is not limited to, ligands such as asialo-seromucoid, asialoglycoprotein, lipoic acid, biotin, apolipoprotein E sequence, insulin-like growth factor II, calcitonin gene-related peptide, thymopoietin, hepatocyte growth factor, endothelin-1, atrial natriuretic factor, RGD-containing cell adhesion peptides, and the like. The ligand may also be a plant virus movement protein or a peptide derived from such a protein. Suitable peptides and proteins are described, for example, in U.S. Pat. No. 10,538,784, the contents of which are hereby incorporated by reference in their entirety.

[0158] Those skilled in the art will readily recognize that the ligand selected will depend on which receptor is bound. Because different types of cells have different receptors, this provides a method for targeting nucleic acids to specific cell types, depending on which cell surface ligand is used. Therefore, the preferred cell surface ligand may depend on the targeted cell type.

[0159] As used herein, the term "nuclear localization agent", "nuclear localization signal" or "nuclear ligand" refers to a ligand, such as a peptide, which will localize an agent covalently or non-covalently attached thereto to the cell nucleus, usually by binding to a nuclear receptor. As used herein, the term "nuclear receptor" refers to a chemical group on the nuclear membrane that will bind a specific ligand and help transport the ligand and the attached linking moiety across the nuclear membrane. Nuclear receptors can be, but are not limited to, those receptors that bind to a nuclear localization sequence. Non-limiting examples of nuclear ligands include GYSTPPKKKRKVEDP (SEQ ID NO. 1), GYSTPPKTRRRP (SEQ ID NO. 2), GYSTPGRKKR (SEQ ID NO. 3), GYSTPRRNRRRRW (SEQ ID NO. 4), PDEVKRKKKPPTSYG (SEQ ID NO. 5), PRRRTKPPTSYG (SEQ ID NO. 6), RKKRGPTSYG (SEQ ID NO. 7), WRRRRNRRPTSYG (SEQ ID NO. 8), and GYGPPKKKRKVEAPYKA (SEQ ID NO. 9). 20-40 K (SEQ ID NO. 584), can be used to transport nucleic acids into the cell nucleus.

[0160] As used herein, the term "polycationic nucleic acid binding moiety" refers to a moiety containing multiple positive charges at physiological pH that allows the moiety to bind negatively charged nucleic acids. The polycationic nucleic acid binding moiety can be attached to, for example, a cell surface ligand, a fusogenic agent, and / or a nuclear localization peptide. The connection can be covalent. Suitable polycationic nucleic acid binding moieties include polyamines and polycyclic peptides containing, for example, multiple lysine residues, ornithine residues, or histidine residues.

[0161] As used herein, the term "lytic agent" or "endosome release agent" refers to a molecule, compound, protein or peptide that is capable of breaking down endosomes and releasing DNA transporters into the cytoplasm of a cell. This term includes, but is not limited to, viruses, synthetic compounds, lytic peptides or derivatives thereof. The term "lytic peptide" refers to any chemical grouping that permeates a membrane so that the structural organization and integrity of the membrane are lost. Due to the presence of a lytic agent, the membrane undergoes lysis, fusion or both. Examples of lytic agents / endosome release agents include chloroquine, polyamines and polyamidoamines. Suitable agents are described, for example, in Ei and Buyanova, Bioconjugate Chem, 30: 273-283 (2009) and Juliano, Nucleic Acid Therapeutics, 28: 166-177 (2018).

[0162] The term "nucleic acid", when not applied to a specific type of molecule such as unmodified DNA or RNA, refers to any type of nucleic acid currently known or prepared or identified in the future, provided that when mixed with any lipid of structure (I), the nucleic acid is sufficiently negatively charged to form lipid aggregates, liposomes or liposome-like complexes. As used herein, nucleic acid refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and their mixtures and polymers. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs). Nucleic acids can be in the form of antisense molecules, such as "spacer oligonucleotides" containing RNA-DNA-RNA structures that activate RNAseH. The nucleic acid can be, for example, DNA or RNA, or an RNA-DNA hybrid, and can be an oligonucleotide, a plasmid, a portion of a plasmid DNA, a precondensed DNA, a product of a polymerase chain reaction (PCR), a vector, an expression cassette, a chimeric sequence, a chromosomal DNA, or a derivative of these groups or other forms of nucleic acid molecules. The nucleic acid can be a double-stranded RNA molecule of the type used to inhibit gene expression by RNA interference. The nucleic acid can be a short interfering double-stranded RNA molecule (siRNA). The nucleic acid molecule can also be a Stealth TM RNAi molecules (Invitrogen Corporation / Life Technologies Corporation, Carlsbad, CA). Advantageously, the nucleic acid is a single-stranded RNA molecule, particularly an mRNA molecule. Alternatively, the nucleic acid is an oRNA, for example, as described in WO 2020 / 0237227A, or a self-amplifying RNA.

[0163] The term "amphipathic peptide" refers to a peptide whose secondary structure places hydrophobic and hydrophilic amino acid residues on different faces of the peptide. Peptides typically adopt a helical secondary structure. In some cases, amphipathic peptides can also function as fusogenic agents. Examples of amphipathic peptides suitable for use in the compositions described herein include, but are not limited to, peptides comprising a sequence selected from the group consisting of FEAALAEALAEALA (SEQ ID NO. 42, Ac-LARLLPRLLARL-NHCH3 (SEQ ID NO. 43), GLLEELLELLEELWEELLEG (SEQ ID NO. 44), GWEGLIEGIEGGWEGLIEG (SEQ ID NO. 45), GLFEALAEFIEGGWEGLIEG (SEQ ID NO. 46), GLFEALLELLESLWELLLEA (SEQ ID NO. 47), GGYCLEKWMIVASELKCFGNTA (SEQ ID NO. 48), GGYCLTRWMLIEAELKCFGNTAV (SEQ ID NO. 49), and WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO. 50). The amphipathic peptide can be optionally linked to a polycationic nucleic acid binding portion, for example, via a covalent bond.

[0164] Specific examples of the compound of structure (I) include compounds having the following structures:

[0165]

[0166]

[0167]

[0168] In these molecules, R 1 or R 2 Each may be, for example, but not limited to, C 14-18 Alkyl or C 14-18 alkenyl; and R 9 Each independently can be but not limited to H, -(CO)C 14 -C 18 Alkyl or -(CO)C 14 -C 18 Alkenyl.

[0169] Those skilled in the art will recognize that although the molecules of the present invention are shown herein for convenience in their neutral (unprotonated) form, these molecules will exist in partially or fully protonated form in solution at an appropriate pH, and that the present invention includes molecules in all their protonated, unprotonated, ionized and non-ionized forms without limitation, unless otherwise specifically noted.

[0170] Preparation of lipids

[0171] Symmetric cationic lipids and asymmetric cationic lipids of general structure (I) can be synthesized using methods known in the art, as shown in, for example, Scheme 1.

[0172] Solution 1

[0173] Synthesis of squaramide analogs

[0174] The synthesis of compounds such as 24 is shown in Scheme 1. Typically, compound 20 is combined with an amine such as 21. The product 22 is then reacted with amine 23 to produce the final product 24.

[0175]

[0176] Synthesis of 1,4,8,11-tetraazacyclotetradecane (cyclam) analogs

[0177] Solution 2

[0178] Alkyl substituted cyclams are well known in the art and can be prepared by methods described, for example, in US Pat. Nos. 3,860,576 and 4,168,265, both of which are incorporated by reference.

[0179] Typically, cyclams such as 26 are alkylated with compounds such as 27 in the presence of a base such as N,N-diisopropylethylamine to give 28. Secondary amines can be protected with boc to give 29. Incubation with hydrazine hydrate in an alcohol such as ethanol under reflux conditions for 16 hours will give primary amines 30, which can be alkylated with 2 equivalents of 31 followed by incubation in a strong acid such as HCL to give compound 32. Alternatively, alkylation with 33 gives olefin 34.

[0180]

[0181] Solution 3

[0182] The synthesis of compound 38 can be accomplished by reacting cyclam 26 with epoxide 35 to form the amino alcohol, followed by hydrazine-mediated deprotection of the amine to give 36. Acylation of the amine, LAH-mediated amide reduction, and salt formation then give 38.

[0183] The synthesis of compound 39 can be accomplished by reacting 36 with an acyl chloride followed by acidification.

[0184]

[0185] Solution 4

[0186] The dimethyl tartrate 4-1 of step a can be treated with an alkylamine at an elevated temperature (e.g., 70° C.) in a sealed pressure reactor to obtain compound 4-2. This compound can be alkylated with an alkyl methanesulfonate as shown in step b to obtain compound 4-3, and then reduced using lithium aluminum hydride in step c to produce diamine 4-4. Compound 4-4 can be reacted with N-(3-bromopropyl)-phthalimide, followed by hydrazinolysis to provide tetramine 4-5, as shown in step d. Compound 4-5 can then be reacted with 3-(methylamino)-4-methoxycyclobut-3-ene-1,2-dione in step e to provide compound 4-6. 3-(methylamino)-4-methoxycyclobut-3-ene-1,2-dione can be obtained by reacting 3,4-dimethoxy-3-cyclobutene-1,2-dione with methylamine.

[0187]

[0188] Alternatively, asymmetric cationic lipids (i.e., compounds lacking a plane of symmetry) can be prepared using, for example, tartaric acid monoesters, which are easily prepared from diacetyltartaric anhydride (see Organic Syntheses, Coll. Vol. 4, p. 242 (1963); Vol. 35, p. 49 (1955)), as shown. See Scheme 5. Protecting the diol (g), followed by DIBAL reduction, reductive amination, and amine protection (h) produces protected 2,3,4-trihydroxybutylamine. Mild oxidation, reductive amination, and protection produce differentially protected 2,3-dihydroxy-1,4-diamine compounds (i). Deprotection of the diol and alkylation produce compound 5-5. Stepwise selective amine deprotection and coupling reactions similar to those in Scheme 4 can then be used to prepare asymmetric compounds.

[0189] Similar synthetic routes shown in Scheme 6, steps k to p, can be used to prepare compounds where the long chain moiety is attached via an ester moiety.

[0190] Solution 5

[0191]

[0192] Solution 6

[0193]

[0194] The compound of structure (I) can be prepared from the intermediate of structure 4 by methods well known in the art. For example, wherein R 2 Compound 4 which is an alkyl group can be reacted with a suitably protected epoxyamine (e.g., epoxyphthalimide) followed by deprotection to provide a β-hydroxyamine such as

[0195]

[0196] Other methods for preparing compounds of structure (I) will be apparent to those skilled in the art. For example, reductive amination of a protected carbohydrate molecule can be used to provide a 2 A compound of structure (I) containing multiple hydroxyl groups.

[0197] The lipid of structure (I) can be combined with one or more nucleic acids to deliver the nucleic acid to a target cell or tissue. However, advantageously, the lipid is combined with additional components and nucleic acids, as described in detail below. In certain embodiments, the lipid of structure (I) is combined with one or more additional components and one or more nucleic acids to form a lipid nanoparticle composition.

[0198] Cationic lipid / ionizable lipid preparations

[0199] Cationic lipid / ionizable lipid compositions provided herein encompass complexes in the form of lipid nanoparticles, liposomes (e.g., lipid vesicles) and liposome complexes. As used herein, the term "liposome" encompasses any compartment encapsulated by a lipid bilayer. The term liposome includes a single-chamber capsule, which is composed of a single lipid bilayer and generally has a diameter in the range of about 20nm to about 400nm. Liposomes can also be multichambers with a diameter in the range of about 1 μm to about 10 μm. Multichamber liposomes can be composed of several (from two to hundreds) single-chamber capsules, forming one inside another, with a gradually reduced size, producing a multichamber structure of concentric phospholipid spheres separated by a water layer. Alternatively, multichamber liposomes can be composed of many smaller non-concentric lipid spheres within large liposomes. In embodiments, liposomes include multichamber capsules (MLV), large single-chamber capsules (LUV) and small single-chamber capsules (SUV). In some embodiments, the composition comprises liposomes containing any suitable ionizable lipids and neutral lipids and peptides provided herein.

[0200] In some embodiments, the composition comprises lipid nanoparticles (LNP). The size of the LNP composition is generally micron-sized or small, and may include a lipid bilayer. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 1 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 900 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 800 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 700 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 600 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 500 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 400 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 300 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 200 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 100 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 20nm to about 50 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 900 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 800 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 700 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 600 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid preparation, wherein the size is about 100nm to about 500 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 400 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 300 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 200 μm. In some embodiments, the lipid nanoparticle composition comprises a lipid formulation, wherein the size is about 100 nm to about 150 μm.

[0201] In embodiments, the composition comprises lipids of structure (I) as provided herein, including embodiments thereof, and also comprises a bioactive agent. As provided herein, a "bioactive agent" refers to a compound that has a detectable effect on the biological function of the cell, tissue or organism when applied to the cell, tissue or organism. In embodiments, the detectable effect is a biological effect. In embodiments, the detectable effect is a therapeutic effect. In embodiments, the detectable effect is a diagnostic effect. The bioactive agent can form lipid aggregates with the composition including its embodiments provided herein. In embodiments, the bioactive agent is a test compound. As provided herein, a "test compound" is a compound that determines its effect on biological function relative to a control compound. As provided herein, a "control compound" refers to a compound with a known effect on biological function. In embodiments, the bioactive agent is a control compound. In embodiments, the bioactive agent is a therapeutic agent or a diagnostic agent. In embodiments, the bioactive agent is a therapeutic agent. In embodiments, the bioactive agent is a diagnostic agent. In embodiments, the bioactive agent includes a protein. In embodiments, the bioactive agent includes a nucleic acid, a ribonucleoprotein or a small molecule. In embodiments, the bioactive agent includes nucleic acid. In embodiments, the bioactive agent includes ribonucleoprotein. In embodiments, the bioactive agent includes small molecules. In embodiments, nucleic acid is mRNA, siRNA, miRNA or guide RNA (gRNA). In embodiments, nucleic acid is mRNA. In embodiments, ribonucleic acid is siRNA. In embodiments, ribonucleic acid is miRNA. In embodiments, nucleic acid is gRNA. In embodiments, the bioactive agent includes nucleic acid and ribonucleoprotein. In embodiments, ribonucleoprotein is CRISPR associated protein 9 (Cas9). "mRNA" as provided herein refers to a ribonucleic acid molecule, which includes one or more expressible nucleic acid sequences encoding one or more proteins or polypeptides or other DNA molecules.

[0202] The lipid of structure (I) is combined with a nucleic acid and / or protein payload to produce a lipoplex formulation. For example, the lipid of structure (I) is combined with a payload selected from one or more of the following: siRNA, miRNA, mRNA, shRNA, self-amplifying RNA, stRNA, oRNA (or non-naturally occurring circular RNA), antisense oligonucleotides (ASOs), gRNA, ribonucleoproteins (e.g., CRISPR complexes), dsDNA, plasmid DNA, etc.

[0203] In the case of delivering nucleic acids, the amount of nucleic acids (e.g., mRNA, self-amplifying RNA, etc.) in the lipoplex formulation may depend on the size, sequence, and other characteristics of the nucleic acids. The amount of nucleic acids in the lipoplex formulation may also depend on the size, composition, desired target, and other characteristics of the lipoplex formulation. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. In some embodiments, the weight / weight ratio of the lipid component to the nucleic acid (such as mRNA) in the lipoplex composition may be about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the weight / weight ratio of the lipid component to the nucleic acid (such as mRNA) may be about 10:1 to about 40:1. The amount of nucleic acid in a lipoplex composition can be measured, for example, using absorption spectroscopy (eg, ultraviolet-visible (UV-vis) spectroscopy).

[0204] The lipoplex formulation may comprise the nucleic acid at a concentration of about 0.1 mg / ml to 2 mg / ml, such as, but not limited to, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, 1.5 mg / ml, 1.6 mg / ml, 1.7 mg / ml, 1.8 mg / ml, 1.9 mg / ml, 2.0 mg / ml or greater than 2.0 mg / ml.

[0205] Preferably, one or more nucleic acids (e.g., mRNA), lipids, and their amounts can be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of the number of nitrogen atoms in one or more lipids to the number of phosphate groups in a nucleic acid (e.g., mRNA). In general, a lower N:P ratio is preferred. One or more mRNAs, lipids, and their amounts can be selected to provide an N:P ratio of about 2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In certain embodiments, the N:P ratio may be about 2:1 to about 5:1. In preferred embodiments, the N:P ratio may be about 4:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. Additional lipid components

[0206] Advantageously, in addition to the lipids of structure (I), the lipoplex formulations also comprise one or more co-lipids, most advantageously neutral co-lipids, although those skilled in the art will recognize that other lipids, including cationic lipids / ionizable lipids, may be used. However, some formulations comprise only lipids of structure (I) in combination with nucleic acids.

[0207] Ionizable lipids as described herein refer to lipids having at least one protonatable group or deprotonatable group, such that the lipid is positively charged at a pH equal to or lower than physiological pH (e.g., pH 7.4), and is neutral at a second pH, preferably at or higher than physiological pH. Preferably, when incorporated into lipid complexes (such as, for example, lipid nanoparticles and liposomes), the ionizable lipids provided herein have a pKa of a protonatable group of about 4 to about 11, for example, about 4 to about 7, for example, between about 5 and 7, such as between about 5.5 and 6.9.

[0208] Thus, in addition to the lipids of structure (I), the lipid complex formulation also comprises neutral lipids (such as phospholipids). Phospholipids that can be used in the compositions disclosed herein include, but are not limited to, DOPE, DPhPE, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoylphosphatidylcholine (DPhPE). Phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (trans-DOPE) or any combination thereof. The phospholipids that can be used in the compositions provided herein can be present, for example, at about 5 mol% to about 20 mol% of the lipid complex preparation. Advantageously, the phospholipids are present in a range of about 1 mol% to about 40 mol%, for example, 1 mol% to about 25 mol%. Preferably, the amount of phospholipid in the lipoplex formulations disclosed herein is at least about 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 12 mol%, 14 mol%, 16 mol%, 18 mol% or 20 mol%, or any amount therebetween, of the total lipoplex formulation.

[0209] Other neutral lipids that may be advantageously included in the lipid complex formulations provided herein include sterols or lipids containing sterol moieties ("sterol derivatives"). As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols. Exemplary sterols and lipids containing sterol moieties that can be used in the lipid complex formulations provided herein include, but are not limited to, poriferosterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, α-tocopherol, hopanes, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. Some lipid complex formulations provided herein contain sterols or sterol derivatives. Sterols or sterol derivatives may be present in about 5 mol% to 60 mol% of the total lipid complex formulation. Advantageously, sterols or sterol derivatives are present in about 15 mol% to 50 mol%, for example, 25 mol% to 40 mol%. Preferably, the amount of sterol (such as cholesterol) or sterol derivative in the lipid composition disclosed herein is at least about 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol% or 60 mol% of the total lipid preparation. Some of the lipid complex formulations provided herein do not contain a sterol or a sterol derivative.

[0210] The lipid complex preparations provided herein may also include stabilizers, such as stable lipids. Stable lipids may be neutral lipids, or they may be charged. The stable lipids that may be advantageously used in the preparations provided herein include, but are not limited to, lipids modified with polyethylene glycol (PEG). Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropane-3-amines. Such lipids are also referred to as PEGylated lipids. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids. Other stable lipids that may be used in the compositions disclosed herein include, for example, polyethylene glycol lipids, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether copolymers, triblock polyoxyethylene alkyl ether copolymers, and amphiphilic branched polymers. In an embodiment, the stabilizer can be polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate ("Myrj52"), poly(propylene glycol) 11-block-poly(ethylene glycol) 16-block-poly(propylene glycol) 11, poly(propylene glycol) 12-block-poly(ethylene glycol) 28-block-poly(propylene glycol) 12, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadecene-9-enoate), Myrj52 (polyoxyethylene (40) stearate), Brij TM S10 (polyoxyethylene (10) stearyl ether), BRIJ TM L4 = polyoxyethylene (4) lauryl ether; BRIJ TM S20 = polyoxyethylene (20) stearyl ether; BRIJ TM S35 = polyoxyethylene (23) lauryl ether; TPGS1000 = D-α-tocopheryl polyethylene glycol 1000 succinate; Tween 20 / polysorbate 80 / tridecyl-D-maltoside in equal proportions and combinations thereof. In certain compositions, the stabilizer is present at about 0.1 mol% to 5 mol% of the lipid complex formulation. For example, in some compositions, the stabilizer is present at about 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol%, 5 mol%, or any value therebetween, of the lipid complex formulation.

[0211] In addition to the lipids of structure (I), the lipoplex formulations may further comprise one or more cationic lipids / ionizable lipids. For example, some lipoplex formulations comprise a cationic lipid / ionizable lipid selected from the group consisting of DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3- Dipalmityloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmityloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, L-spermine-5-carboxy-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine, 3,5-(N,N- L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine, L-lysine-bis(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, -β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl]piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4,-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-myristylamino]-butane-2,3-diol, 1,4,-bis[(3-oleylamino)propyl]piperazine, L-arginine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, bis[(3-(3-aminopropyl)-myristylamino)2-hydroxypropyl]piperazine, L-arginine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-serine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitoylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, cholesteryl-3β-carboxy-amidoethylenetrimethylammonium iodide, 1,4-bis[( 3-Myristylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol carboxylate iodide, cholesteryl-3β-carboxamidoethyleneamine, cholesteryl-3β-oxysuccinamideethylenetrimethylamine iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinic acid iodide, 2-[(2-trimethylammonio)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinic acid iodide, 3β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol and 3β-[N-(polyethyleneimine)-carbamoyl]cholesterol, 1,4-bis[(3-palmitylamino)propyl]piperazine, L-ornithineglycyl-N-(1-heptadecylooctadecyl)glycinamide, N, 2 ,N 5 -bis(3-aminopropyl)-L-ornithineglycyl-N-(1-heptadecanyloctadecyl)glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dioctadecyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine,

[0212] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioctadecyl-L-α-asparagine, N-[N 2 -[N2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-diol-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine,

[0213] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dioleyl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-oleylamino)propyl]piperazine, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-asparagine,

[0214] N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dipalmitoyl-L-glutamine]-L-

[0215] Glutamate, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dimyristyl-L-glutamine,

[0216] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)-2-hydroxypropyl]piperazine, N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5 -Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino)propyl]piperazine, N 2 -[N 2 ,N 5 -bis(3-aminopropyl)-L-ornithine]-N,N-dilauryl-L-glutamine, N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauryl-L-α-glutamine,

[0217] N 2 -[N 2 ,N 5 -bis(aminopropyl)-L-ornithine]-NN-dilauryl-L-α-asparagine, N-[N 2 -[N 2 ,N 5 -bis[(1,1-dimethylethoxy)carbonyl]-N 2 ,N 5-Bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dilauryl-L-glutaminyl]-L-glutamic acid, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-dioctadecyl-9-enylpropionamide, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-bispalmitylpropionamide, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-bispalmitylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl ] piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dioleylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dipalmitylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristylaminoaminopropane, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)propyl]piperazine, [(3-aminopropyl)-bis-(2-tetradecylethoxy)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleylethoxy)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmitylamino)propane Palmitoylethoxy)] methylammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dipalmitoyl-1-N,N-dimethylaminopropane, myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-amino-propyl)-4-(3-amino-propylamino)-4-tetradecylcarbamoyl-butylcarbamoylcholesterol ester, (3-amino-propyl)-4-(3-amino-propylamino)-4-carbamoylbutylcarbamoylcholesterol ester, (3-amino- Spermine-5-carboxyglycine (N'-stearyl-N'-oleyl) amide tetrakis trifluoroacetate, Spermine-5-carboxyglycine (N'-stearyl-N'-antioleyl) amide tetrakis trifluoroacetate, Agmatine carboxycholesterol acetate, Spermine-5-carboxy-β-alanine cholesteryl tetrakis trifluoroacetate, 2,6-diaminohexenyl β-alanine cholesteryl bis trifluoroacetate, 2,4-diaminobutyryl β-alanine cholesteryl bis trifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesteryl tris trifluoroacetate, [N,N-bis(2-hydroxyethyl)-2-aminoethyl] aminocarboxy cholesterol ester, stearyl carnitine ester, palmitoyl carnitine ester, myristyl carnitine ester, stearyl stearoyl carnitine ester hydrochloride, L-stearyl stearoyl carnitine ester, stearyl oleoyl carnitine ester hydrochloride, palmityl palmitoyl carnitine ester hydrochloride, myristyl myristoyl carnitine ester hydrochloride, L-myristyl myristoyl carnitine ester hydrochloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)propyl]piperazine, N-(3-aminopropyl)-N,N'-bis-(dodecylethoxy)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(oleylethoxy)-piperazine bromide, N-( 3-Aminopropyl)-N,N'-bis-(palmitylethoxy)-piperazine bromide, N-(3-aminopropyl)-N,N'-bis-(myristylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-palmitylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristylethoxy)-piperazine bromide, 1,4-bis[(3 -(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxy-propyl]piperazine and 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazine, KL22, KL25, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]dioxolane (DLin-K-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA or MC 3), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA (2R)) and (2S)-2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA (2S)).

[0218] Preferably, the lipid of structure (I) or the combination of a lipid of structure (I) and one or more cationic lipids / ionizable lipids is present at about 5 mol% to 80 mol% of the lipoplex formulation. For example, some lipoplex formulations include less than 50 mol% of structure (I), or a combination of structure (I) and one or more additional cationic lipids / ionizable lipids. Other lipoplex formulations include greater than 50 mol% of structure (I), or a combination of structure (I) and one or more additional cationic lipids / ionizable lipids.

[0219] Thus, some lipoplex formulations include lipids of structure (I), or combinations of structure (I) and one or more cationic lipids / ionizable lipids (15 to 80 mol% of the lipoplex formulation), sterols (20 to 60 mol% of the lipoplex formulation), stabilizers (0.5 to 5 mol% of the lipoplex formulation), and phospholipids (1 to 40 mol% of the lipoplex formulation). Exemplary lipoplex formulations may include about 20 to 60 mol% of lipids of structure (I), or combinations of lipids of structure (I) and one or more additional cationic lipids / ionizable lipids, about 5 to 25 mol% of phospholipids, about 25 to 55 mol% of sterols or sterol derivatives; and about 0.5 to 15 mol% of stabilizers. Another exemplary lipid complex formulation comprises about 50 mol% of a lipid of structure (I), or a combination of a lipid of structure (I) and one or more cationic lipids / ionizable lipids, about 1.5 mol% of a stabilizer, about 38.5 mol% of a sterol or sterol derivative, and about 10 mol% of a phospholipid. Another exemplary lipid complex formulation comprises about 55 mol% of a lipid of structure (I), or a combination of a lipid of structure (I) and one or more cationic lipids / ionizable lipids, about 2.5 mol% of a stabilizer, about 32.5 mol% of a sterol or sterol derivative, and about 10 mol% of a phospholipid.

[0220] Polyamine components

[0221] Other formulations may also include one or more polyamine transfection agents, such as dense star dendrimers, PAMAM dendrimers, NH3 core dendrimers, ethylenediamine core dendrimers, dendrimers of the 5th generation or higher, dendrimers with substituted groups, dendrimers comprising one or more amino acids, grafted dendrimers, activated dendrimers, polyethyleneimine and / or polyethyleneimine conjugates.

[0222] Transfection Enhancer

[0223] Other agents may include transfection enhancers, such as fusogens (such as endosomal release agents), cell surface ligands and / or nuclear localization agents, such as nuclear receptor ligand peptides. Examples of transfection enhancers include, but are not limited to, reovirus-related gene fusion peptides (see WO07 / 130073, which is incorporated herein by reference in its entirety), insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell targeting antibodies, lactoferrin, fibronectin, adenovirus penton bases, knobs, hexon proteins, vesicular stomatitis virus glycoproteins, victoria forest virus core protein, influenza hemagglutinin, hepatitis B core protein, HIV Tat protein, herpes simplex virus VP22 protein, histone protein, arginine-rich cell permeable protein, high mobility group protein and invasin protein, internalization protein protein, endotoxin, diphtheria toxin, Shigella toxin, melittin, magainin, gramicidin, cecropin, defensin, antimicrobial peptide, tachypleus, sulfonin, indolicidin, bovine antimicrobial peptide, drosophilin, bee antimicrobial peptide, cathepsin inhibitor, bactericidal-permeable-increasing protein, nisin, toad aglycone and fragments thereof. Other cell penetrating peptides that can be used in the compositions provided herein include those provided in Table 2 below:

[0224] Table 2

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] The lipid complex compositions provided herein can also be combined with one or more exosomes or biological materials (eg, lipids, proteins, nucleic acids, etc.) derived from or purified from exosomes.

[0240] Exemplary compositions may include, for example, lipids of structure (I) and one or more exosomes; lipids of structure (I), and one or more exosomes and one or more neutral lipids; lipids of structure (I), and one or more exosomes, one or more neutral lipids, and one or more stabilizers; lipids of structure (I), and one or more exosomes and one or more neutral lipids, optionally one or more stabilizers and optionally one or more cell penetrating peptides.

[0241] Other exemplary compositions include, for example: lipids of structure (I), and one or more biological materials derived from or purified from exosomes; lipids of structure (I), and one or more biological materials derived from or purified from exosomes and one or more neutral lipids; lipids of structure (I), and one or more biological materials derived from or purified from exosomes, one or more neutral lipids, and or multiple stabilizers; lipids of structure (I), and one or more biological materials derived from or purified from exosomes and one or more neutral lipids, optionally one or more stabilizers and optionally one or more cell penetrating peptides.

[0242] Methods of use and preparation of lipid preparations

[0243] The use of these compositions in transfection can be carried out by methods known in the art. For example, the compositions described herein can be used to transfect cells in vitro or ex vivo.

[0244] For in vitro delivery, WO07 / 130073 describes the "before" and "after" schemes for transfection at page 54 to page 60, wherein the components of the transfection complex are mixed in different orders before adding cell culture. Usually, the liposome preparation containing or not containing amphoteric auxiliary lipids is prepared, and then mixed with macromolecules, such as DNA molecules or RNA molecules, such as mRNA molecules or RNAi molecules, to form a transfection complex. The complex is then added to the cell culture, and the method known to be used to monitor transfection. Additional components (such as cell surface ligands, fusogenic agents, nuclear localization agents, etc.) can be added to the nucleic acid before mixing with the liposome, or can be added to the liposome before adding the nucleic acid.

[0245] Cells that can be transfected according to these methods include, but are not limited to, almost any eukaryotic cell, including primary cells, cells in culture, continuous cell cultures or cell lines, and cells in cultured tissues. Suitable cells include human cell lines and animal cell lines. The cells can be fibroblasts. The cells can be attached cells or suspended cells (suspension cells). In certain exemplary aspects, the cells are suspension CHO-S cells and suspension 293-F cells. Other cells that can be used include, but are not limited to, 293, 293-S, CHO, Cos, 3T3, Hela, primary fibroblasts, A549, Be2C, SW480, CHOK1, Griptite 293, HepG2, Jurkat, LNCaP, MCF-7, NIH-3T3, PC12, C6, Caco-2, COS-7, HL60, HT-1080, IMR-90, K-562, SK-BR3, PHP1, HUVEC, MJ90, NHFF, NDFF, and primary neurons.

[0246] For in vivo administration, the lipid complex formulation is preferably administered parenterally (i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly). In a specific embodiment, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection. For example, see Stadler et al., U.S. Pat. No. 5,286,634, which is incorporated herein by reference. Intracellular nucleic acid delivery is also discussed in the following literature: Straubringer et al., Methods in Enzymology, Academic Press, New York. 101: 512-527 (1983); Mannino et al., Biotechniques 6: 682-690 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst. 6: 239-271 (1989) and Behr, Acc. Chem. Res. 26: 274-278 (1993). Other methods of administering lipid-based therapeutics are described, for example, in Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

[0247] Another embodiment is a method for producing a protein, comprising contacting a cell with a lipid-nucleic acid complex as described above, wherein the nucleic acid encodes the protein. The cells are incubated to produce the protein and collect the protein. Cells that can be used for protein production are described above. In addition, any composition comprising the lipid of structure (I) can be used for transfecting cells. Such compositions are further discussed herein, and include but are not limited to compositions comprising lipids, co-lipids and optional transfection enhancers (such as gene fusion peptides or proteins) of structure (I). In such methods, contacting a cell with a lipid-nucleic acid complex can occur in vitro, in vitro or in vivo.

[0248] Another embodiment is a method for inhibiting protein production in a cell, comprising contacting the cell with a lipid-nucleic acid complex as described above, wherein the nucleic acid is a double-stranded RNA molecule, such as an RNAi molecule or siRNA molecule designed to inhibit protein expression. The method of designing such RNA molecules is well known in the art. The lipid of structure (I) is particularly suitable for delivering RNAi molecules in this way. Incubate cells and observe the phenotypic results of the production of the selected protein. In such methods, contacting the cell with the lipid-nucleic acid complex can occur in vitro, in vitro or in vivo.

[0249] In other embodiments, a method for delivering a lipid composition to a subject's spleen tissue and / or lung tissue is provided, comprising administering a cationic lipid composition as described above to the subject. In some embodiments, a method for delivering a bioactive agent to a subject's spleen tissue and / or lung tissue is provided, comprising administering a cationic lipid-bioactive agent complex to the subject.

[0250] In some embodiments, a method for delivering a bioactive agent to the spleen tissue of a subject is also provided, the method comprising: (i) mixing the bioactive agent with a composition as described above to form a bioactive agent-lipid complex bioactive agent; and (ii) administering an effective amount of the bioactive agent-lipid complex to the subject, thereby delivering the bioactive agent-lipid complex to the spleen tissue of the subject.

[0251] In some embodiments, a method for delivering a bioactive agent to the lung tissue of a subject is also provided, the method comprising: (i) mixing the bioactive agent with a composition as described above to form a bioactive agent-lipid complex bioactive agent; and (ii) administering an effective amount of the bioactive agent-lipid complex to the subject, thereby delivering the bioactive agent-lipid complex to the lung tissue of the subject.

[0252] In certain embodiments, the administration of the cationic lipid composition or bioactive agent-lipid complex to the subject is via systemic administration. In some embodiments, such cationic lipid composition or bioactive agent lipid complex further comprises at least one neutral lipid. In some embodiments, such cationic lipid composition or bioactive agent lipid complex further comprises at least one neutral lipid and at least one transfection enhancer, such as a cell penetrating peptide and / or a gene fusion peptide. In certain embodiments, the bioactive agent for in vivo delivery is a nucleic acid molecule.

[0253] In certain embodiments, in a method for delivering a cationic lipid composition or a bioactive agent-lipid complex to the spleen and / or lung of a subject, the cationic lipid composition or the bioactive agent-lipid complex comprises a lipid compound of structure (I), wherein only one of X or Y is

[0254]

[0255] The above-mentioned lipids can be formulated for transfection and in vivo administration by various methods. One of the simplest formulation methods is reverse evaporation, as described in U.S. Patent No. 9,259,475, which is incorporated herein by reference in its entirety. In some embodiments, the lipid film can be hydrated with water, and the hydrated lipid film and nucleic acid payload are diluted in a buffer and mechanically mixed by pipetting and / or vortexing to form a liposome colony. Other formulation methods that can be used are ultrasonic treatment and microfluidization. Advantageously, lipids are formulated into lipid nanoparticles using microfluidic mixing, such as described in Roces et al., Pharmaceutics, 12: 1095 (2020). Suitable microfluidic mixing devices can be commercially available from, for example, Precision Nanosystems (Vancouver, BC). Typically, microfluidic mixing combines two fluid streams, one containing nucleic acids and the other containing lipids and other components of structure (I), such as peptides, ligands, and other lipid components as described below.

[0256] In some embodiments, provided herein is a method for preparing a group of lipid complex preparations containing nucleic acid molecules, comprising: (a) transferring an aqueous solution comprising a buffer and nucleic acid molecules to a mixing container; optionally adding other components, such as peptides, ligands or other lipid components described herein; (b) injecting a lipid solution comprising cationic lipids / ionizable lipids and neutral lipids into the aqueous solution, wherein the injection comprises extrusion, mixing in a pipeline, microfluidic mixing, evaporation or vortexing; and (c) generating a population of lipid preparations complexed with nucleic acids.

[0257] For example, in some embodiments, for a lipoplex composition comprising RNA, a solution of RNA in deionized water at a concentration of 0.1 mg / ml is diluted in a 50 mM sodium citrate buffer at a pH between 3 and 4 to form a stock solution. The lipoplex composition can be treated by dialysis to remove ethanol and achieve buffer exchange. The formulation can be dialyzed against phosphate buffered saline (PBS), pH 7.4 using a desired molecular weight cutoff (e.g., 10 kD). The resulting lipoplex suspension can be filtered through a 0.2 μm sterile filter (Sarstedt, Numbrecht, Germany) into a glass vial and sealed.

[0258] Methods for determining particle size in nanoparticle formulations are well known in the art. For example, the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) can be used to determine the particle size, polydispersity index (PDI), and zeta potential of nanoparticle compositions. UV-visible spectroscopy can be used to determine the concentration of nucleic acids (e.g., mRNA) in lipoplex compositions. A certain amount of the composition is diluted in a suitable solvent and the absorption spectrum of the solution is recorded on a spectrophotometer, for example, between 230 nm and 330 nm. The concentration of the therapeutic and / or prophylactic agent in the lipoplex composition can be calculated based on the extinction coefficient of the therapeutic and / or prophylactic agent used in the composition and based on the difference between the absorbance at a wavelength of, for example, 260 nm and a baseline value at a wavelength of, for example, 330 nm.

[0259] For lipid complex compositions containing RNA, QUANT-IT TM RNA determination (Invitrogen Corporation, Carlsbad, CA) can be used to evaluate the encapsulation of RNA by lipid compositions using the method provided by the manufacturer. The fluorescence intensity produced after adding RIBOGREEN reagent can be measured using a fluorescence plate reader at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence value of the reagent blank is subtracted from the fluorescence value of each sample in the sample, and the percentage of free RNA is determined by dividing the fluorescence intensity of the intact sample (without adding Triton X-100) by the fluorescence value of the ruptured sample (caused by adding Triton X-100).

[0260] Reagent test kit

[0261] The components of the above-mentioned transfection and lipid formulation compositions can be provided in a kit. The kit contains lipids of structure (I) and additional components, such as neutral lipids, cationic lipids, cell surface ligands, fusogens and / or nuclear localization agents, etc. The kit components can be separate or can be premixed in any way. For example, lipids of structure (I) (or a combination of lipids of structure (I) and one or more cationic lipids / ionizable lipids) can be mixed with one or more neutral lipids, sterols, stabilizers, transfection enhancers, etc. Additional components may also be present in the same container or may be present in one or more separate containers. The kit typically includes containers, such as vials and / or tubes, which are packaged together, for example, in cardboard boxes. The kit can be shipped from a supplier to a consumer. For example, in one example, a kit comprising a vial is provided herein, the vial comprising a liposome formulation as described above and an optional transfection agent and a transfection enhancing peptide. The kit may also include, for example, a separate container comprising a transfection enhancing agent, such as a transfection enhancing peptide, for example, Plus Reagent TM (Invitrogen Corp., Carlsbad, CA). The kit may also include cells, cell culture medium, and a reporter nucleic acid sequence, such as a plasmid expressing a reporter gene, in separate containers. In some examples, the culture medium may be a reduced serum medium and / or a protein expression medium.

[0262] Also provided are kits containing compounds of structure (I) and additional reagents, such as cationic lipids / ionizable lipids, neutral lipids (e.g., phospholipids), sterols, amphipathic peptides, amphipathic peptides comprising polycationic nucleic acid binding moieties, cell surface ligands, cell surface ligands comprising polycationic nucleic acid binding moieties, fusogenic agents, fusogenic agents comprising polycationic nucleic acid binding moieties, nuclear localization peptides or proteins, and nuclear localization peptides or proteins comprising polycationic nucleic acid binding moieties. The kit may contain one, some, or all of these additional reagents in any possible combination. Advantageously, the additional reagents include cationic lipids, amphipathic peptides, and cell surface ligands comprising polycationic nucleic acid binding moieties. When the cell surface ligand is a peptide or protein, the polycationic nucleic acid binding moiety is a polyvalent amino acid sequence.

[0263] In one embodiment, the kit comprises a cationic lipid (such as a lipid of structure (I)) and a peptide, protein, or fragment thereof, or a modified peptide, protein, or fragment thereof, a single portion or a mixture. In another embodiment, the kit comprises a polycationic polymer and a peptide, protein, or fragment thereof, or a modified peptide, protein, or fragment thereof, a single portion or a mixture. The cationic lipid transfection kit may optionally comprise a neutral lipid and other transfection enhancers or other additives, and the relative amounts of the components in the kit may be adjusted to facilitate the preparation of the transfection composition. The kit components may include suitable media or solvents for the other kit components.

[0264] Nucleic acids that can be transfected by the methods of the invention include DNA and RNA (including mRNA and RNAi / siRNA) of any size from any source containing natural or unnatural bases, and include those that encode and are capable of expressing therapeutic or otherwise useful proteins in cells, those that inhibit the expression of undesirable nucleic acids in cells, those that inhibit undesirable enzyme activities or activate desired enzymes, those that catalyze reactions (ribozymes), and those that play a role in diagnostic assays (e.g., diagnostic nucleic acids). Therapeutic nucleic acids include those that encode or can express therapeutically useful proteins, peptides, or polypeptides in cells, those that inhibit the expression of undesirable nucleic acids in cells, and those that inhibit undesirable enzyme activities or activate desired enzymes in cells.

[0265] In view of the disclosure herein, the compositions and methods provided herein can also be easily applied to the introduction of biologically active macromolecules other than nucleic acids (including polyamines, polyamino acids, polypeptides and proteins, etc.) into eukaryotic cells. Other useful materials, such as therapeutic agents, diagnostic materials, research reagents, can be bound to peptides and modified peptides and introduced into eukaryotic cells by the methods of the present invention.

[0266] Example

[0267]

[0268] Example 1

[0269]

[0270] To a round-bottom flask containing a pyridine solution (15 ml) of dodecylamine hydrochloride (1.4 g, 6.5 mmol, 1.1 equiv) was added butene-1,2-dione (1.0 g, 5.87 mmol), and the resulting solution was heated to 50 ° C and stirred for 4 hours under nitrogen. The reactants were then heated to 60 ° C and stirred for 16 hours. The reactants were cooled to room temperature and then filtered to remove the precipitate. The filtrate was concentrated under reduced pressure to produce an oil, which was purified by flash chromatography using a 24 g normal phase column with (0% to 100%) MeOH / DCM to produce 3-(dodecylamino)-4-ethoxycyclobut-3-ene-1,2-dione (0.75 g, 41.6% yield).

[0271] Example 2

[0272]

[0273] To a dry round-bottom flask containing a solution of HDMS (0.87 g, 1.57 mmol) in anhydrous DMF (10 ml) was added a solution of 3-(dodecylamino)-4-ethoxycyclobut-3-ene-1,2-dione (0.53 g, 1.57 mmol) in anhydrous DMF (5 ml). The solution was heated to 50°C and stirred under nitrogen for 22 hours. The resulting solution was concentrated to dryness under reduced pressure. The resulting oil was purified by flash chromatography using a 25 g normal phase column with (0% to 100%) MeOH / DCM to yield 3-(dodecylamino)-4-((2-hydroxy-3-(tetradecyl(4-(tetradecylamino)butyl)amino)propyl)amino)cyclobut-3-ene-1,2-dione (0.4 g, 0.489 mmol, 31%) and 3-(dodecylamino)-4-((3-((4-((2-(dodecylamino)-3,4-dioxocyclobut-1-en-1-yl)(tetradecyl)amino)butyl)(tetradecyl)amino)-2-hydroxypropyl)amino)cyclobut-3-ene-1,2-dione (0.23 g).

[0274] Example 3

[0275]

[0276] To a round bottom flask charged with a solution of 3-(dodecylamino)-4-((2-hydroxy-3-(tetradecyl(4-(tetradecylamino)butyl)amino)propyl)amino)cyclobut-3-ene-1,2-dione (0.4 g, 0.489 mmol) in anhydrous DMF (10 ml) was added 4.0 M HCl in dioxane (0.49 ml) and stirred at room temperature for 1.5 hours under nitrogen. The resulting solution was concentrated under reduced pressure to give a crude oil, which was purified on RP-HPLC with (95% to 100%) MeOH / water to give mono(N1-(3-((2-(dodecylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)-2-hydroxypropyl)-N1,N4-ditetradecylbutane-1,4-diammonium) monochloride (0.2 g, 48%). (M+H)+=817.7 m / z.

[0277] Example 4

[0278]

[0279] To a round-bottom flask charged with a solution of 3-(dodecylamino)-4-((3-((4-((2-(dodecylamino)-3,4-dioxocyclobut-1-en-1-yl)(tetradecyl)amino)butyl)(tetradecyl)amino)-2-hydroxypropyl)amino)cyclobut-3-ene-1,2-dione (0.23 g, 0.21 mmol) in anhydrous DMF (5 ml) was added 4.0 M HCl in dioxane (0.21 ml) and stirred at room temperature under nitrogen for 1.5 hours. The resulting solution was concentrated under reduced pressure to give a crude oil, which was purified on RP-HPLC with (95% to 100%) MeOH / water to give N1-(2-(dodecylamino)-3,4-dioxocyclobut-1-en-1-yl)-N4-(3-((2-(dodecylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)-2-hydroxypropyl)-N1,N4-ditetradecylbutane-1,4-diammonium chloride (0.13 g, 54%). (M+H)+=1080.95.

[0280] Example 5

[0281]

[0282] 16 was synthesized by the same reaction and procedure as 13.

[0283] Example 6

[0284]

[0285] 2,5,8,11,14,17-hexaoxa nonadecan-19-amine (0.91 g, 3.1 mmol, 1.05 equiv) was added to a round-bottom flask containing an EtOH solution (5 ml) of 3,4-diethoxycyclobut-3-ene-1,2-dione (0.5 g, 2.94 mmol). The reaction solution was stirred at room temperature for 16 hours. The reaction solution was placed in a refrigerator for 16 hours and then concentrated to dryness under reduced pressure to produce an oil. The crude oil was purified on a normal phase 10 g column with (0% to 20%) MeOH / DCM to produce 3-((2,5,8,11,14,17-hexaoxa nonadecan-19-yl)amino)-4-ethoxycyclobut-3-ene-1,2-dione (0.82 g, 60%).

[0286] Example 7

[0287]

[0288] A DMF solution (16 ml) of 3-((2,5,8,11,14,17-hexaoxanonadecan-19-yl)amino)-4-ethoxycyclobut-3-ene-1,2-dione (0.5 g, 1.19 mmol) was added to a round-bottom flask charged with DHDMS (1.03 g, 1.3 mmol, 1.1 eq) and stirred at 50° C. for 16 hours. The resulting solution was concentrated to dryness under reduced pressure to give an oil. The crude oil was purified on a 25 g normal phase column with (0% to 100%) MeOH / DCM to yield 3-((2,5,9,13,17,21-hexaoxatriacosan-23-yl)amino)-4-((2-hydroxy-3-(tetradecyl(4-(tetradecylamino)butyl)amino)propyl)amino)cyclobut-3-ene-1,2-dione (0.24 g, 22%).

[0289] Example 8

[0290]

[0291] To a round bottom flask charged with a solution of 3-((2,5,9,13,17,21-hexaoxatriacosan-23-yl)amino)-4-((2-hydroxy-3-(tetradecyl(4-(tetradecylamino)butyl)amino)propyl)amino)cyclobut-3-ene-1,2-dione (0.24 g, 259 mmol) in DCM (5 ml) was added HCl (4M in dioxane) (0.284 ml) and stirred at room temperature under nitrogen for 1.25 hours. The resulting solution was concentrated to dryness under reduced pressure to give a crude oil, which was purified on RP-HPLC with a C18 column using (75% to 100%) MeOH / water to yield N1-(3-((2-((2,5,9,13,17,21-hexaoxatriacosan-23-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-2-hydroxypropyl)-N1,N4-ditetradecylbutane-1,4-diammonium chloride (236 mg, 91% yield).

[0292] Example 9

[0293]

[0294] To a 100 ml round bottom flask charged with a DMF solution (10 ml) of 3,4-diethoxycyclobut-3-ene-1,2-dione (0.12 g, 0.7 mmol) was added HDMS (0.788 g, 1.42 mmol, 2.0 equiv). The reaction was heated to 50 °C for 5 hours. The reaction was cooled to room temperature and concentrated under reduced pressure to produce an oil, which was purified on a C18 reverse phase column with (2% to 70%) MeOH / chloroform to produce 3,4-bis((2-hydroxy-3-(tetradecyl(4-(tetradecylamino)butyl)amino)propyl)amino)cyclobut-3-ene-1,2-dione (0.41 g, 48% yield).

[0295] Example 10

[0296]

[0297] The synthesis of compound 6 shown in Scheme 3 can be accomplished by reacting cyclam A with an epoxide to form an amino alcohol, followed by hydrazine-mediated deprotection of the amine. The amine is then acylated, LAH-mediated amide reduction, and salt formation.

[0298] Embodiment 11

[0299]

[0300] The synthesis of the alkylated cyclam salts of compounds E and F can be started from 1,8-dimethyl-1,4,8,11-tetraazacyclotetradecane, which will undergo alkylation followed by methylation and hydrazine-mediated deprotection of the amine. The primary amine is then alkylated with a bromoester followed by salt formation.

[0301] Example 12

[0302] Transfection of six different cell types with green fluorescent protein (GFP) expression plasmids was performed using formulations containing cationic lipids / ionizable lipids provided herein. Exemplary formulations and results are provided below.

[0303] Cells were seeded so that the cells were 80% to 90% confluent on the day of transfection in a 96-well tissue culture plate. A lipid solution containing a cationic lipid and a neutral helper lipid, DOPE, was mixed with peptides containing SEQ ID NO: 47 and SEQ ID NO: 540. As shown in Table 3, the formulations tested varied in, for example, the type of cationic lipid (see Table 1) and the ratio of cationic lipid to neutral lipid in the formulation.

[0304] Table 3. Exemplary lipid formulations :

[0305]

[0306] A 2 mg / ml cationic lipid / DOPE lipid aqueous solution was mixed with a peptide solution containing peptides SEQ ID NO: 47 and SEQ ID NO: 540 at a 1: 1.5 (v / v) ratio (lipid solution: peptide solution). The cationic lipid / DOPE / peptide solution contained 0.8 mg / ml cationic lipid / DOPE, 1.2 mg / ml peptide SEQ ID NO: 540, and 0.05 mg / ml peptide SEQ ID NO: 47. A CMV-green fluorescent protein (GFP) expression plasmid was used in these experiments.

[0307] All solutions used to form transfection complexes were transfected at room temperature. 1 μL to 6 μL of lipid preparations of Table 3 were equally divided into six test tubes containing 0.05 mL OptiMEM culture medium. 0.05 mL of OptiMEM solution of 20 μg / mL eGFP plasmid DNA was equally divided into each tube containing lipid preparation aliquots. The lipid preparation and DNA solution were mixed by pipetting up and down twice. The transfection complex was formed at room temperature for 10 minutes. After 10 minutes of compounding, 0.01 mL of transfection complex (0.1 μL to 0.6 μL lipid preparation, 100 ng DNA / well) was added to the cells in each well. HeLa, human primary adult dermal fibroblasts (HDFa), A549, MCF7, HEK293 and CHO-K1 cells were used for these experiments. Cells were incubated at 37 ° C under 5% CO2 for 48 hours. The plate was read on a Perkin-Elmer fluorescent plate reader to measure GFP intensity.

[0308] Exemplary transfection results are shown in Figures 1 to Figure 8 The results show that the lipid compounds described herein provide effective transfection efficiency in all cell types as measured by GFP expression. Cells were also visually examined under a microscope with a fluorescent microscope to assess the degree of transfection (based on the percentage of transfected cells). Equivalent results were obtained when mRNA encoding GFP was used with lipid formulations instead of plasmid DNA.

[0309] Embodiment 13

[0310] Based on a systematic design of experiments (DOE) approach, a composition comprising at least one cationic lipid / ionizable lipid of exemplary structure (I) and one or more neutral lipids was prepared and complexed with mRNA using a previously developed protocol.

[0311] As shown in Table 4, the formulations examined varied in, for example, the type of cationic lipid / ionizable lipid (see Table 1) and the type of lipid complex (ie, lipid nanoparticle (LNP) or liposome).

[0312] Table 4. Exemplary lipid complex formulations :

[0313]

[0314] Lipoplex formulations with at least one cationic lipid / ionizable lipid and at least one neutral lipid were screened and evaluated by in vivo functional testing using RNA payloads of the complexes. Performance and transfection efficiency analyses included payload delivery, biodistribution, and expression of payload-encoded protein.

[0315] The lipid-based nanoparticle complex contains at least one cationic lipid / ionizable lipid of exemplary structure (I) selected from lipid compound 2 free base, lipid compound 2 HCl salt, lipid compound 1 free base and lipid compound 1 HCl salt. The lipid complex is formulated using reverse evaporation or microfluidics instrumentation.

[0316] Lipid nanoparticle complexes were also formulated with DOPE, cholesterol, and 1,2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000). The lipids were weighed and dissolved in ethanol and the RNA payload was encapsulated using a microfluidic instrument.

[0317] The liposome complexes contained (i) Compound 1 free base and DOPE or (ii) Compound 1 HCl salt and DOPE. The lipids were weighed and dissolved in chloroform and then evaporated on a rotary evaporator. The lipid film was hydrated with water and used for liposome preparation. For the liposome complexes, the hydrated lipid film and nucleic acid were diluted in buffer, mechanically mixed by pipetting and / or vortexing, and incubated at room temperature for 10 to 20 minutes before delivery to the rodent model.

[0318] Lipoplexes were incubated overnight and injected into mice the next day or stored at 4°C until use. Female BALB / c mice between 6 and 10 weeks old were purchased from The Jackson Laboratory and acclimated for 7 days prior to the study.

[0319] Firefly luciferase mRNA was complexed with each lipid formulation. Mice were injected with 10 μg of fLuc mRNA formulated lipid complexes using an intravenous tail vein injection in a total volume of 200 μl. Four hours after injection, mice were anesthetized with isoflurane anesthesia and imaged for 10 minutes after an intraperitoneal injection of 100 μL Rediject D-Luciferin (Perkin Elmer). Quantitative bioluminescence imaging was performed in vivo (whole body) and ex vivo (organs) using the IVIS Lumina III imaging system (Perkin Elmer) and analyzed using Living Image software.

[0320] Intravenous administration of various lipid complex formulations was performed in the lungs of injected mice ( Fig. 9 ) and spleen ( Fig.10 ) to achieve mRNA delivery and luciferase expression. Complexes of lipid nanoparticles with compound 1 (HCl or free base) delivered high levels of mRNA payload expression in the lung (see Fig. 9 The lung flux results in LP14, LP15 and LP16 are shown in Figure 2). Liposome complexes with Compound 1 (HCl or free base) delivered high levels of mRNA payload expression in the spleen (see Fig.10 The spleen flux results in LP22 and LP23 were also analyzed.

Claims

1. A compound having structure I in L2 is selected from optionally -OR at up to 2 positions 9 Substituted C3-C 12 Alkylene, C3-C 12 Carbon ring, C3-C 12 Heterocyclic and -(CH2) m CHOR 9 (CH2) n CHOR 9 (CH2) p - the group consisting of, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8; L1 and L3 are selected from the group consisting of: chemical bonds, -CO q -, wherein q is 1 or 2, C2-C8 alkyl optionally interrupted by N, O or -C(O)O-, monounsaturated C4-C8 alkenyl, -CO optionally interrupted by N, O or -C(O)O- q C2-C8 alkyl, and -CO q -monounsaturated C4-C8 alkenyl, or L1X and L3Y are independently -CR 3 =C(R 4 )R 5 ; Each L1 and L3 can be independently -OR'd at up to 2 positions 9 replace; R 1 and R 2 Independently H, C8-C 20 Alkyl or monounsaturated C8-C 20 alkenyl, provided that when L1X and / or L3Y are independently -CR 3 =C(R 4 )R 5 When R 1 and R 2 Not H; R 9 Selected from H, -OH, -M-C8-C 20 Alkyl and -M-C8-C 20 alkenyl, wherein M is selected from the group consisting of a chemical bond, O, -C(=O)-, -C(=O)N(R6)-, -N(R6)C(=O)-, -N(R6)C(=O)N(R7)-, -C(=O)O-, -OC(=O)-, -S-, -SS-, -C=S-, -C(=S)O-, -C(=O)S-, -SC(=O)- and -(R6)P(=O)-; X and Y are independently selected from the group consisting of: L4-Het, wherein L4 is C1-C4 alkylene or -(CH2) m CHOR 9 (CH2) n CHOR 9 (CH2) p -, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8, and Het is a C4-C 12 Heterocyclic rings, Provided that either X or Y, but not both, can be selected from -H, -C1-C 20 Alkyl, -NH2 and -NH-C1-C 20 The group composed of; R 3 and R 4 Each independently selected from H, C1-C 20 Alkyl and C3-C6 cycloalkyl, HDMS, DHDMS, R 12 And by N * , R 3 and R 4 A group consisting of an optionally substituted 3 to 7 membered heterocyclic ring; R 5 Selected from the group consisting of H, C1-C6 alkyl and C3-C6 cycloalkyl; R 6 To R 8 Selected from the group consisting of H and C1-C4 alkyl; Q is selected from C3-C4 alkyl optionally interrupted by -N(H)- or -N(C1-C4 alkyl)- 12 Alkyl, C4-C 12 The group consisting of alkenyl and cycloheteroalkyl; R 10 Selected from the group consisting of H, C1-C4 alkyl or may not exist; R 11 Selected from H, C1-C4 alkyl or -OR 9 The group composed of; When present, R 12 selected from the group consisting of PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinyl pyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide], and poly(amino acids), wherein (i) the PEG or polymer is linear or branched; (ii) the PEG or polymer is polymerized from n subunits; (iii) n is a number average degree of polymerization between 5 and 200 units; and (iv) the compound of structure (I) has at most two R 10 Group.

2. The compound according to claim 1, wherein X or Y is each independently selected from the group consisting of: s = 0, 1, or 2 t=2, 3, 4 or 5.

3. The compound according to claim 1, selected from the group consisting of: The compound according to claim 1 , wherein X and Y are the same. The compound according to claim 1 , wherein X and Y are different. The compound according to claim 1 , wherein R1 and R2 are the same.

7. The compound of claim 1, wherein R1 and R2 are different.

8. The compound according to claim 1, wherein X or Y or both X and Y are 9. The compound according to claim 1, wherein X or Y or both X and Y are 10. The compound of claim 1, wherein L1 is selected from the group consisting of -CO-, -C(=O)-, -OC(=O)-, or -C(=O)O-, and X is L4Het.

11. The compound according to claim 1, wherein L4X is -CH=C(R3)R4.

12. The compound according to claim 1, wherein R3 to R8 are H or C1-C3 alkyl.

13. The compound of claim 1, wherein L2 is -CH2CH(OR9)CH(OR9)CH2-.

14. The compound of claim 1, wherein R9 is H, and R1 and R2 are not H.

15. The compound of claim 1, wherein R9 is not H, and R1 and R2 are H.

16. The compound of claim 1, wherein R9 is selected from the group consisting of -C18 alkyl or -CO-C14-C18 alkyl, and R1 and R2 are H.

17. The compound of claim 1, wherein R1 and R2 are independently selected from the group consisting of H, C8-C20 alkyl, or monounsaturated C8-C20 alkenyl.

18. The compound of claim 1, wherein R3 to R8 are independently selected from the group consisting of H, C1-C6 alkyl and C3-C6 cycloalkyl.

19. The compound according to claim 1, wherein R3 to R8 are independently selected from the group consisting of H or C1-C3 alkyl.

20. The compound of claim 1, wherein R9 is selected from the group consisting of H, -CO-, C8-C20 alkyl, monounsaturated -CO-, C8-C20 alkenyl, C8-C20 alkyl or monounsaturated C8-C20 alkenyl.

21. The compound of claim 1, wherein R9 is selected from the group consisting of C14-C18 alkyl or monounsaturated alkenyl.

22. The compound of claim 1, wherein L2 is C4-C12 alkylene.

23. The compound of claim 1, wherein L2 is -(CH2)mCHOR9(CH2)nCHOR9(CH2)p-, wherein m and p are 1 to 6 and n is 0 to 6, and wherein m+n+p=2 to 8.

24. The compound of claim 1, wherein L2 is -CH2CH(OR9)CH(OR9)CH2-.

25. A composition comprising a compound according to any preceding claim and at least one additional lipid, wherein the additional lipid is a neutral lipid or a cationic lipid / ionizable lipid.

26. The composition of claim 25, wherein the additional lipid is a cationic lipid selected from the group consisting of DOTMA, DOTAP, DMRIE, DC-Chol, DDAB, DOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, TMDOS, DHDMS, HDMS, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-hydroxypropane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmityloxypropyl)-2-hydroxypropane-1,3-diamine, 1,3-diamine, N-1-dimethyl-N-1-(2,3-dioleoyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dimyristyloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, N-1-dimethyl-N-1-(2,3-dipalmityloxypropyl)-2-(3-amino-2-hydroxypropoxy)propane-1,3-diamine, L-spermine-5-carboxy-3-(DL-1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine, 3,5-(N,N-dilysyl)-diaminobenzoyl-glycyl-3-(DL -1,2-dipalmitoyl-dimethylaminopropyl-β-hydroxyethylamine), L-lysine-bis(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, L-lysine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-alkylamino)-2-hydroxypropyl)piperazine, L-lysine-bis-(O,O'-myristoyl-β-hydroxyethyl)amide dihydrochloride, L-ornithine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxypropyl)piperazine, propyl] piperazine, L-ornithine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, 1,4,-bis[(3-amino-2-hydroxypropyl)-oleylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-palmitylamino]-butane-2,3-diol, 1,4,-bis[(3-amino-2-hydroxypropyl)-myristylamino]-butane-2,3-diol, 1,4,-bis[(3-oleylamino)propyl]piperazine, L-arginine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, bis[(3-(3-aminopropyl)-myristylamino)2-hydroxypropyl]piperazine, L-arginine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-serine-bis-(O,O'-oleoyl-β-hydroxyethyl)amide dihydrochloride, 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxypropyl]piperazine, glycine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, sarcosine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide dihydrochloride, L-histidine-bis-(O,O'-palmitoyl-β-hydroxyethyl)amide )amide dihydrochloride, cholesteryl-3β-carboxyl-amidoethylene trimethylamine iodide, 1,4-bis[(3-myristylamino)propyl]piperazine, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterol carboxylate iodide, cholesteryl-3β-carboxylamidoethyleneamine, cholesteryl-3β-oxysuccinamideethylene trimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinic acid iodide, 2-[(2-trimethylamino)propyl]piperazine =The following are the iodine compounds: 1,4-bis[(3-palmitylamino)propyl]piperazine, L-ornithine glycyl-N-(1-heptadecylodecyl)glycinamide, N2,N5-bis(3-aminopropyl)-L ... alkyl) glycinamide, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-alkylamino)-2-hydroxypropyl]piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornithine]-N,N-dioctadecyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dioctadecyl-L-α-glutamine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)2-hydroxypropyl]piperazine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dioctadecyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dioctadecyl-L-glutaminyl]-L-glutamic acid, N2-[N2,N5-bis(3-aminopropyl)-L-ornithine]-N,N-diol-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dioleyl-L-α-glutamine, 4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)-2-hydroxypropyl]piperazine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dioleyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dioleyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3 -aminopropyl)-oleylamino)propyl]piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornithine]-N,N-dipalmitoyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dipalmitoyl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dipalmitoyl-L-glutamine]-L-glutamic acid, N2-[N2,N5-bis(3-aminopropyl)-L-ornithine]-N,N-dimyristyl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dimyristyl-L-α-asparagine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitylamino)-2-hydroxypropyl]piperazine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dimyristyl-L-glutaminyl]-L-glutamic acid, 1,4-bis[(3-(3-aminopropyl)-myristylamino) propyl] piperazine, N2-[N2,N5-bis(3-aminopropyl)-L-ornithine]-N,N-dilauryl-L-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dilauryl-L-α-glutamine, N2-[N2,N5-bis(aminopropyl)-L-ornithine]-NN-dilauryl-L-α-asparagine, N-[N2-[N2,N5-bis[(1,1-dimethylethoxy)carbonyl]-N2,N5-bis[3-[(1,1-dimethylethoxy)carbonyl]aminopropyl]-L-ornithine-NN-dilauryl-L-glutaminyl]-L-glutamic acid, 3-[N',N"-bis(2-tert-butyloxycarbonyl] 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-bispalmitylpropionamide, 3-[N',N"-bis(2-tert-butoxycarbonylaminoethyl)guanidino]-N,N-bispalmitylpropionamide, 1,4-bis[(3-(3-aminopropyl)-palmitylamino)propyl]piperazine, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-oleylamino)propyl]piperazine, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dioleylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dioleylaminopropane Dipalmitylaminopropane, N,N-(2-hydroxy-3-aminopropyl)-N-2-hydroxypropyl-3-N,N-dimyristylaminopropane, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-myristylamino)propyl]piperazine, [(3-aminopropyl)-bis-(2-tetradecylethoxy)]methylammonium bromide, [(3-aminopropyl)-bis-(2-oleylethoxy)]methylammonium bromide, [(3-aminopropyl)-bis-(2-palmitylethoxy)]methylammonium bromide, oleoyl-2-hydroxy-3-N,N-dimethylaminopropane, 2-didecanoyl-1-N,N-dimethylaminopropane, palmitoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-Dipalmitoyl-1-N,N-dimethylaminopropane, Myristoyl-2-hydroxy-3-N,N-dimethylaminopropane, 1,2-dimyristoyl-1-N,N-dimethylaminopropane, (3-amino-propyl)-4-(3-amino-propylamino)-4-tetradecylcarbamoyl-butylcarbamoyl cholesterol ester, (3-amino-propyl)-4-(3-amino-propylamino)-4-carbamoyl-butylcarbamoyl cholesterol ester, (3-amino-propyl)-4-(3-amino-propylamino)-4-(2-dimethylamino-ethylcarbamoyl)-butylcarbamoyl cholesterol ester, spermine-5-carboxyglyceryl Spermine-5-carboxyglycine (N'-stearyl-N'-oleyl) amide tetrakis trifluoroacetate, spermine-5-carboxyglycine (N'-stearyl-N'-oleyl) amide tetrakis trifluoroacetate, agmatine carboxy cholesterol acetate, spermine-5-carboxy-β-alanine cholesterol ester tetrakis trifluoroacetate, 2,6-diaminohexenyl β-alanine cholesterol ester bis trifluoroacetate, 2,4-diaminobutyryl β-alanine cholesterol ester bis trifluoroacetate, N,N-bis(3-aminopropyl)-3-aminopropionyl β-alanine cholesterol ester tris trifluoroacetate, [N,N-bis(2- [(hydroxyethyl)-2-aminoethyl] aminocarboxycholesterol ester, stearyl carnitine ester, palmitoyl carnitine ester, myristyl carnitine ester, stearyl stearoyl carnitine ester hydrochloride, L-stearyl stearoyl carnitine ester, stearyl oleoyl carnitine ester hydrochloride, palmityl palmitoyl carnitine ester hydrochloride, myristyl myristoyl carnitine ester hydrochloride, L-myristyl myristoyl carnitine ester hydrochloride, 1,4-bis[(3-(3-amino-2-hydroxypropyl)-palmitoylamino)propyl]piperazine, N-(3-aminopropyl)-N,N'-bis-(dodecylethoxy)-piperazine bromide 、N-(3-aminopropyl)-N,N'-bis-(oleylethoxy)-piperazine bromide、N-(3-aminopropyl)-N,N'-bis-(palmitylethoxy)-piperazine bromide、N-(3-aminopropyl)-N,N'-bis-(myristylethoxy)-piperazine bromide、N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-dodecylethoxy)-piperazine bromide、N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-oleylethoxy)-piperazine bromide、N-(3-aminopropyl)- N'-Methyl-N,N'-(bis-2-palmitylethoxy)-piperazine bromide, N-(3-aminopropyl)-N'-methyl-N,N'-(bis-2-myristylethoxy)-piperazine bromide, 1,4-bis[(3-(3-aminopropyl)-oleylamino)-2-hydroxy-propyl]piperazine, 1,4-bis[(3-(3-aminopropyl)-myristylamino)-2-hydroxy-propyl]piperazine and 1,4-bis[(3-(3-aminopropyl)-palmitylamino)-2-hydroxy-propyl]piperazine, KL22, KL2 5. 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]dioxolane (DLin-K-DMA), heptathriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA or MC3), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]dioxolane (DLin-KC2-DMA), 1,2-Dilinoleyloxy- N,N-dimethylaminopropane (DODMA), 2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3.β.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propane-1-amine Methyl-3-[(9Z-,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2R)) and (2S)-2-({8-[(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)).

27. The composition of claim 25, wherein the neutral lipid is selected from the group consisting of sterols or sterol derivatives, phospholipids, or combinations thereof.

28. The composition of claim 25, comprising a phosphatidylcholine selected from the group consisting of DOPE, DPhPE, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (POPC), POPE and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (trans-DOPE).

29. The composition of any one of claims 16 to 28, further comprising at least one additional neutral lipid.

30. The composition of claim 29, wherein the additional neutral lipid is a phospholipid selected from the group consisting of DOPE, DPhPE, cholesterol, DOPC, Lyso-PE (1-acyl-2-hydroxy-sn-glycero-3-phosphoethanolamine), Lyso-PC (1-acyl-3-hydroxy-sn-glycero-3-phosphocholine).

31. The composition of any one of claims 25 to 30, further comprising a cell targeting peptide, wherein the peptide optionally comprises a polycationic nucleic acid binding portion.

32. The composition of any one of claims 25 to 30, further comprising a nuclear localization peptide, wherein the peptide optionally comprises a polycationic nucleic acid binding portion.

33. A composition according to any one of claims 25 to 32, further comprising a fusogenic agent, wherein the fusogenic agent optionally comprises a polycationic nucleic acid binding portion.

34. The composition of any one of claims 25 to 33, further comprising a peptide endosomal releasing agent or a non-peptide endosomal releasing agent.

35. A composition according to any one of claims 25 to 34 further comprising a stabilizer.

36. A composition according to any one of claims 25 to 34, further comprising an exosome or a biological material derived from or isolated from an exosome.

37. A composition comprising a compound according to any one of claims 1 to 24 and a nucleic acid.

38. A composition according to any one of claims 25 to 36, further comprising a nucleic acid.

39. The composition of claim 37 or 38, wherein the nucleic acid is an RNA molecule.

40. The composition of claim 39, wherein the RNA molecule is an mRNA molecule.

41. A method of introducing a nucleic acid into a eukaryotic cell, the method comprising contacting the cell with the nucleic acid and a composition according to any one of claims 25 to 40, thereby introducing the nucleic acid into the cell.

42. The method of claim 41, wherein the cell is an animal cell.

43. The method of claim 41, wherein the cell is a human cell.

44. The method of claim 41, wherein the contacting occurs in vivo.

45. The method of claim 41, wherein the contacting occurs in vitro or ex vivo.

46. ​​A method for delivering a lipid composition to spleen tissue and / or lung tissue of a subject, the method comprising administering to the subject a composition according to any one of claims 25 to 40.

47. A method for delivering a bioactive agent to spleen tissue and / or lung tissue of a subject, the method comprising: (i) mixing a bioactive agent with the composition according to any one of claims 25 to 36, thereby forming a bioactive agent-lipid complex; as well as (ii) administering an effective amount of the bioactive agent-lipid complex to a subject, thereby delivering the bioactive agent-lipid complex to the spleen tissue and / or lung tissue of the subject.

48. The method of claim 47, wherein the compound having structure I Contains a single 49. The method of claim 47, wherein the composition comprises a compound selected from the group consisting of compounds of Table 1.

50. The method of claim 47, wherein the bioactive agent-lipid complex comprises lipid nanoparticles.

51. The method of claim 47, wherein the bioactive agent comprises a nucleic acid.

52. The method of claim 51, wherein the nucleic acid comprises an RNA molecule.

53. The method of claim 52, wherein the RNA molecule comprises mRNA, siRNA, miRNA, shRNA, self-amplifying RNA, stRNA, gRNA, or a combination thereof.

54. The method of claim 47, wherein the administering is via systemic administration.

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