Lipids for delivery of charged materials, formulations thereof, and methods of making the same

Abstract

Disclosed herein are lipids having a net charge at physiological pH and covalently linked to a lipid moiety. The lipid moiety comprises a hydrocarbon structure having two or more linked hydrocarbon chains, the chains optionally having cis or trans C=C, at least one of the chains being covalently linked to the head group via a linker region. The hydrocarbon chains are bonded to one another at branching points of interior carbons of the chains linked to the linker region, the branching points comprising functional groups having electronegative atoms. The hydrocarbon chains each have from 1 to 40 carbon atoms, with the hydrocarbon structure comprising a total of from 10 to 150 carbon atoms. Advantageously, the hydrocarbon structure can adopt a generally open shape for improved delivery of cargo molecules. Also provided are delivery vehicles comprising the lipids.

Description

Technical Field

[0001] This article provides information on lipids, formulations of these lipids, and methods for their preparation. Lipids and their formulations are useful for the delivery of charged materials, including (but not limited to) nucleic acids and peptides. Background Technology

[0002] Incorporation of these lipids into delivery systems, such as lipid nanoparticles (LNPs), facilitates intracellular delivery of nucleic acids or other charged molecules, such as peptides. For example, ionizable lipids are the main lipid components responsible for the efficient encapsulation of nucleic acids during LNP production. Furthermore, following up via endocytosis in target cells, ionizable or cationic lipids contribute to the subsequent controlled release of nucleic acids from endosomes.

[0003] It has been proposed that transfection or gene delivery activity depends on the chemical structure of ionizable lipids, such as cationic lipids (Semple, SC et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol, 2010, 28(2): p. 172-6). Specifically, the lipophilic moiety should have a non-cylindrical shape. Examples are lipids with a small ionizable head group and a hydrocarbon moiety that widens or expands outward from the head group.

[0004] Currently, there remains a need for ionizable or charged lipid structures with desired shapes that can be prepared using simple or convenient methods. The ability to provide these lipids could significantly advance the clinical development of therapies that rely on the delivery of nucleic acids or other charged molecules to target cells.

[0005] The compositions and methods disclosed in this invention aim to solve the above-mentioned problems and / or provide useful alternatives to those previously described in the art. Summary of the Invention

[0006] This article provides ionizable or cationic lipids with hydrocarbon structures that facilitate the delivery of charged materials. The charged materials can be negatively charged materials, such as nucleic acids, or positively charged materials. The lipids described herein can be prepared in a modular manner, thereby providing tunable hydrocarbon structures with a variety of non-cylindrical shapes to facilitate the delivery of a variety of negatively or positively charged materials.

[0007] This document discloses lipids comprising a head group having a net charge at a physiological pH and covalently linked to a lipid moiety via an optional linker region. The lipid moiety comprises a hydrocarbon structure having two or more linked hydrocarbon chains, the hydrocarbon chains optionally having a cis or trans C=C configuration, at least one of the chains optionally being covalently linked to an ionizable head group via the linker region. The hydrocarbon chains are bonded to each other at branch points of the internal carbons of the chains linked to the head group via the optional linker region, the branch points comprising X1 functional groups having electronegative atoms, as further described herein. The hydrocarbon chains each have 1 to 40 or 1 to 30 carbon atoms, wherein the hydrocarbon structure comprises a total of 10 to 150 carbon atoms.

[0008] According to one embodiment, a charged lipid is provided, comprising a cationic or anionic lipid comprising: a head group having a net charge at a physiological pH and covalently linked to a lipid moiety via an optional linker region; and a lipid moiety comprising a hydrocarbon structure having two or more linked hydrocarbon chains, said hydrocarbon chains optionally having a cis or trans C=C configuration, at least one of said chains being covalently linked to an ionizable head group via said linker region, and wherein said hydrocarbon chains are bonded to each other at branch points of the inner carbons of the hydrocarbon chains connected to said linker region, said branch points comprising an X1 functional group selected from: -OC(O)-, -C(O)O. -, -O-, -NR1-, -C(O)N(R1)-, N(R1)C(O)-, -OC(O)O-, -OC(O)N(R1)-, -N(R1)C(O)O-, -S-, -SS-, -C(R1)=NNC(O)-, -C(O)-NN=C(R1), -ON=C(R1)- or -C(R1)=NO-, wherein the hydrocarbon chains each have 1 to 30 carbon atoms, wherein the hydrocarbon structures comprise a total of 10 to 150 carbon atoms, and wherein R1 is independently selected from hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic.

[0009] In one embodiment, the two or more linked hydrocarbon chains have a structure of Formula III:

[0010] Chemical Formula III

[0011]

[0012] L1', L1”, L1”' and L1”” are independently selected from hydrocarbon chains having 1 to 30 atoms, which optionally contain one or more cis or trans C=C bonds, and L1' is a hydrocarbon chain covalently attached to the junction region.

[0013] According to other embodiments, the hydrocarbon structure further includes a hydrophobic chain, L2, which is bonded to the head group via a linker region, and wherein L2 is a hydrocarbon chain having 1 to 30 carbon atoms, optionally having cis or trans C=C bonds, or wherein L2 has a structure of formula IIIa.

[0014]

[0015] L2', L2”, L2”' and L2”” are independently selected from hydrocarbon chains having 1 to 30 atoms, which optionally contain one or more cis or trans C=C bonds, and wherein L2' is covalently connected to the junction region.

[0016] In other embodiments, the hydrocarbon further includes a hydrophobic chain, L3, which is bonded to the head group via a linker region, and wherein L3 has 1 to 30 carbon atoms, optionally having cis or trans C=C bonds, or having a structure of formula IIIa.

[0017]

[0018] L3', L3”, L3”' and L3”” are independently selected from hydrocarbon chains having 1 to 30 atoms, which optionally contain one or more cis or trans C=C bonds, and wherein L3' is covalently attached to the junction region.

[0019] In other embodiments, the lipid may further comprise 1 to 10 side chains S bonded to L1 via X1 bonds, each side chain having 1 to 30 atoms, optionally having cis or trans C=C bonds or having a structure of chemical formula IIIb:

[0020]

[0021] S1', S1”, S1”' and S1”” are independently selected from hydrocarbon chains having 1 to 30 atoms, which optionally contain one or more cis or trans C=C bonds, and wherein S1' is bonded to the carbon of L1.

[0022] According to other embodiments, the lipid may also comprise 1 to 10 side chains S bonded to L2 via X1 bonds, each side chain having 1 to 30 atoms, optionally having cis or trans C=C bonds or having a structure of chemical formula IIIb:

[0023]

[0024] S2', S2”, S2”' and S2”” are independently selected from hydrocarbon chains having 1 to 30 atoms, which optionally contain one or more cis or trans C=C bonds, and wherein S2' is bonded to the carbon of L2.

[0025] In other embodiments, the lipid further includes 1 to 10 side chains S bonded to L3 via X1 bonds, each side chain having 1 to 30 atoms, optionally having cis or trans C=C bonds or having a structure of chemical formula IIIb:

[0026]

[0027] S3', S3”, S3”' and S3”” are independently selected from hydrocarbon chains having 1 to 30 atoms, which optionally contain one or more cis or trans C=C bonds, and wherein S3' is bonded to the carbon of L3.

[0028] In an alternative implementation, X1, which appears once or multiple times, is biodegradable.

[0029] In other embodiments, at least the hydrocarbon chain bonded to the head group via the linker region is derived from a lipid having one or more reactive groups selected from hydroxyl, amino, and / or amide, the hydroxyl, amino, and / or amide being bonded to its internal carbon atom to serve as the skeletal carbon chain in the hydrocarbon structure, and at least one other hydrocarbon chain in the hydrocarbon structure is derived from an acyl lipid, wherein the X1 bond is formed by the reaction of the reactive group on the skeletal carbon chain with the carboxylic acid of the acyl chain.

[0030] In other embodiments, the lipid is a dihydroxy lipid.

[0031] The head group can impart a pK value between 5.0 and 9.0 to the lipid. a Or a PK between 5.5 and 8.0 a In an alternative embodiment, the head group has the structure of Formula I.

[0032] In other embodiments, the junction region bonded to the hydrocarbon structure has a structure of chemical formula IIa or IIb.

[0033] In other embodiments, the hydrocarbon structure is non-cylindrical.

[0034] In other embodiments, the lipids can be combined with other lipids in an aqueous solution to assemble into lipid nanoparticles.

[0035] In other embodiments, other vesicle-forming lipids include phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, ceramide, sphingomyelin, or hydrophilic polymer lipid conjugates.

[0036] Also provided are drug delivery vehicle formulations comprising lipids as described in any of the above embodiments incorporated into a lipid bilayer or a monolayer thereof and comprising nucleic acids or peptides.

[0037] In one embodiment, the nucleic acid is a small interfering RNA, a small activating RNA, a messenger RNA, a microRNA, an antisense oligonucleotide, a ribonuclease, an aptamer, a plasmid, circular DNA, linear DNA, antagomir, an anti-miRNA oligonucleotide, or a miRNA mimic.

[0038] In other embodiments, the drug delivery medium formulation comprises lipid nanoparticles (LNPs).

[0039] It also provides a simple method for preparing these lipids.

[0040] This implementation covers a method for preparing hydrocarbon structures for use in delivering molecules of interest, the method comprising:

[0041] (i) providing a hydrocarbon chain having one or more reactive groups on its internal carbon atoms, said reactive groups comprising atoms selected from O, P, N and / or S; and

[0042] (ii) The hydrocarbon chain is conjugated to one or more acyl chains via one or more of the reactive groups to produce the hydrocarbon structure, wherein the shape of the hydrocarbon structure is non-cylindrical.

[0043] In one embodiment, once conjugated with one of the respective acyl chains, one or more of the reactive groups form their respective X1 bonds.

[0044] Lipids produced by the above method are also provided.

[0045] Charged lipids are also provided, which comprise a branched lipid moiety L having the structure of chemical formula I:

[0046] Chemical Formula I:

[0047] A-(V) m -ZL, where

[0048] A is the head group that is physiologically charged at pH;

[0049] (V) m It is optionally -(CR1R2)-, and m is 1 to 10 or 2 to 6, wherein R1 and R2 are independently: hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic or independently selected optionally substituted monocyclic, bicyclic or tricyclic carbocyclic or heterocyclic ring having 4 to 12 ring atoms; and

[0050] ZL has the following chemical formulas II, IIa, or IIb:

[0051] Chemical Formula II direct link head structure:

[0052] X1-L b ,

[0053] Wherein X1 is optional and is selected from ether, ester, and carbamate groups; and

[0054] L b It is a branched-chain lipid with chemical formula IIIc;

[0055] Chemical formula IIa branched linker structure:

[0056]

[0057] W is optional;

[0058] If W exists, W is an X1 bond, NC(O), NC(O)O, or N-OC(O);

[0059] Wherein W is optionally substituted by D, which is optionally substituted alkyl, alkenyl, ynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic;

[0060] Each occurrence of (X) n It is an independently chosen -(CR1R2)-; (X) n n is 0 to 10; and T is optional and is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic, and wherein T is optionally substituted;

[0061] B is a carbon atom connected to L1 and L2 via its own G1 and G2;

[0062] Wherein G1 and G2 are independently selected from X1, and each of G1 and G2 is independently and optionally preceded by (G). u And covalently bonded to (G) u , where G is independently chosen -(CR1R2)-, where R1 and R2 are independently: hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic, and u is 0 to 16;

[0063] Where G3 is optional and selected from X1, and optionally preceded by (G). u And covalently bonded to (G) u ;

[0064] L1 is a branched hydrocarbon with chemical formula IIIc;

[0065] L2 is a hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds or having the chemical formula IIIc.

[0066] If L3 is present, L3 is hydrogen, a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds, or having the structure of chemical formula IIIc.

[0067] Chemical formula IIb ring structure:

[0068]

[0069] The curves represent rings, and E and K show the atoms that partially form the structure of the ring, which is a substituted or unsubstituted ring having 3 to 8 ring atoms;

[0070] At least one of L1, L2, and L3 is optionally bonded to a single atom in the ring via respective G1, G2, and G3, wherein each of G1, G2, and G3 is independently and optionally preceded by (G). u And covalently bonded to (G) u ;

[0071] Wherein L1 of chemical formula IIb and optionally L2 and / or L3 have the structure of chemical formula IIIc:

[0072] Chemical formula IIIc:

[0073]

[0074] The L main chain consists of L1'-L1”-G1-CH-[CH2] q -CH3 indicates that the total number of carbon atoms in the L main chain is between 10 and 30;

[0075] L1' is a straight-chain hydrocarbon chain with 5-20, 6-20, 7-20, 8-20, 5-12, 5-10, 5-9, 6-12, 6-10, 6-9, 7-12, 7-10 or 7-9 carbon atoms and 0-3 cis or trans double bonds;

[0076] L1” is a carbon atom;

[0077] L1”' is represented by G1-CH-CH2-CH3 and where G 1 It is a hydrocarbon chain with 0-4 carbon atoms, optionally having a cis or trans double bond;

[0078] Where n is between 0 and 4;

[0079] Where p ranges from 1 to 4;

[0080] Where n+p ranges from 1 to 4;

[0081] q is between 0 and 20;

[0082] Each X1 is independently selected from ether, ester, and carbamate groups;

[0083] Each S and L1"" hydrocarbon side chain is independently:

[0084] (a) A straight-chain or branched-chain-terminated hydrocarbon chain having 0 to 5 cis or trans C=C and 1 to 30 carbon atoms and being fused to one of the respective X1 at any carbon atom in the hydrocarbon chain; or

[0085] (b) The structure of a branched hydrocarbon of chemical formula IIIc.

[0086] The total number of L1”” and S hydrocarbon chains in chemical formula IIIc is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4 or 1 to 3;

[0087] Each of the L1”” and S hydrocarbon chains in the lipid moiety is optionally replaced by a heteroatom, provided that no more than two heteroatoms are replaced in the hydrocarbon chain.

[0088] In another embodiment, ZL of chemical formula I has the structure of chemical formula II (direct link head structure):

[0089] X1-L b ;

[0090] L1' of chemical formula IIIc has 5 to 9 carbon atoms and 0 to 2 cis or trans double bonds;

[0091] G of chemical formula IIIc 1 It does not exist, CH2 or CH2CH=CH, and the double bond therein is either cis or trans;

[0092] L1”” and S in chemical formula IIIc are independently selected from hydrocarbons having 0-5 cis or trans CH=CH and 2 to 18 carbon atoms;

[0093] The backbone of chemical formula IIIc is represented by CH2-L1”-G1-CH-CH2-CH3 (L1”’ represents 8 to 30 carbon atoms; and

[0094] Where q ranges from 1 to 9.

[0095] In another embodiment, chemical formula I (V) m It is (CH2) m , where m is from 1 to 20;

[0096] ZL has a structure with chemical formula IIa (branched linker structure):

[0097]

[0098] Where W is an ether, ester, or carbamate group (in any orientation) and D is absent, and (X) n It is (CH2) n , where n is from 1 to 10;

[0099] G1 and G2 exist and are each preceded by (G). u And covalently bonded to each (G) u , of which (G) u It is CH2;

[0100] G3-L3 is present and is a hydrocarbon selected from CH3 and CH2CH3; or G3-L3 is CH2X1L3 and L3 is a straight-chain or branched hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds or having the chemical formula IIIc.

[0101] In other embodiments, ZL of chemical formula I has the structure of chemical formula IIb:

[0102] Chemical formula IIb ring structure:

[0103]

[0104] The curves represent rings, and E and K show the atoms that partially form the structure of the ring, which is a substituted or unsubstituted carbon ring having 3 to 6 ring atoms.

[0105] In one embodiment, the ring comprises 3 or 5 carbon atoms. In other embodiments, at least L1 and L2 are present and connected to the ring via respective G1 and G2 groups, and each G1 and G2 group is optionally preceded by Gu, where u is 0 to 10 or 0 to 6.

[0106] The head group A in chemical formula I can be selected from:

[0107] (i) The ionizable cation moiety, selected from:

[0108]

[0109] (ii) Permanently charged portions, selected from:

[0110]

[0111] (iii) The ionizable anionic moiety, selected from:

[0112]

[0113] (iv) The zwitterionic portion, selected from:

[0114]

[0115] In one embodiment, the hydrocarbon structure L of chemical formula I is non-cylindrical.

[0116] In other embodiments, the lipids can be combined with other lipids in an aqueous solution to assemble into lipid nanoparticles. In one embodiment, other vesicle-forming lipids include phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, ceramide, sphingomyelin, or hydrophilic polymer lipid conjugates.

[0117] Also provided are drug delivery vehicle formulations comprising lipids described in any of the above embodiments incorporated into a lipid bilayer or a monolayer thereof and comprising nucleic acids, proteins or peptides.

[0118] In one embodiment, the nucleic acid is a small interfering RNA, a small activating RNA, a messenger RNA, a microRNA, an antisense oligonucleotide, a ribonuclease, an aptamer, a plasmid, circular DNA, linear DNA, antagomir, an anti-miRNA oligonucleotide, or a miRNA mimic.

[0119] In one embodiment, the drug delivery medium comprises a charged peptide.

[0120] In other embodiments, the drug delivery medium is lipid nanoparticles (LNPs).

[0121] This document describes several non-limiting implementations. Attached Figure Description

[0122] Figure 1A The illustration shows an amino lipid as disclosed in this invention, having a simple head group (H) optionally conjugated to a hydrocarbon chain within a lipid moiety L via a linker group. The lipid chain conjugated to the head group is derived from a hydroxy lipid, which in turn conjugates to one or more other lipid chains derived from hydroxy lipids. These lipid chains are further conjugated to one or more hydrocarbon chains derived from acyl lipids.

[0123] Figure 1B The invention discloses an aminolipid prepared by optionally conjugating the head groups of two hydrocarbons within a lipid moiety L via a linker group. The two hydrocarbons conjugated to the head groups are derived from hydroxylipids. Conversely, each of the two lipid chains derived from the hydroxylipids is conjugated to another respective lipid chain derived from the hydroxylipid, and one or more hydrocarbon chains derived from acyllipids are further conjugated to these lipids.

[0124] Figure 2AThe in vitro activity of aminolipids, as evaluated in cultured luciferase-expressing 22Rv1 cells, was demonstrated. Cells were treated with lipid nanoparticles (LNPs) containing the ionizable lipids disclosed in this invention and luciferase siRNA at concentrations of 1.0 (left column), 0.3 (middle column), and 0.1 μg / mL (right column). Luminescence levels were measured after treatment. The ionizable lipids incorporated into LNPs were 2-(dimethylamino)ethyl(±)-syn-9,10-dilinoleic acid oxystearate (INT-A001), 3-(diethylamino)propyl(±)-syn-9,10-dilinoleic acid oxystearate (INT-A002), 3-(dimethylamino)propyl(±)-syn-9,10-dilinoleic acid oxystearate (INT-A003), 2-(diethylamino)ethyl(±)- Syn-9,10-Dilinoleoyloxystearate (INT-A004), 3-(dimethylamino)propyl(12R)-linoleoyloxystearate (INT-A005), 3-(diethylamino)propyl(syn-9,10,12R)-trilinoleoyloxystearate (INT-A006) and 3-(diethylamino)propyl(±)-syn-9,10-bis(2-hexyldecanoyloxy)stearate (INT-A007).

[0125] Figure 2B The in vitro activity of aminolipids at a concentration of 1 μg / mL siRNA is shown in cultured 22Rv1 cells. Luminescence levels were measured 16–24 hours after treatment, and relative luminescence was ranked from high (left panel) to low (right panel).

[0126] Figure 3 The in vivo activity of the aminolipids, as evaluated in a mouse FVII model, was demonstrated. Lipid nanoparticles (LNPs) containing the cationic lipids and FVII siRNA disclosed in this invention were injected via the tail vein into C57BI / 6 wild-type mice, and plasma FVII levels were measured post-injection. Mice were injected with LNP-siRNA at doses of 0.03 (left column), 0.1 (middle column), and 0.3 mg / kg (right column). The cationic lipids incorporated into the LNPs were INT-A001, INT-A002, INT-A003, and INT-A007.

[0127] Figure 4AThe in vitro activity of ionizable lipids, as evaluated in cultured HepG2 hepatocytes, was demonstrated. Cells were treated with an LNP formulation containing the ionizable lipids and luciferase mRNA disclosed in this invention at a concentration of 0.125–1 μg / mL. After treatment, luminescence levels were measured. From left to right, the ionizable lipids incorporating LNP are INT-A001 (blank column), INT-A002 (dotted column), INT-A003 (black column), and INT-A004 (gray column).

[0128] Figure 4B In vivo expression of mRNA in mouse liver is shown. C57BI / 6 wild-type mice were injected via tail vein with a lipid nanoparticle (LNP) formulation containing the cationic lipids and luciferase mRNA disclosed in this invention, and luminescence in the liver was measured after injection. From left to right, the cationic lipids incorporating LNP-mRNA are INT-A001, INT-A002, INT-A003, and INT-A004. Untreated animals were injected with phosphate-buffered saline (PBS). Detailed Implementation

[0129] Structure of lipids for delivery of charged materials

[0130] The lipids described herein have a head group A and a lipid moiety L, said lipid moiety L having a hydrocarbon structure as described below. The head group A is charged at physiological pH and is ionizable in some embodiments, although permanently charged groups are also covered in this disclosure. The head group A may contain other groups that are charged at physiological pH, but have a net overall positive or negative charge at physiological pH. The charged lipids may be monovalent or polyvalent.

[0131] The lipid moiety L typically consists of a hydrocarbon structure having one or more carbon chains that act as a skeleton to bind other hydrocarbon chains or groups within the hydrocarbon structure. In another advantageous embodiment, the lipid moiety L is at least partially composed of a hydrocarbon chain derived from a hydroxyl group or other lipids having functional groups and serves as a skeletal component of the hydrocarbon structure. Conversely, hydroxyl lipids may bind to one or more hydrocarbon chains derived from acyl lipids.

[0132] As used herein, a “skeletal carbon chain” is a carbon chain that provides this skeletal function through covalent bonding with another hydrocarbon chain in the lipid moiety L via a functional group as described herein (e.g., “X1 functional group,” “X1 bond,” or other similar conventions used herein). In one example, the X1 functional group comprises a group having an electronegative atom, such as N, O, S, or P, as an atom in said group, or optionally as the sole atom in said group, and providing covalent bonding of the skeletal carbon chain with one or more hydrocarbon chains, including another skeletal carbon chain. Suitable examples of X1 functional groups are esters, although other groups, including ethers and carbamate groups, will be readily conceived by those skilled in the art.

[0133] The skeletal carbon chain can be derived from precursor lipids having hydroxyl groups as described herein. These lipids are commonly referred to as hydroxylipids and are either naturally occurring or can be synthesized in the laboratory. To generate an ester group, for example, the hydroxyl group of a hydrocarbon chain can be reacted with a carboxylic acid on another hydrocarbon chain via a condensation reaction. However, the methods for preparing hydrocarbon structures are not limited to any specific preparation method, as several different synthetic routes are considered in this paper.

[0134] exist Figure 1A and 1B Examples of structures of charged lipids derived from hydroxylated lipids as a backbone are described. The lipid moiety (L) can be linked to the head group A via one, two, or three hydrocarbon groups within the lipid moiety (L).

[0135] Figure 1A The illustration shows a charged lipid as disclosed in this invention, having a simple head group (A) optionally conjugated to a hydrocarbon within a lipid moiety L via a linker group. In this embodiment, the first lipid is derived from a hydroxy lipid. Conversely, the hydroxy lipid may be conjugated to one or more other lipid chains derived from the hydroxy lipid. These lipid chains are further conjugated to one or more hydrocarbon chains derived from acyl lipids.

[0136] Figure 1B The invention discloses a charged lipid prepared by optionally conjugating the head groups (A) of two hydrocarbons within a lipid moiety L via a linker group. In this example, the two hydrocarbons conjugated to the head groups are derived from hydroxy lipids. Conversely, each of the two lipids is conjugated to another respective lipid derived from a hydroxy lipid, and one or more hydrocarbon chains derived from acyl lipids are further conjugated to these lipids.

[0137] However, it should be understood that Figure 1 is merely an illustration of the selected embodiment and should not be considered as a limitation in any way. For example, as an alternative, the skeletal carbon chain can be generated from fatty acid amines, fatty acid amides, and / or branched fatty acid esters.

[0138] In one embodiment of the present invention, the lipid comprises a head group having a net positive or negative charge at a physiological pH and covalently linked to a lipid moiety via a linker region (also referred to herein as a "linker"), the lipid moiety comprising a hydrocarbon structure having two or more linked hydrocarbon chains, the hydrocarbon chains optionally having a cis or trans C=C configuration, at least one of the chains being covalently linked to an ionizable head group via the linker region, and wherein the hydrocarbon chains are bonded to each other at branch points on the inner carbons of the chains connected to the linker region, the branch points comprising an X1 functional group selected from: -OC(O)-, -C(O)-. -O-, -NR1-, -C(O)N(R1)-, N(R1)C(O)-, -OC(O)O-, -OC(O)N(R1)-, -N(R1)C(O)O-, -S-, -SS-, -C(R1)=NNC(O)-, -C(O)-NN=C(R1), -ON=C(R1)- or -C(R1)=NO-, wherein the hydrocarbon chain has 1 to 30 carbon atoms respectively, wherein the hydrocarbon structure comprises a total of 10 to 150 carbon atoms, and wherein R1 is independently selected from hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic.

[0139] In other embodiments, the hydrocarbon structure of the charged lipid has a non-cylindrical shape. Many phospholipids have hydrocarbon chains (formed by two fatty acid chains) linked to a glycerol backbone that typically forms a cylindrical lipophilic component. However, in some embodiments, the hydrocarbon structures described herein form an "open" structure (also known as a "truncated conical" shape), meaning that the hydrocarbon structure has a diameter at any point along its length that is at least 1.2, 1.5, or 2 times larger than the maximum diameter of the head group. These shapes are advantageous because they can facilitate the transfection of nucleic acids or other charged molecules into cells.

[0140] In another embodiment, the hydrocarbon structure of the charged lipid has three or more hydrocarbon chains, each of which is internally carbon-linked to another chain via the aforementioned X1 bonds. In another embodiment, the lipid moiety has a hydrocarbon structure having 2 to 20 or 3 to 18 conjoined hydrocarbon chains. In one embodiment, the hydrocarbon structure can be described as a lattice or matrix of connected hydrocarbon chains forming an open, non-cylindrical structure. The connection points are optionally either biodegradable functionality or at least 1, 2, 3, 4, or 5 of the connection points are biodegradable, as measured in vivo after administration to a patient.

[0141] While various head groups are considered, in one instance, the head group comprises an ionizable amino group, such as a terminal amine group. In one embodiment, the head group does not contain a phosphate group. In another embodiment, the amine is a primary, secondary, tertiary, or quaternary amine. If a quaternary amine is used in the head group, the lipid may be non-ionizable based on the presence or absence of other ionizable groups in the head group. In another embodiment, the head group is non-zwitterionic.

[0142] It should be understood that each of the X1 bonds in the hydrocarbon chain connecting L can be the same or different. That is, each X1 can be independently selected from the X1 bond groups listed above.

[0143] In one embodiment, the X1 bond is selected from OC(O)-, -C(O)O-, and -O-. In other alternative embodiments, the X1 bond is any biodegradable covalent bond. The X1 bond can also be pH-sensitive, meaning that cleavage depends on the pH of the surrounding solution.

[0144] In other embodiments, the hydrocarbon structure is generated from one or more hydroxy lipids and one or more acyl lipids, wherein the one or more hydroxy lipids act as a skeletal carbon chain to conjugate the one or more acyl lipids.

[0145] In other embodiments, the lipids have an apparent pK between 5.0 and 9.0, or between 5.5 and 8.5, or between 5.0 and 8.0. a .

[0146] In other embodiments, the charged lipids (e.g., cationic or ionizable lipids) described above can be formulated in lipid nanoparticles, including (but not limited to) liposomes.

[0147] In other embodiments, the charged lipid has the structure of Formula I:

[0148] Chemical Formula I:

[0149] A-(V) m -ZL, where

[0150] A is the head group that is physiologically charged at pH;

[0151] (V) m It is optionally -(CR1R2)-, and m is 1 to 10 or 2 to 6, wherein R1 and R2 are independently: hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic or independently selected optionally substituted monocyclic, bicyclic or tricyclic carbocyclic or heterocyclic ring having 4 to 12 ring atoms; and

[0152] ZL has the following chemical formulas: II, IIa, or IIb.

[0153] Chemical formula II has a straight link head structure:

[0154] X1-L b ,

[0155] Wherein X1 is optional and is selected from ether, ester, and carbamate groups; and

[0156] L b It is a branched-chain lipid with the following chemical formula IIIc.

[0157] In another embodiment disclosed in this invention, the charged lipid is an amino lipid and has the following chemical formula Ia:

[0158] Chemical formula Ia:

[0159] R1, R2, and R3 are, independently, hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocyclic.

[0160] Optionally, one of R1, R2 and R3 is either a lone pair or hydrogen.

[0161] Each occurrence of V is independently chosen -(CR1R2)-, and m is 1 to 10 or 2 to 6.

[0162] ZL of chemical formula I or Z-L1 of amino lipids of chemical formula Ia can be represented by the following chemical formulas II, IIa or IIb:

[0163] Chemical Formula II:

[0164] X1-L b ,

[0165] Wherein X1 is optional and, in one embodiment, X1 is selected from ether, ester, and carbamate groups; and

[0166] L b It is a branched lipid with the following chemical formulas III, IIIa, or IIIc.

[0167] Chemical formula IIa:

[0168]

[0169] W is optional;

[0170] If W is present, W is -OC(O)-, -C(O)O-, -O-, -NR1-, -C(O)N(R1)-, N(R1)C(O)-, -NC(O)R1-, -OC(O)O-, -OC (O)N(R1)-, -N(R1)C(O)O-, -S-, -SS-, C(R1)=NNC(O)-, C(O)-NN=C(R1), -ON=C(R1) or C(R1)=NO-;

[0171] Wherein W is optionally substituted by D, which is optionally substituted alkyl, alkenyl, ynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic;

[0172] Each occurrence of (X) n It is an independently chosen -(CR1R2)-, or independently selected from optionally substituted monocyclic, bicyclic, or tricyclic carbon rings or heterocyclic rings having 4 to 12 ring atoms; (X) n n is 0 to 10; and T is optional and optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic;

[0173] B is a carbon, oxygen, or nitrogen atom optionally covalently linked to L1 via G1; and optionally additionally, optionally linked to independently selected L2 and / or L3 via respective G2 and G3 groups;

[0174] Wherein G1, G2, and G3 are independently selected from -OC(O)-, -C(O)O-, -O-, -NR1-, -C(O)N(R1)-, N(R1)C(O)-, -OC(O)O-, -OC(O)N(R1)-, -N(R1)C(O)O-, -S-, -SS-, C(R1)=NNC(O)-, C(O)-NN=C(R1), -ON=C(R1) or C(R1)=NO-, and each of G1, G2, and G3 is independently and optionally preceded by (G). u And covalently bonded to (G) u , where G is independently chosen -(CR1R2)- and u is 0 to 10;

[0175] Chemical formula IIb:

[0176]

[0177] W is optional;

[0178] If W is present, W is -OC(O)-, -C(O)O-, -O-, -NR1-, -C(O)N(R1)-, N(R1)C(O)-, -NC(O)R1-, -OC(O)O-, -OC (O)N(R1)-, -N(R1)C(O)O-, -S-, -SS-, C(R1)=NNC(O)-, C(O)-NN=C(R1), -ON=C(R1) or C(R1)=NO-;

[0179] Wherein W is optionally substituted by D, which is optionally substituted alkyl, alkenyl, ynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocyclic;

[0180] The curves represent rings and E and K show any atoms that partially form the structure of the ring, which is a substituted or unsubstituted monocyclic, bicyclic or tricyclic carbon ring or a monocyclic, bicyclic or tricyclic heteroatom ring having 4 to 12 ring atoms.

[0181] L2 and L3 in chemical formulas Ia and Ib are optional hydrogen atoms or hydrocarbon chains having 1 to 40 carbon atoms and optionally having one or more cis or trans C=C double bonds or structures having chemical formula IIIa (described below);

[0182] Two of L1, L2, and L3 are optionally bonded to a single atom in the ring represented by the curve;

[0183] The above chemical formulas L and L b L1 and optionally L2 and / or L3 are lipid moieties having a structure of Formula III, which will be described shortly below. Alternatively, the lipid moieties may comprise a structure comprising Formula IIIa or Formula IIIc, as described below.

[0184] Chemical Formula III:

[0185]

[0186] L1', L1”, L1”' and L1”” are independently selected from hydrocarbon chains having 1 to 30 atoms, and optionally contain one or more cis or trans C=C;

[0187] where )-, -N(R1)C(O)O-, -S-, -SS-, C(R1)=NNC(O)-, C(O)-NN=C(R1), -ON=C(R1) or C(R1)=NO-; and

[0188] Formula III optionally includes 1 to 10 side chains S, each side chain S being independently selected from a hydrocarbon chain having 1 to 30 atoms, the hydrocarbon chain optionally including one or more cis or trans C=C, or sterols, and wherein at least one S is covalently linked via an X1 bond to a carbon atom of L1', L1”, L1”' and / or L1””.

[0189] The charged lipids (e.g., ionizable lipids) may be a mixture of enantiomers or contain a single optical isomer. As discussed above, the X1 group may be biodegradable, meaning it can be cleaved after administration to a subject. Unrestricted, the ester bond can be hydrolyzed by esterases after administration to a patient, thereby releasing the hydrocarbon chain from the lipid. This disclosure also covers other groups capable of enzymatic hydrolysis or responding to pH changes. However, it will be understood that the X1 group may also be non-biodegradable.

[0190] In one embodiment, the lipid is soluble in a biocompatible alcohol, thereby facilitating its incorporation into a delivery medium and co-encapsulation with nucleic acids such as small interfering RNA, small activating RNA, messenger RNA, microRNA, antisense oligonucleotides, ribonucleases, aptamers, plasmids, circular DNA, straight-chain DNA, antagomir, anti-miRNA oligonucleotides, miRNA mimics, or gene-editing materials.

[0191] Head group (A)

[0192] Lipids are typically conveniently represented as having a head group (referred to herein as "A") covalently linked to one or more hydrocarbon chains. Optionally, head group A is linked to one or more hydrocarbon chains via a linker region. Suitable head groups and linker regions are described below.

[0193] In one embodiment, the head group is selected from ionizable, permanently charged, or zwitterionic moieties. The head group can impart a positive or negative charge to the lipid at a given pH value or range, including pH 7.4 (physiological pH). Examples of chemical groups belonging to each of these categories are shown below:

[0194] (i) Ionizable cation moiety:

[0195]

[0196] (ii) Permanently charged portion:

[0197]

[0198] (iii) Ionizable anionic portion:

[0199]

[0200] (iv) Zwitterionic portion:

[0201]

[0202] Typically, the head group A of the lipid is relatively small and charged at a physiological pH. In one non-limiting embodiment, such a head group comprises a terminal ionizable amino group, although groups such as phosphate and sulfate groups are also considered herein. In another embodiment, the head group has an apparent pK of 5 to 8 that imparts to the ionizable lipid. a Alternatively, impart a pK of 5.5 to 7.5 to the ionizable lipid. a The terminal amino group is ionizable. The pK of the amino group will be understood. a It can be influenced by adjacent atoms in the head group. Head groups containing sulfates or phosphates can impart an overall negative charge to the lipid.

[0203] The amino group can be a primary, secondary, or tertiary amino group. The head group can contain other amino groups, such as two or three identical or different amino groups.

[0204] In one embodiment, the amine moiety of the head group has the following chemical formula:

[0205]

[0206] R1, R2, and R3 are, independently, hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocyclic; wherein one of R1, R2, and R3 is absent (lone pair) or hydrogen.

[0207] In those embodiments where the head group is positively charged, the group does not have a phosphate ester group, because such a group imparts a negative charge and could therefore result in electrically neutral lipids. However, such a group may be included in the head group, provided that the overall charge of the lipid is positive at a physiological pH.

[0208] Optionally, the head group is conjugated to a hydrophilic polymer, including polymers that improve circulatory life after the lipid or its formulation is administered to a patient. A non-limiting example of such a hydrophilic polymer is polyethylene glycol (PEG).

[0209] As those skilled in the art will understand, the lipid can exist as a salt when the head group is charged. Any pharmaceutically acceptable salt is included within the scope of this disclosure.

[0210] Optional connector area

[0211] In addition to ionizable amine groups, the lipid may contain a region having one or more electronegative atoms that allow covalent attachment to one, two, or three lipid chains, at least one of which is a skeletal carbon chain. This region in the lipid may be referred to as a connector region or simply a “connector” and is optional, as one or more hydrocarbon chains may be directly attached to the head group.

[0212] The chemical structure of the connector may depend on the number of lipid chains attached to the head group, but may include ester, ether, glycerol ester connectors, derivatives of glycerol ester connectors, or ring connectors, including multi-rings composed of carbon or heteroatoms.

[0213] The linker region attached to the head group can have the following chemical formula:

[0214]

[0215] Where (V) m -Z shows a junction region that is connected to a head group A at one end and to L1, a hydrocarbon region, at the other end.

[0216] The chemical formula Z-L1 can be represented by the chemical formula II, chemical formula IIa, or chemical formula IIb described above.

[0217] Optionally, the connector region is fitted to a hydrophilic polymer, including polymers that improve cycle life, such as polyethylene glycol (PEG).

[0218] As those skilled in the art will understand, this invention discloses a variety of different combinations of head groups and linker groups. The linker region may be characterized by the number of hydrocarbon chains it is connected to. Alternatively, the linker may be described as straight-chain, branched, or cyclic. Examples of each category are provided below.

[0219] The following are non-limiting examples of head groups having a connector region linking a single hydrocarbon chain:

[0220]

[0221] The following are non-limiting examples of head groups having junction regions connecting two hydrocarbon chains:

[0222]

[0223] The following are non-limiting examples of head groups with junction regions connecting three hydrocarbon chains:

[0224]

[0225] The linker region can also be described as linear, branched, or cyclic. Examples of linear, branched, and cyclic linker regions are described in chemical formulas II, IIa, and IIb described above, respectively.

[0226] lipid fraction L

[0227] The lipid moiety L may include one, two, or three hydrocarbon chains within a hydrocarbon structure that is directly or via a linker region to the head group. In embodiments where only one hydrocarbon chain is connected to the head group or linker region, other hydrocarbon side chains S may be connected to the inner carbon of L1.

[0228] Therefore, in at least one embodiment, the head group has been attached to or via a connector to L1, which is a lipid moiety having a structure of formula III:

[0229] Chemical Formula III:

[0230]

[0231] L1', L1”, L1”', and L1”” are independently selected from hydrocarbon chains having 1 to 30 atoms or 1 to 20 atoms, optionally comprising one or more cis or trans C=C arrangements; and

[0232] where )-, -N(R1)C(O)O-, -S-, -SS-, C(R1)=NNC(O)-, -C(O)-NN=C(R1), -ON=C(R1) or -C(R1)=NO-.

[0233] Optionally, the head group is attached, optionally via a linker, to the L2 or other L3 lipid moiety as a lipid moiety. The optional L2 and L3 lipid moieties may be independently selected from hydrocarbon chains having 1 to 30 atoms or 1 to 20 atoms (the hydrocarbon chain optionally comprising one or more cis or trans C=C), or sterols. L2 and / or L3 may optionally be lipids of the following chemical formula IIIa.

[0234] Chemical formula IIIa:

[0235]

[0236] Ln' is covalently connected to the connector.

[0237] According to the nomenclature of chemical formula IIIa, if L is L2, then n is 2, and if L is L3, then n is 3. For example, for L2 hydrocarbons, chemical formula IIIa is named as follows:

[0238]

[0239] Chemical formula III or IIIa optionally contains 1 to 20 side chains S, described as (S). n , where n is 0 to 20, each independently selected from hydrocarbon chains having 1 to 30 atoms or 1 to 20 atoms, said hydrocarbon chains optionally comprising one or more cis or trans C=C, or sterols, and wherein (S) n The X1 bond is covalently linked to carbon atoms of L1, L2, and / or L3 via an X1 bond, or covalently bonded to another side chain S in the hydrocarbon structure via such a bond. In one embodiment, the X1 bond is biodegradable, such as an ester bond. However, other bonds besides those that are biodegradable may be used in the practice of this invention.

[0240] For example, L1 may have a side chain S1' attached to any one of L1', L1”', or L1”” via an X1 bond. Other side chains S1”, S1”', or S1”” may be attached to the carbon of L1 or any S1 side chain. In another example, if L2 has the structure of the above chemical formula IIIa, then L2 may have a side chain S2' attached to any one of L2', L2”', or L2””. Other side chains S2”, S2”', or S2”” may be attached to the carbon of L2 or any S2 side chain. Similarly, if L3 is present and has the above chemical formula IIIa, then it may have a side chain S3' attached to any one of L3', L3”', or L3””. Other side chains S3”, S3”', or S3”” may be attached to the carbon of L3 or any S3 side chain.

[0241] Table 1 below shows examples of lipids having the structures covered by this disclosure. Structure AF in Table 1 includes a head group having a linker to a single hydrocarbon having an X1 branch point that provides skeletal connection points for other S hydrocarbon chains. Structure GJ in Table 1 includes a head group having a linker connecting two hydrocarbons L1 and L2. The lipid described as structure KM has a linker having connection points to three hydrocarbons L1, L2, and L3.

[0242] The following note, structure A, illustrates the convention used in this text for showing the hydrophobic regions of X1 and L1 in formula III. It will be understood that each occurrence of X1 is independently selected from another X1 in the structure described. As discussed, the X1 group includes any suitable functional group with an electronegative atom.

[0243] The example shown is an aminolipid having a single L1 lipid hydrocarbon chain (L1', L1”, and L1”') according to the above chemical formula III, having a side chain S attached to the carbon atom of L1:

[0244] Structure A (INT-A001):

[0245]

[0246] As can be seen in structure A (INT-A001) above, L1', L1”, and L1”' together form a straight-chain hydrocarbon backbone derived from di-hydroxylipids (referred to herein as the “skeleton carbon chain”). This hydrocarbon chain L1'-L1”-L1”' connects L1”” to the L1” carbon of L1'-L1”-L1”' via an X1 bond, and attaches a second S “side chain” hydrocarbon chain to L1”' via a second X1 bond. In this structure, both X1 bonds are ester groups. However, as mentioned above, the X1 functional group can be independently selected from various functional groups containing O, N, P, and / or S atoms.

[0247] Table 1 below provides examples of lipids having a connector region within the head group that links to one, two, or three hydrocarbon chains. For each lipid, substituents of formula III, L1' / L1” / L1”' / L1””, and optionally L2 and L3 hydrocarbon chains are provided. Structures of head groups and connector regions as defined by formula IIa or IIb are also provided. In each case, the X1 group is an ester that links the hydrocarbon chains together to form an interconnected hydrocarbon lattice structure. As can be seen from the following structures, the interconnected hydrocarbon structures of L form a generally open or truncated conical shape.

[0248] Table 1: Selected amino lipids having open hydrocarbon structures formed by connecting to 1, 2, or 3 hydrocarbon chains L1, L2, and L3.

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] The alternative name used to describe the lipid portion is chemical formula IIIc:

[0257] Chemical formula IIIc:

[0258]

[0259] The L main chain consists of L1'-L1”-G1-CH-[CH2] q -CH3 indicates that the total number of carbon atoms in the L main chain is between 10 and 30;

[0260] L1' is a straight-chain hydrocarbon chain with 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 5-12, 5-10, 5-9, 6-12, 6-10, 6-9, 7-12, 7-10 or 7-9 carbon atoms and 0-3 cis or trans double bonds;

[0261] L1” is a carbon atom;

[0262] L1”' is represented by G1-CH-CH2-CH3;

[0263] G 1 It is a hydrocarbon chain with 0-4 carbon atoms, which optionally has a cis or trans double bond;

[0264] Where n is between 0 and 4;

[0265] Where p ranges from 1 to 4;

[0266] Where n+p is 1 to 6 or 1 to 4;

[0267] q can be 0 to 20, 0 to 10, or 1 to 5;

[0268] Each X1 is any suitable X1 group described above, or is independently selected from ether, ester and carbamate groups;

[0269] Each S and L1"" hydrocarbon side chain is independently:

[0270] A straight-chain or branched-chain-terminated hydrocarbon chain having 0 to 5 cis or trans C=C and 1 to 30 or 2 to 18 carbon atoms and being fused to one of their respective X1s at any carbon atom in the hydrocarbon chain; or

[0271] The branched structure of chemical formula IIIc

[0272] The total number of L1”” and S hydrocarbon chains in chemical formula IIIc is 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4 or 1 to 3;

[0273] Each of the L1”” and S hydrocarbon chains in the lipid moiety is optionally replaced by a heteroatom, provided that no more than 4, 3 or 2 heteroatoms are replaced in the hydrocarbon chain.

[0274] The X1 ester group can be oriented in any position relative to the carbonyl group, as shown below:

[0275]

[0276] *To clarify the description

[0277] G 2 Orientation markers of the middle ester

[0278] In one embodiment, the total number of carbon atoms in chemical formula IIIc does not exceed 150, 125, 100, 90, 80, 70, 60 or 50.

[0279] In another embodiment, the structure of formula IIIc is in a non-conical or open shape, as defined by the region adjacent to the head group or connector region toward the distal carbon atom on the hydrocarbon structure. As mentioned, these structures facilitate the delivery of cargo molecules when lipids are formulated in a delivery medium.

[0280] skeletal carbon chain

[0281] In one embodiment, the main chain hydrocarbon chain of the L1, L2, and / or L3 lipid moieties that provide skeletal function is derived from fatty acids having functional groups for bonding to side chains S. This includes fatty acids substituted with groups having atoms selected from O, N, P, and / or S. These groups facilitate the attachment of one or more side chains to the main chain carbons of the L1, L2, and / or L3 that constitute the skeleton.

[0282] For example, L1, L2, and / or L3 can be derived from hydroxy fatty acids (HFAs), which are fatty acids having an OH group bonded at any position on their carbon chain. Without limitation, the HFA can be a β-hydroxy fatty acid, an ω-hydroxy fatty acid, or any (ω-1)-hydroxy fatty acid, or any other HFA having reactive functionality at an inner carbon position in the carbon backbone. The HFA can be saturated or unsaturated. Two or more hydroxyl functional groups may also be present on the carbon chain.

[0283] Alternatively, L1, L2, and / or L3 are derived from branched-chain fatty acid esters of HFAs known in the art, such as fatty acid esters of hydroxy fatty acids (FAHFA). These fatty acid esters contain branched ester bonds between the fatty acid and the HFA. For example, 9-[(9Z)-oleoyloxy]octadecanoic acid is a fatty acid ester obtained by the condensation reaction of the carboxyl group of oleic acid with the hydroxyl group of 9-hydroxystearic acid.

[0284] In an alternative embodiment, L1, L2, and / or L3 are derived from fatty acid amides, which may contain ethanolamine as an amine component. Alternatively, L1, L2, and / or L3 may be derived from fatty acid amines.

[0285] The carbon skeleton of Formula III can be derived from fatty acids other than those described above. Furthermore, it will be understood that these fatty acids can, in turn, be derived from their corresponding triglycerides.

[0286] In chemical formulas III, IIIa, and IIIb, L1', L1”, and L1”' together form a straight-chain hydrocarbon backbone (referred to herein as the “skeletal carbon chain”). According to the above chemical formula IIIc, through L1'-L1”-G 1 -CH-[CH2] q -CH3 represents a skeletal carbon chain, in which the total number of carbon atoms in the skeleton is between 10 and 30.

[0287] preparation

[0288] Cationic or ionizable lipids facilitate the encapsulation of nucleic acids, including (but not limited to) small interfering RNA, small activating RNA, messenger RNA, microRNA, antisense oligonucleotides, ribonucleases, aptamers, plasmids, circular DNA, linear DNA, antagomir, anti-miRNA oligonucleotides and miRNA mimics and / or gene-editing materials. Alternatively, negatively charged proteins and amino acids may be introduced into the delivery medium.

[0289] The charged lipids described herein can be used to deliver other charged molecules besides nucleic acids. This includes a variety of positively or negatively charged peptides, proteins, polysaccharides, or carbohydrates, including both bioactive agents and prodrugs, examples of which are described below.

[0290] The cationic or ionizable lipids described herein can be administered in free form along with nucleic acids or other negatively or positively charged cargo molecules, or these components can be introduced into a delivery medium. Various delivery systems can be used to prepare pharmaceutical formulations. If the charged lipids and associated charged molecules are in free form, a drug-grade salt or excipient can be included in the pharmaceutical formulation.

[0291] The lipids disclosed in this invention are particularly suitable for incorporation into nanoparticles, such as liposomes, or polymer-based systems containing lipids or other hydrophobic components (referred to herein as "lipid nanoparticles" or "LNPs").

[0292] For example, in some embodiments, the loading efficiency into a given lipid nanoparticle is 60% to 100%, 70% to 100%, or most advantageously 80% to 100%.

[0293] In one embodiment, lipids are loaded into lipid nanoparticles, such as liposomes, by mixing them with lipid formulation components, including vesicle-forming lipids and optionally sterols. Therefore, a variety of well-described formulation methods known to those skilled in the art, including (but not limited to) extrusion, ethanol injection, and in-line mixing, can be used to prepare lipid nanoparticles incorporating ionizable or cationic lipids. These methods are described in Maclachlan, I. and P. Cullis, "Diffusible-PEG-lipid Stabilized Plasmid Lipid Particles", Adv. Genet., 2005, 53PA:157-188; Jeffs, LB et al., "A Scalable, Extrusion-free Method for Efficient Liposomal Encapsulation of Plasmid DNA", Pharm Res, 2005, 22(3):362-72; and Leung, AK et al., "Lipid Nanoparticles Containing siRNA synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core", The Journal of Physical Chemistry. C, Nanomaterials and Interfaces, 2012, 116(34):18440-18450. The entire contents of each of these references are incorporated herein by reference.

[0294] In addition to charged lipids, other lipid components that may be included in the lipid nanoparticles include vesicle-forming lipids such as phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, ceramide, or other lipids. Cholesterol may also be included in the LNP to broaden the phase transition temperature. The LNP may also include lipids conjugated with hydrophilic polymers, such as distearate-phosphatidylethanolamine-PEG. Surface stabilizing functionalities, such as those of the hydrophilic polymer, may be desirable to reduce post-application clearance of the nanoparticles. The LNP formulation may also include fusogenic lipids, which facilitate the fusion of the delivery medium with target cells via endocytosis.

[0295] Suitable LNPs include (but are not limited to) liposomes or multilayer vesicles (MLVs) prepared by extrusion using known methods. The internal space of the liposome may include a trapping agent, such as a drug, or one or more such agents that can be contained and dispensed within a bilayer.

[0296] Another example of a suitable LNP delivery system is a stable nucleic acid lipid particle, known as a SNALP. SNALPs can contain nucleic acids bound to cationic lipids, non-cationic lipids, and optionally hydrophilic polymer lipid conjugates, such as PEGylated lipids and fusion-promoting lipids.

[0297] Lipid nanoparticles may contain a lipophilic core. For example, the delivery medium may also be nanoparticles containing a lipid core stabilized by a surfactant. Vesicle-forming lipids may be used as stabilizers. In another embodiment, the lipid nanoparticles are polymer-lipid hybridization systems containing a polymer nanoparticle core surrounded by stabilized lipids.

[0298] Alternatively, nanoparticles can be prepared from polymers without lipids. These nanoparticles may contain a concentrated drug core surrounded by a polymer shell, or they may have a solid or liquid dispersed throughout the polymer matrix.

[0299] The lipids described herein can also be incorporated into emulsions, which are drug delivery media containing oil droplets or oil cores. The emulsions can be lipid-stabilized. For example, an emulsion can contain an oil-filled core stabilized by a monolayer or bilayer of emulsifying components, such as lipids.

[0300] The lipids described herein can also be formulated in micelles. Micelles are self-assembled particles composed of amphiphilic lipid or polymer components, used for the delivery of reagents present in a hydrophobic core.

[0301] Other types of drug delivery media known to those skilled in the art who can formulate charged lipids include carbon nanotubes.

[0302] Various methods for preparing the above-mentioned delivery media and the incorporation of charged lipids therein are known and can be readily performed by those skilled in the art.

[0303] Certain lipids covered by this invention can form part of a carrier-free system. In these embodiments, lipids associated with negatively charged molecules can self-assemble into particles. An example is the formation of liposome complexes, which are combinations between DNA and cationic lipids. These formulations may optionally include pharmaceutically available salts and / or excipients.

[0304] Delivery media that introduce cationic or ionizable lipids may also include active agents introduced into the media, such as anticancer drugs or other therapeutic agents, including prodrugs.

[0305] The delivery medium may optionally include lipoproteins, such as adipoproteins.

[0306] Delivery of nucleic acids, genetic materials, proteins, peptides or other charged reagents

[0307] As discussed herein, the charged lipids disclosed herein facilitate the introduction of molecules with a net negative or positive charge (referred herein as “cargo” or “cargo molecules”) into a delivery medium and subsequent in vitro or in vivo delivery to target cells.

[0308] In one embodiment, the molecule is genetic material, such as nucleic acid. The nucleic acid includes, without limitation, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), microRNA (miRNA), or DNA, such as plasmid DNA or straight-stranded DNA. The length of the nucleic acid can vary and may include nucleic acids with a length of 5-50,000 nucleotides. The nucleic acid can be in any form, including single-stranded DNA or RNA, double-stranded DNA or RNA, or hybrids thereof. Single-stranded nucleic acids include antisense oligonucleotides.

[0309] In a particularly advantageous embodiment, the cargo is siRNA. siRNA is incorporated into endogenous cellular machinery to cause mRNA disruption, thereby preventing transcription. Since RNA is readily degraded, its incorporation into the delivery medium can reduce or prevent such degradation, thereby facilitating delivery to the target site.

[0310] Gene editing systems can also be introduced into delivery media containing charged lipids. This includes Cas9-CRISPR, TALEN, and zinc finger nuclease gene editing systems. In the case of Cas9-CRISPR, guide RNA (gRNA) can be introduced together with a plasmid or mRNA encoding the Cas9 protein into a delivery media containing cationic lipids as described herein. Optionally, ribonucleoprotein complexes can be introduced into delivery media containing cationic lipids as described herein. Similarly, the present invention discloses embodiments in which gene material encoding the DNA binding and cleavage domains of zinc finger nucleases or TALEN systems are introduced into delivery media together with ionizable or cationic lipids.

[0311] The charged lipids can also facilitate the incorporation of proteins and peptides with an overall charge into the delivery medium. This includes both linear and non-linear peptides. Examples of peptides include bacterial / antibiotic peptides, fungal peptides, invertebrate peptides, amphibian / skin peptides, snake venom peptides, cancer / anti-cancer peptides, vaccine peptides, immune / anti-inflammatory peptides, brain peptides, endocrine peptides, digestive peptides, gastrointestinal peptides, cardiovascular peptides, renal peptides, respiratory peptides, opioid peptides, neurotrophic peptides, and blood-brain peptides. Specific examples of peptides are provided above.

[0312] Specific examples of peptides that can bind to the charged lipids described in this article are interferons and other macrophage activating factors. This includes lymphokines, muramyl dipeptide (MDP), gamma-interferon, alpha-interferon, and beta-interferon, and related antiviral and tumor-killing agents; opioid peptides and neuropeptides, including enkephalin, endorphin, and dynorphin, and other analgesics; renin inhibitors, including (for example) antihypertensive agents; cholecystokinin (CCK) analogs, such as CCK, blue peptide, and octopus toxin, and related cardiovascular and CNS-targeting agents; leukotrienes and prostaglandins, including oxytocin, and other anti-inflammatory, oxytocin, and abortifacient compounds; erythropoietin and its derivatives, and related hematologic agents; LHRH analogs, such as leuprolide, buserrelin, and nafarelin, and related pituitary receptor downregulators; parathyroid hormone and other growth hormone analogs; enzymes, such as DNase, catalytic enzymes, and alpha-I antitrypsin; immunosuppressants, such as cyclosporine; GM-CSF and other immunomodulators; and insulin.

[0313] application

[0314] In some embodiments, charged lipids (which are free or formulated in a drug delivery medium) bound to nucleic acids or other charged molecules are administered to treat, prevent, and / or improve a patient's condition. Specifically, administering charged lipids, either in free form or formulated in a delivery medium, together with nucleic acids or other charged materials can provide preventative, ameliorative, or therapeutic benefits. Pharmaceutical compositions containing said charged lipids are administered at any suitable dose. In one embodiment, the lipids, whether in free form or formulated in a drug delivery medium, are administered parenterally, i.e., intra-arterial, intravenous, subcutaneous, or intramuscular. In other embodiments, the lipids described herein, whether in free form or formulated in a delivery medium, can be administered topically. In other alternative embodiments, the lipids described herein, whether in free form or formulated in a delivery medium, can be administered orally. In other embodiments, the lipids, whether in free form or formulated in a delivery medium, are administered to the lungs via an aerosol or powder dispersion system.

[0315] The compositions described herein can be administered to any subject, including “patients”, which, as used herein, include human or non-human subjects.

[0316] In some embodiments, the lipids described herein are used on cells, including in vitro transfection of stem cells derived from patients and cultured cells. In one embodiment, the transfected cells are stem cells and said cells are administered back to the patient from whom they were previously obtained.

[0317] The following embodiments are provided for illustrative purposes only and not to limit the scope of the invention.

[0318] Example

[0319] Materials and methods

[0320] For the organic synthesis reactions described in Example 1, unless otherwise stated, all reagents and solvents, except for THF (freshly distilled from Na / benzophenone under nitrogen) and Et3N, DMF, and CH2Cl2 (freshly distilled from CaH2 under nitrogen), were purchased from commercial suppliers and used without further purification. USP grade castor oil was purchased from a local pharmacy (LifeBrand) and used directly. NMR chemical shifts are reported in parts per million (ppm) on the δ-scale, and coupling constant J is reported in Hertz (Hz). Spectra are referenced to residual solvent signals. Multiplets are reported as “s” (singleton), “d” (doublet), “t” (triplet), “q” (quartet), “quint” (quintet), “sept” (septet), and “m” (multiplex), and further defined by “app” (apparent) and “br” (broad peak).

[0321] Lipid nanoparticles (LNPs) were prepared using neutral lipids, 1,2-distearate-sn-glycero-3-choline phosphate (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DMPE), sterols, and cholesterol. DSPC and PEG-DMPE were purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol was obtained from Sigma (St. Louis, MO).

[0322] LNPs were characterized by measuring particle size and polydispersity (PdI). Particle size and polydispersity were determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). LNPs were diluted to appropriate concentrations in PBS. Number-weighted size and distribution data were used in the assays, and formulations with PdI > 0.15 were not used for further studies.

[0323] Lipid concentration was determined by measuring total cholesterol using a cholesterol E enzymatic assay kit from Wako Chemicals USA (Richmond, VA).

[0324] Encapsulation efficiency of RNA or antisense oligonucleotides was determined using either Quant-iT Ribogreen RNA or Oligreen ssDNA assays (Life Technologies, Burlington, ON). Briefly, LNPs were incubated at 37°C for 10 min with or without 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO), followed by the addition of Ribogreen or Oligreen reagent. Fluorescence intensity (Ex / Em: 480 / 520 nm) was determined, and samples treated with Triton X-100 represented total nucleic acids, while untreated samples represented unencapsulated nucleic acids.

[0325] According to the manufacturer's instructions, protein / peptide quantification is performed using the BCA protein assay (Pierce) or the CBQCA protein quantification kit (Invitrogen).

[0326] The apparent acid dissociation constant (pK) of the LNP system was determined according to the procedure described in the literature (Jayaraman, M. et al., Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angewandte Chemie, 2012, 51(34): p. 8529-33). a Briefly, 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS, Sigma-Aldrich, St. Louis, MO) and LNP were diluted in buffers (10 mM HEPES, 10 mM MES, and 10 mM ammonium acetate) with a pH range of 2.5 to 11. The final concentration of TNS or total lipids was 6 μM. The samples were then mixed and fluorescence intensity (Ex / Em: 321 / 445 nm) was measured using a Perkin Elmer LS55. S-shaped best-fit analysis was applied and pK... a The pH was measured at half maximum fluorescence intensity.

[0327] Example 1: Synthesis of Ionizable Lipids

[0328]

[0329] Esterification of A-hydroxy fatty acids using conventional procedures

[0330] Hydroxylated fatty acids (1.00 equivalents) were suspended in MeOH (0.4-0.5 M) in a round-bottom flask equipped with a condenser. Concentrated sulfuric acid (0.05 equivalents) was added to the mixture, and the result was refluxed and heated until homogenized within 5-15 min. After 16 h, residual MeOH was removed on a rotary evaporator, and the remaining residue was partitioned between ethyl acetate (EtOAc) and a saturated aqueous solution of NaHCO3. The aqueous layer was extracted twice with EtOAc, and the combined organic layers were washed once with water and brine. The mixture was dried with Na2SO4, filtered by gravity, and concentrated on a rotary evaporator to provide a white powder, which was not further purified before use.

[0331] Acylation of β-hydroxy fatty acid esters using conventional procedures

[0332] Under argon atmosphere, N,N'-dicyclohexylcarbodiimide (DCC; 1.00 equivalent per hydroxyl group + an additional 0.10 equivalent) was added to a cold CH2Cl2 (0.3 M) solution of the desired carboxylic acid (1.00 equivalent per hydroxyl group + an additional 0.10 equivalent) in a round-bottom flask. The ice bath was then removed and the resulting mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath, and a solid hydroxy fatty acid (1.00 equivalent) was added, followed by 4-dimethylaminopyridine (DMAP; 1.00 equivalent per hydroxyl group + an additional 0.50 equivalent). The reaction mixture was heated to room temperature over 16 h, diluted with hexane, stirred for 10 min, and then passed through… Pad filtration. The filtrate is concentrated on a rotary evaporator to obtain a crude mixture, from which the desired acylated material is purified by rapid column chromatography.

[0333] Saponification of C-peracylated fatty acid methyl esters using conventional procedures

[0334] Under argon atmosphere, an aqueous solution of NaOH (2.0 M, 1.00 equivalent) was added to a room-temperature t-BuOH (0.3 M) solution of acylated fatty acid methyl ester (1.10 equivalent) in a round-bottom flask. After stirring for 16 h, the reaction mixture was acidified to pH ≤ 2 by adding an aqueous solution of HCl (2.0 M) and extracted three times with hexane. The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated on a rotary evaporator to provide a colorless, oily crude product. The crude product was purified by rapid column chromatography to provide the desired fatty acid.

[0335] Conventional procedure D-esterification of peracylated fatty acids with amino alcohols

[0336] Under argon atmosphere, DCC (1.10 equivalents) was added to an ice-cold CH2Cl2 (0.2 M) solution of fatty acids (1.00 equivalents) in a round-bottom flask. The ice bath was removed and the mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath, and pure amino alcohol (1.20–2.00 equivalents) was added, followed by DMAP (1.20 equivalents), and the reaction mixture was heated to room temperature over 16 h. The reaction mixture was diluted with Et2O, stirred for 10 min, and then passed through an argon atmosphere. Pad filtration. The filtrate was concentrated on a rotary evaporator to obtain crude oil, which was then purified by rapid column chromatography to provide the desired peracylated amino ester.

[0337]

[0338] Methyl(12R)-linoleoxyoleate

[0339] According to standard procedure B, methyl ricinoleate (500 mg, 1.60 mmol), linoleic acid (538 mg, 1.92 mmol, 1.20 equivalent), DCC (396 mg, 1.20 mmol, 2.20 equivalent) and DMAP (293 mg, 2.40 mmol, 1.50 equivalent) in CH2Cl2 (5 mL) yielded a clear, colorless oily title compound (875 mg, 93% yield).

[0340] 1 H(300MHz, CDCl3):5.55-5.28(m,6H),4.90(quint,J=6.2Hz,1H),3.69(s,3H),2.79(t,J=5.8Hz,2H ),2.40-2.21(m,6H),2.16-1.93(m,6H),1.72-1.46(m,8H),1.46-1.18(m,32H),1.00-0.80(m,6H).

[0341]

[0342] Methyl(±)-syn-9,10-dilinoleic acid oxystearic acid ester

[0343] According to standard procedure B, dihydroxystearate (1.32 g, 4.00 mmol), linoleic acid (2.47 g, 8.80 mmol, 2.20 equivalent), DCC (1.82 g, 8.80 mmol, 2.20 equivalent) and DMAP (1.22 g, 10.0 mmol, 2.50 equivalent) in CH2Cl2 (10 mL) yielded a clear, colorless oily title compound (2.64 g, 77% yield).

[0344] 1 H(300MHz, CDCl3):5.47-5.28(m,8H),5.04-4.94(m,2H),3.68(s,3H),2.79(t,J=5.8Hz,4H),2. 35-2.25(m,6H),2.11-2.00(m,8H),1.69-1.47(m,10H),1.44-1.16(m,48H),0.97-0.84(m,9H).

[0345]

[0346] Methyl(±)-syn-9,10-bis(2-hexyldecanoyloxy)stearate

[0347] According to standard procedure B, dihydroxystearate (2.31 g, 7.00 mmol), (±)-2-hexyldecanoic acid (3.77 g, 14.7 mmol, 2.10 equivalents), DCC (3.03 g, 14.7 mmol, 2.10 equivalents) and DMAP (2.14 g, 17.5 mmol, 2.50 equivalents) in CH2Cl2 (18 mL) provided a clear, colorless oily title compound (4.20 g, 74% yield).

[0348] 1 H(300MHz, CDCl3):5.06-4.96(m,2H),3.68(s,3H),2.31(t,J=7.6Hz,2H),1.69-1.50(m,10H),1.50-1.16(m,64H),0.94-0.84(m,15H).

[0349]

[0350] Methyl (syn-9,10,12R)-trinyl oxystearate

[0351] According to standard procedure B, trihydroxystearate (1.04 g, 3.00 mmol), linoleic acid (2.61 g, 9.30 mmol, 3.10 equivalents), DCC (1.92 g, 9.30 mmol, 3.10 equivalents) and DMAP (1.28 g, 10.5 mmol, 3.50 equivalents) in CH2Cl2 (10 mL) yielded a clear, colorless oily title compound (2.33 g, 68% yield).

[0352] 1H(300MHz, CDCl3):5.49-5.26(m,12H),5.14-4.84(m,3H),3.68(s,3H),2.79(brt,J=6.0Hz,6H),2 .37-2.21(m,8H),2.14-1.99(m,12H),1.92-1.47(m,16H),1.47-1.16(m,56H),0.96-0.84(m,12H).

[0353]

[0354] (12R)-Linoleic acid oxyoleic acid

[0355] Following standard procedure C, after rapid column chromatography (SiO2, 95:5:0→90:10:0→85:12:3 hexane / EtOAc / MeOH), acyl methyl ester (5.97 g, 10.4 mmol, 1.10 equivalent), NaOH aqueous solution (2.0 M, 4.70 mL, 1.00 equivalent), and t-BuOH (26 mL) provided a clear, colorless oily solution of the title compound (4.48 g, 85% yield).

[0356] 1 H(300MHz, CDCl3):5.55-5.28(m,6H),4.90(quint,J=6.2Hz,1H),2.79(t,J=6.0Hz,2H),2.4 3-2.21(m,6H),2.14-1.96(m,6H),1.73-1.47(m,6H),1.46-1.18(m,30H),0.99-0.81(m,6H).

[0357]

[0358] (±)-syn-9,10-dilinoleic oxystearic acid (INT-A008)

[0359] Following standard procedure C, after rapid column chromatography (SiO2, 95:5:0→90:10:0→85:12:3 hexane / EtOAc / MeOH), diacylmethyl ester (1.88 g, 2.20 mmol, 1.10 equivalent), NaOH aqueous solution (2.0 M, 1.00 mL), and t-BuOH (7 mL) provided a clear, colorless oily INT-A008 (1.50 g, 89% yield).

[0360] 1H(300MHz, CDCl3):5.47-5.28(m,8H),5.04-4.94(m,2H),2.79(br t,J=6.0Hz,4H),2.36(t,J=7.0Hz,2H),2.30(t,J=7.5Hz,4H),2.12-1.98(m,8H),1.72-1.46(m,10H),1.44-1.16(m,48H),0.97-0.84(m,9H).

[0361]

[0362] (syn-9,10,12R)-Trinyl oleate oxystearic acid

[0363] Following standard procedure C, after rapid column chromatography (SiO2, 90:10:0 → 80:17:3 hexane / EtOAc / MeOH), triacylmethyl ester (2.41 g, 2.12 mmol, 1.05 equivalent), NaOH aqueous solution (2.0 M, 1.01 mL), and t-BuOH (7 mL) provided a clear, colorless, oily triacylated fatty acid (1.37 g, 60% yield).

[0364] 1 H(300MHz, CDCl3):5.47-5.26(m,12H),5.14-4.84(m,3H),2.79(br t,J=6.0Hz,6H),2.41-2.20(m,8H),2.14-1.99(m,12H),1.92-1.47(m,16H),1.47-1.16(m,56H),0.96-0.84(m,12H).

[0365]

[0366] (±)-syn-9,10-bis(2-hexyldecanoyloxy)stearic acid (INT-A009)

[0367] Following standard procedure C, after rapid column chromatography (SiO2, 95:5:0→90:10:0→85:12:3 hexane / EtOAc / MeOH), diacylmethyl ester (4.20 g, 5.21 mmol, 1.05 equivalents), NaOH aqueous solution (2.0 M, 2.48 mL), and t-BuOH (7 mL) provided a clear, colorless oily INT-A009 (3.12 g, 79% yield).

[0368] 1H(300MHz, CDCl3):5.09-4.93(m,2H),2.41-2.23(m,4H),1.71-1.51(m,10H),1.51-1.15(m,64H),0.95-0.82(m,15H).

[0369]

[0370] 3-(Dimethylamino)propyl(12R)-linoleic acid oxyoleate (INT-A005)

[0371] Following standard procedure D, after rapid column chromatography (SiO2, 88:10:2 → 68:30:2 hexane / EtOAc / Et3N), carboxylic acids (561 mg, 1.00 mmol), amino alcohols (0.18 mL, 1.20 mmol, 1.20 equivalent), DCC (227 mg, 1.10 mmol, 1.10 equivalent), and DMAP (147 mg, 1.20 mmol, 1.20 equivalent) in CH2Cl2 (3.5 mL) provided a clear, colorless oily INT-A005 (639 mg, 95% yield).

[0372] 1 H(300MHz, CDCl3):5.54-5.23(m,6H),4.89(quint,J=6.0Hz,1H),4.12(t,J=6.0Hz,2H),2.78(br t,J=6.0Hz,2H),2.58-2.45(m,6H),2.29(br q,J=6.0Hz,6H),2.12-1.96(m,6H),1.83-1.72(m,2H),1.69-1.47(m,6H),1.44-1.18(m,30H),1.02(t,J=7.1Hz,6H),0.94-0.84(m,6H).

[0373]

[0374] 2-(Dimethylamino)ethyl(±)-syn-9,10-dilinoleic acid oxystearic acid ester (INT-A001)

[0375] Following standard procedure D, after rapid column chromatography (SiO2, 88:20:2→68:30:2→48:50:2 hexane / EtOAc / Et3N), carboxylic acids (336 mg, 0.40 mmol), amino alcohols (0.12 mL, 1.20 mmol, 3.00 equivalents), DCC (91 mg, 0.44 mmol, 1.10 equivalents), and DMAP (59 mg, 0.48 mmol, 1.20 equivalents) in CH2Cl2 (2 mL) provided a clear, colorless oily INT-A001 (255 mg, 70% yield).

[0376] 1 H(300MHz, CDCl3):5.47-5.27(m,8H),5.04-4.94(m,2H),4.18(t,J=5.9Hz,2H),2.79(br t,J=5.90Hz,4H),2.57(t,J=5.8Hz,2H),2.39-2.24(m,9H),2.30(s,3H),2. 13-1.99(m,8H),1.70-1.46(m,10H),1.44-1.16(m,48H),0.97-0.84(m,9H).

[0377]

[0378] 3-(Diethylamino)propyl(±)-syn-9,10-dilinoleic acid oxystearic acid ester (INT-A002)

[0379] Following standard procedure D, after rapid column chromatography (SiO2, 78:20:2 → 68:30:2 hexane / EtOAc / Et3N), carboxylic acids (336 mg, 0.40 mmol), amino alcohols (0.12 mL, 0.80 mmol, 2.00 equivalents), DCC (91 mg, 0.44 mmol, 1.10 equivalents), and DMAP (59 mg, 0.48 mmol, 1.20 equivalents) in CH2Cl2 (2 mL) provided a clear, colorless oily INT-A002 (272 mg, 71% yield).

[0380] 1H(300MHz, CDCl3):5.47-5.27(m,8H),5.04-4.94(m,2H),4.12(t,J=6.5Hz,2H),2.79(br t,J=5.9Hz,4H),2.59-2.46(m,6H),2.30(t,J=7.5Hz,6H),2.12-1.99(m,8H),1.78(quint,J= 6.9Hz,2H),1.70-1.46(m,10H),1.44-1.16(m,48H),1.03(t,J=7.1Hz,6H),0.97-0.84(m,9H).

[0381]

[0382] 3-(Dimethylamino)propyl(±)-syn-9,10-dilinoleic acid oxystearic acid ester (INT-A003)

[0383] Following standard procedure D, after rapid column chromatography (SiO2, 78:20:2 → 68:30:2 hexane / EtOAc / Et3N), carboxylic acids (421 mg, 0.50 mmol), amino alcohols (88 μL, 0.75 mmol, 1.50 equivalents), DCC (113 mg, 0.55 mmol, 1.10 equivalents), and DMAP (73 mg, 0.60 mmol, 1.20 equivalents) in CH2Cl2 (3 mL) provided a clear, colorless oily INT-A003 (323 mg, 70% yield).

[0384] 1 H(300MHz, CDCl3):5.47-5.27(m,8H),5.04-4.94(m,2H),4.13(t,J=6.60Hz,2H),2.79(br t,J=5.90Hz,4H),2.39-2.25(m,8H),2.24(s,3H),2.13-1.99(m,8H),1.81(qu int,J=6.8Hz,2H),1.70-1.46(m,10H),1.44-1.16(m,48H),0.97-0.84(m,9H).

[0385]

[0386] 2-(Diethylamino)ethyl(±)-syn-9,10-dilinoleic acid oxystearic acid ester (INT-A004)

[0387] Following standard procedure D, after rapid column chromatography (SiO2, 78:20:2 → 68:30:2 hexane / EtOAc / Et3N), carboxylic acids (421 mg, 0.50 mmol), amino alcohols (80 μL, 0.60 mmol, 1.20 equivalents), DCC (113 mg, 0.55 mmol, 1.10 equivalents), and DMAP (73 mg, 0.60 mmol, 1.20 equivalents) in CH2Cl2 (3 mL) provided a clear, colorless oily INT-A004 (406 mg, 86% yield).

[0388] 1 H(300MHz, CDCl3):5.47-5.27(m,8H),5.05-4.93(m,2H),4.15(t,J=6.3Hz,2H),2.78(br t,J=5.9Hz,4H),2.70(t,J=6.1Hz,2H),2.59(q,J=7.2Hz,4H),2.36-2.23(m,6H),2.12-1.9 9(m,8H),1.69-1.45(m,10H),1.44-1.16(m,48H),1.05(t,J=7.1Hz,6H),0.97-0.84(m,9H).

[0389]

[0390] 3-(Diethylamino)propyl(±)-syn-9,10-bis(2-hexyldecanoyloxy)stearate (INT-A007)

[0391] Following standard procedure D, after rapid column chromatography (SiO2, 85:15:0→80:15:5→75:20:5 hexane / EtOAc / MeOH), carboxylic acids (595 mg, 0.75 mmol), amino alcohols (0.13 mL, 0.90 mmol, 1.20 equivalents), DCC (170 mg, 0.82 mmol, 1.10 equivalents), and DMAP (110 mg, 0.90 mmol, 1.20 equivalents) in CH2Cl2 (4 mL) provided a clear, colorless oily INT-A007 (577 mg, 85% yield).

[0392] 1H(300MHz, CDCl3):5.06-4.96(m,2H),4.12(t,J=6.5Hz,2H),2.60-2.46(m,6H),2.37-2.23(m,5H),1.79 (quint,J=7.2Hz,2H),1.70-1.50(m,10H),1.50-1.16(m,64H),1.04(t,J=7.1Hz,6H)0.94-0.84(m,15H).

[0393]

[0394] 3-(Diethylamino)propyl (syn-9,10,12R)-Trinyl oleate oxystearate (INT-A006)

[0395] Following standard procedure D, after rapid column chromatography (SiO2, 85:15:0→80:15:5→75:20:5 hexane / EtOAc / MeOH), carboxylic acids (560 mg, 0.50 mmol), amino alcohols (89 μL, 0.60 mmol, 1.20 equivalents), DCC (113 mg, 0.55 mmol, 1.10 equivalents), and DMAP (73 mg, 0.60 mmol, 1.20 equivalents) in CH2Cl2 (3 mL) provided a clear, colorless oily INT-A006 (417 mg, 68% yield).

[0396] 1 H(300MHz, CDCl3):5.49-5.26(m,12H),5.12-4.84(m,3H),4.12(t,J=6.5Hz,2H),2.59-2.45(m,6H),2.36-2.21 (m,8H),2.14-1.99(m,12H),1.92-1.47(m,18H),1.47-1.16(m,56H),1.03(t,J=7.1Hz,6H),0.96-0.84(m,12H).

[0397]

[0398] Isobutyl(10Z)-nonadecanyl sulfonate

[0399] The procedure (M. Xie, T.S. Widlanski, Tetrahedron Lett. 1996, 37, 4443) was modified by adding an n-BuLi solution (6.52 mL of 1.15 M solution in hexane, 7.50 mmol, 1.50 equivalent) to a 9:1 THF / DMPU solution (15 mL) of isobutyl methanesulfonate (1.22 g, 8.00 mmol, 1.60 equivalent) in a round-bottom flask at -78 °C and stirring the result for 30 min. Simultaneously, while still at -78 °C, a 2 mL THF solution of oleyl iodide (1.89 g, 5.00 mmol, 1.00 equivalent) was added to the above solution, and the reaction mixture was heated over 16 h. The reaction mixture was quenched with 10% citric acid aqueous solution, extracted with Et2O (2 × 10 mL), and the combined organic matter was washed with water (1 × 10 mL) and brine (1 × 10 mL). The mixture was then dried over Na2SO4 and concentrated on a rotary evaporator. The crude residue was purified by rapid column chromatography (98:2 → 95:5 hexane / EtOAc) to provide a clear, colorless, oily alkyl sulfonate (1.19 g, 46% yield).

[0400] 1 H(300MHz, CDCl3):5.46-5.27(m,2H),4.00(d,J=6.6Hz,2H),3.14-3.05(m,2H),2.11-1.94(m ,5H),1.94-1.80(m,2H),1.51-1.18(m,26H),1.00(t,J=6.8Hz,6H),0.90(brt,J=6.6Hz,3H).

[0401]

[0402] Isobutyl(±)-syn-10,11-dihydroxynonadecanosulfonate

[0403] Under argon atmosphere, an OsO4 solution (0.11 mL of 4% aqueous solution, 0.02 mmol, 0.01 equivalent) was added to a 4:1 Me2CO / H2O (5.5 mL) solution of isobutyl(10Z)-nonadecanesulfonate (709 mg, 1.76 mmol) and NMO (0.54 mL of 50% aqueous solution, 2.64 mmol, 1.50 equivalent) at room temperature. After stirring for 16 h, a saturated NaHSO3 aqueous solution was added and the reaction mixture was stirred for 1 h. The mixture was then extracted with EtOAc (3 × 10 mL) and washed with water (1 × 10 mL) and brine (1 × 10 mL). The combined organic matter was dried with Na2SO4 and concentrated on a rotary evaporator to provide a white solid diol (768 mg, quantitative yield), which was not further purified before use.

[0404] 1 H(300MHz, CDCl3):4.00(d,J=6.6Hz,2H),3.67-3.65(m,2H),3.14-3.05(m,2H),2.05(se pt,J=6.7Hz,1H),1.95-1.78(m,4H),1.58-1.19(m,28H),1.00(t,J=6.8Hz,6H),0.90(br t,J=6.6Hz,3H).

[0405]

[0406] Isobutyl(±)-syn-10,11-bis(2-hexyldecanoyloxy)nonadecanyl sulfonate

[0407] Under argon atmosphere, solid DCC (388 mg, 1.88 mmol, 2.10 equivalences) was added to a round-bottom flask containing an ice-cold CH2Cl2 solution of (±)-2-hexyldecanoic acid (482 mg, 1.88 mmol, 2.10 equivalences). The ice bath was then removed, and the resulting mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath, and solid dihydroxysulfonate (391 mg, 0.90 mmol, 1.00 equivalences) was added, followed by DMAP (273 mg, 2.23 mmol, 2.50 equivalences). The reaction mixture was heated to room temperature over 16 h, diluted with Et2O, stirred for 10 min, and then passed through a sieve. Pad filtration. The filtrate was concentrated on a rotary evaporator and then purified by rapid column chromatography (SiO2, 95:5 → 90:10 hexane / EtOAc) to provide diacylated sulfonate (574 mg, 70% yield).

[0408] 1H(300MHz, CDCl3):5.08-4.93(m,2H),4.00(d,J=6.6Hz,2H),3.67-3.65(m,2H),3.14-3.05(m,2H),2.38-2.24(m, 2H), 2.05(sept,J=6.7Hz,1H),1.95-1.80(m,2H),1.70-1.15(m,74H),1.00(t,J=6.8Hz,6H),0.96-0.83(m,15H).

[0409]

[0410] (±)-syn-10,11-bis(2-hexyldecanoyloxy)nonadecanosulfonate sodium (INT-A012)

[0411] NaI (82 mg, 0.55 mmol, 2.00 equivalents) was added to a solution containing... A solution of diacylated isobutyl sulfonate (250 mg, 0.27 mmol) in 0.9 mL Me₂CO at room temperature was obtained in a screw-capped, sealed glass tube under argon atmosphere. The reaction mixture was then refluxed and heated for 24 h, diluted with 3 mL Me₂CO, cooled on ice for 2–3 h, and a white precipitate was collected and washed with ice-cold Me₂CO to provide a white powder of INT-A012 (200 mg, 84% yield).

[0412] 1 H(300MHz, CDCl3):5.10-4.93(m,2H),2.90(app br s,2H),2.46-2.22(m,4H),1.70-1.15(m,76H),0.96-0.83(m,15H).

[0413] Example 2: Formulating lipid nanoparticles containing nucleic acids into lipid nanoparticles (LNPs).

[0414] The lipids INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 synthesized as described in Example 1 were formulated together with nucleic acids in lipid nanoparticles. As an example of cargo, the nucleic acid used for LNP incorporation was siRNA targeting factor VII, a protein involved in blood clotting. The level of factor VII in blood plasma can be readily measured by chromatographic assay, and the factor VII level represents a suitable model for determining siRNA-mediated downregulation of this factor.

[0415] Physiological and chemical parameters of nanoparticles incorporating cationic lipids and siRNA targeting factor VII, including epigenetic pK, were measured and reported below. a Particle size, polydispersity (PDI), and encapsulation efficiency were analyzed. The following results show that LNPs are suitable for the encapsulation and delivery of nucleic acids.

[0416] To prepare LNPs, ionizable lipids, DSPC, cholesterol, and PEG-DMPE were dissolved in ethanol. siRNA was dissolved in a buffer of pH 4.0–6.2 consisting of 10–50 mM acetate, succinate, or citrate. In selected examples, 10–50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) or 2-(N-morpholino)ethanesulfonic acid (MES) buffer was used. LNPs were prepared by rapidly mixing the lipid component in ethanol (at a molar ratio of 50 / 10 / 38.5 / 1.5 DSPC / chol / PEG-DMPE) with the nucleic acid in an aqueous buffer at a volumetric flow rate of 1:3 (ethanol to water, combined flow rate >12 mL / min) at room temperature. Typically, the siRNA / lipid ratio was set at 0.056 wt / μmol. The product was then dialyzed against 1× phosphate-buffered saline (PBS) at pH 7.4 for 24 hours to remove residual ethanol and raise the pH. PBS was updated 4 hours later.

[0417] As shown in Table 2, INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 ionizable lipids contribute to the incorporation of FVII siRNA with high encapsulation efficiency and low polydispersity, both of which are desirable physiological and chemical properties for drug delivery systems. The apparent pK values ​​of these ionizable lipids were also measured. a Value, because previously reported apparent pK a Some ionizable lipids with values ​​between 6 and 7 are active in mediating gene silencing (Semple, SC et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol, 2010, 28(2): p. 172-6 and Jayaraman, M et al., Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angewandte Chemie, 2012, 51(34): p. 8529-33).

[0418] Determine that INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 show pK in the appropriate range of 6 to 7. a Values ​​(Table 2).

[0419] Table 2: Physiological and chemical parameters of LNPs containing ionizable lipids and siRNA

[0420]

[0421]

[0422] Cumulatively, this data demonstrates the suitability of these novel ionizable lipids for the encapsulation of nucleic acids in LNPs.

[0423] Example 3: Determination of LNP activity in cultured 22Rv1 cells

[0424] The in vitro activity of ionizable lipids was evaluated in human prostate cells (22Rv1) expressing luciferase. LNPs containing siRNA targeting firefly luciferase were prepared as described in Example 2. Cells were treated with 0.1–1 μg / mL of siRNA-containing LNPs for 16–24 hours, followed by lysis with Glo lysis buffer (Promega). Equal volumes of Steady-Glo reagent (Promega) were added to each sample, and luminescence levels were determined using a Synergy LX microplate reader (BioTek). Figure 2A The luminescence levels of various treatments were shown. Typically, a dose-dependent effect of silencing the firefly luciferase gene was observed. Figure 2B The relative activities of formulations containing ionizable lipids A001-A007 were shown. The results indicate that the ionizable lipids disclosed in this invention can effectively deliver siRNA and induce gene silencing in vitro. In this in vitro gene silencing model, A007 at 1 μg / mL exhibited the strongest activity.

[0425] Example 4: Determination of LNP activity in a mouse factor VII model

[0426] Next, the in vivo activity of the ionizable lipids was evaluated in a mouse FVII model. The results showed that the ionizable lipids disclosed in this invention can effectively deliver nucleic acids in vivo.

[0427] The LNP containing siRNA targeting factor VII (FVII) prepared as described in Example 2 was diluted with PBS to maintain an injection volume of 10 mL / kg body weight and administered intravenously via the tail vein in 6- to 8-week-old female C57BL / 6 mice (Charles River Laboratories, Wilmington, MA) (based on siRNA concentration). Twenty-four hours post-injection, the animals were euthanized and blood was collected via intracardiac sampling. Blood samples were allowed to clot overnight at 4°C, and serum was separated by centrifugation at 12,000 rpm for 15 min. Serum FVII levels were determined using a Biophen VII chromogenic assay (Aniara, Mason, OH) according to the manufacturer's specifications.

[0428] Figure 3 Residual FVII levels in mice injected with formulations of INT-A001, INT-A002, INT-A003, INT-A005, or INT-A007 were shown. These ionizable lipids were determined to be active in mediating gene silencing. INT-A002 was the most active of the formulated lipids tested. The ED50 of INT-A002 was estimated to be less than 0.1 mg / kg (ED50 is the effective dose for achieving 50% gene silencing).

[0429] Example 5: Formulation of LNPs containing mRNA into lipid nanoparticles

[0430] The ability of formulations containing INT-A001, INT-A002, INT-A003, and INT-A004 to deliver mRNA in vitro was evaluated. The results showed that these ionizable lipids can effectively deliver mRNA.

[0431] As described in Example 2, LNPs containing firefly luciferase mRNA were prepared. As shown in Table 3 below, the ionizable lipids INT-A001, INT-A002, INT-A003, and INT-A004 facilitate the incorporation of luciferase mRNA with high encapsulation efficiency and low polydispersity, both of which are desirable physiological and chemical properties for drug delivery systems.

[0432] Table 3: Physiological and chemical parameters of LNPs containing ionizable lipids and mRNA

[0433] Ionizable lipid ID Particle size (nm) Polydispersity Index (PDI) Packaging efficiency (%) INT-A001 43 0.064 96 INT-A002 47 0.053 99 INT-A003 49 0.050 99 INT-A004 42 0.090 96

[0434] The in vitro activity of ionizable lipids was evaluated in cultured HepG2 cells. Firefly luciferase mRNA containing LNPs was prepared as described in Example 2. LNPs were diluted to 0.125–1 μg / mL in DMEM medium containing 10% FBS and incubated with HepG2 cells for 16–24 hours. Cells were then lysed with Glo lysis buffer (Promega). An equal volume of Steady-Glo reagent (Promega) was added to each sample, and the luminescence level was determined using a Synergy LX microplate reader (BioTek). Figure 4A The luminescence levels of various treatments were shown. The INT-A003 formulation exhibited the strongest activity in delivering mRNA and inducing its in vitro expression.

[0435] To evaluate in vivo activity, LNPs containing firefly luciferase mRNA were prepared as described in Example 2. The LNPs were diluted with PBS and administered intravenously at 1 mg / kg via the tail vein to 6- to 8-week-old female C57BL / 6 mice (Charles River Laboratories, Wilmington, MA). Four hours post-injection, the animals were euthanized and the livers were collected. Approximately 100 mg of liver was homogenized in 0.5 mL of Glo lysis buffer (Promega). The homogenate was further diluted 1:4 with lysis buffer, and then 50 μL of the diluted homogenate was added to 50 μL of Steady-Glo reagent (Promega). The luminescence level was determined using a Synergy LX microplate reader (BioTek). Figure 4B The relative luminescence levels are shown as a result of successful in vivo delivery of mRNA. The INT-A002 formulation exhibits the strongest activity in both mRNA delivery and induction of in vivo mRNA expression.

[0436] Example 6: Formulation of LNPs from lipid nanoparticles containing antisense oligonucleotides

[0437] As described in Example 2, LNPs containing antisense oligonucleotides were prepared. As shown in Table 4 below, the ionizable lipids INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 facilitate the incorporation of antisense oligonucleotides with high encapsulation efficiency and low polydispersity, both of which are desirable physiological and chemical properties for drug delivery systems.

[0438] Table 4: Physiological and chemical parameters of LNPs containing ionizable lipids and antisense oligonucleotides

[0439] Ionizable lipid ID Particle size (nm) Polydispersity Index (PDI) Packaging efficiency (%) INT-A001 44 0.103 84 INT-A002 49 0.096 92 INT-A003 54 0.036 89 INT-A004 45 0.065 88 INT-A005 47 0.090 74 INT-A006 55 0.062 88 INT-A007 55 0.070 84

[0440] Example 7: Formulation of LNPs containing hyaluronic acid (HA) lipid nanoparticles.

[0441] Examples of using hyaluronic acid as an anionic cargo. As described in Example 2, LNPs containing HA with a molecular weight of 8-15 kDa were prepared. As shown in Table 5 below, ionizable lipids INT-A002, INT-A003, INT-A005, INT-A006, and INT-A007 facilitate the incorporation of HA.

[0442] Table 5: Physiological and chemical parameters of LNPs containing ionizable lipids and HA

[0443] Ionizable lipid ID Particle size (nm) Polydispersity Index (PDI) HA: Lipids (wt / wt) INT-A002 83 0.027 0.093 INT-A003 64 0.035 0.094 INT-A005 71 0.096 0.114 INT-A006 74 0.037 0.078 INT-A007 58 0.065 0.096

[0444] Example 8: Formulation of LNPs from lipid nanoparticles containing acidic peptides.

[0445] Similar to nucleic acids, cationic ionizable lipids can be used to incorporate proteins or peptides with a net negative charge into LNPs. As described in Example 2, an LNP containing an acidic peptide with a molecular weight of 4.2 kDa and a predicted net charge of -3 was prepared. As shown in Table 6 below, ionizable lipids INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 facilitate the incorporation of this acidic peptide.

[0446] Table 6: Physiological and chemical parameters of LNPs containing ionizable lipids and a 4.2 kDa acidic peptide

[0447] Ionizable lipid ID Particle size (nm) Polydispersity Index (PDI) Peptide:Lipid (wt / wt) INT-A001 48 0.021 0.161 INT-A002 62 0.015 0.157 INT-A003 76 0.031 0.160 INT-A004 40 0.034 0.158 INT-A005 87 0.021 0.193 INT-A006 58 0.070 0.132 INT-A007 52 0.049 0.162

[0448] Example 9: Formulation of LNPs from lipid nanoparticles containing small acidic peptides.

[0449] As described in Example 2, an LNP containing an acidic peptide with a molecular weight of 2.0 kDa and a predicted net charge of -3 was prepared. As shown in Table 7 below, ionizable lipids INT-A003, INT-A004, INT-A006, and INT-A007 facilitate the incorporation of this acidic peptide.

[0450] Table 7: Physiological and chemical parameters of LNPs containing ionizable lipids and 2.0 kDa acidic peptides

[0451] Ionizable lipid ID Particle size (nm) Polydispersity Index (PDI) Peptide:Lipid (wt / wt) INT-A003 142 0.025 0.074 INT-A004 59 0.051 0.073 INT-A006 94 0.054 0.061 INT-A007 79 0.019 0.075

[0452] Example 10: Formulation of LNPs containing cationic cargoes into lipid nanoparticles

[0453] Examples of using basic peptides as cationic cargoes. As described in Example 2, an LNP containing a basic peptide with a molecular weight of 2.0 kDa and a predicted net charge of +4 was prepared. As shown in Table 8 below, ionizable lipids INT-A008 and INT-A009 facilitate the incorporation of this basic peptide.

[0454] Table 8: Physiological and chemical parameters of LNPs containing ionizable lipids and 2.0 kDa basic peptides

[0455] Ionizable lipid ID Particle size (nm) Polydispersity Index (PDI) Peptide:Lipid (wt / wt) INT-A008 39 0.058 0.104 INT-A009 39 0.057 0.121

[0456] The claims appended to this specification should not be limited to any specific embodiments described above, and should be considered to include all possible embodiments and equivalents conferred by these claims.

Claims

1. A lipid having one of the following structures or a pharmaceutically acceptable salt thereof: (INT-A001); (INT-A002); (INT-A003); (INT-A004); (INT-A005); (INT-A006); or (INT-A007)。 2. A drug delivery vehicle formulation comprising the lipid of claim 1 and comprising a cargo molecule or compound, said cargo molecule or compound being a nucleic acid, protein, or peptide.

3. The drug delivery medium formulation according to claim 2, wherein the nucleic acid is small interfering RNA, small activating RNA, messenger RNA, microRNA, antisense oligonucleotide, ribonuclease, aptamer, circular DNA, straight DNA, or miRNA mimic.

4. The drug delivery vehicle formulation according to claim 2, wherein the nucleic acid is a plasmid or a miRNA antagonist.

5. The drug delivery medium formulation according to claim 2, wherein the nucleic acid is an anti-miRNA oligonucleotide.

6. The drug delivery medium formulation according to claim 3, wherein the nucleic acid is messenger RNA.

7. The drug delivery medium formulation according to claim 2, wherein the cargo molecule or compound is a peptide.

8. The drug delivery vehicle formulation according to any one of claims 2 to 7, comprising lipid nanoparticles (LNP).

9. The drug delivery vehicle formulation of claim 8, wherein the lipid nanoparticles comprise vesicle-forming lipids and optionally sterols.

10. The drug delivery medium formulation of claim 8, wherein the lipid nanoparticles comprise phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, ceramide, sphingomyelin, or a hydrophilic polymer lipid conjugate.

11. The drug delivery medium formulation of claim 8, wherein the loading efficiency into the lipid nanoparticles is 70% to 100%.

12. The drug delivery medium formulation of claim 11, wherein the loading efficiency into the lipid nanoparticles is 80% to 100%.

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