Ionizable lipids for the delivery of nucleic acids

Abstract

Novel ionizable lipids, compositions, and methods of using the novel lipids and compositions are disclosed. Lipid nanoparticle compositions include novel lipids as well as additional lipids such as phospholipids, structural lipids, and PEG lipids. Lipid nanoparticle compositions further including biologically active agents such as mRNA are useful in the delivery of therapeutics, diagnostics and / or prophylactics to cells or organs.

Description

IONIZABLE LIPIDS FOR THE DELIVERY OF NUCLEIC ACIDSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 589,422 filed 11 October 2023, the entire contents of which are incorporated herein in their entirety by reference.TECHNICAL FIELD

[0002] This disclosure provides novel compounds, compositions comprising such compounds, and methods to deliver one or more biologically active agents in cells or organs.BACKGROUND

[0003] Delivery of biologically active substances, such as small molecule drugs, proteins, and nucleic acids including mRNA, is a medical challenge. In particular, the delivery of nucleic acids to cells is made difficult by the relative instability and low cell permeability of such molecules. Currently, FDA approved lipid nanoparticle (LNP) compositions require a mixture of four components: phospholipid(s); cholesterol; PEGylated lipid(s); and cationic or ionizable lipid(s), e.g., for delivery of biological materials including but not limited to mRNA vaccines and gene therapies.

[0004] Lipid nanoparticles are a versatile and successful gene delivery system, notably highlighted by their use in vaccines against COVID-19. Yet some issues exist for traditional LNPs, such as long-term stability, selective biodistribution and better performance to increasing global access to mRNA vaccines, especially in low-resource regions. There exists an unmet need to develop LNP compositions comprising improved ionizable lipids that are stable and there exists a need to develop compounds, compositions, and improve delivery of biologically active substances into cells or organs.SUMMARY OF THE INVENTION

[0005] The present disclosure includes novel ionizable lipid compounds and compositions and methods involving the same. Lipid nanoparticle compositions comprising the novel lipids are disclosed. Four-component lipid compositions include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids. Three-component lipid nanoparticle compositions contain a steroidal or structural lipid-containing component, a stabilizing lipid (such as PEGylated lipid-containing component), a novel cationic or ionizable lipid-containingcomponent of the disclosure. Lipid nanoparticle compositions with less than three components, e.g. one component or two components, and lipid nanoparticles with more than four components, e.g. five components or six components, are also contemplated.

[0006] In one aspect, the novel ionizable lipid compound is a compound of Formula I:(I), or a salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected fromwherein each m is independently selected from 0-6; each o is independently selected from 0-7; each R1and R2is, each X is independently selected from

[0007] In certain aspects, compounds of Formula I may include, but are not limited to:

[0008] Synthesis Scheme L General synthesis route for the synthesis of compounds ofFormula I.

[0009] In some aspects, R3and R4correspond to groups such that “Q” is satisfied. In some aspects, compounds of A-1, A-2, A-5, A-7, A-9 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art

[0010] In some aspects, the present disclosure provides a method for synthesizing the compound of PMA or 1(a) comprising performing the following reaction:

[0011] In some aspects, the present disclosure provides a method for synthesizing the compound of EL-396 or 1(b) comprising performing the following reaction:

[0012] In some aspects, the present disclosure provides a method for synthesizing the compound of EL-382 or 1(c) comprising performing the following reaction:

[0013] In some aspects, the present disclosure provides a method for synthesizing the compound of EL-360 or 1(d) comprising performing the following reaction:

[0014] In one aspect, the novel ionizable lipid compound is a compound of Formula II:(II), or a salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected fromwherein each m is independently selected from 0-6 and o is independently selected from 0-7 and;

[0015] In certain aspects, compounds of Formula II may include, but are not limited to:

[0016] Synthesis Scheme IL General synthesis route for the synthesis of compounds of Formula II

[0017] In some aspects, R3and R4correspond to groups such that “Q” is satisfied. In some aspects, compounds of B-1, B-2, B-4, B-6, and B-8 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art

[0018] In some aspects, the present disclosure provides a method for synthesizing the compound of EW-1 or n(a) comprising performing the following reaction:

[0019] A method for synthesizing the compound of EW-2 or 11(b) comprising performing the following reaction:

[0020] In some aspects, the present disclosure provides a method for synthesizing the compound of N1 or 11(c) comprising performing the following reaction:

[0021] In some aspects, the present disclosure provides a method for synthesizing the compound of N4 or 11(d) comprising performing the following reaction:

[0022] In one aspect, the cationic or ionizable lipid-containing component may comprise compounds of Formula (IA):or a salt or isomer thereof, wherein m is 0-9; n is 0-9; o is 0-12; p is 0-12;Ri is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, - OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

[0023] In certain aspects, compounds of Formula IA may include, for example, the following compounds:

[0024] In another aspect, the present disclosure provides compounds of Formula (IB):or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12;R is the side chain of an independently selected amino acid;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or linear C1-12alkyl;R5is the side chain of an independently selected amino acid;X1is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;X2is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;X3is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-; andX4is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-.

[0025] In some aspects, R or R5comprises the side chain of a Serine (S), Threonine (1), Cysteine (C), Selenocysteine (U), Glycine (G), Alanine (A), Isoleucine (I), Leucine (L), Methionine (M), or Valine (V). In some aspects, the carbonyl group in Formula IB is bonded to the amino terminus of the amino acid. In some aspects, the carbonyl group in Formula IB is bonded to the carboxy terminus of the amino acid.

[0026] In certain aspects, compounds of Formula IB may include, for example, the following compounds:

[0027] In another aspect, the present disclosure provides compounds of Formula (IC):or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-5;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is H or CH3;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H; andX is selected from -CH2-, -O-, -S-, or -P(O)(OR)O-.

[0028] In certain aspects, compounds of Formula IC may include, for example, the following compounds:

[0029] In one aspect, the cationic or ionizable lipid-containing component may comprise compounds of Formula (HA):or a salt or isomer thereof, wherein m is selected from 0-5; n is selected from 0-12; o is selected from 0-12; q is selected from 1-3;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andX is selected from C(R)2, N(R), or O, wherein R is independently selected from a methyl and H.

[0030] In certain aspects, compounds of Formula HA may include, for example, the following compound.

[0031] In another aspect, the present disclosure provides compounds of Formula (IB):or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-6;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

[0032] In certain aspects, compounds of Formula HB may include, for example

[0033] In another aspect, the present disclosure provides compounds of Formula (IIC):or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

[0034] In certain aspects, compounds of Formula IIC may include, for example

[0035] In another aspect, the present disclosure provides compounds of Formula (HD):or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 1-7; o is selected from 0-12; p is selected from 0-12;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

[0036] In certain aspects, compounds of Formula IID may include, for example

[0037] In another aspect, the present disclosure provides compounds of Formula (HE):or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

[0038] In certain aspects, compounds of Formula HE may include, for example

[0039] In another aspect, the present disclosure provides compounds of Formula (IIF):or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and Ma are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

[0040] In certain aspects, compounds of Formula IIF may include, for example

[0041] The present disclosure includes lipid nanoparticle (LNP) comprising at least one ionizable lipid compound of the disclosure. In one aspect, the LNP further comprises a biologically active agent In some aspects, the biologically active agent is a nucleic acid. In some aspects, the biologically active agent is a RNA molecule. In some aspects, the biologically active agent is siRNA, miRNA, shRNA, tRNA, mRNA, circRNA and mixtures thereof.

[0042] In one aspect, the LNP further comprises a phospholipid selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-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), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1 -oleoyl-2- cholesteiylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero- 3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin. In some aspect, the phospholipid is DSPC.

[0043] In one aspect, the LNP further comprises a structural lipid selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some aspects, the structural component is cholesterol.

[0044] In one aspect, the LNP further comprises a PEG lipid selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG- modified dialkylglycerol, a PEG-modified myristoyl diglyceride, and mixtures thereof. In some aspects, the PEG lipid is DMG-PEG2000.

[0045] In one aspect, the components in the LNP have the following relative molar percentages:20 mol % to 60 mol % of an ionizable lipid component,25 mol % to 45 mol % of a structural lipid component, 0 mol % to 25 mol % of a phospholipid, or0 mol % to 5 mol % of a PEGylated lipid component.

[0046] In one aspect, the present disclosure provides a three-component LNP composition wherein the three components are:1) a steroidal or structural lipid-containing component;2) a PEGylated lipid-containing component; and3) a cationic or ionizable lipid-containing component.

[0047] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:1) 0.5 to 60 mole % of a steroidal or structural lipid-containing component;2) 0.5 to 20 mole % of a PEGylated lipid-containing component; and3) 25 to 97.2 mole % of a cationic or ionizable lipid-containing component

[0048] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:1 ) 20 to 50 mole % of a steroidal or structural lipid-containing component;2) 0.8 to 10 mole % of a PEGylated lipid-containing component; and3) 40 to 62 mole % of a cationic or ionizable lipid-containing component

[0049] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:1) 25 to 46 mole % of a steroidal or structural lipid-containing component;2) 1 to 7 mole % of a PEGylated lipid-containing component; and3) 44 to 58 mole % of a cationic or ionizable lipid-containing component

[0050] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:1) 35 to 44 mole % of a steroidal or structural lipid-containing component;2) 1.2 to 5 mole % of a PEGylated lipid-containing component; and3) 48 to 57 mole % of a cationic or ionizable lipid-containing component.

[0051] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:1) 20 to 35 mole % of a steroidal or structural lipid-containing component;2) 0.8 to 10 mole % of a PEGylated lipid-containing component; and3) 55 to 79 mole % of a cationic or ionizable lipid-containing component.

[0052] In one aspect, the cationic or ionizable lipid-containing component may comprise MC3, ALC-0315, ALC-0159, SM-102, DOTAP, or similar, or a cationic and / or ionizable lipid disclosed in WO2017049245 A2 (Benenato), which is incorporated herein by reference.

[0053] In one aspect, the LNP has an encapsulation efficiency of at least 70% after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a zeta potential (ZP) of about -30 to +30 mV, -25 to +25 mV, -20 to +20 mV, -15 to +15 mV, -10 to +10 mV, or any range or value in the range of -30 to +30 mV, after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a polydispersity index (PDI) of less than 0.300 after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a particle size of less than 220 nm after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a nitrogen to phosphorus ratio (N:P) range of about 10 to 1.

[0054] In one aspect, the nanoparticle composition has an encapsulation efficiency of at least 70% when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days. In one aspect, the nanoparticle composition according to any one of claims 25-35, wherein the nanoparticle composition has a zeta potential of -30 to + 30 mV when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days. In one aspect, the nanoparticle composition has a particle size of less than 220 nm when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days. In one aspect, the nanoparticle composition has a nitrogen to phosphorus ratio (N:P) of 5.6:1. “Stability,” “stabilized,” and “stable” in the context of the present disclosure refers to the resistance of nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and / or in-use conditions, e.g., when stress is applied such as shear force, freeze / thaw stress, etc.

[0055] In one aspect, the LNP further comprises a cryoprotectant. In some aspects, the cryoprotectant is sucrose. In some aspects, the cryoprotectant has a volume / volume concentration of 5-16% (v / v).

[0056] In one aspect, the LNP comprises an ionizable lipid of the disclosure, a structural lipid, and a PEG lipid.

[0057] In one aspect, the pharmaceutical composition comprising the LNP of the disclosure and a pharmaceutically acceptable carrier.

[0058] In one aspect, the present disclosure includes a method of delivering a biologically active agent to a cell, the method comprising administering to a subject the LNP of the disclosure, said administering comprising contacting the cell with the LNP, whereby the biologically active agent is delivered to the cell.

[0059] In one aspect, the present disclosure includes a method of delivering a biologically active agent to a cell of a subject, the method comprising administering a pharmaceutical composition of comprising the LNP of the disclosure and a pharmaceutically acceptable carrier to the subject.

[0060] In one aspect, the present disclosure includes a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the LNP of the disclosure.

[0061] In another aspect, the present disclosure provides a method of producing a polypeptide of interest in a cell (e.g., a mammalian cell) by contacting the cell with the LNP composition and an mRNA encoding the polypeptide of interest, whereby the mRNA is capable of being translated in the cell to produce the polypeptide.

[0062] In some aspects, an LNP comprising at least one ionizable lipid of the disclosure elicits an in vivo immune response comparable to that of a commercially available LNP, e.g. SMI 02 (Modema, SpikeVax).

[0063] Each of the aspects of the present disclosure can encompass various elements of the present disclosure. It is, therefore, anticipated that each of the aspects of the present disclosure involving any one element or combinations of elements can be included in each aspect of the present disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following detailed description or illustrated in the drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0064] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0065] Fig. 1 show in vivo transfection efficiency of LNPs formulated using ionizable lipids of the disclosure and control lipid SMI 02 in mouse sera 35 days after two intramuscular injections on mouse models with 3 mice per group.

[0066] Fig. 2 show in vivo data of end point titer in mouse sera after 35 days following two intramuscular injections in mouse models, with 3 mice per group, of LNPs formulated using ionizable lipids of the disclosure and control lipid SMI 02.

[0067] Fig. 3 show in vivo data of end point titer in mouse sera after 14 and 35 days following two intramuscular injections of LNPs formulated using ionizable lipids of the disclosure and control lipid SMI 02 encapsulated with mRNA encoding the omicron BA.1 variant of the spike protein of S ARS-CoV-2.

[0068] Fig. 4 show a comparison of the reciprocal IgG endpoint titer of 4-component LNP formulated with SM-102 compared to 3 -component LNPs formulated using either SM-102 or EL- PMA, demonstrating similar performance between all formulations comprising EL-PMA and SM- 102 ionizable lipids in mouse models.

[0069] Fig. 5 show a comparison of 4-component LNP formulated with SM-102 compared to 4-component and 3-component LNPs formulated using ionizable lipids of the disclosure - EW1 , EW2, PMA, and N4. Three ionizable lipids of the disclosure - EW2, PMA, and N4 - have similar in vivo performance relative to SM-102.

[0070] Fig. 6 shows aXH NMR spectrum of an ionizable lipid of the disclosure (EL-396).

[0071] Fig. 7 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EL-396).

[0072] Fig. 8 shows a *H NMR spectrum of an ionizable lipid of the disclosure (EL-382).

[0073] Fig. 9 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EL-382).

[0074] Fig. 10 shows a *H NMR spectrum of an ionizable lipid of the disclosure (EL-360).

[0075] Fig. 11 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EL-360).

[0076] Fig. 12 shows a NMR spectrum of an ionizable lipid of the disclosure (EW-1).

[0077] Fig. 13 shows an HPLC chromatogram of an ionizable lipid of the disclosure (EW- 1).

[0078] Fig. 14 shows a NMR spectrum of an ionizable lipid of the disclosure (EW-2).

[0079] Fig. 15 shows a NMR spectrum of an ionizable lipid of the disclosure (EL-N1).

[0080] Fig. 16 shows a NMR spectrum of an ionizable lipid of the disclosure (EL-N4).

[0081] Fig. 17 shows in vitro results from Table 6 and 7 demonstrating that mLNP transfects well after lyophilization compared to before lyophilization.

[0082] Fig. 18 shows Western Blot data before and after lyophilization (top panel). A graph showing transfection efficiency of mLNP and LNP before and after lyophilization is shown in the bottom panel.

[0083] Fig. 19 shows IgG binding antibody titers after the first (a) and the second (b) administration with mLNP and LNP. Compared with LNP group, mLNP group 1 showed higher IgG titer before lyophilization. The immunogenicity response of mLNP group 1 could be maintained after lyophilization.

[0084] Fig. 20 shows that PMA and N4 under “E” condition (mLNP) do not show in vitro expression, but showed excellent in vivo expression.

[0085] Fig. 21 shows that IgG titer from mouse models indicate excellent expression of the mRNA from the novel lipids under mLNP (E) conditions.

[0086] Fig. 22 shows that EW-2 and PMA have excellent T-Cell response in mouse models under mLNP conditions.

[0087] Fig. 23 shows Western blot analysis of HEK293T cells transfected with mRNA encoding Omicron BA.1 formulated various forms lipids in Table 1. After 48 hrs of transfection, Western Blot analysis was performed using anti-SARS-CoV-2 specific RBD antibody to detect the expression of spike protein.

[0088] Fig. 24 shows a plot of the reciprocal IgG Endpoint titer as a function of dose of various LNP and mLNP formulations.

[0089] Fig. 25 shows transfection test of PEG content screening data.

[0090] Fig. 26 shows transfection efficiency of N / P ratio screening (samples dialyzed with 8% sucrose). The transfection efficiency of mLNP was decreased when the N / P ratio was increased.

[0091] Fig. 27 shows IgG binding antibody titers after the second administration with mLNP and LNP. mLNP produces equivalent immune response with LNP in mice model, which is consistent with the previous results.

[0092] Fig. 28 shows mLNP sample pre frozen to -45°C (a) and collapse at -30°C.

[0093] Fig. 29 shows cake appearance after lyophilization.

[0094] Fig. 30 shows characterization of mLNP after lyophilization. Size (a) and zeta potential (b) of mLNP after 2nd batch lyophilization. Size (c) and zeta potential (d) of mLNP after 12th batch lyophilization.

[0095] Fig. 31 shows TEM images of SM-102 mLNP before and after lyophilization.

[0096] Fig. 32 shows TEM images of SM-102 mLNP before and after freezing.

[0097] Fig. 33 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by particle size over the course of 1 month at 25°C, 4°C, -20°C, and -80°C.

[0098] Fig. 34 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by particle size distribution over the course of 1 month at 25°C, 4°C, -20°C, and -80°C.

[0099] Fig. 35 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by zeta potential over the course of 1 month at 25°C, 4°C, -20°C, and -80°C.[000100] Fig. 36 shows stability of RSV-mLNP under four conditions or RSV-LNP as measured by encapsulation efficiency over the course of 1 month at 25°C, 4°C, -20°C, and -80°C. [000101] Fig. 37 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by particle size over the course of 1 month.[000102] Fig. 38 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by particle size distribution over the course of 1 month.[000103] Fig. 39 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by zeta potential over the course of 1 month.[000104] Fig. 40 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by encapsulation efficiency over the course of 1 month.[000105] Fig. 41 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by particle size over the course of 1 month.[000106] Fig. 42 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by particle size distribution over the course of 1 month.[000107] Fig. 43 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by zeta potential over the course of 1 month.[000108] Fig. 44 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by encapsulation efficiency over the course of 1 month.[000109] Fig. 45 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by particle size over the course of 1 month.[000110] Fig. 46 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by particle size distribution over the course of 1 month.[000111] Fig. 47 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by zeta potential over the course of 1 month.[000112] Fig. 48 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by encapsulation efficiency over the course of 1 month.[000113] Fig. 49 shows Particle size, PDI and encapsulation efficiency change of RSV mLNP after freezing and lyophilization during 6 months.[000114] Fig. 50 shows characterization of mLNP with different mRNA. All size of samples was uniform and the PDI was small. The zeta potential was ± 20 mV.[000115] Fig. 51 shows characterization of mLNP with different ionizable lipids.[000116] Fig. 52 shows characterization of mLNP before and after lyophilization. The size and zeta potential of mLNP was maintained after lyophilization.[000117] Fig. 53 shows a titration curve of moisture content of mLNP after lyophilization via C30s+InMotion KF Flex and C30s.[000118] Fig. 54 shows biodistribution of mRNA expression (dark spots) in mice differences between LNP and mLNP formulations with SM-102.[000119] Fig. 55 shows biodistribution among the lymph nodes, liver, and spleen demonstrating that mLNP has a distinct difference in biodistribution than LNP.[000120] Fig. 56 shows biodistribution of both LNP and mLNP can be controlled by choice of ionizable lipid. EL-396 does not deliver to the liver under either LNP or mLNP conditions.[000121] Fig. 57 shows Cell uptake of Cy5-LNP and Cy5-mLNP. Flow cytometry was used to determine distribution of Cy5 fluorescence (panel a), percentage of Cy5 positive cells (panel b) and mean fluorescence intensity (panel c).[000122] Note that any one of more of the illustrative components of the molecule are optional and the present disclosure includes aspects that contain greater than or fewer than all of the illustrated elements.DETAILED DESCRIPTION[000123] The disclosure relates to novel ionizable lipid compounds and compositions and methods involving the same. The disclosure also provides methods of delivering a biologically active agent to a cell, and treating a disease or disorder in a mammal in need thereof. For example, a method of producing a polypeptide of interest in a cell involves contacting a nanoparticle composition comprising an mRNA, such as modified mRNA, circular mRNA, or self-amplifying mRNA, with a cell, whereby the mRNA may be translated to produce the polypeptide or regulatory structure of interest[000124] In the present specification, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, it being understood that not all isomers may have the same level of activity. In addition, a crystal polymorphism may be present for the compounds represented by the formula. Any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.[000125] 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.[000126] In the reaction schemes described herein, multiple stereoisomers may be produced.When no particular stereoisomer is indicated, it is understood to mean all possible stereoisomers that could be produced from the reaction. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known, for example, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC may be used to separate the isomers.[000127] Lipid nanoparticle compositions comprising the novel lipids are disclosed. Four- component lipid compositions include a novel lipid as well as additional lipids such as phospholipids, structural lipids, and PEG lipids. Three-component lipid nanoparticle compositions contain a steroidal or structural lipid-containing component, a stabilizing lipid (such as PEGylated lipid-containing component), a novel cationic or ionizable lipid-containing component of the disclosure. Lipid nanoparticle compositions with less than three components, e.g. one component or two components, and lipid nanoparticles with more than four components, e.g. five components or six components, are also contemplated.[000128] A three-component LNP composition (also referred to herein as a “modified LNP” or a “mLNP”) may include one or more biologically active agents. salt or isomer thereof, wherein,each n is independently selected from 0-8; each Q is independently selected fromwherein each m is independently selected from 0-6; each o is independently selected from 0-7; each R1and R2is, each X is independently selected from[000129] In certain aspects, compounds of Formula I may include, but are not limited to:[000130] Synthesis Scheme L General synthesis route for the synthesis of compounds ofFormula I.[000131] In some aspects, R3 and R4correspond to groups such that “Q” is satisfied. In some aspects, compounds of A-l, A-2, A-5, A-7, A-9 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art[000132] In some aspects, the present disclosure provides a method for synthesizing the compound of PMA or 1(a) comprising performing the following reaction:[000133] In some aspects, the present disclosure provides a method for synthesizing the compound of EL-396 or 1(b) comprising performing the following reaction:[000134] In some aspects, the present disclosure provides a method for synthesizing the compound of EL-382 or 1(c) comprising performing the following reaction:[000135] In some aspects, the present disclosure provides a method for synthesizing the compound of EL-360 or 1(d) comprising performing the following reaction:[000136] In one aspect, the novel ionizable lipid compound is a compound of Formula II:(II), or a salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected fromwherein each m is independently selected from 0-6 and o is independently selected from 0-7 and;each X is selected from[000137] In certain aspects, compounds of Formula II may include, but are not limited to:[000138] Synthesis Scheme IL General synthesis route for the synthesis of compounds of Formula II[000139] In some aspects, R3 and R4correspond to groups such that “Q” is satisfied. In some aspects, compounds of B-l, B-2, B-4, B-6, and B-8 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art[000140] In some aspects, the present disclosure provides a method for synthesizing the compound of EW-1 or 11(a) comprising performing the following reaction:[000141] A method for synthesizing the compound of EW-2 or 11(b) comprising performing the following reaction:[000142] In some aspects, the present disclosure provides a method for synthesizing the compound of N1 or 11(c) comprising performing the following reaction:[000143] In some aspects, the present disclosure provides a method for synthesizing the compound of N4 or 11(d) comprising performing the following reaction:[000144] The present disclosure discloses novel lipids and lipid nanoparticle compositions comprising such novel lipids.[000145] In one aspect, the present disclosure provides compounds of Formula IA:or a salt or isomer thereof, wherein m is 0-9; n is 0-9; o is 0-12; p is 0-12;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups wherein R is a H or a methyl group.[000146] Synthesis Scheme 1. General synthesis route for the synthesis of compounds of Formula IA.[000147] Scheme 2. Fatty acid tail synthesis of R1and R2, which also applies to synthesis of R3and R4.[000148] Scheme 3a. Fatty Acid tail conversion from hydroxy to thiol[000149] Scheme 3b. Fatty acid tail conversion of hydroxy to amine.[000150] Scheme 4. Fatty acid tail conversion of hydroxy to carboxylic acid.[000151] Scheme 5. Fatty acid tail conversion of hydroxy to isocyanate.[000152] In certain aspects, compounds of Formula IA may include, for example, the following compounds:[000153] In another aspect, the present disclosure provides compounds of Formula IB:or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12;R is the side chain of an independently selected amino acid;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is the side chain of an independently selected amino acid;X1is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;X2is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;X3is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;X4is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-. In some aspects, R or R5comprises the side chain of the amino acid, wherein the amino acid is Serine (S), Threonine (T), Cysteine (C), Selenocysteine (U), Glycine (G), Alanine (A), Isoleucine (I), Leucine (L), Methionine (M), or Valine (V).[000154] In some aspects, the carbonyl group in Formula IB is bonded to the amino terminus of the amino acid. For example, a compound of Formula IB may have the following structure:in which Xi and X2 represent independently an amino acid, wherein the amino acid is Serine (S), Threonine (T), Cysteine (C), Selenocysteine (U), Glycine (G), Alanine (A), Isoleucine (I), Leucine (L), Methionine (M), or Valine (V).[000155] In some aspects, the carbonyl group in Formula IB is bonded to the carboxy terminus of the amino acid.[000156] Synthesis Scheme 6. General synthetic route for the synthesis of compounds ofFormula IB.[000157] Scheme 6a. General synthesis for the protection of the amino acid and orientation of the amino acid with the carbonyl group on the fatty acid tail side.R is the selected amino acid side chain;R6is the protected amino acid side chain;X is a compatible functional group with carboxylic acid;X1is N(H)C(O) or N(H)C(O);X2is C(O)N(H) or C(O)O and;X3 is a compatible functional group with the amine.[000158] Scheme 6b. General synthesis for the orientation of the amino acid so that the carbonyl is on the ionizable head group side of the lipid.R is the selected amino acid side chain;R6is the protected amino acid side chain;X is a compatible functional group with carboxylic acid;X1is C(O)N(H) or C(O)O;X2is N(H)C(O) or N(H)C(O) and;X3 is a compatible functional group with the amine.[000159] Scheme 6c. General synthetic scheme of the lipid with included amino acids.R5is the side chain of an independently selected amino acid side chain and;R7is the protected side chain of an independently selected amino acid side chain.Synthesis of fatty acid tails are shown in Schemes 2 through 5 above.[000160] In certain aspects, compounds of Formula IB may include, for example, the following compounds:[000161] In another aspect, the present disclosure provides compounds of Formula IC:or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-5;R1is a linear C1-12alkyl;R2 is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is H or CH3;M1and M2are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -C(O)S- , -SC(O)-, -OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is a H or a methyl group; and X is selected from -CH2-, -O-, -S-, or -P(O)(OR)O-.[000162] Scheme 7. General synthetic route for the precursor to compounds of Formula IC.[000163] Scheme 8. Synthetic route of the conversion of the precursor hydroxy to thiol group.[000164] Scheme 9. Synthetic route of the conversion of the precursor hydroxy to amine group.[000165] Scheme 10. Synthetic route of the conversion of the precursor hydroxy to phosphine group.[000166] Scheme 11. General synthetic route of the synthesis of compound IC.[000167] In another aspect, the present disclosure provides compounds of Formula ILA:or a salt or isomer thereof, wherein m is selected from 0-5; n is selected from 0-12; o is selected from 0-12; q is selected from 1-3;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andX is selected from C(R)i, N(R), or O, wherein R is independently selected from a methyl and H.[000168] Scheme 12. General synthesis route for the synthesis of compounds of Formula nA.wherein X1, X2, and X3are either carboxylic acid (RC(O)O) functional groups, or they are isocyanate (RNCO) functional groups, and X is as defined above.[000169] Scheme 13. Fatty acid tail synthesis for R1and R2. The route is the same for R3and R4.[000170] In another aspect, the present disclosure provides compounds of Formula IIB:or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-6;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.[000171] Scheme 14. General route for the synthesis of head groups of compounds ofFormula IIB.X is a halide and;PG is a protecting group such as N-tert-butyloxycarbonyl group.[000172] Scheme 15. General synthesis route for the compound IIB.[000173] Scheme 16. Fatty Acid tail conversion from hydroxy to thiol[000174] Scheme 17. Fatty acid tail conversion of hydroxy to amine.[000175] Scheme 18. Fatty acid tail conversion of hydroxy to carboxylic acid.[000176] Scheme 19. Fatty acid tail conversion of hydroxy to isocyanate.[000177] In another aspect, the present disclosure provides compounds of Formula IIC:or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.[000178] Scheme 20. General synthesis for compound IIC.The side chains are synthesized according to Schemes 16-19.[000179] In another aspect, the present disclosure provides compounds of Formula IID:or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 1-7; o is selected from 0-12; p is selected from 0-12;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.[000180] Scheme 21. General route for the synthesis of the head groups for the synthesis of compounds of Formula IID.X1is a functional group (such as an amine, carboxylic acid, isocyante, etc) compatible with X2(an amine, carboxylic acid, isocyanate, etc) to give M1such that it is -C(O)N(R)-, -N(R)C(O)-, - C(O)S-, -SC(O)-, -OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.[000181] In another aspect, the present disclosure provides compounds of Formula HE:or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.[000182] Scheme 21. General route for the synthesis of compound HE.[000183] In another aspect, the present disclosure provides compounds of Formula IIF :or a salt or isomer thereof, whereinm is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.[000184] Scheme 23. General route for the synthesis of compound IIF.[000185] In the reaction schemes described herein, multiple stereoisomers may be produced. When no particular stereoisomer is indicated, it is understood to mean all possible stereoisomers that could be produced from the reaction. A person of ordinary skill in the art will recognize that the reactions can be optimized to give one isomer preferentially, or new schemes may be devised to produce a single isomer. If mixtures are produced, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC may be used to separate the isomers.[000186] The present disclosure further provides nanoparticle compositions comprise a lipid component including at least one compound according to Formulae I, II, III, IA, IB, IC, IIA, IIB, IIC, IID, IIE, IIF, including lAa-IAc, IBa-Ibe, ICa-ICc, IIAa-IIAb, IIBa, IICa, HDa, HEa-IIEb, and IIFa-IIFb, and any combination thereof. Nanoparticle compositions may also include a variety of other components. For example, the lipid component of a nanoparticle composition may includeone or more other lipids in addition to a lipid according to Formula I, II, III, LA, IB, IC, HA, IIB, IIC, IID, IIE, IIF, including lAa-IAc, IBa-Ibe, ICa-ICc, IIAa-IIAb, IIBa, IICa, HDa, HEa-IIEb, and IIFa-IIFb.[000187] Three-component Lipid Nanoparticle Compositions [000188] The disclosure includes three-component LNP compositions containing:1) a steroidal or structural lipid-containing component;2) a PEGylated lipid-containing component; and3) a cationic or ionizable lipid-containing component.[000189] In some embodiments, the largest dimension of a nanoparticle composition is 1 μm or shorter (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter), e.g., when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and / or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels.[000190] Cationic / Ionizable Lipid-containing Component[000191] A three component LNP composition of the present disclosure may include one or more cationic and / or ionizable lipids (e.g., lipids that may have a positive or partial positive charge at physiological pH) including, but not limited to, MC3, ALC-0315, ALC-0159, SM-102, DOTAP, Mol-111, Mol-114, MH-094, or a cationic and / or ionizable lipid disclosed in WO2017049245 A2 (Benenato), lipids of Formulae I, II, III, IA, IB, IC, IIA, IIB, IIC, IID, IIE, IIF, including lAa-IAc, IBa-Ibe, ICa-ICc, IIAa-IIAb, IIBa, IICa, HDa, IIEa-IIEb, and IIFa-IIFb, and any combination thereof.[000192] Stabilizing Lipid-containing Component[000193] A three component LNP composition of the present disclosure may include one or more stabilizing lipids that use steric bulk to minimize or prevent aggregation. For example, a three component LNP composition of the present disclosure may include one or more PEG orPEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group consisting of 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, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.[000194] Steroidal / Structural Lipid-containing Component[000195] A three component LNP composition of the present disclosure may include one or more structural lipids. Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.[000196] In some non-limiting aspects, a three component LNP composition of the present disclosure may be free of phospholipids. For example, the three component LNP composition of the present disclosure may be free of any one or more of the following phospholipids: 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-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), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-gly cero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn-glycero-3- phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3 -phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,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, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.[000197] Adjuvants[000198] In some embodiments, the three-component LNP may be combined in a composition with one or more adjuvants, e.g., Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminum hydroxide, Pam3CSK4, saponin extracts (e.g. Quil-A®), and Lipid A.[000199] The present disclosure includes nanoparticle compositions comprising at least one ionizable lipid compound of the disclosure. In one aspect, the nanoparticle compositions further comprise a biologically active agent. In some aspects, the biologically active agent is a nucleic acid. In some aspects, the biologically active agent is an RNA molecule. In some aspects, the biologically active agent is siRNA, miRNA, shRNA, tRNA, mRNA, circRNA and mixtures thereof.[000200] In one aspect, a nanoparticle composition, e.g., a LNP, further comprises a phospholipid selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-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), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,1.2-diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-diarachidonoy 1- sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1.2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin. In some aspect, the phospholipid is DSPC.[000201] In one aspect, the nanoparticle composition further comprises a structural lipid selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some aspects, the structural component is cholesterol.[000202] In one aspect, a nanoparticle composition further comprises a PEG lipid selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, a PEG-modified myristoyl diglyceride, and mixtures thereof. In some aspects, the PEG lipid is DMG-PEG2000.[000203] In one aspect, the components in the nanoparticle composition have the following relative molar percentages:20 mol % to 60 mol % of an ionizable lipid component,25 mol % to 45 mol % of a structural lipid component, 0 mol % to 25 mol % of a phospholipid, or0 mol % to 5 mol % of a PEGylated lipid component[000204] In one aspect, the nanoparticle composition has an encapsulation efficiency of at least 70% after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a zeta potential of about -30 to +30 mV after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a PDI of less than 0.300 after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a particle size of less than 220 nm after storage at about -80°C to 25°C for at least 28 days. In some aspects, the LNP has a nitrogen to phosphorus ratio (N:P) range of about 10 to 1.[000205] In one aspect, the nanoparticle composition further comprises a cryoprotectant In some aspects, the cryoprotectant is sucrose. In some aspects, the cryoprotectant has a volume / volume concentration of 5-12% (v / v).[000206] In one aspect, the nanoparticle composition comprises an ionizable lipid of the disclosure, a structural lipid, and a PEG lipid.[000207] In one aspect, the present disclosure includes a pharmaceutical composition comprising the nanoparticle composition and a pharmaceutically acceptable carrier.[000208] In one aspect, the present disclosure includes a method of delivering a biologically active agent to a cell, the method comprising administering to a subject the LNP of the disclosure, said administering comprising contacting the cell with the nanoparticle composition, whereby the biologically active agent is delivered to the cell.[000209] In one aspect, the present disclosure includes a method of delivering a biologically active agent to a cell of a subject, the method comprising administering a pharmaceutical composition of comprising the nanoparticle composition of the disclosure and a pharmaceutically acceptable carrier to the subject[000210] In one aspect, the present disclosure includes a method of treating a disease or disorder in a subject in need thereof by administering the nanoparticle composition of the disclosure.[000211] In some aspects, the present disclosure provides a method of making a composition by lyophilizing a solution comprising a nanoparticle composition and a nucleic acid. The lyophilized sample may be reconstituted in water or an aqueous solution for administering to a subject to deliver the payload. In some embodiments, the method comprises freezing a solution comprising a nanoparticle composition and a nucleic acid at a temperature several degree Celsius below the eutectic point of the solution, and applying vacuum to the frozen solution to remove water to produce a sample. The sample may then be placed under vacuum at a warmer temperature (e.g., a temperature around or above room temperature, e.g., 25°C to 35°C, such as 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35°C) to produce a lyophilized sample. The lyophilized sample produced according to the present disclosure may be reconstituted very easily in a small amount of water or an aqueous solution. The nanoparticle composition produced by the method may be capable of expressing the payload nucleic acid (e.g., mRNA) at a higher level (e.g., at least 2-fold) compared to the same nanoparticle composition before lyophilization. After reconstitution, the nanoparticle composition produced by the method may remain stable.[000212] The “stabilized” formulations of the disclosure preferably retain at least 80%, 85%,90%, 95%, 98%, 99%, or 99.5% of the LNP size distribution of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and / or in-use conditions. The “stabilized” formulations of the disclosure preferably retain at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5%of the encapsulation efficiency of a starting, standard, or reference preparation of the LNP formulation (e.g., mRNA-loaded LNP formulation) under given manufacturing, preparation, transportation, storage and / or in-use conditions.[000213] Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art Definitions[000214] Unless otherwise defined, 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 invention belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.[000215] The terms “a” and “an” refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, “a cell” encompasses one or more cells.[000216] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 0.01 to 2.0” should be interpreted to include not only the explicitly recited values of about 0.01 to about 2.0, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from 0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc. Furthermore, such an interpretation should apply regardless of the breadthof the range or the characteristics being described. Additionally, it is noted that all percentages are in weight, unless specified otherwise.[000217] In understanding the scope of the present disclosure, the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of,” as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristics) of features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transition terms (i.e. “comprising,” “consisting,” or “consisting essentially”) provides direct support for replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of’ or “consisting of’ would find direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.[000218] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.[000219] The term “exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more concrete fashion.[000220] Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, disorders, conditions, properties, steps, or the like may be discussed in the context of one specific embodiment or aspect or in a separate paragraph or section of this disclosure. It is understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other embodiments or aspects found anywhere in the present disclosure and claims, which all form the application and claimed invention at the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, molecule, isolated polypeptide, activatable molecule, composition, or method is intended to and does find direct support for embodiments related to constructs, molecules, isolated polypeptides, activatable molecules, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect. [000221] In the present specification, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, it being understood that not all isomers may have the same level of activity. In addition, a crystal polymorphism may be present for the compounds represented by the formula. Any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.[000222] 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.[000223] In the reaction schemes described herein, multiple stereoisomers may be produced. When no particular stereoisomer is indicated, it is understood to mean all possible stereoisomers that could be produced from the reaction. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known,for example, techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC may be used to separate the isomers.[000224] 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. 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.[000225] As used herein, the term “alkyl” or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation “C1-C24 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.[000226] As used herein, the term “alkenyl” or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation “C1-C24 alkenyl” means an optionally substituted linear or branched hydrocarbon including 1-24 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, Cis alkenyl may include one or more double bonds. A Cis alkenyl 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.[000227] 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 carbon-carbon triple bond, which is optionally substituted. The notation “C1-C24 alkynyl" means an optionally substituted linear or branched hydrocarbonincluding 1-24 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. For example, Cis alkynyl may include one or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.[000228] Alkyl, alkenyl, alkynyl, acyl, aryl, heteroaryl and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified.[000229] As used herein, the terms “approximately” and “about,” as applied to one or more values of interest, refer to a value that is ±20% of the recited value, unless otherwise defined. In certain aspects, if so defined by the applicant, the term “approximately” or “about” may refer to a range of values that fall within 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).[000230] As used herein, the terms “carbocycle” and “carbo cyclic group” refer to an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. The notation "Ce 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-dihy dronaphthyl 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.[000231] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a cell with a nanoparticle composition means that the cell and the nanoparticle composition 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 relevant arts. Moreover, more than one cell may be contacted by the nanoparticle composition.[000232] As used herein, the term “delivering” means providing an entity to a destination. For example, delivering a biologically active agent to a subject may involve administering a LNP including the biologically active agent to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of a nanoparticle composition to a subject or cell may involve contacting one or more cells with the nanoparticle composition.[000233] As used herein, “encapsulation efficiency” refers to the amount of a biologically active agent that becomes part of a nanoparticle composition, relative to the initial total amount of biologically active agent used in the preparation of a nanoparticle composition. For example, if 97 mg of biologically active agent are encapsulated in a nanoparticle composition out of a total 100 mg of therapeutic and / or prophylactic initially provided to the composition, the encapsulation efficiency may be given as 97%. The encapsulation efficiency may be measured, for example, by encapsulating mRNA encoding GFP (green fluorescent protein) sequences in the nanoparticle composition and then measuring the encapsulation efficiency of the RFP sequence using flow cytometry. As used herein, “encapsulation” may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement[000234] 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.[000235] 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 phosphorates), alkyl groups, amidates, or glycerols. For example, 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.[000236] As used herein, a “lipid component” is that component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may include one or more ionizable, PEGylated, structural, or phospholipids.[000237] As used herein, “methods of administration” may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to asubject A method of administration may be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body.[000238] As used herein, “modified” means non-natural. For example, 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. For example, a modified nucleobase species may include one or more substitutions that are not naturally occurring.[000239] 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 nanoparticle composition including a lipid component and an RNA.[000240] As used herein, a “nanoparticle composition” is a composition comprising one or more lipids and biologically active agents. Nanoparticle compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition may be a liposome having a lipid bilayer with a diameter of 500 nm or less.[000241] As used herein, “naturally occurring” means existing in nature without artificial aid.[000242] As used herein, a “lipid” refers to a group of organic compounds that include, for example, esters of fatty acids that are characterized by being less soluble in water but more soluble in many organic solvents.[000243] As used herein, the term “subject” refers to any organism to which a composition 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, “patient” refers to an individual who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or under care by a trained professional for a particular disease or condition.[000244] As used herein, a “PEG lipid" or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.[000245] 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). Particular phospholipids may facilitate fusion to a membrane. For example, 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.[000246] As used herein, in some aspects, the term “structural lipid” refers to a compound comprising the following carbon skeleton:[000247] As used herein, “cationic lipid” and “ionizable lipid” refer to compounds that comprise both a polar (hydrophilic) head-group or moiety and a non-polar (hydrophobic or lipophilic) tail-group or moiety and may have a positive or partial positive charge at physiological pH. In certain aspects, such polar head-group and non-polar tail-group are bound (e.g., by one or more of hydrogen-bonds, van der Waals' forces, ionic interactions and covalent bonds) to each other (e.g., by an optionally substituted, variably unsaturated Cl -CIO alkyl or alkenyl). In certain aspects, the head-group or moiety is hydrophilic (e.g., a hydrophilic head-group comprising an optionally-substituted alkyl amino). As used herein, the term “hydrophilic” is used to indicate in qualitative terms that a functional group is water-preferring, and water-soluble. As used herein, the term “hydrophobic” is used to indicate in qualitative terms that a functional group is wateravoiding, and typically such groups are not water soluble.[000248] The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, reasonably suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication.[000249] The phrase “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein 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 (com), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and other species disclosed herein.[000250] The term “pharmaceutically acceptable compositions,” as used herein, 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; such as, nonaqueous media like 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.[000251] 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.[000252] As used herein, an “RNA” refers to a ribonucleic acid that may be naturally or non- naturally occurring. For example, an RNA may include modified and / or non-naturally occurring components such as one or more nucleobases, nucleosides, nucleotides, or linkers. An RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a poly A sequence, and / or a polyadenylation signal. An RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, an RNA may be a messenger RNA (mRNA, such as modified mRNA, circular mRNA, and self-amplifying mRNA), transfer RNA (tRNA), small interference RNA (siRNA), small activating RNA (saRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA)). Translation of an mRNA encoding a particular polypeptide, for example, in vivo translation of an mRNA inside amammalian cell, may produce the encoded polypeptide or regulatory structure. RNAs may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), small activating RNA (saRNA), transfer RNA (tRNA), mRNA (modified mRNA, circular mRNA, and self-amplifying mRNA), and mixtures thereof.[000253] Nucleic acids and polynucleotides useful in the disclosure typically include a first region of linked nucleosides encoding a polypeptide of interest (e.g., a coding region), a first flanking region located at the 5 '-terminus of the first region (e.g., a 5'-UTR), a second flanking region located at the 3'-terminus of the first region (e.g., a 3'-UTR), at least one 5'-cap region, and a 3 '-stabilizing region. In some aspects, a nucleic acid or polynucleotide further includes a poly- A region or a Kozak sequence (e.g., in the 5'-UTR). In some cases, polynucleotides may contain one or more intronic nucleotide sequences capable of being excised from the polynucleotide. In some aspects, a polynucleotide or nucleic acid (e.g., an mRNA) may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a poly A sequence, and / or a polyadenylation signal. Any one of the regions of a nucleic acid may include one or more alternative components (e.g., an alternative nucleoside). For example, the 3 '-stabilizing region may contain an alternative nucleoside such as an L-nucleoside, an inverted thymidine, or a 2'-O-methyl nucleoside and / or the coding region, 5'-UTR, 3'-UTR, or cap region may include an alternative nucleoside such as a 5- substituted uridine (e.g., 5-methoxyuridine), a 1 -substituted pseudouridine (e.g., 1-methyl- pseudouridine or 1 -ethyl-pseudouridine), and / or a 5-substituted cytidine (e.g., 5-methyl-cytidine). [000254] As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.[000255] The term “biologically active 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.[000256] 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 orsusceptible 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. For example, the disease or disorder is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, rare diseases, obesity, diabetes, neurodegenerative diseases, ophthalmic diseases, cardio- and reno-vascular diseases, and metabolic diseases.[000257] Lipid nanoparticles with more than four components, e.g. five, six, or more components are also included in this disclosure. Lipid nanoparticles with fewer than four components, e.g. one, two, or three components are also included in this disclosure.[000258] Any numerical value within the recited ranges and any combination of ranges and specific numerical values within the claimed ranges are contemplated and supported by the foregoing disclosures, i.e., 5 to 60 mole % of a steroidal or structural lipid-containing component includes any numerical value and range within the range of 5 to 60, e.g., 5, 5.01, 5.02, ...59.97, 59.98, 59.99, 60, 5-10, 5-20, 10-30, 15-25, etc. Similarly, 0.5 to 20 mole % of a PEGylated lipid- containing component includes any numerical value and range within the range of 0.5 to 20, e.g., 0.5, 0.501, 0.502, ...19.97, 19.98, 19.99, 20, 0.5-10, 0.52-15, 1-14, 5-13, etc. Similarly, 30 to 70 mole % of a cationic or ionizable lipid-containing component includes any numerical value and range within the range of 30 to 70, e.g., 30, 30.01, 30.02, ...69.97, 69.98, 69.99, 70, 30.5-68, 35- 51, 40-52, 45-63, etc.[000259] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:4) 0.5 to 60 mole % of a steroidal or structural lipid-containing component;5) 0.5 to 20 mole % of a PEGylated lipid-containing component; and6) 25 to 97.2 mole % of a cationic or ionizable lipid-containing component[000260] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:4) 20 to 50 mole % of a steroidal or structural lipid-containing component;5) 0.8 to 10 mole % of a PEGylated lipid-containing component; and6) 40 to 62 mole % of a cationic or ionizable lipid-containing component[000261] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:4) 25 to 46 mole % of a steroidal or structural lipid-containing component;5) 1 to 7 mole % of a PEGylated lipid-containing component; and6) 44 to 58 mole % of a cationic or ionizable lipid-containing component.[000262] In one aspect, the three-component LNP composition contains the three components in the following relative mole percentages:4) 35 to 44 mole % of a steroidal or structural lipid-containing component;5) 1.2 to 5 mole % of a PEGylated lipid-containing component; and6) 48 to 57 mole % of a cationic or ionizable lipid-containing component[000263] In one aspect the three-component LNP composition contains the three components in the following relative mole percentages:4) 20 to 35 mole % of a steroidal or structural lipid-containing component5) 0.8 to 10 mole % of a PEGylated lipid-containing component; and6) 55 to 79 mole % of a cationic or ionizable lipid-containing component[000264] The amount of a biologically active agent in a nanoparticle composition may depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic and / or prophylactic. For example, the amount of a RNA useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a biologically active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some aspects, the wt / wt ratio of the lipid component to a biologically active agent in a nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of the lipid component to a biologically active agent may be from about 10: 1 to about 40: 1. In certain aspects, the wt / wt ratio is about 20:1. The amount of a biologically active agent in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).[000265] In one aspect, the nanoparticle composition has an encapsulation efficiency of at least 80% when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days. In one aspect, thenanoparticle composition has a zeta potential of 5-15 mV when stored at 25°C, 4°C, -20°C, or - 80°C for at least 28 days. In one aspect, the nanoparticle composition has a PDI of less than 0.2 when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days. In one aspect, the nanoparticle composition has a particle size of less than 140 nm when stored at 25°C, 4°C, -20°C, or -80°C for at least 28 days. “Stability," “stabilized,” and “stable" in the context of the present disclosure refers to the resistance of nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing, preparation, transportation, storage and / or in- use conditions, e.g., when stress is applied such as shear force, freeze / thaw stress, etc.[000266] In one aspect, the novel ionizable lipids of the disclosure is characterized using nuclear (or heteronuclear) magnetic resonance (NMR), UV-Vis spectrophotometry, high performance liquid chromatography mass spectrometer (HPLC / MS), and infrared spectrometry, other characterization methods known in the art are also contemplated.[000267] In one aspect, the disclosure includes methods used to determine characteristics of the ionizable lipids of the disclosure and LNPs, including the purity, LNP mean size, encapsulation efficiency, biological activity, immunogenicity, therapeutic index, and number of impurities. For example, dynamic light scattering (DLS) can measure the LNP mean size, polydispersity index; zeta potential, including HPLC / MS, and UV-Vis can be used to determine the concentration of a nucleotide in an LNP; and micro-flow imaging (MFI) can be used for measuring the amount of sub-visible particles. Other methods known in the art for measuring characteristics such as purity, LNP mean size, encapsulation efficiency, biological activity, immunogenicity, therapeutic index, and number of impurities are also contemplated.[000268] In one aspect, the nanoparticle compositions may have a pKa from 5.6 to 7.6.[000269] Nanoparticle compositions may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions may include one or more nanoparticle compositions. For example, a pharmaceutical composition may include one or more nanoparticle compositions including one or more different biologically active agents. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Scienceand Practice of Pharmacy, 21stEdition, A. R Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a nanoparticle composition. An excipient or accessory ingredient may be incompatible with a component of a nanoparticle composition if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.[000270] In some aspects, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a nanoparticle composition. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some aspects, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some aspects, an excipient is approved for use in humans and for veterinary use. In some aspects, an excipient is approved by United States Food and Drug Administration. In some aspects, an excipient is pharmaceutical grade. In some aspects, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.[000271] In one aspect, the nanoparticle composition further comprises a cryoprotectant. In one aspect, the cryoprotectant is sucrose. In one aspect, the concentration of the cryoprotectant can be from 4%-32% v / v. In one aspect, the cryoprotectant is from 5-12% (v / v) of the nanoparticle composition. In one aspect, the LNP is cryoprotected with 8% v / v sucrose. However, any cryoprotectant will suffice (e.g. trehalose). In one aspect, a post formulation sample including a cryoprotectant and a buffer is subjected to dialysis at 2 to 10 °C for 10 - 20 hours.[000272] In certain aspects, the pharmaceutical composition of the disclosure contain the therapeutic or prophylactic agent at a ratio of 0.05 to 25 mg / ml, 0.1 to 20 mg / ml, 0.2 to 18 mg / ml, 0.5 to 15 mg / ml, 0.7 to 12 mg / ml, 0.9 to 10 mg / ml, 1 to 8 mg / ml, 1.5 to 6 mg / ml, 2 to 5 mg / ml, 2.5 to 4 mg / ml, 0.5 to 3.0 mg / ml, 0.2 to 4.0 mg / ml, 0.4 to 2.0 mg / ml, and any numerical value or range within the range of 0.05 to 25 mg / ml.[000273] Relative amounts of the one or more nanoparticle compositions, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceuticalcomposition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more nanoparticle compositions.[000274] Nanoparticle compositions and / or pharmaceutical compositions including one or more nanoparticle compositions may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of a biologically active agent to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions provided herein of nanoparticle compositions and pharmaceutical compositions including nanoparticle compositions are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.[000275] A pharmaceutical composition including one or more nanoparticle compositions may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and / or packaging the product into a desired single- or multi-dose unit.[000276] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., nanoparticle composition). The amount of the active ingredient is generally equal to the dosage of the active ingredient whichwould be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.[000277] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.[000278] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.Methods of Producing Polypeptides in Cells[000279] The present disclosure provides methods of producing a polypeptide of interest in a mammalian cell. Methods of producing polypeptides involve contacting a cell with a nanoparticle composition including an mRNA encoding the polypeptide of interest. Upon contacting the cell with the nanoparticle composition, the mRNA may be taken up and translated in the cell to produce the polypeptide of interest.[000280] In general, the step of contacting a mammalian cell with a nanoparticle composition including an mRNA encoding a polypeptide of interest may be performed in vivo, ex vivo, in culture, or in vitro. The amount of nanoparticle composition contacted with a cell, and / or the amount of mRNA therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the nanoparticle composition and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors. In general, an effective amount of the nanoparticle composition will allow for efficient polypeptide production in the cell. Metrics for efficiency may include polypeptide translation (indicated by polypeptide expression), level of mRNA degradation, and immune response indicators.[000281 ] The step of contacting a nanoparticle composition including an mRNA with a cell may involve or cause transfection. A phospholipid including in the lipid component of a nanoparticle composition may facilitate transfection and / or increase transfection efficiency, for example, by interacting and / or fusing with a cellular or intracellular membrane. Transfection may allow for the translation of the mRNA within the cell.Methods of Delivering Therapeutic Agents to Cells and Organs[000282] The present disclosure provides methods of delivering a biologically active agent to a mammalian cell or organ. Delivery of a biologically active agent to a cell involves administering a nanoparticle composition including the biologically active agent to a subject, where administration of the composition involves contacting the cell with the composition. For example, a protein, cytotoxic agent, radioactive ion, chemotherapeutic agent, or nucleic acid (such as an RNA, e.g., mRNA) may be delivered to a cell or organ. In the instance that a biologically active agent is an mRNA, upon contacting a cell with the nanoparticle composition, a translatable mRNA may be translated in the cell to produce a polypeptide of interest. However, mRNAs that are substantially not translatable may also be delivered to cells. Substantially non-translatable mRNAs may be useful as vaccines and / or may sequester translational components of a cell to reduce expression of other species in the cell.[000283] In some aspects, a nanoparticle composition may target a particular type or class of cells (e.g., cells of a particular organ or system thereof). For example, a nanoparticle composition including a biologically active agent of interest may be specifically delivered to a mammalian liver, kidney, spleen, femur, or lung. Specific delivery to a particular class of cells, an organ, or a system or group thereof implies that a higher proportion of nanoparticle compositions including a biologically active agent are delivered to the destination (e.g., tissue) of interest relative to other destinations, e.g., upon administration of a nanoparticle composition to a mammal. In some aspects, specific delivery may result in a greater than 2 fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in the amount of biologically active agent per 1 g of tissue of the targeted destination (e.g., tissue of interest, such as a liver) as compared to another destination (e.g., the spleen). In some aspects, the tissue of interest is selected from the group consisting of a liver, kidney, a lung, a spleen, a femur, an ocular tissue (e.g., via intraocular, subretinal, or intravitreal injection), vascularendothelium in vessels (e.g., intra-coronary or intra-femoral) or kidney, and tumor tissue (e.g., via intratumoral injection).[000284] As another example of targeted or specific delivery, an mRNA that encodes a protein-binding partner (e.g., an antibody or functional fragment thereof, a scaffold protein, or a peptide) or a receptor on a cell surface may be included in a nanoparticle composition. An mRNA may additionally or instead be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties. Alternatively, other biologically active agents or elements (e.g., lipids or ligands) of a nanoparticle composition may be selected based on their affinity for particular receptors (e.g., low density lipoprotein receptors) such that a nanoparticle composition may more readily interact with a target cell population including the receptors. For example, ligands may include, but are not limited to, members of a specific binding pair, antibodies, monoclonal antibodies, Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents including mono- or bi-specific antibodies such as disulfide stabilized Fv fragments, scFv tandems, diabodies, tribodies, or tetrabodies; and aptamers, receptors, and fusion proteins. [000285] In some aspects, a ligand may be a surface-bound antibody, which can permit tuning of cell targeting specificity. This is especially useful since highly specific antibodies can be raised against an epitope of interest for the desired targeting site. In one aspect, multiple antibodies are expressed on the surface of a cell, and each antibody can have a different specificity for a desired target. Such approaches can increase the avidity and specificity of targeting interactions. [000286] In certain aspects, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg of a biologically active agent(e.g., an mRNA) in a given dose, where a dose of 1 mg / kg (mpk) provides 1 mg of a biologically active agent per 1 kg of subject body weight. In some aspects, a dose of about 0.001 mg / kg to about 10 mg / kg of a biologically active agent (e.g., mRNA) of a nanoparticle composition may be administered. In other aspects, a dose of about 0.005 mg / kg to about 2.5 mg / kg of a biologically active agent may be administered. In certain aspects, a dose of about 0.1 mg / kg to about 1 mg / kg may be administered. In other aspects, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered. A dose may be administered one or more times per day, in the same or a different amount, to obtain a desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered, for example, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain aspects, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In some aspects, a single dose may be administered, for example, prior to or after a surgical procedure or in the instance of an acute disease, disorder, or condition.[000287] Nanoparticle compositions including one or more biologically active agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. For example, one or more nanoparticle compositionsincluding one or more different biologically active agents may be administered in combination. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent. In some aspects, the present disclosure encompasses the delivery of compositions, or imaging, diagnostic, or prophylactic compositions thereof in combination with agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution within the body.[000288] It will further be appreciated that one or more biologically active or imaging agents may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some aspects, the levels utilized in combination may be lower than those utilized individually.[000289] The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a composition useful for treating cancer may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects, such as infusion related reactions).EXAMPLESExample 1: LNP Characterization[000290] The ionizable lipids of the present disclosure were synthesized and formulated into LNPs with cholesterol, DSPC and DMG-PEG2k and characterized. Physical chemical data of EL- 396 and EL-PMA and EL-N4 relative to SM-102 for both 4-component systems (SM-102, EL- 396, EL-N4, and EL-PMA) and 3 -component systems (SM-102-E, EL-396-E, EL-N4-E, and EL- PMA-E) are shown in Table 1.TABLE I[000291] The effects of different molar ratios on physical chemical data for EL-396 in a four- component LNP (LI -L5) and three-component mLNPs (ml -m4) are shown in Table 2.TABLE 2Example 2: mLNP Characterization[000292] The three-component or four-component LNP compositions were prepared for testing by creating 3x or 4x molarity stock solutions in 200 proof ethanol for each lipid to be used in the formulation. These stock solutions were diluted or combined to give the desired final molar ratio and total lipid concentration for each experiment. The mRNA was prepared from GMP-grade stock solutions of mRNA. The aqueous stock solution using either sodium citrate or sodium acetatemaster stock solutions at select pH values. The buffered, aqueous stock solution for the formulation was prepared by adding the appropriate master buffer stock solution to an aqueous aliquot and then adding the stock solution of mRNA to give the desired final mRNA concentration, buffer, and pH value. The lipids ethanolic solution and the mRNA aqueous solutions were mixed by microfluidic mixing by PNI Ignite. Solutions were then dialyzed into the desired final product buffer using Thermofisher dialysis cassettes (MWCO lOkDa, PES). The final product buffer was generally prepared by adding the appropriate cryoprotectant, e.g. sucrose, by weight into the corresponding weight of water to create the desired weight percent of cryoprotectant. The final product buffer was sterilized by sterile filtration through a 0.2um PES bulk filter.[000293] A three-component LNP composition of the present disclosure was made containing 25 mM SM-102 in ethanol, 19 mM cholesterol in ethanol, and 0.75 mM DMG- PEG2000. Equal volumes of each lipid and of a blank ethanol were combined to give lipid mole ratios as follows: SM-102 (55.9%), cholesterol (42.4%), and DMG-PEG2000 (1.7%). The 0.13 mg / g mRNA in 25 mM sodium acetate pH 6.0 was prepared.[000294] The LNP was formulated with a mRNA aqueous solution to lipid ethanolic solution ratio of 3:1 using microfludics to give unimodal peaks. The sample was then dialyzed using 10 kDa MWCO cassettes at 4 °C against 20 mM Tris-HCl, 8% sucrose to produce the final three- component LNP composition. The sample was concentrated to an mRNA concentration over 0.2 mg / mL by UV and filter-sterilization was performed. Surprisingly, the encapsulation was found to be comparable to current 4-component LNP systems.[000295] A control (four-component LNP) composition was formulated using SM-102 (50%), cholesterol (38.5%), DSPC (10%), and DMG-PEG2000 (1.5%) in ethanol mixed by microfluidics with mRNA (0.13 mg / mL) in 25 mM sodium acetate pH 6.0 and dialyzed into 20 mM Tris-HCl pH 7.4, 8% sucrose (“RL-007”). The LNPs were filter-sterilized and concentrated to give an mRNA concentration over 0.2 mg / mL by UV.[000296] To further establish the bounds of good encapsulation and compare the effect of cholesterol and DSPC of the traditional LNP compared to the mLNP, a systematic adjustment of cholesterol was performed. The range of Cholesterol was systematically decreased. The encapsulation was excellent down to 30 mole % cholesterol. However, at the optimized condition of 38.5% cholesterol, when DSPC was switched the encapsulation efficiency was very poor, thePDI and size were very large and unacceptable. Thus, establishing that cholesterol and DSPC serve unique roles in the traditional LNP and that in the mLNP, DPSC does not improve stability, encapsulation efficiency nor size and PDI.TABLE 3: Tabulation of DSPC and cholesterol composition to study the effect on encapsulation efficiency.TABLE 4: Demonstration of the experiment in Table 1 on encapsulation effectTABLE 5: Screening of composition, pH, and total lipid concentration.Example 3: Lyophilization Examples[000297] After lyophilization, the mLNP of the present disclosure possesses good appearance, physicochemical properties, in vitro cell uptake and expression.TABLE 6: Screening of cryoprotectants.TABLE 7: Screening of cryoprotectants - Physical chemical data from Table 6[000298] The in vitro results from Tables 6 and 7 and Fig. 17 demonstrate that mLNP of the disclosure transfects well after lyophilization compared to before lyophilization. Further, combining cryoprotectants like sucrose plus mannitol and sucrose plus trehalose could not stabilize mLNP while changing reconstitution media could be done. The size and PDI of mLNP after reconstituted with NaCl solution could be maintained.TABLE 8: Screening of reconstitution media after lyophilization[000299] The results shown in Table 8 show that the size and PDI of mLNP after reconstituted with NaCl solution could be maintained. The concentration of NaCl (100 mM) was better.Example 4: Screening Lipid Ratio[000300] Different ratios of three component LNPs were screened to produce and characterize three component mLNPs. As shown in Table 9 below and Fig. 18, Group 3 containing SM-102 (59.8%), cholesterol (38.5%), DMG-PEG2000 (1.7%) had better performance in physicochemical parameters before and after lyophilization. The size of Group 4 containing SM- 102 (54.6%), cholesterol (42%), DMG-PEG2000 (3.4%) was smallest.TABLE 9: In Pitro Screening of Lipid Ratios[000301 ] Fig. 18 show the in vitro results from Table 9. The Western Blot data indicates that before and after lyophilization is good. Further, the transfection efficiency of mLNP was decreased observably when the PEG content increasing to 3.4%. The transfection of mLNP and LNP before and after lyophilization could be maintained.[000302] The mLNPs of the disclosure were also tested in vivo. Table 10 shows the results of a mouse immunogenicity study design of lipid ratio screening. The in vivo study was performed by formulating the mLNP by microfluidic mixing. The mRNA was prepared in a 25 mM sodium acetate buffer, pH 5.0. The Lipids were prepared in ethanol at the relative molar ratios given in Table 10. Three formulations were performed, Ml, M3, and LI (Table 10). After formulation, these samples were dialyzed against 20mM Tris-HCl, pH 7.4, 8%w / w sucrose. The mRNA concentrations were tested by the Invitrogen Ribogreen Assay. Each product was divided and half of each sample (Ml, M3, and LI) was lyophilized together to give the corresponding M2, M4, and L2. The lyophilized samples were reconstituted, concentration checked by the Ribogreen Assay,and prepared for in vivo intramuscular injection. Seven groups of 5 balbc mice were injected with the 7 groups in Table 10. The sera was collected and analyzed to give data in Fig. 19.TABLE 10: In Vivo Screening of Lipid Ratios μg μl μg μl μg μl μg μl μg μl μg[000303 J Fig. 19 show IgG binding antibody titers after the first (a) and the second (b) administration with mLNP and LNP. The mice from Table 10 were injected and bled 14 days after after injection. The second immunization was administered 7 days later. The second bleeding was at day 35 from first injection. The second bleeding was 14 days after second immunization. Female BALB / c mice, aged 6 to 8 weeks, were randomly grouped into sets of five mice each. They received intra-muscular vaccinations with SARS-CoV-2 Omicron BA.1 mRNA, which was formulated using groups from Table 10. Each mouse underwent two rounds of immunization, spaced 21 days apart. Blood samples were obtained from all mice prior to each immunization at 14 and 35 days after the initial immunization, using submandibular bleeding. After collection, theblood was allowed to clot overnight at 4°C, followed by serum extraction through centrifugation at 500 g for 15 minutes at 22°C. These serum samples were then stored at 4°C until they were ready for further analysis. MaxiSorp plates (Biolegend) were coated with 100 μl of recombinant Omicron BA.1 SI or RBD (1 μg / ml) in lx Antigen coating buffer overnight at 4°C. The wells were washed 3 times with PBS-T and incubated the plates with 200 μl of blocking buffer for 2 hours at RT. After washing 3 times with PBS-T, the plate was incubated with 100 μl of serially diluted sera for 2 hours at RT. After 2 hours incubation, the plate was washed 5 times with PBS- T and incubated with HRP-conjugated secondary antibody for 1 hour at RT. Finally, the plate was washed 5 times with PBS-T and incubated with 100 μl of lx TMB substrate for 10-20 minutes. The reaction was stopped by adding 100 μl of 1N-HC1 and read at 450nm using the Cytation?.[000304] The results of Table 10 and Fig. 19 show that, compared with LNP group, mLNP group 1 showed higher IgG titer before lyophilization. The immunogenicity response of mLNP group 1 could be maintained after lyophilization.[000305] The mLNPs of the present disclosure were also compared to 4 component LNPs. In Table 11 below, each “condition” represents a LNP composition comprising different lipid ratios. Condition “E” represents a 3 component mLNP of the disclosure. The samples were formulated by microfluidic mixing. The mRNA was prepared in a 25 mM sodium acetate buffer, pH 5.0. The lipids were prepared in 200 proof ethanol at the relative molar ratios given in Table 11. After formulation, these samples were dialyzed against 20mM Tris-HCl, pH 7.4, 8%w / w sucrose. After dialysis, the samples were concentrated by centrifuge. The mRNA concentrations were tested by the Invitrogen Ribogreen Assay.TABLE 11 : Composition conditions for examining potential lipids[000306] The mLNP of condition E was further tested using different ionizable lipids. Using the conditions and formulation technique from Table 11, ionizable lipids (SM-102, EW1, EW2,PMA, and N4) were tested at various conditions. Size and PDI were measured on Malvern Zetasizer. Concentration and encapsulation efficiency were measured using the Invitrogen Ribogreen Assay.TABLE 12: List of lipids and experiments performed for Condition E[000307] Fig. 20 show that PMA and N4 under “E” condition (mLNP) do not show in vitro expression, but showed excellent in vivo expression. The Western blot analysis was performed using lysates of transfected HEK293T cells were prepared in 200 μl RIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed. The BCA assays were performed for quantifying the concentration of proteins. The cell lysates were (10-20 μg per lane) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk / TBST (lx Trisbuffered saline containing 0.1% Tween-20), membranes were incubated with primary antibody in 5% milk / TBST at 4°C overnight After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk / TBST for 1 hour at room temperature. Chemiluminescence (Clarity Western ECL substrate, Bio-Rad) image acquisition and quantitation of band intensities were performed using an Azure Chemidoc™ Imaging System(Azure Biosystem 400). Fig. 21 shows that IgG titer from mouse models indicate excellent expression of the mRNA from the novel lipids under mLNP (E) conditions. The in vivo data from Fig. 21 was collected from female BALB / c mice, aged 6 to 8 weeks. They received intra-muscular vaccinations with SARS-CoV-2 Omicron BA.1 mRNA, which was formulated using groups from Table 12. Each mouse underwent two rounds of immunization, spaced 21 days apart Blood samples were obtained from all mice prior to each immunization at 14 and 35 days after the initial immunization, using submandibular bleeding. After collection, the blood was allowed to clot overnight at 4°C, followed by serum extraction through centrifugation at 500 g for 15 minutes at 22°C. These serum samples were then stored at 4°C until they were ready for further analysis. MaxiSorp plates (Biolegend) were coated with 100 μl of recombinant Omicron BA.1 SI or RBD (1 μg / ml) in lx Antigen coating buffer overnight at 4°C. The wells were washed 3 times with PBS- T and incubated the plates with 200 μl of blocking buffer for 2 hours at RT. After washing 3 times with PBS-T, the plate was incubated with 100 μl of serially diluted sera for 2 hours at RT. After 2 hours incubation, the plate was washed 5 times with PBS-T and incubated with HRP-conjugated secondary antibody for 1 hour at RT. Finally, the plate was washed 5 times with PBS-T and incubated with 100 μl of lx TMB substrate for 10-20 minutes. The reaction was stopped by adding 100 μl of 1N-HC1 and read at 450nm using the Cytation?. Panels A and B of Fig. 22 show that EW-2 and PMA have excellent T-Cell response in mouse models under mLNP conditions. Further, mLNP conditions improve the T-cell response of EW-2 from poor to excellent under EW-2-E. Spleens from mice were collected and processed into single-cell suspensions in RPMI1640 media supplemented with 10% heat-inactivated fetal calf serum and penicillin / streptomycin (R10 media). Red blood cells were lysed using RBC lysis buffer (KD Medical) and resuspended in R10 media to stop the lysis. Splenocytes were counted and 200,000 cells were added to the 96 well plates of the Mouse JFN-g ELISpot PLUS (ALP) kit (Mabtech). Cells were then stimulated for 16 hours at 37°C with 2 μg of PepMix™ SARS-CoV-2 (Spike Glycoprotein B.1.1529 Omicron, GenScript) or media alone as a negative control. Spots were developed as per the manufacturer’s instructions. Spots were quantified by Cytation?.[000308] A dose dependent study was conducted using ionizable lipids PMA and EL-396 based on the results shown above. Fig. 23 shows that EL-396 can produce an in vitro response while EL-PMA does not produce an in vitro response under both 4-component and mLNPconditions. The Western blot analysis was performed using lysates of transfected HEK293T cells were prepared in 200 μ RlIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed. The BCA assays were performed for quantifying the concentration of proteins. The cell lysates were (10-20 μ pger lane) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk / TBST (lx Tris-buffered saline containing 0.1% Tween-20), membranes were incubated with primary antibody in 5% milk / TBST at 4°C overnight. After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk / TBST for 1 hour at room temperature. Chemiluminescence (Clarify Western ECL substrate, Bio-Rad) image acquisition and quantitation of band intensities were performed using an Azure Chemidoc™ Imaging System (Azure Biosystem 400). Fig. 24 shows that EL-396 and PMA perform well in both 4-component and mLNP conditions which is in contrast to in vitro data which shows that only EL-396 can express the spike protein encoded by the payload delivered using the mLNP of the disclosure. The in vivo data from Fig. 24 was collected from female BALB / c mice, aged 6 to 8 weeks. They received intra-muscular vaccinations with SARS-CoV-2 Omicron BA.1 mRNA, which was formulated using 6 formulations. The LNP conditions were at molar ratios of ionizable lipid (SM-102, EL-396, or PMA), DSPC, cholesterol, and DMG-PEG2000 of 50: 10:38.5: 1.5. The mLNP formulation condition was for the ionizable lipid (SM-102, EL-396, or PMA), cholesterol, and DMG-PEG2000 at 59.8:38.5: 1.7. The OmicronBA.1 mRNA was prepared in 25mM sodium acetate pH 5.0. After formulation each of the six formulations was dialyzed into 20mM Tris-HCl, 8% sucrose. The sample concentrations were determined by Invitrogen Ribogreen Assay. The doses (0.5, 1, 2.5, and 5 ug) for the in vivo study were prepared by dilution into PBS. For SM-102 and EL-396, each mouse underwent two rounds of immunization, spaced 21 days apart. Blood samples were obtained from all mice prior to each immunization at 14 and 35 days after the initial immunization, using submandibular bleeding. For PMA, each mouse underwent one round of immunization. Blood samples were obtained 14 days after the immunization, using submandibular bleeding. After collection, the blood was allowed to clotovernight at 4°C, followed by serum extraction through centrifugation at 500 g for 15 minutes at 22°C. These serum samples were then stored at 4°C until they were ready for further analysis. MaxiSorp plates (Biolegend) were coated with 100 of μ rlecombinant Omicron BA.1 SI or RBD (1 μg / ml) in lx Antigen coating buffer overnight at 4°C. The wells were washed 3 times with PBS- T and incubated the plates with 200 of μ bllocking buffer for 2 hours at RT. After washing 3 times with PBS-T, the plate was incubated with 100 of s μelrially diluted sera for 2 hours at RT. After 2 hours incubation, the plate was washed 5 times with PBS-T and incubated with HRP-conjugated secondary antibody for 1 hour at RT. Finally, the plate was washed 5 times with PBS-T and incubated with 100 μ olf lx TMB substrate for 10-20 minutes. The reaction was stopped by adding 100 μl of 1N-HC1 and read at 450nm using the Cytation7.Example 5: Characterization of Sterol and PEG Lipids[000309] The sterol component of the 3 component mLNP of the disclosure was also studied. Table 13 below show that animal derived and plant derived cholesterol have little gap in the physical characterization. Cholesterol was the only registered sterol lipid at DMF and CDE. Of the sterol lipids tested, mLNPs comprising cholesterol showed the most optimal physiochemical parameters. The formulations were performed using SM-102, a sterol lipid according to Table 13, and DMG-PEG2000 at molar ratios of 59.8, 38.5, and 1.7 respectively. After microfluidic formulation with mRNA, the samples were dialyzed against 20mM Tris-HCl, pH 7.4 and concentrated by centrifuge. The particle size and PDI and zeta potential were determined by Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was determined by Invitrogen Ribogreen Assay.TABLE 13: Screening of sterol lipid[000310] The PEG lipid component of the 3 component mLNP of the disclosure was also studied. Table 14 below show that all mLNP groups tested had uniform size, DSG-PEG2000 had lower transfection efficiency and was not registered at DMF or CDE. Of the PEG lipids tested, mLNPs comprising DMG-PEG2000 showed the most optimal physiochemical. The LNP formulations were performed using SM-102, DSPC, cholesterol, and DMG-PEG2000 at molar ratios of 50:10:38.5:1.5. The mLNP formulations were prepared using SM-102, cholesterol, and DMG-PEG2000 at molar ratios according to Table 14. After microfluidic formulation with mRNA, the samples were dialyzed against 20mM Tris-HCl, pH 7.4 and concentrated by centrifuge. The particle size and PDI and zeta potential were determined by Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was determined by Invitrogen Ribogreen Assay.TABLE 14: Screening of PEG mole % (batch 1)[000311J The size of mLNP in Table 14 was decreased when the content of PEG increased.The group with 1% PEG had largest size. This size is above the 220nm sterile filtration limit, indicating that more than 1% DMG-PEG2000 is needed to control the size to a level for sterile filtration. The same experiments were performed on a second batch of mLNPs. Each condition was tested before and after lyophilization. The LNP formulation was prepared using SM-102,DSPC, cholesterol, and DMG-PEG2000 at molar ratios of 50:10:38.5:1.5. The mLNP formulations were prepared using SM-102, cholesterol, and DMG-PEG2000 at molar ratios according to Table15. After microfluidic formulation with mRNA, the samples were divided and aliquots for eachformulation was dialyzed against 20mM Tris-HCl, pH 7.4 with one of three sugars combinations: first, 8% sucrose; second, 8% sucrose+4% mannitol; and, third, 8%sucrose+4% trehalose. Each sample was reconstituted with lOOmM NaCl. The particle size and PDI and zeta potential were determined by Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was determined by Invitrogen Ribogreen Assay.TABLE 15: Screening of PEG mole % (batch 2)[000312] Transfection efficiency of mLNPs containing different PEG lipid content was also tested before and after lyophilization. Fig. 25 shows Western blot data of the formulations in Table 15 under the various conditions. After lyophilization column show three entries compared to the before lyophilization column because three reconstitution conditions were evaluated: one, 8% sucrose; two, 8% sucrose and 4% mannitol; and, three, 8% sucrose and 4% trehalose. The Western blot analysis was performed using lysates of transfected HEK293T cells were prepared in 200 pl RIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed. The BCA assays were performed for quantifying the concentration of proteins. The cell lysates were (10-20 μg per lane) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk / TBST (lx Tris-buffered saline containing 0.1% Tween-20), membranes were incubated with primary antibody in 5% milk / TBST at 4°C overnight. After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk / TBST for 1 hour at room temperature. Chemiluminescence (Clarity Western ECL substrate, Bio-Rad) imageacquisition and quantitation of band intensities were performed using an Azure Chemidoc™ Imaging System (Azure Biosystem 400).[000313] As shown in Fig. 25, PEG content affected the physicochemical parameters and transfection of mLNP. Group 1 containing SM-102 (59.8%), cholesterol (38.5%), DMG-PEG2000 (1.7%) had better performance in physicochemical parameters and transfection efficiency before and after lyophilization.[000314] The N / P ratios of mLNPs containing different lipid ratios were evaluated and shown in Fig. 26. As shown in Table 16 below, each condition possesses a size less than 200 nm, PDI values well below 0.3, zeta potential values less than a magnitude of 30 and encapsulation efficiencies well above 80%. These values indicate that the mLNP is compatible at wide ranges of the N / P ratio from 3:1 to N / P of 8:1. The zeta potential of mLNP with N / P ratio 3:1 is negative charge, which might be due to structural changes of the mLNP at low N / P ratios. The formulations for Table 16 were prepared on microfluidic mixer. The mRNA was diluted in 25mM sodium acetate, pH 5.0 and the lipids (SM-102, cholesterol, and DMG-PEG2000) were diluted in 200 proof ethanol. The microfluidic mixing was performed at a flow rate ration of 3:1 of aqueous to organic phase. The total flow rate was 8mL / min. The samples were dialyzed against 20mM Tris- HC1, pH 7.4, 8% sucrose. The size, PDI, and zeta potential were measured using Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was calculated using the Ribogreen Assay kit from Invitrogen. The Western blot analysis in Fig. 26 was performed using lysates of transfected HEK293T cells were prepared in 200 RI μPl A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed. The BCA assays were performed for quantifying the concentration of proteins. The cell lysates were (10-20 per lan μeg) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk / TBST (lx Tris-buffered saline containing 0.1% Tween-20), membranes were incubated with primary antibody in 5% milk / TBST at 4°C overnight. After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk / TBST for 1 hour at room temperature. Chemiluminescence (ClarityWestern ECL substrate, Bio-Rad) image acquisition and quantitation of band intensities were performed using an Azure Chemidoc™ Imaging System (Azure Biosystem 400).TABLE 16: Screening of N / P ratio (samples dialyzed with 8% sucrose)[000315] The N / P ratio of the same mLNP compositions were studied using a higher amount of cryoprotectant. Table 17 and 18 show that the higher cryoprotectant did not change the physical chemical data significantly. The zeta potential of mLNP with N / P ratio 3:1 is positive charge, which is different with 8% and 10% sucrose groups. Increasing the content of sucrose could protect the microstructure of mLNP better. Each data point was acceptable. The formulations for Table 17 and 18 were prepared on microfluidic mixer. The mRNA was diluted in 25mM sodium acetate, pH 5.0 and the lipids (SM-102, cholesterol, and DMG-PEG2000) were diluted in 200 proof ethanol. The microfluidic mixing was performed at a flow rate ration of 3: 1 of aqueous to organic phase. The total flow rate was 8mL / min. The samples were dialyzed against 20mM Tris-HCl, pH 7.4, and a cryoprotectant. The cryoprotectant for Table 17 was 10% sucrose and the cryoprotectant for Table 18 was 12%. The size, PDI, and zeta potential were measured using Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was calculated using the Ribogreen Assay kit from Invitrogen.TABLE 17: Screening of N / P ratio (samples dialyzed with 10% sucrose)TABLE 18: Screening of N / P ratio (samples dialyzed with 12% sucrose)[000316] As shown in Table 19, a second batch of mLNPs were tested. Delta mLNP withN / P ratio 4.6 and 6.6 had better control after lyophilization. The lyophilized LNP or mLNPstructures were better protected in 12% sucrose buffer (EE%). The formulations for Table 19 was prepared on microfluidic mixer. The mRNA was diluted in 25mM sodium acetate, pH 5.0 and the lipids (SM-102, cholesterol, and DMG-PEG2000) were diluted in 200 proof ethanol. The microfluidic mixing was performed at a flow rate ratio of 3:1 of aqueous to organic phase. The total flow rate was 8mL / min. The samples were dialyzed against 20mM Tris-HCl, pH 7.4, and 8% serose. Each formulation was aliquoted and a portion was lyophilized. The size, PDI, and zeta potential were measured using Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was calculated using the Ribogreen Assay kit from Invitrogen. The in vitro analysis was performed (Fig. 26) and expression appeared to increase as the N / P ratio was lowered. The Western blot analysis in Fig. 26 was performed using lysates of transfected HEK293T cells were prepared in 200 R μlIP A buffer (Thermo Scientific) containing proteinase inhibitor cocktail per well in a 6-well plate. The plate was incubated on ice for 30 min with a gentle mixing. The cell lysates were transferred to 1.5 ml tube and sonicated briefly, followed by centrifugation for 15 minutes at maximum speed. The BCA assays were performed for quantifying the concentration of proteins. The cell lysates were (10-20 per μ lagne) inactivated by heat (95°C for 5 min) were separated in an SDS-PAGE gel (4-12% NuPAGE, Invitrogen) with MES Running buffer for 30 minutes at 200 V and transferred onto PVDF membrane using iBlot2 system (Invitrogen). After blocking with 5% milk / TBST (lx Tris-buffered saline containing 0.1% Tween- 20), membranes were incubated with primary antibody in 5% milk / TBST at 4°C overnight. After washing 3 times with TBST, the secondary antibodies conjugated with HRP were added to the membrane in 5% milk / TBST for 1 hour at room temperature. Chemiluminescence (Clarity Western ECL substrate, Bio-Rad) image acquisition and quantitation of band intensities were performed using an Azure Chemidoc™ Imaging System (Azure Biosystem 400).TABLE 19: Screening of N / P ratio (batch 2)[000317] After lyophilization, the Zeta potential of mLNP tends to switch from positive to negative, but LNP does not switch. As seen in Fig. 27, this switch does not affect the in vivo immunogenicity.[000318] Example 6[000319] A mouse immunogenicity study of N / P ratio screening was performed. Table 20 and Fig. 27 show the data. IgG binding antibody titers after the second administration with mLNP and LNP. The in vivo data from Fig. 27 was collected from female BALB / c mice, aged 6 to 8 weeks. They received intra-muscular vaccinations with SARS-CoV-2 Delta mRNA, which was formulated using groups from Table 19. Each mouse underwent two rounds of immunization, spaced 21 days apart. Blood samples were obtained from all mice prior to each immunization at 14 and 35 days after the initial immunization, using submandibular bleeding. After collection, the blood was allowed to clot overnight at 4°C, followed by serum extraction through centrifugation at 500 g for 15 minutes at 22°C. These serum samples were then stored at 4°C until they were ready for further analysis. MaxiSorp plates (Biolegend) were coated with 100 of re μclombinant Omicron BA.1 SI or RBD (1 μg / ml) in lx Antigen coating buffer overnight at 4°C. The wells were washed 3 times with PBS-T and incubated the plates with 200 of b μllocking buffer for 2 hours at RT. After washing 3 times with PBS-T, the plate was incubated with 100 of se μrliallydiluted sera for 2 hours at RT. After 2 hours incubation, the plate was washed 5 times with PBS- T and incubated with HRP-conjugated secondary antibody for 1 hour at RT. Finally, the plate was washed 5 times with PBS-T and incubated with 100 of μ llx TMB substrate for 10-20 minutes. The reaction was stopped by adding 100 of 1 μNl -HC1 and read at 450nm using the Cytation?. The reciprocal serum endpoint titer was calculated by serial dilution.[000320] mLNP produces equivalent immune response with LNP in mice model, which is consistent with the previous results. N / P ratio of mLNP did not greatly affect the in vivo effect.[000321 ] Table 20. Mouse immunogenicity study design of N / P ratio screeningProcess experiments were performed. As shown in Fig. 28, the process involved Prefreezing to - 45 °C, then turning on the vacuum pump to achieve 0.1 mbar vacuum degree. The sublimationinterface moved toward the center. Applied heating, observed the temperature. The collapse temperature (Tc) is -30 °C.[000322] As shown in Fig. 29, cake appearance after lslbatch (a), 2ndbatch (b) and 12thbatch (c) lyophilization, (d) Reconstitution of 12thbatch mLNP. Cake appearance after lyophilization was excellent and there was no collapse. The lyophilization conditions were optimized as shown in Fig. 29 to give a better cake appearance. The 1stbatch lyophilization condition was cool to -5°C (5min), hold at -5°C (lh), cool to -45°C (30min), hold at -45°C (9h), warm to -35 (50min), hold at -35°C (36h), warm to 30°C (6.25h), hold at 30°C (4h), cool to 2°C (15min), and hold at 2°C (48h). The 2ndbatch lyophilization condition was cool to -5°C (5min), hold at -5°C (lh), cool to -50°C (30min), hold at -50°C (9h), warm to -45 (50min), hold at -45°C (36h), warm to 25°C (6.25h), hold at 25°C (4h), cool to 2°C (15min), and hold at 2°C (48h). The 12thbatch lyophiliation condition was cool to -5°C (40min), hold at -5°C (30min), cool to -45°C (lOOmin), hold at -45°C (9h), warm to -30 (90min), hold at -30°C (36h), warm to 25°C (4.5h), and hold at 25°C (4h). The 12thbatch after reconstitution.[000323] Fig. 30 shows characterization of mLNP after lyophilization. Size (a) and zeta potential (b) of mLNP after 2ndbatch lyophilization. Size (c) and zeta potential (d) of mLNP after 12thbatch lyophilization.[000324] Table. 21. Moisture content test by Karl Fischer method[000325] The moisture content of mLNP after lyophilization was controlled in 2.0%.[000326] The best lyophilization cycle began with a freezing step at -45°C for 9 hours, followed by primary drying at -30 °C and 0.1 ~0.2 mbar for 36 hours, and finishing with secondary drying at 25 °C and 0.1 -0.2 mbar for 4 hours. The freezing rate is 1 °C per min. Ramp rate from primary to secondary drying is 0.2 °C per min. The size was 126.3 nm, the PDI was 0.07081, the zeta potential was -6.159 and the encapsulation was 86%. The moisture content of mLNP after lyophilization was controlled in 2.0%, which was met the quality requirements.[000327] Example 7 - Screening of multiple mRNAs[000328] Table 22. Characterization of mLNP with different mRNA.[000329] .. The encapsulated mRNA length ranges from around 4000 nb to 900 nb. The robustness of the mLNP for encapsulation of a wide range of mRNA sizes was established with different mRNAs (Delta, RSV, Flue, RAS, NYESO1, VEE-RAS, and a bivalent SARS-CoV2 Delta + BA.1. Each mRNA was formulated at an N / P ratio of 5.6. The lipids used were SM-102, cholesterol, and DMG- PEG2000 at molar ratio of 59.8:38.5:1.7. The mixing was performed by microfluidic mixing at a flow rate ratio of 3:1 and a total flow rate of 8mL / min. Each formulation was dialyzed against 20mM Tris-HCl, pH 7.4, 8% sucrose. The samples were concentrated. The size, PDI, and zeta potential were determined by a Malvern Zetasizer using DTS1070 folded cuvettes. The encapsulation efficiency was determined using an Invitrogen Ribogreen Assay kit [000330] Table 23. Delta mLNP and LNP before and after lyophilization.[000331] Negative stained-transmission electron microscopy (TEM). The grid was treated with glow discharge. Particle sample (4 pL, 25 ng / pL of mRNA) in buffer solution was dropped on a 200-mesh copper grid coated with a continuous carbon film and excess solution was removed after 60 s of waiting by blotting with a wipe. Then 4 pL of negative staining solution, phosphotungstic acid as a 1% aqueous solution was dropped on the TEM grid and removed by blotting after 30 s, and the grid is dried at room temperature. The dried grid was mounted on a JEOL single tilt holder equipped in the TEM column. The specimen was cooled down by liquid-nitrogen. Imaging on a JEOL 2100 PEG microscope was conducted using a minimum dose method that is essential to avoid sample damage under the electron beam. The microscope was operated at 200 kV and with a magnification in the ranges of 10,000-60,000 for assessing particle size and distribution. All images were recorded on a Gatan 2kx2k UltraScan CCD camera. The image was analysed by DigitalMicrograph 3.[000332] Fig. 31 shows TEM images of LNP and mLNP before and after lyophilization. Images of LNP before (a) and after (b) lyophilization. Images of mLNP before (c) and after (d) lyophilization.[000333] The microstructure of LNP and mLNP is relatively uniform. There are some bulges on the particle surface. The microstructure of mLNP and LNP is different. A layer is on the surface of mLNP. LNP after lyophilization is not uniform and there are more bulges on the particle surface. mLNP after lyophilization is more uniform than LNP. It is circular in its shape. The mLNP showed great stability after lyophilization.[000334] Fig. 32 shows TEM images of SM-102 mLNP before and after freezing.[000335] The microstructure of SM-102 mLNP is relatively uniform. There were no particulate larger than 500 nm in these samples. Some small black spots in the mLNP, which might be the concavity of surface. The mLNP showed great stability after freezing.[000336] Example 8- Stability screening[000337] A comparison of mLNP and LNP after freezing was performed.[000338] Table 24. Lipid ratio of mLNP for stability test.[000339] Figs. 33-36 show characterization of RSV mLNP and LNP after lyophilization after 1 month storage. Size did not change too much at 4 and -80 °C. At -20 °C, change of mLNP was more than LNP. Zeta potential was a little bit higher than before at 4, -20 , -80 °C.[000340] Comparison of mLNP and LNP before and after lyophilization after storage at different conditions[000341] Table 25. Characterization of Delta mLNP and LNP after lyophilization after 1 month storage.[000342] The physicochemical parameters of mLNP after storage at 25 °C, 4 °C, -20 °C, -80 °C for 1 month were coincident with 0 day. Whereas, the physicochemical parameters of LNP after storage at 25°C, 4 °C, -20 °C, -80 °C for 1 month were larger for size and PDI but coincident for zeta potential and encapsulation efficiency.[000343] N / P ratio screening of mLNP[000344] Table 26. N / P ratio of mLNP for stability test for cold storage[000345] Fig. 37 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by particle size over the course of 1 month.[000346] Fig. 38 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by particle size distribution over the course of 1 month.[000347] Fig. 39 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by zeta potential over the course of 1 month.[000348] Fig. 40 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 8% sucrose as measured by encapsulation efficiency over the course of 1 month.[000349] Fig. 41 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by particle size over the course of 1 month.[000350] Fig. 42 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by particle size distribution over the course of 1 month.[000351] Fig. 43 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by zeta potential over the course of 1 month.[000352] Fig. 44 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 10% sucrose as measured by encapsulation efficiency over the course of 1 month.[000353] Fig. 45 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by particle size over the course of 1 month.[000354] Fig. 46 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by particle size distribution over the course of 1 month.[000355] Fig. 47 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by zeta potential over the course of 1 month.[000356] Fig. 48 shows stability of Delta-mLNP at five N / P ratios when dialyzed with 12% sucrose as measured by encapsulation efficiency over the course of 1 month.[000357] Table 27. Lipid ratio of mLNP for stability test (8% sucrose).[000358] Table 28. Lipid ratio of mLNP for stability test (10% sucrose).[000359J Table 29. Lipid ratio of mLNP for stability test (12% sucrose).[000360] N / P ratio affected the size, when the N / P ratio decrease to 6.6, the size is smallest Zeta potential and encapsulation is almost unchanged. Stability of mLNP could be increased by increasing the sucrose content to 10% and adjusting the N / P ratio to 6.6.[000361] Fig. 49 shows Particle size, PDI and encapsulation efficiency change of RSV mLNP after freezing and lyophilization during 6 months.Particle size, PDI and encapsulation efficiency of mLNP nearly did not change after freezing and lyophilization during 6 months. mLNP showed great stability after freezing and lyophilization.[000362] Fig. 50 shows characterization of mLNP with different mRNA. All size of samples was uniform and the PDI was small. The zeta potential was ± 20 mV.[000363] Fig. 51 shows Characterization of mLNP with different ionizable lipids. All size of samples was uniform and the PDI was small. The zeta potential was ± 20 mV.[000364] Fig. 52 shows Characterization of mLNP before and after lyophilization. The size and zeta potential of mLNP was maintained after lyophilization. The zeta potential was ± 20 mV. [000365] Fig. 53 shows a titration curve of moisture content. The moisture content of mLNP after lyophilization via C30s+InMotion KF Flex and C30s.[000366] Fig. 53 shows a titration curve of moisture content of mLNP after lyophilization via C30s+InMotion KF Flex and C30s.[000367] Biodistribution test[000368] Balb / C female mice were intramuscularly injected (50 p.1 in both hind legs) with LNPs formulated with Flue mRNA at a dose of 0.1 mg kg-1. The biodistribution of different preparations at 6, 10, 24, 48, and 72 hours was observed. At every time point, mice were intraperitoneal injected with D-luciferin (150 mg kg-1). After 4 min, the whole of mice was imaged using imaged using an IVIS Lumina system. After that, organs (hearts, lungs, liver, kidneys, spleens and lymph nodes) were dissected and imaged. Luminescence was quantified as average luminescence of total organ areas (ps-1 cm-2 si^l) using Living image 4.4.[000369] Fig. 54 shows biodistribution of mRNA expression (dark spots) in mice differences between LNP and mLNP formulations with SM-102.[000370] Fig. 55 shows biodistribution among the lymph nodes, liver, and spleen demonstrating that mLNP has a distinct difference in biodistribution than LNP. Robust luciferasesignal was observed both at the injection sites and the liver between 6 and 10 hours after intramuscular administration, which gradually declined within 72 hours. The biodistribution of LNP or mLNP with different lipids was different. Compared to mLNP, LNP had a better secondary lymphoid organ draining ability.[000371 ] Fig. 56 shows biodistribution of both LNP and mLNP can be controlled by choice of ionizable lipid. EL-396 does not deliver to the liver under either LNP or mLNP conditions.[000372] Cell uptake[000373] DC2.5 cells were treated with Cy5-labeled Delta mRNA LNP or mLNP to quantify the percentage of transfection by flow cytometry. Cells were seeded at 7 x 105cells per well in a 6-well plate. After 24 h, cells were treated with LNPs (500 ng and 1 Cy5-l μagbeled Delta mRNA) and 1 mL of fresh DMEM (10% FBS was added). Cells were washed with PBS, trypsinized for 3 min at 37 °C, neutralized with 1 mL of DMEM, centrifuged at 300g for 5 min, washed with cold PBS twice, and diluted in 300 pL of cold PBS. Finally, cells were kept on ice until they were analyzed by a flow cytometer. Fluorescence intensity was quantified by FlowJo VI 0.[000374] Fig. 57 shows Cell uptake of Cy5-LNP and Cy5-mLNP. Flow cytometry was used to determine distribution of Cy5 fluorescence (a), percentage of Cy5 positive cells (b) and mean fluorescence intensity (c). The cell uptake of mLNP was higher than that of LNP.[000375] In addition, it is to be understood that any particular aspect of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such aspects are deemed to be part of the whole of the present disclosure, any part of the whole disclosure may be excluded even if the exclusion is not set forth explicitly herein.[000376] It is to be understood that while the present disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and alterations are within the scope of the following claims.

Claims

CLAIMS1. A lipid compound of Formula I(I), or a salt or isomer thereof, wherein, each n is independently selected from 0-8; each Q is independently selected fromwherein each m is independently selected from 0-6; each o is independently selected from 0-7; each R1and R2is, each X is independently selected from2. The lipid compound of claim 1, wherein the compound is:

3. A method for synthesizing the compound of I comprising performing the following reaction:

4. A lipid compound of Formula II(II), or a salt or isomer thereof, wherein, n is 0-8; each Q is independently selected fromwherein each m is independently selected from 0-6 and o is independently selected from 0-7 and;5. The lipid compound of claim 4, wherein the compound is:

6. A method for synthesizing the compound of n(a) comprising performing the following reaction:

7. A nanoparticle composition comprising at least one lipid compound of any one of claims 1, 2, 4, or 5, or a compound of Formula (IA), (IB), (IC), (HA), (HB), (IIC), (HD), (HE), (HF), or a combination thereof:or a salt or isomer thereof, wherein m is 0-9; n is 0-9; o is 0-12; p is 0-12;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12;R is the side chain of an independently selected amino acid;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or linear C1-12alkyl;R5is the side chain of an independently selected amino acid;X1is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;X2is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;X3is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-; andX4is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-5;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is H or CH3;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H; andX is selected from -CH2-, -O-, -S-, or -P(O)(OR)O-;or a salt or isomer thereof, wherein m is selected from 0-5; n is selected from 0-12; o is selected from 0-12; q is selected from 1-3;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andX is selected from C(R)2, N(R), or O, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-6;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R5is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 1-7; o is selected from 0-12; p is selected from 0-12;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

8. The nanoparticle composition of claim 7, further comprising a biologically active agent9. The nanoparticle composition of claim 8, wherein the biologically active agent is a nucleic acid.

10. The nanoparticle composition of any one of claims 8-9, wherein the biologically active agent is an RNA molecule.

11. The nanoparticle composition of any one of claims 8-10, wherein the biologically active agent is siRNA, miRNA, shRNA, tRNA, mRNA, and mixtures thereof.

12. The nanoparticle composition of any one of claims 8-11, further comprising a phospholipid selected from the group consisting of l,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), l-oleoyl-2-cholesterylhemisuccinoyl-sn- glycero-3 -phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero- 3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphocholine, 1,2- diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-gly cero-3 -phosphoethanolamine, 1 ,2- dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

13. The nanoparticle composition of claim 12, wherein the phospholipid is DSPC.

14. The nanoparticle composition of any one of claims 8-13, further comprising a structural lipid selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

15. The nanoparticle composition of any one of claim 14, wherein the structural component is cholesterol.

16. The nanoparticle composition of any one of claims 8-15, further comprising a PEG lipid selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG- modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, aPEG-modified diacylglycerol, a PEG-modified dialkylglycerol, a PEG-modified myristoyl diglyceride, and mixtures thereof.

17. The nanoparticle composition of any one of claim 16, wherein the PEG lipid is DMG- PEG2000.

18. The nanoparticle composition of any one of claims 7-17, wherein the components in the LNP have the following relative molar percentages:20 mol % to 60 mol % of the lipid compound,25 mol % to 45 mol % of a structural lipid component, 0 mol % to 25 mol % of a phospholipid, or0 mol % to 5 mol % of a PEGylated lipid component.

19. The nanoparticle composition of any one of claims 7-17, wherein the components in the LNP have the following relative molar percentages:1) 0.5 to 60 mole % of a steroidal or structural lipid-containing component;2) 0.5 to 20 mole % of a PEGylated lipid-containing component; and3) 25 to 97.2 mole % of the lipid compound.

20. The nanoparticle composition of any one of claims 7-17, wherein the components in the LNP have the following relative molar percentages:1) 20 to 50 mole % of a steroidal or structural lipid-containing component;2) 0.8 to 10 mole % of a PEGylated lipid-containing component; and3) 40 to 62 mole % of the lipid compound.

21. The nanoparticle composition of any one of claims 7-17, wherein the components in the LNP have the following relative molar percentages:1) 25 to 46 mole % of a steroidal or structural lipid-containing component;2) 1 to 7 mole % of a PEGylated lipid-containing component; and3) 44 to 58 mole % of the lipid compound.

22. The nanoparticle composition of any one of claims 7-17, wherein the components in the LNP have the following relative molar percentages:1) 35 to 44 mole % of a steroidal or structural lipid-containing component;2) 1.2 to 5 mole % of a PEGylated lipid-containing component; and3) 48 to 57 mole % of the lipid compound.

23. The nanoparticle composition of any one of claims 7-17, wherein the components in the LNP have the following relative molar percentages:1) 20 to 35 mole % of a steroidal or structural lipid-containing component;2) 0.8 to 10 mole % of a PEGylated lipid-containing component; and3) 55 to 79 mole % of the lipid compound.

24. The nanoparticle composition according to any one of claims 7-23, wherein the LNP has an encapsulation efficiency of at least 70% after storage at about -80°C to 25°C for at least 28 days.

25. The nanoparticle composition according to any one of claims 7-24, wherein the LNP has a zeta potential of about -30 to +30 mV after storage at about -80°C to 25°C for at least 28 days.

26. The nanoparticle composition according any one of claims 7-25, wherein the LNP has a PDI of less than 0.300 after storage at about -80°C to 25°C for at least 28 days.

27. The nanoparticle composition according any one of claims 7-26, wherein the LNP has a particle size of less than 220 nm after storage at about -80°C to 25°C for at least 28 days.

28. The nanoparticle composition according any one of claims 7-27, wherein the LNP has a nitrogen to phosphorus ratio (N:P) range of about 10 to 1.

29. The nanoparticle composition according to any one of claims 7-28, further comprising a cryoprotectant.

30. The nanoparticle composition of claim 29, wherein the cryoprotectant is sucrose, trehalose, mannose, mannitol, glucose, or a combination thereof.

31. The nanoparticle composition of any one of claims 29-30, wherein the cryoprotectant has a volume / volume concentration of 0-50% (v / v).

32. A three-component LNP composition wherein the three components are: 1) a structural lipid component; 2) a PEGylated lipid component; or 3) a lipid compound of any one of claims 1, 2, 4, or 5, or a compound of Formula (IA), (IB), (IC), (IIA), (IIB), (IIC), (HD), (HE), (HF), or a combination thereof:or a salt or isomer thereof, wherein m is 0-9; n is 0-9; o is 0-12; p is 0-12;R1is a linear Ci-n alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9;n is selected from 0-9; o is selected from 0-12; p is selected from 0-12;R is the side chain of an independently selected amino acid;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R5is a linear C1-12alkyl;R4is H or linear C1-12alkyl;R5is the side chain of an independently selected amino acid;X1is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-;X2is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;X3is -OC(O)N(H)-, -C(O)N(H)-, -N(H)C(O)-, or -OC(O)-; andX4is -C(O)N(H)-, -C(O)O-, -N(H)C(O)-, or -N(H)C(O)-;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-5;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is H or CH3;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H; andX is selected from -CH2-, -O-, -S-, or -P(O)(OR)O-;or a salt or isomer thereof, wherein m is selected from 0-5; n is selected from 0-12; o is selected from 0-12; q is selected from 1-3;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andX is selected from C(R)2, N(R), or O, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9;n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 0-6;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl;R5is a linear C1-4alkyl alcohol;R6is a linear C1-4alkyl alcohol;M1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 1-7; o is selected from 0-12; p is selected from 0-12;R1is a linear C1-12alkyl;R2is H or a linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, whereinm is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H;or a salt or isomer thereof, wherein m is selected from 0-9; n is selected from 0-9; o is selected from 0-12; p is selected from 0-12; q is selected from 2-6;R1is a linear C1-12alkyl;R2is H or linear C1-12alkyl;R3is a linear C1-12alkyl;R4is H or a linear C1-12alkyl; andM1and M2are independently selected from -C(O)N(R)-, -N(R)C(O)-, -C(O)S-, -SC(O)-, - OC(O)O-, -OC(O)N(R)-, or -N(R)C(O)O- groups, wherein R is independently selected from a methyl and H.

33. A pharmaceutical composition comprising the nanoparticle composition of any one of claims 8-32, and a pharmaceutically acceptable carrier.

34. A method of delivering a biologically active agent to a cell, the method comprising administering to a subject the nanoparticle composition of any one of claims 8-32, said administering comprising contacting the cell with the LNP, whereby the biologically active agent is delivered to the cell.

35. A method of delivering a biologically active agent to a cell of a subject, the method comprising administering the pharmaceutical composition of claim 33 to the subject.

36. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition of any one of claims 8-32.

37. A method for synthesizing 1(a) comprising38. A method for synthesizing 1(b) comprising:

39. A method for synthesizing 1(c) comprising40. A method for synthesizing 1(d) comprising41. A method for synthesizing 11(a) comprising42. A method for synthesizing 11(b) comprising43. A method for synthesizing 11(c) comprising:

44. A method for synthesizing the compound of 11(d) comprising

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