Lipids for delivering charged substances, formulations thereof, and methods for producing the same

Abstract

Disclosed herein is a lipid having a net charge at physiological pH and attached to a lipid moiety. The lipid moiety comprises a hydrocarbon structure having two or more attached hydrocarbon chains, optionally with cis or trans C=C, at least one of which is attached to a head group via an optional linker region. The hydrocarbon chains are connected to each other at branch points on internal carbons of the chains attached to the linker region, and the branch points comprise functional groups containing electronegative atoms. Each hydrocarbon chain has 1 to 40 carbon atoms, and the entire hydrocarbon structure comprises 10 to 150 carbon atoms. Advantageously, the hydrocarbon structure can have a generally flared shape to enhance delivery of cargo molecules. Additionally, a delivery vehicle comprising this lipid is provided.

Description

Technical Field

[0001] This specification provides lipids, lipid formulations, and methods for their preparation. The lipids and their formulations are useful for the delivery of charged substances, including but not limited to nucleic acids and peptides.

Background Art

[0002] The intracellular delivery of charged substances such as nucleic acids and peptides is facilitated by incorporation into delivery systems such as lipid nanoparticles (LNP). For example, ionizable lipids are a major lipid component for efficiently encapsulating nucleic acids during the LNP manufacturing process. Furthermore, ionizable or cationic lipids facilitate the controlled release of nucleic acids from endosomes after being taken up by endocytosis of target cells.

[0003] It has been proposed that transfection or gene delivery activity depends on the chemical structure of ionizable lipids such as cationic lipids (Semple, S.C., et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol, 2010,28(2):p.172-6). In particular, it is desirable that the new oily moiety has a non-cylindrical shape. As an example, a lipid having an ionizable small head group and a hydrocarbon moiety that spreads outward from the head group.

[0004] Currently, there is a need for structures of ionizable or charged lipids in a desired shape that can be prepared in a simple or convenient manner. If such lipids can be provided, it may greatly advance the clinical development of therapeutic methods that rely on the delivery of nucleic acids or other charged molecules to target cells.

[0005] The compositions and methods of the present disclosure are aimed at addressing the aforementioned problems and / or providing useful alternatives to those previously described in the art.

Summary of the Invention

[0006] This specification provides ionizable or cationic lipids having a hydrocarbon structure that facilitates the delivery of a charged substance. The charged substance may be a negatively charged substance such as a nucleic acid or a positively charged substance. The lipids described herein can be prepared in a modular fashion, providing hydrocarbon structures prepared in various non-cylindrical shapes and facilitating the delivery of various negatively or positively charged substances.

[0007] This specification discloses a lipid comprising a head group having a net charge at physiological pH and covalently bonded to a lipid moiety via an optional linker region. The lipid moiety consists of a hydrocarbon structure having two or more linked hydrocarbon chains optionally having cis or trans C═C, and at least one of the chains is covalently bonded to an ionizable head group optionally via a linker region. The hydrocarbon chains are bonded to each other at a branch point of the internal carbon of the chain bonded to the head group via an optional linker region, and this branch point contains an X1 functional group further described herein having an electronegative atom. Each of the hydrocarbon chains has 1 to 40 or 1 to 30 carbon atoms, and the hydrocarbon structure as a whole has 10 to 150 carbon atoms.

[0008] According to one embodiment, there is provided a charged lipid comprising a cationic or anionic lipid comprising: a head group having a net charge at physiological pH and covalently bonded to a lipid moiety via an optional linker region. The lipid moiety consists of a hydrocarbon structure having two or more linked hydrocarbon chains, optionally having cis or trans C═C, at least one of said chains being covalently bonded to an ionizable head group via a linker region, and the hydrocarbon chains being bonded to each other at a branching point at an internal carbon of the hydrocarbon chain bonded to the linker region, said branching point containing an X1 functional group, and the X1 functional group being selected from the following: -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-, -S-S-, -C(R1)=N-N-C(O)-, -C(O)-N-N=C(R1), -ON=C(R1)-, or -C(R1)=NO-, where each hydrocarbon chain has 1 to 30 carbon atoms, the hydrocarbon structure contains a total of 10 to 150 carbon atoms, and R1 is independently selected from hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle.

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

Chemical formula

[0010] In a further embodiment, the hydrocarbon structure further comprises a hydrophobic chain L2 bonded to the head group via the linker region, L2 being a hydrocarbon chain having 1 to 30 carbon atoms and optionally having cis or trans C═C bonds, or L2 having the structure of formula IIIa. Formula IIIa: [Chemical formula] In the formula, L2’, L2’’, L2’’’, and L2’’’’ are independently selected from hydrocarbon chains having 1 to 30 atoms, optionally containing one or more cis or trans C=C bonds, and L2’ is bonded to the linker region.

[0011] In yet another embodiment, the hydrocarbon further comprises a hydrocarbon chain L3 bonded to the head group via the linker region, where L3 has 1 to 30 carbon atoms and optionally has a cis or trans C=C bond or has the structure of Formula IIIa. Formula IIIa: [Chemical formula] In the formula, L3’, L3’’, L3’’’, and L3’’’’ are independently selected from hydrocarbon chains having 1 to 30 atoms optionally containing one or more cis or trans C=C bonds, and L3’ is bonded to the linker region.

[0012] In a further embodiment, the lipid may further comprise 1 to 10 side chains S bonded to L1 via an X1 linker, each side chain having 1 to 30 atoms and optionally having a cis or trans C=C bond or having the structure of Formula IIIb. Formula IIIb: [Chemical formula] In the formula, S1’, S1’’, S1’’’, and S1’’’’ are independently selected from hydrocarbon chains having 1 to 30 atoms, optionally containing one or more cis or trans C=C bonds, and S1’ is bonded to the carbon of L1.

[0013] In a further embodiment, the lipid may further comprise 1 to 10 side chains S bonded to L2 via an X1 linker, each side chain having 1 to 30 atoms and optionally having a cis or trans C=C bond or having the structure of Formula IIIb. Formula IIIb: [Chem.] In the formula, S2’, S2’’, S2’’’, and S2’’’’ are independently selected from hydrocarbon chains having 1 to 30 atoms, optionally contain one or more cis or trans C═C bonds, and S2’ is bonded to the carbon of L2.

[0014] In a further embodiment, the lipid further comprises 1 to 10 side chains S bonded to L3 via an X1 linker, each side chain having 1 to 30 atoms and optionally having a cis or trans C═C bond or having the structure of Formula IIIb. Formula IIIb: [Chem.] In the formula, S3’, S3’’, S3’’’, and S3’’’’ are independently selected from hydrocarbon chains having 1 to 30 atoms, optionally contain one or more cis or trans C═C bonds, and S3’ is bonded to the carbon of L3.

[0015] In an alternative embodiment, one or more sites of X1 are biodegradable.

[0016] In yet another embodiment, a hydrocarbon chain bonded to the head group via at least the linker region is derived from a lipid having one or more reactive groups selected from a hydroxy group, an amino group, and / or an amide group bonded to its internal carbon atoms, and at least one other hydrocarbon chain in the hydrocarbon structure is derived from an acyl lipid. The scaffold carbon chain of the hydrocarbon structure functions as a scaffold carbon chain, at least one other hydrocarbon chain of the hydrocarbon structure is derived from an acyl lipid, and the X1 bond is formed by the reaction of a reactive group on the scaffold carbon chain with the carboxylic acid of the acyl chain.

[0017] In a further embodiment, the lipid is a dihydroxy lipid. The head group may impart a pKa between 5.0 and 9.0, or a pKa between 5.5 and 8.0 to the lipid. In an alternative embodiment, the head group has the structure of Formula I.

[0018] In a further embodiment, the linker region attached to the hydrocarbon structure has the structure of Formula IIa or IIb. In a further embodiment, the hydrocarbon structure is non-cylindrical. In a further embodiment, the lipid can form lipid nanoparticles in combination with other lipids in an aqueous solution. In a further embodiment, the lipids that form other vesicles include phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, ceramide, sphingomyelin, or a conjugate of a hydrophilic polymer and a lipid. Furthermore, the lipid according to any one of the above embodiments is incorporated into its lipid bilayer or monolayer to provide a drug delivery carrier formulation containing a nucleic acid or a peptide. In one embodiment, the nucleic acid is a small interfering RNA, a small activating RNA, a messenger RNA, a microRNA, an antisense oligonucleotide, a ribozyme, an aptamer, a plasmid, a circular DNA, a linear DNA, an antagomir, an anti-miRNA oligonucleotide, or a miRNA mimic.

[0019] In a further embodiment, the drug delivery carrier formulation contains lipid nanoparticles (LNP). Furthermore, a convenient method for preparing such lipids is provided. Such an embodiment includes a method for preparing a hydrocarbon structure of a charged lipid for use in delivering a molecule of interest, the method including the following: (i) providing a hydrocarbon chain having a reactive group on its internal carbon, the reactive group including an atom selected from O, P, N, and / or S; and (ii) bonding the hydrocarbon chain to one or more acyl chains via one or more reactive groups to generate a hydrocarbon structure, the hydrocarbon structure being non-cylindrical.

[0020] In one embodiment, the reactive groups are each attached to one of the acyl chains to form each X1 bond. Further provided is a lipid produced by the method.

[0021] Further provided is a charged lipid comprising a branched lipid moiety L having the structure of formula I. Formula I: (Chemical formula 7) A-(V) m -Z-L Wherein A is a head group that is charged at physiological pH; (V) m is any -(CR1R2)-, m is 1 to 10 or 2 to 6, and R1 and R2 are each independently hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle, or independently selected optionally substituted monocyclic, bicyclic or tricyclic carbocyclic or heterocyclic ring having 4 to 12 ring atoms; Z-L has the structure of formula II, IIa or IIb below. Formula II of a linear linker structure: (Chemical formula 8) X1-L b Wherein X1 is optional, and X1 is selected from an ether group, an ester group and a carbamate group, L b is a branched lipid of formula IIIc.

[0022] Formula IIa of a branched linker structure:

Chemical formula

[0023] Ring structure of formula IIb:

Chemical formula

[0024] Formula IIIc:

Chemical formula

[0025] In another embodiment, Z-L of formula I has the structure of formula II (linear linker structure): (Chemical formula 12) X1-L b In the formula, L1' of formula IIIc has 5 to 9 carbon atoms and has 0 to 2 cis or trans double bonds; In the formula, G of formula IIIc 1 is absent, CH2 or CH2CH=CH, and the double bond is cis or trans; In the formula, L1'''' and S of formula IIIc are independently selected from hydrocarbons having 0 to 5 cis or trans CH=CH and 2 to 18 carbon atoms; The scaffold backbone of formula IIIc is CH2-L1''-G1-CH-CH2-CH3 (L1''' is 8 to 30 carbon atoms; and in the formula, q is 1 to 9.

[0026] In another embodiment, (V) of formula I m is (CH2) m and m is 1 to 20; Z-L has the structure of formula IIa (branched linker structure).

Chemical formula

[0027] In a further embodiment, Z-L of formula I has the structure of formula IIb. Formula IIb of the ring structure:

Chemical formula

[0028] The leading group A in formula I can be selected from one of the following (i) to (iv). (i) An ionizable cationic moiety selected from the following group

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0029] In one embodiment, the hydrocarbon structure L of Formula I is non-cylindrical. In a further embodiment, the lipid is capable of forming lipid nanoparticles in combination with other lipids in an aqueous solution. In one embodiment, the other vesicle-forming lipids include phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, ceramide, sphingomyelin, or hydrophilic polymer-lipid conjugates. Furthermore, provided is a drug delivery carrier formulation comprising the lipid according to any one of the above embodiments, which is incorporated into the lipid bilayer or monolayer and contains nucleic acids, proteins or peptides.

[0030] In one embodiment, the nucleic acid is small interfering RNA, small activating RNA, messenger RNA, microRNA, antisense oligonucleotide, ribozyme, aptamer, plasmid, circular DNA, linear DNA, antigomir, anti-miRNA oligonucleotide or miRNA mimic. In one embodiment, the drug delivery carrier contains a charged peptide. In a further embodiment, the drug delivery carrier is a lipid nanoparticle (LNP). It is not limited to the embodiments described herein.

Brief Description of the Drawings

[0031]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 3

Figure 4A

Figure 4B

MODE FOR CARRYING OUT THE INVENTION

[0032] Structure of lipids used for delivering charged substances The lipids described herein have a head group A and a lipid moiety L having a hydrocarbon structure as described hereinafter. The head group A is charged at physiological pH and is ionizable in some embodiments, although permanent charged groups are likewise included in the present disclosure. The head group H can include other charged groups at physiological pH, but has a net overall positive or negative charge at physiological pH. The charged lipid can be monovalent or polyvalent. The lipid moiety L generally consists of a hydrocarbon structure having a carbon chain that functions as a scaffold for conjugating additional 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 hydroxy or other lipid having a functional group and functions as a scaffold component of the hydrocarbon structure. The hydroxy lipid can be bonded to one or more hydrocarbon chains derived from acyl lipids.

[0033] As used herein, a "scaffold carbon chain" refers to a carbon chain that confers such a scaffold function by covalently bonding to another hydrocarbon chain within the lipid moiety L via a functional group (e.g., "X1 functional group", "X1 bond" or other similar rules used herein) as described herein. In one example, the X1 functional group includes a group having an electronegative atom such as N, O, S or P as an atom in the group, or optionally as the only atom in the group, and provides for covalently bonding the scaffold carbon chain to one or more hydrocarbon chains including another scaffold carbon chain. An example of a suitable X1 functional group is an ester, although other groups including ether and carbamate groups can be readily envisioned by those skilled in the art.

[0034] The scaffold carbon chain may be derived from a precursor lipid having a hydroxyl group as described herein. Such lipids are generally referred to as hydroxy lipids and can be naturally occurring or synthesized in the laboratory. To form an ester group, for example, the hydroxyl group of a hydrocarbon chain can be reacted with a carboxylic acid on another hydrocarbon chain by a condensation reaction. However, since various different synthetic routes are envisioned, the method for preparing the hydrocarbon structure is not particularly limited herein. Examples of the structures of charged lipids derived from hydroxylated lipids as scaffolds are shown in FIGS. 1A and 1B. The lipid moiety (L) may be linked to the head group A via one, two or three hydrocarbon groups within the lipid moiety (L).

[0035] FIG. 1A represents a charged lipid encompassed by the present disclosure having a simple head group (A) attached to one hydrocarbon within the lipid moiety L, optionally via a linker group. The first lipid chain in this embodiment is derived from a hydroxy-lipid. The hydroxy-lipid may be attached to one or more additional lipid chains derived from the hydroxy-lipid. Further, these lipid chains are attached to one or more hydrocarbon chains derived from acyl lipids.

[0036] FIG. 1B represents a charged lipid included in the present disclosure prepared with a head group (A) attached to two hydrocarbons in the lipid moiety L, optionally via a linker group. In this example, the two hydrocarbons attached to the head group are derived from hydroxy lipids. Next, each of the two lipids is attached to a respective other lipid derived from a hydroxy lipid, and one or more hydrocarbon chains derived from acyl lipids are further attached to these lipids. However, it will be understood that FIG. 1 is merely illustrative of a selected embodiment and should not be construed in any way as limiting. For example, the scaffold carbon chain can alternatively be synthesized from fatty acid amines, fatty acid amides and / or branched fatty acid esters.

[0037] In one example of the present disclosure, the lipid comprises a head group having a net positive or negative charge at physiological pH, covalently attached 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, optionally with cis or trans C=C, at least one of the chains being attached to the ionizable head group via the linker region, the hydrocarbon chains being attached to each other at a branching point at an internal carbon of the chain attached to the linker region, the branching point comprising an X1 functional group, the X1 functional group being 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-, -S-S-, -C(R1)=N-N-C(O)-, -C(O)-N-N=C(R1), -ON=C(R1)-, or -C(R1)=NO-, the hydrocarbon chains each having from 1 to 30 carbon atoms, the hydrocarbon structure consisting of a total of from 10 to 150 carbon atoms, and R1 being independently selected from hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle.

[0038] In a further embodiment, the hydrocarbon structure of the charged lipid has a shape that is non-cylindrical. Many phospholipids have hydrocarbon chains (formed by two fatty acid chains) attached to a glycerol backbone and generally form a lipophilic component that is cylindrical. However, in certain embodiments, the hydrocarbon structures described herein have a "flare" structure (also referred to as a "frustoconical" shape), i.e., the hydrocarbon structure forms such that at any point along its length it has a diameter that is at least 1.2 times, or at least 1.5 times, or at least 2 times the maximum diameter of the head group. Such a shape is advantageous in that it facilitates the introduction of nucleic acids and other charged molecules into cells.

[0039] In another embodiment, the hydrocarbon structure of the charged lipid has three or more hydrocarbon chains, and each of the three or more hydrocarbon chains is bonded to another chain at its internal carbon via the X1 bond. In another embodiment, the lipid moiety has a hydrocarbon structure having 2 to 20 or 3 to 18 conjugated hydrocarbon chains. The hydrocarbon structure in one embodiment can be described as a lattice or matrix of linked hydrocarbon chains forming a flared non-cylindrical structure. The connection points may be each biodegradable functional group, or at least 1, 2, 3, 4 or 5 of the connection points can be said to be biodegradable as measured in vivo after administration to a patient.

[0040] Although various head groups are conceivable, in one example, the head group is composed of 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. When a quaternary amine is utilized as the head group, the lipid may not be ionizable depending on the presence or absence of other ionizable groups within the head group. In another embodiment, the head group is nonzwitterionic.

[0041] Each of the X1 bonds connecting the hydrocarbon chains in the hydrocarbon structure of L may be the same or different. That is, each X1 can be independently selected from the X1 bonding groups listed above. In one embodiment, the X1 bond is selected from OC(O)-, -C(O)O-, and -O-. In yet another embodiment, the X1 bond is any covalent bond that is biodegradable. The X1 bond may also be pH-sensitive, which means that cleavage depends on the pH of the surrounding solution.

[0042] In a further embodiment, the hydrocarbon structure is generated from one or more hydroxy lipids and one or more acyl lipids, and the one or more hydroxy lipids function as a scaffold carbon chain for conjugating the one or more acyl lipids. In a further embodiment, the lipid has an apparent pK between 5.0 and 9.0, or between 5.5 and 8.5, or between 5.0 and 8.0 aIt has. In yet further embodiments, the charged lipid (e.g., cationic or ionizable lipid) described above can be incorporated into lipid nanoparticles including, but not limited to, liposomes.

[0043] In further embodiments, the charged lipid has the structure of Formula I. Formula I: (Chemical Formula 19) A-(V) m -Z-L Wherein A is a head group that is charged at physiological pH; (V) m is any -(CR1R2)-, m is 1 to 10 or 2 to 6, and R1 and R2 are each independently hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle, or independently selected optionally substituted monocyclic, bicyclic or tricyclic carbocyclic or heterocyclic ring having 4 to 12 ring atoms; Z-L has the structure of Formula II, IIa or IIb below.

[0044] Formula II is a linear linker structure. (Chemical Formula 20) X1-L b Wherein X1 is optional and is selected from ether, ester and carbamate groups; and L b is a branched lipid of Formula IIIc below.

[0045] In another example of the present disclosure, the charged lipid is an amino lipid and has the following Formula Ia. Formula Ia: [Chemical Structure] Wherein R1, R2 and R3 are each independently hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle; Here, optionally, one of R1, R2, and R3 is absent (lone pair) or is hydrogen; Each occurrence of V is independently selected -(CR1R2)-, and m is 1 to 10 or 2 to 6. Z-L of the amino lipid of formula I or Z-L1 of formula Ia can be represented by formula II, formula IIa, or formula IIb represented below.

[0046] Formula II: (Chemical formula 22) X1-L b Wherein X1 is optional, and in one embodiment, X1 is selected from ether, ester, and carbamate groups; and L b is a branched lipid of formula III, IIIa, or IIIc.

[0047] Formula IIa:

Chemical formula

[0048] Formula IIb:

Chemical formula

[0049] Formula III:

Chemical formula

[0050] The charged lipid (e.g., an ionizable lipid) may be a mixture of enantiomers or may contain a single optical isomer. As described above, the X1 group can be biodegradable and can be cleaved after administration to a subject. Without limitation, an ester bond can be hydrolyzed by an esterase after administration to a patient, thereby releasing the hydrocarbon chain from the lipid. Other groups that can be hydrolyzed by an enzyme or by a pH change are similarly included in the present disclosure. However, it will be understood that the X1 group can likewise be a non-biodegradable group.

[0051] In one embodiment, the lipid is soluble in a biocompatible alcohol, thereby facilitating incorporation into a delivery carrier and co-encapsulation of nucleic acids such as small interfering RNA, small activating RNA, messenger RNA, microRNA, antisense oligonucleotide, ribozyme, aptamer, plasmid, circular DNA, linear DNA, antagomir, anti-miRNA oligonucleotide, miRNA mimic or gene editing agent.

[0052] Head group (A) Lipids are often represented for convenience as having a head group (referred to herein as "A") covalently attached to one or more hydrocarbon chains. Optionally, the head group A is attached to the hydrocarbon chain by a linker region. Suitable head groups and linker regions are as described below. In one embodiment, the head group is selected from sites that are ionizable, permanently charged, or zwitterionic. The head group may impart a positive or negative charge to the lipid at a specific pH value or range including pH 7.4 (physiological pH). Examples of substituents classified into each of these categories are as follows.

[0053] (i) Ionizable cationic moiety:

Chemical formula

Chemical formula

Chem.

Chem.

[0054] Typically, the lipid headgroup A is relatively small and charged at physiological pH. In one non-limiting embodiment, such a headgroup consists of a terminal ionizable amine group, although groups such as phosphate or sulfate groups are also included herein. In another embodiment, the headgroup has an apparent pK between 5 and 8, or a pK between 5.5 and 7.5, for the ionizable lipid, and has a terminal ionizable amine group that confers such pK to the ionizable lipid. It will be understood that the pK of the amino group may be affected by adjacent atoms within the headgroup. Headgroups containing sulfate or phosphate may confer an overall negative charge to the lipid. a or a pK between 5.5 and 7.5 for the ionizable lipid a and has a terminal ionizable amine group that confers such pK to the ionizable lipid. The pK of the amino group a may be affected by adjacent atoms within the headgroup. Headgroups containing sulfate or phosphate may confer an overall negative charge to the lipid.

[0055] The amine group may be any of a primary, secondary, or tertiary amine group. The headgroup may further contain, for example, two or three amino groups that are the same or different.

[0056] In one embodiment, the amine portion of the headgroup has the following formula.

Chem.

[0057] In these embodiments where the head group is positively charged, this group may impart a negative charge and thus result in a charge-neutral lipid, so this group does not have a phosphate group. However, if the overall charge of the lipid is positive at physiological pH, such a group may be included in the head group.

[0058] Optionally, the head group is conjugated to a hydrophilic polymer that includes a polymer that improves the blood retention of the lipid or its formulation after administration to a patient. Non-limiting examples of such hydrophilic polymers include polyethylene glycol (PEG). As will be understood by those skilled in the art, when the head group is charged, the lipid may exist as a salt. Pharmaceutically acceptable salts are included within the scope of the present disclosure.

[0059] Any linker region In addition to the ionizable amine group, the lipid may include a region having one or more electronegative atoms that can covalently bond to one, two, or three lipid chains, at least one of which is a scaffold carbon chain. Such a region in the lipid can be a linker region, or simply referred to as a "linker", and is optional because the hydrocarbon chain may be directly linked to the head group. The chemical structure of the linker can include an ester, an ether, a glyceride linker, a derivative of a glyceride linker, or a cyclic linker including a polycyclic ring composed of carbon atoms or heteroatoms, depending on the number of lipid chains bonded to the head group.

[0060] The linker region bonded to the head group can have the following formula.

Chemical formula

[0061] Optionally, the linker region binds to a hydrophilic polymer comprising a polymer that improves blood retention, such as polyethylene glycol (PEG).

[0062] As will be understood by those skilled in the art, various different combinations of head linker groups are encompassed by the present disclosure. The linker region may be characterized by the number of hydrocarbon chains attached thereto. Alternatively, the linker may be described as linear, branched, or cyclic. Examples of each category are provided below.

[0063] An example of a head group having a linker region that attaches a single hydrocarbon chain is shown below, but is not limited thereto.

Chemical formula

[0064] An example of a head group having a linker region that binds two hydrocarbon chains is provided below, but is not limited thereto.

Chemical formula

[0065] An example of a head group having a linker region that attaches three hydrocarbon chains is provided below, but is not limited thereto.

Chemical formula

[0066] Also, the linker region can be described as linear, branched, or cyclic. Examples of linear, branched, and cyclic linker regions are described in Formulas II, IIa, and IIb, respectively, described above.

[0067] Lipid moiety L The lipid moiety L can include one, two, or three hydrocarbon chains within a hydrocarbon structure that is directly or via a linker region attached to the head group. In those embodiments where only one hydrocarbon chain is attached to the head group or linker region, an additional hydrocarbon side chain S may be attached to an internal carbon of L1. Thus, in at least one embodiment, the head group is attached to a lipid moiety L1 having the structure of Formula III, or via a linker.

[0068] Formula III:

Chemical Formula

[0069] Formula IIIa:

Chemical Formula

[0070] [Chemical Formula] Formula III or IIIa optionally contains from 1 to 20 side chains S represented as (S) n where n is from 0 to 20 and each independently is a hydrocarbon chain having from 1 to 30 atoms or from 1 to 20 atoms, optionally having one or more cis or trans C═C, or a sterol, and (S) n is bonded to the carbon atoms of L1, L2 and / or L3 via an X1 bond or is bonded via such a bond to another side chain S within the hydrocarbon structure. In one embodiment, the X1 bond is biodegradable such as an ester bond. However, other bonds other than those that are biodegradable can be used in the practice of the present invention.

[0071] For example, L1 may have a side chain S1' bonded via an X1 bond to any one of L1', L1''' or L1''''. Additional side chains S1'', S1''' or S1'''' may be bonded to the carbon of L1 or any S1 side chain. In a further example, when L2 has the structure of Formula IIIa above, L2 may have a side chain S2' bonded to any one of L2', L2''' or L2''''. Additional side chains S2'', S2''' and S2'''' may be bonded to the carbon of L2 or any S2 side chain. Similarly, when L3 is present and has Formula IIIa above, it may have a side chain S3' bonded to any one of L3', L3''' or L3''''. Additional side chains S3'', S3''' and S3'''' may be bonded to the carbon of L3 or any S3 side chain.

[0072] Examples of lipids having structures encompassed by the present disclosure are depicted in Table 1 (below). Structures A - F shown in Table 1 include a headgroup having a linker attached to a single hydrocarbon having an X1 branch point that provides an anchoring point to an additional S hydrocarbon chain. Structures G - J shown in Table 1 include a headgroup having a linker that binds two hydrocarbons, L1 and L2. Lipids depicted as structures K - M have a linker with attachment points to three hydrocarbons, L1, L2, and L3.

[0073] The following annotated Structure A exemplifies the rules used in Formula III herein to depict the X1 bond and hydrophobic region of L1. It will be understood that the presence of X1 is selected independently from other X1s in the structure. As explained, the X1 group includes any suitable functional group having an electronegative atom. The example shown is an amino lipid having a single L1 lipid hydrocarbon chain (L1’, L1’’, and L1’’’) according to Formula III above with a side chain S attached to the carbon of L1.

[0074] Structure A (INT - A001):

Chemical formula

[0075] Examples of lipids having a linker region within a head group attached to 1, 2 or 3 hydrocarbon chains are shown in Table 1 below. For each lipid shown, the substituents L1’ / L’’ / L’’’ / L’’’’ of L1 of Formula III are provided, and L2 and L3 hydrocarbon chains may also be provided. Also provided is the structure of the head group and linker region defined by Formula IIa or IIb. 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 structure of L generally forms a flared or frustoconical shape.

[0076] Table 1: Selective amino lipids having a linker region attached to 1, 2 or 3 hydrocarbon chains L1, L2 and L3 that form a flared hydrocarbon structure [Table 1] TIFF0007708388000034.tif211170TIFF0007708388000035.tif201170TIFF0007708388000036.tif201170TIFF0007708388000037.tif211170TIFF0007708388000038.tif209170TIFF0007708388000039.tif208170TIFF0007708388000040.tif208170TIFF0007708388000041.tif50170

[0077] An alternative structure for explaining the lipid moiety is Formula IIIc: Formula IIIc: [Chemical formula] wherein the L skeleton is represented by L1’-L1’’-G1-CH-[CH2] q -CH3, and the total number of carbon atoms in the L skeleton is 10 to 30; L1’ is a linear hydrocarbon chain having 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 5 to 12, 5 to 10, 5 to 9, 6 to 12, 6 to 10, 6 to 9, 7 to 12, 7 to 10, or 7 to 9 carbon atoms and 0 to 3 cis or trans double bonds; L1’’ is a carbon atom; L1’’’ is represented by G1-CH-CH2-CH3; G1 is a hydrocarbon chain having 0 to 4 carbon atoms and may have one cis or trans double bond; n is 0 to 4; p is 1 to 4; n + p is 1 to 6 or 1 to 4; q is 0 to 20 or 0 to 10 or 1 to 5; Each X1 is any suitable X1 group as described above or, independently, is selected from ether, ester, and carbamate groups; Each S and L1’’’’ hydrocarbon side chain is independently: (c) having 0 to 5 cis or trans C=C and 1 to 30 or 2 to 18 carbon atoms, a linear or branched terminal hydrocarbon chain bonded to one of each X1 at any carbon atom in the hydrocarbon chain; or (d) a branched structure of formula IIIc, wherein the total number of L1’’’’ and S hydrocarbon chains in 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; here, each of the L1’’’’ and S hydrocarbon chains in the lipid moiety may be substituted with heteroatoms, provided that the number of heteroatoms substituting the hydrocarbon chain is 4, 3, or 2 or less.

[0078] The X1 ester group may be in any orientation with respect to the position of the carbonyl group, as exemplified below.

Chemical formula

[0079] Scaffold carbon chain In one embodiment, the backbone hydrocarbon chains of the L1, L2 and / or L3 lipid moieties that provide the scaffold function are derived from fatty acids having a functional group for linking to the side chain S. This includes fatty acids substituted with a group having an atom selected from O, N, P and / or S. Such groups facilitate the bonding of the side chain and the backbone carbon of L1, L2 and / or L3 that constitute the scaffold.

[0080] For example, L1, L2 and / or L3 may be derived from a hydroxy fatty acid (HFA) which is a fatty acid having a hydroxy group bonded at any position on the carbon chain. The HFA may be a β-hydroxy fatty acid, an ω-hydroxy fatty acid, or any (ω-1)-hydroxy fatty acid, or any other HFA having a reactive functional group on an internal carbon of the carbon skeleton, but is not limited thereto. The HFA may be saturated or unsaturated. Two or more hydroxy functional groups can also be present on the carbon chain.

[0081] Alternatively, L1, L2 and / or L3 are derived from a branched fatty acid ester of HFA known in the art as a fatty acid ester of a hydroxyl fatty acid (FAHFA). These fatty acid esters comprise a branched ester bond between a fatty acid and an HFA. For example, 9-[(9Z)-octadec-9-enoyloxy]octadecanoic acid is a fatty acid ester obtained by condensation of the carboxy group of oleic acid and the hydroxy group of 9-hydroxyoctadecanoic acid.

[0082] In alternative embodiments, L1, L2 and / or L3 are derived from a fatty acid amide, which may be composed of ethanolamine as the amine component. Further, L1, L2 and / or L3 may be derived from a fatty amine.

[0083] The scaffold carbon chain of Formula III may be derived from other fatty acids other than those described above. In addition, it is understood that the fatty acids may in turn be derived from their corresponding triglycerides. In Formulas III, IIIa and IIIb, L1’, L1’’ and L1’’’ together form a linear hydrocarbon backbone (referred to herein as the “scaffold carbon chain”). According to Formula IIIc above, the scaffold carbon chain is L1’-L1’’-G 1 -CH-[CH2] q -CH3, and the total number of carbon atoms in the scaffold is 10 to 30.

[0084] Formulations Cationic or ionizable lipids can readily encapsulate nucleic acids such as small interfering RNAs, small activating RNAs, messenger RNAs, microRNAs, antisense oligonucleotides, ribozymes, aptamers, plasmids, circular DNAs, linear DNAs, antagomers, anti-miRNA oligonucleotides and miRNA mimics, and / or gene editing materials. Alternatively or additionally, negatively charged proteins and amino acids can be incorporated into the delivery carrier.

[0085] The charged lipids described herein may be used to deliver other charged molecules other than nucleic acids. This includes a wide variety of positively or negatively charged peptides, proteins, polysaccharides or carbohydrates, including both bioactive agents and prodrugs, examples of which are described below.

[0086] The cationic or ionizable lipids described herein can be administered in free form together with nucleic acids or other negatively or positively charged cargo molecules, or these components can be incorporated into delivery carriers. Various delivery systems can be used to prepare pharmaceutical formulations. When the charged lipids and associated charged molecules are in free form, pharmaceutically acceptable salts or excipients can be included in the pharmaceutical formulation.

[0087] The lipids of the present disclosure are particularly suitable for incorporation into nanoparticles such as liposomes or polymer-based systems that contain lipids or other hydrophobic components, referred to herein as "lipid nanoparticles" or "LNPs". For example, in some embodiments, the loading efficiency into a given lipid nanoparticle is 60% to 100%, 70% to 100%, or most preferably 80% to 100%.

[0088] In one embodiment, the lipid is carried on lipid nanoparticles such as liposomes by mixing with a lipid formulation component comprising a vesicle-forming lipid and optionally a sterol. As a result, lipid nanoparticles incorporating ionizable or cationic lipids can be prepared using a wide variety of well-reported and well-known formulation methodologies to those skilled in the art, including but not limited to extrusion, ethanol injection, and in-line mixing. Such methods are described in papers such as Maclachlan, I. and P. Cullis, “Diffusible-PEG-lipid Stabilized Plasmid Lipid Particles”, Adv. Genet., 2005. 53PA:157-188, Jeffs, L.B., et al., “A Scalable, Extrusion-free Method for Efficient Liposomal Encapsulation of Plasmid DNA”, Pharm Res, 2005, 22(3):362-72, and Leung, A.K., 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, each of which is hereby incorporated by reference in its entirety.

[0089] As lipid components that can be included in lipid nanoparticles in addition to charged lipids, vesicle-forming lipids such as phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, and ceramide can be mentioned. Also, in order to widen the phase transition temperature, cholesterol may be contained in the LNP. Further, the LNP may contain a lipid conjugated with a hydrophilic polymer such as distearoylphosphatidylethanolamine-PEG. Surface stabilizing functional groups such as hydrophilic polymers may be desirable in order to reduce the clearance of the nanoparticles after administration. The LNP formulation may also contain a fusogenic lipid that promotes the fusion of the delivery carrier via endocytosis with the target cell.

[0090] Suitable LNPs include, but are not limited to, liposomes prepared by extrusion by known methods, or multilamellar vesicles (MLV). The internal space of the liposome may contain an encapsulated agent such as a drug, and the bilayer or double layer may contain such an agent divided therein. Another example of a suitable LNP delivery system is a stable nucleic acid-lipid particle called SNALP. SNALP may contain a nucleic acid with a cationic lipid, a non-cationic lipid, and any hydrophilic polymer-lipid conjugate, such as a PEGylated lipid and a membrane fusion lipid.

[0091] The lipid nanoparticles may be composed of a lipophilic core. For example, the delivery carrier can also be nanoparticles composed of a lipid core stabilized by a surfactant. The vesicle-forming lipid can be used as a stabilizer. The lipid nanoparticles in another embodiment are a polymer-lipid hybrid system composed of a polymer nanoparticle core surrounded by a stabilizing lipid. The nanoparticles can alternatively be prepared from a lipid-free polymer. Such nanoparticles may consist of a concentrated core of the agent surrounded by a polymer shell, or may have a solid or liquid dispersed in the polymer matrix.

[0092] The lipids described herein can also be incorporated into emulsions, which are drug delivery carriers containing oil droplets or an oil core. The emulsions can be stabilized with lipids. For example, an emulsion can be composed of a core filled with oil stabilized by an emulsifying component such as a monolayer or bilayer of lipids. The lipids provided herein can also be formulated into micelles. Micelles are self-assembled particles composed of amphiphilic lipid or polymer components and are utilized for the delivery of agents present in the hydrophobic core.

[0093] A further class of drug delivery carriers known to those skilled in the art that can incorporate charged lipids is carbon nanotubes. Various methods for preparing the aforementioned delivery carriers and incorporating charged lipids therein are known and can be readily implemented by those skilled in the art.

[0094] Certain lipids encompassed by the present disclosure may form part of a carrier-free system. In such embodiments, the lipids associated with negatively charged molecules can self-assemble into particles. An example is the formation of lipoplexes, which is an association between DNA and cationic lipids. Such formulations may optionally contain pharmaceutically acceptable salts and / or excipients. Delivery carriers incorporating cationic or ionizable lipids can also contain active agents incorporated into the carrier, such as anti-cancer agents or other therapeutic agents including prodrugs. The delivery carrier may optionally contain lipoproteins such as apolipoproteins.

[0095] Delivery of nucleic acids, genetic material, proteins, peptides, and other charged particles As described above, the charged lipids disclosed herein facilitate the incorporation of molecules having a net negative or positive charge (also referred to herein as "cargo" or "cargo molecules") into a delivery carrier and subsequent delivery to target cells in vitro or in vivo.

[0096] In one embodiment, the molecule is a genetic material such as nucleic acid. Nucleic acids include, but are not limited to, RNAs such as small interfering RNA (siRNA), small nuclear RNA (snRNA), microRNA (miRNA), or DNAs such as plasmid DNA or linear DNA. The length of the nucleic acid varies, and can include nucleic acids with a length of 5 to 50,000 nucleotides. The nucleic acid can be in any form, such as single-stranded DNA or RNA, double-stranded DNA or RNA, or their hybrids. Single-stranded nucleic acids include antisense oligonucleotides. In one particularly advantageous embodiment, the cargo is siRNA. siRNA is taken up by endogenous cellular machinery, resulting in the degradation of mRNA, thereby blocking transcription. Since RNA is easily degraded, when incorporated into a delivery carrier, such degradation can be reduced or prevented, thereby facilitating delivery to the target site.

[0097] It is also possible to incorporate a gene editing system into a delivery carrier composed of charged lipids. This includes Cas9-CRISPR, TALEN, and zinc finger nuclease gene editing systems. In the case of Cas9-CRISPR, a guide RNA (gRNA) may be incorporated into a delivery carrier containing the cationic lipids described herein, together with a plasmid or mRNA encoding the Cas9 protein. Optionally, a ribonucleoprotein complex may be incorporated into a delivery carrier containing the cationic lipids described herein. Similarly, the present disclosure includes embodiments in which genetic material encoding the DNA binding and cleavage domains of the zinc finger nuclease or TALEN system is incorporated into a delivery carrier together with an ionizable or cationic lipid.

[0098] Charged lipids can also readily incorporate overall charged proteins and peptides 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, venom peptides, cancer / anticancer agent peptides, vaccine peptides, immune / anti-inflammatory peptides, brain peptides, endocrine peptides, feeding peptides, gastrointestinal peptides, cardiovascular peptides, kidney peptides, respiratory peptides, opium peptides, neurotrophic peptides, and blood-brain peptides. Specific examples of peptides are provided above.

[0099] Specific examples of peptides that may be associated with the charged lipids described herein are interferons and other macrophage activating factors. This includes lymphokines, muramyl dipeptide (MDP), gamma-interferon, alpha-interferon, beta-interferon, and related antiviral and antineoplastic agents; opioid peptides and neuropeptides, including enkephalin, endorphin, and dynorphin, and other analgesics; renin inhibitors, including hypotensive agents; cholecystokinin (CCK analogs) such as CCK, ceruletide, and eledoisin, and related cardiovascular and CNS targeting drugs; leukotrienes and prostaglandins such as oxytocin, and anti-inflammatory, oxytocic, and abortifacient compounds; erythropoietin and its derivatives and related hematopoietic agents; LHRH analogs such as leuprolide, buserelin, and nafarelin, and related pituitary receptor downregulators; parathyroid hormone and other growth hormone analogs; enzymes such as Dnase, catalase, and alpha-I antitrypsin; immunosuppressive agents such as cyclosporine; GM-CSF and other immunomodulatory agents; and insulin.

[0100] Administration In certain embodiments, charged lipids associated with nucleic acids or other charged molecules, either free or formulated in a drug delivery carrier, are administered to treat, prevent, and / or ameliorate a patient's condition. In particular, charged lipids formulated in free form or in a delivery carrier together with a nucleic acid or other charged substance can provide a prophylactic, ameliorative, or therapeutic benefit. Pharmaceutical compositions containing charged lipids are administered at any suitable dosage. In one embodiment, the lipids, either free or formulated in a drug delivery carrier, are administered parenterally, i.e., by intraarterial, intravenous, subcutaneous, or intramuscular administration. In other embodiments, the lipids, either free or formulated in the delivery carriers described herein, may be administered topically. In yet another embodiment, the lipids in free form or formulated in a delivery carrier described herein may be administered orally. In still further embodiments, the lipids in free form or formulated in a delivery carrier are for pulmonary administration by aerosol or powder dispersion.

[0101] The compositions described herein can be administered to any subject, including a "patient" as used herein, which includes human or non-human subjects. In some embodiments, the lipids described herein are used for in vitro transfection of cells, including stem cells and cultured cells obtained from a patient. In one embodiment, the transfected cells are stem cells, which are administered back to the patient from whom they were harvested.

[0102] The following examples are presented for illustrative purposes only and are not intended to limit the scope of the invention.

Examples

[0103] Materials and Methods For the organic synthesis reaction described in Example 1, unless otherwise specified, all reagents and solvents were purchased commercially and used without further purification, except for THF (freshly distilled from Na / benzophenone under nitrogen), and Et3N, DMF, and CH2Cl2 (freshly distilled from CaH2 under nitrogen). USP grade castor oil was purchased from a local pharmacy (Life Brand) and used as received. NMR chemical shifts are reported in parts per million (ppm) on the δ scale, and coupling constants J are reported in Hertz (Hz). Spectra are referenced to the signals of the residual solvents. Multiplicities are reported as "s" (singlet), "d" (doublet), "t" (triplet), "q" (quartet), "quint" (quintet), "sept" (septet), "m" (multiplet), and further as "app" (apparent) and "br" (broad).

[0104] Lipid nanoparticles (LNPs) were prepared using sterols, cholesterol-like, the neutral lipid 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG-DMPE). DSPC and PEG-DMPE were purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol was obtained from Sigma (St. Louis, MO).

[0105] The properties of the LNPs were evaluated by measuring the particle size and polydispersity (PdI). The particle size and polydispersity were measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). The LNPs were diluted to an appropriate concentration with PBS. Number-weight size and distribution data were used for determination, and formulations with PdI > 0.15 were not used for further studies. The lipid concentration was determined by measuring the total cholesterol using a cholesterol E enzyme assay kit from Wako Chemicals USA (Richmond, VA).

[0106] The encapsulation efficiency of RNA or antisense oligonucleotides was measured using the Quant-iT Ribogreen RNA or Oligreen ssDNA Assays (Life Technologies, Burlington, ON), respectively. Briefly, the LNPs were incubated at 37 °C for 10 min in the presence or absence of 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO), followed by the addition of Ribogreen reagent or Oligreen reagent. The fluorescence intensity (Ex / Em: 480 / 520 nm) was measured, and the samples treated with Triton X-100 represent total nucleic acids, while the untreated samples represent unencapsulated nucleic acids. Protein / peptide quantification was performed using the BCA Protein Assay (Pierce) or CBQCA Protein Quantitation Kit (Invitrogen) according to the manufacturer's instructions.

[0107] The apparent acid dissociation constant (pKa) 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). Briefly, 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS, Sigma-Aldrich, St. Louis, MO) and LNPs were diluted in buffers (10 mM HEPES, 10 mM MES, and 10 mM ammonium acetate) in the pH range of 2.5 - 11. The final concentration of TNS or total lipids was 6 μM. Next, the samples were mixed and the fluorescence intensity was measured using a Perkin Elmer LS55 (Ex / Em: 321 / 445 nm). A sigmoid best-fit analysis was applied, and the pH at which the fluorescence intensity reached half-maximum was measured as the pKa.

[0108] Example 1: Synthesis of Ionizable Lipids

Chemical Structure

[0109] General Procedure A - Esterification of Hydroxy Fatty Acids The hydroxy fatty acid (1.00 equivalent) was suspended in MeOH (0.4 - 0.5 M) in a round-bottom flask equipped with a condenser. Concentrated sulfuric acid (0.05 equivalent) was added to the above mixture, and the resulting mixture was heated under reflux and became homogeneous in 5 - 15 minutes. After 16 hours, the residual MeOH was removed with a rotary evaporator, and the residue was partitioned between ethyl acetate (EtOAc) and saturated aqueous NaHCO3. The aqueous layer was extracted twice with EtOAc, the combined organic layers were washed once with water and once with physiological saline, dried over Na2SO4, filtered by gravity filtration, and concentrated on a rotary evaporator to obtain a white powder, which was used without further purification.

[0110] General Procedure B - Acylation Reaction of Hydroxy Fatty Acid Esters N,N'-Dicyclohexylcarbodiimide (DCC; 1.00 equivalent per hydroxyl + an additional 0.10 equivalent) was added to a solution of the target carboxylic acid (1.00 equivalent per hydroxyl + an additional 0.10 equivalent) in ice-cooled CH2Cl2 (0.3 M) in a round-bottom flask under argon. Then, the ice bath was removed, and the resulting mixture was stirred for 15 minutes. The reaction mixture was cooled again in an ice bath, solid hydroxy fatty acid (1.00 equivalent) was added thereto, and subsequently 4-dimethylaminopyridine (DMAP; 1.00 equivalent per hydroxyl + an additional 0.50 equivalent) was added. The reaction mixture was warmed to room temperature over 16 hours, diluted with hexane, stirred for 10 minutes, and then filtered through a pad of Celite®. The filtrate was concentrated on a rotary evaporator to obtain a crude mixture, from which the desired acylated substance was purified by flash column chromatography.

[0111] General Procedure C - Saponification Reaction of Peracylated Fatty Acid Methyl Esters Under an argon atmosphere, aqueous NaOH (2.0 M, 1.00 equivalent) was added to a solution of acylated fatty acid methyl ester (1.10 equivalents) in room-temperature t-BuOH (0.3 M) in a round-bottom flask to synthesize the acylated fatty acid methyl ester. After stirring for 16 hours, aqueous hydrochloric acid (2.0 M) was added to the reaction mixture to acidify it to pH ≤ 2, and the mixture was extracted three times with hexane. The combined organic extracts were washed with physiological saline, dried over Na2SO4, and concentrated using a rotary evaporator to obtain the crude product as a colorless oil. This crude product was purified by flash column chromatography to obtain the desired fatty acid.

[0112] General Procedure D - Esterification of Peracylated Fatty Acids with Amino Alcohols Under an argon atmosphere, DCC (1.10 equivalents) was added to a solution of fatty acid (1.00 equivalent) in ice-cooled CH2Cl2 (0.2 M) in a round-bottom flask. The ice bath was removed, and the resulting mixture was stirred for 15 minutes. The reaction mixture was cooled again in an ice bath, pure amino alcohol (1.20 - 2.00 equivalents) was added, followed by DMAP (1.20 equivalents), and the reaction mixture was warmed to room temperature over 16 hours. The filtrate was concentrated using a rotary evaporator to obtain a crude oil, which was purified by flash column chromatography to obtain the desired peracylated amino lipid.

[0113]

Chemical formula

[0114]

Chem.

[0115]

Chem.

[0116]

Chem.

[0117]

Chem.

[0118]

Chem.

[0119]

Chem.

[0120]

Chem.

[0121]

Chem.

[0122]

Chemical formula

[0123]

Chem.

[0124]

Chem.

[0125]

Chemical Structure

[0126]

Chem.

[0127]

Chem.

[0128] [Chemical formula] Isobutyl (10Z)-nonadecene sulfonate According to the procedure of M. Xie, T. S. Widlanski, Tetrahedron Lett. 1996, 37, 4443, n-BuLi solution (6.52 mL of 1.15 M in hexane, 7.50 mmol, 1.5 equiv) was added to a -78 °C solution of isobutyl methanesulfonate (1.22 g, 8.00 mmol, 1.60 equiv) in 9:1 THF / DMPU (15 mL) in a round-bottom flask under argon, and the resulting solution was stirred for 30 min. A THF (2 mL) solution of oleyl iodide (1.89 g, 5.00 mmol, 1.00 equiv) was added to the above solution at -78 °C, and the reaction mixture was warmed for 16 h. The reaction mixture was quenched with 10% aqueous citric acid, extracted with Et2O (2 × 10 mL), and the combined organics were washed with water (1 × 10 mL), brine (1 × 10 mL), dried over Na2SO4, and concentrated on a rotary evaporator. The crude residue was purified by flash column chromatography (98:2→95:5 hexane / EtOAc) to give the alkylated sulfonic acid (1.19 g, 46% yield) as a colorless transparent oil. 1 H (300 MHz, CDCl3): 5.46 - 5.27 (m, 2H), 4.00 (d, J = 6.6 Hz, 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 = 6.8 Hz, 6H), 0.90 (brt, J = 6.6 Hz, 3H).

[0129] [Chemical Structure] (±)-Isobutyl syn-10,11-dihydroxynonadecanesulfonate An OsO4 solution (0.11 mL of 4% aqueous solution, 0.02 mmol, 0.01 equivalent) was added to a solution of isobutyl (10Z)-nonadecene sulfonic acid (709 mg, 1.76 mmol) and NMO (0.54 mL of 50% aqueous solution, 2.64 mmol, 1.50 equivalent) in 4:1 = Me2CO / H2O (5.5 mL) at room temperature in a round-bottom flask under an argon atmosphere. After stirring for 16 h, saturated aqueous NaHSO3 was added and the reaction mixture was stirred for 1 h, at which point it was extracted with EtOAc (3 × 10 mL), and the combined organics were washed with water (1 × 10 mL), brine (1 × 10 mL), dried over Na2SO4, and concentrated on a rotary evaporator to give the diol (768 mg, quantitative yield) as a white solid, which was used without further purification. 1 H (300 MHz, CDCl3): 4.00 (d, J = 6.6 Hz, 2H), 3.67 - 3.65 (m, 2H), 3.14 - 3.05 (m, 2H), 2.05 (sept, J = 6.7 Hz, 1H), 1.95 - 1.78 (m, 4H), 1.58 - 1.19 (m, 28H), 1.00 (t, J = 6.8 Hz, 6H), 0.90 (brt, J = 6.6 Hz, 3H).

[0130]

Chemical Structure

[0131]

Chemical formula

[0132] Example 2: Lipid Nanoparticle (LNP) Formulation Containing Nucleic Acid The lipids synthesized as described in Example 1, INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, INT-A007 were formulated into lipid nanoparticles together with nucleic acid. The nucleic acid incorporated into the LNP as a cargo in the examples was siRNA against Factor VII, a protein involved in blood coagulation. Since the level of Factor VII can be easily measured by a chromogenic assay in plasma, it serves as a convenient model for determining the downregulation by siRNA of this factor. The physicochemical parameters such as the apparent pKa, particle size, polydispersity index (PDI), and encapsulation efficiency of the nanoparticles incorporating the cationic lipid and siRNA against Factor VII were measured and reported below. From the following results, it was shown that the LNP is suitable for the encapsulation and delivery of nucleic acid.

[0133] To prepare the LNP, ionizable lipid, DSPC, cholesterol, and PEG-DMPE were dissolved in ethanol. The siRNA was dissolved in a buffer with a pH of 4.0 to 6.2 consisting of 10 to 50 mM acetic acid, succinic acid, or citric acid. Optionally, 10 to 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) or 2-(N-morpholino)ethanesulfonic acid (MES) buffer was used. The LNP was prepared by rapidly mixing the lipid components in ethanol (molar ratio of DSPC / chol / PEG-DMPE 50 / 10 / 38.5 / 1.5) and the nucleic acid in aqueous buffer at a volume flow rate ratio of 1:3 (ethanol to water, combined flow rate > 12 mL / min) at room temperature. Usually, the siRNA / lipid ratio was targeted at 0.056 wt / μmol. Next, the product was dialyzed against 1× phosphate buffered saline (PBS) at pH 7.4 for 24 hours to remove residual ethanol and raise the pH. The PBS was refreshed after 4 hours.

[0134] As shown in Table 2 below, the ionizable lipids of INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 facilitated the incorporation of factor VII siRNA with high encapsulation efficiency and low polydispersity, which are physicochemical properties desirable for drug delivery systems. A specific ionizable lipid with an apparent pK a value between 6 and 7 has previously been reported to have activity mediating gene silencing, and thus the apparent pKa values of these ionizable lipids were also measured (Semple, S.C., 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). INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006 and INT-007 were confirmed to have pK a values within the appropriate range of 6 - 7 (Table 2).

[0135] Table 2: Physicochemical parameters of LNP containing ionizable lipids and siRNA

Table 2

[0136] Example 3: Measurement of LNP activity in cultured 22Rv1 cells The in vitro activity of ionizable lipids was evaluated using luciferase-expressing human prostate cells (22Rv1). LNP containing siRNA against firefly luciferase was prepared as described in Example 2. Cells were treated with LNP containing 0.1 - 1 μg / mL siRNA for 16 - 24 hours and 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 plate reader (BioTek). Figure 2A shows the luminescence levels in various treatments. Generally, a dose-dependent effect was observed in the silencing of the firefly luciferase gene. Figure 2B shows the relative activities of formulations containing ionizable lipids A001 - A007. This result indicates that the ionizable lipids of the present disclosure can effectively deliver siRNA in vitro and induce gene silencing. A007 had the highest activity at 1 μg / mL in this in vitro model of gene silencing.

[0137] Example 4: Measurement of LNP activity in a mouse factor VII model Next, the activity of the ionizable lipid in vivo was evaluated using a mouse FVII model. As a result, it was shown that the ionizable lipid of the present disclosure can effectively deliver nucleic acids in vivo. The LNP prepared as described in Example 2 containing siRNA against factor VII (FVII) was diluted with PBS so that the injection volume was maintained at 10 mL / kg body weight, and intravenously administered (based on the siRNA concentration) via the tail vein to 6- to 8-week-old female C57Bl / 6 mice (Charles River Laboratories, Wilmington, MA). The animals were euthanized 24 hours after administration, and blood was collected by intracardiac sampling. The blood samples were allowed to clot overnight at 4°C, serum was separated, and then centrifuged at 12,000 rpm for 15 minutes. Serum FVII levels were determined using the Biophen VII chromogenic assay (Aniara, Mason, OH) according to the manufacturer's protocol.

[0138] Figure 3 shows the remaining FVII levels in mice injected with the INT-A001, INT-A002, INT-A003, INT-A005, or INT-A007 formulation. These ionizable lipids were determined to have the activity to mediate gene silencing. INT-A002 was the most active among the formulated lipids tested. The ED50 of INT-002 was estimated to be 0.1 mg / kg or less (ED50 is the effective amount to achieve 50% gene suppression).

[0139] Example 5: Formulation of lipid nanoparticles containing mRNA in LNP The in vitro and in vivo mRNA delivery capabilities of formulations containing INT-A001, INT-A002, INT-A003, and INT-A004 were evaluated. As a result, it was shown that these ionizable lipids can effectively deliver mRNA. LNPs containing firefly luciferase mRNA were prepared as described in Example 2. 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 physicochemical properties for drug delivery systems.

[0140] Table 3: Physicochemical parameters of LNPs containing ionizable lipids and mRNA

Table 3

[0141] The in vitro activity of the ionizable lipids was evaluated in cultured HepG2 cells. LNPs containing firefly luciferase mRNA were prepared as described in Example 2. The LNPs were diluted to 0.125 - 1 μg / mL in DMEM medium containing 10% FBS and incubated with HepG2 cells for 16 - 24 hours. The cells were then lysed with Glo-Lysis buffer (Promega). An equal volume of Steady-Glo reagent (Promega) was added to each sample, and luminescence levels were determined using a Synergy LX plate reader (BioTek). Figure 4A shows the luminescence levels in various treatments. The INT-A003 formulation was the most active in delivering mRNA and inducing its expression in vitro.

[0142] To evaluate in vivo activity, LNP containing firefly luciferase mRNA was prepared as described in Example 2. The LNP was diluted with PBS and intravenously administered at 1 mg / kg via the tail vein to female C57Bl / 6 mice (Charles River Laboratories, Wilmington, MA) aged 6 - 8 weeks. Four hours after administration, the animals were euthanized and the livers were harvested. 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 50 μL of the diluted homogenate was added to 50 μL of Steady-Glo reagent (Promega). The level of luminescence was determined using a Synergy LX plate reader (BioTek). Figure 4B shows the relative levels of luminescence as a result of successful delivery of mRNA in vivo. The INT-A002 formulation had the highest activity in delivering mRNA and inducing mRNA expression in vivo.

[0143] Example 6: Formulation of Antisense Oligonucleotides into Lipid Nanoparticles (LNP) LNP was prepared as described in Example 2 containing antisense oligonucleotides. As shown in Table 4 below, the ionizable lipids INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 promoted the incorporation of antisense oligonucleotides with both high encapsulation efficiency and low polydispersity, which are physicochemical properties desirable for drug delivery systems.

[0144] Table 4: Physicochemical Parameters of LNP Containing Ionizable Lipids and Antisense Oligonucleotides [Table 4]

[0145] Example 7: Formulation of Hyaluronic Acid (HA) into Lipid Nanoparticles (LNP) As an example of anionic cargo, hyaluronic acid was used. The LNPs were prepared as described in Example 2 containing HA with a molecular weight of 8 - 15 kDa. As shown in Table 5 below, the ionizable lipids INT-A002, INT-A003, INT-A005, INT-A006, and INT-A007 facilitated the incorporation of HA.

[0146] Table 5: Physicochemical parameters of LNPs containing ionizable lipids and HA

Table 5

[0147] Example 8: Formulation of acidic peptides into lipid nanoparticles (LNPs) Similar to nucleic acids, proteins and peptides with a net negative charge can also be incorporated into LNPs using cationic ionizable lipids. LNPs were prepared by the method described in Example 2 containing an acidic peptide with a molecular weight of 4.2 kDa and a predicted net charge of -3. As shown in Table 6 below, the ionizable lipids INT-A001, INT-A002, INT-A003, INT-A004, INT-A005, INT-A006, and INT-A007 facilitated the incorporation of this acidic peptide.

[0148] Table 6: Physicochemical parameters of LNPs containing ionizable lipids and a 4.2 kDa acidic peptide

Table 6

[0149] Example 9: Formulation of acidic small peptides into lipid nanoparticles (LNPs) LNPs were prepared as described in Example 2 containing an acidic peptide with a molecular weight of 2.0 kDa and a predicted net charge of -3. As shown in Table 7 below, the ionizable lipids INT-A003, INT-A004, INT-A006, and INT-A007 facilitated the incorporation of this acidic peptide.

[0150] Table 7: Physicochemical parameters of LNP containing ionizable lipid and 2.0 kDa acidic peptide [Table 7]

[0151] Example 10: Formulation of cationic cargo into lipid nanoparticles (LNP) A basic peptide was used as an example of the cationic cargo. LNPs as described in Example 2 containing a basic peptide with a molecular weight of 2.0 kDa and a predicted net charge of +4 were prepared. As shown in Table 8 below, the ionizable lipids INT-A008 and INT-A009 promoted the incorporation of this basic peptide.

[0152] Table 8: Physicochemical parameters of LNP containing ionizable lipid and 2.0 kDa basic peptide [Table 8]

[0153] The claims appended hereto should not be limited by the specific embodiments described above, but should be construed to include all possible embodiments and equivalents to which such claims are entitled.

Claims

1. Formula I: 【Chemical 1】 wherein, in the formula, A is a starting group that is ionizable, permanently charged, or zwitterionic; (V) ｍ is -(CR1R2) ｍ wherein m is 0 to 10 or 2 to 6, and R1 and R2 are each independently hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle; Z-L has a structure of Formula II, IIa or IIb, L is a hydrocarbon structure with a total number of carbon atoms not exceeding 150, of Formula IIIc: [Chemical Formula 2] However, in the formula, the carbon chain scaffold of L is L1’ - [L1’’]n - G １ - [CH]p - [CH ２ ｑ - CH ３ represented by, and the total number of carbon atoms in the carbon skeleton of L is 10 to 30;​ L1' is a straight-chain hydrocarbon chain without heteroatoms, having 5 to 12 carbon atoms and 0 to 3 cis or trans double bonds; L1'' is a carbon atom; each X1 is independently selected from ether, ester, and carbamate groups; The carbon partial skeleton of the scaffold carbon chain L is G １ -CH-[CH ２ < ｑ -CH ３ (hereinafter referred to as L1''') and in the formula, G １ is a hydrocarbon chain having 0 to 4 carbon atoms and may have one cis or trans double bond; n is 0 to 4; p is 1 to 4; n + p is 1 to 4; q is 0 to 20; wherein each S and L1'''' are hydrocarbon side chains, each independently: (a) a straight-chain or branched terminal hydrocarbon chain having 0 to 5 cis or trans C═C and 5 to 30 carbon atoms, and bonding to one of each X1 at any carbon atom in the hydrocarbon chain; or, (b) a branched hydrocarbon structure of Formula IIIc with a total number of L1'''' and S hydrocarbon chains of 1 to 10, wherein each carbon atom of the L1'''' and S hydrocarbon chains of the lipid moiety may be substituted with a heteroatom, provided that no more than two heteroatoms are substituted in the hydrocarbon chain) is represented as a partial structure of, here, the structure of Formula II is a straight-chain linker structure: [Chemical 3] where Z is X1, wherein X1 is arbitrary, and X1 is selected from ether groups, ester groups, and carbamate groups, L ｂ is a branched lipid of formula IIIc, the structure of Formula IIa is a branched linker structure: 【Chemical Formula 4】 wherein Z is 【Chemical Formula 5】 ; W is arbitrary, when W is present, W is an X1 bond, N-C(O), N-C(O)O, or N-OC(O); W may be substituted with D, and D is an optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle; (X) ｎ If exists, (X) ｎ are each independently selected -(CR1R2) ｎ - is; (X) ｎ n is 0-10; T is optional and T is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle, which may be substituted; B is a carbon atom bonded to L1 and L2 via G1 and G2 respectively; G1 and G2 are independently selected from X1; Each of G1 and G2 may be independently bonded to B via a group (G) intervening as B-(G) ｕ -G1 or B-(G) ｕ -G2; ｕ and may be bonded to B via a group (G) intervening as B-(G); (G) ｕ is independently selected - (CR1R2) ｕ -, wherein R1 and R2 are each independently hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle, and u is from 0 to 16; G3 is optional, G3 is selected from X1, and intervenes as B-(G) ｕ -G3 (where G intervenes) ｕ may be bonded to B via a group; L1 is a branched hydrocarbon of Formula IIIc; L2 is a hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds, or has a structure of Formula IIIc; When L3 is present, L3 has a straight-chain or branched hydrocarbon chain having hydrogen, 1 to 20 carbon atoms, and 0 to 2 cis or trans double bonds, or has the structure of formula IIIc, The structure of formula IIb is a ring structure: ​ wherein Z is 【Chemical Formula 7】 and The curve represents a ring, and E and K represent atoms that partially constitute the ring structure, and the ring is a substituted or unsubstituted ring having 3 to 8 ring atoms; At least one of L1, L2, and L3 is bonded to a single atom within an arbitrary ring via G1, G2, and G3, respectively, and G1, G2, and G3 are each independently G1-(G) ｕ -L1, G2-(G) ｕ -L2 or G3-(G) ｕ -L3, with intervening (G) ｕ is optionally bonded to each one of L1, L2, and L3 via L1 and any L2 and / or L3 of formula IIb have the structure of formula IIIc, The A moiety is one of the following (i) to (iv): (i) An ionizable cationic moiety selected from the following group: 【Chemical Formula 8】 (ii) A permanently charged moiety selected from the following group: 【Chemical Formula 9】 (iii) An ionizable anionic moiety selected from the following group: 【Chemical Formula 10】 (iv) A zwitterionic moiety selected from the following group: 【Chemical 11】 A charged lipid containing a branched lipid moiety L selected from **Claim 2** Z-L has the structure of formula II of a linear linker structure: 【Chemical 12】 wherein L1' of formula IIIc has 5 to 12 or 5 to 9 carbon atoms and has 0 to 3 or 0 to 2 cis or trans double bonds; When L1'''' of formula IIIc is present, L1'''' and S are independently selected from linear or branched hydrocarbons having 0 to 5 cis or trans CH=CH and 5 to 18 carbon atoms; and G of formula IIIc １ is absent, CH ２ or CH = CHCH ２ wherein the double bond is cis or trans; q of formula IIIc is 1 to 9 or 1 to 5, The charged lipid according to claim 1. **Claim 3** The charged lipid according to claim 1 or 2, wherein n + p is 2, 3 or 4. **Claim 4** The charged lipid according to claim 2 or 3, wherein each X1 of formula IIIc has an ester orientation with an oxygen of an ester bonded to the L carbon skeleton. **Claim 5** Z-L has the structure of formula IIa of a branched linker structure: (V) of Formula I ｍ is (CH ２ ), ｍ where m is from 1 to 20; wherein 【Chemical Formula 13】 **Claim 6** In the formula, W is an ether, ester or carbamate group, D is absent, and (X) ｎ is (CH ２ ) ｎ and n is from 1 to 10. G1 and G2 exist, and each of G1 and G2 is independently B-(G) ｕ -L1 or B-(G) ｕ -L2, where (G) ｕ is coupled to B via (G) ｕ is (CH ２ ) ｕ and; G3-L3 exists, and G3-L3 is CH ３ and CH ２ CH ３ is a hydrocarbon selected from, or G3-L3 is CH ２ X1L3, where 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 has the structure of formula IIIc, the charged lipid according to claim 1. Z-L has the structure of formula IIb of a ring structure: wherein 【Chemical Formula 14】 The curve represents a ring, and E and K represent atoms that partially constitute the ring structure, and the ring is a substituted or unsubstituted carbocyclic ring having 3 to 6 ring atoms. The charged lipid according to claim 1. **Claim 7** The charged lipid according to claim 6, wherein the ring has 3 or 5 carbon atoms. **Claim 8** **Claim 9** At least L1 and L2 are present, and L1 and L2 are each bonded to the ring via G1 and G2 groups, respectively, and each of the G1 and G2 groups is optionally bonded to a ring atom via an intervening (G) ｕ and may be optionally bonded to a ring atom via (G) ｕ where (G) ２ is (CH ｕ ) and u is from 0 to 10 or from 0 to 6, the charged lipid according to claim 6 or 7 **Claim 10** (V) ｍ The charged lipid according to any one of claims 1 to 8, wherein R1 or R2 of (V) is each independently a cycloalkyl which may be substituted with a monocyclic, bicyclic or tricyclic carbocyclic ring. **Claim 11** (V) The charged lipid according to any one of claims 1 to 8, wherein R1 or R2 of m is a hydrogen atom. (V) ｍ The charged lipid according to any one of claims 1 to 8, wherein R1 or R2 is each independently selected from heteroatom rings having 4 to 12 ring atoms. **Claim 12** The charged lipid according to any one of claims 1 to 8, wherein m = 0. ​ **Claim 13**: The A moiety is one of the following (i) to (ii): (i) An ionizable cationic moiety selected from the following group: 【Chemical Formula 15】 (ii) A permanently charged moiety selected from the following group: 【Chemical 16】 The charged lipid according to any one of claims 1 to 12, selected from the above. **Claim 14**: The A moiety is -NR₂, where each R is independently a hydrogen atom, methyl, ethyl, propyl or butyl, and the charged lipid according to any one of claims 1 to 12. **Claim 15**: The A moiety is -NR₂, where each R is independently methyl or ethyl, and the charged lipid according to claim 14. **Claim 16**: Any of the following structures: 【Chemical 17】 The charged lipid according to claim 1 or a pharmaceutically acceptable salt thereof, having the above structure. **Claim 17** A drug delivery vehicle formulation comprising the charged lipid according to any one of claims 1 to 16 and a cargo molecule or compound which is a nucleic acid, protein or peptide. **Claim 18** The drug delivery vehicle formulation according to claim 17, wherein the nucleic acid is small interfering RNA, small activating RNA, messenger RNA, microRNA, antisense oligonucleotide, ribozyme, aptamer, plasmid, circular DNA, linear DNA, antigomir, anti-miRNA oligonucleotide or miRNA mimic. **Claim 19**: The drug delivery vehicle formulation according to claim 18, wherein the nucleic acid is messenger RNA. **Claim 20** The drug delivery vehicle formulation according to claim 17, wherein the cargo molecule or compound is a peptide. **Claim 21**: The drug delivery vehicle formulation according to any one of claims 17 to 20, comprising lipid nanoparticles (LNP). **Claim 22**: The drug delivery vehicle formulation according to claim 21, wherein the lipid nanoparticles are vesicle-forming lipids. **Claim 23**: The drug delivery vehicle formulation according to claim 22, wherein the lipid nanoparticles further contain a sterol. **Claim 24**: The drug delivery vehicle formulation according to claim 21 or 22, wherein the lipid nanoparticles further contain a hydrophilic polymer-lipid conjugate. **Claim 25**: The drug delivery vehicle formulation according to any one of claims 21 to 24, wherein the lipid nanoparticles (LNP) contain phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, ceramide, sphingomyelin or a hydrophilic polymer-lipid conjugate. **Claim 26** The drug delivery vehicle formulation according to any one of claims 21 to 25, wherein the polydispersity of the lipid nanoparticles is less than 0.

15. **Claim 27** The drug delivery vehicle formulation according to any one of claims 21 to 26, wherein the encapsulation efficiency of the lipid nanoparticles is 70% to 100%. **Claim 28** The drug delivery vehicle formulation according to claim 27, wherein the encapsulation efficiency of the lipid nanoparticles is 80% to 100%.

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