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WO2025217299A1 - Novel lipids containing diketopiperazine core, lipid nanoparticle containing thereof, and methods of using thereof - Google Patents

Novel lipids containing diketopiperazine core, lipid nanoparticle containing thereof, and methods of using thereof

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Publication number
WO2025217299A1
WO2025217299A1 PCT/US2025/023900 US2025023900W WO2025217299A1 WO 2025217299 A1 WO2025217299 A1 WO 2025217299A1 US 2025023900 W US2025023900 W US 2025023900W WO 2025217299 A1 WO2025217299 A1 WO 2025217299A1
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WO
WIPO (PCT)
Prior art keywords
cells
independently
lipid
compound
lung
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023900
Other languages
French (fr)
Inventor
Kun Zhao
Marine Z.C. Hatit
James DAHLMAN
Kalina PAUNOVSKA
Curtis Dobrowolski
Peter Edwards
Zhongguo Wang
Alejandro DA SILVA SANCHEZ
Benjamin Elliot MEAD
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nava Therapeutics
Georgia Tech Research Institute
Georgia Tech Research Corp
Original Assignee
Nava Therapeutics
Georgia Tech Research Institute
Georgia Tech Research Corp
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Application filed by Nava Therapeutics, Georgia Tech Research Institute, Georgia Tech Research Corp filed Critical Nava Therapeutics
Publication of WO2025217299A1 publication Critical patent/WO2025217299A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/06—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members
    • C07D241/08—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • lipid nanoparticles LNPs
  • FDA Food and Drug Administration
  • RNA vaccines and liver therapies have been FDA approved or been given Emergency Use Authorization to vaccinate against coronavirus disease of 2019.
  • efficacy of approved RNA vaccines and liver therapies underscores the potential clinical impact of LNPs with tropism to new cell types.
  • this challenge is stark; no systemically administered LNP carrying an RNA drug has yet reached phase III clinical trials, let alone been FDA approved.
  • CKK-E12 is a potent lipid that was proven useful in delivering siRNA and mRNA [PMID: 24516150].
  • RNA to non-hepatocytes has remained challenging in large part due to the anatomy and physiology of the liver. Specifically, the hepatic sinusoids contain a discontinuous vasculature as well as slow blood flow; both increase nanoparticle extravasation and subsequent interactions with hepatocytes.
  • an LNP with tropism to hepatocytes is retargeted with an active targeting ligand.
  • LNPs made with DLin-MC3-DMA an ionizable lipid that is FDA approved for hepatocyte siRNA delivery, have been retargeted to immune cells using a lipid-bound antibody.
  • Y is H; aryl; heteroaryl; branched or unbranched C 1 -C 20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl; wherein: each t is independently 1, 2, 3, or 4; each X is independently -O-, -S-, or -N(R′′)-; Z is absent, -C(O)-, -C(O)O-, -C(O)N(R′′)-, or -S(O)(O)-; each L is independently a C 1 -C 6 alkylene optionally substituted by OH; R, R′, and R′′ each are independently H, OR 14 , branched or unbranched C 1 -C 6 alkyl, or C 3 -C 7 cycloalkyl; R a and R b
  • the compounds also include the a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds disclosed herein, [0009]
  • Another aspect of the invention relates to a method of making a compound of formula reacting with one or more deprotecting reagents to remove the carboxyl protecting group and amine protecting group, and react the resulting intermediate under conditions sufficient to couple and cyclize the intermediate to form the compound of formula (IIA), wherein: each X is independently -O-, -S-, or -N(R′′)-; each t is independently 1, 2, 3, or 4; each L is independently a C 1 -C 6 alkylene optionally substituted by OH; R, R′′, R a , and R b each are independently H, branched or unbranched C 1 -C 6 alkyl, or C 3 -C 7 cycloalkyl; and R 1 and R 2 each are independently branched or unbranched, saturated or unsaturated C 1 -C 20 monovalent hydrocarbon chain,
  • lipid nanoparticles comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein.
  • Another aspect of the invention relates to a pharmaceutical composition comprising the lipid nanoparticle, comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, and a pharmaceutically acceptable carrier.
  • FIGS.1A-1E illustrate the processes and parameters with which diketopiperazine- based lipids were used to formulate stable lipid nanoparticles (LNPs).
  • FIG.1A illustrates how stereopure lipids were formulated with two cholesterol variants (cholesterol or 20 -OH cholesterol), two PEG-lipid variants (C14PEG2K or C18PEG2K), and DOPE.
  • FIG.1B provides how LNPs were formulated using four molar ratios, thereby formulating 96 total LNPs, as shown in FIG.1C.
  • FIG.1D illustrates hydrodynamic diameters of the 64 individual LNPs and the 64 LNP pool.
  • FIG.1E provides hydrodynamic diameter as a function of stereopure structure, average +/- SEM.
  • FIGS.2A-2I provide the results of a study quantifying delivery of mRNA by a lipid nanoparticle according to the present disclosure.
  • FIG.2A depicts the chemical structure and molar ratio of SPC-T LNP.
  • FIG.2B provides the biophysical properties of SPC-T LNP.
  • FIG.2C illustrates how SPC-T LNP was formulated to carry chemically modified mRNA encoding Cre recombinase and was intravenously administered to Ai14 mice at a total nucleic dose of 1 mg / kg.
  • FIG.2E shows delivery as a function of SPC-T: mRNA mass ratio.
  • FIG.2F provides a ribogreen assay revealing that increasing the SPC-T: mRNA mass ratio increased the amount of RNA encapsulated.
  • FIGS.3A-3D provide the results of an investigation of SPC-T LNP tropism at the cell type level, and quantification of the aVHH expression.
  • FIG.3A provides cell populations in mice treated with PBS or SPC-T, showing no substantial changes.
  • FIG.3D provides the percentage of aVHH+ cells measured with scRNA-Seq in each spleen and lymph node cell types.
  • FIGS.4A-4D illustrate the flow cytometry results for three high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 18 in the liver (FIG.4A), spleen (FIG.4B), lung (FIG.4B), or bone marrow (FIG.4D).
  • FIGS.5A-5B show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.5A) and spleen (FIG.5B).
  • FIGS.6A-6B show the flow cytometry results for individual LNP formulations selected from Screen 3 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.6A) and spleen (FIG.6B).
  • FIGS.7A-7D show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 26, Compound 29, Compound 30, and Compound 31 in the liver (FIG.7A), lung (FIG.7B), spleen (FIG. 7C), and bone marrow (FIG.7D).
  • FIGS.8A-8F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 39 and Compound 42 in the liver (FIG.8A), heart (FIG.8B), bone marrow (FIG.8C), kidney (FIG.
  • FIGS.9A-9F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 49 and Compound 50 in the liver (FIG.9A), lung (FIG.9B), bone marrow (FIG.9C), spleen (FIG. 9D), heart (FIG.9E), and kidney (FIG.9F).
  • FIGS.10A-10C show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs.9A-9F) in mouse using the LNP formulations containing Compound 50 in the liver (FIG.10A), lung (FIG.10B), and bone marrow (FIG. 10C).
  • FIGS.11A-11F show the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 55 in the liver (FIG.11A), lung (FIG.11B), bone marrow (FIG.11C), spleen (FIG.11D), kidney (FIG. 11E), and heart (FIG.11F).
  • FIGS.12A-12F show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs.11A-11F) in mouse using the LNP formulations containing Compound 55 in the liver (FIG.12A), lung (FIG.12B), bone marrow (FIG.12C), spleen (FIG.12D), kidney (FIG.12E), and heart (FIG.12F).
  • FIGS.13A-13F show the flow cytometry results for a high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 60 and Compound 63 in the liver (FIG.13A), lung (FIG.13B), bone marrow (FIG.13C), spleen (FIG.13D), kidney (FIG.13E), and heart (FIG.13F).
  • FIGS.14A-14B show the flow cytometry results for a LNP formulation containing Compound 18 (e.g., LNP-156) selected from a previous screening (discussed in FIGs.5A- 5B) in primate at a dosage of 0.45 mg/kg, in the liver (FIG.14A) and spleen (FIG.14B).
  • Compound 18 e.g., LNP-156
  • FIGS.14A-14B show the flow cytometry results for a LNP formulation containing Compound 18 (e.g., LNP-156) selected from a previous screening (discussed in FIGs.5A- 5B) in primate at a dosage of 0.45 mg/kg, in the liver (FIG.14A) and spleen (FIG.14B).
  • a reference to “a compound” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth.
  • a certain chemical moiety “may be” X, Y, or Z it is not necessarily intended by such usage to exclude other choices for the moiety; for example, a statement to the effect that R 1 “may be alkyl, aryl, or amino” does not necessarily exclude other choices for R 1 , such as halo, aralkyl, and the like.
  • “about X” (where X is a numerical value) preferably refers to ⁇ 10% of the recited value, inclusive.
  • the phrase “about 8” may refer to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” may refer to a value of 7.2% to 8.8%, inclusive.
  • the term “about” precedes a range it is understood that the term modifies both recited endpoints and all points embraced within the range.
  • the phrase “about 1-10” is understood to mean “about 1 to about 10”, as well as “about x”, wherein x refers to any value between 1 and 10. Where present, all ranges are inclusive and combinable.
  • the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.”
  • a listing of possible substituents including “hydrogen, alkyl, and aryl” is provided, the recited listing may be construed as including situations whereby any of “hydrogen, alkyl, and aryl” is negatively excluded; thus, a recitation of “hydrogen, alkyl, and aryl” may be construed as “hydrogen and aryl, but not alkyl”, or simply “wherein the substituent is not alkyl”.
  • the terms “component”, “compound”, “drug”, “pharmacologically active agent”, “active agent”, “therapeutic”, “therapeutic agent”, “therapy”, “treatment”, or “medicament” may be used herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action.
  • alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C 1 -C 12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it.
  • alkyl generally refers to C 1 -C 24 alkyl (e.g., C 1 -C 12 alkyl, C 1 -C 8 alkyl, or C 1 -C 4 alkyl).
  • alkenyl refers to a straight or branched hydrocarbon chain containing 2- 8 carbon atoms and characterized in having one or more double bonds.
  • alkenyl generally refers to C 2 -C 8 alkenyl (e.g., C 2 -C 6 alkenyl, C 2 -C 4 alkenyl, or C 2 -C 3 alkenyl).
  • alkenyl examples include, but not limited to, allyl, propenyl, 2- butenyl, 3-hexenyl and 3-octenyl groups.
  • alkoxy refers to an -O-alkyl radical.
  • alkylene refers to a divalent alkyl (i.e., -R-).
  • aminoalkyl refers to an alkyl substituted with an amino.
  • mercapto refers to an -SH radical.
  • thioalkoxy refers to an -S-alkyl radical.
  • alkylene refers to abivalent form of an alkyl group.
  • alkylene chain is a polymethylene group, i.e., —(CH 2 ) n —, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
  • a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below.
  • alkenylene refers to a bivalent alkenyl group.
  • a substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below.
  • aryl refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
  • aryl may be used interchangeably with the term “aryl ring.” Examples of aryl groups include phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents.
  • aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
  • arylalkyl or the term “aralkyl” refers to alkyl substituted with an aryl.
  • arylalkoxy refers to an alkoxy substituted with aryl.
  • cycloalkyl or “cyclyl” as employed herein includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted.
  • Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
  • heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzofuranyl, di
  • heterocyclyl refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent.
  • nitrogen When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl).
  • heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like.
  • heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
  • a divalent radical of an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, heterocyclyl is formed by removal of a hydrogen atom from an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radical, respectively (or by removal of two hydrogen atoms from an alkane, alkene, arene, heteroarene, cycloalkane, or heterocycle, respectively).
  • substituted refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamin
  • the substituent can be further substituted.
  • the compounds described herein or their pharmaceutically acceptable salts may include all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like.
  • the compounds can contain one or more stereocenters and may thus give rise to geometic isomers (e.g., double bond causing geometric E/Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or ( ⁇ )) or cis/trans isomers), and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- such as for sugar anomers, or as (D)- or (L)- such as for amino acids.
  • the present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms.
  • Optically active (+) and (- ), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization.
  • Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • a “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable.
  • the present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.
  • the term “pharmaceutically acceptable salt” include the salts derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI alkyl ⁇ salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.
  • Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.
  • an appropriate electrophile e.g., an alkyl halide
  • Disclosed herein are diketopiperazine-containing ionizable lipids that can, among other things, preferentially deliver mRNA to immune cells or stem cells in various tissues in vivo without targeting ligands.
  • the inventive lipids were synthesized and characterized, and high-throughput DNA barcoding was used to quantify how a collection of chemically distinct LNPs functionally delivered mRNA (i.e., mRNA translated into functional, gene-editing protein) in multiple cell types directly in vivo.
  • lipid traits that increase delivery in vivo were identified.
  • LNPs that preferentially delivers mRNA to liver or non-liver cells at clinically relevant doses were prepared and characterized.
  • inventive nanoparticles with natural non-hepatocyte tropism and demonstrated that the presently disclosed lipids with bioactive small-molecule motifs successfully deliver mRNA in vivo.
  • novel lipids [0050] Accordingly, disclosed are compounds of Formula (I): wherein: Y is H; aryl; heteroaryl; branched or unbranched C 1 -C 20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl; wherein: each t is independently 1, 2, 3, or 4; each X is independently -O-, -S-, or -N(R′′)-; Z is absent, -C(O)-, -C(O)O-, -C(O)N(R′′)-, or -S(O)(O)-; each L is independently a C 1 -C 6 alkylene optionally substituted by OH; R, R′, and R′′ each are independently H, OR 14 , branched or unbranched C 1 -C 6 alkyl, or C 3 -C 7 cycloalkyl; R a and R b each are independently H
  • the lipid compound has the formula of: All the variables have the same definitions as those defined above. [0052] In certain embodiments, in any of the formulas disclosed herein (each of the below variables, if present): Y is H; aryl; branched or unbranched C 1 -C 6 alkyl; C 1 -C 6 alkyl interrupted with a N, O, or S atom; C 1 -C 3 alkyl substituted with an aryl; ; or each R, R′, R a , and R b are H; each t is independently 1 or 2; each X is independently -O-, -S-, -NH-, or -N(OH)-; Z is absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)
  • the compound the formula of: variables have the same definitions as those defined above.
  • at least one X is -O-, -S-, -NH-, or -N(OH)-.
  • at least one X is O or S.
  • at least one X is -N(R′′)- (e.g., -NH- or -N(OH)-.
  • each R, R′, and R′′ are independently H, OR 14 , branched or unbranched C 1 -C 6 alkyl, or C 3 -C 7 cycloalkyl. In some embodiments, R, R′, and R′′ each may be independently H or C 1 -C 3 alkyl. In some embodiments, R, R′, and R′′ each may be independently a C 3 -C 7 cycloalkyl. In some embodiments, R′′ is OR 14 (e.g., OH, or OCH 3 ). In some embodiments, each R, R′, and R′′ are H.
  • each L is independently a C2-C4 alkylene optionally substituted by OH.
  • each L is independently a C 2 -C 4 alkylene, or -CH 2 -CH(OH)-CH 2 - (e.g., including both R and S isomers for the carbon atom that OH is attached to) when the X variable next to the L variable is -O-.
  • L is independently a C 2 -C 4 alkylene (e.g., a C 2 , C 3 , or C 4 alkylene).
  • L is independently -CH 2 -CH(OH)-CH 2 - (e.g., including both R and S isomers for the carbon atom that OH is attached to) when the X variable next to the L variable is -O-.
  • Y is H; aryl; branched or unbranched C 1 -C 6 alkyl; C 1 -C 6 alkyl interrupted with a N, O, or S atom; or C 1 -C 3 alkyl substituted with an aryl.
  • Y is H.
  • Y is branched or unbranched C 1 -C 4 alkyl, a C 1 -C 3 alkyl interrupted with an S atom, or benzyl. [0059] In some embodiment, Y is . In some embodiments, Z is absent, -C(O)-, -C(O)O-, -C(O)N(R′′)-, or -S(O)(O)-. In some embodiments, Z is absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)-. In some embodiments, Z is -C(O)-.
  • Z is absent, -C(O)O-, -C(O)NH-, or -S(O)(O)-.
  • R a and R b each are independently H, branched or unbranched C 1 -C 6 alkyl, or C 3 -C 7 cycloalkyl.
  • R a and R b each are independently H, or C 1 -C 3 alkyl.
  • R a and/or R b is C 3 -C 7 cycloalkyl.
  • R a and R b are H.
  • each of the X groups within the compound are the same, each of the R 1 groups within the compound are the same, and each of the R 2 groups within the compound are the same.
  • the X groups within the compound may be independently selected, i.e., may be different, the R 1 groups within the compound may be independently selected, i.e., may be different, and the R 2 groups within the compound may be independently selected, i.e., may be different.
  • each R 1 and R 2 may be the same, e.g., each R 1 may be the same as each R 2 .
  • at least one, two, or three of the R 1 and R 2 variables within the compound are different than the other(s).
  • a particular R 1 group may be the same as one or two R 2 groups within the compound.
  • the compound contains two R 1 groups, and both R 1 groups are the same as one or two R 2 groups within the compound.
  • each R 1 and R 2 is independently branched or unbranched C 1- 20 alkyl or branched or unbranched C 2 -C 20 alkenyl.
  • each R 1 and R 2 may independently be C 1-20 linear or branched alkyl; each R 1 and R 2 may independently be C 1-20 linear or branched alkenyl.
  • each R1 and R2 are independently C9-C16 alkyl.
  • each R 1 and R 2 are independently C 9 -C 12 alkyl.
  • each R 1 and R 2 may independently be C 9 alkyl, C 10 alkyl, C 11 alkyl, C 12 alkyl, C 13 alkyl, C 14 alkyl, C 15 alkyl, or C 16 alkyl. In some embodiments, each R 1 and R 2 are independently C 9 -C 16 alkenyl. In some embodiments, each R 1 and R 2 are independently C 9 - C 12 alkenyl. [0069] In some embodiments, R 1 and/or R 2 may be represented by –(C(R 11 )(R 12 )) m -Q- (C(R 11 )(R 12 )) n H.
  • Non-limiting examples of the lipid compounds are set forth below.
  • exemplary lipid compounds are: pound 123),
  • these exemplary lipid compounds provide targeted delivery to specific cells, tissues, and/or organs such as the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject. In one embodiment, these exemplary lipid compounds provide targeted delivery to the cells or tissues of lung. In one embodiment, these exemplary lipid compounds provide targeted delivery to the cells or tissues of spleen. Preparation of the novel lipids [0075] Certain aspects of the invention relate to methods of making the novel lipids described herein. [0076] All above descriptions and all embodiments regarding the novel lipid compounds discussed in the above aspects of the invention are applicable to these aspect of the invention relating to the method of making the compounds.
  • one aspect of the invention relates to a method of making a compound of formula reacting with one or more deprotecting reagents to remove the carboxyl protecting group and amine protecting group, and react the resulting intermediate under conditions sufficient to couple and cyclize the intermediate to form the compound of formula (IIA), wherein: each X is independently -O-, -S-, or -N(R′′)-; each t is independently 1, 2, 3, or 4; each L is independently a C 1 -C 6 alkylene optionally substituted by OH; R, R′′, R a , and R b each are independently H, branched or unbranched C 1 -C 6 alkyl, or C 3 -C 7 cycloalkyl; and R 1 and R 2 each are independently branched or unbranched, saturated or unsaturated C 1 -C 20 monovalent hydrocarbon chain, or represented by –(C(R 11 )(R 12 )) m -Q-(C(R 11
  • the preparation further employs one or more coupling reagents couple and cyclize the intermediate to form the compound of formula (IIA).
  • the coupling reagent comprises EDCI, DMAP, HATU, and/or Hunig’s base.
  • the reactan X is -O-, -S-, or -NH-,
  • the deprotecting agent is an acid.
  • the acid is trifluoroacetic acid (TFA).
  • the acid is an inorganic acid such as HCl.
  • the reaction conditions comprises adding acetonitrile and triethylamine.
  • the organic base is DIPEA. Other organic bases are also suitable for use herein.
  • the solvent is DMF or its mixture with DCM.
  • the reactant i form corresponding the reaction reagents further comprises a benzotriazole or its derivative.
  • the reactant is the reaction reagents further comprises a benzotriazole or its derivative.
  • the use of the benzotriazole or its derivative could eliminate or avoid the racemization of single-enantiomer chiral molecules and to result in a chiral enantiomer.
  • the benzotriazole or its derivative is HOBt, HATU, or a mixture thereof.
  • the benzotriazole or its derivative is PyBOP.
  • the reagents further comprise a coupling agent, such as EDCI.
  • Another aspect of the invention relates to a method of making a compound, comprising r s
  • Another aspect of the invention relates to a method of making a compound, comprising reacting f aryl; heteroaryl; branched or unbranched C 1 -C 20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl.
  • the organic solvent is an alcohol (e.g., isopropyl alcohol).
  • the organic solvent is ACN.
  • the lipid nanoparticles according to the present disclosure that include a compound of Formula (I) deliver a therapeutic agent, such as a nucleic acid, preferentially to liver cells, lung cells, splenic cells, heart cells, kidney cells, marrow cells, or lymphatic cells of the subject.
  • a therapeutic agent such as a nucleic acid
  • the presently disclosed lipid nanoparticles can deliver a therapeutic agent preferentially to liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, or other immune cells within the liver.
  • the lipid nanoparticles can deliver a therapeutic agent preferentially to spleen dendritic cells, spleen neutrophils, spleen macrophages, spleen B cells, spleen T cells, spleen natural killer (NK) cells, or other immune cells within the spleen.
  • the lipid nanoparticles can deliver a therapeutic agent preferentially to lymphatic dendritic cells, lymphatic neutrophils, lymphatic macrophages, lymphatic B cells, lymphatic T cells, lymphatic natural killer (NK) cells, or other immune cells within the lymph nodes.
  • the lipid nanoparticles can deliver a therapeutic agent preferentially to lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung macrophages, lung T cells, lung natural killer (NK) cells, or other immune cells within the lung.
  • the lipid nanoparticles can deliver a therapeutic agent preferentially to heart endothelial cells, heart epithelial cells, heart dendritic cells, heart macrophages, heart B cells, heart T cells, heart natural killer (NK) cells, or other immune cells within the heart.
  • the lipid nanoparticles can deliver a therapeutic agent preferentially to kidney endothelial cells, kidney epithelial cells, kidney macrophages, kidney dendritic cells, kidney B cells, kidney T cells, kidney natural killer (NK) cells, or other immune cells within the kidney.
  • the lipid nanoparticles can deliver a therapeutic agent preferentially to marrow dendritic cells, marrow macrophages, marrow B cells, marrow T cells, other immune cells within marrow, or marrow stem-like cells, or marrow hematopoietic stem cells (HSCs).
  • preferential delivery to a particular class of cells or cell type refers to delivery at a higher rate than to non-targeted cells.
  • the preferential delivery can mean delivery at or above a rate that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than to non-targeted cells, i.e., cells not within the particular class of cells or of the particular cell type.
  • the delivery is to a particular targeted class of cells or cell type, and there is no delivery or only minimal delivery to non-targeted cells.
  • helper lipids for lipid nanoparticles are known among those skilled in the art and may be any compound that contributes to the stability and delivery efficiency of the LNP, or to the stable encapsulation of a therapeutic agent within the LNP.
  • the helper lipid is a cationic lipid.
  • the helper lipid is DDAB (Didodecyldimethylammonium bromide), DOTAP (1,2-Dioleoyl-3-trimethylammonium propane), or DOTMA (1, 2-di-O- octadecenyl-3-trimethylammonium propane).
  • the helper lipid is a phospholipid.
  • Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and a sphingomyelin.
  • a fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Particular phospholipids can facilitate fusion to a membrane.
  • a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.
  • a membrane e.g., a cellular or intracellular membrane.
  • elements e.g., a therapeutic agent
  • a lipid-containing composition e.g., LNPs
  • Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated.
  • a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond).
  • alkynes e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond.
  • an alkyne group can undergo a copper- catalyzed cycloaddition upon exposure to an azide.
  • Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).
  • Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.
  • the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccin
  • the lipid nanoparticle further comprises one or more helper lipids selected from the group consisting of 1,2-dioeoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), didodecyldimethylammonium bromide (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3- trimethylammonium propane (DOTAP), 18:1 PA, N-methyldioctadecylamine (MDOA), N,N-dicotadecylaniline, sn-(3-myristoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'- dimyristoyl
  • helper lipids selected
  • the one or more helper lipids are 1,2-dioeoyl-sn-glycero-3- phosphoethanolamine (DOPE), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), or a combination thereof.
  • DOPE 1,2-dioeoyl-sn-glycero-3- phosphoethanolamine
  • DDAB dimethyldioctadecylammonium
  • DOTAP 1,2-dioleoyl-3- trimethylammonium-propane
  • DSPC 2-distearoyl-sn-glycero-3-phosphocholine
  • DOTMA 1,2-di-O-octadecen
  • a combination of two or more cholesterols or derivatives thereof are used.
  • the inclusion of a cholesterol in nanoparticle formulations can improve efficacy, potentially due to enhanced membrane fusion.
  • Exemplary cholesterol or derivatives thereof include cholesterol (C 27 H 46 O), 20 ⁇ -OH cholesterol, 20 ⁇ -hydroxycholesterol (5-cholestene-3 ⁇ ,20 ⁇ - diol), and DC-Cholesterol (N,N-dimethyl- N-ethylcarboxamidocholesterol). Any natural sterols may also be used.
  • Examples of natural sterols include, for example, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, 14-demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)- dehydrocholesterol, FF-MAS, diosgenin, DHEA sulfate, DHEA, sitosterol, lanosterol-95, cholesterol (plant), dihydro FF-MAS-d6, dihydro T-MAS-d6, zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol, pregnenolone, dihydro T-MAS, delta 5- avenasterol, brassicasterol, dihydro FF-MAS, and 24-methylene cholesterol.
  • the lipid nanoparticles may also include a PEG-lipid.
  • PEG lipid refers to polyethylene glycol (PEG)-modified lipids.
  • Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG- ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids.
  • a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • the PEG lipid includes, but are not limited to, 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA).
  • PEG-DMG 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG-DSPE 1,2-distearoyl-sn-g
  • the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG- modified dialkylglycerol, and mixtures thereof.
  • the lipid moiety of the PEG lipids includes those having lengths of from about C 14 to about C 22 . In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C 14 to about C 16 .
  • a PEG moiety for example an mPEG-NH 2 , has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.
  • the PEG lipid is PEG 2k -DMG.
  • the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG.
  • Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE.
  • the PEG-modified lipid is PEG-DEG or PEG-PE.
  • the PEG-modified lipid is 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000 (DMPE-PEG2K; 14:0 PEG2K PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)- 2000 (DSPE-PEG2K; 18:0 PEG2K PE), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG 2K), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC- 0159), distearoyl-rac-glycerol-PEG2K (DSG-PEG 2K), or a combination thereof.
  • DMG-PEG 2K 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-
  • the LNP in addition to the compound of Formula (I), also comprises a helper lipid (e.g., DOPE, DDAB, DOTAP, DSPC, or a mixture thereof), a sterol (e.g., cholesterol (C 27 H 46 O), 20 ⁇ -OH cholesterol, DC-cholesterol, or a mixture thereof), and a PEG lipid (e.g., 14:0 PEG2K PE, 18:0 PEG2K PE, or a mixture thereof).
  • a helper lipid e.g., DOPE, DDAB, DOTAP, DSPC, or a mixture thereof
  • a sterol e.g., cholesterol (C 27 H 46 O), 20 ⁇ -OH cholesterol, DC-cholesterol, or a mixture thereof
  • PEG lipid e.g., 14:0 PEG2K PE, 18:0 PEG2K PE, or a mixture thereof.
  • the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid in the lipid nanoparticles may be about 20-60 : 7- 50 : 5-70 : 0.5-3. In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid in the lipid nanoparticles may be about 30-60 : 7-50 : 15-50 : 1-3.
  • the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid in the lipid nanoparticles may be about 30-55: 10-20 : 30-50 : 1-3. In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid is about 32-50 : 12-18 : 32-48 : 1-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid is about 35-50 : 12-16 : 35-47 : 2-3.
  • the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid is about 25 : 33 : 40 : 2; is about 30 : 22.5 : 45 : 2.5; is about 30 : 39 : 30 : 1; is about 30 : 50 : 18 : 2; is about 35 : 35 : 27.5 : 2.5; is about 35 : 43 : 20 : 2; is about 35 : 44.5 : 18 : 2.5; is about 35 : 16: 46.5 : 2.5; is about 45 : 13 : 39.5 : 2.5; is about 50 : 40 : 7.5 : 2.5; is about 50 : 17.5 : 30 : 2.5; is about 50 : 12.5 : 35 : 2.5; is about 50 : 10 : 38.5 : 1.5; or is about 35 : 15 : 47.5 : 2.5.
  • the sterol component (e.g., cholesterol or its derivative) may be absent from the LNP.
  • the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid in the lipid nanoparticles may be about 20-60 : 10-60 : 0.5-3.
  • the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 25-55 : 15-60 : 0.5-3.
  • the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 30-55 : 30-55 : 1-3.
  • the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 32-50 : 40- 55 : 1-3. [00122] In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 45 : 52.5 : 2.5; is about 45 : 53: 2; or is about 50 : 47.5 : 2.5.
  • the molar concentration of the compound according to Formula (I) in the LNPs may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 percent of the total lipids in the LNP.
  • the molar concentration of the helper lipid in the LNPs may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the molar concentration of the cholesterol or its derivative in the LNPs may be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 percent of the total lipids in the LNP.
  • the molar concentration of the PEG lipid in the LNPs may be about 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, or 3.5 percent of the total lipids in the LNP.
  • the lipid nanoparticle may have a diameter of about 20-250, 20-225, 20-200, 30- 250, 30-225, 30-200, 40-250, 40-225, 40-200, 45-250, 45-225, 45-200, 50-200, 50-180, or 75-170 nm.
  • the diameter of the lipid nanoparticle may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250.
  • the population may include individual members of respectively different sizes.
  • the particle size distribution of a given population of LNPs according to the present disclosure may be characterized by a D90 of about 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, or 60 nm, and/or a D10 of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.
  • the lipid nanoparticles according to the present disclosure can be used for the delivery of therapeutic agents to a living organism, such as to a human subject.
  • the therapeutic agent may be encapsulated within the lipid nanoparticle.
  • the therapeutic agent may be a nucleic acid molecule (e.g., oligonucleotide), protein or peptide, carbohydrate or glycoprotein, lipid, small molecule, or any combination thereof.
  • the therapeutic agent is a small molecule.
  • the therapeutic agent is a protein or peptide.
  • the therapeutic agent is a nucleic acid molecule (e.g., DNA or RNA).
  • the therapeutic agent is a DNA, e.g., in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • PCR polymerase chain reaction
  • vectors e.g., PI, PAC, BAC, YAC, artificial chromosomes
  • expression cassettes e.g., chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • the therapeutic agent is an RNA, e.g., in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis- natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (ta) of messenger RNA (mRNA
  • the therapeutic agent is an mRNA.
  • the lipid nanoparticle (LNP) formulations include (i) a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any one of the embodiments described herein, (ii) a helper lipid, (iii) a PEG lipid, and optionally (iv) a sterol (e.g., cholesterol).
  • compound (i) is compound KC-34 (e.g., S,S or R,R), compound 123, compound 125, or compound 126; the helper lipid (ii) is DDAB; the PEG lipid (iii) is C14PEG2K PE, C18PEG2K PE, or DSG- PEG2K; and the sterol (iv) is cholesterol.
  • compound (i) is compound KC-34 (e.g., S,S or R,R), compound 123, compound 125, or compound 126
  • the helper lipid (ii) is DDAB
  • the PEG lipid (iii) is C14PEG2K PE, C18PEG2K PE, or DSG- PEG2K
  • the sterol (iv) is cholesterol.
  • exemplary LNP formulations include: [00135]
  • the above exemplary LNP formulations provide targeted delivery to specific cells, tissues, and/or organs such as the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject.
  • the above exemplary lipid compounds provide targeted delivery to the cells or tissues of lung.
  • compound (i) is compound KC-34 (e.g., S,S or R,R); the helper lipid (ii) is DSPC or DOPE; the PEG lipid (iii) is C14PEG2K PE or DMG-PEG2K; and the sterol (iv) is cholesterol.
  • compound (i) is compound KC-34 (e.g., S,S or R,R); the helper lipid (ii) is DSPC or DOPE; the PEG lipid (iii) is C14PEG2K PE or DMG-PEG2K; and the sterol (iv) is cholesterol.
  • exemplary LNP formulations include: Helper Helper Lipid (i) Cholesterol PEG Lipid Lipid (ii) Lipid (i) PEG Lipid Lipid Mol % (iv) Mol % (iii) Mol % Mol % KC-34 (S,S) DMG-PEG2K DOPE 30 30 1 39 KC-34 (S,S) C14PEG2K PE DSPC 30 30 1 39 KC-34 (S,S) DMG-PEG2K DSPC 30 30 1 39 KC-34 (S,S) C14PEG2K DSPC 30 30 1 39 KC-34 (S,S) C14PEG2K DSPC 30 30 1 39 KC-34 (S,S) C14PEG2K DSPC 30 30 1 39 KC-34 (S,S) C14PEG2K DSPC 30 30 1 39 KC-34 (S,S) C14PEG2K DSPC 30 30 1 39 KC-34 (S,S) C14PEG
  • the above exemplary lipid compounds provide targeted delivery to the cells or tissues of spleen.
  • Additional non-limiting examples of the lipid nanoparticles formulations are further illustrated in Examples 2-3 and 5-6.
  • the present disclosure also provides a pharmaceutical composition comprising the lipid nanoparticle comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any of the embodiments described herein, and a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable carrier” preferably refers to a material that can be incorporated into a composition and administered to a patient without causing unacceptable biological effects or interacting in an unacceptable manner with other components of the composition.
  • Such pharmaceutically acceptable materials typically have met the required standards of toxicological and manufacturing testing, and include those materials identified as suitable inactive ingredients by the U.S. Food and Drug Administration.
  • All above descriptions and all embodiments regarding the novel lipid compounds and lipid nanoparticles discussed in the above aspects of the invention are applicable to this aspect of the invention relating to the pharmaceutical composition.
  • the LNPs according to the present disclosure may be provided in a composition that is formulated for any type of administration.
  • compositions may be formulated for administration orally, topically, parenterally, enterally, or by inhalation (e.g., intranasally).
  • the active agent may be formulated for neat administration, or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid.
  • the applicable solid carrier, diluent, or excipient may function as, among other things, a binder, disintegrant, filler, lubricant, glidant, compression aid, processing aid, color, sweetener, preservative, suspensing/dispersing agent, tablet-disintegrating agent, encapsulating material, film former or coating, flavoring agent, or printing ink.
  • any material used in preparing any dosage unit form is preferably pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the LNPs may be incorporated into sustained-release preparations and formulations.
  • Administration in this respect includes administration by, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol, and rectal systemic.
  • the carrier, diluent, or excipient may be a finely divided solid that is in admixture with the finely divided active ingredient.
  • the LNPs are mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the LNPs may be incorporated with the carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the amount of LNP in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained.
  • Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like.
  • the LNPs may be suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat.
  • a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat.
  • the liquid carrier, excipient, or diluent can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo-regulators.
  • Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar-agar, tragacanth, or mixtures thereof.
  • liquid carriers, diluents, and excipients for example, for oral, topical, or parenteral administration, include water (particularly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil), or mixtures thereof.
  • the carrier, diluent, or excipient can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • sterile liquid carriers diluents, or excipients
  • Solutions of the LNPs can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • a dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form is preferably sterile and fluid to provide easy syringability. It is preferably stable under the conditions of manufacture and storage and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier, diluent, or excipient may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the antimicrobial peptides themselves may be sufficient to prevent contamination by microorganisms.
  • Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the LNPs in the pharmaceutically appropriate amounts, in the appropriate solvent, with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions may be prepared by incorporating the LNPs into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation may include vacuum drying and freeze drying techniques that yield a powder of the LNPs or ingredients, plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • All above descriptions and all embodiments regarding the novel lipid compounds and lipid nanoparticles discussed in the above aspects of the invention are applicable to this aspect of the invention relating to the pharmaceutical composition.
  • the inventive nanoparticles preferentially target human lung, heart, kidney, liver, splenic, lymphatic, or marrow cells, and can thereby preferentially deliver the therapeutic agent to such cells.
  • the cells to which the present LNPs deliver the therapeutic agent can include, for example, lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung T cells, lung macrophages, lung natural killer (NK) cells, or other immune cells within the lung; heart endothelial cells, heart epithelial cells, heart dendritic cells, heart B cells, heart T cells, heart natural killer (NK) cells, heart macrophages, or other immune cells within the heart; kidney endothelial cells, kidney epithelial cells, kidney dendritic cells, kidney B cells, kidney T cells, kidney natural killer (NK) cells, kidney macrophages, or other immune cells within the kidney; liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, other immune cells within the liver; spleen dendritic cells, spleen neutrophils, spleen
  • the present disclosure also provides methods for delivering a therapeutic agent to lung, heart, kidney, liver, splenic, lymphatic, or marrow cells of a subject, comprising administering to the subject a lipid nanoparticle according to any of the embodiments disclosed herein.
  • the lipid nanoparticles can deliver the therapeutic agent to the subject at clinically relevant doses. In some embodiments, that dose at which the present LNPs deliver the therapeutic agent is about 0.01 to about 3.0 mg/kg.
  • the dose at which the therapeutic agent is delivered may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.51.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2., 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, 2.9, or 3.0 mg/kg.
  • FIGS.1A-1E illustrate the formulation of the inventive stereopure lipids into stable LNPs.
  • stereopure lipids were formulated with two cholesterol variants (cholesterol or 20 -OH cholesterol), two PEG-lipid variants (C14PEG2K or C18PEG2K), and DOPE.
  • the LNPs were formulated using four molar ratios (FIG.1B), thereby formulating 96 total LNPs (FIG.1C), of which 64 met inclusion criteria.
  • FIG.1D illustrates hydrodynamic diameters of the 64 individual LNPs (gray dots) and the 64 LNP pool (purple dot designated by arrow). The pool diameter was within the range of the LNPs comprising the pool.
  • FIG.1E provides hydrodynamic diameter as a function of stereopure structure, average +/- SEM.
  • each lipid was formulated with C14PEG2K or C18PEG2K as well as unmodified cholesterol or 20 -OH (FIG.1A).
  • the helper lipid 1-2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)
  • DOPE 1-2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • LNPs were then barcoded for use in Fast Identification of Nanoparticle Delivery.
  • 96 LNPs were formulated so that LNP 1, with chemical composition 1, carried Cre mRNA and DNA barcode 1; LNP 96, with chemistry 96, was formulated to carry Cre mRNA and DNA barcode 96.
  • the total lipid to nucleic acid mass ratio was 10:1.
  • the hydrodynamic diameter and polydispersity of the 96 individual LNPs was then investigated with dynamic light scattering.
  • Nanoparticle Formulation Nanoparticles were formulated with a microfluidic device. Nucleic acids (DNA barcodes and mRNA) were diluted in 10mM citrate buffer (Teknova).
  • Lipid-amine compounds PEG-lipids (1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] and 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]), cholesterols (cholesterol and 20 ⁇ -hydroxycholesterol), and helper lipid dioleoylphosphatidylethanolamine were diluted in 100% ethanol.
  • Cre mRNA and DNA barcodes were mixed at a 10:1 mass ratio. All PEG-lipids, cholesterols, and helper lipid were purchased from Avanti Polar Lipids (Alabaster, AL).
  • nucleotides on the 5’ and 3’ ends were modified with phosphorothioates to reduce exonuclease degradation
  • ii) universal forward and reverse primer regions were included to ensure equal amplification of each sequence
  • iii) 7 random nucleotides were include to monitor PCR bias
  • iv) a droplet digital PCR (ddPR) probe site was include for ddPCR compatibility
  • v) a unique 8-nt barcode An 8-nucleotide sequence can generate over 4 8 (65,536) distinct barcodes. We used only the 8 nucleotide sequences designed to prevent sequence bleaching and reading errors on the Illumina MiniSeq TM sequencing machine.
  • LNP hydrodynamic diameter was measured using high throughput dynamic light scattering (DLS) (DynaPro Plate Reader II, Wyatt). LNPs were diluted in sterile 1X PBS and analyzed. To avoid using unstable LNPs, and to enable sterile purification using a 0.22 ⁇ m filter, LNPs were included only if they met three criteria: diameter >20 nm, diameter ⁇ 200 nm, and correlation function with one inflection point. Particles that met these criteria were pooled and dialyzed in a 20 kD dialysis cassettes (Thermo Scientific) and a 100kD cassette (Thermo Scientific) in 1X PBS.
  • DLS high throughput dynamic light scattering
  • FIGS.2A-2I illustrates how an inventive nanoparticle, designated SPC-T LNP (formed from SPC-A9), delivers mRNA at doses as low as 0.01 mg/kg without noticeable toxicity at 1 mg/kg.
  • FIG.2A depicts the chemical structure and molar ratio of SPC-T LNP.
  • FIG.2B provides the biophysical properties of SPC-T LNP.
  • SPC-T LNP was formulated to carry chemically modified mRNA encoding Cre recombinase and was intravenously administered to Ai14 mice at a total nucleic dose of 1 mg / kg.
  • Cells within Ai14 mice contain a CAG-Lox-Stop-Lox-tdTomato construct. These cells become tdTomato + (tdTom + ) when Cre mRNA is delivered into the cytoplasm and translated into the functional Cre protein, which edits the genome by excising the Stop cassette.
  • tdTom + tdTomato +
  • SPC-T-mediated mRNA delivery in liver and 5 cell types in the spleen. (N 3 / group, average +/- SEM).
  • FIG.2E shows delivery as a function of SPC-T: mRNA mass ratio.
  • FIG.2F a ribogreen assay revealed that increasing the SPC-T: mRNA mass ratio increased the amount of RNA encapsulated.
  • FIGS.2H and 2I depict serum cytokines after a 0.5 or 1.0 mg / kg dose. Mice treated with low dose LPS is used as a positive control, whereas mice treated with PBS are used as a negative control. Only cytokine CCL2 was observe with SPC-T LNP, though in lower concentration than for LPS positive control.
  • LNPs co-localized with cells where functional mRNA has occurred were identified.
  • FACS fluorescence activated cell sorting
  • a LNP was identified that was subsequently named SPC-T and that was formed with the lipid SPC-A9, cholesterol, C 14 PEG 2k -, and DOPE.
  • FIGS.2A, 2B Functional delivery mediated by SPC-T carrying Cre mRNA in Ai14 mice was measured (FIG.2C). It was found that cells across the liver and spleen were transfected (FIG.2D).
  • FIGS.3A-3D illustrate how single-cell RNA sequencing was used to identify SPC- T LNP tropism at the cell type level as well as quantifying the aVHH expression.
  • FIG.3A provides cell populations in mice treated with PBS or SPC-T, showing no substantial changes.
  • the expression of aVHH protein was measured in each cell and overlaid on the t- SNE plots in FIG.3B, spleen and FIG.3C, lymph nodes. Small gray dots represent cells with no aVHH protein.
  • FIG.3D provides the percentage of aVHH+ cells measured with scRNA- Seq in each spleen and lymph node cell types.
  • scRNA-seq single-cell RNA-sequencing
  • FRG KO on NOD mice were repopulated with cryopreserved hepatocytes from a human donor (HHM18029) and later injected with human PBMC.
  • N 3-5 mice / group were used. All mice were injected intravenously via the lateral tail vein with LNPs or 1X PBS.
  • LNPs LNPs
  • 1X PBS 1X PBS.
  • Cell Isolation & Staining Cells were isolated 24 or 72 hours after injection with LNPs, unless otherwise noted. Mice were perfused in the liver portal vein with 5 mL of Krebs Ringer buffer (pH 7.3). Tissues were finely minced, and then placed in Collagenase XI (Sigma Aldrich) at 37 oC at 550 rpm for 30 minutes.
  • PCR Amplification All samples were amplified and prepared for sequencing using nested PCR. More specifically, 1 ⁇ L of primers (5 uM for Final Reverse / Forward) were added to 5 ⁇ L of Kapa HiFi 2X master mix (Roche), and 4 ⁇ L template DNA / water. During the second PCR Nextera XT chemistry, indices and i5/i7 adapter regions were added. Dual- indexed samples were run on a 2% agarose gel to ensure that PCR reaction occurred before being pooled and gel purified. [00180] Deep Sequencing. PCR samples were purified by AMPure XP beads. Final library QC was conducted using the Agilent Bioanalyzer 2100.
  • Cytokines were measured using Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D Systems).
  • stereopure lipids can be formulated into stable nanoparticles, and that these nanoparticles can deliver nucleic acids to a variety of cell types in vivo.
  • the nanoparticles delivered mRNA preferentially to liver, spleen, or lymphatic cells at a dose as low as 0.01 mg/kg, was identified directly using an in vivo barcoding approach, demonstrating the utility of direct to in vivo high-throughput nanoparticle studies.
  • reaction completion Upon reaction completion, the reaction was diluted with 30 mL DCM, washed with 10 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (10g column, 0-10% MeOH/DCM) yielded product as a colorless oil that solidified upon standing.
  • reaction completion Upon reaction completion, the reaction was diluted with 5 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated to a colorless wax. Purification via Flash chromatography (10g column, 0-20% MeOH/DCM) yielded product as a sticky colorless gel.
  • reaction completion Upon reaction completion, the reaction was diluted with 5 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated to a colorless wax. Purification via Flash chromatography (10g column, 0-20% MeOH/DCM) yielded product as a white waxy solid.
  • reaction completion Upon reaction completion, the reaction was diluted with 5 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated to a colorless wax. Purification via Flash chromatography (10g column, 0-20% MeOH/DCM) yielded product as a white waxy solid.
  • reaction completion Upon reaction completion, the reaction was diluted with 75 mL DCM, washed with 25 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (25g column, 0-20% MeOH/DCM) yielded product as a colorless oil.
  • N 1 ,N 1 -didecylethane-1,2-diamine [00191] To a vial containing tert-butyl (2-(didecylamino)ethyl)carbamate (1.38 g, 3.14 mmol) was added HCl in dioxane (15.69 mL, 62.76 mmol, 4M), and the resulting solution was stirred at rt for 4 hours. Upon reaction completion, the reaction was concentrated in vacuo, dissolved in 60 mL DCM, washed with 10 mL each 1M NaOH, water, and brine, dried over MgSO 4 , and concentrated in vacuo to a dark yellow oil.
  • N 1 ,N 1 -didecylethane-1,2-diamine (91.5 mg, 0.269 mmol) was added and the resulting clear, brown solution was stirred overnight at rt. Upon reaction completion, the reaction was concentrated in vacuo to a brown oil. Purification via Flash chromatography (5g HC column, 0-20% MeOH/DCM, 0.1% NH3), yielded product.
  • N 1 ,N 1 -didecylethane-1,2-diamine (98.9 mg, 0.290 mmol) was then added, and the reaction was stirred at rt overnight. Upon reaction completion, the reaction was concentrated in vacuo to a brown oil. Diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution., water, and brine, dried over MgSO 4 , and concentrated to a brown oil. Purification via Flash chromatography (5g HC column, 0-30% MeOH/DCM w/0.1% NH3) yielded product as a sticky yellow solid.
  • reaction completion Upon reaction completion, the reaction was diluted with 8 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (0-20% MeOH/DCM, 0.1% NH3) yielded product.
  • reaction completion Upon reaction completion, the reaction was diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (5g HC column, 3-6% MeOH/DCM) yielded product.
  • reaction completion Upon reaction completion, the reaction was diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO 4 , and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (5g HC column, 3-10% MeOH/DCM over 20 CVs) yielded product.
  • reaction completion Upon reaction completion, the reaction was diluted with 10 mL water, extracted with 3 x 10 mL DCM, washed combined organic phases with 10 mL brine, dried over MgSO 4 , and concentrated in vacuo to a yellow oil. Purification via Flash chromatography (0-10% EtOAc/DCM) yielded product as a yellow liquid.
  • reaction completion Upon reaction completion, the reaction was diluted with 10 mL saturated sodium bicarbonate solution, extracted with 3 x 10 mL DCM, washed combined organic phases with 10 mL each water x 3 and brine, dried over MgSO 4 , and concentrated in vacuo to an orange oil. Purification via Flash chromatography (6g C18 column, 50-100% ACN/water w/0.1% TFA). Yielded product as a TFA salt. Dissolved in 2 mL DCM, washed with 2 mL saturated sodium bicarbonate solution, dried over MgSO 4 , and concentrated in vacuo to a yellow gum.
  • N 1 ,N 1 -didecylethane-1,2-diamine 150 mg, 0.440 mmol
  • DMF 2.2 mL
  • the resulting solution was stirred at rt overnight.
  • the reaction was diluted with DCM (30 mL), washed with 10 mL each saturated sodium bicarbonate solution, water x3, and brine, dried over MgSO4, and concentrated in vacuo to a yellow oil. Purification via flash chromatography (5 g HC column, 0-10% MeOH/DCM, 10 CV ramp) yielded product.
  • Example 5 Delivery of mRNA to Mouse Using Lipid Nanoparticle Formulated With Exemplary Diketopiperazine Lipids Formulation of Lipid Nanoparticles Using Diketopiperazine Lipids
  • Exemplary lipid nanoparticle formulations were prepared using the diketopiperazine lipids synthesized according to Examples 1 and 4. These lipid nanoparticle formulations were formulated with various combinations of various exemplified diketopiperazine lipids, cholesterol, helper lipid, and PEG lipid at various ratios, to encapsulate mRNA, using procedures similar to those described in Example 2.
  • LNPs were prepared using the NanoAssemblr Ignite system or the NanoAssemblr Spark system by combining i) an aqueous phase with mRNA and 25 mM acetic acid buffer at pH 5.0, and ii) an organic phase containing a combination of four lipids dissolved in ethanol. After mixing, resulting LNPs were dialyzed in 1X PBS with a 20 kDa dialysis cassette for 2-3 hours and filtered using a 0.22 ⁇ m pore size PES filter. Where the nanoparticles contained a DNA barcode in addition to mRNA, LNPs were not analyzed until a second dialysis step.
  • Nanoparticles were then analyzed in the Unchained Stunner to determine whether they meet the quality control criteria: diameter (20 nm - 200 nm), polydispersity index ( ⁇ 0.3), concentration, intensity of peak of interest (> 75%), and turbidity (A260 ⁇ 3.0 and A330 ⁇ 1.25).
  • the nanoparticles contained a DNA barcode in addition to the mRNA, and passed the quality control criteria were pooled and dialyzed again in 1X PBS for 1 hour in a 100 kDa dialysis cassette.
  • Dose Administration Doses were calculated based on animal weight the day of administration. LNPs were administered as a bolus via a tail vein using an insulin syringe with a 28 gauge.
  • Tissue Collection Animals injected with LNPs containing reporter mRNA were sacrificed in a carbon dioxide chamber 16 - 24 hours after nanoparticle administration. Relevant tissues were then isolated, minced finely on top of Petri dishes into ⁇ 1 cm 3 cubes (with the exception of bone marrow) and placed into individual aliquots containing specific digestive enzyme cocktails depending on the organ. [0212] Tissue Digestion: Samples were digested as discussed below: i.
  • Liver Digestion Digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (200 U/mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 oC for 30 minutes at 750 rpm. Then, they were transferred through a 70 ⁇ m mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed.
  • Collagenase I 450 U/mL
  • Collagenase XI 125 U/mL
  • Hyaluronidase 60 U/mL
  • DNAse I 200 U/mL
  • RPMI-1640 solvent RPMI-1640 solvent
  • Kidney Digestion Digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (200 U/mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 oC for 30 minutes at 750 rpm. Then, they were transferred through a 70 ⁇ m mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed.
  • Liver Panel anti-mouse CD3 (17A2), anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD68 (FA-11), anti-mouse CD31 (390), anti-mouse TER-119 (TER- 119), anti-mouse NK1.1 (PK136), anti-mouse CD11c (N418), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. ii.
  • Lung Panel anti-mouse CD8a (53-6.7), anti-mouse CD326 (G8.8), anti- mouse/human CD49f (GoH3), anti-mouse CD4 (RM4-5), anti-mouse CD3 (17A2), anti- mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD31 (390), anti-mouse TER- 119 (TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iii.
  • Kidney Panel anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD31 (390), anti-mouse TER-119 (TER-119), anti-mouse CD309 (89B3A5), anti-mouse Podocalyxin (10B9), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iv.
  • Heart Panel anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD31 (390), anti-mouse TER-119 (TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. v.
  • Spleen Panel anti-mouse CD8a (53-6.7), anti-mouse CD19 (6D5), anti-mouse CD11b (M1/70), anti-mouse CD3 (17A2), anti-mouse TER-119 (TER-119), anti-mouse NK1.1 (PK136), anti-mouse CD4 (RM4-5), anti-mouse CD11c (N418), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. vi.
  • Bone Marrow Panel anti-mouse CD4 (RM4-5), anti-mouse CD117 (2B8), anti- mouse CD8a (53-6.7), anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse Sca1 (D7), anti-mouse TER-119 (TER-119), anti-mouse CD3 (17A2), anti-mouse CD34 (SA376A4), anti-mouse CD11c (), Lineage Cocktail (CD3 / GR-1 / CD11b / CD45R / B220 / TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered.
  • Flow Cytometry Readouts Different cell populations were analyzed for reporter protein expression relative to an untreated primate. The results were expressed as the percentage of cells that were positive for the reporter protein. Where LNPs contained DNA barcodes in addition to the mRNA, populations expressing reporter protein were sorted into QuickExtract buffer to isolate intracellular DNA. [0215] DNA Sequencing of Sorted Samples: DNA barcodes of sorted cells were amplified under two rounds of PCR and Illumina handles were attached for compatibility with Next Generation Sequencing. Amplified samples were pooled, cleaned, diluted to 1 nM, and loaded into an iSeq cartridge for sequencing. Samples were analyzed for relative DNA barcode counts for selection of LNP candidates to advance in the pipeline.
  • FIGS.4-14 The results of LNP screening in mouse for LNP formulations containing various exemplary diketopiperazine lipids as disclosed herein are shown in FIGS.4-14.
  • certain LNP formulations were selected from high-throughput, barcoding based, screens.
  • each LNP formulation was numbered as LNP-1 through LNP-XYZ in database, referred to as the sample number for that LNP formulation.
  • LNP-151 indicates the 151st LNP formulation for a screen.
  • FIGS.4A-4D illustrate the flow cytometry results for three high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 18 (also referred to as KC-34 in Example 1) in the liver (FIG.4A), spleen (FIG.4B), lung (FIG. 4B), or bone marrow (FIG.4D).
  • LNPs were formulated with Compound 18 (Lipomer), cholesterol, a PEG lipid, and a helper lipid (HL).
  • HL helper lipid
  • the total lipid to nucleic acid mass ratio was 10:1.
  • the PEG lipid included C14PEG2K PE, C18PEG2K PE, DMG-PEG2K, or DSG-PEG2K; and the helper lipid included DOPE or DSPC.
  • the ratios for the lipid components were: [0219]
  • the PEG lipid included C14PEG2K PE, C18PEG2K PE, DMG-PEG2K, or DSG-PEG2K; and the helper lipid included 14:0 PA, 18:1 PA, 14:0 Hemi BMP, or 18:1 Hemi BMP.
  • the molar ratios for the lipid components were: [0220] In Screen 3, the PEG lipid included C14PEG2K PE or DMG-PEG; and the helper lipid included DOPE or DSPC. The molar ratios for the lipid components were: [0221]
  • the results in FIGS.4A-4D show that the LNP delivery was found in multiple cell populations in the liver of mouse, including endothelial cells, and immune cells; and in multiple cell populations in the spleen of mouse, including some delivery to B cells, macrophages, and dendritic cells.
  • FIGS.5A-5B show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.5A) and spleen (FIG.5B).
  • Screen 1 a number of LNPs that performed well in Screen 1 (a high-throughput, barcoding based, screen disclosed in FIGS.4A-4D) were selected and individually injected one by one in mice.
  • LNP formulations (Lipomer: Compound 18) for FIGS.5A-5B are characterized below: [0223] As shown in FIG.5A, certain LNP formulations (e.g., LNP-156) performed well across all hepatic populations, while certain LNP formulations (e.g., LNP-185, LNP-121, and LNP-122) performed well in immune cell populations in the liver in mouse. FIG.5B shows significant levels of delivery in the spleen in mouse, such as in dendritic cells in the spleen, for certain LNP formulations (e.g., LNP-185, LNP-121, LNP-122, and LNP-156).
  • LNP-185 e.g., LNP-185, LNP-121, LNP-122, and LNP-156
  • FIGS.6A-6B show the flow cytometry results for individual LNP formulations selected from Screen 3 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.6A) and spleen (FIG.6B).
  • FIGS.6A-6B show the flow cytometry results for individual LNP formulations selected from Screen 3 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.6A) and spleen (FIG.6B).
  • a number of LNPs that performed well in Screen 3 were selected and individually injected one by one in mice.
  • the LNP formulations (Lipomer: Compound 18) for FIGS.6A-6B are characterized below:
  • FIG.6A shows that many LNP formulations performed well in the liver in mouse.
  • FIG.6B shows that certain LNP formulations that did not perform as well in the liver had high levels of delivery in multiple populations across the spleen in mouse.
  • FIGS.7A-7D show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 26, Compound 29, Compound 30, and Compound 31 in the liver (FIG.7A), lung (FIG.7B), spleen (FIG. 7C), and bone marrow (FIG.7D).
  • LNPs were formulated with a Lipomer (Compound 26, Compound 29, Compound 30, or Compound 31), cholesterol, a PEG lipid, and a helper lipid (HL).
  • a Lipomer Compound 26, Compound 29, Compound 30, or Compound 31
  • cholesterol a PEG lipid
  • HL helper lipid
  • the lipid components LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced.
  • the total lipid to nucleic acid mass ratio was 10:1.
  • the PEG lipid included ALC-0159, PEG2K-C-DMG, or C18PEG2K PE; and the helper lipid included DSPC.
  • the molar ratios for the lipid components were: [0228] In Screen 2, the PEG lipid included ALC-0159, PEG2K-C-DMG, or C18PEG2K PE; and the helper lipid included DDAB or DOTAP. The molar ratios for the lipid components were: [0229] The results in FIGS.7A-7D show that the LNP delivery was found in immune cell populations in the liver in mouse, including macrophages.
  • FIGS.8A-8F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 39 and Compound 42 in the liver (FIG.8A), heart (FIG.8B), bone marrow (FIG.8C), kidney (FIG. 8D), spleen (FIG.8E), and lung (FIG.8F).
  • LNPs were formulated with a Lipomer (Compound 39 or Compound 42), cholesterol, a PEG lipid, and a helper lipid (HL).
  • HL helper lipid
  • the total lipid to nucleic acid mass ratio was 10:1.
  • the PEG lipid included C14-PEG2K PE, DSG-PEG2K, DMG-PEG2K, or C18-PEG2K PE; and the helper lipid included DDAB or DSPC.
  • the molar ratios for the lipid components were: [0232]
  • the results in FIGS.8A-8F show that LNP delivery was found in basal and stem populations in the lung in mouse.
  • FIGS.9A-9F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 49 and Compound 50 in the liver (FIG.9A), lung (FIG.9B), bone marrow (FIG.9C), spleen (FIG. 9D), heart (FIG.9E), and kidney (FIG.9F).
  • LNPs were formulated with a Lipomer (Compound 49 or Compound 50), cholesterol, a PEG lipid, and a helper lipid (HL).
  • HL helper lipid
  • the total lipid to nucleic acid mass ratio was 10:1.
  • the PEG lipid included C14-PEG2K PE, DSG-PEG2K, DMG-PEG2K, or C18-PEG2K PE; and the helper lipid included DSPC.
  • the molar ratios for the lipid components were: [0235]
  • the PEG lipid included C14-PEG2K PE, DSG-PEG2K, DMG-PEG2K, or C18-PEG2K PE; and the helper lipid included DDAB.
  • FIGS.9A-9F show that LNP delivery was found in the liver, lung, and bone marrow in mouse.
  • FIGS.10A-10C show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGS.9A-9F) in mouse using the LNP formulations containing Compound 50 in the liver (FIG.10A), lung (FIG.10B), and bone marrow (FIG. 10C).
  • a number of LNPs that performed well in Screen 2 were selected and individually injected one by one in mice.
  • FIGS.11A-11F show the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 55 in the liver (FIG.11A), lung (FIG.11B), bone marrow (FIG.11C), spleen (FIG.11D), kidney (FIG. 11E), and heart (FIG.11F).
  • LNPs were formulated with Compound 55 (Lipomer), cholesterol, a PEG lipid, and a helper lipid (HL).
  • the molar ratios for the lipid components were:
  • FIGS.11A-11F show that LNP delivery was found in the liver, lung, bone marrow, spleen, kidney, and heart; and significant levels of LNP delivery were found in progenitor cells in the bone marrow in mouse (FIG.11C), in macrophage and monocyte populations in the spleen in mouse (FIG.11D), and in endothelial cells and macrophages in the kidney in mouse (FIG.11E).
  • FIGS.12A-12F show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs.11A-11F) in mouse using the LNP formulations containing Compound 55 in the liver (FIG.12A), lung (FIG.12B), bone marrow (FIG.12C), spleen (FIG.12D), kidney (FIG.12E), and heart (FIG.12F).
  • FIGS.12A-12F show the LNP formulations (Lipomer: Compound 55) for FIGS.12A-12F.
  • the results in FIGS.12A-12F show that some LNP delivery was found in the liver, lung, bone marrow, spleen, kidney, and heart; although the levels of delivery in these tissues or populations were not significant.
  • FIGS.13A-13F show the flow cytometry results for a high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 60 and Compound 63 in the liver (FIG.13A), lung (FIG.13B), bone marrow (FIG.13C), spleen (FIG.13D), kidney (FIG.13E), and heart (FIG.13F).
  • LNPs were formulated with a Lipomer (Compound 60 or Compound 63), cholesterol, a PEG lipid, and a helper lipid (HL).
  • a Lipomer Compound 60 or Compound 63
  • cholesterol a PEG lipid
  • HL helper lipid
  • the total lipid to nucleic acid mass ratio was 10:1.
  • the PEG lipid included C14-PEG2K PE or C18-PEG2K PE; and the helper lipid included DDAB or DSPC.
  • the molar ratios for the lipid components were: [0244] As shown in FIGS.13A-13F, high levels of LNP delivery were found in cell populations of the liver in mouse, and in endothelial cells of the lung in mouse. Moreover, these figures show that significant levels of LNP delivery were found in progenitor cells in the bone marrow of mouse, in macrophage and monocyte populations in the spleen of mouse, in endothelial cells and macrophages in the kidney of mouse, and in endothelial cells in the heart of mouse.
  • Example 6 Delivery of mRNA to non-human Primate (NHP) Using Lipid Nanoparticles Formulated With the Exemplary Diketopiperazine Lipids Formulation of Lipid Nanoparticles Using Diketopiperazine Lipids
  • Exemplary lipid nanoparticle formulations were prepared using the diketopiperazine lipids synthesized according to Examples 1 and 4. These lipid nanoparticle formulations were formulated with various combinations of various exemplified diketopiperazine lipids, cholesterol, helper lipid, and PEG lipid at various ratios, to encapsulate mRNA, using procedures similar to those described in Example 2.
  • Predose Biopsy Collections Blood (8 to 10 mL) were collected via a femoral vein into tubes containing K 2 EDTA from each animal predose (Day -20 to -22 based upon each animal’s dosing date). Additionally, bone marrow and liver biopsy samples were collected from all animals on Day -20 to -22 based upon the animal’s date of dosing. Animals underwent laparoscopic liver biopsy procedures while bone marrow were collected by perfusion of 30 mL of phosphate buffered saline (PBS) through one femur bone.
  • PBS phosphate buffered saline
  • LNPs were prepared using the NanoAssemblr Ignite system by combining i) an aqueous phase with mRNA and 25 mM acetic acid buffer at pH 5.0, and ii) an organic phase containing a combination of four lipids dissolved in ethanol.
  • Nanoparticles were dialyzed in 1X PBS with a 20 kDa dialysis cassette for 2-3 hours and filtered using a 0.22 ⁇ m pore size PES filter. Nanoparticles were then analyzed to determine whether they meet the quality control criteria: using DLS for diameter (20 nm - 200 nm), polydispersity index ( ⁇ 0.3), concentration, intensity of peak of interest (> 75%), and turbidity (A260 ⁇ 3.0 and A330 ⁇ 1.25). The nanoparticles that met these criteria were diluted to a solution of 1X PBS and 10% w/v trehalose, frozen at -20 oC, transferred to -80 oC, and shipped in dry ice the next day.
  • LNPs were allowed to thaw at room temperature for at least 45 minutes. Once thawed, they were filtered using 0.22 ⁇ m pore size, 33 mm diameter PES filters. Nanodrop RNA concentration measurements were taken before and after filtering to calculate dose retention from the original LNP concentration.
  • Dose Administration Doses were calculated based on animal weight the day of administration. LNPs were administered as a 30-minute infusion via a saphenous vein using a catheter set.
  • Post-dose Procedures Body weight and temperature were monitored after infusion until sacrifice. Additionally, hematology values and clinical chemistry were tracked several times after administration to monitor the response of the animal to the infused LNP.
  • Tissue Collection Animals injected with LNPs containing reporter mRNA were sacrificed by exsanguination under anesthesia 16 - 24 hours after nanoparticle administration. Relevant tissues were then isolated, minced finely into ⁇ 3 cm 3 cubes (with the exception of bone marrow, blood, and spleen) and placed into individual aliquots of a solution containing 90% FBS and 10% DMSO. All samples will be stored in Mr. Frosty for control temperature change up to 72 hours at -70 to -90°C and then frozen in liquid nitrogen or at approximately - 80°C until shipped. [0253] Tissue Digestion: All samples were allowed to thaw at room temperature prior to processing.
  • tissue digestion solutions were processed as indicated below: i. Liver Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (400 U/mL), and RPMI-1640 solvent. Tissues were incubated at 37 oC for 30 minutes at 1,000 rpm.
  • Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (400 U/mL), and RPMI-1640 solvent. Tissues were incubated at 37 oC for 45 minutes at 1,000 rpm. Then, they were transferred through a 70 ⁇ m mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed.
  • Collagenase I 450 U/mL
  • Collagenase XI 125 U/mL
  • Hyaluronidase 60 U/mL
  • DNAse I 400 U/mL
  • RPMI-1640 solvent RPMI-1640 solvent
  • Kidney Digestion Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (400 U/mL), and RPMI-1640 solvent. Tissues were incubated at 37 oC for 30 minutes at 1,000 rpm.
  • Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase II (1 mg/mL), Dispase (0.6 U/mL), DNAse I (400 U/mL), FBS (2% v/v), and RPMI-1640 solvent. Tissues were incubated at 37 oC for 1 hour at 1,000 rpm. Then, they were transferred through a 70 ⁇ m mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed.
  • Liver Panel anti-human CD56 (REA196), anti-human CD31 (WM59), anti-human CD4 (OKT4), anti-NHP CD45 (D058-1283), anti-human CD3 (SP34-2), anti-mouse CD11b (M1/70), anti-human CD68 (REA886), anti-human CD11c (S-HCL-3), anti-human CD8 (BW135/80), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. ii.
  • Lung Panel anti-human CD31 (WM59), anti-human CD326 (MH99), anti-mouse CD11b (M1/70), anti-mouse/human CD49f (GoH3), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered.
  • Kidney Panel anti-human CD31 (WM59), anti-mouse CD11b (M1/70), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered.
  • Heart Panel anti-human CD31 (WM59), anti-mouse CD11b (M1/70), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. v.
  • Spleen Panel anti-human CD56 (REA196), anti-human CD4 (OKT4), anti-human CD3 (SP34-2), anti-mouse CD11b (M1/70), anti-human CD20 (2H7), anti-human CD11c (S- HCL-3), anti-human CD8 (BW135/80), anti-human CD31 (WM59), anti-mouse CD11b (M1/70), anti-human CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. vi.
  • Bone Marrow Panel anti-human CD123 (7G3), and-human CD117 (104D2), anti- human CD4 (OKT4), anti-mouse CD11b (M1/70), anti-human CD34 (561 and 563), anti- human CD45RA (5H9), anti-human CD90 (REA897), anti-human CD8 (BW135/80), anti- human CD45RO (UCHL1), anti-human CD20 (2H7), anti-human CD2 (RPA-2.10), anti- human CD14 (M5E2), anti-human CD16 (3G8), anti-human CD23 (M-L233), anti-human CD21 (B-ly4), anti-human CD56 (REA196), anti-human CD3 (SP34-2), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered.
  • FIGS.7A-7B The results of LNP screening in primate for a LNP formulation containing an exemplary diketopiperazine lipid as disclosed herein are shown in FIGS.7A-7B.
  • certain LNP formulations were selected from a high-throughput, barcoding based, screen. During high-throughput screening, each LNP formulation was numbered as LNP-1 through LNP-XYZ in database, referred to as the sample number for that LNP formulation. For instance, LNP-156 indicates the 156th LNP formulation for a screen.
  • FIGS.14A-14B show the flow cytometry results for a LNP formulation containing Compound 18 (e.g., LNP-156) selected from a previous screening (discussed in FIGs.5A- 5B) in primate at a dosage of 0.45 mg/kg, in the liver (FIG.14A) and spleen (FIG.14B).
  • the LNP formulations (Lipomer: Compound 18) for FIGS.14A-14B are characterized below: [0258]
  • FIG.14A shows that LNP delivery was found in most cell populations in the liver of primate, although the delivery in hepatocytes was not as significant as other cell populations.
  • FIG.14B shows that LNP delivery was found in all cell populations in the spleen of primate assessed, and similar delivery levels were found across every population in the spleen of primate assessed.

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Abstract

The present disclosure provides ionizable lipids containing a diketopiperazine moiety, lipid nanoparticles formed using such ionizable lipids, as well as their use in delivering nucleic acids and other therapeutic agents to certain cell types.

Description

NOVEL LIPIDS CONTAINING DIKETOPIPERAZINE CORE, LIPID NANOPARTICLE CONTAINING THEREOF, AND METHODS OF USING THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of priority to U.S. Provisional App. No. 63/632,381, filed April 10, 2024, the entire contents of which are incorporated herein by reference. GOVERNMENT RIGHTS [0002] This invention was made with government support under Contract No. UG3- TR002855 awarded by the National Institutes of Health, and Contract No. HR00111920008 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention. TECHNICAL FIELD [0003] The present disclosure concerns ionizable lipids that can be used to form lipid nanoparticles (LNPs). BACKGROUND [0004] In humans, lipid nanoparticles (LNPs) have safely delivered therapeutic RNA to hepatocytes after systemic administration and to antigen-presenting cells after intramuscular injection. The Food and Drug Administration (FDA) approved its first lipid nanoparticle (LNP)-based siRNA drug to treat an inherited genetic disease in 2018. Since then, systemically administered siRNA therapeutics have been approved to treat three additional liver diseases and generated promising earlier-stage mRNA clinical data. Similarly, intramuscularly administered mRNA therapies have been FDA approved or been given Emergency Use Authorization to vaccinate against coronavirus disease of 2019. Unfortunately, there have also been clinical failures driven by insufficient delivery. Taken together, the efficacy of approved RNA vaccines and liver therapies underscores the potential clinical impact of LNPs with tropism to new cell types. However, this challenge is stark; no systemically administered LNP carrying an RNA drug has yet reached phase III clinical trials, let alone been FDA approved. [0005] CKK-E12 is a potent lipid that was proven useful in delivering siRNA and mRNA [PMID: 24516150]. However, the presence of β-OH groups introduced four uncontrollable stereocenters, which was hypothesized to lead to batch-to-batch inconsistency. Assuming a 0.3 mg mRNA / kg injection in a 100 kg human, a 20:1 lipid:RNA mass ratio, and ignoring loss during formulation, one clinical LNP-mRNA injection requires hundreds of milligrams of lipid. Thus, an ideal synthetic route would generate lipid at gram scales without uncontrolled stereocenters, which make purification challenging and can lead to enantiomer- specific biological interactions. It is an unmet need to design a series of lipids stereopure lipids lacking any uncontrollable stereocenters, and discover novel LNP formulations with stereopure lipid materials. [0006] In addition, delivering RNA to non-hepatocytes has remained challenging in large part due to the anatomy and physiology of the liver. Specifically, the hepatic sinusoids contain a discontinuous vasculature as well as slow blood flow; both increase nanoparticle extravasation and subsequent interactions with hepatocytes. To target non-hepatocytes, scientists have used two approaches. In the first approach, an LNP with tropism to hepatocytes is retargeted with an active targeting ligand. For example, LNPs made with DLin-MC3-DMA, an ionizable lipid that is FDA approved for hepatocyte siRNA delivery, have been retargeted to immune cells using a lipid-bound antibody. One potential limitation of this approach is that actively targeted nanoparticles containing RNA drugs have led to adverse events in clinical trials. In a second approach, scientists identify nanoparticles that interact with natural trafficking pathways, thereby leading to endogenous targeting. Although these approaches have led to an FDA approval and promising phase 1 clinical data, this second approach also has a key limitation. After synthesizing a large, chemically diverse lipid library, scientists must evaluate how each nanoparticle delivers its payload into cells. Since injecting and sacrificing thousands of mice per library is unethical, this screening is performed in vitro (i.e., in cell culture). For example, across three representative papers, labs tested 4,736 nanoparticles in vitro, using the data to select 14 nanoparticles for in vivo studies. However, this screening method is likely inefficient, given that in vitro nanoparticle delivery can be a poor predictor of in vivo nanoparticle delivery. [0007] Systemic RNA delivery to non-hepatocytes remains challenging, especially without targeting ligands such as antibodies, peptides, or aptamers. SUMMARY [0008] One aspect of the invention relates to a compound of Formula (I): wherein: Y is H; aryl; heteroaryl; branched or unbranched C1-C20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl; wherein: each t is independently 1, 2, 3, or 4; each X is independently -O-, -S-, or -N(R′′)-; Z is absent, -C(O)-, -C(O)O-, -C(O)N(R′′)-, or -S(O)(O)-; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′, and R′′ each are independently H, OR14, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; Ra and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl or heterocyclic, each R11, R12, and R14 are independently H, branched or unbranched alkyl or alkenyl, and each R13 is independently H, alkyl, or OR14; provided that when X is O or S or when Z is C(O), Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-. The compounds also include the a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds disclosed herein, [0009] Another aspect of the invention relates to a method of making a compound of formula reacting with one or more deprotecting reagents to remove the carboxyl protecting group and amine protecting group, and react the resulting intermediate under conditions sufficient to couple and cyclize the intermediate to form the compound of formula (IIA), wherein: each X is independently -O-, -S-, or -N(R′′)-; each t is independently 1, 2, 3, or 4; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′′, Ra, and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14 are independently H, branched or unbranched alkyl or alkenyl. [0010] Another aspect of the invention relates to a method of making a compound of formula organic base and a solvent to form the compound, wherein: each t is independently 1, 2, 3, or 4; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′, Ra, and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14 are independently H, branched or unbranched alkyl or alkenyl. [0011] Also disclosed are lipid nanoparticles comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein. [0012] Another aspect of the invention relates to a pharmaceutical composition comprising the lipid nanoparticle, comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, and a pharmaceutically acceptable carrier. [0013] Another aspect of the invention relates to methods of delivering a therapeutic agent to a subject comprising administering to the subject the presently disclosed lipid nanoparticles, wherein the lipid nanoparticles comprise a therapeutic agent which may be optionally encapsulated within the lipid nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS [0014] FIGS.1A-1E illustrate the processes and parameters with which diketopiperazine- based lipids were used to formulate stable lipid nanoparticles (LNPs). FIG.1A illustrates how stereopure lipids were formulated with two cholesterol variants (cholesterol or 20 -OH cholesterol), two PEG-lipid variants (C14PEG2K or C18PEG2K), and DOPE. FIG.1B provides how LNPs were formulated using four molar ratios, thereby formulating 96 total LNPs, as shown in FIG.1C. FIG.1D illustrates hydrodynamic diameters of the 64 individual LNPs and the 64 LNP pool. FIG.1E provides hydrodynamic diameter as a function of stereopure structure, average +/- SEM. [0015] FIGS.2A-2I provide the results of a study quantifying delivery of mRNA by a lipid nanoparticle according to the present disclosure. FIG.2A depicts the chemical structure and molar ratio of SPC-T LNP. FIG.2B provides the biophysical properties of SPC-T LNP. FIG.2C illustrates how SPC-T LNP was formulated to carry chemically modified mRNA encoding Cre recombinase and was intravenously administered to Ai14 mice at a total nucleic dose of 1 mg / kg. FIG.2D illustrates SPC-T-mediated mRNA delivery in liver and 5 cell types in the spleen. (N = 3 / group, average +/- SEM). FIG.2E shows delivery as a function of SPC-T: mRNA mass ratio. FIG.2F provides a ribogreen assay revealing that increasing the SPC-T: mRNA mass ratio increased the amount of RNA encapsulated. FIG.2G illustrates mRNA delivery at doses as low as 0.01 mg / kg. (N = 3 / group, average +/- SEM). FIGS.2H and 2I depict serum cytokines after a 0.5 or 1.0 mg / kg dose. [0016] FIGS.3A-3D provide the results of an investigation of SPC-T LNP tropism at the cell type level, and quantification of the aVHH expression. FIG.3A provides cell populations in mice treated with PBS or SPC-T, showing no substantial changes. The expression of aVHH protein was measured in each cell and overlaid on the t-SNE plots in FIG.3B, spleen and FIG.3C, lymph nodes. FIG.3D provides the percentage of aVHH+ cells measured with scRNA-Seq in each spleen and lymph node cell types. [0017] FIGS.4A-4D illustrate the flow cytometry results for three high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 18 in the liver (FIG.4A), spleen (FIG.4B), lung (FIG.4B), or bone marrow (FIG.4D). [0018] FIGS.5A-5B show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.5A) and spleen (FIG.5B). [0019] FIGS.6A-6B show the flow cytometry results for individual LNP formulations selected from Screen 3 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.6A) and spleen (FIG.6B). [0020] FIGS.7A-7D show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 26, Compound 29, Compound 30, and Compound 31 in the liver (FIG.7A), lung (FIG.7B), spleen (FIG. 7C), and bone marrow (FIG.7D). [0021] FIGS.8A-8F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 39 and Compound 42 in the liver (FIG.8A), heart (FIG.8B), bone marrow (FIG.8C), kidney (FIG. 8D), spleen (FIG.8E), and lung (FIG.8F). [0022] FIGS.9A-9F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 49 and Compound 50 in the liver (FIG.9A), lung (FIG.9B), bone marrow (FIG.9C), spleen (FIG. 9D), heart (FIG.9E), and kidney (FIG.9F). [0023] FIGS.10A-10C show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs.9A-9F) in mouse using the LNP formulations containing Compound 50 in the liver (FIG.10A), lung (FIG.10B), and bone marrow (FIG. 10C). [0024] FIGS.11A-11F show the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 55 in the liver (FIG.11A), lung (FIG.11B), bone marrow (FIG.11C), spleen (FIG.11D), kidney (FIG. 11E), and heart (FIG.11F). [0025] FIGS.12A-12F show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs.11A-11F) in mouse using the LNP formulations containing Compound 55 in the liver (FIG.12A), lung (FIG.12B), bone marrow (FIG.12C), spleen (FIG.12D), kidney (FIG.12E), and heart (FIG.12F). [0026] FIGS.13A-13F show the flow cytometry results for a high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 60 and Compound 63 in the liver (FIG.13A), lung (FIG.13B), bone marrow (FIG.13C), spleen (FIG.13D), kidney (FIG.13E), and heart (FIG.13F). [0027] FIGS.14A-14B show the flow cytometry results for a LNP formulation containing Compound 18 (e.g., LNP-156) selected from a previous screening (discussed in FIGs.5A- 5B) in primate at a dosage of 0.45 mg/kg, in the liver (FIG.14A) and spleen (FIG.14B). DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [0028] The present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific products, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. [0029] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in their entirety. [0030] As employed above and throughout the disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings. [0031] In the present disclosure the singular forms “a”, “an”, and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. Furthermore, when indicating that a certain chemical moiety “may be” X, Y, or Z, it is not necessarily intended by such usage to exclude other choices for the moiety; for example, a statement to the effect that R1 “may be alkyl, aryl, or amino” does not necessarily exclude other choices for R1, such as halo, aralkyl, and the like. [0032] When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10% of the recited value, inclusive. For example, the phrase “about 8” may refer to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” may refer to a value of 7.2% to 8.8%, inclusive. Also, when the term “about” precedes a range, it is understood that the term modifies both recited endpoints and all points embraced within the range. For example, the phrase “about 1-10” is understood to mean “about 1 to about 10”, as well as “about x”, wherein x refers to any value between 1 and 10. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” In another example, when a listing of possible substituents including “hydrogen, alkyl, and aryl” is provided, the recited listing may be construed as including situations whereby any of “hydrogen, alkyl, and aryl” is negatively excluded; thus, a recitation of “hydrogen, alkyl, and aryl” may be construed as “hydrogen and aryl, but not alkyl”, or simply “wherein the substituent is not alkyl”. [0033] Protective groups are abbreviated according to the system disclosed in Greene, T.W. and Wuts, P.G.M., Protective Groups in Organic Synthesis 2d. Ed., Wiley & Sons, 1991, which is incorporated in its entirety herein. For example, “CBZ” or “Cbz” or “Z” stands for carbobenzyloxy or benzyloxycarbonyl, “Boc” or “BOC” represents t-butoxycarbonyl, “Alloc” denotes allyloxycarbonyl, Bz means benzoyl, and “Fmoc” stands for 9- fluorenylmethoxycarbonyl. [0034] As used herein, the terms “component”, “compound”, “drug”, “pharmacologically active agent”, “active agent”, “therapeutic”, “therapeutic agent”, “therapy”, “treatment”, or “medicament” may be used herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action. [0035] The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. Unless otherwise indicated, “alkyl” generally refers to C1-C24 alkyl (e.g., C1-C12 alkyl, C1-C8 alkyl, or C1-C4 alkyl). [0036] The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2- 8 carbon atoms and characterized in having one or more double bonds. Unless otherwise indicated, “alkenyl” generally refers to C2-C8 alkenyl (e.g., C2-C6 alkenyl, C2-C4 alkenyl, or C2-C3 alkenyl). Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2- butenyl, 3-hexenyl and 3-octenyl groups. [0037] The term “alkoxy” refers to an -O-alkyl radical. The term “alkylene” refers to a divalent alkyl (i.e., -R-). The term “aminoalkyl” refers to an alkyl substituted with an amino. The term “mercapto” refers to an -SH radical. The term “thioalkoxy” refers to an -S-alkyl radical. [0038] The term “alkylene” refers to abivalent form of an alkyl group. An “alkylene chain” is a polymethylene group, i.e., —(CH2)n—, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below. [0039] The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below. [0040] The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term “aryl” may be used interchangeably with the term “aryl ring.” Examples of aryl groups include phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl. The term “arylalkoxy” refers to an alkoxy substituted with aryl. [0041] The term “cycloalkyl” or “cyclyl” as employed herein includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. [0042] The term “heteroaryl” or “heteroar-” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H- quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one and the like. [0043] The term “heterocyclyl,” “heterocycle,” “heterocyclic radical,” or “heterocyclic ring” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted. [0044] A divalent radical of an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, heterocyclyl is formed by removal of a hydrogen atom from an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radical, respectively (or by removal of two hydrogen atoms from an alkane, alkene, arene, heteroarene, cycloalkane, or heterocycle, respectively). [0045] The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted. [0046] “Isomers.” The compounds described herein or their pharmaceutically acceptable salts may include all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like. For instance, the compounds can contain one or more stereocenters and may thus give rise to geometic isomers (e.g., double bond causing geometric E/Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/trans isomers), and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- such as for sugar anomers, or as (D)- or (L)- such as for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (- ), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. [0047] A "stereoisomer" refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes "enantiomers", which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another. [0048] The term “pharmaceutically acceptable salt” include the salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or rnalonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI alkyl^ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt. [0049] Disclosed herein are diketopiperazine-containing ionizable lipids that can, among other things, preferentially deliver mRNA to immune cells or stem cells in various tissues in vivo without targeting ligands. The inventive lipids were synthesized and characterized, and high-throughput DNA barcoding was used to quantify how a collection of chemically distinct LNPs functionally delivered mRNA (i.e., mRNA translated into functional, gene-editing protein) in multiple cell types directly in vivo. By analyzing the relationships between lipid structure and cellular targeting, lipid traits that increase delivery in vivo were identified. In addition, LNPs that preferentially delivers mRNA to liver or non-liver cells at clinically relevant doses were prepared and characterized. The obtained data highlighted inventive nanoparticles with natural non-hepatocyte tropism and demonstrated that the presently disclosed lipids with bioactive small-molecule motifs successfully deliver mRNA in vivo. The novel lipids [0050] Accordingly, disclosed are compounds of Formula (I): wherein: Y is H; aryl; heteroaryl; branched or unbranched C1-C20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl; wherein: each t is independently 1, 2, 3, or 4; each X is independently -O-, -S-, or -N(R′′)-; Z is absent, -C(O)-, -C(O)O-, -C(O)N(R′′)-, or -S(O)(O)-; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′, and R′′ each are independently H, OR14, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; Ra and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl or heterocyclic, each R11, R12, and R14 are independently H, branched or unbranched alkyl or alkenyl, and each R13 is independently H, alkyl, or OR14; provided that when X is O or S or when Z is C(O), Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-. The compounds also include the a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the compounds disclosed herein, [0051] In some embodiments, the lipid compound has the formula of: All the variables have the same definitions as those defined above. [0052] In certain embodiments, in any of the formulas disclosed herein (each of the below variables, if present): Y is H; aryl; branched or unbranched C1-C6 alkyl; C1-C6 alkyl interrupted with a N, O, or S atom; C1-C3 alkyl substituted with an aryl; ; or each R, R′, Ra, and Rb are H; each t is independently 1 or 2; each X is independently -O-, -S-, -NH-, or -N(OH)-; Z is absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)-; each L is independently a C2-C4 alkylene optionally substituted by OH; and R1 and R2 are each independently represented by –(CH2)m-Q-(CH2)nH, wherein Q is absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -N(OH)-, -C(O)NH-, -C(O)N(OH)-, - - [0053] In some embodiments, the compound has the formula of wherein each u is independently 2, 3, or 4. All the other variables have the same definitions as those defined above. [0054] In some embodiments, the compound the formula of: variables have the same definitions as those defined above. [0055] In some embodiments, in any of the formulas disclosed herein (if X is present), at least one X is -O-, -S-, -NH-, or -N(OH)-. In some embodiments, at least one X is O or S. In some embodiments, at least one X is -N(R′′)- (e.g., -NH- or -N(OH)-. [0056] In some embodiments, each R, R′, and R′′ are independently H, OR14, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl. In some embodiments, R, R′, and R′′ each may be independently H or C1-C3 alkyl. In some embodiments, R, R′, and R′′ each may be independently a C3-C7 cycloalkyl. In some embodiments, R′′ is OR14 (e.g., OH, or OCH3). In some embodiments, each R, R′, and R′′ are H. [0057] In some embodiments, in any of the formulas disclosed herein (if L is present), each L is independently a C2-C4 alkylene optionally substituted by OH. In some embodiments, each L is independently a C2-C4 alkylene, or -CH2-CH(OH)-CH2- (e.g., including both R and S isomers for the carbon atom that OH is attached to) when the X variable next to the L variable is -O-. In one embodiment, L is independently a C2-C4 alkylene (e.g., a C2, C3, or C4 alkylene). In one embodiment, L is independently -CH2-CH(OH)-CH2- (e.g., including both R and S isomers for the carbon atom that OH is attached to) when the X variable next to the L variable is -O-. [0058] In some embodiments, in any of the formulas disclosed herein (if Y is present), Y is H; aryl; branched or unbranched C1-C6 alkyl; C1-C6 alkyl interrupted with a N, O, or S atom; or C1-C3 alkyl substituted with an aryl. In one embodiment, Y is H. In one embodiment, Y is branched or unbranched C1-C4 alkyl, a C1-C3 alkyl interrupted with an S atom, or benzyl. [0059] In some embodiment, Y is . In some embodiments, Z is absent, -C(O)-, -C(O)O-, -C(O)N(R′′)-, or -S(O)(O)-. In some embodiments, Z is absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)-. In some embodiments, Z is -C(O)-. In some embodiments, Z is absent, -C(O)O-, -C(O)NH-, or -S(O)(O)-. [0060] In some embodiment, [0061] In some embodiments, in any of the formulas disclosed herein (if Ra/Rb is present), Ra and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl. In some embodiments, Ra and Rb each are independently H, or C1-C3 alkyl. In some embodiments, Ra and/or Rb is C3-C7 cycloalkyl. In one embodiment, Ra and Rb are H. [0062] In some embodiments, in any of the formulas disclosed herein (if t is present), each t is independently 1, 2, 3, or 4. In some embodiments, each t is independently 1 or 2. [0063] In some embodiments, the compound has the formula of: All the other variables have the same definitions as those defined above. [0064] In some embodiments, the compound has the formula of: All the variables have the same definitions as those defined above. [0065] In some embodiments, Y may be -(CH2)tC(=O)X-L-N(R1)(R2). In certain instances wherein Y is -(CH2)tC(=O)X-L-N(R1)(R2), each of the X groups within the compound are the same, each of the R1 groups within the compound are the same, and each of the R2 groups within the compound are the same. In other embodiments in which Y is -(CH2)tC(=O)X-L- N(R1)(R2), the X groups within the compound may be independently selected, i.e., may be different, the R1 groups within the compound may be independently selected, i.e., may be different, and the R2 groups within the compound may be independently selected, i.e., may be different. [0066] In some embodiments, in any of the formulas disclosed herein (if R1/R2 is present), each R1 and R2 may be the same, e.g., each R1 may be the same as each R2. In some embodiments, at least one, two, or three of the R1 and R2 variables within the compound are different than the other(s). In some embodiments, a particular R1 group may be the same as one or two R2 groups within the compound. In other instances, the compound contains two R1 groups, and both R1 groups are the same as one or two R2 groups within the compound. [0067] In some embodiments, each R1 and R2 is independently branched or unbranched C1- 20 alkyl or branched or unbranched C2 -C20 alkenyl. For example, each R1 and R2 may independently be C1-20 linear or branched alkyl; each R1 and R2 may independently be C1-20 linear or branched alkenyl. In some embodiments, each R1 and R2 are independently C9-C16 alkyl. [0068] In some embodiments, each R1 and R2 are independently C9-C12 alkyl. In some embodiments, each R1 and R2 may independently be C9 alkyl, C10alkyl, C11alkyl, C12alkyl, C13alkyl, C14alkyl, C15alkyl, or C16alkyl. In some embodiments, each R1 and R2 are independently C9-C16 alkenyl. In some embodiments, each R1 and R2 are independently C9- C12 alkenyl. [0069] In some embodiments, R1 and/or R2 may be represented by –(C(R11)(R12))m-Q- (C(R11)(R12))nH. In this formula, each m is independently 0 to 10; each n is independently 1 to 10; each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl or heterocyclic; each R11, R12, and R14 are independently H, branched or unbranched alkyl or alkenyl; and each R13 is independently H, alkyl, or OR14. In some embodiments, when X is O or S or when Z is C(O), Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-. [0070] In some embodiments, Q in each R1 and R2 variable is independently -CH=CH-, -C≡C-, or -S-S-. In some embodiments, Q in each R1 and R2 variable is independently -C(O)O- or -OC(O)-. In some embodiments, Q in each R1 and R2 variable is independently -C(O)NH-, -C(O)N(OH)-, -N(OH)C(O)-, or-NHC(O)-. [0071] In some embodiments, one of R1 and R2 is C1 -C16 branched or unbranched alkyl; and in the other R1 or R2, Q is -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(OH)-, -N(OH)C(O)-, or -NHC(O)-. [0072] Non-limiting examples of the lipid compounds are set forth below.
[0073] In some embodiments, exemplary lipid compounds are: pound 123),
). [0074] In some embodiments, these exemplary lipid compounds provide targeted delivery to specific cells, tissues, and/or organs such as the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject. In one embodiment, these exemplary lipid compounds provide targeted delivery to the cells or tissues of lung. In one embodiment, these exemplary lipid compounds provide targeted delivery to the cells or tissues of spleen. Preparation of the novel lipids [0075] Certain aspects of the invention relate to methods of making the novel lipids described herein. [0076] All above descriptions and all embodiments regarding the novel lipid compounds discussed in the above aspects of the invention are applicable to these aspect of the invention relating to the method of making the compounds. [0077] Accordingly, one aspect of the invention relates to a method of making a compound of formula reacting with one or more deprotecting reagents to remove the carboxyl protecting group and amine protecting group, and react the resulting intermediate under conditions sufficient to couple and cyclize the intermediate to form the compound of formula (IIA), wherein: each X is independently -O-, -S-, or -N(R′′)-; each t is independently 1, 2, 3, or 4; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′′, Ra, and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14 are independently H, branched or unbranched alkyl or alkenyl. prepared by [0079] In some embodiments, the preparation further employs one or more coupling reagents couple and cyclize the intermediate to form the compound of formula (IIA). In some embodiments, the coupling reagent comprises EDCI, DMAP, HATU, and/or Hunig’s base. [0080] In some embodiments, the reactan X is -O-, -S-, or -NH-, [0081] In some embodiments, the deprotecting agent is an acid. In some embodiments, the acid is trifluoroacetic acid (TFA). In some embodiment, the acid is an inorganic acid such as HCl. In some embodiments, the reaction conditions comprises adding acetonitrile and triethylamine. [0082] Another aspect of the invention relates to a method of making a compound of formula organic base and a solvent to form the compound, wherein: each t is independently 1, 2, 3, or 4; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′, Ra, and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14 are independently H, branched or unbranched alkyl or alkenyl. [0083] In some embodiments, the organic base is DIPEA. Other organic bases are also suitable for use herein. In some embodiments, the solvent is DMF or its mixture with DCM. [0084] In some embodiments, the reactant i form corresponding the reaction reagents further comprises a benzotriazole or its derivative. In some embodiments, the reactant is the reaction reagents further comprises a benzotriazole or its derivative. [0085] The use of the benzotriazole or its derivative could eliminate or avoid the racemization of single-enantiomer chiral molecules and to result in a chiral enantiomer. In some embodiments, the benzotriazole or its derivative is HOBt, HATU, or a mixture thereof. In some embodiments, the benzotriazole or its derivative is PyBOP. [0086] In some embodiments, the reagents further comprise a coupling agent, such as EDCI. [0087] Another aspect of the invention relates to a method of making a compound, comprising r s [0088] Another aspect of the invention relates to a method of making a compound, comprising reacting f aryl; heteroaryl; branched or unbranched C1-C20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or heteroaryl. [0089] In some embodiments, the organic solvent is an alcohol (e.g., isopropyl alcohol). In some embodiments, the organic solvent is ACN. [0090] These above aspects of the invention relating to the synthesis of the compounds are further illustrated in Examples 1 and 4. Lipid Nanoparticles [0091] Also disclosed herein are lipid nanoparticles comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any one of the embodiments described herein. The lipid nanoparticles may further comprise one or more of a helper lipid, a sterol, and a polyethylene glycol (PEG)-modified lipid. Advantageously, the lipid nanoparticles according to the present disclosure that include a compound of Formula (I) deliver a therapeutic agent, such as a nucleic acid, preferentially to liver cells, lung cells, splenic cells, heart cells, kidney cells, marrow cells, or lymphatic cells of the subject. Such preferential delivery occurs without the requirement for a specific targeting ligand. [0092] For example, the presently disclosed lipid nanoparticles can deliver a therapeutic agent preferentially to liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, or other immune cells within the liver. [0093] In other embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to spleen dendritic cells, spleen neutrophils, spleen macrophages, spleen B cells, spleen T cells, spleen natural killer (NK) cells, or other immune cells within the spleen. [0094] In other embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to lymphatic dendritic cells, lymphatic neutrophils, lymphatic macrophages, lymphatic B cells, lymphatic T cells, lymphatic natural killer (NK) cells, or other immune cells within the lymph nodes. [0095] In some embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung macrophages, lung T cells, lung natural killer (NK) cells, or other immune cells within the lung. [0096] In some embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to heart endothelial cells, heart epithelial cells, heart dendritic cells, heart macrophages, heart B cells, heart T cells, heart natural killer (NK) cells, or other immune cells within the heart. [0097] In some embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to kidney endothelial cells, kidney epithelial cells, kidney macrophages, kidney dendritic cells, kidney B cells, kidney T cells, kidney natural killer (NK) cells, or other immune cells within the kidney. [0098] In some embodiments, the lipid nanoparticles can deliver a therapeutic agent preferentially to marrow dendritic cells, marrow macrophages, marrow B cells, marrow T cells, other immune cells within marrow, or marrow stem-like cells, or marrow hematopoietic stem cells (HSCs). [0099] As used herein, preferential delivery to a particular class of cells or cell type refers to delivery at a higher rate than to non-targeted cells. For example, the preferential delivery can mean delivery at or above a rate that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than to non-targeted cells, i.e., cells not within the particular class of cells or of the particular cell type. In some instances, the delivery is to a particular targeted class of cells or cell type, and there is no delivery or only minimal delivery to non-targeted cells. As a result, the preferential delivery can be at a rate that is hundreds of times, thousands of times, or, theoretically infinitely greater than to the non-targeted cells. [00100] The role and identity of exemplary helper lipids for lipid nanoparticles are known among those skilled in the art and may be any compound that contributes to the stability and delivery efficiency of the LNP, or to the stable encapsulation of a therapeutic agent within the LNP. [00101] In certain embodiments, the helper lipid is a cationic lipid. [00102] In certain embodiments, the helper lipid is DDAB (Didodecyldimethylammonium bromide), DOTAP (1,2-Dioleoyl-3-trimethylammonium propane), or DOTMA (1, 2-di-O- octadecenyl-3-trimethylammonium propane). [00103] In certain embodiments, the helper lipid is a phospholipid. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2- lysophosphatidyl choline, and a sphingomyelin. A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. [00104] Particular phospholipids can facilitate fusion to a membrane. In some embodiments, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. [00105] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. In some embodiments, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper- catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). [00106] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin. [00107] In certain embodiments, the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoetha nolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and derivatives thereof. [00108] In some embodiments, the lipid nanoparticle further comprises one or more helper lipids selected from the group consisting of 1,2-dioeoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), didodecyldimethylammonium bromide (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3- trimethylammonium propane (DOTAP), 18:1 PA, N-methyldioctadecylamine (MDOA), N,N-dicotadecylaniline, sn-(3-myristoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'- dimyristoyl)-glycerol (14:0 Hemi BMP), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA; 14:0 PA), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA; 18:1 PA), 1,2-distearoyl-sn-glycero- 3-phosphate (DSPA; 18:0 PA), and a combination thereof. [00109] In some embodiments, the one or more helper lipids are 1,2-dioeoyl-sn-glycero-3- phosphoethanolamine (DOPE), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), or a combination thereof. [00110] The lipid nanoparticles may also include a sterol or derivative thereof. In some embodiments, the sterol or derivative thereof is cholesterol or derivative thereof. In some embodiments, a combination of two or more cholesterols or derivatives thereof are used. The inclusion of a cholesterol in nanoparticle formulations can improve efficacy, potentially due to enhanced membrane fusion. Exemplary cholesterol or derivatives thereof include cholesterol (C27H46O), 20α-OH cholesterol, 20α-hydroxycholesterol (5-cholestene-3β,20α- diol), and DC-Cholesterol (N,N-dimethyl- N-ethylcarboxamidocholesterol). Any natural sterols may also be used. Examples of natural sterols include, for example, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, 14-demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)- dehydrocholesterol, FF-MAS, diosgenin, DHEA sulfate, DHEA, sitosterol, lanosterol-95, cholesterol (plant), dihydro FF-MAS-d6, dihydro T-MAS-d6, zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol, pregnenolone, dihydro T-MAS, delta 5- avenasterol, brassicasterol, dihydro FF-MAS, and 24-methylene cholesterol. A large diversity of structural analogs of cholesterol exist as natural products (e.g., phytosterols that are plant-based sterols, which provide stability to the plant cell wall). Exemplary cholesterol analogs include, for example, Vitamin D derivatives (such as 9,10-secosteroids, Vitamin D2, Vitamin D3, Calcipotriol), alkyl-substituted steroids (such as C-24 alkyl steroids), and cholesterol analogs wherein the tail is modified into a fifth ring (such as pentacyclic steroids). [00111] The lipid nanoparticles may also include a PEG-lipid. As used herein, the term “PEG lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG- ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. [00112] In some embodiments, the PEG lipid includes, but are not limited to, 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). [00113] In some embodiment, the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG- modified dialkylglycerol, and mixtures thereof. [00114] In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C14 to about C22. In some embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG lipid is PEG2k-DMG. [00115] In one embodiment, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. [00116] In some embodiment, the PEG-modified lipid is PEG-DEG or PEG-PE. [00117] In some embodiment, the PEG-modified lipid is 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000 (DMPE-PEG2K; 14:0 PEG2K PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)- 2000 (DSPE-PEG2K; 18:0 PEG2K PE), 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (DMG-PEG 2K), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC- 0159), distearoyl-rac-glycerol-PEG2K (DSG-PEG 2K), or a combination thereof. [00118] In some embodiments, in the presently disclosed LNPs, in addition to the compound of Formula (I), the LNP also comprises a helper lipid (e.g., DOPE, DDAB, DOTAP, DSPC, or a mixture thereof), a sterol (e.g., cholesterol (C27H46O), 20α-OH cholesterol, DC-cholesterol, or a mixture thereof), and a PEG lipid (e.g., 14:0 PEG2K PE, 18:0 PEG2K PE, or a mixture thereof). [00119] The molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid in the lipid nanoparticles may be about 20-60 : 7- 50 : 5-70 : 0.5-3. In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid in the lipid nanoparticles may be about 30-60 : 7-50 : 15-50 : 1-3. In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid in the lipid nanoparticles may be about 30-55: 10-20 : 30-50 : 1-3. In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid is about 32-50 : 12-18 : 32-48 : 1-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative : the PEG lipid is about 35-50 : 12-16 : 35-47 : 2-3. [00120] In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the cholesterol or its derivative: the PEG lipid is about 25 : 33 : 40 : 2; is about 30 : 22.5 : 45 : 2.5; is about 30 : 39 : 30 : 1; is about 30 : 50 : 18 : 2; is about 35 : 35 : 27.5 : 2.5; is about 35 : 43 : 20 : 2; is about 35 : 44.5 : 18 : 2.5; is about 35 : 16: 46.5 : 2.5; is about 45 : 13 : 39.5 : 2.5; is about 50 : 40 : 7.5 : 2.5; is about 50 : 17.5 : 30 : 2.5; is about 50 : 12.5 : 35 : 2.5; is about 50 : 10 : 38.5 : 1.5; or is about 35 : 15 : 47.5 : 2.5. [00121] In some embodiments, the sterol component (e.g., cholesterol or its derivative) may be absent from the LNP. In these embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid in the lipid nanoparticles may be about 20-60 : 10-60 : 0.5-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 25-55 : 15-60 : 0.5-3. In certain embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 30-55 : 30-55 : 1-3. In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 32-50 : 40- 55 : 1-3. [00122] In some embodiments, the molar ratio of the compound according to Formula (I) : the helper lipid : the PEG lipid is about 45 : 52.5 : 2.5; is about 45 : 53: 2; or is about 50 : 47.5 : 2.5. [00123] The molar concentration of the compound according to Formula (I) in the LNPs may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 percent of the total lipids in the LNP. The molar concentration of the helper lipid in the LNPs may be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. The molar concentration of the cholesterol or its derivative in the LNPs may be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 percent of the total lipids in the LNP. The molar concentration of the PEG lipid in the LNPs may be about 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, or 3.5 percent of the total lipids in the LNP. [00124] The lipid nanoparticle may have a diameter of about 20-250, 20-225, 20-200, 30- 250, 30-225, 30-200, 40-250, 40-225, 40-200, 45-250, 45-225, 45-200, 50-200, 50-180, or 75-170 nm. For example, the diameter of the lipid nanoparticle may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250. In a given population of lipid nanoparticles according to the present disclosure, the population may include individual members of respectively different sizes. The particle size distribution of a given population of LNPs according to the present disclosure may be characterized by a D90 of about 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, or 60 nm, and/or a D10 of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. [00125] The lipid nanoparticles according to the present disclosure can be used for the delivery of therapeutic agents to a living organism, such as to a human subject. The therapeutic agent may be encapsulated within the lipid nanoparticle. In some embodiments, the therapeutic agent may be a nucleic acid molecule (e.g., oligonucleotide), protein or peptide, carbohydrate or glycoprotein, lipid, small molecule, or any combination thereof. [00126] In some embodiments, the therapeutic agent is a small molecule. [00127] In some embodiments, the therapeutic agent is a protein or peptide. [00128] In some embodiment, the therapeutic agent is a nucleic acid molecule (e.g., DNA or RNA). [00129] In some embodiments, the therapeutic agent is a DNA, e.g., in the form of antisense DNA, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), vectors (e.g., PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. [00130] In some embodiments, the therapeutic agent is an RNA, e.g., in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis- natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 73K RNA, retrotransposons, a viral genome, a viroid, satellite RNA, or derivatives of these groups. [00131] In some embodiments, the therapeutic agent is an mRNA. [00132] In some embodiments, the lipid nanoparticle (LNP) formulations include (i) a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any one of the embodiments described herein, (ii) a helper lipid, (iii) a PEG lipid, and optionally (iv) a sterol (e.g., cholesterol). [00133] In some embodiments, in the exemplary LNP formulations, compound (i) is compound KC-34 (e.g., S,S or R,R), compound 123, compound 125, or compound 126; the helper lipid (ii) is DDAB; the PEG lipid (iii) is C14PEG2K PE, C18PEG2K PE, or DSG- PEG2K; and the sterol (iv) is cholesterol. The ratios for these lipid components are: [00134] In some embodiment, exemplary LNP formulations include: [00135] In some embodiments, the above exemplary LNP formulations provide targeted delivery to specific cells, tissues, and/or organs such as the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject. In one embodiment, the above exemplary lipid compounds provide targeted delivery to the cells or tissues of lung. [00136] In some embodiments, in the exemplary LNP formulations, compound (i) is compound KC-34 (e.g., S,S or R,R); the helper lipid (ii) is DSPC or DOPE; the PEG lipid (iii) is C14PEG2K PE or DMG-PEG2K; and the sterol (iv) is cholesterol. The ratios for these lipid components are: Cholesterol (iv) PEG (iii) Lipid Helper Lipid (ii) Lipid (i) Mol % Mol % Mol % Mol % 25 40 2 33 30 30 1 39 30 45 2.5 22.5 35 46.5 2.5 16 45 0 2.5 52.5 45 39.5 2.5 13 50 0 2.5 47.5 50 30 2.5 17.5 50 35 2.5 12.5 50 38.5 1.5 10 [00137] In some embodiment, exemplary LNP formulations include: Helper Helper Lipid (i) Cholesterol PEG Lipid Lipid (ii) Lipid (i) PEG Lipid Lipid Mol % (iv) Mol % (iii) Mol % Mol % KC-34 (S,S) DMG-PEG2K DOPE 30 30 1 39 KC-34 (S,S) C14PEG2K PE DSPC 30 30 1 39 KC-34 (S,S) DMG-PEG2K DSPC 30 30 1 39 KC-34 (S,S) C14PEG2K PE DOPE 30 30 1 39 KC-34 (S,S) DMG-PEG2K DSPC 30 45 2.5 22.5 KC-34 (R,R) C14PEG2K PE DSPC 30 45 2.5 22.5 KC-34 (S,S) C14PEG2K PE DSPC 30 45 2.5 22.5 KC-34 (R,R) DMG-PEG2K DSPC 30 45 2.5 22.5 KC-34 (S,S) DMG-PEG2K DSPC 35 46.5 2.5 16 KC-34 (R,R) DMG-PEG2K DSPC 35 46.5 2.5 16 KC-34 (R,R) C14PEG2K PE DOPE 45 0 2.5 52.5 KC-34 (S,S) C14PEG2K PE DOPE 45 39.5 2.5 13 KC-34 (R,R) C14PEG2K PE DSPC 45 39.5 2.5 13 KC-34 (S,S) C14PEG2K PE DSPC 45 39.5 2.5 13 KC-34 (R,R) C14PEG2K PE DOPE 45 39.5 2.5 13 KC-34 (R,R) C14PEG2K PE DOPE 50 0 2.5 47.5 [00138] In some embodiments, the above exemplary LNP formulations provide targeted delivery to specific cells, tissues, and/or organs such as the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject. In one embodiment, the above exemplary lipid compounds provide targeted delivery to the cells or tissues of spleen. [00139] Additional non-limiting examples of the lipid nanoparticles formulations are further illustrated in Examples 2-3 and 5-6. [00140] The present disclosure also provides a pharmaceutical composition comprising the lipid nanoparticle comprising a compound of Formula (I), a compound of the subgenus formulas of formula (I), or any of the compounds belonging to any subgenus or species of these formulas according to any of the embodiments described herein, and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” preferably refers to a material that can be incorporated into a composition and administered to a patient without causing unacceptable biological effects or interacting in an unacceptable manner with other components of the composition. Such pharmaceutically acceptable materials typically have met the required standards of toxicological and manufacturing testing, and include those materials identified as suitable inactive ingredients by the U.S. Food and Drug Administration. [00141] All above descriptions and all embodiments regarding the novel lipid compounds and lipid nanoparticles discussed in the above aspects of the invention are applicable to this aspect of the invention relating to the pharmaceutical composition. [00142] Thus, the LNPs according to the present disclosure may be provided in a composition that is formulated for any type of administration. For example, the compositions may be formulated for administration orally, topically, parenterally, enterally, or by inhalation (e.g., intranasally). The active agent may be formulated for neat administration, or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. The applicable solid carrier, diluent, or excipient may function as, among other things, a binder, disintegrant, filler, lubricant, glidant, compression aid, processing aid, color, sweetener, preservative, suspensing/dispersing agent, tablet-disintegrating agent, encapsulating material, film former or coating, flavoring agent, or printing ink. Any material used in preparing any dosage unit form is preferably pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the LNPs may be incorporated into sustained-release preparations and formulations. Administration in this respect includes administration by, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol, and rectal systemic. [00143] In powders, the carrier, diluent, or excipient may be a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the LNPs are mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired. For oral therapeutic administration, the LNPs may be incorporated with the carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of LNP in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained. [00144] Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like. The LNPs may be suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat. The liquid carrier, excipient, or diluent can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo-regulators. [00145] Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar-agar, tragacanth, or mixtures thereof. [00146] Suitable examples of liquid carriers, diluents, and excipients, for example, for oral, topical, or parenteral administration, include water (particularly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil), or mixtures thereof. [00147] For parenteral administration, the carrier, diluent, or excipient can also be an oily ester such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents, or excipients, which are used in sterile liquid form compositions for parenteral administration. Solutions of the LNPs can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. A dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. [00148] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and fluid to provide easy syringability. It is preferably stable under the conditions of manufacture and storage and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier, diluent, or excipient may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some instances, the antimicrobial peptides themselves may be sufficient to prevent contamination by microorganisms. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. [00149] Sterile injectable solutions may be prepared by incorporating the LNPs in the pharmaceutically appropriate amounts, in the appropriate solvent, with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the LNPs into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation may include vacuum drying and freeze drying techniques that yield a powder of the LNPs or ingredients, plus any additional desired ingredient from the previously sterile-filtered solution thereof. [00150] Also provided herein are methods of delivering a therapeutic agent to a subject, comprising administering to the subject a lipid nanoparticle according to any of the presently disclosed embodiments, wherein the lipid nanoparticle comprises a therapeutic agent. [00151] All above descriptions and all embodiments regarding the novel lipid compounds and lipid nanoparticles discussed in the above aspects of the invention are applicable to this aspect of the invention relating to the pharmaceutical composition. [00152] It has surprisingly been discovered that the inventive nanoparticles preferentially target human lung, heart, kidney, liver, splenic, lymphatic, or marrow cells, and can thereby preferentially deliver the therapeutic agent to such cells. The cells to which the present LNPs deliver the therapeutic agent can include, for example, lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung T cells, lung macrophages, lung natural killer (NK) cells, or other immune cells within the lung; heart endothelial cells, heart epithelial cells, heart dendritic cells, heart B cells, heart T cells, heart natural killer (NK) cells, heart macrophages, or other immune cells within the heart; kidney endothelial cells, kidney epithelial cells, kidney dendritic cells, kidney B cells, kidney T cells, kidney natural killer (NK) cells, kidney macrophages, or other immune cells within the kidney; liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, other immune cells within the liver; spleen dendritic cells, spleen neutrophils, spleen macrophages, spleen B cells, spleen T cells, spleen natural killer (NK) cells, other immune cells within the spleen; lymphatic dendritic cells, lymphatic neutrophils, lymphatic macrophages, lymphatic B cells, lymphatic T cells, or lymphatic natural killer (NK) cells; marrow dendritic cells, marrow macrophages, marrow B cells, marrow T cells, other immune cells within marrow, or marrow stem-like cells, or marrow hematopoietic stem cells (HSCs). [00153] Accordingly, the present disclosure also provides methods for delivering a therapeutic agent to lung, heart, kidney, liver, splenic, lymphatic, or marrow cells of a subject, comprising administering to the subject a lipid nanoparticle according to any of the embodiments disclosed herein. [00154] Beneficially, the lipid nanoparticles can deliver the therapeutic agent to the subject at clinically relevant doses. In some embodiments, that dose at which the present LNPs deliver the therapeutic agent is about 0.01 to about 3.0 mg/kg. For example the dose at which the therapeutic agent is delivered may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.51.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2., 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, 2.9, or 3.0 mg/kg. EXAMPLES [00155] The present invention is further defined in the following Examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and should not be construed as limiting the appended claims. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Example 1 – Design and Characterization of Ionizable Lipids [00156] Cyclo(Glu-Glu) 1 and bis-amine 2 were synthesized according to literature procedures: Langmuir 2017, 33, 13821−13827 and Colloid Polym Sci, 2017, 295, 1549– 1561, European Journal of Medicinal Chemistry, 2014, 83, 433-447, and WO2009142892. [00157] To a solution of cyclo(Glu-Glu) 1 (0.15 mmol) in DMF (1.5 mL) was added DIPEA (0.375 mmol), EDCI (0.375 mmol), HOBt (0.375 mmol). The resulting suspension was stirred for 10 min, and a solution of bis-amine 2 (0.293 mmol) in DMF (0.5 mL) was slowly added. The reaction mixture was stirred at room temperature for 24 hours, then quenched with 5 mL of saturated sodium bicarbonate, and extracted with EtOAc (2×10 mL). The organic phase was combined and washed with 10 mL of brine, and dried over sodium sulfate. After removing the solvent in vacuo, the residue was purified by column chromatography (DCM/MeOH =30:1 to DCM/MeOH=15:1) to yield the desired products. Large Scale Synthesis. [00158] A 250 mL flask equipped with a stirring bar was charged with cyclo(Glu-Glu) 1 (9.0 mmol), PyBOP (19.8 mmol), DIPEA (19.8 mmol) and DMF (100 mL). The resulting suspension was stirred at 0°C for 0.5 h, and a solution of bis-amine 2 (18.45 mmol) in DMF (30 mL) was slowly added. The reaction mixture was stirred at room temperature for 50 hours, then quenched with 100 mL of saturated sodium bicarbonate, and extracted with EtOAc (3×200 mL). The organic phase was combined and washed with 200 mL of brine, and dried over sodium sulfate. After removing the solvent in vacuo, the residue was purified by column chromatography (DCM/MeOH =40:1 to DCM/MeOH/NH3(aq.) =15:1:0.16) to yield SPC-A9 as a colorless wax (5.10 g, 67% yield). The analytical data of the gram scale reaction were consistent with those of the 0.15 mmol scale experiment. Characterization Data: [ Yield: 63%, 80.08 mg.1H-NMR (500 MHz, CDCl3): δ 7.76 (s, 1H), 6.72–6.70 (m, 1H), 4.00 (t, J = 5.8 Hz, 2H), 3.28–3.24 (m, 4H), 2.56–2.51 (m, 4H), 2.44–2.36 (m, 12H), 2.28–2.21 (m, 2H), 2.13-2.09 (m, 2H), 1.41–1.37 (m, 8H), 1.28–1.20 (m, 48H), 0.85 (t, J = 6.9 Hz, 12H). 13C-NMR (125 MHz, CDCl3): δ 172.5, 168.6, 77.2, 54.4, 53.5, 52.8, 36.7, 31.9, 31.9, 29.6, 29.6, 29.5, 29.5, 29.3, 29.0, 27.4, 26.0, 22.7,14.1. HRMS(ESI): m/z [M+H]+ calculated for C50H98N6O4: 847.7722, found 847.7711 [ . Yield: 66%, 95.0 mg, 1H-NMR (500 MHz, CDCl3): δ 7.70 (brs, 1H), 7.05 (m, 1H), 4.00 (t, J = 5.8 Hz, 2H), 3.38–3.27 (m, 4H), 2.68–2.62 (m, 4H), 2.55–2.51 (m, 8H), 2.40–2.36 (m, 4H), 2.26–2.22 (m, 2H), 2.15–2.10 (m, 2H), 1.47–1.41 (m, 8H), 1.29–1.23 (m, 64H), 0.85 (t, J = 6.8 Hz, 12H). 13C-NMR (125 MHz, CDCl3): δ 172.7, 168.5, 54.5, 53.5, 52.9, 36.7, 32.1, 32.0, 29.8, 29.8, 29.7, 29.7, 29.6, 29.5, 28.7, 27.5, 26.0, 22.8, 14.2. HRMS(ESI): m/z [M+H]+ calculated for C58H114N6O4: 959.8974, found 959.8958. Yield: 49%, 78.78 mg, 1H-NMR (500 MHz, CDCl3): δ 7.56 (s, 1H), 7.53 (s, 1H), 4.01 (t, J = 5.8 Hz, 2H), 3.46–3.32 (m, 4H), 2.83–2.63 (m, 12H), 2.42–2.39 (m, 4H), 2.30–2.24 (m, 2H), 2.20–2.13 (m, 2H), 1.53–1.47 (m, 8H), 1.28–1.24 (m, 64H), 0.86 (t, J = 6.9 Hz, 12H).13C- NMR (125 MHz, CDCl3): δ 172.8, 168.4, 54.7, 53.3, 53.0, 36.2, 32.1, 32.0, 29.8, 29.8, 29.8, 29.7, 29.6, 29.5, 28.5, 27.4, 25.4, 22.8, 14.2. HRMS(ESI): m/z [M+H]+ calculated for C66H130N6O4: 1072.0226, found 1072.0191. Yield: 52%, 96.73 mg, 1H-NMR (500 MHz, CDCl3): δ 7.72 (brs, 1H), 7.85 (s, 1H), 4.03 (t, J = 5.8 Hz, 2H), 3.36–3.28 (m, 4H), 2.63–2.60 (m, 4H), 2.51–2.46 (m, 8H) 2.43–2.39 (m, 4H), 2.28–2.24 (m, 2H), 2.18–2.12 (m, 2H), 1.46–1.42 (m, 8H), 1.27–1.25 (m, 104H), 0.88 (t, J = 6.9 Hz, 12H). 13C-NMR (125 MHz, CDCl3): δ 172.5, 168.5, 53.7, 52.8, 36.9, 32.0, 29.8, 29.8, 29.8, 29.7, 29.7, 29.5, 27.6, 26.4, 22.8, 14.2. HRMS(ESI): m/z [M+H]+ calculated for C78H154N6O4: 1240.2104, found 1240.2083. Yield: 60%, 98.79 mg, 1H-NMR (500 MHz, CDCl3): δ 7.80 (s, 1H), 7.70 (t, J = 5.7 Hz, 1H), 4.00 (t, J = 5.8 Hz, 2H), 3.29–3.24 (m, 4H), 2.68–3.62 (m, 4H), 2.44–2.33 (m, 12H), 2.28– 2.20 (m, 2H), 2.13–2.07 (m, 2H), 1.65–1.60 (m, 4H), 1.43–1.38 (m, 8H), 1.29–1.24 (m, 48H), 0.86 (t, J = 6.9 Hz, 12H).13C-NMR (125 MHz, CDCl3): δ 172.4, 168.6, 54.6, 54.0, 53.3, 39.7, 32.3, 32.0, 29.7, 29.7, 29.4, 28.2, 27.7, 26.7, 25.8, 22.8, 14.2. HRMS(ESI): m/z [M+H]+ calculated for C52H102N6O4: 875.8035, found 875.8024. Yield: 56%, 82.96 mg, 1H-NMR (500 MHz, CDCl3): δ 7.77 (s, 1H), 7.70 (t, J = 5.7 Hz, 1H), 4.00 (t, J = 5.8 Hz, 2H), 3.3–3.25 (m, 4H), 2.52–3.48 (m, 4H), 2.43–2.34 (m, 12H), 2.28–2.22 (m, 2H), 2.13–2.06 (m, 2H), 1.64–1.59 (m, 4H), 1.43–1.37 (m, 8H), 1.32–1.21 (m, 64H), 0.86 (t, J = 6.9 Hz, 12H). 13C-NMR (125 MHz, CDCl3): δ 172.4, 168.6, 54.6, 54.1, 53.5, 39.9, 32.4, 32.0, 29.8, 29.8, 29.8, 29.7, 29.7, 29.5, 28.0, 27.8, 26.9, 25.7, 22.8, 14.2. HRMS(ESI): m/z [M+H]+ calculated for C60H118N6O4: 987.9287, found 987.9275. Yield: 70%, 121.38 mg, 1H-NMR (500 MHz, CDCl3): δ 7.95 (s, 1H), 7.73 (t, J = 5.7 Hz, 1H), 4.00 (t, J = 5.8 Hz, 2H), 3.26–3.21 (m, 4H), 2.57–3.50 (m, 4H), 2.46–2.38 (m, 8H), 2.37–2.32 (m, 4H), 2.25–2.18 (m, 2H), 2.14–2.09 (m, 2H), 1.66–1.61 (m, 4H), 1.44–1.38 (m, 8H), 1.28–1.22 (m, 88H), 0.86 (t, J = 6.9 Hz, 12H). 13C-NMR (125 MHz, CDCl3): δ 172.5, 168.6, 54.5, 53.8, 32.0, 29.8, 29.8, 29.8, 29.7, 29.7, 29.7, 29.5, 27.7, 26.4, 25.9, 22.8, 14.2. HRMS(ESI): m/z [M+H]+ calculated for C72H142N6O4: 1156.1165, found 1156.1147. Yield: 56%, 106.53 mg , 1H-NMR (500 MHz, CDCl 3 ): δ 7.82 (s, 1H), 7.74 (t, J = 5.7 Hz, 1H), 4.00 (t, J = 5.8 Hz, 2H), 3.29–3.23 (m, 4H), 2.58–3.53 (m, 4H), 2.46–2.41 (m, 8H), 2.37–2.34 (m, 4H), 2.27–2.23 (m, 2H), 2.14–2.08 (m, 2H), 1.68–1.63 (m, 4H), 1.44–1.20 (m, 112H), 0.87 (t, J = 6.9 Hz, 12H). 13C-NMR (125 MHz, CDCl3): δ 172.5, 168.6, 54.6, 53.8, 53.0, 39.3, 32.2, 32.0, 29.8, 29.8, 29.8, 29.8, 29.8, 29.7, 29.7, 29.5, 28.3, 27.7, 27.7, 26.4, 25.7, 22.8, 14.2. HRMS(ESI): m/z [M+H]+ calculated for C80H158N6O4: 1268.2417, found 1268.2406. Example 2 – Formulation of Lipid Nanoparticles Using Diketopiperazine Lipids [00167] FIGS.1A-1E illustrate the formulation of the inventive stereopure lipids into stable LNPs. As shown in FIG.1A, stereopure lipids were formulated with two cholesterol variants (cholesterol or 20 -OH cholesterol), two PEG-lipid variants (C14PEG2K or C18PEG2K), and DOPE. The LNPs were formulated using four molar ratios (FIG.1B), thereby formulating 96 total LNPs (FIG.1C), of which 64 met inclusion criteria. FIG.1D, illustrates hydrodynamic diameters of the 64 individual LNPs (gray dots) and the 64 LNP pool (purple dot designated by arrow). The pool diameter was within the range of the LNPs comprising the pool. FIG.1E provides hydrodynamic diameter as a function of stereopure structure, average +/- SEM. [00168] Since PEG-lipid and cholesterol structure can influence LNP delivery, each lipid was formulated with C14PEG2K or C18PEG2K as well as unmodified cholesterol or 20 -OH (FIG.1A). The helper lipid, 1-2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), was kept constant, since helper lipid charge can override endogenous tropism; it was reasoned that a constant, neutrally charged helper lipid would assist in the study of endogenous stereopure lipid tropism. Finally, to control for changes as a function of molar ratio, the components were mixed in three molar ratios (FIG.1B). By formulating eight stereopure lipids with two PEG-lipids, two cholesterol variants, and three molar ratios, a library of 96 chemically distinct LNPs was created. [00169] The LNPs were then barcoded for use in Fast Identification of Nanoparticle Delivery. Using microfluidics, 96 LNPs were formulated so that LNP 1, with chemical composition 1, carried Cre mRNA and DNA barcode 1; LNP 96, with chemistry 96, was formulated to carry Cre mRNA and DNA barcode 96. The total lipid to nucleic acid mass ratio was 10:1. The hydrodynamic diameter and polydispersity of the 96 individual LNPs was then investigated with dynamic light scattering. Of the 96 formulations, 64 formed small (20 < diameter < 200 nm) and monodisperse LNPs (FIGS.1C, 1D), providing one line of evidence that stereopure lipids could be formulated into stable LNPs. The average hydrodynamic diameter was then measured as a function of the lipids, which were found to be less than 200 nm, providing a second line of evidence that these LNPs could form stable structures (FIG.1E). [00170] Nanoparticle Formulation. Nanoparticles were formulated with a microfluidic device. Nucleic acids (DNA barcodes and mRNA) were diluted in 10mM citrate buffer (Teknova). Lipid-amine compounds, PEG-lipids (1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] and 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]), cholesterols (cholesterol and 20α-hydroxycholesterol), and helper lipid dioleoylphosphatidylethanolamine were diluted in 100% ethanol. For mRNA screens, Cre mRNA and DNA barcodes were mixed at a 10:1 mass ratio. All PEG-lipids, cholesterols, and helper lipid were purchased from Avanti Polar Lipids (Alabaster, AL). Citrate and ethanol phases were combined in a microfluidic device by syringes (Hamilton Company) at a flow rate of 3:1. [00171] DNA Barcoding. Each LNP was formulated to carry its own unique DNA barcode. DNA barcodes were designed rationally with several characteristics. All DNA barcodes were 91 nt long, single stranded DNA sequences purchased from Integrated DNA Technologies. Briefly, the following modification were on all barcodes: i) nucleotides on the 5’ and 3’ ends were modified with phosphorothioates to reduce exonuclease degradation ii) universal forward and reverse primer regions were included to ensure equal amplification of each sequence, iii) 7 random nucleotides were include to monitor PCR bias, iv) a droplet digital PCR (ddPR) probe site was include for ddPCR compatibility, and v) a unique 8-nt barcode. An 8-nucleotide sequence can generate over 48 (65,536) distinct barcodes. We used only the 8 nucleotide sequences designed to prevent sequence bleaching and reading errors on the Illumina MiniSeqTM sequencing machine. [00172] Nanoparticle Characterization. LNP hydrodynamic diameter was measured using high throughput dynamic light scattering (DLS) (DynaPro Plate Reader II, Wyatt). LNPs were diluted in sterile 1X PBS and analyzed. To avoid using unstable LNPs, and to enable sterile purification using a 0.22 μm filter, LNPs were included only if they met three criteria: diameter >20 nm, diameter <200 nm, and correlation function with one inflection point. Particles that met these criteria were pooled and dialyzed in a 20 kD dialysis cassettes (Thermo Scientific) and a 100kD cassette (Thermo Scientific) in 1X PBS. The nanoparticle concentration was determined using NanoDrop (Thermo Scientific). LNP encapsulation was measure using a Quant-iT RiboGreen assay (Thermo Fisher). Example 3 – Delivery of mRNA From Lipid Nanoparticle Formed With Diketopiperazine Lipids [00173] FIGS.2A-2I illustrates how an inventive nanoparticle, designated SPC-T LNP (formed from SPC-A9), delivers mRNA at doses as low as 0.01 mg/kg without noticeable toxicity at 1 mg/kg. FIG.2A depicts the chemical structure and molar ratio of SPC-T LNP. FIG.2B provides the biophysical properties of SPC-T LNP. As shown in FIG.2C, SPC-T LNP was formulated to carry chemically modified mRNA encoding Cre recombinase and was intravenously administered to Ai14 mice at a total nucleic dose of 1 mg / kg. Cells within Ai14 mice contain a CAG-Lox-Stop-Lox-tdTomato construct. These cells become tdTomato+ (tdTom+) when Cre mRNA is delivered into the cytoplasm and translated into the functional Cre protein, which edits the genome by excising the Stop cassette. As illustrated in FIG.2D, SPC-T-mediated mRNA delivery in liver and 5 cell types in the spleen. (N = 3 / group, average +/- SEM). FIG.2E shows delivery as a function of SPC-T: mRNA mass ratio. In FIG.2F, a ribogreen assay revealed that increasing the SPC-T: mRNA mass ratio increased the amount of RNA encapsulated. FIG.2G illustrates mRNA delivery at doses as low as 0.01 mg / kg. (N = 3 / group, average +/- SEM). FIGS.2H and 2I depict serum cytokines after a 0.5 or 1.0 mg / kg dose. Mice treated with low dose LPS is used as a positive control, whereas mice treated with PBS are used as a negative control. Only cytokine CCL2 was observe with SPC-T LNP, though in lower concentration than for LPS positive control. [00174] Accordingly, following preparation of the LNPs as described in Example 2, an in vivo high throughput DNA barcoding screen was performed. The 64 pooled LNPs were intravenously injected into Ai14 mice at a total nucleic acid dose of 1.5 mg / kg (i.e., an average of 0.023 mg / kg / LNP, for all 64 LNPs). Ai14 mice have a Lox-Stop-Lox- tdTomato construct downstream a CAG promoter. If Cre mRNA is delivered into the cell and translated into Cre protein that translocates into the nucleus and functions, cells express tdTomato (tdTom+). By isolating tdTom+ cells using fluorescence activated cell sorting (FACS) and sequencing the DNA barcodes therein, LNPs co-localized with cells where functional mRNA has occurred were identified. Using this approach, a LNP was identified that was subsequently named SPC-T and that was formed with the lipid SPC-A9, cholesterol, C14PEG2k -, and DOPE. (FIGS.2A, 2B). Functional delivery mediated by SPC-T carrying Cre mRNA in Ai14 mice was measured (FIG.2C). It was found that cells across the liver and spleen were transfected (FIG.2D). A measurement was then made of delivery after varying the SPC-T lipid: mRNA mass ratio from 5:1 to 15:1 (FIG.2E), and it was found that increasing the mass ratio also increased encapsulation (FIG.2F). Notably, SPC-T delivered mRNA to cells across the liver at doses as low as 0.01 mg / kg (FIG.2D), even though the LNP did not elicit a strong cytokine response at doses as high as 1.0 mg / kg (FIGS.2H, 2I). [00175] FIGS.3A-3D illustrate how single-cell RNA sequencing was used to identify SPC- T LNP tropism at the cell type level as well as quantifying the aVHH expression. FIG.3A provides cell populations in mice treated with PBS or SPC-T, showing no substantial changes. The expression of aVHH protein was measured in each cell and overlaid on the t- SNE plots in FIG.3B, spleen and FIG.3C, lymph nodes. Small gray dots represent cells with no aVHH protein. FIG.3D provides the percentage of aVHH+ cells measured with scRNA- Seq in each spleen and lymph node cell types. [00176] Accordingly, SPC-T delivery in the spleen was then quantified using single-cell RNA-sequencing (scRNA-seq), which enables the measurement of delivery in heterogeneous cell populations. It was first evaluated whether the cell populations in the spleen changed in mice treated with SPC-T, compared to mice treated with PBS. No evidence of substantial cell population shifts was found, providing a second line of evidence that SPC-T was well tolerated (FIG.3A). Delivery in immune cells was observed across the spleen (FIG.3B) and lymph nodes (FIG.3C). Delivery in both organs was then quantified, and delivery to many cell types was observed (FIG.3D). [00177] Animal Experiments. Human PBMC and hepatocytes dual repopulated FRG mice were generated and provided by Yecuris (Portland, Oregon). FRG KO on NOD mice were repopulated with cryopreserved hepatocytes from a human donor (HHM18029) and later injected with human PBMC. In all experiments, N=3-5 mice / group were used. All mice were injected intravenously via the lateral tail vein with LNPs or 1X PBS. [00178] Cell Isolation & Staining. Cells were isolated 24 or 72 hours after injection with LNPs, unless otherwise noted. Mice were perfused in the liver portal vein with 5 mL of Krebs Ringer buffer (pH 7.3). Tissues were finely minced, and then placed in Collagenase XI (Sigma Aldrich) at 37 ºC at 550 rpm for 30 minutes. Cell suspension was filtered through 70^m mesh and washed with 1X PBS. Cells were stained to identify specific cell populations and sorted using the BD FacsFusion cell sorter. The antibody clones used were: Live/dead blue fluorescent dye (Invitrogen), anti-TER-119 (TER-119, BioLegend), Pacific Blue Annexin V (BioLegend), anti-mouse CD31 (390, BioLegend), anti-mouse CD45.2 (104, BioLegend), anti-mouse CD68 (FA11, Biolegend), anti-mouse CD11c (N418, Biolegend), anti-mouse CD3 (17A2, Biolegend), anti-mouse CD19 (6D5, Biolegend), anti-human CD45 (2D1, BioLegend), anti-human CD3 (HIT3a, BioLegend), anti-human CD19 (HIB19, BioLegend), anti-human HLA-ABC (W6/32, Invitrogen) and PE anti-mouse CD47 (miap301, BioLegend), and MonoRab^^ Anti-Camelid VHH (96A3F5, GenScript). [00179] PCR Amplification. All samples were amplified and prepared for sequencing using nested PCR. More specifically, 1 μL of primers (5 uM for Final Reverse / Forward) were added to 5 μL of Kapa HiFi 2X master mix (Roche), and 4 μL template DNA / water. During the second PCR Nextera XT chemistry, indices and i5/i7 adapter regions were added. Dual- indexed samples were run on a 2% agarose gel to ensure that PCR reaction occurred before being pooled and gel purified. [00180] Deep Sequencing. PCR samples were purified by AMPure XP beads. Final library QC was conducted using the Agilent Bioanalyzer 2100. Illumina deep sequencing was conducted on an Illumina MiniseqTM. Primers were designed based on Nextera XT adapter sequences. [00181] Nanoparticle Data Analysis & Statistics. Sequencing results were processed using a custom python-based tool to extract raw barcode counts for each tissue. These raw counts were then normalized with an R script prior for further analysis. Counts for each particle, per tissue, were normalized to the barcoded LNP mixture we injected into the mouse. This ‘input’ DNA provided the DNA counts and was used to normalize DNA counts from the cells and tissues. Statistical analysis was done using GraphPad Prism 8. Data is plotted as mean ^ standard error mean unless otherwise stated. [00182] Cytokine Assay. Cytokines were measured using Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D Systems). [00183] By designing, synthesizing, and characterizing 96 novel LNPs, it was found that stereopure lipids can be formulated into stable nanoparticles, and that these nanoparticles can deliver nucleic acids to a variety of cell types in vivo. Notably, the nanoparticles delivered mRNA preferentially to liver, spleen, or lymphatic cells at a dose as low as 0.01 mg/kg, was identified directly using an in vivo barcoding approach, demonstrating the utility of direct to in vivo high-throughput nanoparticle studies. More broadly, the stereopure lipids and LNPs prepared using such lipids generate compelling evidence that bioactive motifs can be added to LNPs without compromising delivery. Example 4 – Synthesis and Characterization of Exemplary Ionizable Lipids Compound 29 & Compound 26 2-(didecylamino)ethan-1-ol [00184] To a round bottom flask containing a solution of ethanolamine (98.8 uL, 1.64 mmol) in 1,2-dichloroethane (16.4 mL) was added decanal (1.23 mL, 6.55 mmol), followed by sodium triacetoxyborohydride (1.04g, 4.91 mmol), and the resulting suspension was stirred overnight at room temperature. Upon reaction completion, the reaction was diluted with 30 mL DCM, washed with 10 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (10g column, 0-10% MeOH/DCM) yielded product as a colorless oil that solidified upon standing. Yield – 541 mg, 97% yield Mass Spec – 342 (m+1) 1H NMR (400 MHz, CDCl3) δ 3.79 (t, J = 5.0 Hz, 2H), 2.89 (t, J = 5.0 Hz, 2H), 2.79 (t, J = 8.1 Hz, 4H), 1.66 (s, 2H), 1.36 – 1.26 (m, 32H), 0.90 (t, J = 6.7 Hz, 6H). 2-(didecylamino)ethyl (S)-2-(3,6-dioxopiperazin-2-yl)acetate – Compound 29 [00185] To a vial containing a solution of cyclo(Asp-Gly) (25 mg, 0.145 mmol) in DCM (1.45 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (61 mg, 0.320 mmol) and 4-dimethylaminopyridine (1.8 mg, 14.5 uM), and the resulting suspension was stirred at room temperature for 45 minutes.2-(didecylamino)ethan-1-ol (99 mg, 0.29 mmol) was then added and the solution was stirred overnight. Upon reaction completion, the reaction was diluted with 5 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated to a colorless wax. Purification via Flash chromatography (10g column, 0-20% MeOH/DCM) yielded product as a sticky colorless gel. Yield – 30 mg (100% purity, 42% yield) Mass Spec – 496.8 (m+1) 1H NMR (400 MHz, CDCl3) δ 7.25 (s, 0H), 6.06 (s, 0H), 4.44 – 4.10 (m, 1H), 4.06 (d, J = 13.7 Hz, 0H), 3.56 (t, J = 5.3 Hz, 2H), 2.80 – 2.68 (m, 1H), 2.62 (t, J = 5.4 Hz, 2H), 2.56 – 2.40 (m, 4H), 1.45 (q, J = 8.4 Hz, 5H), 1.29 (d, J = 3.6 Hz, 31H), 0.90 (t, J = 6.7 Hz, 6H). 2-(didecylamino)ethyl (S)-3-(3,6-dioxopiperazin-2-yl)propanoate – Compound 26 [00186] To a vial containing a solution of cyclo(Glu-Gly) (25 mg, 0.134 mmol) in DCM (1.34 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (57 mg, 0.295 mmol) and 4-dimethylaminopyridine (1.6 mg, 13.4 uM), and the resulting suspension was stirred at room temperature for 45 minutes.2-(didecylamino)ethan-1-ol (91 mg, 0.27 mmol) was then added, and the reaction was stirred overnight. Diluted with 5 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated to a colorless wax. Purification via Flash chromatography (10g column, 0- 20% MeOH/DCM) yielded product as a white waxy solid. Yield: 84 mg (95% purity, 95% yield) Mass Spec - 510.6 (m+1) 1H NMR (400 MHz, CDCl3) δ 4.31 – 4.00 (m, 2H), 3.55 (t, J = 5.4 Hz, 1H), 2.71 (q, J = 6.3 Hz, 1H), 2.66 – 2.14 (m, 6H), 1.54 – 1.38 (m, 5H), 1.29 (d, J = 3.2 Hz, 32H), 0.90 (t, J = 6.7 Hz, 6H). Compound 30 & Compound 31 2-(didecylamino)ethane-1-thiol [00187] To a vial containing 2-aminoethanethiol HCl (300 mg, 2.64 mmol) in DCM (13.2 mL) was added decanal (1.99 mL, 10.56 mmol), followed by sodium triacetoxyborohydride (1.68 g, 7.92 mmol), and the resulting suspension was stirred over the weekend at room temperature. Upon reaction completion, the reaction was quenched with 30 mL DCM, washed with 10 mL each saturated sodium bicarbonate solution., water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (30g column, 0-10% MeOH/DCM) yielded product as a colorless oil. Characterization Yield – 825 mg (83% yield, 95% purity) Mass Spec – 358.4 (m+1) 1H NMR (400 MHz, CDCl3) δ 3.66 (t, J = 6.6 Hz, 2H), 3.24 – 2.69 (m, 1H), 1.74 (s, 2H), 1.59 (p, J = 6.7 Hz, 2H), 1.45 – 1.17 (m, 30H), 0.90 (t, J = 6.7 Hz, 6H). S-(2-(didecylamino)ethyl) (S)-2-(3,6-dioxopiperazin-2-yl)ethanethioate – Compound 30 [00188] To a solution of cyclo(Asp-Gly) (25 mg, 0.145 mmol) in DCM (1 mL) was added EDCI (61 mg, 0.320 mmol) and DMAP (1.8 mg, 14.5 umol), and the resulting solution was stirred at rt for 45 minutes. A solution of 2-(didecylamino)ethane-1-thiol (104 mg, 0.290 mmol) in DCM (0.45 mL) was then added, and the solution was stirred at rt overnight. Upon reaction completion, the reaction was diluted with 5 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated to a colorless wax. Purification via Flash chromatography (10g column, 0-20% MeOH/DCM) yielded product as a white waxy solid. Yield: 29 mg (100% purity, 31% yield) 1H NMR (400 MHz, CDCl3) δ 6.75 (s, 1H), 6.49 (s, 1H), 4.37 (dt, J = 8.6, 2.7 Hz, 1H), 4.21 – 4.04 (m, 6H), 3.08 (dd, J = 17.6, 3.2 Hz, 2H), 2.82 (dd, J = 17.7, 9.2 Hz, 2H), 2.44 (d, J = 7.8 Hz, 1H), 1.65 (p, J = 6.9 Hz, 4H), 1.46 (d, J = 8.3 Hz, 1H), 1.40 – 1.06 (m, 30H), 0.90 (t, J = 6.6 Hz, 6H). S-(2-(didecylamino)ethyl) (S)-3-(3,6-dioxopiperazin-2-yl)propanethioate – Compound 31 [00189] To a solution of cyclo(Glu-Gly) (25 mg, 0.134 mmol) in DCM (1 mL) was added EDCI (57 mg, 0.295 mmol) and DMAP (1.6 mg, 13.4 umol), and the resulting solution was stirred at rt for 45 minutes. A solution of 2-(didecylamino)ethane-1-thiol (96 mg, 0.269 mmol) in DCM (0.34 mL) was then added, and the solution was stirred at rt overnight. Upon reaction completion, the reaction was diluted with 5 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated to a colorless wax. Purification via Flash chromatography (10g column, 0-20% MeOH/DCM) yielded product as a white waxy solid. Characterization Yield: 19 mg (100% purity, 29% yield) 1H NMR (400 MHz, CDCl3) δ 6.34 (s, 1H), 5.94 (s, 1H), 4.16 – 3.99 (m, 2H), 2.79 (s, 3H), 2.54 (td, J = 7.1, 2.5 Hz, 1H), 2.45 (t, J = 7.5 Hz, 3H), 2.35 – 2.15 (m, 1H), 1.75 – 1.54 (m, 2H), 1.45 (t, J = 7.3 Hz, 4H), 1.29 (d, J = 5.2 Hz, 30H), 0.90 (t, J = 6.7 Hz, 6H). Compound 39 & Compound 42 tert-butyl (2-(didecylamino)ethyl)carbamate [00190] To a flask containing N-Boc-ethylenediamine (600 mg, 3.74 mmol) in DCM (37.4 mL) was added decanal (2.8 mL, 14.98 mmol), followed by sodium triacetoxyborohydride (2.38 g, 11.24 mmol), and the resulting suspension was stirred over the a few days at room temperature. Upon reaction completion, the reaction was diluted with 75 mL DCM, washed with 25 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (25g column, 0-20% MeOH/DCM) yielded product as a colorless oil. Yield: 1.383 g (100% purity, 88% yield) Mass Spec – 441.6 (m+1) (400 MHz, CDCl3) δ 3.50 (d, J = 8.6 Hz, 2H), 2.91 (d, J = 52.8 Hz, 4H), 1.64 (d, J = 43.8 Hz, 4H), 1.46 (s, 9H), 1.41 – 1.08 (m, 30H), 0.90 (t, J = 6.7 Hz, 6H). N 1 ,N 1 -didecylethane-1,2-diamine [00191] To a vial containing tert-butyl (2-(didecylamino)ethyl)carbamate (1.38 g, 3.14 mmol) was added HCl in dioxane (15.69 mL, 62.76 mmol, 4M), and the resulting solution was stirred at rt for 4 hours. Upon reaction completion, the reaction was concentrated in vacuo, dissolved in 60 mL DCM, washed with 10 mL each 1M NaOH, water, and brine, dried over MgSO4, and concentrated in vacuo to a dark yellow oil. Yield: 1.006g (94% yield) Mass Spec – 341.1 (m+1) 1H NMR (400 MHz, CDCl3) δ 3.22 (dt, J = 30.4, 6.3 Hz, 4H), 2.90 – 2.77 (m, 4H), 1.76 – 1.52 (m, 4H), 1.30 (d, J = 13.0 Hz, 28H), 0.90 (t, J = 6.7 Hz, 6H). (S)-N-(2-(didecylamino)ethyl)-3-(3,6-dioxopiperazin-2-yl)propenamide – Compound 39 [00192] To a vial containing a 0 oC solution of cyclo(gly,glu) (25 mg, 0.134 mmol) in DMF (0.5 mL) was added Hunig’s base (28.0 uL, 0.161 mmol), followed by HBTU (61.1 mg, 0.161 mmol) and HOBt (25.3 mg, 0.161 mmol), and the resulting clear, colorless solution was stirred at rt for 15 minutes. N1,N1-didecylethane-1,2-diamine (91.5 mg, 0.269 mmol) was added and the resulting clear, brown solution was stirred overnight at rt. Upon reaction completion, the reaction was concentrated in vacuo to a brown oil. Purification via Flash chromatography (5g HC column, 0-20% MeOH/DCM, 0.1% NH3), yielded product. Yield: 24 mg (35% yield) MS: m/z = 509.8 (m+1) 1H NMR (400 MHz, CDCl3) δ 7.25 (s, 1H), 6.62 (s, 0H), 6.02 (s, 1H), 4.17 – 3.90 (m, 2H), 3.66 (t, J = 6.7 Hz, 1H), 3.52 – 3.24 (m, 1H), 2.63 (d, J = 45.6 Hz, 3H), 2.43 (t, J = 6.4 Hz, 1H), 2.37 – 2.12 (m, 1H), 1.59 (p, J = 6.7 Hz, 1H), 1.48 (s, 3H), 1.30 (d, J = 6.7 Hz, 30H), 0.90 (t, J = 6.6 Hz, 6H). (S)-N-(2-(didecylamino)ethyl)-2-(3,6-dioxopiperazin-2-yl)acetamide – Compound 42 [00193] To a 0 oC solution of cyclo(gly,asp) (25 mg, 0.145 mmol) in DMF (0.5 mL) was added hunig’s base (30.3 uL, 0.174 mmol), followed by HBTU (66.1 mg, 0.174 mmol) and HOBt (27.4 mg, 0.174 mmol), and the resulting solution was stirred at rt for 15 minutes. N1,N1-didecylethane-1,2-diamine (98.9 mg, 0.290 mmol) was then added, and the reaction was stirred at rt overnight. Upon reaction completion, the reaction was concentrated in vacuo to a brown oil. Diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution., water, and brine, dried over MgSO4, and concentrated to a brown oil. Purification via Flash chromatography (5g HC column, 0-30% MeOH/DCM w/0.1% NH3) yielded product as a sticky yellow solid. Yield: 24.9 mg (~95% purity, 35% yield) Mass Spec – 495.6 (m+1) 1H NMR (400 MHz, CDCl3) δ 7.01 (s, 1H), 6.55 (s, 1H), 5.95 (s, 1H), 4.47 – 4.36 (m, 1H), 4.06 (s, 2H), 3.51 – 3.25 (m, 2H), 3.01 (dd, J = 16.0, 3.5 Hz, 1H), 2.64 (q, J = 9.8 Hz, 2H), 2.52 (s, 4H), 1.67 (s, 2H), 1.46 (s, 3H), 1.30 (d, J = 6.3 Hz, 28H), 0.91 (t, J = 6.6 Hz, 6H). Compound 49 & Compound 50 bis(2-(didecylamino)ethyl) 2,2’-((2S,5S)-3,6-dioxopiperazine-2,5-diyl)diacetate – Compound 49 [00194] To a vial containing a suspension of cyclo(Asp,Asp) (25.0 mg, 0.109 mmol) in DCM (1 mL) was added EDCI (62.5 mg, 0.326 mmol) and DMAP (1.3 mg, 0.011 mmol), and the resulting suspension was stirred at rt for 45 minutes.2-(didecylamino)ethan-1-ol (111.3 mg, 0.326 mmol) was then added, and the reaction was stirred overnight at rt. Upon reaction completion, the reaction was concentrated in vacuo to a yellow oil. Purification via Flash chromatography (5g HC column, 0-30% MeOH/DCM) yielded product as a sticky, colorless gum. Yield: 42.8 mg (100% purity, 45% yield) Mass spec – 877.8 (m+1) 1H NMR (400 MHz, CDCl3) δ 7.20 (s, 1H), 4.35 (t, J = 11.1 Hz, 3H), 4.11 (dd, J = 11.7, 5.8 Hz, 1H), 3.57 (s, 1H), 3.17 (d, J = 17.0 Hz, 1H), 2.70 (d, J = 7.1 Hz, 4H), 2.48 (t, J = 7.8 Hz, 6H), 1.59 (s, 10H), 1.42 (d, J = 8.2 Hz, 5H), 1.26 (s, 58H), 0.88 (t, J = 6.6 Hz, 12H). bis(2-(didecylamino)ethyl) 3,3’-((2S,5S)-3,6-dioxopiperazine-2,5-diyl)dipropionate – Compound 50 [00195] To a suspension of cyclo(glu,glu) (25 mg, 0.097 mmol) in DCM (1 mL) was added EDCI (55.7 mg, 0.290 mmol) and DMAP (1.18 mg, 8.68 umol), and the resulting suspension was stirred at rt for 45 minutes.2-(didecylamino)ethan-1-ol (99.2 mg, 0.290 mmol) was then added, and the resulting solution was stirred at rt overnight. Upon reaction completion, the reaction was concentrated in vacuo to a yellow oil. Purification via Flash chromatography (5g HC column, 0-30% MeOH/DCM) yielded product as a colorless oil. Yield: 90.3 mg (95% purity, 97% yield) Mass Spec – 905.81H NMR (400 MHz, CDCl3) δ 4.27 (td, J = 11.2, 6.5 Hz, 3H), 3.88 (s, 2H), 3.80 (s, 1H), 3.01 (s, 2H), 2.91 (s, 4H), 2.77 (s, 2H), 2.50 (dq, J = 17.6, 6.8 Hz, 4H), 2.45 – 2.14 (m, 3H), 1.73 (s, 4H), 1.45 (d, J = 7.8 Hz, 4H), 1.29 (d, J = 6.1 Hz, 60H), 0.90 (t, J = 6.7 Hz, 12H). Compound 54 2-(didecylamino)ethyl 2-((2S,5S)-5-isopropyl-3,6-dioxopiperazin-2-yl)acetate – Compound 54 [00196] To a solution of cyclo(valine,aspartic acid) (25 mg, 0.117 mmol) in DMF (1.17 mL) was added EDCI (26.9 mg, 0.140 mmol) and DMAP (1.4 mg, 11.7 umol), and the resulting solution was stirred at rt for 45 minutes.2-(didecylamino)ethan-1-ol (79.7 mg, 0.233 mmol) was then added, and the resulting solution was stirred at rt overnight. Upon reaction completion, the reaction was diluted with 8 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (0-20% MeOH/DCM, 0.1% NH3) yielded product. Yield: 24.1 mg (38.4% yield) Mass Spec – 538.5 (m+1) 1H NMR (400 MHz, CDCl3) δ 5.82 (s, 1H), 4.42 (dd, J = 23.6, 8.9 Hz, 1H), 4.22 – 4.08 (m, 1H), 3.95 (d, J = 13.2 Hz, 1H), 3.58 (s, 1H), 3.29 (d, J = 17.2 Hz, 1H), 2.79 – 2.55 (m, 2H), 2.50 (t, J = 7.8 Hz, 2H), 1.50 (d, J = 51.6 Hz, 6H), 1.29 (s, 28H), 1.06 (dd, J = 7.2, 3.5 Hz, 3H), 1.01 – 0.81 (m, 9H). Compound 55 [00197] To a solution of 2-(dinonylamino)ethan-1-ol (383 mg, 2.5 mmol) in DMF (10 mL) was added 3,3’-((2S,5S)-3,6-dioxopiperazine-2,5-diyl)dipropionic acid (258 mg, 1 mmol), EDCI (477mg, 2.5 mmol), HOBt (377 mg, 2.5 mmol), and DIPEA (6 mmol, 1 ml), and the solution was stirred at RT for overnight. Upon reaction completion, the solution was concentrated in vacuo, the residue was dissolved in DCM, washed with water and sodium bicarbonate solution, dried over Na2SO4 and concentrated. Reverse phase flash chromatography (0 to 100% acetonitrile in water w/0.1% TFA) followed by desalting with 3.5% K2CO3 solution/chloroform afforded 29.1 mg of the desired product (3.42% yield).1H NMR (400 MHz, CDCl3) δ 4.30 – 3.89 (m, 6H), 2.71 (m, 4H), 2.59 – 2.39 (m, 8H), 2.38 – 2.04 (m, 4H), 1.65(m, 8H), 1.43 (m, 4H), 1.27 (m, 48H), 0.94 – 0.77 (m, 12H). Compound 58 2-(didecylamino)ethyl 2-((2S,5S)-5-benzyl-3,6-dioxopiperazin-2-yl)acetate – Compound 58 [00198] To a solution of cyclo(Asp,Phe) (50 mg, 0.191 mmol) in DMF (2 mL) was added EDCI (73.1 mg, 0.381 mmol) and DMAP (2.33 mg, 0.019 mmol), and the resulting solution was stirred at rt for 45 minutes.2-(didecylamino)ethan-1-ol (130.3 mg, 0.381 mmol) was then added, and the resulting solution was stirred overnight at rt. Upon reaction completion, the reaction was diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (5g HC column, 3-6% MeOH/DCM) yielded product. Yield: 8.8 mg (99% pure, 8% yield) Mass Spec – 586.4 (m+1) 1H NMR (400 MHz, CDCl3) δ 7.45 – 7.29 (m, 4H), 7.24 (d, J = 6.6 Hz, 2H), 6.26 (d, J = 18.3 Hz, 1H), 4.40 – 4.19 (m, 3H), 4.07 (dt, J = 11.6, 5.9 Hz, 1H), 3.71 (s, 1H), 3.20 (t, J = 6.1 Hz, 2H), 2.82 (ddd, J = 17.8, 11.1, 2.7 Hz, 1H), 2.69 (t, J = 6.1 Hz, 1H), 2.50 (t, J = 7.8 Hz, 3H), 1.60 – 1.40 (m, 4H), 1.30 (d, J = 5.9 Hz, 28H), 0.90 (t, J = 6.6 Hz, 6H). Compound 59 2-(didecylamino)ethyl 2-((2S,5S)-5-methyl-3,6-dioxopiperazin-2-yl)acetate – Compound 59 [00199] To a solution of cyclo(Asp,Ala) (50 mg, 0.269 mmol) in DMF (2 mL) was added EDCI (103.0 mg, 0.537 mmol) and DMAP (3.28 mg, 0.027 mmol), and the resulting solution was stirred at rt for 45 minutes.2-(didecylamino)ethan-1-ol (183.5 mg, 0.537 mmol) was then added, and the resulting solution was stirred overnight at rt. Upon reaction completion, the reaction was diluted with 10 mL DCM, washed with 3 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (5g HC column, 3-10% MeOH/DCM over 20 CVs) yielded product. Yield: 29.2 mg (98% purity, 21% yield) Mass Spec – 510.4 (m+1) 1H NMR (400 MHz, CDCl3) δ 7.14 (s, 1H), 6.54 (s, 1H), 6.18 (d, J = 10.4 Hz, 1H), 4.48 – 4.28 (m, 2H), 4.15 (tt, J = 14.9, 6.6 Hz, 2H), 3.64 (s, 1H), 3.21 (ddd, J = 17.9, 9.5, 2.7 Hz, 1H), 2.82 – 2.41 (m, 6H), 1.71 – 1.39 (m, 5H), 1.29 (d, J = 7.7 Hz, 29H), 0.90 (t, J = 6.6 Hz, 6H). Compound 60 & Compound 63 (S)-N-decyl-N-(oxiran-2-ylmethyl)decan-1-amine [00200] To a vial containing didecylamine (1.00 g, 3.36 mmol) was added water (9 uL) and s-epichlorohydrin (0.316 mL, 4.03 mmol). The reaction was then heated to 40 oC and the reaction was stirred overnight. Upon full consumption of didecylamine starting material, 2 mL 36% NaOH was added and vigorously stirred at rt overnight. Upon reaction completion, the reaction was diluted with 10 mL water, extracted with 3 x 10 mL DCM, washed combined organic phases with 10 mL brine, dried over MgSO4, and concentrated in vacuo to a yellow oil. Purification via Flash chromatography (0-10% EtOAc/DCM) yielded product as a yellow liquid. Yield: 490.5 mg (98% purity, 40% yield) Mass Spec – 354.4 (m+1) 1H NMR (400 MHz, CDCl3) δ 3.07 (dq, J = 6.9, 3.7 Hz, 1H), 2.82 – 2.70 (m, 2H), 2.52 (dtt, J = 14.9, 12.4, 6.6 Hz, 6H), 1.47 (t, J = 7.1 Hz, 5H), 1.30 (d, J = 5.6 Hz, 29H), 0.90 (t, J = 6.6 Hz, 6H). bis((S)-3-(didecylamino)-2-hydroxypropyl) 2,2’-((2S,5S)-3,6-dioxopiperazine-2,5- diyl)diacetate – Compound 60 [00201] To a suspension of cyclo(ser,ser) (25 mg, 0.144 mmol) in isopropanol (1 mL) was added (S)-N-decyl-N-(oxiran-2-ylmethyl)decan-1-amine (111.7 mg, 0.316 mmol), and the resulting suspension was stirred at 80 oC overnight. Upon reaction completion, the reaction was diluted with 10 mL saturated sodium bicarbonate solution, extracted with 3 x 10 mL DCM, washed combined organic phases with 10 mL each water x 3 and brine, dried over MgSO4, and concentrated in vacuo to an orange oil. Purification via Flash chromatography (6g C18 column, 50-100% ACN/water w/0.1% TFA). Yielded product as a TFA salt. Dissolved in 2 mL DCM, washed with 2 mL saturated sodium bicarbonate solution, dried over MgSO4, and concentrated in vacuo to a yellow gum. Yield: 9.2 mg (100% purity, 9% yield) Mass Spec – 937.8 (m+1) 1H NMR (400 MHz, CDCl3) δ 5.02 – 3.91 (m, 11H), 3.57 – 2.68 (m, 16H), 1.70 (s, 7H), 1.31 (d, J = 22.7 Hz, 57H), 0.90 (t, J = 6.6 Hz, 12H). bis((S)-3-(didecylamino)-2-hydroxypropyl) 3,3’-((2S,5S)-3,6-dioxopiperazine-2,5- diyl)dipropionate – Compound 63 [0200] To a suspension of cyclo(Glu,Glu) (25.0 mg, 0.097 mmol) in isopropanol (1 mL) was added (S)-N-decyl-N-(oxiran-2-ylmethyl)decan-1-amine (75.3 mg, 0.213 mmol), and the resulting suspension was stirred at 80 oC overnight. Upon reaction completion, 0.1 mL DBU was added then the reaction was concentrated in vacuo to a yellow oil. Purification via Flash chromatography (5g HC column, 3-13% MeOH/DCM) yielded product. Yield: 30.0 mg (32% yield) Mass Spec – 965.8 (m+1) 1H NMR (400 MHz, CDCl3) δ 3.78 (ddd, J = 14.8, 10.1, 4.1 Hz, 3H), 3.52 (dd, J = 11.1, 3.9 Hz, 1H), 2.81 – 2.16 (m, 20H), 1.50 (d, J = 9.3 Hz, 9H), 1.29 (d, J = 5.6 Hz, 64H), 0.90 (t, J = 6.7 Hz, 12H). Compound 66 (S)-3-(didecylamino)-2-hydroxypropyl 2-((S)-3,6-dioxopiperazin-2-yl)acetate – Compound 66 [0201] To a vial containing (S)-N-decyl-N-(oxiran-2-ylmethyl)decan-1-amine (123.3 mg, 0.349 mmol) was added acetonitrile (2 mL), followed by cyclo (Gly,Asp) (50 mg, 0.290 mmol), and the resulting suspension was stirred at 80 oC over the weekend. After 24 hours, DMSO (1 mL) was added and the reaction was continued heating for 24 hours. Upon reaction completion, water (10 mL) was added to the reaction and the solution was lyophilized. Purification of the resulting oil via Flash chromatography (5g HC column, 0-20% MeOH/DCM) yielded product. Yield: 73.5 mg (95% purity, 45% yield) Mass Spec – 526.4 (m+1) 1H NMR (400 MHz, CDCl3) δ 6.67 (d, J = 88.1 Hz, 1H), 6.15 (s, 1H), 4.46 – 3.67 (m, 6H), 3.51 (dd, J = 11.4, 4.3 Hz, 1H), 3.11 (ddd, J = 20.9, 17.4, 3.4 Hz, 1H), 2.95 – 2.33 (m, 7H), 1.48 (d, J = 30.5 Hz, 4H), 1.27 (d, J = 7.4 Hz, 31H), 0.88 (t, J = 6.7 Hz, 6H). Compound 77 (S)-3-(didecylamino)-2-hydroxypropyl 3-((S)-3,6-dioxopiperazin-2-yl)propanoate – Compound 77 [0202] To a solution of cyclo(Gly,Glu) (50 mg, 0.268 mmol) in isopropanol (2.7 mL) was added (S)-N-decyl-N-(oxiran-2-ylmethyl)decan-1-amine (142 mg, 0.402 mmol), and the resulting solution was stirred overnight at 80 C. Upon reaction completion, the reaction was concentrated in vacuo. Purification on Flash chromatography (5g HC column, 0-25% MeOH/DCM) yielded product. Yield: 62.4 mg (43% yield) Mass Spec – 540.6 (m+1) NMR (400 MHz, CDCl3) δ 7.04 (d, J = 15.2 Hz, 1H), 6.93 (s, 1H), 5.15 – 4.99 (m, J = 6.3 Hz, 1H), 4.31 – 3.94 (m, 1H), 3.85 – 3.58 (m, 1H), 3.59 – 3.45 (m, 1H), 2.83 – 2.19 (m, 6H), 1.48 (s, 4H), 1.29 (q, J = 3.1 Hz, 36H), 1.00 – 0.80 (m, 6H). Compound 87 3-(didecylamino)propan-1-ol [0203] To a solution of 3-amino-1-propanol (0.305 mL, 3.99 mmol) in dichloroethane (20 mL) was added decanal (2.26 mL, 11.98 mmol) and sodium triacetoxy borohydride (2.54 g, 11.98 mmol), and the resulting solution was stirred at rt overnight. Upon reaction completion, the reaction was diluted with 75 mL dichloromethane, washed with 25 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a colorless oil. Purification via Flash chromatography (25g column, 10-30% MeOH/DCM) yielded product as a colorless oil. Yield: 1.304 g (85% purity, 78% yield) Mass Spec – 356.2 (m+1) 1H NMR (400 MHz, CDCl3) δ 3.82 (t, J = 5.2 Hz, 2H), 2.84 (t, J = 6.0 Hz, 2H), 2.72 – 2.53 (m, 4H), 1.80 (p, J = 5.7 Hz, 2H), 1.59 (dp, J = 13.7, 6.7 Hz, 4H), 1.30 (d, J = 10.5 Hz, 28H), 0.90 (t, J = 6.8 Hz, 6H). 5-(3-(didecylamino)propyl) 1-methyl (tert-butoxycarbonyl)-L-glutamate [0204] To a solution of Boc-Glu-Ome (261 mg, 1.00 mmol) in DCM (10 mL) was added 3- (didecylamino)propan-1-ol (427 mg, 1.2 mmol), EDCI (211 mg, 1.10 mmol, and Hunig’s base (348 uL, 2.00 mmol), and the resulting solution was stirred overnight at rt. Upon reaction completion, the reaction was diluted with 30 mL DCM, washed with 10 mL each saturated sodium bicarbonate solution, water, and brine, dried over MgSO4, and concentrated in vacuo to a yellow oil. Purification via Flash chromatography (10 g HC column, 0-5% MeOH/DCM, 20 CV ramp) yielded product. Characterization: Yield: 265 mg (43% yield) Mass Spec – 599.5 (m+1) 1H NMR (400 MHz, CDCl3) δ 5.11 (d, J = 8.4 Hz, 1H), 4.39 – 4.25 (m, 1H), 4.21 – 4.04 (m, 2H), 3.75 (s, 2H), 2.78 (d, J = 83.2 Hz, 5H), 2.40 (q, J = 7.6 Hz, 1H), 2.28 – 1.83 (m, 2H), 1.72 – 1.47 (m, 3H), 1.44 (s, 7H), 1.27 (d, J = 8.9 Hz, 30H), 0.88 (t, J = 6.7 Hz, 6H). bis(3-(didecylamino)propyl) 3,3’-((2S,5S)-3,6-dioxopiperazine-2,5-diyl)dipropionate – Compound 87 [0205] To a solution of 5-(3-(didecylamino)propyl) 1-methyl (tert-butoxycarbonyl)-L- glutamate) (50 mg, 0.0835 mmol) in DCM (334 uL) was added TFA (83.5 uL), and the resulting solution was stirred at rt for 3 hours. After 3 hours, the reaction was concentrated in vacuo and the excess TFA was removed through azeotropic distillation with toluene. The resulting oil was dissolved in acetonitrile (334 uL) and triethylamine (83.5 uL), and the solution was heated overnight at 60 C. Upon reaction completion, the reaction was concentrated in vacuo to an orange oil. Purification via flash chromatography (5g HC column, 0-15% MeOH/DCM) yielded product. Yield: 33.2 mg (85% yield, 95% purity) Mass Spec – 933.8 (m+1) 1H NMR (400 MHz, CDCl3) δ 5.94 (s, 2H), 4.31 – 4.10 (m, 2H), 3.13 (d, J = 7.6 Hz, 3H), 3.02 – 2.83 (m, 8H), 2.57 – 2.17 (m, 8H), 1.92 (p, J = 6.3 Hz, 3H), 1.63 (q, J = 8.0 Hz, 9H), 1.29 (d, J = 24.1 Hz, 63H), 0.88 (t, J = 6.7 Hz, 12H). Compound 88 methyl N 2 -(tert-butoxycarbonyl)-N 5 -(2-(didecylamino)ethyl)-L-glutaminate [0206] To a solution of Boc-Glu-Ome (173 mg, 0.661 mmol) in DMF (2.2 mL) was added HATU (234 mg, 0.616 mmol) and Hunig’s base (153 uL, 0.881 mmol), and the resulting yellow solution was stirred at rt for 15 minutes. N1,N1-didecylethane-1,2-diamine (150 mg, 0.440 mmol) in DMF (2.2 mL) was then added, and the resulting solution was stirred at rt overnight. Upon reaction completion, the reaction was diluted with DCM (30 mL), washed with 10 mL each saturated sodium bicarbonate solution, water x3, and brine, dried over MgSO4, and concentrated in vacuo to a yellow oil. Purification via flash chromatography (5 g HC column, 0-10% MeOH/DCM, 10 CV ramp) yielded product. Yield: 189.2 mg (74% yield) MS: 584.52 (m+1) 1H NMR (400 MHz, CDCl3) δ 5.34 (d, J = 8.4 Hz, 1H), 4.39 – 4.18 (m, 1H), 3.74 (d, J = 11.5 Hz, 4H), 3.59 – 3.37 (m, 2H), 2.80 (s, 3H), 2.34 (t, J = 7.2 Hz, 2H), 2.21 (dt, J = 31.8, 6.7 Hz, 1H), 1.92 (td, J = 17.1, 7.4 Hz, 1H), 1.55 (d, J = 32.8 Hz, 0H), 1.43 (d, J = 2.2 Hz, 12H), 1.29 (d, J = 22.7 Hz, 31H), 0.88 (t, J = 6.7 Hz, 6H). 3,3’-((2S,5S)-3,6-dioxopiperazine-2,5-diyl)bis(N-(2- (didecylamino)ethyl)- 65 -ropenamide) – Compound 88 [0207] To a solution of methyl N2-(tert-butoxycarbonyl)-N5-(2-(didecylamino)ethyl)-L- glutaminate (50 mg, 0.086 mmol) in DCM (0.344 mL) was added TFA (0.086 mL), and the resulting solution was stirred at rt for 3 hours. The reaction was then concentrated in vacuo, and the residue was dissolved in acetonitrile (0.344 mL) and Hunig’s base (0.086 mL). The reaction was then heated to 60C for overnight. Upon reaction completion, the solution was concentrated in vacuo and purified via flash chromatography (5 g HC column, 0-15% MeOH/DCM) to yield product as a single peak. Yield: 10.5 mg (27% yield) Mass Spec – 903.8 (m+1) 1H NMR (400 MHz, CDCl3) δ 5.83 (s, 5H), 4.28 (dd, J = 8.7, 5.1 Hz, 4H), 3.73 (dt, J = 12.7, 6.3 Hz, 1H), 3.14 (q, J = 7.4 Hz, 1H), 2.98 – 2.88 (m, 1H), 2.59 – 2.22 (m, 19H), 1.53 – 1.17 (m, 50H), 0.90 (t, J = 6.7 Hz, 12H). Example 5 – Delivery of mRNA to Mouse Using Lipid Nanoparticle Formulated With Exemplary Diketopiperazine Lipids Formulation of Lipid Nanoparticles Using Diketopiperazine Lipids [0208] Exemplary lipid nanoparticle formulations were prepared using the diketopiperazine lipids synthesized according to Examples 1 and 4. These lipid nanoparticle formulations were formulated with various combinations of various exemplified diketopiperazine lipids, cholesterol, helper lipid, and PEG lipid at various ratios, to encapsulate mRNA, using procedures similar to those described in Example 2. Experimental Protocols for Mouse Data [0209] Dose Preparation: LNPs were prepared using the NanoAssemblr Ignite system or the NanoAssemblr Spark system by combining i) an aqueous phase with mRNA and 25 mM acetic acid buffer at pH 5.0, and ii) an organic phase containing a combination of four lipids dissolved in ethanol. After mixing, resulting LNPs were dialyzed in 1X PBS with a 20 kDa dialysis cassette for 2-3 hours and filtered using a 0.22 µm pore size PES filter. Where the nanoparticles contained a DNA barcode in addition to mRNA, LNPs were not analyzed until a second dialysis step. Nanoparticles were then analyzed in the Unchained Stunner to determine whether they meet the quality control criteria: diameter (20 nm - 200 nm), polydispersity index (< 0.3), concentration, intensity of peak of interest (> 75%), and turbidity (A260 < 3.0 and A330 < 1.25). The nanoparticles contained a DNA barcode in addition to the mRNA, and passed the quality control criteria were pooled and dialyzed again in 1X PBS for 1 hour in a 100 kDa dialysis cassette. [0210] Dose Administration: Doses were calculated based on animal weight the day of administration. LNPs were administered as a bolus via a tail vein using an insulin syringe with a 28 gauge. Maximum volume for administrations was set to 20 µL / gram based on approved IACUC protocols. Body weight was monitored after administration until sacrifice. [0211] Tissue Collection: Animals injected with LNPs containing reporter mRNA were sacrificed in a carbon dioxide chamber 16 - 24 hours after nanoparticle administration. Relevant tissues were then isolated, minced finely on top of Petri dishes into ~1 cm3 cubes (with the exception of bone marrow) and placed into individual aliquots containing specific digestive enzyme cocktails depending on the organ. [0212] Tissue Digestion: Samples were digested as discussed below: i. Liver Digestion: Digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (200 U/mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 ºC for 30 minutes at 750 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Mouse Monoclonal (0.5% v/v) FcX block and transferred to microcentrifuge tubes to stain. ii. Lung Digestion: Digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (200 U/mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 ºC for 20 minutes at 750 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Mouse Monoclonal (0.5% v/v) FcX block and transferred to microcentrifuge tubes to stain. iii. Kidney Digestion: Digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (200 U/mL), and RPMI-1640 solvent was used. Tissues were incubated at 37 ºC for 30 minutes at 750 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Mouse Monoclonal (0.5% v/v) FcX block and transferred to microcentrifuge tubes to stain. iv. Heart Digestion: Digestive enzyme cocktail containing a mix of Collagenase II (1 mg/mL), Dispase (0.6 U/mL), DNAse I (200 U/mL), FBS (2% v/v), and RPMI-1640 solvent was used. Tissues were incubated at 37 ºC for 1 hour at 1,000 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant was removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Mouse Monoclonal (0.5% v/v) FcX block and transferred to microcentrifuge tubes to stain. v. Spleen Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (200 U/mL) in RPMI-1640 solvent. Tissues were incubated at 37 ºC for 10 minutes at 750 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Mouse Monoclonal (5% v/v) FcX block and transferred to microcentrifuge tubes to stain. vi. Bone Marrow Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (200 U/mL) in RPMI-1640 solvent. Tissues were incubated at 37 ºC for 10 minutes at 750 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Mouse Monoclonal (5% v/v) FcX block and transferred to microcentrifuge tubes to stain. [0213] Cell Population Staining: After an incubation period of 15 minutes in FcX block reagent at 4 ºC, each tissue was stained with a cocktail containing a combination of the following antibodies for 45 minutes at 4 ºC or 30 minutes at room temperature. i. Liver Panel: anti-mouse CD3 (17A2), anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD68 (FA-11), anti-mouse CD31 (390), anti-mouse TER-119 (TER- 119), anti-mouse NK1.1 (PK136), anti-mouse CD11c (N418), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. ii. Lung Panel: anti-mouse CD8a (53-6.7), anti-mouse CD326 (G8.8), anti- mouse/human CD49f (GoH3), anti-mouse CD4 (RM4-5), anti-mouse CD3 (17A2), anti- mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD31 (390), anti-mouse TER- 119 (TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iii. Kidney Panel: anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD31 (390), anti-mouse TER-119 (TER-119), anti-mouse CD309 (89B3A5), anti-mouse Podocalyxin (10B9), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iv. Heart Panel: anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse CD31 (390), anti-mouse TER-119 (TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. v. Spleen Panel: anti-mouse CD8a (53-6.7), anti-mouse CD19 (6D5), anti-mouse CD11b (M1/70), anti-mouse CD3 (17A2), anti-mouse TER-119 (TER-119), anti-mouse NK1.1 (PK136), anti-mouse CD4 (RM4-5), anti-mouse CD11c (N418), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. vi. Bone Marrow Panel: anti-mouse CD4 (RM4-5), anti-mouse CD117 (2B8), anti- mouse CD8a (53-6.7), anti-mouse CD45.2 (104), anti-mouse CD11b (M1/70), anti-mouse Sca1 (D7), anti-mouse TER-119 (TER-119), anti-mouse CD3 (17A2), anti-mouse CD34 (SA376A4), anti-mouse CD11c (), Lineage Cocktail (CD3 / GR-1 / CD11b / CD45R / B220 / TER-119), Annexin V, LiveDead Fixable Violet Dye or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. [0214] Flow Cytometry Readouts: Different cell populations were analyzed for reporter protein expression relative to an untreated primate. The results were expressed as the percentage of cells that were positive for the reporter protein. Where LNPs contained DNA barcodes in addition to the mRNA, populations expressing reporter protein were sorted into QuickExtract buffer to isolate intracellular DNA. [0215] DNA Sequencing of Sorted Samples: DNA barcodes of sorted cells were amplified under two rounds of PCR and Illumina handles were attached for compatibility with Next Generation Sequencing. Amplified samples were pooled, cleaned, diluted to 1 nM, and loaded into an iSeq cartridge for sequencing. Samples were analyzed for relative DNA barcode counts for selection of LNP candidates to advance in the pipeline. [0216] The results of LNP screening in mouse for LNP formulations containing various exemplary diketopiperazine lipids as disclosed herein are shown in FIGS.4-14. In these figures, certain LNP formulations were selected from high-throughput, barcoding based, screens. During high-throughput screening, each LNP formulation was numbered as LNP-1 through LNP-XYZ in database, referred to as the sample number for that LNP formulation. For instance, LNP-151 indicates the 151st LNP formulation for a screen. [0217] FIGS.4A-4D illustrate the flow cytometry results for three high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 18 (also referred to as KC-34 in Example 1) in the liver (FIG.4A), spleen (FIG.4B), lung (FIG. 4B), or bone marrow (FIG.4D). For each screen, LNPs were formulated with Compound 18 (Lipomer), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. [0218] In Screen 1, the PEG lipid included C14PEG2K PE, C18PEG2K PE, DMG-PEG2K, or DSG-PEG2K; and the helper lipid included DOPE or DSPC. The ratios for the lipid components were: [0219] In Screen 2, the PEG lipid included C14PEG2K PE, C18PEG2K PE, DMG-PEG2K, or DSG-PEG2K; and the helper lipid included 14:0 PA, 18:1 PA, 14:0 Hemi BMP, or 18:1 Hemi BMP. The molar ratios for the lipid components were: [0220] In Screen 3, the PEG lipid included C14PEG2K PE or DMG-PEG; and the helper lipid included DOPE or DSPC. The molar ratios for the lipid components were: [0221] The results in FIGS.4A-4D show that the LNP delivery was found in multiple cell populations in the liver of mouse, including endothelial cells, and immune cells; and in multiple cell populations in the spleen of mouse, including some delivery to B cells, macrophages, and dendritic cells. [0222] FIGS.5A-5B show the flow cytometry results for individual LNP formulations selected from Screen 1 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.5A) and spleen (FIG.5B). To conduct this experiment, a number of LNPs that performed well in Screen 1 (a high-throughput, barcoding based, screen disclosed in FIGS.4A-4D) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 18) for FIGS.5A-5B are characterized below: [0223] As shown in FIG.5A, certain LNP formulations (e.g., LNP-156) performed well across all hepatic populations, while certain LNP formulations (e.g., LNP-185, LNP-121, and LNP-122) performed well in immune cell populations in the liver in mouse. FIG.5B shows significant levels of delivery in the spleen in mouse, such as in dendritic cells in the spleen, for certain LNP formulations (e.g., LNP-185, LNP-121, LNP-122, and LNP-156). [0224] FIGS.6A-6B show the flow cytometry results for individual LNP formulations selected from Screen 3 (discussed in FIGs.4A-4D) in mouse using the LNP formulations containing Compound 18 in the liver (FIG.6A) and spleen (FIG.6B). To conduct this experiment, a number of LNPs that performed well in Screen 3 (a high-throughput, barcoding based, screen disclosed in FIGS.4A-4D) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 18) for FIGS.6A-6B are characterized below:
[0225] As shown in FIG.6A, many LNP formulations performed well in the liver in mouse. FIG.6B shows that certain LNP formulations that did not perform as well in the liver had high levels of delivery in multiple populations across the spleen in mouse. [0226] FIGS.7A-7D show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 26, Compound 29, Compound 30, and Compound 31 in the liver (FIG.7A), lung (FIG.7B), spleen (FIG. 7C), and bone marrow (FIG.7D). For each screen, LNPs were formulated with a Lipomer (Compound 26, Compound 29, Compound 30, or Compound 31), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. [0227] In Screen 1, the PEG lipid included ALC-0159, PEG2K-C-DMG, or C18PEG2K PE; and the helper lipid included DSPC. The molar ratios for the lipid components were: [0228] In Screen 2, the PEG lipid included ALC-0159, PEG2K-C-DMG, or C18PEG2K PE; and the helper lipid included DDAB or DOTAP. The molar ratios for the lipid components were: [0229] The results in FIGS.7A-7D show that the LNP delivery was found in immune cell populations in the liver in mouse, including macrophages. [0230] FIGS.8A-8F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 39 and Compound 42 in the liver (FIG.8A), heart (FIG.8B), bone marrow (FIG.8C), kidney (FIG. 8D), spleen (FIG.8E), and lung (FIG.8F). For each screen, LNPs were formulated with a Lipomer (Compound 39 or Compound 42), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. [0231] In Screen 1, the PEG lipid included C14-PEG2K PE, DSG-PEG2K, DMG-PEG2K, or C18-PEG2K PE; and the helper lipid included DDAB or DSPC. The molar ratios for the lipid components were: [0232] The results in FIGS.8A-8F show that LNP delivery was found in basal and stem populations in the lung in mouse. [0233] FIGS.9A-9F show the flow cytometry results for two high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 49 and Compound 50 in the liver (FIG.9A), lung (FIG.9B), bone marrow (FIG.9C), spleen (FIG. 9D), heart (FIG.9E), and kidney (FIG.9F). For each screen, LNPs were formulated with a Lipomer (Compound 49 or Compound 50), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. [0234] In Screen 1, the PEG lipid included C14-PEG2K PE, DSG-PEG2K, DMG-PEG2K, or C18-PEG2K PE; and the helper lipid included DSPC. The molar ratios for the lipid components were: [0235] In Screen 2, the PEG lipid included C14-PEG2K PE, DSG-PEG2K, DMG-PEG2K, or C18-PEG2K PE; and the helper lipid included DDAB. The molar ratios for the lipid components were: [0236] The results in FIGS.9A-9F show that LNP delivery was found in the liver, lung, and bone marrow in mouse. [0237] FIGS.10A-10C show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGS.9A-9F) in mouse using the LNP formulations containing Compound 50 in the liver (FIG.10A), lung (FIG.10B), and bone marrow (FIG. 10C). To conduct this experiment, a number of LNPs that performed well in Screen 2 (a high-throughput, barcoding based, screen disclosed in FIGS.9A-9F) were selected and individually injected one by one in mice. The LNP formulations (Lipomer: Compound 50) for FIGS.10A-10C are characterized below: [0238] Figure 10C shows some levels of LNP delivery in progenitor cells of the bone marrow. [0239] FIGS.11A-11F show the flow cytometry results for a high-throughput, barcoding based, screen in mouse using the LNP formulations containing Compound 55 in the liver (FIG.11A), lung (FIG.11B), bone marrow (FIG.11C), spleen (FIG.11D), kidney (FIG. 11E), and heart (FIG.11F). For the screen, LNPs were formulated with Compound 55 (Lipomer), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. The PEG lipid included C14-PEG2K PE or C18-PEG2K PE; and the helper lipid included DSPC or DDAB. The molar ratios for the lipid components were:
[0240] The results in FIGS.11A-11F show that LNP delivery was found in the liver, lung, bone marrow, spleen, kidney, and heart; and significant levels of LNP delivery were found in progenitor cells in the bone marrow in mouse (FIG.11C), in macrophage and monocyte populations in the spleen in mouse (FIG.11D), and in endothelial cells and macrophages in the kidney in mouse (FIG.11E). [0241] FIGS.12A-12F show the flow cytometry results for individual LNP formulations selected from Screen 2 (discussed in FIGs.11A-11F) in mouse using the LNP formulations containing Compound 55 in the liver (FIG.12A), lung (FIG.12B), bone marrow (FIG.12C), spleen (FIG.12D), kidney (FIG.12E), and heart (FIG.12F). The LNP formulations (Lipomer: Compound 55) for FIGS.12A-12F are characterized below: [0242] The results in FIGS.12A-12F show that some LNP delivery was found in the liver, lung, bone marrow, spleen, kidney, and heart; although the levels of delivery in these tissues or populations were not significant. [0243] FIGS.13A-13F show the flow cytometry results for a high-throughput, barcoding based, screens in mouse using the LNP formulations containing Compound 60 and Compound 63 in the liver (FIG.13A), lung (FIG.13B), bone marrow (FIG.13C), spleen (FIG.13D), kidney (FIG.13E), and heart (FIG.13F). For the screen, LNPs were formulated with a Lipomer (Compound 60 or Compound 63), cholesterol, a PEG lipid, and a helper lipid (HL). For the lipid components, LNPs for the four lipid components at all possible combinations, at various ratio combinations shown in the chart below, were produced. For each LNP formulation, the total lipid to nucleic acid mass ratio was 10:1. The PEG lipid included C14-PEG2K PE or C18-PEG2K PE; and the helper lipid included DDAB or DSPC. The molar ratios for the lipid components were: [0244] As shown in FIGS.13A-13F, high levels of LNP delivery were found in cell populations of the liver in mouse, and in endothelial cells of the lung in mouse. Moreover, these figures show that significant levels of LNP delivery were found in progenitor cells in the bone marrow of mouse, in macrophage and monocyte populations in the spleen of mouse, in endothelial cells and macrophages in the kidney of mouse, and in endothelial cells in the heart of mouse. Example 6 – Delivery of mRNA to non-human Primate (NHP) Using Lipid Nanoparticles Formulated With the Exemplary Diketopiperazine Lipids Formulation of Lipid Nanoparticles Using Diketopiperazine Lipids [0245] Exemplary lipid nanoparticle formulations were prepared using the diketopiperazine lipids synthesized according to Examples 1 and 4. These lipid nanoparticle formulations were formulated with various combinations of various exemplified diketopiperazine lipids, cholesterol, helper lipid, and PEG lipid at various ratios, to encapsulate mRNA, using procedures similar to those described in Example 2. Experimental Protocols for non-human Primate [0246] Predose Biopsy Collections: Blood (8 to 10 mL) were collected via a femoral vein into tubes containing K2EDTA from each animal predose (Day -20 to -22 based upon each animal’s dosing date). Additionally, bone marrow and liver biopsy samples were collected from all animals on Day -20 to -22 based upon the animal’s date of dosing. Animals underwent laparoscopic liver biopsy procedures while bone marrow were collected by perfusion of 30 mL of phosphate buffered saline (PBS) through one femur bone. [0247] Animal Pretreatment: Primates were pretreated with a dose of 1.0 mg/kg of dexamethasone administered intravenously, 0.5 mg/kg of famotidine administered intravenously, and 1.5 mg/kg diphenhydramine administered intramuscularly both 16-24 hours and 1 hour before LNP-mRNA administration. [0248] Dose Preparation: LNPs were prepared using the NanoAssemblr Ignite system by combining i) an aqueous phase with mRNA and 25 mM acetic acid buffer at pH 5.0, and ii) an organic phase containing a combination of four lipids dissolved in ethanol. After mixing, resulting LNPs were dialyzed in 1X PBS with a 20 kDa dialysis cassette for 2-3 hours and filtered using a 0.22 µm pore size PES filter. Nanoparticles were then analyzed to determine whether they meet the quality control criteria: using DLS for diameter (20 nm - 200 nm), polydispersity index (< 0.3), concentration, intensity of peak of interest (> 75%), and turbidity (A260 < 3.0 and A330 < 1.25). The nanoparticles that met these criteria were diluted to a solution of 1X PBS and 10% w/v trehalose, frozen at -20 ºC, transferred to -80 ºC, and shipped in dry ice the next day. [0249] At the administration site, frozen LNPs were allowed to thaw at room temperature for at least 45 minutes. Once thawed, they were filtered using 0.22 µm pore size, 33 mm diameter PES filters. Nanodrop RNA concentration measurements were taken before and after filtering to calculate dose retention from the original LNP concentration. [0250] Dose Administration: Doses were calculated based on animal weight the day of administration. LNPs were administered as a 30-minute infusion via a saphenous vein using a catheter set. [0251] Post-dose Procedures: Body weight and temperature were monitored after infusion until sacrifice. Additionally, hematology values and clinical chemistry were tracked several times after administration to monitor the response of the animal to the infused LNP. [0252] Tissue Collection: Animals injected with LNPs containing reporter mRNA were sacrificed by exsanguination under anesthesia 16 - 24 hours after nanoparticle administration. Relevant tissues were then isolated, minced finely into ~3 cm3 cubes (with the exception of bone marrow, blood, and spleen) and placed into individual aliquots of a solution containing 90% FBS and 10% DMSO. All samples will be stored in Mr. Frosty for control temperature change up to 72 hours at -70 to -90°C and then frozen in liquid nitrogen or at approximately - 80°C until shipped. [0253] Tissue Digestion: All samples were allowed to thaw at room temperature prior to processing. Once thawed, tissues were transferred to a complete media solution containing FBS and centrifuged to remove any traces of DMSO. After centrifugation samples were resuspended in tissue digestion solutions and processed as indicated below: i. Liver Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (400 U/mL), and RPMI-1640 solvent. Tissues were incubated at 37 ºC for 30 minutes at 1,000 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Human Monoclonal (5% v/v) and Polyclonal (15% v/v) FcX block and transferred to microcentrifuge tubes to stain. ii. Lung Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (400 U/mL), and RPMI-1640 solvent. Tissues were incubated at 37 ºC for 45 minutes at 1,000 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Human Monoclonal (5% v/v) and Polyclonal (15% v/v) FcX block and transferred to microcentrifuge tubes to stain. iii. Kidney Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase I (450 U/mL), Collagenase XI (125 U/mL), Hyaluronidase (60 U/mL), DNAse I (400 U/mL), and RPMI-1640 solvent. Tissues were incubated at 37 ºC for 30 minutes at 1,000 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Human Monoclonal (5% v/v) and Polyclonal (15% v/v) FcX block and transferred to microcentrifuge tubes to stain. iv. Heart Digestion: Tissues were minced finely on top of a Petri dish and placed into a digestive enzyme cocktail containing a mix of Collagenase II (1 mg/mL), Dispase (0.6 U/mL), DNAse I (400 U/mL), FBS (2% v/v), and RPMI-1640 solvent. Tissues were incubated at 37 ºC for 1 hour at 1,000 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Human Monoclonal (5% v/v) and Polyclonal (15% v/v) FcX block and transferred to microcentrifuge tubes to stain. v. Spleen Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (400 U/mL) in RPMI-1640 solvent. Tissues were incubated at 37 ºC for 10 minutes at 1,000 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Human Monoclonal (5% v/v) and Polyclonal (15% v/v) FcX block and transferred to microcentrifuge tubes to stain. vi. Bone Marrow Digestion: Tissues were placed into a digestive enzyme cocktail containing DNAse I (400 U/mL) in RPMI-1640 solvent. Tissues were incubated at 37 ºC for 10 minutes at 1,000 rpm. Then, they were transferred through a 70 µm mesh filter into 50 mL conical and washed with 7 mL PBS. Suspensions were then centrifuged at 800g for 7 min and the supernatant removed. Finally, cells were resuspended in 250-300 µL of MojoSort buffer containing Human Monoclonal (5% v/v) and Polyclonal (15% v/v) FcX block and transferred to microcentrifuge tubes to stain. [0254] Cell Population Staining: After an incubation period of 15 minutes in FcX block reagent at 4 ºC, each tissue was stained with a cocktail containing a combination of the following antibodies for 45 minutes at 4 ºC or 30 minutes at room temperature. i. Liver Panel: anti-human CD56 (REA196), anti-human CD31 (WM59), anti-human CD4 (OKT4), anti-NHP CD45 (D058-1283), anti-human CD3 (SP34-2), anti-mouse CD11b (M1/70), anti-human CD68 (REA886), anti-human CD11c (S-HCL-3), anti-human CD8 (BW135/80), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. ii. Lung Panel: anti-human CD31 (WM59), anti-human CD326 (MH99), anti-mouse CD11b (M1/70), anti-mouse/human CD49f (GoH3), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iii. Kidney Panel: anti-human CD31 (WM59), anti-mouse CD11b (M1/70), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. iv. Heart Panel: anti-human CD31 (WM59), anti-mouse CD11b (M1/70), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. v. Spleen Panel: anti-human CD56 (REA196), anti-human CD4 (OKT4), anti-human CD3 (SP34-2), anti-mouse CD11b (M1/70), anti-human CD20 (2H7), anti-human CD11c (S- HCL-3), anti-human CD8 (BW135/80), anti-human CD31 (WM59), anti-mouse CD11b (M1/70), anti-human CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. vi. Bone Marrow Panel: anti-human CD123 (7G3), and-human CD117 (104D2), anti- human CD4 (OKT4), anti-mouse CD11b (M1/70), anti-human CD34 (561 and 563), anti- human CD45RA (5H9), anti-human CD90 (REA897), anti-human CD8 (BW135/80), anti- human CD45RO (UCHL1), anti-human CD20 (2H7), anti-human CD2 (RPA-2.10), anti- human CD14 (M5E2), anti-human CD16 (3G8), anti-human CD23 (M-L233), anti-human CD21 (B-ly4), anti-human CD56 (REA196), anti-human CD3 (SP34-2), anti-NHP CD45 (D058-1283), Annexin V, Zombie R718 or a similar viability dye, and a monoclonal antibody against the protein produced by the mRNA delivered. [0255] Flow Cytometry Readouts: Different cell populations were analyzed for reporter protein expression relative to an untreated primate. The results were expressed as the percentage of cells that were positive for the reporter protein. [0256] The results of LNP screening in primate for a LNP formulation containing an exemplary diketopiperazine lipid as disclosed herein are shown in FIGS.7A-7B. In These figures, certain LNP formulations were selected from a high-throughput, barcoding based, screen. During high-throughput screening, each LNP formulation was numbered as LNP-1 through LNP-XYZ in database, referred to as the sample number for that LNP formulation. For instance, LNP-156 indicates the 156th LNP formulation for a screen. [0257] FIGS.14A-14B show the flow cytometry results for a LNP formulation containing Compound 18 (e.g., LNP-156) selected from a previous screening (discussed in FIGs.5A- 5B) in primate at a dosage of 0.45 mg/kg, in the liver (FIG.14A) and spleen (FIG.14B). The LNP formulations (Lipomer: Compound 18) for FIGS.14A-14B are characterized below: [0258] FIG.14A shows that LNP delivery was found in most cell populations in the liver of primate, although the delivery in hepatocytes was not as significant as other cell populations. FIG.14B shows that LNP delivery was found in all cell populations in the spleen of primate assessed, and similar delivery levels were found across every population in the spleen of primate assessed.

Claims

What is claimed is: 1. A compound of Formula (I): wherein: Y is H; aryl; heteroaryl; branched or unbranched C1-C20 alkyl, optionally interrupted with one or more N, O, or S atoms, or optionally substituted with one or more aryl or wherein: each t is independently 1,
2,
3, or 4; each X is independently -O-, -S-, or -N(R′′)-; Z is absent, -C(O)-, -C(O)O-, -C(O)N(R′′)-, or -S(O)(O)-; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′, and R′′ each are independently H, OR14, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; Ra and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl or heterocyclic, each R11, R12, and R14 are independently H, branched or unbranched alkyl or alkenyl, and each R13 is independently H, alkyl, or OR14; provided that when X is O or S or when Z is C(O), Q is not -CH(OR14)-, -C(O)O-, or -OC(O)-. 2. The compound of claim 1, having the formula of: 3. The compound of claim 1 or 2, wherein: Y is H; aryl; branched or unbranched C1-C6 alkyl; C1-C6 alkyl interrupted with a N, O, or S atom; C1-C3 alkyl substituted with an ary each R, R′, Ra, and Rb are H; each t is independently 1 or 2; each X is independently -O-, -S-, -NH-, or -N(OH)-; Z is absent, -C(O)-, -C(O)O-, -C(O)NH-, or -S(O)(O)-; each L is independently a C2-C4 alkylene optionally substituted by OH; and R1 and R2 are each independently represented by –(CH2)m-Q-(CH2)nH, wherein Q is absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -N(OH)-, -C(O)NH-, -C(O)N(OH)-,
4. The compound of claim 1, having the formula of: (IIIB), wherein each u is independently 2, 3, or 4.
5. The compound of claim 4, having the formula of:
6. The compound according to any one of claims 1-5, wherein X is O or S.
7. The compound according to any one of claims 1-5, wherein at least one X is N(R′′).
8. The compound according to claim 3, wherein each L is independently a C2-C4 alkylene, or -CH2-CH(OH)-CH2- when the X variable next to the L variable is -O-.
9. The compound according to any one of claims 1-8, wherein Y is .
10. The compound of claim 9, having the formula of:
11. The compound of claim 10, having the formula of: (
12. The compound according to any one of claims 1-8, wherein Y is H.
13. The compound according to any one of claims 1-8, wherein Y is branched or unbranched C1-C4 alkyl, a C1-C3 alkyl interrupted with an S atom, or benzyl.
14. The compound according to any one of claims 1-8, wherein Y is .
15. The compound according to any one of claims 1-14, wherein each R1 and R2 are the same.
16. The compound according to any one of claims 1-14, wherein at least one, two, or three of the R1 and R2 variables are different than the other(s).
17. The compound according to any one of claims 1-14, wherein each R1 and R2 are independently branched or unbranched C1 -C20 alkyl or branched or unbranched C2 -C20 alkenyl.
18. The compound according to claim 17, wherein each R1 and R2 are independently C9- C16 alkyl or C9-C16 alkenyl.
19. The compound according to claim 17, wherein each R1 and R2 are independently C9- C12 alkyl or C9-C12 alkenyl.
20. The compound according to any one of claims 1-14, wherein Q in each R1 and R2 variable is independently -CH=CH-, -C≡C-, or -S-S-.
21. The compound according to any one of claims 1-14, wherein Q in each R1 and R2 variable is independently -C(O)O- or -OC(O)-.
22. The compound according to any one of claims 1-14, wherein Q in each R1 and R2 variable is independently -C(O)NH-, -C(O)N(OH)-, -N(OH)C(O)-, or-NHC(O)-.
23. The compound according to any one of claims 1-14, wherein one of R1 and R2 is C1 - C16 branched or unbranched alkyl; and in the other R1 or R2, Q is -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)NH-, -C(O)N(OH)-, -N(OH)C(O)-, or -NHC(O)-.
24. A method of making a compound of formula
reacting with one or more deprotecting reagents to remove the carboxyl protecting group and amine protecting group, and react the resulting intermediate under conditions sufficient to couple and cyclize the intermediate to form the compound of formula (IIA), wherein: each X is independently -O-, -S-, or -N(R′′)-; each t is independently 1, 2, 3, or 4; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′′, Ra, and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14 are independently H, branched or unbranched alkyl or alkenyl.
25. The method of claim 24, wherein the reactant prepared .
26. The method of claim 25, wherein the preparation further employs one or more coupling reagents comprising EDCI, DMAP, HATU, and/or Hunig’s base.
27. The method of claim 24, 25, or 26, wherein the reactant is , wherein X is -O-, -S-, or -NH-.
28. The method of claim 27, wherein: the deprotecting agent is an acid, and the conditions comprises adding acetonitrile and triethylamine.
29. A method of making a compound of formula comprising: reacting the presence of an organic base and a solvent to form the compound, wherein: each t is independently 1, 2, 3, or 4; each L is independently a C1-C6 alkylene optionally substituted by OH; R, R′, Ra, and Rb each are independently H, branched or unbranched C1-C6 alkyl, or C3-C7 cycloalkyl; and R1 and R2 each are independently branched or unbranched, saturated or unsaturated C1 -C20 monovalent hydrocarbon chain, or represented by –(C(R11)(R12))m-Q-(C(R11)(R12))nH, wherein: each m is independently 0 to 10, each n is independently 1 to 10, each Q is independently absent, -CH=CH-, -C≡C-, -S-S-, -C(O)O-, -OC(O)-, -C(O)N(R13)-, -N(R13)C(O)-, -C(O)S-, -SC(O)-, -CH(OR14)-, -CH(R11)-, a divalent cycloalkyl, or heterocyclic, each R11, R12, R13, and R14 are independently H, branched or unbranched alkyl or alkenyl.
30. The method of claim 29, wherein the organic base is DIPEA, and the solvent is DMF or its mixture with DCM.
31. The method of claim 29, wherein:
c the reaction reagents further comprises a benzotriazole or its derivative.
32. The method of claim 31, wherein the benzotriazole or its derivative is HOBt, HATU, or a mixture thereof.
33. The method of claim 32, wherein the reagents further comprise EDCI.
34. The method of claim 29, wherein the benzotriazole or its derivative is PyBOP.
35. A lipid nanoparticle comprising the compound according to any one of claims 1-23.
36. The lipid nanoparticle according to claim 35, further comprising a helper lipid.
37. The lipid nanoparticle according to claim 36, wherein the helper lipid is a phospholipid.
38. The lipid nanoparticle according to claim 36, wherein the helper lipid is 1,2-dioeoyl- sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), didodecyldimethylammonium bromide (DDAB), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2- dioleoyl-3-trimethylammonium propane (DOTAP), 18:1 PA, N-methyldioctadecylamine (MDOA), N,N-dicotadecylaniline, sn-(3-myristoyl-2-hydroxy)-glycerol-1-phospho-sn-3'- (1',2'-dimyristoyl)-glycerol (14:0 Hemi BMP), sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho- sn-3′-(1′,2′-dioleoyl)-glycerol (18:1 Hemi BMP), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA; 14:0 PA), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA; 18:1 PA), 1,2-distearoyl-sn- glycero-3-phosphate (DSPA; 18:0 PA), or a combination thereof.
39. The lipid nanoparticle according to claim 38, wherein the helper lipid is 1,2-dioeoyl- sn-glycero-3-phosphoethanolamine (DOPE), dimethyldioctadecylammonium (DDAB), 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), or a combination thereof.
40. The lipid nanoparticle according to any one of claims 35-39, further comprising a sterol.
41. The lipid nanoparticle according to claim 40, wherein the sterol is cholesterol, a cholesterol derivative, or a combination thereof.
42. The lipid nanoparticle according to any one of claims 35-41, further comprising a PEG-modified lipid.
43. The lipid nanoparticle according to claim 42, wherein the PEG-modified lipid is PEG- DMG or PEG-PE.
44. The lipid nanoparticle according to claim 42, wherein the PEG-modified lipid is 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000 (DMPE-PEG2K; 14:0 PEG2K PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethyleneglycol)-2000 (DSPE-PEG2K; 18:0 PEG2K PE), 1,2-dimyristoyl-sn- glycero-3-methoxypolyethylene glycol (DMG-PEG 2K), 2-[(polyethylene glycol)-2000]- N,N-ditetradecylacetamide (ALC-0159), distearoyl-rac-glycerol-PEG2K (DSG-PEG 2K), or a combination thereof.
45. The lipid nanoparticle according to claim 35, further comprising a helper lipid, a cholesterol, and a PEG-modified lipid, wherein the molar ratio of the compound : the helper lipid : the cholesterol : the PEG lipid is about 20-60 : 7-50 : 5-70 : 0.5-3, about 32-50 : 12-18 : 32-48 : 1-3, or about 35-50 : 12-16 : 35-47 : 2-3.
46. The lipid nanoparticle according to claim 45, wherein the molar ratio of the compound: the helper lipid : the cholesterol : the PEG lipid is about 25 : 33 : 40 : 2, about 30 : 22.5 : 45 : 2.5, about 30:39:30:1, about 30 : 50 : 18 : 2, about 35 : 35 : 27.5 : 2.5, about 35 : 43 : 20 : 2, about 35:44.5:18:2.5, about 35:16:46.5:2.5, about 45:13:39.5:2.5, about 50 : 40 : 7.5 : 2.5, about 50 : 17.5 : 30 : 2.5, about 50 : 12.5 : 35 : 2.5, about 50 : 10 : 38.5 : 1.5, or about 35:15:47.5:2.5.
47. The lipid nanoparticle according to claim 35, further comprising a helper lipid and a PEG-modified lipid, wherein the molar ratio of the compound: the helper lipid: the PEG lipid is about 20-60 : 10-60 : 0.5-3, about 30-55 : 30-55 : 1-3, or about 32-50 : 40-55 : 1.5-2.5.
48. The lipid nanoparticle according to any one of claims 35-47, having a diameter of about 20 to about 200 nm.
49. The lipid nanoparticle according to any one of claims 35-48, further comprising a therapeutic agent.
50. The lipid nanoparticle according to claim 49, wherein the therapeutic agent is a nucleic acid molecule, protein or peptide, carbohydrate or glycoprotein, small molecule, or any combination thereof.
51. The lipid nanoparticle according to claim 49, wherein the therapeutic agent is DNA or RNA.
52. The lipid nanoparticle according to claim 51, wherein the therapeutic agent is an mRNA.
53. The lipid nanoparticle according to any one of claims 35-52, wherein the lipid nanoparticle is suitable for delivery of a therapeutic agent to the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of a subject.
54. The lipid nanoparticle according to claim 53, wherein the lipid nanoparticle is suitable for delivery of a therapeutic agent to lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung macrophages, lung dendritic cells, lung B cells, lung T cells, lung natural killer (NK) cells, or other immune cells within the lung.
55. The lipid nanoparticle according to any one of claims 35-52, wherein the lipid nanoparticle is suitable for delivery of a therapeutic agent to stem-like cells and/or hematopoietic stem cells (HSCs).
56. A pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 35-55 and a pharmaceutically acceptable carrier.
57. A method of delivering a therapeutic agent to a subject, comprising administering to the subject the lipid nanoparticle according to any one of claims 35-55.
58. The method according to claim 57, wherein the therapeutic agent is delivered to the lung, heart, kidney, liver, splenic, lymphatic cells, or marrow cells of the subject.
59. The method according to claim 57, wherein the therapeutic agent is delivered to one or more of lung endothelial cells, lung basal cells, lung stem cells, lung epithelial cells, lung dendritic cells, lung B cells, lung T cells, lung macrophages, lung natural killer (NK) cells, or other immune cells within the lung; heart endothelial cells, heart macrophages, heart epithelial cells, heart dendritic cells, heart B cells, heart T cells, heart natural killer (NK) cells, or other immune cells within the heart; kidney endothelial cells, kidney epithelial cells, kidney dendritic cells, kidney B cells, kidney T cells, kidney macrophages, kidney natural killer (NK) cells, or other immune cells within the kidney; liver endothelial cells, hepatocytes, liver macrophages, liver dendritic cells, liver Kupffer cells, liver B cells, liver T cells, or other immune cells within the liver; spleen dendritic cells, spleen neutrophils, spleen macrophages, spleen B cells, spleen T cells, spleen natural killer (NK) cells, or other immune cells within the spleen; lymphatic dendritic cells, lymphatic neutrophils, lymphatic macrophages, lymphatic B cells, lymphatic T cells, or lymphatic natural killer (NK) cells of the subject.
60. The method according to claim 57, wherein the therapeutic agent is delivered to bone marrow stem-like cells, and/or bone marrow hematopoietic stem cells (HSCs).
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