Stable lipid nanoparticle compositions prepared without polymeric lipids and uses thereof

Abstract

Empty lipid nanoparticles free of PEG lipids are disclosed, along with methods of producing empty lipid nanoparticles. Loaded lipid nanoparticles produced using the empty lipid nanoparticles are disclosed, along with methods of producing the same. Lipid nanoparticles with no core polymeric lipid (e.g, PEG lipid) achieve stability, encapsulation efficiency, and in vitro protein expression on par with lipid nanoparticles having core polymeric lipids (e.g, PEG lipids).

Description

[0001] STABLE LIPID NANOPARTICLE COMPOSITIONS PREPARED WITHOUT POLYMERIC LIPIDS AND USES THEREOF CROSS-REFERENCE This application claims the benefit of, and priority to, U.S. Provisional Application No. 63 / 683,953, filed August 16, 2024, the entire content of which is hereby incorporated by reference herein, in its entirety. BACKGROUND The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto. Delivery of therapeutic nucleic acids (e.g., mRNA) to cells remains a technological challenge due to the relative instability and low cell permeability of nucleic acid molecules. Lipid nanoparticles (LNPs), liposomes, lipoplexes, and other delivery vehicles have been shown to effectively deliver nucleic acids into cells and / or into intracellular compartments. However, there exists a need for delivery vehicles (e.g., lipid nanoparticles) that achieve improved stability, efficacy, safety, and expression of nucleic acid-encoded proteins, along with the flexibility to accommodate and deliver a variety of different types of therapeutic agents. It is against this backdrop that the compositions and methods of the present application were developed. SUMMARY To enhance their stability, prevent aggregation, reduce immunogenicity, and modulate protein adsorption to the LNP surface, polymeric lipids, including polyethylene glycol (PEG) lipids, are conventionally included in LNP compositions. However, in the context of post hoc loading (PHL), whereby mRNA is loaded into an empty LNP (eLNP), the presence of PEG- lipids within the composition was surprisingly discovered to detrimentally affect the properties of the eLNP drug substance. Embodiments of eLNPs described herein exclude PEG-lipids and related steric stabilizers or use very low concentrations of PEG-lipids or related steric stabilizers. In such embodiments, PEG-lipids instead may be added in downstream process steps, following mRNA encapsulation and charge neutralization. Lipid nanoparticles according to the present disclosure have unexpectedly high stability, encapsulation efficiency, and robustness to stress (e.g., freeze-thaw cycling). In one aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a lipid nanoparticle (LNP), comprising: an ionizable amino lipid (IAL); a structural lipid; and a phospholipid, wherein the lipid nanoparticle comprises 5 mol% or less of polymeric lipids. In some embodiments, the LNP comprises 3 mol% or less of polymeric lipids. In some embodiments, the LNP comprises 1 mol% or less of polymeric lipids. In some embodiments, the LNP comprises 0.1 mol% or less of polymeric lipids. In some embodiments, the LNP does not comprise any polymeric lipids. In some embodiments, the LNP does not comprise any PEG lipids. In some embodiments, the IAL is a compound of Formula (IL*): or a salt thereof, wherein: R1is -OH, -NRN-C4-10cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN’’); RNis H or C1-6 alkyl; RN’is H or C1-6alkyl; RN’’is H or C1-6 alkyl; o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8; m is 4, 5, 6, 7, or 8; M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2; M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3; R2is or –(C1-6alkylene)-(C3-8cycloalkyl)-C1-6alkyl; R2ais -H or C1-10 alkyl; R2bis -H or C1-10 alkyl; R2cis C1-8alkyl or C2-8alkenyl; R3is ; R3ais H or C1-10 alkyl; R3bis H or C1-8 alkyl; and R3cis C1-10alkyl or C2-8alkenyl. In some embodiments, the IAL is a compound of Formula (IL**-I): or a salt thereof, wherein: R1is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2cis C4-8alkyl; R3ais C7-10alkyl; and R3cis C3-5 alkyl. In some embodiments, the IAL is a compound of Formula (IL**-III): or a salt thereof, wherein: R1is NRN-C4-10cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN’’); RNis H; RN’is C1-2 alkyl; RN’’is H; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2ais C7-10 alkyl; R2cis C4-6alkyl; R3ais C1-3 alkyl; and R3cis C4-6 alkyl. In some embodiments, the IAL is a compound of Formula (IL**-IV): or a salt thereof, wherein: R1is OH; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2bis C3-5 alkyl; R2cis C2-4alkyl; R3ais C7-10alkyl; and R3cis C4-6 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-I): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; and R3ais C1-8 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-Ia): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8alkyl. In some embodiments, the IAL is a compound of Formula (IL*-Ia’): or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais C1-8alkyl. In some embodiments, the IAL is a compound of Formula (IL*-IIa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-II’): or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais C1-8 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-III): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-IIIa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8alkyl; and R3ais C1-8 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-IIIa’): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8alkyl; and R3ais C1-8 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-IIIb): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8alkyl. In some embodiments, the IAL is a compound of Formula (IL*-IIIb’): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8alkyl. In some embodiments, the IAL is a compound of Formula (IL*-IV): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8alkyl; and R3ais C1-8 alkyl. In some embodiments, the IAL is a compound of Formula (IL*-IVa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8alkyl; and R3ais C1-8alkyl. In some embodiments, the IAL is a compound of Formula (IL*-Iva’): 2b or a salt thereof, wherein: o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8alkyl; and R3ais C1-8alkyl. In some embodiments, the IAL is a compound selected from: In some embodiments, the phospholipid comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some embodiments, the phospholipid comprises DSPC. In some embodiments, the structural lipid is selected from the group consisting of: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, derivatives thereof, and mixtures thereof. In some embodiments, the structural lipid comprises cholesterol or a derivative thereof. In some embodiments, the lipid nanoparticle comprises 30-60 mol% of the IAL, 15-50 mol% of the structural lipid, and 0-30 mol% of the phospholipid. In some embodiments, the lipid nanoparticle comprises 45-50 mol.% of the ionizable amino lipid, 8-12 mol.% of the phospholipid, 35-40 mol.% of the structural lipid. In some embodiments, the LNP has a mean diameter of about 100 nm to about 500 nm. In some embodiments, the LNP has a mean diameter of about 10 nm to about 200 nm, preferably about 15 nm to about 150 nm, preferably about 90 nm to about 130 nm. In some embodiments, the LNP is suitable to achieve a polynucleotide encapsulation efficiency of at least about 80%. In some embodiments, the LNP is suitable to achieve a polynucleotide encapsulation efficiency of at least about 97%. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a lipid nanoparticle (LNP), consisting of: one or more ionizable amino lipids (IALs); one or more structural lipids; and one or more phospholipids. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to an empty lipid nanoparticle solution (eLNP solution), comprising: the lipid nanoparticle according to any of the above-discussed embodiments; and a carrier. In some embodiments, the eLNP solution comprises a cryoprotectant. In some embodiments, the cryoprotectant comprises a sugar. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the eLNP solution comprises an organic solvent. In some embodiments, the eLNP solution comprises a buffering agent. In some embodiments, the buffering agent comprises a citrate buffer, an acetate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the buffering agent is present at a concentration about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, eLNP composition has a pH of about 4.8 to about 5.8, about 4.9 to about 5.75, or about 5.0 to about 5.5. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a loaded lipid nanoparticle (LLNP), comprising: the lipid nanoparticle according to any of the above-discussed embodiments; and a nucleic acid encapsulated within the lipid nanoparticle. In some embodiments, the nucleic acid comprises a ribonucleic acid. In some embodiments, the ribonucleic acid is at least one selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (lncRNA). In some embodiments, the nucleic acid is a messenger RNA (mRNA). In some embodiments, the mRNA is at least 30 nucleotides in length. In some embodiments, the mRNA is at least 300 nucleotides in length. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to loaded lipid nanoparticle solution, comprising: the loaded lipid nanoparticle of any of the embodiments disclosed herein; and a carrier. In some embodiments, loaded lipid nanoparticle further comprises 0.1 mol% to 5 mol% of a polymeric lipid. In some embodiments, the polymeric lipid comprises a PEG lipid. In some embodiments, the LLNP solution comprises a cryoprotectant. In some embodiments, the cryoprotectant comprises a sugar. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the LLNP solution comprises a buffering agent. In some embodiments, the buffering agent in the loaded lipid nanoparticle solution comprises a citrate buffer, an acetate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the buffering agent is present at a concentration about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, the loaded lipid nanoparticle solution has a pH of about 4.5 to about 5.5, about 4.6 to about 5.4, about 4.7 to about 5.3, or about 4.8 to about 5.2. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a method of producing an empty lipid nanoparticle (eLNP), the method comprising: mixing a lipid solution comprising an ionizable amino lipid, a phospholipid, and a structural lipid with a first aqueous buffer solution comprising a first buffering agent, to obtain an eLNP solution comprising the eLNP, wherein the eLNP comprises 5 mol% or less of polymeric lipids. In some embodiments, the eLNP comprises 3 mol% or less of polymeric lipids. In some embodiments, the eLNP comprises 1 mol% or less of polymeric lipids. In some embodiments, the eLNP comprises 0.1 mol% or less of polymeric lipids. In some embodiments, the eLNP does not comprise any PEG lipids. In some embodiments, the eLNP does not comprise any polymeric lipids. In some embodiments, the eLNP consists of one or more IALs, one or more phospholipids, and one or more structural lipids. In some embodiments, the first buffering agent comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the first buffering agent is present in the first aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, the eLNP solution has a pH of about 4.8 to about 5.8, about 4.9 to about 5.75, or about 5.0 to about 5.5. In some embodiments, the method comprises adding a cryoprotectant to the eLNP solution. In some embodiments, eLNP solution comprises about 1 mg / mL to about 1000 mg / mL of the cryoprotectant. In some embodiments, the cryoprotectant comprises a sugar. In some embodiments, the sugar comprises sucrose. In some embodiments, the method further comprises processing the eLNP solution. In some embodiments, the processing the eLNP solution comprises adjusting the pH of the eLNP solution. In some embodiments, the adjusting the pH of the eLNP solution comprises adding a second aqueous buffer solution comprising a second buffering agent to the eLNP solution. In some embodiments, the second aqueous buffer solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the second buffering agent is present in the second aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, the processing the eLNP solution comprises filtering, buffer exchanging, lyophilizing, diluting, or storing the eLNP solution, or any combination thereof. In some embodiments, the processing the eLNP solution comprises filtering the eLNP solution by tangential flow filtration. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a method of producing a loaded lipid nanoparticle, the method comprising: mixing a lipid solution comprising an ionizable amino lipid, a phospholipid, and a structural lipid with a first aqueous buffer solution comprising a first buffering agent, to obtain an eLNP solution comprising the eLNP, wherein the eLNP comprises 5 mol% or less of polymeric lipids; processing the eLNP solution; loading the eLNP with a nucleic acid by mixing a nucleic acid solution comprising the nucleic acid with the processed eLNP solution to obtain a loaded LNP composition comprising the loaded LNP. In some embodiments, the eLNP comprises 3 mol% or less of polymeric lipids. In some embodiments, the eLNP comprises 1 mol% or less of polymeric lipids. In some embodiments, the eLNP comprises 0.1 mol% or less of polymeric lipids. In some embodiments, the eLNP does not comprise any PEG lipids. In some embodiments, the eLNP does not comprise any polymeric lipids. In some embodiments, the eLNP consists of one or more IALs, one or more phospholipids, and one or more structural lipids. In some embodiments, the first buffering agent comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the first buffering agent is present in the first aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, the eLNP solution has a pH of about 4.8 to about 5.8, about 4.9 to about 5.75, or about 5.0 to about 5.5. In some embodiments, the processing the eLNP solution comprises adjusting the pH of the eLNP solution. In some embodiments, the adjusting the pH of the eLNP solution comprises adding a second aqueous buffer solution comprising a second buffering agent to the eLNP solution. In some embodiments, the second aqueous buffer solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the second buffering agent is present in the second aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, the processing the eLNP solution comprises filtering, buffer exchanging, lyophilizing, diluting, or storing the eLNP solution, or any combination thereof. In some embodiments, the processing the eLNP solution comprises filtering the eLNP solution by tangential flow filtration. In some embodiments, the nucleic acid comprises a ribonucleic acid. In some embodiments, the ribonucleic acid is at least one selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (lncRNA). In some embodiments, the nucleic acid is a messenger RNA (mRNA). In some embodiments, the mRNA is at least 30 nucleotides in length. In some embodiments, the mRNA is at least 300 nucleotides in length. In some embodiments, the nucleic acid solution comprises about 0.01 to about 1.0 mg / mL of the nucleic acid, about 0.05 to about 0.5 mg / mL of the nucleic acid, or about 0.1 to about 0.25 mg / mL of the nucleic acid. In some embodiments, the nucleic acid solution comprises a buffer selected from the group consisting of an acetate buffer, a citrate buffer, a phosphate buffer, and a tris buffer. In some embodiments, the nucleic acid solution comprises from about 1 mM to about 200 mM acetate buffer, from about 2 mM to about 180 mM acetate buffer, from about 3 mM to about 160 mM acetate buffer, from about 4 mM to about 150 mM acetate buffer, from about 4 mM to about 140 mM acetate buffer, from about 5 mM to about 130 mM acetate buffer, from about 6 mM to about 120 mM acetate buffer, from about 7 mM to about 110 mM acetate buffer, from about 8 mM to about 100 mM acetate buffer, from about 9 mM to about 90 mM acetate buffer, from about 10 mM to about 80 mM acetate buffer, from about 15 mM to about 70 mM acetate buffer, from about 20 mM to about 60 mM acetate buffer, from about 25 mM to about 50 mM acetate buffer, or from about 30 mM to about 40 mM acetate buffer. In some embodiments, the nucleic acid solution and the empty-LNP solution are mixed at a volumetric flow ratio of from about 5:1 to about 7:1, from about 4:1 to about 6:1, from about 3:1 to about 5:1, or from about 2:1 to about 4:1 during the loading. In some embodiments, the method further comprises processing the loaded LNP solution to obtain a LNP formulation. In some embodiments, the processing the loaded LNP solution comprises adding a polymeric lipid to the loaded LNP. In some embodiments, the polymeric lipid comprises a PEG lipid. In some embodiments, the adding the PEG lipid comprises mixing a PEG lipid solution comprising the PEG lipid with the loaded LNP solution. In some embodiments, the adding the PEG lipid comprises adding the PEG lipid in an amount of from about 0.1 mol% to about 3.0 mol% PEG lipid, from about 0.2 mol% to about 2.5 mol% PEG lipid, from about 0.5 mol% to about 2.0 mol% PEG lipid, from about 0.75 mol% to about 1.5 mol% PEG lipid, from about 1.0 mol% to about 1.25 mol% PEG lipid, from about 0.2 mol% to about 0.8 mol%, 5 mol% or less, 3 mol% or less, 1 mol% or less, or 0.1 mol% or less PEG lipid to the loaded LNP. In some embodiments, the PEG lipid is PEG-DMG or a compound of Formula (VI): wherein r is 40-50, preferably 45; In some embodiments, the processing further comprises buffer exchanging, filtering, lyophilizing, or storing the loaded LNP solution. In some embodiments, the loaded LNP solution or the LNP formulation further comprises a cryoprotectant. In some embodiments, the cryoprotectant comprises a sugar. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the loaded LNP solution or the LNP formulation further comprises a buffering agent. In some embodiments, the buffering agent in the loaded LNP solution or the LNP formulation comprises a citrate buffer, an acetate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the buffering agent in the loaded LNP solution or the LNP formulation is present at a concentration about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, the loaded LNP solution or the LNP formulation has a pH of about 4.5 to about 7.5, about 5.0 to about 7.4, about 5.5 to about 7.3, or about 6.0 to about 7.0. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the loaded LNP of any one of the embodiments disclosed herein, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the loaded LNP solution of any one of the embodiments disclosed herein, wherein the loaded LNP solution comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the LNP formulation of any one of the embodiments disclosed herein, wherein the LNP formulation comprises a therapeutically effective amount of a therapeutic mRNA. In some embodiments, the administering is performed parenterally. In some embodiments, the administering is performed intramuscularly, intradermally, subcutaneously, and / or intravenously. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to the loaded LNP of any one of the embodiments disclosed herein, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to the loaded LNP solution of any one of the embodiments disclosed herein, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to the LNP formulation of any one of the embodiments disclosed herein, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a use of the loaded LNP of any one of the embodiments disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a use of the loaded LNP solution of any one of the embodiments disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a use of the LNP formulation of any one of the embodiments disclosed herein in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a kit, comprising the empty LNP of any one of the embodiments disclosed herein, the empty LNP solution of any one of the embodiments disclosed herein, the loaded LNP of any one of the embodiments disclosed herein, the loaded LNP solution of any one of the embodiments disclosed herein, or the LNP formulation of any one of the embodiments disclosed herein. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a kit, comprising: a first container comprising the empty LNP of any one of the embodiments disclosed herein or the empty LNP solution of any one of the embodiments disclosed herein; and a second container comprising a therapeutic agent. In some embodiments, the kit comprises instructions for combining contents of the first container with contents of the second container. In some embodiments, the therapeutic agent comprises a therapeutic mRNA. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to an empty lipid nanoparticle, wherein the empty lipid nanoparticle is obtained by any of the methods disclosed herein. In another aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a loaded lipid nanoparticle, wherein the loaded lipid nanoparticle is obtained by any of the methods disclosed herein. The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS Various objects, aspects, features, and advantages of the disclosure will become more apparent and better understood by referring to the detailed description taken in conjunction with the accompanying figures. FIG. 1A is a process schematic for producing an empty lipid nanoparticle (eLNP) solution. FIG. 1B is a process schematic for producing a loaded lipid nanoparticle solution. FIG. 1C is a process schematic for producing a LNP formulation from a loaded LNP solution. FIG. 2 is a schematic illustration of LNPs having core PEG lipids and surface PEG lipids (left) and an LNP having no core PEG lipids and only surface PEG lipids (right). FIG. 3 is a process schematic showing two different addition points of PEG lipids in producing LNP formulations: “core PEG” and “post-addition (PA).” FIG. 4 shows plots of LNP stability (rate of diameter change per day, %) versus concentration of PEG lipid for LNPs in which the PEG lipid was added during (A) core mixing (Core); and (B) post-addition (PA). FIG. 5 shows capillary zone electrophoresis (CZE) profiles for loaded lipid nanoparticle solutions having various PEG concentrations, where PEG lipid was added during core mixing. FIG. 6 is a plot showing mRNA encapsulation efficiency (EE, %) versus PEG lipid concentration for LNPs in which PEG lipid was introduced during core mixing or PA. FIG. 7A and FIG.7B show plots of liquid stability of PEG-less eLNPs and eLNPs comprising 0.5 mol% PEG lipid over time (days) at 5℃ (FIG.7A) and at room temperature (FIG.7B). FIG. 8 is a bar chart comparing protein expression for LNPs comprising 0.5 mol% PEG lipid added during core mixing and a similar LNP comprising no PEG lipid. FIG. 9A and FIG.9B are plots showing the effect of freeze-thaw (FT) cycling on (FIG. 9A) eLNP particle size and (FIG.9B) polydispersity index (PDI) for PEG-less eLNPs and eLNPs having 0.5 mol% core PEG. FIG. 10A and FIG.10B show freeze-thaw (FT) stability (via particle size) of (FIG> 10A) eLNPs comprising no core PEG lipid and (FIG.10B) comprising 0.5 mol% core PEG lipid, for different total lipid concentrations. FIG. 11 is a bar chart showing in vitro protein expression achieved using LNPs according to the present disclosure (with 0% core PEG) versus LNPs prepared using 0.5 mol% core PEG. FIG. 12 shows degradation of PEG-DMG over time for LNPs having 0% core PEG lipid or LNPs having 5 mol% core PEG lipid. Reference will now be made in detail to some specific embodiments contemplated by the present disclosure. While various embodiments are described herein, it will be understood that it is not intended to limit the present technology to the described embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the technology as defined by the appended claims. DETAILED DESCRIPTION Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Methods of Producing Empty Nanoparticles (eLNPs) In an aspect, which may be combined with any other aspect or embodiment, the present disclosure relates to a method of producing an empty lipid nanoparticle (eLNP), the method comprising: mixing a lipid solution comprising an ionizable amino lipid, a phospholipid, and a structural lipid with a first aqueous buffer solution comprising a first buffering agent, to obtain an eLNP solution comprising the eLNP. In some embodiments, the eLNP comprises polymeric lipids at a concentration of 5 mol% or less, 3 mol% or less, 1 mol% or less, or 0.1 mol% or less. In some embodiments, the eLNP does not comprise any PEG lipid. In some embodiments, the eLNP does not comprise any polymeric lipids. Referring to FIG.1A, methods of making eLNPs may include mixing a combination of lipids in a lipid solution to form a “core” LNP. In some embodiments, the lipid solution comprises an ionizable amino lipid (IAL), a phospholipid, and a structural lipid, as further defined herein. In some embodiments, the lipid solution comprises an organic solvent (e.g., ethanol, methanol, or isopropanol), water, or a combination thereof. In some embodiments, the lipid solution is mixed with a first aqueous buffer solution to produce an empty lipid nanoparticle solution (eLNP solution). In some embodiments, any of the IAL, the phospholipid, or the structural lipid may be present in the lipid solution, individually or collectively, at a concentration of greater than or equal to about 1 mM, greater than or equal to about 2 mM, greater than or equal to about 3 mM, greater than or equal to about 4 mM, greater than or equal to about 5 mM, greater than or equal to about 6 mM, greater than or equal to about 7 mM, greater than or equal to about 8 mM, greater than or equal to about 9 mM, greater than or equal to about 10 mM, greater than or equal to about 15 mM, greater than or equal to about 20 mM, greater than or equal to about 25 mM, greater than or equal to about 30 mM, greater than or equal to about 35 mM, greater than or equal to about 40 mM, greater than or equal to about 45 mM, greater than or equal to about 50 mM, greater than or equal to about 55 mM, greater than or equal to about 60 mM, greater than or equal to about 65 mM, greater than or equal to about 70 mM, greater than or equal to about 75 mM, greater than or equal to about 80 mM, greater than or equal to about 85 mM, greater than or equal to about 90 mM, greater than or equal to about 95 mM, greater than or equal to about 100 mM, or any range or value including and / or in between any two of these values. In some embodiments, any of the IAL, the phospholipid, or the structural lipid may be present in the lipid solution, individually or collectively, at a concentration of less than or equal to about 100 mM, less than or equal to about 95 mM, less than or equal to about 90 mM, less than or equal to about 85 mM, less than or equal to about 80 mM, less than or equal to about 75 mM, less than or equal to about 70 mM, less than or equal to about 65 mM, less than or equal to about 60 mM, less than or equal to about 55 mM, less than or equal to about 50 mM, less than or equal to about 45 mM, less than or equal to about 40 mM, less than or equal to about 35 mM, less than or equal to about 30 mM, less than or equal to about 25 mM, less than or equal to about 20 mM, less than or equal to about 15 mM, less than or equal to about 10 mM, less than or equal to about 9 mM, less than or equal to about 8 mM, less than or equal to about 7 mM, less than or equal to about 6 mM, less than or equal to about 5 mM, less than or equal to about 4 mM, less than or equal to about 3 mM, less than or equal to about 2 mM, less than or equal to about 1 mM, or any range or value including and / or in between any two of these values. In some embodiments, any of the IAL, the phospholipid, or the structural lipid may be present in the lipid solution, individually or collectively, at a concentration of greater than or equal to about 1 mg / mL, greater than or equal to about 2 mg / mL, greater than or equal to about 3 mg / mL, greater than or equal to about 4 mg / mL, greater than or equal to about 5 mg / mL, greater than or equal to about 6 mg / mL, greater than or equal to about 7 mg / mL, greater than or equal to about 8 mg / mL, greater than or equal to about 9 mg / mL, greater than or equal to about 10 mg / mL, greater than or equal to about 15 mg / mL, greater than or equal to about 20 mg / mL, greater than or equal to about 25 mg / mL, greater than or equal to about 30 mg / mL, greater than or equal to about 35 mg / mL, greater than or equal to about 40 mg / mL, greater than or equal to about 45 mg / mL, greater than or equal to about 50 mg / mL, greater than or equal to about 55 mg / mL, greater than or equal to about 60 mg / mL, greater than or equal to about 65 mg / mL, greater than or equal to about 70 mg / mL, greater than or equal to about 75 mg / mL, greater than or equal to about 80 mg / mL, greater than or equal to about 85 mg / mL, greater than or equal to about 90 mg / mL, greater than or equal to about 95 mg / mL, greater than or equal to about 100 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, any of the IAL, the phospholipid, or the structural lipid may be present in the lipid solution, individually or collectively, at a concentration of less than or equal to about 100 mg / mL, less than or equal to about 95 mg / mL, less than or equal to about 90 mg / mL, less than or equal to about 85 mg / mL, less than or equal to about 80 mg / mL, less than or equal to about 75 mg / mL, less than or equal to about 70 mg / mL, less than or equal to about 65 mg / mL, less than or equal to about 60 mg / mL, less than or equal to about 55 mg / mL, less than or equal to about 50 mg / mL, less than or equal to about 45 mg / mL, less than or equal to about 40 mg / mL, less than or equal to about 35 mg / mL, less than or equal to about 30 mg / mL, less than or equal to about 25 mg / mL, less than or equal to about 20 mg / mL, less than or equal to about 15 mg / mL, less than or equal to about 10 mg / mL, less than or equal to about 9 mg / mL, less than or equal to about 8 mg / mL, less than or equal to about 7 mg / mL, less than or equal to about 6 mg / mL, less than or equal to about 5 mg / mL, less than or equal to about 4 mg / mL, less than or equal to about 3 mg / mL, less than or equal to about 2 mg / mL, less than or equal to about 1 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the lipid solution may comprise one or more organic solvents (including a mixture of organic solvents) (e.g., ethanol, isopropanol, methanol) at a concentration of greater than or equal to about 60 vol.%, greater than or equal to about 65 vol.%, greater than or equal to about 70 vol.%, greater than or equal to about 75 vol.%, greater than or equal to about 80 vol.%, greater than or equal to about 85 vol.%, greater than or equal to about 90 vol.%, greater than or equal to about 95 vol.%, about 100 vol.%, or any range or value including and / or in between any two of these values. In some embodiments, the lipid solution may comprise an organic solvent (e.g., ethanol, isopropanol, methanol) at a concentration of less than or equal to about 100 vol.%, less than or equal to about 95 vol.%, less than or equal to about 90 vol.%, less than or equal to about 85 vol.%, less than or equal to about 80 vol.%, less than or equal to about 75 vol.%, less than or equal to about 70 vol.%, less than or equal to about 65 vol.%, less than or equal to about 60 vol.%, or any range or value including and / or in between any two of these values. In some embodiments, the mixing comprises adding a first aqueous buffer solution comprising a first buffering agent to the lipid solution. In some embodiments, the first buffering agent comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer (e.g., ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, potassium citrate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), etc.). In some embodiments, the first buffering agent is present in the first aqueous buffer solution at a concentration of greater than or equal to about 0.001 mM, greater than or equal to about 0.002 mM, greater than or equal to about 0.003 mM, greater than or equal to about 0.004 mM, greater than or equal to about 0.005 mM, greater than or equal to about 0.006 mM, greater than or equal to about 0.007 mM, greater than or equal to about 0.008 mM, greater than or equal to about 0.009 mM, greater than or equal to about 0.01 mM, greater than or equal to about 0.02 mM, greater than or equal to about 0.03 mM, greater than or equal to about 0.04 mM, greater than or equal to about 0.05 mM, greater than or equal to about 0.06 mM, greater than or equal to about 0.07 mM, greater than or equal to about 0.08 mM, greater than or equal to about 0.09 mM, greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, greater than or equal to about 0.6 mM, greater than or equal to about 0.7 mM, greater than or equal to about 0.8 mM, greater than or equal to about 0.9 mM, greater than or equal to about 1.0 mM, greater than or equal to about 2.0 mM, greater than or equal to about 3.0 mM, greater than or equal to about 4.0 mM, greater than or equal to about 5.0 mM, greater than or equal to about 6.0 mM, greater than or equal to about 7.0 mM, greater than or equal to about 8.0 mM, greater than or equal to about 9.0 mM, greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, greater than or equal to about 110 mM, greater than or equal to about 120 mM, greater than or equal to about 130 mM, greater than or equal to about 140 mM, greater than or equal to about 150 mM, greater than or equal to about 160 mM, greater than or equal to about 170 mM, greater than or equal to about 180 mM, greater than or equal to about 190 mM, greater than or equal to about 200 mM, or any range or value including and / or in between any two of these values. In some embodiments, the first buffering agent is present in the first aqueous buffer solution at a concentration of less than or equal to about 200 mM, less than or equal to about 190 mM, less than or equal to about 180 mM, less than or equal to about 170 mM, less than or equal to about 160 mM, less than or equal to about 150 mM, less than or equal to about 140 mM, less than or equal to about 130 mM, less than or equal to about 120 mM, less than or equal to about 110 mM, less than or equal to about 100 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, less than or equal to about 60 mM, less than or equal to about 50 mM, less than or equal to about 40 mM, less than or equal to about 30 mM, less than or equal to about 20 mM, less than or equal to about 10 mM, less than or equal to about 9.0 mM, less than or equal to about 8.0 mM, less than or equal to about 7.0 mM, less than or equal to about 6.0 mM, less than or equal to about 5.0 mM, less than or equal to about 4.0 mM, less than or equal to about 3.0 mM, less than or equal to about 2.0 mM, less than or equal to about 1.0 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, less than or equal to about 0.6 mM, less than or equal to about 0.5 mM, less than or equal to about 0.4 mM, less than or equal to about 0.3 mM, less than or equal to about 0.2 mM, less than or equal to about 0.1 mM, less than or equal to about 0.09 mM, less than or equal to about 0.08 mM, less than or equal to about 0.07 mM, less than or equal to about 0.06 mM, less than or equal to about 0.05 mM, less than or equal to about 0.04 mM, less than or equal to about 0.03 mM, less than or equal to about 0.02 mM, less than or equal to about 0.01 mM, less than or equal to about 0.009 mM, less than or equal to about 0.008 mM, less than or equal to about 0.007 mM, less than or equal to about 0.006 mM, less than or equal to about 0.005 mM, less than or equal to about 0.004 mM, less than or equal to about 0.003 mM, less than or equal to about 0.002 mM, less than or equal to about 0.001 mM, or any range or value including and / or in between any two of these values. In some embodiments, the first aqueous buffer solution has a pH of greater than or equal to about 2.5, greater than or equal to about 2.6, greater than or equal to about 2.7, greater than or equal to about 2.8, greater than or equal to about 2.9, greater than or equal to about 3.0, greater than or equal to about 3.1, greater than or equal to about 3.2, greater than or equal to about 3.3, greater than or equal to about 3.4, greater than or equal to about 3.5, greater than or equal to about 3.6, greater than or equal to about 3.7, greater than or equal to about 3.8, greater than or equal to about 3.9, greater than or equal to about 4.0, greater than or equal to about 4.1, greater than or equal to about 4.2, greater than or equal to about 4.3, greater than or equal to about 4.4, greater than or equal to about 4.5, greater than or equal to about 4.6, greater than or equal to about 4.7, greater than or equal to about 4.8, greater than or equal to about 4.9, greater than or equal to about 5.0, greater than or equal to about 5.1, greater than or equal to about 5.2, greater than or equal to about 5.3, greater than or equal to about 5.4, greater than or equal to about 5.5, greater than or equal to about 5.6, greater than or equal to about 5.7, greater than or equal to about 5.8, greater than or equal to about 5.9, greater than or equal to about 6.0, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7.0, or any range or value including and / or in between any two of these values. In some embodiments, the first aqueous buffer solution has a pH of less than or equal to about 7.0, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6.0, less than or equal to about 5.9, less than or equal to about 5.8, less than or equal to about 5.7, less than or equal to about 5.6, less than or equal to about 5.5, less than or equal to about 5.4, less than or equal to about 5.3, less than or equal to about 5.2, less than or equal to about 5.1, less than or equal to about 5.0, less than or equal to about 4.9, less than or equal to about 4.8, less than or equal to about 4.7, less than or equal to about 4.6, less than or equal to about 4.5, less than or equal to about 4.4, less than or equal to about 4.3, less than or equal to about 4.2, less than or equal to about 4.1, less than or equal to about 4.0, less than or equal to about 3.9, less than or equal to about 3.8, less than or equal to about 3.7, less than or equal to about 3.6, less than or equal to about 3.5, less than or equal to about 3.4, less than or equal to about 3.3, less than or equal to about 3.2, less than or equal to about 3.1, less than or equal to about 3.0, less than or equal to about 2.9, less than or equal to about 2.8, less than or equal to about 2.7, less than or equal to about 2.6, less than or equal to about 2.5, or any range or value including and / or in between any two of these values. In some embodiments, the first aqueous buffer solution is an acetate buffer or phosphate buffer wherein the buffering agent is present at a concentration of about 1 mM to about 50 mM (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM), wherein the first aqueous buffer solution has a pH of about 4 to 8 (e.g., about 4, 4.5, 5, 5.5, 6, 6., 7, 7.5 or 8). For instance, in some embodiments, the first aqueous buffer solution may comprise about 5 mM of an acetate buffering agent and have a pH of about 5. In some embodiments, the first aqueous buffer solution may comprise about 5 mM of a phosphate buffering agent and have a pH of about 8. In some embodiments, the first aqueous buffer solution may comprise about 45 mM of an acetate buffering agent and have a pH of about 4. In some embodiments, the mixing is performed using a T-junction, confined impinging jets, microfluidic mixer, or vortex mixer. In some embodiments, the mixing is performed using an apparatus disclosed in WIPO Publication No. WO 2023 / 193002A1 or WIPO Publication No. 2020 / 160430, the entireties of which are hereby incorporated by reference herein. In some embodiments, the mixing is performed at a temperature of less than or equal to about 55 ℃, less than or equal to about 50 ℃, less than or equal to about 45 ℃, less than or equal to about 40 ℃, less than or equal to about 35 ℃, less than or equal to about 30 ℃, less than or equal to about 25 ℃, less than or equal to about 20 ℃, less than or equal to about 15 ℃, less than or equal to about 10 ℃, less than or equal to about 9 ℃, less than or equal to about 8 ℃, less than or equal to about 7 ℃, less than or equal to about 6 ℃, less than or equal to about 5 ℃, less than or equal to about 4 ℃, less than or equal to about 3 ℃, less than or equal to about 2 ℃, less than or equal to about 1 ℃, or any range or value including and / or in between any two of these values. In some embodiments, the eLNP solution has a pH of greater than or equal to about 2.5, greater than or equal to about 2.6, greater than or equal to about 2.7, greater than or equal to about 2.8, greater than or equal to about 2.9, greater than or equal to about 3.0, greater than or equal to about 3.1, greater than or equal to about 3.2, greater than or equal to about 3.3, greater than or equal to about 3.4, greater than or equal to about 3.5, greater than or equal to about 3.6, greater than or equal to about 3.7, greater than or equal to about 3.8, greater than or equal to about 3.9, greater than or equal to about 4.0, greater than or equal to about 4.1, greater than or equal to about 4.2, greater than or equal to about 4.3, greater than or equal to about 4.4, greater than or equal to about 4.5, greater than or equal to about 4.6, greater than or equal to about 4.7, greater than or equal to about 4.8, greater than or equal to about 4.9, greater than or equal to about 5.0, greater than or equal to about 5.1, greater than or equal to about 5.2, greater than or equal to about 5.3, greater than or equal to about 5.4, greater than or equal to about 5.5, greater than or equal to about 5.6, greater than or equal to about 5.7, greater than or equal to about 5.8, greater than or equal to about 5.9, greater than or equal to about 6.0, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7.0, greater than or equal to about 7.1, greater than or equal to about 7.2, greater than or equal to about 7.3, greater than or equal to about 7.4, greater than or equal to about 7.5, greater than or equal to about 7.6, greater than or equal to about 7.7, greater than or equal to about 7.8, greater than or equal to about 7.9, greater than or equal to about 8.0 , greater than or equal to about 8.1, greater than or equal to about 8.2, greater than or equal to about 8.3, greater than or equal to about 8.4, greater than or equal to about 8.5, or any range or value including and / or in between any two of these values. In some embodiments, the eLNP solution has a pH of less than or equal to about 8.5, less than or equal to about 8.4, less than or equal to about 8.3, less than or equal to about 8.2, less than or equal to about 8.1, less than or equal to about 8.0, less than or equal to about 7.9, less than or equal to about 7.8, less than or equal to about 7.7, less than or equal to about 7.6, less than or equal to about 7.5, less than or equal to about 7.4, less than or equal to about 7.3, less than or equal to about 7.2, less than or equal to about 71, less than or equal to about 7.0, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6.0, less than or equal to about 5.9, less than or equal to about 5.8, less than or equal to about 5.7, less than or equal to about 5.6, less than or equal to about 5.5, less than or equal to about 5.4, less than or equal to about 5.3, less than or equal to about 5.2, less than or equal to about 5.1, less than or equal to about 5.0, less than or equal to about 4.9, less than or equal to about 4.8, less than or equal to about 4.7, less than or equal to about 4.6, less than or equal to about 4.5, less than or equal to about 4.4, less than or equal to about 4.3, less than or equal to about 4.2, less than or equal to about 4.1, less than or equal to about 4.0, less than or equal to about 3.9, less than or equal to about 3.8, less than or equal to about 3.7, less than or equal to about 3.6, less than or equal to about 3.5, less than or equal to about 3.4, less than or equal to about 3.3, less than or equal to about 3.2, less than or equal to about 3.1, less than or equal to about 3.0, less than or equal to about 2.9, less than or equal to about 2.8, less than or equal to about 2.7, less than or equal to about 2.6, less than or equal to about 2.5, or any range or value including and / or in between any two of these values. In some embodiments, the method comprises adding a cryoprotectant to the eLNP solution, the aqueous buffer solution, or the lipid solution. In some embodiments, the cryoprotectant is added to the eLNP solution or loaded LNP solution prior to lyophilization. In some embodiments, the cryoprotectant comprises one or more cryoprotective agents , and each of the one or more cryoprotective agents is independently a polyol (e.g., a diol or a triol such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / -)-2-methyl-2,4- pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3-butanediol, ethylene glycol, or diethylene glycol), a nondetergent sulfobetaine (e.g., NDSB-201 (3-(1-pyridino)-1-propane sulfonate), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P 400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D- (+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant consists of sucrose. In some embodiments, the cryoprotectant is present in the LNP formulation, loaded lipid nanoparticle solution, eLNP solution, the first aqueous buffer solution, or the lipid solution at a concentration of greater than or equal to about 1 mg / mL, greater than or equal to about 2 mg / mL, greater than or equal to about 3 mg / mL, greater than or equal to about 4 mg / mL, greater than or equal to about 5 mg / mL, greater than or equal to about 6 mg / mL, greater than or equal to about 7 mg / mL, greater than or equal to about 8 mg / mL, greater than or equal to about 9 mg / mL, greater than or equal to about 10 mg / mL, greater than or equal to about 15 mg / mL, greater than or equal to about 20 mg / mL, greater than or equal to about 25 mg / mL, greater than or equal to about 30 mg / mL, greater than or equal to about 35 mg / mL, greater than or equal to about 40 mg / mL, greater than or equal to about 45 mg / mL, greater than or equal to about 50 mg / mL, greater than or equal to about 55 mg / mL, greater than or equal to about 60 mg / mL, greater than or equal to about 65 mg / mL, greater than or equal to about 70 mg / mL, greater than or equal to about 75 mg / mL, greater than or equal to about 80 mg / mL, greater than or equal to about 85 mg / mL, greater than or equal to about 90 mg / mL, greater than or equal to about 95 mg / mL, greater than or equal to about 100 mg / mL, greater than or equal to about 150 mg / mL, greater than or equal to about 200 mg / mL, greater than or equal to about 250 mg / mL, greater than or equal to about 300 mg / mL, greater than or equal to about 350 mg / mL, greater than or equal to about 400 mg / mL, greater than or equal to about 450 mg / mL, greater than or equal to about 500 mg / mL, greater than or equal to about 550 mg / mL, greater than or equal to about 600 mg / mL, greater than or equal to about 650 mg / mL, greater than or equal to about 700 mg / mL, greater than or equal to about 750 mg / mL, greater than or equal to about 800 mg / mL, greater than or equal to about 850 mg / mL, greater than or equal to about 900 mg / mL, greater than or equal to about 950 mg / mL, greater than or equal to about 1000 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the cryoprotectant is present in the LNP formulation, loaded LNP solution, eLNP solution, the first aqueous buffer solution, or the lipid solution at a concentration of less than or equal to about 1000 mg / mL, less than or equal to about 950 mg / mL, less than or equal to about 900 mg / mL, less than or equal to about 850 mg / mL, less than or equal to about 800 mg / mL, less than or equal to about 750 mg / mL, less than or equal to about 700 mg / mL, less than or equal to about 650 mg / mL, less than or equal to about 600 mg / mL, less than or equal to about 550 mg / mL, less than or equal to about 500 mg / mL, less than or equal to about 450 mg / mL, less than or equal to about 400 mg / mL, less than or equal to about 350 mg / mL, less than or equal to about 300 mg / mL, less than or equal to about 250 mg / mL, less than or equal to about 200 mg / mL, less than or equal to about 150 mg / mL, less than or equal to about 100 mg / mL, less than or equal to about 95 mg / mL, less than or equal to about 90 mg / mL, less than or equal to about 85 mg / mL, less than or equal to about 80 mg / mL, less than or equal to about 75 mg / mL, less than or equal to about 70 mg / mL, less than or equal to about 65 mg / mL, less than or equal to about 60 mg / mL, less than or equal to about 55 mg / mL, less than or equal to about 50 mg / mL, less than or equal to about 45 mg / mL, less than or equal to about 40 mg / mL, less than or equal to about 35 mg / mL, less than or equal to about 30 mg / mL, less than or equal to about 25 mg / mL, less than or equal to about 20 mg / mL, less than or equal to about 15 mg / mL, less than or equal to about 10 mg / mL, less than or equal to about 9 mg / mL, less than or equal to about 8 mg / mL, less than or equal to about 7 mg / mL, less than or equal to about 6 mg / mL, less than or equal to about 5 mg / mL, less than or equal to about 4 mg / mL, less than or equal to about 3 mg / mL, less than or equal to about 2 mg / mL, less than or equal to about 1 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the cryoprotectant is present in the LNP formulation, loaded lipid nanoparticle solution, eLNP solution, the first aqueous buffer solution, or the lipid solution at a concentration of about 1 mg / mL to about 1000 mg / mL, about 5 mg / mL to about 950 mg / mL, about 10 mg / mL to about 900 mg / mL, about 20 mg / mL to about 800 mg / mL, about 50 mg / mL to about 700 mg / mL, about 100 mg / mL to about 500 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the method comprises processing the eLNP solution. In some embodiments, the processing the eLNP solution comprises adjusting the pH of the eLNP solution. In some embodiments, the adjusting the pH of the eLNP solution comprises adding a second aqueous buffer solution comprising a second buffering agent to the eLNP solution. In some embodiments, adjusting the pH of the eLNP solution comprises a buffer exchange. In some embodiments, the second aqueous buffer solution comprises a second buffering agent. In some embodiments, the second buffering agent comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer (e.g., ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, potassium citrate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), etc.). In some embodiments, the second buffering agent is present in the second aqueous buffer solution at a concentration of greater than or equal to about 0.001 mM, greater than or equal to about 0.002 mM, greater than or equal to about 0.003 mM, greater than or equal to about 0.004 mM, greater than or equal to about 0.005 mM, greater than or equal to about 0.006 mM, greater than or equal to about 0.007 mM, greater than or equal to about 0.008 mM, greater than or equal to about 0.009 mM, greater than or equal to about 0.01 mM, greater than or equal to about 0.02 mM, greater than or equal to about 0.03 mM, greater than or equal to about 0.04 mM, greater than or equal to about 0.05 mM, greater than or equal to about 0.06 mM, greater than or equal to about 0.07 mM, greater than or equal to about 0.08 mM, greater than or equal to about 0.09 mM, greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, greater than or equal to about 0.6 mM, greater than or equal to about 0.7 mM, greater than or equal to about 0.8 mM, greater than or equal to about 0.9 mM, greater than or equal to about 1.0 mM, greater than or equal to about 2.0 mM, greater than or equal to about 3.0 mM, greater than or equal to about 4.0 mM, greater than or equal to about 5.0 mM, greater than or equal to about 6.0 mM, greater than or equal to about 7.0 mM, greater than or equal to about 8.0 mM, greater than or equal to about 9.0 mM, greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, greater than or equal to about 110 mM, greater than or equal to about 120 mM, greater than or equal to about 130 mM, greater than or equal to about 140 mM, greater than or equal to about 150 mM, greater than or equal to about 160 mM, greater than or equal to about 170 mM, greater than or equal to about 180 mM, greater than or equal to about 190 mM, greater than or equal to about 200 mM, or any range or value including and / or in between any two of these values. In some embodiments, the second buffering agent is present in the second aqueous buffer solution at a concentration of less than or equal to about 200 mM, less than or equal to about 190 mM, less than or equal to about 180 mM, less than or equal to about 170 mM, less than or equal to about 160 mM, less than or equal to about 150 mM, less than or equal to about 140 mM, less than or equal to about 130 mM, less than or equal to about 120 mM, less than or equal to about 110 mM, less than or equal to about 100 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, less than or equal to about 60 mM, less than or equal to about 50 mM, less than or equal to about 40 mM, less than or equal to about 30 mM, less than or equal to about 20 mM, less than or equal to about 10 mM, less than or equal to about 9.0 mM, less than or equal to about 8.0 mM, less than or equal to about 7.0 mM, less than or equal to about 6.0 mM, less than or equal to about 5.0 mM, less than or equal to about 4.0 mM, less than or equal to about 3.0 mM, less than or equal to about 2.0 mM, less than or equal to about 1.0 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, less than or equal to about 0.6 mM, less than or equal to about 0.5 mM, less than or equal to about 0.4 mM, less than or equal to about 0.3 mM, less than or equal to about 0.2 mM, less than or equal to about 0.1 mM, less than or equal to about 0.09 mM, less than or equal to about 0.08 mM, less than or equal to about 0.07 mM, less than or equal to about 0.06 mM, less than or equal to about 0.05 mM, less than or equal to about 0.04 mM, less than or equal to about 0.03 mM, less than or equal to about 0.02 mM, less than or equal to about 0.01 mM, less than or equal to about 0.009 mM, less than or equal to about 0.008 mM, less than or equal to about 0.007 mM, less than or equal to about 0.006 mM, less than or equal to about 0.005 mM, less than or equal to about 0.004 mM, less than or equal to about 0.003 mM, less than or equal to about 0.002 mM, less than or equal to about 0.001 mM, or any range or value including and / or in between any two of these values. In some embodiments, the second aqueous buffer solution has a pH of greater than or equal to about 2.5, greater than or equal to about 2.6, greater than or equal to about 2.7, greater than or equal to about 2.8, greater than or equal to about 2.9, greater than or equal to about 3.0, greater than or equal to about 3.1, greater than or equal to about 3.2, greater than or equal to about 3.3, greater than or equal to about 3.4, greater than or equal to about 3.5, greater than or equal to about 3.6, greater than or equal to about 3.7, greater than or equal to about 3.8, greater than or equal to about 3.9, greater than or equal to about 4.0, greater than or equal to about 4.1, greater than or equal to about 4.2, greater than or equal to about 4.3, greater than or equal to about 4.4, greater than or equal to about 4.5, greater than or equal to about 4.6, greater than or equal to about 4.7, greater than or equal to about 4.8, greater than or equal to about 4.9, greater than or equal to about 5.0, greater than or equal to about 5.1, greater than or equal to about 5.2, greater than or equal to about 5.3, greater than or equal to about 5.4, greater than or equal to about 5.5, greater than or equal to about 5.6, greater than or equal to about 5.7, greater than or equal to about 5.8, greater than or equal to about 5.9, greater than or equal to about 6.0, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7.0, or any range or value including and / or in between any two of these values. In some embodiments, the second aqueous buffer solution has a pH of less than or equal to about 7.0, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6.0, less than or equal to about 5.9, less than or equal to about 5.8, less than or equal to about 5.7, less than or equal to about 5.6, less than or equal to about 5.5, less than or equal to about 5.4, less than or equal to about 5.3, less than or equal to about 5.2, less than or equal to about 5.1, less than or equal to about 5.0, less than or equal to about 4.9, less than or equal to about 4.8, less than or equal to about 4.7, less than or equal to about 4.6, less than or equal to about 4.5, less than or equal to about 4.4, less than or equal to about 4.3, less than or equal to about 4.2, less than or equal to about 4.1, less than or equal to about 4.0, less than or equal to about 3.9, less than or equal to about 3.8, less than or equal to about 3.7, less than or equal to about 3.6, less than or equal to about 3.5, less than or equal to about 3.4, less than or equal to about 3.3, less than or equal to about 3.2, less than or equal to about 3.1, less than or equal to about 3.0, less than or equal to about 2.9, less than or equal to about 2.8, less than or equal to about 2.7, less than or equal to about 2.6, less than or equal to about 2.5, or any range or value including and / or in between any two of these values. In some embodiments, the second aqueous buffer solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the second buffering agent is present in the second aqueous buffer solution at about 1 mM to about 200 mM, about 1 mM to about 150 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. In some embodiments, the second aqueous buffer solution is an acetate buffer or phosphate buffer wherein the buffering agent is present at a concentration of about 1 mM to about 100 mM (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM), wherein the second aqueous buffer solution has a pH of about 4 to 8 (e.g., about 4, 4.5, 5, 5.5, 6, 6., 7, 7.5 or 8). For instance, in some embodiments, the second aqueous buffer solution may comprise about 5 mM of an acetate buffering agent and have a pH of about 5. In some embodiments, the second aqueous buffer solution may comprise about 5 mM of a phosphate buffering agent and have a pH of about 8. In some embodiments, the first aqueous buffer solution may comprise about 55 mM of an acetate buffering agent and have a pH of about 4 to 6 (e.g., 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In some embodiments, the processing the eLNP solution comprises filtering, buffer exchanging, lyophilizing, diluting, concentrating, freezing, thawing or storing the eLNP solution, or any combination thereof. In some embodiments, the processing the eLNP solution comprises filtering the eLNP solution by tangential flow filtration, dialysis (e.g., centrifugal dialysis, dialysis cassette, etc.). In some embodiments, the filtering removes an organic solvent (e.g., an alcohol such as ethanol) from the eLNP solution. In some embodiments, the eLNP solution is sterilized before storage or use. In some embodiments, after adding the second aqueous buffer solution, the eLNP solution is processed by tangential flow filtration (e.g., to remove ethanol) into an aqueous buffer solution comprising an acetate, phosphate, or tris buffer, wherein the buffering agent is present at a concentration of about 1 mM to about 100 mM (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM), wherein the second aqueous buffer solution has a pH of about 4 to 8 (e.g., about 4, 4.5, 5, 5.5, 6, 6., 7, 7.5 or 8). For instance, after adding the second aqueous buffer solution, the eLNP solution may be filtered by TFF into an aqueous buffer comprising about 5 mM of an acetate buffering agent having a pH of about 5. In some embodiments, after adding the second aqueous buffer solution, the eLNP solution may be filtered by TFF into an aqueous buffer comprising about 5 mM of an phosphate buffering agent having a pH of about 8. In some embodiments, the processing the eLNP solution comprises buffer exchanging. In some embodiments (e.g., upon removal of the organic solvent), the eLNP solution is buffered at a neutral pH (e.g., 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or any range or value including and / or in between any two of these values). In some embodiments, the buffer exchanging comprises addition of a third aqueous buffer solution comprising a third buffering agent. In some embodiments, the third aqueous buffer solution comprises a third buffering agent to the lipid solution. In some embodiments, the third buffering agent comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer (e.g., ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, potassium citrate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), etc.). In some embodiments, the third buffering agent is present in the third aqueous buffer solution at a concentration of greater than or equal to about 0.001 mM, greater than or equal to about 0.002 mM, greater than or equal to about 0.003 mM, greater than or equal to about 0.004 mM, greater than or equal to about 0.005 mM, greater than or equal to about 0.006 mM, greater than or equal to about 0.007 mM, greater than or equal to about 0.008 mM, greater than or equal to about 0.009 mM, greater than or equal to about 0.01 mM, greater than or equal to about 0.02 mM, greater than or equal to about 0.03 mM, greater than or equal to about 0.04 mM, greater than or equal to about 0.05 mM, greater than or equal to about 0.06 mM, greater than or equal to about 0.07 mM, greater than or equal to about 0.08 mM, greater than or equal to about 0.09 mM, greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, greater than or equal to about 0.6 mM, greater than or equal to about 0.7 mM, greater than or equal to about 0.8 mM, greater than or equal to about 0.9 mM, greater than or equal to about 1.0 mM, greater than or equal to about 2.0 mM, greater than or equal to about 3.0 mM, greater than or equal to about 4.0 mM, greater than or equal to about 5.0 mM, greater than or equal to about 6.0 mM, greater than or equal to about 7.0 mM, greater than or equal to about 8.0 mM, greater than or equal to about 9.0 mM, greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, greater than or equal to about 110 mM, greater than or equal to about 120 mM, greater than or equal to about 130 mM, greater than or equal to about 140 mM, greater than or equal to about 150 mM, greater than or equal to about 160 mM, greater than or equal to about 170 mM, greater than or equal to about 180 mM, greater than or equal to about 190 mM, greater than or equal to about 200 mM, or any range or value including and / or in between any two of these values. In some embodiments, the third buffering agent is present in the third aqueous buffer solution at a concentration of less than or equal to about 200 mM, less than or equal to about 190 mM, less than or equal to about 180 mM, less than or equal to about 170 mM, less than or equal to about 160 mM, less than or equal to about 150 mM, less than or equal to about 140 mM, less than or equal to about 130 mM, less than or equal to about 120 mM, less than or equal to about 110 mM, less than or equal to about 100 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, less than or equal to about 60 mM, less than or equal to about 50 mM, less than or equal to about 40 mM, less than or equal to about 30 mM, less than or equal to about 20 mM, less than or equal to about 10 mM, less than or equal to about 9.0 mM, less than or equal to about 8.0 mM, less than or equal to about 7.0 mM, less than or equal to about 6.0 mM, less than or equal to about 5.0 mM, less than or equal to about 4.0 mM, less than or equal to about 3.0 mM, less than or equal to about 2.0 mM, less than or equal to about 1.0 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, less than or equal to about 0.6 mM, less than or equal to about 0.5 mM, less than or equal to about 0.4 mM, less than or equal to about 0.3 mM, less than or equal to about 0.2 mM, less than or equal to about 0.1 mM, less than or equal to about 0.09 mM, less than or equal to about 0.08 mM, less than or equal to about 0.07 mM, less than or equal to about 0.06 mM, less than or equal to about 0.05 mM, less than or equal to about 0.04 mM, less than or equal to about 0.03 mM, less than or equal to about 0.02 mM, less than or equal to about 0.01 mM, less than or equal to about 0.009 mM, less than or equal to about 0.008 mM, less than or equal to about 0.007 mM, less than or equal to about 0.006 mM, less than or equal to about 0.005 mM, less than or equal to about 0.004 mM, less than or equal to about 0.003 mM, less than or equal to about 0.002 mM, less than or equal to about 0.001 mM, or any range or value including and / or in between any two of these values. In some embodiments, the third aqueous buffer solution has a pH of about 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, or any range or value including and / or in between any two of these values. In some embodiments, the processing the eLNP solution further comprises diluting the eLNP solution. In some embodiments, the processing the eLNP solution comprises dialyzing the eLNP solution. In some embodiments, the processing the eLNP solution comprises concentrating the eLNP solution. In some embodiments, the processing the eLNP solution comprises freezing the eLNP solution. In some embodiments, the processing the eLNP solution comprises lyophilizing the eLNP solution. In some embodiments, the lyophilizing comprises freezing the eLNP solution at a temperature from about -100℃. to about 0℃, about -80℃ to about -10℃, about -60℃ to about - 20℃, about -50℃ to about -25℃, or about -40℃ to about -30℃. In some embodiments, the lyophilizing further comprises drying (e.g., under vacuum of from about 50 mTorr to about 150 mTorr at about 25℃) the frozen eLNP solution to form a lyophilized eLNP. In some embodiments of the methods of the disclosure, the drying is performed at a vacuum ranging from about 50 mTorr to about 150 mTorr. In some embodiments, the processing the eLNP solution comprises storing the eLNP solution (e.g., at a temperature of about -50℃, -45℃, -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, or any range or value including and / or in between any two of these values). Empty Lipid Nanoparticles (eLNPs) In one aspect, the present disclosure relates to a lipid nanoparticle (LNP), comprising: an ionizable amino lipid (IAL); a structural lipid; and a phospholipid, wherein the lipid nanoparticle comprises 5 mol% or less, 3 mol% or less, 1 mol% or less, 0.1 mol% or less, or does not comprise any PEG lipids. In some embodiments, the LNP does not comprise any polymeric lipids. In some embodiments, the LNP consists of: one or more IALs; one or more structural lipids; and one or more phospholipids. When the lipid nanoparticle does not encapsulate a therapeutic agent or prophylactic agent (e.g., a therapeutic polynucleotide, an active pharmaceutical ingredient, etc.), the lipid nanoparticle is referred to as an “empty lipid nanoparticle” (“eLNP”). As used herein, “empty nanoparticle,” “empty lipid nanoparticle,” “empty LNP,” or “eLNP” may refer to a lipid nanoparticle that comprises less than less than 5 wt.%, less than 4 wt.%, less than 3 wt.%, less than 2 wt.%, less than 1 wt.%, less than 0.9 wt.%, less than 0.8 wt.%, less than 0.7 wt.%, less than 0.6 wt.%, less than 0.5 wt.%, less than 0.4 wt.%, less than 0.3 wt.%, less than 0.2 wt.%, or less than 0.1% of a therapeutic or prophylactic agent. As used herein, an “empty nanoparticle,” “empty lipid nanoparticle,” “empty LNP,” or “eLNP” may refer to a nanoparticle that is substantially free of a nucleic acid. As used herein, the term “substantially free of a nucleic acid” means that the nanoparticle contains no significant amount of nucleic acid (e.g., an mRNA). As used herein, an “empty nanoparticle” may refer to a nanoparticle that consists substantially of only lipid components. As used herein, “empty nanoparticle,” “empty lipid nanoparticle,” “empty LNP,” or “eLNP” may refer to a lipid nanoparticle that comprises less than less than 5 wt.%, less than 4 wt.%, less than 3 wt.%, less than 2 wt.%, less than 1 wt.%, less than 0.9 wt.%, less than 0.8 wt.%, less than 0.7 wt.%, less than 0.6 wt.%, less than 0.5 wt.%, less than 0.4 wt.%, less than 0.3 wt.%, less than 0.2 wt.%, or less than 0.1% of a nucleic acid (e.g., an mRNA). As used herein, an “empty nanoparticle,” “empty lipid nanoparticle,” “empty LNP,” or “eLNP” may refer to a nanoparticle that is substantially free of a nucleotide or a polypeptide. As used herein, an “empty nanoparticle,” “empty lipid nanoparticle,” “empty LNP,” or “eLNP” may refer to a nanoparticle that consists substantially of only lipid components. As used herein, “empty nanoparticle,” “empty lipid nanoparticle,” “empty LNP,” or “eLNP” may refer to a nanoparticle that does not comprise any therapeutic agent or prophylactic agent (e.g., a nucleic acid such as an mRNA). Ionizable Amino Lipids (IALs) In some embodiments, the ionizable lipid is a compound of Formula (IL*) or a salt thereof, wherein: R1is -OH, -NRN-C4-10 cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN’’); RNis H or C1-6alkyl; RN’is H or C1-6 alkyl; RN’’is H or C1-6 alkyl; o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8; m is 4, 5, 6, 7, or 8; M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2; M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3; R2aR2b2c2R R is or –(C1-6alkylene)-(C3-8cycloalkyl)-C1-6alkyl; R2ais -H or C1-10 alkyl; R2bis -H or C1-10 alkyl; R2cis C1-8alkyl or C2-8alkenyl; R3cR3isR3a R3b; R3ais H or C1-10 alkyl; R3bis H or C1-8 alkyl; and R3cis C1-10alkyl or C2-8alkenyl. In some embodiments, the ionizable lipid is of Formula (IL**-I):

[0002] or a salt thereof, wherein: R1is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2cis C4-8 alkyl; R3ais C7-10alkyl; and R3cis C3-5alkyl. In some embodiments, the ionizable lipid is of Formula (IL**-III): or a salt thereof, wherein: R1is NRN-C4-10 cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN’’); RNis H; RN’is C1-2 alkyl; RN’’is H; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2ais C7-10 alkyl; R2cis C4-6alkyl; R3ais C1-3alkyl; and R3cis C4-6 alkyl. In some embodiments, the ionizable lipid is of Formula (IL**-IV): or a salt thereof, wherein: R1is OH; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2bis C3-5 alkyl; R2cis C2-4alkyl; R3ais C7-10alkyl; and R3cis C4-6 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-I):

[0003] or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; and R3ais C1-8alkyl. In some embodiments, ionizable lipid is of Formula (IL*-Ia): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-Ia’): or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais C1-8alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-II’): or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-III): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8alkyl; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8alkyl; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIa’): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIb): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IIIb’): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IV): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8alkyl; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-IVa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8alkyl; and R3ais C1-8 alkyl. In some embodiments, the ionizable lipid is of Formula (IL*-Iva’): or a salt thereof, wherein: o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8 alkyl. Variables o, R1, RN, RN’, RN’’of Ionizable Lipid In some embodiments of the ionizable lipid, o is 1. In some embodiments of the ionizable lipid, o is 2. In some embodiments of the ionizable lipid, o is 3. In some embodiments of the ionizable lipid, o is 4. In some embodiments of the ionizable lipid, R1is -OH. In some embodiments of the ionizable lipid, RNis H. In some embodiments of the ionizable lipid, RNis methyl. In some embodiments of the ionizable lipid, RNis ethyl. In some embodiments of the ionizable lipid, R1is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RN’RN’’). In some embodiments of the ionizable lipid, RN’is H. In some embodiments of the ionizable lipid, RN’is methyl. In some embodiments of the ionizable lipid, RN’is ethyl. In some embodiments of the ionizable lipid, RN’’is H. In some embodiments of the ionizable lipid, RN’’is methyl. In some embodiments of the ionizable lipid, RN’’is ethyl. In some embodiments of the ionizable lipid, RN’is H and RN’’is methyl. In some embodiments of the ionizable lipid, R1is In some embodiments of the ionizable lipid, R1is Variables m and n of the Ionizable Lipid In some embodiments of the ionizable lipid, m is 4. In some embodiments of the ionizable lipid, m is 5. In some embodiments of the ionizable lipid, m is 6. In some embodiments of the ionizable lipid, m is 7. In some embodiments of the ionizable lipid, m is 8. In some embodiments of the ionizable lipid, m is 4. In some embodiments of the ionizable lipid, n is 5. In some embodiments of the ionizable lipid, n is 6. In some embodiments of the ionizable lipid, n is 7. In some embodiments of the ionizable lipid, n is 8. In some embodiments of the ionizable lipid, n is 5 and m is 7. In some embodiments of the ionizable lipid, n is 7 and m is 7. In some embodiments of the ionizable lipid, m is 6 and n is 6. Variables M and M’ In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2. In some embodiments of the ionizable lipid, M is -C(=O)-O-* wherein * indicates attachment to R2. In some embodiments of the ionizable lipid, M’ is -O-C(=O)-*, wherein * indicates attachment to R3. In some embodiments of the ionizable lipid, M’ is -C(=O)-O-* wherein * indicates attachment to R3. In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2, and M’ is -C(=O)-O-* wherein * indicates attachment to R3Variables R2, R2a, R2b, R2cIn some embodiments of the ionizable lipid, R2is In some embodiments of the ionizable lipid, R2ais hydrogen. In some embodiments of the ionizable lipid, R2ais methyl. In some embodiments of the ionizable lipid, R2ais ethyl. In some embodiments of the ionizable lipid, R2ais propyl. In some embodiments of the ionizable lipid, R2ais butyl. In some embodiments of the ionizable lipid, R2ais pentyl. In some embodiments of the ionizable lipid, R2ais hexyl. In some embodiments of the ionizable lipid, R2ais heptyl. In some embodiments of the ionizable lipid, R2ais octyl. In some embodiments of the ionizable lipid, R2bis hydrogen. In some embodiments of the ionizable lipid, R2bis methyl. In some embodiments of the ionizable lipid, R2bis ethyl. In some embodiments of the ionizable lipid, R2bis propyl. In some embodiments of the ionizable lipid, R2bis butyl. In some embodiments of the ionizable lipid, R2bis pentyl. In some embodiments of the ionizable lipid, R2bis hexyl. In some embodiments of the ionizable lipid, R2bis heptyl. In some embodiments of the ionizable lipid, R2bis octyl. In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis hydrogen. In some embodiments of the ionizable lipid, R2ais hexyl and R2bis hydrogen. In some embodiments of the ionizable lipid, R2ais octyl and R2bis hydrogen. In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis butyl. In some embodiments of the ionizable lipid, R2cis methyl. In some embodiments of the ionizable lipid, R2cis ethyl. In some embodiments of the ionizable lipid, R2cis propyl. In some embodiments of the ionizable lipid, R2cis butyl. In some embodiments of the ionizable lipid, R2cis pentyl. In some embodiments of the ionizable lipid, R2cis hexyl. In some embodiments of the ionizable lipid, R2cis heptyl. In some embodiments of the ionizable lipid, R2cis octyl. In some embodiments of the ionizable lipid, R2is –(C1-6 alkylene)-(C3-8 cycloalkyl)-C1-6 alkyl. In some embodiments of the ionizable lipid, R2is –(C1-6alkylene)-(cyclohexyl)-C1-6alkyl. In some embodiments of the ionizable lipid, R2is –(C1-6 alkylene)-(cyclopentyl)-C1-6 alkyl. Variables R3, R3a, R3b, and R3cIn some embodiments of the ionizable lipid, R3 is In some embodiments of the ionizable lipid, R3ais hydrogen. In some embodiments of the ionizable lipid, R3ais methyl. In some embodiments of the ionizable lipid, R3ais ethyl. In some embodiments of the ionizable lipid, R3ais propyl. In some embodiments of the ionizable lipid, R3ais butyl. In some embodiments of the ionizable lipid, R3ais pentyl. In some embodiments of the ionizable lipid, R3ais hexyl. In some embodiments of the ionizable lipid, R3ais heptyl. In some embodiments of the ionizable lipid, R3ais octyl. In some embodiments of the ionizable lipid, R3bis hydrogen. In some embodiments of the ionizable lipid, R3bis methyl. In some embodiments of the ionizable lipid, R3bis ethyl. In some embodiments of the ionizable lipid, R3bis propyl. In some embodiments of the ionizable lipid, R3bis butyl. In some embodiments of the ionizable lipid, R3bis pentyl. In some embodiments of the ionizable lipid, R3bis hexyl. In some embodiments of the ionizable lipid, R3bis heptyl. In some embodiments of the ionizable lipid, R3bis octyl. In some embodiments of the ionizable lipid, R3ais octyl and R3bis hydrogen. In some embodiments of the ionizable lipid, R3ais ethyl and R3bis hydrogen. In some embodiments of the ionizable lipid, R3ais hexyl and R3bis hydrogen. In some embodiments of the ionizable lipid, R3cis methyl. In some embodiments of the ionizable lipid, R3cis ethyl. In some embodiments of the ionizable lipid, R3cis propyl. In some embodiments of the ionizable lipid, R3cis butyl. In some embodiments of the ionizable lipid, R3cis pentyl. In some embodiments of the ionizable lipid, R3cis hexyl. In some embodiments of the ionizable lipid, R3cis heptyl. In some embodiments of the ionizable lipid, R3cis octyl. It is understood that, for an ionizable lipid, variables o, R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3ccan each be, where applicable, selected from the groups described herein, and any group described herein for any of variables o,.R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3ccan be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R1, RN, RN’, RN’, m, n, M, M’, R2, R2a, R2b, R2c, R3, R3a, R3b, and R3c. In some embodiments, the ionizable lipid is a compound selected from: In some embodiments, the ionizable lipid is In some embodiments, the ionizable lipid is In some embodiments, the ionizable lipid is In some embodiments, the ionizable lipid is Without being bound by any particular theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge. Phospholipids The eLNPs and LLNPs of the present technology optionally include a phospholipid. Phospholipids, as defined herein, are lipids that comprise a phosphate group. The lipid component of a lipid nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Non-natural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper- catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). In some embodiments, the lipid nanoparticle compositions described herein can comprise about 5 mol% to about 25 mol% of phospholipid, including about 5 mol% to about 15 mol%, or about 8 mol% to about 13 mol%. In some embodiments, the nanoparticle described herein comprises about 5 mol%, about 7.5 mol%, about 10 mol%, about 12.5 mol%, about 15 mol%, about 20 mol%, or about 25 mol%, of phospholipid, or a range between and including any two of the foregoing values. In some embodiments, the lipid nanoparticle composition comprises about 8 mol% to about 13 mol% of phospholipid. In some embodiments, the lipid nanoparticle composition comprises about 10 mol% to about 12 mol% of phospholipid. Suitable phospholipids include: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0 PC), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (4ME 16:0 PG), 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0 PS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. Each possibility represents a separate embodiment. In some embodiments, the phospholipid is selected from the group consisting of DOMG, DMG, DLPE, DMPE, DPPC, DSPE and a combination of any two or more thereof.In some embodiments, the phospholipid is DSPC. In certain embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid includes both DSPC and DOPE. Examples of suitable phospholipids include, but are not limited to, the following:

[0004] In some embodiments, a phospholipid is a compound of Formula (PHL-I): or a salt thereof, wherein: each R41is independently H or optionally substituted alkyl; or optionally two R41are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R41are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m4 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the formula: each instance of L42is independently a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with –O–, –N(RN)–, –S–, –C(O)–, –C(O)N(RN)–, –NRNC(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, – OC(O)N(RN)–, –NRNC(O)O–, or –NRNC(O)N(RN)–; each instance of R42is independently optionally substituted C1-30 alkyl, optionally substituted C1-30alkenyl, or optionally substituted C1-30alkynyl; optionally wherein one or more methylene units of R42are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, –N(RN)–, –O–, –S–, –C(O)–, –C(O)N(RN)–, –NRNC(O)–, –NRNC(O)N(RN)–, – C(O)O–, –OC(O)–, –OC(O)O–, –OC(O)N(RN)–, –NRNC(O)O–, –C(O)S–, –SC(O)–, – C(=NRN)–, –C(=NRN)N(RN)–, –NRNC(=NRN)–, –NRNC(=NRN)N(RN)–, –C(S)–, –C(S)N(RN)–, –NRNC(S)–, –NRNC(S)N(RN)–, –S(O)–, –OS(O)–, –S(O)O–, –OS(O)O–, –OS(O)2–, –S(O)2O–, –OS(O)2O–, –N(RN)S(O)–, –S(O)N(RN)–, –N(RN)S(O)N(RN)–, –OS(O)N(RN)–, –N(RN)S(O)O–, –S(O)2–, –N(RN)S(O)2–, –S(O)2N(RN)–, –N(RN)S(O)2N(RN)–, –OS(O)2N(RN)–, or – N(RN)S(O)2O–; each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p4 is 1 or 2; provided that the compound is not of the formula: wherein each instance of R42is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl. In some embodiments, a suitable phospholipid is an analog or variant of DSPC such as a compound of Formula (PHL-I): or a salt thereof, wherein: each R41is independently optionally substituted alkyl; or optionally two R41are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R41are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n4 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m4 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the formula: each instance of L42is independently a bond or optionally substituted C1-6 alkylene, wherein one methylene unit of the optionally substituted C1-6 alkylene is optionally replaced with –O–, –N(RN)–, –S–, –C(O)–, –C(O)N(RN)–, –NRNC(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, – OC(O)N(RN)–, –NRNC(O)O–, or –NRNC(O)N(RN)–; each instance of R42is independently optionally substituted C1-30 alkyl, optionally substituted C1-30alkenyl, or optionally substituted C1-30alkynyl; optionally wherein one or more methylene units of R42are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, –N(RN)–, –O–, –S–, –C(O)–, –C(O)N(RN)–, –NRNC(O)–, –NRNC(O)N(RN)–, – C(O)O–, –OC(O)–, –OC(O)O–, –OC(O)N(RN)–, –NRNC(O)O–, –C(O)S–, –SC(O)–, – C(=NRN)–, –C(=NRN)N(RN)–, –NRNC(=NRN)–, –NRNC(=NRN)N(RN)–, –C(S)–, –C(S)N(RN)–, –NRNC(S)–, –NRNC(S)N(RN)–, –S(O)–, –OS(O)–, –S(O)O–, –OS(O)O–, –OS(O)2–, –S(O)2O–, –OS(O)2O–, –N(RN)S(O)–, –S(O)N(RN)–, –N(RN)S(O)N(RN)–, –OS(O)N(RN)–, –N(RN)S(O)O–, –S(O)2–, –N(RN)S(O)2–, –S(O)2N(RN)–, –N(RN)S(O)2N(RN)–, –OS(O)2N(RN)–, or – N(RN)S(O)2O–; each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p4 is 1 or 2. provided that the compound is not of the formula: wherein each instance of R42is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl. In some embodiments, the compound is not of the formula: wherein each instance of R42is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl. In certain embodiments, a suitable phospholipid comprises a modified phospholipid head (e.g., a modified choline group). In certain embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine. For example, in embodiments of Formula (PHL-I), at least one of R41is not methyl. In certain embodiments, at least one of R41is not hydrogen or methyl. In certain embodiments, the compound of Formula (PHL-I) is of one of the following formulae:

[0005] or a salt thereof, wherein: each t4 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each u4 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and each v4 is independently 1, 2, or 3. In certain embodiments, the compound of Formula (PHL-I) is of one of the following formulae: or a salt thereof. In some embodiments, a compound of Formula (PHL-I) is one of the following:

[0006] or a salt thereof. In certain embodiments, a compound of Formula (PHL-I) is of Formula (PHL-I-a): or a salt thereof. In certain embodiments, suitable phospholipids comprise a modified core. In certain embodiments, a phospholipid with a modified core described herein is DSPC, or analog thereof, with a modified core structure. For example, in certain embodiments of Formula (PHL-I-a), group A is not of the following formula: In certain embodiments, the compound of Formula (PHL-I-b-4) is one of the following formulae: or a salt thereof. In certain embodiments, a suitable phospholipid comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n4 is not 2). Therefore, in certain embodiments, a phospholipid is a compound of Formula (PHL-I), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, a compound of Formula (PHL-I) is of one of the following formulae: or a salt thereof. In certain embodiments, a compound of Formula (PHL-I) is one of the following:

[0007] or a salt thereof. Structural Lipids The eLNPs and LLNPs of the present technology may include one or more structural lipids. As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol. Examples of structural lipids include, but are not limited to, the following: In some embodiments, the structural lipid is selected from the group consisting of a sterol, a tocopherol, and mixtures of two or more thereof. In some embodiments, the structural lipid is selected from the group consisting of cholesterol and alpha-tocopherol. In some embodiments, the lipid nanoparticle compositions described herein can comprise about 20 mol% to about 60 mol% structural lipid, including about 30 mol% to about 50 mol%. In some embodiments, the lipid nanoparticle compositions comprise about 15 mol% to about 45 mol% of structural lipid. In some embodiments, the lipid nanoparticle compositions comprise about 35 mol% to about 45 mol% of structural lipid. In some embodiments, the lipid nanoparticle compositions comprise about 37 mol% to about 42 mol% of structural lipid. In some embodiments, the lipid nanoparticle compositions comprise about 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol%, of structural lipid, or a range between and including any two of the foregoing values. In some embodiments, the lipid nanoparticle compositions comprise about 35, about 36, about 37, about 38, about 39, or about 40 mol% of structural lipid. In some embodiments, the nanoparticle comprises about 39 to about 40 mol% structural lipid. In some embodiments, the structural lipid is cholesterol or a compound having the following structure: Polymeric Lipids The LLNPs and eLNPs of the present technology optionally include one or more polymeric lipids. As used herein, a “polymeric lipid” refers to a lipid comprising repeating subunits in its chemical structure. In some embodiments, the polymeric lipid is a lipid comprising a polymer component. In some embodiments, the polymeric lipid is a PEG lipid. In some embodiments, the polymeric lipid is not a PEG lipid. PEG Lipids The LLNPs and eLNPs of the present technology optionally include a PEG lipid (e.g., a PEG-lipid described herein). PEG-lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG-lipid is a lipid modified with polyethylene glycol. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.15 mol% to about 15 mol% of PEG-lipid, including about 0.15 mol% to about 10 mol%, about 0.15 mol% to about 5 mol%, about 0.15 mol% to about 3 mol%, or about 1 mol% to about 3 mol%. In some embodiments, the nanoparticle described herein comprises about 0.15 mol%, about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, about 10 mol%, or about 15 mol% of PEG-lipid, or a range between and including any two of the foregoing values. In some embodiments, the lipid nanoparticle compositions described herein comprise about 0.15 mol% to about 5 mol% of PEG-lipid. In some embodiments, the lipid nanoparticle compositions described herein comprise about 1 mol% to about 3 mol% of PEG-lipid. A PEG-lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG-lipid may be PEG-c-DOMG, PEG-DMG (e.g., PEG- DMG 2000 or DMG-PEG 2000), PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid is PEG-DMG (DMG-PEG or 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol). In some embodiments, the PEG-lipid is PEG-DMG 2000 (or DMG-PEG 2000), where the 2000 represents an average molecular weight. Representative PEG-DMG structures are below. In some embodiments, PEG-lipids can be PEGylated lipids such as described in International Publication Nos. WO 2012 / 099755 and WO 2017 / 099823, the contents of each of which is herein incorporated by reference in its entirety. Any of these exemplary PEG-lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG-lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy- PEGylated lipid comprises an –OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment. In certain embodiments, a PEG-lipid is a PEGylated fatty acid. In certain embodiments, a PEG-lipid is a compound of Formula (PGL-I). Provided herein are compounds of Formula (PGL-I): or a salts thereof, wherein: R33is–ORO; ROis hydrogen, optionally substituted alkyl or an oxygen protecting group; rr is an integer between 1 and 100, inclusive; R35is optionally substituted C10-40alkyl, optionally substituted C10-40alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R35are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, –N(RN)–, –O–, –S–, –C(O)–, –C(O)N(RN)–, –NRNC(O)–, –NRNC(O)N(RN)–, –C(O)O–, –OC(O)–, –OC(O)O–, –OC(O)N(RN)–, –NRNC(O)O–, –C(O)S–, –SC(O)–, –C(=NRN)–, –C(=NRN)N(RN)–, –NRNC(=NRN)–, –NRNC(=NRN)N(RN)–, –C(S)–, –C(S)N(RN)–, –NRNC(S)–, –NRNC(S)N(RN)–, –S(O)–, –OS(O)–, –S(O)O–, –OS(O)O–, –OS(O)2–, –S(O)2O–, –OS(O)2O–, –N(RN)S(O)–, –S(O)N(RN)–, –N(RN)S(O)N(RN)–, –OS(O)N(RN)–, –N(RN)S(O)O–, –S(O)2–, –N(RN)S(O)2–, –S(O)2N(RN)–, –N(RN)S(O)2N(RN)–, –OS(O)2N(RN)–, or –N(RN)S(O)2O–; and each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound of Formula (PGL-I) is of Formula (PGL-I-OH): or a salt thereof. In certain embodiments, a compound of Formula (PGL-I) is of one of the following formulae: or a salt thereof. In some embodiments, rr is 43, 44, 45, or 46. In some embodiments, rr is 45. In yet other embodiments the compound of Formula (PGL-I) has the formula: In some embodiments, the compound of Formula (PGL-I) is In certain embodiments, certain embodiments, the PEG-lipid is one of the following formula: or a salt thereof. In some embodiments, rr is 45. Suitable additional PEG-lipids are described in WO 2017 / 099823 which is herein incorporated by reference in its entirety. In some embodiments, the eLNP does not comprise a PEG lipid (i.e., is free of PEG lipids). In some embodiments, the eLNP comprises PEG lipids at a concentration, relative to the total moles of lipids present in the eLNP, of less than or equal to about 0.5 mol%, less than or equal to about 0.45 mol%, less than or equal to about 0.4 mol%, less than or equal to about 0.35 mol%, less than or equal to about 0.3 mol%, less than or equal to about 0.25 mol%, less than or equal to about 0.2 mol%, less than or equal to about 0.15 mol%, less than or equal to about 0.1 mol%, less than or equal to about 0.09 mol%, less than or equal to about 0.08 mol%, less than or equal to about 0.07 mol%, less than or equal to about 0.06 mol%, less than or equal to about 0.05 mol%, less than or equal to about 0.04 mol%, less than or equal to about 0.03 mol%, less than or equal to about 0.02 mol%, less than or equal to about 0.01 mol%, or about 0 mol%, or any range or value including and / or in between any two of these values. In some embodiments, the eLNP does not comprise a PEG lipid. In some embodiments, the eLNP comprises PEG lipids at a concentration, relative to the total weight of the eLNP, of less than or equal to about 5.0 wt.%, less than or equal to about 4.5 wt.%, less than or equal to about 4.0 wt.%, less than or equal to about 3.5 wt.%, less than or equal to about 3.0 wt.%, less than or equal to about 2.5 wt.%, less than or equal to about 2.0 wt.%, less than or equal to about 1.5 wt.%, less than or equal to about 1.0 wt.%, less than or equal to about 0.95 wt.%, less than or equal to about 0.9 wt.%, less than or equal to about 0.85 wt.%, less than or equal to about 0.8 wt.%, less than or equal to about 0.75 wt.%, less than or equal to about 0.7 wt.%, less than or equal to about 0.65 wt.%, less than or equal to about 0.6 wt.%, less than or equal to about 0.55 wt.%, less than or equal to about 0.5 wt.%, less than or equal to about 0.45 wt.%, less than or equal to about 0.4 wt.%, less than or equal to about 0.35 wt.%, less than or equal to about 0.3 wt.%, less than or equal to about 0.25 wt.%, less than or equal to about 0.2 wt.%, less than or equal to about 0.15 wt.%, less than or equal to about 0.1 wt.%, less than or equal to about 0.09 wt.%, less than or equal to about 0.08 wt.%, less than or equal to about 0.07 wt.%, less than or equal to about 0.06 wt.%, less than or equal to about 0.05 wt.%, less than or equal to about 0.04 wt.%, less than or equal to about 0.03 wt.%, less than or equal to about 0.02 wt.%, less than or equal to about 0.01 wt.%, or any range or value including and / or in between any two of these values. eLNP Compositions and Features In some embodiments, the eLNP comprises 25-75 mol% of the IAL, 0-60 mol% of the structural lipid, and 0-30 mol% of the phospholipid. In some embodiments, the eLNP comprises 30-60 mol% of the IAL, 15-50 mol% of the structural lipid, and 0-30 mol% of the phospholipid. In some embodiments, the lipid nanoparticle comprises 45-50 mol.% of the IAL, 8-12 mol.% of the phospholipid, and 35-40 mol.% of the structural lipid. In some embodiments, the eLNP has an average diameter of greater than or equal to about 10 nm, greater than or equal to about 15 nm, greater than or equal to about 20 nm, greater than or equal to about 25 nm, greater than or equal to about 30 nm, greater than or equal to about 35 nm, greater than or equal to about 40 nm, greater than or equal to about 45 nm, greater than or equal to about 50 nm, greater than or equal to about 60 nm, greater than or equal to about 70 nm, greater than or equal to about 80 nm, greater than or equal to about 90 nm, greater than or equal to about 100 nm, greater than or equal to about 110 nm, greater than or equal to about 120 nm, greater than or equal to about 130 nm, greater than or equal to about 140 nm, greater than or equal to about 150 nm, greater than or equal to about 160 nm, greater than or equal to about 170 nm, greater than or equal to about 180 nm, greater than or equal to about 190 nm, greater than or equal to about 200 nm, or any range or value including and / or in between any two of these values. In some embodiments, the eLNP has an average diameter of less than or equal to about 200 nm, less than or equal to about 190 nm, less than or equal to about 180 nm, less than or equal to about 170 nm, less than or equal to about 160 nm, less than or equal to about 150 nm, less than or equal to about 140 nm, less than or equal to about 130 nm, less than or equal to about 120 nm, less than or equal to about 110 nm, less than or equal to about 100 nm, less than or equal to about 90 nm, less than or equal to about 80 nm, less than or equal to about 70 nm, less than or equal to about 60 nm, less than or equal to about 50 nm, less than or equal to about 45 nm, less than or equal to about 40 nm, less than or equal to about 35 nm, less than or equal to about 30 nm, less than or equal to about 25 nm, less than or equal to about 20 nm, less than or equal to about 15 nm, less than or equal to about 10 nm, or any range or value including and / or in between any two of these values. In some embodiments, the eLNP has an average diameter of about 10 nm to about 500 nm. In some embodiments, the eLNP has an average diameter of about 10 nm to about 200 nm (e.g., 30-150 nm, 50-130 nm, 80-120 nm, or any range or value therein between). Methods of Producing Loaded Lipid Nanoparticles (LLNPs) Referring now to FIG.1B, in an aspect, the present disclosure relates to a method of producing a loaded lipid nanoparticle (LLNP), the method comprising: mixing a lipid solution comprising an ionizable amino lipid (IAL), a phospholipid, and a structural lipid with a first aqueous buffer solution comprising a first buffering agent, to obtain an eLNP solution comprising the eLNP, wherein the eLNP comprises 5 mol% or less, 3 mol% or less, 1 mol% or less, 0.1 mol% or less, or does not comprise a polymeric lipid (e.g., a PEG lipid); optionally processing the eLNP solution; and loading the eLNP with a nucleic acid by mixing a nucleic acid solution comprising the nucleic acid with the eLNP solution to obtain a loaded LNP composition comprising the LLNP. In some embodiments, the nucleic acid comprises a ribonucleic acid. In some embodiments, the ribonucleic acid is at least one selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (lncRNA). In some embodiments, the nucleic acid is a messenger RNA (mRNA). In some embodiments, the mRNA has a number of nucleotides greater than or equal to about 10, greater than or equal to about 20, greater than or equal to about 30, greater than or equal to about 40, greater than or equal to about 50, greater than or equal to about 60, greater than or equal to about 70, greater than or equal to about 80, greater than or equal to about 90, greater than or equal to about 100, greater than or equal to about 150, greater than or equal to about 200, greater than or equal to about 250, greater than or equal to about 300, greater than or equal to about 350, greater than or equal to about 400, greater than or equal to about 450, greater than or equal to about 500, greater than or equal to about 550, greater than or equal to about 600, greater than or equal to about 650, greater than or equal to about 700, greater than or equal to about 750, greater than or equal to about 800, greater than or equal to about 850, greater than or equal to about 900, greater than or equal to about 950, greater than or equal to about 1000, greater than or equal to about 1500, greater than or equal to about 2000, greater than or equal to about 2500, greater than or equal to about 3000, greater than or equal to about 3500, greater than or equal to about 4000, greater than or equal to about 4500, greater than or equal to about 5000, greater than or equal to about 5500, greater than or equal to about 6000, greater than or equal to about 6500, greater than or equal to about 7000, greater than or equal to about 7500, greater than or equal to about 8000, greater than or equal to about 8500, greater than or equal to about 9000, greater than or equal to about 9500, greater than or equal to about 10000, or any range or value including and / or in between any two of these values. In some embodiments, the nucleic acid is present in the nucleic acid solution at a concentration of greater than or equal to about 0.01 mg / mL, greater than or equal to about 0.02 mg / mL, greater than or equal to about 0.03 mg / mL, greater than or equal to about 0.04 mg / mL, greater than or equal to about 0.05 mg / mL, greater than or equal to about 0.06 mg / mL, greater than or equal to about 0.07 mg / mL, greater than or equal to about 0.08 mg / mL, greater than or equal to about 0.09 mg / mL, greater than or equal to about 0.1 mg / mL, greater than or equal to about 0.2 mg / mL, greater than or equal to about 0.3 mg / mL, greater than or equal to about 0.4 mg / mL, greater than or equal to about 0.5 mg / mL, greater than or equal to about 0.6 mg / mL, greater than or equal to about 0.7 mg / mL, greater than or equal to about 0.8 mg / mL, greater than or equal to about 0.9 mg / mL, greater than or equal to about 1.0 mg / mL, greater than or equal to about 1.5 mg / mL, greater than or equal to about 2.0 mg / mL, greater than or equal to about 2.5 mg / mL, greater than or equal to about 3.0 mg / mL, greater than or equal to about 3.5 mg / mL, greater than or equal to about 4.0 mg / mL, greater than or equal to about 4.5 mg / mL, greater than or equal to about 5.0 mg / mL, greater than or equal to about 5.5 mg / mL, greater than or equal to about 6.0 mg / mL, greater than or equal to about 6.5 mg / mL, greater than or equal to about 7.0 mg / mL, greater than or equal to about 7.5 mg / mL, greater than or equal to about 8.0 mg / mL, greater than or equal to about 8.5 mg / mL, greater than or equal to about 9.0 mg / mL, greater than or equal to about 9.5 mg / mL, greater than or equal to about 10.0 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the nucleic acid is present in the nucleic acid solution at a concentration of less than or equal to about 10.0 mg / mL, less than or equal to about 9.5 mg / mL, less than or equal to about 9.0 mg / mL, less than or equal to about 8.5 mg / mL, less than or equal to about 8.0 mg / mL, less than or equal to about 7.5 mg / mL, less than or equal to about 7.0 mg / mL, less than or equal to about 6.5 mg / mL, less than or equal to about 6.0 mg / mL, less than or equal to about 5.5 mg / mL, less than or equal to about 5.0 mg / mL, less than or equal to about 4.5 mg / mL, less than or equal to about 4.0 mg / mL, less than or equal to about 3.5 mg / mL, less than or equal to about 3.0 mg / mL, less than or equal to about 2.5 mg / mL, less than or equal to about 2.0 mg / mL, less than or equal to about 1.5 mg / mL, less than or equal to about 1.0 mg / mL, less than or equal to about 0.9 mg / mL, less than or equal to about 0.8 mg / mL, less than or equal to about 0.7 mg / mL, less than or equal to about 0.6 mg / mL, less than or equal to about 0.5 mg / mL, less than or equal to about 0.4 mg / mL, less than or equal to about 0.3 mg / mL, less than or equal to about 0.2 mg / mL, less than or equal to about 0.1 mg / mL, less than or equal to about 0.09 mg / mL, less than or equal to about 0.08 mg / mL, less than or equal to about 0.07 mg / mL, less than or equal to about 0.06 mg / mL, less than or equal to about 0.05 mg / mL, less than or equal to about 0.04 mg / mL, less than or equal to about 0.03 mg / mL, less than or equal to about 0.02 mg / mL, less than or equal to about 0.01 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the nucleic acid solution comprises about 0.01 to about 10.0 mg / mL of the nucleic acid, about 0.05 to about 5.0 mg / mL of the nucleic acid, about 0.1 to about 2.5 mg / mL of the nucleic acid, or about 1.0 mg / mL to about 2.0 mg / mL of the nucleic acid. In some embodiments, the nucleic acid solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer (e.g., ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, potassium citrate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), etc.). In some embodiments, the nucleic acid solution comprises a buffer selected from the group consisting of an acetate buffer, a citrate buffer, a phosphate buffer, and a tris buffer. In some embodiments, the nucleic acid solution comprises a buffering agent at a concentration of greater than or equal to about 0.001 mM, greater than or equal to about 0.002 mM, greater than or equal to about 0.003 mM, greater than or equal to about 0.004 mM, greater than or equal to about 0.005 mM, greater than or equal to about 0.006 mM, greater than or equal to about 0.007 mM, greater than or equal to about 0.008 mM, greater than or equal to about 0.009 mM, greater than or equal to about 0.01 mM, greater than or equal to about 0.02 mM, greater than or equal to about 0.03 mM, greater than or equal to about 0.04 mM, greater than or equal to about 0.05 mM, greater than or equal to about 0.06 mM, greater than or equal to about 0.07 mM, greater than or equal to about 0.08 mM, greater than or equal to about 0.09 mM, greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, greater than or equal to about 0.6 mM, greater than or equal to about 0.7 mM, greater than or equal to about 0.8 mM, greater than or equal to about 0.9 mM, greater than or equal to about 1.0 mM, greater than or equal to about 2.0 mM, greater than or equal to about 3.0 mM, greater than or equal to about 4.0 mM, greater than or equal to about 5.0 mM, greater than or equal to about 6.0 mM, greater than or equal to about 7.0 mM, greater than or equal to about 8.0 mM, greater than or equal to about 9.0 mM, greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, greater than or equal to about 110 mM, greater than or equal to about 120 mM, greater than or equal to about 130 mM, greater than or equal to about 140 mM, greater than or equal to about 150 mM, greater than or equal to about 160 mM, greater than or equal to about 170 mM, greater than or equal to about 180 mM, greater than or equal to about 190 mM, greater than or equal to about 200 mM, or any range or value including and / or in between any two of these values. In some embodiments, the nucleic acid solution comprises a buffering agent at a concentration of less than or equal to about 200 mM, less than or equal to about 190 mM, less than or equal to about 180 mM, less than or equal to about 170 mM, less than or equal to about 160 mM, less than or equal to about 150 mM, less than or equal to about 140 mM, less than or equal to about 130 mM, less than or equal to about 120 mM, less than or equal to about 110 mM, less than or equal to about 100 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, less than or equal to about 60 mM, less than or equal to about 50 mM, less than or equal to about 40 mM, less than or equal to about 30 mM, less than or equal to about 20 mM, less than or equal to about 10 mM, less than or equal to about 9.0 mM, less than or equal to about 8.0 mM, less than or equal to about 7.0 mM, less than or equal to about 6.0 mM, less than or equal to about 5.0 mM, less than or equal to about 4.0 mM, less than or equal to about 3.0 mM, less than or equal to about 2.0 mM, less than or equal to about 1.0 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, less than or equal to about 0.6 mM, less than or equal to about 0.5 mM, less than or equal to about 0.4 mM, less than or equal to about 0.3 mM, less than or equal to about 0.2 mM, less than or equal to about 0.1 mM, less than or equal to about 0.09 mM, less than or equal to about 0.08 mM, less than or equal to about 0.07 mM, less than or equal to about 0.06 mM, less than or equal to about 0.05 mM, less than or equal to about 0.04 mM, less than or equal to about 0.03 mM, less than or equal to about 0.02 mM, less than or equal to about 0.01 mM, less than or equal to about 0.009 mM, less than or equal to about 0.008 mM, less than or equal to about 0.007 mM, less than or equal to about 0.006 mM, less than or equal to about 0.005 mM, less than or equal to about 0.004 mM, less than or equal to about 0.003 mM, less than or equal to about 0.002 mM, less than or equal to about 0.001 mM, or any range or value including and / or in between any two of these values. In some embodiments, the nucleic acid solution has pH of greater than or equal to about 2.5, greater than or equal to about 2.6, greater than or equal to about 2.7, greater than or equal to about 2.8, greater than or equal to about 2.9, greater than or equal to about 3.0, greater than or equal to about 3.1, greater than or equal to about 3.2, greater than or equal to about 3.3, greater than or equal to about 3.4, greater than or equal to about 3.5, greater than or equal to about 3.6, greater than or equal to about 3.7, greater than or equal to about 3.8, greater than or equal to about 3.9, greater than or equal to about 4.0, greater than or equal to about 4.1, greater than or equal to about 4.2, greater than or equal to about 4.3, greater than or equal to about 4.4, greater than or equal to about 4.5, greater than or equal to about 4.6, greater than or equal to about 4.7, greater than or equal to about 4.8, greater than or equal to about 4.9, greater than or equal to about 5.0, greater than or equal to about 5.1, greater than or equal to about 5.2, greater than or equal to about 5.3, greater than or equal to about 5.4, greater than or equal to about 5.5, greater than or equal to about 5.6, greater than or equal to about 5.7, greater than or equal to about 5.8, greater than or equal to about 5.9, greater than or equal to about 6.0, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7.0, greater than or equal to about 7.1, greater than or equal to about 7.2, greater than or equal to about 7.3, greater than or equal to about 7.4, greater than or equal to about 7.5, or any range or value including and / or in between any two of these values. In some embodiments, the nucleic acid solution has a pH of less than or equal to about 7.5, less than or equal to about 7.4, less than or equal to about 7.3, less than or equal to about 7.2, less than or equal to about 7.1, less than or equal to about 7.0, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6.0, less than or equal to about 5.9, less than or equal to about 5.8, less than or equal to about 5.7, less than or equal to about 5.6, less than or equal to about 5.5, less than or equal to about 5.4, less than or equal to about 5.3, less than or equal to about 5.2, less than or equal to about 5.1, less than or equal to about 5.0, less than or equal to about 4.9, less than or equal to about 4.8, less than or equal to about 4.7, less than or equal to about 4.6, less than or equal to about 4.5, less than or equal to about 4.4, less than or equal to about 4.3, less than or equal to about 4.2, less than or equal to about 4.1, less than or equal to about 4.0, less than or equal to about 3.9, less than or equal to about 3.8, less than or equal to about 3.7, less than or equal to about 3.6, less than or equal to about 3.5, less than or equal to about 3.4, less than or equal to about 3.3, less than or equal to about 3.2, less than or equal to about 3.1, less than or equal to about 3.0, less than or equal to about 2.9, less than or equal to about 2.8, less than or equal to about 2.7, less than or equal to about 2.6, less than or equal to about 2.5, or any range or value including and / or in between any two of these values. In some embodiments, the nucleic acid solution comprises from about 1 mM to about 200 mM acetate buffer, from about 2 mM to about 180 mM acetate buffer, from about 3 mM to about 160 mM acetate buffer, from about 4 mM to about 150 mM acetate buffer, from about 4 mM to about 140 mM acetate buffer, from about 5 mM to about 130 mM acetate buffer, from about 6 mM to about 120 mM acetate buffer, from about 7 mM to about 110 mM acetate buffer, from about 8 mM to about 100 mM acetate buffer, from about 9 mM to about 90 mM acetate buffer, from about 10 mM to about 80 mM acetate buffer, from about 15 mM to about 70 mM acetate buffer, from about 20 mM to about 60 mM acetate buffer, from about 25 mM to about 50 mM acetate buffer, or from about 30 mM to about 40 mM acetate buffer. In some embodiments, the nucleic acid solution comprises an acetate buffering agent, phosphate buffering agent, or tris buffering agent at about 20 mM to about 50 mM (e.g., 20 mM, 22.5 mM, 25 mM, 27.5 mM, 30 mM, 32.5 mM, 35 mM, 37.5 mM, 40 mM, 42.5 mM, 45 mM, 47.5 mM, or 50 mM) and has a pH of about 4 to about 8 (e.g., 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8). In some embodiments, the loading step performed using a T-junction, confined impinging jets, microfluidic mixer, or vortex mixer. In some embodiments, the mixing for the loading step is performed using an apparatus disclosed in WIPO Publication No. WO 2023 / 193002A1 or WIPO Publication No.2020 / 160430, the entireties of which are hereby incorporated by reference herein. In some embodiments, the loading comprises mixing the nucleic acid solution and the eLNP solution at a volumetric flow ratio greater than or equal to about 1:1, greater than or equal to about 2:1, greater than or equal to about 3:1, greater than or equal to about 4:1, greater than or equal to about 5:1, greater than or equal to about 6:1, greater than or equal to about 7:1, greater than or equal to about 8:1, greater than or equal to about 9:1, greater than or equal to about 10:1, greater than or equal to about 12:1, greater than or equal to about 15:1, greater than or equal to about 20:1, or any or any range or value including and / or in between any two of these values. In some embodiments, the loading comprises mixing the nucleic acid solution and the eLNP solution at a volumetric flow ratio of less than or equal to about 20:1, less than or equal to about 15:1, less than or equal to about 12:1, less than or equal to about 10:1, less than or equal to about 9:1, less than or equal to about 8:1, less than or equal to about 7:1, less than or equal to about 6:1, less than or equal to about 5:1, less than or equal to about 4:1, less than or equal to about 3:1, less than or equal to about 2:1, less than or equal to about 1:1, or any or any range or value including and / or in between any two of these values. In some embodiments, the loading is performed at a temperature of less than or equal to about 55 ℃, less than or equal to about 50 ℃, less than or equal to about 45 ℃, less than or equal to about 40 ℃, less than or equal to about 35 ℃, less than or equal to about 30 ℃, less than or equal to about 25 ℃, less than or equal to about 20 ℃, less than or equal to about 15 ℃, less than or equal to about 10 ℃, less than or equal to about 9 ℃, less than or equal to about 8 ℃, less than or equal to about 7 ℃, less than or equal to about 6 ℃, less than or equal to about 5 ℃, less than or equal to about 4 ℃, less than or equal to about 3 ℃, less than or equal to about 2 ℃, less than or equal to about 1 ℃, or any range or value including and / or in between any two of these values. In some embodiments, the eLNP solution or the eLNP are subjected to the loading after storage or holding. In some embodiments, the eLNP solution or the eLNP are subjected to the loading without storage or holding. In some embodiments, the eLNP solution or the eLNP are subjected to the loading after holding for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, or about 24 hours. In some embodiments, the eLNP solution or the eLNP are subjected to the loading after storage for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years. In some embodiments, the storage is performed at a temperature of about -70℃, -65℃, - 60℃, -55℃, -50℃, -45℃, -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, or any range or value including and / or in between any two of these values. In some embodiments, the eLNP solution or the eLNP are subjected to holding or storing for about 2 days at about 5℃, for about 3 days at about 5℃, for about 4 days at about 5℃, for about 5 days at about 5℃, for about 6 days at about 5℃, for about 7 days at about 5℃, for about 8 days at about 5℃, for about 9 days at about 5℃, for about 10 days at about 5℃, for about 1 month at -70℃, about 2 months at about -70℃, about 3 months at about -70℃, about 4 months at about -70℃, about 5 months at about -70℃, about 6 months at about -70℃, about 7 months at about -70℃, about 8 months at about -70℃, about 9 months at about -70℃, about 10 months at about -70℃, about 11 months at about -70℃, about 12 months at -70℃. In some embodiments, the eLNP solution is stored in a container suitable for freezing, shipment, and ambient storage. In some embodiments, the container is a PTFE bag. In some embodiments, the LLNP has an average diameter of greater than or equal to about 10 nm, greater than or equal to about 15 nm, greater than or equal to about 20 nm, greater than or equal to about 25 nm, greater than or equal to about 30 nm, greater than or equal to about 35 nm, greater than or equal to about 40 nm, greater than or equal to about 45 nm, greater than or equal to about 50 nm, greater than or equal to about 60 nm, greater than or equal to about 70 nm, greater than or equal to about 80 nm, greater than or equal to about 90 nm, greater than or equal to about 100 nm, greater than or equal to about 110 nm, greater than or equal to about 120 nm, greater than or equal to about 130 nm, greater than or equal to about 140 nm, greater than or equal to about 150 nm, greater than or equal to about 160 nm, greater than or equal to about 170 nm, greater than or equal to about 180 nm, greater than or equal to about 190 nm, greater than or equal to about 200 nm, greater than or equal to about 210 nm, greater than or equal to about 220 nm, greater than or equal to about 230 nm, greater than or equal to about 240 nm, greater than or equal to about 250 nm, or any range or value including and / or in between any two of these values. In some embodiments, the loaded LNP has an average diameter of less than or equal to about 250 nm, less than or equal to about 240 nm, less than or equal to about 230 nm, less than or equal to about 220 nm, less than or equal to about 210 nm, less than or equal to about 200 nm, less than or equal to about 190 nm, less than or equal to about 180 nm, less than or equal to about 170 nm, less than or equal to about 160 nm, less than or equal to about 150 nm, less than or equal to about 140 nm, less than or equal to about 130 nm, less than or equal to about 120 nm, less than or equal to about 110 nm, less than or equal to about 100 nm, less than or equal to about 90 nm, less than or equal to about 80 nm, less than or equal to about 70 nm, less than or equal to about 60 nm, less than or equal to about 50 nm, less than or equal to about 45 nm, less than or equal to about 40 nm, less than or equal to about 35 nm, less than or equal to about 30 nm, less than or equal to about 25 nm, less than or equal to about 20 nm, less than or equal to about 15 nm, less than or equal to about 10 nm, or any range or value including and / or in between any two of these values. In some embodiments, the LLNP has an average diameter of about 10 nm to about 500 nm. In some embodiments, the LLNP has an average diameter of about 10 nm to about 200 nm (e.g., 30-150 nm, 50-130 nm, 80-120 nm, or any range or value therein between). In some embodiments, the LLNP solution has pH of greater than or equal to about 2.5, greater than or equal to about 2.6, greater than or equal to about 2.7, greater than or equal to about 2.8, greater than or equal to about 2.9, greater than or equal to about 3.0, greater than or equal to about 3.1, greater than or equal to about 3.2, greater than or equal to about 3.3, greater than or equal to about 3.4, greater than or equal to about 3.5, greater than or equal to about 3.6, greater than or equal to about 3.7, greater than or equal to about 3.8, greater than or equal to about 3.9, greater than or equal to about 4.0, greater than or equal to about 4.1, greater than or equal to about 4.2, greater than or equal to about 4.3, greater than or equal to about 4.4, greater than or equal to about 4.5, greater than or equal to about 4.6, greater than or equal to about 4.7, greater than or equal to about 4.8, greater than or equal to about 4.9, greater than or equal to about 5.0, greater than or equal to about 5.1, greater than or equal to about 5.2, greater than or equal to about 5.3, greater than or equal to about 5.4, greater than or equal to about 5.5, greater than or equal to about 5.6, greater than or equal to about 5.7, greater than or equal to about 5.8, greater than or equal to about 5.9, greater than or equal to about 6.0, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7.0, or any range or value including and / or in between any two of these values. In some embodiments, the LLNP solution has a pH of less than or equal to about 7.0, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6.0, less than or equal to about 5.9, less than or equal to about 5.8, less than or equal to about 5.7, less than or equal to about 5.6, less than or equal to about 5.5, less than or equal to about 5.4, less than or equal to about 5.3, less than or equal to about 5.2, less than or equal to about 5.1, less than or equal to about 5.0, less than or equal to about 4.9, less than or equal to about 4.8, less than or equal to about 4.7, less than or equal to about 4.6, less than or equal to about 4.5, less than or equal to about 4.4, less than or equal to about 4.3, less than or equal to about 4.2, less than or equal to about 4.1, less than or equal to about 4.0, less than or equal to about 3.9, less than or equal to about 3.8, less than or equal to about 3.7, less than or equal to about 3.6, less than or equal to about 3.5, less than or equal to about 3.4, less than or equal to about 3.3, less than or equal to about 3.2, less than or equal to about 3.1, less than or equal to about 3.0, less than or equal to about 2.9, less than or equal to about 2.8, less than or equal to about 2.7, less than or equal to about 2.6, less than or equal to about 2.5, or any range or value including and / or in between any two of these values. In some embodiments, the LLNP solution has a pH of about 2.5 to about 7.5, about 2.5 to about 7.0, about 2.5 to about 6.5, about 4.5 to about 5.5, about 4.6 to about 5.4, about 4.7 to about 5.3, or about 4.8 to about 5.2. In some embodiments, the eLNPs have an encapsulation efficiency of greater than or equal to about 60%, greater than or equal to about 65%, greater than or equal to about 70%, greater than or equal to about 71%, greater than or equal to about 72%, greater than or equal to about 73%, greater than or equal to about 74%, greater than or equal to about 75%, greater than or equal to about 76%, greater than or equal to about 77%, greater than or equal to about 78%, greater than or equal to about 79%, greater than or equal to about 80%, greater than or equal to about 81%, greater than or equal to about 82%, greater than or equal to about 83%, greater than or equal to about 84%, greater than or equal to about 85%, greater than or equal to about 86%, greater than or equal to about 87%, greater than or equal to about 88%, greater than or equal to about 89%, greater than or equal to about 90%, greater than or equal to about 91%, greater than or equal to about 92%, greater than or equal to about 93%, greater than or equal to about 94%, greater than or equal to about 95%, greater than or equal to about 96%, greater than or equal to about 97%, greater than or equal to about 98%, greater than or equal to about 99%, about 100%, or any range or value including and / or in between any two of these values. In some embodiments, the eLNPs have an encapsulation efficiency of less than or equal to about 100%, less than or equal to about 99%, less than or equal to about 98%, less than or equal to about 97%, less than or equal to about 96%, less than or equal to about 95%, less than or equal to about 94%, less than or equal to about 93%, less than or equal to about 92%, less than or equal to about 91%, less than or equal to about 90%, less than or equal to about 89%, less than or equal to about 88%, less than or equal to about 87%, less than or equal to about 86%, less than or equal to about 85%, less than or equal to about 84%, less than or equal to about 83%, less than or equal to about 82%, less than or equal to about 81%, less than or equal to about 80%, less than or equal to about 79%, less than or equal to about 78%, less than or equal to about 77%, less than or equal to about 76%, less than or equal to about 75%, less than or equal to about 74%, less than or equal to about 73%, less than or equal to about 72%, less than or equal to about 71%, less than or equal to about 70%, less than or equal to about 65%, less than or equal to about 60%, or any range or value including and / or in between any two of these values. Referring now to FIG.1C, in some embodiments, the method comprises processing the LLNP solution to obtain a LNP formulation. In some embodiments, the processing the LLNP solution comprises pH adjustment and / or buffer exchange, followed by adding a PEG lipid to the LLNP. In some embodiments, the adding the PEG lipid comprises mixing a PEG lipid solution comprising the PEG lipid with the loaded LNP solution. For instance, in some embodiments, the processing the LLNP solution comprises pH adjustment and / or buffer exchange using an aqueous buffer solution comprising an acetate, phosphate, or tris buffer, wherein the buffering agent is present at 100 mM to 200 mM (e.g., 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM) and having a pH of 4 to 8.5 (e.g., 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, .6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5). The aqueous buffer solution may further comprise a cryoprotectant (e.g., sucrose) at a concentration of 100- 500 g / L. In some embodiments, after the pH adjustment and / or buffer exchange, the processing further comprises adding PEG by mixing the LLNP solution with a PEG solution comprising 1-100 g / L (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / L) of a PEG lipid (e.g., PEG-DMG) further comprising an acetate, phosphate, or tris buffer, wherein the buffering agent is present at 10-200 mM (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mM) and having a pH of 4 to 8.5 (e.g., 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, .6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5). For instance, in some embodiments, the processing the LLNP solution comprises pH adjustment and / or buffer exchange by mixing the LLNP solution with a 120 mM tris buffer having a pH of about 8.3 and about 300-350 g / L sucrose, followed by mixing the pH-adjusted or buffer-exchanged LLNP solution with a PEG lipid solution comprising 5-15 g / L PEG-DMG in a 10-30 mM tris buffer having a pH of about 7.5. In some embodiments, the processing the loaded LNP solution comprises pH adjusting, buffer exchanging, filtering, sterilizing, lyophilizing, concentrating, diluting, a first adding step comprising adding a polymeric lipid (e.g., a PEG lipid) to the loaded LNP, a second adding step comprising adding a polymeric lipid (e.g., a PEG lipid) to the loaded LNP, or any combination thereof. In some embodiments, the processing the loaded LNP solution comprises pH adjusting (e.g., neutralization), followed by adding a PEG lipid. In some embodiments, the processing the loaded LNP solution comprises pH adjusting (e.g., neutralization), followed by adding a PEG lipid, followed by filtration (e.g., TFF) into an aqueous buffer solution to obtain the LNP formulation. In some embodiments, the first adding comprises adding one or more polymeric lipids (e.g., PEG lipids) in an amount of from about about 0.1 mol% to about 5.0 mol%, about 0.1 mol% to about 4.5 mol%, about 0.1 mol% to about 4.0 mol%, about 0.1 mol% to about 3.5 mol%, 0.1 mol% to about 3.0 mol%, from about 0.2 mol% to about 2.5 mol%, from about 0.5 mol% to about 2.0 mol%, from about 0.75 mol% to about 1.5 mol%, from about 1.5 mol% to about 3.0 mol% PEG lipid, from about 1.0 mol% to about 1.25 mol%, from about 0.2 mol% to about 0.8 mol%, 5 mol% or less, 3 mol% or less, 1 mol% or less, or 0.1 mol% or less, to the loaded LNP. In some embodiments, the second adding comprises adding one or more polymeric lipids (e.g., PEG lipids) in an amount of from about 0.1 mol% to about 3.0 mol%, from about 0.2 mol% to about 2.5 mol%, from about 0.5 mol% to about 2.0 mol%, from about 0.75 mol% to about 1.5 mol%, from about 1.0 mol% to about 1.25 mol%, or from about 0.2 mol% to about 0.8 mol% to the loaded LNP. In some embodiments the first adding or second adding comprises mixing the loaded LNP solution with a PEG lipid solution having about 1g / L to 100 gL, about 2 g / L to about 90 g / L, about 3 g / L to about 80 g / L, about 4 g / L to about 70 g / L, about 5 g / L to about 60 g / L, about 6 g / L to about 50 g / L, about 7 g / L to about 40 g / L, about 8 g / L to about 30 g / L about 9 g / L to about 20 g / L, or about 10 g / L to about 15 g / L PEG lipid. In some embodiments the PEG lipid solution comprise about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, about 50 g / L, about 55 g / L, about 60 g / L, about 65 g / L, about 70 g / L, about 75 g / L, about 80 g / L, about 85 g / L, about 90 g / L, about 95 g / L, or about 100 g / L, of one or more PEG lipids (e.g., PEG-DMG). In some embodiments, the PEG lipid solution comprises an acetate, phosphate, or tris buffering agent, wherein the buffering agent is present at a concentration of about 1 mM to about 500 mM (e.g., 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM,250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM), wherein the PEG solution has a pH of about 4 to 8 (e.g., about 4, 4.5, 5, 5.5, 6, 6., 7, 7.5 or 8). For instance, after the loading step, the LLNP solution may be processed by mixing the LLNP solution with a PEG lipid solution comprising 1-10 g / L PEG lipid (e.g., PEG-DMG) in a 10 mM to 50 mM (e.g., 20 mM) tris buffer. In some embodiments, the processing the loaded LNP comprises a first adding step and a second adding step after the first adding step. In some embodiments, the processing the loaded LNP solution comprises pH adjusting, a first adding step after the pH adjusting, and a second adding step after the first adding step. In some embodiments, the processing the loaded LNP solution comprises a first adding step, a second adding step after the first adding step, and a filtering step after the second adding step. In some embodiments the processing the loaded LNP solution comprises a first adding step, a buffer exchanging step after the first adding step, a second adding step after the buffer exchanging step, and a filtering step after the second adding step. In some embodiments, the processing the loaded LNP solution comprises pH adjusting, a first adding step after the pH adjusting, a buffer exchanging step after the first adding step, a second adding step after the buffer exchanging step, and a filtering step after the second adding step. In some embodiments, the processing the loaded LNP solution further comprises a sterilizing step performed before, after, or simultaneously with the filtering step. In some embodiments, a filtering step is performed on eLNPs before and / or after a freeze-thaw cycle, on the LLNPs before and / or after a freeze-thaw cycle, or an the LNP formulation before and / or after a freeze-thaw cycle. In some embodiments, the pH adjusting comprises mixing the loaded LNP solution with a fourth aqueous buffer solution comprising a fourth buffering agent. In some embodiments, the fourth aqueous buffer solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer (e.g., ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, potassium citrate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), etc.). In some embodiments, the fourth aqueous buffer solution comprises a buffer selected from the group consisting of an acetate buffer, a citrate buffer, a phosphate buffer, and a tris buffer. In some embodiments, the fourth aqueous buffer solution further comprises a cryoprotective agent (e.g., sucrose), at any of the above-discussed concentrations for cryprotective agents. In some embodiments, the fourth aqueous buffer solution comprises a buffering agent at a concentration of greater than or equal to about 0.001 mM, greater than or equal to about 0.002 mM, greater than or equal to about 0.003 mM, greater than or equal to about 0.004 mM, greater than or equal to about 0.005 mM, greater than or equal to about 0.006 mM, greater than or equal to about 0.007 mM, greater than or equal to about 0.008 mM, greater than or equal to about 0.009 mM, greater than or equal to about 0.01 mM, greater than or equal to about 0.02 mM, greater than or equal to about 0.03 mM, greater than or equal to about 0.04 mM, greater than or equal to about 0.05 mM, greater than or equal to about 0.06 mM, greater than or equal to about 0.07 mM, greater than or equal to about 0.08 mM, greater than or equal to about 0.09 mM, greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, greater than or equal to about 0.6 mM, greater than or equal to about 0.7 mM, greater than or equal to about 0.8 mM, greater than or equal to about 0.9 mM, greater than or equal to about 1.0 mM, greater than or equal to about 2.0 mM, greater than or equal to about 3.0 mM, greater than or equal to about 4.0 mM, greater than or equal to about 5.0 mM, greater than or equal to about 6.0 mM, greater than or equal to about 7.0 mM, greater than or equal to about 8.0 mM, greater than or equal to about 9.0 mM, greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, greater than or equal to about 200 mM, greater than or equal to about 300 mM, greater than or equal to about 400 mM, greater than or equal to about 500 mM, greater than or equal to about 600 mM, greater than or equal to about 700 mM, greater than or equal to about 800 mM, greater than or equal to about 900 mM, greater than or equal to about 1M, greater than or equal to about 2 M, greater than or equal to about 3 M, greater than or equal to about 4 M, greater than or equal to about 5 M, or any range or value including and / or in between any two of these values. In some embodiments, the fourth aqueous buffer solution comprises a buffering agent at a concentration of less than or equal to about 5 M, less than or equal to about 4 M, less than or equal to about 3 M, less than or equal to about 2 M, less than or equal to about 1M, less than or equal to about 900 mM, less than or equal to about 800 mM, less than or equal to about 700 mM, less than or equal to about 600 mM, less than or equal to about 500 mM, less than or equal to about 400 mM, less than or equal to about 300 mM, less than or equal to about 200 mM, less than or equal to about 100 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, less than or equal to about 60 mM, less than or equal to about 50 mM, less than or equal to about 40 mM, less than or equal to about 30 mM, less than or equal to about 20 mM, less than or equal to about 10 mM, less than or equal to about 9.0 mM, less than or equal to about 8.0 mM, less than or equal to about 7.0 mM, less than or equal to about 6.0 mM, less than or equal to about 5.0 mM, less than or equal to about 4.0 mM, less than or equal to about 3.0 mM, less than or equal to about 2.0 mM, less than or equal to about 1.0 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, less than or equal to about 0.6 mM, less than or equal to about 0.5 mM, less than or equal to about 0.4 mM, less than or equal to about 0.3 mM, less than or equal to about 0.2 mM, less than or equal to about 0.1 mM, less than or equal to about 0.09 mM, less than or equal to about 0.08 mM, less than or equal to about 0.07 mM, less than or equal to about 0.06 mM, less than or equal to about 0.05 mM, less than or equal to about 0.04 mM, less than or equal to about 0.03 mM, less than or equal to about 0.02 mM, less than or equal to about 0.01 mM, less than or equal to about 0.009 mM, less than or equal to about 0.008 mM, less than or equal to about 0.007 mM, less than or equal to about 0.006 mM, less than or equal to about 0.005 mM, less than or equal to about 0.004 mM, less than or equal to about 0.003 mM, less than or equal to about 0.002 mM, less than or equal to about 0.001 mM, or any range or value including and / or in between any two of these values. In some embodiments, the fourth aqueous buffer solution has a pH of greater than or equal to about 6, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7, greater than or equal to about 7.1, greater than or equal to about 7.2, greater than or equal to about 7.3, greater than or equal to about 7.4, greater than or equal to about 7.5, greater than or equal to about 7.6, greater than or equal to about 7.7, greater than or equal to about 7.8, greater than or equal to about 7.9, greater than or equal to about 8, greater than or equal to about 8.1, greater than or equal to about 8.2, greater than or equal to about 8.3, greater than or equal to about 8.4, greater than or equal to about 8.5, greater than or equal to about 8.6, greater than or equal to about 8.7, greater than or equal to about 8.8, greater than or equal to about 8.9, greater than or equal to about 9, greater than or equal to about 9.1, greater than or equal to about 9.2, greater than or equal to about 9.3, greater than or equal to about 9.4, greater than or equal to about 9.5, greater than or equal to about 9.6, greater than or equal to about 9.7, greater than or equal to about 9.8, greater than or equal to about 9.9, greater than or equal to about 10, or any range or value including and / or in between any two of these values. In some embodiments, the fourth aqueous buffer solution has a pH of less than or equal to about 10, less than or equal to about 9.9, less than or equal to about 9.8, less than or equal to about 9.7, less than or equal to about 9.6, less than or equal to about 9.5, less than or equal to about 9.4, less than or equal to about 9.3, less than or equal to about 9.2, less than or equal to about 9.1, less than or equal to about 9, less than or equal to about 8.9, less than or equal to about 8.8, less than or equal to about 8.7, less than or equal to about 8.6, less than or equal to about 8.5, less than or equal to about 8.4, less than or equal to about 8.3, less than or equal to about 8.2, less than or equal to about 8.1, less than or equal to about 8, less than or equal to about 7.9, less than or equal to about 7.8, less than or equal to about 7.7, less than or equal to about 7.6, less than or equal to about 7.5, less than or equal to about 7.4, less than or equal to about 7.3, less than or equal to about 7.2, less than or equal to about 7.1, less than or equal to about 7, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6, or any range or value including and / or in between any two of these values. In some embodiments, after the pH adjusting, the LNP formulation has a pH of greater than or equal to about 6.0, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7.0, greater than or equal to about 7.1, greater than or equal to about 7.2, greater than or equal to about 7.3, greater than or equal to about 7.4, greater than or equal to about 7.5, greater than or equal to about 7.6, greater than or equal to about 7.7, greater than or equal to about 7.8, greater than or equal to about 7.9, greater than or equal to about 8.0, greater than or equal to about 8.1, greater than or equal to about 8.2, greater than or equal to about 8.3, greater than or equal to about 8.4, greater than or equal to about 8.5, greater than or equal to about 8.6, greater than or equal to about 8.7, greater than or equal to about 8.8, greater than or equal to about 8.9, greater than or equal to about 9.0, or any range or value including and / or in between any two of these values. In some embodiments, after the pH adjusting, the LNP formulation has a pH of less than or equal to about 9.0, less than or equal to about 8.9, less than or equal to about 8.8, less than or equal to about 8.7, less than or equal to about 8.6, less than or equal to about 8.5, less than or equal to about 8.4, less than or equal to about 8.3, less than or equal to about 8.2, less than or equal to about 8.1, less than or equal to about 8.0, less than or equal to about 7.9, less than or equal to about 7.8, less than or equal to about 7.7, less than or equal to about 7.6, less than or equal to about 7.5, less than or equal to about 7.4, less than or equal to about 7.3, less than or equal to about 7.2, less than or equal to about 7.1, less than or equal to about 7.0, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6.0, or any range or value including and / or in between any two of these values. In some embodiments, the buffer exchanging comprises mixing the loaded LNP solution or the LNP formulation with a fifth aqueous buffer solution comprising a fifth buffering agent. In some embodiments, the fifth aqueous buffer solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer (e.g., ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, potassium citrate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), etc.). In some embodiments, the fifth aqueous buffer solution comprises a buffer selected from the group consisting of an acetate buffer, a citrate buffer, a phosphate buffer, and a tris buffer. In some embodiments, the fifth aqueous buffer solution comprises a buffering agent at a concentration of greater than or equal to about 0.001 mM, greater than or equal to about 0.002 mM, greater than or equal to about 0.003 mM, greater than or equal to about 0.004 mM, greater than or equal to about 0.005 mM, greater than or equal to about 0.006 mM, greater than or equal to about 0.007 mM, greater than or equal to about 0.008 mM, greater than or equal to about 0.009 mM, greater than or equal to about 0.01 mM, greater than or equal to about 0.02 mM, greater than or equal to about 0.03 mM, greater than or equal to about 0.04 mM, greater than or equal to about 0.05 mM, greater than or equal to about 0.06 mM, greater than or equal to about 0.07 mM, greater than or equal to about 0.08 mM, greater than or equal to about 0.09 mM, greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, greater than or equal to about 0.6 mM, greater than or equal to about 0.7 mM, greater than or equal to about 0.8 mM, greater than or equal to about 0.9 mM, greater than or equal to about 1.0 mM, greater than or equal to about 2.0 mM, greater than or equal to about 3.0 mM, greater than or equal to about 4.0 mM, greater than or equal to about 5.0 mM, greater than or equal to about 6.0 mM, greater than or equal to about 7.0 mM, greater than or equal to about 8.0 mM, greater than or equal to about 9.0 mM, greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, greater than or equal to about 200 mM, greater than or equal to about 300 mM, greater than or equal to about 400 mM, greater than or equal to about 500 mM, greater than or equal to about 600 mM, greater than or equal to about 700 mM, greater than or equal to about 800 mM, greater than or equal to about 900 mM, greater than or equal to about 1M, greater than or equal to about 2 M, greater than or equal to about 3 M, greater than or equal to about 4 M, greater than or equal to about 5 M, or any range or value including and / or in between any two of these values. In some embodiments, the fifth aqueous buffer solution comprises a buffering agent at a concentration of less than or equal to about 5 M, less than or equal to about 4 M, less than or equal to about 3 M, less than or equal to about 2 M, less than or equal to about 1M, less than or equal to about 900 mM, less than or equal to about 800 mM, less than or equal to about 700 mM, less than or equal to about 600 mM, less than or equal to about 500 mM, less than or equal to about 400 mM, less than or equal to about 300 mM, less than or equal to about 200 mM, less than or equal to about 100 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, less than or equal to about 60 mM, less than or equal to about 50 mM, less than or equal to about 40 mM, less than or equal to about 30 mM, less than or equal to about 20 mM, less than or equal to about 10 mM, less than or equal to about 9.0 mM, less than or equal to about 8.0 mM, less than or equal to about 7.0 mM, less than or equal to about 6.0 mM, less than or equal to about 5.0 mM, less than or equal to about 4.0 mM, less than or equal to about 3.0 mM, less than or equal to about 2.0 mM, less than or equal to about 1.0 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, less than or equal to about 0.6 mM, less than or equal to about 0.5 mM, less than or equal to about 0.4 mM, less than or equal to about 0.3 mM, less than or equal to about 0.2 mM, less than or equal to about 0.1 mM, less than or equal to about 0.09 mM, less than or equal to about 0.08 mM, less than or equal to about 0.07 mM, less than or equal to about 0.06 mM, less than or equal to about 0.05 mM, less than or equal to about 0.04 mM, less than or equal to about 0.03 mM, less than or equal to about 0.02 mM, less than or equal to about 0.01 mM, less than or equal to about 0.009 mM, less than or equal to about 0.008 mM, less than or equal to about 0.007 mM, less than or equal to about 0.006 mM, less than or equal to about 0.005 mM, less than or equal to about 0.004 mM, less than or equal to about 0.003 mM, less than or equal to about 0.002 mM, less than or equal to about 0.001 mM, or any range or value including and / or in between any two of these values. In some embodiments, the fifth aqueous buffer solution has a pH of greater than or equal to about 6, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7, greater than or equal to about 7.1, greater than or equal to about 7.2, greater than or equal to about 7.3, greater than or equal to about 7.4, greater than or equal to about 7.5, greater than or equal to about 7.6, greater than or equal to about 7.7, greater than or equal to about 7.8, greater than or equal to about 7.9, greater than or equal to about 8, greater than or equal to about 8.1, greater than or equal to about 8.2, greater than or equal to about 8.3, greater than or equal to about 8.4, greater than or equal to about 8.5, greater than or equal to about 8.6, greater than or equal to about 8.7, greater than or equal to about 8.8, greater than or equal to about 8.9, greater than or equal to about 9, greater than or equal to about 9.1, greater than or equal to about 9.2, greater than or equal to about 9.3, greater than or equal to about 9.4, greater than or equal to about 9.5, greater than or equal to about 9.6, greater than or equal to about 9.7, greater than or equal to about 9.8, greater than or equal to about 9.9, greater than or equal to about 10, or any range or value including and / or in between any two of these values. In some embodiments, the fifth aqueous buffer solution has a pH of less than or equal to about 10, less than or equal to about 9.9, less than or equal to about 9.8, less than or equal to about 9.7, less than or equal to about 9.6, less than or equal to about 9.5, less than or equal to about 9.4, less than or equal to about 9.3, less than or equal to about 9.2, less than or equal to about 9.1, less than or equal to about 9, less than or equal to about 8.9, less than or equal to about 8.8, less than or equal to about 8.7, less than or equal to about 8.6, less than or equal to about 8.5, less than or equal to about 8.4, less than or equal to about 8.3, less than or equal to about 8.2, less than or equal to about 8.1, less than or equal to about 8, less than or equal to about 7.9, less than or equal to about 7.8, less than or equal to about 7.7, less than or equal to about 7.6, less than or equal to about 7.5, less than or equal to about 7.4, less than or equal to about 7.3, less than or equal to about 7.2, less than or equal to about 7.1, less than or equal to about 7, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6, or any range or value including and / or in between any two of these values. In some embodiments, after the buffer exchanging, the LNP formulation has a pH of greater than or equal to about 6.0, greater than or equal to about 6.1, greater than or equal to about 6.2, greater than or equal to about 6.3, greater than or equal to about 6.4, greater than or equal to about 6.5, greater than or equal to about 6.6, greater than or equal to about 6.7, greater than or equal to about 6.8, greater than or equal to about 6.9, greater than or equal to about 7.0, greater than or equal to about 7.1, greater than or equal to about 7.2, greater than or equal to about 7.3, greater than or equal to about 7.4, greater than or equal to about 7.5, greater than or equal to about 7.6, greater than or equal to about 7.7, greater than or equal to about 7.8, greater than or equal to about 7.9, greater than or equal to about 8.0, greater than or equal to about 8.1, greater than or equal to about 8.2, greater than or equal to about 8.3, greater than or equal to about 8.4, greater than or equal to about 8.5, greater than or equal to about 8.6, greater than or equal to about 8.7, greater than or equal to about 8.8, greater than or equal to about 8.9, greater than or equal to about 9.0, or any range or value including and / or in between any two of these values. In some embodiments, after the buffer exchanging, the LNP formulation has a pH of less than or equal to about 9.0, less than or equal to about 8.9, less than or equal to about 8.8, less than or equal to about 8.7, less than or equal to about 8.6, less than or equal to about 8.5, less than or equal to about 8.4, less than or equal to about 8.3, less than or equal to about 8.2, less than or equal to about 8.1, less than or equal to about 8.0, less than or equal to about 7.9, less than or equal to about 7.8, less than or equal to about 7.7, less than or equal to about 7.6, less than or equal to about 7.5, less than or equal to about 7.4, less than or equal to about 7.3, less than or equal to about 7.2, less than or equal to about 7.1, less than or equal to about 7.0, less than or equal to about 6.9, less than or equal to about 6.8, less than or equal to about 6.7, less than or equal to about 6.6, less than or equal to about 6.5, less than or equal to about 6.4, less than or equal to about 6.3, less than or equal to about 6.2, less than or equal to about 6.1, less than or equal to about 6.0, or any range or value including and / or in between any two of these values. LNP Formulations In some embodiments, the LLNP formulation is processed to produce a LNP formulation. In some embodiments, the LNP formulation comprises a LLNP, wherein the LLNP comprises one or more PEG lipids. Referring to FIG.2, when a LLNP is produced by mixing an eLNP solution with a nucleic acid solution, thereby encapsulating a nucleic acid in the LNP, the loaded LNP comprises 5 mol% or less, 3 mol% or less, 1 mol% or less, 0.1 mol% or less, or does not comprise any “core” polymeric lipid (e.g., PEG lipid). When a polymeric lipid (e.g., PEG lipid) is added to the loaded LNP, the polymeric lipid is located at the surface of the loaded LNP. In some embodiments, the fraction of the polymeric lipid (e.g., PEG lipid) at the surface of the LNP, relative to the total polymeric lipid in the LNP is greater than or equal to about 50%, greater than or equal to about 55%, greater than or equal to about 60%, greater than or equal to about 65%, greater than or equal to about 70%, greater than or equal to about 75%, greater than or equal to about 80%, greater than or equal to about 85%, greater than or equal to about 90%, greater than or equal to about 91%, greater than or equal to about 92%, greater than or equal to about 93%, greater than or equal to about 94%, greater than or equal to about 95%, greater than or equal to about 96%, greater than or equal to about 97%, greater than or equal to about 98%, greater than or equal to about 99%, greater than or equal to about 99.1%, greater than or equal to about 99.2%, greater than or equal to about 99.3%, greater than or equal to about 99.4%, greater than or equal to about 99.5%, greater than or equal to about 99.6%, greater than or equal to about 99.7%, greater than or equal to about 99.8%, greater than or equal to about 99.9%, about 100%, or any range or value including and / or in between any two of these values. In some embodiments, the fraction of the polymeric lipid (e.g., PEG lipid) at the surface of the LNP, relative to the total polymeric lipid in the LNP is less than or equal to about 100%, less than or equal to about 99.9%, less than or equal to about 99.8%, less than or equal to about 99.7%, less than or equal to about 99.6%, less than or equal to about 99.5%, less than or equal to about 99.4%, less than or equal to about 99.3%, less than or equal to about 99.2%, less than or equal to about 99.1%, less than or equal to about 99%, less than or equal to about 98%, less than or equal to about 97%, less than or equal to about 96%, less than or equal to about 95%, less than or equal to about 94%, less than or equal to about 93%, less than or equal to about 92%, less than or equal to about 91%, less than or equal to about 90%, less than or equal to about 85%, less than or equal to about 80%, less than or equal to about 75%, less than or equal to about 70%, less than or equal to about 65%, less than or equal to about 60%, less than or equal to about 55%, less than or equal to about 50%, or any range or value including and / or in between any two of these values. In some embodiments, the LNP formulations comprises a cryoprotectant. In some embodiments, the cryoprotectant comprises or consists of a sugar. In some embodiments, the sugar is sucrose. In some embodiments, the cryoprotectant is present in the LNP formulation at a concentration of greater than or equal to about 1 mg / mL, greater than or equal to about 2 mg / mL, greater than or equal to about 3 mg / mL, greater than or equal to about 4 mg / mL, greater than or equal to about 5 mg / mL, greater than or equal to about 6 mg / mL, greater than or equal to about 7 mg / mL, greater than or equal to about 8 mg / mL, greater than or equal to about 9 mg / mL, greater than or equal to about 10 mg / mL, greater than or equal to about 15 mg / mL, greater than or equal to about 20 mg / mL, greater than or equal to about 25 mg / mL, greater than or equal to about 30 mg / mL, greater than or equal to about 35 mg / mL, greater than or equal to about 40 mg / mL, greater than or equal to about 45 mg / mL, greater than or equal to about 50 mg / mL, greater than or equal to about 55 mg / mL, greater than or equal to about 60 mg / mL, greater than or equal to about 65 mg / mL, greater than or equal to about 70 mg / mL, greater than or equal to about 75 mg / mL, greater than or equal to about 80 mg / mL, greater than or equal to about 85 mg / mL, greater than or equal to about 90 mg / mL, greater than or equal to about 95 mg / mL, greater than or equal to about 100 mg / mL, greater than or equal to about 150 mg / mL, greater than or equal to about 200 mg / mL, greater than or equal to about 250 mg / mL, greater than or equal to about 300 mg / mL, greater than or equal to about 350 mg / mL, greater than or equal to about 400 mg / mL, greater than or equal to about 450 mg / mL, greater than or equal to about 500 mg / mL, greater than or equal to about 550 mg / mL, greater than or equal to about 600 mg / mL, greater than or equal to about 650 mg / mL, greater than or equal to about 700 mg / mL, greater than or equal to about 750 mg / mL, greater than or equal to about 800 mg / mL, greater than or equal to about 850 mg / mL, greater than or equal to about 900 mg / mL, greater than or equal to about 950 mg / mL, greater than or equal to about 1000 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the cryoprotectant is present in the LNP formulation at a concentration of less than or equal to about 1000 mg / mL, less than or equal to about 950 mg / mL, less than or equal to about 900 mg / mL, less than or equal to about 850 mg / mL, less than or equal to about 800 mg / mL, less than or equal to about 750 mg / mL, less than or equal to about 700 mg / mL, less than or equal to about 650 mg / mL, less than or equal to about 600 mg / mL, less than or equal to about 550 mg / mL, less than or equal to about 500 mg / mL, less than or equal to about 450 mg / mL, less than or equal to about 400 mg / mL, less than or equal to about 350 mg / mL, less than or equal to about 300 mg / mL, less than or equal to about 250 mg / mL, less than or equal to about 200 mg / mL, less than or equal to about 150 mg / mL, less than or equal to about 100 mg / mL, less than or equal to about 95 mg / mL, less than or equal to about 90 mg / mL, less than or equal to about 85 mg / mL, less than or equal to about 80 mg / mL, less than or equal to about 75 mg / mL, less than or equal to about 70 mg / mL, less than or equal to about 65 mg / mL, less than or equal to about 60 mg / mL, less than or equal to about 55 mg / mL, less than or equal to about 50 mg / mL, less than or equal to about 45 mg / mL, less than or equal to about 40 mg / mL, less than or equal to about 35 mg / mL, less than or equal to about 30 mg / mL, less than or equal to about 25 mg / mL, less than or equal to about 20 mg / mL, less than or equal to about 15 mg / mL, less than or equal to about 10 mg / mL, less than or equal to about 9 mg / mL, less than or equal to about 8 mg / mL, less than or equal to about 7 mg / mL, less than or equal to about 6 mg / mL, less than or equal to about 5 mg / mL, less than or equal to about 4 mg / mL, less than or equal to about 3 mg / mL, less than or equal to about 2 mg / mL, less than or equal to about 1 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the cryoprotectant is present in the LNP formulation, at a concentration of about1 mg / mL to about 1000 mg / mL, about 5 mg / mL to about 950 mg / mL, about 10 mg / mL to about 900 mg / mL, about 20 mg / mL to about 800 mg / mL, about 50 mg / mL to about 700 mg / mL, about 100 mg / mL to about 500 mg / mL, or any range or value including and / or in between any two of these values. In some embodiments, the LNP formulation comprises a buffering agent. In some embodiments, the buffering agent in the LNP formulation comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer (e.g., ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, potassium citrate, tris(hydroxymethyl)aminomethane (tris), sodium phosphate, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), etc.). In some embodiments, the buffering agent is present in the LNP formulation at a concentration of greater than or equal to about 0.001 mM, greater than or equal to about 0.002 mM, greater than or equal to about 0.003 mM, greater than or equal to about 0.004 mM, greater than or equal to about 0.005 mM, greater than or equal to about 0.006 mM, greater than or equal to about 0.007 mM, greater than or equal to about 0.008 mM, greater than or equal to about 0.009 mM, greater than or equal to about 0.01 mM, greater than or equal to about 0.02 mM, greater than or equal to about 0.03 mM, greater than or equal to about 0.04 mM, greater than or equal to about 0.05 mM, greater than or equal to about 0.06 mM, greater than or equal to about 0.07 mM, greater than or equal to about 0.08 mM, greater than or equal to about 0.09 mM, greater than or equal to about 0.1 mM, greater than or equal to about 0.2 mM, greater than or equal to about 0.3 mM, greater than or equal to about 0.4 mM, greater than or equal to about 0.5 mM, greater than or equal to about 0.6 mM, greater than or equal to about 0.7 mM, greater than or equal to about 0.8 mM, greater than or equal to about 0.9 mM, greater than or equal to about 1.0 mM, greater than or equal to about 2.0 mM, greater than or equal to about 3.0 mM, greater than or equal to about 4.0 mM, greater than or equal to about 5.0 mM, greater than or equal to about 6.0 mM, greater than or equal to about 7.0 mM, greater than or equal to about 8.0 mM, greater than or equal to about 9.0 mM, greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, greater than or equal to about 200 mM, greater than or equal to about 300 mM, greater than or equal to about 400 mM, greater than or equal to about 500 mM, greater than or equal to about 600 mM, greater than or equal to about 700 mM, greater than or equal to about 800 mM, greater than or equal to about 900 mM, greater than or equal to about 1M, greater than or equal to about 2 M, greater than or equal to about 3 M, greater than or equal to about 4 M, greater than or equal to about 5 M, or any range or value including and / or in between any two of these values. In some embodiments, the buffering agent is present in the LNP formulation at a concentration of less than or equal to about 5 M, less than or equal to about 4 M, less than or equal to about 3 M, less than or equal to about 2 M, less than or equal to about 1M, less than or equal to about 900 mM, less than or equal to about 800 mM, less than or equal to about 700 mM, less than or equal to about 600 mM, less than or equal to about 500 mM, less than or equal to about 400 mM, less than or equal to about 300 mM, less than or equal to about 200 mM, less than or equal to about 100 mM, less than or equal to about 90 mM, less than or equal to about 80 mM, less than or equal to about 70 mM, less than or equal to about 60 mM, less than or equal to about 50 mM, less than or equal to about 40 mM, less than or equal to about 30 mM, less than or equal to about 20 mM, less than or equal to about 10 mM, less than or equal to about 9.0 mM, less than or equal to about 8.0 mM, less than or equal to about 7.0 mM, less than or equal to about 6.0 mM, less than or equal to about 5.0 mM, less than or equal to about 4.0 mM, less than or equal to about 3.0 mM, less than or equal to about 2.0 mM, less than or equal to about 1.0 mM, less than or equal to about 0.9 mM, less than or equal to about 0.8 mM, less than or equal to about 0.7 mM, less than or equal to about 0.6 mM, less than or equal to about 0.5 mM, less than or equal to about 0.4 mM, less than or equal to about 0.3 mM, less than or equal to about 0.2 mM, less than or equal to about 0.1 mM, less than or equal to about 0.09 mM, less than or equal to about 0.08 mM, less than or equal to about 0.07 mM, less than or equal to about 0.06 mM, less than or equal to about 0.05 mM, less than or equal to about 0.04 mM, less than or equal to about 0.03 mM, less than or equal to about 0.02 mM, less than or equal to about 0.01 mM, less than or equal to about 0.009 mM, less than or equal to about 0.008 mM, less than or equal to about 0.007 mM, less than or equal to about 0.006 mM, less than or equal to about 0.005 mM, less than or equal to about 0.004 mM, less than or equal to about 0.003 mM, less than or equal to about 0.002 mM, less than or equal to about 0.001 mM, or any range or value including and / or in between any two of these values. In some embodiments, the LNP formulation is lyophilized. In some embodiments, the lyophilizing comprises freezing the LNP formulation at a temperature from about -100℃. to about 0℃, about -80℃ to about -10℃, about -60℃ to about -20℃, about -50℃ to about -25℃, or about -40℃ to about -30℃. In some embodiments, the lyophilizing further comprises drying (e.g., under vacuum of from about 50 mTorr to about 150 mTorr at about 25℃) the frozen LNP formulation to form a lyophilized eLNP. In some embodiments of the methods of the disclosure, the drying is performed at a vacuum ranging from about 50 mTorr to about 150 mTorr. In some embodiments, the LNP formulation is stored (e.g., at a temperature of about - 50℃, -45℃, -40℃, -35℃, -30℃, -25℃, -20℃, -15℃, -10℃, -5℃, 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, or any range or value including and / or in between any two of these values). In some embodiments, the LNP formulation is stored for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years. Kits In an aspect, the present disclosure relates to a kit, comprising: a first container comprising the eLNP of any one of the embodiments disclosed herein or the eLNP solution of any one of the embodiments disclosed herein; and a second container comprising a therapeutic agent. In some embodiments, the kit comprises instructions for combining contents of the first container with contents of the second container and / or instructions for administering the product obtained by combining the contents of the first container with the contents of the second container. In some embodiments, the combining comprises loading the therapeutic agent into the eLNP to produce a LLNP. In some embodiments the first container is a polytetrafluoroethylene (PTFE) bag. In some embodiments the second container is a polytetrafluoroethylene (PTFE) bag. In some embodiments, the therapeutic agent comprises a therapeutic nucleic acid. In some embodiments, the therapeutic agent comprises a therapeutic ribonucleic acid. In some embodiments, the ribonucleic acid comprises a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), a long non-coding RNA (lncRNA), or any combination thereof. In some embodiments, the therapeutic agent comprises a therapeutic mRNA. Methods of Treatment In an aspect, the present disclosure relates to a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the loaded LNP of any one of the embodiments disclosed herein, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In an aspect, the present disclosure relates to a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the loaded LNP solution of any one of the embodiments disclosed herein, wherein the loaded LNP solution comprises a therapeutically effective amount of a therapeutic mRNA. In an aspect, the present disclosure relates to a method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the LNP formulation of any one of the embodiments disclosed herein, wherein the LNP formulation comprises a therapeutically effective amount of a therapeutic mRNA. In some embodiments, the administering is performed by any route that results in a therapeutically effective outcome, such as intravenous (into a vein) administration. These also include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration that is then covered by a dressing that occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. In specific embodiments, compositions can be administered in a way that allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier. In some embodiments, a formulation for a route of administration can include at least one inactive ingredient. In some embodiments, the administration is a single administration. In some embodiments, the administration is a repeated administration. In some embodiments, the administration is about once per day, about once per week, about once per two weeks, or about once per month. In some embodiments, the pharmaceutical composition is administered intravenously, intramuscularly, or subcutaneously. In an aspect, the present disclosure relates to a loaded LNP of any one of the embodiments disclosed herein, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In an aspect, the present disclosure relates to a loaded LNP solution of any one of the embodiments disclosed herein, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In an aspect, the present disclosure relates to a LNP formulation of any one of the embodiments disclosed herein, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In an aspect, the present disclosure relates to a use of the loaded LNP of any one of the embodiments disclosed herein, in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In an aspect, the present disclosure relates to a use of the loaded LNP solution of any one of the embodiments disclosed herein, in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. In an aspect, the present disclosure relates to a use of the LNP formulation of any one of the embodiments disclosed herein, in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. Definitions In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. In this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple." Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the present disclosure. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an present disclosure is disclosed as having a plurality of alternatives, examples of that present disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an present disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed. The term "about" as used in connection with a numerical value throughout the specification and the claims refers to an interval of accuracy, familiar and acceptable to a person skilled in the art such as, for example, an interval of accuracy of ± 10 %, unless otherwise specified. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. As used herein, the term "administered in combination" or "combined administration" or “combination therapy” means that two or more agents are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved. As used herein, the term “compound,” is meant to include all stereoisomers and isotopes of the structure depicted. As used herein, the term “stereoisomer” means any geometric isomer (e.g., cis- and trans- isomer), enantiomer, or diastereomer of a compound. The present disclosure encompasses any and all stereoisomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. Further, a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods. As used herein, the term “delivering” means providing an entity to a destination. For example, delivering a polynucleotide to a subject can involve administering a nanoparticle composition including the polynucleotide to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a nanoparticle composition to a mammal or mammalian cell can involve contacting one or more cells with the lipid nanoparticle composition. As used herein, "delivery agent" refers to any substance that facilitates, at least in part, the in vivo, in vitro, or ex vivo delivery of a polynucleotide to targeted cells. As used herein, the term "effective amount" of an agent is that amount sufficient to effect beneficial or desired results, and, as such, an "effective amount" depends upon the context in which it is being applied. For example, in the context of administering an agent that increases protein expression in a target tissue, an effective amount of an agent is, for example, an amount of mRNA expressing sufficient amount of said protein to increase protein expression in the target tissue, as compared to the protein expression observed without administration of the agent. The term "effective amount" can be used interchangeably with "effective dose." As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template from a DNA sequence (e.g., by transcription); (2) processing of an mRNA transcript (e.g., by splicing, editing, 5′ cap formation, and / or 3′ end processing); (3) translation of an mRNA into a polypeptide or protein; and (4) post- translational modification of a polypeptide or protein. As used herein, a "linker" refers to a group of atoms, e.g., 10-1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be attached to a modified nucleoside or nucleotide on the nucleobase or sugar moiety at a first end, and to a payload, e.g., a detectable or therapeutic agent, at a second end. The linker can be of sufficient length as to not interfere with incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as to form polynucleotide multimers (e.g., through linkage of two or more chimeric polynucleotides molecules or IVT polynucleotides) or polynucleotides conjugates, as well as to administer a payload, as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers and derivatives thereof., Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (-S-S-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bond include an amido bond can be cleaved for example by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and / or photolysis, as well as an ester bond can be cleaved for example by acidic or basic hydrolysis. As used herein, the term “lipid nanoparticle” or “LNP” refers to a nanoparticle comprising one or more lipids. In some embodiments, the LNP has a size of about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, or about 100 nm or less. In some embodiments, the LNP has a size ranging from about 1 nm to about 500 nm or about 1 nm to about 100 nm. As used herein, the term “liposome” refers to a composite having at least one lipid bilayer. In some embodiments, the liposome has a size of about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, or about 100 nm or less. In some embodiments, the liposome has a size ranging from about 1 nm to about 500 nm or 1 nm to about 100 nm. As used herein, the term “total lipids” refers to the collection of ionizable lipids, structural lipids, and phospholipids, and PEG lipids (to the extent of their existence) in a given composition (e.g., a population of lipid assemblies). In some embodiments, when a population of lipid assemblies is free of PEG lipid, the total lipids in the population is the total amount of the ionizable lipid, the structural lipid, and the phospholipid in the population. In some embodiments, when a population of lipid assemblies comprises a PEG lipid, the total lipids in the population is the total amount of the ionizable lipid, the structural lipid, the phospholipid, and the PEG lipid in the population. As used herein, the term “lipid amine” refers to a lipid molecule having one or more amine functional groups appended thereto. The amine functional group can include one or more primary (NH2), secondary (NHR), or tertiary amine groups (NR2), where R denotes a non- hydrogen group such as an alkyl group, carbocyclic group, heterocyclic group, or substituted derivatives of the same. The lipid amine include sterol amines, where the lipid portion of the molecule is a steroid, such as cholesterol or a related moiety. As used herein, the phrase, “moiety cleavable under physiological conditions” refers to, for example, an ester, amide, carbonate, carbamate, or urea moiety. As used herein, "patient" refers to a subject (e.g., a human subject) who seeks or is in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable excipient," as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients can include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. The present disclosure also includes salts of the compounds described herein. As used herein, "salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In some embodiments, the salt is a pharmaceutically acceptable salt. Lists of pharmaceutically acceptable salts are found in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety. The term "polynucleotide" as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and single-stranded ribonucleic acid ("RNA"). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In particular aspects, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one unnatural nucleobase. In some aspects, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some aspects, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T (thymidine) in the case of a synthetic DNA, or A, C, G, and U (uridine) in the case of a synthetic RNA. The skilled artisan will appreciate that the T bases in the codon maps disclosed herein are present in DNA, whereas the T bases would be replaced by U bases in corresponding RNAs. For example, a codon-nucleotide sequence disclosed herein in DNA form, e.g., a vector or an in-vitro translation (IVT) template, would have its T bases transcribed as U based in its corresponding transcribed mRNA. In this respect, both codon-optimized DNA sequences (comprising T) and their corresponding mRNA sequences (comprising U) are considered codon-optimized nucleotide sequence of the present disclosure. A skilled artisan would also understand that equivalent codon-maps can be generated by replaced one or more bases with non-natural bases. Thus, e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map), which in turn would correspond to a ΨΨC codon (RNA map in which U has been replaced with pseudouridine). Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3 and C4-NH2, of cytidine and the C2- NH2, N′—H and C6-oxy, respectively, of guanosine. Thus, for example, guanosine (2-amino-6- oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D- ribofuranosyl-purine). Such modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (1-β-D- ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4- oxy-pyrimidine-) results in a modified nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine (U.S. Pat. No.5,681,702 to Collins et al.). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2′-deoxy-5-methyl-isocytidine can be prepared by the method of Tor et al., 1993, J. Am. Chem. Soc.115:4461-4467 and references cited therein; and isoguanine nucleotides can be prepared using the method described by Switzer et al., 1993, supra, and Mantsch et al., 1993, Biochem. 14:5593-5601, or by the method described in U.S. Pat. No.5,780,610 to Collins et al. Other nonnatural base pairs can be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37, for the synthesis of 2,6-diaminopyrimidine and its complement (1- methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotide units which form unique base pairs are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc.114:3675-3683 and Switzer et al., supra. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a monomer or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid. In some embodiments, a "peptide" can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. In other embodiments, a subject is a human patient. As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical characteristics rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical characteristics. The term "therapeutic agent" refers to an agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. For example, in some embodiments, an mRNA encoding a polypeptide can be a therapeutic agent. As used herein, the term “alkyl” or “alkyl group” means a linear or branched, saturated hydrocarbon including one to twenty carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty carbon atoms), which is optionally substituted. The notation “C1-14 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups. As used herein, the term “alkylene” refers to a linking alkyl group. As used herein, the term “alkenyl” or “alkenyl group” means a linear or branched hydrocarbon including two to twenty carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty carbon atoms) and at least one double bond, which is optionally substituted. The notation “C2-14 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. In some embodiments, C18 alkenyl may include one or more double bonds, e.g., one, two, or three double bonds. A C18alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups. As used herein, the term “carbocycle,” “carbocyclyl” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms, e.g., one, two, three, or four rings. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. In some embodiments, the rings may be three, four, five, six, seven, eight membered rings. The notation “C3-6 carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2- dihydronaphthyl groups. The term “cycloalkyl” as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles. As used herein, the term “alkynyl” or “alkynyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one triple bond. As used herein, the term “carbocyclylene” refers to a linking carbocyclyl group. As used herein, the term “carbocyclylalkyl” refers to an alkyl group substituted by a carbocyclyl group. An example carbocyclylalkyl group is benzyl. As used herein, the term “heteroalkyl” or “heteroalkyl group” means an optionally substituted alkyl group in which one, two, three, four, five, six, or seven of the alkyl carbons has been replaced by one or more heteroatoms selected from O, S, or N, provided that not more than two consecutive alkyl carbons are replaced. A heteroalkyl group may be linear or branched. Examples of heteroalkyl groups include but are not limited to CH2CH2OCH3, OCH2CH2OCH2CH2CH3, CH2NHCH3, CH2CH2N(CH3)CH2CH3, and CH2CH2OCH3. As used herein, the term “heteroalkylene” refers to a linking heteroalkyl group. As used herein, the term “heterocycle,” “heterocyclyl,” or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings. Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles. As used herein, the term “heterocycloalkyl” refers to a non-aromatic heterocycle, and represents a subset of heterocycles. Example heterocycloalkyl groups include azetidinyl, pyrolidinyl, piperidinyl, morpholinyl, and the like. As used herein, the term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl group. As used herein, the term “heterocyclylene” refers to a linking heterocyclyl group. As used herein, an “aryl group” is a carbocyclic group including one or more carbocyclic aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, the term “arylene” refers to a linking aryl group. As used herein, a “heteroaryl group” is a heterocyclic group including one or more heterocyclic aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted. As used herein, the term “heteroarylene” refers to a linking heteroaryl group. As used herein, the term “oxygen protecting group” refers to an oxo substituent that can be selectively removed under certain conditions (e.g., acidic or basic conditions). Example oxygen protecting groups can include optionally substituted alkyl, carbocyclyl, heterocyclyl, carbocyclylalkyl, and heterocyclylalkyl groups. As used herein, the term “nitrogen protecting group” refers to a nitrogen substituent (e.g., an amino substituent) that can be selectively removed under certain conditions (e.g., acidic of basic conditions). In some embodiments, the nitrogen protecting group is 9- fluorenylmethoxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc). Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Optional substituents may be selected from the group consisting of, but are not limited to, a halogen atom (e.g., a chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl, -OH), an ester (e.g., -C(O)OR or -OC(O)R), an aldehyde (e.g.,-C(O)H), a carbonyl (e.g., -C(O)R, alternatively represented by C=O), an acyl halide (e.g.,-C(O)X, in which X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy (e.g., -OR), an acetal (e.g.,-C(OR)2R””, in which each OR are alkoxy groups that can be the same or different and R”” is an alkyl or alkenyl group), a phosphate (e.g., P(O)43-), a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulfinic acid (e.g., -S(O)OH), a sulfonic acid (e.g., -S(O)2OH), a thial (e.g., -C(S)H), a sulfate (e.g., S(O)42-), a sulfonyl (e.g., -S(O)2-), an amide (e.g., -C(O)NR2, or -N(R)C(O)R), an azido (e.g., -N3), a nitro (e.g., -NO2), a cyano (e.g., -CN), an isocyano (e.g., -NC), an acyloxy (e.g.,-OC(O)R), an amino (e.g., -NR2, -NRH, or -NH2), a carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), a sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)S(O)2H, or -N(H)S(O)2H), an alkyl group, an alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R is an alkyl or alkenyl group, as defined herein. In some embodiments, the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. In some embodiments, a C1-6alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein. As used herein, the terms “approximately” and “about”, as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). In some embodiments, when used in the context of an amount of a given compound in a lipid component of a lipid assembly, “about” may mean + / - 10% of the recited value. For instance, a lipid assembly including a lipid component having about 40% of a given compound may include 30-50% of the compound. As used herein, the term “compound,” is meant to include all isomers and isotopes of the structure depicted. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. In some embodiments, isotopes of hydrogen include tritium and deuterium. Further, a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods. As used herein, the term “upon” intends to refer to the time point being after an action happens. For example, “upon administration” refers to the time point being after the action of administration. As used herein, the term “contacting” means establishing a physical connection between two or more entities. In some embodiments, contacting a mammalian cell with a TLA means that the mammalian cell and a nanoparticle are made to share a physical connection. Methods of contacting cells with external entities both in vivo and ex vivo are well known in the biological arts. In some embodiments, contacting a TLA and a mammalian cell disposed within a mammal may be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and may involve varied amounts of lipid assemblies. Moreover, more than one mammalian cell may be contacted by a TLA. As used herein, the term “comparable method” refers to a method with comparable parameters or steps, as of the method being compared (e.g., the producing the TLA formulation of the present disclosure). In some embodiments, the “comparable method” is a method with one or more of steps i), ia), iaa), ib), ii), iia), iib), iic), iid), and iie) of the method being compared. In some embodiments, the “comparable method” is a method without one or more of steps i), ia), iaa), ib), ii), iia), iib), iic), iid), and iie) of the method being compared. In some embodiments, the “comparable method” is a method without one or more of steps ia) and ib) of the method being compared. In some embodiments, the “comparable method” is a method employing a water- soluble salt of a nucleic acid. In some embodiments, the “comparable method” is a method employing an organic solution that does not comprise an organic solvent-soluble nucleic acid. In some embodiments, the “comparable method” is a method comprising processing the TLA prior to administering the TLA formulation. As used herein, the term “delivering” means providing an entity to a destination. In some embodiments, delivering a therapeutic and / or prophylactic to a subject may involve administering a TLA including the therapeutic and / or prophylactic to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a TLA to a mammal or mammalian cell may involve contacting one or more cells with the TLA. As used herein, the term “enhanced delivery” means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to the level of delivery of a therapeutic and / or prophylactic by a control nanoparticle to a target tissue of interest (e.g., MC3, KC2, or DLinDMA). The level of delivery of a nanoparticle to a particular tissue may be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of therapeutic and / or prophylactic in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of therapeutic and / or prophylactic in a tissue to the amount of total therapeutic and / or prophylactic in said tissue. It will be understood that the enhanced delivery of a nanoparticle to a target tissue need not be determined in a subject being treated, it may be determined in a surrogate such as an animal model (e.g., a rat model). As used herein, the term “specific delivery,” “specifically deliver,” or “specifically delivering” means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3- fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to an off-target tissue (e.g., mammalian spleen). The level of delivery of a nanoparticle to a particular tissue may be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of therapeutic and / or prophylactic in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of therapeutic and / or prophylactic in a tissue to the amount of total therapeutic and / or prophylactic in said tissue. In some embodiments, for renovascular targeting, a therapeutic and / or prophylactic is specifically provided to a mammalian kidney as compared to the liver and spleen if 1.5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold more therapeutic and / or prophylactic per 1 g of tissue is delivered to a kidney compared to that delivered to the liver or spleen following systemic administration of the therapeutic and / or prophylactic. It will be understood that the ability of a nanoparticle to specifically deliver to a target tissue need not be determined in a subject being treated, it may be determined in a surrogate such as an animal model (e.g., a rat model). As used herein, “encapsulation efficiency” refers to the amount of a therapeutic (e.g., a therapeutic polynucleotide) and / or prophylactic (e.g., a prophylactic polynucleotide) that becomes part of a TLA (e.g., a LNP), relative to the initial total amount of therapeutic and / or prophylactic used in the preparation of a TLA. For example, if 97 mg of therapeutic and / or prophylactic are encapsulated in a TLA out of a total 100 mg of therapeutic and / or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. As used herein, “encapsulation”, “encapsulated”, “loaded”, and “associated” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement. As used herein, “encapsulation” or “association” may refer to the process of confining an individual nucleic acid molecule within a nanoparticle and / or establishing a physiochemical relationship between an individual nucleic acid molecule and a nanoparticle. As used herein, an “empty nanoparticle” may refer to a nanoparticle that is substantially free of a therapeutic or prophylactic agent. As used herein, an “empty nanoparticle” may refer to a nanoparticle that is substantially free of a nucleic acid. As used herein, an “empty nanoparticle” may refer to a nanoparticle that consists substantially of only lipid components. As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide or protein and / or post-translational modification of a polypeptide or protein. As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe). As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof). As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment. As used herein, the term “isomer” means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds may include one or more chiral centers and / or double bonds and may thus exist as stereoisomers, such as double-bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). The present disclosure encompasses any and all isomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known. As used herein, a “lipid component” is that component of a TLA that includes one or more lipids. In some embodiments, the lipid component may include one or more cationic / ionizable, PEGylated, structural, or other lipids, such as phospholipids. As used herein, a “linker” is a moiety connecting two moieties, for example, the connection between two nucleosides of a cap species. A linker may include one or more groups including but not limited to phosphate groups (e.g., phosphates, boranophosphates, thiophosphates, selenophosphates, and phosphonates), alkyl groups, amidates, or glycerols. In some embodiments, two nucleosides of a cap analog may be linked at their 5’ positions by a triphosphate group or by a chain including two phosphate moieties and a boranophosphate moiety. As used herein, “methods of administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. A method of administration may be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body. As used herein, “modified” means non-natural. In some embodiments, an RNA may be a modified RNA. That is, an RNA may include one or more nucleobases, nucleosides, nucleotides, or linkers that are non-naturally occurring. A “modified” species may also be referred to herein as an “altered” species. Species may be modified or altered chemically, structurally, or functionally. In some embodiments, a modified nucleobase species may include one or more substitutions that are not naturally occurring. As used herein, the “N:P ratio” is the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in a lipid to phosphate groups in an RNA, e.g., in a TLA including a lipid component and an RNA. As used herein, “naturally occurring” means existing in nature without artificial aid. As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition. As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component. As used herein, a “polymeric lipid” refers to a lipid comprising repeating subunits in its chemical structure. In some embodiments, the polymeric lipid is a lipid comprising a polymer component. In some embodiments, the polymeric lipid is a PEG lipid. In some embodiments, the polymeric lipid is not a PEG lipid. In some embodiments, the polymeric lipid is Brij or OH-PEG- stearate. The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, composition, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complication, commensurate with a reasonable benefit / risk ratio. The phrase “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending, complexing, or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein. Compositions may also include salts of one or more compounds. Salts may be pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting an existing acid or base moiety to its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. In some embodiments, the nonaqueous media are ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., 1985, p.1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety. As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). A phospholipid or an analog or derivative thereof may include choline. A phospholipid or an analog or derivative thereof may not include choline. Particular phospholipids may facilitate fusion to a membrane. In some embodiments, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell. As used herein, an amphiphilic “polymer” is an amphiphilic compound that comprises an oligomer or a polymer. In some embodiments, an amphiphilic polymer can comprise an oligomer fragment, such as two or more PEG monomer units. In some embodiments, an amphiphilic polymer described herein can be PS 20. As used herein, the term “polypeptide” or “polypeptide of interest” refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically. As used herein, an “RNA” refers to a ribonucleic acid that may be naturally or non- naturally occurring. In some embodiments, an RNA may include modified and / or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. In some embodiments, an RNA may be a messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. RNAs may be selected from the non-liming group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, long non-coding RNA (lncRNA) and mixtures thereof. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event. As used herein, a “split dose” is the division of a single unit dose or total daily dose into two or more doses. As used herein, a “total daily dose” is an amount given or prescribed in a 24 hour period. It may be administered as a single unit dose. As used herein, the term “subject” refers to any organism to which a composition or formulation in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. As used herein, “targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in the tissue or organ of an organism. The organism may be an animal. In some embodiments, the organism is a mammal. In some embodiments, the organism is a human. In some embodiments, the organism is a patient. As used herein, “target tissue” refers to any one or more tissue types of interest in which the delivery of a therapeutic and / or prophylactic would result in a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. In particular applications, a target tissue may be a kidney, a lung, a spleen, vascular endothelium in vessels (e.g., intra-coronary or intra-femoral), or tumor tissue (e.g., via intratumoral injection). An “off-target tissue” refers to any one or more tissue types in which the expression of the encoded protein does not result in a desired biological and / or pharmacological effect. In particular applications, off-target tissues may include the liver and the spleen. The term “therapeutic agent” or “prophylactic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents are also referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids. As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition. As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. In some embodiments, “treating” cancer may refer to inhibiting survival, growth, and / or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. As used herein, the term “zeta potential” refers to the electrokinetic potential of a lipid, e.g., in a particle composition. As used herein, the term “polydispersity”, “polydispersity index”, or “PDI” refers to a measurement of the distribution of molecular mass in a given sample. The polydispersity is calculated as Mw / Mn, in which Mw is the mass-average molar mass (or molecular weight) and Mn is the number-average molar mass (or molecular weight). As used herein, the “polydispersity index” is a ratio that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.3, indicates a narrow particle size distribution. It is understood that some properties of LNPs disclosed herein may be characterized by capillary zone electrophoresis (CZE). Capillary zone electrophoresis (CZE) refers to a separation technique which uses high voltage across a capillary to separate charged species based on their electrophoretic mobility. In some embodiments, the CZE is conducted with an acetate buffer (e.g., 50mM sodium acetate at pH 5). In some embodiments, the CZE is conducted with a reverse voltage of about 10kV across a 75um capillary of 20cm effective length. In some embodiments, the capillary is coated with polyethyleneimine. The term “mobility peak”, as used herein, refers to a peak representing the distribution of a substance (e.g., a population of LNPs) as measured by CZE. In some embodiments, the intensity of the mobility peak is detected by scattered light. It is understood that the intensity of the peak may indicate the amount of the portion of the substance at the position of the peak. In some embodiments, the position of the peak is calculated against a neutral reference standard (e.g., DMSO) being characterized by a mobility peak at 0, and a charged reference standard (e.g., benzylamine) being characterized by a mobility peak at 1.0. In some embodiments, a population of LNPs may exhibit more than one peak as measured by CZE, and unless indicated otherwise, the mobility peak refers to the peak having the greatest peak area among the more than one peaks. The term “free of”, as used herein, means not comprising the referenced component. For example, when a population, solution, or formulation is described as being “free of PEG lipid”, the population, solution, or formulation does not comprise PEG lipid (e.g., does not comprise a PEG lipid described herein (e.g., does not comprise PEG-DMG)). Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art can be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they can be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the present disclosure (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art. It is further appreciated that certain features, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control. Reference will now be made in detail to some specific embodiments contemplated by the present disclosure. While various embodiments are described herein, it will be understood that it is not intended to limit the present technology to the described embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the technology as defined by the appended claims. EXAMPLES Example 1. Effect of Downstream PEG Addition on LNP Stability To assess the effect of downstream PEG addition on LNP stability, LNPs were prepared according to the process illustrated in FIG. 3, wherein PEG lipid (PEG-DMG) was added in varied molar amounts at two selected points during the production process: (A) lipid solution before mixing (“Core”); and (B) after neutralization (post-addition (“PA”)). The rate of LNP diameter change per day (%) was determined using dynamic light scattering to measure particle diameter over the course of two weeks, followed by plotting the [((final diameter – initial diameter) / initial diameter) / days] at room temperature versus the molar amount of PEG-DMG added. As shown in FIG.4, the further downstream the addition of PEG-DMG, the less total PEG that was needed to achieve a physically stable formulation in terms of particle diameter. Thus, addition of PEG after nucleic acid addition (i.e., loading) appears to afford LNPs with greater stability, despite having no core PEG lipid. Referring to FIG.5, capillary zone electrophoresis (CZE) characterization of the obtained LNPs reveals that addition of only core PEG lipid results in relatively high mobility and broader mobility peaks (higher polydispersity) for a given PEG-DMG amount, relative to downstream PEG lipid addition. Without being bound to any theory, it is hypothesized that this enhanced stability, less mobility and uniformity for LNPs with no core PEG lipid stems from a more significant population of surface PEG, relative to core PEG. Example 2. Effect of Downstream PEG Addition on mRNA Encapsulation Efficiency Referring to FIG.6, the encapsulation efficiency of an mRNA construct was analyzed as a function of molar amount of PEG-DMG added at specific time points during the LNP production process. LNPs were made by mixing pre-formed eLNP with an aqueous buffer containing mRNA-1273 construct at pH 5, at a ratio of 1:1, using a microTee. The mixed product was then pH adjusted using Tris buffer to pH 7.5. For the PA PEG arms, PEG buffer was added at different amounts post-pH adjustment. The encapsulation efficiency was measured by ribogreen. As shown in FIG.6, the encapsulation efficiency decreases with increasing molar amount of PEG-DMG. Without being bound to any particular theory, it is hypothesized that the decreased encapsulation efficiency results from the blocking effect of increased amounts of PEG-DMG at the LNP surface. Additionally, the encapsulation efficiency increases, for a given PEG-DMG concentration, the further downstream the PEG addition in the LNP production process. Example 3. Effect of Core PEG Lipid on Stability of Empty Lipid Nanoparticles Made at a Representative Scale. To assess the effect of adding core PEG lipid on stability of empty lipid nanoparticles at a representative scale, 0 mol% core PEG and 0.5 mol% core PEG LNPs were synthesized. TFF was performed to remove ethanol from the mixed product, buffer exchange into 5 mM Sodium Acetate, and concentrated. After TFF, the eLNP were diluted to target 74.5 mg / mL with cryoprotectant buffer and physical stability was monitored at RT and 5C post-F / T. LNP mixes were performed using a 1 mm Zepheros batch-mode unit operation utilizing mRNA-1273 construct. Post mix, the LNPs were neutralized using 120mM Tris, 323 g / L Sucrose, pH 8.3 (20% v / v) buffer which increases the buffer pH above the pKa of the ionizable lipid. A bolus (20% v / v) of PEG Buffer (20 mM Tris, 9 g / L PEG2000-DMG, pH 7.5) was then added to attain a desirable final target LNP PEG composition. For the 0% core PEG formulation, 2.5% PEG- DMG was added compared to our control of 2% PEG-DMG PA, for a final PEG content of 2.5% in both formulations. FIG. 7A and FIG.7B compare the stability (size) at 5℃ and room temperature, respectively, of eLNPs prepared with 0% and 0.5 mol% PEG-DMG at 5℃ over time. At 5℃, the obtained eLNPs showed comparable liquid stability. However, at room temperature, the eLNPs comprising 0.5 mol% PEG-DMG showed slightly better liquid stability after 15 days. The results indicate that eLNPs prepared with 0 mol% and 0.5 mol% PEG lipid have comparable stability, and eLNPs with 0% PEG are viable candidates as intermediate materials that can be held or stored and later loaded with an active agent (e.g., a therapeutic mRNA). To assess the robustness of the LNPs prepared with 0 mol% and 0.5 mol% core PEG, LNP formulations were prepared by subjecting the eLNP solutions to one freeze-thaw cycle at - 70℃ for 1 week. A first batch of eLNP was thawed then stored at room temperature and 5℃ for 28 days and size was measured and recorded at the 28-day mark. The second batch of eLNP which was subjected to one freeze-thaw cycle was loaded with nucleic acid immediately after thawing with no hold time. The loading step involved mixing the eLNPs with a nucleic acid solution comprising an mRNA construct, followed by neutralization and addition of 2 or 2.5 mol% PEG-DMG for a final PEG content of 2.5% in the final LNP formulation. The stability of the eLNPs and loaded LNPs is summarized in Table 1. Table 1. Average LNP Size for LNPs with 0 mol% and 5 mol% Core PEG FIG. 8 shows protein expression results achieved using the LNPs having 0 mol% core PEG is equivalent, within experimental error, to that achieved using LNPs having 0.5 mol% core PEG. This assay involved transfecting LNPs to HeLa cells at 40ng / well dose in 96 well plates. After 20-24h incubation, cells were lysed to extract the proteins. The proteins were reduced with DTT, alkylated by IAM, buffer exchanged into ammonium acetate, and digested into peptides with trypsin by overnight incubation at 37C. After the reaction is quenched by formic acid, the mixture was injected to HPLC-MS / MS for quantification of a fragment ion from the signature peptide that is specific to mRNA-1273 encoded spike protein. Moreover, eLNPs having 0 mol% and 0.5 mol% PEG-DMG achieved encapsulation efficiency of >97%. Thus, LNPs without core PEG lipid achieve stability, protein expression and encapsulation efficiency on par with LNPs having 0.5 mol% PEG lipid. Example 4. Evaluating PEG-less eLNP Robustness Across Different Stress Models To assess the robustness of PEG-less eLNPs prepared according to the methods of the present disclosure, eLNPs were produced by mixing lipid solutions comprising 0 mol% and 0.5 mol% PEG-DMG, followed by tangential flow filtration, to produce an eLNP solution similar to the method used in Example 3. The eLNP solutions where then subjected to a series of F / T cycles (up to 6 cycles) to determine the effect of core PEG concentration on particle size and polydispersity index (PDI). FIG.9A and FIG.9B show the variation in LNP size and PDI, respectively, with the number of F / T cycles. The data indicates that eLNPs with 0 mol% PEG lipid have better particle size and PDI stability across six freeze-thaw cycles than eLNPs with 0.5 mol% PEG lipid. FIG. 10A and FIG.10B show the effect of total lipid concentration on the robustness of eLNPs with 0 mol% PEG and 0.5 mol% PEG. The data shows that PEG-less eLNPs and eLNPs with 0.5 mol% PEG show better F / T stability at lower lipid concentrations than at higher concentrations. To further assess eLNP robustness, eLNPs were subjected to two different stress models: (1) a single freeze-thaw cycle, followed by holding for 3 days before mRNA loading; or (2) two freeze-thaw cycles before mRNA loading. The size data are summarized in Table 2. Table 2. eLNP Size and PDI Stability Across Two Different F / T Stress Models Lastly, loaded LNPs were produced from the eLNPs after the two different F / T treatments discussed above, using a method similar to that described in Example 2. The eLNPs were mixed with a nucleic acid solution comprising an mRNA construct, followed by a freeze- thaw cycle and clarification. Table 3 summarizes the diameter, PDI, and encapsulation efficiency observed for the loaded LNPs, as a function of PEG concentration and stress model. Table 3. Diameter, PDI, and Encapsulation Efficiency for LNPs Produced From eLNPs Subjected to F / T Stress The data shows that particle size and PDI are similar for LNPs produced using eLNPs with 0 mol% and 0.5 mol% PEG lipid. The LNPs with 0 mol% core PEG had larger sizes and lower PDIs than LNPs with 0.5 mol% core PEG. Comparable encapsulation efficiency was observed across all four treatment conditions. The effect on in vitro protein expression of core PEG concentration and stress treatment was compared for LNPs with 0 mol% core PEG and 0.5 mol% core PEG. FIG.11 shows expression for four different LNP lots. LNPs with 0 mol% core PEG afforded higher expression than the 0.5 mol% core PEG samples across both stress models. Further, the 0 mol% core PEG formulation outperformed the GMP control for both 2 × F / T and 3 day post F / T hold stress models. 2 × F / T stress model condition resulted in higher in vitro expression than the 3 day post- thaw hold condition. Lastly, the rate of PEG-DMG degradation was measured over time for four different LNP samples (0 mol% core PEG and 0.5 mol% core PEG, each with two different stress models). This assay was carried out using a stock solution of esterase from porcine liver prepared at a concentration of 0.5 U / µL in 20 mM Tris-HCl buffer. The LNP samples were mixed with the esterase stock solution and Tris-HCl buffer to achieve final concentrations of 0.2 mg / mL PEG- DMG and 0.05 U / µL esterase. The samples were incubated at room temperature and monitored at 0, 1, 2, 3, 4, and 24 hours. At each time point, a 100 µL aliquot from the reaction mixture was quenched with 400 µL of ethanol to inhibit the enzyme activity. Following completion of the enzymatic digestion, the samples were prepared for analysis by HPLC-CAD to obtain remaining PEG content. PEG-DMG degradation kinetics were analyzed using one-phase decay nonlinear regression in GraphPad to determine the rate constant (K) for comparison of PEG-DMG accessibility across samples. As shown in FIG. 12, LNP samples for which the eLNPs had undergone the F / T + hold exhibited a higher PEG-DMG degradation rate than LNPs for which the eLNPs had undergone multiple F / T cycles. Thus, there appears to be more PEG shedding from the surface after multiple F / T cycles. Further, LNPs with 0 mol% core PEG showed a higher degradation rate than LNPs with 0.5 mol% core PEG, which may stem from a higher amount of accessible surface PEG for LNPs with 0 mol% core PEG when compared to LNPs with 0.5 mol% core PEG. NUMBERED EMBODIMENTS 1. A lipid nanoparticle (LNP), comprising: an ionizable amino lipid (IAL); a structural lipid; and a phospholipid, wherein the lipid nanoparticle comprises 5 mol% or less of polymeric lipids. 2. The lipid nanoparticle of embodiment 1, wherein the LNP comprises 3 mol% or less of polymeric lipids. 3. The lipid nanoparticle of embodiment 1, wherein the LNP comprises 1 mol% or less of polymeric lipids. 4. The lipid nanoparticle of embodiment 1, wherein the LNP comprises 0.1 mol% or less of polymeric lipids. 5. The lipid nanoparticle of embodiment 1, wherein the LNP does not comprise any polymeric lipids. 6. The lipid nanoparticle of any one of embodiments 1-4, wherein the LNP does not comprise any PEG lipids. 7. The lipid nanoparticle of any one of embodiments 1-6, wherein the IAL is a compound of Formula (IL*): or a salt thereof, wherein: R1is -OH, -NRN-C4-10cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN’’); RNis H or C1-6 alkyl; RN’is H or C1-6 alkyl; RN’’is H or C1-6alkyl; o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8; m is 4, 5, 6, 7, or 8; M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2; M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3; R2is or –(C1-6 alkylene)-(C3-8 cycloalkyl)-C1-6 alkyl; R2ais -H or C1-10alkyl; R2bis -H or C1-10alkyl; R2cis C1-8 alkyl or C2-8 alkenyl; R3is 3 Rais H or C1-10alkyl; R3bis H or C1-8 alkyl; and R3cis C1-10 alkyl or C2-8 alkenyl. 8. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL**-I): or a salt thereof, wherein: R1is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2cis C4-8alkyl; R3ais C7-10alkyl; and R3cis C3-5 alkyl. 9. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL**-III): or a salt thereof, wherein: R1is NRN-C4-10cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN’’); RNis H; RN’is C1-2 alkyl; RN’’is H; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2ais C7-10alkyl; R2cis C4-6alkyl; R3ais C1-3 alkyl; and R3cis C4-6 alkyl. 10. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL**-IV): or a salt thereof, wherein: R1is OH; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2bis C3-5alkyl; R2cis C2-4 alkyl; R3ais C7-10 alkyl; and R3cis C4-6 alkyl. 11. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-I): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; and R3ais C1-8 alkyl. 12. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-Ia): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8 alkyl. 13. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-Ia’): or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais C1-8alkyl. 14. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-IIa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais C1-8 alkyl. 15. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-II’): or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais C1-8 alkyl.

[0008] 16. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-III): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8alkyl. 17. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-IIIa): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8 alkyl; and R3ais C1-8 alkyl. 18. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-IIIa’): (IL*-IIIa’) or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8 alkyl. 19. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-IIIb): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8alkyl. 20. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-IIIb’): or a salt thereof, wherein: R1, o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8 alkyl; and R3ais C1-8 alkyl. 21. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-IV): or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8alkyl; and R3ais C1-8alkyl. 22. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-IVa): or a salt thereof, wherein : R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; R2bis a C1-8 alkyl; and R3ais C1-8 alkyl. 23. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound of Formula (IL*-Iva’): or a salt thereof, wherein: o, M, M’, R2c, and R3care as defined for variable IL*; R2ais a C1-8alkyl; and R3ais C1-8 alkyl. 24. The lipid nanoparticle of any one of embodiments 1-7, wherein the IAL is a compound selected from: 25. The lipid nanoparticle of any one of embodiments 1-24, wherein the phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl- sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3- phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero- 3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1- glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. 26. The lipid nanoparticle of any one of embodiments 1-25, wherein the phospholipid comprises DSPC. 27. The lipid nanoparticle of any one of embodiments 1-26, wherein the structural lipid is selected from the group consisting of: cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, derivatives thereof, and mixtures thereof. 28. The lipid nanoparticle of any one of embodiments 1-27, wherein the structural lipid comprises cholesterol or a derivative thereof. 29. The lipid nanoparticle of any one of embodiments 1-28, wherein the lipid nanoparticle comprises 30-60 mol% of the IAL, 15-50 mol% of the structural lipid, and 0-30 mol% of the phospholipid. 30. The lipid nanoparticle of any one of embodiments 1-29, wherein the lipid nanoparticle comprises 45-50 mol.% of the ionizable amino lipid, 8-12 mol.% of the phospholipid, 35-40 mol.% of the structural lipid. 31. The lipid nanoparticle of any one of embodiments 1-30, wherein the LNP has a mean diameter of about 100 nm to about 500 nm. 32. The lipid nanoparticle of any one of embodiments 1-31, wherein the LNP has a mean diameter of about 10 nm to about 200 nm, preferably about 15 nm to about 150 nm, preferably about 90 nm to about 130 nm. 33. The lipid nanoparticle of any one of embodiments 1-32, wherein the LNP is suitable to achieve a polynucleotide encapsulation efficiency of at least about 80%. 34. The lipid nanoparticle of any one of embodiments 1-33, wherein the LNP is suitable to achieve a polynucleotide encapsulation efficiency of at least about 97%. 35. A lipid nanoparticle (LNP), consisting of: one or more ionizable amino lipids (IALs); one or more structural lipids; and one or more phospholipids. 36. An empty lipid nanoparticle solution, comprising: the lipid nanoparticle according to any one of embodiments 1-35; and a carrier. 37. The empty lipid nanoparticle solution according to embodiment 36, further comprising a cryoprotectant. 38. The empty lipid nanoparticle solution according to embodiment 37, wherein the cryoprotectant comprises a sugar. 39. The empty lipid nanoparticle solution according to embodiment 37 or 38, wherein the cryoprotectant comprises sucrose. 40. The empty lipid nanoparticle solution according to any one of embodiments 36-39, further comprising an organic solvent. 41. The empty lipid nanoparticle solution according to any one of embodiments 36-40, further comprising a buffering agent. 42. The empty lipid nanoparticle solution according to embodiment 41, wherein the buffering agent comprises a citrate buffer, an acetate buffer, a phosphate buffer, or a tris buffer. 43. The empty lipid nanoparticle solution according to embodiment 41 or 42, wherein the buffering agent is present at a concentration about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. 44. The empty lipid nanoparticle solution according to any one of embodiments 36-43, wherein the eLNP composition has a pH of about 4.8 to about 5.8, about 4.9 to about 5.75, or about 5.0 to about 5.5. 45. A loaded lipid nanoparticle, comprising: the lipid nanoparticle according to any one of embodiments 1-35; and a nucleic acid encapsulated within the lipid nanoparticle. 46. The loaded lipid nanoparticle of embodiment 45, wherein the nucleic acid comprises a ribonucleic acid. 47. The loaded lipid nanoparticle of embodiment 46, wherein the ribonucleic acid is at least one selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (lncRNA). 48. The loaded lipid nanoparticle of any one embodiments 45-47, wherein the nucleic acid is a messenger RNA (mRNA). 49. The loaded lipid nanoparticle of embodiment 48, wherein the mRNA is at least 30 nucleotides in length. 50. The loaded lipid nanoparticle of embodiment 48, wherein the mRNA is at least 300 nucleotides in length. 51. A loaded lipid nanoparticle solution, comprising: the loaded lipid nanoparticle of any one of embodiments 45-50; and a carrier. 52. The loaded lipid nanoparticle solution of embodiment 51, wherein the loaded lipid nanoparticle further comprises 0.1 mol% to 5 mol% of a polymeric lipid. 53. The loaded lipid nanoparticle solution of embodiment 52, wherein the polymeric lipid comprises a PEG lipid. 54. The loaded lipid nanoparticle solution of any one of embodiments 51-53, further comprising a cryoprotectant. 55. The loaded lipid nanoparticle solution of embodiment 54, wherein the cryoprotectant comprises a sugar. 56. The loaded lipid nanoparticle solution of embodiment 54 or 55, wherein the cryoprotectant comprises sucrose. 57. The loaded lipid nanoparticle solution according to any one of embodiments 51-56, further comprising a buffering agent. 58. The loaded lipid nanoparticle solution according to embodiment 57, wherein the buffering agent in the loaded lipid nanoparticle solution comprises a citrate buffer, an acetate buffer, a phosphate buffer, or a tris buffer. 59. The loaded lipid nanoparticle solution according to embodiment 57 or 58, wherein the buffering agent is present at a concentration about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. 60. The loaded lipid nanoparticle solution according to any one of embodiments 51-59, wherein the loaded LNP composition has a pH of about 4.5 to about 5.5, about 4.6 to about 5.4, about 4.7 to about 5.3, or about 4.8 to about 5.2. 61. A method of producing an empty lipid nanoparticle (eLNP), the method comprising: mixing a lipid solution comprising an ionizable amino lipid, a phospholipid, and a structural lipid with a first aqueous buffer solution comprising a first buffering agent, to obtain an eLNP solution comprising the eLNP, wherein the eLNP comprises 5 mol% or less of polymeric lipids. 62. The method of embodiment 61, wherein the eLNP comprises 3 mol% or less of polymeric lipids. 63. The method of embodiment 61, wherein the eLNP comprises 1 mol% or less of polymeric lipids. 64. The method of embodiment 61, wherein the eLNP comprises 0.1 mol% or less of polymeric lipids. 65. The method of any one of embodiments 61-64, wherein the eLNP does not comprise any PEG lipids. 66. The method of any one of embodiments 61-65, wherein the eLNP does not comprise any polymeric lipids. 67. The method of any one of embodiments 61-66, wherein the eLNP consists of one or more IALs, one or more phospholipids, and one or more structural lipids. 68. The method of any one of embodiments 61-67, wherein the first buffering agent comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. 69. The method of any one of embodiments 61-68, wherein the first buffering agent is present in the first aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. 70. The method of any one of embodiments 61-69, wherein the eLNP solution has a pH of about 4.8 to about 5.8, about 4.9 to about 5.75, or about 5.0 to about 5.5. 71. The method of any one of embodiments 61-70, further comprising adding a cryoprotectant to the eLNP solution. 72. The method of embodiment 71, wherein the eLNP solution comprises about 1 mg / mL to about 1000 mg / mL of the cryoprotectant. 73. The method of embodiment 71 or 72, wherein the cryoprotectant comprises a sugar. 74. The method of embodiment 73, wherein the sugar comprises sucrose. 75. The method of any one of embodiments 61-74, further comprising processing the eLNP solution. 76. The method of embodiment 75, wherein the processing the eLNP solution comprises adjusting the pH of the eLNP solution. 77. The method of embodiment 76, wherein the adjusting the pH of the eLNP solution comprises adding a second aqueous buffer solution comprising a second buffering agent to the eLNP solution. 78. The method of embodiment 77, wherein the second aqueous buffer solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. 79. The method of embodiment 77 or 78, wherein the second buffering agent is present in the second aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. 80. The method of any one of embodiments 75-79, wherein the processing the eLNP solution comprises filtering, buffer exchanging, lyophilizing, diluting, or storing the eLNP solution, or any combination thereof. 81. The method of any one of embodiments 75-80, wherein the processing the eLNP solution comprises filtering the eLNP solution by tangential flow filtration. 82. A method of producing a loaded lipid nanoparticle, the method comprising: mixing a lipid solution comprising an ionizable amino lipid, a phospholipid, and a structural lipid with a first aqueous buffer solution comprising a first buffering agent, to obtain an eLNP solution comprising the eLNP, wherein the eLNP comprises 5 mol% or less of polymeric lipids; processing the eLNP solution; loading the eLNP with a nucleic acid by mixing a nucleic acid solution comprising the nucleic acid with the processed eLNP solution to obtain a loaded LNP composition comprising the loaded LNP. 83. The method of embodiment 82, wherein the eLNP comprises 3 mol% or less of polymeric lipids. 84. The method of embodiment 82, wherein the eLNP comprises 1 mol% or less of polymeric lipids. 85. The method of embodiment 82, wherein the eLNP comprises 0.1 mol% or less of polymeric lipids. 86. The method of any one of embodiments 82-85, wherein the eLNP does not comprise any PEG lipids. 87. The method of any one of embodiments 82-86, wherein the eLNP does not comprise any polymeric lipids. 88. The method of any one of embodiments 82-87, wherein the eLNP consists of one or more IALs, one or more phospholipids, and one or more structural lipids. 89. The method of any one of embodiments 82-88, wherein the first buffering agent comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. 90. The method of any one of embodiments 82-89, wherein the first buffering agent is present in the first aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. 91. The method of any one of embodiments 82-90, wherein the eLNP solution has a pH of about 4.8 to about 5.8, about 4.9 to about 5.75, or about 5.0 to about 5.5. 92. The method of any one of embodiments 82-91, wherein the processing the eLNP solution comprises adjusting the pH of the eLNP solution. 93. The method of embodiment 92, wherein the adjusting the pH of the eLNP solution comprises adding a second aqueous buffer solution comprising a second buffering agent to the eLNP solution. 94. The method of embodiment 93, wherein the second aqueous buffer solution comprises an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. 95. The method of embodiment 93 or 94, wherein the second buffering agent is present in the second aqueous buffer solution at about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. 96. The method of any one of embodiments 82-95, wherein the processing the eLNP solution comprises filtering, buffer exchanging, lyophilizing, diluting, or storing the eLNP solution, or any combination thereof. 97. The method of any one of embodiments 82-96, wherein the processing the eLNP solution comprises filtering the eLNP solution by tangential flow filtration. 98. The method of any one of embodiments 82-98, wherein the nucleic acid comprises a ribonucleic acid. 99. The method of embodiment 98, wherein the ribonucleic acid is at least one selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (lncRNA). 100. The method of any one of embodiments 82-99, wherein the nucleic acid is a messenger RNA (mRNA). 101. The method of embodiment 100, wherein the mRNA is at least 30 nucleotides in length. 102. The method of embodiment 100, wherein the mRNA is at least 300 nucleotides in length. 103. The method of any one of embodiments 82-102, wherein the nucleic acid solution comprises about 0.01 to about 1.0 mg / mL of the nucleic acid, about 0.05 to about 0.5 mg / mL of the nucleic acid, or about 0.1 to about 0.25 mg / mL of the nucleic acid. 104. The method of any one of embodiments 82-103, wherein the nucleic acid solution comprises a buffer selected from the group consisting of an acetate buffer, a citrate buffer, a phosphate buffer, and a tris buffer. 105. The method of any one of embodiments 82-104, wherein the nucleic acid solution comprises from about 1 mM to about 200 mM acetate buffer, from about 2 mM to about 180 mM acetate buffer, from about 3 mM to about 160 mM acetate buffer, from about 4 mM to about 150 mM acetate buffer, from about 4 mM to about 140 mM acetate buffer, from about 5 mM to about 130 mM acetate buffer, from about 6 mM to about 120 mM acetate buffer, from about 7 mM to about 110 mM acetate buffer, from about 8 mM to about 100 mM acetate buffer, from about 9 mM to about 90 mM acetate buffer, from about 10 mM to about 80 mM acetate buffer, from about 15 mM to about 70 mM acetate buffer, from about 20 mM to about 60 mM acetate buffer, from about 25 mM to about 50 mM acetate buffer, or from about 30 mM to about 40 mM acetate buffer. 106. The method of any one of embodiments 82-105, wherein the nucleic acid solution and the empty-LNP solution are mixed at a volumetric flow ratio of from about 5:1 to about 7:1, from about 4:1 to about 6:1, from about 3:1 to about 5:1, or from about 2:1 to about 4:1 during the loading. 107. The method of any one of embodiments 82-106, further comprising processing the loaded LNP solution to obtain a LNP formulation. 108. The method of embodiment 107, wherein the processing the loaded LNP solution comprises adding a polymeric lipid to the loaded LNP. 109. The method of embodiment 108, wherein the polymeric lipid comprises a PEG lipid. 110. The method of embodiment 109, wherein the adding the PEG lipid comprises mixing a PEG lipid solution comprising the PEG lipid with the loaded LNP solution. 111. The method of embodiment 109, wherein the adding the PEG lipid comprises adding the PEG lipid in an amount of from about 0.1 mol% to about 3.0 mol% PEG lipid, from about 0.2 mol% to about 2.5 mol% PEG lipid, from about 0.5 mol% to about 2.0 mol% PEG lipid, from about 0.75 mol% to about 1.5 mol% PEG lipid, from about 1.0 mol% to about 1.25 mol% PEG lipid, from about 0.2 mol% to about 0.8 mol%, 5 mol% or less, 3 mol% or less, 1 mol% or less, or 0.1 mol% or less PEG lipid to the loaded LNP. 112. The method of any one of embodiments 109-111, wherein the PEG lipid is PEG-DMG or a compound of Formula (VI): wherein r is 40-50, preferably 45; 113. The method of any one of embodiments 107-112, wherein the processing further comprises buffer exchanging, filtering, lyophilizing, or storing the loaded LNP solution. 114. The method of any one of embodiments 82-113, wherein the loaded LNP solution or the LNP formulation further comprising a cryoprotectant. 115. The method of embodiment 114, wherein the cryoprotectant comprises a sugar. 116. The method of embodiment 114 or 115, wherein the cryoprotectant comprises sucrose. 117. The method of any one of embodiments 82-116, wherein the loaded LNP solution or the LNP formulation further comprises a buffering agent. 118. The method of embodiment 117 wherein the buffering agent in the loaded LNP solution or the LNP formulation comprises a citrate buffer, an acetate buffer, a phosphate buffer, or a tris buffer. 119. The method of embodiment 117 or 118, wherein the buffering agent in the loaded LNP solution or the LNP formulation is present at a concentration about 1 mM to about 30 mM, about 2 mM to about 20 mM, about 3 mM to about 10 mM, about 4 mM to about 8 mM, or about 5 mM to about 6 mM. 120. The method of any one of embodiments 82-120, wherein the loaded LNP solution or the LNP formulation has a pH of about 4.5 to about 7.5, about 5.0 to about 7.4, about 5.5 to about 7.3, or about 6.0 to about 7.0. 121. An empty lipid nanoparticle, wherein the empty lipid nanoparticle is obtained by the method according to any one of embodiments 61-81. 122. A loaded lipid nanoparticle, wherein the loaded lipid nanoparticle is obtained by the method according to any one of embodiments 82-120. 123. A method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the loaded LNP of any one of embodiments 45-50 or 122, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. 124. A method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the loaded LNP solution of any one of embodiments 51-60, wherein the loaded LNP solution comprises a therapeutically effective amount of a therapeutic mRNA. 125. A method of treating or preventing a disease or disorder, the method comprising administering to a subject in need thereof the LNP formulation of any one of embodiments 107-120, wherein the LNP formulation comprises a therapeutically effective amount of a therapeutic mRNA. 126. The method of any one of embodiments 123-125, wherein the administering is performed parenterally. 127. The method of any one of embodiments 123-125, wherein the administering is performed intramuscularly, intradermally, subcutaneously, and / or intravenously. 128. The loaded LNP of any one of embodiments 45-50 or 122, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. 129. The loaded LNP solution of any one of embodiments 51-60, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. 130. The LNP formulation of any one of embodiments 107-120, for use in treating or preventing a disease or disorder in a subject, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. 131. Use of the loaded LNP of any one of embodiments 45-50 or 122 in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. 132. Use of the loaded LNP solution of any one embodiments 51-60 in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. 133. Use of the LNP formulation of any one embodiments 107-120 in the manufacture of a medicament for treating or preventing a disease or disorder, wherein the loaded LNP comprises a therapeutically effective amount of a therapeutic mRNA. 134. A kit, comprising the empty LNP of any one of embodiments 1-35 or 121, the empty LNP solution of any one of embodiments 36-44, the loaded LNP of any one of embodiments 45-50 or 122, the loaded LNP solution of any one of embodiments 51-60, or LNP formulation of any one of embodiments 107-120. 135. A kit, comprising: a first container comprising the empty LNP of any one of embodiments 1-35 or 121 or the empty LNP solution of any one of embodiments 36-44; and a second container comprising a therapeutic agent. 136. The kit of embodiment 133, further comprising instructions for combining contents of the first container with contents of the second container. 137. The kit of embodiment 133 or 134, wherein the therapeutic agent comprises a therapeutic mRNA. * * * * Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set for...

Claims

1. WHAT IS CLAIMED IS:

1. A lipid nanoparticle (LNP), comprising: an ionizable amino lipid (IAL); a structural lipid; and a phospholipid, wherein the lipid nanoparticle comprises 5 mol% or less of polymeric lipids.

2. The lipid nanoparticle of claim 1, wherein the LNP comprises 3 mol% or less of polymeric lipids.

3. The lipid nanoparticle of claim 1, wherein the LNP comprises 1 mol% or less of polymeric lipids.

4. The lipid nanoparticle of claim 1, wherein the LNP comprises 0.1 mol% or less of polymeric lipids.

5. The lipid nanoparticle of claim 1, wherein the LNP does not comprise any polymeric lipids.

6. The lipid nanoparticle of any one of claims 1-4, wherein the LNP does not comprise any PEG lipids.

7. The lipid nanoparticle of any one of claims 1-6, wherein the IAL is a compound of Formula (IL*): or a salt thereof, wherein: R1is -OH, -NRN-C4-10cycloalkenyl optionally substituted with one or more oxo or - N(RN’RN’’); RNis H or C1-6 alkyl; RN’is H or C1-6alkyl; RN’’is H or C1-6alkyl; o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8; m is 4, 5, 6, 7, or 8; M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2; M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3; R2is or –(C1-6 alkylene)-(C3-8 cycloalkyl)-C1-6 alkyl; R2ais -H or C1-10 alkyl; R2bis -H or C1-10alkyl; R2cis C1-8 alkyl or C2-8 alkenyl; R3is 3a R is H or C1-10 alkyl; R3bis H or C1-8alkyl; and R3cis C1-10alkyl or C2-8alkenyl.

8. The lipid nanoparticle of any one of claims 1-7, wherein the IAL is a compound of Formula (IL**-I): or a salt thereof, wherein: R1is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2cis C4-8 alkyl; R3ais C7-10alkyl; and R3cis C3-5 alkyl.

9. The lipid nanoparticle of any one of claims 1-7, wherein the IAL is a compound of Formula (IL**-III): or a salt thereof, wherein: R1is NRN-C4-10cycloalkenyl optionally substituted with one or more oxo or -N(RN’RN’’); RNis H; RN’is C1-2 alkyl; RN’’is H; o is 2, 3, or 4; n is 6, 7, or 8; M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8; M’ is -C(=O)-O-*, wherein * indicates attachment to R3; R2ais C7-10 alkyl; R2cis C4-6alkyl; R3ais C1-3alkyl; and R3cis C4-6 alkyl.

10. The lipid nanoparticle of any one of claims 1-7, wherein the IAL is a compound selected from:

11. A loaded lipid nanoparticle, comprising: the lipid nanoparticle according to any one of claims 1-10; and a nucleic acid encapsulated within the lipid nanoparticle.

12. The loaded lipid nanoparticle of claim 11 wherein the nucleic acid is a messenger RNA (mRNA).

13. The loaded lipid nanoparticle of claim 12, wherein the mRNA is at least 30 nucleotides in length.

14. The loaded lipid nanoparticle of claim 12, wherein the mRNA is at least 300 nucleotides in length.

15. The loaded lipid nanoparticle solution of claim 11, wherein the loaded lipid nanoparticle further comprises 0.1 mol% to 5 mol% of a polymeric lipid.

16. The loaded lipid nanoparticle solution of claim 15, wherein the polymeric lipid comprises a PEG lipid.

17. A method of producing an empty lipid nanoparticle (eLNP), the method comprising: mixing a lipid solution comprising an ionizable amino lipid, a phospholipid, and a structural lipid with a first aqueous buffer solution comprising a first buffering agent, to obtain an eLNP solution comprising the eLNP, wherein the eLNP comprises 5 mol% or less of polymeric lipids.

18. The method of claim 17, wherein the eLNP comprises 1 mol% or less of polymeric lipids.

19. The method of any one of claims 17-18, wherein the eLNP does not comprise any PEG lipids.

20. The method of any one of claims 17-19, wherein the eLNP does not comprise any polymeric lipids.

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