Modular lipid compounds and two-to-three-component lipid nanoparticle compositions

Abstract

Novel delivery nanoparticles consisting of a two-component or three-component lipid compound. Compositions comprising such lipid compounds and related methods of use are disclosed. Nanoparticle compositions comprise at least one novel modular lipid and additional lipids such as ionizable lipids and phospholipids. Nanoparticle compositions further comprising a bioactive agent, such as siRNA or mRNA, can be used to deliver the bioactive agent to a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of priority to U.S. Provisional Application No. 63 / 477,252, filed on December 27, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure provides novel lipids, lipid nanoparticle compositions, and related methods of synthesis and use. Background Art

[0004] The use of biologically active substances, such as small molecule drugs, proteins, and nucleic acids, including DNA and RNA, to treat disease represents a promising alternative to conventional vaccination or disease treatment approaches due to its potential for greater efficacy, rapid development, low-cost manufacturing, and safe administration.

[0005] The latest advances in this field use bipolar lipids (such as phospholipids or amine lipids) assembled into monolayers or spheres to deliver drugs (such as therapeutic proteins, compounds or nucleic acids). Over the past two decades, many bipolar lipids and helper lipids (called lipid nanoparticles (LNPs)) have been developed for the delivery of nucleic acids, especially ribonucleic acids (RNA). Currently, various RNA vaccine platforms for infectious diseases, genetic diseases and several types of cancer have shown encouraging results in animal models and humans. It is worth noting that such platforms, including mRNA COVID vaccines, require the use of a four-component LNP delivery system to achieve preventive effects in humans.

[0006] However, although the latest progress in this field has solved some problems such as relative instability and low cell permeability that lead to reduced expression in vivo, the delivery of these bioactive substances still faces safety issues. For example, safety issues using conventional four-component lipid nanoparticle (LNP) delivery systems include local and systemic inflammation, biodistribution and persistence of expressed immunogens, stimulation of autoreactive antibodies, and potential toxic effects of delivery system components. In addition, using four-component LNPs for delivering therapeutic mRNA to achieve antigen expression sufficient for protection or immunomodulation remains a medical challenge and an unmet need.

[0007] Therefore, there is a need to develop compounds, compositions and methods that improve stability, promote internalization, increase target affinity and reduce safety concerns of nucleic acid therapeutics. Summary of the Invention

[0008] The present disclosure provides novel lipids, LNP compositions, methods of synthesis, and methods of using the novel lipids and compositions of the present disclosure.

[0009] In one aspect, the present disclosure provides a nanoparticle composition comprising an ionizable lipid component and a glycolipid component.

[0010] In one aspect, the present disclosure provides a two-component LNP composition, wherein the two lipid components are a modular lipid component and a stabilizer lipid component, wherein the stabilizer lipid component is a glycolipid compound or a PEG lipid, wherein the modular lipid component comprises from about 0.5 mol% to about 99.5 mol% of the total lipids present in the particle, or any subrange thereof, e.g., 0.5-9.5, 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-99, 2-20, 21-40, 41-60, 61-80, 2-98, 5-25, 26-45, 46-65, 70-95, 3-30, 31-60, 61-90, 10-80, 15- %, or any range or value within any range within the recited ranges, and wherein the stabilizer lipid component comprises from about 0.5 mol% to about 99.5 mol% or any subrange thereof, such as 0.5-9.5, 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-99, 2-20, 21-40, 41-60, 61-80, 2-98, 5-25, 26-45, 46-65, 70-95, 3-30, 31-60, 61-90, 10-80, 15-85, 55.5-99.5 mol%, or any range or value within any range within the recited ranges.

[0011] In one aspect, the present disclosure provides a three-component LNP composition, wherein the three lipid components are a modular lipid or ionizable lipid component, a stabilizer lipid component, and a phospholipid component, wherein the stabilizer lipid component is a glycolipid compound or a PEG lipid, wherein the phospholipid component comprises about 5 to 60 mol% or any subrange thereof, e.g., 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 45 to 55, 50 to 60, 5 to 25, 15 to 35, 25 to 45, 35 to 55, or 10 to 50 mol% of the total lipids present in the particle, wherein the stabilizer lipid component comprises about 0.2 to 80 mol% or any subrange thereof, e.g., 0.2 to 5.5, 5.5 to 10.75, 5.5 to 15. to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 38 to 50, 42 to 55, 45 to 60, 48 to 65, 50 to 70, 55 to 75, 60 to 80, or 70 to 80 mol%, wherein the modular lipid or ionizable lipid component constitutes about 5 to 80 mol% of the total lipid present in the particle, or any subrange thereof, For example, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 45 to 55, 50 to 60, 55 to 65, 60 to 70, 65 to 75, 70 to 80, 5 to 25, 15 to 35, 25 to 45, 35 to 55, 10 to 50, 20 to 60, 30 to 70 or 40 to 80 mol %.

[0012] In one aspect, the present disclosure provides a three-component LNP composition, wherein the three lipid components are a modular lipid component, an ionizable lipid component, and a phospholipid component, wherein the stabilizer lipid component is a glycolipid compound or a PEG lipid, wherein the phospholipid component comprises from about 15 mol% to about 55 mol% of the total lipids present in the particle, or any subrange thereof, e.g., 15 to 20, 18 to 25, 20 to 30, 22 to 35, 25 to 40, 28 to 45, 30 to 50, 33 to 55, 35 to 40, 40 to 45, 45 to 50, or 50 to 55 mol%, wherein the modular lipid component comprises from about 25 mol% to about 30 mol% of the total lipids present in the particle. mol % or any subrange thereof, e.g., 3 to 10, 5 to 15, 8 to 20, 12 to 25, 15 to 30, 20 to 35, 25 to 40, 30 to 45, or 35 to 50 mol % wherein the ionizable lipid component comprises about 30 mol % to about 80 mol % of the total lipid present in the particle, or any subrange thereof, e.g., 30 to 40, 35 to 45, 38 to 50, 42 to 55, 45 to 60, 48 to 65, 50 to 70, 55 to 75, 60 to 80, 32 to 42, 38 to 48, 40 to 50, 45 to 55, 48 to 58, 55 to 65, 58 to 68, 62 to 72, or 70 to 80 mol %.

[0013] In one aspect, the present disclosure provides modular LNP (mLNP) compositions comprising a compound of formula (I):

[0014] in

[0015] an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any subrange selected from the range of 0 to 10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0016] R 1 and R 2 Each of which is 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,

[0017]

[0018] a, b and c are each independently an integer from 1 to 24;

[0019] Each X is independently selected from CH or N;

[0020] Each Y is independently selected from CH2, NH, O or S; and

[0021] Each Z is independently selected from CH2, NH, O or S.

[0022] In certain aspects, compounds of Formula II may include, for example, the following compounds:

[0023]

[0024] or a salt or isomer thereof.

[0025] In one aspect, the present disclosure provides mLNP compositions comprising a compound of formula (II):

[0026]

[0027] or a salt or isomer thereof, wherein

[0028] R 1 、R 4 and R 5 Each of which is independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0029]

[0030] R 2 and R 3 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0031]

[0032]

[0033] a, b and c are each independently an integer from 0 to 24;

[0034] R 6 、R 7 、R 8 、R9 Each of the following is 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,

[0035]

[0036] a, b and c are each independently an integer from 0 to 24;

[0037] Each X is independently selected from CH, N;

[0038] Each Y is independently selected from CH2, NH, O or S; and

[0039] Each Z is independently selected from CH or N.

[0040] In certain aspects, compounds of Formula II may include, for example, the following compounds:

[0041]

[0042]

[0043] In one aspect, the present disclosure provides novel glycolipids.

[0044] In one aspect, the present disclosure provides mLNP compositions comprising a compound of formula (III):

[0045]

[0046] or a salt or isomer thereof, wherein

[0047] R 1 、R 2 、R 3 、R 4 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0048]

[0049] a, b and c are each independently an integer from 0 to 24;

[0050] R 6 、R 7 、R 8 、R9 Each of the following is 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,

[0051]

[0052] a, b and c are each independently an integer from 0 to 24;

[0053] Each X is independently selected from CH, N;

[0054] Each Y is independently selected from CH2, NH, O or S;

[0055] Each Z is independently selected from CH or N;

[0056] And wherein the sugar is selected from monosaccharides, disaccharides, oligosaccharides and polysaccharides.

[0057] In certain aspects, compounds of Formula III may include, for example, the following compounds:

[0058]

[0059]

[0060]

[0061] In one aspect, the present disclosure provides compounds of formula (IV):

[0062]

[0063] or a salt or isomer thereof, wherein

[0064] R 1 、R 2 、R 3 、R 4 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0065]

[0066] a, b and c are each independently an integer from 0 to 24;

[0067] R 6 、R7 、R 8 、R 9 Each of the following is 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,

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

[0069] Each X is independently selected from CH, N;

[0070] Each Y is independently selected from CH2, NH, O or S;

[0071] Each Z is independently selected from CH or N; and

[0072] Each sugar is independently selected from monosaccharides, disaccharides, oligosaccharides and polysaccharides.

[0073] In certain aspects, compounds of Formula IV may include, for example, the following compounds:

[0074]

[0075] Monosaccharides that can be used in the compositions of the present disclosure include trioses (ketotriose, aldotetraose), tetroses (ketotetraose, aldotetraose), pentoses (ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose), hexoses (psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, fucose, fucose, rhamnose, heptose, octose, nonose, gulose, idose, galactose, talose), and sedoheptulose.

[0076] Disaccharides include sucrose, lactose, maltose, trehalose, turanose, and cellobiose.

[0077] Oligosaccharides include raffinose, melezitose, maltotriose, acarbose, stachyose, fructooligosaccharides, galacto-oligosaccharides, and mannose oligosaccharides.

[0078] Polysaccharides include polyglycitol, n-acetylglucosamine, and chitin.

[0079] In one aspect, the present disclosure provides modular lipids comprising two or more functional groups and at least one linker between at least two functional groups. In some aspects, the functional group is selected from a cationic or ionizable lipid, a phospholipid, a glycolipid, a lipid raft, a stabilizer lipid, a bipolar compound with a hydrophobic and hydrophilic end, a sterically hindered group, a sterol group, a folic acid group, an N-acetylgalactosamine (GalNAc) group, an oligopeptide group, an oligonucleotide group, or a combination thereof.

[0080] In one aspect, the present disclosure provides a method for synthesizing a modular lipid comprising a cationic ionizable group and a sterol derivative or a sterol group-containing lipid, the method comprising performing a four-component reaction of an acid compound, an amine compound, an aldehyde / ketone compound, and an isocyanate compound as follows:

[0081]

[0082] where R 1 、R 4 and R 5 Each of which is independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0083]

[0084]

[0085] where R 2 and R 3 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0086]

[0087]

[0088] Each R 6 、R 7 、R 8 and R 9independently 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,

[0089] a, b, c and d are each independently an integer from 0 to 24;

[0090] Each X is independently selected from CH, N;

[0091] Each Y is independently selected from CH2, NH, O or S; and

[0092] Each Z is independently selected from CH or N.

[0093] In one aspect, the present disclosure provides modular lipids of Formula V, VI, VII, VIII, IX or X:

[0094]

[0095] or a salt or isomer thereof, wherein

[0096] Each R 1 、R 4 and R 10 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0097]

[0098]

[0099] Each R 2 、R 2' 、R 3 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0100]

[0101] Each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0102]

[0103] 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,

[0104]

[0105] a, b, c and d are each independently an integer from 0 to 24;

[0106] Each E is independently selected from CH2, NH, O or S;

[0107] Each X is independently selected from CH, N;

[0108] Each Y is independently selected from CH2, NH, O or S; and

[0109] Each Z is independently selected from CH or N.

[0110] In one aspect, the present disclosure provides methods for synthesizing modular lipids of Formulas V, VI, and VII comprising performing the following four-component reaction:

[0111]

[0112]

[0113] or a salt or isomer thereof, wherein

[0114] Each R 1 and R 4 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0115]

[0116] Each 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0117]

[0118]

[0119] Each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0120]

[0121] R 6 、R 7 、R 8 and R 9 Each is 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,

[0122]

[0123] a, b, c and d are each independently an integer from 0 to 24;

[0124] Each E is independently selected from CH2, NH, O or S;

[0125] Each X is independently selected from CH, N;

[0126] Each Y is independently selected from CH2, NH, O or S; and

[0127] Each Z is independently selected from CH or N.

[0128] In one aspect, the present disclosure provides methods for synthesizing modular lipids of Formulas VIII, IX, and X, comprising performing the following four-component reactions, respectively:

[0129]

[0130] or a salt or isomer thereof, wherein

[0131] Each R 1 、R 4 and R 10 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0132]

[0133]

[0134] Each R 2 、R 2' 、R 3 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0135]

[0136] Each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0137]

[0138] R 6 、R 7 、R 8 and R 9 Each is 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,

[0139]

[0140]

[0141] a, b, c and d are each independently an integer from 0 to 24;

[0142] Each E is independently selected from CH2, NH, O or S;

[0143] Each X is independently selected from CH, N;

[0144] Each Y is independently selected from CH2, NH, O or S; and each Z is independently selected from CH or N.

[0145] A modular lipid wherein the lipid is:

[0146]

[0147]

[0148]

[0149]

[0150] In one aspect, the present disclosure provides a kind of nanoparticle composition, this nanoparticle composition comprises modular lipid component, stabilizer lipid component and / or phospholipid component, optionally wherein nanoparticle composition is two-component composition or three-component composition.In some respects, stabilizer lipid component is glycolipid compound or PEG lipid of the present disclosure.In some respects, two-component LNP contains modular lipid and PEG lipid, and this PEG lipid can be glycolipid (comprising PEG group).

[0151] In one aspect, the present disclosure provides a nanoparticle composition comprising a modular lipid component, a phospholipid component, and a glycolipid component.

[0152] In one aspect, the present disclosure provides two-component or three-component nanoparticle compositions and uses thereof, which include a novel sugar LNP (mLNP) composition and a nanoparticle composition comprising at least one modular lipid, and provide better nucleic acid transfection efficiency compared to traditional four-component LNP systems.

[0153] In one aspect, the present disclosure provides nanoparticle compositions and uses thereof or methods of delivering a payload (e.g., a bioactive agent nucleic acid) to a cell (e.g., a cell) by administering a nanoparticle composition comprising at least one glycolipid or modular lipid of the present disclosure.

[0154] In one aspect, the present disclosure provides a nanoparticle composition and its use or a method of delivering a payload (e.g., a bioactive agent nucleic acid) to a cell (e.g., a cell) by administering the nanoparticle composition, wherein the nanoparticle composition comprises at least one glycolipid, a modular lipid of the present disclosure, and at least one ionizable lipid.

[0155] In one aspect, the present disclosure provides a nanoparticle composition and its use or a method of delivering a payload (e.g., a bioactive agent nucleic acid) to a cell (e.g., a cell) by administering a nanoparticle composition, wherein the nanoparticle composition comprises at least one glycolipid, or a modular lipid of the present disclosure, at least one ionizable lipid, and at least one phospholipid.

[0156] In one aspect, the present disclosure provides uses and methods for delivering at least one payload to a subject in need thereof or to a biological sample using a two-component or three-component composition. In some aspects, the uses or methods of the present disclosure are prophylactic, diagnostic, or therapeutic uses or methods. In some aspects, when administered to a subject in a therapeutically effective amount, a therapeutic benefit is provided to the subject.

[0157] In one aspect, the present disclosure provides a two-component composition comprising two components in the following relative mole percentages: 1 to 99 mole % of a glycolipid; and 1 to 99 mole % of an ionizable lipid.

[0158] In one aspect, the present disclosure provides a nanoparticle composition comprising a modular lipid component, a phospholipid component, and a glycolipid component. In some aspects, the modular lipid component comprises a linker, a cationically ionizable group, and a lipid raft group.

[0159] In one aspect, the present disclosure provides a three-component composition comprising the following relative mole percentages of three components: 0.2 to 80 mole % of glycolipids; 5 to 80 mole % of ionizable lipids; and 5 to 60 mole % of phospholipids.

[0160] In one aspect, the payload is a conventional mRNA or self-amplification mRNA encoding a target polypeptide, whereby the mRNA can be translated in the cell to produce a polypeptide. In another aspect, the payload is a DNA encoding a target gene, whereby the cell becomes capable of expressing the introduced gene. In another aspect, the payload is an siRNA or antisense RNA capable of regulating the expression of the target gene, whereby the cell reduces the expression of the target gene. In one aspect, at least one lipid in the ionizable lipid is an ionizable lipid of the present disclosure, such as a compound of formula I, including any species and derivatives thereof. In one aspect, at least one lipid in the glycolipid is a compound of formula III or IV, including any species and derivatives thereof. In one aspect, the nanoparticle composition comprises at least two different glycolipids. In one aspect, the nanoparticle composition comprises both glycolipids of formula III and IV. In one aspect, the nanoparticle composition comprises modular lipids of the present disclosure.

[0161] In another aspect, the present disclosure provides a nanoparticle composition comprising (i) at least one ionizable lipid, (ii) a phospholipid moiety, (iii) a glycolipid, (iv) a payload, or any combination thereof. In one aspect, the phospholipid moiety 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-henicosanoyl-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-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diamidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In one aspect, at least one of the ionizable lipids is a compound of Formula I, including species thereof such as Ia, Ib, etc. In one aspect, at least one of the ionizable lipids is a compound of Formula II, including species thereof such as IIa, IIb, IIc, IId, IIe, etc. In one aspect, at least one of the glycolipids is a compound of Formula III or IV, including species thereof. In one aspect, the nanoparticle composition comprises at least two different glycolipids. In one aspect, the nanoparticle composition comprises both glycolipids of Formula III or IV, including species thereof.

[0162] In another aspect, the present disclosure provides methods of synthesizing compounds of Formula (I), (II), (III) and / or (IV) (including species thereof such as Ia, Ib, IIa, IIb, IIc, IId, IIe, etc.).

[0163] In another aspect, the ionizable lipid is:

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210] In some aspects, the nanoparticle compositions of the present invention are used together with another therapeutic compound that is separate from the nanoparticles to treat the same indication in a subject. In certain instances, the LNP and the therapeutic agent are delivered separately or together. When delivered together, they may or may not be in the same formulation, and they may or may not be delivered by the same route.

[0211] In another aspect, the present disclosure provides methods of preparing nanoparticle compositions comprising lipid components comprising a compound of Formula (I), (II), (III), and / or (IV). BRIEF DESCRIPTION OF THE DRAWINGS

[0212] Figure 1 Shown is a heat map of a high-throughput screen of the percentage transfection of GFP self-amplifying mRNA (GFP-saRNA) in 293T cells in a 96-well plate using mLNPs formulated with glycolipids and ionizable lipids of formula (Ib) for delivery of GFP-saRNA compared to the percentage transfection of GFP-saRNA delivered using a 4-component LNP comprising an ionizable lipid of formula (Ib), a modular lipid, a phospholipid, and a PEG-lipid. The glycolipid of formula (III) (a) corresponds to position E2 of the 96-well plate, the glycolipid of formula (III) (b) corresponds to position F11, and the glycolipid of formula (III) (c) corresponds to position C6.

[0213] Figure 2 Shown is the in vitro expression of GFP-saRNA in 293T cells transfected using mLNPs formulated with glycolipids and ionizable lipids of Formula (Ib), including glycolipids of Formula (III) (a) (also known as P1_E2), (III) (b) (also known as P1_F11), and (III) (c) (also known as P1_C6).

[0214] Figure 3 Shown is a heat map of a high-throughput screen of the percentage transfection of GFP self-amplifying mRNA (GFP-saRNA) in 293T cells in a 96-well plate using mLNPs formulated with glycolipids and ionizable lipids of formula (IIe) for delivery of GFP-saRNA compared to the percentage transfection of GFP-saRNA delivered using a 4-component LNP comprising an ionizable lipid of formula (IIe), a structural lipid, a phospholipid, and a PEG-lipid. The glycolipid of formula (III) (d) corresponds to position E8 of the 96-well plate, the glycolipid of formula (III) (e) corresponds to position D8, and the glycolipid of formula (III) (f) corresponds to position D9.

[0215] Figure 4Shown is the in vitro expression of GFP-saRNA in 293T cells transfected using mLNPs formulated with glycolipids and ionizable lipids of formula (IIe), including glycolipids of formula (III)(d) (also known as P1_E8), (III)(e) (also known as P1_D8), and (III)(f) (also known as P1_D9).

[0216] Figure 5 A schematic diagram of a four-component nanoparticle containing a nucleic acid payload comprising a bipolar compound, a cationic ionizable lipid, a lipid raft, and a stabilizer is shown. Bipolar compounds (such as phospholipids or glycolipids) help form nanoparticles in water or under physiological conditions, thereby improving the encapsulation of the payload and facilitating cellular delivery. Cationic ionizable lipids promote nucleic acid encapsulation and mediate endosomal membrane disruption to enable nucleic acid release into the cytosol. Lipid rafts (such as cholesterol or squalene) mediate nanoparticle formation and regulate membrane fluidity. Stabilizers (such as PEG-lipids) prevent particle aggregation; improve particle stability during preparation and storage; and regulate immune responses to nanoparticles. In addition, some nanoparticles have functional ligands on the surface of the nanoparticle.

[0217] Figure 6 Shown is a schematic diagram of a nanoparticle comprising a bipolar compound; modular lipids comprising a cationically ionizable group and a lipid raft group; a sugar stabilizer, such as a glycolipid; a nucleic acid payload; and a functional ligand on the nanoparticle surface.

[0218] Figure 7 A schematic diagram of a modular lipid comprising a linker covalently linked to five functional groups is shown. Functional groups include, but are not limited to, lipid groups, cationically ionizable groups, sterically hindered groups, lipid raft groups, sterol groups, sugar groups, folate groups, GalNAc groups, oligopeptide groups, and oligonucleotide groups.

[0219] Figure 8 The luminous intensity of the LNP prepared using MC3 and the modular lipid of the present disclosure comprising a cation ionizable group and a lipid raft group is shown in the HEK293 cells shown in 96 well plates (upper figure) or in the graph (lower figure). Modular lipid (ionizable group and sterol derivative group), DOPE, cholesterol and DMG-PEG2000 (50 / 38.5 / 10 / / 1.5 mol ratio) are used to prepare the LNP of the self-amplification mRNA (SamRNA-LUC) of the encapsulated coding reporter gene firefly luciferase. HEK293 cells are processed with the SamRNA-LUC of 100ng for 24 hours. Every kind of LNP preparation is tested in triplicate, and is expressed as mean ± SD.

[0220] Figure 9 Figure 2 shows GFP expression in HEK293T cells treated with LNPs of the present disclosure for 24 hours. Cells were treated with LNPs encapsulating sAMRNA-GFP formulated with modular lipids of the present disclosure containing cationic ionizable groups, DOPE, and DMG-PEG2000. Four-component LNPs containing SM102 were used as a control group. Each LNP formulation was tested in triplicate and expressed as mean ± SD.

[0221] Figure 10 The luminous intensity of the LNP prepared using the modular lipids of the present disclosure comprising a sterol derivative group, a sugar group and a PEG group in the HEK293T cells shown in the 96-well plate (upper figure) or the curve graph (control group, lower figure) is shown. Cationic ionizable lipids (P54B6, P38D8 or E6), DOPE and modular lipids (35 / 40 / 15 mol ratio) are used to prepare the LNPs encapsulating SamRNA-LUC. HEK293 cells were processed with 100ng of SamRNA-LUC for 24 hours. Each LNP formulation was tested in triplicate and is expressed as mean ± SD.

[0222] Figure 11 GFP expression in HEK293 cells treated with LNPs of the present disclosure for 24 hours is shown. Three-component LNPs encapsulating SamRNA-GFP were prepared using a cationic ionizable lipid (P38D8), DOPE, and a modular lipid (SP11H3, SP11A6, SP11A11, SP11A12, or SP11H6) (35 / 40 / 15 molar ratio). Four-component LNPs containing P38D8, E6, or MC3 were used as a control group. Each LNP formulation was tested in triplicate and expressed as mean ± SD. 4-Com LNP: four-component LNP. 3-Com LNP: three-component LNP.

[0223] Figure 12 GFP expression in HEK293 cells treated with LNPs of the present disclosure for 24 hours is shown. LNPs encapsulating samRNA-GFP were formulated using modular lipids P161F5, P161F6, or P161F10; DOPE; and DMG-PEG2000 (40 / 10 / 2, molar ratio). 100 ng of RNA was incubated with HEK293 cells at 60-70% confluence in wells of a 96-well plate. Each LNP formulation was tested in triplicate and expressed as mean ± SD.

[0224] Figure 13GFP expression in HEK293T cells is shown. LNPs of the present disclosure were prepared using P38D8, P40D7 or P1D4, DOPE and modular lipid SP1F11 (40 / 60 / 15, molar ratio). LNPs of the present disclosure and a four-component LNP control group (ionizable lipid / DOPE / cholesterol / DMP-PEG2000, 30 / 15 / 50 / 1.5 molar ratio) encapsulating SamRNA-GFP were applied to HEK293T cells for 24 hours. 100 ng mRNA was incubated with HEK293 cells at 60-70% confluence into wells of 96-well plates. Each LNP formulation was tested in triplicate and expressed as mean ± SD.

[0225] Figure 14 GFP expression in HEK293T cells treated with LNP of the present invention for 24 hours is shown. Ionizable lipids P54B6, SM102, MC3 or ALC0315 are used; DOPE; and modular lipids SP2B12, SP11A6 or SP11A12 (35 / 40 / 20, molar ratio) are used to prepare LNPs encapsulating SamRNA-GFP. Four-component LNPs containing P54B6, SM102, MC3 or ALC0315 are used as a control group. The preparation containing 100ng mRNA is incubated with cells for 24 hours. Each LNP preparation is tested in triplicate and expressed as mean ± SD. From left to right, four bars in each group show 4-component LNP, 3-component LNP_SP2B12, 3-component LNP_SP11A6 and 3-component LNP_SP11A12. 4-Com LNP: four-component LNP. 3-Com LNP: three-component LNP.

[0226] Figures 15A-15B GFP expression in HEK293 cells treated with LNPs of the present disclosure for 24 hours is shown. LNPs encapsulating samRNA-GFP or modified mRNA-GFP were prepared using P287A12, DOPE, DMG-PEG2000 (40 / 10 / 2, molar ratio) or P287A12 and DMG-PEG2000 (40 / 2, molar ratio) and were prepared in acidic buffer (citrate buffer, pH 4.5) and / or neutral buffer (PBS, pH 7.4). Figure 15A ) or 100 ng of modified mRNA-GFP ( Figure 15B ) were incubated with HEK293 cells at 60-70% confluence in 96-well plates. Each LNP formulation was tested in triplicate and expressed as mean ± SD. 3-Com LNP: three-component LNP. 2-Com LNP: two-component LNP.

[0227] Figure 16 The in vivo bioluminescence intensity of the metastatic sites in mice treated with LNPs of the present invention measured using an IVIS imaging system is shown. LNPs were prepared using ionizable lipids P54B6, DOPE, cholesterol, and DMG-PEG2000 (30 / 15 / 50 / 1.5, molar ratio); or ionizable lipids P54B6, DOPE, and modular lipids SP2B12 (35 / 40 / 20, molar ratio). Four-component LNPs containing MC3 were used as a control group. Each mouse was treated with lug samRNA-LUC. Each LNP formulation was tested in 5 replicates and expressed as mean ± SD. 4-Com LNP: four-component LNP. 3-Com LNP: three-component LNP.

[0228] Figure 17 Shown is the in vivo bioluminescence intensity of the metastatic sites in mice treated with LNPs of the present invention measured using an IVIS imaging system. LNPs were prepared using P161F5, DOPE, DMG-PEG2000 (40 / 10 / 2, molar ratio); or P161F5, DOPE, and SP1E2K (40 / 10 / 2, molar ratio). Four-component LNPs containing MC3 were used as a control group. Each mouse was treated with 1 ug of samRNA-LUC. Each LNP formulation was tested in 5 replicates and expressed as mean ± SD. 4-Com LNP: four-component LNP. 3-Com LNP: three-component LNP.

[0229] Figure 18 Shown is the in vivo bioluminescence intensity of the metastatic sites in mice treated with LNPs of the present invention measured using an IVIS imaging system. LNPs were prepared using P38D8, DOPE, cholesterol, and DMG-PEG2000 (30 / 15 / 50 / 1.5, molar ratio); or P38D8, DOPE, and SP1F11 (40 / 60 / 15, molar ratio). Four-component LNPs containing MC3 were used as a control group. Each mouse was treated with lug SamRNA-LUC. Each LNP formulation was tested in 5 replicates and expressed as mean ± SD. 4-Com LNP: four-component LNP. 3-Com LNP: three-component LNP.

[0230] Figures 19A-19B The nanoparticle size of the LNPs of the present disclosure is shown ( Figure 19A ) and polydispersity index (PDI) ( Figure 19B ). 4-Com LNP: four-component LNP. 3-Com LNP: three-component LNP. DETAILED DESCRIPTION

[0231] The present disclosure relates to novel lipids and lipid nanoparticle compositions comprising at least one novel lipid of the present disclosure. The present disclosure also provides methods for delivering a bioactive agent to a cell, particularly a bioactive agent to an organ, and 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 mRNA with the cell, whereby the mRNA can be translated to produce the target polypeptide. A method for delivering a bioactive agent to a cell or organ can involve administering a nanoparticle composition comprising the bioactive agent to a subject, wherein administration involves contacting the cell or organ with the composition, thereby delivering the bioactive agent to the cell or organ.

[0232] The present disclosure provides novel glycolipids. Viral particles have a lipid envelope in which envelope proteins or lipids are modified with sugars. Disaccharides have been shown to influence the lateral organization of the lipid membrane, as discussed in J. Am. Chem. Soc., 2014, Vol. 136, No. 46: pp. 16167-16175, which is incorporated herein by reference in its entirety. The novel glycolipids mimic the characteristics of viral particles and improve transfection efficiency compared to four-component LNPs.

[0233] 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. One of ordinary skill in the art will recognize that the reaction can be optimized to preferentially obtain one isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

[0234] 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). 1-14 "Alkyl" means an optionally substituted straight or branched chain saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise specified, alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

[0235] 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 "C 2-14"Alkenyl" means an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. An 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 stated, alkenyl groups described herein refer to both unsubstituted alkenyl and substituted alkenyl groups.

[0236] 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 "C 2-14 "Alkynyl" means an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group 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.

[0237] Unless otherwise specified, alkyl, alkenyl, and cyclic groups (eg, carbocyclyl and heterocyclyl) may be optionally substituted.

[0238] About, approximately: As used herein, when applied to one or more target values, the terms "about" and "approximately" refer to values ​​that are approximately the same as the reference value. In certain aspects, unless otherwise specified or otherwise obvious from the context (unless such a number would exceed 100% of the possible value), the terms "about" or "approximately" refer to values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "about" can mean + / - 10% of the stated value.

[0239] 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 different mass numbers due to differences in the number 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.

[0240] 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 share a physical connection. Methods for contacting a cell with an external entity in vivo and in vitro are well known in the field of biology. For example, contacting a nanoparticle composition with a cell disposed in a mammal can be performed by different routes of administration (e.g., intravenous, intranasal, intratracheal, intraperitoneal, intramuscular, intradermal, and subcutaneous) and can involve different amounts of nanoparticle composition. In addition, more than one cell can be contacted by the nanoparticle composition.

[0241] As used herein, the term "delivery" means providing an entity to a target. For example, delivering a bioactive agent to a subject can involve administering a nanoparticle composition comprising the bioactive agent to the subject (e.g., via intravenous, intranasal, intratracheal, intraperitoneal, intramuscular, intradermal, or subcutaneous routes). Administering a nanoparticle composition to a mammal or cell can involve contacting one or more cells with the nanoparticle composition.

[0242] As used herein, "encapsulation efficiency" refers to the amount of bioactive agent that becomes part of a nanoparticle composition relative to the initial total amount of bioactive agent used to prepare the nanoparticle composition. For example, if the total amount of bioactive agent initially provided to the composition is 100 mg, of which 97 mg of bioactive agent is encapsulated in the nanoparticle composition, the encapsulation efficiency can be 97%. As used herein, "encapsulation" can refer to completely, substantially, or partially enclosing, confining, surrounding, or enclosing.

[0243] 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.

[0244] 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 and stereoisomeric mixtures, such as racemates. Enantiomers and stereoisomeric mixtures of compounds and methods of resolving them into their component enantiomers or stereoisomers are well known.

[0245] Those skilled in the art will recognize that the reaction can be optimized to preferentially obtain one isomer, or new schemes can be designed to produce a single isomer or an isomer mixture comprising any ratio of multiple isomer ratios. For example, in the case of combining only two isomers, mixtures comprising 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1 or 100:0 isomer ratios are contemplated by the present invention. Those skilled in the art will readily appreciate that similar ratios are contemplated for more complex isomer mixtures. If one isomer is preferred, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC or preparative SFC.

[0246] As used herein, a "lipid component" is a component of a nanoparticle composition that comprises one or more lipids. For example, the lipid component can include one or more cationic / ionizable, pegylated, structural or other lipids, such as phospholipids.

[0247] As used herein, "stabilizer lipid" or "stabilizer" refers to a lipid component that prevents particle aggregation; improves particle stability during preparation and storage; and modulates immune responses to nanoparticles. Stabilizers include glycolipids and PEGylated lipids of the present disclosure. In some aspects, if a modular lipid of the present disclosure contains PEG and / or sugar groups, it can be a stabilizer lipid.

[0248] 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 (e.g., specifically deliver) to a particular area or system of the body.

[0249] As used herein, "modified" means non-natural. For example, RNA can be modified RNA. That is, the RNA can include one or more nucleobases, nucleosides, nucleotides, or linkers that do not occur naturally. "Modified" species can also be referred to herein as "altered" species. A species can be chemically, structurally, or functionally modified or altered. For example, a modified nucleobase species can include one or more substitutions that do not occur naturally.

[0250] As used herein, "naturally occurring" means occurring in nature without human assistance.

[0251] As used herein, "patient" refers to a subject who may seek or need treatment, is in need of treatment, is receiving treatment, will be receiving treatment, or is under the care of a trained professional for a particular disease or condition.

[0252] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with an appropriate benefit / risk ratio.

[0253] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending, complexing, or dissolving the active compound) that is substantially non-toxic and non-inflammatory in 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 water of hydration. 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.

[0254] The composition may also include one or more salts of the compound. 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 modified 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 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, for example, 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. Generally speaking, 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 in an organic solvent or in a mixture of the two; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.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, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is herein incorporated by reference in its entirety.

[0255] As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, typically linked by peptide bonds, which can be produced naturally (eg, isolated or purified) or synthetically.

[0256] As used herein, "RNA" refers to ribonucleic acid, which may be naturally occurring or non-naturally occurring. For example, RNA may comprise modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may comprise 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). Translation of an mRNA encoding a specific polypeptide (e.g., in vivo translation of an mRNA within a cell) can produce the encoded polypeptide. RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, and mixtures thereof.

[0257] As used herein, the term "PEG lipid" or "PEG-modified lipid" or "PEGylated lipid" refers to a lipid modified with polyethylene glycol. The PEG lipid 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, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0258] As used herein, the term "structural lipid" is a steroid, and structural lipids suitable for use in four-component LNPs include cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, α-tocopherol, and mixtures thereof. In some aspects, the two-component or three-component mLNPs of the present disclosure do not contain structural lipids. For example, the two-component or three-component mLNPs of the present disclosure may not contain steroid / structural lipids, such as cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, α-tocopherol, and mixtures thereof.

[0259] As used herein, "size" or "average size" in the context of a nanoparticle composition refers to the average diameter of the nanoparticle composition.

[0260] As used herein, the term "subject" or "patient" refers to any organism that can receive administration of a composition according to the present disclosure, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0261] The term "biologically active agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect. Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0262] As used herein, the term "therapeutically effective amount" or "effective amount" means an amount of an agent (e.g., a nucleic acid, a drug, a composition, a therapeutic agent, a diagnostic agent, a prophylactic agent, etc.) to be delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, ameliorate the symptoms of, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0263] Nanoparticle compositions

[0264] The present disclosure provides novel nanoparticle compositions. As used herein, a "nanoparticle composition" is a composition comprising one or more lipids. The size of the nanoparticle composition is typically micron-sized or smaller and may comprise a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. Nanoparticle compositions comprising a lipid component comprising at least one compound according to formula (I), (II), (III), or (IV) are described herein. In some aspects, the LNPs of the present disclosure do not include lipid compositions having a core-shell structure.

[0265] In one aspect, the present disclosure provides a compound of formula (I) or a salt or isomer thereof:

[0266]

[0267] wherein each n is independently an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any sub-range selected from the range of 0 to 10, such as 2-9, 3-8, 4-7, 1-5, 1-4, 5-9, etc.;

[0268] R 1 and R 2 Each of which is 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,

[0269]

[0270]

[0271] a, b and c are each independently an integer from 1 to 24;

[0272] Each X is independently selected from CH or N;

[0273] Each Y is independently selected from CH2, NH, O or S; and each Z is independently selected from CH2, NH, O or S.

[0274]

[0275]

[0276] or a salt or isomer thereof.

[0277] Synthesis Scheme 1.

[0278] In one aspect, the present disclosure provides general synthetic routes for synthesizing compounds of Formula I.

[0279]

[0280] or

[0281]

[0282] In one aspect, the present disclosure provides compounds of formula (II):

[0283]

[0284] or a salt or isomer thereof, wherein

[0285] R 1 、R 4 and R 5 Each of which is independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0286]

[0287] R 2 and R 3 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0288]

[0289]

[0290] a, b and c are each independently an integer from 0 to 24;

[0291] R 6 、R 7 、R 8 、R 9 Each of the following is 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,

[0292]

[0293] a, b and c are each independently an integer from 0 to 24;

[0294] Each X is independently selected from CH, N;

[0295] Each Y is independently selected from CH2, NH, O or S;

[0296] Each Z is independently selected from CH or N;

[0297] In certain aspects, compounds of Formula II may include, for example, the following compounds:

[0298]

[0299]

[0300] Synthesis Scheme 2.

[0301] General synthetic routes for the synthesis of compounds of formula II.

[0302]

[0303] In one aspect, the present disclosure provides compounds of formula (III):

[0304]

[0305] or a salt or isomer thereof, wherein

[0306] R 1 、R 2 、R 3 and R 4 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0307]

[0308] a, b and c are each independently an integer from 0 to 24;

[0309] R 6 、R 7 、R 8 and R 9 Each is 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,

[0310]

[0311] a, b and c are each independently an integer from 0 to 24;

[0312] Each X is independently selected from CH, N;

[0313] Each Y is independently selected from CH2, NH, O or S;

[0314] Each Z is independently selected from CH or N;

[0315] And wherein the sugar is selected from monosaccharides, disaccharides, oligosaccharides and polysaccharides.

[0316] In certain aspects, compounds of Formula III may include, for example, the following compounds:

[0317]

[0318]

[0319]

[0320] Synthesis Scheme 3.

[0321] In one aspect, the present disclosure provides general synthetic routes for synthesizing compounds of Formula III.

[0322]

[0323] In one aspect, is one of the following:

[0324]

[0325] In one aspect, is one of the following:

[0326]

[0327]

[0328] In one aspect, is one of the following:

[0329]

[0330]

[0331] In one aspect, is one of the following:

[0332]

[0333] In one aspect, the present disclosure provides compounds of formula (IV):

[0334]

[0335] or a salt or isomer thereof, wherein

[0336] R 1 、R2 、R 3 and R 4 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0337]

[0338] a, b and c are each independently an integer from 0 to 24;

[0339] R 6 、R 7 、R 8 and R 9 Each of the following is 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,

[0340]

[0341] a, b and c are each independently an integer from 0 to 24;

[0342] Each X is independently selected from CH, N;

[0343] Each Y is independently selected from CH2, NH, O or S;

[0344] Each Z is independently selected from CH or N;

[0345] Each sugar is independently selected from monosaccharides, disaccharides, oligosaccharides and polysaccharides.

[0346] In certain aspects, compounds of Formula IV may include, for example, the following compounds:

[0347]

[0348] Synthesis Scheme 4.

[0349] In one aspect, the present disclosure provides general synthetic routes for synthesizing compounds of Formula IV.

[0350]

[0351] In one aspect, is one of the following:

[0352]

[0353]

[0354] In one aspect, is one of the following:

[0355]

[0356] In one aspect, is one of the following:

[0357]

[0358]

[0359] In one aspect, is one of the following:

[0360]

[0361] lipid nanoparticles

[0362] In some aspects, the nanoparticle composition has a size of 1 μm or shorter (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 shorter), for example, when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy or another method. The nanoparticle composition includes, for example, lipid nanoparticles (LNPs), liposomes, lipid vesicles and lipid complexes. In some aspects, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain aspects, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayer can be functionalized and / or cross-linked. The lipid bilayer can comprise one or more ligands, proteins or channels.

[0363] The nanoparticle composition comprises a lipid component comprising at least one compound according to Formula (I), (II), (III), or (IV). For example, the lipid component of the nanoparticle composition may comprise one or more of compounds (I)-(IV). The nanoparticle composition may also comprise a variety of other components. For example, in addition to the lipids according to Formula (I), (II), (III), and (IV), the lipid component of the nanoparticle composition may include one or more other lipids.

[0364] Typically, LNPs used as delivery systems in the research and development of new drugs (including FDA-approved mRNA vaccines such as mRNA COVID vaccines and FDA-approved siRNA therapies such as siRNA therapies for treating polyneuropathy in people with hereditary transthyretin-mediated amyloidosis) use a 4-component LNP delivery system. In the four-component LNP delivery system, phospholipids play a role in increasing the efficacy of nucleic acid transfection; cationic / ionizable lipids play a role in stabilizing the nucleic acids within lipid nanoparticles; stabilizing lipids act as "lipid rafts" to stabilize the integrity of LNPs; and PEG-lipids inhibit aggregation and prevent clearance of macrophages, monocytes or other phagocytes in the body. The LNPs disclosed herein are 2-3 component LNPs. 2-component LNPs include ionizable lipids and glycolipids. 3-component LNPs include ionizable lipids, phospholipids and glycolipids.

[0365] In one aspect, the present disclosure provides a nanoparticle composition comprising a modular lipid component, a stabilizer lipid component and / or a phospholipid component, optionally wherein the nanoparticle composition is a two-component composition or a three-component composition. In some aspects, the nanoparticle composition comprises a modular lipid component, a phospholipid component and a glycolipid component. In some aspects, the modular lipid component comprises a linker, a cationic ionizable group and a lipid raft group. In some aspects, the phospholipid component comprises 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-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- 2-henicosanoyl-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-cholestyryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 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-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or a mixture thereof. In some aspects, the nanoparticle composition of the present disclosure further comprises a bioactive agent.

[0366] Cationic / ionizable lipids

[0367] As used herein, the term "ionizable lipid" or "cationic lipid" refers to a lipid that can have a positive charge or a partial positive charge at physiological pH. In addition to the lipids according to formula (I)-(X), the nanoparticle composition may also contain one or more ionizable lipids.

[0368] phospholipids

[0369] 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. Generally speaking, the phospholipids can include a phospholipid portion and one or more fatty acid portions.

[0370] The phospholipids useful in the compositions and methods of the present disclosure may 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 (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-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-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-diole ... Acyl-sn-glycero-3-phosphocholine (DPPC), 1,2-heneicosanoyl-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-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diamidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-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.

[0371] Modular lipids

[0372] In one aspect, the present disclosure provides a kind of modular lipid, this modular lipid comprises at least one joint between two or more functional groups and at least two functional groups.In some aspects, one functional group in described two or more functional groups is lipid group, lipid raft group, cation ionizable group, steric hindered group, sterol group, sugar group, folic acid group, GalNAc group, oligopeptide group or oligonucleotide group.In some aspects, joint is covalently linked to two or more functional groups.In some aspects, described two or more functional groups comprise lipid raft group and cation ionizable group.In one aspect, described two or more functional groups comprise sterol group and cation ionizable group.In one aspect, described two or more functional groups comprise at least one of sugar group and sterol group and PEG group.

[0373] In one aspect, the present disclosure provides a method for synthesizing modular lipids comprising a cationic ionizable group and a sterol group, the method comprising performing a four-component reaction of an acid compound, an amine compound, a ketone compound, and an isocyanate compound as follows:

[0374]

[0375] Each R 1 、R 4 and R 5 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0376]

[0377]

[0378] Each 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0379]

[0380]

[0381]

[0382] 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,

[0383] a, b, c and d are each independently an integer from 0 to 24;

[0384] Each E is independently selected from CH2, NH, O or S;

[0385] Each X is independently selected from CH, N;

[0386] Each Y is independently selected from CH2, NH, O or S; and each Z is independently selected from CH or N.

[0387] In some aspects, the acid is:

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396] In some aspects, the amine is:

[0397]

[0398]

[0399]

[0400] In some aspects, the aldehyde or ketone is:

[0401]

[0402]

[0403]

[0404] In some forms, isocyanate is:

[0405]

[0406]

[0407] In one aspect, the present disclosure provides modular lipids of Formula V, VI, VII, VIII, IX or X:

[0408]

[0409]

[0410] or a salt or isomer thereof, wherein

[0411] Each R 1 、R 4 and R 10 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0412]

[0413]

[0414] Each R 2 、R 2' 、R 3 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0415]

[0416]

[0417] Each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0418]

[0419] R 6 、R 7 、R 8 and R 9 Each is 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,

[0420]

[0421] a, b, c and d are each independently an integer from 0 to 24;

[0422] Each E is independently selected from CH2, NH, O or S;

[0423] Each X is independently selected from CH, N;

[0424] Each Y is independently selected from CH2, NH, O or S;

[0425] Each Z is independently selected from CH or N.

[0426] In one aspect, the present disclosure provides methods for synthesizing modular lipids of Formulas V, VI, and VII comprising performing the following four-component reaction:

[0427]

[0428]

[0429] or a salt or isomer thereof, wherein

[0430] Each R 1 and R 4 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0431]

[0432] Each 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0433]

[0434]

[0435] Each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0436]

[0437]

[0438] R 6 、R 7 、R 8 and R 9 Each is 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,

[0439]

[0440] a, b, c and d are each independently an integer from 0 to 24;

[0441] Each E is independently selected from CH2, NH, O or S;

[0442] Each X is independently selected from CH, N;

[0443] Each Y is independently selected from CH2, NH, O or S;

[0444] Each Z is independently selected from CH or N.

[0445] In one aspect, the present disclosure provides methods for synthesizing modular lipids of Formulas VIII, IX, and X, comprising performing the following four-component reaction:

[0446]

[0447] or a salt or isomer thereof, wherein

[0448] Each R 1 、R 4 and R 10 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG),

[0449]

[0450]

[0451] Each R 2 、R 2' 、R 3 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0452]

[0453]

[0454] Each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and

[0455]

[0456] R 6 、R 7 、R 8 and R 9 Each is 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,

[0457]

[0458]

[0459] a, b, c and d are each independently an integer from 0 to 24;

[0460] Each E is independently selected from CH2, NH, O or S;

[0461] Each X is independently selected from CH, N;

[0462] Each Y is independently selected from CH2, NH, O or S;

[0463] Each Z is independently selected from CH or N.

[0464] In one aspect, the present disclosure provides a modular lipid, wherein the lipid is:

[0465]

[0466]

[0467]

[0468]

[0469]

[0470] Glycolipids

[0471] As used herein, the term "glycolipid" or "glycostabilizer lipid" refers to novel lipids of the present disclosure that mimic the lipid envelope of certain viral particles. Glycolipids are viral envelope lipids with sugar modifications. Glycolipids can be used in the compositions and methods of the present disclosure that include compounds of formula (III) and (IV). Other glycolipids with similar structures to compounds of formula (III) and (IV) are also contemplated, for example, for use in forming Figure 1 、 Figure 2 、 Figure 3 and Figure 4 In one aspect, the glycolipid is a modular lipid. In one aspect, the glycolipid is a bipolar lipid.

[0472] Monosaccharides that can be used in the compositions of the present disclosure include trioses (ketotriose, aldotetraose), tetroses (ketotetraose, aldotetraose), pentoses (ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose), hexoses (psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, fucose, fucose, rhamnose, heptose, octose, nonose, gulose, idose, galactose, talose), and sedoheptulose.

[0473] Disaccharides useful in the compositions of the present disclosure include sucrose, lactose, maltose, trehalose, turanose, cellobiose.

[0474] Oligosaccharides that can be used in the compositions of the present disclosure include raffinose, melezitose, maltotriose, acarbose, stachyose, fructooligosaccharides, galacto-oligosaccharides, and mannose oligosaccharides.

[0475] Polysaccharides that can be used in the compositions of the present disclosure include polyols, n-acetylglucosamine, and chitin.

[0476] adjuvant

[0477] 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.

[0478] bioactive agents

[0479] Nanoparticle compositions can include a payload. The payload can include one or more bioactive agents. The present disclosure features methods for delivering a bioactive agent to a cell or organ and treating a disease or condition in a subject in need thereof, comprising administering to the subject and / or contacting the cell with a nanoparticle composition comprising the bioactive agent.

[0480] Bioactive agents can be substances that, once delivered to a cell or organ, cause a desired change in a cell, organ, or other body tissue or system. Such substances can be used to treat one or more diseases, conditions, or disorders. In some aspects, bioactive agents are small molecule drugs that can be used to treat a specific disease, condition, or disorder.

[0481] 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), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), Antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifen, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, and imaging agents.

[0482] Polynucleotides and nucleic acids

[0483] In some aspects, the bioactive agent delivered in the LNP of the present disclosure is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide" includes any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain in its broadest sense. Exemplary polynucleotides used according to the present disclosure include, but are not limited to, one or more of: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrid, RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, nucleic acid ligands, carriers, etc. In some aspects, the bioactive 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: short polymer, antagomir, antisense, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and mixtures thereof. In some aspects, the RNA is mRNA.

[0484] In some aspects, the bioactive agent is mRNA. The mRNA can encode any target polypeptide, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can have any size and any secondary structure or activity. In some aspects, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0485] In other aspects, the bioactive agent is siRNA. siRNA may be able to selectively regulate the expression of a target gene. For example, siRNA can be selected to knock down or downregulate a gene associated with a specific disease, condition or illness when administered to a subject in need. siRNA may comprise a sequence complementary to the mRNA sequence encoding the target protein. In some aspects, siRNA may be an immunomodulatory siRNA.

[0486] In some aspects, the bioactive agent is a shRNA or a vector or plasmid encoding shRNA. After the appropriate construct is delivered to the nucleus, shRNA can be produced in the target cell. The construct and mechanism associated with shRNA are well known in the relevant art.

[0487] The nucleic acids and polynucleotides that can be used in the present disclosure generally 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) at the 5'-end of the first region, a second flanking region (e.g., a 3'-UTR) 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 includes 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 uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and / or 5-substituted cytidines (e.g., 5-methyl-cytidine).

[0488] preparation

[0489] Nanoparticle compositions can include a lipid component and one or more additional components, such as a bioactive agent. Nanoparticle compositions can be designed for one or more specific applications or targets. The elements of a nanoparticle composition can be selected based on the specific application or target, and / or based on 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 a specific application or target based on, for example, the efficacy and toxicity of a particular combination of elements.

[0490] The lipid component of the nanoparticle composition may include, for example, an ionizable lipid according to formula (I) or (II), a glycolipid according to formula (III) or (IV), and optionally a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC). The elements of the lipid component may be provided in specific fractions.

[0491] In the nanoparticle composition, the amount of bioactivator can depend on the size, composition, desired target and / or application or other characteristics of the nanoparticle composition, and the characteristic of the bioactivator. For example, the amount of the nucleic acid that can be used for the nanoparticle composition can depend on the size, sequence and other characteristics of the nucleic acid. The relative amount of bioactivator and other key elements (for example, lipid) can also change in the nanoparticle composition. In some aspects, the wt / wt ratio of lipid component and bioactivator in the nanoparticle composition can 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 wt / wt ratio of the lipid component to the bioactive agent can be about 1: 1 to about 4: 1. In certain aspects, the wt / wt ratio is about 20: 1. In certain aspects, the wt / wt ratio is about 60: 1. The amount of bioactive agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0492] In one aspect, the present disclosure provides a method for preparing nanoparticles of the present disclosure, wherein the nucleic acid is dissolved in a first solution comprising an acidic buffer or a neutral buffer, and the lipid component is dissolved in a second solution comprising ethanol, thereby forming nanoparticles by mixing the first solution with the second solution. In some aspects, the acidic buffer is a citrate buffer. In some aspects, the acidic buffer has a pH of 3-6. In some aspects, the acidic buffer has a pH of 4.5. In some aspects, the neutral buffer is PBS. In some aspects, the neutral buffer has a pH of 7-8. In some aspects, the neutral buffer has a pH of 7.4.

[0493] Pharmaceutical composition

[0494] Nanoparticle compositions can be formulated in whole or in part as pharmaceutical compositions. A pharmaceutical composition can comprise one or more nanoparticle compositions. For example, a pharmaceutical composition can comprise one or more nanoparticle compositions comprising one or more different bioactive agents. Pharmaceutical compositions can also comprise one or more pharmaceutically acceptable excipients or adjunct ingredients, such as those described herein. General guidance for formulating and manufacturing pharmaceutical compositions and dosage forms can be found, for example, in Remington's The Science and Practice of Pharmacy, 21st ed., A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and adjuvants can be used in any pharmaceutical composition, except where any conventional excipient or adjuvant may be incompatible with one or more components of the nanoparticle composition. An excipient or adjuvant may be incompatible with a component of the nanoparticle composition if the combination of the excipient or adjuvant with the component could result in any undesirable biological effect or other deleterious effect.

[0495] In some aspects, one or more excipients or adjuvants can 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 can 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 U.S. 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.

[0496] 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.

[0497] In some aspects, the nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and / or transport (e.g., at 4°C or lower, such as 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), and / or (IV) is a solution that is refrigerated for storage and / or transport at, 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 pharmaceutical composition comprising a compound of any of Formula (I), (II), (III) and / or (IV) by storing the nanoparticle composition and / or pharmaceutical composition at a temperature of 4°C or lower, such as between about -150°C and about 0°C or between about -80°C and about -20°C, for example, 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, e.g., at a temperature of 4° C. or less (e.g., between about 4° C. and −20° C.). In one embodiment, the formulation is stable for at least 4 weeks at about 4° C. In some aspects, the pharmaceutical compositions disclosed herein comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, an acetate (e.g., sodium acetate), a citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the concentration of the carrier may be 1-100 mM (e.g., including but not limited to any number or range within the range of 1-100 mM, such as 1, 2, 3, 4, ... 97, 98, 99, 100, 10-90 mM, 20-80 mM, 30-70 mM, etc.).

[0498] In certain aspects, the pharmaceutical compositions of the present disclosure have a % saturation of 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,

[0499] 7.9 or 8.0, or a pH between 7.5 and 8 or between 7 and 7.8). For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, suitable for storage and / or transportation at, for example, about -20°C. For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, and has 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 conditions of manufacture, preparation, transport, storage, and / or use (e.g., when stresses such as shear forces, freeze / thaw stress, etc. are applied).

[0500] In certain embodiments, the pharmaceutical compositions of the present disclosure contain a therapeutic or prophylactic agent at a rate of any number or range between 0.05 and 25 mg / mL, 0.1 and 20 mg / mL, 0.2 and 18 mg / mL, 0.5 and 15 mg / mL, 0.7 and 12 mg / mL, 0.9 and 10 mg / mL, 1 and 8 mg / mL, 1.5 and 6 mg / mL, 2 and 5 mg / mL, 2.5 and 4 mg / mL, 0.5 and 3.0 mg / mL, 0.2 and 4.0 mg / mL, 0.4 and 2.0 mg / mL, and 0.05 to 25 mg / mL.

[0501] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including patients or subjects who may benefit from the therapeutic effects provided by delivering a bioactive agent to one or more specific cells, tissues, organs or systems or groups thereof (such as the renal system). Although the descriptions of the nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein are primarily directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. It is well known to modify compositions suitable for administration to humans to make them suitable for administration to various animals, and veterinary pharmacologists of ordinary skill can design and / or perform such modifications with only ordinary experimentation (if any). The subjects to whom the envisioned compositions are administered 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.

[0502] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods involve 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 multi-dose units.

[0503] 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 a subject and / or a conventional fraction of such a dose, such as, for example, half or one-third of such a dose.

[0504] Injectable preparations, such as sterile aqueous or oily suspensions for injection, can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Sterile injection preparations can be sterile injection solutions, suspensions and / or emulsions dissolved in nontoxic parenteral acceptable diluents and / or solvents, such as solutions dissolved 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 suspending 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 injections.

[0505] 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.

[0506] Methods for producing polypeptides in cells

[0507] The present disclosure provides a method for producing a target polypeptide in a cell. The method for producing the polypeptide involves contacting the cell with a nanoparticle composition that contains an mRNA (including self-amplifying mRNA) encoding the target polypeptide. After contacting the cell with the nanoparticle composition, the mRNA can be taken up by the cell and translated to produce the target polypeptide.

[0508] In general, the step of contacting a cell with a nanoparticle composition comprising an mRNA encoding a polypeptide of interest 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 cell or tissue contacted, mode of administration, nanoparticle composition, and the mRNA therein. In general, an effective amount of the nanoparticle composition will allow polypeptide to be effectively produced in the cell. Efficiency indicators can include polypeptide translation (indicated by polypeptide expression), mRNA degradation levels, and immune response indicators.

[0509] The step of contacting the nanoparticle composition comprising the mRNA with the cell can involve or cause transfection. The phospholipids contained 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 or intracellular membrane. Transfection can allow translation of the mRNA within the cell.

[0510] Methods for delivering therapeutic agents to cells and organs

[0511] The present disclosure provides a method for delivering a bioactive agent to a cell or organ. The bioactive agent is delivered to the cell and is related to the nanoparticle composition comprising the bioactive agent used to the experimenter, wherein the use of the composition is related to contacting the cell with the composition. For example, protein, cytotoxic agent, radioactive ion, chemotherapeutic agent or nucleic acid (such as RNA, for example mRNA) can be delivered to the cell or organ. When the bioactive agent is mRNA, when the cell is contacted with the nanoparticle composition, translatable mRNA can be translated in the cell to produce the target polypeptide. However, the mRNA that can not be translated basically also can be delivered to the cell. The mRNA that can not be translated basically 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.

[0512] In some respects, nanoparticle compositions can target cells of specific types or categories (for example, cells of specific organs or their systems).For example, the nanoparticle compositions comprising target bioactivator can be specifically delivered to liver, kidney, spleen, femur or lung.For example, when applying nanoparticle compositions to mammals, the specific delivery to cells of specific categories, organs or systems or their groups means that relative to other destinations, the nanoparticle compositions comprising bioactivator of a higher ratio are delivered to target destination (for example, tissue). In some respects, specific delivery can result in, compared with another destination (for example, spleen), the amount of the bioactivator of every 1g tissue of the target destination (for example, target tissue, such as liver) increases by more than 2 times, 5 times, 10 times, 15 times or 20 times. In some respects, target tissue is selected from the group consisting of the following: liver, kidney, lung, spleen, femur, eye tissue (for example, via intraocular injection, subretinal injection or intravitreal injection), vascular endothelium in blood vessel (for example, in coronary artery or femoral artery) or kidney and tumor tissue (for example, via intratumoral injection).

[0513] As another example of targeted 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 moieties. 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 the 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, diabodies, triabodies or tetrabodies; and aptamers, receptors and fusion proteins.

[0514] In some aspects, the ligand can be a surface-bound antibody that can allow for the regulation of cell targeting specificity. This is particularly useful because highly specific antibodies can be generated for the target epitope of the desired target site. In one embodiment, multiple antibodies are expressed on the surface of the cell, and each antibody can have different specificities for the desired target. Such methods can increase the avidity and specificity of the targeted interaction.

[0515] In certain aspects, the compositions according to the present disclosure can be delivered at dosage levels sufficient to deliver 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, about 5 mg / kg to about 10 mg / kg, or about 10 mg / kg to about 10 mg / kg in a given dose. 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, 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 0.05 mg / kg to about 1 mg / kg. In some aspects, the bioactivator (for example, mRNA) of the nanoparticle composition of about 0.001mg / kg to about 10mg / kg dosage can be used. In other aspects, the bioactivator of the dosage of about 0.005mg / kg to about 2.5mg / kg can be used. In some aspects, the dosage of about 0.1mg / kg to about 1mg / kg can be used. In other aspects, a dose of about 0.05 mg / kg to about 0.25 mg / kg can be administered.Doses may be administered once or multiple times daily in the same or varying amounts to achieve the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic or imaging effect.

[0516] 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, condition, or illness.

[0517] Nanoparticle compositions comprising one or more bioactive agents can be used in combination with one or more other therapeutic agents, prophylactics, diagnostics, or imaging agents. By "in combination with," it is not intended that the agents must be administered simultaneously and / or formulated for delivery together, but these methods of delivery are within the scope of this disclosure. For example, one or more nanoparticle compositions comprising one or more different bioactive 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 in accordance with a dosage and / or schedule determined for the agent. In some aspects, this disclosure encompasses the delivery of a combination of a composition or its imaging agent, diagnostic, or prophylactic composition with an agent that improves its bioavailability, reduces and / or alters its metabolism, inhibits its excretion, and / or alters its distribution in the body.

[0518] It should also be understood that the bioactive agents or imaging agents used in combination can be administered together in a single composition or separately in different compositions. Generally speaking, it is expected that the level of the agent used in combination will not exceed the level at which it is used alone. In some aspects, the level of the combined agent may be lower than the level at which it is used alone.

[0519] The specific combination of therapies (therapeutics or procedures) used in a combination regimen will take into account the compatibility of the desired therapeutic agents 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 concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., to control any adverse effects, such as infusion-related reactions).

[0520] Example

[0521] Example 1: Glycolipids

[0522] The glycolipids disclosed herein were synthesized using a four-component chemical reaction as part of a library of over 10,000 glycolipids. 96 different glycolipids (including (III) (a)-(IV) (c)) were formulated into mLNPs by mixing the lipid-containing ethanolic phase with the mRNA-containing aqueous phase using a pipette. The ethanolic phase was prepared by mixing the ionizable lipid (I) b (or (II) e), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and the sugar-lipid in a predetermined molar ratio of 20 (or 30):30 (or 15):40 (or 50) in ethanol. The aqueous phase was prepared in citrate buffer (100 mM, pH 4.5) with GFP-mRNA. The aqueous and ethanolic phases were mixed in a 3:1 ratio with an N:P ratio of 8.4:1 (or 4.2:1). The transfection efficacy and cytotoxicity of the LNPs were determined in 293T cells. The LNPs of the present disclosure were found to be effective in vitro in transfection and expression of GFP in 293T cells. The data showed that their transfection efficiency was better or comparable to that of the currently FDA-approved LNPs.

[0523] Figure 1 Shown is a heat map of a high-throughput screen of the percentage transfection of GFP self-amplifying mRNA (GFP-saRNA) in 293T cells in a 96-well plate using LNPs formulated with glycolipids and ionizable lipids of formula (Ib) for delivery of GFP-saRNA compared to the percentage transfection of GFP-saRNA delivered using a 4-component LNP comprising an ionizable lipid of formula (I), a glycolipid, a phospholipid, and a PEG-lipid. The glycolipid of formula (III) (a) corresponds to position E2 of the 96-well plate, the glycolipid of formula (III) (b) corresponds to position F11, and the glycolipid of formula (III) (c) corresponds to position C6.

[0524] Figure 2 Shown is the in vitro expression of GFP-saRNA in 293T cells transfected with LNPs formulated with glycolipids and ionizable lipids of Formula (Ib), including glycolipids of Formula (III) (a) (P1_E2), (III) (b) (P1_F11), and (III) (c) (P1_C6).

[0525] Figure 3Shown is a heat map of a high-throughput screen of the percentage transfection of GFP self-amplifying mRNA (GFP-saRNA) in 293T cells in a 96-well plate using LNPs formulated with glycolipids and ionizable lipids of formula (IIe) for delivery of GFP-saRNA compared to the percentage transfection of GFP-saRNA delivered using a 4-component LNP comprising an ionizable lipid of formula (IIe), a glycolipid, a phospholipid, and a PEG-lipid. The glycolipid of formula (III) (c) corresponds to position E8 of the 96-well plate, the glycolipid of formula (III) (e) corresponds to position D8, and the glycolipid of formula (III) (f) corresponds to position D9.

[0526] Figure 4 Shown is the in vitro expression of GFP-saRNA in 293T cells transfected using LNPs formulated with glycolipids and ionizable lipids of formula (IIe), including glycolipids of formula (III)(d) (also known as P1_E8), (III)(e) (also known as P1_D8), and (III)(f) (also known as P1_D9).

[0527] Example 2: Nanoparticles containing modular lipids

[0528] The LNPs of the present disclosure comprise modular lipids that integrate two or more functions of traditional lipid components of LNPs, such as e.g. Figure 5 The modular lipids P161F5, P161F6, P161F10, P161F12, P287A12 and P287C12 disclosed herein comprise a sterol or sterol derivative group and a cationic ionizable group. Figure 6 The three-component LNPs shown contain modular lipids (P161F5, P161F6, P161F10, P161F12, P287A12, or P287C12); a bipolar compound and a sugar stabilizer lipid. In addition, two-component LNPs were prepared using the modular lipid P287A12 and DMG-PEG2000. As described below, both types of LNPs demonstrated efficient transfection in vivo and in vitro.

[0529] The ionizable lipids for preparing LNPs of the present invention are synthesized by a two-step / one-pot reaction, wherein the molar ratio of acid / amine / aldehyde / isocyanate is 1:1:1:1. Aldehyde (1 mmol) and amine (1 mmol) are mixed and stirred for one hour in 3 mL of MeOH at room temperature (rt), followed by the addition of isocyanate (1 mmol) and acid (1 mmol). The resulting mixture is further stirred overnight. The solvent is removed on a rotary evaporator. Ethyl acetate (100 mL) is added and washed with saline (2 × 50 mL), dried over MgSO. Ethyl acetate is removed, and the Combi-Flash Rf system with a silica gel column (Redisep Gold Resolution, Teledyne, Isco) is used, and the residue is purified by column chromatography using a gradient elution of CH2Cl2 / methanol (0%-40% methanol). The ionizable lipid structures were confirmed by 1H nuclear magnetic resonance spectroscopy (Bruker AVANCE-400 NMR, Custom NMR Services, Inc.) and / or LC-mass spectrometry (Agilent 1100 & 1200 HPLC / MS, Organix, Inc.).

[0530] The modular lipids of the present invention are mixed with at least one of ionizable lipids, DOPE, cholesterol (Chol) and / or DMG-PEG2000 in ethanol at a predetermined molar ratio. The organic phase is prepared using an ionizable lipid:SamRNA molar ratio of 6: 1. SamRNA-LUC or SamRNA-GFP is diluted in 50mM citrate buffer (pH 4.5, Fisher) or PBS (pH 7.4, Fisher) to prepare an aqueous phase. SamRNA is stored at -80°C and thawed on ice before use. Ethanol and aqueous phase are mixed at a ratio of 1: 3 by pipette. The elution is carried out at 4°C by ultrafiltration (100,000 molecular weight cutoff, MilliporeSigma) before injection. TM Amicon TM The resulting LNPs were purified using an Ultra-15 centrifugal filter device.

[0531] Reagents used in the above reactions: Fetal bovine serum (FBS) and Ribogreen were purchased from Fisher Scientific. DOPE, 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, BLDpharm, Aaron Chemicals, AAblocks, 1clickchemistry, Enamine, Aurum, Achemblock, BroadPharm, ChemShuttle, and Biopharma PEG.

[0532] In Example 1, it was demonstrated that LNPs comprising modular lipids having sugar groups have improved transfection efficiency, and LNPs comprising ligand receptor functional groups such as GalNAc have improved targeting function.

[0533] Example 3: Modular lipid library

[0534] A multifunctional modular lipid library has been designed. The modular lipids of the library combine two or more components in the traditional LNP lipid components into a modular lipid. One or two additional components are used to formulate LNPs comprising the modular lipids of the modular lipid library to deliver nucleic acids. The modular lipids of the present disclosure contain two or more functional groups (FGs), which include but are not limited to lipid groups, cationic ionizable groups, steric hindered groups, lipid raft groups, sterol groups, sugar groups, folic acid groups, GalNAc groups, oligopeptide groups and oligonucleotide groups, such as Figure 7 The modular lipids of the multifunctional modular lipid library include more than 1,000,000 modular lipids synthesized using the four-component reaction of the present disclosure:

[0535]

[0536] in It's acid. It is an amine. is an aldehyde or a ketone, and It's isocyanide.

[0537] The synthesis reaction was carried out in a 96-well plate (Analytical Sales and Services) with a glass insert. First, the amine and aldehyde were mixed and stirred at room temperature (rt), and then the acid and isocyanate were added. The reactants were stirred overnight at rt. The lipid mixture was directly used for in vitro high-throughput screening of LNP delivery as described below.

[0538] Example 4: In vitro transfection efficiency

[0539] LNPs containing SamRNA-GFP or SamRNA-LUC were added to 96-well plates pre-seeded with HEK293, C2C12, or MC38 cells at 100 ng / well. After incubation at 37°C and 5% CO2 for 24 hours, the transfection efficiency of SamRNA-LUC was measured by a plate reader (Perkin Elmer Envision 2104) and by flow cytometry (BD FACS phony TM A5 SE cell analyzer) was used to measure the GFP SamRNA transfection efficiency.

[0540] Compared with LNPs formulated with ionizable lipids used in FDA-approved therapies ("FDA-approved lipids"), LNPs containing modular lipids showed increased or comparable in vitro transfection efficiency. HEK293 cells were used to identify the transfection efficacy of LNPs formulated with modular lipids comprising cholesterol groups and cationic ionizable groups in 96-well plates. LNP (ionizable lipid-Chol / DOPE / DMG-PEG2000, 50-38.5 / 10 / 1.5 molar ratio) encapsulated SamRNA-LUC or SamRNA-GFP. HEK293 cells were treated with 100ng of SamRNA-LUC or 100ng of SamRNA-GFP for 24 hours. The transfection efficiency of LNP-SamRNA-GFP ( Figure 9 ) and LNP-SamRNA-LUC( Figure 8 ) transfection efficiency. Figure 8 ) and SM102( Figure 9 ) prepared LNPs were used as controls. Data were collected in triplicate and expressed as mean ± standard deviation.

[0541] SamRNA encoding firefly luciferase (SamRNA-LUC) and GFP (SamRNA-GFP) was synthesized by SunVax mRNA Therapeutics.

[0542] The transfection efficacy of the LNP prepared using the modular lipid comprising a sterol derivative group, a sugar group and a PEG group is added to a 96-well plate in which HEK293 cells are pre-seeded. LNP is prepared using cation ionizable lipids (P54B6, P38D8 or E6), DOPE and modular lipids (35 / 40 / 15 mol ratio), encapsulating SamRNA-LUC or SamRNA-GFP. HEK293 cells are processed with 100ng of SamRNA-LUC or SamRNA-GFP, continuing 24 hours. The structure of two kinds of lipids in the ionizable lipid for preparing LNP is as follows:

[0543]

[0544] LNP-SamRNA-GFP ( Figure 11 ) and LNP-SamRNA-LUC( Figure 10 ) transfection efficiency. LNPs formulated with the FDA-approved ionizable lipid MC3 were used as controls. Data were collected in triplicate and expressed as mean ± SD.

[0545] Modular lipids P161F5, P161F6, or P161F10; DOPE and DMG-PEG were used in 96-well plates using HEK293T cells. 2000 (Modular lipids / DOPE / DMG-PEG 2000 Transfection efficacy of LNPs encapsulating LNP-SamRNA-GFP (40 / 10 / 2 molar ratio). 100 ng RNA was incubated with HEK293T cells at 60-70% confluence in 96-well plates for 24 hours. 2000 The in vitro delivery efficiency of LNPs containing MC3 and SM102 (molar ratio of 40 / 10 / 2) showed comparable or higher transfection efficiency than that of four-component LNPs containing MC3 and SM102, as shown in Figure 2. Figure 12 The data were collected in triplicate and expressed as mean ± SD.

[0546] The transfection efficiency of the LNPs encapsulated with LNP-SamRNA-GFP prepared with modular lipids SP1F11; ionizable lipids P38D8, P40D7 or P1D4; and DOPE (ionizable lipids / DOPE / SP1F11, 40 / 60 / 15 molar ratio) was compared with that of the four-component LNP control group (ionizable lipids / DOPE / cholesterol / DMP-PEG2000, 30 / 15 / 50 / 1.5 molar ratio). 100 ng RNA was incubated in 96-well plates with HEK293T cells at a confluence of 60-70% for 24 hours. The structures of the two lipids in the ionizable lipids used to prepare LNPs are shown below:

[0547]

[0548] About 50% of HEK293 cells were transfected with both four-component and three-component LNPs, as shown in Figure 2. Figure 13 The data were collected in triplicate and expressed as mean ± SD.

[0549] HEK293T cells were used to study the transfection efficacy of LNPs encapsulating SamRNA-GFP formulated with ionizable lipids P54B6, SM102, MC3, or ALC0315; DOPE; and modular lipids SP2B12, SP11A6, or SP11A12 (35 / 40 / 20, molar ratio). 100 ng of RNA was incubated with HEK293T cells for 24 hours. The structure of P54B6 is shown below:

[0550]

[0551] Modular lipid containing three-component LNPs showed comparable or higher in vitro delivery efficiency than four-component LNPs, such as Figure 14 The data were collected in triplicate and expressed as mean ± SD.

[0552] The transfection efficacy of LNPs encapsulating SamRNA-GFP or modified mRNA-GFP formulated with P287A12, DOPE, DMG-PEG2000 (40 / 10 / 2, molar ratio) or P287A12 and DMG-PEG2000 (40 / 2, molar ratio) in acidic buffer (citrate buffer, pH 4.5) and / or neutral buffer (PBS, pH 7.4) was studied using HEK293 cells in 96-well plates.

[0553] LNPs were prepared in an acidic buffer (citrate buffer, pH 4.5) or a neutral buffer (PBS, pH 7.4) and ethanol. The nucleic acid payload was dissolved in the buffer while the lipid component was dissolved in ethanol and the LNPs of the present disclosure were formed by mixing the two phases. 100 ng of RNA was incubated with HEK293 cells for 24 hours using a neutral (PBS) or acidic (citric acid) buffer. Figure 15A and 15B As shown, there is no significant difference in the in vitro delivery efficiency between LNPs prepared in acidic and neutral buffers. Citrate buffer is traditionally used in LNP formulations and has a low pH of about 4.5. PBS is a neutral buffer with a pH of about 7.4. Data were collected in triplicate and expressed as mean ± standard deviation.

[0554] Example 5: Modular lipids

[0555] Modular lipids P161F5, P161F6, P161F10, P161F12, P287A12, P287C12, SP1E2, SP1F2, SP1F11, SP1E8, SP1E2K, SP1E2KI, SP2B12, SP2A3, SP11H3, SP11A6, SP11H6, SP11A12, and SP1E2-PEG3 were synthesized and confirmed by mass spectrometry.

[0556] 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]phenanthren-17-yl)-N-(2-octyldodecyl)pentanamide (P161F10): Yield (51%). MS (APCI) m / z 974.9 [M+H].

[0557] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-N-(2-decyltetradecyl)-4-(3,6-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (P161F6): Yield (57%). MS (APCI) m / z 1031.0 [M+H]+.

[0558] N-(2-(((1s,3s)-adamantan-1-yl)amino)-1-(1-ethylpiperidin-4-yl)-2-oxoethyl)-4-(3,6-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N-(tricosan-12-yl)pentanamide (P161F5): Yield (61%). MS (APCI) m / z 1017.9 [M+H].

[0559] N-(1-(((3s,5s,7s)-adamantan-1-yl)amino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-decyltetradecyl)-4-(3,6-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (P161F5): Yield (49%). MS (APCI) m / z 1030.9 [M+H].

[0560] N-(1-(Benzylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-decyltetradecyl)-4-(3,12-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (P287A12): Yield (42%). MS (APCI) m / z 986.8 [M+H]+.

[0561] (4R)-N-(1-(Benzylamino)-3-(1-methylpiperidin-4-yl)-1-oxopropan-2-yl)-N-(2-decyltetradecyl)-4-((3R,5S,7R,8R,9S,10S,13R,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (P287C12): Yield (40%). MS (APCI) m / z 986.8 [M+H].

[0562] (3S,4R,5S,6R)-N-(57-(((3R,5R,7R)-adamantan-1-yl)amino)-57-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53-octadecaoxaheptadecane-56-yl)-N-((8S,9S,10R,13R, (14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxamide (SP2B12): Yield (53%). MS (APCI) m / z 780.5 [M+2H]2+.

[0563] (141-((Z)-heptadeca-8-en-1-yl)-140-((2S,3S,4S,5R,6R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carbonyl)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,6 5,68,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137-tetrahexaoxa-140-azatetradodec-142-yl)glycine ethyl ester (SP1E2K): yield (33%). MS (MALDI-TOF) m / z 2657.6 [M+K]+.

[0564] (3S,4R,5S,6R)-3,4,5,6-tetrahydroxy-N-((Z)-1-((4-methoxyphenyl)amino)-1-oxonadecan-10-en-2-yl)-N-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,6 8,71,74,77,80,83,86,89,92,95,98,101,104,107,110,113,116,119,122,125,128,131,134,137-tetrahexadecanoic acid nonaconic acid-139-yl)tetrahydro-2H-pyran-2-carboxamide (SP1E2KI): Yield (40%). MS (MALDI-TOF) m / z 2657.6 [M+K]+.

[0565] N-(Cyclohex-1-en-1-yl)-52-((2R,3S,4R,5R)-N-((8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-2,3,4,5,6-pentahydroxyhexanoylamino)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatriacontanoate-53-carboxamide (SP11H6): Yield (54%). MS(MALDI-TOF)m / z 1473.9[M+Na]+.

[0566] N-Cycloheptyl-52-((2R,3S,4R,5R)-N-((8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)-2,3,4,5,6-pentahydroxyhexanoylamino)-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-heptadecaoxatriacontane-53-carboxamide (SP11A12): Yield (54%). MS(MALDI-TOF)m / z 1489.9[M+Na]+.

[0567] Example 6: In vivo transfection efficiency

[0568] Compared with traditional four-component LNPs, LNPs containing modular lipids showed higher or comparable in vivo transfection efficiency. The transfection efficacy of LNPs encapsulating SamRNA-LUC formulated with P54B6 or P38D8; DOPE; cholesterol; and DMG-PEG2000 (30 / 15 / 50 / 1.5, molar ratio); P54B6, DOPE and modular lipid SP2B12 (35 / 40 / 20, molar ratio); P161F5, DOPE, DMG-PEG2000 (40 / 10 / 2, molar ratio); P161F5, DOPE and SP1E2K (40 / 10 / 2, molar ratio); or P38D8, DOPE and SP1F11 (40 / 60 / 15, molar ratio) was studied in mice.

[0569] All animal procedures were performed under ethical compliance and with approval from the Institutional Animal Care and Use Committee (IACUC). Female Balb / c mice (6-8 weeks) were obtained from Charles River Laboratories Inc.

[0570] 1 μg of LNP was injected intramuscularly into mice. Subsequently, luciferin (Perkin Elmer, 6 mg / mouse) was injected intraperitoneally into mice, and the bioluminescence intensity was measured using an IVIS imaging system (IVIS, Perkin Elmer). Figure 16 、 17 As shown in Figures 1 and 18, the in vivo bioluminescence intensity of metastatic sites in mice treated with four-component LNPs was lower than or equivalent to that of LNPs containing modular lipids of the present disclosure. Notably, LNPs containing P38D8, SP1F11, and DOPE showed higher transfection efficiency than four-component LNPs containing MC3. Data were collected in quintuples and are presented as mean ± standard deviation.

[0571] Example 7: Characterization of modular lipids

[0572] The LNPs of the present disclosure were characterized and the nanoparticle size ( Figure 19A ) and PDI( Figure 19B ).

[0573] Size and polydispersity index (PDI) were measured by dynamic light scattering (SZ-100-Z2 (MTS), Horiba Scientific). Diameters were reported as intensity average peak mean. Nucleic acid encapsulation efficiency was calculated using a modified Quant-iT RiboGreen RNA assay (Invitrogen).

[0574] Furthermore, it should be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such aspects are considered to be known to those of ordinary skill in the art, they may be excluded even if not explicitly stated herein.

[0575] It should be understood that although the present disclosure has been described in conjunction with specific embodiments, 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 modifications are within the scope of the following claims.

Claims

1. A nanoparticle composition comprising an ionizable lipid component and a glycolipid component.

2. A nanoparticle composition having two lipid components, wherein the two lipid components are a modular lipid component and a stabilizer lipid component, wherein the stabilizer lipid component is a glycolipid compound or a PEG lipid, wherein the modular lipid component accounts for about 0.5 mol% to about 99.5 mol% of the total lipid components present in the nanoparticle, and wherein the stabilizer lipid component accounts for about 0.5 mol% to about 99.5 mol% of the total lipid components present in the nanoparticle.

3. A nanoparticle composition having three lipid components and a phospholipid component, wherein the stabilizer lipid component is a glycolipid compound or a PEG lipid, wherein the phospholipid component accounts for about 5 to 60 mol% of the total lipid components present in the nanoparticle, wherein the stabilizer lipid component accounts for about 0.2 to 80 mol% of the total lipids present in the nanoparticle, and wherein the modular lipid or ionizable lipid component accounts for about 5 to 80 mol% of the total lipid components present in the nanoparticle.

4. A nanoparticle composition having three lipid components, wherein the three lipid components are modular lipids, ionizable lipid components, and phospholipid components, wherein the stabilizer lipid component is a glycolipid compound or a PEG lipid, wherein the phospholipid component accounts for about 15 mol% to about 55 mol% of the total lipid components present in the nanoparticle, wherein the modular lipid component accounts for about 3 mol% to about 25 mol% of the total lipid components present in the nanoparticle, and wherein the ionizable lipid component accounts for about 30 mol% to about 80 mol% of the total lipid components present in the nanoparticle.

5. The nanoparticle composition of any one of claims 1 to 4, wherein the stabilizer lipid component is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

6. The nanoparticle composition of any one of claims 1-5, further comprising a bioactive agent.

7. The nanoparticle composition of any one of claims 3-4, wherein the phospholipid component comprises 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-distearoyl-sn-glycero-3-phosphocholine (DPPC), Choline (DSPC), 1,2-heneicosanoyl-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-cholestyryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 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-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or a mixture thereof.

8. A compound of formula III or a salt or isomer thereof, wherein R 1 、R 2 、R 3 and R 4 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), R 6 、R 7 、R 8 and R 9 Each of the following is 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, 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; Each Z is independently selected from CH or N; and Each sugar is independently selected from monosaccharides, disaccharides, oligosaccharides and polysaccharides.

9. The compound of claim 8, wherein the sugar is a monosaccharide.

10. The compound of claim 9, wherein the monosaccharide is independently selected from ketotriose, aldotetraose, ketotetraose, aldotetraose, ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, fucose, fucose, rhamnose, heptose, octose, nonose, gulose, idose, galactose, talose, sedoheptulose, or a combination thereof.

11. The compound of claim 8, wherein the sugar is a disaccharide.

12. The compound of claim 11, wherein the disaccharide is independently selected from sucrose, lactose, maltose, trehalose, turanose, cellobiose, or a combination thereof.

13. The compound of claim 8, wherein the sugar is an oligosaccharide.

14. The compound of claim 13, wherein the oligosaccharides are independently selected from raffinose, melezitose, maltotriose, acarbose, stachyose, fructooligosaccharides, galacto-oligosaccharides, manno-oligosaccharides, or a combination thereof.

15. The compound of claim 8, wherein the sugar is a polysaccharide.

16. The compound of claim 15, wherein the polysaccharide is independently selected from polyhydroxylated sugar alcohols, n-acetylglucosamine, and chitin.

17. The compound of claim 8, wherein the compound is: or a salt or isomer thereof.

18. A method for synthesizing a compound as claimed in any one of claims 8 to 17, comprising performing the following four-component reaction:

19. The method of claim 18, wherein said yes 20. The method of any one of claims 18-19, wherein the yes 21. The method of any one of claims 18 to 20, wherein the yes 22. The method of any one of claims 18 to 21, wherein the yes 23. A compound of formula IV or a salt or isomer thereof, wherein R 1 、R 2 、R 3 and R 4 Each of which is independently selected from H, Cl-C 24 Alkyl, C1-C 24 Alkenyl, C1-C 24 Alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), a, b and c are each independently an integer from 0 to 24; R 6 、R 7 、R 8 and R 9 Each of the following is 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, 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; Each Z is independently selected from CH or N; Each sugar is independently selected from monosaccharides, disaccharides, oligosaccharides and polysaccharides.

24. The compound of claim 23, wherein the sugar is a monosaccharide.

25. The compound of claim 24, wherein the monosaccharide is independently selected from ketotriose, aldotetraose, ketotetraose, aldotetraose, ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, fucose, fucose, rhamnose, heptose, octose, nonose, gulose, idose, galactose, talose, and sedoheptulose.

26. The compound of claim 23, wherein the sugar is a disaccharide.

27. The compound of claim 26, wherein the disaccharides are independently selected from sucrose, lactose, maltose, trehalose, turanose, cellobiose.

28. The compound of claim 23, wherein the sugar is an oligosaccharide.

29. The compound of claim 28, wherein the oligosaccharides are independently selected from raffinose, melezitose, maltotriose, acarbose, stachyose, fructooligosaccharides, galacto-oligosaccharides and manno-oligosaccharides.

30. The compound of claim 23, wherein the sugar is a polysaccharide.

31. The compound of claim 30, wherein the polysaccharide is independently selected from polyhydroxylated sugar alcohols, n-acetylglucosamine, and chitin.

32. The compound of claim 23, wherein the compound is: or a salt or isomer thereof.

33. A compound selected from:

34. A method for synthesizing a compound as claimed in any one of claims 23 to 32, comprising performing the following four-component reaction:

35. The method of claim 34, wherein said yes 36. The method of any one of claims 34-35, wherein the yes 37. The method of any one of claims 34-36, wherein the yes 38. The method of any one of claims 34-37, wherein the yes 39. A method for synthesizing a modular lipid comprising a cation ionizable group and / or a sterol derivative group, the method comprising performing a four-component reaction of an acid compound, an amine compound, an aldehyde or ketone compound, and an isocyanate compound as follows: Each R 1 、R 4 and R 5 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), Each 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and 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, a, b, c and d 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 CH or N.

40. The method of claim 39, wherein the acid compound is:

41. The method of any one of claims 39-40, wherein the amine compound is:

42. The method of any one of claims 39-41, wherein the aldehyde or ketone compound is:

43. The method of any one of claims 39-42, wherein the isocyanate compound is:

44. A modular lipid of Formula V, VI, VII, VIII, IX or X: or a salt or isomer thereof, wherein Each R 1 、R 4 and R 10 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), Each R 2 、R 2' 、R 3 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and wherein each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and where R 6 、R 7 、R 8 and R 9 Each is 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, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, a, b, c and d are each independently an integer from 0 to 24; Each E is independently selected from CH2, NH, O or S; 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 CH or N.

45. A method for synthesizing modular lipids of Formulas V, VI, and VII, comprising performing the following four-component reaction: or a salt or isomer thereof, wherein Each R 1 and R 4 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), Each 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and wherein each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and where R 6 、R 7 、R 8 and R 9 Each is 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, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, a, b, c and d are each independently an integer from 0 to 24; Each E is independently selected from CH2, NH, O or S; Each X is independently selected from CH or N; Each Y is independently selected from CH2, NH, O or S; Each Z is independently selected from CH or N.

46. ​​A method for synthesizing modular lipids of Formula VIII, IX, and X, comprising performing the following four-component reaction: or a salt or isomer thereof, wherein Each R 1 、R 4 and R 10 Independently selected from C2-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, substituted C2-C 24 Alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), Each R 2 、R 2' 、R 3 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 carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and Each L is independently selected from alkyl, alkenyl, alkynyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted acyl, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, poly(ethylene glycol) (PEG), and where R 6 、R 7 、R 8 and R 9 Each is 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, substituted carbocyclyl, substituted heterocyclyl, substituted aryl, substituted heteroaryl, a, b, c and d are each independently an integer from 0 to 24; Each E is independently selected from CH2, NH, O or S; 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 CH or N.

47. A modular lipid comprising two or more functional groups and at least one linker between at least two functional groups, wherein the functional groups are selected from cationic or ionizable lipids, phospholipids, glycolipids, lipid rafts, stabilizer lipids, bipolar compounds with hydrophobic and hydrophilic ends, steric hindering groups, sterol groups, sterol-containing groups, sterol derivative groups, folate-containing groups, N-acetylgalactosamine (GalNAc)-containing groups, oligopeptide groups, oligonucleotide groups, or combinations thereof.

48. The modular lipid of claim 47, wherein the lipid is:

49. A nanoparticle composition comprising the lipid of any one of claims 8-17 and 23-33, or any combination thereof.

50. The nanoparticle composition of claim 49, further comprising the modular lipid component of any one of claims 44 and 47-48.

51. The nanoparticle composition of claim 49 or claim 50, comprising a modular lipid component, a stabilizer lipid component, and / or a phospholipid component, optionally wherein the nanoparticle composition is a bi-lipid composition or a tri-lipid composition.

52. The nanoparticle composition of claim 51, wherein the modular lipid component comprises a linker, a cationic ionizable group, and a lipid raft group.

53. The nanoparticle composition of any one of claims 49-52, wherein the stabilizer lipid component is a glycolipid compound or a PEG lipid as described in any one of claims 8-17 and 23-32.

54. The nanoparticle composition of any one of claims 49-53, wherein the stabilizer lipid component is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

55. The nanoparticle composition of any one of claims 49-54, wherein the phospholipid component comprises 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-distearoyl-sn-glycero-3- Phosphocholine (DSPC), 1,2-heneicosanoyl-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-cholestyryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 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-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or a mixture thereof.

56. The nanoparticle composition of any one of claims 1-7 or 49-55, wherein the nanoparticle composition further comprises a bioactive agent.

57. A pharmaceutical composition comprising the nanoparticle composition of any one of claims 1-7 or 49-56 and a pharmaceutically acceptable carrier.

58. A method of delivering a bioactive agent to a cell, the method comprising administering to a subject the nanoparticle composition of any one of claims 1-7 or 49-56, the administration comprising contacting the cell with the nanoparticle composition, thereby delivering the bioactive agent to the cell.

59. A method of delivering a bioactive agent to a cell, the method comprising administering to a subject the pharmaceutical composition of claim 57.

60. A method of producing a polypeptide of interest in a cell, the method comprising contacting the cell with the nanoparticle composition of claim 56, wherein the bioactive agent is an mRNA encoding the polypeptide of interest, whereby the mRNA can be translated in the cell to produce the polypeptide of interest.

61. A method for modulating expression of an endogenous nucleic acid in a cell, the method comprising contacting the cell with a nanoparticle composition as described in claim 56, wherein the bioactive agent is an siRNA capable of binding to the endogenous nucleic acid, thereby the siRNA is capable of modulating the expression of the endogenous nucleic acid.

62. A method for modulating expression of an endogenous nucleic acid in a cell, the method comprising contacting the cell with a nanoparticle composition as described in claim 56, wherein the bioactive agent is an antisense RNA capable of binding to the endogenous nucleic acid, thereby the antisense RNA is capable of modulating the expression of the endogenous nucleic acid.

63. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nanoparticle composition of any one of claims 1-7 or 49-56.

64. A method of formulating a nanoparticle composition as described in claim 56, wherein the nucleic acid is dissolved in a first solution comprising an acidic buffer or a neutral buffer, and the lipid component is dissolved in a second solution comprising ethanol, thereby forming the nanoparticles by mixing the first solution with the second solution.

65. The method of claim 64, wherein the acidic buffer is a citrate buffer.

66. The method of any one of claims 64-65, wherein the acidic buffer has a pH of 3-6.

67. The method of any one of claims 64-66, wherein the acidic buffer has a pH of 4.

5.

68. The method of claim 64, wherein the neutral buffer is PBS.

69. The method of claim 64 or claim 68, wherein the neutral buffer has a pH of 7-8.

70. The method of any one of claims 64 or 68-69, wherein the neutral buffer has a pH of 7.4.

Images