Ionizable lipid compounds and lipid nanoparticle compositions

Abstract

Disclosed are novel ionizable lipids, compositions, and methods of using the novel ionizable lipids and compositions as described in Formula (I). The lipid nanoparticle compositions comprise novel ionizable lipids and additional lipids, such as phospholipids, structural lipids, and PEG lipids. The lipid nanoparticle compositions further comprise a bioactive agent, such as mRNA or DNA, which can be used to deliver the bioactive agent to mammalian cells or organs. # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 329,200 filed on April 8, 2022 and U.S. Provisional Application No. 63 / 429,468 filed on December 1, 2022, the disclosures of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present disclosure provides novel compounds, compositions comprising such compounds, and methods of delivering one or more biologically active agents to mammalian cells or organs. Background Art

[0004] The delivery of bioactive substances such as small molecule drugs, proteins and nucleic acids (including DNA and mRNA) is a medical challenge. Particularly, due to the relative instability and low cell permeability of nucleic acid molecules, it becomes difficult to deliver such molecules to cells. In addition, lipid nanoparticles, the components of the lipid nanoparticle delivery system (LNP) approved by FDA are currently synthesized using a multi-step method. Therefore, it is necessary to develop compounds, compositions and methods for improving the delivery of bioactive molecules (including nucleic acids) to cells or organs and to synthesize such compounds in a time-efficient, cost-effective and high-throughput manner. Summary of the invention

[0005] The present disclosure provides novel compounds and compositions and methods involving the same.

[0006] In one aspect, the present disclosure provides compounds of formula (I):

[0007]

[0008] or a salt or isomer thereof, wherein

[0009] m and n are each independently 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range within the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0010] Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0011]

[0012] R 1 or R 2 Each a, b and c in is independently an integer selected from 0-24, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or any sub-range selected from the range of 0-24.

[0013] Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0014]

[0015] R 3 , R 4 and R 5 wherein a, b and c are each independently 0-24, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0016] Each X is independently selected from CH2, NH, O or S;

[0017] Each Y is independently selected from CH2, NH, O or S;

[0018] Each Z is independently selected from CH or N; and

[0019] Each L is independently selected from CH2, NH, O or S.

[0020] In certain aspects, the compound of Formula I may comprise, for example, the following formula:

[0021]

[0022] or a salt or isomer thereof, wherein each R 1 and R 2 As defined above.

[0023] General synthetic route for synthesizing compounds of formula 1-1:

[0024]

[0025] In certain aspects, compounds of Formula I may include, for example, the following compounds:

[0026] (Ia), also known as C7;

[0027] (Ib), also known as D7.

[0028] In another aspect, the present disclosure provides a compound of formula (II):

[0029]

[0030] or a salt or isomer thereof, wherein

[0031] Each n is independently 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range within the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0032] Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0033]

[0034] R 1 or R 2 a, b and c are each independently an integer from 0 to 24;

[0035] Each R 3 , R 4 and R 5 independently selected from C1-C24 alkyl, C1-C24 alkenyl, C1-C24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl,

[0036]

[0037] R 3 , R 4 or R 5wherein a, b and c are each independently 0-24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0038] Each X is independently selected from CH or N;

[0039] Each Y is independently selected from CH2, NH, O or S; and

[0040] Each Z is independently selected from CH2, NH, O or S.

[0041] In certain aspects, the compound of formula II may comprise, for example, the following formula:

[0042]

[0043] or a salt or isomer thereof, wherein each R 1 and R 2 As defined above.

[0044] General synthetic route for synthesizing compounds of formula II-1:

[0045]

[0046] In certain aspects, compounds of Formula II may include, for example, the following compounds:

[0047] Also known as TAb-9 or E2;

[0048] Also known as TAb-13 or E6;

[0049] or a salt or isomer thereof.

[0050] In another aspect, the present disclosure provides a compound of formula (III):

[0051]

[0052] or a salt or isomer thereof, wherein

[0053] Each R 1 , R 1’ , R 2 and R 3 Independently selected from H, C1-C24 alkyl, C1-C 24 Alkenyl, C1-C 24Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0054]

[0055] a, b and c are each independently 0-24, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0056] Each R 9 Independently selected from

[0057] Each R 10 , R 10’ , R 10” , R 11 , R 11’ , R 11” and R 12 Independently selected from H, C1-C 24 Alkyl, 1-C 24 Alkenyl, 1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0058]

[0059] a, b and c are each independently 0-24, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0060] R 3” , R 4” and R 5” Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0061]

[0062] a, b and c are each independently 0-24, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24.

[0063] In certain aspects, the compound of formula III may comprise, for example, the following formula:

[0064]

[0065] or a salt or isomer thereof,

[0066] Each R 1 , R 1’ , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0067]

[0068] a, b and c are each independently an integer from 0 to 24;

[0069] Each R 3” , R 4” and R 5” Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0070]

[0071] a, b and c are each independently an integer from 0 to 24;

[0072] Or each of R 4 , R 5 , R 6 , R 7 and R 8 Independently for:

[0073]

[0074] Where R 1”’ , R 1”” , R 2”’ and R 3”’ Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0075]

[0076] Each a, b and c is independently an integer selected from 0-24;

[0077] Each X is independently CH, N, O or S;

[0078] Each Y is independently CH, N, O or S

[0079] Each Z is independently CH, N, O or S

[0080] Each F is independently CH, N, O or S, and

[0081] Each E is independently CH, N, O or S.

[0082] General synthetic route for synthesizing compounds of formula III-1:

[0083]

[0084] In certain aspects, the compound of formula III may include, for example, the following compounds:

[0085]

[0086]

[0087]

[0088] Also known as TP4G11;

[0089]

[0090]

[0091] Also known as TP1A11;

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Also known as TP3G11; Also known as TP3F11;

[0098] Also known as TP5A1;

[0099] or a salt or isomer thereof.

[0100] In another aspect, the present disclosure provides a compound of formula (IV):

[0101]

[0102] or a salt or isomer thereof, wherein

[0103] Each R 1 , R 2 , R 3 , R 4 and R 5 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0104]

[0105]

[0106] a, b and c are each independently an integer from 0 to 24;

[0107] Each R 6 , R 7 , R 8 and R 9 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0108]

[0109] a, b and c are each independently an integer from 0 to 24;

[0110] Each X is independently selected from CH, N;

[0111] Each Y is independently selected from CH2, NH, O or S;

[0112] Each Z is independently selected from CH or N.

[0113] In certain aspects, compounds of Formula IV may include, for example, the following compounds:

[0114] Also known as P53F12;

[0115] Also known as P38C2;

[0116] Also known as P55H12;

[0117] Also known as P54A6;

[0118] Also known as P6A2; Also known as P1C1;

[0119] Also known as P30A1;

[0120] Also known as P51C12;

[0121] Also known as P54B4;

[0122] Also known as P38D1;

[0123] Also known as P56A1; Also known as P40D7;

[0124] Also known as P56B1;

[0125] Also known as P14A2;

[0126] Also known as P16B1;

[0127] Also known as P53A5;

[0128] Also known as P26D4;

[0129] Also known as P1D4;

[0130] Also known as P30B7;

[0131] Also known as P53A6;

[0132] Also known as P30C7;

[0133] Also known as P38D7;

[0134] Also known as P38D8;

[0135] Also known as P40B10;

[0136] Also known as P40C11;

[0137] Also known as P40C10;

[0138] Also known as P38D4;

[0139] Also known as P56A2;

[0140] Also known as P149A2;

[0141] Also known as P159C10;

[0142] Also known as P161F10;

[0143] or a salt or isomer thereof.

[0144] In another aspect, the present disclosure provides a compound of formula (V):

[0145]

[0146] or a salt or isomer thereof, wherein

[0147] Each R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Independently selected from H, C1-C24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0148]

[0149] a, b and c are each independently an integer from 0 to 24;

[0150] Each R 3’ , R 4’ and R 5’ Independently selected from H, C1-C24 alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0151]

[0152] a, b and c are each independently 0-24, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24.

[0153] In certain aspects, compounds of Formula V may include, for example, the following compounds:

[0154]

[0155]

[0156] or a salt or isomer thereof.

[0157] In another aspect, the present disclosure provides a compound of formula (VI):

[0158]

[0159] or a salt or isomer thereof, wherein

[0160] each n is independently 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range of the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0161] Each R 1 and R 2 Independently selected from C1-C24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0162]

[0163] a, b and c are each independently an integer from 0 to 24;

[0164] Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0165]

[0166] a, b and c are each independently 0-24, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0167] Each Y is independently selected from CH or N;

[0168] Each Z is independently selected from CH2, NH, O or S.

[0169] In certain aspects, the compound of formula VI may comprise, for example, the following formula:

[0170]

[0171] or a salt or isomer thereof, wherein each R 1 and R 2 As defined above.

[0172] General synthetic route for synthesizing compounds of formula VI-1:

[0173]

[0174] In certain aspects, compounds of Formula VI may include, for example, the following compounds:

[0175]

[0176]

[0177] or a salt or isomer thereof.

[0178] In another aspect, the present disclosure provides a compound of formula (VII):

[0179]

[0180] or a salt or isomer thereof, wherein

[0181] each n is independently 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range of the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0182] Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0183]

[0184] a, b and c are each independently an integer from 0 to 24;

[0185] Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0186]

[0187] a, b and c are each independently an integer from 0 to 24;

[0188] Each X is independently selected from CH2, NH, O or S;

[0189] Each Y is independently selected from CH, N;

[0190] Each Z is independently selected from CH2, NH, O or S; and

[0191] Each E is independently selected from CH2, NH, O or S.

[0192] In certain aspects, the compound of formula VII may comprise, for example, the following formula:

[0193]

[0194] Each R 1 and R 2 As defined above.

[0195] General synthetic route for synthesizing compounds of formula VII-1:

[0196]

[0197] In certain aspects, the compound of formula VII may include, for example, the following compounds:

[0198] (VIIa), also known as T7;

[0199] (VIIb), also known as P7;

[0200] or a salt or isomer thereof.

[0201] In another aspect, the present disclosure provides a composition comprising a lipid component comprising a compound of formula (I), (II), (III), (IV), (V), (VI) and / or (VII). In some aspects, the composition may comprise one or more cationic and / or ionizable lipids, anionic lipids, phospholipids including polyunsaturated lipids, PEG lipids, structural lipids and / or biologically active molecules.

[0202] In another aspect, the present disclosure provides a method of preparing a nanoparticle composition comprising a lipid component comprising a compound of Formula (I), (II), (III), (IV), (V), (VI) and / or (VII).

[0203] In another aspect, the present disclosure provides a method of delivering a bioactive agent (e.g., mRNA) to a cell (e.g., a mammalian cell) by administering to a subject a nanoparticle composition comprising (i) a lipid component comprising a phospholipid, a PEG lipid, a structural lipid, and at least one of an ionizable lipid of Formula (I), (II), (III), (IV), (V), (VI), and (VII); and (ii) the bioactive agent, wherein the administration involves contacting the cell with the nanoparticle composition such that the bioactive agent is delivered to the cell.

[0204] In another aspect, the present disclosure provides a method for producing a target polypeptide in a cell (e.g., a mammalian cell) by contacting the cell with a nanoparticle composition comprising (i) a lipid component comprising a phospholipid, a PEG lipid, a structural lipid, and at least one of an ionizable lipid of Formula (I), (II), (III), (IV), (V), (VI), and (VII); and (ii) an mRNA encoding the target polypeptide, whereby the mRNA can be translated in the cell to produce the polypeptide.

[0205] In another aspect, the present disclosure provides a method for introducing a gene into a cell (e.g., a mammalian cell) by contacting the cell with a nanoparticle composition comprising (i) a lipid component comprising a phospholipid, a PEG lipid, a structural lipid, and at least one of an ionizable lipid of formula (I), (II), (III), (IV), (V), (VI), and (VII); and (ii) a DNA encoding a target gene, whereby the cell is capable of expressing the introduced gene.

[0206] In some aspects, the nanoparticles of the present invention are used together with another therapeutic compound that is different from the nanoparticles for the treatment of the same indication of an individual. In certain cases, the nanoparticles and the therapeutic compound are delivered individually or together. When delivered together, they may or may not be in the same preparation, and they may or may not be delivered by the same approach.

[0207] In another aspect, the present disclosure provides methods of synthesizing compounds of Formula (I), (II), (III), (IV), (V), (VI), and (VII). BRIEF DESCRIPTION OF THE DRAWINGS

[0208] Figure 1 Shown are the structures of TAb-9 and TAb-13 and fluorescence activated cell sorting (FACS) of TAb-9 and TAb-13 formulated LNPs encapsulating self-amplifying mRNA encoding GFP, which were transfected in C2C12 cells and expressed GFP-mRNA.

[0209] Figure 2 Shown are FACS of TAb-9 and TAb-13 formulated LNPs encapsulating self-amplifying mRNA encoding GFP, which were transfected in 293T cells and expressed GFP-mRNA.

[0210] Figure 3 Shown are FACS of TAb-9 and TAb-13 formulated LNPs encapsulating self-amplifying mRNA encoding GFP, which were transfected and expressed in MC38 cells.

[0211] Figure 4 High-throughput screening of ionizable lipids synthesized by a three-component reaction is shown for the formulation of LNPs encapsulating self-amplifying mRNA encoding GFP, which transfect GFP-mRNA into C2C12, 293T, and MC38 cells in 96-well plates. Formulated LNPs with more than 2% GFP expression in the above cells are highlighted.

[0212] Figure 5 Molecular structures of 4 individual ionizable lipids synthesized via a three-component reaction are shown. The formulated LNPs showed better or comparable transfection efficiency than control ionizable lipids used in FDA-approved LNPs.

[0213] Figure 6 Shown are FACS plots of LNPs encapsulating self-amplifying mRNA encoding GFP formulated with six separate ionizable lipids synthesized by a three-component reaction, which were transfected in C2C12 cells and expressed GFP-mRNA.

[0214] Figure 7 Shown are FACS of 11 separate ionizable lipid formulated LNPs encapsulating self-amplifying mRNA encoding GFP synthesized by a three-component reaction, which were transfected and expressed GFP-mRNA in 293T cells.

[0215] Figure 8 Shown are FACS of seven separate ionizable lipid formulated LNPs synthesized by a three-component reaction encapsulating self-amplifying mRNA encoding GFP, which were transfected and expressed GFP-mRNA in MC38 cells.

[0216] Fig. 9 The FACS of the ionizable lipid-prepared LNP and the control LNP prepared by TAb-9 (IIa) encapsulating the self-amplified mRNA encoding GFP is shown, and the LNP is transfected and expresses GFP-mRNA in C2C12 cells. The bottom is a graph of GFP (X axis) and live / dead (7-AAD, Y axis). GFP positive living cells are shown in Q3.

[0217] Fig.10 FACS of TAb-13 (IIb) ionizable lipid formulated LNPs and control LNPs encapsulated with self-amplifying mRNA encoding GFP, which were transfected and expressed in C2C12 cells. Above is a merged image of the GFP and bright field channels. GFP-positive live cells are shown in Q3.

[0218] Fig.11Shown is FACS of (VIIa) ionizable lipid formulated LNPs encapsulating self-amplifying mRNA encoding GFP and control LNPs transfected in 293T cells and expressing GFP-mRNA on day 1 post-transfection. GFP-positive live cells are shown in Q3.

[0219] Fig.12 Shown is the FACS of (Ia) ionizable lipid-formulated LNP and control LNP encapsulated with self-amplified mRNA encoding GFP, which is transfected and expresses GFP-mRNA in C2C12 cells. Bottom is a graph of GFP (X axis) and live / dead (7-AAD, Y axis). GFP positive live cells are shown in Q3.

[0220] Fig.13 Shown is FACS of (Ib) ionizable lipid formulated LNPs encapsulating self-amplifying mRNA encoding GFP and control LNPs transfected in 293T cells and expressing GFP-mRNA. GFP-positive live cells are shown in Q3.

[0221] Fig.14 FACS of ionizable lipid formulated LNPs (C7 and D7) encapsulating self-amplifying mRNA encoding GFP and control LNPs transfected in C2C12 cells and expressing GFP-mRNA are shown. GFP positive live cells are shown in Q3.

[0222] Fig.15 Shown is FACS of ionizable lipid formulated LNPs (E2 and E6) encapsulating self-amplifying mRNA encoding GFP and control LNPs transfected in 293T cells and expressing GFP-mRNA. GFP positive live cells are shown in Q3.

[0223] Fig.16 A heat map is shown showing the relative luminescence intensity of ionizable lipids synthesized using the four-component reaction of the present disclosure in high throughput, formulated into LNPs encapsulating self-amplifying mRNA encoding LUC, transfected into 293T cells and incubated overnight. Data were repeated three times and are expressed as mean ± SD.

[0224] Fig.17 The percentage of GFP expression in 293T cells treated for 24 hours by LNPs formulated with ionizable lipids of the present disclosure and encapsulating self-amplifying mRNA encoding GFP is shown. The data were repeated three times and are expressed as mean ± standard deviation (SD).

[0225] Fig.18FACS of LNPs formulated with E2 ionizable lipids and DOPE, DSPC or POPE phospholipids and control LNPs transfected in 293T cells and expressing GFP-mRNA are shown. GFP-positive live cells are shown in Q3.

[0226] Fig.19 GFP expression in 293T cells treated with P6A2-LNP for 24 hours by sixteen combinations (A-1 to A-16) is shown. Cells were treated with 50 ng, 25 ng or 12.5 ng of self-amplified mRNA encoding GFP. Data were repeated three times and are expressed as mean ± SD.

[0227] Fig. 20 The average GFP expression of LNP formulations containing different molar ratios of cholesterol, DOPE, P6A2 and DMG-PEG2000 and covering self-amplifying mRNA encoding GFP is shown. GFP delivery efficiency increases with increasing molar ratios of cholesterol and DOPE. Increasing molar ratios of P6A2 and DMG-PEG2000 will reduce GFP delivery efficiency. Data were repeated three times and are expressed as mean ± SD.

[0228] Fig.21 GFP expression in 293T cells treated with P6A2-LNP for 24 hours by sixteen combinations (B-1 to B-16) is shown. Cells were treated with 50 ng or 12.5 ng of self-amplified mRNA encoding GFP. Data were repeated three times and are expressed as mean ± SD.

[0229] Fig. 22 The average GFP expression of LNP formulations containing cholesterol, DOPE, P6A2 and DMG-PEG2000 at different molar ratios and covering self-amplifying mRNA encoding GFP is shown. The formulation combination of the present disclosure is P6A2 / DOPE / cholesterol / DMG-PEG200030 / 15 / 50 / 1.5. The data were repeated three times and are expressed as mean ± SD.

[0230] Fig.23 The encapsulation efficiency, nanoparticle size and polydispersity index (PDI) of P6A2 LNPs are shown. The data were repeated three times and expressed as mean ± SD.

[0231] Fig.24 GFP expression in 293T cells treated for 24 hours by LNPs formulated with ionizable lipid P6A2 is shown. Cells were treated with 12.5 ng of self-amplified mRNA-GFP. Data were repeated three times and are presented as mean ± SD.

[0232] Fig.25The in vivo bioluminescence intensity of the metastatic sites of mice treated with LNPs formulated with ionizable lipids C7, E2, and E6 encapsulating SamRNA-LUC is shown. Each mouse was treated with 1 ug of self-amplified mRNA-LUC. The data represent the mean ± SD of 5 mice.

[0233] Fig.26 Shown is the in vivo bioluminescence intensity of the mouse metastasis site processed with P1D4-LNP, P30B7-LNP, P30C7-LNP, P30A1-LNP, P54B6-LNP, P38D1-LNP and P6A2-LNP (upper left) encapsulated with SamRNA-LUC; P38D7-LNP, P38D8-LNP, P40B10-LNP, P40C11-LNP, P56A1-LNP, P56B1-LNP and P28D10 (upper right); P40C10, P40D7, P53A6, P53A5 and P51C12 (bottom), MC3-LNP is used as a control group. Each mouse is processed with 1ug SamRNA-LUC. Data represent the mean ± SD of 5 mice.

[0234] Fig. 27 Shown are FACS of ionizable lipid formulated LNPs (P7 and T7) encapsulating self-amplifying mRNA encoding GFP and control LNPs transfected in 293T cells and expressing GFP-mRNA. GFP positive live cells are shown in Q3.

[0235] Fig.28 Shown is a FACS of (E6) ionizable lipid formulated LNPs encapsulated with DNA encoding CMV-EGFP, transfected and expressing GFP in HEK293 cells. The bottom shows a graph of GFP (X-axis) versus live / dead (7-AAD, Y-axis). GFP-positive live cells are shown in Q3. DETAILED DESCRIPTION

[0236] The present disclosure relates to novel ionizable lipids and lipid nanoparticle compositions comprising novel ionizable lipids. The present disclosure also provides methods for delivering a bioactive agent to a cell and methods for treating a disease or condition in a mammal in need thereof. For example, a method for producing a target polypeptide in a cell involves contacting a nanoparticle composition comprising an mRNA (such as a modified mRNA, a circular mRNA, or a self-amplifying mRNA) with the cell, whereby the mRNA can be translated to produce a target polypeptide or a regulatory structure.

[0237] In this specification, the structural formula of the compound conveniently represents a certain isomer in some cases, but the present disclosure includes all isomers, and it should be understood that not all isomers have the same activity level. In addition, the compound represented by the formula may have crystal polymorphism. Any crystal form, crystal form mixture or its anhydride or hydrate is included in the scope of the present disclosure.

[0238] As used herein, the term "isomer" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. A compound may contain one or more chiral centers and / or double bonds, and may therefore 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 stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomers as well as stereoisomeric mixtures, such as racemates.

[0239] In the reaction schemes described herein, a variety of stereoisomers may be produced. When no specific stereoisomer is indicated, it is understood to mean all possible stereoisomers that may be produced in the reaction. Enantiomers and stereoisomer mixtures of compounds and methods of separating 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 can be used to separate isomers.

[0240] As used herein, the term "compound" is intended to include all isomers and isotopes of the depicted structure. "Isotopes" refer to atoms having the same atomic number but having different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts or complexes of the present disclosure can be prepared by combining with solvents or water molecules to form solvates and hydrates by conventional methods.

[0241] As used herein, the term "alkyl" or "alkyl group" means an optionally substituted straight or branched chain saturated hydrocarbon containing 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). The symbol "C1-C24 alkyl" means an optionally substituted straight or branched chain saturated hydrocarbon containing 1-14 carbon atoms. Unless otherwise specified, the alkyl described herein refers to both unsubstituted alkyl and substituted alkyl.

[0242] As used herein, the term "alkenyl" or "alkenyl group" means an optionally substituted straight or branched chain hydrocarbon containing 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. The symbol "C1-C 24 "Alkenyl" means an optionally substituted straight or branched chain hydrocarbon containing 1 to 24 carbon atoms and at least one carbon-carbon double bond. The alkenyl group may contain one, two, three, four or more carbon-carbon double bonds. For example, C 18 An alkenyl group may contain one or more double bonds. 18 The alkenyl group may be a linoleyl group. Unless otherwise specified, the alkenyl groups described herein refer to both unsubstituted alkenyl groups and substituted alkenyl groups.

[0243] As used herein, the term "alkynyl" or "alkynyl group" means an optionally substituted straight or branched chain hydrocarbon containing 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. The symbol "C1-C 24 "Alkynyl" means an optionally substituted straight or branched chain hydrocarbon containing 1 to 24 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups may contain one, two, three, four or more carbon-carbon triple bonds. For example, C 18 Alkynyl groups may contain one or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

[0244] Unless otherwise specified, alkyl, alkenyl, alkynyl, acyl, aryl, heteroaryl, and cyclic groups (eg, carbocyclyl and heterocyclyl) may be optionally substituted.

[0245] About, approximately: As used herein, the terms "about" and "approximately" when applied to one or more values ​​of interest refer to values ​​± 20% of the stated value.

[0246] 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 share a physical connection. Methods for contacting a cell with an external entity both in vivo and in vitro are well known in the relevant art. In addition, the nanoparticle composition can contact more than one cell.

[0247] As used herein, the term "delivery" means providing an entity to an endpoint. For example, delivering a bioactive agent to a subject may involve administering to the subject an LNP comprising the bioactive agent (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a subject or cell may involve contacting one or more cells with the nanoparticle composition.

[0248] As used herein, "encapsulation efficiency" refers to the amount of the bioactive agent that becomes a part of the nanoparticle composition relative to the initial total amount of the bioactive agent used to prepare the nanoparticle composition. For example, if 97 mg of the bioactive agent is encapsulated in the nanoparticle composition out of a total of 100 mg of therapeutic and / or prophylactic agent initially provided to the composition, the encapsulation efficiency can be given as 97%. The encapsulation efficiency can be measured by, for example, encapsulating an mRNA encoding a GFP (green fluorescent protein) sequence in the nanoparticle composition, and then measuring the encapsulation efficiency of the RFP sequence using flow cytometry. As used herein, "encapsulation efficiency" can refer to complete, substantial or partial closure, restriction, encirclement or encapsulation.

[0249] As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or post-translational modification of the polypeptide or protein.

[0250] As used herein, a "lipid component" is a component of a nanoparticle composition that includes one or more lipids. For example, the lipid component may include one or more ionizable lipids, PEGylated lipids, structural lipids, or phospholipids.

[0251] As used herein, "administration method" may include intravenous, intramuscular, intradermal, subcutaneous or other methods of delivering the composition to a subject. The administration method may be selected to target delivery (eg, specifically deliver) to a specific area or system of the body.

[0252] As used herein, "modified" means non-natural. For example, the RNA can be a modified RNA. That is, the RNA can contain one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. "Modified" species may also be referred to as "altered" species herein. A species may be modified or altered chemically, structurally, or functionally. For example, a modified nucleobase species may contain one or more non-naturally occurring substitutions.

[0253] As used herein, "naturally occurring" means occurring in nature without human assistance.

[0254] As used herein, a "nanoparticle composition" is a composition comprising one or more lipids and a bioactive agent. The size of the nanoparticle composition is typically micrometer-sized or smaller and may include a lipid bilayer. The nanoparticle composition encompasses lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, the nanoparticle composition may be a liposome having a lipid bilayer having a diameter of 500 nm or less.

[0255] As used herein, "lipid" refers to a group of organic compounds, including, for example, fatty acid esters, that are characterized by being relatively poorly soluble in water but relatively highly soluble in many organic solvents.

[0256] As used herein, the term "subject" refers to any organism to which a composition according to the present disclosure can be administered, e.g., for experimental, diagnostic, preventive 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 seeks or needs treatment for a particular disease or condition, requires treatment, is receiving treatment, will receive treatment, or is receiving care from a trained professional.

[0257] As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol component.

[0258] As used herein, "phospholipid" is a lipid comprising a phosphoric acid moiety and one or more carbon chains (such as unsaturated fatty acid chains). Phospholipids may comprise one or more multiple bonds (e.g., double bonds or triple bonds) (e.g., one or more unsaturated). Specific phospholipids may contribute to membrane fusion. For example, cationic phospholipids may interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). Phospholipids may allow one or more elements of a lipid composition to pass through the membrane, thereby allowing, for example, one or more elements to be delivered to a cell.

[0259] As used herein, in some aspects, the term "structured lipid" refers to a compound comprising the following carbon skeleton:

[0260]

[0261] As used herein, "cationic lipids" and "ionizable lipids" refer to compounds comprising a polar (hydrophilic) head group or moiety and a non-polar (hydrophobic or lipophilic) tail group or moiety and may carry a positive charge or a partial positive charge at physiological pH. In some aspects, such polar head groups and non-polar tail groups are bound to each other (e.g., by one or more of hydrogen bonds, van der Waals forces, ionic interactions, and covalent bonds) (e.g., by optionally substituted, variable unsaturated C1-C10 alkyl or alkenyl). In some aspects, the head group or moiety is hydrophilic (e.g., a hydrophilic head group comprising an optionally substituted alkylamino group). As used herein, the term "hydrophilic" is used to qualitatively indicate that the energy group prefers water and is soluble in water. As used herein, the term "hydrophobic" is used to qualitatively indicate that the energy group avoids water, and generally such groups are insoluble in water.

[0262] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are reasonably suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications.

[0263] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein, and has substantially non-toxic, non-inflammatory properties to the patient. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, cross-linked carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked povidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium carboxymethyl starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, xylitol, and others disclosed herein.

[0264] A "pharmaceutically acceptable composition" may also include a salt of one or more compounds. The salt may be a pharmaceutically acceptable salt. 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 into 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 salts or organic acid salts of basic residues such as amines; alkali metal salts or organic salts of acidic residues such as carboxylic acids; etc. Examples of 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, heptanoate, 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, and the like. Representative alkali metal or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound, such as those formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two; such as a non-aqueous medium, such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; Pharmaceutical Salts: Properties, Selection, and Use, PH Stahl and CG 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.

[0265] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically linked by peptide bonds, which may be produced naturally (eg, isolated or purified) or synthetically.

[0266] As used herein, "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or joints. RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a target polypeptide. For example, RNA may be a messenger RNA (mRNA, such as modified mRNA, circular mRNA, and self-amplifying mRNA), a transfer RNA (tRNA), a small interfering RNA (siRNA), a small activating RNA (saRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA (dsRNA), a small hairpin RNA (shRNA)). Translation of mRNA encoding a specific polypeptide, such as in vivo translation of mRNA in a mammalian cell, can produce an encoded polypeptide or regulatory structure. The RNA can be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (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.

[0267] As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0268] The term "biologically active agent" refers to any agent that has a therapeutic, diagnostic and / or prophylactic effect and / or causes a desired biological and / or pharmacological effect when administered to a subject. Therapeutic agents are also referred to as "active substances" or "active agents". Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0269] As used herein, the term "therapeutically effective amount" means the amount of an agent to be delivered (e.g., a nucleic acid, drug, composition, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient to treat, ameliorate the symptoms of, diagnose, prevent and / or delay the onset of an infection, disease, disorder and / or condition when administered to a subject suffering from or susceptible to an infection, disease, disorder and / or condition.

[0270] Nanoparticle composition

[0271] The disclosure also features nanoparticle compositions comprising a lipid component comprising a compound according to Formula (I), (II), (III), (IV), (V), (VI), and (VII) as described herein.

[0272] In one aspect, the present disclosure provides compounds of formula (I):

[0273]

[0274] or a salt or isomer thereof, wherein

[0275] each m and n is independently 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any subrange within the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0276] Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0277]

[0278] Each a, b and c is independently selected from 0-24, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0279] Each R 3 , R 4 and R 5 independently selected from C1-C24 alkyl, C1-C24 alkenyl, C1-C24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl,

[0280]

[0281] a, b and c are each independently 0-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0282] Each X is independently selected from CH2, NH, O or S;

[0283] Each Y is independently selected from CH2, NH, O or S;

[0284] Each Z is independently selected from CH or N; and

[0285] Each L is independently selected from CH2, NH, O or S.

[0286] Synthesis Scheme 1.

[0287] General synthetic route for the synthesis of compounds of formula I:

[0288]

[0289] For example, the α,β-unsaturated Michael acceptor may be

[0290]

[0291] For example, the amine may be selected from

[0292]

[0293] In certain aspects, the compound of Formula I may comprise, for example, the following formula:

[0294]

[0295] or a salt or isomer thereof, wherein each R 1 and R 2 As defined above.

[0296] General synthetic route for synthesizing compounds of formula 1-1:

[0297]

[0298] In one example, the compound of formula I-1 is synthesized as follows:

[0299] Tris (2-acryloyloxyethyl) isocyanurate (0.2 mmol) was mixed with amine (0.66 mmol) in 10 mL toluene / ACN (5 / 1) at a molar ratio of 1: 3.3, and the mixture was stirred in a glass screw vial in a 95°C oil bath for two days. The solvent can be removed using methods known in the art, such as a rotary evaporator. The product can be purified using methods known in the art, such as column chromatography with a hexane / ethyl acetate gradient elution. The structure is confirmed using techniques known in the art, such as 1H NMR spectroscopy and LC-mass spectrometry.

[0300] (2,4,6-Trioxo-1,3,5-triazinane-1,3,5-triyl)tris(ethane-2,1-diyl)tris(3-(ditetradecylamino)propionate) (C7): Yield (20%). 1 H-NMR (400MHz, CDCl3, δ) 4.29 (t, J = 8Hz, 3H), 4.16 (t, J = 8Hz, 3H), 2.75 (t, J = 8Hz, 3H ), 2.60 (t, J=8Hz, 3H), 2.42-2.34 (m, 12H), 1.50-1.26 (m, 84H), 0.87 (t, J=8Hz, 18H). MS(APCI)m / z 1148.3[M+H] + .

[0301] (2,4,6-Trioxo-1,3,5-triazinane-1,3,5-triyl)tris(ethane-2,1-diyl)tris(3-(dioctylamino)propionate) (D7): Yield (23%). 1 H-NMR (400MHz, CDCl3, δ) 4.29 (t, J = 8Hz, 3H), 4.17 (t, J = 8Hz, 3H), 2.75 (t, J = 8Hz, 3H) , 2.60 (t, J=8Hz, 3H), 2.39-2.34 (m, 12H), 1.50-1.26 (m, 156H), 0.89 (t, J=8Hz, 18H). MS(APCI)m / z 1653.5[M+H] + .

[0302] In certain aspects, compounds of Formula I may include, for example, the following compounds:

[0303]

[0304] Also known as C7;

[0305]

[0306] Also known as D7.

[0307] In another aspect, the present disclosure provides a compound of formula (II):

[0308]

[0309] or a salt or isomer thereof, wherein

[0310] Each n is independently 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range within the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0311] Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0312]

[0313] a, b and c are each independently an integer from 0 to 24;

[0314] Each R 3 , R 4 and R 5 independently selected from C1-C24 alkyl, C1-C24 alkenyl, C1-C24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl,

[0315]

[0316] a, b and c are each independently 0-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0317] Each X is independently selected from CH or N;

[0318] Each Y is independently selected from CH2, NH, O or S; and

[0319] Each Z is independently selected from CH2, NH, O or S.

[0320] Synthesis Scheme 2.

[0321] General synthetic route for the synthesis of compounds of formula II:

[0322]

[0323] For example, the α,β-unsaturated Michael acceptor may be

[0324]

[0325] For example, the amine may be selected from DA8-DA14, DA18, AP1-AP8, as indicated above.

[0326] In certain aspects, the compound of formula II may comprise, for example, the following formula:

[0327]

[0328] or a salt or isomer thereof, wherein each R 1 and R 2 As defined above.

[0329] General synthetic route for synthesizing compounds of formula II-1:

[0330]

[0331] In some examples, the compound of formula I-1 is synthesized as follows:

[0332] 1,3,5-triacryloyl hexahydro-1,3,5-triazine (0.2 mmol) was mixed with amine (0.66 mmol) in 10 mL toluene / ACN (5 / 1) at a molar ratio of 1:3.3, and the mixture was stirred in a glass screw vial in a 95°C oil bath for two days. Techniques known in the art, such as a rotary evaporator, can be used to remove the solvent, and column chromatography, for example, can be used to purify the product. Techniques known in the art, such as 1H nuclear magnetic resonance spectroscopy and LC-mass spectroscopy can be used to confirm the structure.

[0333] 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tris(3-(dinonylamino)propan-1-one) (E2): Yield (25%). 1H-NMR (400 MHz, CDCl3, δ) 5.26 (s, 6H), 2.79 (t, J=8 Hz, 6H), 2.65 (t, J=8 Hz, 6H), 2.41 (t, J=8 Hz, 12H), 1.42-1.26 (m, 84H), 0.88 (t, J=8 Hz, 18H). MS (ESI) m / z 529.6 [M+2H] 2 +.

[0334] 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tris(3-(dinonylamino)propan-1-one) (E6): Yield (22%). 1H-NMR (400 MHz, CDCl3, δ) 5.26 (s, 6H), 2.81 (t, J=8 Hz, 6H), 2.67 (t, J=8 Hz, 6H), 2.43 (t, J=8 Hz, 12H), 1.44-1.23 (m, 132H), 0.88 (t, J=8 Hz, 18H). MS (ESI) m / z 697.9 [M+2H] 2 +.

[0335] In certain aspects, compounds of Formula II may include, for example, the following compounds:

[0336] Also known as TAb-9, E2 or (II)-A;

[0337] Also known as TAb-13, E6 or (II)-B;

[0338] or a salt or isomer thereof.

[0339] In another aspect, the present disclosure provides a compound of formula (III):

[0340]

[0341] or a salt or isomer thereof, wherein

[0342] Each R 1 , R 1’ , R 2 and R 3 Independently selected from H, C1-C24 alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0343]

[0344] a, b and c are each independently 0-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0345] Each R 9 Independently selected from

[0346] Each R 10 , R 10’ , R 10” , R 11 , R 11’ , R 11” and R 12 Independently selected from H, C1-C24 alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0347]

[0348] a, b and c are each independently 0-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0349] R 3” , R 4” and R 5” Independently selected from H, C1-C24 alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0350]

[0351] a, b and c are each independently 0-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0352] Synthesis Scheme 3.

[0353] General synthetic route for the synthesis of compounds of formula III:

[0354]

[0355] For example, the boric acid may be selected from:

[0356]

[0357] For example, the amine may be selected from DA8-DA14, DA18, AP1-AP8, as shown above.

[0358] For example, the aldehyde or ketone may be selected from:

[0359]

[0360] In some examples, the compound of formula III is synthesized as follows:

[0361] Ionizable lipids were synthesized in 96-well plates with glass inserts at a molar ratio of boronic acid / amine / aldehyde of 1:1:1. Structures were confirmed using techniques known in the art, such as 1H NMR spectroscopy and LC-mass spectroscopy.

[0362] 2,2′-(1,4-phenylenebis((dioctylamino)methylene))diphenol (TP1A11): Yield (17%). 1H-NMR (400 MHz, CDCl3, δ) 7.37 (s, 4H), 7.10-7.14 (t, J=8 Hz, 2H), 6.83-6.85 (d, J=8 Hz, 2H), 6.77-6.80 (m, 2H), 6.64-6.69 (t, J=12 Hz, 2H), 4.88 (d, J=8 Hz, 2H), 2.42-2.65 (m, 8H), 1.21-1.25 (m, 40H), 0.84-0.89 (m, 12H). MS (APCI) m / z 769.7 [M+H]+.

[0363] 2-((Bis-tetradecylamino)(thiophen-2-yl)methyl)phenol (TP4G11): Yield 23%. HNMR (400 MHz, CDCl3, δ) 7.39 (d, J = 8 Hz, 1H), 7.08-7.14 (m, 2H), 7.02-7.05 (m, 1H), 6.86-6.89 (d, J = 12 Hz, 1H), 6.75-6.78 (m, 1H), 6.66-6.72 (m, 1H), 5.44 (s, 1H), 2.45-2.69 (m, 4H), 1.29 (m, 48H), 0.84-0.90 (t, 6H). MS (APCI) m / z 769.7 [M+H]+.

[0364] 2-((Ditridecylamino)(4-dodecylphenyl)methyl)phenol (TP3F11): yield 25%. HNMR (400 MHz, CDCl3, δ) 7.30 (d, J = 12 Hz, 2H), 7.14 (d, J = 12 Hz, 3H), 6.85 (t, J = 12 Hz, 2H), 6.69 (t, J = 12 Hz, 1H), 4.92 (s, 1H), 2.60-2.55 (m, 4H), 2.39-2.44 (m, 2H), 1.59-1.51 (m, 6H), 1.25 (m, 60H), 0.88 (t, J = 12 Hz, 9H). MS (APCI) m / z 732.7 [M+H]+.

[0365] 2-((Bis-tetradecylamino)(4-dodecylphenyl)methyl)phenol (TP3G11): Yield 26%. HNMR (400 MHz, CDCl3, δ) 7.30 (d, J = 12 Hz, 2H), 7.14 (d, J = 12 Hz, 3H), 6.82 (t, J = 12 Hz, 2H), 6.66 (t, J = 12 Hz, 1H), 4.92 (s, 1H), 2.60-2.55 (m, 4H), 2.39-2.44 (m, 2H), 1.59-1.51 (m, 6H), 1.25 (m, 64H), 0.88 (t, J = 12 Hz, 9H). MS (APCI) m / z 732.7 [M+H]+.

[0366] In certain aspects, the compound of formula III may comprise, for example, the following formula:

[0367]

[0368] or a salt or isomer thereof, wherein

[0369] Each R 1 , R 1’ , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0370]

[0371] a, b and c are each independently an integer from 0 to 24;

[0372] R 3” , R 4” and R 5” Independently selected from H, C1-C24 alkyl, C1-C 24 Alkyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0373]

[0374] a, b and c are each independently an integer from 0 to 24; or

[0375] Each R 4 , R 5 , R 6 , R 7 and R 8 Independently for:

[0376]

[0377] Where R 1”’ , R 1”” , R 2”’ and R 3”’ Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0378]

[0379] Each a, b and c is independently an integer selected from 0-24;

[0380] Each X is independently CH, N, O or S;

[0381] Each Y is independently CH, N, O or S

[0382] Each Z is independently CH, N, O or S

[0383] Each F is independently CH, N, O or S, and

[0384] Each E is independently CH, N, O or S.

[0385] General synthetic route for synthesizing compounds of formula III-1:

[0386]

[0387] In certain aspects, compounds of formula III may include, for example, the following compounds:

[0388]

[0389]

[0390]

[0391] Also known as TP4G11;

[0392] Also known as TP1A11;

[0393]

[0394]

[0395]

[0396]

[0397] Also known as TP3G11;

[0398] Also known as TP3F11;

[0399] Also known as TP5A1;

[0400] or a salt or isomer thereof.

[0401] In another aspect, the present disclosure provides a compound of formula (IV):

[0402]

[0403] or a salt or isomer thereof, wherein

[0404] Each R 1 , R 2 , R 3 , R 4 and R 5 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0405]

[0406] a, b and c are each independently an integer from 0 to 24;

[0407] Each R 6 , R 7 , R 8 and R 9 independently selected from H, C1-C24 alkyl, C1-C24 alkenyl, C1-C24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl,

[0408]

[0409] a, b and c are each independently an integer from 0 to 24;

[0410] Each X is independently selected from CH, N;

[0411] Each Y is independently selected from CH2, NH, O or S;

[0412] Each Z is independently selected from CH or N.

[0413] Synthesis Scheme 4.

[0414] General synthetic route for the synthesis of compounds of formula IV:

[0415]

[0416] For example, the aldehyde or ketone may be selected from:

[0417]

[0418]

[0419] For example, the amine may be selected from:

[0420]

[0421]

[0422] For example, the acid may be selected from:

[0423]

[0424]

[0425] For example, the isonitrile may be selected from:

[0426]

[0427] In some examples, the compound of formula IV is synthesized as follows:

[0428] The ionizable lipids were synthesized via a two-step / one-pot reaction with a 1:1:1:1 molar ratio of acid / amine / aldehyde / isocyanide.

[0429] At room temperature aldehyde (1mmol) and amine (1mmol) are mixed in 3mL MeOH, and stirred for one hour, then add isocyanide (1mmol) and acid (1mmol). The gained mixture is further stirred overnight. Remove solvent with rotary evaporator. Add ethyl acetate (100mL) and wash with saline (2X 50mL), via MgSO Dry. Remove ethyl acetate and use the CombiFlash Rf system with silica gel column (Redisep Gold Resolution, Teledyne, Isco) by column chromatography, by CH Cl / methanol (0%-40% methanol) gradient elution to purify residue. Use technology known in the art, for example H NMR spectrum and LC-mass spectrum confirm structure.

[0430] (2-(1-ethylpiperidin-4-yl)-2-(N-(pentadecan-8-yl)tridecanoyl)acetyl)glycine ethyl ester (P1C1): yield 16%. 1H-NMR (400 MHz, Me-OD, δ) 4.34 (d, J = 8 Hz, 1H), 4.21-4.14 (m, 4H), 3.41 (t, J = 8 Hz, 1H), 3.01 (q, J = 8 Hz, 2H), 2.47-2.40 (m, 5H), 2.01-1.83 (t, J = 12 Hz, 2H), 1.65-1.56 (m, 6H), 1.29-1.17 (m, 42H), 1.17-1.10 (m, 6H), 0.93-0.89 (m, 9H). MS (APCI) m / z 678.7 [M+H]+.

[0431] N-(2-(Cyclohex-1-en-1-ylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)palmitamide (P6A2): Yield (45%). 1H-NMR (400 MHz, Me-OD, δ) 6.05 (t, J=8 Hz, 1H), 4.26 (s, 1H), 3.26-3.17 (m, 3H), 2.49-2.42 (m, 5H), 2.19-2.01 (m, 6H), 1.67-1.57 (m, 14H), 1.31-1.16 (m, 48H), 0.92-0.87 (m, 12H). MS (APCI) m / z 742.7 [M+H]+.

[0432] N-(2-(Cyclohexylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)palmitamide (P30A1): Yield (58%). 1H-NMR (400 MHz, CDCl3, δ) 8.41 (d, J = 8 Hz, 1H), 3.70-3.63 (m, 1H), 3.57 (t, J = 8 Hz, 1H), 3.18-3.09 (m, 3H), 2.80-2.24 (m, 3H), 2.44-2.24 (m, 4H), 1.79-1.20 (m, 67H), 0.89-0.85 (m, 9H). MS (APCI) m / z 716.8 [M+H]+.

[0433] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-decyltetradecyl)undecylamide (P54B6): yield (48%). MS (APCI) m / z 824.8 [M+H]+.

[0434] N-(2-(Cycloheptylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)tetradecamide (P38D1): Yield (56%). MS (APCI) m / z 702.7 [M+H]+.

[0435] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)palmitamide (P56A1): Yield (53%). MS (APCI) m / z 768.7 [M+H]+.

[0436] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)heptadecanamide (P56B 1): Yield (59%). MS (APCI) m / z 782.8 [M+H]+.

[0437] N-(2-(Butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)octanamide (P14A2): Yield (40%). MS (APCI) m / z 606.6 [M+H]+.

[0438] N-(2-(Butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)undecanoic acid amide (P16B1): yield (43%). MS (APCI) m / z 620.6 [M+H]+.

[0439] N-(2-(Cyclohexylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-octyldodecyl)tetradecamide (P26D4): Yield (23%). MS (APCI) m / z 758.8 [M+H]+.

[0440] (2-(1-ethylpiperidin-4-yl)-2-(N-(2-octyldodecyl)tetradecylamide)acetyl)glycine ethyl ester (P1D4): yield (30%). MS (APCI) m / z 762.8 [M+H]+.

[0441] N-(1-(Cyclohexylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)heptadecanamide (P30B7): Yield (56%). MS (APCI) m / z 730.8 [M+H]+.

[0442] N-(1-(Cyclohexylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)stearamide (P30C7): yield (53%). MS (APCI) m / z 744.8 [M+H]+.

[0443] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)tetradecamide (P38D7): Yield (45%). MS (APCI) m / z 702.7 [M+H]+.

[0444] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(heptadecan-9-yl)tetradecanoic acid amide (P38D8): Yield (52%). MS (APCI) m / z 730.7 [M+H]+.

[0445] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-octyldodecyl)undecylamide (P40B10): Yield (42%). MS (APCI) m / z 730.7 [M+H]+.

[0446] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(tricosan-12-yl)dodecanoamide (P40C11): Yield (57%). MS (APCI) m / z 786.8 [M+H]+.

[0447] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-octyldodecyl)dodecylamide (P40C10): Yield (70%). MS (APCI) m / z 744.8 [M+H]+.

[0448] N-(1-(tert-Butylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-decyltetradecyl)tridecamide (P51C12): Yield (70%). MS (APCI) m / z 774.8 [M+H]+.

[0449] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)pentadecanamide (P40D7): Yield (61%). MS (APCI) m / z 716.7 [M+H]+.

[0450] N-(2-(tert-Butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(tricosan-12-yl)decanamide (P53A5): Yield (61%). MS (APCI) m / z 718.7 [M+H]+.

[0451] N-(2-(tert-butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-decyltetradecyl)decanamide (P53A6): yield (61%). MS (APCI) m / z 732.7 [M+H]+

[0452] N-(2-(Cycloheptylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-decyltetradecyl)tetradecamide (P38D4): yield (51%). MS (APCI) m / z 828.7 [M+H]+

[0453] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)palmitamide (P5 6A2): yield (46%). MS (APCI) m / z 796.7 [M+H]+

[0454] N-(2-(Cycloheptylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)oleamide (P149A2): yield (42%). MS (APCI) m / z 784.8 [M+H]+

[0455] 4-((2-(Cycloheptylamino)-1-(1-ethylpiperidin-3-yl)-2-oxoethyl)(2-octyldodecyl)amino)-4-oxobutanoic acid dodecyl ester (P159C10): yield (43%). MS (APCI) m / z 830.8 [M+H]+

[0456] N-(1-(((1s,3s)-adamantan-1-yl)amino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-4-(3,6-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-N-(2-octyldodecyl)pentanamide (P161F10): yield (53%). MS (APCI) m / z 974.9 [M+H]+.

[0457] In certain aspects, compounds of Formula IV may include, for example, the following compounds:

[0458] Also known as P53F12;

[0459] Also known as P38C2;

[0460] Also known as P55H12;

[0461] Also known as P54A6;

[0462] Also known as P6A2;

[0463] Also known as P1C1;

[0464] Also known as P30A1;

[0465] Also known as P51C12;

[0466] Also known as P54B1;

[0467] Also known as P38D1;

[0468] Also known as P56A1;

[0469] Also known as P40D7;

[0470] Also known as P56B1;

[0471] Also known as P14A2;

[0472] Also known as P16B1;

[0473] Also known as P53A5;

[0474] Also known as P26D4; Also known as P1D4;

[0475] Also known as P30B7;

[0476] Also known as P53A6;

[0477] Also known as P30C7;

[0478] Also known as P38D7;

[0479] Also known as P38D8;

[0480] Also known as P40B10;

[0481] Also known as P40C11;

[0482] Also known as P40C10;

[0483] Also known as P38D4;

[0484] Also known as P56A2;

[0485] Also known as P149A2;

[0486] Also known as P159C10;

[0487] Also known as P161F10;

[0488] or a salt or isomer thereof.

[0489] In another aspect, the present disclosure provides a compound of formula (V):

[0490]

[0491] or a salt or isomer thereof, wherein

[0492] Each R1 , R 2 , R 3 , R 4 , R 5 and R 6 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0493]

[0494] a, b and c are each independently an integer from 0 to 24;

[0495] Each R 3’ , R 4’ and R 5’ Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0496]

[0497] a, b and c are each independently 0-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24.

[0498] Synthesis Scheme 5.

[0499] General synthetic route for synthesizing compounds of formula V:

[0500]

[0501] For example, the aldehyde or ketone may be selected from:

[0502]

[0503] For example, the amine may be selected from:

[0504]

[0505] For example, the boc amino acid may be selected from:

[0506]

[0507] In certain aspects, compounds of Formula V may include, for example, the following compounds:

[0508]

[0509] or a salt or isomer thereof.

[0510] In another aspect, the present disclosure provides a compound of formula (VI):

[0511]

[0512] or a salt or isomer thereof, wherein

[0513] Each n is independently 0-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range within the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0514] Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0515]

[0516] a, b and c are each independently an integer from 0 to 24;

[0517] Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0518]

[0519] a, b and c are each independently 0-24, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or an integer selected from any sub-range within the range of 0-24;

[0520] Each Y is independently selected from CH or N;

[0521] Each Z is independently selected from CH2, NH, O or S.

[0522] Synthesis Scheme 6.

[0523] General synthetic route for the synthesis of compounds of formula VI.

[0524]

[0525] Wherein X is independently selected from F, Cl, Br, Ts,

[0526] And wherein each n is independently 0-10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range within the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.

[0527] For example, the amine may be selected from DA8-DA14, DA18, AP1-AP8, as indicated above.

[0528] In certain aspects, the compound of formula VI may comprise, for example, the following formula:

[0529]

[0530] or a salt or isomer thereof, wherein each R 1 and R 2 As defined above.

[0531] General synthetic route for synthesizing compounds of formula VI-1:

[0532]

[0533] In certain aspects, compounds of Formula VI may include, for example, the following compounds:

[0534]

[0535]

[0536] or a salt or isomer thereof.

[0537] In another aspect, the present disclosure provides a compound of formula (VII):

[0538]

[0539] or a salt or isomer thereof, wherein

[0540] Each n and m is independently 0-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or an integer selected from any sub-range within the range of 0-10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0541] Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0542]

[0543] a, b and c are each independently an integer from 0 to 24;

[0544] Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl,

[0545]

[0546] a, b and c are each independently an integer from 0 to 24;

[0547] Each X is independently selected from CH2, NH, O or S;

[0548] Each Y is independently selected from CH, N;

[0549] Each Z is independently selected from CH2, NH, O or S; and

[0550] Each E is independently selected from CH2, NH, O or S.

[0551] Synthesis Scheme 7.

[0552] General synthetic route for the synthesis of compounds of formula VII:

[0553]

[0554] In certain aspects, the compound of formula VII may comprise, for example, the following formula:

[0555]

[0556] or a salt or isomer thereof, wherein each R 1 and R 2 As defined above.

[0557] General synthetic route for synthesizing compounds of formula VII-1:

[0558]

[0559] In some aspects, the compound of formula VII-1 can be synthesized as follows:

[0560] At 0 ° C under N2, a solution of acryloyl chloride (56 mmol) in anhydrous CHCl (50 mL) was added dropwise to a mixture of cyclohexane-1,3,5-triol (14 mmol) and EtN (84 mmol) in anhydrous CHCl (60 mL). After about 1 hour, the mixture was returned to room temperature and reacted for 4 hours. The mixture was then treated with saturated sodium bicarbonate solution (150 mL) and brine (150 mL) and extracted with CHCl (150 mL×3). The combined organic layer was dried over anhydrous magnesium sulfate, and the solvent was removed. The crude product was purified by column chromatography using hexane / ethyl acetate to obtain a compound cyclohexane-1,3,5-triyl triacrylate as a white solid. 1H-NMR (400MHz, Me-OD, δ) 6.40 (dd, J=16, 4Hz, 3H), 6.08 (dd, J=20, 8Hz, 3H), 5.83 (dd, J=16, 4Hz, 3H), 4.98-4.87 (m, 3H), 1.24 (s, 6H). MS (ESI) m / z 317.1[M+Na]+.

[0561] Cyclohexane-1,3,5-triyl triacrylate (0.1 mmol) was mixed with an amine in 5 mL of ethanol at a molar ratio of 1:3.3, and the mixture was stirred in a glass screw vial in a 50°C oil bath for two days. The solvent was removed by a rotary evaporator, and the product can be purified by column chromatography. Techniques known in the art, such as 1H NMR and LC-mass spectrometry, can be used to confirm the structure.

[0562] In certain aspects, the compound of formula VII may include, for example, the following compounds:

[0563] Also known as T7;

[0564] (VIIb), also known as P7;

[0565] or a salt or isomer thereof.

[0566] Lipid Nanoparticles

[0567] In some aspects, the nanoparticle composition has a size of 1 μm or less (e.g., 1 μm, 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 less) and encapsulates mRNA ranging in size from 1,000 to 20,000 nucleotides, for example when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or other methods. In some aspects, the size of the encapsulated mRNA is 1,000, 1,500, 1,700, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000 or 20,000 nucleotides, and any number of nucleotides within the disclosed range are included in this specification, as clearly stated herein. Nanoparticle compositions include, for example, lipid nanoparticles (LNP), liposomes, lipid vesicles and lipid complexes. In some aspects, nanoparticle compositions are vesicles comprising one or more lipid bilayers. In some aspects, nanoparticle compositions include two or more concentric bilayers separated by aqueous compartments. The lipid bilayer may be functionalized and / or cross-linked to each other.The lipid bilayer may contain one or more ligands, proteins or channels.

[0568] The nanoparticle composition comprises a lipid component comprising at least one compound according to Formula (I), (II), (III), (IV), (V), (VI) and (VII). For example, the lipid component of the nanoparticle composition may comprise one or more of Compounds Ia-VIIb. The nanoparticle composition may also comprise a variety of other components. For example, the lipid component of the nanoparticle composition may comprise one or more other lipids in addition to the lipids according to Formula (I), (II), (III), (IV), (V), (VI) and (VII).

[0569] Ionizable lipids

[0570] The lipid component of the nanoparticle composition may include one or more ionizable lipids. The nanoparticle composition may include one or more ionizable lipids in addition to lipids according to formula (I), (II), (III), (IV), (V), (VI) and (VII). As used herein, the term "ionizable lipid of the present disclosure" includes lipid compounds according to formula (I), (II), (III), (IV), (V), (VI) and (VII), such as but not limited to compounds Ia-VIIb. The ionizable lipids of the present disclosure can be synthesized in one step.

[0571] PEG lipids

[0572] The lipid component of nanoparticle composition can comprise one or more PEG lipids or PEG modified lipids. Such kind can also be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids can be selected from the non-limiting group consisting of: PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide, PEG modified dialkylamine, PEG modified diacylglycerol, PEG modified dialkylglycerol and mixtures thereof. For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipid.

[0573] Structured lipids

[0574] The lipid component of the nanoparticle composition may include one or more structural lipids. The structural lipid may be selected from the following group consisting of, but not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some aspects, the structural lipid is cholesterol. In some aspects, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0575] Phospholipids

[0576] The lipid component of the nanoparticle composition can include one or more phospholipids, such as one or more (poly) unsaturated lipids. The phospholipids can be assembled into one or more lipid bilayers. In general, the phospholipids can include a phospholipid portion and one or more fatty acid portions.

[0577] The phospholipids useful in the compositions and methods can be selected from the non-limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diondecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 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-diundecanoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocho -2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and sphingomyelin. In some aspects, the nanoparticle composition comprises DSPC. In some aspects, the nanoparticle composition comprises DOPE. In some aspects, the nanoparticle composition comprises both DSPC and DOPE.

[0578] Adjuvant

[0579] In some aspects, the nanoparticle compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxynucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0580] Bioactive Agents

[0581] The nanoparticle composition may include one or more bioactive agents. The disclosure features methods of delivering a bioactive agent to a mammalian cell or organ, producing a target polypeptide in a mammalian cell, and treating a disease or condition in a mammal in need thereof, comprising administering to the mammal a nanoparticle composition comprising a bioactive agent and / or contacting a mammalian cell with the nanoparticle composition.

[0582] A bioactive agent may be a substance that, once delivered to a cell or organ, brings about a desired change in a cell, organ, or other body tissue or system. Such species may be used to treat one or more diseases, disorders, or conditions.

[0583] In some aspects, the bioactive agent is a small molecule drug that can be used to treat a particular disease, disorder or condition. Examples of drugs that can be used in nanoparticle compositions include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin); antineoplastic agents (e.g., actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, compounds), antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs); anti-infectives; local anesthetics (e.g., dibucaine and chlorpromazine); beta-adrenergic blockers (e.g., propranolol, timolol, and labetolol), antihypertensives (e.g., clonidine and hydralazine); antidepressants (e.g., imipramine, amitriptyline, and doxepin); anticonvulsants (e.g., phenytoin); antihistamines (e.g., diphenhydramine, chlorphenirimine, and promethazine); antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin); antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B); antiparasitics; hormones;Hormone antagonists; immunomodulators; neurotransmitter antagonists; antiglaucoma agents; vitamins; anesthetics; and imaging agents.

[0584] In some aspects, the bioactive agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "nucleic acid" in its broadest sense includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary nucleic acids used in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger mRNA (mRNA), hybrids thereof, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, vectors, etc. In some aspects, the therapeutic and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described herein can be selected from the group consisting of, but not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNAs (siRNAs), asymmetric interfering RNAs (aiRNAs), small activating mRNAs (saRNAs), microRNAs (miRNAs), Dicer-substrate RNAs (dsRNAs), small hairpin RNAs (shRNAs), transfer RNAs (tRNAs), messenger RNAs (mRNAs), and mixtures thereof. In certain aspects, the RNA is mRNA.

[0585] In some aspects, the bioactive agent is mRNA. The mRNA can encode any target polypeptide or regulatory structure, including any natural or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some aspects, the regulatory structure or polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0586] In one aspect, the bioactive agent is a self-amplifying mRNA. Self-amplifying mRNA can be derived from modified alphavirus species, including but not limited to Venezuelan equine encephalitis virus (VEEV). Self-amplifying mRNA can encode both target polypeptides or regulatory structures, including any natural or non-naturally occurring or otherwise modified polypeptides and RNA-dependent RNA polymerases, which can replicate the entire self-amplifying mRNA or target polypeptides or regulatory structures after being delivered to the target cell. After appropriate constructs are delivered to the nucleus, self-amplifying mRNA can be produced in the target cell. Compared with non-self-amplifying RNA, self-amplifying mRNA can produce a larger amount of mRNA templates, and therefore enhance expression with lower dosages.

[0587] In other aspects, the bioactive agent is siRNA. siRNA can selectively knock down or down-regulate the expression of a target gene. For example, siRNA can be selected to silence genes associated with a specific disease, disorder or illness after administering a nanoparticle composition comprising siRNA to a subject in need. siRNA can include a sequence complementary to an mRNA sequence encoding a target gene or protein. In some aspects, the siRNA can be an immunomodulatory siRNA.

[0588] In some aspects, the bioactive agent is shRNA or a vector or plasmid encoding it. After the appropriate construct is delivered to the nucleus, the shRNA can be produced in the target cell. The construct and mechanism associated with shRNA are well known in the relevant art.

[0589] Nucleic acids and polynucleotides that can be used in the present disclosure may include a first region (e.g., a coding region) encoding a linked nucleoside of a target polypeptide, a first flanking region (e.g., a 5'-UTR) located at the 5'-end of the first region, a second flanking region (e.g., a 3'-UTR) located at the 3'-end of the first region, at least one 5'-cap region and a 3'-stabilizing region. In some aspects, the nucleic acid or polynucleotide further comprises a poly-A region or a Kozak sequence (e.g., in a 5'-UTR). In some cases, the polynucleotide may contain one or more intronic nucleotide sequences that can be excised from the polynucleotide. In some aspects, the polynucleotide or nucleic acid (e.g., mRNA) may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence and / or a polyadenylation signal. Any region of the nucleic acid may include one or more alternative components (e.g., alternative nucleosides). For example, the 3′-stabilizing region may contain alternative nucleosides, such as L-nucleosides, trans-thymidine, or 2′-O-methyl nucleosides, and / or the coding region, 5′-UTR, 3′-UTR, or cap region may comprise alternative nucleosides, such as 5-substituted uridine (e.g., 5-methoxyuridine), 1-substituted pseudouridine (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and / or 5-substituted cytidine (e.g., 5-methyl-cytidine).

[0590] preparation

[0591] Nanoparticle compositions can include lipid components and one or more additional components, such as bioactive agents. Nanoparticle compositions can be designed for one or more specific applications or targets. The elements of nanoparticle compositions can be selected based on specific applications or targets, and / or based on the efficacy, toxicity, cost, ease of use, availability or other characteristics of one or more elements. Similarly, specific formulations of nanoparticle compositions can be selected for specific applications or targets based on, for example, the efficacy and toxicity of a particular combination of elements.

[0592] The lipid component of the nanoparticle composition can include, for example, lipids according to formula (I), (II), (III), (IV), (V), (VI) and (VII), phospholipids (such as unsaturated lipids, e.g., DOPE or DSPC), PEG lipids and structural lipids. The elements of the lipid component can be provided in specific fractions.

[0593] The amount of the therapeutic and / or preventive agent in the nanoparticle composition may depend on the size, composition, desired target and / or application or other characteristics of the nanoparticle composition and the characteristics of the therapeutic and / or preventive agent. For example, the amount of RNA that can be used in the nanoparticle composition may depend on the size, sequence and other characteristics of the RNA. The relative amounts of the therapeutic and / or preventive agent elements and other elements (e.g., lipids) in the nanoparticle composition may also vary. In some aspects, the weight / weight ratio of the lipid component to the bioactive agent in the nanoparticle composition may be about 1:1 to about 60:1, such as 1:1, 2:1, 3:1, 4:1, 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 weight / weight ratio of the lipid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain aspects, the weight / weight ratio is about 20: 1. For example, the amount of the therapeutic diagnostic and / or prophylactic agent in the nanoparticle composition can be measured using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0594] The nanoparticle composition can be formulated in whole or in part as a pharmaceutical composition. A pharmaceutical composition may comprise one or more nanoparticle compositions. For example, a pharmaceutical composition may comprise one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or adjuvants, such as those described herein. General guidelines for the preparation and manufacture of pharmaceutical compositions and agents can be obtained, for example, in Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and adjuvants can be used in any pharmaceutical composition, unless any conventional excipient or adjuvant may be incompatible with one or more components of the nanoparticle composition. If the combination of an excipient or adjuvant with a component of the nanoparticle composition may result in any undesirable biological effect or other adverse effect, the excipient or adjuvant may be incompatible with the component.

[0595] In some aspects, one or more excipients or adjuvants may account for more than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, one or more excipients or adjuvants may account for 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In some aspects, the purity of the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%. In some aspects, the excipient is approved for human and veterinary use. In some aspects, the excipient has been approved by the United States Food and Drug Administration. In some aspects, the excipient is pharmaceutical grade. In some aspects, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0596] In accordance with the present disclosure, the relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition will vary depending on the identity, size, and / or condition of the subject being treated and also depending on the route of administration of the composition.

[0597] In some aspects, nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or transport (e.g., stored at 4°C or lower, such as at a temperature between about -150°C and about 0°C or between about -80°C and about -20°C). For example, a pharmaceutical composition comprising a compound of any of Formula (I), (II), (III), (IV), (V), (VI), and (VII) is a solution that is refrigerated for storage and / or transport at a temperature of, for example, about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C. In certain aspects, the present disclosure is also directed to a method of increasing the stability of a nanoparticle composition and / or a pharmaceutical composition comprising a compound of any of Formula (I), (II), (III), (IV), (V), (VI), and (VII) by storing the nanoparticle composition and / or the pharmaceutical composition at a temperature of 4°C or less, such as between about -150°C and about 0°C or between about -80°C and about -20°C, for example, at a temperature of about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C. For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months at a temperature of, for example, 4°C or less (e.g., between about 4°C and -20°C). In one aspect, the formulation is stable for at least 4 weeks at about 4°C. In certain aspects, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain aspects, the pH of the pharmaceutical compositions of the present disclosure is between about 5 and 8 (e.g., 5, 5.5, 6, 6.5, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8 or between 7 and 7.8). For example, the pharmaceutical compositions of the present disclosure comprise the nanoparticle compositions disclosed herein, Tris, saline, and sucrose, and have a pH of about 7.5-8, suitable for storage and / or transportation at, for example, about -20°C. For example, the pharmaceutical compositions of the present disclosure comprise the nanoparticle compositions disclosed herein and PBS, and have a pH of about 7-7.8, suitable for storage and / or transportation at, for example, about 4°C or lower."Stability," "stabilized," and "stable" in the context of the present disclosure refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size changes, aggregation, encapsulation changes, etc.) under given manufacturing, preparation, transportation, storage, and / or use conditions (e.g., when stresses such as shear forces, freeze / thaw stresses, etc. are applied).

[0598] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be applied to any patient or subject, including those patients or subjects who can benefit from the therapeutic effect provided by delivering therapeutic agents and / or preventive agents to one or more specific cells, tissues, organs or systems or groups thereof (such as the renal system). Although the description of the nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein is mainly directed to compositions suitable for application to humans, it will be understood by those skilled in the art that such compositions are generally suitable for application to any other mammal. It is well known that compositions suitable for application to humans are modified to make them suitable for application to various animals, and veterinary pharmacologists of ordinary skill can design and / or perform such modifications only by ordinary experiments (if any). Subjects contemplated for application of the composition 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.

[0599] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include mixing the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then if desired or necessary, dividing, shaping and / or packaging the product into desired single or multiple dose units.

[0600] Pharmaceutical compositions according to the present disclosure can be prepared, packaged and / or sold in batches, as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to the subject and / or a conventional fraction of such a dose, such as, for example, half or one-third of such a dose.

[0601] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared using suitable dispersants, wetting agents and / or suspending agents according to known techniques. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution (Ringer's solution, USP) and isotonic sodium chloride solution. Sterile fixed oils are usually used as solvents or suspension media. To achieve this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids (such as oleic acid) can be used to prepare injectables.

[0602] The injectable formulations can be sterilized, for example, by filtration through a bacteria-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.

[0603] The present disclosure provides a method for producing a target polypeptide or a regulatory structure in a mammalian cell. The method for producing a polypeptide involves contacting a cell with a nanoparticle composition comprising an mRNA encoding the target polypeptide or the regulatory structure. After contacting the cell with the nanoparticle composition, the mRNA can be taken up in the cell and translated to produce the target polypeptide.

[0604] In general, the step of contacting mammalian cells with a nanoparticle composition comprising an mRNA encoding a target polypeptide or a regulatory structure can be carried out in vivo, in vitro, in culture or in vitro. The amount of the nanoparticle composition contacted with the cell and / or the amount of the mRNA therein can depend on the biochemical characteristics (e.g., size, charge and chemical composition) and other factors of the type of the cell or tissue contacted, the mode of administration, the nanoparticle composition and the mRNA therein. In general, the nanoparticle composition of an effective amount will allow polypeptides to be effectively produced in cells. Efficiency indicators can include polypeptide translation (indicated by polypeptide expression), mRNA degradation levels and immune response indicators.

[0605] The step of contacting the nanoparticle composition comprising mRNA with the cell can involve or cause transfection. The phospholipids included in the lipid component of the nanoparticle composition can promote transfection and / or increase transfection efficiency, for example, by interacting and / or fusing with the cell membrane or intracellular membrane. Transfection can allow translation of mRNA in the cell.

[0606] The present disclosure provides a method for delivering a bioactive agent to a mammalian cell or organ. Delivery of a bioactive agent to a cell involves administering a nanoparticle composition comprising a bioactive agent to a subject, wherein the administration of the composition involves contacting the cell with the composition. For example, a protein, a cytotoxic agent, a radioactive ion, a chemotherapeutic agent or a nucleic acid (such as RNA, for example mRNA) can be delivered to a cell or an organ. In the case where a therapeutic agent and / or a preventive agent is mRNA, after contacting the cell with the nanoparticle composition, translatable mRNA can be translated in the cell to produce a target polypeptide. However, mRNA that is substantially intranslatable can also be delivered to the cell. Substantially intranslatable mRNA can be used as a vaccine and / or can isolate the translation component of the cell to reduce the expression of other types in the cell.

[0607] In some aspects, nanoparticle compositions can target cells of a specific type or category (e.g., cells of a specific organ or its system). For example, nanoparticle compositions comprising a target bioactive agent can be specifically delivered to the liver, kidney, spleen, femur or lung of a mammal. Specific delivery to cells, organs or systems or groups thereof of a specific category means that, for example, after applying nanoparticle compositions to a mammal, the ratio of nanoparticle compositions comprising therapeutic agents and / or preventive agents delivered to the target endpoint (e.g., tissue) is higher relative to other endpoints. In some aspects, specific delivery can result in an increase of more than 2 times, 5 times, 10 times, 15 times or 20 times in the amount of therapeutic agents and / or preventive agents in each 1g of tissue of the target endpoint (e.g., target tissue, such as the liver) compared to another endpoint (e.g., spleen). In some aspects, the target tissue is selected from the group consisting of: liver, kidney, lung, spleen, femur, eye tissue (e.g., via intraocular, subretinal or intravitreal injection), vascular endothelium in blood vessels (e.g., intracoronary or femoral artery) or kidney and tumor tissue (e.g., via intratumoral injection).

[0608] As another example of targeted delivery or specific delivery, mRNA encoding a protein binding partner (e.g., an antibody or a functional fragment thereof, a scaffold protein or peptide) or a receptor on the cell surface may be included in the nanoparticle composition. mRNA may be used in addition or instead to direct the synthesis and extracellular localization of lipids, carbohydrates or other biological parts. Alternatively, other bioactive agents or elements (e.g., lipids or ligands) of the nanoparticle composition may be selected based on affinity for a specific receptor (e.g., a low-density lipoprotein receptor) so that the nanoparticle composition can more easily interact with a target cell population containing a receptor. For example, ligands may include, but are not limited to, members of specific binding pairs, 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 agents, including monospecific or bispecific antibodies, such as disulfide-stabilized Fv fragments, scFv tandems, double antibodies, three antibodies or four antibodies; and aptamers, receptors and fusion proteins.

[0609] In some aspects, the ligand can be a surface-bound antibody, which can allow for the regulation of cell targeting specificity. This is particularly useful because highly specific antibodies can be generated for the desired targeting site of the target epitope. In one aspect, multiple antibodies are expressed on the cell surface, and each antibody can have different specificities for the desired target. Such methods can increase the affinity and specificity of the targeted interaction.

[0610] In certain aspects, the compositions according to the present disclosure can be administered at a dose level sufficient to deliver the following amounts of therapeutic and / or prophylactic agents (e.g., mRNA): about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg. kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.001 mg / kg to about 2.5 mg / kg. g / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.0 5mg / kg to about 1mg / kg, about 0.1mg / kg to about 1mg / kg, about 0.0001mg / kg to about 0.25mg / kg, about 0.001mg / kg to about 0.25mg / kg, about 0.005mg / kg to about 0.25mg / kg, about 0.01mg / kg to about 0.25mg / kg, about 0.05mg / kg to about 0.25mg / kg, or about 0.1mg / kg to about 0.25mg / kg, wherein a dosage of 1mg / kg (mpk) provides 1mg of a therapeutic and / or preventive agent per 1kg of subject body weight. In some aspects, a therapeutic and / or preventive agent (e.g., mRNA) of a nanoparticle composition of about 0.001mg / kg to about 10mg / kg dosage can be administered. In other aspects, a therapeutic and / or preventive agent of about 0.005mg / kg to about 2.5mg / kg dosage can 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 dosage of about 0.05 mg / kg to about 0.25 mg / kg may be administered. The dosage may be administered once or more times per day in the same or different amounts to obtain the desired level of mRNA expression and / or therapeutic, diagnostic, preventive or imaging effect.

[0611] The desired dose can be delivered, for example, three times a day, twice a day, once a day, every other day, every three days, once a week, every two weeks, every three weeks, or every four weeks. In some aspects, the desired dose can 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 can be administered, for example, before or after surgery, or in the case of an acute disease, disorder, or illness.

[0612] Nanoparticle compositions comprising one or more therapeutic and / or prophylactic agents can be used in combination with one or more other therapeutic, prophylactic, diagnostic or imaging agents. "Combination" is not intended to imply that the agents must be administered simultaneously and / or formulated to be delivered together, although these delivery methods are within the scope of the present disclosure. For example, one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents can be administered in combination. The composition can be administered simultaneously with, before or after one or more other desired therapeutic agents or medical procedures. In general, each agent will be administered according to a dose and / or schedule determined for the agent. In some aspects, the present disclosure encompasses the delivery of a combination of a composition or its imaging, diagnostic or prophylactic composition with an agent that improves its bioavailability, reduces and / or changes its metabolism, inhibits its excretion and / or changes its distribution in the body.

[0613] It should also be understood that the treatment, prevention, diagnosis or imaging active agent used in combination can be used together in a single composition or used separately in different compositions. In general, it is expected that the use level of the agent used in combination will not exceed the level used alone. In some aspects, the level used in combination may be lower than the level used alone.

[0614] The particular combination of therapies (therapeutics or procedures) used in a combination regimen will take into account the compatibility of the desired therapeutics and / or procedures with the desired therapeutic effect to be achieved. It will also be understood that the therapies used may achieve the desired effect for the same condition (e.g., a composition useful for treating cancer may be administered simultaneously with a chemotherapeutic agent), or they may achieve different effects (e.g., controlling any adverse effects, such as infusion-related reactions).

[0615] Example

[0616] The following examples are provided to help further understand the present invention, but are not intended to limit the reasonable scope of the present invention.

[0617] Example 1: One-step synthesis scheme

[0618] At present, the synthesis of ionizable lipids generally includes multiple steps, which is time-efficient and cost-effective. The ionizable lipids (formula (I-VI)) disclosed herein are designed to be synthesized using a one-step synthesis scheme, which enables the synthesis of ionizable lipids in a high-throughput manner. A multi-step synthesis scheme can be used to synthesize the same formula. For example, a two-component reaction can be used to synthesize compounds of formulas I, II, and VI; a three-component reaction is used to synthesize formula III; and a four-component reaction is used to synthesize formulas IV and V. 16,824 ionizable lipids are synthesized using formulas I, II, III, and IV, however, formulas I-VI can be used as the basis for synthesizing unlimited ionizable lipids.

[0619] Ionizable lipids of formula I, such as C7(2,4,6-trioxo-1,3,5-triazinane-1,3,5-triyl)tris(ethane-2,1-diyl)tris(3-(ditetradecylamino)propionate) ( Fig.14 ) can be prepared via the following two-component reaction.

[0620] Tri(2-acryloyloxyethyl)isocyanurate (0.2 mmol) and dioctylamine (0.66 mmol) were mixed in 10 mL of toluene / acetonitrile (v / v=5 / 1) and stirred at 95° C. for two days in a capped glass vial. The mixture was purified by flash column chromatography to obtain Compound C7.

[0621] Ionizable lipids of formula II, such as TAa-9(2,4,6-trioxo-1,3,5-triazinane-1,3,5-triyl)tris(ethane-2,1-diyl)tris(3-(dinonylamino)propionate) ( Figure 1 ) can be prepared via the following two-component reaction.

[0622] 1,3,5-Triacryloylhexahydro-1,3,5-triazine (0.2 mmol) and dinonylamine (0.66 mmol) were mixed in 10 mL of toluene / acetonitrile (v / v=5 / 1) and stirred at 95° C. for three days in a capped glass vial. The mixture was purified by flash column chromatography to obtain compound TAa-9.

[0623] An ionizable lipid of formula III, such as TP3G11 (2-((ditetradecylamino)(4-dodecylphenyl)methyl)phenol) Figure 5 ) can be prepared via the following three-component reaction.

[0624] Ditetradecylamine (0.11 mmol in 2.5 mL MeOH) was added to a solution of 2-hydroxybenzaldehyde (0.11 mmol in 2.5 mL toluene), and then (4-dodecylphenyl)boronic acid (0.1 mmol) was added to the same mixture. The reaction mixture was stirred at 95° C. for three days. The mixture was purified by flash column chromatography to obtain compound TP3G11.

[0625] The synthesis of ionizable lipids of Formula IV, such as IVe (N-(2-(cyclohex-1-en-1-ylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)palmitamide), is prepared via the following four component reaction.

[0626] 1-Ethylpiperidine-4-carboxaldehyde (0.1mmol) was added to a solution of heptadecan-9-amine (0.1mmol in 2mL MeOH) at room temperature. After 2 hours, palmitic acid (0.1mmol) and 1-isocyanatocyclohex-1-ene (0.1mmol) were added to the reaction mixture, and the mixture was stirred at room temperature overnight. The mixture was purified by flash column chromatography to obtain compound IVe.

[0627] The synthesis of ionizable lipids of Formula V, such as Va (ethyl 4-(3-(dimethylamino)propyl)-5,5-di((E)-heptadeca-8-en-1-yl)-3,6-dioxopiperazine-2-carboxylate) is prepared via the following four component reaction.

[0628] Pentatriacontane-9,26-diene-18-one (0.1 mmol) was added to a solution of N',N'-dimethylpropane-1,3-diamine (0.1 mmol in 2 mL CH2Cl2) at room temperature. Two hours later, 2-((tert-butyloxycarbonyl)amino)-3-ethoxy-3-oxopropionic acid (0.1 mmol) and 1-isocyanatocyclohex-1-ene (0.1 mmol) were added to the reaction mixture, and the mixture was stirred at room temperature overnight. Then, 0.1 mL HCl (1 M) was added to the reaction mixture, and stirred at room temperature overnight. The mixture was purified by flash column chromatography to obtain compound Va.

[0629] The synthesis of ionizable lipids of Formula VI, such as VIa N,N',N"-(cyclohexane-1,3,5-triyltri(methylene))tris(N-tridecyltridec-1-amine) can be prepared via the following two component reaction.

[0630] 1,3,5-Tris(bromomethyl)cyclohexane (0.2 mmol) and ditridecylamine (0.66 mmol) were mixed in 10 mL MeOH / DMF (v / v=5 / 1) and stirred at room temperature for four days in a capped glass vial. The mixture was purified by flash column chromatography to obtain Compound VIa.

[0631] Example 2: Multi-step synthesis scheme

[0632] Synthesis of the ionizable lipid of formula VII. The ionizable lipid was synthesized in two steps as follows.

[0633] At 0 ℃ under N2, a solution of acryloyl chloride (56mmol) in anhydrous CH2Cl2 (50mL) was added dropwise to a mixture of cyclohexane-1,3,5-triol (14mmol) and Et3N (84mmol) in anhydrous CH2Cl2 (60mL). After about 1 hour, the mixture was allowed to warm to room temperature and reacted for 4 hours. The mixture was then treated with saturated sodium bicarbonate solution (150mL) and brine (150mL) and extracted with CH2Cl2 (150mL×3). The combined organic layers were dried over anhydrous magnesium sulfate and the solvent was removed by a rotary evaporator. The crude product was purified by column chromatography using a CombiFlash Rf system with a silica gel column (Redisep Gold Resolution, Teledyne, Isco) by hexane / ethyl acetate (5%-100% ethyl acetate) to obtain a compound cyclohexane-1,3,5-triyl triacrylate (0.92g, 22.4%) as a white solid. 1H-NMR (400MHz, Me-OD, δ) 6.40 (dd, J=16, 4Hz, 3H), 6.08 (dd, J=20, 8Hz, 3H), 5.83 (dd, J=16, 4Hz, 3H), 4.98-4.87 (m, 3H), 1.24 (s, 6H). MS (ESI) m / z 317.1[M+Na]+.

[0634] Cyclohexane-1,3,5-triyl triacrylate (0.1mmol) was mixed with amine (0.33mmol) in 5mL ethanol in a molar ratio of 1:3.3, and the mixture was stirred in a glass screw vial in a 50°C oil bath for two days. The solvent was removed by a rotary evaporator, and the product was purified by hexane / ethyl acetate (5%-100% ethyl acetate) using a CombiFlash Rf system with a silica gel column (Redisep Gold Resolution, Teledyne, Isco) by column chromatography. The structure was confirmed by 1H nuclear magnetic resonance spectroscopy (Bruker AVANCE-400NMR, Custom NMR Services, Inc.) and LC-mass spectrometry (Agilent1100&1200 HPLC / MS, Organix, Inc).

[0635] Synthesis of ionizable lipids of Formula I. The ionizable lipids were synthesized as follows.

[0636] Tris (2-acryloyloxyethyl) isocyanurate (0.2mmol) was mixed with amine (0.66mmol) in 10mL toluene / ACN (5 / 1) in a molar ratio of 1: 3.3, and the mixture was stirred in a glass screw vial in a 95°C oil bath for two days. The solvent was removed by a rotary evaporator, and the product was purified by column chromatography using a CombiFlash Rf system with a silica gel column (Redisep Gold Resolution, Teledyne, Isco) by a gradient elution of hexane / ethyl acetate (5%-100% ethyl acetate). The structure was confirmed by 1H nuclear magnetic resonance spectroscopy (Bruker AVANCE-400NMR, Custom NMR Services, Inc.) and LC-mass spectrometry (Agilent 1100&1200HPLC / MS, Organix, Inc).

[0637] (2,4,6-Trioxo-1,3,5-triazinane-1,3,5-triyl)tris(ethane-2,1-diyl)tris(3-(ditetradecylamino)propionate) (C7): Yield (20%). 1H-NMR (400 MHz, CDCl3, δ) 4.29 (t, J = 8 Hz, 3H), 4.16 (t, J = 8 Hz, 3H), 2.75 (t, J = 8 Hz, 3H), 2.60 (t, J = 8 Hz, 3H), 2.42-2.34 (m, 12H), 1.50-1.26 (m, 84H), 0.87 (t, J = 8 Hz, 18H). MS (APCI) m / z 1148.3 [M+H]+.

[0638] (2,4,6-Trioxo-1,3,5-triazinane-1,3,5-triyl)tri(ethane-2,1-diyl)tri(3-(dioctylamino)propionate) (D7): Yield (23%). 1H-NMR (400 MHz, CDCl3, δ) 4.29 (t, J = 8 Hz, 3H), 4.17 (t, J = 8 Hz, 3H), 2.75 (t, J = 8 Hz, 3H), 2.60 (t, J = 8 Hz, 3H), 2.39-2.34 (m, 12H), 1.50-1.26 (m, 156H), 0.89 (t, J = 8 Hz, 18H). MS (APCI) m / z 1653.5 [M+H]+.

[0639] Synthesis of Ionizable Lipids of Formula II The ionizable lipids were synthesized as follows.

[0640] 1,3,5-triacryloyl hexahydro-1,3,5-triazine (0.2mmol) was mixed with amine (0.66mmol) in 10mL toluene / ACN (5 / 1) in a molar ratio of 1:3.3, and the mixture was stirred in a glass screw vial in a 95°C oil bath for two days. The solvent was removed by a rotary evaporator, and the product was purified by column chromatography using a CombiFlash Rf system with a silica gel column (Redisep Gold Resolution, Teledyne, Isco) by a gradient elution of CH2Cl2 / methanol (0%-40% methanol). The structure was confirmed by 1H nuclear magnetic resonance spectroscopy (Bruker AVANCE-400NMR, Custom NMR Services, Inc.) and LC-mass spectrometry (Agilent 1100&1200HPLC / MS, Organix, Inc).

[0641] 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tris(3-(dinonylamino)propan-1-one) (E2): Yield (25%). 1H-NMR (400 MHz, CDCl3, δ) 5.26 (s, 6H), 2.79 (t, J=8 Hz, 6H), 2.65 (t, J=8 Hz, 6H), 2.41 (t, J=8 Hz, 12H), 1.42-1.26 (m, 84H), 0.88 (t, J=8 Hz, 18H). MS (ESI) m / z 529.6 [M+2H]2+.

[0642] 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tris(3-(dinonylamino)propan-1-one) (E6): Yield (25%). 1H-NMR (400 MHz, CDCl3, δ) 5.26 (s, 6H), 2.81 (t, J=8 Hz, 6H), 2.67 (t, J=8 Hz, 6H), 2.43 (t, J=8 Hz, 12H), 1.44-1.23 (m, 132H), 0.88 (t, J=8 Hz, 18H). MS (ESI) m / z 697.9 [M+2H]2+.

[0643] Synthesis of ionizable lipid of formula III The ionizable lipid was synthesized as follows.

[0644] Ionizable lipids are synthesized with a molar ratio of boronic acid / amine / aldehyde of 1:1:1. Library reactions are performed on 96-well plates (Analytical Sales and Services) with glass inserts. The reaction is allowed to occur overnight at 95°C in toluene / methanol (10 / 1). The product mixture is directly used for in vitro high-throughput screening. In addition, ionizable lipids are purified by column chromatography using a CombiFlash Rf system with a silica gel column (Redisep Gold Resolution, Teledyne, Isco) by gradient elution with CH2Cl2 / methanol (0%-40% methanol). The structure is confirmed by 1H nuclear magnetic resonance spectroscopy (BrukerAVANCE-400 NMR, Custom NMR Services, Inc.) and LC-mass spectrometry (Agilent 1100&1200 HPLC / MS, Organix, Inc).

[0645] 2,2′-(1,4-phenylenebis((dioctylamino)methylene))diphenol (TP1A11): Yield (17%). 1H-NMR (400 MHz, CDCl3, δ) 7.37 (s, 4H), 7.10-7.14 (t, J=8 Hz, 2H), 6.83-6.85 (d, J=8 Hz, 2H), 6.77-6.80 (m, 2H), 6.64-6.69 (t, J=12 Hz, 2H), 4.88 (d, J=8 Hz, 2H), 2.42-2.65 (m, 8H), 1.21-1.25 (m, 40H), 0.84-0.89 (m, 12H). MS (APCI) m / z 769.7 [M+H]+.

[0646] 2-((Bis-tetradecylamino)(thiophen-2-yl)methyl)phenol (TP4G11): Yield 23%. HNMR (400 MHz, CDCl3, δ) 7.39 (d, J = 8 Hz, 1H), 7.08-7.14 (m, 2H), 7.02-7.05 (m, 1H), 6.86-6.89 (d, J = 12 Hz, 1H), 6.75-6.78 (m, 1H), 6.66-6.72 (m, 1H), 5.44 (s, 1H), 2.45-2.69 (m, 4H), 1.29 (m, 48H), 0.84-0.90 (t, 6H). MS (APCI) m / z 769.7 [M+H]+.

[0647] 2-((Ditridecylamino)(4-dodecylphenyl)methyl)phenol (TP3 F11): yield 25%. HNMR (400 MHz, CDCl3, δ) 7.30 (d, J = 12 Hz, 2H), 7.14 (d, J = 12 Hz, 3H), 6.85 (t, J = 12 Hz, 2H), 6.69 (t, J = 12 Hz, 1H), 4.92 (s, 1H), 2.60-2.55 (m, 4H), 2.39-2.44 (m, 2H), 1.59-1.51 (m, 6H), 1.25 (m, 60H), 0.88 (t, J = 12 Hz, 9H). MS (APCI) m / z 732.7 [M+H]+.

[0648] 2-((Bis-tetradecylamino)(4-dodecylphenyl)methyl)phenol (TP3G11): Yield 26%. HNMR (400 MHz, CDCl3, δ) 7.30 (d, J = 12 Hz, 2H), 7.14 (d, J = 12 Hz, 3H), 6.82 (t, J = 12 Hz, 2H), 6.66 (t, J = 12 Hz, 1H), 4.92 (s, 1H), 2.60-2.55 (m, 4H), 2.39-2.44 (m, 2H), 1.59-1.51 (m, 6H), 1.25 (m, 64H), 0.88 (t, J = 12 Hz, 9H). MS (APCI) m / z 732.7 [M+H]+.

[0649] Synthesis of ionizable lipid of formula IV. The ionizable lipid was synthesized in one pot as follows.

[0650] Ionizable lipids were synthesized by a four-component reaction with a molar ratio of acid / amine / aldehyde / isocyanide of 1:1:1:1. Library reactions were performed in 96-well plates with glass inserts (Analytical Sales and Services). Amine and aldehyde were first mixed and stirred at room temperature (rt), then acid and isocyanide were added. The reaction was stirred overnight at rt.

[0651] Synthesis of exemplary ionizable lipids.At room temperature, aldehyde (1mmol) and amine (1mmol) are mixed and stirred for one hour in 3mL MeOH, then isocyanide (1mmol) and acid (1mmol) are added. The resulting mixture is further stirred overnight. Remove the solvent with a rotary evaporator. Add ethyl acetate (100mL) and wash with saline (2X 50mL), via MgSO4 dry. Remove ethyl acetate and use the CombiFlash Rf system with a silica gel column (Redisep Gold Resolution, Teledyne, Isco) by column chromatography, by CH2Cl2 / methanol (0%-40% methanol) gradient elution to purify residue. By1H nuclear magnetic resonance spectrum (Bruker AVANCE-400NMR, Custom NMR Services, Inc.) and / or LC-mass spectrum (Agilent 1100&1200HPLC / MS, Organix, Inc) to confirm the structure.

[0652] (2-(1-ethylpiperidin-4-yl)-2-(N-(pentadecan-8-yl)tridecanoyl)acetyl)glycine ethyl ester (P1C1): yield 16%. 1H-NMR (400 MHz, Me-OD, δ) 4.34 (d, J = 8 Hz, 1H), 4.21-4.14 (m, 4H), 3.41 (t, J = 8 Hz, 1H), 3.01 (q, J = 8 Hz, 2H), 2.47-2.40 (m, 5H), 2.01-1.83 (t, J = 12 Hz, 2H), 1.65-1.56 (m, 6H), 1.29-1.17 (m, 42H), 1.17-1.10 (m, 6H), 0.93-0.89 (m, 9H). MS (APCI) m / z 678.7 [M+H]+.

[0653] N-(2-(Cyclohex-1-en-1-ylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)palmitamide (P6A2): Yield (45%). 1H-NMR (400 MHz, Me-OD, δ) 6.05 (t, J=8 Hz, 1H), 4.26 (s, 1H), 3.26-3.17 (m, 3H), 2.49-2.42 (m, 5H), 2.19-2.01 (m, 6H), 1.67-1.57 (m, 14H), 1.31-1.16 (m, 48H), 0.92-0.87 (m, 12H). MS (APCI) m / z 742.7 [M+H]+.

[0654] N-(2-(Cyclohexylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)palmitamide (P30A1): Yield (58%). 1H-NMR (400 MHz, CDCl3, δ) 8.41 (d, J = 8 Hz, 1H), 3.70-3.63 (m, 1H), 3.57 (t, J = 8 Hz, 1H), 3.18-3.09 (m, 3H), 2.80-2.24 (m, 3H), 2.44-2.24 (m, 4H), 1.79-1.20 (m, 67H), 0.89-0.85 (m, 9H). MS (APCI) m / z 716.8 [M+H]+.

[0655] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-decyltetradecyl)undecylamide (P54B6): yield (48%). MS (APCI) m / z 824.8 [M+H]+.

[0656] N-(2-(Cycloheptylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)tetradecamide (P38D1): Yield (56%). MS (APCI) m / z 702.7 [M+H]+.

[0657] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)palmitamide (P56A1): Yield (53%). MS (APCI) m / z 768.7 [M+H]+.

[0658] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)heptadecanamide (P56B 1): Yield (59%). MS (APCI) m / z 782.8 [M+H]+.

[0659] N-(2-(Butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)octanamide (P14A2): Yield (40%). MS (APCI) m / z 606.6 [M+H]+.

[0660] N-(2-(Butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(pentadecan-8-yl)undecanoic acid amide (P16B1): yield (43%). MS (APCI) m / z 620.6 [M+H]+.

[0661] N-(2-(Cyclohexylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-octyldodecyl)tetradecamide (P26D4): Yield (23%). MS (APCI) m / z 758.8 [M+H]+.

[0662] (2-(1-ethylpiperidin-4-yl)-2-(N-(2-octyldodecyl)tetradecylamide)acetyl)glycine ethyl ester (P1D4): yield (30%). MS (APCI) m / z 762.8 [M+H]+.

[0663] N-(1-(Cyclohexylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)heptadecanamide (P30B7): Yield (56%). MS (APCI) m / z 730.8 [M+H]+.

[0664] N-(1-(Cyclohexylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)stearamide (P30C7): yield (53%). MS (APCI) m / z 744.8 [M+H]+.

[0665] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)tetradecamide (P38D7): Yield (45%). MS (APCI) m / z 702.7 [M+H]+.

[0666] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(heptadecan-9-yl)tetradecanoic acid amide (P38D8): Yield (52%). MS (APCI) m / z 730.7 [M+H]+.

[0667] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-octyldodecyl)undecylamide (P40B 10): Yield (42%). MS (APCI) m / z 730.7 [M+H]+.

[0668] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(tricosan-12-yl)dodecanoamide (P40C11): Yield (57%). MS (APCI) m / z 786.8 [M+H]+.

[0669] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-octyldodecyl)dodecylamide (P40C10): Yield (70%). MS (APCI) m / z 744.8 [M+H]+.

[0670] N-(1-(tert-Butylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-decyltetradecyl)tridecamide (P51C12): Yield (70%). MS (APCI) m / z 774.8 [M+H]+.

[0671] N-(1-(Cycloheptylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(pentadecan-8-yl)pentadecanamide (P40D7): Yield (61%). MS (APCI) m / z 716.7 [M+H]+.

[0672] N-(2-(tert-Butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(tricosan-12-yl)decanamide (P53A5): Yield (61%). MS (APCI) m / z 718.7 [M+H]+.

[0673] N-(2-(tert-butylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-decyltetradecyl)decanamide (P53A6): yield (61%). MS (APCI) m / z 732.7 [M+H]+

[0674] N-(2-(Cycloheptylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-decyltetradecyl)tetradecamide (P38D4): yield (51%). MS (APCI) m / z 828.7 [M+H]+

[0675] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)palmitamide (P5 6A2): yield (46%). MS (APCI) m / z 796.7 [M+H]+

[0676] N-(2-(Cycloheptylamino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(heptadecan-9-yl)oleamide (P1 49A2): yield (42%). MS (APCI) m / z 784.8 [M+H]+

[0677] 4-((2-(Cycloheptylamino)-1-(1-ethylpiperidin-3-yl)-2-oxoethyl)(2-octyldodecyl)amino)-4-oxobutanoic acid dodecyl ester (P159C10): yield (43%). MS (APCI) m / z 830.8 [M+H]+

[0678] N-(1-(((1s,3s)-adamantan-1-yl)amino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-4-(3,6-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)-N-(2-octyldodecyl)pentanamide (P 161F 10): yield (53%). MS (APCI) m / z 974.9 [M+H]+.

[0679] Example 3: LNP Synthesis

[0680] Self-amplified mRNA (samRNA) encoding firefly luciferase (SamRNA-LUC) and GFP (SamRNA-GFP) was prepared by SunVax mRNA 7herapeutics. Fetal bovine serum (FBS) and Ribogreen reagent were purchased from Fisher Scientific. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and cholesterol were purchased from Avanti Polar Lipids. Chemical reagents were purchased from Sigma-Aldrich, Millipore Sigma, Fisher Scientific, TCI America, Ambeed, CaymanChem, A2B Chem, BLD pharm, Aaron Chemicals, AAb1ocks, Ic1ickchemistry, Enamine, Aurum, Achemblock, BroadPharm, ChemShuttle, BiopharmaPEG.

[0681] The ionizable lipids of the present disclosure were synthesized and formulated into LNPs with DOPE, cholesterol and DMG-PEG2000.

[0682] More than 50 LNPs made using the lipids of the present disclosure have shown effective transfection ( Figure 4 The results showed that four separate ionizable lipids synthesized via a two-component reaction ( Figures 1 to 3 , Fig.14 ) and twelve individual ionizable lipids synthesized via a three-component reaction ( Figures 5 to 8 ) formulated LNPs encapsulating self-amplifying mRNA encoding GFP-mRNA transfection showed better or comparable transfection efficiency than those of FDA-approved LNPs. Figures 9 to 13 Additional examples of effective transfection using LNPs made with additional lipids of the present disclosure are shown. The LNPs of the present disclosure were found to effectively transfect C2C12, MC38 and 293T cells in vitro. The data show that their transfection efficiency is better or comparable to that of currently FDA-approved LNPs.

[0683] The LNPs with ionizable lipids of the present disclosure are prepared by mixing the ethanol phase containing lipids with the aqueous phase containing mRNA using a pipette. The ethanol phase is prepared in a predetermined molar ratio of 50:10:38.5:1.5 by mixing ionizable lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), cholesterol (Avanti) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000, Broadpharm) in ethanol. The aqueous phase is prepared in a citrate buffer (10mM, pH 3.0, MilliporeSigma) with GMP-mRNA. The aqueous phase and the ethanol phase are mixed in a 1:1 ratio, and N:P is mixed in a 6:1 ratio. The formulated LNPs are dialyzed with PBS for 2 hours at 4°C in a 20,000MWCO box (Fisher) and stored at 4°C before use. Specifically, DLS was used to measure the size, polydispersity index and zeta potential of the formulated LNPs (AnoPartica SZ-100-Z2, Horiba).

[0684] Size and polydispersity index (PDI) were measured by dynamic light scattering (SZ-100-Z2 (MTS), Horiba Scientific). Diameters were reported as intensity mean peak average. Nucleic acid encapsulation efficiency was calculated by a modified Quant-iT RiboGreenRNA assay (Invitrogen).

[0685] Example 4: Bioactive Agent Synthesis

[0686] Figure 8 The GFP-mRNA transfection in the present invention is a self-amplifying mRNA encoding GFP, which is synthesized de novo from the Venezuelan equine encephalitis (VEE) virus strain TC-83. The sequence encoding GFP mRNA in the subgenomic region of VEE replaces the structural protein. The self-amplifying mRNA is transcribed in vitro from the synthetic plasmid and capped by the vaccinia virus capping system. The synthesized mRNA is then purified for in vitro or in vivo determination.

[0687] The delivery system used to deliver GFP-mRNA transfection was LNP formulated using 12 individually ionizable lipids synthesized via a three-component reaction. Figure 8The LNPs encapsulated with self-amplifying mRNA used in the present invention are dissolved in ethanol and directly added to an ethanol solution containing DOPE, cholesterol and DMG-PEG2000 at a predetermined molar ratio. The self-amplifying mRNA encoding the GFP sequence is mixed with the aqueous solution, and the LNP mixture (100ng mRNA) is added to a 96-well plate pre-seeded with MC38, C2C12 or 293T cells. The cells are incubated overnight at 5% CO2 and 37°C. The GFP mRNA transfection efficiency is measured by flow cytometry.

[0688] Example 5: Transfection efficiency

[0689] The ionizable lipids disclosed herein are formulated with DOPE, cholesterol and DMG-PEG2000 to encapsulate larger self-amplifying mRNAs encoding reporter genes such as green fluorescent protein (GFP) or luciferase (LUC). The ionizable lipids disclosed herein can form LNPs and encapsulate larger nucleic acids such as self-amplifying mRNAs, and current LNPs are mainly developed for small-sized mRNAs, such as mRNAs encoding a portion of the COVID spike protein. The ionizable lipids disclosed herein, DOPE, cholesterol and DMG-PEG2000 are formulated into LNPs and transfection efficiency is tested.

[0690] The ionizable lipid E6 disclosed in the present invention is mixed with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), methoxy polyethylene glycol (DMG-PEG 2000 ) with 50 / 10 / 38.5 / 1.5 (ionizable lipid / DOPE / Chol / DMG-PEG 2000 ) was formulated into LNPs with a molar ratio of 1:1, which encapsulated self-amplifying mRNA or DNA encoding a reporter gene (green fluorescent protein (samRNA-GFP or DNA-CMV-EGFP) or firefly luciferase (samRNA-LUC)). The formulation containing 100 ng of self-amplifying mRNA was incubated in wells of a 96-well plate, where HEK293, C2C12 or MC38 cells had a fusion rate of 60%-70% ( Fig.10 , Fig.15 , Fig.28 ). Transfection efficiency was determined by flow cytometry for GFP or by plate reader for luciferase. LNPs formulated with the FDA-approved ionizable lipid MC3 were used as controls ( Fig.10 , Fig.15 ).

[0691] LNP containing SamRNA-GFP (or SamRNA-LUC, 100ng / well) is added to 96-well plates pre-seeded with 293T, C2C12 or MC38 cells. After incubation for 24 hours at 37°C and 5% CO2, the luciferase SamRNA transfection efficiency is measured by a plate reader (PerkinElmer Envision 2104). GFP SamRNA transfection efficiency is measured by flow cytometry (BDFACSymphonyTM A5 SE cell analyzer).

[0692] Compared with the FDA-approved ionizable lipid MC3, the synthetic C7 and D7 ( Figure 12-14 ); E2 and E6 of Formula II ( Fig. 9 , Fig.10 , Fig.15 ) LNP; and LNP-SamRNA-GFP prepared by TP1A2, TP1H5, TP3A5, TP3G11, TP3G12, TP4G4, TP4G11, TP1A11, TP3F11 of formula III can more effectively deliver mRNA-GFP ( Figures 4 to 8 LNP-SamRNA-Luc formulated with P1C1, P6A2, P30A1, P54B6, P38D1, P56A1, P56B1, P14A2, P16B1, P26D4, P1D4, P30B7, P30C7, P38D7, P38D8, P40B10, P40C11, P40C10, P51C12, P40D7, P53A5 and P53A6 of Formula IV showed a stronger bioluminescent signal than MC3-LNP-SamRNA-Luc ( Fig.16 ). Other leading ionizable lipids showed very high samRNA-GFP delivery efficiency in 239T cells, such as Fig.17 The high-throughput synthesis of ionizable lipids by three components is shown in Figure 4 As shown, and the four-component reaction part is as follows Fig.16 shown.

[0693] Among the ionizable lipids tested, 117 ionizable lipids showed high transfection efficiency, as indicated by the percentage of GFP-positive cells after transfection or by luciferase luminescence ( Figure 16 to Figure 17 ). Orthogonal experiments using 22 ionizable lipids of the present disclosure were performed to find a better ratio between ionizable lipids, DOPE, cholesterol and DMG-PEG2000, thereby achieving compensation optimization of LNP. LNP was applied to mice, and 15 LNPs formulated using the tested ionizable lipids of the present disclosure showed higher in vivo delivery efficiency than the FDA-approved ionizable lipid D-Lin-MC3-DMA (MC3).

[0694] Example 6: Characterization of ionizable lipids

[0695] Ionizable lipids C7, D7, E2, E6, TP1A1 1, TP3G11, TP4G11, TP3F11, TP5A1, P4G11, P1C1, P6A2, P30A1, P54B6, P38D1, P56A1, P56B1, P14A2, P16B1, P26D4, P1D4, P30B7, P30C7, P38D7, P38D8, P40B10, P40C11, P40C10, P51C12, P40D7, P53A5, P53A6, P56A2, and P38D4 were purified and the structures were confirmed by 1H-NMR spectroscopy, mass spectrometry, or both.

[0696] LNPs prepared with E6, Chol, DMG-PEG2000, and DOPC, DSPC, or DOPE. In 293T cells, the delivery efficiency of LNPs prepared with DOPE was higher than that of DSPC and POPE ( Fig.18 ). DOPE was used for further studies.

[0697] An orthogonal experimental design was applied to optimize the formulations of synthetic ionizable lipids, DOPE, Chol, and DMG-PEG2000 encapsulating samRNA-GFP. Library A contained 16 formulations with four factors and four levels.

[0698] Table 2: Orthogonal arrays of P6A2-LNP combinatorial optimization

[0699]

[0700]

[0701] In library A, 293T cells were treated with different amounts of LNP (50ng, 25ng and 2.5ng SamRNA-GFP). When 293T cells were transfected with 50ng, 25ng and 12.5ng mRNA, the delivery efficiency of LNP-SamRNA-GFP of A-6, A-9, A-10 and A-13 all showed more than 60% GFP expression ( Fig.19 ). The samRNA-GFP delivery efficiency increased with the increase of the molar ratio of Chol and DOPE, while increasing the molar ratio of P6A2 and DMG-PEG2000 decreased the samRNA-GFP delivery efficiency ( Fig. 20 ).

[0702] A second orthogonal experimental design was applied to optimize the formulation of LNPs. In library B, 293T cells were treated with 50 ng or 12.5 ng samRNA-GFP ( Fig.21 ).

[0703] Table 2: Orthogonal arrays of P6A2-LNP combination optimization Table B

[0704]

[0705] The influence trends of P6A2, DOPE, Chol and DMG-PEG2000 are as follows Fig. 22 As shown. In library B, LNPs of B-4, B-6, B-7 and B-15 were the best performing formulations in the 50ng and 12.5ng mRNA treatment groups. The predicted optimal formulation combination (C-1) was P6A2 / DOPE / Chol / DMG-PEG2000=30 / 15 / 50 / 1.5. The particle size, PDI and encapsulation efficiency of the P6A2-LNP formulation candidates were measured by dynamic light scattering and Ribogreen assay ( Fig.23 The C-1 formulation of P6A2-LNP showed delivery efficiency comparable to that of B-7 and B-15, and higher than that of B-4 and B-6 ( Fig.24 ).

[0706] As described above, the LNP formulations of ionizable lipid C7 in Formula I, E2 and E6 in Formula II, and TP4G11 in Formula III were optimized. The nanoparticle size, PDI, and mRNA encapsulation efficiency of the optimized C7, D7, E2, E6, TP4G11, P4G11, P1D4, P6A2, P30B7, P30C7, P30A1, P54B6, P38D1, P38D7, P38D8, P40B10, P40C11, P56A1, P56B1, P28D10, P53A5, P40C10, P51C12, P53A6, and P40D7 LNPs were determined by dispersive light scanner and Ribogreen assay, respectively.

[0707] Table 3: LNP characterization

[0708]

[0709]

[0710] Example 7: In vivo verification of transfection efficiency

[0711] The in vivo delivery efficiency of LNPs formulated with C7, E2, E6, P1D4, P30B7, P30C7, P30A1, P54B6, P38D1, P6A2, P38D7, P38D8, P40B10, P40C11, P56A1, P56B1, P28D10, P40C10, P40D7, P53A6, P51C12, and P53A5 encapsulating self-amplifying mRNA encoding luciferase was tested ( Fig.25 and Fig.26 ). 1ug of LNPs encapsulating samRNA-LUC, including controls (MC3-LNP-samRNA-LUC), were injected intramuscularly into mice, and after intraperitoneal injection of luciferin (PerkinElmer, 6 mg / mouse), the expression of luciferase (luminescence) was measured by an in vivo imaging system (IVIS, Perkin Elmer). LNPs formulated with 13 screened ionizable lipids showed better delivery efficiency than MC3-LNP-SamRNA-Luc. LNPs formulated with 9 screened ionizable lipids showed comparable delivery efficiency to MC3-LNP ( Fig.25 and Fig.26 ).

[0712] All animal procedures were performed in accordance with ethical requirements and approved by the Institutional Animal Care and Use Committee (IACUC). Female Balb / c mice (6-8 weeks) were obtained from Charles River Laboratories Inc.

[0713] Example 8: Design and Synthesis of Formula VII

[0714] The initial design of Formula VII required a multi-step synthesis scheme for the Formula VII compound. LNPs formulated with ionizable lipids of Formula VII (P7 and T7) showed better delivery efficiency than the FDA-approved ionizable lipid MC3 ( Fig. 27 ). Therefore, using the information obtained from designing the one-step synthetic scheme for Formulas I-VI, a multi-step synthetic scheme for Formula VII was redesigned, thereby allowing for high-throughput production of the compound of Formula VII by creating a method for synthesizing the compound of Formula VII using a two-step synthetic scheme.

[0715] Therefore, the information generated in Formula I-VII and Library A and B of the present disclosure can be used as a database for manual redesign, machine learning or artificial intelligence of known ionizable lipids. In addition, the database library can be used to design and synthesize ionizable lipids with better or comparable transfection efficiency than currently commercially available ionizable lipids.

[0716] It should be understood that any specific embodiment of the present disclosure that belongs to the prior art may be explicitly excluded from any one or more claims. Since such embodiments are deemed to be known to those skilled in the art, they may be excluded even if the exclusion is not explicitly stated herein.

[0717] It should be understood that although the present disclosure has been described in conjunction with the detailed description of the present disclosure, the foregoing description is intended to illustrate rather than limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages and changes are within the scope of the appended claims.

Claims

1. A compound of formula I or a salt or isomer thereof, wherein Each m and n is independently an integer from 0 to 10; Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24 Each X is independently CH2, NH, O or S; Each Y is independently CH2, NH, O or S; each Z is independently CH or N; and Each L is independently CH2, NH, O or S.

2. The compound of claim 1, wherein the compound is: or a salt or isomer thereof.

3. A method for synthesizing a compound as claimed in any one of claims 1 to 2, comprising reacting an α,β-unsaturated Michael acceptor with an amine:

4. The method of claim 3, wherein the α,β-unsaturated Michael acceptor is 5. The method of claim 3 or claim 4, wherein the amine is selected from 6. A compound of formula II or a salt or isomer thereof, wherein Each n is independently an integer from 0 to 10; Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each X is independently selected from CH or N; Each Y is independently selected from CH2, NH, O or S; and Each Z is independently selected from CH2, NH, O or S.

7. The compound of claim 6, wherein the compound is:

8. A method for synthesizing a compound as claimed in any one of claims 6 to 7, comprising reacting an α,β-unsaturated Michael acceptor with an amine:

9. The method of claim 8, wherein the α,β-unsaturated Michael acceptor is 10. The method of claim 8 or claim 9, wherein the amine is selected from 11. A compound of formula III or a salt or isomer thereof, wherein Each n is independently an integer from 0 to 10; Each R 1 , R 1’ , R 2 and R 3 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 9 Independently selected from Each R 10 , R 10’ , R 10” , R 11 , R 11’ , R 11” and R 12 Independently selected from H, C1-C24 alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; R 3” , R 4” and R 5” Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

12. The compound of claim 11, wherein the compound is: or a salt or isomer thereof.

13. A method for synthesizing a compound as claimed in any one of claims 11 to 12, comprising carrying out the following reaction:

14. The method of claim 13, wherein the boric acid is selected from:

15. The method of claim 13 or claim 14, wherein the amine is selected from 16. The method of any one of claims 13 to 15, wherein the aldehyde or ketone is selected from:

17. A compound of formula IV or a salt or isomer thereof, wherein Each R 1 , R 2 , R 3 , R 4 and R 5 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 6 , R 7 , R 8 and R 9 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each X is independently selected from CH, N; Each Y is independently selected from CH2, NH, O or S; Each Z is independently selected from CH or N.

18. The compound of claim 17, wherein the compound is: or a salt or isomer thereof.

19. A method for synthesizing a compound as claimed in any one of claims 17 to 18, comprising carrying out the following reaction:

20. The method of claim 19, wherein the aldehyde or ketone is selected from:

21. The method of claim 19 or claim 20, wherein the amine is selected from:

22. The method of any one of claims 19 to 21, wherein the acid is selected from:

23. The method of any one of claims 19 to 22, wherein the isocyanide is selected from:

24. A compound of formula V or a salt or isomer thereof, wherein Each R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3’ , R 4’ and R 5’ Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

25. The compound of claim 24, wherein the compound is:

26. A method for synthesizing a compound or a salt thereof as claimed in any one of claims 24 to 25, comprising performing the following reaction:

27. The method of claim 26, wherein the ketone or aldehyde is selected from 28. The method of claim 26 or claim 27, wherein the amine is selected from:

29. The method of any one of claims 26 to 28, wherein the boc amino acid is selected from:

30. A compound of formula VI or a salt or isomer thereof, wherein Each n is independently an integer from 0 to 10; Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each Y is independently selected from CH or N; Each Z is independently selected from CH2, NH, O or S.

31. The compound of claim 30, wherein the compound is: or a salt or isomer thereof.

32. A method for synthesizing a compound as described in any one of claims 30 to 31, comprising performing the following reaction: Wherein X is selected from: F, Cl, Br, Ts, wherein each n is independently an integer from 0 to 10, Each Y is independently selected from CH or N, Each Z is independently selected from CH2, NH, O or S, Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

33. The method of claim 32, wherein the amine is selected from 34. A method for synthesizing a compound as described in any one of claims 32 to 33, comprising performing the following reaction: Wherein X is selected from: F, Cl, Br, Ts, wherein each n is independently an integer from 0 to 10, Each Y is independently selected from CH or N, Each Z is independently selected from CH2, NH, O or S, Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

35. A compound of formula VII or a salt or isomer thereof, wherein Each m and n is independently an integer from 0 to 10; Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each X is independently selected from CH2, NH, O or S; Each Y is independently selected from CH, N; Each Z is independently selected from CH2, NH, O or S; and Each E is independently selected from CH2, NH, O or S.

36. The compound of claim 35, wherein the compound is: or a salt or isomer thereof.

37. A method for synthesizing a compound as described in any one of claims 35 to 36, comprising performing the following reaction:

38. The method of claim 37, wherein the amine is selected from 39. A compound of formula I-1 or a salt or isomer thereof, wherein Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, Each a is independently an integer selected from 0-24; Each b and c is independently an integer selected from 0-24; Each R 3 , R 4 and R 5 independently selected from C1-C24 alkyl, C1-C24 alkenyl, C1-C24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

40. A method for synthesizing the compound of claim 39, comprising performing the following reaction:

41. A compound of formula II-1 or a salt or isomer thereof, wherein Each R 1 and R 2 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 independently selected from C1-C24 alkyl, C1-C24 alkenyl, C1-C24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

42. A method for synthesizing a compound as claimed in claim 41, comprising carrying out the following reaction:

43. A compound of formula III-1 or a salt or isomer thereof, wherein Each R 1 , R 1’ , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; R 3” , R 4” and R 5” Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Or each of R 4 , R 5 , R 6 , R 7 and R 8 Independently for: Where R 1”’ , R 1”” , R 2”’ and R 3”’ Independently selected from H, C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, Each a, b and c is independently an integer selected from 0-24; Each X is independently CH, N, O or S; Each Y is independently CH, N, O or S Each Z is independently CH, N, O or S Each F is independently CH, N, O or S, and Each E is independently CH, N, O or S.

44. A method for synthesizing a compound as claimed in claim 43, comprising carrying out the following reaction:

45. A compound of formula VI-1 or a salt or isomer thereof, wherein Each n is independently an integer from 0 to 10; Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

46. ​​A method for synthesizing the compound as claimed in claim 45, comprising carrying out the following reaction:

47. A compound of formula VII-1 Each R 1 and R 2 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer from 0 to 24; Each R 3 , R 4 and R 5 Independently selected from C1-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclic group, substituted heterocyclic group, substituted aryl, substituted heteroaryl, a, b and c are each independently an integer of 0-24.

48. A method for synthesizing a compound as claimed in claim 47, comprising carrying out the following reaction:

49. A nanoparticle composition comprising an ionizable lipid component comprising a compound as described in any one or a combination of claims 1, 2, 6, 7, 11, 12, 17, 18, 24, 25, 30, 31, 35, 36, 39, 41, 43, 45 and 47.

50. The nanoparticle composition of claim 49, further comprising a bioactive agent.

51. The nanoparticle composition of claim 49 or claim 50, further comprising one or more phospholipids.

52. The nanoparticle composition of claim 51, wherein the one or more phospholipids are selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC ... Palmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) and sphingomyelin.

53. The nanoparticle composition of any one of claims 49 to 52, further comprising one or more steroids.

54. The nanoparticle composition of claim 53, wherein the one or more steroids are selected from the group consisting of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, lycopene, ursolic acid, alpha-tocopherol, and mixtures thereof.

55. The nanoparticle composition of any one of claims 49 to 54, further comprising one or more PEG lipids.

56. The nanoparticle composition of claim 55, wherein the one or more PEG lipids are selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

57. The nanoparticle composition of any one of claims 49 to 56, further comprising a cationic lipid and / or an ionizable lipid in addition to the ionizable lipid comprising the compound described in any one or combination of claims 1, 2, 6, 7, 11, 12, 17, 18, 24, 25, 30, 31, 35, 36, 39, 41, 43, 45, and 47.

58. A pharmaceutical composition comprising the nanoparticle composition of any one of claims 49 to 57 and a pharmaceutically acceptable carrier.

59. A method of delivering a bioactive agent to a cell, the method comprising administering to a subject a nanoparticle composition as described in any one of claims 49 to 57, the administration comprising contacting the cell with the nanoparticle composition, thereby delivering the bioactive agent to the cell.

60. A method of delivering a bioactive agent to a subject, the method comprising administering to the subject the pharmaceutical composition of claim 58.

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