Ionizable lipids for drug delivery

Abstract

This article provides ionizable lipids, nanoparticles containing ionizable lipids, compositions containing nanoparticles, and methods for delivering therapeutic agents (e.g., mRNA) to specific cells, tissues, or organs (e.g., the lungs) using ionizable lipids, nanoparticles, and compositions.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 620,430, filed January 12, 2024. The disclosure of the earlier application is considered part of the disclosure of this application (and is incorporated herein by reference).

[0003] Statement on Federally Funded Research

[0004] This invention was developed with government support from the Defense Advanced Research Projects Agency (DARPA) under registration number HR.0011-19-2-0007 and the United States Department of the Interior (DOI) under registration number Dl6AP00143. The U.S. government holds certain rights to this invention. Technical Field

[0005] This document relates to ionizable lipids, nanoparticles comprising ionizable lipids, compositions comprising nanoparticles, and methods for delivering therapeutic agents (e.g., mRNA) to specific cells, tissues, or organs (e.g., the lungs) using ionizable lipids, nanoparticles, and compositions. Background Technology

[0006] Messenger RNA (mRNA) is a versatile drug that can be used in protein replacement therapy, antiviral and anticancer vaccines, gene editing and regulation, CAR-T therapy, and tissue engineering. However, a major challenge hindering the clinical translation of mRNA therapies is its delivery to target cells. This is because mRNA is an unstable molecule that is rapidly degraded by nucleases in the blood and lacks a mechanism for entering cells to be translated into proteins (Phua et al., J. Controlled Release 2013, 166:227; and Tavernier et al., J. Controlled Release 2011, 150:238). The most advanced mRNA delivery system in the clinical field is lipid nanoparticles (LNPs), which are non-viral vehicles containing multiple lipids (Sun et al., Adv. Drug Delivery Rev. 2023, 200:115042). LNPs can protect mRNA from RNase, inhibit phagocytic clearance, facilitate cell entry via endocytosis, and mediate escape from endosomes (Kiaie et al., J. Nanobiotechnol. 2022, 20:276; and Maugeri et al., Nat. Commun. 2019, 10:4333). Furthermore, LNPs can attract protein coronas after injection into the biological environment, and these coronas can guide the uptake and delivery of nanoparticles to specific cell types (Ritz et al., Biomacromolecules 2015, 16:1311). LNPs offer several advantages over viral delivery systems, including their larger payload capacity and lower immunogenicity, the latter of which allows for repeated administration (Kiaie et al., ibid.).

[0007] LNPs are multi-component carriers that can be generated by co-precipitating mRNA with a mixture of natural and synthetic lipids (Gindy et al., Langmuir 2014, 30:4613; and Fenton et al., Adv. Mater. 2017, 29:1606944). The lipid components typically consist of four parts: synthetic ionizable lipids or lipidoids, phospholipids (referred to as “helper lipids”), cholesterol, and polyethylene glycol-conjugated lipids (PEG-lipids) (Fenton et al., ibid.; and Kauffman et al., Nano Lett. 2015, 15:7300). Each component of the LNP plays a role in the delivery process. Ionizable lipids or lipidoids can impart a neutral charge to the nanoparticle surface in the bloodstream, which is converted to a positive charge in the acidic early endosomes, thereby facilitating endosome escape (Maugeri et al., ibid.; and Sahay et al., Nat. Biotechnol. 2013, 31:653). Ionizable lipids can also influence the protein crown that forms around LNPs and can control particle orientation (Cai and Chen, Adv. Mater. 2019, 31:1805740; and Wang et al., Nat. Protoc. 2023, 18:265). Auxiliary lipids are structurally important in both cellular and extracellular vesicle membranes, and they have similar value in optimizing LNP lipid stacking and increasing stability (Kaie et al., ibid.). Furthermore, the charge of auxiliary lipids can influence nanoparticle orientation, with neutral, negative, and positive charges guiding LNPs to the liver, spleen, or lungs, respectively (Cheng et al., Nat. Nanotechnol. 2020, 15:313; and LoPresti et al., Controlled Release 2022, 345:819). PEG-lipids bind to the particle surface, stabilizing the exterior, preventing aggregation, and providing some protection from the immune system (Kiaie et al., ibid.; and Ryals et al., PLoS One 2020, 15:e0241006).

[0008] When delivered systemically via intravenous (iv) injection, many LNPs are strongly liver-tropic, while others transfect the spleen (Hajj et al., Nano Lett. 2020, 20:5167; Kiaie et al., ibid.; and Loughrey and Dahlman, Acc. Chem. Res. 2022, 55:13). Several strategies have been described to reduce hepatic tropism, including adding anti-hepatic microRNA sequences to mRNA (Fenton et al., ibid.), altering LNP helper lipids (Cheng et al., ibid.; and LoPresti et al., ibid.), and introducing active targets such as antibodies (Li et al., ACSChem. Biol. 2020, 15:830; Torchilin et al., Proc. Natl. Acad. Sci. USA 2001, 98:8786; and Abd Elwakil et al., Adv. Funct. Mater. 2019, 29:1807677).

[0009] The next frontier in RNA delivery involves transfection into other organs such as the lungs, heart, bone marrow, and muscle tissue. Delivery to the lungs, in particular, holds the potential to create new treatments for many refractory diseases. For example, cystic fibrosis, a lung-specific symptom originating from epithelial cells, is difficult to treat because these tumors suppress natural killer cells and dendritic cells in the lungs (Engelhardt et al., J. Clin. Invest. 1994, 93:737; and Ahluwalia et al., Cancers 2021, 13:4037). For pulmonary delivery, changing the administration route to inhaled liquid or nebulized LNP can lead to transfection of epithelial cells in vivo (Li et al., Nat. Biotechnol. 2023, 41:1410; Lokugamage et al., Nat. Biomed. Eng. 2021, 5:1059; and Tam et al., Eur. J. Pharm. Sci. 2022, 176:106234). Endogenous targeting to the lungs can also be improved by modulating ionizable lipid chemistry or introducing positively charged co-operating lipids (Qiu et al., Proc. Natl. Acad. Sci. USA 2022, 119:e2116271119; and Sago et al., Proc. Natl. Acad. Sci. USA 2018, 115:E9944). However, some cationic lipids (e.g., 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP)) can cause toxicity. Furthermore, most lung-tropic nanoparticles are limited to endothelial cell delivery (Wei et al., Cell Res. 2015, 25:237; and Pei et al., Chem. Phys. Lipids 2022, 243:105178). Therefore, new systems that safely target other clinically significant cell populations in the lungs remain needed. Summary of the Invention

[0010] Ionizable lipids include those that, when incorporated into lipid nanoparticles, have a neutral charge at physiological pH (7.0) and a positive charge at endosomal pH (4.5–6.5). As described herein, a combinatorial library of 580 ionizable materials was generated for incorporation into lipid nanoparticles. Specifically, libraries of ionizable lipids were created and their efficacy as organ-targeting functions was investigated to identify LNPs that efficiently deliver mRNA to the lungs with and without cationic assist lipids. Also as described herein, LNPs incorporating neutral assist lipids primarily target natural killer cells and dendritic cells, while those formulated with cationic assist lipids enhance delivery to endothelial cells (lymphatics and blood vessels) and epithelial cells (bronchioles and alveoli). The data described herein demonstrate that endogenous transfection of non-endothelial lung cell targets following intravenous injection is feasible, thus enabling the treatment of cystic fibrosis, non-small cell lung cancer, and other lung diseases.

[0011] This document provides ionizable lipids, lipid nanoparticles comprising ionizable lipids, and methods of using them (e.g., for drug delivery). In some cases, the ionizable lipids described herein comprise an acrylate tail and a novel carbon backbone. Such lipids can be prepared by reacting an amine head and an acrylate tail via a Michael addition reaction, during which the hydrogen atom of the primary amine on the head is replaced by the acrylate tail. The ionizable lipids provided herein can have tail carbon lengths of various lengths, including lengths exceeding 10-14. The lipids provided herein can be used, for example, to generate LNPs. In some cases, the LNPs described herein can be formulated with therapeutic agents (e.g., nucleic acids, including but not limited to mRNA and / or siRNA) and can be cargoed into the cytoplasm of target cells. This document also provides efficient delivery carriers. In some cases, the methods and materials described herein can be used to unlock therapies for diseases (including but not limited to diseases in the lungs and / or liver). In some cases, the LNPs described herein can be used for delivery to the liver. In some cases, the LNPs described herein can be used for delivery to the lungs. In some cases, the LNPs described herein can be used to facilitate RNA delivery to the lungs (e.g., via intravenous (iv) injection). In some cases, the LNPs described herein can be administered intravenously. In some cases, the LNPs described herein may comprise neutral formulations (e.g., without cationic lipids that may be associated with toxicity). In some cases, the LNPs described herein can target immune cells. In some cases, the LNPs described herein can target dendritic cells. In some cases, the LNPs described herein can target natural killer cells.

[0012] In a first aspect, this document features a lipid-containing particle comprising, substantially comprising, or consisting of: a lipid; cholesterol or a derivative thereof; an accessory lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipid comprises an alkylamine head and one or more alkyl acrylate tails, each of the one or more alkyl acrylate tails comprising an alkyl chain having a first branch and a second branch at a second carbon atom, and wherein the first branch of each tail has n carbons, and the second branch has n+1, n+2, or n+3 carbons. The lipid may have two, three, four, or more than four alkyl acrylate tails. Each alkyl acrylate tail may be identical, or the lipid may comprise two or more different alkyl acrylate tails. The lipid-containing particle may be a lipid nanoparticle (LNP). The accessory lipid may be a neutral lipid or a zwitterionic lipid. Neutral or zwitterionic lipids may include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids can include 1,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearyl-sn-glycerol-3-trimethylammonium-propane (DSTAP), and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl The lipid-containing particles are selected from one or more of the following: [1-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The lipid particles may be lung-oriented. Lipids may include: N... 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. Lipid-containing particles can target natural killer (NK) and / or dendritic cells. Lipid-containing particles may include approximately 10-40% neutral or zwitterionic helper lipids. Neutral or zwitterionic helper lipids may include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. Lipid-containing particles may include approximately 16% DOPE. Lipid-containing particles may be hepatotactic. Lipids may include: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1-methylethane-1,2-diamine)alkylamine head, 4-(2-aminopropan-2-yl)-1-methylcyclohexane-1-amine alkylamino head, 2-(piperazin-1-yl)ethane-1-amine head or N 1 -((4-)2-aminoethyl)piperazin-1-yl)methyl)ethane-1,2-diamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. Lipids may include a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head and a 2-octyldodecyl acrylate tail. Cholesterol or a derivative thereof may be cholesterol. PEG-based compounds may be PEG-lipids. PEG may have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0013] On the other hand, this document features a composition comprising, substantially composed of, or consisting of lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein the one or more alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. The therapeutic agent may be a nucleic acid. The nucleic acid may be RNA. RNA may include one or more of mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and RNA aptamers. The RNA may be mRNA encoding luciferase. The lipids may have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each alkyl acrylate tail can be identical, or the lipid can include two or more different alkyl acrylate tails. The lipid-containing particles can be lipid nanoparticles (LNPs). The auxiliary lipids can be neutral lipids or zwitterionic lipids. Neutral or zwitterionic lipids can include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids may include one or more of the following: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxypropyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxypropyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Lipid-containing particles may be lung-oriented. Lipids can include: N. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. Lipid-containing particles can target natural killer (NK) and / or dendritic cells. Lipid-containing particles may include approximately 10-40% neutral or zwitterionic helper lipids. Neutral or zwitterionic helper lipids may include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. Lipid-containing particles may contain approximately 16% DOPE. Lipid-containing particles may be hepatotactic. Lipids may include: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1-Methylethane-1,2-diamine)alkylamine head or 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. Lipids may include a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head and a 2-octyldodecyl acrylate tail. Cholesterol or a derivative thereof may be cholesterol. PEG-based compounds may be PEG-lipids. PEG may have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0014] On the other hand, this document is characterized by a method for delivering a therapeutic agent to a mammal. The method may include, consist of, or substantially consist of: administering to a mammal a composition comprising lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. Administration may include intravenous, intramuscular, or subcutaneous administration. The lipid-containing particles may be lipid nanoparticles (LNPs). The therapeutic agent may be a nucleic acid. The nucleic acid may be RNA. RNA may include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. RNA can be administered at a dose of about 0.01 mg / kg to about 10 mg / kg or at a dose of about 0.5 mg / kg. The lipids can have two, three, four, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails can be identical, or the lipids can include two or more different alkyl acrylate tails. The accessory lipids can be neutral or zwitterionic lipids. Neutral or zwitterionic lipids may include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids may include one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Cholesterol or its derivatives may be cholesterol. PEG-based compounds may be PEG-lipids. PEG can have a molecular weight of approximately 300 g / mol to approximately 5000 g / mol. Mammalian animals may have been identified as having lung disorders, and the lipid-containing particles may be lung-oriented. Lung disorders may include cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infections (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. Lipids may include: N. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(propane-1,3-diyl)bis(N 1(-Ethylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. The lipid-containing particles can target natural killer (NK) and / or dendritic cells. The lipid-containing particles may include approximately 10-40% neutral or zwitterionic helper lipids. Neutral or zwitterionic helper lipids may include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The lipid-containing particles may include approximately 16% DOPE. Mammalians may have been identified as having liver disease, and the lipid-containing particles may be liver-oriented. Liver diseases can include alpha-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or combinations thereof. Lipids can include: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 -Methylethane-1,2-diamine)alkylamine head or 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. Lipids may include a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head and a 2-octyldodecyl acrylate tail. Mammalians may include humans.

[0015] On the other hand, this document is characterized by a method for treating mammals suffering from lung diseases or their symptoms, or liver diseases or their symptoms. The method may include, consist of, or substantially consist of: administering to the mammal a composition comprising lipid-containing particles, the lipid-containing particles containing a therapeutic agent, wherein the lipid-containing particles further comprise: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. Administration may include intravenous, intramuscular, or subcutaneous administration. The lipid-containing particles may be lipid nanoparticles (LNPs). The therapeutic agent may be a nucleic acid. The nucleic acid may be RNA. RNA may include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. RNA can be administered at a dose of about 0.01 mg / kg to about 10 mg / kg or at a dose of about 0.5 mg / kg. The lipids can have two, three, four, or more than four alkyl acrylate tails. Each alkyl acrylate tail can be identical, or the lipids can include two or more different alkyl acrylate tails. The accessory lipids can be neutral or zwitterionic lipids. Neutral or zwitterionic lipids may include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids may include one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Cholesterol or its derivatives may be cholesterol. PEG-based compounds may be PEG-lipids. PEG can have a molecular weight of approximately 300 g / mol to approximately 5000 g / mol. Mammalian organisms may have been identified as having lung diseases, and the lipid-containing particles may be lung-oriented. Lung diseases may include cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesis, viral lung infections (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. Lipids may include: N. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(propane-1,3-diyl)bis(N 1(-Ethylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. The lipid-containing particles can target natural killer (NK) and / or dendritic cells. The lipid-containing particles may include approximately 10-40% neutral or zwitterionic helper lipids. Neutral or zwitterionic helper lipids may include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The lipid-containing particles may contain approximately 16% DOPE. Mammalians may have been identified as having liver disease, and the lipid-containing particles may be hepatotropic. Liver diseases can include alpha-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or combinations thereof. Lipids can include: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 -Methylethane-1,2-diamine)alkylamine head or 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. Lipids may include a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head and a 2-octyldodecyl acrylate tail. Mammalians may include humans.

[0016] In another aspect, this document features a method for delivering a therapeutic agent to lung cells. The method may include, consist of, or substantially consist of: contacting lung cells with a composition comprising lipid-containing particles, said lipid-containing particles comprising a therapeutic agent and: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein said lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein said alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon atom, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. The lipid-containing particles may be lipid nanoparticles (LNPs). The lipids may have two, three, four, or more than four alkyl acrylate tails. Each of said alkyl acrylate tails may be identical, or said lipids may comprise two or more different alkyl acrylate tails. The cofactor lipids may be neutral lipids or zwitterionic cofactor lipids. Neutral lipids or zwitterionic co-lipids may include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (…). The accessory lipids are one or more of the following: POPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids may include one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Cholesterol or its derivatives may be cholesterol. PEG-based compounds may be PEG-lipids. PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol. Lung cells can be in mammals. Mammals can be humans. The therapeutic agent can be a nucleic acid. Nucleic acid can be RNA. RNA can include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. RNA can be administered at a dose of about 0.01 mg / kg to about 10 mg / kg or at a dose of about 0.5 mg / kg. Administration can include intravenous, intramuscular, or subcutaneous administration. Lung cells can be in mammals identified as having a lung disease. Lung diseases can include cystic fibrosis, non-small cell lung cancer, another type of lung cancer, α-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infections (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. Lipids can include: N. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail. Lipids may include N 1 N1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head and 2-hexyldecyl acrylate tail. The lipid-containing particles can target natural killer (NK) and / or dendritic cells. The lipid-containing particles may include approximately 10-40% neutral or zwitterionic helper lipids. Neutral or zwitterionic helper lipids may include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The lipid-containing particles may include approximately 16% DOPE.

[0017] On the other hand, this document is characterized by a lipid-containing particle comprising, consisting of, or substantially consisting of: lipids; cholesterol or a derivative thereof; accessory lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N-terminated. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1The lipid-containing particles are a -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head, and wherein the one or more alkyl acrylate tails comprise one or more of a 2-octyldecyl acrylate tail and a 9-methyldecyl acrylate tail. The lipid particles may be lipid nanoparticles (LNPs). The lipids may have two, three, four, or more than four alkyl acrylate tails. Each alkyl acrylate tail may be identical, or the lipids may comprise two or more different alkyl acrylate tails. The accessory lipids may be neutral or zwitterionic lipids. Neutral or zwitterionic lipids may include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids. Cationic lipids may include one or more of the following: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-octyldecyl acrylate tail. Lipids may include N1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 9-methyldecyl acrylate tail. Lipids may include N 1 -((4-)2-aminoethyl)piperazin-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail. Cholesterol or a derivative thereof may be cholesterol. PEG-based compounds may be PEG-lipids. PEG may have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0018] In another aspect, this document is characterized by a composition comprising, consisting of, or substantially consisting of lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N-terminated. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1The alkyl acrylate head is 1,2-((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine, and the one or more alkyl acrylate tails comprise one or more of a 2-octyldecyl acrylate tail and a 9-methyldecyl acrylate tail. The therapeutic agent may be a nucleic acid. The nucleic acid may be RNA. The RNA may include one or more of mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and RNA aptamers. The RNA may be mRNA encoding luciferase. The lipid may have two, three, four, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails may be identical, or the lipid may include two or more different alkyl acrylate tails. The lipid-containing particles may be lipid nanoparticles (LNPs). The accessory lipids may be neutral lipids or zwitterionic lipids. Neutral or zwitterionic lipids may include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids may include one or more of the following: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxypropyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxypropyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Lipids may include N. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-octyldecyl acrylate tail. Lipids may include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 9-methyldecyl acrylate tail. Lipids may include N 1 -((4-)2-aminoethyl)piperazin-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail. Cholesterol or a derivative thereof may be cholesterol. PEG-based compounds may be PEG-lipids. PEG may have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0019] On the other hand, this document is characterized by a method for delivering a therapeutic agent to a mammal, wherein the method comprises, consists of, or substantially consists of: administering to a mammal a composition comprising lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; coenzyme lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1The alkyl acrylate head is 1,2-((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine, and the one or more alkyl acrylate tails comprise one or more of a 2-octyldecyl acrylate tail and a 9-methyldecyl acrylate tail. Administration may include intravenous, intramuscular, or subcutaneous administration. The lipid-containing particles may be lipid nanoparticles (LNPs). The therapeutic agent may be a nucleic acid. The nucleic acid may be RNA. RNA may include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. RNA may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg or at a dose of about 0.5 mg / kg. The lipid may have two, three, four, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails may be identical, or the lipid may include two or more different alkyl acrylate tails. The auxiliary lipids can be neutral lipids or zwitterionic lipids. Neutral or zwitterionic lipids can include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids may include one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Cholesterol or its derivatives may be cholesterol. PEG-based compounds may be PEG-lipids. PEG can have a molecular weight of approximately 300 g / mol to approximately 5000 g / mol. Mammals may have been identified as having lung diseases. Lung diseases can include cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infections (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. Mammals may have been identified as having liver diseases. Liver diseases can include alpha-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or combinations thereof. Lipids may include nitrogen (N). 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-octyldecyl acrylate tail. Lipids may include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 9-methyldecyl acrylate tail. Lipids may include N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail. Mammalian can be human.

[0020] On the other hand, this document is characterized by a method for treating mammals suffering from lung diseases or their symptoms, or liver diseases or their symptoms, wherein the method comprises, consists of, or substantially consists of: administering to the mammal a composition comprising lipid-containing particles, said lipid-containing particles comprising a therapeutic agent, wherein said lipid-containing particles further comprise: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein said lipids comprise an alkylamino head and one or more alkyl acrylate tails, wherein said alkylamino head is N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1The alkyl acrylate head is 1,2-((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine, and the one or more alkyl acrylate tails comprise one or more of a 2-octyldecyl acrylate tail and a 9-methyldecyl acrylate tail. Administration may include intravenous, intramuscular, or subcutaneous administration. The lipid-containing particles may be lipid nanoparticles (LNPs). The therapeutic agent may be a nucleic acid. The nucleic acid may be RNA. RNA may include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. RNA may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg or at a dose of about 0.5 mg / kg. The lipid may have two, three, four, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails may be identical, or the lipid may include two or more different alkyl acrylate tails. The auxiliary lipids can be neutral lipids or zwitterionic lipids. Neutral or zwitterionic lipids can include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (P... One or more of the following: OPC, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. The accessory lipids may be cationic lipids.Cationic lipids may include one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Cholesterol or its derivatives may be cholesterol. PEG-based compounds may be PEG-lipids. PEG can have a molecular weight of approximately 300 g / mol to approximately 5000 g / mol. Lung conditions may include cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infections (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. Liver conditions may include alpha-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or combinations thereof. Lipids may include nitrogen (N). 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-octyldecyl acrylate tail. Lipids may include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 9-methyldecyl acrylate tail. Lipids may include N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail. Mammalian can be human.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials may be used to practice this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including the definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0022] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the invention will become apparent from the detailed description and the drawings, as well as from the claims. Attached Figure Description

[0023] Figure 1A-1C This diagram shows the chemical reactions and functional groups used to generate a library of 580 ionizable lipids containing straight-chain, branched, and unsaturated tails. This was achieved through 29 alkylamine heads (…). Figure 1B ) and 20 kinds of alkyl acrylate tails ( Figure 1C Combinatorial Michael addition reaction ( Figure 1A Synthetic lipids. For tail nomenclature, "O" and "X" represent fully saturated and partially unsaturated tails, respectively.

[0024] Figure 2A-2C This demonstrates that a portion of the ionizable lipid library efficiently delivers mRNA in mice. Figure 2A The results of the initial screening are shown, which was performed using a cell-free 2-(p-toluidine)naphthalene-6-sulfonic acid (TNS) assay at pH 5. Higher levels of TNS fluorescence correspond to darker shadows. Figure 2B The TNS fluorescence of 18 lipids with high levels, 18 lipids with medium levels, and 13 lipids with low levels was plotted. Each lipid was formulated with firefly luciferase mRNA and intravenously injected into mice at a total mRNA dose of 0.75 mg / kg. Total intracellular luminescence was measured and quantified by IVIS 3 hours later. Figure 2C Includes a pair of plots showing that TNS fluorescence measurements correlated more strongly with in vivo efficacy (left plot) than with efficacy in HeLa cells (right plot). In these plots, in vivo luminescence scales logarithmically, while in vitro luminescence and TNS fluorescence scale linearly. For TNS assays, N=12, showing the mean; for in vitro luminescence, N=4; for in vivo luminescence, N=3. Error bars represent standard deviations.

[0025] Figure 3This includes a series of plots depicting LNP characterization data. The encapsulation was measured using RiboGreen™ (left plot), and the number-average diameter (center plot) and zeta potential (right plot) were measured using dynamic light scattering. N=4, and error bars represent sd.

[0026] Figures 4A-4C The results showed that LNPs are primarily liver-oriented, with several types effectively delivering mRNA to the lungs and spleen. Three hours after intravenous injection of LNPs containing 0.75 mg / kg mLuc LNPs, mice were sacrificed, organs were harvested, and chemiluminescence imaging was performed. Figure 4A The diagram shows the luminescence in the lungs, liver, and spleen, revealing that although a subset of lipids induces protein expression in the spleen and lungs, delivery primarily occurs in the liver. Figure 4B This includes a pie chart showing the organ distribution of the luminescent signals of the first three liver-oriented compounds. Figure 4C Includes a pie chart illustrating the organ distribution of the luminescent signals of the top three compounds, dominated by signals from the lungs and spleen. N=3, and the error bars represent the standard deviation.

[0027] Figures 5A-5E The auxiliary lipid charge and molar percentage changed by 514O. 6,10 Organ tropism of lipid nanoparticles. Containing 514O 6,10 LNPs were prepared using 16 mol% DOPE, 40% DOPE, or 40% DOTAP. Mice were intravenously injected with 0.75 mg / kg of mRNA encoding luciferase (mLuc) or green fluorescent protein (mGFP). Figure 5A Includes a pie chart showing the luminescent signal quantified by IVIS in the mLuc experiment, in which mice were sacrificed 3 hours after injection. Figure 5B These are images from a GFP experiment, showing the right lung lobe of mice collected for immunohistochemistry, where GFP expression is visualized in green. H&E staining of the lungs was performed immediately before injection (0h) and between 1 and 48 hours after injection. Figure 5C Liver function tests Figure 5D ) and cytokine analysis ( Figure 5E The study investigated safety. For all subplots, N=3, and the error bars represent standard errors. *, **, ***, and **** indicate p<0.05, 0.01, 0.001, and 0.0001, respectively, according to the two-way Dunnett multiple comparisons ANOVA.

[0028] Figures 6A-6C Display using lipid 514O 6,10The prepared LNPs disproportionately transfected several rare lung cell types. 514O was prepared using 16 mol% DOPE (16PE), 40% DOPE (40PE), or 40% DOTAP (40TAP). 6,10 LNP was administered to mice via tail vein injection at a dose of 0.75 mg / kg of mGFP. Animals were euthanized 24 hours after injection, and organs were processed into single-cell suspensions for analysis by flow cytometry. Figure 6A Includes pie charts showing the influence of helper lipids on LNP tropism in lymphoid cells, endothelial cells, and epithelial cells. Figure 6B This includes plotting graphs of GFP expression in individual cell types, quantified by flow cytometry using panels of lymphoid cells, endothelial cells, and epithelial cells. The y-axis in each graph represents the percentage of GFP+ cells for each cell type and is normalized to autofluorescence in unstained control samples. The raw data before normalization are available... Figures 7A-7D Obtained from. Untr = Untreated, DC = Dendritic cells, NK = Natural killer cells. Figure 6C This includes a series of bars showing the specificity of transfected cells, illustrated by comparing the overall cell type distribution in the lung sample (top bar) with the transfected (GFP+) cell type (bottom bar). N=3, error bars represent standard deviations, and *, **, and **** correspond to p < 0.05, 0.005, or 0.0001 respectively based on two-way ANOVA supplemented by Dunnet post-hoc analysis.

[0029] Figures 7A-7D Includes plots of raw GFP transfection efficacy data as a function of cell type before normalization. 514O was prepared using 16 mol% DOPE (16PE), 40% DOPE (40PE), or 40% DOTAP (40TAP). 6,10 LNP was administered to mice via tail vein injection at a dose of 0.75 mg / kg mGFP. Animals were euthanized 24 hours post-injection, and organs were processed into single-cell suspensions for analysis by flow cytometry. Lymphoid tissue was analyzed. Figure 7A ), endothelium ( Figure 7B Epithelium Figure 7C ) and medullary ( Figure 7D Cell set. The y-axis represents the percentage of GFP+ cells for each cell type. Untr = untreated, DC = dendritic cells, NK = natural killer cells. N = 3, error bars represent standard deviation, *, **, **** correspond to p < 0.05, 0.005, or 0.0001 respectively based on two-way ANOVA with Dunnet multiple comparisons. Figure 6BIt was generated from these data by normalizing them based on untreated controls. Figure 6B Myeloid cells are not shown because their overall number is low and the background fluorescence is too high.

[0030] Figures 8A-8C This demonstrates a guillotine-based strategy for defining cell populations in the lungs using fluorescent antibodies from Table 3. Figure 8A Lymphoid group set; Figure 8B Myeloid group set; Figure 8C The endothelium and epithelium are combined. Detailed Implementation

[0031] This article provides ionizable lipids, lipid particles containing ionizable lipids, and methods for delivering one or more therapeutic agents (e.g., nucleic acids, such as mRNA) to mammals using particles containing ionizable lipids.

[0032] Lipid-containing particles are small particles or structures comprising lipids and / or lipid-like materials (e.g., lipidoids). Lipid-containing particles exist in various biological forms, such as LNPs (which can be used in drug delivery systems), lipoproteins (which can transport lipids in the bloodstream), lipid droplets (which are intracellular organelles), exosomes (which participate in intercellular communication), vesicles (which are small membrane-bound sacs within cells), and so on. Typically, the lipid-containing particles (e.g., LNPs) described herein comprise mixtures of: (i) ionizable and / or cationic lipids or lipidoids; (ii) cholesterol, cholesterol analogs, or cholesterol derivatives; (iii) accessory lipids; and (iv) polyethylene glycol-lipid conjugates (PEG-lipids) or PEG-cholesterol conjugates (PEG-cholesterol).

[0033] Any suitable ionizable lipid or lipid-like substance may be included in the lipid-containing particles (e.g., LNPs) provided herein. Suitable lipids include, for example, fats, waxes, sterols, fat-soluble vitamins, and other similar substances. Lipid-like substances are a class of lipid-like materials commonly used in biotechnology and nanomedicine, particularly for delivering nucleic acids such as RNA and DNA. Typically, the ionizable lipid-like substances provided herein have an alkylamine head and one or more alkyl acrylate tails. Non-limiting examples of suitable alkylamine heads are depicted in... Figure 1B In, for example, the alkylamine head can be N. 1 N 1 '-(propane-1,3-diyl)bis(N 1 -Methylethane-1,2-diamine)alkylamine head (503), N 1 N 1 '-(propane-1,3-diyl)bis(N 1 -Ethylethane-1,2-diamine)alkylamine head (514), N 1 N1 '-(ethane-1,2-diyl)bis(N 1 -Methylethane-1,2-diamine)alkylamine head (500), 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-amine alkylamino head (402), 2-(piperazin-1-yl)ethane-1-amine head (205) or N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head (200).

[0034] The lipids provided herein can have any suitable number of alkyl acrylate tails. For example, the lipids provided herein can have one, two, three, four, or more than four (e.g., five, six, seven, or eight) alkyl acrylate tails. In some cases, when the lipid molecules provided herein have more than one alkyl acrylate tail, all alkyl acrylate tails may be identical. In some cases, when the lipid molecules provided herein have more than one alkyl acrylate tail, the lipids may include two or more (e.g., two, three, four, five, or more than five) different alkyl acrylate tails. Non-limiting examples of suitable alkyl acrylate tails are depicted in Figure 1C In some cases, the alkyl acrylate tail may include an alkyl chain having a first branch and a second branch at the second carbon atom. In some cases, the lengths of the first and second branches may be different. For example, the first branch of the alkyl acrylate tail may have n carbons, and the second branch may have n+1, n+2, or n+3 carbons. For example, the alkyl acrylate tail may be a 2-butylheptyl acrylate tail (O... 4,7 ), 2-Butyloctyl acrylate tail (O) 4,8 ), 2-Butylnonyl acrylate tail (O) 4,9 ), 2-hexyl nonyl acrylate tail (O) 6,9 ), 2-hexyldecyl acrylate tail (O) 6,10 ), 2-hexyl undecyl acrylate tail (O) 6,11 ), 2-octyl undecyl acrylate tail (O) 8,11 ), 2-octyl dodecyl acrylate tail (O) 8,12 ) or the tail of 2-octyltridecyl acrylate (O 8,13 In some cases, the first and second branches can be of the same length. For example, the alkyl acrylate tail can be the 2-octyldecyl acrylate tail (O... 8,10 ), 2-Nonyl undecyl acrylate tail (O) 9,11 ), 2-heptylnonyl acrylate tail (O) 7,9 ) or the tail of 2-hexyl octyl acrylate (O) 6,8In some cases, the alkyl acrylate tail may not contain branches. For example, in some cases, the alkyl acrylate tail may be a 9-methyldecyl acrylate tail (O... i10 ).

[0035] Suitable examples of lipids include, but are not limited to, 503O. i10 514O 6,10 500O 6,10 402O 8,12 503O 8,10 503O 6,10 205O 6,10 200O i10 402O 4,8 and 402O 4,8 In some cases, the lipids in the lipid-containing particles provided herein may include 503O. i10 514O 6,10 500O 6,10 402O 8,12 Or any combination thereof. For example, the lipids contained in LNPs could be 503O. i10 514O 6,10 500O 6,10 Or 402O 8,12 .

[0036] The lipids provided herein can be generated using any suitable method. For example, the alkylamine head can be combined with one or more alkyl acrylate tails in any suitable ratio (e.g., a stoichiometric ratio of about 1:2 to about 1:10 (e.g., about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10)) and can be mixed together at any suitable temperature and for any suitable duration. For example, the alkylamine head and one or more alkyl acrylate tails can be mixed at about 60°C to about 120°C (e.g., about 60°C, about 70°C, about 80°C, about 90°C, about 100°C, about 110°C, or about 120°C) for about 12 hours to about 5 days (e.g., about 12 to 24 hours, about 1 to 2 days, about 2 to 3 days, about 3 to 4 days, or about 4 to 5 days). Figure 1A As shown, such procedures can generate the lipids presented herein via a combinatorial Michael addition reaction.

[0037] Any suitable cholesterol-related molecule (e.g., cholesterol, cholesterol analogs, or cholesterol derivatives) may be included in the lipid-containing particles (e.g., LNPs) provided herein. Examples of suitable cholesterol derivatives (or analogs) include, but are not limited to, oxidized cholesterol, sterols, 7-dehydrocholesterol, ergosterol, lanosterol, ketosterone, cholesterol sulfate, dehydroergosterol, cholesteroltratrienol, 5-cholesterol, and pregnenolone. In some cases, the lipid-containing particles (e.g., LNPs) provided herein may contain cholesterol.

[0038] Any suitable assisting lipid may be included in the lipid-containing particles (e.g., LNPs) provided herein. The assisting lipids may be cationic, anionic, neutral, or zwitterionic amphiphilic lipids, and together with cholesterol or its derivatives (e.g., cholesterol analogs), may contribute to the molecular stacking and stability of the lipid-containing particles (e.g., LNPs). The assisting lipids may also enhance the efficacy of lipid nanoparticles by promoting fusion with both cell membranes and endosome membranes, and by promoting cellular uptake and endosome release. In some cases, the lipid-containing particles provided herein may include one or more zwitterionic assisting lipids. Non-limiting examples of suitable zwitterionic-assisted lipids include 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl- 2-Oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoyl sphingomyelin (PSM), and sterol sphingomyelin (SSM). In some cases, the lipid-containing particles provided herein may include one or more neutral cofactor lipids. Non-limiting examples of suitable neutral cofactor lipids include triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides. In some cases, the lipid-containing particles provided herein may include one or more cationic cofactor lipids. Non-limiting examples of suitable cationic co-adjuvant lipids include 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0039] Any suitable PEG-based compound may be included in the lipid-containing particles (e.g., LNPs) provided herein. PEG is a petroleum-derived polyether compound. PEG and PEG-based compounds can be used in a variety of applications, such as drug delivery agents, solvents, adhesives, adsorbents, and tissue engineering scaffolds. PEG-based compounds that may be used in the lipid-containing particles provided herein include, but are not limited to, PEG-lipids (PEGylated lipids) and PEG-cholesterols (PEGylated cholesterol). PEG-lipids include PEG moieties linked to one or more lipid moieties (e.g., ceramide, succinyl, or carbamate moieties). PEG-cholesterols include PEG moieties linked to one or more cholesterol moieties. PEG-lipids and / or PEG-cholesterols may form a protective, non-aggregating, non-immunogenic shell around the surface of the LNP. Depending on the final delivery pathway of the LNP, the lipid groups (e.g., in length) can be altered to determine the duration for which the PEG-lipid will associate with the LNP. Longer lipid chains tend to remain associated with the LNP for a longer period of time, while shorter lipid chains are often used to provide “diffusionable” PEG lipids that diffuse rapidly from the lipid nanoparticles to produce LNPs with an increased transfection rate. The PEG portion of PEG-lipids or PEG-cholesterol may have a molecular weight ranging from about 300 g / mol to about 5000 g / mol (e.g., about 300 g / mol to about 500 g / ml, about 500 g / mol to about 1000 g / mol, about 1000 g / mol to about 2000 g / mol, about 1500 g / mol to about 2500 g / mol, about 2000 g / mol to about 3000 g / mol, about 2500 g / mol to about 3500 g / mol, about 3000 g / mol to about 4000 g / mol, about 3500 g / mol to about 4500 g / mol, about 4000 g / mol to about 5000 g / mol, about 1000 g / mol, about 2000 g / mol, about 3000 g / mol, about 4000 g / mol, or about 5000 g / mol). For example, the PEG portion of PEG-lipids or PEG-cholesterol can have a molecular weight of about 2000, which is called PEG 2000.Non-limiting examples of suitable PEG-lipids include 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000], N-octanoyl-sphingosine-1-{succinoyl[methoxy(polyethylene glycol)2000]}, N-palmitoyl-sphingosine-1-{succinoyl[methoxy(polyethylene glycol)5000]}, 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-1000] (ammonium salt), and PEG-cholesterol, such as cholesterol-(polyethylene glycol-600). In some cases, the lipid-containing particles (e.g., LNPs) in the compositions provided herein may contain PEG-lipids. In some cases, PEG-lipids or PEG-cholesterols can be modified with a targeting moiety (such as N-acetylgalactosamine (GalNAc) for liver targeting) or with another ligand or binding agent (such as peptides (e.g., antibodies or antibody fragments), apolipoproteins (e.g., ApoE), peptides and / or small molecule ligands similar to GalNAc).

[0040] Lipid-containing particles (e.g., LNPs) can effectively deliver therapeutic agents (e.g., nucleic acids and nucleic acid analogs) to cells, typically in vivo (see, for example, Kulkarni et al., Nucleic Acid Therapeutics, 28(3):146-157, 2018; Hajj and Whitehead, Nature Reviews Materials, 2:17056, 2017; US Publication No. 20130245107; and US Patent No. 8,754,062). As used herein, a “therapeutic agent” is any compound or composition that can be delivered to a patient to achieve a desired effect such as a beneficial effect, therapeutic effect, or curative effect. Therapeutic agents include, but are not limited to, nucleic acids, nucleic acid analogs, proteins, peptides, small molecule drugs, antibiotics, antiviral drugs, and cell-based therapies (e.g., CAR-T cell therapy).

[0041] In some cases, the lipid-containing particles provided herein may include therapeutic agents that are nucleic acids or nucleic acid analogs, or include nucleic acids or nucleic acid analogs. As used herein, the term "nucleic acid" includes any compound and / or substance comprising a nucleotide polymer (also known as a "polynucleotide" or "oligonucleotide"). Exemplary nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization) or hybrids thereof. Naturally occurring nucleic acids typically have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0042] Nucleic acids and nucleic acid analogs have a backbone and a nucleobase sequence. In the context of this document, backbone monomer residues can be any suitable nucleic acid backbone monomer residue with a negative charge, such as ribose or deoxyribose linked to another ribose or deoxyribose via a phosphodiester bond, or backbone residues of nucleic acid analog monomers. Backbone monomers can include both structural “residue” components (such as ribose in RNA) and any active groups that are modified when the monomers are linked together (such as the 5' triphosphate group and 3' hydroxyl group of a ribonucleotide, which are modified to produce a negatively charged phosphodiester bond when polymerized into RNA).

[0043] For example, therapeutic agents can be nucleic acids or nucleic acid analogs with a negatively charged backbone, including but not limited to single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified forms of any of the foregoing (e.g., forms including one or more alterations to nucleotide components), or analogs of any of the foregoing (e.g., synthetic molecules mimicking the original structure and function). Regarding the overall structure and function of the nucleic acid or nucleic acid analog, the nucleic acid or nucleic acid analog can be, but is not limited to: mRNA (messenger RNA), siRNA (small interfering RNA), miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), lncRNA (long non-coding RNA), circRNA (circular RNA), antisense RNA, ncRNA (non-coding RNA), telomerase RNA, piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNA (small Cajalosome RNA), Y... RNA, eRNA (enhancer RNA), shRNA (hairpin RNA), stRNA (hour sequence RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multi-copy single-stranded DNA), XNA (heterogeneous nucleic acid), ethylene glycol nucleic acid, threonine nucleic acid, hexonine nucleic acid, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholine oligomers, antisense oligonucleotides, ribozymes, deoxyribozymes, aptamers, cloning vectors, phage particles, plasmids, λ phage, cosmids, fosmids, or artificial chromosomes.

[0044] RNA therapy is a class of RNA-based treatments that target specific genes or genetic pathways with high specificity. The use of RNA therapy can allow for transient expression or inhibition, which can reduce long-term side effects. RNA therapy utilizes various forms of RNA to treat diseases such as, but not limited to, infectious diseases, cancer, genetic disorders, cardiovascular diseases, and neurological disorders. Research has been ongoing since the 1990s, with significant success in cancer therapy in the early 2010s (see, for example, Sahin et al., Nature Reviews Drug Discovery, 13:759-780, 2014). RNA used in RNA therapy can include, for example, mRNA, siRNA, short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, gRNA, long non-coding RNA, transfer RNA, ribosomal RNA, double-stranded RNA (dsRNA), and / or RNA aptamers. In some cases, for example, mRNA-based therapies can be used.

[0045] mRNA-based therapies can trigger protein synthesis by delivering the encoding mRNA into cells, making such therapies particularly useful in vaccine development (see, for example, DeFrancesco, Nature Biotechnology, 35:193-197, 2017). The encoding mRNA can be programmed as a blueprint to generate target proteins (e.g., reporter proteins, functional proteins, or antigens). In some implementations, the target protein can be an antigen produced by a pathogen (e.g., a virus) or by cancer cells. Such protein molecules can stimulate an adaptive immune response that teaches the body to recognize and destroy the corresponding pathogen or cancer cells (see, for example, Bae and Park, Advanced Drug Delivery Reviews, 158:4-16, 2020). mRNA vaccines (e.g., Pfizer-BioNTech COVID-19 vaccine and Moderna COVID-19 vaccine) have been developed to combat coronavirus disease during the COVID-19 pandemic (see, Noor, Current Clinical Microbiology Reports, 8(3):178-185, 2021). In some cases, the designed mRNA can encode a reporter, such as firefly luciferase. The mRNA encoding the desired protein (e.g., the mRNA encoding luciferase) can be delivered into cells using lipid-containing particles (e.g., LNPs) to produce the protein (e.g., luciferase) in vitro (via cell culture) and in vivo (e.g., in mouse models or in any other suitable mammal (e.g., human, non-human primates, rats, rabbits, cattle, pigs, sheep, dogs, and / or cats)). The mammal can be healthy or may suffer from disease, symptom, or clinical condition.

[0046] The lipid-containing particles provided herein can be generated using any suitable method. For example, lipid-containing particles (e.g., LNPs) can be generated by combining ionizable lipids or lipid-like substances, cholesterol or cholesterol derivatives, cofactor lipids, and PEG-based compounds provided herein in any suitable amounts or proportions. In some cases, lipid-containing particles (e.g., LNPs) can be prepared by combining the following:

[0047] The lipids or lipid-like substances provided herein (e.g., 503O) comprise approximately 30 mol% to approximately 70 mol% (e.g., approximately 30 to 40 mol%, approximately 35 to 45 mol%, approximately 40 to 50 mol%, approximately 45 to 55 mol%, approximately 50 to 60 mol%, approximately 55 to 65 mol%, approximately 60 to 70 mol%, approximately 30 mol%, approximately 40 mol%, approximately 50 mol%, approximately 60 mol%, or approximately 70 mol%).i10 514O 6,10 500O 6,10 Or 402O 8,12 );

[0048] The auxiliary lipids (e.g., DSPC, DOPE or DOTAP) are about 2 mol% to about 25 mol% (e.g., about 2 to 10 mol%, about 5 to 15 mol%, about 10 to 20 mol%, about 5 mol%, about 10 mol% or about 15 mol%).

[0049] About 20 to about 60 mol% (e.g., about 20 to 30 mol%, about 25 to 35 mol%, about 30 to 40 mol%, about 35 to 45 mol%, about 40 to 50 mol%, about 45 to 55 mol%, about 50 to 60 mol%, about 30 mol%, about 35 mol%, about 38.5 mol%, about 40 mol%, about 45 mol%, or about 50 mol%) of cholesterol or cholesterol derivatives (e.g., cholesterol); and

[0050] About 0.2 to about 3 mol% (e.g., about 0.2 to 1 mol%, about 0.5 to 1.5 mol%, about 1 to 2 mol%, about 1.5 to 2.5 mol%, about 2 to 3 mol%, about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, or about 2.5 mol%) of PEG-based compounds (e.g., PEG or PEG-lipids).

[0051] In some cases, lipid-containing particles can be prepared by combining the above components in a molar ratio of about 35:46.5:16:2.5 (lipids:cholesterol:coenzymes:PEG), or about 35:22.5:40:2.5 (lipids:cholesterol:coenzymes:PEG), or in any other suitable molar ratio.

[0052] The lipid-containing particles (e.g., LNPs) prepared as described herein can be combined with any suitable cargo (e.g., nucleic acids or other therapeutic agents, and / or biomarkers). In some cases, the concentration of mRNA as cargo in the lipid-containing particles (e.g., for in vitro cell culture) can be from about 0.001 mg / mL to about 2 mg / mL (e.g., about 0.001 mg / mL to about 1.5 mg / mL, about 0.003 mg / mL to about 1.5 mg / mL, about 0.005 mg / mL to about 1.5 mg / mL, about 0.003 mg / mL to about 1 mg / mL, about 0.003 mg / mL to about 0.5 mg / mL, about 0.005 mg / mL to about 1.5 mg / mL). Approximately 0.005 to approximately 1 mg / mL, approximately 0.005 to approximately 0.5 mg / mL, approximately 0.001 mg / mL, approximately 0.003 mg / mL, approximately 0.005 mg / mL, approximately 0.01 mg / mL, approximately 0.03 mg / mL, approximately 0.05 mg / mL, approximately 0.08 mg / mL, approximately 0.1 mg / mL, approximately 0.2 mg / mL, approximately 0.3 mg / mL, approximately 0.4 mg / mL, approximately 0.5 mg / mL, approximately 1 mg / mL, approximately 1.5 mg / mL, or approximately 2 mg / mL). In some cases, the concentration of mRNA as cargo in lipid-containing particles (e.g., for in vivo use) can be from about 0.01 mg / mL to about 10 mg / mL (e.g., about 0.01 mg / mL to about 1 mg / mL, about 0.03 mg / mL to about 3 mg / mL, about 0.05 mg / mL to about 5 mg / mL, about 0.1 mg / mL to about 2 mg / mL, about 0.3 mg / mL to about 3 mg / mL, about 0.5 mg / mL to about 5 mg / mL). Approximately 1 mg / mL to approximately 3 mg / mL, approximately 3 mg / mL to approximately 5 mg / mL, approximately 0.01 mg / mL, approximately 0.03 mg / mL, approximately 0.05 mg / mL, approximately 0.1 mg / mL, approximately 0.3 mg / mL, approximately 0.5 mg / mL, approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, or approximately 10 mg / mL. In some cases, when the lipid-containing particles include mRNA, the lipid-to-mRNA ratio (by weight) in the lipid-containing particles can be approximately 5:1 to approximately 30:1 (e.g., approximately 5:1, approximately 8:1, approximately 10:1, approximately 12:1, approximately 15:1, approximately 20:1, approximately 25:1, or approximately 30:1).

[0053] This document also provides compositions comprising lipid-containing particles (e.g., LNPs) and lipid components as described herein, in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicle for delivering a therapeutic agent to a subject. Pharmaceutically acceptable carriers can be liquid or solid and can be selected with regard to the planned mode of administration to provide the desired volume, consistency, and other associated delivery and chemical properties when combined with one or more therapeutic compounds and any other components of a given pharmaceutical composition. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: water; saline solutions; binders (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose or dextran and other sugars, gelatin or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrants (e.g., starch or sodium carboxymethyl starch); and wetting agents (e.g., sodium lauryl sulfate). In some cases, the compositions provided herein may include one or more sugars (e.g., sucrose and / or trehalose) that can act as LNP stabilizers / preservatives.

[0054] The efficacy of the lipid-containing particles (e.g., LNPs) provided herein can be determined using any suitable method. For example, any suitable method can be used to determine the efficacy of lipid-containing particles (e.g., LNPs) in delivering cargo (e.g., therapeutic agents or nucleic acids encoding detectable markers) to cells in vitro or in vivo. For example, in vitro efficacy can be evaluated by contacting cells in a culture (e.g., primary cells obtained from mammals or cells derived from cell lines) with an LNP formulated with a nucleic acid (e.g., mRNA) encoding a marker that can detect its expression (e.g., luciferase or green fluorescent protein (GFP)). After incubating the LNP with cells, the cells can be assessed by measuring fluorescence or luminescence, for example, to determine whether the marker has been expressed in the cells (and to what extent). In some cases, in vivo efficacy can be assessed by administering an LNP formulated with nucleic acid (e.g., mRNA) encoding a biomarker (e.g., luciferase or GFP) that can be detected for expression to mammals (e.g., mice or rats) or zebrafish, and, after an appropriate time period (e.g., 12 hours to 3 days after administration), the expression of the biomarker from one or more tissues and / or organs (e.g., liver, spleen, lung, heart, pancreas, kidney, brain, muscle, and / or intestine) can be assessed by, for example, measuring the fluorescence or luminescence of the tissue or organ. In some cases, an LNP formulated with nucleic acid (e.g., mRNA) encoding a biomarker (e.g., luciferase or GFP) that can be detected for expression can be administered to a mammal, and, after an appropriate time period, one or more tissues and / or organs can be harvested from that mammal for flow cytometry analysis to determine whether a specific type of cell within the tissue and / or organ contains the biomarker.

[0055] In some cases, the lipid-containing particles provided herein may be specific (also referred to as “tendency” or “targeting”) to one or more specific organ, tissue, or cell types within an organ or tissue. As used herein, the terms “specific,” “tendency,” or “targeting” in relation to a particular lipid-containing particle (e.g., the lipid-containing LNP provided herein) mean that the lipid-containing particle is more likely to interact with (e.g., deliver cargo to) its specific organ, tissue, and / or cell type, and less likely to interact with other organs, tissues, and / or cell types. In some cases, for example, a “lung-tendency” (or “specific” or “targeting” to the lungs and / or cells therein) lipid-containing particle is more likely to interact with the lungs and cells therein than with other organs and cells. In some cases, lipid-containing particles may have a preference for certain cell types within an organ or tissue. As described in the embodiments herein, for example, the lipid-containing particles provided herein may target the lungs, or the lipid-containing particles provided herein may target the liver. In some cases, lipid-containing particles can target NK cells (e.g., NK cells in lung tissue). In other cases, the organ, tissue, or cell specificity of lipid-containing particles can be conferred by the lipids contained within them. For example, 514O... 6,10 And / or 503O i10 Lipid-containing particles can be lung-oriented, and those including 500O 6,10 402O 8,12 503O 8,10 503O 6,10 205O 6,10 200O i10 402O 4,8 402O 4,8 Lipid-containing particles, or combinations thereof, can be liver-oriented. In some cases, when lipid-containing particles are referred to as "lung-oriented," at least 15% (e.g., at least 20%, at least 25%, or at least 30%) of a detectable signal originating from the lipid-containing particles is present in the lungs or lung tissue, while the remaining percentage of a detectable signal is present in other tissues or organs. In some cases, when lipid-containing particles are referred to as "liver-oriented," at least 15% (e.g., at least 20%, at least 25%, or at least 30%) of a detectable signal originating from the liver or liver tissue is present, while the remaining percentage of a detectable signal is present in other tissues or organs.

[0056] Methods for delivering RNA into cells can be used for research and therapeutic applications, including gene silencing, gene editing, and mRNA-based therapies. As described herein, RNA delivery can be achieved via lipid-containing particles (LNPs), which avoids the problems associated with delivering naked single-stranded RNA (naked single-stranded RNA is susceptible to nuclease degradation, can activate the immune system, and is too large and negatively charged to passively cross the cell membrane). Therefore, this document provides methods for delivering lipid-containing particles (e.g., LNPs) containing one or more therapeutic agents as described herein to mammals in need. In some cases, one or more therapeutic agents may include nucleic acids (e.g., mRNA). The methods may include administering lipid-containing particles (e.g., LNPs) encapsulating a therapeutic agent (e.g., mRNA) to a mammal.

[0057] The methods provided herein can be used to deliver therapeutic agents to subjects (e.g., mammals such as humans, non-human primates, mice, rats, rabbits, dogs, cats, horses, cattle, pigs, or sheep). The methods may include administering to the subject a lipid-containing particle (e.g., LNP) containing the therapeutic agent. The lipid-containing particle (e.g., LNP) may be administered to the subject via any suitable route. For example, the lipid-containing particle (e.g., LNP) may be administered intravenously, intramuscularly, or subcutaneously. The lipid-containing particle (e.g., LNP) may be administered to the subject at a dose of about 0.01 to about 10 mg / kg of mRNA. For example, lipid-containing particles (e.g., LNP) can be present in amounts from about 0.01 to about 0.05 mg / kg, from about 0.05 mg / kg to about 0.1 mg / kg, from about 0.1 mg / kg to about 0.2 mg / kg, from about 0.2 mg / kg to about 0.3 mg / kg, from about 0.3 mg / kg to about 0.5 mg / kg, from about 0.5 mg / kg to about 1 mg / kg, from about 1 mg / kg to about 2 mg / kg, from about 2 mg / kg to about 5 mg / kg. The mRNA may be administered at doses of approximately 0.01 mg / kg, approximately 0.05 mg / kg, approximately 0.1 mg / kg, approximately 0.2 mg / kg, approximately 0.3 mg / kg, approximately 0.4 mg / kg, approximately 0.5 mg / kg, approximately 0.6 mg / kg, approximately 0.7 mg / kg, approximately 0.8 mg / kg, approximately 0.9 mg / kg, approximately 1 mg / kg, approximately 1.5 mg / kg, approximately 2 mg / kg, approximately 2.5 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 7.5 mg / kg, approximately 8 mg / kg, or approximately 9 mg / kg. In some cases, lipid-containing particles (e.g., LNP) may be administered at a dose of approximately 0.5 mg / kg of mRNA.

[0058] As described herein, in some cases, the therapeutic agent contained within the lipid-containing particle (e.g., LNP) used in the methods provided herein may be a nucleic acid (e.g., RNA). The RNA as a therapeutic agent may be mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer. In some cases, RNA encoding a biomarker (e.g., mRNA encoding a luciferase polypeptide or a fluorescent polypeptide such as GFP, a yellow fluorescent polypeptide, or a red fluorescent polypeptide) may be used in the methods described herein. Luciferase is an enzyme that catalyzes a bioluminescent reaction, producing light as a byproduct. This reaction can be used for various biological and medical research applications, particularly for reporter assays to study gene expression and cellular processes. Bioluminescence generated by luciferase encoded by mRNA encapsulated in a lipid-containing particle (e.g., LNP), or fluorescence generated by a fluorescent biomarker encoded by mRNA encapsulated in a lipid-containing particle (e.g., LNP), can be used to assess the efficacy of mRNA delivery.

[0059] In some cases, the methods described herein can be used to treat clinical conditions in subjects (e.g., mammals). As used herein, “treatment” means improvement (e.g., reduction or elimination) of at least one symptom of the condition. Administration of a therapeutically effective amount of the lipid-containing particles described herein can result in more targeted LNP-mediated mRNA delivery. Therefore, the methods described herein provide a way to enhance the effectiveness of RNA therapies.

[0060] Conditions treatable according to the methods provided herein include, but are not limited to, lung conditions, liver conditions, and diseases involving one or more gene or protein disorders. Lung conditions treatable as described herein include, but are not limited to, cystic fibrosis, non-small cell lung cancer, other forms of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinetics, viral lung infections (such as pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, allergic pneumonia, and combinations thereof. For example, lung conditions in mammals can be treated by administering the lung-tendant lipid-containing particles (e.g., containing N...) provided herein. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1The lipid-containing particles (LNPs) are a type of lipid-containing compound that can be treated with a head of 2-butylheptaacrylate, 2-butyloctyl acrylate, 2-butylnonyl acrylate, 2-hexylnonyl acrylate, 2-hexyldecyl acrylate, 2-hexylundecyl acrylate, 2-octylundecyl acrylate, 2-octyldodecyl acrylate, or 2-octyltridecyl acrylate, and optionally also contain neutral auxiliary lipids such as DSPC or DOPE. These lipid-containing particles target cells in the lungs or one or more cell types within the lungs. Liver conditions that can be treated as described herein include, but are not limited to, α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, and combinations thereof. For example, liver diseases in mammals can be treated by administering liver-oriented lipid particles (e.g., containing N-type lipids) as described herein. 1 N 1 '-(ethane-1,2-diyl)bis(N 1 The lipid-containing particles are used for treatment with a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-amine alkylamine head or a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-amine alkylamine head, and one or more lipid-like particles (LNPs) with tails of 2-butylheptyl acrylate, 2-butyloctyl acrylate, 2-butylnonyl acrylate, 2-hexylnonyl acrylate, 2-hexyldecyl acrylate, 2-hexylundecyl acrylate, 2-octylundecyl acrylate, 2-octyldodecyl acrylate, or 2-octyltridecyl acrylate, wherein the lipid-containing particles target cells in the liver, or target one or more types of cells within the liver. In some cases, the clinical condition may be an inflammatory condition, an infectious disease, an autoimmune disease, a respiratory disease, cancer, a genetic disease, a metabolic disease, or any combination thereof.

[0061] Typically, the methods described herein may include administering a therapeutically effective amount of a lipid-containing particle (e.g., LNP) as described herein to a subject who requires or has been identified as requiring such treatment. The lipid-containing particle (e.g., LNP) encapsulates a therapeutic agent intended for the desired treatment. The therapeutic agent may be mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer.

[0062] Effective dosage can vary depending on the severity of the condition, route of administration, age and general health of the subject, excipient use, the possibility of co-administration with other therapeutic treatments, and the judgment of the treating clinician. An effective amount of a composition comprising one or more HMOs and / or lipid-containing particles (e.g., LNPs) as described herein can be any amount that reduces one or more symptoms of the condition in a subject (e.g., a mammal) without causing serious toxicity in mammals. As described herein, the effective dose of lipid-containing particles (e.g., LNPs) can be an mRNA dose of about 0.01 to about 10 mg / kg (e.g., about 0.01 to about 0.05 mg / kg, about 0.05 to about 0.1 mg / kg, about 0.1 to about 0.5 mg / kg, about 0.5 to about 1 mg / kg, about 1 to about 3 mg / kg, about 3 to about 5 mg / kg, about 5 to about 7.5 mg / kg, about 7.5 to about 10 mg / kg, about 0.05, about 0.1, about 0.3, about 0.5, about 0.75, about 1, about 2.5, about 3, about 5, or about 10 mg / kg). The effective dose can be kept constant or adjusted to a sliding scale or variable dose based on the mammalian response to treatment. Various factors can affect the actual effective dose for a particular application. For example, the frequency of administration, duration of treatment, use of multiple therapeutic agents, route of administration, severity of the condition, or risk level of the same or another condition in the treated mammal may require increasing or decreasing the actual effective dose.

[0063] If a particular mammal fails to respond to a specific amount of lipid-containing particles (e.g., LNP), the amount of lipid-containing particles can be increased, for example, by a factor of two. After receiving a higher dose, both the mammal's responsiveness to treatment and symptoms of toxicity can be monitored, and adjustments can be made accordingly. The effective dose can be kept constant or adjusted to a sliding scale or variable dose based on the mammal's response to treatment. Various factors can affect the actual effective dose used for a particular application. For example, the frequency of administration, duration of treatment, route of administration, and severity of symptoms may necessitate increasing or decreasing the actual effective dose administered.

[0064] The frequency at which one or more lipid-containing particles (e.g., LNPs) are administered to a subject (e.g., a mammal) can be any frequency that reduces symptoms of a condition in the subject without causing significant toxicity. For example, the frequency of administration of lipid-containing particles (e.g., LNPs) can be from about four times daily to about once daily, from about once daily to three times weekly, from about three times weekly to about twice weekly, from about twice weekly to about once weekly, or from about once weekly to about once monthly (e.g., from about once weekly to about once every other week or about once every three weeks). The frequency of administration can remain constant or can be variable during the duration of treatment. Treatment with lipid-containing particles (e.g., LNPs) as described herein may include rest periods. For example, LNPs may be administered daily for a period of week, followed by a week of rest, and such regimens may be repeated multiple times. As with effective doses, various factors can influence the actual frequency of administration for a particular application. For example, effective doses, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the condition may necessitate increasing or decreasing the frequency of administration.

[0065] The effective duration of administration of one or more lipid-containing particles (e.g., LNPs) to a subject (e.g., a mammal) can be any duration during which symptoms of a disease are reduced in the mammal without significant toxicity to the mammal. In some cases, the effective duration can vary from days to months to years or longer. Many factors can influence the actual effective duration for a particular treatment. For example, the effective duration can vary with the effective dose, frequency of administration, use of multiple therapeutic agents, route of administration, and severity of the disease being treated.

[0066] In some cases, it may be possible to monitor the progression of a condition in a subject (e.g., a mammal) or the severity of one or more symptoms associated with the condition in a treated subject. Any suitable method may be used to determine whether a subject with a condition is being treated effectively.

[0067] In some cases, the progression of a disease in a mammal or the severity of one or more symptoms associated with the disease in the treated mammal can be monitored. Any suitable method can be used to determine whether the mammal has been effectively treated. For example, clinical scanning techniques (e.g., computed tomography (CT), positron emission tomography (PET) / CT, bone scans, magnetic resonance imaging (MRI)) and / or measurements of one or more biomarkers in biological samples can be used to determine the presence or absence of a disease such as cancer in the treated mammal (e.g., a human). In such cases, a reduction in the number of tumor cells, a decrease in tumor size, or a reduction in tumor markers can indicate effective treatment.

[0068] Exemplary Implementation

[0069] Implementation Scheme 1 is a lipid-containing particle comprising: a lipid; cholesterol or a derivative thereof; an accessory lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipid comprises an alkylamine head and one or more alkyl acrylate tails, wherein each of the one or more alkyl acrylate tails comprises an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch of each tail has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0070] Implementation scheme 2 is a lipid-containing particle of implementation scheme 1, wherein the lipid has two alkyl acrylate tails.

[0071] Implementation scheme 3 is a lipid-containing particle of implementation scheme 1, wherein the lipid has three alkyl acrylate tails.

[0072] Implementation scheme 4 is a lipid-containing particle of implementation scheme 1, wherein the lipid has four alkyl acrylate tails.

[0073] Implementation scheme 5 is a lipid-containing particle of implementation scheme 1, wherein the lipid-like particle has more than four alkyl acrylate tails.

[0074] Implementation scheme 6 is a lipid-containing particle of any of implementation schemes 2-5, wherein each alkyl acrylate tail is identical.

[0075] Implementation scheme 7 is a lipid-containing particle of any one of implementation schemes 2-5, wherein the lipid comprises two or more different alkyl acrylate tails.

[0076] Implementation scheme 8 is a lipid-containing particle of any one of implementation schemes 1-7, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0077] Implementation scheme 9 is a lipid-containing particle of any one of implementation schemes 1-8, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0078] Implementation Scheme 10 is a lipid-containing particle of Implementation Scheme 9, wherein the neutral lipid or zwitterionic lipid includes 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (DPPC), etc. One or more of the following: oleoyl-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterolsphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0079] Implementation scheme 11 is a lipid-containing particle of any one of implementation schemes 1-8, wherein the auxiliary lipid is a cationic lipid.

[0080] Implementation Scheme 12 is a lipid-containing particle of Implementation Scheme 11, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0081] Implementation scheme 13 is a lipid-containing particle of any one of implementation schemes 1-12, wherein the lipid-containing particle is lung-oriented.

[0082] Implementation scheme 14 is a lipid-containing particle of implementation scheme 13, wherein the lipids include: N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail.

[0083] Implementation scheme 15 is a lipid-containing particle of implementation scheme 13, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0084] Implementation scheme 16 is a lipid-containing particle of implementation scheme 15, wherein the lipid-containing particle targets natural killer (NK) and / or dendritic cells.

[0085] Implementation scheme 17 is the lipid-containing particle of implementation scheme 16, wherein the lipid-containing particle comprises about 10-40% neutral auxiliary lipids or zwitterionic auxiliary lipids.

[0086] Implementation Scheme 18 is a lipid-containing particle of Implementation Scheme 17, wherein the neutral or zwitterionic auxiliary lipid includes one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0087] Implementation scheme 19 is a lipid-containing particle of implementation scheme 17, wherein the lipid-containing particle comprises about 16% DOPE.

[0088] Implementation scheme 20 is a lipid-containing particle of any one of implementation schemes 1-12, wherein the lipid-containing particle is liver-oriented.

[0089] Implementation scheme 21 is a lipid-containing particle of implementation scheme 20, wherein the lipids include: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 -methylethane-1,2-diamine)alkylamine head, 4-(2-aminopropan-2-yl)-1-methylcyclohexane-1-amine alkylamino head, 2-(piperazin-1-yl)ethane-1-amine head or N 1-((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail.

[0090] Implementation scheme 22 is a lipid-containing particle of implementation scheme 20, wherein the lipids include N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-Methylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0091] Implementation scheme 23 is a lipid-containing particle of implementation scheme 20, wherein the lipid comprises a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head and a 2-octyldodecyl acrylate tail.

[0092] Implementation scheme 24 is a lipid-containing particle of any one of implementation schemes 1-23, wherein the cholesterol or its derivative is cholesterol.

[0093] Implementation scheme 25 is a lipid-containing particle of any one of implementation schemes 1-24, wherein the PEG-based compound is a PEG-lipid.

[0094] Embodiment 26 is a lipid-containing particle of Embodiment 25, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0095] Embodiment 27 is a composition comprising lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein the one or more alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0096] Implementation scheme 28 is the composition of implementation scheme 27, wherein the therapeutic agent is a nucleic acid.

[0097] Implementation Scheme 29 is a composition of Implementation Scheme 28, wherein the nucleic acid comprises RNA.

[0098] Implementation scheme 30 is a composition of implementation scheme 29, wherein the RNA includes one or more of mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA and RNA aptamers.

[0099] Embodiment 31 is a composition of Embodiment 29 or Embodiment 30, wherein the RNA is an mRNA encoding luciferase.

[0100] Embodiment 32 is a composition of any one of Embodiments 27-31, wherein the lipid has two alkyl acrylate tails.

[0101] Embodiment 33 is a composition of any one of embodiments 27-31, wherein the lipid has three alkyl acrylate tails.

[0102] Embodiment 34 is a composition of any one of embodiments 27-31, wherein the lipid has four alkyl acrylate tails.

[0103] Embodiment 35 is a composition of any one of Embodiments 27-31, wherein the lipid has more than four alkyl acrylate tails.

[0104] Embodiment 36 is a composition of any one of embodiments 32-35, wherein each alkyl acrylate tail is identical.

[0105] Embodiment 37 is a composition of any one of embodiments 32-35, wherein the lipid comprises two or more different alkyl acrylate tails.

[0106] Embodiment 38 is a composition of any one of Embodiments 27-37, wherein the lipid-containing particles are lipid nanoparticles (LNPs).

[0107] Embodiment 39 is a composition of any one of Embodiments 27-38, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0108] Embodiment 40 is a composition of Embodiment 39, wherein the neutral lipid or zwitterionic lipid comprises 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (DPPC), etc. One or more of the following: oleoyl-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterolsphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0109] Embodiment 41 is a composition of any one of Embodiments 27-38, wherein the auxiliary lipid is a cationic lipid.

[0110] Embodiment 42 is a composition of Embodiment 41, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0111] Implementation scheme 43 is a composition of any one of implementation schemes 27-42, wherein the lipid-containing particles are lung-oriented.

[0112] Embodiment 44 is the composition of Embodiment 43, wherein the lipid comprises: N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail.

[0113] Embodiment 45 is the composition of Embodiment 43, wherein the lipids comprise N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0114] Embodiment 46 is the composition of Embodiment 45, wherein the lipid-containing particles target natural killer (NK) and / or dendritic cells.

[0115] Embodiment 47 is the composition of Embodiment 46, wherein the lipid-containing particles comprise about 10-40% neutral or zwitterionic auxiliary lipids.

[0116] Embodiment 48 is a composition of Embodiment 47, wherein the neutral or zwitterionic auxiliary lipid comprises one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0117] Embodiment 49 is the composition of Embodiment 47, wherein the lipid-containing particles comprise about 16% DOPE.

[0118] Embodiment 50 is a composition of any one of Embodiments 27-42, wherein the lipid-containing particles are liver-oriented.

[0119] Embodiment 51 is the composition of Embodiment 50, wherein the lipid comprises: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1-Methylethane-1,2-diamine)alkylamine head or 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail or 2-octyltridecyl acrylate tail.

[0120] Embodiment 52 is the composition of Embodiment 50, wherein the lipids comprise N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-Methylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0121] Embodiment 53 is the composition of Embodiment 50, wherein the lipid comprises a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-amine alkylamino head and a 2-octyldodecyl acrylate tail.

[0122] Implementation scheme 54 is a composition of any one of implementation schemes 27-53, wherein the cholesterol or a derivative thereof is cholesterol.

[0123] Embodiment 55 is a composition of any one of Embodiments 27-54, wherein the PEG-based compound is a PEG-lipid.

[0124] Embodiment 56 is the composition of Embodiment 55, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0125] Embodiment 57 is a method for delivering a therapeutic agent to a mammal, wherein the method includes administering to the mammal a composition comprising lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0126] Implementation scheme 58 is the method of implementation scheme 57, wherein the administration includes intravenous administration, intramuscular administration or subcutaneous administration.

[0127] Implementation scheme 59 is the method of implementation scheme 57 or implementation scheme 58, wherein the lipid-containing particles are lipid nanoparticles (LNP).

[0128] Implementation scheme 60 is a method of any one of implementation schemes 57-59, wherein the therapeutic agent is a nucleic acid.

[0129] Implementation scheme 61 is the method of implementation scheme 60, wherein the nucleic acid includes RNA.

[0130] Implementation scheme 62 is the method of implementation scheme 61, wherein the RNA includes mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA or RNA aptamer.

[0131] Embodiment 63 is a method of Embodiment 61 or Embodiment 62, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0132] Implementation scheme 64 is the method of implementation scheme 61 or implementation scheme 62, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0133] Implementation scheme 65 is a method of any one of implementation schemes 57-64, wherein the lipid has two alkyl acrylate tails.

[0134] Implementation scheme 66 is a method of any one of implementation schemes 57-64, wherein the lipid has three alkyl acrylate tails.

[0135] Implementation scheme 67 is a method of any one of implementation schemes 57-64, wherein the lipid has four alkyl acrylate tails.

[0136] Implementation scheme 68 is a method of any one of implementation schemes 57-64, wherein the lipid has more than four alkyl acrylate tails.

[0137] Implementation scheme 69 is the method of any one of implementation schemes 65-68, wherein each alkyl acrylate tail is identical.

[0138] Implementation scheme 70 is a method of any one of implementation schemes 65-68, wherein the lipid comprises two or more different alkyl acrylate tails.

[0139] Implementation scheme 71 is a method of any one of implementation schemes 57-70, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0140] Implementation Scheme 72 is the method of Implementation Scheme 71, wherein the neutral lipid or zwitterionic lipid comprises 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1-palmitoyl-2-oleoyl-sn-glycerol One or more of the following: 3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0141] Implementation scheme 73 is a method of any one of implementation schemes 57-72, wherein the auxiliary lipid is a cationic lipid.

[0142] Implementation Scheme 74 is the method of Implementation Scheme 73, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0143] Implementation scheme 75 is a method of any one of implementation schemes 57-74, wherein the cholesterol or a derivative thereof is cholesterol.

[0144] Implementation scheme 76 is a method of any one of implementation schemes 57-75, wherein the PEG-based compound is a PEG-lipid.

[0145] Implementation scheme 77 is the method of implementation scheme 76, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0146] Implementation scheme 78 is a method of any one of implementation schemes 57-77, wherein the mammal has been identified as having a lung disease, and wherein the lipid-containing particles are lung-oriented.

[0147] Implementation scheme 79 is the method of implementation scheme 78, wherein the lung condition includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0148] Implementation scheme 80 is the method of implementation scheme 78 or 79, wherein the lipids comprise: N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail.

[0149] Implementation scheme 81 is the method of implementation scheme 80, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0150] Implementation scheme 82 is the method of implementation scheme 80, wherein the lipid-containing particles target natural killer (NK) and / or dendritic cells.

[0151] Implementation scheme 83 is the method of implementation scheme 82, wherein the lipid-containing particles comprise about 10-40% neutral auxiliary lipids or zwitterionic auxiliary lipids.

[0152] Implementation scheme 84 is the method of implementation scheme 83, wherein the neutral assist lipid or zwitterionic assist lipid includes one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols and ceramides.

[0153] Implementation scheme 85 is the method of implementation scheme 83, wherein the lipid-containing particles comprise about 16% DOPE.

[0154] Implementation scheme 86 is a method of any one of implementation schemes 57-77, wherein the mammal has been identified as having a liver disease, and wherein the lipid-containing particles are liver-oriented.

[0155] Implementation scheme 87 is the method of implementation scheme 86, wherein the liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0156] Implementation scheme 88 is the method of implementation scheme 86 or implementation scheme 87, wherein the lipids comprise: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 -Methylethane-1,2-diamine)alkylamine head or 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail or 2-octyltridecyl acrylate tail.

[0157] Implementation scheme 89 is the method of implementation scheme 88, wherein the lipids include N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-Methylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0158] Implementation scheme 90 is the method of implementation scheme 88, wherein the lipid comprises a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head and a 2-octyldodecyl acrylate tail.

[0159] Implementation scheme 91 is a method of any one of implementation schemes 57-90, wherein the mammal is a human.

[0160] Implementation scheme 92 is a method for treating a mammal suffering from a lung disease or its symptoms, or a liver disease or its symptoms, wherein the method includes administering to the mammal a composition comprising lipid-containing particles, the lipid-containing particles comprising a therapeutic agent, wherein the lipid-containing particles further comprise: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0161] Implementation scheme 93 is the method of implementation scheme 92, wherein the administration includes intravenous administration, intramuscular administration or subcutaneous administration.

[0162] Implementation scheme 94 is the method of implementation scheme 92 or implementation scheme 93, wherein the lipid-containing particles are lipid nanoparticles (LNP).

[0163] Implementation scheme 95 is a method of any one of implementation schemes 92-94, wherein the therapeutic agent is a nucleic acid.

[0164] Implementation scheme 96 is the method of implementation scheme 95, wherein the nucleic acid includes RNA.

[0165] Implementation scheme 97 is the method of implementation scheme 96, wherein the RNA includes mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA or RNA aptamer.

[0166] Implementation scheme 98 is a method of implementation scheme 96 or implementation scheme 97, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0167] Implementation scheme 99 is the method of implementation scheme 96 or implementation scheme 97, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0168] Implementation scheme 100 is a method of any one of implementation schemes 92-99, wherein the lipid has two alkyl acrylate tails.

[0169] Implementation scheme 101 is a method of any one of implementation schemes 92-99, wherein the lipid has three alkyl acrylate tails.

[0170] Implementation scheme 102 is a method of any one of implementation schemes 92-99, wherein the lipid has four alkyl acrylate tails.

[0171] Implementation scheme 103 is a method of any one of implementation schemes 99-99, wherein the lipid has more than four alkyl acrylate tails.

[0172] Implementation scheme 104 is the method of any one of implementation schemes 100-103, wherein each alkyl acrylate tail is identical.

[0173] Implementation scheme 105 is a method of any one of implementation schemes 100-103, wherein the lipid comprises two or more different alkyl acrylate tails.

[0174] Implementation scheme 106 is a method of any one of implementation schemes 92-105, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0175] Implementation scheme 107 is the method of implementation scheme 106, wherein the neutral lipid or zwitterionic lipid includes 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (DPPC), etc. One or more of the following: oleoyl-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterolsphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0176] Implementation scheme 108 is a method of any one of implementation schemes 92-105, wherein the auxiliary lipid is a cationic lipid.

[0177] Implementation scheme 109 is the method of implementation scheme 108, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0178] Implementation scheme 110 is a method of any one of implementation schemes 92-109, wherein the cholesterol or a derivative thereof is cholesterol.

[0179] Implementation scheme 111 is a method of any one of implementation schemes 92-110, wherein the PEG-based compound is a PEG-lipid.

[0180] Implementation scheme 112 is the method of implementation scheme 111, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0181] Implementation scheme 113 is a method of any one of implementation schemes 92-112, wherein the mammal has been identified as having a lung disease, and wherein the lipid-containing particles are lung-oriented.

[0182] Implementation scheme 114 is the method of implementation scheme 113, wherein the lung condition includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0183] Implementation scheme 115 is the method of implementation scheme 113 or implementation scheme 114, wherein the lipids comprise: N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1(-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail.

[0184] Implementation scheme 116 is the method of implementation scheme 115, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0185] Implementation scheme 117 is the method of implementation scheme 115, wherein the lipid-containing particles target natural killer (NK) and / or dendritic cells.

[0186] Implementation scheme 118 is the method of implementation scheme 117, wherein the lipid-containing particles comprise about 10-40% neutral assist lipids or zwitterionic assist lipids.

[0187] Implementation scheme 119 is the method of implementation scheme 118, wherein the neutral assist lipid or zwitterionic assist lipid includes one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols and ceramides.

[0188] Implementation scheme 120 is the method of implementation scheme 118, wherein the lipid-containing particles comprise about 16% DOPE.

[0189] Implementation scheme 121 is a method of any one of implementation schemes 92-112, wherein the mammal has been identified as having a liver disease, and wherein the lipid-containing particles are liver-oriented.

[0190] Implementation scheme 122 is the method of implementation scheme 121, wherein the liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0191] Implementation scheme 123 is a method of implementation scheme 121 or implementation scheme 122, wherein the lipids include: N 1 N1 '-(ethane-1,2-diyl)bis(N 1 -Methylethane-1,2-diamine)alkylamine head or 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail or 2-octyltridecyl acrylate tail.

[0192] Implementation scheme 124 is the method of implementation scheme 123, wherein the lipids include N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-Methylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0193] Implementation scheme 125 is the method of implementation scheme 123, wherein the lipid comprises a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head and a 2-octyldodecyl acrylate tail.

[0194] Implementation scheme 126 is a method of any one of implementation schemes 92-125, wherein the mammal is a human.

[0195] Embodiment 127 is a method for delivering a therapeutic agent to lung cells, wherein the method includes contacting the lung cells with a composition comprising lipid-containing particles, the lipid-containing particles comprising the therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having a first branch and a second branch at a second carbon, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0196] Implementation scheme 128 is the method of implementation scheme 127, wherein the lipid-containing particles are lipid nanoparticles (LNP).

[0197] Implementation scheme 129 is the method of implementation scheme 127 or implementation scheme 128, wherein the lipid has two alkyl acrylate tails.

[0198] Implementation scheme 130 is the method of implementation scheme 127 or implementation scheme 128, wherein the lipid has three alkyl acrylate tails.

[0199] Implementation scheme 131 is the method of implementation scheme 127 or implementation scheme 128, wherein the lipid has four alkyl acrylate tails.

[0200] Implementation scheme 132 is the method of implementation scheme 127 or implementation scheme 128, wherein the lipid has more than four alkyl acrylate tails.

[0201] Implementation scheme 133 is the method of any one of implementation schemes 129-132, wherein each alkyl acrylate tail is identical.

[0202] Implementation scheme 134 is a method of any of implementation schemes 129-132, wherein the lipid comprises two or more different alkyl acrylate tails.

[0203] Implementation scheme 135 is a method of any one of implementation schemes 127-134, wherein the auxiliary lipid is a neutral lipid or a zwitterionic auxiliary lipid.

[0204] Implementation scheme 136 is the method of implementation scheme 135, wherein the neutral lipid or zwitterionic auxiliary lipid includes 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn- One or more of the following: glycerol-3-phosphate choline (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0205] Implementation scheme 137 is a method of any one of implementation schemes 127-134, wherein the auxiliary lipid is a cationic lipid.

[0206] Implementation scheme 138 is the method of implementation scheme 137, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0207] Implementation scheme 139 is a method of any one of implementation schemes 127-138, wherein the cholesterol or a derivative thereof is cholesterol.

[0208] Implementation scheme 140 is a method of any of implementation schemes 127-139, wherein the PEG-based compound is a PEG-lipid.

[0209] Implementation scheme 141 is the method of implementation scheme 140, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0210] Implementation scheme 142 is a method of any one of implementation schemes 127-141, wherein the lung cells are in a mammal.

[0211] Implementation scheme 143 is the method of implementation scheme 142, wherein the mammal is a human.

[0212] Implementation scheme 144 is a method of any one of implementation schemes 127-143, wherein the therapeutic agent is a nucleic acid.

[0213] Implementation scheme 145 is the method of implementation scheme 144, wherein the nucleic acid includes RNA.

[0214] Implementation scheme 146 is the method of implementation scheme 145, wherein the RNA includes mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA or RNA aptamer.

[0215] Implementation scheme 147 is a method of implementation scheme 145 or implementation scheme 146, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0216] Implementation scheme 148 is a method of implementation scheme 145 or implementation scheme 146, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0217] Implementation scheme 149 is a method of any one of implementation schemes 127-148, wherein the administration includes intravenous administration, intramuscular administration or subcutaneous administration.

[0218] Implementation scheme 150 is a method of any of implementation schemes 127-149, wherein the lung cells are in a mammal identified as having a lung disease.

[0219] Implementation scheme 151 is the method of implementation scheme 150, wherein the lung condition includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0220] Implementation scheme 152 is a method of implementation scheme 150 or implementation scheme 151, wherein the lipids comprise: N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-butylheptyl acrylate tail, 2-butyloctyl acrylate tail, 2-butylnonyl acrylate tail, 2-hexylnonyl acrylate tail, 2-hexyldecyl acrylate tail, 2-hexylundecyl acrylate tail, 2-octylundecyl acrylate tail, 2-octyldodecyl acrylate tail, or 2-octyltridecyl acrylate tail.

[0221] Implementation scheme 153 is the method of implementation scheme 152, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

[0222] Implementation scheme 154 is the method of implementation scheme 152, wherein the lipid-containing particles target natural killer (NK) and / or dendritic cells.

[0223] Implementation scheme 155 is the method of implementation scheme 154, wherein the lipid-containing particles comprise about 10-40% neutral assist lipids or zwitterionic assist lipids.

[0224] Implementation scheme 156 is the method of implementation scheme 155, wherein the neutral assist lipid or zwitterionic assist lipid includes one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols and ceramides.

[0225] Implementation scheme 157 is the method of implementation scheme 155, wherein the lipid-containing particles comprise about 16% DOPE.

[0226] Implementation scheme 158 is a lipid-containing particle comprising: a lipid; cholesterol or a derivative thereof; a cofactor lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipid comprises an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N- 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head, and wherein the one or more alkyl acrylate tails include one or more of 2-octyldecyl acrylate tails and 9-methyldecyl acrylate tails.

[0227] Implementation scheme 159 is the lipid-containing particle of implementation scheme 158, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0228] Implementation scheme 160 is a lipid-containing particle of implementation scheme 158, wherein the lipid has two alkyl acrylate tails.

[0229] Implementation scheme 161 is a lipid-containing particle of implementation scheme 158, wherein the lipid has three alkyl acrylate tails.

[0230] Implementation scheme 162 is a lipid-containing particle of implementation scheme 158, wherein the lipid has four alkyl acrylate tails.

[0231] Implementation scheme 163 is a lipid-containing particle of implementation scheme 158, wherein the lipid has more than four alkyl acrylate tails.

[0232] Implementation scheme 164 is a lipid-containing particle of any of implementation schemes 160-163, wherein each alkyl acrylate tail is identical.

[0233] Implementation scheme 165 is a lipid-containing particle of any one of implementation schemes 160-163, wherein the lipid comprises two or more different alkyl acrylate tails.

[0234] Implementation scheme 166 is a lipid-containing particle of any one of implementation schemes 158-165, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0235] Implementation scheme 167 is a lipid-containing particle of implementation scheme 166, wherein the neutral lipid or zwitterionic lipid includes 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn One or more of the following: glycerol-3-phosphate choline (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0236] Implementation scheme 168 is a lipid-containing particle of any one of implementation schemes 158-165, wherein the auxiliary lipid is a cationic lipid.

[0237] Implementation Scheme 169 is a lipid-containing particle of Implementation Scheme 168, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0238] Implementation scheme 170 is a lipid-containing particle of any one of implementation schemes 158-169, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine) alkylamine head and 2-octyldecyl acrylate tail.

[0239] Implementation scheme 171 is a lipid-containing particle of any one of implementation schemes 158-169, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine) alkylamine head and 9-methyldecyl acrylate tail.

[0240] Implementation scheme 172 is a lipid-containing particle of any one of implementation schemes 158-169, wherein the lipids include N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail.

[0241] Implementation scheme 173 is a lipid-containing particle of any one of implementation schemes 158-172, wherein the cholesterol or its derivative is cholesterol.

[0242] Implementation scheme 174 is a lipid-containing particle of any one of implementation schemes 158-173, wherein the PEG-based compound is a PEG-lipid.

[0243] Embodiment 175 is a lipid-containing particle of Embodiment 174, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0244] Embodiment 176 is a composition comprising lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N-terminated. 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head, and wherein the one or more alkyl acrylate tails include one or more of 2-octyldecyl acrylate tails and 9-methyldecyl acrylate tails.

[0245] Implementation scheme 177 is the composition of implementation scheme 176, wherein the therapeutic agent is a nucleic acid.

[0246] Implementation scheme 178 is a composition of implementation scheme 177, wherein the nucleic acid comprises RNA.

[0247] Implementation scheme 179 is a composition of implementation scheme 178, wherein the RNA includes one or more of mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA and RNA aptamers.

[0248] Embodiment 180 is a composition of Embodiment 178 or Embodiment 179, wherein the RNA is mRNA encoding luciferase.

[0249] Embodiment 181 is a composition of any one of Embodiments 176-180, wherein the lipid has two alkyl acrylate tails.

[0250] Embodiment 182 is a composition of any one of Embodiments 176-180, wherein the lipid has three alkyl acrylate tails.

[0251] Embodiment 183 is a composition of any one of embodiments 176-180, wherein the lipid has four alkyl acrylate tails.

[0252] Embodiment 184 is a composition of any one of Embodiments 176-180, wherein the lipid has more than four alkyl acrylate tails.

[0253] Embodiment 185 is a composition of any one of Embodiments 181-184, wherein each alkyl acrylate tail is identical.

[0254] Embodiment 186 is a composition of any one of embodiments 181-184, wherein the lipid comprises two or more distinct alkyl acrylate tails.

[0255] Embodiment 187 is a composition of any one of embodiments 176-186, wherein the lipid-containing particles are lipid nanoparticles (LNPs).

[0256] Embodiment 188 is a composition of any one of Embodiments 176-187, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0257] Embodiment 189 is a composition of Embodiment 188, wherein the neutral lipid or zwitterionic lipid comprises 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn- One or more of the following: glycerol-3-phosphate choline (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0258] Embodiment 190 is a composition of any one of Embodiments 176-187, wherein the auxiliary lipid is a cationic lipid.

[0259] Embodiment 191 is a composition of Embodiment 41, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0260] Embodiment 192 is a composition of any one of embodiments 176-191, wherein the lipids comprise N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine) alkylamine head and 2-octyldecyl acrylate tail.

[0261] Embodiment 193 is a composition of any one of embodiments 176-191, wherein the lipids comprise N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine) alkylamine head and 9-methyldecyl acrylate tail.

[0262] Embodiment 194 is a composition of any one of embodiments 176-191, wherein the lipids comprise N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail.

[0263] Implementation scheme 195 is a composition of any one of implementation schemes 176-194, wherein the cholesterol or a derivative thereof is cholesterol.

[0264] Embodiment 196 is a composition of any one of Embodiments 176-195, wherein the PEG-based compound is a PEG-lipid.

[0265] Embodiment 197 is the composition of Embodiment 196, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0266] Implementation Scheme 198 is a method for delivering a therapeutic agent to a mammal, wherein the method includes administering to the mammal a composition comprising lipid-containing particles, wherein the lipid-containing particles comprise a therapeutic agent and the following: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head, and wherein the one or more alkyl acrylate tails include one or more of 2-octyldecyl acrylate tails and 9-methyldecyl acrylate tails.

[0267] Implementation scheme 199 is the method of implementation scheme 198, wherein the administration includes intravenous administration, intramuscular administration or subcutaneous administration.

[0268] Implementation scheme 200 is the method of implementation scheme 198 or implementation scheme 199, wherein the lipid-containing particles are lipid nanoparticles (LNP).

[0269] Implementation scheme 201 is a method of any one of implementation schemes 198-200, wherein the therapeutic agent is a nucleic acid.

[0270] Implementation scheme 202 is the method of implementation scheme 201, wherein the nucleic acid includes RNA.

[0271] Implementation scheme 203 is the method of implementation scheme 202, wherein the RNA includes mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA or RNA aptamer.

[0272] Implementation scheme 204 is a method of implementation scheme 202 or implementation scheme 203, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0273] Implementation scheme 205 is a method of implementation scheme 202 or implementation scheme 203, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0274] Implementation scheme 206 is a method of any one of implementation schemes 198-205, wherein the lipid has two alkyl acrylate tails.

[0275] Implementation scheme 207 is a method of any one of implementation schemes 198-205, wherein the lipid has three alkyl acrylate tails.

[0276] Implementation scheme 208 is a method of any one of implementation schemes 198-205, wherein the lipid has four alkyl acrylate tails.

[0277] Implementation scheme 209 is a method of any one of implementation schemes 198-205, wherein the lipid has more than four alkyl acrylate tails.

[0278] Implementation scheme 210 is the method of any one of implementation schemes 206-209, wherein each alkyl acrylate tail is identical.

[0279] Implementation scheme 211 is a method of any of implementation schemes 206-209, wherein the lipid comprises two or more different alkyl acrylate tails.

[0280] Implementation scheme 212 is a method of any one of implementation schemes 198-211, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0281] Implementation scheme 213 is the method of implementation scheme 212, wherein the neutral lipid or zwitterionic lipid includes 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (DPPC), etc. One or more of the following: oleoyl-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterolsphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0282] Implementation scheme 214 is a method of any one of implementation schemes 198-211, wherein the auxiliary lipid is a cationic lipid.

[0283] Implementation scheme 215 is the method of implementation scheme 214, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0284] Implementation scheme 216 is a method of any one of implementation schemes 198-215, wherein the cholesterol or a derivative thereof is cholesterol.

[0285] Implementation scheme 217 is a method of any one of implementation schemes 198-216, wherein the PEG-based compound is a PEG-lipid.

[0286] Implementation scheme 218 is the method of implementation scheme 217, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0287] Implementation scheme 219 is the method of any one of implementation schemes 198-218, wherein the mammal has been identified as having a lung disease.

[0288] Implementation scheme 220 is the method of implementation scheme 219, wherein the lung condition includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0289] Implementation scheme 221 is a method of any of implementation schemes 198-218, wherein the mammal has been identified as having a liver disease.

[0290] Implementation scheme 222 is the method of implementation scheme 221, wherein the liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0291] Implementation scheme 223 is a method of any one of implementation schemes 198-222, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine) alkylamine head and 2-octyldecyl acrylate tail.

[0292] Implementation scheme 224 is a method of any one of implementation schemes 198-222, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine) alkylamine head and 9-methyldecyl acrylate tail.

[0293] Implementation scheme 225 is a method of any one of implementation schemes 198-222, wherein the lipids include N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail.

[0294] Implementation scheme 226 is the method of any one of implementation schemes 198-225, wherein the mammal is a human.

[0295] Implementation Scheme 227 is a method for treating a mammal suffering from a lung disease or its symptoms, or a liver disease or its symptoms, wherein the method includes administering to the mammal a composition comprising lipid-containing particles, the lipid-containing particles comprising a therapeutic agent, wherein the lipid-containing particles further comprise: lipids; cholesterol or a derivative thereof; cofactor lipids; and a polyethylene glycol (PEG)-based compound, wherein the lipids comprise an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head, and wherein the one or more alkyl acrylate tails include one or more of 2-octyldecyl acrylate tails and 9-methyldecyl acrylate tails.

[0296] Implementation scheme 228 is the method of implementation scheme 227, wherein the administration includes intravenous administration, intramuscular administration or subcutaneous administration.

[0297] Implementation scheme 229 is the method of implementation scheme 227 or implementation scheme 228, wherein the lipid-containing particles are lipid nanoparticles (LNP).

[0298] Implementation scheme 230 is a method of any one of implementation schemes 227-229, wherein the therapeutic agent is a nucleic acid.

[0299] Implementation scheme 231 is the method of implementation scheme 230, wherein the nucleic acid includes RNA.

[0300] Implementation scheme 232 is the method of implementation scheme 231, wherein the RNA includes mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA or RNA aptamer.

[0301] Implementation scheme 233 is a method of implementation scheme 231 or implementation scheme 232, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0302] Implementation scheme 234 is a method of implementation scheme 231 or implementation scheme 232, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0303] Implementation scheme 235 is a method of any one of implementation schemes 227-234, wherein the lipid has two alkyl acrylate tails.

[0304] Implementation scheme 236 is a method of any one of implementation schemes 227-234, wherein the lipid has three alkyl acrylate tails.

[0305] Implementation scheme 237 is a method of any one of implementation schemes 227-234, wherein the lipid has four alkyl acrylate tails.

[0306] Implementation scheme 238 is a method of any one of implementation schemes 227-234, wherein the lipid has more than four alkyl acrylate tails.

[0307] Implementation scheme 239 is the method of any one of implementation schemes 235-238, wherein each alkyl acrylate tail is identical.

[0308] Implementation scheme 240 is a method of any of implementation schemes 235-238, wherein the lipid comprises two or more different alkyl acrylate tails.

[0309] Implementation scheme 241 is a method of any one of implementation schemes 227-240, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid.

[0310] Implementation scheme 242 is the method of implementation scheme 241, wherein the neutral lipid or zwitterionic lipid includes 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (DPPC), etc. One or more of the following: oleoyl-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-diapalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterolsphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

[0311] Implementation scheme 243 is a method of any one of implementation schemes 227-240, wherein the auxiliary lipid is a cationic lipid.

[0312] Implementation scheme 244 is the method of implementation scheme 243, wherein the cationic lipid comprises one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), 1,2-distearate-sn-glycerol-3-trimethylammonium-propane (DSTAP), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxy-propyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0313] Implementation scheme 245 is a method of any one of implementation schemes 227-244, wherein the cholesterol or a derivative thereof is cholesterol.

[0314] Implementation scheme 246 is a method of any of implementation schemes 227-245, wherein the PEG-based compound is a PEG-lipid.

[0315] Implementation scheme 247 is the method of implementation scheme 246, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0316] Implementation scheme 248 is the method of any one of implementation schemes 227-247, wherein the lung condition includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, α-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0317] Implementation scheme 249 is the method of any one of implementation schemes 227-247, wherein the liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0318] Implementation scheme 250 is a method of any one of implementation schemes 227-249, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N1 (-methylethane-1,2-diamine) alkylamine head and 2-octyldecyl acrylate tail.

[0319] Implementation scheme 251 is a method of any one of implementation schemes 227-249, wherein the lipids include N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine) alkylamine head and 9-methyldecyl acrylate tail.

[0320] Implementation scheme 252 is a method of any one of implementation schemes 227-249, wherein the lipids include N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail.

[0321] Implementation scheme 253 is the method of any one of implementation schemes 227-252, wherein the mammal is a human.

[0322] The invention will be further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0323] Example

[0324] Methods and Materials

[0325] Lipid library synthesis: Various ionizable lipids were synthesized by combining alkylamine heads with alkyl acrylate tails. Figure 1A A stoichiometric ratio of 1:4 was used. The reagents were combined in scintillation flasks and kept at 90°C for 3 days with stirring (see, Hajj et al., Small 2019, 15(6):e1805097; and Strelkova Petersen et al., Eur.J. Pharm. Biopharm. 2023, 192:126). Twenty-nine (29) amine heads (80 to 602) were each combined with one of 20 acrylate tails to produce a library of 580 ionizable lipid members. For detailed structures and sources of the amine heads and acrylate tails, see [reference needed]. Figure 1B-1C And Tables 1 and 2.

[0326] Table 1: List of amine heads used in the synthesis of ionizable lipid libraries

[0327]

[0328]

[0329] Table 2: List of acrylate tails used in the synthesis of ionizable lipid libraries

[0330]

[0331] LNP formulation: LNPs are formulated, as described elsewhere (Hajj et al. 2020, ibid.; and Strelkova Petersen et al., ibid.) by combining a lipid solution with an aqueous solution of mRNA. The lipid solution consists of ionizable lipids (prepared in the laboratory), cholesterol (Sigma-Aldrich, St Louis, MO), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine) from Avanti PolarLipids, Alabaster, AL, and PEG2000 (14:0 PEG2000 PE) from AvantiPolarLipids. These four compounds are combined in a molar ratio of 35:46.5:16:2.5 (lipids:cholesterol:coenzyme:PEG) for the 16PE formulation, or in a molar ratio of 35:22.5:40:2.5 for the 40PE and 40TAP formulations. The lipids were then dissolved in an ethanol / citrate buffer solution (9:1 ethanol and citrate buffer v / v). An aqueous solution containing 5-methoxyuridine (5 molU) base-modified mRNA dissolved in 10 mL citrate buffer (pH 4) was prepared, ensuring a lipid-to-mRNA ratio of 10:1 w / w. LNPs were prepared at a concentration of 0.09 mg / mL mRNA. To form LNPs, the lipid solution was added to the mRNA solution at a 1:1 volume ratio using a pipette. Immediately after addition, the LNPs were vortexed at 3200 rpm for 5 seconds. The LNPs used in animal experiments were then dialyzed for 1 hour in a dialysis cassette with a 3.5 kDa molecular weight cutoff membrane (Thermo Fisher, Waltham, MA) relative to phosphate-buffered saline (PBS, VWR, Randor, PA).

[0332] Encapsulation: To measure RNA encapsulation, the RNA content of intact and lysed nanoparticles was measured using the Quant-iT RiboGreen RNA Assay Kit (ThermoFisher) according to the manufacturer's instructions. Lipid nanoparticles were generated at a concentration of 0.005 mg / mL (firefly luciferase) and then diluted tenfold. Briefly, LNP was diluted in an equal volume of Tris-EDTA buffer or 2% Triton X-100 in DI aqueous buffer, and an equal volume of RiboGreen reagent was added to each sample, followed by incubation at 37°C for 15 min. Fluorescence was read on a Tecan Spark Multimode microplate reader. The excitation signal was 480 nm and the emission signal was 520 nm.

[0333] Zeta potential: The zeta potential was determined using a Malvern Zetasizer Nano ZSP (Malvern, UK). Lipid nanoparticles were diluted 1:100 from 0.005 mg / mL for testing. Three technical replicates were performed for each sample to measure the surface zeta potential.

[0334] Nanoparticle tracking analysis: LNP size was analyzed using a NanoSight LM10 system (NanoSight, Ltd., Amesbury, UK) equipped with a 405nm laser and a high-sensitivity digital camera system (OrcaFlash2.8, Hamamatsu C11440; NanoSight, Ltd., Malvern, UK). The camera shutter speed was fixed at 30.01ms, and the camera gain was set to 500. Video was collected and analyzed using NTA software (version 2.3), with the minimum expected particle size, minimum trajectory length, and blur settings all set to automatic.

[0335] TNS assay: LNP ionization at pH 5.0 was assessed using a 2-(p-toluidine)naphthalene-6-sulfonic acid (TNS) fluorescent probe (Sigma-Aldrich, St. Louis, MO), following the protocol described elsewhere (Hajj et al., 2019, ibid.). Each well of a black 96-well plate received 250 μL of TNS buffer (containing 150 mm sodium chloride, 20 mm sodium phosphate, 20 mm ammonium acetate, and 25 mm ammonium citrate; pH adjusted to 5.0 after mixing). Subsequently, 5 μL of LNP (0.09 mg / mL mRNA concentration) and 10 μL of 0.16 mm TNS stock solution were added to each well. The resulting fluorescence signal was rapidly measured using a Synergy H1 plate reader (BioTek, Winooski, VT).

[0336] In vitro LNP efficacy: HeLa cells were seeded at a concentration of 15,000 cells per well in white 96-well plates (Greiner Bio-One, Monroe, NC). LNPs containing 0.09 mg / mL mRNA encoding firefly luciferase mRNA (TriLink BioTechnologies, San Diego, CA) were prepared and diluted with PBS to a concentration of 0.005 mg / mL mRNA. Twenty (20) μL of LNP was added to each well. Twenty-four (24) hours after LNP addition to the cells, Dulbecco modified Eagle medium (DMEM) was aspirated from the wells, and 50 μL of a PBS / BrightGlo reagent mixture (Promega, Madison, WI) (1:1 v:v) was added to each well. The plates were incubated at room temperature in the dark for 5 minutes, and the luminescence was detected using a Spark plate reader (TECAN, Switzerland). Each LNP was tested in quadruple biological replicates.

[0337] Animal testing: All animal testing was conducted in accordance with all state and local regulations in the United States.

[0338] LNP efficacy in mouse organs: LNP efficacy in mice was measured using a firefly luciferase D-luciferin reporter system. The prepared LNP contained firefly luciferase mRNA at a total mRNA concentration of 0.09 mg / mL (TriLink Biotechnologies). Six- to eight-week-old (6- to 8-week-old) female C57BL / 6 mice (Charles River Laboratories, Wilmington, MA) were injected with LNP via the tail vein at a total mRNA dose of 0.75 mg / kg. Three hours after LNP injection, 130 μL of 30 mg / mL D-luciferin (PerkinElmer, Waltham, MA) was injected intraperitoneally (IP), and the mice were euthanized 15 minutes later using CO2 gas and cervical dislocation. Target organs (liver, spleen, lung, heart, pancreas, and kidney) were transferred to black construction paper, and their luminescence signals were measured using an IVIS imaging device (PerkinElmer).

[0339] LNP efficacy in lung cell types: GFP was used to assess which lung cell types were transfected. For these experiments, LNPs were formulated with GFP mRNA (TriLink Biotechnologies) at a total mRNA concentration of 0.09 mg / mL. LNPs were administered via tail vein to 6-8 week old female C57BL / 6 mice (Charles River Laboratories). Mice received a total dose of 0.75 mg mRNA / kg. Mice were euthanized 24 hours after LNP injection using CO2 gas and cervical dislocation. Lungs were harvested; the left lobe was placed in 4°C DMEM for flow cytometry, and the right lobe was fixed in 4% formaldehyde solution for histology.

[0340] Flow cytometry: The harvested lungs were washed in DMEM and placed in 1.27 mL of enzyme-PBS solution (1000 U / mL type 1 collagenase and 16 U / mL DNase in Ca). 2+ and Mg 2+The cells were incubated in the enzyme solution at 37°C and 1200 rpm for 25 minutes on a heated shaker (VWR, International). The cells were then extruded through an 18G needle (BD Biosciences, Franklin Lakes, NJ) and returned to the shaker for an additional 25 minutes of incubation. The resulting suspension was vigorously mixed with a pipette and transferred through a 70 μm cell filter (Avantor, VWR, International) to a 50 mL tube (Corning, Falcon, Corning, NY). The cell filter contents were then washed with an additional 1 mL of PBS. The cells were centrifuged at 300 g with gentle acceleration and deceleration for 5 minutes and resuspended in 3 mL of R&D System's erythrocyte lysis buffer. After incubating in the dark at room temperature for 5 minutes, 15 mL of PBS was added to the RBC lysis buffer cell suspension, and the cells were centrifuged at 300 g with gentle acceleration and deceleration for 5 minutes. The cell pellet was then resuspended in 2 mL of PBS, and the resulting suspension was separated into two tubes. Stain the contents of one tube with a fixative yellow viability dye (Thermo Fisher Scientific) according to the manufacturer's instructions. Centrifuge both tubes at 300g with gentle acceleration and deceleration for 5 minutes, and resuspend the cells in 1 mL of 4% formaldehyde solution. After incubating at room temperature for 10 minutes, add 5 mL of flow cytometry buffer (5% FBS in PBS) and centrifuge the cells at 400g with regular acceleration and deceleration for 5 minutes. Wash the cells twice with 5 mL of flow cytometry buffer. After the final wash, resuspend the cells in 1 mL of flow cytometry buffer and transfer the sample to a 96-well plate (200 μL per well for fully stained samples and 50 μL per well for single-stained controls), and centrifuge at 400g for 5 minutes. Then resuspend the cells in a suitable antibody staining solution, where the resulting concentration of each antibody is equal to 1 μg of antibody per 1 mL of permeabilization buffer (R&D Systems). For a complete list of antibodies and staining kits, see Table 3. After incubating the samples at 4°C in the dark for 45 minutes, 100 μL of permeation buffer was added, and the cells were centrifuged at 400 g for 5 minutes. The samples were then washed once with permeation buffer, followed by once with flow cytometry buffer, and finally resuspended in 100 μL of flow cytometry buffer per sample. The samples were analyzed using a Nova-Cyte 3000 flow cytometer (ACEA Biosciences, Inc., San Diego, CA) with gating from Figure 8 and NovaExpress software, using internal automatic compensation based on a single staining control.

[0341] Lung histology: Histology was performed as described elsewhere (LoPresti et al., ibid.). Specifically, the right lung lobe was incubated in 50 mL of 4% formaldehyde PBS solution for 48 hours, washed in PBS, and then transferred to 30% sucrose for an additional 48 hours. The fixed tissue was cryoprotected in 30% sucrose. After cryoprotection, the tissue was embedded in an OCT (optimal cutting temperature) compound from Tissue-Tek, and 25 μm sections were prepared using a cryostat. The sections were permeabilized in 0.1% Triton X 100 solution (Millipore-Sigma) at 25 °C for 10 min. Nonspecific binding was minimized by blocking with 10% goat serum at 25 °C for 20 min. The sections were then washed in wash buffer (PBS, 0.1% BSA) and incubated overnight at 4 °C with Alexa Fluor 488 anti-GFP antibody (#338 007, BioLegend) diluted 1:100 (PBS with 0.1% BSA). The slides were then washed three times with washing buffer and stained with DyLight 594-phalloidin (#12877, Cell Signaling Technology) at 25°C for 15 minutes. Each slide was washed three times with PBS buffer, stained with Hoechst (Thermo Fisher, 1:1000 in PBS) for 5 minutes, and then washed three more times with PBS. The slides were then mounted with ProLong Gold mounting solution (Thermo Fisher) and allowed to dry overnight before imaging with Keyence fluorescence microscopy. For H&E staining, the slides were washed with PBS between steps. The following steps were used: First, the samples were stained with hematoxylin for 4 minutes, then rinsed with 0.3% HCl in 70% EtOH and then with Scott's tap water. The samples were then stained with eosin for 2 minutes. After staining, each slide was incubated sequentially in 95% EtOH, 100% EtOH, and 100% xylene for 2 minutes each. The samples were then mounted onto coverslips.

[0342] In vivo toxicity assessment: Male and female C57BL / 6 mice (N=3) received 514O via intravenous injection through the tail vein. 6,12LNPs were prepared with luciferase mRNA (0.5 mg / kg) and 16% DOPE, 40% DOPE, or 40% DOTAP assist lipids. For cytokine analysis, blood was collected via the submandibular vein before injection and at 1, 3, 6, 24, and 48 hours after injection, and serum was separated. ELISA was performed using a 1:25 serum dilution (TNF-α, IL-6, and IL-2) according to the manufacturer's (BioLegend) instructions. For blood chemistry analysis, male and female mice (N=4) were intravenously injected with LNPs carrying luciferase mRNA (0.5 mg / kg). Five days after injection, mice were sacrificed, and blood samples were collected via cardiac puncture and briefly centrifuged at 3000 rpm for 15 minutes to obtain plasma. Plasma was frozen at -80°C, and alkaline phosphatase (ALP) and alanine aminotransferase (ALT) levels were measured using a DRI-CHEMNX700V animal blood chemistry analyzer (Fujifilm, Tokyo) within 24 hours of collection.

[0343] Data Analysis and Statistics: Statistical analysis and graph generation were performed using GraphPad Prism software (GraphPad, San Diego, CA) version 9.4.1. Figures 6B-6C The data were normalized to exclude autofluorescence. The original data before normalization can be found in [the database / source]. Figures 7A-7D The sample size varies depending on the type of experiment. Typically, for animal experiments, N=3 replicates; for in vitro experiments, N=4 replicates; for TNS assays, N=12 replicates; and for flow cytometry analysis, N=3 replicates. See the accompanying figures for more detailed information, which also indicates the error bar types. Where applicable, one-way or two-way ANOVA with Dunnet multiple comparisons or parametric two-tailed t-tests is performed to determine statistical significance.

[0344] Table 3: Kits and markers used to define cell populations in mouse lungs in flow cytometry experiments

[0345]

[0346]

[0347] result

[0348] Lipid library synthesis

[0349] To identify novel ionizable lipids that offer unique functionality, a library of 580 materials was generated using Michael addition chemistry. This reaction ( Figure 1AThis occurs between the primary or secondary amine group on the hydrophilic head group and the acrylate group on the hydrophobic tail group. For this study, 29 alkylamines were selected. Figure 1B This is at least in part based on materials for siRNA delivery as described elsewhere (Whitehead et al., Nat. Commun. 2014, 5:4277; and Knapp et al., Bioeng. Transl. Med. 2018, 3:138). These amine heads are coupled with 20 alkyl acrylate tails ( Figure 1C The tail is a combination reaction, with a length between 8 and 18 carbons, containing straight-chain, branched, saturated, and partially unsaturated chemistries. These properties can each affect mRNA delivery efficiency (see Knapp et al., Cell Mol. Bioeng. 2016, 9:305; Kon et al., Curr. Opin. Biotechnol. 2022, 73:329; and Eygeris et al., Acc. Chem. Res. 2022, 55:2).

[0350] Using TNS to screen for the in vivo efficacy of new LNPs

[0351] Efficient LNPs for siRNA and mRNA delivery typically possess surface pKa values ​​in the pH range of 6.1–6.5 (Kon et al., ibid.; and Patel et al., Trends Pharmacol. Sci. 2021, 42:448). Therefore, studies were conducted to assess how the pH of LNP buffers affects LNP surface properties and biological effects. pKa values ​​were determined indirectly by measuring LNP surface ionization as a function of pH using 2-(p-toluidine)naphthalene-6-sulfonic acid (TNS) fluorescence assays (Whitehead et al., ibid.; Knapp et al. 2016, ibid.; and Patel et al., ibid.). Studies described elsewhere have shown that higher levels of surface ionization at pH 5 are associated with the potency of a small class of lipid materials in mice (Hajj et al. 2019, ibid.), possibly because some ionized LNPs mediate late endosome escape from pH around 5 (Maugeri et al., ibid.). Therefore, a TNS assay at pH 5 was chosen for the initial screening of lipid libraries to identify those most likely to provide efficacy in mice. In these experiments, LNPs containing polyU mRNA were generated, and the molar ratios used were optimized for the mRNA lipid nanoparticle formulation: 35 mol% lipids, 16 mol% DOPE, 46.5 mol% cholesterol, and 2.5 mol% C14-PEG2000, as described elsewhere (Hajj 2019, ibid.; and Kauffman et al., ibid.). Fluorescence was measured on a plate reader after incubation with TNS buffer at pH 5.

[0352] Figure 2A The heatmaps show significant variations in the ionization potential of LNPs, with darker colors corresponding to higher degrees of ionization. Some structure dependence was observed, with a longer tail (O...) 14 -O 18 It produces LNPs with the lowest TNS fluorescence and a medium-length tail (O). 10 O i10 and O 8,12 The LNPs that produce the highest TNS fluorescence. The low TNS fluorescence observed with long-chain lipids may be related to their melting temperature, which in turn may affect their solubility in ethanol compared to lipids prepared with shorter chain tails.

[0353] Based on TNS assessment, 49 LNPs were selected for in vivo studies. Characterization data for some of these LNPs are available in [link to TNS analysis]. Figure 3For these experiments, LNPs containing mRNA encoding firefly luciferase were generated and intravenously (IV) injected into mice at a total mRNA dose of 0.75 mg / kg. Three hours later, luminescence in six major organs (liver, spleen, lung, pancreas, heart, and kidney) was measured using IVIS imaging. The total signal was plotted on... Figure 2B In addition to the 306O described elsewhere. i10 In addition to (Hajj 2020, ibid.; and LoPresti et al., ibid.), two new high-efficiency LNPs (402O) were identified. 8,12 and 500O 6,10 Its total luminous efficacy exceeds 10 9 p / s. Thirteen additional LNPs were identified, with a total luminescence exceeding 10. 8 p / s. To better understand the ability of different assays to predict in vivo potency, the correlation between in vivo performance and TNS fluorescence or in vitro potency measurements was investigated. Figure 2C Although neither correlation was strong, TNS assays were more advantageous because they were a necessary but not sufficient condition for in vivo efficacy in these experiments. Furthermore, TNS assays were much faster and more economical than cell-based efficacy assays.

[0354] Neutral helper lipids enable mRNA to be delivered to extracellular targets in hepatocytes.

[0355] When selecting a suitable LNP for a specific application, in addition to potency, it is crucial to consider target specificity, as most treatments require delivery to a specific organ or cell population. To determine LNP tropism, the same 49 LNPs discussed above were investigated, and luciferase protein expression in each major organ was quantified (Figure 4). Most LNPs are liver-tropy, which is typical for LNP formulations containing neutral helper lipids. This includes the three most potent LNPs ( Figure 4B Interestingly, three potent LNPs exhibited significant spleen and lung targeting. Figure 4C This is unusual because the cholesterol content is high and there are no charged lipids such as DOTAP or DOPS, which have been shown to direct LNP tropism toward the lungs and spleen, respectively (Cheng et al., ibid.; and LoPresti et al., ibid.). For example, 514O 6,10 Signals generated in the lungs and spleen each account for 35%, while only 30% are generated in the liver. LNP 503O i10 and 503O 8,12 It is spleen-oriented, and the former also produces significant signals in the lungs. Although there are other LNPs with reduced fractional liver-targeting (e.g., 211O)10 However, their overall potency is low, and they are excluded from further research. Notably, approximately 75% of the most potent LNPs are formulated with ionizable lipids containing branched unsaturated tails. Without being bound by a specific mechanism, the effectiveness of branched-tail lipids can be enhanced by their enhanced pyramidal structure, which, when ionized and paired with anionic phospholipids in the endosome membrane, can more effectively disrupt endosomes for intracellular RNA release (Han et al., Nat. Commun. 2021, 12:7233; Buschmann et al., Vaccines 2021, 9:65; and Witzigmann et al., Adv. Drug Delivery Rev. 2020, 159:344).

[0356] LNPs, with their superior ability, deliver mRNA to lung cells in response to changes in helper lipids.

[0357] Given that ionizable lipids 514O 6,10 The unusual lung-targeting behavior prompted further investigation. Typically, transfection of lung cells after intravenous injection is accomplished by substantially altering other components of the LNP formulation. For example, neutral cofactor lipids (e.g., DOPE) can be replaced with positively charged cofactor lipids such as DOTAP, and the fraction of cofactor lipids in the formulation can be increased from 16 mol% to 40 mol% (Cheng et al., ibid.; and LoPresti et al., ibid.). Therefore, studies were conducted to determine the appropriate use of 514O. 6,10 And whether adjusting the auxiliary lipids to 40% DOTAP would further improve lung targeting. Figure 5A As shown, the introduction of DOTAP did indeed increase lung expression from 35% to 94%, while simply increasing the molar percentage of DOPE to 40% reduced lung tropism. The total luminescent signal in the lungs... Figure 5A The pie chart shown below illustrates that the efficacy of each auxiliary lipid variant is on the same order of magnitude.

[0358] To further understand lung expression in response to changes in accessory lipid chemistry, 514O was generated using mRNA encoding green fluorescent protein (GFP). 6,10 LNP was administered intravenously at a dose of 0.75 mg / kg. Lung sections were harvested 24 hours later. The left lung lobe was analyzed by flow cytometry, and the right lung lobe was fixed for immunohistochemical imaging. Right lung lobe sections were stained with anti-GFP fluorescent antibody, as well as hematoxylin and eosin (H&E). All three preparations produced GFP throughout the lung. Figure 5BAlthough the relative efficacy of the formulation differs slightly compared to luciferase, this is likely due to differences in expression kinetics and half-life between luciferase (quantified at 3 hours) and GFP (imaging at 24 hours). For both DOTAP and 40% DOPE formulations, the signal was more pronounced in and around the cells that make up the bronchi and blood vessels.

[0359] Regarding safety, H&E staining did not reveal any significant tissue toxicity or immunogenicity of any of the formulations in the lungs. Figure 5C The levels of liver enzymes ALT and ALP were also assessed, showing that DOTAP particles caused a significant increase compared to the DOPE formulation, while the DOPE formulation was comparable to that in untreated animals. Figure 5D Because nanoparticles may induce inflammation, serum cytokine levels were also analyzed within 48 hours after administration. Figure 5E All three LNPs increased the levels of pro-inflammatory (Th1) cytokines IL-1β, TNF-α, and IFN-γ between 1 and 6 hours post-injection, resolving by 24 hours. The 40% DOTAP and 16% DOPE formulations showed the greatest and least inflammation, respectively. These increases are consistent with those observed elsewhere (Fang et al., Int. J. Nanomed. 2015, 2015:371; Anderson et al., N. Engl. J. Med. 2020, 383:NEJMoa2028436; and Radmand et al., Nano Lett. 2023, 23:993), including significantly higher levels of inflammation in positively charged particles (here, DOTAP LNPs). Cytokine levels returned to baseline within 24 hours, indicating a lack of tissue damage-induced inflammation. In summary, liver function and acute time-point cytokine data suggest that neutral lipid nanoparticles targeting the lungs offer advantages over cationic formulations.

[0360] Next, research will be conducted to determine the use of 514O containing these different cofactors. 6,10 Which cell types in the lungs are transfected by LNPs? This could also elucidate how DOTAP directs signaling to the lungs and whether it increases uptake by all cell types or targets new cell types. To answer this question, samples were stained with several antibody kits: lymphoid, endothelial, epithelial, and myeloid. The signal distribution among these aggregates was plotted... Figure 6A Interestingly, all formulations transfected lymphoid immune cells, while only the DOTAP and DOPE 40 mol% formulations transfected epithelial and endothelial cell populations. No formulation delivered mRNA to a significant proportion of myeloid cells (e.g., macrophages).

[0361] Then, the GFP signal in these cell populations was analyzed in more detail. Figure 6B The percentage of GFP+ cells for each cell type is shown. Since animal cells tend to exhibit autofluorescence, a single-stain control and compensation were used to normalize the data. Data before normalization are available... Figures 7A-7D Obtained from. Figure 6B The bars in the graphs are stacked for visual simplicity and are not intended to add up to 100%. For example, in the lymphoid group, the 16PE formulation transfected approximately 15% of the natural killer (NK) cells and approximately 20% of the dendritic cells in the sample. The 40 mol% formulation transfected 25% each of dendritic cells and NK cells, and a small percentage of cytotoxic T cells. No LNPs were transfected into helper T or B cells. Only the 40 mol% formulation transfected endothelial cells, with approximately twice the efficacy in lymphoid endothelial cells compared to vascular endothelial cells. The 40% DOTAP formulation transfected endothelial cells most effectively. Overall, epithelial cells were the least likely to be transfected, with approximately 7% to approximately 12% of type I (barrier-forming) epithelial cells expressing GFP. Regardless of the formulation used, only a very small percentage of bronchial and type II (secreting and stem cell-like) epithelial cells were transfected.

[0362] Figure 6C Different presentations of data from the same experiments are shown to better understand LNP cell type specificity. Here, the distribution of cell types within each sample is compared to that of GFP+ cell types. For example, in the lymphoid set, untreated samples contained approximately 40%, 20%, 20%, 2%, and 5% B cells, T helper cells, T killer cells, NK cells, and dendritic cells, respectively. The relative percentages of these cells did not change significantly in the treated samples, indicating that these treatments did not induce immune cell entry or exit from lung tissue. When this cell type distribution was compared to that seen in GFP+ cells, it was evident that NK cells and dendritic cells were enriched in the total transfected cell population. This suggests that mRNA delivery is relatively selective for NK cells and dendritic cells. The endothelial cell set showed that vascular and lymphoid endothelial cells comprised approximately 85% and 15% of the total endothelial cells in the samples, respectively. Furthermore, all treatment groups showed equivalent efficacy in both cell types. For epithelial cells, there were modest, non-statistically significant changes in the relative percentages of each cell type in the samples. Figure 6C (See bottom bar chart). Among epithelial cells, bronchial cells and type 2 alveolar cells are more easily transfected than type 1 alveolar cells.

[0363] In summary, these data indicate that LNP composition not only influences the organ location of protein expression but also the transfected cell types within those organs. The 40 mol% formulation exhibits similar tropism for dendritic, NK, and endothelial lung cells. If endothelial cells are the desired target, the 40 mol% formulation must be used. Furthermore, the cationic formulation containing DOTAP provides optimal overall selectivity for these lung cells because it is more lung-tropist than the 40 mol% DOPE formulation. Figure 5A However, this greater degree of lung transfection was driven by a disproportionate increase in mRNA delivery to endothelial cells. Figure 6B These data suggest that improving overall tropism for a specific organ can actually reduce tropism at the cellular level.

[0364] Interestingly, the 16 mol% DOPE formulation provided excellent selectivity for lung dendritic and NK cells, which is exciting because most lung tropism formulations have been shown to transfect endothelial cells (Wei et al., ibid.; and Pei et al., ibid.). Since dendritic and NK cells are associated with many lung diseases, the ability to deliver mRNA to dendritic and NK cells suggests an expanded range of conditions that could benefit from RNA therapy. For example, applications of transfecting these cell types include cancer therapy (Ahluwalia et al., ibid.), antiviral or anticancer vaccine development (Zhang et al., Oncol. Lett. 2016, 11:2605; Mohsenzadegan et al., J. Cell. Physiol. 2020, 235;74; and Soleimanian and Yaghobi, Front. Pharmacol. 2020, 11:1309), CAR-T therapy (Hung et al., Adv. Exp. Med. Biol. 2020, 1292:113; and H. Ebrahimiyan et al., Int. Immunopharmacol. 2022, 106:108587), and allergy treatment (Akdis et al., Sci. Transl. Med. 2023, 15:eadd2563). Furthermore, these studies demonstrate that delivery to these cell types can be achieved without the use of cationic assist lipids, given the liver enzyme and cytokine data ( Figure 5D-5E This could be advantageous, given the issues of toxicity associated with positively charged lipids (Wei et al., ibid.).

[0365] Other implementation plans

[0366] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A lipid-containing particle, comprising: Lipids; Cholesterol or its derivatives; Support lipids; and Compounds based on polyethylene glycol (PEG), The lipid comprises an alkylamine head and one or more alkyl acrylate tails, each of the one or more alkyl acrylate tails comprising an alkyl chain having a first branch and a second branch at a second carbon atom, and wherein the first branch of each tail has n carbon atoms and the second branch has n+1, n+2, or n+3 carbon atoms.

2. The lipid-containing particle according to claim 1, wherein the lipid has two, three or four alkyl acrylate tails.

3. The lipid-containing particles of claim 2, wherein each of the alkyl acrylate tails is identical.

4. The lipid-containing particle of claim 2, wherein the lipid comprises two or more distinct alkyl acrylate tails.

5. The lipid-containing particles according to claim 1, wherein the lipid-containing particles are lipid nanoparticles (LNPs).

6. The lipid-containing particles according to claim 1, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid, optionally wherein the neutral lipid or zwitterionic lipid comprises 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1- ... Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides, or one or more of these.

7. The lipid-containing particle according to claim 1, wherein the auxiliary lipid is a cationic lipid, optionally wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), or 1,2-distearateoyl-sn-glycerol-3-trimethylammonium-propane (DSTAP). One or more of the following: 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxypropyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxypropyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

8. The lipid-containing particles according to claim 1, wherein the lipid-containing particles are lung-oriented.

9. The lipid-containing particles according to claim 8, wherein the lipids comprise: N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine)alkylamine head; and 2-Butylheptyl acrylate tail, 2-Butyl octyl acrylate tail, The tail of 2-butylnonyl acrylate The tail of 2-hexylnonyl acrylate, The tail of 2-hexyldecyl acrylate, The tail of 2-hexyl undecyl acrylate The tail of 2-octyl undecyl acrylate The tail of 2-octyl dodecyl acrylate, or The tail of 2-octyltridecyl acrylate.

10. The lipid-containing particles according to claim 8, wherein the lipids comprise N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-Ethylethane-1,2-diamine) alkylamine head and 2-hexyldecyl acrylate tail.

11. The lipid-containing particles of claim 8, wherein the lipid-containing particles target natural killer (NK) and / or dendritic cells.

12. The lipid-containing particles of claim 11, wherein the lipid-containing particles comprise about 10-40% neutral assist lipids or zwitterionic assist lipids, optionally wherein the neutral assist lipids or zwitterionic assist lipids comprise one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides.

13. The lipid-containing particles according to claim 1, wherein the lipid-containing particles are liver-oriented.

14. The lipid-containing particle of claim 13, wherein the lipids comprise: N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head, 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-aminealkylamino head, 2-(piperazin-1-yl)ethane-1-amine head, or N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head; and 2-Butylheptyl acrylate tail, 2-Butyl octyl acrylate tail, The tail of 2-butylnonyl acrylate The tail of 2-hexylnonyl acrylate, The tail of 2-hexyldecyl acrylate, The tail of 2-hexyl undecyl acrylate The tail of 2-octyl undecyl acrylate The tail of 2-octyl dodecyl acrylate, or The tail of 2-octyltridecyl acrylate.

15. The lipid-containing particles of claim 14, wherein the lipids comprise N 1 N 1 '-(ethane-1,2-diyl)bis(N 1 The lipid comprises a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-amine alkylamine head and a 2-hexyldecyl acrylate tail, or the lipid comprises a 4-(2-aminopropane-2-yl)-1-methylcyclohexane-1-amine alkylamine head and a 2-octyldodecyl acrylate tail.

16. The lipid-containing particles according to claim 1, wherein the cholesterol or a derivative thereof is cholesterol.

17. The lipid-containing particles of claim 1, wherein the PEG-based compound is a PEG-lipid, optionally wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

18. A composition comprising lipid-containing particles according to any one of claims 1-17, wherein the lipid-containing particles further comprise a therapeutic agent.

19. The composition of claim 18, wherein the therapeutic agent comprises RNA, wherein the RNA comprises mRNA, optionally wherein the mRNA encodes luciferase, or wherein the RNA comprises siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, and RNA aptamer.

20. A method for delivering a therapeutic agent to a mammal, wherein the method comprises administering to the mammal a composition comprising lipid-containing particles according to any one of claims 1-17, wherein the lipid-containing particles further comprise a therapeutic agent.

21. The method of claim 20, wherein the therapeutic agent comprises RNA, optionally wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer.

22. The method of claim 20, wherein the mammal has been identified as having a lung disease, optionally wherein the lung disease includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

23. The method of claim 20, wherein the mammal has been identified as having a liver disease, optionally said liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or combinations thereof.

24. The method of claim 20, wherein the mammal is a human.

25. A method for treating a mammal suffering from a lung disease or its symptoms, or a liver disease or its symptoms, wherein the method comprises administering to the mammal a composition comprising lipid-containing particles according to any one of claims 1-17, wherein the lipid-containing particles further comprise a therapeutic agent.

26. The method of claim 25, wherein the lipid-containing particles are lipid nanoparticles (LNPs).

27. The method of claim 25, wherein the therapeutic agent comprises RNA, optionally wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer.

28. The method of claim 25, wherein the mammal has been identified as having a lung disease, optionally wherein the lung disease includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

29. The method of claim 25, wherein the mammal has been identified as having a liver disease, optionally said liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or combinations thereof.

30. The method of claim 25, wherein the mammal is a human.

31. A method for delivering a therapeutic agent to lung cells, wherein the method comprises contacting the lung cells with a composition comprising lipid-containing particles according to any one of claims 1-17, wherein the lipid-containing particles further comprise the therapeutic agent.

32. The method of claim 31, wherein the lung cells are in a mammal.

33. The method of claim 32, wherein the mammal is a human.

34. The method of claim 31, wherein the therapeutic agent comprises RNA, optionally wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer.

35. The method of claim 31, wherein the lung cells are in a mammal identified as having a lung disease, optionally wherein the lung disease includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

36. A lipid-containing particle, comprising: Lipids; Cholesterol or its derivatives; Support lipids; and Compounds based on polyethylene glycol (PEG), The lipid comprises an alkylamino head and one or more alkyl acrylate tails, wherein the alkylamino head is N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head or N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head, and wherein the one or more alkyl acrylate tails include one or more of 2-octyldecyl acrylate tails and 9-methyldecyl acrylate tails.

37. The lipid-containing particles according to claim 36, wherein the lipid-containing particles are lipid nanoparticles (LNPs).

38. The lipid-containing particle of claim 36, wherein the lipid has two, three, or four alkyl acrylate tails.

39. The lipid-containing particles of claim 38, wherein each of the alkyl acrylate tails is identical.

40. The lipid-containing particle of claim 38, wherein the lipid comprises two or more distinct alkyl acrylate tails.

41. The lipid-containing particles of claim 36, wherein the auxiliary lipid is a neutral lipid or a zwitterionic lipid, optionally wherein the neutral lipid or zwitterionic lipid comprises 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1- Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), triglycerides, triacylglycerols, diacylglycerols, diacylglycerols, and ceramides, or one or more of these.

42. The lipid-containing particles of claim 36, wherein the auxiliary lipid is a cationic lipid, optionally wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dimyristoyl-sn-glycerol-3-trimethylammonium-propane (DMTAP), 1,2-dipalmitoyl-sn-glycerol-3-trimethylammonium-propane (DPTAP), or 1,2-distearateoyl-sn-glycerol-3-trimethylammonium-propane (DSTAP). P), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyl dimethylammonium bromide (DDAB), dioctadecyloxypropyl-glycerol (DOGS), dimethyl dioctadecyl ammonium bromide (DODAB), dioleenoyloxypropyl-trimethylammonium (DOSPA), and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) 43. The lipid-containing particles of claim 36, wherein the lipids comprise N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 2-octyldecyl acrylate tail, or said lipid includes N 1 N 1 '-(propane-1,3-diyl)bis(N 1 (-methylethane-1,2-diamine)alkylamine head and 9-methyldecyl acrylate tail, or said lipid comprising N 1 -((4-)2-aminoethyl)piperazine-1-yl)methyl)ethane-1,2-diamine head and 9-methyldecyl acrylate tail.

44. The lipid-containing particles of claim 36, wherein the cholesterol or a derivative thereof is cholesterol.

45. The lipid-containing particles of claim 36, wherein the PEG-based compound is a PEG-lipid, optionally wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

46. ​​A composition comprising lipid-containing particles according to any one of claims 36-45, wherein the lipid-containing particles return Including therapeutic agents.

47. The composition of claim 46, wherein the therapeutic agent comprises RNA, wherein the RNA comprises mRNA, optionally wherein the mRNA encodes luciferase, or wherein the RNA comprises siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, and RNA aptamer.

48. A method for delivering a therapeutic agent to a mammal, wherein the method comprises administering to the mammal a composition comprising lipid-containing particles according to any one of claims 36-45, wherein the lipid-containing particles further comprise the therapeutic agent.

49. The method of claim 48, wherein the therapeutic agent comprises RNA, optionally wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer.

50. The method of claim 48, wherein the mammal has been identified as having a lung disease, optionally wherein the lung disease includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

51. The method of claim 48, wherein the mammal has been identified as having a liver disease, optionally said liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

52. The method of claim 48, wherein the mammal is a human.

53. A method for treating a mammal suffering from a lung disease or its symptoms, or a liver disease or its symptoms, wherein the method comprises administering to the mammal a composition comprising lipid-containing particles according to any one of claims 36-45, wherein the lipid-containing particles further comprise a therapeutic agent.

54. The method of claim 53, wherein the therapeutic agent comprises RNA, optionally wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer.

55. The method of claim 53, wherein the lung condition includes cystic fibrosis, non-small cell lung cancer, another type of lung cancer, alpha-1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disease (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

56. The method of claim 53, wherein the liver disease includes α-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson's disease, hereditary tyrosinemia, glycogen storage disease, viral hepatitis, familial hypercholesterolemia, non-alcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or combinations thereof.

57. The method of claim 53, wherein the mammal is a human.

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