Ionized lipids and nanoparticles containing the same

Abstract

One or more ionized lipids and lipid nanoparticles comprising the same are provided, as well as lipid nanoparticles encapsulating polynucleic acids. Pharmaceutical compositions are also provided, including a therapeutically effective amount of lipid nanoparticles encapsulating therapeutically active polynucleic acids. Pharmaceutical compositions are also provided for use in delivering polynucleic acids to the lung tissue of a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 437,810, entitled "Ionized Lipids and Nanoparticles Comprising Same," filed January 9, 2023, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to ionized lipids and lipid nanoparticles comprising same, and their use in pharmaceutical compositions. [Background technology]

[0003] New delivery methods for therapeutic and diagnostic compounds are constantly being developed. Lipid-based nanoparticles are a well-known delivery vehicle, but these agents are also constantly being improved. In particular, the ability of therapeutic carriers to effectively load active agents and subsequently deliver them to the target site is crucial for reducing dosage and improving therapeutic efficacy.

[0004] Although various ionizable lipids capable of encapsulating hydrophilic drugs such as DNA and / or RNA are known, there is a continuing need for new and improved lipid nanoparticles (LNPs). In particular, there is a great need for the development of new and improved LNPs that can improve drug delivery to specific sites in the body. In particular, there is a need for lipid nanoparticles that are characterized by improved lung specificity and are suitable for the administration of lung-specific drugs. Summary of the Invention

[0005] The present invention provides novel compounds suitable for use as ionizable lipids. It also provides nanoparticles containing the same. It also provides compositions containing the nanoparticles that are useful for delivering active agents to a subject, such as for treating or preventing a disease or disorder in the subject.

[0006] In one embodiment, there is provided a lipid nanoparticle comprising a compound, a salt of the compound, or both, wherein the compound has Formula 1: [ka] wherein each X independently represents -O-, -S-, CH2, or X is absent; each Z independently represents -OH or -SH; each A independently represents O or S; each n independently represents an integer from 0 to 5, provided that at least one n is not 0; each R independently represents H or an optionally substituted C5-C30 alkyl; the lipid nanoparticle further comprises an active agent, a helper lipid, a structural lipid, and a modified lipid; the helper lipid is a cationic lipid; the ratio of the compound to the total lipid content of the lipid nanoparticle is 10 to 50 mol%; the ratio of the structural lipid to the total lipid content of the lipid nanoparticle is 5 to 50 mol%; and the lipid nanoparticle is characterized by having an average zeta potential in the range of -5 to +40 mV at pH 6 to 8.

[0007] In one embodiment, the molar ratio of the helper lipid to the modified lipid is 1:0.2 to 1:0.01.

[0008] In one embodiment, the molar ratio of the compound to the helper lipid is from 0.2:1 to 5:1.

[0009] In one embodiment, the molar ratio of the structured lipid to the modified lipid is 1:0.01 to 1:0.2.

[0010] In one embodiment, the active agent comprises a polynucleic acid; the N:P ratio within the LNP is 1-20.

[0011] In one embodiment, the weight ratio of (i) the total amount of the compound, the helper lipid, the structural lipid, and the modified lipid to (ii) the polynucleic acid in the lipid nanoparticle is 0.001:1 to 10:1.

[0012] In one embodiment, the lipid nanoparticles have a particle size distribution in the range of 50 to 500 nm; and the cationic lipid is selected from DOTAP, DDAB, 18:1EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine), and 18:0EPC (1,2-distearoyl-sn-glycero-3-ethylphosphocholine), including any salt thereof and any combination thereof.

[0013] In one embodiment, the lipid nanoparticles are characterized by any one of the following: an average zeta potential of 0 to +5 mV at pH 6 to 8; an N:P ratio of 3 to 9; the structural lipid is cholesterol; the modified lipid is a PEG-lipid; the ratio of the helper lipid to the total lipid content of the lipid nanoparticle is 35 to 45 mol %; and the ratio of PEG-lipid to the total lipid content of the lipid nanoparticle is 0.1 to 3 mol %.

[0014] In one embodiment, the lipid nanoparticles are characterized by a pKa of 5 to 9, and the compound has the following structure: [ka] It contains either MB-212 or MB-222.

[0015] In one embodiment, upon administration to a subject, the LNP is characterized by at least 10-fold greater expression in the lung compared to any one of the liver, spleen, and kidney of said subject.

[0016] In one embodiment, the N:P ratio is about 5, the cationic lipid is DOTAP, and the proportion of said compound relative to the total lipid content of said lipid nanoparticles is about 15 to about 25 mol %.

[0017] In another aspect, there is provided a pharmaceutical composition comprising a plurality of lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0018] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the active agent.

[0019] In another aspect, a method of delivering an active agent to pulmonary tissue of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention, thereby delivering an active agent to the pulmonary tissue.

[0020] In one embodiment, the therapeutically effective amount is 0.01 to 5 mg / kg per day.

[0021] In the methods of the present invention, the administration is intravenous, intratracheal, intranasal, or by inhalation.

[0022] In another embodiment, lipid nanoparticles are provided that include a compound, a salt of the compound, or both, wherein the compound has Formula I: [ka] wherein each L independently represents R1, [ka] and; Each L1 independently selects R1, [ka] and [ka] represents a single bond, triple bond, or double bond; Z independently represents -OH or -SH; A independently represents O or S; each k is independently from 0 to 10; each Y independently includes absent, CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, -C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-, or phosphate (as allowed by valences); each T independently represents an optionally substituted C5-C30 alkyl or an optionally substituted C5-C30 alkenyl; each R' is independently H or comprises an optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each X independently represents a heteroatom, CH, optionally substituted C-C alkyl, or X is absent; each n and p is independently 0 to 5, and at least one n is not 0; m is 1 to 3; each R is independently H or comprises an optionally substituted C5-C30 alkyl; Each R1 is an optionally substituted C1-C24 alkyl, and at least one L or L1 is [ka] is or contains; The lipid nanoparticles further comprise an active agent, a helper lipid, a structural lipid, and a modified lipid, wherein the ratio of the compound to the total lipid content of the lipid nanoparticles is 15 to 55 mol %, and the ratio of the structural lipid to the total lipid content of the lipid nanoparticles is 20 to 60 mol %.

[0023] In some embodiments, the molar ratio of helper lipid to modified lipid ranges from 1:0.2 to 1:0.01.

[0024] In some embodiments, the molar ratio of compound to helper lipid ranges from 0.5:1 to 5:1.

[0025] In some embodiments, the molar ratio of structured lipid to modified lipid ranges from 1:0.01 to 1:0.2.

[0026] In some embodiments, the active agent comprises a polynucleic acid.

[0027] In some embodiments, the weight ratio of (i) the total amount of compound, helper lipid, structural lipid, and modified lipid to (ii) the polynucleic acid in the lipid nanoparticle is between 0.001:1 and 10:1.

[0028] In some embodiments, the size distribution of the lipid nanoparticles ranges from 50 to 500 nm.

[0029] In some embodiments, the lipid nanoparticles are characterized by a zeta potential in the range of -5 to 40 mV at pH 6-8.

[0030] In some embodiments, the lipid nanoparticles are characterized by a pKa of 5-9.

[0031] In some embodiments, the N:P ratio within the lipid nanoparticles ranges from 3-20.

[0032] In some embodiments, the N:P ratio is about 12.

[0033] In another aspect, there is provided a pharmaceutical composition comprising a plurality of lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0034] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the active agent.

[0035] In some embodiments, the pharmaceutical composition is used to deliver an active agent to pulmonary tissue.

[0036] In some embodiments, the pharmaceutical composition is used to treat a pulmonary disease or disorder.

[0037] In another aspect, a method of delivering an active agent to pulmonary tissue of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention, thereby delivering the active agent to the pulmonary tissue.

[0038] In some embodiments, the therapeutically effective amount is 0.01 to 1 mg / kg per day.

[0039] Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. Reference will now be made in detail to the drawings, and it is emphasized in particular that the details shown therein are by way of example and are for illustrative purposes only. In this regard, reading the description in conjunction with the drawings will make apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief explanation of the drawings]

[0040] [Figure 1] 1 is a bar graph showing in vivo expression analysis of three LNP compositions (FMB-1143, FMB-748, and FMB-745), showing fluorescent signal intensity in lung tissue compared to heart, spleen, kidney, and liver tissue. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention is based, at least in part, on the surprising discovery that certain lipid nanoparticles (LNPs) of the invention exhibit lung-specific in vivo activity. Accordingly, the LNPs of the invention can be used for the specific delivery of active agents to the lung tissue of a subject in need thereof.

[0042] The compounds disclosed in the present invention were discovered through computational screening methods. A large number of LNP compositions were generated in silico and ranked based on predicted activity using a machine learning algorithm. After multiple in silico optimization cycles, a library containing multiple LNP compositions was obtained. RNA was encapsulated in the LNP compositions, and RNA activity in vivo was investigated. The disclosed LNP compositions were selected based on the results obtained from in vivo experiments described below.

[0043] lipid nanoparticles In one aspect of the invention, there is provided a lipid nanoparticle (LNP) comprising: (i) a compound and any salt thereof; (ii) a helper lipid; (iii) a structural lipid; and (iv) a modified lipid; the molar ratio of the compound to the total lipid content of the composition is about 15-55 mol %, or about 10-55 mol %, and the compound has Formula I: [ka] wherein each L independently represents R1, [ka] and each L1 independently represents R1, [ka] and [ka] represents a single bond, a triple bond, or a double bond; Z independently represents -OH or -SH; A independently represents O or S; each k independently ranges from 0 to 10; and each Y independently represents none, CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S )O—, —NC(═S)N—, —C(═O)—, —C(═O)O—, —OC(═O)O—, —OC(═O)N—, —OC(═S)O—, —OC(═S)N—, or phosphate (where allowed by valence); each T independently represents an optionally substituted C5-C30 alkyl or an optionally substituted C5-C30 alkenyl; each R′ independently is H or any each X independently represents a heteroatom, CH, or an optionally substituted C-C alkyl, or X is absent; each n and p independently ranges from 0 to 5, with at least one n being different from 0; m ranges from 1 to 3; each R independently represents H, an optionally substituted C-C alkyl, or a C-C alkyl; each R is an optionally substituted C-C alkyl, or a C-C alkyl; [ka] is or contains

[0044] In some embodiments, a composition is provided comprising a plurality of LNPs, each LNP comprising (i) a compound and any salt thereof, (ii) a helper lipid, (iii) a structural lipid, and (iv) a modifying lipid; the molar ratio of the compound to the total lipid content of the composition is about 105-55 mol %, and the compound has Formula I: [ka] wherein each L independently represents R1, [ka] and each L1 independently represents R1, [ka] and [ka] represents a single bond, a triple bond, or a double bond; Z independently represents -OH or -SH; A independently represents O or S; each k is independently a value from 0 to 10; and each Y is independently absent, CH, CHR', NR', NH, O, S, -CONH-, -CONR'-, C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N- , -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-, or phosphate (where allowed by valence); each T independently represents an optionally substituted C5-C30 alkyl or an optionally substituted C5-C30 alkenyl, and each R' independently represents H or or optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or combinations thereof; each X independently represents a heteroatom, CH2, or optionally substituted C1-C10 alkyl, or X is absent; each n and p independently ranges from 0 to 5, with at least one n being different from 0; m ranges from 1 to 3; each R independently represents H, optionally substituted C5-C30 alkyl, or C1-C30 alkyl; each R1 is optionally substituted C1-C24 alkyl, and at least one L or L1 is [ka] is or contains

[0045] In some embodiments, the compositions of the invention are LNP compositions (e.g., solid compositions comprising dried LNPs or liquid dispersions comprising LNPs dispersed or suspended in a solvent, such as an aqueous solvent), comprising (i) a compound and any salt thereof, (ii) a helper lipid, (iii) a structural lipid, and (iv) a modifying lipid; the molar ratio of the compound to the total lipid content in the composition is about 15-55 mol %, or about 10-55 mol %, and the compound has Formula I: [ka] wherein each L independently represents R1, [ka] and each L1 independently represents R1, [ka] and [ka] represents a single bond, a triple bond, or a double bond; Z independently represents -OH or -SH; A independently represents O or S; each k is independently 0 to 10; and each Y is independently absent, or CH, CHR', NR', NH, O, S, -CONH-, -CONR'-, C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, - each T independently represents an optionally substituted C5-C30 alkyl or an optionally substituted C5-C30 alkenyl; each R' independently represents H or or optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or combinations thereof; each X independently represents a heteroatom, CH2, or optionally substituted C1-C10 alkyl, or X is absent; each n and p independently ranges from 0 to 5, with at least one n not being 0; m ranges from 1 to 3; each R independently ranges from H or includes optionally substituted C5-C30 alkyl or C1-C30 alkyl; each R1 is optionally substituted C1-C24 alkyl, and at least one L or L1 is [ka] is or contains

[0046] In some embodiments, the compound has Formula II: [ka] Each L and L1 is expressed as [ka] wherein X, Z, A, and R are as defined above, and each n is independently 0 to 5, and at least one n is not 0. In some embodiments, the compound is represented by Formula II, wherein each X is selected from -O-, -S-, and CH2; and at least two R represent optionally substituted C1-C30 alkyl.

[0047] In some embodiments, the compound is represented by Formula II or Formula I, and the total number of C atoms in the R groups is 5-50, 10-50, 40, 20-40, 20-35, 10-35, and any range therebetween.

[0048] In some embodiments, the compound has Formula II or Formula I, and at least one R represents an optionally substituted alkyl of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, or any range therebetween.

[0049] In some embodiments, the compound has Formula II or Formula I, and at least two R's represent optionally substituted alkyl of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, or any range therebetween.

[0050] In some embodiments, the compound has Formula II or Formula I, wherein each R represents an optionally substituted alkyl of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, or any range therebetween.

[0051] In some embodiments, the compound has Formula 1: [ka] wherein Z, A, R, and n are as defined above; each X independently represents -O-, -S-, methylene, or X is not present; and at least one n is not 0. In some embodiments, the compound has Formula 1, wherein at least one X is not present and at least one R is not H. In some embodiments, the compound has Formula 1, wherein at least two R are not H.

[0052] In some embodiments, the compound has Formula 1 and the total number of C atoms in the R groups is 10-60, 5-50, 5-60, 510-40, 20-40, 20-35, 10-35, and any range therebetween. In some embodiments, the compound has Formula 1 and at least one, or at least two R groups represent an optionally substituted alkyl of 1-30, 5-30, 5-20, 1-20, 1-10, 5-10 carbon atoms in length, or any range therebetween.

[0053] In some embodiments, the compound is represented by Formula 1, wherein each A is -O-, each Z is -OH, and each R is an optionally substituted alkyl of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, or any range therebetween.

[0054] In some embodiments, the compound has Formula 2: [ka] wherein X is as defined above, each n is 1-5, m is 0-3, and each R is an optionally substituted alkyl of 1-30, 5-30, 5-20, 1-20, 1-10, 5-10 carbon atoms in length, or any range therebetween. In some embodiments, the compound has Formula 2, wherein each R is an optionally substituted C5-C10 alkyl. In some embodiments, the compound has the following structure: [ka] It contains either one or both of MB-212 and MB-222.

[0055] In some embodiments, each LNP comprises (i) a compound represented by Formula 1 or 2 (including salts thereof), (ii) a helper lipid, (iii) a structural lipid, and (iv) a modifying lipid, wherein the helper lipid is a cationic lipid; the structural lipid is a sterol; the modifying lipid is a PEG-lipid; the plurality of LNPs are characterized by a positive or neutral average zeta potential; and each LNP encapsulates an active agent comprising a polynucleic acid.

[0056] In some embodiments, the molar concentration of one or more compounds of the present invention in an LNP is 10-55 mol%, 10-25, 10-20, 20-25, 15-30, 20-35, 30-40, 30-35, or 20-55 mol%, including any range therebetween. As used herein, the term "concentration" or "molarity" refers to the molar ratio relative to the total lipid content of the nanoparticle. Those skilled in the art will understand that the molar ratios of essential components (i.e., compounds of the present invention, helper lipids, structural lipids, and modified lipids) in an LNP and in compositions of the present invention are identical. Thus, for example, molar concentrations and molar ratios disclosed herein with respect to an LNP encompass the corresponding molar concentrations and molar ratios in compositions of the present invention, and vice versa.

[0057] In some embodiments, total lipid content refers to the combined content of the compound of the present invention, structural lipids, modified lipids, and helper lipids.

[0058] In some embodiments, the molar ratio of structured lipid to the total lipid content of the composition is 5-50 mol%, including any range or value therebetween. In some embodiments, the molar ratio of compound (represented by any of Formulas I-2) to the total lipid content of the lipid nanoparticle is 10-55 mol%.

[0059] In some embodiments, the molar ratio of the compound of the present invention to the total lipid content of the composition is 10-55 mol%, 15-25 mol%, 15-20 mol%, 20-25 mol%, 15-30 mol%, 20-35 mol%, 30-40 mol%, 30-35 mol%, 20-55 mol%, 20-55 mol%, or any range therebetween. In some embodiments, the molar ratio of the compound of the present invention to the total lipid content of the composition is about 20 to about 40 mol%.

[0060] In some embodiments, the molar concentration of the helper lipid relative to the total lipid content of the composition is 5 to about 60 mol%, 5 to 10 mol%, 5 to 15 mol%, 10 to 40 mol%, 10 to 30 mol%, 30 to 40 mol%, 40 to 45 mol%, 30 to 55 mol%, 30 to 60 mol%, including any range therebetween.

[0061] In some embodiments, the molar concentration of the helper lipid in the LNP is about 35 to about 45, about 38 to about 45 mol%, about 38 to about 42 mol%, or about 40 mol%, including any range therebetween.

[0062] In some embodiments, the molar concentration of structured lipid relative to the total lipid content of the composition is 5-50, 15-60, 15-50, 20-25, 20-30, 20-40, 25-45, 35-40, 35-45, 35-50, 40-50, 45-50, 35-50 mol%, including any range or value therebetween.

[0063] In some embodiments, the molar concentration of structural lipids within the LNP is 20-40 mol%, 20-23 mol%, 20-25 mol%, 35-40 mol%, 37-40 mol%, 25-35 mol%, including any range therebetween.

[0064] In some embodiments, the molar concentration of modified lipids (e.g., PEG-lipids) within the LNPs is 0.5-10 mol%, 0.1-10 mol%, 0.1-0.5 mol%, 0.5-1 mol%, 1-5 mol%, 0.5-2 mol%, 5-10 mol%, 5-7 mol%, 7-10 mol%, including any range therebetween.

[0065] In some embodiments, the LNP or composition comprises (i) about 20 to about 40 mol % molar concentration of structured lipids, and (ii) about 1.5 to about 2.5 mol % molar concentration of modified lipids, and (iii) about 35 to about 40 mol % molar concentration of helper lipids, and (iv) about 20 to 35 mol % molar concentration of a compound of the invention.

[0066] In some embodiments, the weight ratio of compound to polynucleic acid in the LNP / composition is 0.001:1 to 10:1, 0.001:1 to 0.1:1, 0.1:1 to 1:1, 1:1 to 10:1, and any range therebetween.

[0067] In some embodiments, the N:P ratio in an LNP or composition of the invention is in the range of 1 to 20, 3 to 6, 3 to 9, 6 to 8, 6 to 8, 8 to 10, 10 to 12, 6 to 12, 6 to 13, and 10 to 20, 8 to 20, 8 to 15, and 4 to 5, including any range therebetween. The term "N:P ratio" refers to the ratio of N atoms of a compound of the invention to P atoms of a polynucleotide within a lipid nanoparticle or composition of the invention.

[0068] In some embodiments, the N:P ratio is selected from about 5, or 3, 4, 5, and 6, or 3 to 9, including any value or range therebetween.

[0069] In some embodiments, the LNPs consist essentially of a compound of the invention, a helper lipid, a structural lipid, and a modified lipid. In some embodiments, the LNPs further encapsulate an active agent. In some embodiments, the LNPs comprise a shell containing an active agent and an aqueous core. In some embodiments, the shell of the LNP comprises a compound of the invention, a helper lipid, a structural lipid, and a modified lipid. In some embodiments, the active agent is incorporated within the shell, within the aqueous core, or within the interface between the shell and the core. As used herein, the term "shell" refers to the outer portion of the particle, which differs in composition from the core.

[0070] In some embodiments, the LNPs consist essentially of a compound of Formula 1, a cationic helper lipid, a sterol, and a PEG-lipid; the LNPs further encapsulate an active agent; the molar ratio of the compound to the total lipid content of the composition is about 15-40 mol %; the molar ratio of the cationic helper lipid to the total lipid content of the composition is about 30-50 mol %, and the LNPs in the composition are characterized by an average zeta potential in the range of -5 to +40 mV when measured at pH 6-8 (e.g., about 7).

[0071] In some embodiments, the combined molar concentration of the helper lipid and the compound of the present invention within the LNP is about 40 to about 75 mol %, or about 60 to about 75 mol %; the N:P ratio is 3 to 9; the average particle size of the LNP in the composition is about 50 to 300 nm; and the LNP in the composition is characterized by an average zeta potential in the range of -5 to +40 mV, or 0 to +10 mV, when measured at pH 6 to 8 (e.g., about 7).

[0072] In some embodiments, the compound of the present invention is of Formula 1 or Formula 2; the total molar concentration of the helper lipid and the compound of the present invention in the LNP is about 40 to about 75 mol % or about 60 to about 75 mol %; the ratio of the helper lipid to the total lipid content of the lipid nanoparticle is 35 to 45 mol %; the ratio of PEG-lipid to the total lipid content of the lipid nanoparticle is 1 to 5 mol %; the N:P ratio is 8 to 12; the average particle size of the LNPs in the composition is about 50 to 300 nm; and the LNPs in the composition are characterized by an average zeta potential in the range of -1 to +5 mV when measured at pH 6 to 8 (e.g., about 7).

[0073] In some embodiments, the compound of the present invention is of Formula 1 or Formula 2; the total molar concentration of the helper lipid and the compound of the present invention in the LNP is about 40 to about 75 mol %; the ratio of the helper lipid to the total lipid content of the lipid nanoparticle is 35 to 45 mol %; the ratio of PEG-lipid to the total lipid content of the lipid nanoparticle is 1 to 5 mol %; the N:P ratio is about 12; the average particle size of the LNPs in the composition is about 50 to 300 nm; and the LNPs in the composition are characterized by an average zeta potential in the range of 0 to +5 mV when measured at pH 6 to 8 (e.g., about 7).

[0074] In some embodiments, the compound of the present invention is of Formula 1 or 2; the helper lipid is DOTAP; the total molar concentration of the helper lipid and the compound of the present invention in the LNP is about 40 to about 75 mol%; the ratio of the helper lipid to the total lipid content of the lipid nanoparticle is 30 to 50 mol%; the ratio of the compound to the total lipid content of the lipid nanoparticle is about 10 to about 30 mol%; the ratio of PEG-lipid to the total lipid content of the lipid nanoparticle is 0.1 to 5 mol%; the N:P ratio is between about 4 and about 5; the average particle size of the LNPs in the composition is 50 to 300 nm; and the LNPs in the composition are characterized by an average zeta potential in the range of 0 to +5 mV when measured at pH 6 to 8 (e.g., about 7).

[0075] In some embodiments, under appropriate conditions, at least one compound of the present invention, an active agent, a helper lipid, a structural lipid, and a modified lipid undergo self-assembly in aqueous solution to form LNPs. In some embodiments, the lipid nanoparticles are formulated to deliver one or more drugs to one or more target cells.

[0076] In some embodiments, the LNPs of the present invention have a spherical shape or form. In some embodiments, the LNPs have an expanded or contracted shape. In some embodiments, the core-shell particles do not have a distinctive shape or form. In some embodiments, the LNPs have a spherical, quasi-spherical, quasi-ellipsoidal, contracted, concave, irregular shape, or any combination thereof.

[0077] In some embodiments, the plurality of core-shell particles in the compositions of the present invention are substantially spherical, where "substantially" is as defined herein. In some embodiments, the plurality of core-shell particles are substantially ellipsoidal, where "substantially" is as defined herein. Those skilled in the art will understand that the exact shape of each of the plurality of core-shell particles may vary from particle to particle. Furthermore, because the exact shape of an LNP can be derived from any of the geometric shapes described above, the shape of the particle will not perfectly conform to a particular geometric shape. Those skilled in the art will understand that the exact shape of an LNP may have a substantial deviation (e.g., deviation of at least 5%, at least 10%, at least 20%) from a particular geometric shape (e.g., sphere or ellipsoid).

[0078] In some embodiments, the ratio of compound to total lipid content of the LNPs of the present invention is 10-55%, 15-25%, 25-30%, 25-35%, 30-35%, 35-45%, and 45-55%, including any range therebetween.

[0079] In some embodiments, the ratio of structural lipids to the total lipid content of the LNPs of the present invention is in the range of 5-50%, 20-30%, 25-30%, 25-35%, 30-35%, 35-45%, 45-50%, 35-50%, including any range therebetween.

[0080] In some embodiments, the molar ratio of helper lipid to modified lipid ranges from 1:0.2 to 1:0.01, 1:0.15 to 1:0.01, 1:0.1 to 1:0.01, 1:0.05 to 1:0.01, including any range therebetween.

[0081] In some embodiments, the molar ratio of compound to helper lipid is in the range of 1:0.5 to 1:5, 1:0.5 to 4:1, 1:0.5 to 3:1, 1:0.5 to 2:1, 1:0.5 to 1:1, 1:0.1 to 5:1, 1:0.25 to 5:1, 1:0.5 to 2:1, 1:1 to 1:2, 1:1 to 1:5, including any range therebetween.

[0082] In some embodiments, the molar ratio of structured lipid to modified lipid ranges from 200:1 to 2:1, 100:1 to 5:1, 100:1 to 10:1, 100:1 to 30:1, 100:1 to 50:1, 100:1 to 70:1, 100:1 to 90:1, 100:1 to 100:3, 100:1 to 20:1, including any range therebetween.

[0083] Lipid Nanoparticle Composition In some embodiments, the compounds of the present invention are represented by Formula I as described above. In some embodiments, the compounds of the present invention are ionizable lipids. In some embodiments, the terms "compound," "compound of the present invention," and "ionizable lipid" are used interchangeably herein. In some embodiments, the heteroatom comprises O, N, NH, NR1, or S. In some embodiments, each X is independently O or absent.

[0084] In some embodiments, one of R and R1 each independently represents a straight-chain or branched alkyl.

[0085] In some embodiments, L is [ka] and R is as described herein. In some embodiments, each R represents the same or different alkyl.

[0086] In some embodiments, L is [ka] and R is as described herein. In some embodiments, each R represents the same or different alkyl.

[0087] In some embodiments, L is [ka] and R is as described herein. In some embodiments, each R represents the same or different alkyl.

[0088] In some embodiments, L is [ka] and R is as described herein. In some embodiments, each R represents the same or different alkyl.

[0089] As used herein, "(C3-C 10 The term "cycloalkyl" refers to an optionally substituted C, C, C, C, C, C, C, or C ring. In some embodiments, (C-C 10 ) ring includes optionally substituted cyclopropane, cyclobutene, cyclopentane, cyclohexane, or cycloheptane.

[0090] As used herein, "C3-C 10 The term "heterocyclyl" refers to a C3, C4, C5, C6, C7, C8, C9 or C10 heterocyclic aromatic and / or aliphatic, or unsaturated ring.

[0091] The term "alkyl," as used herein, refers to an aliphatic hydrocarbon, including straight-chain and branched-chain groups. In some embodiments, an alkyl group has 1 to 10 carbon atoms, 1 to 30 carbon atoms, or 5 to 30 carbon atoms. When a numerical range (e.g., "5-30") is recited herein, the group (in this case, the alkyl group) may contain 5 carbon atoms, 6 carbon atoms, 10 carbon atoms, 5 to 20 carbon atoms, 5 to 25 carbon atoms, 5 to 30 carbon atoms, 10 to 20 carbon atoms, 10 to 25 carbon atoms, 10 to 30 carbon atoms, or any range therebetween, up to 30 carbon atoms. Alkyl can be substituted or unsubstituted, as defined herein.

[0092] As used herein, the term "alkyl" encompasses saturated or unsaturated hydrocarbons, and thus the term further includes alkenyl and alkynyl. In some embodiments, an alkyl group is a C1-C10 alkyl.

[0093] The term "C1-C10 alkyl," as used herein, refers to a straight or branched alkyl chain containing 1 to 6, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10 carbon atoms, or any range therebetween, including C1-C10 alkyl related compounds. In some embodiments, C1-C10 alkyl includes any one of methyl, ethyl, propyl, butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, and tert-butyl, or any combination thereof. In some embodiments, the C1-C10 alkyl described herein further includes an unsaturated bond, where the unsaturated bond is at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th position of the C1-C10 alkyl.

[0094] The term "alkenyl" refers to an unsaturated alkyl, as defined herein, having from 2 to 30 carbon atoms and having at least one carbon-carbon double bond. The alkenyl may be unsubstituted or substituted with one or more substituents, as described herein above.

[0095] The term "alkynyl," as defined herein, is an unsaturated alkyl having from 2 to 30 carbon atoms and having at least one carbon-carbon triple bond. Alkynyl may be unsubstituted or substituted with one or more substituents, as described herein above.

[0096] In some embodiments, ionizable lipids can be ionized (protonated or positively ionized) in a solution having a pH value less than the pKa value of the ionizable lipid. In some embodiments, ionizable lipids can be protonated in a solution having a pH value less than the pKa value of the ionizable lipid. In some embodiments, at least 50 mol% of the ionizable moieties are positively charged (or protonated) in the compositions of the present invention having a pH value less than the pKa value of the ionizable lipid.

[0097] In some embodiments, the compounds of the present invention have a MW of 100-2,000 Da, 100-300 Da, 100-500 Da, 100-800 Da, 300-500 Da, 100-1,000 Da, 500-800 Da, 500-1,000 Da, 800-1,200 Da, 1,000-2,000 Da, including any range therebetween.

[0098] In some embodiments, the compounds of the present invention comprise substantially a single enantiomer of any one of the compounds described herein, where "substantially" comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% by weight, including any value therebetween.

[0099] In some embodiments, the compounds of the present invention further include structurally similar functional derivatives of any of the compounds disclosed herein, where structurally similar means at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% structural similarity, including any range of structural similarity therebetween.

[0100] In some embodiments, the term "structural similarity" refers to the fingerprint similarity of two molecules. The term "fingerprint similarity" is well understood by those skilled in the art. In some embodiments, fingerprint similarity is calculated based on circular fingerprints, substructure key-based fingerprints, and / or topology or path-based fingerprints.

[0101] Exemplary circular fingerprints include, but are not limited to: Molprint2D, ECFP (or Morgan fingerprint), FCFP, etc. In some embodiments, the term "structural similarity" as used herein is calculated by a Morgan fingerprint.

[0102] In some embodiments, the functional derivative is an ionizable lipid with a pKa value of 6.2-6.8, which can self-assemble in water to stably bind and / or encapsulate the polynucleic acid. In some embodiments, the functional derivative is further configured to internalize the polynucleic acid into a cell (e.g., by forming lipid nanoparticles as described herein). Cellular internalization can be determined as described below.

[0103] In some embodiments, the compound is MB-212, described below, or a salt thereof.

[0104] In some embodiments, the compound is MB-222, described below, or a salt thereof.

[0105] In some embodiments, the active agent is a small molecule and / or biomolecule, such as a polypeptide or polynucleotide. In some embodiments, the active agent is selected from a therapeutic agent, a prophylactic agent, and a diagnostic agent, or any combination thereof. In some embodiments, one or more active agents are selected from the group consisting of a protein, a peptide, a nucleic acid, a small molecule, and an antibody.

[0106] In some embodiments, the active agent is a pulmonary therapeutic agent. In some embodiments, the active agent is an anti-cancer agent. In some embodiments, the active agent is an immunotherapeutic agent. In some embodiments, the active agent is an anti-infective agent. In some embodiments, the active agent is an anti-inflammatory agent.

[0107] In some embodiments, the active agent is a polynucleic acid.

[0108] In some embodiments, the terms "polynucleic acid" and "polynucleotide" are used interchangeably herein. In some embodiments, a polynucleotide comprises 60 to 15,000 nucleic acid bases, 15,000 to 10,000 nucleic acid bases, 10,000 to 4,700 nucleic acid bases, 200 to 5,000 nucleic acid bases, 300 to 5,000 nucleic acid bases, 400 to 5,000 nucleic acid bases, 400 to 2,500 nucleic acid bases, 200 to 3,000 nucleic acid bases, 400 to 2,000 nucleic acid bases, 400 to 1,000 nucleic acid bases, or any range therebetween.

[0109] In some embodiments, the polynucleotide comprises at least 10 nucleobases, at least 250 nucleobases, at least 300 nucleobases, at least 350 nucleobases, at least 400 nucleobases, at least 450 nucleobases, at least 475 nucleobases, or at least 500 nucleobases, each possibility representing a separate embodiment of the present invention.

[0110] In some embodiments, the polynucleotide comprises up to 500 nucleobases, up to 750 nucleobases, up to 1,000 nucleobases, up to 1,250 nucleobases, up to 1,750 nucleobases, up to 2,500 nucleobases, up to 3,000 nucleobases, up to 4,000 nucleobases, or up to 5,000 nucleobases, with each possibility representing a separate embodiment of the present invention.

[0111] In some embodiments, the polynucleotide comprises multiple polynucleotide types. In some embodiments, the LNP comprises multiple polynucleotide types. In some embodiments, the composition comprises multiple nanoparticle types, each type of LNP comprising a specific polynucleotide.

[0112] In some embodiments, a particular polynucleotide comprises multiple polynucleotide molecules having the same or identical nucleic acid sequence, hi some embodiments, a particular polynucleotide comprises multiple polynucleotide molecules having substantially the same nucleic acid sequence.

[0113] As used herein, the term "plurality" includes integers greater than or equal to 2. In some embodiments, plurality includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0114] As used herein, the term "polynucleotide type" refers to a plurality of polynucleotides, each of which comprises a nucleic acid sequence that differs from any one of the other polynucleotides in the plurality by at least one nucleobase, at least one nucleobase, at least one nucleobase, at least one nucleobase, at least one nucleobase, at least 10 nucleobases, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0115] In some embodiments, the polynucleotide comprises RNA, DNA, a synthetic analog of RNA, a synthetic analog of DNA, a DNA / RNA hybrid, or any combination thereof. In some embodiments, the LNPs of the invention comprise a polynucleotide selected from RNA, DNA, a synthetic analog of RNA, a synthetic analog of DNA, a DNA / RNA hybrid, or any combination thereof.

[0116] In some embodiments, the polynucleotide comprises or consists of RNA. The polynucleotide comprises or consists of messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to a polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a polyA tail. A polynucleotide can function as an mRNA but can be distinguished from wild-type mRNA by functional and / or structural design features that overcome existing problems in effective polypeptide expression using nucleic acid-based therapeutics.

[0117] The mRNAs provided herein comprise at least one ribonucleic acid (RNA) polynucleotide(s) and have an open reading frame encoding at least one polypeptide of interest. In some embodiments, the RNA polynucleotides of the mRNA encode 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, or 9 to 10 polypeptides. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 polypeptides. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 100 or at least 200 polypeptides.

[0118] In some embodiments, the nucleic acid is a therapeutic polynucleotide. As used herein, the term "therapeutic polynucleotide" refers to a polynucleotide encoding a therapeutic protein. Therapeutic proteins mediate various effects in host cells or subjects to treat diseases or alleviate the signs and symptoms of diseases. For example, therapeutic proteins can replace missing or abnormal proteins, enhance the function of endogenous proteins, and confer new functions to cells (e.g., inhibit or activate endogenous cellular activity, or act as a delivery agent for another therapeutic compound (e.g., antibody-drug conjugate)). Therapeutic mRNAs can be useful in treating the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic diseases, and autoimmune diseases.

[0119] In some embodiments, the therapeutic polynucleotide is mRNA. In some embodiments, the mRNA is for treating pulmonary disease. In some embodiments, the mRNA encodes a therapeutic protein for treating pulmonary disease. In some embodiments, the mRNA has a sequence complementary to a mutated gene associated with pulmonary disease.

[0120] Thus, the polynucleotides of the present invention can be used as therapeutic or prophylactic agents. They are provided for medical use. For example, mRNA of the structure described herein can be administered to a subject, and the polynucleotide is translated in vivo to produce a therapeutic peptide.

[0121] In some embodiments, the polynucleotide comprises an inhibitory nucleic acid. In some embodiments, the polynucleotide comprises an antisense oligonucleotide.

[0122] As used herein, "antisense oligonucleotide" refers to a nucleic acid sequence that is the reverse of and complementary to a DNA or RNA sequence.

[0123] As used herein, a "reverse complementary nucleic acid sequence" is a nucleic acid sequence that can hybridize to another nucleic acid sequence consisting of complementary nucleotide bases. "Hybridize" means the formation of a duplex molecule between complementary nucleotide bases (e.g., adenine (A) base pairs with thymine (T) (or uracil (U) in the case of RNA), and guanine (G) base pairs with cytosine (C)) under appropriate stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). For purposes of this method, the inhibitory nucleic acid need not be complementary to the entire sequence, but only to enough of the sequence to provide specific inhibition; for example, in some embodiments, the sequence is 100% complementary to at least nucleotides (nt) 2-7 or 2-8, e.g., nt 2-7 or 20, of the 5' end of the microRNA itself (e.g., the "seed sequence").

[0124] In some embodiments, the inhibitory nucleic acid has one or more chemical modifications in the backbone or side chain, hi some embodiments, the inhibitory nucleic acid has at least one locked nucleotide and / or has a phosphorothioate backbone.

[0125] Non-limiting examples of inhibitory nucleic acids useful in accordance with the present disclosure include, but are not limited to, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds (e.g., siRNA compounds), modified base / locked nucleic acids (LNAs), antagomir, peptide nucleic acids (PNAs), ribozymes (catalytic RNA molecules capable of cleaving specific sequences in other RNA molecules), and other oligomeric compounds or oligonucleotide mimetics that hybridize with at least a portion of a target nucleic acid and modulate its function. In some embodiments, inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides containing modified linkages, interfering RNA (RNAi), small interfering RNA (siRNA), microRNA (miRNA), small RNA (stRNA), or short hairpin RNA (shRNA), small RNA-guided gene activators (RNAa), small activating RNA (saRNA), or combinations thereof.

[0126] In some embodiments, the inhibitory nucleic acid is an RNA interference molecule (RNAi). In some embodiments, the RNAi is or comprises double-stranded RNA (dsRNA).

[0127] As used herein, "interfering RNA" refers to any double-stranded or single-stranded RNA sequence that can directly or indirectly (i.e., after translation) inhibit or down-regulate gene expression by mediating RNA interference.Interfering RNA includes, but is not limited to, small interfering RNA ("siRNA") and short hairpin RNA ("shRNA")."RNA interference" refers to the selective degradation of messenger RNA transcripts that are compatible with the sequence.

[0128] In some embodiments, the polynucleotide is chemically modified. In some embodiments, the chemical modification is a modification of the backbone of the polynucleotide. In some embodiments, the chemical modification is a modification of the sugar of the polynucleotide. In some embodiments, the chemical modification is a modification of the nucleobase of the polynucleotide. In some embodiments, the chemical modification increases the stability of the polynucleotide within a cell. In some embodiments, the chemical modification increases the stability of the polynucleotide in vivo. In some embodiments, the chemical modification increases the stability of the polynucleotide in vitro, such as in open air, outdoors, or on an air-exposed surface. In some embodiments, the chemical modification increases the ability of the polynucleotide to induce silencing of a target gene or target sequence, including, but not limited to, an RNA molecule from a pathogen or an RNA from a plant cell, as described herein. In some embodiments, the chemical modification is selected from a phosphate ribose backbone, a phosphate deoxyribose backbone, a phosphorothioate-deoxyribose backbone, a 2'-O-methyl-phosphorothioate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, a constrained ethyl backbone, an alternating locked nucleic acid backbone, a phosphorothioate backbone, an N3'-P5' phosphoamidate, a 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid, a cyclohexene nucleic acid backbone nucleic acid, a tricyclo-DNA (tcDNA) nucleic acid backbone, a ligand-linked antisense, and combinations thereof.

[0129] In some embodiments, the structured lipid is or comprises a sterol. In some embodiments, the term "structured lipid" includes non-liposome-forming lipids, as described herein. In some embodiments, the term "non-liposome-forming lipid" is understood to refer to lipids that do not spontaneously form vesicles when introduced into an aqueous medium. There are various types of lipids that do not spontaneously vesicle but can be used or incorporated into vesicles. In some embodiments, the non-liposome-forming lipid is or comprises a sterol.

[0130] In some embodiments, the structured lipid comprises any one of avenasterol, betulin, brassicasterol, calcipotriol, campesterol, campestanol, cholesterol, cholesterol hemisuccinate, cholesterol sulfate, daucosterol, DC-cholesterol, dehydroergosterol, DMAPC-Chol, DMHAPC-Chol, ergosterol, fucosterol, HAPC-Chol, lupeol, MHAPC-Chol, OH-C-Chol, OH-Chol, oleanolic acid, stigmastanol, stigmasterol, ursolic acid, hydrophobic vitamins (e.g., vitamin D2, vitamin D3, vitamin E, etc.), β-sitosterol, β-sitosterol acetate, β-sitosterol arginine, β-sitosterol cysteine, β-sitosterol glycine, β-sitosterol histidine, β-sitosterol serine, or steroids (including any salts or combinations thereof).

[0131] In some embodiments, the structural lipid is cholesterol.

[0132] In some embodiments, the helper lipid is or comprises a phospholipid. In some embodiments, the helper lipid is or comprises a liposome-forming lipid. As used herein, the term "liposome-forming lipid" includes lipids (e.g., phospholipids) that self-assemble to form stable vesicles (e.g., lipid nanoparticles) when dispersed or dissolved in an aqueous solution at a temperature above their transition temperature (Tm). As used herein, Tm refers to the temperature at which a lipid undergoes a phase transition from a solid (ordered phase, also called a gel phase) to a fluid (disordered phase, also called a fluid crystalline phase). Tm also refers to the temperature (or temperature range) at which the maximum change in heat capacity occurs during the phase transition.

[0133] In some embodiments, the phospholipids include a single phospholipid species or multiple chemically distinct phospholipids.

[0134] In some embodiments, the liposome-forming lipids are phospholipids with one or two C12 to C24 hydrocarbon tails (usually acyl, alkyl, or alkenyl chains) and varying degrees of unsaturation, ranging from fully saturated to fully, partially, or non-hydrogenated lipids (the degree of saturation can affect the rigidity of the formed liposomes; typically, liposomes formed from lipids with saturated chains are more rigid than liposomes formed from lipids of the same chain length that have unsaturated chains, particularly cis double bonds). In some embodiments, at least one of the liposome-forming lipids is a phospholipid with one or two C12-C20, C16-C20, or C16-C18 hydrocarbon tails (including any value or range therebetween). In some embodiments, the liposome-forming lipids are fully saturated, straight-chain, or branched-chain.

[0135] Furthermore, the phospholipids may be of natural origin (e.g., naturally occurring phospholipids), semi-synthetic, or fully synthetic, and may be electrically neutral (e.g., zwitterionic), negatively charged, or positively charged.

[0136] Non-limiting examples of neutral phospholipids include, but are not limited to, diacylphosphatidylcholine, dialkylphosphatidylcholine, sphingomyelin, and diacylphosphatidylethanolamine. Phosphatidylcholines (PCs) derived from eggs, soybeans, or other plants, or partially or completely synthetic, with varying lipid chain lengths and degrees of unsaturation, are suitable for use in the present compositions. Synthetic, semi-synthetic, and naturally occurring phosphatidylcholines, including, but not limited to, POPC, DOPC, DMPC, distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), soybean phosphatidylcholine (soybean PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), and dipalmitoylphosphatidylcholine (DPPC), are suitable as phosphatidylcholines for use in preparing liposomes. Charged phospholipids can include phosphatidylglycerol, cardiolipin, or headgroup-modified lipids such as N-succinyl-phosphatidylethanolamine, N-glutaryl-phosphatidylethanolamine, and PEG-derivatized phosphatidylethanolamine.

[0137] In some embodiments, the helper lipid is a cationic lipid. In some embodiments, the helper lipid does not include a non-cationic lipid.

[0138] In some embodiments, the cationic lipid comprises at least one permanent positive charge (i.e., at least one non-ionized cationic moiety). In some embodiments, the cationic lipid is a trialkylammonium-based lipid. In some embodiments, the cationic lipid comprises 1 to 5, or 1 to 3, positive charges.

[0139] Non-limiting examples of cationic lipids include, but are not limited to, 5-carboxyspermylglycine dioctadecylamide or "DOGS," N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA," 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanoaminium or "DOSPA," 1,2-dioleoyl-3-dimethylammonium propane or "DODAP," 1,2-dioleoyl-3-trimethylammonium propane or "DOTAP."Additionally, cationic lipids under investigation include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA," 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA," 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA," 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA," N-dioleyl-N,N-dimethylammonium chloride or "DODAC," N,N ...-3-aminopropane or "DODAC," N,N-dioleyl-N,N-dimethyl-3-aminopropane or "DODAC," N,N-dioleyl-N,N-dimethyl-3-aminopropane or "DODAC," N,N Stearyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadenoxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentoxy)-3-dimethyl-1-1-(cis ,cis-9',1-2'-octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane or "DLincarbDAP", 1,2-dilinoleoylcarbamyl These include rubamyl-3-dimethylaminopropane or "DLinCDAP," 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin-K-DMA," 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA," and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA).

[0140] In some embodiments, the helper lipid is or comprises a non-cationic lipid. As used herein, the term "non-cationic lipid" refers to any neutral or zwitterionic lipid. Non-cationic lipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), or mixtures thereof.

[0141] In some embodiments, helper lipid is cationic lipid.In some embodiments, cationic lipid is or comprises DOTAP, DDAB, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 18:1 EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and 18:0 EPC (1,2-distearoyl-sn-glycero-3-ethylphosphocholine) or combinations thereof.

[0142] In some embodiments, the modified lipid is a PEG-modified lipid. In some embodiments, the modified lipid comprises a single PEG moiety covalently attached to the lipid head group. In some embodiments, the modified lipid comprises multiple PEG moieties covalently attached to the lipid head group. In some embodiments, the PEG moiety comprises an alkylated PEG, such as methoxypoly(ethylene glycol) (mPEG). The PEG group has a head group molecular weight of about 750 Da to about 10,000 Da, sometimes about 750 Da to about 6,000 Da, usually about 1,000 Da to about 5,000 Da, or about 2,000 Da, including any range therebetween.

[0143] In some embodiments, the modified lipid is or comprises DMG-PEG2000.

[0144] Lipid nanoparticle properties In some embodiments, the pKa values ​​of the LNPs of the present invention are between 5 and 9, including any range therebetween. In some embodiments, the pKa values ​​of the LNPs are between 5 and 8, 5 and 6, 6 and 8, 6 and 7, 7 and 9, including any range therebetween.

[0145] In some embodiments, the pKa values ​​of the LNPs of the present invention are between 6 and 7, between 6 and 6.2, between 6.2 and 6.4, between 6.4 and 6.6, between 6.6 and 6.8, and between 6.8 and 7, including any range therebetween.

[0146] In some embodiments, the LNPs are characterized by an average particle size of less than 500 nm to facilitate their passage through the extracellular matrix and into cells, and in one embodiment, the carriers are characterized by an average particle size of less than 300 nm to facilitate their passage through the extracellular matrix and into cells.

[0147] In some embodiments, the LNPs are characterized by an average particle size of less than 500 nm, less than 400 nm, less than 300 nm, less than 350 nm, less than 200 nm, less than 100 nm, less than 50 nm, and any range therebetween, including:

[0148] In some embodiments, the LNPs are characterized by an average particle size of 50-500, 50-250 nm, 50-400 nm, 100-300 nm, 200-400 nm, 250-350 nm, and any range therebetween.

[0149] In some embodiments, the LNPs are characterized by a positive zeta potential (when measured at a pH of about 7, e.g., about 6.5-7.5). In some embodiments, the LNPs are characterized by a zeta potential of -5 mV to +40 mV, and any range therebetween. In some embodiments, the LNPs are characterized by a zeta potential of -3 mV to +20 mV, -3 mV to +10 mV, -2 mV to +10 mV, -1 mV to +10 mV, 0 mV to +20 mV, -0 mV to +10 mV, -0 mV to +5 mV, and any range therebetween, when measured at physiological pH (e.g., including pH 6.5-7.5 and any range therebetween).

[0150] In some embodiments, the LNPs are stable for a period of time, including from one day to one year or longer, and any range therebetween. In some embodiments, the term "stable" refers to the physical and chemical stability of the dried nanoparticles under appropriate storage conditions (e.g., substantial absence of phase separation, aggregation, or disintegration, and / or substantial retention of the initial amount of active agent). In some embodiments, the term "stable" refers to the physical and chemical stability (e.g., dispersion stability) of the nanoparticles in aqueous solution or in compositions of the invention.

[0151] The inventors have made the surprising discovery that, upon administration to a subject (i.e., an animal subject), the LNP compositions of the invention are characterized by a distinct activity profile in vivo compared to similar LNP compositions that do not contain structural lipids and / or helper lipids, or similar LNP compositions that have different ratios between the LNP components. As shown in the Examples section, the LNPs of the invention are characterized by enhanced specificity, and consequently, enhanced activity, in lung tissue relative to other organs of the subject (such as the kidney, liver, or heart).

[0152] In some embodiments, the LNPs of the present invention are characterized by enhanced specificity for the lungs (e.g., lung tissue, lung cells) of a subject. In some embodiments, the LNPs of the present invention are characterized by enhanced specificity for the lungs compared to similar LNPs containing a commercially available ionized lipid (Dlin-MC3-DMA). As used herein, the term "enhanced specificity" encompasses the property of an LNP to be differentially expressed in the lungs of a subject relative to other organs (e.g., kidneys, liver, heart, etc.). In some embodiments, enhanced specificity includes at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, or at least 1,000-fold greater expression of mRNA in the lungs relative to other organs (e.g., kidneys, spleen, liver, heart, etc.). In some embodiments, upon administration to a subject, the LNPs of the invention are characterized by at least 10-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1000-fold, or 10-fold to 500-fold, 10-fold to 200-fold (including any range therebetween) greater expression in the lungs compared to any one of the liver, spleen, or kidneys of the subject.

[0153] The specificity of the LNPs can be determined, for example, by performing in vivo tissue expression analysis (e.g., utilizing LNPs encapsulating signal-emitting probes or by using appropriate imaging techniques such as luminescence).

[0154] Pharmaceutical Composition In another aspect, the LNPs of the present invention can be used as therapeutic or prophylactic agents. In some embodiments, the LNPs are included in a composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises multiple LNPs and a pharmaceutically acceptable carrier, where the LNPs are the same or different. In some embodiments, "different LNPs" refer to lipid nanoparticles having different active agents. In some embodiments, the pharmaceutically acceptable carrier for the active agent is an LNP of the present invention.

[0155] In some embodiments, a pharmaceutically acceptable carrier is also referred to as an excipient or adjuvant. As used herein, the term "carrier," "excipient," or "adjuvant" refers to any component of a pharmaceutical composition other than the active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium such as physiological saline. Some examples of materials which can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol, phosphate buffers, and other non-toxic, compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can function as carriers herein include sucrose, starch, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer, cocoa butter (suppository base), emulsifiers, and other pharmaceutically compatible non-toxic materials used in other pharmaceutical preparations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavorings, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein.In this regard, suitable pharmaceutically acceptable carrier, excipient and diluent are well known to those skilled in the art, for example, those listed in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th Edition (2004); and "Inactive Ingredient Guide", U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Management Office, all of which are incorporated herein by reference.The examples of pharmaceutically acceptable carrier, carrier and diluent that are useful in the present composition include distilled water, physiological saline, Hartmann's solution, Ringer's solution, dextrose solution, Hank's solution and DMSO. These additional inactive ingredients, as well as effective formulation and administration procedures, are well known in the art and are described in standard texts such as Goodman and Gillman: The Pharmacological Bases of Therapeutics, 8th Edition, Gilman et al., Eds., Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), which are incorporated herein by reference in their entireties. The presently described compositions may also be contained in artificially engineered structures such as liposomes, ISCOMS, sustained-release particles, and other vehicles that extend the serum half-life of peptides or polypeptides.The liposomes used with the peptides described herein are formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and a sterol such as cholesterol. The choice of lipid is generally determined by considerations such as liposome size and stability in the blood. Various methods available for preparing liposomes are described, for example, in Coligan, J.E. et al., *Current Protocols in Protein Science*, 1999, John Wiley & Sons, Inc., New York. See also U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0156] In some embodiments, the LNPs of the present invention, comprising the compounds of the present invention, structural lipids, helper lipids, and modified lipids in the relative molar ratios as described in the embodiments above, are based on the active agent to be delivered and its target. In some embodiments, the relative molar ratios as described in the embodiments above, allow the LNPs to be specifically targeted to the lung.

[0157] The carriers may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions described herein.

[0158] In some embodiments, pharmaceutical compositions comprising the LNPs of the invention further comprise an effective amount of an active agent described herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an active agent described herein. In some embodiments, the therapeutically effective amount is sufficient to alleviate at least one symptom or to significantly reduce the severity and / or inhibit the progression of the disease, disorder, or condition described above.

[0159] In some embodiments, the pharmaceutical composition is used to treat or prevent a pulmonary disease. In some embodiments, the pulmonary disease is selected from lung cancer, ARDS, pulmonary fibrosis, viral infection, bacterial infection, COPD, asthma, bronchiectasis, bronchiolitis, bronchitis, cystic fibrosis, emphysema, mesothelioma, pleural effusion, pleuritis, pneumonia, pneumothorax, RSV, SARS, SARS-CoV-2, silicosis, tuberculosis, whooping cough, and influenza. Lung cancers are well known in the art and include, for example, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma, Pancoast tumor, and carcinoid tumor. The term "effective amount" or "therapeutically effective amount" refers to an amount effective at the dosage and for the duration required to achieve the desired therapeutic effect. Those skilled in the art will appreciate that a therapeutically effective amount of a molecule according to the present invention will depend, among other factors, on the administration schedule, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the patient's immune and health status, the therapeutic activity of the administered molecule, and the judgment of the treating physician. In some embodiments, the therapeutically effective amount of an active agent administered daily to a subject is reduced compared to intravenous administration of a pharmaceutical composition comprising the same active agent not encapsulated in the LNPs of the present invention. In some embodiments, the therapeutically effective amount of an active agent administered daily to a subject is reduced compared to a similar pharmaceutical composition comprising the same active agent encapsulated in commercially available LNPs (e.g., Dlin-MC3-DMA). In some embodiments, the reduction in the therapeutically effective amount is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold (including any range therebetween).

[0160] In some embodiments, the LNPs of the invention are comprised of pharmaceutically acceptable components (e.g., phospholipids and sterols) or pharmaceutically acceptable salts thereof. In some embodiments, pharmaceutical compositions comprise the LNPs of the invention, pharmaceutically acceptable salts thereof, or both.

[0161] In some embodiments, the pharmaceutical composition materials are characterized as pharmaceutical grade, which in some embodiments is characterized by a chemical purity of at least 90%, at least 95%, at least 99%, including any range therebetween.

[0162] The term "pharmaceutically acceptable" can mean approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans.

[0163] In some embodiments, the pharmaceutical composition of the present invention is used as a drug. In some embodiments, the pharmaceutical composition is formulated for administration to the lung, lung tissue, or lung cells. In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for intravenous (IV) administration. In some embodiments, the pharmaceutically acceptable carrier comprises a buffer.

[0164] In some embodiments, administration to the lung (e.g., tissue, cells) is by IV. In some embodiments, administration to the lung (e.g., tissue, cells) is systemic.

[0165] As used herein, the terms "administering," "administration," and the like refer to any method, in sound medical practice, of delivering a composition comprising an active agent to a subject in a manner that provides a therapeutic effect. One aspect of the present subject matter provides intravenous administration of a therapeutically effective amount of a subject composition to a patient in need thereof. Other suitable routes of administration can include parenteral, intravenous, subcutaneous, oral, intramuscular, intrathecal, inhalation, intracerebroventricular, intravitreal, transdermal, or intraperitoneal administration. In some embodiments, the pharmaceutical composition is used in a method of treatment. In some embodiments, the method of treatment is a method of therapy. In some embodiments, the pharmaceutical composition is used in the treatment of a medical condition. In some embodiments, the medical condition is a condition, disease, or disorder. In some embodiments, the pharmaceutical composition is used in a method of diagnosis.

[0166] In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, a method comprises administering a pharmaceutical composition of the present invention to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is in need of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is used to treat or prevent a disease or condition in humans and other mammals. In some embodiments, the subject is in need of treatment. In some embodiments, the subject is a volunteer for a diagnostic method. In some embodiments, the subject is in need of a diagnosis.

[0167] The active therapeutic agent of the present invention comprises a lipid nanoparticle or a protein translated from a polynucleotide contained in the lipid nanoparticle.

[0168] In some embodiments, the mammal is a laboratory animal. Examples of laboratory animals include, but are not limited to, mice, rats, rabbits, hamsters, dogs, pigs, monkeys, etc. In some embodiments, the mammal is a mouse or a rat.

[0169] In some embodiments, the pharmaceutical compositions of the present invention are in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, gel, cream, ointment, foam, paste, sustained-release formulation, etc. In some embodiments, the pharmaceutical compositions of the present invention can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, tragacanth gum, or gelatin. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0170] How to use In another aspect, a method for delivering an active agent to the lung, lung tissue, or lung cells of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the invention as described above, thereby delivering the active agent to the lung. In some embodiments, the pharmaceutical composition is used to deliver an active agent to the lung (e.g., lung tissue, lung cells) of a subject.

[0171] In some embodiments, the pharmaceutical composition is used to treat a pulmonary disease, pulmonary disorder, or pulmonary condition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an active agent.

[0172] In some embodiments, the pharmaceutical composition is a lung-targeting composition. In some embodiments, the pharmaceutical composition is used to treat a lung disease. In some embodiments, the lung disease comprises lung inflammation. Examples of lung diseases include, but are not limited to, lung cancer, pulmonary fibrosis, viral infection, bacterial infection, COPD, asthma, bronchiectasis, bronchiolitis, bronchitis, cystic fibrosis, emphysema, mesothelioma, pleural effusion, pleuritis, pneumonia, pneumothorax, RSV, SARS, SARS-CoV-2, silicosis, tuberculosis, whooping cough, and influenza. Lung cancers are well known in the art and include, for example, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, squamous cell carcinoma of the lung, large cell carcinoma of the lung, Pancoast tumor, and carcinoid tumor.

[0173] In some embodiments, the method includes administering an effective amount of the pharmaceutical composition. In some embodiments, the effective amount is the human equivalent of a mouse dose of 0.01-5 mg / kg body weight per day. In some embodiments, the human equivalent of a mouse dose varies depending on the route of administration. In some embodiments, the effective amount is the human equivalent of a mouse dose of 0.01-5, 0.1-5, 0.1-2, 0.01-2, 0.01-3, 0.01-1, or 0.5-5 mg / kg body weight (including any value therebetween).

[0174] The dosage administered will depend upon the age, health, and weight of the recipient, type of concurrent treatment (if any), frequency of treatment, and the nature of the effect desired.

[0175] definition As used herein, the term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, provided that the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0176] As used herein, the term "substantially" refers to at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or in the ranges of 60% to 99.9%, 70% to 80%, 70% to 90%, 80% to 90%, 90% to 95%, 95% to 99.9% (including any range or value therebetween).

[0177] As used herein, the term "substituents" means hydrogen, halogen, -NO, -CN, -OH, oxo, imino, -CONH, -CONR', -CNNR', -CSNR', -CONH-OH, -CONH-NH, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-SOR', -SOR', -SR', -SOR', -SON(R'), -NHNR', -NNR', C-C haloalkyl, optionally substituted C-C alkyl, -NH, -NR', -NH(C-C alkyl), -N(C-C and R' independently represents hydrogen or an optionally substituted C-C alkyl. 10 Alkyl, optionally substituted C-C 10 Cycloalkyl, optionally substituted C-C 10 selected from the group comprising heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH2, -NR'2-NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NR'2, C1-C6 alkyl-SR', or a combination thereof.

[0178] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon, including straight-chain and branched-chain groups. As used herein, the term "alkyl" also encompasses saturated or unsaturated hydrocarbons, and thus the term further includes alkenyl and alkynyl.

[0179] The term "alkenyl" refers to an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be unsubstituted or substituted with one or more substituents, as described herein above.

[0180] The term "alkynyl," as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be unsubstituted or substituted with one or more substituents, as described herein above.

[0181] The term "cycloalkyl" refers to an all-carbon monocyclic or fused-ring (i.e., rings that share adjacent pairs of carbon atoms) group in which one or more rings do not have a completely conjugated pi-electron system. Cycloalkyl groups can be substituted or unsubstituted as indicated herein.

[0182] The term "aryl" refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a completely conjugated pi-electron system. Aryl groups can be substituted or unsubstituted as indicated herein.

[0183] The term "alkoxy" refers to both an -O-alkyl and an -O-cycloalkyl group as defined herein. The term "aryloxy" refers to an -O-aryl group as defined herein.

[0184] Each alkyl, cycloalkyl, and aryl group in the general formula herein may be substituted with one or more substituents, each of which may independently be, for example, halide, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxy, thiol, thioalkoxy, carboxy, amido, aryl, and aryloxy, depending on the substituent and its position in the molecule. Additional substituents are also contemplated.

[0185] The terms "halide," "halogen," or "halo" refer to fluorine, chlorine, bromine, or iodine. The term "haloalkyl" refers to an alkyl group, as defined herein, further substituted with one or more halides. The term "haloalkoxy" refers to an alkoxy group, as defined herein, further substituted with one or more halides. The term "hydroxy" or "hydroxy group" refers to an -OH group. The term "mercapto" or "thiol" refers to an -SH group. The term "thioalkoxy" refers to both an -S-alkyl group and an -S-cycloalkyl group, as defined herein. The term "thioaryloxy" refers to both an -S-aryl and an -S-heteroaryl group, as defined herein. The term "amino" refers to an -NR'R'' group, with R' and R'' as defined herein, or a salt thereof.

[0186] The term "heterocyclyl" refers to a monocyclic or fused ring group containing one or more atoms, such as nitrogen, oxygen, and sulfur, within the ring. The ring may contain one or more double bonds. However, the ring must not have a completely conjugated pi-electron system. Representative examples include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, and the like.

[0187] The term "carboxy" refers to the group -C(O)OR', or a carboxylate thereof, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (attached through a ring carbon), or heterocyclyl (attached through a ring carbon), or "carboxylate," as defined herein.

[0188] The term "carbonyl" refers to the group -C(O)R', where R' is as defined above. The above term also includes thio derivatives thereof (thiocarboxy and thiocarbonyl).

[0189] The term "thiocarbonyl" refers to a -C(S)R' group, where R' is as defined above. A "thiocarboxy" group refers to a -C(S)OR' group, where R' is as defined herein. A "sulfinyl" group refers to a -S(O)R' group, where R' is as defined herein. A "sulfonyl" or "sulfonic acid" group refers to a -S(O)R' group, where R' is as defined herein.

[0190] A "carbamyl" or "carbamate" group refers to the -O-C(O)NR'R" group, where R' is as defined herein and R" is as defined for R'. A "nitro" group refers to the -NO2 group. As used herein, the term "amido" includes C-amido and N-amido. The term "C-amido" refers to a -C(O)NR'R" terminal group or a -C(O)NR'- linking group, where these terms are as defined above and R' and R" are as defined herein. The term "N-amido" refers to a -NR"C(O)R' terminal group or a -NR'C(O)- linking group, where these terms are as defined above and R' and R" are as defined herein.

[0191] The term "cyano" or "nitrile" refers to a -CN group. The terms "azo" or "diazo" refer to an -N=NR' terminal group or an -N=N- linking group, as these terms are defined above, where R' is as defined above. The term "guanidine" refers to an -R'NC(N)NR"R"' terminal group or an -R'NC(N)NR"- linking group, as these terms are defined above, where R', R" and R'" are as defined herein. As used herein, the term "azide" refers to an -N3 group. The term "sulfonamide" refers to a -S(O)2NR'R" group, where R' and R" are as defined herein.

[0192] The term "phosphonyl" or "phosphonate" refers to the group -OP(O)-(OR')2, where R' is as defined above. The term "phosphinyl" refers to the group -PR'R" where R' and R" are as defined above. The term "alkylaryl" refers to an alkyl, as defined herein, substituted with an aryl, as described herein. An example of an alkylaryl is benzyl.

[0193] The term "heteroaryl" refers to a monocyclic or fused ring (i.e., rings sharing adjacent pairs of atoms) group having one or more atoms within the ring, such as nitrogen, oxygen, and sulfur, and further having a fully conjugated π-electron system. As used herein, the term "heteroaryl" refers to an aromatic ring in which at least one atom forming the aromatic ring is a heteroatom. A heteroaryl ring can be formed from 3, 4, 5, 6, 7, 8, 9, or more than 9 atoms. Heteroaryl groups may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C3-C8 heterocyclic groups containing one oxygen or sulfur atom, or two oxygen atoms, or two sulfur atoms, or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and substituted versions thereof, as well as benzo- and pyrido-fused derivatives thereof, such as those bonded through one of the ring-forming carbon atoms. In certain embodiments, heteroaryl is selected from oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrimidinal, pyrazinyl, indolyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl.

[0194] In some embodiments, the heteroaryl group is selected from the group consisting of pyrrolyl, furanyl (furyl), thiophenyl (thienyl), imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3-oxazolyl (oxazolyl), 1,2-oxazolyl (isoxazolyl), oxadiazolyl, 1,3-thiazolyl (thiazolyl), 1,2-thiazolyl (isothiazolyl), tetrazolyl, pyridinyl (pyridyl), pyridazinyl, pyrimidinyl, pyrazinyl, 1, Heteroaryl groups are selected from 2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, indazolyl, indolyl, benzothiophenyl, benzofuranyl, benzothiazolyl, benzimidazolyl, benzodioxolyl, acridinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, thienothiophenyl, 1,8-naphthyridinyl, other naphthyridinyl, pteridinyl, or phenothiazinyl. When a heteroaryl group contains multiple rings, each additional ring is saturated (perhydro) or partially unsaturated (e.g., dihydro or tetrahydro) or maximally unsaturated (non-aromatic). Thus, the term heteroaryl includes bicyclic radicals in which two rings are aromatic and bicyclic radicals in which only one ring is aromatic. Such examples of heteroaryl include 3H-indolinyl, 2(1H)-quinolinonyl, 4-oxo-1,4-dihydroquinolinyl, 2H-1-oxoisoquinolyl, 1,2-dihydroquinolinyl, (2H)quinolinyl. N-oxide, 3,4-dihydroquinolinyl, 1,2-dihydroisoquinolinyl, 3,4-dihydro-isoquinolinyl, chromonyl, 3,4-dihydroiso-quinoxalinyl, 4-(3H)quinazolinonyl, 4H-chromenyl, 4-chromanonyl, oxindolyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydro-quinolinyl, 1H-2,3-dihydroisoindolyl, 2,3-dihydrobenzo[f]isoindolyl, 1,2,3,4-tetrahydrobenzo-[g]isoquinolinyl, 1,2,3,4-tetrahydro-benzo[g]isoquinolinyl, chromanyl, isochromanonyl, 2,3-dihydrochromonyl, 1,4-benzo-dioxanyl, 1,2,3,4-Tetrahydro-quinoxalinyl, 5,6-dihydro-quinolyl, 5,6-dihydroiso-quinolyl, 5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-1H-benzimidazolyl, 4,5-dihydro-benzoxazolyl, 1,4-naphthoquinolyl, 5,6,7,8-tetrahydro-quinolinyl, 5,6,7,8-tetrahydro-isoquinolyl, 5,6,7,8-tetrahydroquinoxalinyl, 5,6,7,8-tetrahydroquinazolyl, 4,5,6,7-tetrahydro-1H-benzimidazolyl, 4,5 ,6,7-Tetrahydro-benzoxazolyl, 1H-4-oxa-1,5-diaza-naphthalen-2-onyl, 1,3-dihydroimidizolo-[4,5]-pyridin-2-onyl, 2,3-dihydro-1,4-dinaphtho-quinonyl, 2,3-dihydro-1H-pyrrole[3,4-b]quinolinyl, 1,2,3,4-tetrahydrobenzo[b]-[1,7]naphthyridinyl, 1,2,3,4-tetrahydrobenzo[b][1,6]-naphthyridinyl, 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indolyl, 1,2,3,4-tetrahydrobenzo[b]-[1,7]naphthyridinyl 1H-tetrahydro-9H-pyrido[4,3-b]indolyl, 2,3-dihydro-1H-pyrrolo-[3,4-b]indolyl, 1H-2,3,4,5-tetrahydro-azepino[3,4-b]indolyl, 1H-2,3,4,5-tetrahydroazepino-[4,3-b]indolyl, 1H-2,3,4,5-tetrahydro-azepino[4,5-b]indolyl, 5,6,7,8-tetrahydro[1,7]napthyridinyl, 1,2,3,4-tetrahydro-[2,7]-naphthyridyl, 2,3-dihydro[1,4]dioxino[2,3-b]pyridyl, 2,3- Dihydro[1,4]-dioxino[2,3-b]pyridyl, 3,4-dihydro-2H-1-oxa[4,6]diazanaphthalenyl, 4,5,6,7-tetrahydro-3H-imidazo-[4,5-c]pyridyl, 6,7-dihydro[5,8]diazanaphthalenyl, 1,2,3,4-tetrahydro[1,5]-napthyridinyl, 1,2,3,4-tetrahydro[1,6]napthyridinyl, 1,2,3,4-tetrahydro[1,7]napthyridinyl, 1,2,3,4-tetrahydro-[1,8]napthyridinyl or 1,2,3,4-tetrahydro[2,In some embodiments, the heteroaryl group may be optionally substituted. In one embodiment, the one or more substituents are each independently halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, C, 1-6 -Alkyl, C 1-6 -haloalkyl, C 1-6 -hydroxyalkyl, C 1-6 -aminoalkyl, C 1-6 - selected from alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl.

[0195] Examples of heteroaryl groups include, but are not limited to, unsubstituted and mono- or di-substituted derivatives of furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline and quinoxaline. In some embodiments, the substituents are halo, hydroxy, cyano, OC 1-6 -Alkyl, C 1-6 -Alkyl, Hydroxy-C 1-6 -Alkyl and Amino-C 1-6 - alkyl.

[0196] As used herein, the terms "halo" and "halide" are used interchangeably herein and refer to a halogen atom, i.e., fluorine, chlorine, bromine, or iodine, and are also referred to herein as fluoride, chloride, bromide, and iodide.

[0197] As used herein, the term "substituted" or "substituent" refers to one or more (e.g., 2, 3, 4, 5, or 6) substituents, wherein the substituents are as described herein.

[0198] General terms As used herein, the term "about" when combined with a value refers to ±10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ±100 nm.

[0199] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. Thus, for example, the term "polynucleotide" is intended to include a plurality of such polynucleotides, the term "polypeptide" is intended to include one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should further be noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a prerequisite for using exclusive terminology, such as "only," "only," and the like, in connection with the recitation of claim elements or the use of "negative" limitations.

[0200] When a convention similar to "at least one of A, B, and C," etc., is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes systems including, but not limited to, A only, B only, C only, a combination of A and B, a combination of A and C, a combination of B and C, and / or a combination of A, B, and C, etc.). One of ordinary skill in the art will understand that almost all alternative words and / or phrases in the specification or claims that present two or more alternative terms contemplate the possibility of including either one term, either term, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0201] It is understood that certain features of the invention, which are, for clarity, described in separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the present invention are expressly embraced by the present invention and are disclosed herein as if each combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the present invention and are disclosed herein as if all such subcombinations were individually and expressly disclosed herein.

[0202] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0203] Various embodiments and aspects of the present invention, as delineated hereinabove and as claimed in the claims section below, find experimental support in the following examples.

[0204] Example Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques, which are fully explained in the literature. For example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methods as set forth in USPat.Nos.4,666,828;4,683,202;4,801,531;5,192,659 and 5,272,057;”Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, JE, ed. (1994); “Culture of Animal Cells-A Manual of Basic Technique” by Freshney, Wiley-Liss, NY (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference. Other general references are provided throughout this specification.

[0205] Example 1 Representative compounds of the present invention (MB-212 and MB-222 shown below) were synthesized according to the synthesis scheme shown below. The inventors successfully formed lipid nanoparticles of the present invention using MB-212 or MB-222 and tested their ability to deliver active agents to the lungs in vivo. Representative compounds (ionized lipids) of the present invention are shown below. [ka]

[0206] General synthetic schemes for some representative compounds of the present invention are shown below: Other possible synthetic strategies will be familiar to those skilled in the art. [ka]

[0207] Some exemplary compositions of the present invention are characterized by enhanced specificity for lung cells, as determined in in vivo mouse studies. For example, LNPs of the present invention containing MB-212 or MB-222 as the ionizable lipid exhibited enhanced specificity for lung tissue compared to similar compositions containing Dlin-MC3-DMA as the ionizable lipid (Figure 1). Surprisingly, the inventors discovered that exemplary LNPs containing about 30 to about 50% helper lipid (e.g., DOTAP), about 20 to about 50% structural lipid (cholesterol), and about 0.1 to about 5% modified lipid (e.g., PEG-lipid) exhibited enhanced lung specificity, as assessed by in vivo mRNA expression analysis, when containing about 15 to about 50% ionizable lipid (e.g., MB-212 or MB-222). Exemplary LNP compositions of the present invention exhibited enhanced lung specificity, with significantly increased expression of encapsulated polynucleic acid (mRNA F-LUC) in the lung compared to commercially available controls (Figure 1). Furthermore, a particular LNP composition of the present invention (FMB-1143), which contains about 15 to about 30% ionized lipids, demonstrated excellent lung specificity (see Figure 1).

[0208] Preparation of FMB-745 40% DOTAP, 22.5% cholesterol, 2.5% DMG-PEG2000, and 35% commercially available ionized lipid (Dlin-MC3-DMA) were dissolved in ethanol (EtOH) at 55–60°C. mRNA, F-LUC, was added to a citrate buffer solution at pH 5.0 (range 4.5–5.5). Lipid mixing was performed using microfluidic mixing or by injecting EtOH into mRNA firefly luciferase (F-LUC)-containing citrate buffer under constant mixing conditions. The pH of the mixture was then increased by dilution with PBS, and residual EtOH was removed prior to injection using a dialysis system prepared by the inventors. This mixture was characterized by an average particle size ranging from approximately 60 to approximately 130 nm.

[0209] Preparation of FMB-748 40% DOTAP, 22.5% cholesterol, 2.5% DMG PEG2000, and 35% ionized MB-222 were dissolved in ethanol (EtOH) at 55–60°C. mRNA and F-LUC were added to a citrate buffer solution at pH 5.0 (range 4.5–5.5). Lipid mixing was performed using microfluidic mixing or by injecting EtOH into the citrate buffer solution containing mRNA and F-LUC under constant mixing conditions. The pH of the mixture was then increased by dilution with PBS, and residual EtOH was removed prior to injection using a dialysis system prepared by the inventors. This mixture was characterized by an average particle size ranging from approximately 60 to approximately 130 nm, an N:P ratio of 8–9, and an average Z of approximately +4 mV.

[0210] Preparation of FMB-1143 40% DOTAP, 38.5% cholesterol, 1.5% DMG-PEG2000, and 20% ionized MB-212 were dissolved in ethanol (EtOH) at 55–60°C. mRNA and F-LUC were added to a citrate buffer solution at pH 5.0 (range 4.5–5.5). Lipid mixing was performed using microfluidic mixing or by injecting EtOH into the citrate buffer solution containing mRNA and F-LUC under constant mixing conditions. The pH of the mixture was then increased by dilution with PBS, and residual EtOH was removed prior to injection using a dialysis system prepared by the inventors. This mixture was characterized by an average particle size ranging from approximately 60 to approximately 130 nm, an N:P ratio of 4–5, and an average Z of approximately +3 mV.

[0211] The inventors determined the in vivo expression distribution of exemplary LNPs by evaluating the expression of encapsulated mRNA (mRNA F-LUC). LNPs encapsulating the present mRNA F-LUC were intravenously injected into BALB / c mice at a dose of 13 μg / mouse (0.52-0.365 mg / kg). In vivo imaging was performed using IVIS imaging. Ex vivo tissue analysis was performed using IVIS on the lungs, heart, spleen, kidneys, and liver. To assess toxicity, histological evaluation using H&E staining was performed based on a pathologist's report. The histological evaluation revealed no treatment-related pathological changes and concluded that the morphology was normal.

[0212] The expression distribution profile shown in Figure 1 shows that the luciferase signal in the lung is over 100-fold greater than in the heart, liver, spleen, and kidney, demonstrating the excellent lung specificity of the exemplary LNPs of the present invention.

[0213] Furthermore, a similar formulation (FMB-393) containing a zwitterionic helper lipid (DOPE) and characterized by a negative Z potential (approximately -22 mV) showed expression / accumulation primarily in the liver (in contrast to the cationic helper lipid and positive Z potential of the exemplary formulations of the present invention).

[0214] FMB-393 was prepared as described above, and its chemical composition is disclosed in Table 1. [Table 1]

[0215] FMB-393 was administered intravenously to BALB / c mice and the in vivo expression distribution profile was assessed as disclosed above.

[0216] The expression distribution profile of FMB-393 is shown in Table 2. [Table 2]

[0217] As is clear from Table 2, FMB-393 accumulated mainly in the liver, with luciferase signals in the liver being more than 100-fold higher than those in the lungs.

[0218] The inventors determined the in vivo expression distribution of exemplary LNPs by assessing the expression of encapsulated mRNA (mCherry mRNA). LNPs of the present invention encapsulating mCherry mRNA were intravenously injected into cynomolgus monkeys at a dose of 0.6 mg / kg. Twenty-four hours after administration, tissue samples were collected from the following tissues: lung, brain, liver, heart, spleen, kidney, testis, and mesenteric lymph node. The samples were analyzed for expressed mCherry signal by immunohistochemistry, compared with similar samples collected from control animals administered PBS / sucrose. Histological analysis revealed moderate to strong positive mCherry staining in lung tissue samples.

[0219] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. 1. A lipid nanoparticle comprising a compound, a salt of said compound, or both, said compound having Formula 1: 【Chemistry 1】 wherein: each X independently represents —O—, —S—, CH2, or X is absent; each Z independently represents —OH or —SH; Each A independently represents O or S; each n is independently 0 to 5, and at least one n is not 0; each R is independently H or comprises an optionally substituted C5-C30 alkyl; The lipid nanoparticles further comprise an active agent, a helper lipid, a structural lipid, and a modified lipid; the helper lipid is a cationic lipid; the ratio of said compound to the total lipid content of said lipid nanoparticles is 10 to 50 mol %; the ratio of the structural lipid to the total lipid content of the lipid nanoparticle is 5 to 50 mol %; The lipid nanoparticles are characterized in that they have an average zeta potential in the range of -5 to +40 mV at pH 6 to 8. Lipid nanoparticles.

2. The lipid nanoparticle of claim 1, wherein the molar ratio of the helper lipid to the modified lipid is 1:0.2 to 1:0.

01.

3. The lipid nanoparticle of claim 1 or 2, wherein the molar ratio of the compound to the helper lipid is 0.2:1 to 5:

1.

4. The lipid nanoparticle according to any one of claims 1 to 3, wherein the molar ratio of the structured lipid to the modified lipid is 1:0.01 to 1:0.

2.

5. The lipid nanoparticle of any one of claims 1 to 4, wherein the active agent comprises a polynucleic acid; and the N:P ratio within the LNP is 1 to 20.

6. The lipid nanoparticle according to any one of claims 1 to 5, wherein the weight ratio of (i) the total amount of the compound, the helper lipid, the structural lipid, and the modified lipid to (ii) the polynucleic acid within the lipid nanoparticle is 0.001:1 to 10:

1.

7. The lipid nanoparticles according to any one of claims 1 to 6, wherein the lipid nanoparticles have a particle size distribution in the range of 50 to 500 nm, and the cationic lipid is selected from DOTAP, DDAB, 18:1 EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine), and 18:0 EPC (1,2-distearoyl-sn-glycero-3-ethylphosphocholine), including salts thereof and any combination thereof.

8. an average zeta potential of 0 to +5 mV at pH 6 to 8; N:P ratio is 3-9; the structural lipid is cholesterol; the modified lipid is a PEG-lipid; The ratio of the helper lipid to the total lipid content of the lipid nanoparticle is 35 to 45 mol %; and The lipid nanoparticle according to any one of claims 5 to 7, characterized in that the ratio of the PEG-lipid to the total lipid content of the lipid nanoparticle is 0.1 to 3 mol%.

9. the lipid nanoparticles are characterized by a pKa of 5 to 9; The compounds are MB-212 and MB222: 【Chemistry 2】 including any one of The lipid nanoparticle according to any one of claims 1 to 8.

10. The lipid nanoparticle of any one of claims 1 to 9, wherein when administered to a subject, the LNP is expressed at least 10 times more in the lung than in any one of the liver, spleen, and kidney of the subject.

11. The lipid nanoparticle of claim 10, wherein the N:P ratio is about 5; the cationic lipid is DOTAP; and the ratio of the compound to the total lipid content of the lipid nanoparticle is about 15 to about 25 mol%.

12. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. 13. The pharmaceutical composition of claim 12, comprising a therapeutically effective amount of the active agent.

14. 14. A pharmaceutical composition according to claim 12 or 13 for use in delivering the active agent to pulmonary tissue.

15. 14. A pharmaceutical composition according to claim 12 or 13 for use in the treatment of a pulmonary disease or disorder.

16. 14. A method of delivering an active agent to the pulmonary tissue of a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 12 or 13, thereby delivering the active agent to the pulmonary tissue.

17. 17. The method of claim 16, wherein the therapeutically effective amount is 0.01 to 5 mg / kg / day.

18. 18. The method of claim 16 or 17, wherein the administration is via intravenous, intratracheal, intranasal administration, or via inhalation.

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