PADILLA MARSHALL SCOTT (US)
CLAIMS What is claimed is: 1. An ionizable lipid compound of Formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof: wherein: R1a and R1b are each independently ; R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are each independently selected from the group consisting of H, optionally substituted C1-C12 alkyl, optionally substituted C2-C12 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl; each occurrence of R3a, R3b, and R3c is independently selected from the group consisting of H, and , wherein no more than one of R3a, R3b, and R3c is H; each occurrence of R4a, R4b, R4c, and R4d, if present, is independently selected from the group consisting of optionally substituted C1-C12 alkyl, halogen, CN, and NO2; each occurrence of R5 is independently selected from the group consisting of optionally substituted C1-C3 alkyl, optionally substituted C3-C12 cycloalkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl; each occurrence of R6 is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C12 cycloalkyl, C2-C12 heterocycloalkyl, optionally substituted C6-C12 aralkyl, optionally substituted C6-C12 aryl, optionally substituted C2-C12 heteroaryl, C(=O)Ra, C(=O)ORa, and C(=O)N(Ra)(Rb); each occurrence of L is independently selected from the group consisting of a bond, - (optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocyloalkylenyl; each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R3c)-, and -O-; each occurrence of Y, if present, is independently selected from the group consisting of a bond, -N(Ra)-, and -O-; each occurrence of Z is C1-C24 alkylenyl, wherein the C1-C24 alkylenyl in each occurrence of Z is independently substituted with at least one substituent selected from the group consisting of C1- C12 alkyl and C1-C12 haloalkyl, and wherein the C1-C24 alkylenyl in each occurrence of Z is independently optionally further substituted; each occurrence of Ra and Rb is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C12 aralkyl, optionally substituted phenyl, optionally substituted C2-C8 heteroaryl, C(=O)Rc, C(=O)ORc, and C(=O)N(Rc)(Rd); each occurrence of Rc and Rd is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C12 aralkyl, optionally substituted phenyl, and optionally substituted C2-C8 heteroaryl; and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4. 2. The compound of claim 1, wherein at least one of the following applies: (a) at least one selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h is H; (b) at least two selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (c) at least three selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (d) at least four selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (e) at least five selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (f) at least six selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (g) at least seven selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; and (h) each of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H. 3. The compound of claim 1 or 2, wherein each occurrence of L is independently selected from the group consisting of L is independently selected from the group consisting of -(CH2)1-10-, -(CH2)2-10NR3c-, -(CH2)2-10O-, -(CH2)1-3-CH(ORa)-(CH2)1-3-, piperazinylenyl, and cyclohexylenyl. 4. The compound of any one of claims 1-3, wherein each occurrence of L is independently selected from the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)10-, - (CH2)2O-, -(CH2)3O-, -CH2CH(ORa)CH2-, -(CH2)2NR3c-, , and 5. The compound of any one of claims 1-4, wherein the compound of Formula (I) is selected from the group consisting of: and 6. The compound of any one of claims 1-5, wherein each occurrence of R4a, R4b, R4c, and R4d is independently H. 7. The compound of any one of claims 1-6, wherein each occurrence of R5 is independently methyl. 8. The compound of any one of claims 1-7, wherein each occurrence of R6 is independently H. 9. The compound of any one of claims 1-8, wherein each occurrence of Z is independently: , wherein: each occurrence of R7a, R7b, R7c, and R7d is independently selected from the group consisting of H, C1-C6 alkyl, and C1-C6 haloalkyl, wherein at least one of R7a, R7b, R7c, and R7d is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. 10. The compound of claim 9, wherein each occurrence of R7a, R7b, R7c, and R7d is selected from the group consisting of H and methyl. 11. The compound of any one of claims 1-10, wherein each occurrence of Z is independently selected from the group consisting of -(CH2)4-10-CH(CH3)-*, -(CH2)4-10- C(CH3)2-*, and -(CH2)4-10-CH(CH3)-CH2-*. 12. The compound of any one of claims 1-11, wherein each occurrence of R3a, R3b, and R3c is independently selected from the group consisting of and . 13. The compound of any one of claims 1-12, wherein each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent selected from the group consisting of C1- C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C3 haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R’)(R’’), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O)2N(R’)(R’’), N(R’)C(=O)R’’, N(R’)S(=O)2R’’, C2-C8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, benzyl, and phenyl. 14. The compound of any one of claims 1-13, which is selected from the group consisting of: , and . 15. A lipid nanoparticle (LNP) composition comprising: (f) at least one ionizable lipid compound or a salt, solvate, stereoisomer, or isotopologue thereof having the structure of Formula (I): wherein: R1a and R1b are each independently R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are each independently selected from the group consisting of H, optionally substituted C1-C12 alkyl, optionally substituted C2-C12 heteroalkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C2-C12 alkenyl, optionally substituted C2-C12 alkynyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl; each occurrence of R3a, R3b, and R3c is independently selected from the group consisting of H, , , , and , wherein no more than one of R3a, R3b, and R3c is H; each occurrence of R4a, R4b, R4c, and R4d, if present, is independently selected from the group consisting of optionally substituted C1-C12 alkyl, halogen, CN, and NO2; each occurrence of R5 is independently selected from the group consisting of optionally substituted C1-C3 alkyl, optionally substituted C3-C12 cycloalkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl; each occurrence of R6 is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C12 cycloalkyl, C2-C12 heterocycloalkyl, optionally substituted C6-C12 aralkyl, optionally substituted C6-C12 aryl, optionally substituted C2-C12 heteroaryl, C(=O)Ra, C(=O)ORa, and C(=O)N(Ra)(Rb); each occurrence of L is independently selected from the group consisting of a bond, - (optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, optionally substituted C3-C8 cycloalkylenyl, and optionally substituted C2-C8 heterocyloalkylenyl; each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R3c)-, and -O-; each occurrence of Y, if present, is independently selected from the group consisting of a bond, -N(Ra)-, and -O-; each occurrence of Z is C1-C24 alkylenyl, wherein the C1-C24 alkylenyl in each occurrence of Z is independently substituted with at least one substituent selected from the group consisting of C1- C12 alkyl and C1-C12 haloalkyl, and wherein the C1-C24 alkylenyl in each occurrence of Z is independently optionally further substituted; each occurrence of Ra and Rb is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C12 aralkyl, optionally substituted phenyl, optionally substituted C2-C8 heteroaryl, C(=O)Rc, C(=O)ORc, and C(=O)N(Rc)(Rd); each occurrence of Rc and Rd is independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 haloalkyl, optionally substituted C6-C12 aralkyl, optionally substituted phenyl, and optionally substituted C2-C8 heteroaryl; and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4; (g) at least one neutral lipid; (h) cholesterol; and (i) at least one conjugated lipid. 16. The LNP of claim 15, wherein the LNP further comprises: (j) at least one nucleic acid and/or therapeutic agent cargo, wherein the cargo is at least partially encapsulated therein. 17. The LNP of claim 15 or 16, wherein at least one of the following applies: (a) at least one selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h is H; (b) at least two selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (c) at least three selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (d) at least four selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (e) at least five selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (f) at least six selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; (g) at least seven selected from the group consisting of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H; and (h) each of R2a, R2b, R2c, R2d, R2e, R2f, R2g, and R2h are H. 18. The LNP of any one of claims 15-17, wherein each occurrence of L is independently selected from the group consisting of L is independently selected from the group consisting of -(CH2)1-10-, -(CH2)2-10NR3c-, -(CH2)2-10O-, -(CH2)1-3-CH(ORa)-(CH2)1-3-, piperazinylenyl, and cyclohexylenyl. 19. The LNP of any one of claims 15-18, wherein each occurrence of L is independently selected from the group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)10-, -(CH2)2O-, - (CH2)3O-, -CH2CH(ORa)CH2-, -(CH2)2NR3c-, and 20. The LNP of any one of claims 15-19, wherein the compound of Formula (I) is selected from the group consisting of: and 21. The LNP of any one of claims 15-20, wherein each occurrence of R4a, R4b, R4c, and R4d is independently H. 22. The LNP of any one of claims 15-21, wherein each occurrence of R5 is independently methyl. 23. The LNP of any one of claims 15-22, wherein each occurrence of R6 is independently H. 24. The LNP of any one of claims 15-23, wherein each occurrence of Z is independently: wherein: each occurrence of R7a, R7b, R7c, and R7d is independently selected from the group consisting of H, C1-C6 alkyl, and C1-C6 haloalkyl, wherein at least one of R7a, R7b, R7c, and R7d is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. 25. The LNP of claim 24, wherein each occurrence of R7a, R7b, R7c, and R7d is selected from the group consisting of H and methyl. 26. The LNP of any one of claims 15-25, wherein each occurrence of Z is independently selected from the group consisting of -(CH2)4-10-CH(CH3)-*, -(CH2)4-10-C(CH3)2-*, and - (CH2)4-10-CH(CH3)-CH2-*. 27. The LNP of any one of claims 15-26, wherein each occurrence of R3a, R3b, and R3c is independently selected from the group consisting of and . 28. The LNP of any one of claims 15-27, wherein each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent selected from the group consisting of C1- C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C3 haloalkoxy, phenoxy, halogen, CN, NO2, OH, N(R’)(R’’), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O)2N(R’)(R’’), N(R’)C(=O)R’’, N(R’)S(=O)2R’’, C2-C8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, benzyl, and phenyl. 29. The LNP of any one of claims 15-28, which is selected from the group consisting of: , and . 30. The LNP of any one of claims 15-29, wherein the at least one ionizable lipid of Formula (I) comprises about 10 mol% to about 90 mol% of the LNP. 31. The LNP of any one of claims 15-30, wherein the at least one ionizable lipid of Formula (I) comprises about 35 mol% or about 40 mol% of the LNP. 32. The LNP of any one of claims 15-31, wherein the at least one neutral lipid comprises about 1 mol% to about 40 mol% of the LNP. 33. The LNP of any one of claims 15-32, wherein the at least one neutral lipid comprises about 16 mol% or about 30 mol% of the LNP. 34. The LNP of any one of claims 15-33, wherein the at least one neutral lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC). 35. The LNP of any one of claims 15-34, wherein the at least one neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE). 36. The LNP of any one of claims 15-35, wherein the cholesterol comprises about 20 mol% to about 75 mol% of the LNP. 37. The LNP of any one of claims 15-36, wherein the cholesterol comprises about 25 mol% or about 46.5 mol% of the LNP. 38. The LNP of any one of claims 15-37, wherein the at least one conjugated lipid comprises about 0.1 mol% to about 15 mol% of the LNP. 39. The LNP of any one of claims 15-38, wherein the at least one conjugated lipid comprises about 2.5 mol% of the LNP. 40. The LNP of any one of claims 15-39, wherein the at least one conjugated lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). 41. The LNP of any one of claims 15-40, wherein the LNP has a molar ratio of (a) : (b) : (c) : (d) of about 35:16:46.5:2.5 or about 40:30:25:2.5 42. The LNP of any one of claims 16-41, wherein the nucleic acid molecule is a therapeutic agent. 43. The LNP of any one of claims 16-42, wherein the nucleic acid molecule is at least one selected from the group consisting of RNA and DNA. 44. The LNP of any one of claims 16-43, wherein the nucleic acid molecule is at least one selected from the group consisting of mRNA, cDNA, miRNA, siRNA, and modified RNA. 45. The LNP of any one of claims 42-44, wherein the nucleic acid is mRNA. 46. The LNP of claim 45, wherein the LNP has a mass ratio of (a) : mRNA of about 20:1 to about 5:1 (w/w), optionally wherein the LNP has a mass ratio of (a) : mRNA of about 10:1. 47. The LNP of claim 45 or 46, wherein the mRNA encodes a chimeric antigen receptor (CAR). 48. The LNP of claim 47, wherein the CAR is specific for binding to a surface antigen of a pathogenic cell or a tumor cell. 49. The LNP of claim 48, wherein the surface antigen is selected from the group consisting of CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD123, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, CCR7, k light chain, ROR1, ErbB2, ErbB3, ErbB4, EGFR vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL13R-α2, MUC1, VEGF-A, Tem8, FAP, EphA2, HER2, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CALX, HLA-AI MAGE A1, HAL-A2 NY-ESO-1, PSC1, folate receptor-α, 8H9, NCAM, VEGF, 5T4, Fetal AchR, NKG2D ligands, TEM1, and TEM8. 50. The LNP of claim 45 or 46, wherein the mRNA encodes an enzyme. 51. The LNP of any one of claims 45-46 and 49, wherein the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9. 52. A pharmaceutical composition comprising the lipid nanoparticle (LNP) of any one of claims 15-51 and at least one pharmaceutically acceptable carrier. 53. The pharmaceutical composition of claim 52, wherein the composition further comprises at least one adjuvant. 54. A method of treating, preventing, and/or ameliorating a disease in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of claims 16-51 and/or at least one pharmaceutical composition of claim 52 or 53. 55. The method of claim 54, wherein the disease is selected from the group consisting of cancer, an autoimmune disorder, cardiovascular disease, and neurological disease. 56. The method of claim 55, wherein the cancer is at least one selected from the group consisting of oral cancer, pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer. 57. The method of claim 54 or 56, wherein the subject is further administered at least one additional agent or therapy useful for treating, preventing, and/or ameliorating cancer in the subject. 58. The method of any one of claims 54-57, wherein the subject is a mammal. 59. The method of claim 58, wherein the mammal is a human. 60. A method of delivering a nucleic acid or therapeutic agent to the liver of a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of claims 16-51 and/or at least one pharmaceutical composition of claim 52 or 53. 61. A method of preparing a modified immune cell or precursor thereof, the method comprising contacting an immune cell or precursor thereof with the lipid nanoparticle (LNP) of any one of claims 16-51 and/or at least one pharmaceutical composition of claim 52 or 53. 62. The method of claim 61, wherein the modified immune cell or precursor thereof is αβ T cell, a γδ T cell, a CD8+ T cell, a CD4+ helper T cell, a CD4+ regulatory T cell, an NK T cell, an NK cell, and any combination thereof. 63. The method of claim 62, wherein the modified immune cell or precursor thereof is a T cell, optionally wherein the T cell is a CD4+ T cell. 64. The method of claim 62, wherein the modified immune cell or precursor thereof is a NK cell. |
Lipid Nanoparticles (LNPs) In another aspect, the present disclosure provides a lipid nanoparticle (LNP) composition. In certain embodiments, the LNP composition comprises at least one ionizable lipid compound or a salt, solvate, stereoisomer, or isotopologue thereof having the structure of Formula (I): wherein: R 1a and R 1b are each independently ; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are each independently selected from the group consisting of H, optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 heteroalkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 2 -C 8 heterocycloalkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 6 -C10 aryl, and optionally substituted C 2 -C10 heteroaryl; each occurrence of R 3a , R 3b , and R 3c is independently selected from the group consisting of H, , and , wherein no more than one of R 3a , R 3b , and R 3c is H; each occurrence of R 4a , R 4b , R 4c , and R 4d , if present, is independently selected from the group consisting of optionally substituted C 1 -C 12 alkyl, halogen, CN, and NO 2 ; each occurrence of R 5 is independently selected from the group consisting of optionally substituted C 1 -C 3 alkyl, optionally substituted C 3 -C 12 cycloalkyl, optionally substituted C 6 -C 10 aryl, and optionally substituted C 2 -C 10 heteroaryl; each occurrence of R 6 is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 12 cycloalkyl, C 2 -C 12 heterocycloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted C 6 -C 12 aryl, optionally substituted C 2 -C 12 heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b ); each occurrence of L is independently selected from the group consisting of a bond, - (optionally substituted C 1 -C 12 alkylenyl)-X-, -(optionally substituted C 2 -C 12 alkenylenyl)-X-, -(optionally substituted C 1 -C 12 alkynylenyl)-X-, -(optionally substituted C 1 -C 12 heteroalkylenyl)-X-, optionally substituted C 3 -C 8 cycloalkylenyl, and optionally substituted C 2 -C8 heterocyloalkylenyl; each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R 3c )-, and -O-; each occurrence of Y, if present, is independently selected from the group consisting of a bond, -N(R a )-, and -O-; each occurrence of Z is C 1 -C 24 alkylenyl, wherein the C 1 -C 24 alkylenyl in each occurrence of Z is independently substituted with at least one substituent selected from the group consisting of C 1 - C 12 alkyl and C 1 -C 12 haloalkyl, and wherein the C 1 -C 24 alkylenyl in each occurrence of Z is independently optionally further substituted; each occurrence of R a and R b is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted phenyl, optionally substituted C 2 -C8 heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d ); each occurrence of R c and R d is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted phenyl, and optionally substituted C 2 -C 8 heteroaryl; and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4; In certain embodiments, the LNP composition comprises at least one neutral lipid. In certain embodiments, the LNP composition comprises cholesterol. In certain embodiments, the LNP composition comprises at least one conjugated lipid. In certain embodiments, the LNP comprises at least one nucleic acid and/or therapeutic agent cargo, wherein the cargo is at least partially encapsulated therein. In certain embodiments, at least one selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h is H. In certain embodiments, at least two selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. In certain embodiments, at least three selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. In certain embodiments, at least four selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. In certain embodiments, at least five selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. In certain embodiments, at least six selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. In certain embodiments, at least seven selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. In certain embodiments, each of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. In certain embodiments, L is -(CH 2 ) 1-10 -. In certain embodiments, L is -(CH 2 ) 2-10 NR 3c - . In certain embodiments, L is -(CH 2 ) 2 -10O-. In certain embodiments, L is -(CH 2 ) 1-3 -CH(OR a )- (CH 2 ) 1-3 -. In certain embodiments, L is piperazinylenyl. In certain embodiments, L is cyclohexylenyl. In certain embodiments, L is -CH 2 -. In certain embodiments, L is -(CH 2 ) 2 -. In certain embodiments, L is -(CH 2 ) 3 -. In certain embodiments, L is -(CH 2 )10-. In certain embodiments, L is -(CH 2 ) 2 O-. In certain embodiments, L is -(CH 2 ) 3 O-. In certain embodiments, L is - CH 2 CH(OR a )CH 2 -. In certain embodiments, L is -(CH 2 ) 2 NR 3c -. In certain embodiments, L is . In certain embodiments, L is . In certain embodiments, L is In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, the compound of Formula (I) is: In certain embodiments, R 4a is H. In certain embodiments, R 4b is H. In certain embodiments, R 4c is H. In certain embodiments, R 4d is H. In certain embodiments, R 5 is methyl. In certain embodiments, R 6 is H. In certain embodiments, each occurrence of Z is independently: wherein: each occurrence of R 7a , R 7b , R 7c , and R 7d is independently selected from the group consisting of H, C 1 -C 6 alkyl, and C 1 -C 6 haloalkyl, wherein at least one of R 7a , R 7b , R 7c , and R 7d is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, R 7a is H. In certain embodiments, R 7b is H. In certain embodiments, R 7c is H. In certain embodiments, R 7d is H. In certain embodiments, R 7a is methyl. In certain embodiments, R 7b is methyl. In certain embodiments, R 7c is methyl. In certain embodiments, R 7d is methyl. In certain embodiments, Z is -(CH 2 ) 4-10 -CH(CH 3 )-*. In certain embodiments, Z is - (CH 2 ) 4-10 -C(CH 3 ) 2 -*. In certain embodiments, Z is -(CH 2 ) 4-10 -CH(CH 3 )-CH 2 -*. In certain embodiments, R 3a , is . In certain e 3a mbodiments, R , is . In certain em 3a bodiments, R , is . In certain embodiments, R 3a , is . In certain embodiments, R 3a , is . In certain embodiments, R 3a , is . In certain embodime 3a nts, R , is . In certain embodiments, R 3a , is . In certain embodiments, R 3a , is . In certain embodiments, R 3a , is . In certain embodiments, R 3a , is . In certain e 3a mbodiments, R , is In certain embodiments, R 3b , is . In certain embodiments, R 3b , is . In certain embodiments, 3b R , is . In certain embodiments, R 3b , is . In certain embodiments, R 3b , is . In certain embodiments, R 3b , is . In certain embod 3b iments, R , is . In certain embodiments, R 3b , is . In certain embodiments, R 3b , is . In certain embodiments, R 3b , is . In certain embodiments, R 3b , is . In certain embodiments, R 3b , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodi 3c ments, R , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, R 3c , is . In certain embodiments, each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent selected from the group consisting of C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 1 - C 6 haloalkyl, C 1 -C 3 haloalkoxy, phenoxy, halogen, CN, NO 2 , OH, N(R’)(R’’), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O) 2 N(R’)(R’’), N(R’)C(=O)R’’, N(R’)S(=O) 2 R’’, C 2 -C 8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 6 haloalkyl, benzyl, and phenyl. In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the compound of Formula (I) is . In certain embodiments, the at least one ionizable lipid of Formula (I) comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises about 35 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises less than about 35 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises greater than about 35 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises about 40 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises less than about 40 mol% of the LNP. In certain embodiments, the at least one ionizable lipid of Formula (I) comprises greater than about 40 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises about 16 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises less than about 16 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 16 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises about 30 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises less than about 30 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 30 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE). In certain embodiments, the at least one neutral lipid comprises distearoylphosphatidylcholine (DSPC). In certain embodiments, the at least one neutral lipid comprises dioleoylphosphatidylcholine (DOPC). In certain embodiments, the cholesterol comprises about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or about 75 mol% of the LNP. In certain embodiments, the cholesterol comprises less than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or about 75 mol% of the LNP. In certain embodiments, the cholesterol comprises greater than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or about 75 mol% of the LNP. In certain embodiments, the cholesterol comprises about 25 mol% of the LNP. In certain embodiments, the cholesterol comprises less than about 25 mol% of the LNP. In certain embodiments, the cholesterol comprises more than about 25 mol% of the LNP. In certain embodiments, the cholesterol comprises about 46.5 mol% of the LNP. In certain embodiments, the cholesterol comprises less than about 46.5 mol% of the LNP. In certain embodiments, the cholesterol comprises more than about 46.5 mol% of the LNP. In certain embodiments, the at least one conjugated lipid comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0 or about 15.0 mol% of the LNP. In certain embodiments, the at least one conjugated lipid comprises less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0 or about 15.0 mol% of the LNP. In certain embodiments, the at least one conjugated lipid comprises greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0 or about 15.0 mol% of the LNP. In certain embodiments, the at least one conjugated lipid comprises 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). In certain embodiments, the LNP has a molar ratio of (a) : (b) : (c) : (d) of about 35:16:46.5:2.5. In certain embodiments, the LNP has a molar ratio of (a) : (b) : (c) : (d) of about 40:30:25:2.5. In certain embodiments, the nucleic acid molecule is a therapeutic agent. In certain embodiments, the nucleic acid molecule comprises RNA. In certain embodiments, the nucleic acid molecule comprises DNA. In certain embodiments, the nucleic acid molecule comprises mRNA. In certain embodiments, the nucleic acid molecule comprises cDNA. In certain embodiments, the nucleic acid molecule comprises miRNA. In certain embodiments, the nucleic acid molecule comprises siRNA. In certain embodiments, the nucleic acid molecule comprises modified RNA. In certain embodiments, the nucleic acid is mRNA. In certain embodiments, the LNP has a mass ratio of (a) : mRNA of about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, or about 5:1 (w/w). In certain embodiments, the LNP has a mass ratio of (a) : mRNA of about 10:1. In certain embodiments, mRNA encodes a chimeric antigen receptor (CAR). In certain embodiments, CAR is specific for binding to a surface antigen of a pathogenic cell or a tumor cell. In certain embodiments, the surface antigen is selected from the group consisting of CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD123, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, CCR7, k light chain, ROR1, ErbB2, ErbB3, ErbB4, EGFR vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL13R-α2, MUC1, VEGF-A, Tem8, FAP, EphA2, HER2, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CALX, HLA-AI MAGE A1, HAL-A2 NY-ESO-1, PSC1, folate receptor-α, 8H9, NCAM, VEGF, 5T4, Fetal AchR, NKG2D ligands, TEM1, and TEM8. In certain embodiments, the mRNA encodes an enzyme. In certain embodiments, the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9. In certain embodiments, the mRNA further encodes a small-guiding RNA (sgRNA). Methods In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating a disease in a subject. In certain embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the present disclosure and/or at least one pharmaceutical composition of the present disclosure. In certain embodiments, the LNPs of the present disclosure are useful for the treatment, prevention, and/or amelioration of any of a number of diseases and/or disorders. In certain embodiments, the LNPs of the present disclosure are suitable for treatment, prevention, and/or amelioration of diseases and/or disorders for which mRNA delivery and/or gene therapy is useful (e.g., cancer immunotherapy). In certain embodiments, the LNPs of the present disclosure are suitable for the treatment, prevention, and/or amelioration of diseases and/or disorders including autoimmune disoders, cardiovascular diseases, and neurological disorders, inter alia. In certain embodiments the LNPs of the present disclosure are suitable for gene editing applications, including gene editing of the liver, brain, lung, and/or hematopoietic cells for monogenic diseases. In certain embodiments, the disease is cancer. In certain embodiments, the cancer is at least one selected from the group consisting of pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer. In certain embodiments, the subject is further administered at least one additional agent or therapy useful for treating, preventing, and/or ameliorating cancer in the subject. In certain embodiments, the subject is a mammal. In certain embodiments, mammal is a human. In another aspect, the present disclosure provides a method of delivering a nucleic acid or therapeutic agent to the liver of a subject. In certain embodiments, the method comprises administering to the subject at least one lipid nanoparticle (LNP) of the present disclosure and/or at least one pharmaceutical composition of the present disclosure. In another aspect, the present disclosure provides a method of preparing a modified immune cell or precursor thereof. In certain embodiments, the method comprises contacting an immune cell or precursor thereof with at least one lipid nanoparticle (LNP) of the present disclosure and/or at least one pharmaceutical composition of the present disclosure. In certain embodiments, the modified immune cell or precursor thereof is αβ T cell, a γδ T cell, a CD8+ T cell, a CD4+ helper T cell, a CD4+ regulatory T cell, an NK T cell, an NK cell, and any combination thereof. In certain embodiments, the modified immune cell or precursor thereof is a T cell, optionally wherein the T cell is a CD4+ T cell. In certain embodiments, the modified immune cell or precursor thereof is a NK cell. In another aspect, the present disclosure provides a method of preparing a compound of Formula (2-2), or a salt, solvate, stereoisomer, or isotopologue thereof: In certain embodiments, the method comprises contacting a compound of formula (1- 4): and a compound of formula (2-1): wherein: each occurrence of L is independently selected from the group consisting of a bond, - (optionally substituted C 1 -C 12 alkylenyl)-X-, -(optionally substituted C 2 -C 12 alkenylenyl)-X-, -(optionally substituted C 1 -C 12 alkynylenyl)-X-, -(optionally substituted C 1 -C 12 heteroalkylenyl)-X-, optionally substituted C 3 -C 8 cycloalkylenyl, and optionally substituted C 2 -C 8 heterocyloalkylenyl; each occurrence of R’ is independently optionally substituted C 1 -C 12 alkyl, wherein each occurrence of R’ comprises at least one tertiary carbon; each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4; and each occurrence of s is independently an integer selected form the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, the contacting occurs in the presence of a solvent, optionally wherein the solvent comprises ethanol (EtOH). In certain embodiments, the contacting occurs at a temperature of about 70 °C to about 90 °C, optionally wherein the contacting occurs at a temperature of about 80 °C. In certain embodiments, the contacting occurs for a period of about 1 h to about 72 h, optionally wherein the contacting occurs for a period of about 48 h. In certain embodiments, the compound of formula (1-4) is prepared by contacting a compound of formula (1-3): and an organomagnesium compound of formula R’-MgX 3 , wherein X 3 is selected from the group consisting of Cl, Br, and I. In certain embodiments, the contacting occurs in the presence of a suitable epoxidation reagent, optionally wherein the epoxidation reagent is m-chloroperoxy benzoic acid. In certain embodiments, the contacting occurs in the presence of a solvent, optionally wherein the solvent comprises dichloromethane. In certain embodiments, the compound of formula (1-3) is prepared by contacting a compound of formula (1-2): and an organomagnesium compound of formula R’-MgX 3 , wherein X 1 and X 3 are each independently selected from the group consisting of Cl, Br, and I. In certain embodiments, the contacting occurs in the presence of a suitable catalyst, optionally wherein the catalyst comprises Li 2 CuCl 4 . In certain embodiments, the contacting occurs in the presence of a solvent, optionally wherein the solvent comprises at least one selected from the group consisting of tetrahydrofuran (THF) and N-methyl pyrrolidone (NMP). In certain embodiments, the compound of formula (1-2) is prepared by contacting a compound of formula (1-1): and an alkoxide base, wherein X 1 , X 2 , and X 3 are each independently selected from the group consisting of Cl, Br, and I. In certain embodiments, the contacting occurs in the presence of a solvent, optionally wherein the solvent comprises THF. In certain embodiments, the alkoxide base comprises a tert-butoxide base, optionally wherein the tert-butoxide base is potassium tert-butoxide. In certain embodiments, the contacting occurs at a temperature of about 60 ℃ to about 80 ℃. Pharmaceutical Compositions In another aspect, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticle (LNP) of the present disclosure and at least one pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises at least one adjuvant. Such a pharmaceutical composition may consist of at least one composition of the invention, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one composition of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art. In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day. The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient. Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations. In certain embodiments, the compositions of the invention are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like. The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN ® ), solubilized gelatins (e.g., GELOFUSINE ® ), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and/or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, "additional ingredients" include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier. The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid. The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alpha- tocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art. Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents. Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art. Administration/Dosing The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of therapeutic (i.e., composition) necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular therapeutic employed; the time of administration; the rate of excretion of the composition; the duration of the treatment; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic composition of the disclosure is from about 0.01 mg/kg to 100 mg/kg of body weight/per day of active agent (i.e., nucleic acid). One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic composition without undue experimentation. The composition may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of composition dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic composition to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic composition and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic composition for the treatment of a disease or disorder in a patient. In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account. The amount of active agent of the composition(s) of the disclosure for administration may be in the range of from about 1 µg to about 7,500 mg, about 20 µg to about 7,000 mg, about 40 µg to about 6,500 mg, about 80 µ g to about 6,000 mg, about 100 µ g to about 5,500 mg, about 200 µ g to about 5,000 mg, about 400 µ g to about 4,000 mg, about 800 µ g to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between. In some embodiments, the dose of active agent (i.e., nucleic acid) present in the composition of the disclosure is from about 0.5 µg and about 5,000 mg. In some embodiments, a dose of active agent present in the composition of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof. In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of the composition of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient. The term "container" includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound's ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient. Administration Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration. Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein. Parenteral Administration As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non- toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. EXAMPLES Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein. Materials and Methods Materials All non-IL LNP lipid excipients as well as lipids to make the artificial endosomes were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cas9 and firefly luciferase mRNA were purchased from TriLink Biotechnologies (San Diego, CA, USA) with 5- methoxyuridine substitutions. TTR sgRNA was synthesized by Axolabs (Kulmbach, Germany), using the following sequence: 5’- ususasCAGCCACGUCUACAGCAGUUUUAGAgcuagaaauagcAAGUUAAAAUAAGGCU AGUCCGUUAUCAacuugaaaaaguggcaccgagucggugcusususu-3’ (SEQ ID NO:1), where N refers to RNA residues, n are 2’-O-methyl residues, and s are phosphorothioate backbone modifications. The chemicals 1,2-epoxyoctane, 1,2-epoxydecane, 1,2-epoxydodecane, 1,2- epoxytetradecane, and 8-bromo-1-octene were purchased from TCI (Montgomeryville, PA, USA); Triton X-100 was purchased from Alfa Aesar (Haverhill, MA, USA), 1,12- dibromododecane and N1-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diami ne were purchased from AmBeed (Arlington Heights, IL, USA); anhydrous 1-methyl-2-pyrrolidinone and chloroform-d were purchased from Acros Organics (Geel, Belgium); all non-anhydrous solvents as well as anhydrous magnesium sulfate, and 1 N hydrochloric acid were purchased from Fisher Scientific (Waltham, MA, USA); 10-bromo-1-decene was purchased from Oakwood Chemicals (Estill, SC, USA); 6-bromo-1-hexene was purchased from Asta Tech (Bristol, PA, USA); 2-{2-[4-(2-{[2-(2-aminoethoxy)ethyl]amino}ethyl)piperazin-1- yl]ethoxy}ethan-1-amine was purchased from Enamine (Kiev, Ukraine). All other chemical reagents were purchased from MilliporeSigma (St. Louis, MO, USA). Synthesis All flash chromatography was performed on a Teledyne Isco (Lincoln, NE, USA) CombiFlash NextGen 300+ equipped with evaporative light scattering detection using RediSep Gold® silica gel disposable flash columns. Solvent evaporation was performed using a Büchi (New Castle, DE, USA) Rotavapor® R-300 System Professional. 1 H and 13 C NMR spectra were acquired in d-chloroform using an Avance Neo 400 MHz spectrometer (Bruker, Billerica, MA, USA). LC-MS spectra were acquired in ethanol using an SQD equipped with an Acquity UPLC (Milford, MA, USA), using a C8 column with a 2 min wash followed by a gradient mobile phase from 50% water (1% trifluoroacetic acid) and 50% acetonitrile (1% trifluoroacetic acid) to 100% acetonitrile (1% trifluoroacetic acid). 12-bromododec-1-ene To a 250 mL round bottom flask was added 1,12-dibromododecane (8.00 g, 24.4 mmol, 2.0 equiv.) and anhydrous tetrahydrofuran (20 mL). Then, potassium tert-butoxide (2.74 g, 24.4 mmol, 1.0 equiv.) in anhydrous THF (50 mL) was added dropwise. The reaction stirred at 70 °C for 16 h. The reaction was then quenched with deionized water (40 mL), and the was extracted with hexanes (3x35 mL). The organic fractions were combined, dried with magnesium sulfate, filtered, and concentrated in vacuo. The crude product was further purified via flash chromatography assisted by a CombiFlash using a liquid injection into an 80 g column. The mobile phase was isocratic hexanes for 10 min using a 20 mL/min flow. The product was isolated as a clear oil at a 25.8% yield. 1 H NMR (400 MHz, CDCl 3 ) δ 5.82 (ddt, J = 16.9, 10.1, 6.7 Hz, 1H), 5.18 – 4.81 (m, 2H), 3.41 (td, J = 6.8, 1.0 Hz, 2H), 2.25 – 1.96 (m, 2H), 1.87 (dt, J = 14.5, 7.0 Hz, 2H), 1.64 – 1.01 (m, 14H). 13 C NMR (101 MHz, CDCl 3 ) δ 139.06, 114.12, 33.83, 33.77, 32.89, 29.52, 29.48, 29.46, 29.15, 28.96, 28.80, 28.21. General Procedure “A”: Branched Alkene Synthesis A 100 mL Schlenk flask was purged with nitrogen. To the flask was added anhydrous tetrahydrofuran (5 or 10 mL), N-methylpyrrolidinone (48.0 mmol, 4.0 equiv.), dilithium tetrachlorocuprate (0.1 M tetrahydrofuran; 0.36 mmol, 0.03 equiv.), and the corresponding bromoalkene (12.0 mmol, 1.0 equiv.). The solution stirred at room temperature and under nitrogen for 5 min. Then, the flask was put on a water bath at room temperature. The corresponding Grignard reagent (13.2 mmol, 1.1 equiv.) was then added dropwise. After 5 min, the flask was removed from the water bath, and then stirred at room temperature for 1 h. The flask was then cooled to 0 °C and then slowly quenched with hydrochloric acid (1 M; 40 mL). The aqueous phase was extracted with hexanes (3 x 20 mL), and the organic layers were combined, washed with hydrochloric acid (1 M; 1 x 40 mL), washed with brine (2 x 40 mL), dried with magnesium sulfate, filtered, and concentrated in vacuo. The crude product was further purified via flash chromatography assisted by a CombiFlash using a liquid injection into a 40 g column. The mobile phase was isocratic hexanes using a flow rate of 7 mL/min. The products were isolated as clear oils. See the Supplementary Information for the specific conditions for each reaction. General Procedure “B”: Branched Epoxide Synthesis To a 100 mL round bottom flask was added the corresponding branched alkene (1.0 equiv.) and dichloromethane (5 mL). The flask was mixed for 1 min and then cooled to 0 °C. Then, to the flask was added dropwise half of a solution of meta-chloroperoxybenzoic acid (70% pure; 2.0 equiv.) dissolved in dichloromethane (30 mL). The mixture stirred for 1 h after which the other half of the meta-chloroperoxybenzoic acid solution in dichloromethane was added dropwise. After 1 h, the reaction flask was removed from the 0 °C bath and stirred at room temperature for 14 h. The reaction was quenched by adding 20 mL of a 1:1 solution of sat. sodium bicarbonate and sat. sodium thiosulfate. The layers were separated, and the organic layer was washed with brine (1 x 30 mL). The aqueous layers were then combined and extracted with DCM (3 x 15 mL). The organic layers were combined, dried with magnesium sulfate, filtered, and concentrated in vacuo. The crude product was further purified via flash chromatography assisted by a CombiFlash using a liquid injection into a 24 g column. The mobile phase had a gradient of 100% hexanes to 90% hexanes and 10% ethyl acetate over 15 min using a flow rate of 35 mL/min. The products were isolated as clear oils. See the Supplementary Information for the specific conditions for each reaction. General Procedure “C”: Ionizable Lipid Synthesis To a 1 dram vial was added the corresponding polyamine core (1.0 equiv.), the corresponding epoxide (6.0 or 7.0 equiv.), and ethanol (0.3 mL). The reaction stirred at 80 °C for 48 h. Afterwards, the solution was diluted with dichloromethane (0.7 mL). The solution was purified via flash chromatography assisted by a CombiFlash using a liquid injection into a 12 g column. The mobile phase had a gradient of 95% dichloromethane and 5% Ultra solution (75% dichloromethane, 22% methanol, and 3% aqueous ammonium hydroxide) to 80% dichloromethane and 20% Ultra solution over 35 min using a flow rate of 7 mL/min. The products were isolated as yellow to clear viscous oils. See the Supplementary Information for the specific conditions for each reaction. LNP Formulation An ethanol phase containing each lipid used in the formulation (i.e., ionizable lipid, neutral lipid, and lipid conjugate) and cholesterol, and an aqueous phase containing mRNA were mixed using a microfluidic device to formulate the LNPs. The ethanol phase consisted of the corresponding ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000] (DMG-PEG2000) with a fixed molar ratio of 35%, 16%, 46.5%, and 2.5%, respectively. The aqueous phase was composed of mRNA dissolved in 10 mM citrate buffer. The aqueous and ethanol phases were mixed at flow rates of 1.8 mL/min and 0.6 mL/min, respectively, using Pump33DS syringe pumps. LNPs were dialyzed in 1X phosphate buffered saline using a microdialysis cassette (20,000 MWCO, Thermo Fisher Scientific, Waltham, MA) for 2 h and then filtered through a 0.22 μm filter. LNP Encapsulation Efficiency mRNA encapsulation efficiencies of each LNP formulation were calculated using the Quant-iT-RiboGreen (Thermo Fisher Scientific, Waltham, MA) assay as previously described (Heyes et al., 2005, J. Controlled Release.107:276-287). Each LNP sample was diluted to approximately 2 ng/µL in two microcentrifuge tubes containing 1X TE buffer or 0.1% (v/v) Triton X-100 (Sigma-Aldrich). After 20 min, LNPs in TE buffer and Triton X-100 as well as mRNA standards were plated in triplicate in black 96-well plates and the fluorescent RiboGreen reagent was added per manufacturer’s instructions. Fluorescence intensity was read on an Infinite 200 Pro plate reader (Tecan) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. RNA content was estimated by comparison to a standard curve estimated using least squares linear regression (LSLR). Encapsulation efficiency was calculated as (B-A)/B⋅100 where A is the RNA content in TE buffer and B is the RNA content in Triton X-100. Encapsulation efficiencies are reported as mean ± standard deviation (n=3). Dynamic Light Scattering and Zeta Potential For baseline dynamic light scattering (DLS) measurements, 10 µL of each LNP solution was diluted 100X in 1X PBS in 4 mL disposable cuvettes. For baseline zeta potential measurements, 20 µL of each LNP solution was diluted 50X in deionized water in DTS1070 zeta potential cuvettes (Malvern Panalytical, Malvern, UK). Four measurements each with at least 10 runs were recorded for each sample using a Zetasizer Nano (Malvern Instruments, Malvern, UK). Data are reported as mean ± standard deviation (n=3 to 4 measurements). LNP pKa Measurements Surface ionization measurements to calculate the pKa of each LNP formulation were performed as previously described (Hajj et al., 2019, Small, 15:1805097). Buffered solution containing 150 mM sodium chloride, 20 mM sodium phosphate, 20 mM ammonium acetate, and 25 mM ammonium citrate was adjusted to pH 2 to 12 in increments of 0.5.125 µL of each pH-adjusted solution and 5 µL of each LNP formulation were plated in triplicate in black 96-well plates.6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS) was then added to each well to a final TNS concentration of 6 µM. The fluorescence intensity was read on an Infinite 200 Pro plate reader (Tecan, Morrisville, NC) at an excitation wavelength of 322 nm and an emission wavelength of 431 nm. Using least squares regression, the pKa was taken as the pH corresponding to half-maximum fluorescence intensity, i.e., 50% protonation. In Vitro LNP-mediated Luciferase mRNA Delivery to HeLa Cells HeLa cells (ATCC no. CCL-2) were cultured in DMEM with L-glutamine (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were plated at 20,000 cells per well in 100 µL of medium in tissue culture treated 96-well plates and were left to adhere overnight. LNP formulations were used to treat cells at a dose of 20 ng of mRNA (i.e., TriLink Luciferase) per 20,000 cells. One group of cells was designated as untreated and was treated with media only. 24 hours after treatment with LNPs, the media was removed. 50 µL of 1X lysis buffer (Promega, Madison, WI) followed by 100 µL of luciferase assay substrate (Promega) was added to each well. After 10 minutes of incubation, luminescence was quantified using an Infinite 200 Pro plate reader (Tecan). The luminescence signal for each condition was normalized by dividing by the luminescence signal from an LNP designated as a control (i.e., C14-494). To evaluate cytotoxicity, additional plates were prepared as described elsewhere herein. After 24 hours, 100 µL of CellTiter-Glo (Promega) was added to each well and the luminescence corresponding to ATP production was quantified using a plate reader following 10 minutes of incubation. Luminescence for each group was normalized by dividing by the luminescence signal from untreated control cells. Luciferase expression and percent viability are reported as mean ± standard deviation (n=3-4 biological replicates and at least 4 technical replicates per plate). For luciferase expression, a 2-way ANOVA with a Dunnett’s multiple comparisons test was used to compare means across formulation and treatment condition. Luciferase Imaging and Quantification Luciferase signal was assessed in Black 6 mice following intravenous tail vein injection containing luciferase mRNA. Specifically, mice were imaged 12 h after I.V. injection of LNPs or PBS. Luciferase imaging was performed using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA). Ten minutes before sacrifice and imaging, the mice were injected intraperitoneally with D-luciferin at 150 mg/kg and potassium salt (Biotium, Fremont, CA). The mouse liver, spleen, lungs, kidneys, and heart were subsequently removed and imaged by IVIS. Image analysis was conducted using the Living Image software (PerkinElmer). Reported total body and organ bioluminescence represent the mean ± standard deviation (SD) (n≥3). The representative organ IVIS images shown are those that have the highest luminescence values for each treatment condition. In vivo biodistribution studies C57BL/6J female mice of 6-8 weeks old with an average weight of 20 g were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were injected with LNPs encapsulating fluc mRNA via the lateral tail vein at a dose of 0.1 mg of mRNA per kg of body mass (mg/kg). After 12 h, the hair on the ventral side of the mice was removed with Veet Gel Cream Hair Remover (Reckitt Benckiser, Slough, UK). The mice were then administered an intraperitoneal injection of D-luciferin (0.2 mL, 15 mg/mL; Biotium, Fremont, CA). After 5 min, full body luminescence images were obtained using an In Vivo Imaging System (IVIS; PerkinElmer, Waltham, MA). Afterwards. The mice were euthanized, and the heart, lungs, liver, kidneys, and spleen were removed and imaged for luminescence using IVIS. Luminescence flux was quantified by the Living Image Software (PerkinElmer) by placing rectangular region of interests (ROI) around the full body or organ images, keeping the same ROI sizes among each body or organ. Total flux was reported as mean ± SEM of n = 3 biological replicates. TTR gene editing One day prior to injections, blood was collected from mice via retro-orbital bleeding. Serum was isolated by centrifuging the blood in Microtainer blood collection tubes containing serum separator gel (BD, Franklin Lakes, NJ, USA) for 15 min at 3,500 rpm. Mice were injected with LNPs encapsulating Cas9 mRNA and TTR sgRNA at a 1:3 molar ratio via the lateral tail vein at a dose of 1.0 mg/kg. After 7 days, serum was isolated as described previously, the mice were euthanized, and the livers were removed. Serum TTR levels were measured using a mouse Prealbumin ELISA Kit (Aviva Systems Biology, San Diego, CA, USA) according to the manufacturer’s instructions. For indel analysis, DNA was extracted from the liver using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and quantified using a nanodrop plate attachment on an Infinite 200 Pro plate reader (Tecan). PCR amplification of the TTR target site was carried out using Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) and the following primer sequences: mTTR-exon2-F, 5’-CGGTTTACTCTGACCCATTTC- 3’ (SEQ ID NO:2) and mTTR-exon2-R, 5’-GGGCTTTCTACAAGCTTACC-3’ (SEQ ID NO:3). Deep sequencing of the TTR amplicons and determination of the on-target indel frequency was performed essentially as described except that 150 bp pair end reads were produced. Primary T Cell Luminescence In certain embodiments the present disclosure provides primary T cell luminescence data obtained using health human donor T cells to evaluate the LNPs of the present disclosure. Cells were plated at 60,000 cells per well with 200 ng of mRNA (i.e., TriLink Luciferase) per well and treated with the LNPs of the present disclosure. One group of cells was designated as untreated and was treated with medium only.24 hours after treatment with LNPs, the cells were centrifuged for 5 min at 700 g and the media was removed.50 µL of 1X lysis buffer (Promega, Madison, WI) followed by 100 µL of luciferase assay substrate (Promega) was added to each well. After 10 minutes of incubation, luminescence was quantified using an Infinite 200 Pro plate reader (Tecan). The luminescence signal for each condition was normalized by dividing by the luminescence signal from an LNP designated as a control (i.e., C14-494). For luciferase expression, a 1- way ANOVA multiple comparisons test with Sidak multiple comparison correction was performed using the B10 formulation was the control group. Additionally, a 2-way ANOVA with a Dunnett’s multiple comparisons test was used to compare means across formulation and treatment condition. To evaluate cytotoxicity, additional plates were prepared as described elsewhere herein. After 24 hours, the cells were centrifuged for 5 min at 700 g, and 100 µL of CellTiter- Glo (Promega) was added to each well and the luminescence corresponding to ATP production was quantified using a plate reader following 10 minutes of incubation. Luminescence for each group was normalized by dividing by the luminescence signal from untreated control cells. Cryogenic Transmission Electron Microscopy Morphology and size was analyzed by cryo-TEM by adding 3 μL of the LNPs at an mRNA concentration of 50 ng/μL were applied to a Quantifoil holey carbon grid which had been glow discharged. Grids were blotted and plunge frozen in liquid ethane using a Vitrobot Mark IV. Imaging was performed at the Beckman Center for cryo-EM on a Titan Krios equipped with a K3 Bioquantum. LNP stability analysis LNPs were diluted 10-fold in either 1X PBS or supplemented DMEM (Gibco). The hydrodynamic diameter and PDI of the LNPs were measured every hour at 37 °C using a DynaPro Plate Reader III (Wyatt Technology), as described above. All samples were run in duplicate. Supplemented DMEM was measured as a control. Kupffer cell knockdown Mouse macrophage cells were depleted by administering 0.2 mL of clodronate liposomes (Liposoma, Amsterdam, Netherlands) at a dose of 5 mg/mL via the lateral tail vein. After 24 h, the mice were reinjected via the lateral tail vein with LNPs encapsulated fluc at a dose of 0.1 mg/kg. After 12 h, liver luminescence was quantified as described above. To verify that Kupffer cells were depleted, a subset of mice were injected with clodronate liposomes as described above. After 24 h, the livers were perfused with 10 mL of 1X PBS followed by 10 mL of supplemented DMEM containing 5 mg/mL collagenase IV (STEMCELL Technologies, Vancouver, Canada). The livers were then dissected and placed in 5 mL of RPMI with 5 mg/mL of collagenase IV for 1 h at room temperature. The livers lobes were then separated, crushed using a syringe slider, and passed through a 70 µm filter. The suspension was then centrifuged for 3 min at 100 rpm to pellet the hepatocytes. The supernatant was transferred to a new tube and up to 30 mL of supplemented DMEM was added. The centrifugation process was repeated twice more. Afterwards, the three separate supernatants were spun down at 200 rpm for 10 min, and afterwards, the top portion of the supernatant was discarded, leaving ~5 mL left in each tube. The fractions were combined into a single tube, spun at 25 g for 5 min, the cell pellet was washed with 6 mL of supplemented DMEM, recentrifuged, and the top half of the supernatant was collected. The cells were counted as described above, and then analyzed via flow cytometry. LNP accumulation LNPs were reformulated with fluc mRNA as described above. Afterwards, the LNPs were mixed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR; 5 µM in DMSO; Thermo Fisher Scientific) at a volume ratio of 50:1, respectively. The solution was placed on a plate shaker at 200 rpm for 25 min at room temperature. Then, the LNPs were administered into mice and dissected as described above. Organ fluorescence was obtained using the specific “DiR” setting on Living Image. ROIs were obtained as described above. Total radiant efficiency was reported as mean ± SEM of n = 3 biological replicates. Artificial endosome disruption assay Artificial endosomes were generated via a lipid film hydration method. To a 3 mL vial was added DOPS, DOPC, DOPE, NBD-PE, and Rho-PE at a molar ratio of 25:25:48:1:1. The lipids were concentrated in vacuo with aluminum foil wrapped around the rotary evaporator to prevent photobleaching. After 2 h of concentrating, the samples were rehydrated with 1X PBS (pH 7.4) at a final concentration of 1 mM using a Branson 3800 Ultrasonic Cleaner (Brookfield, CT, USA) at room temperature for 20 min. The assays were performed in black bottom 96-well plates. To each well were added 0.1 mL PBS (pH 5.5, 0.1 M), 1 µL of the artificial endosome, and an amount of LNP corresponding to 400 ng. As a negative control, LNP was substituted with more PBS, and as a positive control, the LNP was replaced with 2% Triton-X100. The plate was wrapped in aluminum foil and incubated at 37 °C. Fluorescence was measured at various timepoints over the course of 24 h. Example 1: Synthesis of certain exemplary ionizable lipids, preparation of lipid nanoparticle (LNP) formulations, and selected properties thereof Ionizable lipids (ILs) can be generated in one step by reacting a monoamine or polyamine with lipids containing electrophilic functional groups (e.g., epoxides). This allows for the rapid production of combinatorial libraries of ILs by simply mixing different amine cores with epoxides of various lengths. Therefore, a synthetic approach was developed to synthesize epoxides with any desired length and terminal branching that could then be further reacted with most polyamine or monoamine reagents to produce ILs with greater structural variability. To achieve the flexible synthesis of structurally diverse epoxides, primary bromoalkenes were coupled with branched halomagnesiumalkyls via copper-catalyzed Grignard C-C coupling to produce branched alkenes (FIG.1). Primary bromoalkenes are common reagents and thus are used here to establish the different lengths of the epoxides. Longer bromoalkenes can be generated by monoselective elimination of dibromoalkanes with tert-butoxide. Numerous Grignard functionalized alkyls are also commercially available due to their wide-use in other synthetic avenues and are utilized here to establish the terminal branched group. The terminally branched alkenes were converted into the corresponding branched epoxides via mCBPA-mediated epoxidation. The C-C coupling and epoxidation steps can be completed and purified in less than 24 h, demonstrating the ease of this method. In this study, twelve unique branched epoxides were synthesized, non-limiting examples including isopropyl, tert-butyl, and sec-butyl branching groups at four different lipid lengths. To form the ILs, the epoxides were initially reacted via an SN2 reaction with 2- (2-aminoethoxy)-N-(2-(4-(2-(2-aminoethoxy)ethyl)piperazin-1- yl)ethyl)ethan-1-amine (494) or N-(2-(4-(2-aminoethyl)piperazin-1-yl)ethyl)ethane-1,2-diamin e (200) (FIGs.2A-2B). The 494 and 200 polyamine cores have been previously used to generate ILs and the corresponding LNPs for several applications, including CAR T-cell therapy, in utero delivery, and delivery to the placenta, and thus this core represents an interesting structural motif to explore these preliminary studies. As controls, four linear ILs of the same relative lipid length as the branched library were also synthesized using the same SN2 reaction. In total, using 494, sixteen exemplary ILs were synthesized. The nomenclature used herein to described the branched lipids of the present disclosure utilizes the formula “AXb-C”, where A is the reactive moiety of the lipid tail, b is the branching type, C is the lipid core number, and X is the linker length or number of methylene units between A and B. The branching abbreviations are “i” for isopropyl, “t” for tert-butyl, and “s” for sec-butyl. Non-branched ILs utilize their historical name. In one aspect, the present disclosure relates to lipid nanoparticles comprising one or more ionizable lipid compounds of Formula (I), one or more neutral lipids, cholesterol, and one or more conjugated lipids. In certain embodiments, the one or more conjugated lipids inhibit aggregation of two or more lipid nanoparticles. In certain embodiments, the lipid nanoparticles further comprise one or more nucleic acid cargos. The preparation and properties of such lipid nanoparticles is provided herein in greater detail. In certain embodiments, LNPs of the present disclosure were prepared using microfluidic devices, to prepare LNPs having the following molar ratio of components: (a) ionizable lipid of Formula (I) (35 mol%); (b) DOPE (16 mol%); (c) cholesterol (46.5 mol%), and DMG-PEG 2000 (2.5 mol%). In certain embodiments, the ionizable lipid of Formula (I) is selected from the group consisting of E4i-494, E6i-494, E8i-494, E10i-494, E4t-494, E6t- 494, E8t-494, E10t-494, E4s-494, E6s-494, E8s-494, E10s-494, E4i-200, E8i-200, E4t-200, E8t-200, E4s-200, and E8s-200 (Table 1). LNPs comprising each of the aforementioned ionizable lipids of Formula (I) and having the aforementioned molar ratio have been exemplified herein, and such LNP formulations are disclosed herein with reference to the identifier assigned to each ionizable lipid used to prepare the LNP (e.g., E4i-494 ionizable lipid was used to prepare the LNP labeled E4i-494). Since the present exemplary LNPs used in several of the exemplary applications and/or studies described herein are formulating using the same, non-limiting excipient molar ratio as well as using the same phospholipid and PEGylated lipid, the difference between each LNP formulation is the structure of the IL. For consistency, each LNP is named after the corresponding IL, as described herein. The LNPs were initially encapsulated with firefly luciferase (fluc) mRNA at a 10:1 weight ratio of IL and mRNA, and were characterized for encapsulation efficiency, hydrodynamic diameter, ζ-potential, and pKa (Table 2). Although multiple parameters were evaluated, there were no noticeable differences between the LNPs with linear, isopropyl, tert-butyl, and sec- butyl ILs. All exemplary LNPs have >80% encapsulation efficiency and sizes ranging from 70-160 nm with average PDIs of approximately 0.2. The ζ-potentials of the LNPs also ranged from 5.57 to −28.1, indicating that most have neutral or weakly negative charge. Lastly, the majority of the LNPs have a pKa around 6. Table 2. Selected properties of exemplary LNPs of the present disclosure and controls
LNPs C8-494, C10-494, C12-494, and C12-200 correspond to LNPs having the identical formulation to those of the present disclosure, but which differ with respect to the ionizable lipid component, wherein the ionizable lipid was prepared using either polyamine core 494 or polyamine core 200 with an epoxide having a linear alkyl group with a linker length indicated by the C integer (i.e., C10-494 corresponds to an ionizable lipid prepared using polyamine core 494 with 2-undecyloxirane). Example 2: LNPs comprising branched ionizable lipids enhance in vivo, ex vivo, and in vitro mRNA delivery The present disclosure provides data relating to the in vivo, ex vivo, and in vitro delivery of exemplary LNPs of the present disclosure. The efficacy of exemplary LNPs of the present disclosure were evaluated in both in vitro and in vivo models. Each LNP was evaluated for transfection efficacy in HeLa cells by incubating the LNPs at a dose of 20 ng of mRNA per 20,000 cells for 24 h. The luminescence results indicated that branching significantly enhanced LNP transfection at the lowest and highest lipid lengths by 10-fold, whereas in the middle lengths, branching induced no significant change or greatly decreased transfection (FIG.3A). When examined for toxicity using a CellTiter-Glo assay, seven LNPs demonstrated statistically significant toxicity; however, none of the LNPs had viability below 80% (FIG.3B). To verify the consistency of this trend, the LNPs were administered intravenously into C57BL/6J mice at a dose of 0.1 mg/kg. DLin-MC3-DMA (MC3), an FDA-approved LNP formulation for the delivery of siRNA, and C12-200, a more potent LNP for mRNA delivery with a similar polyamine core as 494, were also administered to serve as positive controls. After 12 h, full body and organ luminescence were obtained (FIGs.3C-3D). Contrary to the in vitro results, all LNPs with branched ILs (branched LNPs) performed as well as or better than the corresponding LNPs with linear ILs (linear LNPs) for total RNA delivery and liver delivery. For full body delivery, six branched LNPs and only one linear LNP induced luciferase expression as well as C12-200, whereas E4t-494 and E6t-494, and no linear LNPs, outperformed C12-200. All LNPs transfected the liver preferentially over other organs, and in terms of liver luminescence, six LNPs with branched ILs performed as good as or better than C12-200, with E4i-494 facilitating 1.5-fold greater luminescence (FIGs.3E-3G). Notably, no linear LNPs achieved statistically equal or greater signal than C12-200. When compared against MC3 and not C12-200, four branched LNPs and no linear LNPs had significantly increased luminescence than MC3. To further investigate the branched lipid paradigm, it was investigated whether lipid branching can enhance the delivery of a liver-trophic amine core, specifically the 200 core exemplified herein. As such, six of the branched epoxides were coupled with the 200 core to form six new branched ILs comprising the 200 core (FIG.2B). The successful synthesis of the branched 200 core IL underscores the robust nature of the initial branched epoxide design as it can be applied to many different amine cores. The three branching groups were each evaluated at relative lipid lengths of eight carbons and twelve carbons, although the present disclosure is not limited to such chain lengths, and the corresponding linear versions, C8-200 and C12-200, were utilized as controls. While the C12-200 is considered a standard liver- trophic IL, C8-200 performs much poorer and thus represents an important test as to whether branching can also convert this IL into a potent liver targeting LNP. These eight ILs were then formulated into LNPs with fluc mRNA and characterized using the same methodology as described above (FIG.4A). Akin to the studies with the 494 LNPs, testing in HeLa cells revealed conflicting themes, as C8-200 performed better or the same as the branched equivalents while two of the longer chain branched LNPs strongly outperformed C12-200 (FIG.4B). None of the LNPs demonstrated toxicity in vitro at the concentration tested for the luciferase assay. Afterwards, the 200 core LNPs were intravenously injected into C57BL/6J mice at 0.1 mg/kg of fluc mRNA. All branched LNPs performed similar to or better than C12-200 for full body luminescence and liver delivery (FIGs.4C-4E), including the C8-200 branched lipids, with E4s-200 inducing five-fold greater liver luminescence. These results demonstrate that branching at lipid termini enhance liver delivery irrespective of the lipid core, providing an essential design criterium for future ILs. In certain embodiments, the present disclosure provides delivery data comprising luminescence data (e.g., full body, liver, and/or combined organ luminescence data) obtained by administration of exemplary LNPs comprising TriLink Luciferase to Black 6 mice. In certain embodiments, the mice were also administered one or more controls (e.g., LNPs comprising MC3 and/or unbranched ionizable lipids). In certain embodiments, a subset of LNPs comprising an ionizable lipid prepared from the polyamine core 494 were evaluated in this manner, and the data demonstrated enhanced luminescence with several of the LNPs of the present disclosure as compared to LNPs comprising unbranched ionizable lipids, with respect to organ luminescence (FIG.5A). In certain embodiments, a subset of LNPs comprising an ionizable lipid prepared from the polyamine core 200 were evaluated in this manner, and the data demonstrated enhanced luminescence with several of the LNPs of the present disclosure as compared to LNPs comprising unbranched ionizable lipids, with respect to combined organ luminescence (FIG.5B). Further, the experiments provided herein indicate that the LNPs of the present disclosure were selective for distribution to the liver, as compared to the spleen, lungs, kidneys, and/or heart, with little to no luminescence observed in non-liver organs (FIGs.5A- 5D). Example 3: Branched LNPs induce potent gene editing Next, LNP delivery was evaluated in a clinically-relevant murine model for transthyretin (TTR)-mediated amyloidosis. TTR is a protein that when overexpressed in the liver, will develop into amyloid fibrils that can induce restrictive cardiomyopathy and heart failure. LNP-based siRNA therapies, such as the MC3-based ONPATTRO® have been utilized to silence TTR translation in the liver; however, this can require multiple transfusions as siRNA has a limited therapeutic window. Instead, delivering gene editing machinery can offer a single dose therapy that would permanently reduce TTR to safe levels. As such, the eight 200 core LNPs were reformulated to encapsulate Cas9 mRNA and single guide RNA (sgRNA) targeting TTR. C57BL/6J mice were then injected with 0.3 mg/kg or 1.0 mg/kg of combined Cas9 and TTR sgRNA. After seven days, blood was removed, serum was isolated, and the levels of TTR protein were evaluated compared to serum isolated from the same mice 24 h before the study (FIG.6A). The mice were sacrificed following blood withdrawal, and the livers were removed, underwent DNA isolation, and analyzed for indels using next-generation sequencing (NGS). While the C12-200 LNP reduced TTR levels by 70%, several of the LNPs with branched ILs reduced TTR levels by 80 – 90% (FIG.6B). Additionally, these latter LNPs induced 50 – 60% indels, while the C12-200 LNP only induced 40% indels (FIG.6C). These results demonstrate that lipid branching can enhance delivery regardless of the mRNA, showcasing the universality of this design. To evaluate toxicity, the mice were reinjected with the same formulation. At 12 h, blood was withdrawn and after 24 h, the major organs were removed, stained for H&E, and analyzed for toxic markers via an independent expert. Upon analyzing the blood for liver damage markers, E4i-200, E4s-200, and E8t-200 had significantly higher AST levels compared to the control group, while C12-200 do not. However, for ALT, E4s-200, C12-200, E8i-200, and E8t-200 all had greater levels than PBS. Example 4: Branched ILs facilitate greater endosomal escape To determine the mechanism of the increased efficacy of branched LNPs, several possible explanations were evaluated. Initially, it was evaluated whether lipid branching results in LNPs with optimal physicochemical parameters, by correlating the resulting liver luminescence of each LNP from the studies with fluc mRNA with the particle’s hydrodynamic diameter, PDI, ζ-potential, and pKa (FIGs.7A-7E). The the success in liver delivery was further correlated with HeLa cell transfection (FIG.7F). Intriguingly, after fitting the data with a cubic least squares regression, no parameter was predictive of successful liver delivery. Moreover, HeLa cell transfection was also a very poor predictor of in vivo delivery. Following this study, it was analyzed whether the morphology of branched LNPs differed significantly from linear LNPs; however, upon analyzing the structure via cryo-TEM, all LNPs had a similar egg-shaped morphology (FIGs.7G-7J). This suggests that this form of lipid branching may have minimal impact on the global arrangement of LNPs, although this does not exclude the possibility of finer changes in bilayer and monolayer arrangement. The stability of the LNPs was next evaluated by incubating exemplary LNPs in PBS and Dulbecco's Modified Eagle Medium (DMEM) substituted with 10% fetal bovine serum (FBS) at 37 °C. The size and PDI of the LNPs were measured every hour for 24 h by dynamic light scattering immediately after incubation (FIGs.7K-7N). Similar to the characterization results, no significant destabilization was observed in either PBS or DMEM, and in the latter, all LNPs formed a similar sized protein corona. Since particle physicochemical characteristics, morphology, and stability seemed to be unaffected by lipid branching, the role of liver trafficking and uptake was investigated. Several studies have demonstrated that IL structure can impact the specific liver cells that take up LNPs. For example, Kupffer cells are liver macrophages that more favorably capture certain types LNPs, which can result in different liver biodistributions. As such, C57BL/6J mice were injected with clodronate liposomes, which deplete macrophages, and after 24 h, reinjected the mice with the eight 200-core LNPs encapsulating fluc mRNA (FIG.8E). After a total of 36 h, liver luminescence was imaged, revealing that all LNPs except for C8-200 and E4i-200 had significant decreases in luminescence (FIG.8A). These data suggest that branching is likely not a factor in the prevalence of macrophage uptake. Still, C8-200 and E4i-200, the two LNPs with ILs of the smallest lipid lengths, transfect macrophages at a lower frequency compared to non-macrophage cells; although, a larger library would need to be screened to validate this trend. Next, the role of protein corona and liver interactions was investigated by injecting E4i-200, E4t-200, E4s-200, and C12-200 fluc LNPs into apolipoprotein E (APOE) knockout mice at 0.1 mg/kg. Upon measuring liver luminescence after 12 h, all four LNPs had dramatic decreases of 100–1000 fold luminescence compared to wild type mice (FIG.8B). This suggests that both branched and unbranched LNPs target the liver via an APOE-mediated mechanism, as has been demonstrated with many other liver-trophic LNPs. Next, particle accumulation in the liver was analyzed. LNPs containing fluc were reformulated with 1 mol% DiR, a lipophilic carbocyanine near IR fluorescent dye. The fluorescent LNPs were injected into C57BL/6J mice at 0.1 mg/kg of fluc mRNA, and the major organs were dissected and imaged for fluorescence after 12 h. Here, fluorescence is a marker of LNP accumulation. While mRNA transfection and translation had occurred exclusively in the liver, LNP fluorescence was observed in the liver and spleen (FIGs.8C- 8D). More interestingly, all LNPs showed similar fluorescence in both organs. Without wishing to be bound by any theory, if all eight LNPs are accumulating in the liver at relatively the same amounts, but the branched LNPs are facilitating greater mRNA translation, it was hypothesized that this result is likely due to the branched ILs allowing a higher number of mRNA molecules to escape the endosome and enter the cytosol. While there are other reports of certain IL structural moieties influencing endosomal escape, the specifical role of terminal branching remains unknown in the art. To verify this result, the interactions of the eight LNPs with artificial endosomes was studied, which is a validated strategy to study the role of endosomal escape. Exemplary endosomes were formulated via thin film hydration of DOPE, 18:1 Δ9-Cis phosphocholine (DOPC), 18:1 phospho-L-serine (DOPS), NBD-conjugated 18:1 phosphoethanolamine (NBD-PE), and Lissamine-Rhodamine-B-conjugated 18:1 phosphoethanolamine (Liss-Rhod- PE) at molar ratios of 48:25:25:1:1, respectively. The latter two lipids are a FRET pair that was used to monitor the integrity of the endosome. Increases in the fluorescence of the donor fluorophore corresponds to endosomal disruption as the FRET pair is separated. In this study, each LNP was diluted 6X in pH 5.5 buffer to represent endosomal conditions and mixed with the artificial endosomes. At various timepoints, the fluorescence of the donor fluorophore was measured. The branched LNPs induced 2-to-3-fold greater fluorescence than the linear LNPs for both the C8-200 (FIG.8F) and C12-200 (FIG.8G) groups, supporting the idea that lipid branching facilitates more pronounced endosomal escape and the linear versions. Example 5: Oral cancer tumor suppressor therapy The present disclosure further provides exemplary data relating to the use of the LNPs described herein for treating and/or ameliorating oral cancer tumors. Exemplary LNPs of the present disclosure utilized LNPs comprising ionizable lipids prepared from the 494 polyamine core, including C8-C14-, E4i-E10i-, E4t-E10t-, and E4s-E10s-494 ionizable lipids, whereas C12-200 was utilized as a positive control. In the present study, CAL-27 cells were used as a model to evaluate the utility of LNPs of the present disclosure for delivery of certain mRNA cargo. CAL-27 cells are a human HPV-negative tongue squamous cell carcinoma which are considered a gold standard cell line for studies of HPV-negative oral cancer. Exemplary LNPs were incubated with luciferase mRNA, and the resultant LNPs were subsequently administered to the cells at a dose of 20 ng mRNA per 20,000 cells. After 24 h, a luciferase assay (FIG.9A) and cell viability assay (FIG.9B) were performed to analyze luciferase expression and toxicity, respectively. Luciferase assay data was normalized to C12-200. Several of the evaluated LNPs outperformed the C12-200 LNP (e.g., E10i-494 and E10s-494), while two LNPs comprising branched ionizable lipids significantly outperformed the C12-200 LNP. The present disclosure further describes the use of a mouse tumor model, wherein Nu/J mice were inoculated on the right flank with CAL-27 cells, and tumors were permitted to grow for 2 weeks. Subsequently, five exemplary LNPs encapsulating luciferase mRNA, including two LNPs comprising branched ionizable lipids and three LNPs comprising linear ionizable lipids, were injected intratumorally at a dose of 0.1 mg/kg, and certain mice were administered PBS as a control. The mice were sacrificed, the major organs and tumor were harvested, and imaged for fluorescence (FIGs.10A-10B). The results provided herein indicate that the top performing LNP (i.e., E10i-494), which comprises a branched ionizable lipid, had over 10-fold greater signal than was observed for the LNP comprising C12-200. The present disclosure further describes the use of the LNPs of the present disclosure in viability assays (i.e., killing assays) utilizing CAL-27 and OECM-1 cell lines, wherein OECM-1 cells represent a HPV-negative squamous cell carcinoma cell line. Exemplary LNPs utilized in the studies described herein comprise p53 cargo. In one aspect, p53 represents an ideal target for LNP-mediated suppressor therapy, as over 70% of oral squamous cell carcinomas have mutated or downregulated p53. A range of mRNA doses were utilized (i.e., 5, 20, 50, 100, and 250 ng/µL) at varying time intervals (i.e., 24 h or 48 h) and overall viability was measured (FIGs.11A-11D). Low viability (i.e., cell killing) was observed with administration of exemplary LNPs comprising p53 mRNA at least partially encapsulated therein after both 24 and 48 h. Example 6: Stem cell reprogramming The present disclosure further provides exemplary data relating to the use of the LNPs described herein for reprogramming stem cells (e.g., induced pluripotent stem cells (iPSCs)). iPSCs are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of a source of any type of human cell needed for therapeutic purposes. For example, an iPSC can be differentiated into a blood cell to create new blood free of cancer cells for a leukemia patient. Transfection of iPSCs with mRNA cargo permits expression of certain biomolecules of interest and may permit differentiation to a cell line of interest (e.g., neuron and/or muscle cells), as desired. In the experiments described herein, iPSCs were transfected with mRNA at least partially encapsulated in exemplary LNPs of the present disclosure, said LNPs comprising ionizable lipids prepared from the 494 and 200 polyamine cores, whereas LNPs comprising C12-200 were utilized as a positive control. An initial potency screen of exemplary LNPs was performed with LNPs of the present disclosure comprising luciferase mRNA (20 ng/15,000 cells) and luciferase expression (FIG. 12A) and viability were measured (FIG.12B). Comparable viability was observed with each exemplary LNP, whereas certain LNPs (i.e., E4i-200, E4t-200, and E4s-200) exhibited high relative fluorescence. Dose response luciferase luminescence (FIG.13A) and viability (FIG.13B) experiments were performed with top performing LNPs identified in the screening studies. LNP E4i-200 was identified as a top performer, with respect to relative luminescence. E4i- 200 LNP was reformulated with mCherry. iPSC-SV20 cells were plated in 24-well plates overnight and subsequently treated with E4i-LNP comprising mCherry. Flow cytometry was performed to measure the percentage of cells that were successfully transfected and cells were imaged by fluorescence microscopy to visualize the mCherry signal. (FIGs.14A-14H). A time-point study was performed, wherein iPSCs transfected with mCherry (or controls) were harvested after 24 h (FIGs.15A and 15E), 48 h (FIGs.15B and 15F), 72 h (FIGs.15C and 15G), and 96 h (FIGs.15D and 15H). The results provided herein demonstrate that the mCherry signal remained at high intensity for 48 hours, as determined by FACs analysis. Example 7: CAR-T cell and CAR-Natural Killer (NK) cell therapy In another aspect, the present disclosure relates to the use of the LNPs of the present disclosure for use in CAR-T and CAR-NK therapy. The experiments described herein utilize LNPs comprising ionizable lipids prepared from the 494 and 200 polyamine cores. An initial screen of LNPs was performed utilizing exemplary LNPs comprising luciferase mRNA were incubated in activated primary T cells (1:1 CD4+:CD8+) from health human donors, wherein luciferase was present at a concentration of 200 ng mRNA/60,000 cells. Luciferase expression (FIG.16A) and cell viability (FIG.16B) was measured after 24 h, wherein E8i-200 and E10s-200 LNPs were found to outperform the B10 LNP formulation, with minimal toxicity. The formulation of LNP components of the top performing LNPs identified in the screen described herein was modified to match the formulation used for the B10 LNP (i.e., ionizable lipid:DOPE:cholesterol:C14PEG2000 of 40:30:25:2.5), and relative luminescence (FIG.17A) and viability (FIG.17B) of the exemplary LNPs in primary T cells were measured. LNP E8i-200 was identified as a top performer in this experiment, demonstrating almost 6-fold greater luminescence than B10, with negligible change in viability. The present disclosure further describes evaluation of exemplary LNPs for transfection of NK-92MI cells (i.e., immortalized human natural killer cells which self- express IL-2). In these experiments, LNPs comprising luciferase mRNA (200 ng mRNA/60,000 cells) were incubated with NK-92MI cells and luciferase expression was measured after 24 h. Certain exemplary LNPs comprising branched ionizable lipids (e.g., E6i-200 and E8i-200) significantly outperformed the LNP comprising C12-200, thereby demonstrating utility of the LNPs of the present disclosure for transfection of immune cells (e.g., T cells and NK cells) (FIG.18). Sequence Listing Enumerated Embodiments The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance: Embodiment 1 provides an ionizable lipid compound of Formula (I), or a salt, solvate, stereoisomer, or isotopologue thereof: wherein: R 1a and R 1b are each independently ; R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are each independently selected from the group consisting of H, optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 heteroalkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 2 -C 8 heterocycloalkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 6 -C 10 aryl, and optionally substituted C 2 -C 10 heteroaryl; each occurrence of R 3a , R 3b , and R 3c is independently selected from the group consisting of H, , and , wherein no more than one of R 3a , R 3b , and R 3c is H; each occurrence of R 4a , R 4b , R 4c , and R 4d , if present, is independently selected from the group consisting of optionally substituted C 1 -C 12 alkyl, halogen, CN, and NO 2 ; each occurrence of R 5 is independently selected from the group consisting of optionally substituted C 1 -C 3 alkyl, optionally substituted C 3 -C 12 cycloalkyl, optionally substituted C 6 -C10 aryl, and optionally substituted C 2 -C 10 heteroaryl; each occurrence of R 6 is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 12 cycloalkyl, C 2 -C 12 heterocycloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted C 6 -C 12 aryl, optionally substituted C 2 -C 12 heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b ); each occurrence of L is independently selected from the group consisting of a bond, - (optionally substituted C 1 -C 12 alkylenyl)-X-, -(optionally substituted C 2 -C 12 alkenylenyl)-X-, -(optionally substituted C 1 -C 12 alkynylenyl)-X-, -(optionally substituted C 1 -C 12 heteroalkylenyl)-X-, optionally substituted C 3 -C 8 cycloalkylenyl, and optionally substituted C 2 -C 8 heterocyloalkylenyl; each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R 3c )-, and -O-; each occurrence of Y, if present, is independently selected from the group consisting of a bond, -N(R a )-, and -O-; each occurrence of Z is C 1 -C 24 alkylenyl, wherein the C 1 -C 24 alkylenyl in each occurrence of Z is independently substituted with at least one substituent selected from the group consisting of C 1 - C 12 alkyl and C 1 -C 12 haloalkyl, and wherein the C 1 -C 24 alkylenyl in each occurrence of Z is independently optionally further substituted; each occurrence of R a and R b is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted phenyl, optionally substituted C 2 -C8 heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d ); each occurrence of R c and R d is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted phenyl, and optionally substituted C 2 -C8 heteroaryl; and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4. Embodiment 2 provides the compound of Embodiment 1, wherein at least one of the following applies: (a) at least one selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h is H; (b) at least two selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (c) at least three selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (d) at least four selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (e) at least five selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (f) at least six selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (g) at least seven selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; and (h) each of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. Embodiment 3 provides the compound of Embodiment 1 or 2, wherein each occurrence of L is independently selected from the group consisting of L is independently selected from the group consisting of -(CH 2 ) 1-10 -, -(CH 2 ) 2-10 NR 3c -, -(CH 2 ) 2-10 O-, -(CH 2 ) 1-3 - CH(OR a )-(CH 2 ) 1-3 -, piperazinylenyl, and cyclohexylenyl. Embodiment 4 provides the compound of any one of Embodiments 1-3, wherein each occurrence of L is independently selected from the group consisting of -CH 2 -, -(CH 2 ) 2 -, - (CH 2 )3-, -(CH 2 )10-, -(CH 2 ) 2 O-, -(CH 2 ) 3 O-, -CH 2 CH(OR a )CH 2 -, -(CH 2 ) 2 NR 3c -, , and Embodiment 5 provides the compound of any one of Embodiments 1-4, wherein the compound of Formula (I) is selected from the group consisting of:
, and Embodiment 6 provides the compound of any one of Embodiments 1-5, wherein each occurrence of R 4a , R 4b , R 4c , and R 4d is independently H. Embodiment 7 provides the compound of any one of Embodiments 1-6, wherein each occurrence of R 5 is independently methyl. Embodiment 8 provides the compound of any one of Embodiments 1-7, wherein each occurrence of R 6 is independently H. Embodiment 9 provides the compound of any one of Embodiments 1-8, wherein each occurrence of Z is independently: wherein: each occurrence of R 7a , R 7b , R 7c , and R 7d is independently selected from the group consisting of H, C 1 -C 6 alkyl, and C 1 -C 6 haloalkyl, wherein at least one of R 7a , R 7b , R 7c , and R 7d is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Embodiment 10 provides the compound of Embodiment 9, wherein each occurrence of R 7a , R 7b , R 7c , and R 7d is selected from the group consisting of H and methyl. Embodiment 11 provides the compound of any one of Embodiments 1-10, wherein each occurrence of Z is independently selected from the group consisting of -(CH 2 ) 4-10 - CH(CH 3 )-*, -(CH 2 ) 4-10 -C(CH 3 ) 2 -*, and -(CH 2 ) 4-10 -CH(CH 3 )-CH 2 -*. Embodiment 12 provides the compound of any one of Embodiments 1-11, wherein each occurrence of R 3a , R 3b , and R 3c is independently selected from the group consisting of and Embodiment 13 provides the compound of any one of Embodiments 1-12, wherein each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent selected from the group consisting of C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 6 haloalkyl, C 1 -C3 haloalkoxy, phenoxy, halogen, CN, NO 2 , OH, N(R’)(R’’), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O) 2 N(R’)(R’’), N(R’)C(=O)R’’, N(R’)S(=O) 2 R’’, C 2 -C 8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 6 haloalkyl, benzyl, and phenyl. Embodiment 14 provides the compound of any one of Embodiments 1-13, which is selected from the group consisting of:
, and Embodiment 15 provides a lipid nanoparticle (LNP) composition comprising: (a) at least one ionizable lipid compound or a salt, solvate, stereoisomer, or isotopologue thereof having the structure of Formula (I): wherein: R 1a and R 1b are each independently R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are each independently selected from the group consisting of H, optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 heteroalkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 2 -C8 heterocycloalkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 6 -C10 aryl, and optionally substituted C 2 -C10 heteroaryl; each occurrence of R 3a , R 3b , and R 3c is independently selected from the group consisting of H, , and wherein no more than one of R 3a , R 3b , and R 3c is H; each occurrence of R 4a , R 4b , R 4c , and R 4d , if present, is independently selected from the group consisting of optionally substituted C 1 -C 12 alkyl, halogen, CN, and NO 2 ; each occurrence of R 5 is independently selected from the group consisting of optionally substituted C 1 -C 3 alkyl, optionally substituted C 3 -C 12 cycloalkyl, optionally substituted C 6 -C 10 aryl, and optionally substituted C 2 -C 10 heteroaryl; each occurrence of R 6 is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 12 cycloalkyl, C 2 -C 12 heterocycloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted C 6 -C 12 aryl, optionally substituted C 2 -C 12 heteroaryl, C(=O)R a , C(=O)OR a , and C(=O)N(R a )(R b ); each occurrence of L is independently selected from the group consisting of a bond, - (optionally substituted C 1 -C 12 alkylenyl)-X-, -(optionally substituted C 2 -C 12 alkenylenyl)-X-, -(optionally substituted C 1 -C 12 alkynylenyl)-X-, -(optionally substituted C 1 -C 12 heteroalkylenyl)-X-, optionally substituted C 3 -C 8 cycloalkylenyl, and optionally substituted C 2 -C 8 heterocyloalkylenyl; each occurrence of X, if present, is independently selected from the group consisting of a bond, -N(R 3c )-, and -O-; each occurrence of Y, if present, is independently selected from the group consisting of a bond, -N(R a )-, and -O-; each occurrence of Z is C 1 -C 24 alkylenyl, wherein the C 1 -C 24 alkylenyl in each occurrence of Z is independently substituted with at least one substituent selected from the group consisting of C 1 - C 12 alkyl and C 1 -C 12 haloalkyl, and wherein the C 1 -C 24 alkylenyl in each occurrence of Z is independently optionally further substituted; each occurrence of R a and R b is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted phenyl, optionally substituted C 2 -C 8 heteroaryl, C(=O)R c , C(=O)OR c , and C(=O)N(R c )(R d ); each occurrence of R c and R d is independently selected from the group consisting of H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 3 -C 8 cycloalkyl, optionally substituted C 1 -C 6 haloalkyl, optionally substituted C 6 -C 12 aralkyl, optionally substituted phenyl, and optionally substituted C 2 -C 8 heteroaryl; and each occurrence of m is independently an integer selected from the group consisting of 1, 2, 3, and 4; (b) at least one neutral lipid; (c) cholesterol; and (d) at least one conjugated lipid. Embodiment 16 provides the LNP of Embodiment 15, wherein the LNP further comprises: (e) at least one nucleic acid and/or therapeutic agent cargo, wherein the cargo is at least partially encapsulated therein. Embodiment 17 provides the LNP of Embodiment 15 or 16, wherein at least one of the following applies: (a) at least one selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h is H; (b) at least two selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (c) at least three selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (d) at least four selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (e) at least five selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (f) at least six selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; (g) at least seven selected from the group consisting of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H; and (h) each of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , and R 2h are H. Embodiment 18 provides the LNP of any one of Embodiments 15-17, wherein each occurrence of L is independently selected from the group consisting of L is independently selected from the group consisting of -(CH 2 ) 1-10 -, -(CH 2 ) 2 -10NR 3c -, -(CH 2 ) 2-10 O-, -(CH 2 ) 1-3 - CH(OR a )-(CH 2 ) 1-3 -, piperazinylenyl, and cyclohexylenyl. Embodiment 19 provides the LNP of any one of Embodiments 15-18, wherein each occurrence of L is independently selected from the group consisting of -CH 2 -, -(CH 2 ) 2 -, - (CH 2 ) 3 -, -(CH 2 ) 10 -, -(CH 2 ) 2 O-, -(CH 2 ) 3 O-, -CH 2 CH(OR a )CH 2 -, -(CH 2 ) 2 NR 3c -, , and Embodiment 20 provides the LNP of any one of Embodiments 15-19, wherein the compound of Formula (I) is selected from the group consisting of:
and Embodiment 21 provides the LNP of any one of Embodiments 15-20, wherein each occurrence of R 4a , R 4b , R 4c , and R 4d is independently H. Embodiment 22 provides the LNP of any one of Embodiments 15-21, wherein each occurrence of R 5 is independently methyl. Embodiment 23 provides the LNP of any one of Embodiments 15-22, wherein each occurrence of R 6 is independently H. Embodiment 24 provides the LNP of any one of Embodiments 15-23, wherein each occurrence of Z is independently: wherein: each occurrence of R 7a , R 7b , R 7c , and R 7d is independently selected from the group consisting of H, C 1 -C 6 alkyl, and C 1 -C 6 haloalkyl, wherein at least one of R 7a , R 7b , R 7c , and R 7d is not H; and each occurrence of o is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Embodiment 25 provides the LNP of Embodiment 24, wherein each occurrence of R 7a , R 7b , R 7c , and R 7d is selected from the group consisting of H and methyl. Embodiment 26 provides the LNP of any one of Embodiments 15-25, wherein each occurrence of Z is independently selected from the group consisting of -(CH 2 )4-10-CH(CH 3 )- *, -(CH 2 ) 4-10 -C(CH 3 ) 2 -*, and -(CH 2 ) 4-10 -CH(CH 3 )-CH 2 -*. Embodiment 27 provides the LNP of any one of Embodiments 15-26, wherein each occurrence of R 3a , R 3b , and R 3c is independently selected from the group consisting of and Embodiment 28 provides the LNP of any one of Embodiments 15-27, wherein each occurrence of optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, and optionally substituted heterocycloalkylenyl, if present, is independently optionally substituted with at least one substituent selected from the group consisting of C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 6 haloalkyl, C 1 -C3 haloalkoxy, phenoxy, halogen, CN, NO 2 , OH, N(R’)(R’’), C(=O)R’, C(=O)OR’, OC(=O)OR’, C(=O)N(R’)(R’’), S(=O) 2 N(R’)(R’’), N(R’)C(=O)R’’, N(R’)S(=O) 2 R’’, C 2 -C 8 heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R’’ is independently selected from the group consisting of H, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 1 -C 6 haloalkyl, benzyl, and phenyl. Embodiment 29 provides the LNP of any one of Embodiments 15-28, which is selected from the group consisting of:
, and . Embodiment 30 provides the LNP of any one of Embodiments 15-29, wherein the at least one ionizable lipid of Formula (I) comprises about 10 mol% to about 90 mol% of the LNP. Embodiment 31 provides the LNP of any one of Embodiments 15-30, wherein the at least one ionizable lipid of Formula (I) comprises about 35 mol% or about 40 mol% of the LNP. Embodiment 32 provides the LNP of any one of Embodiments 15-31, wherein the at least one neutral lipid comprises about 1 mol% to about 40 mol% of the LNP. Embodiment 33 provides the LNP of any one of Embodiments 15-32, wherein the at least one neutral lipid comprises about 16 mol% or about 30 mol% of the LNP. Embodiment 34 provides the LNP of any one of Embodiments 15-33, wherein the at least one neutral lipid comprises at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC). Embodiment 35 provides the LNP of any one of Embodiments 15-34, wherein the at least one neutral lipid comprises dioleoylphosphatidylethanolamine (DOPE). Embodiment 36 provides the LNP of any one of Embodiments 15-35, wherein the cholesterol comprises about 20 mol% to about 75 mol% of the LNP. Embodiment 37 provides the LNP of any one of Embodiments 15-36, wherein the cholesterol comprises about 25 mol% or about 46.5 mol% of the LNP. Embodiment 38 provides the LNP of any one of Embodiments 15-37, wherein the at least one conjugated lipid comprises about 0.1 mol% to about 15 mol% of the LNP. Embodiment 39 provides the LNP of any one of Embodiments 15-38, wherein the at least one conjugated lipid comprises about 2.5 mol% of the LNP. Embodiment 40 provides the LNP of any one of Embodiments 15-39, wherein the at least one conjugated lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000 ). Embodiment 41 provides the LNP of any one of Embodiments 15-40, wherein the LNP has a molar ratio of (a) : (b) : (c) : (d) of about 35:16:46.5:2.5 or about 40:30:25:2.5 Embodiment 42 provides the LNP of any one of Embodiments 16-41, wherein the nucleic acid molecule is a therapeutic agent. Embodiment 43 provides the LNP of any one of Embodiments 16-42, wherein the nucleic acid molecule is at least one selected from the group consisting of RNA and DNA. Embodiment 44 provides the LNP of any one of Embodiments 16-43, wherein the nucleic acid molecule is at least one selected from the group consisting of mRNA, cDNA, miRNA, siRNA, and modified RNA. Embodiment 45 provides the LNP of any one of Embodiments 42-44, wherein the nucleic acid is mRNA. Embodiment 46 provides the LNP of Embodiment 45, wherein the LNP has a mass ratio of (a) : mRNA of about 20:1 to about 5:1 (w/w), optionally wherein the LNP has a mass ratio of (a) : mRNA of about 10:1. Embodiment 47 provides the LNP of Embodiment 45 or 46, wherein the mRNA encodes a chimeric antigen receptor (CAR). Embodiment 48 provides the LNP of Embodiment 47, wherein the CAR is specific for binding to a surface antigen of a pathogenic cell or a tumor cell. Embodiment 49 provides the LNP of Embodiment 48, wherein the surface antigen is selected from the group consisting of CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD19, CD20, CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD123, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6, CCR7, k light chain, ROR1, ErbB2, ErbB3, ErbB4, EGFR vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligands, B7-H6, IL13R-α2, MUC1, VEGF-A, Tem8, FAP, EphA2, HER2, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CALX, HLA-AI MAGE A1, HAL-A2 NY-ESO-1, PSC1, folate receptor-α, 8H9, NCAM, VEGF, 5T4, Fetal AchR, NKG2D ligands, TEM1, and TEM8. Embodiment 50 provides the LNP of Embodiment 45 or 46, wherein the mRNA encodes an enzyme. Embodiment 51 provides the LNP of any one of Embodiments 45-46 and 49, wherein the mRNA encodes a clustered regularly interspaced short palindrome repeats (CRISPR) associated protein, optionally wherein the CRISPR associated protein is Cas9. Embodiment 52 provides a pharmaceutical composition comprising the lipid nanoparticle (LNP) of any one of Embodiments 15-51 and at least one pharmaceutically acceptable carrier. Embodiment 53 provides the pharmaceutical composition of Embodiment 52, wherein the composition further comprises at least one adjuvant. Embodiment 54 provides a method of treating, preventing, and/or ameliorating a disease in a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of Embodiments 16-51 and/or at least one pharmaceutical composition of Embodiment 52 or 53. Embodiment 55 provides the method of Embodiment 54, wherein the disease is selected from the group consisting of cancer, an autoimmune disorder, cardiovascular disease, and neurological disease. Embodiment 56 provides the method of Embodiment 55, wherein the cancer is at least one selected from the group consisting of oral cancer, pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer. Embodiment 57 provides the method of Embodiment 54 or 56, wherein the subject is further administered at least one additional agent or therapy useful for treating, preventing, and/or ameliorating cancer in the subject. Embodiment 58 provides the method of any one of Embodiments 54-57, wherein the subject is a mammal. Embodiment 59 provides the method of Embodiment 58, wherein the mammal is a human. Embodiment 60 provides a method of delivering a nucleic acid or therapeutic agent to the liver of a subject, the method comprising administering to the subject at least one lipid nanoparticle (LNP) of any one of Embodiments 16-51 and/or at least one pharmaceutical composition of Embodiment 52 or 53. Embodiment 61 provides a method of preparing a modified immune cell or precursor thereof, the method comprising contacting an immune cell or precursor thereof with the lipid nanoparticle (LNP) of any one of Embodiments 16-51 and/or at least one pharmaceutical composition of Embodiment 52 or 53. Embodiment 62 provides the method of Embodiment 61, wherein the modified immune cell or precursor thereof is αβ T cell, a γδ T cell, a CD8+ T cell, a CD4+ helper T cell, a CD4+ regulatory T cell, an NK T cell, an NK cell, and any combination thereof. Embodiment 63 provides the method of Embodiment 62, wherein the modified immune cell or precursor thereof is a T cell, optionally wherein the T cell is a CD4+ T cell. Embodiment 64 provides the method of Embodiment 62, wherein the modified immune cell or precursor thereof is a NK cell. The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.