Ionizable lipids for drug delivery

Abstract

Ionizable lipids, nanoparticles containing the ionizable lipids, compositions containing the nanoparticles, and methods for using the ionizable lipids, nanoparticles, and compositions to deliver therapeutic agents (e.g., mRNAs) to particular cells, tissues, or organs (e.g., the lungs) are provided herein.

Description

[0001] IONIZABLE LIPIDS FOR DRUG DELIVERY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from U.S. Provisional Application Serial No. 63 / 620,430, filed January 12, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under HR.0011-19-2-0007 awarded by the Defense Advanced Research Projects Agency (DARPA) and D16AP00143 awarded by the United States Department of the Interior (DOI). The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] This document relates to ionizable lipids, nanoparticles containing the ionizable lipids, compositions containing the nanoparticles, and methods for using the ionizable lipids, nanoparticles, and compositions to deliver therapeutic agents (e.g., mRNAs) to particular cells, tissues, or organs (e.g., the lungs).

[0008] BACKGROUND

[0009] Messenger RNA (mRNA) is a versatile drug that can be used for protein replacement therapy, antiviral and anticancer vaccines, gene editing and modulation, CAR-T therapy, and tissue engineering. A major challenge impeding the clinical translation of mRNA therapies, however, is delivery into target cells, because mRNA is a labile molecule that is rapidly degraded by nucleases in the blood, and because mRNA lacks mechanisms to enter the cell for translation into protein (Phua et al., J. Controlled Release 2013, 166:227; and Tavernier et al., J. Controlled Release 2011, 150:238). The most clinically advanced mRNA delivery systems are lipid nanoparticles (LNPs), which are nonviral vehicles containing multiple lipids (Sun et al., Adv. Drug Delivery Rev. 2023, 200: 115042). LNPs can shield mRNA from RNases, hinder clearance by phagocytes, 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). Further, LNPs can attract a protein corona following injection into biological milieu, and this corona can direct nanoparticle uptake and delivery into specific cell types (Ritz et al., Biomacromolecules 2015, 16: 1311). LNPs provide several advantages over viral delivery systems, including their larger cargo capacity and lower immunogenicity, the latter of which can confer repeat dosing ability (Kiaie et al., supra).

[0010] LNPs are multicomponent vehicles that can be generated by coprecipitating mRNA and 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 ingredients usually include four components: a synthetic ionizable lipid or lipidoid, a phospholipid (referred to as a “helper lipid”), cholesterol, and a lipid conjugated to polyethylene glycol (PEG-lipid) (Fenton et al., supra and Kauffman et al., Nano Lett. 2015, 15:7300). Each component of the LNP plays a role during the delivery process. Ionizable lipids or lipidoids can impart a neutral charge to the nanoparticle surface while in the bloodstream, which switches to a positive charge in the acidic early endosome, promoting endosomal escape (Maugeri et al., supra,' and Sahay et al., Nat. Biotechnol. 2013, 31:653). The ionizable lipid also can influence the protein corona that forms around the LNP and that can govern particle tropism (Cai and Chen, Adv. Mater. 2019, 31: 1805740; and Wang et al., Nat. Protoc. 2023, 18:265). Helper lipids are structurally important in cell and extracellular vesicle membranes, and they have similar value in optimizing LNP lipid packing and increasing stability (Kaie et al., supra). Further, the helper lipid’s charge can affect nanoparticle tropism, with neutral, negative, and positive charges directing 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). The PEG-lipid incorporates on the particle surface, stabilizing the exterior, preventing aggregation, and providing some protection from the immune system (Kiaie et al., supra,' and Ryals et al., PLoS One 2020, 15:e0241006).

[0011] When delivered systemically through intravenous (i.v.) injection, many LNPs are strongly liver-tropic, with others transfecting the spleen (Hajj et al., Nano Lett. 2020, 20:5167; Kiaie et al., supra,' and Loughrey and Dahlman, Acc. Chem. Res. 2022, 55: 13). Several strategies have been described to reduce liver tropism, including the addition of antiliver microRNAs sequences to mRNA (Fenton et al., supra), changing the LNP helper lipid (Cheng et al., supra, and LoPresti et al., supra), and incorporating active targeting agents such as antibodies (Li et al., ACS Chem. 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).

[0012] The next frontier of RNA delivery involves the transfection of other organs, such as the lungs, heart, bone marrow, and muscle tissue. Delivery to the lungs in particular has the potential to create new treatments for a number of intractable diseases. For example, lungspecific symptoms of cystic fibrosis originate in epithelial cells, and non-small cell lung cancer is difficult to treat because these tumors suppress natural killer and dendritic cells in the lung (Engelhardt et al., J. Clin. Invest. 1994, 93:737; and Ahluwalia et al., Cancers 2021, 13:4037). For lung delivery, changing the route of administration to inhalation of LNPs in liquid form or nebulized form 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 lung also can be improved by modulating ionizable lipid chemistry or incorporating positively charged helper 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 (such as l,2-di-(9Z- octadecenoyl)-3-trimethylammonium-propane (DOTAP), for example) can cause toxicity. Further, 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). As such, there remains a need for new systems that safely target other clinically significant cell populations in the lung.

[0013] SUMMARY

[0014] Ionizable lipids include lipids that, when incorporated into a lipid nanoparticle, 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. In particular, a library of ionizable lipidoids was created and examined for efficacy as a function of organ targeting, leading to the identification of an LNP that potently delivered mRNA to the lungs with and without the use of a cationic helper lipid. Also as described herein, LNPs incorporating a neutral helper lipid primarily targeted natural killer and dendritic cells, while those formulated with a cationic helper lipid enhanced delivery to endothelial (lymphatic and vascular) and epithelial (bronchial and alveolar) cells. The data described herein demonstrated that endogenous transfection of non-endothelial lung cell targets following i.v. injection is possible, thus enabling treatment of cystic fibrosis, non-small cell lung cancer, and other diseases of the lungs.

[0015] This document provides ionizable lipids, lipid nanoparticles containing the ionizable lipids, and methods for their use (e.g., for drug delivery). In some cases, the ionizable lipids described herein include an acrylate tail and a novel carbon backbone. Such lipids can be made by reacting an amine head and acrylate tails through a Michael addition reaction, during which primary amine hydrogens on the heads are substituted with the acrylate tail. The ionizable lipids provided herein can have tail carbon lengths of various lengths, including lengths outside of the 10-14 range. The lipids provided herein can be used, for example, to generate LNPs. In some cases, LNPs described herein can be formulated with therapeutic agents (e.g., nucleic acids including, but not limited to, mRNA and / or siRNA), and can deliver that cargo into the cytoplasm of target cells. This document also provides effective delivery vehicles. In some cases, the methods and materials described herein can be used to unlock treatments for diseases (including, but not limited to, diseases in the lung and / or liver). In some cases, LNPs described herein can be used for delivery to the liver. In some cases, LNPs described herein can be used for delivery to the lungs. In some cases, LNPs described herein can be used form RNA delivery to the lungs (e.g., via intravenous (i.v.) injection). In some cases, LNPs described herein can be injected intravenously. In some cases, LNPs described herein can include neutral formulations (e.g., without a cationic lipid, which may be associated with toxicity). In some cases, LNPs described herein can target immune cells. In some cases, LNPs described herein can target dendritic cells. In some cases, LNPs described herein can target natural killer cells.

[0016] In a first aspect, this document features a lipid-containing particle that contains, consists essentially of, or consists of: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amine head and one or more alkyl acrylate tails, wherein each of said one or more alkyl acrylate tails includes an alkyl chain having, at the second carbon, a first branch and a second branch, 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 lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails can be the same, or the lipidoid can include two or more different alkyl acrylate tails. The lipid-containing particle can be a lipid nanoparticle (LNP). The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), l,2-dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The lipid-containing particle can be lung-tropic. The lipidoid can include: an N1,Nll-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head or an N1,Nll-(propane-l,3-diyl)bis(N1- ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,Nll-(propane-l,3- diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail. The lipid-containing particle can target natural killer (NK) and / or dendritic cells. The lipid- containing particle can include about 10-40% neutral helper lipid or zwitterionic helper lipid. The neutral helper lipid or zwitterionic helper lipid can include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The lipid-containing particle can include about 16% DOPE. The lipid-containing particle can be liver-tropic. The lipidoid can include: an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head, a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head, a 2-(piperazin-l- yl)ethan-l -amine head, or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail. The lipidoid can include a 4-(2-aminopropan-2- yl)-l-methylcyclohexan-l -amine alkyl amino head and a 2-octyldodecyl acrylate tail. The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0017] In another aspect, this document features a composition that contains, consists essentially of, or consists of a lipid-containing particle, wherein said lipid-containing particle includes a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amine head and one or more alkyl acrylate tails, wherein said one or more alkyl acrylate tails include an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. The therapeutic agent can be a nucleic acid. The nucleic acid can be an RNA. The RNA can include one or more of a mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer. The RNA can be an mRNA encoding luciferase. The lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails can be the same, or the lipidoid can include two or more different alkyl acrylate tails. The lipid-containing particle can be a lipid nanoparticle (LNP). The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l,2-di-myristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), l,2-dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), didodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The lipid-containing particle can be lung-tropic. The lipidoid can include: an N1,Nll-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head or an N1,Nll-(propane-l,3-diyl)bis(N1- ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,Nll-(propane-l,3- diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail. The lipid-containing particle can target natural killer (NK) and / or dendritic cells. The lipid- containing particle can include about 10-40% neutral helper lipid or zwitterionic helper lipid. The neutral helper lipid or zwitterionic helper lipid can include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The lipid-containing particle can contain about 16% DOPE. The lipid-containing particle can be liver-tropic. The lipidoid can include: an N1,Nll-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head; and a 2- butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2- hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2- octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail. The lipidoid can include a 4-(2-aminopropan-2- yl)-l-methylcyclohexan-l -amine alkyl amino head and a 2-octyldodecyl acrylate tail. The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0018] In another aspect, this document features a method for delivering a therapeutic agent to a mammal. The method can include, consist of, or consist essentially of administering to the mammal a composition containing a lipid-containing particle, wherein said lipid- containing particle includes a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amine head and one or more alkyl acrylate tails, wherein said alkyl acrylate tails include an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. The administering can include intravenous administration, intramuscular administration, or subcutaneous administration. The lipid-containing particle can be a lipid nanoparticle (LNP). The therapeutic agent can be a nucleic acid. The nucleic acid can be RNA. The RNA can include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. The 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 lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of said alkyl acrylate tails can be the same, or the lipidoid can include two or more different alkyl acrylate tails. The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero- 3 -phosphoethanolamine (DPPE), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3 -trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol. The mammal can have been identified as having a lung disorder, and the lipid-containing particle can be lungtropic. The lung disorder can include cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. The lipidoid can include: an N^N1'-(propane- 1,3- diyl)bis(N1-methylethane- 1,2-diamine) alkyl amine head or an N1,N1'-(propane-l,3- diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2- butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2- hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2- octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail. The lipid-containing particle can target natural killer (NK) and / or dendritic cells. The lipid-containing particle can include about 10-40% neutral helper lipid or zwitterionic helper lipid. The neutral helper lipid or zwitterionic helper lipid can include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The lipid-containing particle can include about 16% DOPE. The mammal can have been identified as having a liver disorder, and the lipid-containing particle can be liver-tropic. The liver disorder can include alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof. The lipidoid can include: an N1,N1'-(ethane-l,2-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head or a 4-(2-aminopropan-2-yl)-l- methylcyclohexan-1 -amine alkyl amino head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,N1'-(ethane-l,2- diyl)bis(N1-methylethane- 1,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail.

[0019] The lipidoid can include a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head and a 2-octyldodecyl acrylate tail. The mammal can be a human.

[0020] In another aspect, this document features a method for treating a mammal having a lung disorder or a symptom thereof, or having a liver disorder or a symptom thereof. The method can include, consist of, or consist essentially of administering to the mammal a composition containing a lipid-containing particle containing a therapeutic agent, where the lipid-containing particle further includes: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amine head and one or more alkyl acrylate tails, wherein said alkyl acrylate tails include an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. The administering can include intravenous administration, intramuscular administration, or subcutaneous administration. The lipid-containing particle can be a lipid nanoparticle (LNP). The therapeutic agent can be a nucleic acid. The nucleic acid can be RNA. The RNA can include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. The 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 lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails can be the same, or said lipidoid can include two or more different alkyl acrylate tails. The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l,2-di-myristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), l,2-dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol. The mammal can have been identified as having a lung disorder, and the lipid-containing particle can be lungtropic. The lung disorder can include cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. The lipidoid can include: an N^N1'-(propane- 1,3- diyl)bis(N1-methylethane- 1,2-diamine) alkyl amine head or an N1,N1'-(propane-l,3- diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2- butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2- hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2- octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail. The lipid-containing particle can target natural killer (NK) and / or dendritic cells. The lipid-containing particle can include about 10-40% neutral helper lipid or zwitterionic helper lipid. The neutral helper lipid or zwitterionic helper lipid can include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The lipid-containing particle can contain about 16% DOPE. The mammal can have been identified as having a liver disorder, and the lipid-containing particle can be liver-tropic. The liver disorder can include alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof. The lipidoid can include: an N1,N1'-(ethane-l,2-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head or a 4-(2-aminopropan-2-yl)-l- methylcyclohexan-1 -amine alkyl amino head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,N1'-(ethane-l,2- diyl)bis(N1-methylethane- 1,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail.

[0021] The lipidoid can include a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head and a 2-octyldodecyl acrylate tail. The mammal can be a human.

[0022] In still another aspect, this document features a method for delivering a therapeutic agent to a lung cell. The method can include, consist of, or consist essentially of contacting the lung cell with a composition containing a lipid-containing particle that includes a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amine head and one or more alkyl acrylate tails, and wherein said alkyl acrylate tails include an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. The lipid- containing particle can be a lipid nanoparticle (LNP). The lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of said alkyl acrylate tails can be the same, or said lipidoid can include two or more different alkyl acrylate tails. The helper lipid can be a neutral lipid or a zwitterionic helper lipid. The neutral lipid or zwitterionic helper lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3 -trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol. The lung cell can be within a mammal. The mammal can be a human. The therapeutic agent can be a nucleic acid. The nucleic acid can be RNA. The RNA can include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. The 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 administering can include intravenous administration, intramuscular administration, or subcutaneous administration. The lung cell can be within a mammal identified as having a lung disorder. The lung disorder can include cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. The lipidoid can include: an N1,N1'-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head or an N1,N1'-(propane-l,3-diyl)bis(N1- ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail. The lipidoid can include an N1,N1'-(propane-l,3- diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail. The lipid-containing particle can target natural killer (NK) and / or dendritic cells. The lipid- containing particle can include about 10-40% neutral helper lipid or zwitterionic helper lipid. The neutral helper lipid or zwitterionic helper lipid can include one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The lipid-containing particle can include about 16% DOPE.

[0023] In another aspect, this document features a lipid-containing particle that includes, consists of, or consists essentially of: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amino head and one or more alkyl acrylate tails, wherein said alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2- aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head, and wherein said one or more alkyl acrylate tails include one or more of a 2-octyldecyl acrylate tail and a 9-methyldecyl acrylate tail. The lipid-containing particle can be a lipid nanoparticle (LNP). The lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of the alkyl acrylate tails can be the same, or said lipidoid can include two or more different alkyl acrylate tails. The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), l,2-dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3 P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), Dimethyldioctadecylammonium Bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The lipidoid can include a N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail. The lipidoid can include a N1,Nll-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail. The lipidoid can include a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head and a 9- methyldecyl acrylate tail. The cholesterol or derivative thereof can be cholesterol. The PEG- based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0024] In yet another aspect, this document features a composition that includes, consists of, or consists essentially of a lipid-containing particle, wherein said lipid-containing particle includes a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amino head and one or more alkyl acrylate tails, wherein said alkyl amino head is a N^N1'- (propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2- aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head, and wherein said one or more alkyl acrylate tails include one or more of a 2-octyldecyl acrylate tail and a 9-methyldecyl acrylate tail. The therapeutic agent can be a nucleic acid. The nucleic acid can be an RNA. The RNA can include one or more of a mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer. The RNA can be an mRNA encoding luciferase. The lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of said alkyl acrylate tails can be the same, or the lipidoid can include two or more different alkyl acrylate tails. The lipid-containing particle can be a lipid nanoparticle (LNP). The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3-trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3 -trimethylammoniumpropane (DPTAP), 1,2- distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP), 30-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The lipidoid can include a N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2- diamine) alkyl amine head and a 2-octyldecyl acrylate tail. The lipidoid can include a N^N11- (propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail. The lipidoid can include a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane- 1,2-diamine head and a 9-methyldecyl acrylate tail. The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol.

[0025] In another aspect, this document features a method for delivering a therapeutic agent to a mammal, where the method includes, consists of, or consists essentially of administering to the mammal a composition containing a lipid-containing particle, wherein said lipid- containing particle includes a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amino head and one or more alkyl acrylate tails, wherein said alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head, and wherein said one or more alkyl acrylate tails include eone or more of a 2-octyldecyl acrylate tail and a 9-methyldecyl acrylate tail. The administering can include intravenous administration, intramuscular administration, or subcutaneous administration. The lipid-containing particle can be a lipid nanoparticle (LNP). The therapeutic agent can be a nucleic acid. The nucleic acid can be RNA. The RNA can include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. The 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 lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of said alkyl acrylate tails can be the same, or the lipidoid can include two or more different alkyl acrylate tails. The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3-trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3 -trimethylammoniumpropane (DPTAP), 1,2- distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP), 3 -[N-(N',N'- dimethylaminoethane)-carbamoyl] cholesterol (DC-cholesterol), didodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol. The mammal can have been identified as having a lung disorder. The lung disorder can include cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post- viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. The mammal can have been identified as having a liver disorder. The liver disorder can include alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof. The lipidoid can include a N1,Nll-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail. The lipidoid can include a N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail. The lipidoid can include a N1-((4-)2-aminoethyl)piperazin- l-yl)methyl)ethane-l,2-diamine head and a 9-methyldecyl acrylate tail. The mammal can be a human.

[0026] In another aspect, this document features a method for treating a mammal having a lung disorder or a symptom thereof, or having a liver disorder or a symptom thereof, wherein said method includes, consists of, or consists essentially of administering to the mammal a composition containing a lipid-containing particle that includes a therapeutic agent, wherein the lipid-containing particle further includes: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid includes an alkyl amino head and one or more alkyl acrylate tails, wherein said alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head, and wherein said one or more alkyl acrylate tails include one or more of a 2-octyldecyl acrylate tail and a 9- methyldecyl acrylate tail. The administering can include intravenous administration, intramuscular administration, or subcutaneous administration. The lipid-containing particle can be a lipid nanoparticle (LNP). The therapeutic agent can be a nucleic acid. The nucleic acid can be RNA. The RNA can include mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers. The 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 lipidoid can have two alkyl acrylate tails, three alkyl acrylate tails, four alkyl acrylate tails, or more than four alkyl acrylate tails. Each of said alkyl acrylate tails can be the same, or said lipidoid can include two or more different alkyl acrylate tails. The helper lipid can be a neutral lipid or a zwitterionic lipid. The neutral lipid or zwitterionic lipid can include one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. The helper lipid can be a cationic lipid. The cationic lipid can include one or more of l,2-dileoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3-trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3 -trimethylammoniumpropane (DPTAP), 1,2- distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP), 30-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The cholesterol or derivative thereof can be cholesterol. The PEG-based compound can be a PEG-lipid. The PEG can have a molecular weight of about 300 g / mol to about 5000 g / mol. The lung disorder can include cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post- viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. The liver disorder can include alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof. The lipidoid can include a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail. The lipidoid can include a N1,N1'-(propane-l,3- diyl)bis(N1-methylethane- 1,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail. The lipidoid can include a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane- 1,2-diamine head and a 9-methyldecyl acrylate tail. The mammal can be a human.

[0027] 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. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0028] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0029] DESCRIPTION OF DRAWINGS

[0030] FIGS. 1A-1C show the chemical reaction and functional groups used to generate a library of 580 ionizable lipidoids that incorporated linear, branched, and unsaturated tails. Lipidoids were synthesized through the combinatorial Michael addition reaction (FIG. 1 A) of 29 alkyl amine heads (FIG. IB) and 20 alkyl acrylate tails (FIG. 1C). For tail nomenclature, “O” and “X” represent fully saturated and partially unsaturated tails, respectively.

[0031] FIGS. 2A-2C show that a fraction of the ionizable lipidoid library potently delivered mRNA in mice. FIG. 2A shows the results of initial screening, which was performed using a cell-free 2-(p-toluidinyl)naphtthalene-6-sulphonic acid (TNS) assay at pH 5. Higher levels of TNS fluorescence correspond with darker shading. FIG. 2B is a graph plotting TNS fluorescence for 18 lipids with high levels of TNS fluorescence, 18 lipids with medium levels of TNS fluorescence, and 13 lipids with low levels of TNS fluorescence, each of which was formulated with firefly luciferase mRNA and IV-injected into mice at a total mRNA dose of 0.75 mg / kg. Three hours later, total in vivo luminescence was measured and quantified by IVIS. FIG. 2C includes a pair of graphs showing that TNS fluorescence measurements correlated with in vivo efficacy (left graph) to a greater degree than efficacy in HeLa cells (right graph). In these graphs, in vivo luminescence is on a log scale, while in vitro luminescence and TNS fluorescence are on a linear scale. For TNS assay N = 12, with average shown; for in vitro luminescence N = 4; for in vivo luminescence N = 3. Error bars represent standard deviation.

[0032] FIG. 3 includes a series of graphs plotting LNP characterization data. Entrapment (left graph) was measured using the RiboGreen™ assay, and number mean diameter (center graph) and zeta potential (right graph) were measured using dynamic light scattering. N = 4, error bars represent s.d.

[0033] FIGS. 4A-4C show that LNPs were primarily liver tropic, with several potently delivering mRNA to the lungs and spleen. Three hours post IV- injection of LNPs containing 0.75 mg / kg mLuc LNP, mice were sacrificed, and organs were excised and imaged for luminescence. FIG. 4A is a graph plotting luminescence in the lungs, liver, and spleen, showing that delivery occurred primarily to the liver although a subset of lipidoids induced protein expression in the spleen and lungs. FIG. 4B includes pie charts illustrating the organ distribution of luminescent signal for the top three liver-tropic compounds. FIG. 4C includes pie charts illustrating the organ distribution of luminescent signal for the top three compounds dominated by lung and spleen signal. N = 3, error bars represent standard deviation.

[0034] FIGS. 5A-5E show that helper lipid charge and molar percentage shifted the organ tropism of 5140e,io lipid nanoparticles. LNPs containing 5140e,io LNPs were formulated with either 16 mol% DOPE, 40% DOPE, or 40% DOTAP. Mice were IV-injected with 0.75 mg / kg mRNA encoding luciferase (mLuc) or green fluorescent protein (mGFP). FIG. 5A includes pie charts showing luminescent signal quantified as a function of organ by IVIS in mLuc experiments, where mice were sacrificed three hours postinjection. FIG. 5B images from GFP experiments in which the right lobes of mice were collected for immunohistochemistry, with GFP expression visualized in green. Safety was examined through H&E staining of the lung (FIG. 5C), liver function testing (FIG. 5D), and cytokine analysis (FIG. 5E) immediately prior to injection (0 h), and between 1 and 48 hours postinjection. For all panels, N = 3, and error bars represent standard error. *, **, ***, and ****represent p < 0.05, 0.01, 0.001, and 0.0001 according to a two-way Dunnett’s multiple comparison ANOVA.

[0035] FIGS. 6A-6C show that LNPs formulated with the lipidoid 5140e,io disproportionately transfected several rare lung cell types. 5140e,io LNPs were formulated with either 16 mol% DOPE (16PE), 40% DOPE (40PE), or 40% DOTAP (40TAP) and injected via tail vein into mice at an mGFP dose of 0.75 mg / kg. At 24 hours post injection, animals were euthanized and organs were processed into single cell suspensions for analysis by flow cytometry. FIG. 6A includes pie charts showing that helper lipids affected LNP tropism to lymphoid cells, endothelial cells, and epithelial cells. FIG. 6B includes graphs plotting GFP expression in individual cell types, quantified by flow cytometry using lymphoid, endothelial, and epithelial cell panels. The y-axis in each graph represents the percentage of each cell type that was GFP+ normalized to autofluorescence in unstained control samples. Raw data before normalization are available in FIGS. 7A-7D. Untr = Untreated, DC = dendritic cells, NK = Natural Killer cells. FIG. 6C includes a series of bars showing the cell specificity of transfection, illustrated by comparing the overall cell type distribution in the lung sample (top bars) with the transfected (GFP+) cell types (bottom bars). N = 3, error bars represent standard deviation, *, **, **** correspond to p < 0.05, 0.005, or 0.0001, respectively, according to the two-way ANOVA followed by a Dunnet’s post-hoc analysis.

[0036] FIGS. 7A-7D includes graphs plotting raw GFP transfection efficacy data as a function of cell type prior to normalization. 5140e,io LNPs were formulated with either 16 mol% DOPE (16PE), 40% DOPE (40PE) or 40% DOTAP (40TAP) and injected via tail vein into mice at an mGFP dose of 0.75 mg / kg. At 24 hours post-injection, animals were euthanized and organs were processed into single cell suspensions for analysis by flow cytometry. Lymphoid (FIG. 7A), endothelial (FIG. 7B), epithelial (FIG. 7C), and myeloid (FIG. 7D) cell panels were analyzed. The y-axes represent the percentage of each cell type that was GFP+. 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, according to the two-way ANOVA followed by a Dunnet multiple comparison. FIG. 6B was generated from these data by normalizing to the untreated controls. Myeloid cells were not shown in FIG. 6B because of their overall low numbers and prohibitively high background fluorescence.

[0037] FIGS. 8A-8C show the gating strategy for defining cell populations in the lung based on fluorescent antibodies from TABLE 3. FIG. 8A, lymphoid panel; FIG. 8B, myeloid panel; FIG. 8C, endothelial and epithelial panel.

[0038] DETAILED DESCRIPTION

[0039] Provided herein are ionizable lipids, lipid particles containing the ionizable lipids, and methods for using the ionizable lipid-containing particles for delivery of one or more therapeutic agents (e.g., a nucleic acid, such as an mRNA) to mammals.

[0040] Lipid-containing particles are small particles or structures that include lipids and / or lipid-like materials (e.g., lipidoids). Lipid-containing particles are found 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 storage organelles), exosomes (which are involved in cell-to-cell communication), vesicles (which are small membrane-bound sacs within cells), and others. In general, the lipid-containing particles (e.g., LNPs) provided herein include a mixture of: (i) an ionizable and / or cationic lipid or lipidoid; (ii) cholesterol, a cholesterol analogue, or a cholesterol derivative; (iii) a helper lipid; and (iv) a polyethylene glycol-lipid conjugate (PEG-lipid) or PEG-cholesterol conjugate (PEG-cholesterol).

[0041] Any appropriate ionizable lipid or lipidoid can 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. Lipidoids are a class of lipid-like materials often used in biotechnology and nanomedicine, particularly for the delivery of nucleic acids such as RNA and DNA. In general, the ionizable lipidoids provided herein have an alkyl amine head and one or more alkyl acrylate tails. Non-limiting examples of suitable alkyl amine heads are depicted in FIG. IB. For example, an alkyl amine head can be a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head (503), an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head (514), an N^N1'- (ethane-l,2-diyl)bis(N1-methylethane- 1,2-diamine) alkyl amine head (500), a 4-(2- aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head (402), a 2-(piperazin- 1 - yl)ethan-l -amine head (205), or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane- 1,2- diamine head (200).

[0042] The lipidoids provided herein can have any appropriate number of alkyl acrylate tails. For example, a lipidoid 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 a lipidoid molecule provided herein has more than one alkyl acrylate tail, all of the alkyl acrylate tails can be the same. In some cases, when a lipidoid molecule provided herein has more than one alkyl acrylate tails, the lipidoid can 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 FIG. 1C. In some cases, an alkyl acrylate tail can include an alkyl chain having, at the second carbon, a first branch and a second branch. In some cases, the lengths of the first and second branches can differ. For example, the first branch of an alkyl acrylate tail can have n carbons and the second branch can have n+1, n+2, or n+3 carbons. For example, an alkyl acrylate tail can be a 2-butylheptyl acrylate tail (04,?), a 2-butyloctyl acrylate tail (O4,s), a 2-butylnonyl acrylate tail (04,9), a 2-hexylnonyl acrylate tail (Oe,9), a 2-hexyldecyl acrylate tail (Oe, io), a 2-hexylundecyl acrylate tail (Oe,n), a 2-octylundecyl acrylate tail (Os,n), a 2- octyldodecyl acrylate tail (Os, 12), or a 2-octyltridecyl acrylate tail (Os, 13). In some cases, the lengths of the first and second branches can be the same. For example, an alkyl acrylate tail can be a 2-octyldecyl acrylate tail (Os,io), a 2-nonylundecyl acrylate tail (O9.11), a 2- heptylnonyl acrylate tail (67,9), or a 2-hexyloctyl acrylate tail (Oe.s). In some cases, an alkyl acrylate tail may not contain a branch. For example, in some cases an alkyl acrylate tail can be a 9-methyldecyl acrylate tail (Oiio).

[0043] Examples of suitable lipidoids include, without limitation, 5030iio, 51406, 10,

[0044] 50006.10, 40208,12, 5030s,io, 5030e,io, 2050e,io, 2000iio, 40204,8, and 40204,8. In some cases, the lipidoid(s) in a lipid-containing particle provided herein can include 5030iio,

[0045] 51406.10, 50006,IO, 40208,12, or any combination thereof. For example, the lipidoid contained in a LNP can be 5030iio, 51406, 10, 5000e,io, or 40208,12.

[0046] The lipidoids provided herein can be generated using any appropriate methods. For example, an alkyl-amine head can be combined with one or more alkyl-acrylate tails in any appropriate 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 appropriate temperature and for any appropriate length of time. For example, an alkyl amine 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). As depicted in FIG. 1A, such a procedure can generate a lipidoid provided herein via a combinatorial Michael addition reaction.

[0047] Any appropriate cholesterol-related molecule (e.g., cholesterol, a cholesterol analogue, or a cholesterol derivative) can be included in the lipid-containing particles (e.g., LNPs) provided herein. Examples of suitable cholesterol derivatives (or analogues) include, without limitation, oxidized cholesterol, desmosterol, 7-dehydrocholesterol, ergosterol, lanosterol, ketosterone, cholesterol sulfate, dehydroergosterol, cholestratrienol, 5-cholestene, and pregnenolone. In some cases, the lipid-containing particles (e.g., LNPs) provided herein can contain cholesterol.

[0048] Any appropriate helper lipid can be included in the lipid-containing particles (e.g., LNPs) provided herein. Helper lipids can be cationic, anionic, neutral, or zwitterionic amphiphilic lipids, and along with cholesterol or a derivative thereof (e.g., a cholesterol analog), can aid in the molecular packing and stability of a lipid-containing particle (e.g., a LNP). Helper lipids also can enhance lipid nanoparticle efficacy by promoting fusion with both cell and endosomal membranes, facilitating cell uptake and endosomal release. In some cases, a lipid-containing particle provided herein can include one or more zwitterionic helper lipids. Non-limiting examples of suitable zwitterionic helper lipids include 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero- 3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), and sterol sphingomyelin (SSM). In some cases, a lipid-containing particle provided herein can include one or more neutral helper lipids. Non-limiting examples of suitable neutral helper lipids include triglycerides, triacylglycerols, diglycerides, diacylglycerols, and ceramides. In some cases, a lipid-containing particle provided herein can include one or more cationic helper lipids. Nonlimiting examples of suitable cationic helper lipids include l,2-dileoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3-trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3 -trimethylammoniumpropane (DPTAP), 1,2- distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP), 30-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0049] Any appropriate PEG-based compound can be included in the lipid-containing particles (e.g., LNPs) provided herein. PEG is a poly ether compound derived from petroleum. PEG and PEG-based compounds can be used for various applications, such as drug delivery agents, solvents, adhesives, adsorbents, and tissue engineering scaffolds. PEG- based compounds that can be used in the lipid-containing particles provided herein include, without limitation, PEG-lipids (PEGylated lipids) and PEG-cholesterols (PEGylated cholesterols). PEG-lipids include a PEG moiety attached to one or more lipid moieties (e.g., a ceramide, succinoyl, or carbamate moiety). PEG-cholesterols include a PEG moiety attached to one or more cholesterol moieties. PEG-lipids and / or PEG-cholesterols can form a protective, non-aggregating, non-immunogenic shell around the surface of LNPs. Depending on the ultimate delivery route of the LNPs, the lipid group may be varied (e.g., in length) to dictate how long the PEG-lipid will be associated with the LNP, with longer lipid chains tending to remain associated with the LNP for longer time periods, and shorter lipid chains typically being useful for providing “diffusible” PEG lipids that diffuse from the lipid nanoparticle quickly to produce an LNP with increased transfection rates. The PEG moiety of a PEG-lipid or PEG-cholesterol can 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 moiety of a PEG-lipid or PEG-cholesterol can have a molecular weight of about 2000, which is referred to as PEG 2000. Non-limiting examples of suitable PEG-lipids include 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(poly ethylene glycol)-2000], N-octanoyl-sphingosine-1-

[0050] { succinyl [methoxy (poly ethylene glycol)2000]}, N-palmitoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)5000]}, 1,2- dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (ammonium salt), and PEG-cholesterol, such as cholesterol-(poly ethylene glycol-600). In some cases, the lipid-containing particles (e.g., LNPs) in the compositions provided herein can contain a PEG-lipid. In some cases, the PEG-lipid or PEG-cholesterol can be modified with a targeting moiety, such as N-acetylgalactosamine (GalNAc) for liver targeting, or with another ligand or binding reagent, such as a polypeptide (e.g., an antibody or antibody fragment), an apolipoprotein (e.g., ApoE), a peptide, and / or a small molecule ligand similar to GalNAc.

[0051] Lipid-containing particles (e.g., LNPs) can be effective for delivering therapeutic agents (e.g., nucleic acids and nucleic acid analogs) to cells, often in vivo (see, e.g., Kulkarni et al., Nucleic Acid Therapeutics, 28(3): 146-157, 2018; Hajj and Whitehead, Nature Reviews Materials, 2: 17056, 2017; U.S. Publication No. 20130245107; and U.S. Patent No.

[0052] 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, treatment, or curative effect. Therapeutic agents include, without limitation, nucleic acids, nucleic acid analogs, proteins, polypeptides, small molecule drugs, antibiotics, antivirals, and cell-based therapies (e.g., CAR-T cell therapies).

[0053] In some cases, the lipid-containing particles provided herein can include a therapeutic agent that is, or comprises, a nucleic acid or nucleic acid analog. As used herein, the term “nucleic acid” includes any compound and / or substance that comprises a polymer of nucleotides (also referred to as “polynucleotides” or “oligonucleotides”). Exemplary nucleic acids include, without limitation, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an oc-L-ribo configuration (a diastereomer of LNA), 2’-amino-LNA having a 2’-amino functionalization, and 2’-amino-a-LNA having a 2’-amino functionalization) or hybrids thereof. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).

[0054] Nucleic acids and nucleic acid analogs have a backbone and a sequence of nucleobases. In the context of this document, the backbone monomer residues can be any suitable nucleic acid backbone monomer residues having a negative charge, such as a ribose or deoxyribose connected to another ribose or deoxyribose by a phosphodiester bond, or a backbone residue of a nucleic acid analog monomer. The backbone monomer can include both the structural “residue” component, such as the ribose in RNA, and any active groups that are modified in linking monomers together, such as the 5’ triphosphate and 3’ hydroxyl groups of a ribonucleotide, which are modified when polymerized into RNA to result in a negatively-charged phosphodiester linkage.

[0055] For example, a therapeutic agent can be a nucleic acid or nucleic acid analog having a negatively-charged backbone, including but not limited to single-stranded DNA, singlestranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding (e.g., versions that include one or more changes to the nucleotide components), or analogs of any of the preceding (e.g., synthetic molecules that mimic the structure and function of the original). With regard to overall structure and function of the nucleic acid or nucleic acid analog, the nucleic acid or nucleic acid analog can be, without limitation: mRNA (messenger RNA), siRNA (small interfering RNA), miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), IncRNA (long non-coding RNA), circRNA (circular RNA), antisense RNA, ncRNA (noncoding RNA), telomerase RNA, piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, hexose nucleic acid, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino oligomer, antisense oligonucleotide, ribozyme, deoxyribozyme, aptamer, cloning vector, phagemid, plasmid, lambda phage, cosmid, fosmid, or artificial chromosome.

[0056] RNA therapeutics are a class of RNA-based treatments that target specific genes or genetic pathways with high specificity. The use of RNA therapeutics can allow for transient expression or inhibition, which can reduce long-term side effects. RNA therapeutics utilize various forms of RNA to treat diseases such as, without limitation, infectious diseases, cancer, genetic disorders, cardiovascular diseases, and neurological diseases. Research has been ongoing since the 1990s, with significant success in cancer therapy in the early 2010s (see, e.g., Sahin et al., Nature Reviews Drug Discovery, 13:759-780, 2014). The RNA used in an RNA therapeutic 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 an RNA aptamer. In some cases, for example, an mRNA-based therapy can be used. mRNA-based therapies can trigger synthesis of proteins by delivering coding mRNA into cells, making such therapies particularly useful in vaccine development (see, e.g., DeFrancesco, Nature Biotechnology, 35: 193-197, 2017). The coding mRNA can be designed as a blueprint to generate a protein of interest (e.g., a reporter protein, a functional protein, or an antigen). In some embodiments, a protein of interest can be an antigen produced by a pathogen (e.g., a virus) or by a cancer cell. Such protein molecules can stimulate an adaptive immune response that teaches the body to identify and destroy the corresponding pathogen or cancer cells (see, e.g., Bae and Park, Advanced Drug Delivery Reviews, 158:4-16, 2020). mRNA vaccines (e.g., the Pfizer-BioNTech COVID-19 vaccine and the Moderna COVID-19 vaccine) were developed for use in combating the coronavirus disease during the COVID- 19 pandemic (see, Noor, Current Clinical Microbiology Reports, 8(3):178-185, 2021). In some cases, a designed mRNA can encode a reporter such as firefly luciferase. An mRNA encoding a desired protein (e.g., an mRNA encoding luciferase) can be delivered into cells using lipid-containing particles (e.g., LNPs) to produce the protein (e.g., luciferase) in vitro, through cell culture, and in vivo, such as in mouse models or in any other appropriate mammal (e.g., humans, non-human primates, rats, rabbits, cows, pigs, sheep, dogs, and / or cats). The mammal can be healthy or can have a disease, disorder, or clinical condition.

[0057] The lipid-containing particles provided herein can be generated using any appropriate method. For example, lipid-containing particles (e.g., LNPs) can be generated by combining an ionizable lipid or lipidoid provided herein, cholesterol or a cholesterol derivative, a helper lipid, and a PEG-based compound in any appropriate amounts or ratios. In some cases, lipid- containing particles (e.g., LNPs) can be prepared by combining: about 30 mol% to about 70 mol% (e.g., 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 55 to 65 mol%, about to 60 to 70 mol%, about 30 mol%, about 40 mol%, about 50 mol%, about 60 mol%, or about 70 mol%) of a lipid or lipidoid provided herein (e.g., 5030iio, 51406,IO, 500Oe, io, or 402Os,i2); 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%) of a helper lipid (e.g., DSPC, DOPE, or DOTAP); 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%) cholesterol or a cholesterol derivative (e.g., cholesterol); and 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 a PEG-based compound (e.g., PEG or a PEG-lipid).

[0058] In some cases, lipid-containing particles can be prepared by combining the above components at a molar ratio of about 35:46.5: 16:2.5 (lipidoid: cholesterol: helper lipid:PEG), or at a molar ratio of about 35:22.5:40:2.5 (lipidoid: cholesterol: helper lipid:PEG), or at any other appropriate molar ratio.

[0059] Lipid-containing particles (e.g., LNPs) prepared as described herein can be combined with any appropriate cargo (e.g., a nucleic acid or other therapeutic agent, and / or a marker). In some cases, the concentration of mRNAas a cargo in a lipid-containing particle (e.g., for in vitro cell culture) can be from about 0.001 mg / mL to about 2 mg / mL (e.g., from about 0.001 mg / mL to about 1.5 mg / mL, from about 0.003 mg / mL to about 1.5 mg / mL, from about 0.005 mg / mL to about 1.5 mg / mL, from about 0.003 mg / mL to about 1 mg / mL, from about 0.003 mg / mL to about 0.5 mg / mL, from about 0.005 to about 1.5 mg / mL, from about 0.005 to about 1 mg / mL, from about 0.005 to about 0.5 mg / mL, about 0.001 mg / mL, about 0.003 mg / mL, about 0.005 mg / mL, about 0.01 mg / mL, about 0.03 mg / mL, about 0.05 mg / mL, about 0.08 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 1.5 mg / mL, or about 2 mg / mL). In some cases, the concentration of mRNA as a cargo in a lipid-containing particle (e.g., for in vivo use) can be from about 0.01 mg / mL to about 10 mg / mL (e.g., from about 0.01 mg / mL to about 1 mg / mL, from about 0.03 mg / mL to about 3 mg / mL, from about 0.05 mg / mL to about 5 mg / mL, from about 0.1 mg / mL to about 2 mg / mL, from about 0.3 mg / mL to about 3 mg / mL, from about 0.5 mg / mL to about 5 mg / mL, from about 1 mg / mL to about 3 mg / mL, from about 3 to about 5 mg / mL, about 0.01 mg / mL, about 0.03 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.3 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, or about 10 mg / mL). In some cases, when a lipid-containing particle includes mRNA, the ratio of lipidoid:mRNA (weight / weight) in the lipid-containing particle can be from about 5: 1 to about 30: 1 (e.g., about 5: 1, about 8: 1, about 10: 1, about 12:1, about 15:1, about 20:1, about 25: 1, or about 30:1). This document also provides compositions that include lipid-containing particles (e.g., LNPs) with lipidoid components as provided herein, in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicles for delivering therapeutic agents to a subject. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport 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, without limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose or dextrose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate). In some cases, a composition provided herein can include one or more sugars (e.g., sucrose and / or trehalose) that can act as LNP stabilization / preservation agents.

[0060] Any appropriate method can be used to determine the effectiveness of a lipid- containing particle (e.g., LNP) provided herein. For example, any appropriate method can be used to determine the effectiveness of a lipid-containing particle (e.g., a LNP) to deliver a cargo (e.g., a therapeutic agent or a nucleic acid encoding a detectable marker) to a cell in vitro or in vivo. For example, in vitro efficacy can be evaluated by contacting cells in culture (e.g., primary cells obtained from a mammal, or cells from a cell line) with LNPs formulated with nucleic acid (e.g., mRNA) encoding a marker whose expression can be detected (e.g., luciferase or green fluorescent protein (GFP)). After incubating the LNPs with the cells, the cells can be assessed by measuring fluorescence or luminescence, for example, to determine whether (and the extent to which) the marker has been expressed in the cells. In some cases, in vivo efficacy can be assessed by administering LNPs formulated with nucleic acid (e.g., mRNA) encoding a marker whose expression can be detected (e.g., luciferase or GFP) to a mammal (e.g., a mouse or a rat) or a zebrafish, and after an appropriate length of time (e.g., 12 hours to 3 days after administration) assessing one or more tissues and / or organs (e.g., liver, spleen, lungs, heart, pancreas, kidneys, brain, muscle, and / or intestine) from the mammal for expression of the marker by, for example, measuring fluorescence or luminescence of the tissue(s) or organ(s). In some cases, a mammal can be administered LNPs formulated with nucleic acid (e.g., mRNA) encoding a marker whose expression can be detected (e.g., luciferase or GFP), and after an appropriate length of time, one or more tissues and / or organs from the mammal can be harvested for flow cytometry analysis to determine whether particular types of cells within the tissue(s) and / or organ(s) contain the marker.

[0061] In some cases, the lipid-containing particles provided herein can have specificity (also referred to as “tropism” or “targeting”) for one or more particular organs, tissues, or types of cells within an organ or tissue. As used herein, the terms “specificity,” “tropic” or “tropism,” and “targeting,” with regard to a particular lipid-containing particle (e.g., a LNP containing a lipidoid provided herein), mean that the lipid-containing particle is more likely to interact with (e.g., deliver a cargo to) the organ(s), tissue(s), and / or cell type(s) for which it has specificity, and is less likely to interact with other organs, tissues, and / or cell types. In some cases, For example, a lipid-containing particle that is “lung-tropic” (or that has “specificity” for or “targets” the lung and / or cells therein) is more likely to interact with the lung and cells therein than with other organs and cells. In some cases, a lipid-containing particle can have a preference for certain cell types within an organ or tissue. As described in the Example herein, for example, lipid-containing particles provided herein can target the lung, or lipid- containing particles provided herein can target the liver. In some cases, lipid-containing particles can target NK cells (e.g., NK cells within lung tissue). In some cases, the organ, tissue, or cell specificity of a lipid-containing particle can be conferred by the lipidoid(s) contained in the lipid-containing particle. For example, a lipid-containing particle that includes 5140e,io and / or 5030iio may be lung-tropic, while a lipid-containing particle that includes 50006, 10, 40208,12, 5030s,io, 50306, 10, 20506, 10, 2000iio, 40204,8, 40204,8, or a combination thereof may be liver-tropic. In some cases, when a lipid-containing particle is said to be “lung-tropic,” at least 15% (e.g., at least 20%, at least 25%, or at least 30%) of a detectable signal derived from the lipid-containing particle is found in the lung or lung tissue, with the remaining percentage of the detectable signal being found in other tissues or organs. In some cases, when a lipid-containing particle is said to be “liver-tropic,” at least 15% (e.g., at least 20%, at least 25%, or at least 30%) of a detectable signal derived from the lipid- containing particle is found in the liver or liver tissue, with the remaining percentage of the detectable signal being found in other tissues or organs.

[0062] Methods that include delivering RNA into cells can be useful in research and therapeutic applications, including gene silencing, gene editing, and mRNA-based therapeutics. As described herein, RNA delivery can be achieved via LNPs, which can avoid issues encountered with delivery of naked, single-stranded RNA (which is prone to nuclease degradation, can activate the immune system, and is too large and negatively charged to passively cross the cell membrane). Thus, this document provides methods that include delivering, to a mammal in need thereof, a lipid-containing particle (e.g., a LNP) described herein containing one or more therapeutic agents. In some cases, the one or more therapeutic agents can include nucleic acid (e.g., mRNA). The methods can include administering to a mammal a lipid-containing particle (e.g., LNP) that encapsulates the therapeutic agent (e.g., mRNA).

[0063] The methods provided herein can be used for delivering a therapeutic agent to a subject (e.g., a mammal such as a human, non-human primate, mouse, rat, rabbit, dog, cat, horse, cow, pig, or sheep). The methods can include administering to the subject a lipid- containing particle (e.g., a LNP) containing the therapeutic agent, the lipid-containing particle (e.g., LNP) can be administered to the subject via any appropriate route. For example, a lipid-containing particle (e.g., a LNP) can be administered intravenously, intramuscularly, or subcutaneously. A lipid-containing particle (e.g., a LNP) can be administered to a subject at a mRNA dose of about 0.01 to about 10 mg / kg. For example, a lipid-containing particle (e.g., a LNP) can be administered at a mRNA dose of about 0.01 to about 0.05 mg / kg, about 0.05 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.2 mg / kg to about 0.3 mg / kg, about 0.3 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 1 mg / kg to about 2 mg / kg, about 2 mg / kg to about 5 mg / kg, about 5 mg / kg to about 8 mg / kg, or about 8 mg / kg to about 10 mg / kg, or at a dose of about 0.01, about 0.05, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 4, about 5, about 6, about 7, about 7.5, about 8, about 9, or about 10 mg / kg). In some cases, a lipid-containing particle (e.g., a LNP) can be administered at a mRNA dose of about 0.5 mg / kg. As described herein, in some cases, the therapeutic agent contained within the lipid- containing particle (e.g., LNP) used in the methods provided herein can be a nucleic acid (e.g., an RNA). The RNA as the therapeutic agent can be a mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer. In some cases, an RNA encoding a marker (e.g., an mRNA encoding a luciferase polypeptide or a fluorescent polypeptide such as GFP, a yellow fluorescent polypeptide, or a red fluorescent polypeptide) can 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 utilized in various biological and medical research applications, particularly in reporter assays to study gene expression and cellular processes. The bioluminescence produced by luciferase encoded by the mRNA encapsulated in the lipid-containing particle (e.g., LNP), or the fluorescence produced by a fluorescent marker encoded by the mRNA encapsulated in the lipid-containing particle (e.g., LNP) can be used to assess the efficacy of mRNA delivery.

[0064] In some cases, the methods described herein can be used to treat a clinical disorder in a subject (e.g., mammal). As used in this context, to “treat” means to ameliorate (e.g., reduce or eliminate) at least one symptom of a disorder. Administration of a therapeutically effective amount of a lipid-containing particle described herein can result in more targeted LNP- mediate mRNA delivery. Thus, the methods described herein provide an approach to enhance the effectiveness of RNA therapeutics.

[0065] Disorders that can be treated according to methods provided herein include, without limitation, lung disorders, liver disorders, and diseases in which one or more genes or proteins is dysregulated. Lung disorders that can be treated as described herein include, without limitation, cystic fibrosis, non-small cell lung cancer, other forms of lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (such as pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, and combinations thereof. For example, a lung disorder in a mammal can be treated by administering a lipid-containing particle provided herein that is lung-tropic (e.g., a LNP containing a lipidoid having an N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2- diamine) alkyl amine head or an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head, and one or more 2-butylheptyl acrylate, 2-butyloctyl acrylate, 2-butylnonyl acrylate, 2-hexylnonyl acrylate, 2-hexyldecyl acrylate, 2-hexylundecyl acrylate, 2- octylundecyl acrylate, 2-octyldodecyl acrylate, 2-octyltridecyl acrylate tails, and optionally also containing a neutral helper lipid such as DSPC or DOPE) that targets cells in the lung, or that targets one or more types of cells within the lung. Liver disorders that can be treated as described herein include, without limitation, alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, and combinations thereof. For example, a liver disorder in a mammal can be treated by administering a lipid-containing particle provided herein that is liver-tropic (e.g., a LNP containing a lipidoid having an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2- diamine) alkyl amine head or a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head, and one or more 2-butylheptyl acrylate, 2-butyloctyl acrylate, 2-butylnonyl acrylate, 2-hexylnonyl acrylate, 2-hexyldecyl acrylate, 2-hexylundecyl acrylate, 2- octylundecyl acrylate, 2-octyldodecyl acrylate, 2-octyltridecyl acrylate tails) that targets cells in the liver, or that targets one or more types of cells within the liver. In some cases, the clinical disorder can be an inflammatory condition, an infectious disease, an autoimmune disease, a respiratory disease, a cancer, a genetic disorder, a metabolic disease, or any combination thereof.

[0066] Generally, the methods provided herein can include administering a therapeutically effective amount of a lipid-containing particle (e.g., LNP) as described herein, to a subject that is in need thereof or has been determined to be in need of, such treatment. The lipid- containing particle (e.g., LNP) encapsulates a therapeutic agent for the treatment needed. The therapeutic agent can be mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

[0067] Effective doses can vary depending on the severity of the disorder, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating clinician. An effective amount of a composition containing one or more HMOs and / or a lipid-containing particle (e.g., LNP) as described herein can be any amount that reduces one or more symptoms of the disorder (e.g., by at least 10, 25, 35, 45, 50, 55, 65, 75, 80, 90, or 100 percent) within a subject (e.g., a mammal), without producing severe toxicity in the mammal. As described herein, and an effective dose of a lipid-containing particle (e.g., LNP) 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 amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amounts used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple therapeutic agents, route of administration, severity of disorder, or risk level for development of the same or another disorder in the mammal being treated may require an increase or decrease in the actual effective amount administered.

[0068] If a particular mammal fails to respond to a particular amount of a lipid-containing particle (e.g., LNP), then the amount of the lipid-containing particle can be increased by, for example, two-fold. After receiving the higher amount, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments can be made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, route of administration, and severity of the disorder may require an increase or decrease in the actual effective amount administered.

[0069] The frequency of administration of one or more lipid-containing particles (e.g., LNPs) to a subject (e.g., a mammal) can be any frequency that reduces a symptom of a disorder in the subject, without producing significant toxicity to the subject. For example, the frequency of administration of a lipid-containing particle (e.g., LNP) can be from about four times daily to about once a day, from about once daily to three times a week, from about three times a week to about twice a week, from about twice a week to about once a week, or from about once a week to about once a month (e.g., from about once a week 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. A course of treatment with a lipid- containing particle (e.g., LNP) described herein can include rest periods. For example, a LNP can be administered daily over a one-week period followed by a one-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the disorder may require an increase or decrease in administration frequency.

[0070] An effective duration for administering one or more lipid-containing particles (e.g., LNPs) to a subject (e.g., a mammal) can be any duration that reduces a symptom of a disorder in the mammal, without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several days to several months to several years or more. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the effective amount, frequency of administration, use of multiple therapeutic agents, route of administration, and severity of the disorder being treated.

[0071] In some cases, the progression of a disorder in a subject (e.g., a mammal) or the severity of one or more symptoms related to the disorder in the subject being treated can be monitored. Any appropriate method can be used to determine whether or not a subject having a disorder is effectively being treated.

[0072] In some cases, the progression of a disorder in a mammal or the severity of one or more symptoms related to the disorder in the mammal being treated can be monitored. Any appropriate method can be used to determine whether or not a mammal is effectively being treated. For example, clinical scanning techniques (e.g., computed tomography (CT), positron emission tomography (PET) / CT, bone scan, magnetic resonance imaging (MRI)), and / or measurement of one or more markers in a biological sample can be used to determine the presence or absence of a disorder such as cancer within a mammal (e.g., a human) being treated. In such cases, a reduced number of tumor cells, reduced tumor size, or a reduction in a tumor marker can indicate effective treatment. Exemplary Embodiments

[0073] Embodiment 1 is a lipid-containing particle comprising: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipidoid comprises an alkyl amine head and one or more alkyl acrylate tails, wherein each of the one or more alkyl acrylate tails comprises an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch of each tail has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0074] Embodiment 2 is the lipid-containing particle of embodiment 1, wherein the lipidoid has two alkyl acrylate tails.

[0075] Embodiment 3 is the lipid-containing particle of embodiment 1, wherein the lipidoid has three alkyl acrylate tails.

[0076] Embodiment 4 is the lipid-containing particle of embodiment 1, wherein the lipidoid has four alkyl acrylate tails.

[0077] Embodiment 5 is the lipid-containing particle of embodiment 1, wherein the lipidoid has more than four alkyl acrylate tails.

[0078] Embodiment 6 is the lipid-containing particle of any one of embodiments 2-5, wherein each of the alkyl acrylate tails is the same.

[0079] Embodiment 7 is the lipid-containing particle of any one of embodiments 2-5, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0080] Embodiment 8 is the lipid-containing particle of any one of embodiments 1 -7, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0081] Embodiment 9 is the lipid-containing particle of any one of embodiments 1-8, wherein the helper lipid is a neutral lipid or a zwitterionic lipid.

[0082] Embodiment 10 is the lipid-containing particle of embodiment 9, wherein the neutral lipid or zwitterionic lipid comprises one or more of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero- 3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0083] Embodiment 11 is the lipid-containing particle of any one of embodiments 1-8, wherein the helper lipid is a cationic lipid.

[0084] Embodiment 12 is the lipid-containing particle of embodiment 11, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), l,2-dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0085] Embodiment 13 is the lipid-containing particle of any one of embodiments 1-12, wherein the lipid-containing particle is lung-tropic.

[0086] Embodiment 14 is the lipid-containing particle of embodiment 13, wherein the lipidoid comprises: an N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or an N1,Nll-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0087] Embodiment 15 is the lipid-containing particle of embodiment 13, wherein the lipidoid comprises an N1,Nll-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail.

[0088] Embodiment 16 is the lipid-containing particle of embodiment 15, wherein the lipid- containing particle targets natural killer (NK) and / or dendritic cells.

[0089] Embodiment 17 is the lipid-containing particle of embodiment 16, wherein the lipid- containing particle comprises about 10-40% neutral helper lipid or zwitterionic helper lipid.

[0090] Embodiment 18 is the lipid-containing particle of embodiment 17, wherein the neutral helper lipid or zwitterionic helper lipid comprises one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0091] Embodiment 19 is the lipid-containing particle of embodiment 17, wherein the lipid- containing particle comprises about 16% DOPE.

[0092] Embodiment 20 is the lipid-containing particle of any one of embodiments 1-12, wherein the lipid-containing particle is liver-tropic.

[0093] Embodiment 21 is the lipid-containing particle of embodiment 20, wherein the lipidoid comprises: an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head, a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head, a 2- (piperazin-l-yl)ethan-l -amine head, or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane- 1,2-diamine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2- butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2- hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0094] Embodiment 22 is the lipid-containing particle of embodiment 20, wherein the lipidoid comprises an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail.

[0095] Embodiment 23 is the lipid-containing particle of embodiment 20, wherein the lipidoid comprises a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l-amine alkyl amino head and a 2-octyldodecyl acrylate tail.

[0096] Embodiment 24 is the lipid-containing particle of any one of embodiments 1-23, wherein the cholesterol or derivative thereof is cholesterol.

[0097] Embodiment 25 is the lipid-containing particle of any one of embodiments 1-24, wherein the PEG-based compound is a PEG-lipid.

[0098] Embodiment 26 is the lipid-containing particle of embodiment 25, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0099] Embodiment 27 is a composition comprising a lipid-containing particle, wherein the lipid-containing particle comprises a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipidoid comprises an alkyl amine head and one or more alkyl acrylate tails, wherein the one or more alkyl acrylate tails comprise an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0100] Embodiment 28 is the composition of embodiment 27, wherein the therapeutic agent is a nucleic acid.

[0101] Embodiment 29 is the composition of embodiment 28, wherein the nucleic acid comprises an RNA.

[0102] Embodiment 30 is the composition of embodiment 29, wherein the RNA comprises one or more of a mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer.

[0103] Embodiment 31 is the composition of embodiment 29 or embodiment 30, wherein the RNA is an mRNA encoding luciferase.

[0104] Embodiment 32 is the composition of any one of embodiments 27-31, wherein the lipidoid has two alkyl acrylate tails.

[0105] Embodiment 33 is the composition of any one of embodiments 27-31, wherein the lipidoid has three alkyl acrylate tails.

[0106] Embodiment 34 is the composition of any one of embodiments 27-31, wherein the lipidoid has four alkyl acrylate tails.

[0107] Embodiment 35 is the composition of any one of embodiments 27-31, wherein the lipidoid has more than four alkyl acrylate tails.

[0108] Embodiment 36 is the composition of any one of embodiments 32-35, wherein each of the alkyl acrylate tails is the same.

[0109] Embodiment 37 is the composition of any one of embodiments 32-35, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0110] Embodiment 38 is the composition of any one of embodiments 27-37, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0111] Embodiment 39 is the composition of any one of embodiments 27-38, wherein the helper lipid is a neutral lipid or a zwitterionic lipid.

[0112] Embodiment 40 is the composition of embodiment 39, wherein the neutral lipid or zwitterionic lipid comprises one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0113] Embodiment 41 is the composition of any one of embodiments 27-38, wherein the helper lipid is a cationic lipid.

[0114] Embodiment 42 is the composition of embodiment 41, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2- dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn- glycero-3 -trimethylammoniumpropane (DPTAP), 1 ,2-distearoy 1-sn-gly cero-3 - trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0115] Embodiment 43 is the composition of any one of embodiments 27-42, wherein the lipid-containing particle is lung-tropic.

[0116] Embodiment 44 is the composition of embodiment 43, wherein the lipidoid comprises: an N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or an N1,Nll-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2- butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2- hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2- octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0117] Embodiment 45 is the composition of embodiment 43, wherein the lipidoid comprises an N1,Nll-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail.

[0118] Embodiment 46 is the composition of embodiment 45, wherein the lipid-containing particle targets natural killer (NK) and / or dendritic cells. Embodiment 47 is the composition of embodiment 46, wherein the lipid-containing particle comprises about 10-40% neutral helper lipid or zwitterionic helper lipid.

[0119] Embodiment 48 is the composition of embodiment 47, wherein the neutral helper lipid or zwitterionic helper lipid comprises one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0120] Embodiment 49 is the composition of embodiment 47, wherein the lipid-containing particle comprises about 16% DOPE.

[0121] Embodiment 50 is the composition of any one of embodiments 27-42, wherein the lipid-containing particle is liver-tropic.

[0122] Embodiment 51 is the composition of embodiment 50, wherein the lipidoid comprises: an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head; and a 2- butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2- hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2- octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0123] Embodiment 52 is the composition of embodiment 50, wherein the lipidoid comprises an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail.

[0124] Embodiment 53 is the composition of embodiment 50, wherein the lipidoid comprises a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head and a 2- octyldodecyl acrylate tail.

[0125] Embodiment 54 is the composition of any one of embodiments 27-53, wherein the cholesterol or derivative thereof is cholesterol.

[0126] Embodiment 55 is the composition of any one of embodiments 27-54, wherein the PEG-based compound is a PEG-lipid.

[0127] 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.

[0128] Embodiment 57 is a method for delivering a therapeutic agent to a mammal, wherein the method comprises administering to the mammal a composition comprising a lipid- containing particle, wherein the lipid-containing particle comprises a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)- based compound, wherein the lipidoid comprises an alkyl amine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0129] Embodiment 58 is the method of embodiment 57, wherein the administering comprises intravenous administration, intramuscular administration, or subcutaneous administration.

[0130] Embodiment 59 is the method of embodiment 57 or embodiment 58, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0131] Embodiment 60 is the method of any one of embodiments 57-59, wherein the therapeutic agent is a nucleic acid.

[0132] Embodiment 61 is the method of embodiment 60, wherein the nucleic acid comprises RNA.

[0133] Embodiment 62 is the method of embodiment 61, wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

[0134] Embodiment 63 is the 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.

[0135] Embodiment 64 is the method of embodiment 61 or embodiment 62, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0136] Embodiment 65 is the method of any one of embodiments 57-64, wherein the lipidoid has two alkyl acrylate tails.

[0137] Embodiment 66 is the method of any one of embodiments 57-64, wherein the lipidoid has three alkyl acrylate tails.

[0138] Embodiment 67 is the method of any one of embodiments 57-64, wherein the lipidoid has four alkyl acrylate tails.

[0139] Embodiment 68 is the method of any one of embodiments 57-64, wherein the lipidoid has more than four alkyl acrylate tails.

[0140] Embodiment 69 is the method of any one of embodiments 65-68, wherein each of the alkyl acrylate tails is the same. Embodiment 70 is the method of any one of embodiments 65-68, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0141] Embodiment 71 is the method of any one of embodiments 57-70, wherein the helper lipid is a neutral lipid or a zwitterionic lipid.

[0142] Embodiment 72 is the method of embodiment 71, wherein the neutral lipid or zwitterionic lipid comprises one or more of 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0143] Embodiment 73 is the method of any one of embodiments 57-72, wherein the helper lipid is a cationic lipid.

[0144] Embodiment 74 is the method of embodiment 73, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2- dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn- glycero-3 -trimethylammoniumpropane (DPTAP), 1 ,2-distearoy 1-sn-gly cero-3 - trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0145] Embodiment 75 is the method of any one of embodiments 57-74, wherein the cholesterol or a derivative thereof is cholesterol.

[0146] Embodiment 76 is the method of any one of embodiments 57-75, wherein the PEG- based compound is a PEG-lipid.

[0147] Embodiment 77 is the method of embodiment 76, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol. Embodiment 78 is the method of any one of embodiments 57-77, wherein the mammal has been identified as having a lung disorder, and wherein the lipid-containing particle is lung-tropic.

[0148] Embodiment 79 is the method of embodiment 78, wherein the lung disorder comprises cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post- viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0149] Embodiment 80 is the method of embodiment 78 or embodiment 79, wherein the lipidoid comprises: an N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0150] Embodiment 81 is the method of embodiment 80, wherein the lipidoid comprises an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail.

[0151] Embodiment 82 is the method of embodiment 80, wherein the lipid-containing particle targets natural killer (NK) and / or dendritic cells.

[0152] Embodiment 83 is the method of embodiment 82, wherein the lipid-containing particle comprises about 10-40% neutral helper lipid or zwitterionic helper lipid.

[0153] Embodiment 84 is the method of embodiment 83, wherein the neutral helper lipid or zwitterionic helper lipid comprises one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0154] Embodiment 85 is the method of embodiment 83, wherein the lipid-containing particle comprises about 16% DOPE.

[0155] Embodiment 86 is the method of any one of embodiments 57-77, wherein mammal has been identified as having a liver disorder, and wherein the lipid-containing particle is liver-tropic. Embodiment 87 is the method of embodiment 86, wherein the liver disorder comprises alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0156] Embodiment 88 is the method of embodiment 86 or embodiment 87, wherein the lipidoid comprises: an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head; and a 2- butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2- hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2- octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0157] Embodiment 89 is the method of embodiment 88, wherein the lipidoid comprises an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail.

[0158] Embodiment 90 is the method of embodiment 88, wherein the lipidoid comprises a 4- (2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head and a 2-octyldodecyl acrylate tail.

[0159] Embodiment 91 is the method of any one of embodiments 57-90, wherein the mammal is a human.

[0160] Embodiment 92 is a method for treating a mammal having a lung disorder or a symptom thereof, or having a liver disorder or a symptom thereof, wherein the method comprises administering to the mammal a composition comprising a lipid-containing particle comprising a therapeutic agent, wherein the lipid-containing particle further comprises: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)- based compound, wherein the lipidoid comprises an alkyl amine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons. Embodiment 93 is the method of embodiment 92, wherein the administering comprises intravenous administration, intramuscular administration, or subcutaneous administration.

[0161] Embodiment 94 is the method of embodiment 92 or embodiment 93, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0162] Embodiment 95 is the method of any one of embodiments 92-94, wherein the therapeutic agent is a nucleic acid.

[0163] Embodiment 96 is the method of embodiment 95, wherein the nucleic acid comprises RNA.

[0164] Embodiment 97 is the method of embodiment 96, wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

[0165] Embodiment 98 is the method of embodiment 96 or embodiment 97, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0166] Embodiment 99 is the method of embodiment 96 or embodiment 97, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0167] Embodiment 100 is the method of any one of embodiments 92-99, wherein the lipidoid has two alkyl acrylate tails.

[0168] Embodiment 101 is the method of any one of embodiments 92-99, wherein the lipidoid has three alkyl acrylate tails.

[0169] Embodiment 102 is the method of any one of embodiments 92-99, wherein the lipidoid has four alkyl acrylate tails.

[0170] Embodiment 103 is the method of any one of embodiments 99-99, wherein the lipidoid has more than four alkyl acrylate tails.

[0171] Embodiment 104 is the method of any one of embodiments 100-103, wherein each of the alkyl acrylate tails is the same.

[0172] Embodiment 105 is the method of any one of embodiments 100-103, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0173] Embodiment 106 is the method of any one of embodiments 92-105, wherein the helper lipid is a neutral lipid or a zwitterionic lipid. Embodiment 107 is the method of embodiment 106, wherein the neutral lipid or zwitterionic lipid comprises one or more of 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0174] Embodiment 108 is the method of any one of embodiments 92-105, wherein the helper lipid is a cationic lipid.

[0175] Embodiment 109 is the method of embodiment 108, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2- dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn- glycero-3 -trimethylammoniumpropane (DPTAP), 1 ,2-distearoy 1-sn-gly cero-3 - trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0176] Embodiment 110 is the method of any one of embodiments 92-109, wherein the cholesterol or derivative thereof is cholesterol.

[0177] Embodiment 111 is the method of any one of embodiments 92-110, wherein the PEG- based compound is a PEG-lipid.

[0178] Embodiment 112 is the method of embodiment 111, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0179] Embodiment 113 is the method of any one of embodiments 92-112, wherein the mammal has been identified as having a lung disorder, and wherein the lipid-containing particle is lung-tropic. Embodiment 114 is the method of embodiment 113, wherein the lung disorder comprises cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post- viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0180] Embodiment 115 is the method of embodiment 113 or embodiment 114, wherein the lipidoid comprises: an N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0181] Embodiment 116 is the method of embodiment 115, wherein the lipidoid comprises an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail.

[0182] Embodiment 117 is the method of embodiment 115, wherein the lipid-containing particle targets natural killer (NK) and / or dendritic cells.

[0183] Embodiment 118 is the method of embodiment 117, wherein the lipid-containing particle comprises about 10-40% neutral helper lipid or zwitterionic helper lipid.

[0184] Embodiment 119 is the method of embodiment 118, wherein the neutral helper lipid or zwitterionic helper lipid comprises one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0185] Embodiment 120 is the method of embodiment 118, wherein the lipid-containing particle comprises about 16% DOPE.

[0186] Embodiment 121 is the method of any one of embodiments 92-112, wherein mammal has been identified as having a liver disorder, and wherein the lipid-containing particle is liver-tropic.

[0187] Embodiment 122 is the method of embodiment 121, wherein the liver disorder comprises alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0188] Embodiment 123 is the method of embodiment 121 or embodiment 122, wherein the lipidoid comprises: an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head; and a 2- butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2- hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2- octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0189] Embodiment 124 is the method of embodiment 123, wherein the lipidoid comprises an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail.

[0190] Embodiment 125 is the method of embodiment 123, wherein the lipidoid comprises a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head and a 2-octyldodecyl acrylate tail.

[0191] Embodiment 126 is the method of any one of embodiments 92-125, wherein the mammal is a human.

[0192] Embodiment 127 is a method for delivering a therapeutic agent to a lung cell, wherein the method comprises contacting the lung cell with a composition comprising a lipid- containing particle that comprises a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipidoid comprises an alkyl amine head and one or more alkyl acrylate tails, wherein the alkyl acrylate tails comprise an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch has n carbons and the second branch has n+1, n+2, or n+3 carbons.

[0193] Embodiment 128 is the method of embodiment 127, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0194] Embodiment 129 is the method of embodiment 127 or embodiment 128, wherein the lipidoid has two alkyl acrylate tails.

[0195] Embodiment 130 is the method of embodiment 127 or embodiment 128, wherein the lipidoid has three alkyl acrylate tails. Embodiment 131 is the method of embodiment 127 or embodiment 128, wherein the lipidoid has four alkyl acrylate tails.

[0196] Embodiment 132 is the method of embodiment 127 or embodiment 128, wherein the lipidoid has more than four alkyl acrylate tails.

[0197] Embodiment 133 is the method of any one of embodiments 129-132, wherein each of the alkyl acrylate tails is the same.

[0198] Embodiment 134 is the method of any one of embodiments 129-132, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0199] Embodiment 135 is the method of any one of embodiments 127-134, wherein the helper lipid is a neutral lipid or a zwitterionic helper lipid.

[0200] Embodiment 136 is the method of embodiment 135, wherein the neutral lipid or zwitterionic helper lipid comprises one or more of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero- 3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0201] Embodiment 137 is the method of any one of embodiments 127-134, wherein the helper lipid is a cationic lipid.

[0202] Embodiment 138 is the method of embodiment 137, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2- dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn- glycero-3 -trimethylammoniumpropane (DPTAP), 1 ,2-distearoy 1-sn-gly cero-3 - trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Embodiment 139 is the method of any one of embodiments 127-138, wherein the cholesterol or derivative thereof is cholesterol.

[0203] Embodiment 140 is the method of any one of embodiments 127-139, wherein the PEG-based compound is a PEG-lipid.

[0204] Embodiment 141 is the method of embodiment 140, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0205] Embodiment 142 is the method of any one of embodiments 127-141, wherein the lung cell is within a mammal.

[0206] Embodiment 143 is the method of embodiment 142, wherein the mammal is a human.

[0207] Embodiment 144 is the method of any one of embodiments 127-143, wherein the therapeutic agent is a nucleic acid.

[0208] Embodiment 145 is the method of embodiment 144, wherein the nucleic acid comprises RNA.

[0209] Embodiment 146 is the method of embodiment 145, wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

[0210] Embodiment 147 is the method of embodiment 145 or embodiment 146, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0211] Embodiment 148 is the method of embodiment 145 or embodiment 146, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0212] Embodiment 149 is the method of any one of embodiments 127-148, wherein the administering comprises intravenous administration, intramuscular administration, or subcutaneous administration.

[0213] Embodiment 150 is the method of any one of embodiments 127-149, wherein the lung cell is within a mammal identified as having a lung disorder.

[0214] Embodiment 151 is the method of embodiment 150, wherein the lung disorder comprises cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof. Embodiment 152 is the method of embodiment 150 or embodiment 151, wherein the lipidoid comprises: an N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

[0215] Embodiment 153 is the method of embodiment 152, wherein the lipidoid comprises an N1,N1'-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2- hexyldecyl acrylate tail.

[0216] Embodiment 154 is the method of embodiment 152, wherein the lipid-containing particle targets natural killer (NK) and / or dendritic cells.

[0217] Embodiment 155 is the method of embodiment 154, wherein the lipid-containing particle comprises about 10-40% neutral helper lipid or zwitterionic helper lipid.

[0218] Embodiment 156 is the method of embodiment 155, wherein the neutral helper lipid or zwitterionic helper lipid comprises one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0219] Embodiment 157 is the method of embodiment 155, wherein the lipid-containing particle comprises about 16% DOPE.

[0220] Embodiment 158 is a lipid-containing particle comprising: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipidoid comprises an alkyl amino head and one or more alkyl acrylate tails, wherein the alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,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.

[0221] Embodiment 159 is the lipid-containing particle of embodiment 158, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0222] Embodiment 160 is the lipid-containing particle of embodiment 158, wherein the lipidoid has two alkyl acrylate tails. Embodiment 161 is the lipid-containing particle of embodiment 158, wherein the lipidoid has three alkyl acrylate tails.

[0223] Embodiment 162 is the lipid-containing particle of embodiment 158, wherein the lipidoid has four alkyl acrylate tails.

[0224] Embodiment 163 is the lipid-containing particle of embodiment 158, wherein the lipidoid has more than four alkyl acrylate tails.

[0225] Embodiment 164 is the lipid-containing particle of any one of embodiments 160-163, wherein each of the alkyl acrylate tails is the same.

[0226] Embodiment 165 is the lipid-containing particle of any one of embodiments 160-163, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0227] Embodiment 166 is the lipid-containing particle of any one of embodiments 158-165, wherein the helper lipid is a neutral lipid or a zwitterionic lipid.

[0228] Embodiment 167 is the lipid-containing particle of embodiment 166, wherein the neutral lipid or zwitterionic lipid comprises one or more of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero- 3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0229] Embodiment 168 is the lipid-containing particle of any one of embodiments 158-165, wherein the helper lipid is a cationic lipid.

[0230] Embodiment 169 is the lipid-containing particle of embodiment 168, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3 -trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3-trimethylammoniumpropane (DPTAP), 1 ,2-distearoyl-sn-glycero- 3 -trimethylammoniumpropane (DSTAP), 3 P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), Dimethyldioctadecylammonium Bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0231] Embodiment 170 is the lipid-containing particle of any one of embodiments 158-169, wherein the lipidoid comprises a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail.

[0232] Embodiment 171 is the lipid-containing particle of any one of embodiments 158-169, wherein the lipidoid comprises a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail.

[0233] Embodiment 172 is the lipid-containing particle of any one of embodiments 158-169, wherein the lipidoid comprises a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2- diamine head and a 9-methyldecyl acrylate tail.

[0234] Embodiment 173 is the lipid-containing particle of any one of embodiments 158-172, wherein the cholesterol or derivative thereof is cholesterol.

[0235] Embodiment 174 is the lipid-containing particle of any one of embodiments 158-173, wherein the PEG-based compound is a PEG-lipid.

[0236] Embodiment 175 is the lipid-containing particle of embodiment 174, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0237] Embodiment 176 is a composition comprising a lipid-containing particle, wherein the lipid-containing particle comprises a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein the lipidoid comprises an alkyl amino head and one or more alkyl acrylate tails, wherein the alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,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.

[0238] Embodiment 177 is the composition of embodiment 176, wherein the therapeutic agent is a nucleic acid.

[0239] Embodiment 178 is the composition of embodiment 177, wherein the nucleic acid comprises an RNA. Embodiment 179 is the composition of embodiment 178, wherein the RNA comprises one or more of a mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long noncoding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer.

[0240] Embodiment 180 is the composition of embodiment 178 or embodiment 179, wherein the RNA is an mRNA encoding luciferase.

[0241] Embodiment 181 is the composition of any one of embodiments 176-180, wherein the lipidoid has two alkyl acrylate tails.

[0242] Embodiment 182 is the composition of any one of embodiments 176-180, wherein the lipidoid has three alkyl acrylate tails.

[0243] Embodiment 183 is the composition of any one of embodiments 176-180, wherein the lipidoid has four alkyl acrylate tails.

[0244] Embodiment 184 is the composition of any one of embodiments 176-180, wherein the lipidoid has more than four alkyl acrylate tails.

[0245] Embodiment 185 is the composition of any one of embodiments 181-184, wherein each of the alkyl acrylate tails is the same.

[0246] Embodiment 186 is the composition of any one of embodiments 181-184, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0247] Embodiment 187 is the composition of any one of embodiments 176-186, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0248] Embodiment 188 is the composition of any one of embodiments 176-187, wherein the helper lipid is a neutral lipid or a zwitterionic lipid.

[0249] Embodiment 189 is the composition of embodiment 188, wherein the neutral lipid or zwitterionic lipid comprises one or more of 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide. Embodiment 190 is the composition of any one of embodiments 176-187, wherein the helper lipid is a cationic lipid.

[0250] Embodiment 191 is the composition of embodiment 41, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2- dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn- glycero-3 -trimethylammoniumpropane (DPTAP), 1 ,2-distearoy 1-sn-gly cero-3 - trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0251] Embodiment 192 is the composition of any one of embodiments 176-191, wherein the lipidoid comprises a N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail.

[0252] Embodiment 193 is the composition of any one of embodiments 176-191, wherein the lipidoid comprises a N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail.

[0253] Embodiment 194 is the composition of any one of embodiments 176-191, wherein the lipidoid comprises a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head and a 9-methyldecyl acrylate tail.

[0254] Embodiment 195 is the composition of any one of embodiments 176-194, wherein the cholesterol or derivative thereof is cholesterol.

[0255] Embodiment 196 is the composition of any one of embodiments 176-195, wherein the PEG-based compound is a PEG-lipid.

[0256] 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.

[0257] Embodiment 198 is a method for delivering a therapeutic agent to a mammal, wherein the method comprises administering to the mammal a composition comprising a lipid- containing particle, wherein the lipid-containing particle comprises a therapeutic agent and: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)- based compound, wherein the lipidoid comprises an alkyl amino head and one or more alkyl acrylate tails, wherein the alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2-aminoethyl)piperazin-l- yl)methyl)ethane-l,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.

[0258] Embodiment 199 is the method of embodiment 198, wherein the administering comprises intravenous administration, intramuscular administration, or subcutaneous administration.

[0259] Embodiment 200 is the method of embodiment 198 or embodiment 199, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0260] Embodiment 201 is the method of any one of embodiments 198-200, wherein the therapeutic agent is a nucleic acid.

[0261] Embodiment 202 is the method of embodiment 201, wherein the nucleic acid comprises RNA.

[0262] Embodiment 203 is the method of embodiment 202, wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

[0263] Embodiment 204 is the method of embodiment 202 or embodiment 203, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0264] Embodiment 205 is the method of embodiment 202 or embodiment 203, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0265] Embodiment 206 is the method of any one of embodiments 198-205, wherein the lipidoid has two alkyl acrylate tails.

[0266] Embodiment 207 is the method of any one of embodiments 198-205, wherein the lipidoid has three alkyl acrylate tails.

[0267] Embodiment 208 is the method of any one of embodiments 198-205, wherein the lipidoid has four alkyl acrylate tails.

[0268] Embodiment 209 is the method of any one of embodiments 198-205, wherein the lipidoid has more than four alkyl acrylate tails.

[0269] Embodiment 210 is the method of any one of embodiments 206-209, wherein each of the alkyl acrylate tails is the same. Embodiment 211 is the method of any one of embodiments 206-209, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0270] Embodiment 212 is the method of any one of embodiments 198-211, wherein the helper lipid is a neutral lipid or a zwitterionic lipid.

[0271] Embodiment 213 is the method of embodiment 212, wherein the neutral lipid or zwitterionic lipid comprises one or more of 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0272] Embodiment 214 is the method of any one of embodiments 198-211, wherein the helper lipid is a cationic lipid.

[0273] Embodiment 215 is the method of embodiment 214, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2- dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn- glycero-3 -trimethylammoniumpropane (DPTAP), 1 ,2-distearoy 1-sn-gly cero-3 - trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0274] Embodiment 216 is the method of any one of embodiments 198-215, wherein the cholesterol or derivative thereof is cholesterol.

[0275] Embodiment 217 is the method of any one of embodiments 198-216, wherein the PEG-based compound is a PEG-lipid.

[0276] Embodiment 218 is the method of embodiment 217, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol. Embodiment 219 is the method of any one of embodiments 198-218, wherein the mammal has been identified as having a lung disorder.

[0277] Embodiment 220 is the method of embodiment 219, wherein the lung disorder comprises cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post- viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0278] Embodiment 221 is the method of any one of embodiments 198-218, wherein mammal has been identified as having a liver disorder.

[0279] Embodiment 222 is the method of embodiment 221, wherein the liver disorder comprises alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0280] Embodiment 223 is the method of any one of embodiments 198-222, wherein the lipidoid comprises a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail.

[0281] Embodiment 224 is the method of any one of embodiments 198-222, wherein the lipidoid comprises a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail.

[0282] Embodiment 225 is the method of any one of embodiments 198-222, wherein the lipidoid comprises a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head and a 9-methyldecyl acrylate tail.

[0283] Embodiment 226 is the method of any one of embodiments 198-225, wherein the mammal is a human.

[0284] Embodiment 227 is a method for treating a mammal having a lung disorder or a symptom thereof, or having a liver disorder or a symptom thereof, wherein the method comprises administering to the mammal a composition comprising a lipid-containing particle comprising a therapeutic agent, wherein the lipid-containing particle further comprises: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)- based compound, wherein the lipidoid comprises an alkyl amino head and one or more alkyl acrylate tails, wherein the alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head or a N1-((4-)2-aminoethyl)piperazin-l- yl)methyl)ethane-l,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.

[0285] Embodiment 228 is the method of embodiment 227, wherein the administering comprises intravenous administration, intramuscular administration, or subcutaneous administration.

[0286] Embodiment 229 is the method of embodiment 227 or embodiment 228, wherein the lipid-containing particle is a lipid nanoparticle (LNP).

[0287] Embodiment 230 is the method of any one of embodiments 227-229, wherein the therapeutic agent is a nucleic acid.

[0288] Embodiment 231 is the method of embodiment 230, wherein the nucleic acid comprises RNA.

[0289] Embodiment 232 is the method of embodiment 231, wherein the RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

[0290] Embodiment 233 is the method of embodiment 231 or embodiment 232, wherein the RNA is administered at a dose of about 0.01 mg / kg to about 10 mg / kg.

[0291] Embodiment 234 is the method of embodiment 231 or embodiment 232, wherein the RNA is administered at a dose of about 0.5 mg / kg.

[0292] Embodiment 235 is the method of any one of embodiments 227-234, wherein the lipidoid has two alkyl acrylate tails.

[0293] Embodiment 236 is the method of any one of embodiments 227-234, wherein the lipidoid has three alkyl acrylate tails.

[0294] Embodiment 237 is the method of any one of embodiments 227-234, wherein the lipidoid has four alkyl acrylate tails.

[0295] Embodiment 238 is the method of any one of embodiments 227-234, wherein the lipidoid has more than four alkyl acrylate tails. Embodiment 239 is the method of any one of embodiments 235-238, wherein each of the alkyl acrylate tails is the same.

[0296] Embodiment 240 is the method of any one of embodiments 235-238, wherein the lipidoid comprises two or more different alkyl acrylate tails.

[0297] Embodiment 241 is the method of any one of embodiments 227-240, wherein the helper lipid is a neutral lipid or a zwitterionic lipid.

[0298] Embodiment 242 is the method of embodiment 241, wherein the neutral lipid or zwitterionic lipid comprises one or more of 1 ,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

[0299] Embodiment 243 is the method of any one of embodiments 227-240, wherein the helper lipid is a cationic lipid.

[0300] Embodiment 244 is the method of embodiment 243, wherein the cationic lipid comprises one or more of l,2-dileoyl-3-trimethylammonium-propane (DOTAP), 1,2- dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn- glycero-3 -trimethylammoniumpropane (DPTAP), 1 ,2-distearoy 1-sn-gly cero-3 - trimethylammoniumpropane (DSTAP), 3P-[N-(N',N'-dimethylaminoethane)- carbamoyl] cholesterol (DC-cholesterol), di dodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

[0301] Embodiment 245 is the method of any one of embodiments 227-244, wherein the cholesterol or derivative thereof is cholesterol.

[0302] Embodiment 246 is the method of any one of embodiments 227-245, wherein the PEG-based compound is a PEG-lipid. Embodiment 247 is the method of embodiment 246, wherein the PEG has a molecular weight of about 300 g / mol to about 5000 g / mol.

[0303] Embodiment 248 is the method of any one of embodiments 227-247, wherein the lung disorder comprises cystic fibrosis, non-small cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

[0304] Embodiment 249 is the method of any one of embodiments 227-247, wherein the liver disorder comprises alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

[0305] Embodiment 250 is the method of any one of embodiments 227-249, wherein the lipidoid comprises a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail.

[0306] Embodiment 251 is the method of any one of embodiments 227-249, wherein the lipidoid comprises a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail.

[0307] Embodiment 252 is the method of any one of embodiments 227-249, wherein the lipidoid comprises a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head and a 9-methyldecyl acrylate tail.

[0308] Embodiment 253 is the method of any one of embodiments 227-252, wherein the mammal is a human.

[0309] The invention will be further described in the following example, which does not limit the scope of the invention described in the claims. EXAMPLE

[0310] Methods and Materials

[0311] Lipidoid Library Synthesis'. Each of the ionizable lipidoids was synthesized by combining an alkyl-amine head with alkyl-acrylate tails (FIG. 1A). Stoichiometric ratio of 1:4 was used. Reagents were combined in a scintillation vial kept at 90°C for 3 days while stirring (see, Hajj et al., Small 2019, 15(6):el805097; and Strelkova Petersen et al., Eur. J. Pharm. Biopharm. 2023, 192: 126). Twenty-nine (29) amine heads (80 through 602) were each combined with one of 20 acrylate tails, resulting in a library of 580 ionizable lipid members. For detailed structures and origins of amine heads and acrylate tails, see FIGS. 1B-1C and TABLES 1 and 2

[0312] TABLE 1: List of amine heads used for ionizable lipidoid library synthesis TABLE 2: List of acrylate tails used for ionizable lipidoid library synthesis

[0313] LNP Fabrication'. LNPs were formulated by combining a lipid solution with an aqueous mRNA solution, as described elsewhere (Hajj et al. 2020, supra,' and Strelkova Petersen et al., supra). The lipid solution consisted of ionizable lipidoid (made in the lab), cholesterol (Sigma- Aldrich, St Louis, MO), DOPE (l,2-dioleoyl-sn-glycero-3- phosphoethanolamine) from Avanti Polar Lipids, Alabaster, AL, and PEG2000 (14:0 PEG2000 PE) from Avanti Polar Lipids. These four compounds were combined at a molar ratio of 35:46.5:16:2.5 (lipidoid: cholesterol: helper lipid: PEG) for 16PE formulation, or at a molar ratio of 35:22.5:40:2.5 for 40PE and 40TAP formulations. Lipids were then dissolved in ethanol / citrate buffer solution (9: 1 ethanol to citrate buffer v / v). An aqueous solution was prepared containing 5-methoxyuridine (5moU) base modified mRNA dissolved in 10 mm citrate buffer (pH 4), ensuring a resulting lipidoid to mRNA ratio of 10:1 w / w. LNPs were formulated at 0.09 mg / mL mRNA concentration. To form LNPs, the lipid solution was added to the mRNA solution using a pipette at a volumetric ratio of 1 : 1. Immediately following addition, the LNPs were vortexed at 3200 rpm for 5 seconds. LNPs employed in animal experiments were subsequently dialyzed against phosphate buffered saline (PBS, VWR, Randor, PA) in a dialysis cassette (Thermo Fisher, Waltham, MA) with a 3.5 kDa molecular weight cut off membrane for 1 hour.

[0314] Entrapment. To measure RNA entrapment, intact and lysed nanoparticles were measured for RNA content using a Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher) 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, LNPs were diluted in equal volumes of Tris-EDTA buffer or 2% Triton X-100 in DI water buffer, and then an equal volume of RiboGreen reagent was added to each sample and incubated at 37°C for 15 minutes. The fluorescence was read on a Tecan SparkMultimode Microplate Reader. The excitation signal was 480 nm and emission 520 nm.

[0315] Zeta Potential'. Zeta potentials were determined using Malvern Zetasizer Nano ZSP (Malvern, UK). Lipid nanoparticles were diluted 1 : 100 from 0.005 mg / mL for testing. Three technical replicates were conducted on each sample to measure surface zeta potential.

[0316] Nanoparticle Tracking Analysis'. LNP size was analyzed using the NanoSight LM10 system (NanoSight, Ltd., Amesbury, UK), configured with a 405 nm laser and a high- sensitivity digital camera system (OrcaFlash2.8, Hamamatsu Cl 1440; NanoSight, Ltd., Malvern, UK). The camera shutter speed was fixed at 30.01 ms, and camera gain was set to 500. Videos were collected and analyzed using the NTA software (version 2.3), with the minimal expected particle size, minimum track length, and blur setting all set to automatic.

[0317] TNS Assay: 2-(ptoluidinyl) naphthalene-6-sulphonic acid (TNS) fluorescent probe (Sigma-Aldrich, St. Louis, MO) was utilized to assess LNP ionization at pH 5.0, following a protocol described elsewhere (Hajj et al. 2019, supra). Each well of a black 96 flat well plate received 250 pL 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 postmixing). Subsequently, 5 pL of LNP (0.09 mg / mL mRNA concentration) and 10 pL of 0.16 mm TNS stock solution were added to each well. The resulting fluorescence signal was promptly measured using a Synergy Hl plate reader (BioTek, Winooski, VT). LNP Efficacy In Vitro'. HeLa cells were seeded at a concentration of 15,000 cells per well of a white 96 flat well plate (Greiner Bio-One, Monroe, NC). LNPs were formulated with mRNA encoding firefly luciferase (TriLink BioTechnologies, San Diego, CA) at 0.09 mg / mL mRNA concentration and diluted with PBS to 0.005 mg / mL mRNA concentration. Twenty (20) pL of LNP was added per well. Twenty-four (24) hours after LNPs were added to cells, Dulbecco’s Modified Eagle Medium (DMEM) media was aspirated from the wells, and 50 pL of a PBS / BrightGlo reagent (Promega, Madison, WI) mixture (1 : 1 v:v) was then added per well. The plate was incubated in the dark at room temperature for 5 minutes, and resulting luminescence was detected with a Spark plate reader (TEC AN, Switzerland). Each LNP was tested in 4 biological replicates.

[0318] Animal Experiments'. All animal experiments were conducted in accordance with all state and local regulations.

[0319] LNP Efficacy in Murine Organs'. A firefly luciferase D-Luciferin reporter system was used to measure LNP efficacy in mice. LNPs were made with firefly luciferase mRNA (TriLink Biotechnologies) at a total mRNA concentration of 0.09 mg / mL. Six-to-eight (6 to 8) week old C57BL / 6 female mice (Charles River Laboratories, Wilmington, MA) were injected with LNPs via tail vein at a total mRNA dose of 0.75 mg / kg. 130 pL of 30 mg / mL D-Luciferin (PerkinElmer, Waltham, MA) was injected IP 3 hours after LNP injection, and mice were sacrificed fifteen minutes later using CO2 gas and cervical dislocation. Organs of interest (liver, spleen, lungs, heart, pancreas, and kidneys) were transferred to black construction paper and their luminescence signal was measured using an IVIS imaging device (PerkinElmer).

[0320] LNP Efficacy in Lung Cell Types'. GFP was used to assess which lung cell types were transfected. For these experiments, LNPs were formulated with GFPmRNA (TriLink Biotechnologies) at a total mRNA concentration of 0.09 mg / mL. LNPs were administered through the tail vein into 6-8 week-old C57BL / 6 female mice (Charles River Laboratories). Mice received a total dose of 0.75 mg mRNA per kg. 24 hours after LNP injection, mice were sacrificed 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 to for histology. Flow Cytometry. Harvested lungs were washed in DMEM and placed into 1.27 mL of enzyme PBS solution (1000 U / mL Type 1 Collagenase and 16 U / mL DNAse in Ca2+and Mg2+depleted PBS). Lungs were incubated in the enzyme solution on a thermal shaker (VWR, International) at 37°C and 1200 rpm for 25 minutes, extruded through an 18G needle (BD Biosciences, Franklin Lakes, NJ), and returned to the shaker for an additional 25 minute incubation. The resulting suspension was vigorously mixed with a pipette and transferred to a 50 mL tube (Corning, Falcon, Corning, NY) through a 70 pm cell strainer (Avantor, VWR, International). The cell strainer contents were then washed with an additional 1 mL of PBS. Cells were spun down for 5 minutes at 300 g with gentle acceleration and deceleration, and resuspended in 3 mL of R&D Systems’ Red Blood Cell lysis buffer. After a 5 minute incubation in the dark at room temperature, 15 mL of PBS was added to the RBC lysis buffer cell suspension, and cells were spun down for 5 minutes at 300 g with gentle acceleration and deceleration. The cell pellet was then resuspended in 2 mL of PBS and the resulting suspension was separated into two tubes. The contents of one tube were stained with fixable yellow viability dye (Thermo Fisher Scientific) according to the manufacturer’s instructions. Both tubes were spun down for 5 minutes at 300 g with gentle acceleration and deceleration, and cells were resuspended in 1 mL of 4% formaldehyde solution. After incubation for 10 minutes at room temperature, 5 mL of flow buffer (5% FBS in PBS) were added, and cells were spun down for 5 minutes at 400 g with regular acceleration and deceleration. Cells were washed with 5 mL of flow buffer two more times. After the last wash, cells were resuspended in 1 mL of flow buffer, and samples were transferred to a 96 well plate (200 pL per full stained sample and 50 pL per single stain control) and spun down for 5 minutes at 400 g. Cells were then resuspended in an appropriate antibody staining solution with the resulting concentration of each antibody equal to 1 pg of antibody per 1 mL of permeability buffer (R&D Systems). For the full list of antibodies and staining panels, see TABLE 3. After samples were incubated in the dark for 45 minutes at 4°C, 100 pL of permeability buffer was added and cells were spun down for 5 minutes at 400 g. Samples were then washed once with permeability buffer and once with flow buffer to be finally resuspended in 100 pL of flow buffer per sample. Resulting samples were analyzed with the Nova-Cyte 3000 flow cytometer (ACEA Biosciences, Inc., San Diego, CA) using gating from FIG. 8 and NovaExpress software, using internal auto-compensation based on single stained controls. Lung Histology. Histology was performed as described elsewhere (LoPresti et al., supra). Specifically, right lobes were incubated in 50 mL of 4% formaldehyde PBS solution for 48 hours, washed in PBS, and then transferred to 30% sucrose for another 48 hours. Fixed tissue was cryoprotected in 30% sucrose. After cryoprotection, tissue was embedded in the OCT (optimal cutting temperature) compound from Tissue-Tek, and 25 pm sections were produced using a cryotome. Sections were permeabilized in a 0.1% Triton X 100 solution (Millipore-Sigma) for 10 minutes at 25°C. Nonspecific binding was minimized by blocking with 10% goat serum for 20 minutes at 25°C. Sections were then rinsed in wash buffer (PBS, 0.1% BSA) and incubated with a 1 : 100 dilution (PBS with 0.1% BSA) of Alexa Fluor 488 anti-GFP antibody (#338 007, BioLegend) overnight at 4°C. Sections were then washed three times with wash buffer and stained with DyLight 594-Phalloidin (#12 877, Cell Signaling Technology) for 15 minutes at 25°C. Each section was washed three times with PBS buffer and then stained with Hoechst (Thermo Fisher, 1 : 1000 in PBS) for 5 minutes, followed by additional washing with PBS three times. Sections were then mounted using ProLong Gold mounting solution (Thermo Fisher) and allowed to dry overnight before imaging using a Keyence fluorescence microscope. For H&E staining, slides were washed with PBS between each step. The following steps were used: samples were first stained with hematoxylin for 4 minutes, followed by rinsing with 0.3% HC1 in 70% EtOH and rinsing in Scott’s tap water. 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. Samples were then mounted onto cover slides.

[0321] In Vivo Toxicity Assessment'. Male and female C57BL / 6 mice (N = 3) received intravenous injections by tail vein of LNPs formulated with 51406,12, luciferase mRNA (0.5 mg / kg), and 16% DOPE, 40% DOPE, or 40% DOTAP helper lipid. For cytokine analysis, blood was drawn via the submandibular vein before injection and at 1, 3, 6, 24, and 48 hours after injection, and serum was isolated. ELIS As were performed according to the instructions provided by the manufacturer (BioLegend) using serum dilutions of 1 :25 (TNF-cr, IL-6, and IL- 2). For blood chemistry analyses, male and female mice (N = 4) were i.v. injected with LNPs carrying luciferase mRNA (0.5 mg / kg). Five days postinjection, mice were sacrificed and blood samples were drawn 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 determined within 24 hours post-collection using a DRI-CHEM NX700V veterinary blood chemistry analyzer (Fujifilm, Tokyo).

[0322] Data Analysis and Statistics'. GraphPad Prism software (GraphPad, San Diego, CA) version 9.4.1 was used to perform statistical analysis and generate graphs. Data in FIGS. 6B- 6C were normalized to exclude autofluorescence. Raw data before normalization is available in FIGS. 7A-7D. Sample size varied based on the type of experiment. Typically, replicates were N = 3 for animal experiments, N = 4 for in vitro experiments, N = 12 for TNS assay, and N = 3 for flow cytometry analysis. For more detailed information see the Description of the Drawings, which also indicates error bar types. One or two way ANOVAs followed with a Dunnet multiple comparison or a parametric two-tailed t-test was performed where applicable to determine statistical significance.

[0323] TABLE 3: Panels and markers used to define cell populations in mouse lungs in flow cytometry experiments Results

[0324] Lipid library synthesis

[0325] To identify new ionizable lipidoids that provide unique functionality, a library of 580 materials was generated using Michael addition chemistry. This reaction (FIG. 1A) occurs between primary or secondary amines on a hydrophilic head group and the acrylate groups on hydrophobic tails. For this study, 29 alkyl-amines (FIG. IB) were selected, based at least in part on materials useful 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 were reacted combinatorially with 20 alkyl-acrylate tails (FIG. 1C) of lengths between 8 and 18 carbons that incorporated linear, branched, saturated, and partially unsaturated chemistries. Each of these properties can affect mRNA delivery potency (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).

[0326] Screening novel LNPs for in vivo efficacy using TNS

[0327] Potent LNPs for siRNA and mRNA delivery typically have surface pKa values within the 6.1-6.5 pH range (Kon et al., supra,' and Patel et al., Trends Pharmacol. Sci. 2021, 42:448). As such, studies were conducted to assess how the pH value of the LNP buffer influenced LNP surface properties and biological effects. pKa values were determined through the indirect measurement of LNP surface ionization as a function of pH using the 2- (p-toluidinyl)naphthalene-6-sulphonic acid (TNS) fluorescence assay (Whitehead et al., supra,' Knapp et al. 2016, supra,' and Patel et al., supra). Studies described elsewhere showed that greater levels of surface ionization at pH 5 were correlated with potency in mice for a small family of lipidoid materials (Hajj et al. 2019, supra), possibly because some ionized LNPs mediate escape from the late endosome, which has a pH of ~5 (Maugeri et al., supra). Therefore, the TNS assay at pH 5 was chosen for preliminary screening of the lipid library to identify those most likely to offer potency in mice. In these experiments, LNPs were generated with polyU mRNA using molar ratios optimized for mRNA lipidoid nanoparticle formulations: 35 mol% lipidoid, 16 mol% DOPE, 46.5 mol% cholesterol, and 2.5 mol% Cl 4- PEG2000, as described elsewhere (Hajj 2019, supra,' and Kauffman et al., supra). After incubation with TNS buffer at pH 5, resultant fluorescence was measured on a plate reader. The heat map in FIG. 2A indicates that LNPs varied substantially in their ionization potential, with darker colors corresponding to higher degrees of ionization. Some structural dependencies were observed, with longer tails (Ou-Ois) producing LNPs with the lowest TNS fluorescence and medium length tails (Oio, Oiio, and Os, 12) producing the highest. The low TNS fluorescence observed with long-chain lipidoids may have been connected to their melting temperatures, which in turn could have impacted their solubility in ethanol compared to lipidoids made with shorter chain tails.

[0328] Based on the TNS assessment, 49 LNPs for were selected for in vivo study. Characterization data for a subset of these is found in FIG. 3. For these experiments, LNPs were generated with firefly luciferase-encoding mRNA and IV-injected into mice at a total mRNA dose of 0.75 mg / kg. Three hours later, the luminescence of six major organs (liver, spleen, lungs, pancreas, heart, and kidneys) was measured using IVIS imaging. The total signal is plotted in FIG. 2B. In addition to 3060iio, which is described elsewhere (Hajj 2020, supra, and LoPresti et al., supra), two new highly potent LNPs were identified (402Os,i2 and 50006, 10) with total luminescence efficacy over 109p / s. An additional 13 LNPs were identified with total luminescence over 108p / s. To better understand the ability of different assays to predict in vivo potency, the correlation between in vivo performance and either TNS fluorescence or in vitro efficacy measurements was examined (FIG. 2C). Although neither correlation was strong, the TNS assay was superior because it was a necessary but insufficient condition for in vivo efficacy in these experiments. Further, the TNS assay was much faster and more economical than cell-based potency assays.

[0329] Neutral helper lipids enabled mRNA delivery to extrahepatocellular targets

[0330] When selecting an LNP that is appropriate for a specific application, it is crucial to consider targeting specificity in addition to potency given that most therapeutics require delivery to a specific organ or cell population. To determine LNP tropism, the same 49 LNPs discussed above were examined, and luciferase protein expression was quantified for each major organ (FIG. 4A). Most LNPs were liver-tropic, as is typical for LNP formulations containing a neutral helper lipid. This included the three most potent LNPs (FIG. 4B). Interestingly, there were three effective LNPs that demonstrated greater spleen and lung targeting (FIG. 4C). This was unusual, given the high cholesterol content and absence of charged helper lipids such as DOTAP or DOPS, which have been shown to shift LNP tropism to the lungs and spleen, respectively (Cheng et al., supra, and LoPresti et al., supra). For example, 5140e,io produced 35% of the signal in the lung and spleen each, with only 30% in the liver. The LNPs 5030iio and 503Os,i2 were spleen-tropic, with the former also producing significant signal in the lungs. Although there were other LNPs with reduced fractional liver-targeting (e.g., 21 IO10), their overall potency was low and they were excluded from further study. Notably, about 75% of the most efficacious LNPs were formulated with ionizable lipids containing branched, unsaturated tails. Without being bound by a particular mechanism, the efficaciousness of branched tail lipids may have been facilitated by their enhanced cone-like structure that, when ionized and paired with anionic phospholipids in the endosomal membrane, can more effectively disrupt the endosome 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).

[0331] A lead LNP delivered mRNA to lung cells as a function of helper lipid

[0332] Given its unusual lung -targeting behavior, the ionizable lipidoid 5140e,io was further examined. Typically, the transfection of lung cells following an i.v. injection is accomplished by substantially altering the other components of an LNP formulation. For example, the neutral helper lipid (e.g., DOPE) can be swapped out for a positively charged helper lipid such as DOTAP, and the fraction of helper lipid in the formulation can be increased from 16 to 40 mol% (Cheng et al., supra, and LoPresti et al., supra). Because of this, studies were conducted to determine whether the use of 5140e,io together with a helper lipid adjustment to 40% DOTAP would further improve lung targeting. As shown in FIG. 5A, the inclusion of DOTAP did increase lung expression from 35% to 94%, while simply increasing the DOPE molar percentage to 40 reduced lung tropism. The total luminescent signal in the lung is indicated below the pie charts in FIG. 5A, showing that efficacy was the same order of magnitude for each helper lipid variation.

[0333] To further understand lung expression as a function of helper lipid chemistry, 51406,IO LNPs were generated with green fluorescent protein (GFP) encoding mRNA and injected i.v. at a dose of 0.75 mg / kg. After 24 hours, the lungs were harvested. The left lobe was analyzed by flow cytometry and the right lobe was fixed for immunohistochemistry imaging. Right lung lobe sections were stained with anti-GFP fluorescent antibody and hematoxylin and eosin (H&E). All three formulations produced GFP throughout the lung (FIG. 5B), although the relative efficacy of the formulations differed slightly compared to luciferase. This may have been due to differences in expression kinetics and half lives of luciferase (quantified at 3 hours) and GFP (imaged at 24 hours). For DOTAP and 40% DOPE formulations, the signal was more prominent in and around cells that make up bronchial tubes and blood vessels.

[0334] Regarding safety, H&E staining did not reveal any overt tissue toxicity or immunogenicity in the lung for any formulation (FIG. 5C). ALT and ALP liver enzyme levels were also assessed, revealing that DOTAP particles caused significant increases compared to DOPE formulations, which were on par with untreated animals (FIG. 5D). Because nanoparticles can instigate inflammation, serum cytokine levels also were analyzed over a period of 48 hours after dosing (FIG. 5E). All three LNPs elevated the pro- inflammatory (Thl) cytokines IL-1 / ?, TNF-cr, and IFN-y between 1 and 6 hours postinjection, with resolution occurring by 24 hours. 40% DOTAP and 16% DOPE formulations were the most and least inflammatory, respectively. These increases were in line with those observed within several hours post- injection as described 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 for positively charged particles (here, DOTAP LNPs). Cytokine levels returned to baseline within 24 hours, suggesting a lack of tissue damage- induced inflammation. Taken together, the liver function and acute timepoint cytokine data suggested that neutral lungtargeting lipid nanoparticles offered an advantage over cationic formulations.

[0335] Next, studies were conducted to determine what cell types within the lung were being transfected using 5140e,io LNPs containing these distinct helper lipids, which could also shed light on how DOTAP shifts the signal to the lung and whether it increases uptake by all cell types or targets new cell types. To answer this question, samples were stained with several antibody panels: lymphoid, endothelial, epithelial, and myeloid. Signal distribution among those aggregate populations is depicted in FIG. 6A. Interestingly, all formulations transfected lymphoid immune cells, whereas only DOTAP and DOPE 40 mol% formulations transfected epithelial and endothelial cell populations. No formulation delivered mRNA to a significant portion of myeloid cells (e.g., macrophages).

[0336] A more detailed analysis of GFP signal in these cell populations was then performed. FIG. 6B displays the percentage of each cell type that was GFP+. Because animal cells tend to autofluoresce, single stain controls and compensation were used to normalize the data. Data before normalization is available in FIGS. 7A-7D. The bars in FIG. 6B are stacked for visual simplicity and are not intended to add up to 100%. For example, in the lymphoid panel, the 16PE formulation transfected about 15% of natural killer (NK) cells in the sample and about 20% of dendritic cells. The 40 mol% formulations transfected 25% each of dendritic and NK cells, and a small percentage of cytotoxic T cells. None of the LNPs transfected helper T or B cells. Only the 40 mol% formulations transfected endothelial cells, with efficacy in lymphatic endothelial cells approximately twice that in vascular endothelial cells. 40% DOTAP formulations transfected endothelial cells most efficiently. Overall, epithelial cells were the least likely to be transfected, with about 7% to about 12% of Type I (barrier-forming) epithelial cells expressing GFP. Only minute fractions of bronchial and Type II (secretory and stem-like) epithelial cells were transfected with any formulation.

[0337] FIG. 6C shows a different representation of data from the same experiment to better understand LNP cell-type specificity. Here, the distribution of cell types within each sample was compared with GFP+ cell types. For example, in the lymphoid panel, the untreated sample contained about 40%, 20%, 20%, 2%, and 5% B, T helper, T killer, NK, and dendritic cells, respectively. The relative percentages of these cells across the treated samples did not vary appreciably, suggesting that the treatments did not incur immune cell trafficking into or out of lung tissue. When comparing this cell type distribution with the distribution seen among GFP+ cells, it was clear that NK and dendritic cells were enriched in the total transfected cell population. This demonstrated that mRNA delivery was relatively selective for NK and dendritic cells. The endothelial cell panel showed that vascular and lymphatic endothelial cells comprise about 85% and 15% of total endothelial cells in the sample. Further, all treatment groups had equivalent efficacy in both cell types. For epithelial cells, there were modest, nonstatistically significant changes in the relative percentages of each cell type in the sample (FIG. 6C, bottom bar). Among epithelial cells, bronchial cells and Type 2 alveolar cells were more readily transfected than Type 1 alveolar cells. Taken together, these data show that LNP composition affected not only the organ location of protein expression, but also the transfected cell types within those organs. The 40 mol% formulations were similarly tropic for dendritic, NK, and endothelial lung cells. If endothelial cells are a desirable target, then 40 mol% formulations must be used. Additionally, the cationic formulation containing DOTAP offered the best overall selectively for these lung cells, given that it was more lung-tropic than the 40 mol% DOPE formulation (FIG. 5A). However, this greater degree of lung transfection was driven by disproportionate increases in mRNA delivery to endothelial cells (FIG. 6B). These data demonstrated that improved overall tropism to a particular organ may actually reduce cell-level tropism.

[0338] Interestingly, the 16 mol% DOPE formulation offered excellent selectivity for lung dendritic and NK cells, which was exciting because most lung-tropic formulations have been shown to transfect endothelial cells (Wei et al., supra, and Pei et al., supra). The ability to deliver mRNA to dendritic and NK cells suggested an expanded scope of conditions that may benefit from RNA therapy, given that they are implicated in numerous lung diseases. For example, applications for transfection of these cell types include cancer therapy (Ahluwalia et al., supra), antiviral or anti cancer 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 treatments (Akdis et al., Sci. Transl. Med. 2023, 15:eadd2563). Further, these studies showed that delivery to these cell types can be accomplished without the use of a cationic helper lipid, which may be advantageous given liver enzyme and cytokine data (FIGS. 5D-5E) and toxicity concerns associated with positively charged lipids (Wei et al., supra).

[0339] OTHER EMBODIMENTS

[0340] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A lipid-containing particle comprising: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound, wherein said lipidoid comprises an alkyl amine head and one or more alkyl acrylate tails, wherein each of said one or more alkyl acrylate tails comprises an alkyl chain having, at the second carbon, a first branch and a second branch, and wherein the first branch of each tail has n carbons and the second branch has n+1, n+2, or n+3 carbons.

2. The lipid-containing particle of claim 1, wherein said lipidoid has two, three, or four alkyl acrylate tails.

3. The lipid-containing particle of claim 2, wherein each of said alkyl acrylate tails is the same.

4. The lipid-containing particle of claim 2, wherein said lipidoid comprises two or more different alkyl acrylate tails.

5. The lipid-containing particle of claim 1, wherein said lipid-containing particle is a lipid nanoparticle (LNP).

6. The lipid-containing particle of claim 1, wherein said helper lipid is a neutral lipid or a zwitterionic lipid, optionally wherein said neutral lipid or zwitterionic lipid comprises one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1 ,2-di-myristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), palmitoylsphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

7. The lipid-containing particle of claim 1, wherein said helper lipid is a cationic lipid, optionally wherein said cationic lipid comprises one or more of l,2-dileoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3-trimethylammonium- propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3 -trimethylammoniumpropane (DPTAP), 1,2- distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP), 3 -[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), dimethyldioctadecylammonium bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

8. The lipid-containing particle of claim 1, wherein said lipid-containing particle is lungtropic.

9. The lipid-containing particle of claim 8, wherein said lipidoid comprises: an N1,Nll-(propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head or an N1,Nll-(propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

10. The lipid-containing particle of claim 8, wherein said lipidoid comprises an N^N1'- (propane-l,3-diyl)bis(N1-ethylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail.

11. The lipid-containing particle of claim 8, wherein said lipid-containing particle targets natural killer (NK) and / or dendritic cells.

12. The lipid-containing particle of claim 11, wherein said lipid-containing particle comprises about 10-40% neutral helper lipid or zwitterionic helper lipid, optionally wherein said neutral helper lipid or zwitterionic helper lipid comprises one or more of DOPE, DSPC, DOPC, DSPE, DPPC, POPC, DMPC, DPPE, POPE, DMPE, PSM, SSM, a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

13. The lipid-containing particle of claim 1, wherein said lipid-containing particle is liver-tropic.

14. The lipid-containing particle of claim 13, wherein said lipidoid comprises: an N1,N1'-(ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head, a 4-(2-aminopropan-2-yl)-l-methylcyclohexan-l -amine alkyl amino head, a 2-(piperazin-l-yl)ethan-l -amine head, or a N1-((4-)2-aminoethyl)piperazin-l -yl)methyl)ethane-l ,2-diamine head; and a 2-butylheptyl acrylate tail, a 2-butyloctyl acrylate tail, a 2-butylnonyl acrylate tail, a 2-hexylnonyl acrylate tail, a 2-hexyldecyl acrylate tail, a 2-hexylundecyl acrylate tail, a 2-octylundecyl acrylate tail, a 2-octyldodecyl acrylate tail, or a 2-octyltridecyl acrylate tail.

15. The lipid-containing particle of claim 14, wherein said lipidoid comprises an N^N1'- (ethane-l,2-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-hexyldecyl acrylate tail, or wherein said lipidoid comprises a 4-(2-aminopropan-2-yl)-l- methylcyclohexan-1 -amine alkyl amino head and a 2-octyldodecyl acrylate tail.

16. The lipid-containing particle of claim 1, wherein the cholesterol or a derivative thereof is cholesterol.

17. The lipid-containing particle 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 the lipid-containing particle of any one of claims 1-17, wherein said lipid-containing particle further comprises a therapeutic agent.

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

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

21. The method of claim 20, wherein said therapeutic agent comprises an RNA, optionally wherein said RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

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

23. The method of claim 20, wherein mammal has been identified as having a liver disorder, optionally wherein said liver disorder comprises alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

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

25. A method for treating a mammal having a lung disorder or a symptom thereof, or having a liver disorder or a symptom thereof, wherein said method comprises administering to the mammal a composition comprising the lipid-containing particle of any one of claims 1- 17, wherein said lipid-containing particle further comprises a therapeutic agent.

26. The method of claim 25, wherein said lipid-containing particle is a lipid nanoparticle (LNP).

27. The method of claim 25, wherein said therapeutic agent comprises an RNA, optionally wherein said RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

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

29. The method of claim 25, wherein mammal has been identified as having a liver disorder, optionally wherein said liver disorder comprises alpha- 1 antitrypsin deficiency,hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

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

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

32. The method of claim 31, wherein said lung cell is within a mammal.

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

34. The method of claim 31 , wherein said therapeutic agent comprises an RNA, optionally wherein said RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

35. The method of claim 31, wherein said lung cell is within a mammal identified as having a lung disorder, optionally wherein said lung disorder comprises cystic fibrosis, nonsmall cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

36. A lipid-containing particle comprising: a lipidoid; cholesterol or a derivative thereof; a helper lipid; and a polyethylene glycol (PEG)-based compound,wherein said lipidoid comprises an alkyl amino head and one or more alkyl acrylate tails, wherein said alkyl amino head is a N1,N1'-(propane-l,3-diyl)bis(N1-methylethane-l,2- diamine) alkyl amine head or a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2- diamine head, and wherein said one or more alkyl acrylate tails comprise one or more of a 2- octyldecyl acrylate tail and a 9-methyldecyl acrylate tail.

37. The lipid-containing particle of claim 36, wherein said lipid-containing particle is a lipid nanoparticle (LNP).

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

39. The lipid-containing particle of claim 38, wherein each of said alkyl acrylate tails is the same.

40. The lipid-containing particle of claim 38, wherein said lipidoid comprises two or more different alkyl acrylate tails.

41. The lipid-containing particle of claim 36, wherein said helper lipid is a neutral lipid or a zwitterionic lipid, optionally wherein said neutral lipid or zwitterionic lipid comprises one or more of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l,2-di-myristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), palmitoyl sphingomyelin (PSM), sterol sphingomyelin (SSM), a triglyceride, a triacylglycerol, a diglyceride, a diacylglycerol, and a ceramide.

42. The lipid-containing particle of claim 36, wherein said helper lipid is a cationic lipid, optionally wherein said cationic lipid comprises one or more of l,2-dileoyl-3- trimethylammonium-propane (DOTAP), 1 ,2-dimyristoyl-sn-glycero-3-trimethylammonium-propane (DMTAP), 1,2 dipalmitoyl-sn-glycero-3 -trimethylammoniumpropane (DPTAP), 1,2- distearoyl-sn-glycero-3-trimethylammoniumpropane (DSTAP), 30-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), didodecyldimethylammonium bromide (DDAB), dioctadecyloxy-propyl-glycerol (DOGS), Dimethyldioctadecylammonium Bromide (DODAB), dioleyloxy-propyl-trimethylammonium (DOSPA), and N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).

43. The lipid-containing particle of claim 36, wherein said lipidoid comprises a N^N1'- (propane-l,3-diyl)bis(N1-methylethane-l,2-diamine) alkyl amine head and a 2-octyldecyl acrylate tail, or wherein said lipidoid comprises a N1,N1'-(propane-l,3-diyl)bis(N1- methylethane-l,2-diamine) alkyl amine head and a 9-methyldecyl acrylate tail, or wherein said lipidoid comprises a N1-((4-)2-aminoethyl)piperazin-l-yl)methyl)ethane-l,2-diamine head and a 9-methyldecyl acrylate tail.

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

45. The lipid-containing particle 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 the lipid-containing particle of any one of claims 36-45, wherein said lipid-containing particle further comprises a therapeutic agent.

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

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

49. The method of claim 48, wherein said therapeutic agent comprises an RNA, optionally wherein said RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

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

51. The method of claim 48, wherein mammal has been identified as having a liver disorder, optionally wherein said liver disorder comprises alpha- 1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic 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 having a lung disorder or a symptom thereof, or having a liver disorder or a symptom thereof, wherein said method comprises administering to the mammal a composition comprising the lipid-containing particle of any one of claims 36-45, wherein said lipid-containing particle further comprises a therapeutic agent.

54. The method of claim 53, wherein said therapeutic agent comprises an RNA, optionally wherein said RNA comprises mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

55. The method of claim 53, wherein said lung disorder comprises cystic fibrosis, nonsmall cell lung cancer, another lung cancer, alpha- 1 antitrypsin deficiency, idiopathic pulmonary fibrosis, asthma, primary ciliary dyskinesia, a viral lung infection (e.g., pneumonia), post-viral immune dysfunction, bronchiectasis, chronic obstructive pulmonary disorder (COPD), sarcoidosis, hypersensitivity pneumonitis, or any combination thereof.

56. The method of claim 53, wherein said liver disorder comprises alpha-1 antitrypsin deficiency, hereditary hemochromatosis, Wilson’s disease, hereditary tyrosinemia, glycogen storage diseases, viral hepatitis, familial hypercholesterolemia, nonalcoholic fatty liver disease, primary hyperoxaluria, acute intermittent porphyria, hepatocellular carcinoma, paroxysmal nocturnal hemoglobinuria, or a combination thereof.

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

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