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WO2025228979A1 - Lipids nanoparticle formulations suitable for lung targeting - Google Patents

Lipids nanoparticle formulations suitable for lung targeting

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Publication number
WO2025228979A1
WO2025228979A1 PCT/EP2025/061706 EP2025061706W WO2025228979A1 WO 2025228979 A1 WO2025228979 A1 WO 2025228979A1 EP 2025061706 W EP2025061706 W EP 2025061706W WO 2025228979 A1 WO2025228979 A1 WO 2025228979A1
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Prior art keywords
mol
lipid
formulation
alkyl
group
Prior art date
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Pending
Application number
PCT/EP2025/061706
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French (fr)
Inventor
Ramishetti Srinivas
Eran Eilat
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Neovac Ltd
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Neovac Ltd
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Publication of WO2025228979A1 publication Critical patent/WO2025228979A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • 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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • 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

  • the present invention provides lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications.
  • the present formulation is specifically directed to compositions comprising ionizable lipids and a lipid, which comprises a quaternary ammonium group as a permanently charged lipid.
  • nucleic acids including small interfering RNA (siRNA), micro RNA (miRNA), antisense oligonucleotides, messenger RNA (mRNA), ribozymes, pDNA and immune stimulating nucleic acids act via a variety of mechanisms. Specific proteins can be downregulated by siRNA or miRNA through RNA interference (RNAi). Hematopoietic cells, such as leukocytes in general, and primary T lymphocytes and B- cells in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs).
  • RNA interference RNA interference
  • siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against a target protein.
  • siRNA constructs have shown the ability to specially silence target proteins in both in vitro and in vivo models. These are currently being evaluated in clinical studies.
  • Messenger RNA mRNA is the family of large RNA molecules which transport the genetic information from DNA to ribosome. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products.
  • nucleic acids would be useful in the treatment to the of diseases related deficiency of a protein or enzyme.
  • problems associated with nucleic acids are the stability of the phosphodiester inter nucleotide link and its susceptibility to nucleases. Apart from that these nucleic acids have limited ability to cross the cell membrane.
  • Various lipids, e.g., cationic lipids have proved to be excellent carriers of nucleic acids to treat different diseases in gene therapy applications.
  • Dedmi et al. (Biomaterials 31 (2001, 6867-6875) report the systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation.
  • Ramesh (Methods Mol Biol. 2008;433:301-31) discloses a DOTAP:cholesterol-based nanoparticle - mediated gene delivery to the lung.
  • WO 2020/051223 discloses a composition comprising: (A) a therapeutic agent; and (B) a lipid nanoparticle composition comprising: (1) a selective organ targeting compound; (2) an ionizable cationic lipid; and (3) a phospholipid; wherein the composition preferentially delivers the nucleic acid to a target organ selected from the lungs, the heart, the brain, the spleen, the lymph nodes, the bones, the skeletal muscles, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, or the skin.
  • WO 2020051220 discloses a composition comprising: (A) a therapeutic agent; and (B) a lipid nanoparticle composition comprising: (1) a selective organ targeting compound: (2) an ionizable cationic lipid; and (3) a phospholipid; wherein the composition preferentially delivers the nucleic acid to a target organ selected from the lungs, the heart, the brain, the spleen, the lymph nodes, the bone marrow, the bones, the skeletal muscles, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the liver, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, or the skin
  • WO 2022/204043 discloses a method for potent delivery to a non-liver basal cell of a subject, comprising: intravenously administering to said subject a composition comprising a
  • WO 2022/216619 discloses a method for enhancing an expression or activity of cystic fibrosis transmembrane conductance regulator (CFTR) protein in a cell, the method comprising: (a) contacting said cell with a nucleic acid editing system assembled with a lipid composition, which nucleic acid editing system comprises (i) a guide nucleic acid, (ii) a heterologous polypeptide comprising an endonuclease or a heterologous polynucleotide encoding said heterologous polypeptide, and (iii) a donor template nucleic acid, to yield a complex of said heterologous endonuclease with said guide nucleic acid in said cell; (b) cleaving a CFTR gene or transcript in said cell with said complex at a cleavage site to yield a cleaved CFTR gene or transcript; and (c) using said donor template nucleic acid to repair said cleaved CFTR gene or transcript to yield
  • WO 2022/201167 discloses lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications.
  • Sun et al. (AAPS PharmSciTech. 2022 May 9;23(5):135) describe optimization of 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP)/cholesterol cationic lipid nanoparticles for mRNA, pDNA, and oligonucleotide delivery. Nevertheless, there remains a need in the art for suitable and efficient delivery platforms for targeted delivery of nucleic acid and therapeutic agents to specifically the lungs.
  • DOTAP 1,2-dioleoyl-3- trimethylammonium-propane
  • the present invention relates to novel lipid nanoparticle (LNP) formulations.
  • LNP lipid nanoparticle
  • These lipid nanoparticles protect nucleic acids from degradation, clearance from circulation and intracellular release.
  • the nucleic acid encapsulated lipid nanoparticles advantageously are well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient.
  • the present LNP formulations were surprisingly found to specifically target the lung with significantly higher lung expression compared to other organs, including the heart, liver, spleen and kidney.
  • the results provided herein indicate that given similar systemic exposure, the formulations in accordance with the principles of the invention is unexpectedly more effective in delivering biological agents specifically to the lungs than hitherto known formulations.
  • the high specificity of the present lipid nanoparticle formulations in lung targeting is highly advantageous, as it reduces the side effects that accompany the delivery of active agents to undesired sites and organs.
  • this high specificity also enables use of lower dosages, as less active agent is lost, which, in its turn, also contributes to side effect reduction.
  • the present lipid nanoparticle formulations comprise at least one ionizable lipid which is represented by the structure of Formula (IA), Formula (IB) and/or Formula (II) and at least one permanently charged lipid.
  • the permanently charged lipid includes a quaternary ammonium moiety.
  • the quaternary ammonium moiety comprises a tetra-alkyl ammonium group.
  • the permanently charged lipid is selected from the group consisting of:1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DORI), 1,2-dimyristoyl- 3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), Dimethyldioctadecylammonium (DDAB) salts and combinations thereof.
  • DOTAP 1,2- dioleoyl-3-trimethylammonium-propane
  • the permanently charged lipid is DOTAP.
  • DOTAP is used in the formulations described herein as a permanently charged lipid. Surprisingly, in contrast with previous neutral lipids, permanently charged lipids were found to exhibit remarkable properties that enable the present formulations to target the lungs.
  • 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride salt is represented by the chemical structure drawn below: As specified herein, DOTAP is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof.
  • DOTAP and “DOTAP salt” as used herein are intended to include any DOTAP salts, including any counter anion.
  • Suitable counter anions for DOTAP include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H 2 PO 4 -, etc.) and the like.
  • 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), chloride salt is represented by the chemical structure drawn below: DOTMA-chloride
  • DOTMA is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof.
  • DOTMA and “DOTMA salt” as used herein are intended to include any DOTMA salts, including any counter anion.
  • Suitable counter anions for DOTMA include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like.
  • DORI-bromide N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DORI), bromide salt is represented by the chemical structure drawn below: DORI-bromide As specified herein, DORI is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof. The terms “DORI” and “DORI salt” as used herein are intended to include any DORI salts, including any counter anion.
  • Suitable counter anions for DORI include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like.
  • 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane chloride salt is represented by the chemical structure drawn below: DMTAP -chloride
  • DMTAP is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof.
  • DMTAP and “DMTAP salt” as used herein are intended to include any DMTAP salts, including any counter anion.
  • Suitable counter anions for DMTAP include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H 2 PO 4 -, etc.) and the like.
  • DSTAP chloride salt 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) chloride salt is represented by the chemical structure drawn below: DSTAP-chloride
  • DSTAP is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof.
  • DSTAP and DSTAP salt as used herein are intended to include any DSTAP salts, including any counter anion.
  • Suitable counter anions for DSTAP include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H 2 PO 4 -, etc.) and the like.
  • Dimethyldioctadecylammonium (DDAB) bromide salt is represented by the chemical structure drawn below: DDAB As specified herein, DDAB is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof.
  • DDAB dimethyldioctadecylammonium salt
  • DDAB salt any dimethyldioctadecylammonium salt, including any counter anion.
  • Suitable counter anions for dimethyldioctadecylammonium include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like.
  • halides e.g., chloride, bromide, etc.
  • sulfate derivatives e.g., triflate, etc.
  • borate derivatives e.g., tetrafluoroborate, etc.
  • phosphate derivatives e.g. H2PO4-, etc.
  • the present invention provides a lipid nanoparticle formulation comprising: at least one ionizable lipid which is represented by the structure of Formula (IA), Formula (IB) or Formula (II), or salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof, wherein the structures of Formula (IA), Formula (IB) and Formula (II) are represented below; and at least one permanently charged lipid having a quaternary ammonium moiety or a salt thereof; Formula (II): , wherein R 1C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alken
  • R 1A is selected from the group consisting of: OH, -(CH 2 CH 2 O) 2-6 H, C 1-6 hydroxyalkyl and C 1-6 haloalkyl and C 1-3 alkylene-NR NA R NA’ , wherein each one of R NA and R NA’ is individually C 1-4 alkyl or R NA and R NA’ together with the nitrogen to which they are bound, form a ring;
  • R 2A is selected from the group consisting of: C 5 - 25 alkyl, C 5 - 25 alkenyl, C 5 - 15 alkylene-CO 2 -C 5 - 15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-
  • the lipid nanoparticle formulation comprises one ionizable lipid represented be Formula (IA), Formula (IB) or Formula (II). According to some embodiments, the lipid nanoparticle formulation comprises a plurality of ionizable lipids represented be Formula (IA), Formula (IB) and/or Formula (II).
  • the term “at least one” means a single element or object or a plurality of elements and/or objects.
  • the term “plurality” means two or more elements or objects. It is to be understood that “one ionizable lipid” relates to a single species of and ionizable lipid, which may include a plurality of molecules of the same kind.
  • the ionizable lipid is represented by the structure of Formula (IA). It is to be understood that specifying that the lipid nanoparticle formulation comprises one ionizable lipid represented be Formula (IA), Formula (IB) or Formula (II) and that the ionizable lipid is represented by the structure of Formula (IA), broadly cover both the option that the lipid nanoparticle formulation comprises only ionizable lipid(s) of Formula (IA) (i.e., not of Formulae (IB) and/or (II)), and the option that the lipid nanoparticle formulation comprises ionizable lipids of Formulae (IB)/(II), as long as the lipid nanoparticle formulation comprises at least one ionizable lipid of Formula (IA).
  • this embodiment comprises lipid nanoparticle formulations that comprise ionizable lipid(s) that are not covered in any of Formulae (IA)/(IB)/(II), as long as the lipid nanoparticle formulation comprises at least one ionizable lipid of Formula (IA).
  • Similar embodiments which state that the ionizable lipid is represented by the structure of Formula (IB) or (II) have similar breadth.
  • the ionizable lipid is represented by the structure of Formula (IA) or Formula (IB).
  • the ionizable lipid is represented by the structure of Formula (IA) or Formula (II).
  • the ionizable lipid is represented by the structure of Formula (IB) or Formula (II).
  • R 1A is selected from the group consisting of: -CH2CH2OH, -CH 2 CH 2 OCH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 OH and OH. Each possibility represents a separate embodiment of the invention.
  • R 2A is selected from the group consisting of: C 6 - 18 alkyl and C 12 - 24 alkenyl. Each possibility represents a separate embodiment of the invention.
  • n A is selected from the group consisting of: 8, 9 and 10.
  • n A is selected from the group consisting of: 8, 9 and 10, and j A is 0 or 2, or a combination thereof.
  • X A is selected from the group consisting of: -COO-, -OOC-, -NHCO- and -CONH. According to some embodiments, X A is selected from the group consisting of: -COO- and -OOC-. According to some embodiments, X A is -COO- and the lipid is represented by Formula (IA 1 ) . According to some embodiments, j A is 0 or 2. Each possibility represents a separate embodiment of the invention.
  • Y A is selected from the group consisting of: absent, -COO-, -OOC-, - NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-.
  • Y A is selected from the group consisting of: absent, -COO- and -OOC-.
  • Y A is absent or -OOC-.
  • Y A is absent, j A is 0 and the lipid of Formula (IA) is represented by Formula (IA 2 ): .
  • Y A is absent, j A is 0, X A is -COO- and the lipid of Formula (IA) is represented by Formula (IA 3 ): .
  • m A is selected from the group consisting of: 8, 9, and 10. Each possibility represents a separate embodiment of the invention.
  • R 3A is selected from the group consisting of: -CH 2 CH 2 OH, -CH 2 CH 2 OCH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 OH and OH. Each possibility represents a separate embodiment of the invention.
  • the ionizable lipid is selected from the group consisting of: lipid IA-4, lipid IA-5, lipid IA-8, lipid IA-10, lipid IA-11, lipid IA-12, lipid IA-13, lipid IA-14, and lipid IA-15 and a combination thereof. Each possibility represents a separate embodiment of the invention.
  • the ionizable lipid is IA-10 or IA-12.
  • the ionizable lipid is IA-10.
  • the ionizable lipid is IA-12.
  • the ionizable lipid is represented by the structure of Formula (IB).
  • R 1B is selected from the group consisting of: -CH2CH2OCH2CH2OH, -CH2CH2Cl, -CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2OH and -CH2CH2NMe2.
  • R 1B is selected from the group consisting of: CH 2 CH 2 OCH 2 CH 2 OH, -CH 2 CH 2 Cl or -CH 2 CH 2 OH.
  • R 2B is selected from the group consisting of: C 6 - 18 alkyl and C 12 - 24 alkenyl. Each possibility represents a separate embodiment of the invention.
  • W B is a C5-9 unsubstituted alkylene.
  • W B selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH 2 ) 5 -. Each possibility represents a separate embodiment of the invention.
  • Y B is selected from the groups consisting of: absent, -CH 2 CH 2 OCH 2 CH 2 - and -CH 2 CH 2 -. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y B is absent and the lipid of Formula (IB) is represented by Formula (IB 1 ): . According to some embodiments, R 3B is selected from the group consisting of: C 6 - 18 alkyl and C 12 - 24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R 3B is a C 4-16 alkyl. According to some embodiments, R 4B is selected from the group consisting of: C 6 - 18 alkyl and C 12 - 24 alkenyl.
  • R 4B is a C4-16 alkyl.
  • the ionizable lipid is selected from the group consisting of: lipid IB-4, lipid IB-6, lipid IB-9, lipid IB-10, lipid IB-13, lipid IB-24 and a combination thereof.
  • the ionizable lipid is selected from the group consisting of: IB-4, IB-6, IB-10 and a combination thereof.
  • the ionizable lipid is IB-4.
  • the ionizable lipid is represented by the structure of Formula (II).
  • R 1C is selected from the group consisting of: C 4 - 18 alkyl and C 12 - 24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R 1C is a C 4-10 alkyl. According to some embodiments, R 1C is C 6 H 13 . According to some embodiments, R 1C is n- C6H13. According to some embodiments, R 2C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R 2C is a C4-10 alkyl. According to some embodiments, R 2C is C6H13.
  • R 2C is n- C6H13.
  • Y 1C is selected from the groups consisting of: absent, -CH 2 CH 2 OCH 2 CH 2 - and -CH 2 CH 2 -. Each possibility represents a separate embodiment of the invention.
  • each one of Y 1C and Y 2C is absent and the lipid of Formula (II) is represented by Formula (II 1 ): .
  • W 1C is a C 5-9 unsubstituted alkylene.
  • W 1C is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-.
  • each one of W 1C and W 2C is a C4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II 2 ): .
  • each one of Y 1C and Y 2C is absent, each one of W 1C and W 2C is a C 4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II 3 ): .
  • R 5C is selected from the group consisting of: OH, -(CH2CH2O)2-3H, C1-4 hydroxyalkyl and C1-4 haloalkyl and C1-3 alkylene-NR NII R NII’ , wherein each one of R NII and R NII’ is individually C 1-4 alkyl or R NII and R NII’ together with the nitrogen to which they are bound, form a 5-6 membered ring.
  • R NII and R NII’ is individually C 1-4 alkyl or R NII and R NII’ together with the nitrogen to which they are bound, form a 5-6 membered ring.
  • R 5C is selected from the group consisting of: -CH 2 CH 2 OH, -CH 2 CH 2 OCH 2 CH 2 OH, - CH 2 CH 2 CH 2 CH 2 OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2N(CH2)5, -CH2CH2N(CH2)5, -CH2CH2Cl, -OH and -CH2CH2NMe2.
  • R 5C is -CH2CH2OH, or -CH2CH2OCH2CH2OH.
  • W 2C is a C5-9 unsubstituted alkylene.
  • W 2C is selected from the group consisting of: -(CH 2 ) 9 -, -(CHMe)-(CH 2 ) 4 - and -(CH 2 ) 5 -.
  • Y 2C is selected from the groups consisting of: absent, -CH 2 CH 2 OCH 2 CH 2 - and -CH 2 CH 2 -.
  • R 3C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl.
  • R 3C is a C4-10 alkyl.
  • R 3C is C6H13.
  • R 4C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention.
  • R 4C is a C 4-10 alkyl.
  • R 4C is C 6 H 13 .
  • the ionizable lipid is lipid II-1 or II-5.
  • the ionizable lipid is lipid II-.
  • the ionizable lipid is lipid II-5.
  • the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB-22, IB-23, IB-24, II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II- 15, II-16, II-17, II-18, II-19, II-20, II-21, IB-22, IB-23, IB-24, II-1, II
  • the permanently charged lipid is selected from the group consisting of:1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DORI), 1,2-dimyristoyl- 3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), dimethyldioctadecylammonium (DDAB), salts and combinations thereof.
  • DOTAP 1,2- dioleoyl-3-trimethylammonium-propane
  • DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane
  • DORI 1,2-di-O-octadecenyl-3-tri
  • the permanently charged lipid is DOTAP or DOTMA. According to some embodiments, the permanently charged lipid is DOTAP. According to some embodiments, the permanently charged lipid is DOTMA. According to some embodiments, the lipid nanoparticle formulation comprises 10 to 50 mol% of the ionizable lipid, including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 40 mol% of the ionizable lipid. According to some embodiments, the lipid nanoparticle formulation comprises 15 to 30 mol% of the ionizable lipid.
  • the lipid nanoparticle formulation comprises 10 to 40 mol% permanently charged lipid(s), including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation comprises 15 to 35 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises about 20 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation further comprises at least one neutral lipid. According to some embodiments, the neutral lipid comprises a sterol, a phospholipid or both. According to some embodiments, the sterol comprises cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 40 mol% cholesterol, including each value and sub–range within the specified range.
  • the lipid nanoparticle formulation comprises 30 to 40 mol% cholesterol.
  • the phospholipid comprises a phosphatidylethanolamine.
  • the phosphatidylethanolamine is selected from the group consisting of: 1,2-dilauroyl- L-phosphatidyl-ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) 1,3-Dipalmitoyl-sn-glycero-2- phosphoethanolamine (1,3-DPPE), 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), biotin-phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Dipalmitoylphosphatidy
  • DLPE 1,2-dilauroyl-
  • the phosphatidylethanolamine comprises 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
  • the phospholipid comprises 1,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), Distearoylphosphatidylcholine (DSPC) or both.
  • DOPE 1,2-Dioleoyl-sn-glycero-3- phosphoethanolamine
  • DSPC Distearoylphosphatidylcholine
  • the phospholipid comprises DOPE.
  • the phospholipid comprises DSPC.
  • the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE.
  • the lipid nanoparticle formulation comprises 5 to 15 mol% DSPC.
  • the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE, DSPC or both. It is to be understood that the phrase “the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE, DSPC or both” is intended to mean that the lipid nanoparticle formulation comprises one of the following: 5 to 15 mol% DOPE; 5 to 15 mol% DSPC or 5 to 15 mol% of DOPE and DSPC combined. According to some embodiments, the lipid nanoparticle formulation further comprises a PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises 1 to 5 mol% DMG-PEG 2000.
  • the lipid nanoparticle formulation is selected from the group consisting of Formulation 1 to 5 and 11-26, wherein the compositions of Formulations 1 to 5 and 11-26 are described below:
  • Formulation 1 lipid IA-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%;
  • Formulation 2 lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%;
  • Formulation 3 Formula
  • the lipid nanoparticle formulation is Formulation 15 or Formulation 23. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation is Formulation 1 or Formulation 2. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation has average nanoparticle size (Z average) in the range of 50 to 200 nanometers, including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation has average nanoparticle size (Z average ) in the range of 50 to 150 nanometers. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 25 mV.
  • the lipid nanoparticle formulation has Zeta potential in the range of 28 to 40 mV, including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation has polydispersity index (PDI) of no more than 0.25. According to some embodiments, the lipid nanoparticle formulation is devoid of a lung targeting moiety. According to some embodiments, the lipid nanoparticle formulation further comprises a nucleic acid encapsulated within at least one particle thereof.
  • PDI polydispersity index
  • the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
  • siRNA small interfering RNA
  • miRNA micro RNA
  • mRNA messenger RNA
  • ribozymes pDNA
  • CRISPR mRNA CRISPR mRNA
  • gRNA circular RNA
  • immune stimulating nucleic acids a separate embodiment of the invention.
  • the lipid nanoparticle formulation further comprises a therapeutic agent encapsulated within at least one particle thereof.
  • the lipid nanoparticle formulation further comprises a pharmaceutically acceptable carrier, diluent or excipient.
  • the lipid nanoparticle formulation is liposomal composition.
  • the lipid nanoparticle formulation is a liver bypass formulation. According to some embodiments, the lipid nanoparticle formulation is formulated for intravenous (IV) administration. According to some embodiments, the lipid nanoparticle formulation is selectively targeting the lung upon systemic administration. According to some embodiments, the lipid nanoparticle formulation is selectively targeting the lung upon intravenous (IV) administration. According to some embodiments, the lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration in mammals. According to some embodiments, the lipid nanoparticle formulation is for use in the treatment of a lung and/or respiratory disease or disorder.
  • the lipid nanoparticle formulation is use in the treatment of a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection.
  • the lipid nanoparticle formulation is for use in the treatment of a hereditary lung disorder or a genetic lung disease.
  • a method of treating lung and/or respiratory disease or disorder comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the method is for treating a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection.
  • the method is for the treatment of a hereditary lung disorder or a genetic lung disease.
  • the method comprises a step of systemically administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • the method comprises a step of step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • a method of delivery of a therapeutic agent or a nucleic acid to the lung comprising the step of systemically administering to a subject in need thereof the lipid nanoparticle formulation disclosed herein and a pharmaceutically acceptable carrier, diluent or excipient.
  • the method comprises the step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • a lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB-24.
  • the lipid is lipid IA-4.
  • the lipid is lipid IA-13.
  • the lipid is lipid IA-14.
  • the lipid is lipid IA-15.
  • the lipid is lipid IB-4.
  • the lipid is lipid IB-9.
  • the lipid is lipid IB-24.
  • a lipid nanoparticle formulation comprising the lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB-24.
  • lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB-24 each possibility represents a separate embodiment of the invention.
  • a pharmaceutical composition comprising at least one lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB- 24 or the lipid nanoparticle formulation comprising one or more of said lipid.
  • a pharmaceutical composition comprising at least one lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB- 24.
  • a pharmaceutical composition comprising a lipid nanoparticle formulation comprising at least one lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB-24 .
  • Figure 1A is a 1 H NMR spectrum of lipid IA-12 (NV3-006).
  • Figure 1B is an ESI-MS spectrum of lipid IA-12 (NV3-006).
  • Figure 1C is a 1 H NMR spectrum of lipid IA-11 (NV3-004).
  • Figure 1D is an ESI-MS spectrum of lipid IA-11 (NV3-004).
  • Figure 1E is a 1 H NMR spectrum of lipid IA-10 (NV3-002).
  • Figure 1F is an ESI-MS spectrum of lipid IA-10 (NV3-002).
  • Figures 2A-E are IVIS imaging analyses for Luciferase expression for Formulation 1 (Figure 2A), Formulation 2 ( Figure 2B), Formulation 3 ( Figure 2C), Formulation 4 ( Figure 2D) and Formulation 5 ( Figure 2E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figures 3A-E are IVIS imaging analyses for Luciferase expression for Formulation 6 (Figure 3A), Formulation 7 (Figure 3B), Formulation 8 ( Figure 3C), Formulation 9 ( Figure 3D) and Formulation 10 ( Figure 3E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • the lung is not targeted and the white appearance of the lungs does not indicate fluorescence, but rather is a result of the B&W limitation.
  • Figure 4A is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 5 (left group), Formulation 1 (second from left group), Formulation 2 (middle group), Formulation 3 (second from right group) and Formulation 4 (right group).
  • Figure 4B is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 10 (left group), Formulation 6 (second from left group), Formulation 7 (middle group), Formulation 8 (second from right group) and Formulation 9 (right group).
  • Figure 5 is a histogram analysis of Luciferase expression comparing between Lung (circles), liver (squares) and spleen (triangles) for Formulation 5 (left group), Formulation 2 (second from left group), Formulation 4 (middle group), Formulation 3 (second from right group) and Formulation 1 (right group).
  • Figures 6A-F are IVIS imaging analysis for Luciferase expression of Formulation 11 ( Figure 6A), Formulation 22 ( Figure 6B), Formulation 18 ( Figure 6C), Formulation 19 ( Figure 6D), Formulation 20 ( Figure 6E) and Formulation 21 ( Figure 6F) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 7 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group).
  • Figure 8 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group).
  • Figure 9 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group).
  • Figures 10A-E are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 10A), Formulation 17 (Figure 10B), Formulation 18 (Figure 10C), Formulation 26 ( Figure 10D), and Formulation 25 ( Figure 10E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 11 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group), and Formulation 25 (right group).
  • Figure 12 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group).
  • Figure 13 is bar chart showing luciferase expression in the lungs were compared between DOTAP/DOTMA formulations; Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group).
  • Figures 14A-C are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 14A), Formulation 16 ( Figure 14B), and Formulation 24 ( Figure 10C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 15 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 11 (left group), Formulation 16 (middle group) and Formulation 24 (right group).
  • Figure 16 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group).
  • Figure 17 is bar chart showing luciferase expression in the lungs were compared between DSPC vs DOPE formulations; Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group).
  • Figures 18A-E are IVIS imaging analysis for Luciferase expression of Formulation 12 (Figure 18A), Formulation 15 (Figure 18B), Formulation 11 (Figure 18C), Formulation 13 ( Figure 18D), and Formulation 14 ( Figure 18C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 19 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 12 (left group), Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group).
  • Figure 20 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group).
  • Figure 21 is bar chart showing luciferase expression in the lungs were compared between different formulations with increasing DOTAP amount; Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group).
  • Figures 22A-B are IVIS imaging analysis for Luciferase expression of Formulation 15 (Figure 22A), and Formulation 23 ( Figure 22B) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 23 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 15 (left group), and Formulation 23 (right group).
  • Figure 24 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (left group), and Formulation 23 (right group).
  • Figure 25 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 15 (left group), and Formulation 23 (right group).
  • Figure 26A is a 1 H NMR spectrum of lipid IB-4 (NV2-004).
  • Figure 26B is an ESI-MS spectrum of lipid IB-4 (NV2-004).
  • Figure 27A is a 1 H NMR spectrum of lipid IB-13 (NV2-015).
  • Figure 27B is an ESI-MS spectrum of lipid IB-13 (NV2-015).
  • Figure 28A is a 1 H NMR spectrum of lipid IA-8 (NV3-009).
  • Figure 28B is an ESI-MS spectrum of lipid IA-8 (NV3-009).
  • Figure 29A is a 1 H NMR spectrum of lipid IB-24 (NV2-027).
  • Figure 29B is an ESI-MS spectrum of lipid IB-24 (NV2-027).
  • Figure 30A is a 1 H NMR spectrum of lipid IA-13 (NV3-013).
  • Figure 30B is an ESI-MS spectrum of lipid IA-13 (NV3-013).
  • Figure 31A is a 1 H NMR spectrum of lipid IA-14 (NV3-014).
  • Figure 31B is an ESI-MS spectrum of lipid IA-14 (NV3-014).
  • Figure 32A is a 1 H NMR spectrum of lipid IA-15 (NV3-016).
  • Figure 32B is an ESI-MS spectrum of lipid IA-15 (NV3-016).
  • DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention is based on the discovery of lipid nanoparticle compositions useful in selectively delivering active agents to the lungs.
  • the lipid nanoparticle compositions of the present invention are useful comprises ionizable lipid(s) represented by Formula (IA), Formula (IB) and/or Formula (II), and a permanently charged lipid.
  • Ionizable lipids - Formula (IA) As contemplated herein, the present invention relates to an ionizable lipid(s) represented by the structure of Formula (IA): including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.
  • R 1A is selected from the group consisting of: OH, -(CH 2 CH 2 O) 2-6 H, C 1-6 hydroxyalkyl and C 1-6 haloalkyl and C 1-3 alkylene-NR NA R NA’ , wherein each one of R NA and R NA’ is individually C1-4 alkyl or R NA and R NA’ together with the nitrogen to which they are bound, form a ring.
  • the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl.
  • R 1A is selected from the group consisting of: OH, -(CH2CH2O)2-6H and C 1-6 hydroxyalkyl. According to some embodiments, R 1A is selected from the group consisting of: OH, - (CH2CH2O)2-4H and C1-4 hydroxyalkyl. According to some embodiments, R 1A is -(CH2CH2O)1-4H or OH. According to some embodiments, R 1A is selected from the group consisting of: -CH 2 CH 2 OH, - CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH. According to some embodiments, R 1A is OH.
  • R 1A is -CH2CH2OH. According to some embodiments, R 1A is, - CH2CH2OCH2CH2OH. According to some embodiments, R 1A is the same as R 3A . It is the be understood that the term “the same” in the previous paragraph means that the two specified R substituents have the same chemical definition.
  • R 2A is selected from the group consisting of: C 5 - 25 alkyl, C 5 - 25 alkenyl, C 5 - 15 alkylene-CO 2 -C 5 - 15 alkyl, C 5 - 15 alkylene-CO 2 -C 5 - 15 alkenyl, C 5 - 15 alkylene-O 2 C-C 5 - 15 alkyl, C 5 - 15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl.
  • R 2A is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 2A is -C12H25.
  • R 2A is the same as R 4A .
  • n A is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.
  • n A is selected from the group consisting of: 8, 9 and 10.
  • n A is 9 or 10.
  • n A is 9.
  • n A is 10.
  • X A is selected from the group consisting of:-COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-.
  • X A is selected from the group consisting of: -COO-, -OOC-, -NHCO- and -CONH.
  • X A is -COO- or -OOC-.
  • X A is -COO- and the lipid is represented by Formula (IA 1 ) .
  • j A is selected from the group consisting of: 0, 1, 2, 3 and 4. Each possibility represents a separate embodiment of the invention.
  • j A is selected from the group consisting of: 0, 1 and 2. According to some embodiments, j A is 0 or 2. According to some embodiments, j A is 0. According to some embodiments, Y A is selected from the group consisting of: absent, -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y A is absent, -COO- or -OOC-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y A is absent or -OOC-. According to some embodiments, Y A is absent.
  • Y A is absent, j A is 0 and the lipid of Formula (IA) is represented by Formula (IA 2 ): .
  • Y A is absent, j A is 0, X A is -COO- and the lipid of Formula (IA) is represented by Formula (IA 3 ) .
  • m A is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. Each possibility represents a separate embodiment of the invention.
  • m A is selected from the group consisting of: 6, 7, 8, 9, 10, 11, 12 and 13.
  • m A is selected from the group consisting of: 8, 9, and 10.
  • m A is 9 or 10. According to some embodiments, m A is 9. According to some embodiments, m A is 10. According to some embodiments, R 3A is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C 1-6 haloalkyl and C 1-3 alkylene-NR NA’’ R NA’’’ , wherein each one of R NA’’ and R NA’’’’ is individually C 1-4 alkyl or R NA’’ and R NA’’’ together with the nitrogen to which they are bound, form a ring.
  • R NA’ is individually C 1-4 alkyl or R NA’’ and R NA’’’ together with the nitrogen to which they are bound, form a ring.
  • the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl.
  • R 3A is selected from the group consisting of: OH, -(CH 2 CH 2 O) 2-6 H and C1-6 hydroxyalkyl. According to some embodiments, R 3A is selected from the group consisting of: OH, - (CH2CH2O)2-4H and C1-4 hydroxyalkyl. According to some embodiments, R 3A is -(CH2CH2O)1-4H or OH.
  • R 3A is selected from the group consisting of: -CH2CH2OH, - CH 2 CH 2 OCH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 OH and OH. According to some embodiments, R 3A is OH. According to some embodiments, R 3A is -CH2CH2OH. According to some embodiments, R 1A is, -CH2CH2OCH2CH2OH.
  • R 4A is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O 2 C-C 5 - 15 alkyl, C 5 - 15 alkenylene-O 2 C-C5- 15 alkenyl.
  • R 4A is selected from the group consisting of: C 6 - 18 alkyl and C 12 - 24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 4A is -C12H25.
  • the ionizable lipid of Formula (IA) is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14 and IA-15 including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention.
  • the lipid of Formula (IA) is selected from the group consisting of: lipid IA-4, lipid IA-5, lipid IA-8, lipid IA-10, lipid IA-11, lipid IA-12, lipid IA-13, lipid IA-14, and lipid IA-15.
  • the lipid of Formula (IA) is IA-5.
  • the lipid of Formula (IA) is IA-10.
  • the lipid of Formula (IA) is IA-11.
  • the lipid of Formula (IA) is IA-12.
  • Formula (IB) As contemplated herein, the present invention relates to an ionizable lipid(s) represented by the structure of Formula (IB):
  • R 1B is selected from the group consisting of: OH, -(CH 2 CH 2 O) 2-6 H, C 1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NR NB R NB’ , wherein each one of R NA and R NA’ is individually C1-4 alkyl or R NA and R NA’ together with the nitrogen to which they are bound, form a ring.
  • R NA and R NA’ is individually C1-4 alkyl or R NA and R NA’ together with the nitrogen to which they are bound, form a ring.
  • the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. According to some embodiments, R 1B is selected from the group consisting of: -CH 2 CH 2 OCH 2 CH 2 OH, -CH 2 CH 2 Cl, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 N(CH 2 ) 4 , -CH 2 CH 2 CH 2 CH 2 OH and -CH 2 CH 2 NMe 2 .
  • R 1B is selected from the group consisting of: CH2CH2OCH2CH2OH, -CH2CH2Cl or -CH2CH2OH. According to some embodiments, R 1B is -CH2CH2OCH2CH2OH or -CH2CH2Cl. According to some embodiments, R 1B is -CH2CH2OCH2CH2OH. According to some embodiments, R 1B is -CH2CH2Cl. It is to be understood that the group -CH2CH2CH2N(CH2)4, which is specified herein refers to propyl pyrrolidine, i.e., the group drawn below:
  • R 1B is the same as R 3B .
  • R 2B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C 5 - 15 alkylene-CO 2 -C 5 - 15 alkyl, C 5 - 15 alkylene-CO 2 -C 5 - 15 alkenyl, C 5 - 15 alkylene-O 2 C-C 5 - 15 alkyl, C 5 - 15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O 2 C-C 5 - 15 alkyl, C 5 - 15 alkenylene-O 2 C-C5- 15 alkenyl.
  • R 2B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 2B is -C12H25.
  • R 2A is the same as R 4A .
  • R 2A is the same as R 3A .
  • W B is a C 4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen.
  • W B is a C 4-12 alkylene.
  • W B is a straight chain alkylene.
  • W B is a C5-9 alkylene.
  • W B is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention.
  • W B is -(CH2)9-.
  • Y B is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene. According to some embodiments, Y B is selected from the groups consisting of: absent, -CH 2 CH 2 OCH 2 CH 2 - and -CH 2 CH 2 -. It is to be understood by the person having ordinary skill in the art that when a variable is said to be “absent”, no chemical group will appear in the specified place, and the atoms drawn as bonded to the variable, will be bonded to each other. For example, Formula (IB 1 ) is an embodiment of Formula (IB), wherein the variable Y B is absent.
  • Y B is drawn as bonded to an oxygen atom and to a nitrogen atom.
  • the oxygen will be directly bonded to the nitrogen via a single (signa) bond.
  • Y B is absent and the lipid of Formula (IB) is represented by Formula (IB 1 ): .
  • R 3B is selected from the group consisting of: C 5 - 25 alkyl, C 5 - 25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O 2 C-C 5 - 15 alkenyl, C 5 - 15 alkenylene-CO 2 -C 5 - 15 alkyl, C 5 - 15 alkenylene-CO 2 -C 5 - 15 alkenyl, C 5 - 15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl.
  • R 3B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 3B is a C4-16 alkyl.
  • R 3B is selected from the group consisting of: -C12H25, -C6H13 and -C8H17.
  • R 3B is -C6H13 or -C8H17. According to some embodiments, R 3B is -C6H13. According to some embodiments, R 3B is -C8H17. According to some embodiments, R 3B is the same as R 4B .
  • R 4B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O 2 C-C 5 - 15 alkyl, C 5 - 15 alkenylene-O 2 C-C5- 15 alkenyl.
  • R 4B is selected from the group consisting of: C 6 - 18 alkyl and C 12 - 24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 4B is a C4-16 alkyl.
  • R 4B is selected from the group consisting of: -C12H25, -C6H13 and -C8H17.
  • R 4B is -C6H13 or -C8H17. According to some embodiments, R 4B is -C 6 H 13 . According to some embodiments, R 4B is -C 8 H 17 .
  • the ionizable lipid of Formula (IB) is selected from the group consisting of: IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB-22, IB-23 and IB-24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.
  • the ionizable lipid of Formula (IB) is selected from the group consisting of: lipid IB-4, lipid IB-6, lipid IB-9, lipid IB-10, lipid IB- 13, lipid IB-24 and a combination thereof.
  • the lipid of Formula (IB) is selected from the group consisting of: IB-4, IB-6 and IB-10.
  • the lipid of Formula (IB) is IB-4.
  • the lipid of Formula (IB) is IB-6.
  • the lipid of Formula (IB) is IB-10.
  • Formula (II) As contemplated herein, the present invention relates to an ionizable lipid(s) represented by the structure of Formula (II):
  • R 1C is selected from the group consisting of: C 5 - 25 alkyl, C 5 - 25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl.
  • R 1C is selected from the group consisting of: C 4 - 18 alkyl and C 12 - 24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 1C is a C4-16 alkyl.
  • R 1C is a C4-10 alkyl.
  • R 1C is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R 1C is -C6H13 or -C8H17. According to some embodiments, R 1C is -C6H13. According to some embodiments, R 1C is -C8H17. According to some embodiments, R 1C is the same as R 2C . According to some embodiments, R 1C is the same as R 3C . According to some embodiments, R 1C is the same as R 4C . According to some embodiments, R 1C , R 2C , R 3C , and R 4C are the same.
  • R 2C is selected from the group consisting of: C 5 - 25 alkyl, C 5 - 25 alkenyl, C 5 - 15 alkylene-CO 2 -C 5 - 15 alkyl, C 5 - 15 alkylene-CO 2 -C 5 - 15 alkenyl, C 5 - 15 alkylene-O 2 C-C 5 - 15 alkyl, C 5 - 15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl.
  • R 2C is selected from the group consisting of: C 4 - 18 alkyl and C 12 - 24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 2C is a C4-16 alkyl.
  • R 2C is a C4-10 alkyl.
  • R 2C is selected from the group consisting of: -C 12 H 25 , -C 6 H 13 and -C 8 H 17 . According to some embodiments, R 2C is -C 6 H 13 or -C 8 H 17 . According to some embodiments, R 2C is -C 6 H 13 . According to some embodiments, R 2C is -C 8 H 17 . According to some embodiments, Y 1C is selected from the groups consisting of: absent, -(CH 2 CH 2 O) 1-5 CH 2 CH 2 - and C 1-6 alkylene. Each possibility represents a separate embodiment of the invention.
  • Y 1C is selected from the groups consisting of: absent, - CH2CH2OCH2CH2- and -CH2CH2-. According to some embodiments, Y 1C is absent. According to some embodiments, Y 1C is the same as Y 2C . According to some embodiments, each one of Y 1C and Y 2C is absent and the lipid of Formula (II) is represented by Formula (II 1 ): .
  • W 1C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According to some embodiments, W 1C is a C4- 12 alkylene.
  • W 1C is a straight chain alkylene. According to some embodiments, W 1C is a C 5-9 alkylene. According to some embodiments, W 1C is selected from the group consisting of: -(CH 2 ) 9 -, -(CHMe)-(CH 2 ) 4 - and -(CH 2 ) 5 -. Each possibility represents a separate embodiment of the invention. According to some embodiments, W 1C is -(CH2)9-. According to some embodiments, W 1C and W 2C are the same. According to some embodiments, each one of W 1C and W 2C is a C 4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II 2 ): .
  • each one of Y 1C and Y 2C is absent, each one of W 1C and W 2C is a C 4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II 3 ): .
  • R 5C is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C 1-6 haloalkyl and C 1-3 alkylene-NR NII R NII’ , wherein each one of R NII and R NII’ is individually C 1-4 alkyl or R NII and R NII’ together with the nitrogen to which they are bound, form a ring.
  • the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl.
  • R 5C is selected from the group consisting of: OH, -(CH2CH2O)2-3H, C1-4 hydroxyalkyl and C1-4 haloalkyl and C1-3 alkylene-NR NII R NII’ , wherein each one of R NII and R NII’ is individually C1-4 alkyl or R NII and R NII’ together with the nitrogen to which they are bound, form a 5-6 membered ring.
  • R NII and R NII’ is individually C1-4 alkyl or R NII and R NII’ together with the nitrogen to which they are bound, form a 5-6 membered ring.
  • R 5C is selected from the group consisting of: -CH 2 CH 2 OH, -CH 2 CH 2 OCH 2 CH 2 OH, - CH 2 CH 2 CH 2 CH 2 OH, -CH 2 CH 2 CH 2 N(CH 2 ) 4 , -CH 2 CH 2 CH 2 N(CH 2 ) 5 , -CH 2 CH 2 N(CH 2 ) 5 , -CH 2 CH 2 Cl, -OH and -CH2CH2NMe2.
  • R 5C is -CH2CH2OH or -CH2CH2OCH2CH2OH.
  • W 2C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According to some embodiments, W 2C is a C4- 12 alkylene. According to some embodiments, W 2C is a straight chain alkylene. According to some embodiments, W 2C is a C5-9 alkylene. According to some embodiments, W 2C is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, W 2C is -(CH 2 ) 9 -.
  • Y 2C is selected from the groups consisting of: absent, -(CH 2 CH 2 O) 1-5 CH 2 CH 2 - and C 1-6 alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y 2C is selected from the groups consisting of: absent, - CH 2 CH 2 OCH 2 CH 2 - and -CH 2 CH 2 -. According to some embodiments, Y 2C is absent.
  • R 3C is selected from the group consisting of: C 5 - 25 alkyl, C 5 - 25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl.
  • Each possibility represents a separate embodiment of the invention.
  • R 3C is selected from the group consisting of: C 4 - 18 alkyl and C 12 - 24 alkenyl.
  • the alkyl is a straight chain alkyl.
  • the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 3C is a C4-16 alkyl.
  • R 3C is a C4-10 alkyl.
  • R 3C is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R 3C is -C6H13 or -C8H17. According to some embodiments, R 3C is -C6H13. According to some embodiments, R 3B is -C 8 H 17 . According to some embodiments, R 4C is selected from the group consisting of: C 4 - 18 alkyl and C 12 - 24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl.
  • the alkyl is an unsubstituted alkyl.
  • the alkenyl is an unsubstituted alkenyl.
  • R 4C is a C4-16 alkyl.
  • R 4C is a C4-10 alkyl.
  • R 4C is selected from the group consisting of: -C12H25, -C6H13 and -C8H17.
  • R 4C is -C6H13 or -C8H17.
  • R 4C is -C 6 H 13 .
  • R 4C is -C 8 H 17 .
  • the ionizable lipid of Formula (II) is selected from the group consisting of: II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II-15, II-16, II-17, II-18, II- 19, II-20, II-21, II-22, II-23 and II-24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention.
  • the lipid of Formula (II) is selected from the group consisting of: II-1, and II-5.
  • the lipid of Formula (II) is II- 1. According to some embodiments, the lipid of Formula (II) is II-5.
  • the chemical structures of each of the specific lipids are detailed below in the “Exemplary Ionizable Lipids” Section and in the claims. Exemplary Ionizable Lipids Exemplary lipids according to Formula (IA), Formula (IB) and Formula (II) of the present invention are shown below. It is to be understood that, according to some embodiments, the invention is not limited to any one or more of the following exemplary lipids.
  • the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB-22, IB-23, IB-24, II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II- 15, II-16, II-17, II-18, II-19, II-20, II-21, IB-22, IB-23, IB-24, II-1, II
  • the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14 and IA-15.
  • the ionizable lipid is selected from the group consisting of: IB-1, IB-2, IB-3, IB-4, IB-5, IB- 6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB- 22, IB-23 and IB-24.
  • the ionizable lipid is selected from the group consisting of: II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II-15, II-16, II- 17, II-18, II-19, II-20, II-21, II-22, II-23 and II-24.
  • the ionizable lipid is selected from the group consisting of: IA-4, IA-5, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-4, IB-9, IB-10, IB-13, II-1 and II-5.
  • the ionizable lipid is selected from the group consisting of: IA-10, IA-11, IA-12, IB-4, IB-10, II-1 and II-5. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-110 IA-12, IB-4 and IB-5. According to some embodiments, the lipid of is selected from the group consisting of: IA-5, IA-10, IA-11 and IA-12. According to some embodiments, the lipid is IA-10. According to some embodiments, the lipid is selected from the group consisting of: IB-4, IB-6 and IB-10. According to some embodiments, the lipid is IB-4.
  • the lipid is IB-6. According to some embodiments, the lipid is IB-10. According to some embodiments, the lipid is selected from the group consisting of: II-1 and II-5. According to some embodiments, the lipid is II-5. According to some embodiments, the present invention provides an ionizable lipid represented by the structure of Formula (IA), Formula (IB) or Formula (II). Each possibility represents a separate embodiment of the invention.
  • the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB- 18, IB-19, IB-20, IB-21, IB-22, IB-23, IB-24, II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II- 12, II-13, II-14, II-15, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-23 and II-24, including salt
  • the ionizable lipid is selected from the group consisting of: IA-4, IA-13, IA-14, IA-15, IB-4, IB-9 and IB-24.
  • the following exemplary lipids are portrayed as non-limiting examples of the ionizable lipids of the present invention (designation below the chemical structure).
  • Lipid IA-4 is also referred herein as NV3-015.
  • IA-5 Lipid IA-5 is also referred herein as NV3-005.
  • IA-8 Lipid IA-8 is also referred herein as NV3-009.
  • IA-10 Lipid IA-10 is also referred herein as NV3-002.
  • IA-11 Lipid IA-11 is also referred herein as NV3-004.
  • IA-12 Lipid IA-12 is also referred herein as NV3-006.
  • IA-13 Lipid IA-13 is also referred herein as NV3-013.
  • IA-14 Lipid IA-14 is also referred herein as NV3-014.
  • Lipid IB-4 is also referred herein as NV2-004.
  • IB-8 IB-9 Lipid IB-9 is also referred herein as NV2-009.
  • IB-10 Lipid IB-10 is also referred herein as NV2-011.
  • IB-13 Lipid IB-13 is also referred herein as NV2-015.
  • IB-17 is also referred herein as NV2-015.
  • IB-24 Lipid IB-24 is also referred herein as NV2-027.
  • II-1 Lipid II-1 is also referred herein as NV1-001.
  • Lipid II-5 is also referred herein as NV1-005.
  • the lipids of Formulae (IA), (IB) and (II) may be considered as cationic lipids, since they bear 2 or more nitrogen atoms, where these atoms are typically basic and protonizable at the selected pH, so that the lipids may carry a net positive charge.
  • the lipid of the present invention is a cationic lipid.
  • the term “permanently charged lipid”, as used herein refers to lipid species that carries a charge at on one or more of its atoms and that has a total net (positive or negative) charge. The charged atom may not be de- charged in the permanently charged lipid.
  • the charged atom may not be de-charged at different pH environments.
  • the charge of the charged atom may not be altered at different pH environments.
  • the term “permanently charged lipid” includes both charged lipids coupled to a counterion and zwitterionic lipids. Zwitterionic permanently charged lipids may require unequal number of positively charged atoms and negatively charged atom in the lipid, and also include a counterion not bonded to the lipid.
  • DSPC distearoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • Charged atoms of the permanently charged lipid include, but are not limited to nitrogen (e.g., ammonium groups) and/or oxygen (e.g., sulfate groups, carboxylate groups, phosphate groups, etc.).
  • the permanently charged lipid comprises a quaternary ammonium group. Specifically, quaternary ammonium groups are considered to have permanently charged nitrogen atom, due to the three-valent nitrogen bonded to 4 groups (e.g., alkyl groups). According to some embodiments, the permanently charged lipid have a net charge.
  • a permanently charged lipid which has a net charge has a permanently charged atom as part of or chemically bonded to the lipid backbone, and a counterion, which is not covalently bonded to the lipid backbone.
  • the term “quaternary ammonium group” as used herein refers to a chemical functional group containing at least one quaternized nitrogen wherein the nitrogen atom is attached to four organic groups.
  • a permanently charged lipid according to the present invention may comprise one or more quaternized nitrogen atoms.
  • the quaternary ammonium group is a tetra-alkyl ammonium.
  • tetra-alkyl ammonium refers to a group or compound (e.g., lipid) which contain such group, that has a nitrogen atom bonded to four alkyl groups.
  • alkyl is defined below.
  • An “alkyl” group refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain alkyl groups.
  • the alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
  • C n-m alkyl refers to an alkyl group having n to m carbon atoms.
  • An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound.
  • alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, 3-pentyl, hexyl, 1,2,2-trimethylpropyl and the like.
  • alkylene employed alone or in combination with other terms, refers to a divalent alkyl linking group.
  • An alkylene group formally corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkylene group to the remainder of the compound.
  • C n-m alkylene refers to an alkylene group having n to m carbon atoms.
  • alkylene groups include, but are not limited to, ethan-l,2-diyl, ethan-l,l-diyl, propan-l,3-diyl, propan-l,2-diyl, propan-l,l-diyl, butan-l,4-diyl, butan-l,3-diyl, butan-1,2- diyl, 2-methyl-propan-l,3-diyl, -(CHMe)-(CH2)4-, -(CHMe)-(CH2)4- and the like. It is to be understood that for branched alkylene groups the total number of carbon atoms is counted.
  • the substituent -(CHMe)-(CH 2 ) 4 - is a C 6 alkylene. It is to be understood that C 0 -alkylene means that the specified substituent is absent.
  • ranges of alkyl chains are presented, e.g., C 4-16 alkyl, C 6-18 alkyl etc. It is to be understood that such ranges include any sub range thereof.
  • C6-18 alkyl may include and/or be directed to: C6-12 alkyl, C8-14 alkyl, C12-18 alkyl, C8 alkyl etc.
  • alkenyl refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond including straight-chain, branched-chain and cyclic alkenyl groups.
  • alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl- butenyl and decenyl.
  • the alkenyl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl.
  • Alkenyls according to the present invention may include more than one carbon-carbon double bond.
  • dienes see e.g., lipid IA-11, NV3-004, substituent R 4A
  • trienes are within the definition of alkenyl.
  • the alkenyl is a dienyl.
  • Cn-m alkenyl refers to an alkyl group having n to m carbon atoms.
  • An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound.
  • alkenyl moieties include, but are not limited to, chemical groups such as ethenyl, propenyl, isopropenyl, n- butenyl, sec-butenyl the like.
  • alkenylene employed alone or in combination with other terms, refers to a divalent alkenyl linking group.
  • An alkenylene group formally corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkenylene group to the remainder of the compound.
  • C n-m alkenylene refers to an alkenylene group having n to m carbon atoms.
  • alkenyl chains are presented, e.g., C2-8 alkenyl, C4-20 alkenyl etc. It is to be understood that such ranges include any sub range thereof.
  • C4-14 alkenyl may include and/or be directed to: C4-8 alkenyl, C8-14 alkenyl, C6-12 alkenyl, C9 alkenyl etc.
  • each one of the alkenyl double bond has a cis configuration.
  • One or more of the lipids of the invention may be present as a salt.
  • salt encompasses both basic and acid addition salts, including but not limited to, carboxylate salts or salts with amine nitrogen atoms, and include salts formed with the organic and inorganic anions and cations discussed below. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and organic or inorganic acids.
  • Such acids include hydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D- camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.
  • organic or inorganic cation refers to counter-ions for the anion of a salt.
  • the counter-ions include, but are not limited to, alkali and alkaline earth metals (such as lithium, sodium, potassium, barium, aluminum and calcium); ammonium and mono-, di- and tri-alkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2- hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations. See, for example, Berge et al., J. Pharm. Sci. (1977), 66:1-19, which is incorporated herein by reference.
  • the present invention provides a particle comprising the present ionizable lipid and the permanently charged lipid.
  • a composition comprising a plurality of particles as discloses herein and a pharmaceutically acceptable carrier, diluent or excipient.
  • the composition is a liposomal composition.
  • the particles of the present invention are in the form of liposomes.
  • the composition further comprises one or more components selected from the group consisting of a neutral lipid, a charged lipid, a steroid, and a polymer-conjugated lipid. Each possibility represents a separate embodiment of the present invention.
  • the lipid nanoparticle formulation of the present invention comprises 10 to 50 mol% of the ionizable lipid(s) according to Formula (IA), Formula (IB) and or Formula (II). It is to be understood that according to embodiments wherein the lipid nanoparticle formulation comprises a combination of different ionizable lipids according to Formula (IA), Formula (IB) and or Formula (II), the mol% refers to the total mol% of all said different ionizable lipids. According to some embodiments, the lipid nanoparticle formulation of the present invention comprises 15 to 45 mol% of the ionizable lipid(s).
  • the lipid nanoparticle formulation of the present invention comprises 20 to 40 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 15 to 30 mol% of the ionizable lipid. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 30 mol% of the ionizable lipid. According to some embodiments, the lipid nanoparticle formulation of the present invention comprises 25 to 35 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises about 30 mol% of the ionizable lipid(s).
  • the lipid nanoparticle formulation of the present invention comprises at least 10 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises at least 15 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises at least 20 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises at least 25 mol% of the ionizable lipid(s).
  • the lipid nanoparticle formulation of the present invention comprises no more than 60 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 50 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 40 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 35 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 30 mol% of the ionizable lipid(s).
  • the lipid nanoparticle formulation of the present invention comprises no more than 25 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 10 to 40 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 15 to 35 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 15 to 30 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 20 to 30 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises about 25 mol% permanently charged lipid(s).
  • the lipid nanoparticle formulation comprises about 20 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 5 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 10 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 15 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 60 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% permanently charged lipid(s).
  • the lipid nanoparticle formulation comprises no more than 45 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 40 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 35 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 30 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 25 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation further comprises at least one neutral lipid.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH
  • such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn- glyccro-3- phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glyccro-3-phosphocholine (DMPC), 1-Palmitoyl- 2-olcoyl-sn-glyccro-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidyl ethanolamines such as 1,2-Diolcoyl-sn-glyccro-3-phosphoethanolamine (DOPE), sphingomyelins (SM
  • Neutral lipids may be synthetic or naturally derived.
  • the lipid nanoparticle formulation further comprises at least one sterol, at least one phospholipid or both.
  • the neutral lipid comprises a sterol, a phospholipid or both.
  • the sterol comprises cholesterol.
  • the lipid nanoparticle formulation further comprises cholesterol.
  • the lipid nanoparticle formulation comprises 15 to 45 mol% of the sterol.
  • the lipid nanoparticle formulation comprises 20 to 40 mol% of the sterol.
  • the lipid nanoparticle formulation comprises 25 to 37 mol% of the sterol.
  • the lipid nanoparticle formulation comprises 30 to 40 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 30 to 35 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises about 32.5 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 25 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 30 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% of the sterol.
  • the lipid nanoparticle formulation comprises no more than 40 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 35 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 15 to 45 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 40 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 25 to 37 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 30 to 35 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises about 32.5 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% cholesterol.
  • the lipid nanoparticle formulation comprises at least 25 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 30 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 40 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 35 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation further comprises at least one phosphatidylethanolamine. According to some embodiments, the phospholipid comprises a phosphatidylethanolamine.
  • the phosphatidylethanolamine is selected from the group consisting of: 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) 1,3-Dipalmitoyl-sn-glycero-2- phosphoethanolamine (1,3-DPPE), 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), biotin-phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or combinations thereof.
  • DLPE 1,2-dilauroyl
  • the phosphatidylethanolamine comprises DOPE.
  • the phospholipid comprises 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Distearoylphosphatidylcholine (DSPC) or both.
  • the nanoparticle formulation further comprises DOPE.
  • 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is the neutral phospholipid shown below: DOPE.
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • the lipid nanoparticle formulation comprises 5 to 20 mol% of the phospholipid.
  • the lipid nanoparticle formulation comprises 5 to 15 mol% of the phospholipid.
  • the lipid nanoparticle formulation comprises about 10 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 3 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 5 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 7 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 25 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 20 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 15 mol% of the phospholipid.
  • the lipid nanoparticle formulation comprises 5 to 20 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises about 10 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises at least 3 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises at least 5 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises at least 7 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises no more than 25 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises no more than 20 mol% DOPE.
  • the lipid nanoparticle formulation comprises no more than 15 mol% DOPE.
  • the lipid nanoparticle formulation further comprises a PEGylated lipid.
  • PEG refers to polyethylene glycol.
  • PEGylated lipid means a lipid that is bonded to PEG.
  • the PEGylated lipid comprises a PEG moiety having a molecular weight in the range of 1000 gr/mol to 3000 gr/mol, including each value and sub-range within the specified range.
  • the PEGylated lipid comprises a PEG moiety having a molecular weight in the range of 1000 gr/mol to 2000 gr/mol.
  • the PEG moiety has a molecular weight of about 2000 gr/mol.
  • the PEGylated lipid comprises DMG-PEG.
  • the PEGylated lipid comprises DMG-PEG-2000.
  • DMG-PEG means 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol, or ,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol.
  • the term “DMG-PEG-2000” means 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol, or ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol, wherein the polyethylene glycol has a molecular weight of about 2000 gr/mol.
  • the lipid nanoparticle formulation comprises 1 to 5 mol% PEGylated lipid.
  • the lipid nanoparticle formulation comprises 1.5 to 4 mol% PEGylated lipid.
  • the lipid nanoparticle formulation comprises about 2.5 mol% PEGylated lipid.
  • the lipid nanoparticle formulation comprises at least 1 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 2 mol% PEG. According to some embodiments, the lipid nanoparticle formulation comprises no more than 10 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 5 mol% PEGylated lipid. It is to be understood that by the phrase “the molar percentage of the component is at least x mol% of the particle” it is meant that at least x% of the particle molecules are of the component.
  • the particle comprises the membrane stabilizing lipid and a lipid membrane comprising the lipid.
  • the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids.
  • the membrane stabilizing lipid comprises cholesterol.
  • the membrane stabilizing lipids may be selected from, but not limited to: cholesterol, phospholipids (such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols), cephalins, sphingolipids (sphingomyelins and glycosphingolipids), glycoglycerolipids, and combinations thereof.
  • phospholipids such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols
  • cephalins such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidy
  • the phosphatidylethanolamines may be selected from, but not limited to: 1,2-dilauroyl-L-phosphatidyl- ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn- glycero-3-phosphoethanolamine (DPhPE) 1,3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3- DPPE), 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), Biotin- Phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or combinations thereof.
  • DLPE
  • the Phosphatidylethanolamines may be conjugated to a PEG-Amine derivative.
  • the lipid nanoparticle formulation is selected from the group consisting of Formulation 1 to 5 and 11-26, wherein the compositions of Formulations 1 to 5 and 11-26 are described below.
  • Formulation 1 comprises: lipid IA-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%.
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • Formulation 2 comprises: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%.
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • Formulation 3 comprises: lipid IB-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and polyethylene glycol (PEG): 1.5 mol% to 4 mol%.
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • PEG polyethylene glycol
  • Formulation 4 comprises: lipid IB-6: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%.
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • Formulation 5 comprises: lipid II-5: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%.
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • Formulation 11 comprises: lipid II-1: 25 mol% to 35 mol%; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%;
  • Formulation 12 comprises: lipid II-1: 25 mol% to 35 mol%; DOTAP: 5 mol% to 15 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 50 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%;
  • Formulation 13 comprises: lipid II-1: 20 mol%
  • Formulation 19 comprises: lipid IA-12: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%;
  • Formulation 19 comprises: lipid IA-12: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%;
  • Formulation 20 comprises: lipid IA-8: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-s
  • Formulation 23 comprises: lipid II-5: 15 mol% to 25 mol%; DOTAP: 15 mol% to 25 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 55 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%;
  • Formulation 24 comprises: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; Distearoylphosphatidylcholine (DSPC): 7 mol% to 13
  • the lipid nanoparticle formulation is Formulation 1. According to some embodiments, the lipid nanoparticle formulation is Formulation 2. According to some embodiments, the lipid nanoparticle formulation is Formulation 3. According to some embodiments, the lipid nanoparticle formulation is Formulation 4. According to some embodiments, the lipid nanoparticle formulation is Formulation 5. According to some embodiments, the lipid nanoparticle formulation is Formulation 6. According to some embodiments, the lipid nanoparticle formulation is Formulation 7. According to some embodiments, the lipid nanoparticle formulation is Formulation 8. According to some embodiments, the lipid nanoparticle formulation is Formulation 9. According to some embodiments, the lipid nanoparticle formulation is Formulation 10. According to some embodiments, the lipid nanoparticle formulation is Formulation 11.
  • the lipid nanoparticle formulation is Formulation 12. According to some embodiments, the lipid nanoparticle formulation is Formulation 13. According to some embodiments, the lipid nanoparticle formulation is Formulation 14. According to some embodiments, the lipid nanoparticle formulation is Formulation 15. According to some embodiments, the lipid nanoparticle formulation is Formulation 16. According to some embodiments, the lipid nanoparticle formulation is Formulation 17. According to some embodiments, the lipid nanoparticle formulation is Formulation 18. According to some embodiments, the lipid nanoparticle formulation is Formulation 19. According to some embodiments, the lipid nanoparticle formulation is Formulation 20. According to some embodiments, the lipid nanoparticle formulation is Formulation 21. According to some embodiments, the lipid nanoparticle formulation is Formulation 22.
  • the lipid nanoparticle formulation is Formulation 23. According to some embodiments, the lipid nanoparticle formulation is Formulation 25. According to some embodiments, the lipid nanoparticle formulation is Formulation 25. According to some embodiments, the lipid nanoparticle formulation is Formulation 26. According to some embodiments, the lipid nanoparticle formulation is Formulation 1 or Formulation 2. According to some embodiments, the lipid nanoparticle formulation is Formulation 15 or Formulation 23. According to some embodiments, the lipid nanoparticle formulation comprises a targeting moiety. According to some embodiments, the lipid nanoparticle formulation comprises at least one nanoparticle, which is conjugated to a targeting moiety. According to some embodiments, the targeting moiety is a lung targeting moiety.
  • the lipid nanoparticle formulation is devoid of lung targeting moieties. Specifically, it was found that the present lipid nanoparticles are highly effective in targeting the lungs, even in the absence of lung targeting moieties. Accordingly, the incorporation of such moieties, which are typically employed for lung-targeting purposes, may be avoided. This enables a simplified formulation process at significantly reduced costs. According to some embodiments, the lipid nanoparticle formulation further comprises a nucleic acid encapsulated within at least one particle thereof.
  • the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
  • the particle further comprises a nucleic acid.
  • the nucleic acid is encapsulated within a particle of the lipid nanoparticle formulation.
  • the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
  • the composition may further comprise a nucleic acid.
  • nucleic acids include small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
  • the weight ratio between the nucleic acid and the lipid mixture may be adjusted so as to achieve maximal biological effect by the nucleic acid on the target site.
  • the ratio between the nucleic acid and the lipid phase may be 1:1.
  • the weight ratio between the nucleic acid and the lipid phase may be 1:2.
  • the weight ratio between the nucleic acid and the lipid phase may be 1:5.
  • the weight ratio between the nucleic acid and the lipid phase may be 1:10.
  • the weight ratio between the nucleic acid and the lipids phase may be 1:16.
  • the weight ratio between the nucleic acid and the lipid phase may be 1:20.
  • the weight ratio between the nucleic acid and the lipid phase is about 1:1 to 1:20 (w:w).
  • the particle further comprises a therapeutic agent.
  • the therapeutic agent is encapsulated within a particle comprising the lipid.
  • the lipid nanoparticle formulation comprises a therapeutic agent encapsulated within at least one particle thereof.
  • the present lipid nanoparticle formulation may include a plurality of nanoparticle. The following section related to the diameter of the nanoparticles of the present lipid nanoparticle formulation.
  • the lipid nanoparticle formulation has average nanoparticle size (Z average ) in the range of 10 to 500 nanometers.
  • the lipid nanoparticle formulation has average nanoparticle size in the range of 25 to 400 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Z average) in the range of 50 to 200 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Z average) in the range of 50 to 150 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Z average ) in the range of 60 to 120 nanometers. In some embodiments, the particles (including any nucleic acid, therapeutic agent and the like encapsulated within and any targeting moiety conjugated thereto) have a particle size (diameter) in the range of about 10 to about 500 nm.
  • the particles have a particle size (diameter) in the range of about 10 to about 350 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 40 to about 270 nm. In some embodiments, the particles have a particle size (diameter) in the range of over about 10 nm. In some embodiments, the particles have a particle size (diameter) of over about 20 nm. In some embodiments, the particles have a particle size (diameter) of over about 30 nm. In some embodiments, the particles have a particle size (diameter) of over about 40 nm.
  • the particles have a particle size (diameter) of over about 45 nm. In some embodiments, the particles have a particle size (diameter) of over about 50 nm. In some embodiments, the particles have a particle size (diameter) of over about 60 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 500 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 400 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 300 nm. In some embodiments, the size is a hydrodynamic diameter.
  • the lipid nanoparticle formulation has Zeta potential of at least 10mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 15mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 20mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 25mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 27mV.According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 29mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential in the range of 28 to 40 mV.
  • zeta potential refers to a physical measurement of a colloidal system by electrophoresis. It gives the value of the potential (in mV) of a colloid in a suspension at the boundary between the Stern layer and the diffuse layer.
  • the zeta potential in a colloidal system is the difference in potential between the immovable layer attached to the surface of the dispersed phase and the dispersion medium.
  • the zeta potential is related to stability of suspensions of particles. Zeta potential may be adjusted, in part, for example, by adjusting the concentration of an electrolyte in the buffer system.
  • the lipid nanoparticle formulation has polydispersity index (PDI) of no more than 0.75.
  • the lipid nanoparticle formulation has PDI of no more than 0.5. According to some embodiments, the lipid nanoparticle formulation has PDI of no more than 0.25. According to some embodiments, the lipid nanoparticle formulation has PDI of no more than 0.2.
  • a composition suitable for administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation. Each possibility represents a separate embodiment of the invention.
  • IV intravenous
  • a liquid composition suitable for IV administration wherein the liquid composition comprises the present lipid nanoparticle formulation. It was found that the present lipid nanoparticle formulations are suitable for IV delivery.
  • the composition is a liquid composition.
  • a liquid composition suitable for IV administration wherein the liquid composition comprises the present lipid nanoparticle formulation.
  • the liquid composition is in the form of an aqueous solution, emulsion or suspension.
  • the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
  • compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like.
  • Aqueous injection suspensions may also contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • parenteral formulations can be present in unit dose or multiple dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as, for example, water for injection, immediately prior to use.
  • parenteral administration includes intravenous administration.
  • intravenous administration refers to one or more boluses injections as well as a continuous intravenous infusion. Each possibility represents a separate embodiment.
  • intravenous bolus refers to administration into a vein of an animal or human in a duration of several minutes or less.
  • intravenous infusion refers to administration into the vein of an animal or human patient over a period of time greater than 5 minutes, for example between about 30 to about 120 minutes, including each value within the specified range.
  • the present lipid nanoparticle formulation is selectively targeting the lung upon systemic administration.
  • the present lipid nanoparticle formulation is selectively targeting the lung upon administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation.
  • the present lipid nanoparticle formulation is selectively targeting the lung upon administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation.
  • the present lipid nanoparticle formulation is selectively targeting the lung upon intravenous (IV) administration.
  • the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney upon administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation.
  • IV intravenous
  • the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney upon intravenous (IV) administration.
  • Each possibility represents a separate embodiment of the invention.
  • the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration. According to some embodiments, the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration in mammals. According to some embodiments, the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration in humans.
  • the present lipid nanoparticle formulation comprises a nucleic acid and/or protein encapsulated within at least one particle thereof, wherein administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation, in mammals of the formulation results in higher expression in the lungs of the mammal compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney.
  • IV intravenous
  • organs selected from the group consisting of: heart, liver, spleen and kidney.
  • the present lipid nanoparticle formulation comprises a nucleic acid and/or protein encapsulated within at least one particle thereof, wherein intravenous (IV) administration in mammals of the formulation results in higher expression in the lungs of the mammal compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney.
  • IV intravenous
  • the lung expression is higher than each one of the heart, liver, spleen and kidney.
  • the compositions of the present invention may be used as a delivery system to administer a therapeutic agent to its target location in the body, wherein the target location is the lungs.
  • the present invention relates to a method for administering a therapeutic agent, by preparing a composition comprising a lipid as described herein and a biologically active agent (e.g., a therapeutic agent, a protein, a nucleic acid and the like), and administering the composition to a subject in need thereof.
  • a biologically active agent e.g., a therapeutic agent, a protein, a nucleic acid and the like
  • the present invention relates to a method for administering a therapeutic agent, by preparing a particle as described herein comprising an active agent, and administering the composition to a subject in need thereof.
  • the method further comprises encapsulating the active agent within a particle comprising the lipid.
  • these lipid nanoparticle formulations are useful for expression of protein encoded by mRNA.
  • these improved lipid nanoparticles formulations are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA. According to some embodiments, these improved lipid nanoparticle formulations are useful for down- regulating (e.g., silencing) the protein levels and/or mRNA levels of target genes. According to some embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes.
  • the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antibody.
  • a method of delivery of an active agent to the lung comprising the step of systemically administering to a subject in need thereof the lipid nanoparticle formulation of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the active agent is selected from the group consisting of: a protein, a therapeutic agent and a nucleic acid.
  • the active agent is a therapeutic agent.
  • the active agent is a nucleic acid. Exemplary nucleic acids are discussed above.
  • the method comprises the step of administering intravenously (IV), intranasally or via inhalation to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • the method comprises the step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • the administration is systemic.
  • the administration is selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation.
  • IV intravenous
  • the administration is IV.
  • the lipid nanoparticle formulation is for use in the treatment of a lung disease or disorder.
  • the lipid nanoparticle formulation is for use in the treatment of a respiratory disease or disorder.
  • the lipid nanoparticle formulation is for use in the treatment of a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection.
  • a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection.
  • the respiratory infection is caused by a genetic disease or disorder.
  • the lipid nanoparticle formulation is for use in the treatment of a hereditary lung disorder or a genetic lung disease.
  • the present invention provides a method of treating a lung disease or disorder, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides a method of treating a respiratory disease or disorder, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides a method of treating a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation and a pharmaceutically acceptable carrier, diluent or excipient.
  • the respiratory infection is caused by a genetic disease or disorder.
  • the method is for the treatment of a hereditary lung disorder or a genetic lung disease.
  • the method comprises the step of systemically administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • the method comprises the step of administering intravenously (IV), intranasally or via inhalation to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • the method comprises the step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient.
  • the administration in any one of the methods disclosed herein is in the liquid phase.
  • the lipid nanoparticle formulations are administered IV as solutions, emulsions or suspensions.
  • the lipid nanoparticle formulations are administered IV as solutions.
  • the lipid nanoparticle formulations are administered via inhalation as aerosols.
  • the present invention provides a dosage form suitable for administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation, wherein the dosage form comprises at least one container, which contains the present lipid nanoparticle formulation.
  • IV intravenous
  • the dosage form comprises at least one container, which contains the present lipid nanoparticle formulation.
  • the present invention provides a dosage form suitable for IV administration, wherein the dosage form comprises at least one container, which contains the present lipid nanoparticle formulation.
  • the dosage form comprises at least two containers, wherein the first container comprises the present lipid nanoparticle formulation in solid form and the second container comprises a biocompatible solvent suitable for reconstitution prior to IV administration.
  • the biocompatible solvent is an aqueous medium, for example water or saline to maintain electrolyte balance.
  • IV administration can be performed by bolus injections, for example using a hypodermic syringe.
  • IV administration can be performed by continuous infusion using a needle or a plastic or silicone catheter.
  • nanoparticle refers to a nanostructure that is generally or substantially spherical or spheroidal.
  • each dimension of a nanoparticle is in a range of about 1 nm to about 1000 nm
  • the terms “nucleic acid”, “nucleic acid molecules” “oligonucleotide”, “polynucleotide”, and “nucleotide” may interchangeably be used herein.
  • the terms are directed to polymers of deoxy ribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof.
  • the term also encompasses RNA/DNA hybrids.
  • the polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA.
  • the DNA or RNA molecules may be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, Antisense RNA, CRISPR/Cas and the like. Each possibility represents a separate embodiment of the present invention.
  • the terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent inter nucleoside linkages, as well as oligonucleotides having non- naturally occurring portions, which function similarly to respective naturally occurring portions.
  • polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • construct refers to an artificially assembled or isolated nucleic acid molecule which may include one or more nucleic acid sequences, wherein the nucleic acid sequences may include coding sequences (that is, sequence which encodes an end product), regulatory sequences, non-coding sequences, or any combination thereof.
  • the term construct includes, for example, vector but should not be seen as being limited thereto.
  • “Expression vector” refers to constructs that have the ability to incorporate and express heterologous nucleic acid fragments (such as, for example, DNA), in a foreign cell.
  • an expression vector comprises nucleic acid sequences/fragments (such as DNA, mRNA, tRNA, rRNA), capable of being transcribed.
  • the expression vector may encode for a double stranded RNA molecule in the target site.
  • expression refers to the production of a desired end-product molecule in a target cell.
  • the end-product molecule may include, for example an RNA molecule; a peptide or a protein; and the like; or combinations thereof.
  • introducing and “transfection” may interchangeably be used and refer to the transfer of molecules, such as, for example, nucleic acids, polynucleotide molecules, vectors, and the like into a target cell(s), and more specifically into the interior of a membrane-enclosed space of a target cell(s).
  • the molecules can be "introduced” into the target cell(s) by any means known to those of skill in the art, for example as taught by Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001), the contents of which are incorporated by reference herein.
  • Means of "introducing" molecules into a cell include, for example, but are not limited to: heat shock, calcium phosphate transfection, PEI transfection, electroporation, lipofection, transfection reagent(s), viral-mediated transfer, and the like, or combinations thereof.
  • the transfection of the cell may be performed on any type of cell, of any origin, such as, for example, human cells, animal cells, plant cells, virus cell, and the like.
  • the cells may be selected from isolated cells, tissue cultured cells, cell lines, cells present within an organism body, and the like.
  • treating refers to abrogating, inhibiting, slowing or reversing the progression of a disease or condition, ameliorating clinical symptoms of a disease or condition or preventing the appearance of clinical symptoms of a disease or condition.
  • preventing is defined herein as barring a subject from acquiring a disorder or disease or condition.
  • treatment of cancer is directed to include one or more of the following: a decrease in the rate of growth of the cancer (i.e. the cancer still grows but at a slower rate); cessation of growth of the cancerous growth, i.e., stasis of the tumor growth, and, the tumor diminishes or is reduced in size.
  • the term also includes reduction in the number of metastases, reduction in the number of new metastases formed, slowing of the progression of cancer from one stage to the other and a decrease in the angiogenesis induced by the cancer. In most preferred cases, the tumor is totally eliminated. Additionally included in this term is lengthening of the survival period of the subject undergoing treatment, lengthening the time of diseases progression, tumor regression, and the like.
  • the cancer is a blood cancer.
  • liver bypass refers to a property of a lipid nanoparticle (LNP) formulation wherein the accumulation of the LNPs and/or the expression of a delivered payload (e.g., mRNA) in the liver is substantially reduced relative to other organs, such as the lungs or spleen.
  • LNP lipid nanoparticle
  • mRNA delivered payload
  • liver bypass may be reflected by a liver-to-target organ (e.g., lung or spleen) expression ratio of less than about 30%, 20%, 10%, 5%, or even less than about 1%, as determined by normalized luciferase activity or other quantitative expression assays.
  • the liver-to-lung expression ratio is less than about 30%, 20%, 10%, 5%, or 1%, indicating preferential accumulation or expression in the lungs relative to the liver.
  • the extent of liver bypass may be determined experimentally, for example by administering an LNP formulation comprising an mRNA encoding a reporter protein such as luciferase, and subsequently quantifying organ-specific expression using bioluminescent imaging or tissue-based luciferase assays.
  • a formulation is considered to exhibit liver bypass when the liver shows minimal or significantly lower expression levels compared to one or more target organs, such as the lungs.
  • Liver bypass may result from various formulation parameters, including but not limited to lipid composition, charge ratio, helper lipids, PEG-lipid content, and overall particle size or surface properties.
  • lung targeting formulation refers to a lipid nanoparticle (LNP) composition that, upon systemic administration, preferentially delivers its payload (e.g., mRNA) to the lung tissue relative to other organs.
  • Such formulations result in enhanced accumulation of the LNPs and/or increased expression of the delivered nucleic acid in the lungs, as determined by appropriate in vivo or ex vivo assays, including but not limited to luciferase-based bioluminescence imaging or tissue lysate quantification.
  • Lung targeting may be achieved by modifying one or more characteristics of the LNP formulation, such as lipid composition, molar ratios, helper lipid identity, PEG-lipid content, particle size, surface charge, or other physicochemical properties.
  • lung targeting formulations also exhibit liver bypass properties, such that the liver receives minimal expression relative to the lung. Examples EXAMPLE 1: Synthesis of ionizable lipids Example 1A: Synthesis of lipid IA-12:
  • Linoleic aldehyde (3.20 g, 12.12 mmol, 1 equiv.) and ethanolamine (0.89 ml, 14.54 mmol, 1.2 equiv.) were dissolved in dry CH 2 Cl 2 (60 mL) under nitrogen atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (5.10 g, 24.24 mmol, 2 equiv.) was added portion wise over a period of 15 min, and stirred it for 16 hr at the same temperature. Later, the reaction mixture was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times).
  • FIG. 1A is a 1H NMR spectrum of lipid IA-12 (NV3-006).
  • Figure 1B is an ESI-MS spectrum of lipid IA- 12 (NV3-006).
  • FIG. 1C is a 1H NMR spectrum of lipid IA-11 (NV3-004).
  • Figure 1D is an ESI-MS spectrum of lipid IA- 11 (NV3-004).
  • Example 1C Synthesis of lipid IA-10:
  • reaction mixture was quenched with sat. NaHCO 3 and extracted with CH 2 Cl 2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na 2 SO 4 . The solvent was evaporated on rotary evaporator and the crude product was purified by column chromatography using 0-5% isopropanol in chloroform to obtain the 13 (564 mg, 89%) as a pale yellow color viscus liquid.
  • FIG. 1E is a 1 H NMR spectrum of lipid IA-10 (NV3-002).
  • Figure 1F is an ESI-MS spectrum of lipid IA- 10 (NV3-002).
  • Examples 1D and 1E Syntheses of lipids II-1 and II-5: To a solution of hexanol (4.0 g, 39.2 mmol, 1 equiv.) in dry CH2Cl2 (100 mL), molecular sieves (4 ⁇ MS) were added under argon atmosphere.
  • PCC (12.64 g, 58.8 mmol, 1.5 equiv.) was added portion wise over a period of 10 min and stirred for 2 hr at room temperature. After completing the reaction filtered it through a silica gel pad using CH 2 Cl 2 to remove PCC. The solvent was evaporated on rotary evaporator with low vacuum to get a solution of hexanal (20 mL). The aldehyde solution was dried over anhydrous sodium sulfate and subjected to the next step.
  • the filtrate was diluted with water and extracted with diethyl ether (4 x 10 mL).
  • the organic layer dried over anhydrous Na 2 SO 4 and the solvent was removed on rotary evaporator.
  • the crude product was dried and subject to the next step.
  • the crude product was dissolved in dry CH2Cl2 (20 mL) and ethanolamine (27 ⁇ L, 0.45 mmol, 0.475 equiv.) was added under argon atmosphere and stirred for 2 hr. at room temperature.
  • sodium triacetoxyborohydride (298 mg, 1.41 mmol, 1.5 equiv.) was added portion wise and stirred for 24 hr at the same temperature.
  • Example 1G Synthesis of lipid IB-4 (NV2-004): To a clean flask were charged 10-((dihexylamino)oxy)-10-oxodecan-1-ol (1 wt, 1.0 equiv) and PCC, 98% (2 wt, 2.0 equiv). The reagents were suspended in anhydrous DCM (5 vol) and celite (3 wt) was added to the mixture. The reaction mixture was stirred at 18 to 25 °C for at least 3 h or until complete by TLC analysis in 20% (v/v) EtOAc in hexane.
  • FIG. 26A is a 1H NMR spectrum of lipid IB-4 (NV2-004).
  • Figure 26B is an ESI-MS spectrum of lipid IB- 4 (NV2-004).
  • Example 1H Synthesis of lipid IB-13 (NV2-015):
  • NV2-015 2-(didodecylamino) ethyl 6-(dodecyl(2-(2-hydroxyethoxy) ethyl) amino)hexanoate (NV2-015) as a pale-yellow liquid in pure and 0.20g of NV2-015 as yellow liquid in less pure.
  • Figure 27A is a 1H NMR spectrum of lipid IB-13 (NV2-015).
  • Figure 27B is an ESI-MS spectrum of lipid IB-13 (NV2-015).
  • Example 1I Synthesis of lipid IB-9 (NV2-009): Experimental procedure for NV2-009 Synthesis of 10-oxodecanoic acid (2) To a stirred solution of IBX (2.602g, 9.295mmol, 1.75equiv.) in DMSO (13 mL) was added a solution of 10-hydroxydecanoic acid (1.0g, 5.311 mmol, 1.0 equiv.) in THF (10 mL) at rt, stirred for 4-5h. The progress of the reaction monitored by TLC analysis (60% EtOAc in Hexane). After completion of the reaction, the reaction mixture quenched with the addition of H 2 O (20ml), solids were precipitated out which were collected by filtration.
  • TLC analysis 50% EtOAc in Hexane
  • FIG. 29A is a 1H NMR spectrum of lipid IB-24 (NV2-027).
  • Figure 29B is an ESI-MS spectrum of lipid IB-24 (NV2-027).
  • Example 1K Synthesis of lipid IA-13 (NV3-013):
  • Figure 30B is an ESI-MS spectrum of lipid IA-13 (NV3-013).
  • Example 1L Synthesis of lipid IA-14 (NV3-014): Experimental procedure for NV3-014 Synthesis of ethane-1,2-diyl bis(10-bromodecanoate) (2): To a stirred solution of ethylene glycol (0.3 g, 4.8 mmol, 1.0 equiv.) and 10-bromodecanoic acid (3.61 g, 14.4 mmol,1.2 equiv.) in DCM (60 mL) was added DMAP (0.238g, 1.900 mmol, 0.4 equiv.) at room temperature, stirred for 10 min.
  • Figure 31B is an ESI-MS spectrum of lipid IA-14 (NV3-014).
  • Example 1M Synthesis of lipid IA-15 (NV3-016): Experimental procedure for NV3-016 Synthesis of (Z)-1-bromooctadec-9-ene (1) To a stirred solution of Oleyl alcohol (5.0g, 18.622 mmol, 1.0 equiv.) in DCM (100 mL) was added triphenyl phosphine (5.85g, 22.347 mmol, 1.2 equiv.), stirred for 10 min then carbon tetra bromide (7.41g, 22.347 mmol, 1.2 equiv.) added in one portion. Then the reaction mixture stirred at room temperature for overnight.
  • FIG. 32A is a 1H NMR spectrum of lipid IA-15 (NV3-016).
  • Figure 32B is an ESI-MS spectrum of lipid IA-15 (NV3-016).
  • EXAMPLE 2 Preparation and characterization of LNPs comprising ionizable lipids Ionizable lipids were synthesized as detailed in Example 1. Other lipids DSPC, DOPE, DOTAP, Cholesterol and DMG-PEG were purchased from Avanti polar lipids.
  • Example 2A LNPs comprising ionizable lipids, including a permanently charge lipid: Lipid nanoparticles were synthesized by microfluidic mixing device. Briefly, one volume of lipid mix (Ionizable lipid, DOTAP, DOPE, DMG-PEG, Cholesterol at 30:25:10:32.5:2.5 mol ratio) in ethanol solution and three volumes of mRNA (lipid to mRNA ratio at 40:1w/w) in citrate buffer (pH 4.5) were mixed through a microfluidic mixing device Ignite (Precision Nanosystems Inc) at total flow rate of 12 ml/min.
  • Ignite Precision Nanosystems Inc
  • Example 2B LNPs comprising ionizable lipids, without a permanently charge lipid: Lipid nanoparticles were synthesized by microfluidic mixing device.
  • EXAMPLE 3 RNA encapsulation and quantification
  • the Quant-iT RiboGreen RNA assay kit (Life Technologies) was used to measure the mRNA encapsulation in LNPs.
  • 0.5 ⁇ L of LNP was diluted in a final volume of 100 ⁇ L of TE buffer (20 m admir EDTA, 10 m Understand Tris-HCL) with or without Triton X-100 (1%, Sigma-Aldrich). Samples were loaded in a 96-well black plate (Costar, Corning). The plate was incubated for 5 min at 37 °C before adding 100 ⁇ L of RiboGreen in TE buffer (1:200 v/v) to each well.
  • EXAMPLE 4 Size and ⁇ -potential analysis of LNPs Nano size and ⁇ -potential of mRNA-LNPs were analyzed by dynamic light scattering (DLS) using a Malvern nano ZS ⁇ -sizer (Malvern Instruments). Briefly, mRNA-LNPs were diluted in double-distilled water (1:50, volume ratio) and PBS (1:50, volume ratio) for ⁇ potential and size measurements, respectively. The physico-chemical properties - particle size, polydispersity index (PDI), Zeta potential and encapsulation efficiency (EE%) - of Formulations 1, 2, 3, 4 and 5 are presented in Table 3.
  • PDI polydispersity index
  • EE% Zeta potential and encapsulation efficiency
  • FIG. 3A-E Luciferase expression was not observed mostly in lungs for all the liquid nanoparticles (LNPs) which are devoid of permanently charged lipids.
  • Figure 4A is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 5 (left group), Formulation 1 (second from left group), Formulation 2 (middle group), Formulation 3 (second from right group) and Formulation 4 (right group).
  • Figure 4B is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 10 (left group), Formulation 6 (second from left group), Formulation 7 (middle group), Formulation 8 (second from right group) and Formulation 9 (right group).
  • Figure 5 is a histogram analysis of Luciferase expression comparing between Lung (circles), liver (squares) and spleen (triangles) for Formulation 5 (left group), Formulation 2 (second from left group), Formulation 4 (middle group), Formulation 3 (second from right group) and Formulation 1 (right group).
  • EXAMPLE 6 Preparation and characterization of LNPs comprising ionizable lipids Ionizable lipids were synthesized as detailed in Example 1. Other lipids DSPC, DOPE, DOTAP, Cholesterol and DMG-PEG were purchased from Avanti polar lipids. Lipid nanoparticles were synthesized as described in Example 2A. The details of Formulations 11-26 are presented in Table 6: Table 6: Formulation details (with permanently charge lipid)
  • RNA encapsulation and quantification and Size and ⁇ -potential analysis of LNPs RNA encapsulation and quantification of Formulations 11-26 were performed as described in Example 3. Size and ⁇ -potential analysis of LNPs were performed as described in Example 4. The physico-chemical properties - particle size, polydispersity index (PDI), Zeta potential and encapsulation efficiency (EE%) - of Formulations 1126 are presented in Table 7. Table 7: Physico-chemical properties of formulations with permanently charge lipid
  • EXAMPLE 8 In vivo luciferase assay & organ distribution study - Formulations 11 and 18-22 LNPs composed of iL, DOTAP, DOPE, Cholesterol & DMG-PEG at 30:25:10:32.5:2.5 mole ratios were formulated with Luc-mRNA respectively. Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analyzed for Luciferase expression by IVIS imaging system.
  • Figure 7 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group).
  • Figure 8 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21 (right group).
  • Figure 9 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21 (right group).
  • EXAMPLE 9 In vivo luciferase assay & organ distribution study – Formulations 11, 17-18 and 25-26 LNPs composed of iL, DOTAP or DOTMA, DOPE, Cholesterol & DMG-PEG at 30:25:10:32.5:2.5 mole ratios were formulated with Luc-mRNA respectively (F3.1 or F3.7). Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analyzed for Luciferase expression by IVIS imaging system.
  • Figures 10A-E are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 10A), Formulation 17 (Figure 10B), Formulation 18 (Figure 10C), Formulation 26 ( Figure 10D), and Formulation 25 ( Figure 10E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 11 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group), and Formulation 25 (right group).
  • Figure 12 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group).
  • Figure 13 is bar chart showing luciferase expression in the lungs were compared between DOTAP/DOTMA formulations; Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group).
  • the effect of cationic lipid DOTAP or DOTMA in the formulation has no significant effect on lung distribution and expression.
  • the lipid NV1-001 composed of DOTMA has lowered liver distribution
  • the lipid NV3-004 has no difference.
  • EXAMPLE 10 In vivo luciferase assay & organ distribution study – Formulations 11, 16 and 24 LNPs composed of iL, DOTAP, DOPE or DSPC, Cholesterol & DMG-PEG at 30:25:10:32.5:2.5 mole ratios were formulated with Luc-mRNA respectively (F3.1 or F3.6). Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analysed for Luciferase expression by IVIS imaging system.
  • Figures 14A-C are IVIS imaging analysis for Luciferase expression of Formulation 11 ( Figure 14A), Formulation 16 ( Figure 14B), and Formulation 24 (Figure 10C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 15 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 11 (left group), Formulation 16 (middle group) and Formulation 24 (right group).
  • Figure 16 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group).
  • Figure 17 is bar chart showing luciferase expression in the lungs were compared between DSPC vs DOPE formulations; Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group).
  • Formulations composed of either DOPE or DSPC helper lipids have no significant effect on lung distribution. The protein expression was similar in the lungs and other organs as well.
  • EXAMPLE 11 In vivo luciferase assay & organ distribution study – Formulations 11-15 LNPs composed of iL (NV1-001), DOTAP, DOPE, Cholesterol & DMG-PEG at various DOTAP mole ratios were formulated with Luc-mRNA. Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analysed for Luciferase expression by IVIS imaging system.
  • Figures 18A-E are IVIS imaging analysis for Luciferase expression of Formulation 12 (Figure 18A), Formulation 15 (Figure 18B), Formulation 11 (Figure 18C), Formulation 13 ( Figure 18D), and Formulation 14 (Figure 18C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 19 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 12 (left group), Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group).
  • Figure 20 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group).
  • Figure 21 is bar chart showing luciferase expression in the lungs were compared between different formulations with increasing DOTAP amount; Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group).
  • the bio-distribution of NV1-001 LNPs varied on DOTAP amount.
  • Significant protein expression in the lungs compared to liver was observed with 20% DOTAP.
  • EXAMPLE 12 In vivo luciferase assay & organ distribution study – Formulations 15 and 23 LNPs composed of iL (1-001 or 1-005), DOTAP, DOPE, Cholesterol & DMG-PEG at 20:20:10:38.5:2.5 mole ratios were formulated with Luc-mRNA respectively (F3.1 or F3.6). Mice were administered Luc- mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analysed for Luciferase expression by IVIS imaging system.
  • Figures 22A-B are IVIS imaging analysis for Luciferase expression of Formulation 15 ( Figure 22A), and Formulation 23 (Figure 22B) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row).
  • Figure 23 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “ ⁇ ” diagonal lines) for Formulation 15 (left group), and Formulation 23 (right group).
  • Figure 24 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (left group), and Formulation 23 (right group).
  • Figure 25 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 15 (left group), and Formulation 23 (right group). The lung distribution between the two different ionizable lipids formulations each containing equal amount of DOTAP amount were similar.

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Abstract

The present invention provides lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications. The present formulation is specifically directed to compositions comprising ionizable lipids and a lipid, which comprises a quaternary ammonium group as a permanently charged lipid.

Description

LIPIDS NANOPARTICLE FORMULATIONS SUITABLE FOR LUNG TARGETING FIELD OF THE INVENTION The present invention provides lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications. The present formulation is specifically directed to compositions comprising ionizable lipids and a lipid, which comprises a quaternary ammonium group as a permanently charged lipid. BACKGROUND OF THE INVENTION Therapeutic nucleic acids including small interfering RNA (siRNA), micro RNA (miRNA), antisense oligonucleotides, messenger RNA (mRNA), ribozymes, pDNA and immune stimulating nucleic acids act via a variety of mechanisms. Specific proteins can be downregulated by siRNA or miRNA through RNA interference (RNAi). Hematopoietic cells, such as leukocytes in general, and primary T lymphocytes and B- cells in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Modulating immune cells function, such as T cells and B cells, by downregulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity and viral infections. The therapeutic applications of RNAi are extremely broad, since siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against a target protein. To date, siRNA constructs have shown the ability to specially silence target proteins in both in vitro and in vivo models. These are currently being evaluated in clinical studies. Messenger RNA (mRNA) is the family of large RNA molecules which transport the genetic information from DNA to ribosome. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products. Such nucleic acids would be useful in the treatment to the of diseases related deficiency of a protein or enzyme. However, there are many problems associated with nucleic acids in therapeutic contexts. One of the major problems with therapeutic nucleic acids is the stability of the phosphodiester inter nucleotide link and its susceptibility to nucleases. Apart from that these nucleic acids have limited ability to cross the cell membrane. Various lipids, e.g., cationic lipids, have proved to be excellent carriers of nucleic acids to treat different diseases in gene therapy applications. Lipid nanoparticles formed from cationic lipids and other co-lipids 1 such as cholesterol, DSPC and PEGylated lipids encapsulated oligonucleotides which protect them from degradation and facilitate the cellular uptake. Dedmi et al. (Biomaterials 31 (2001, 6867-6875) report the systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation. Ramesh (Methods Mol Biol. 2008;433:301-31) discloses a DOTAP:cholesterol-based nanoparticle - mediated gene delivery to the lung. WO 2018/087753 discloses a cationic lipid comprising a functional group represented by the structure: -W- (T=O)m-X-(CH2)z-Y, wherein X and Y are each independently O, N or NH, wherein X and Y cannot both be O; W is a bond, O, NH or S; T is C or S; m is 0 or 1; and z is 0 or 2, wherein said functional group is linked to at least one saturated or unsaturated fatty acid residue. Cheng et al. (Nat Nanotechnol. 2020 April ; 15(4): 313-320) discloses selective organ targeting nanoparticles for tissue specific mRNA delivery and CRISPR/Cas gene editing. WO 2020/051223 discloses a composition comprising: (A) a therapeutic agent; and (B) a lipid nanoparticle composition comprising: (1) a selective organ targeting compound; (2) an ionizable cationic lipid; and (3) a phospholipid; wherein the composition preferentially delivers the nucleic acid to a target organ selected from the lungs, the heart, the brain, the spleen, the lymph nodes, the bones, the skeletal muscles, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, or the skin. WO 2020051220 discloses a composition comprising: (A) a therapeutic agent; and (B) a lipid nanoparticle composition comprising: (1) a selective organ targeting compound: (2) an ionizable cationic lipid; and (3) a phospholipid; wherein the composition preferentially delivers the nucleic acid to a target organ selected from the lungs, the heart, the brain, the spleen, the lymph nodes, the bone marrow, the bones, the skeletal muscles, the stomach, the small intestine, the large intestine, the kidneys, the bladder, the breast, the liver, the testes, the ovaries, the uterus, the spleen, the thymus, the brainstem, the cerebellum, the spinal cord, the eye, the ear, the tongue, or the skin WO 2022/204043 discloses a method for potent delivery to a non-liver basal cell of a subject, comprising: intravenously administering to said subject a composition comprising a therapeutic agent assembled with a lipid composition which comprises: (i) an ionizable cationic lipid; and (ii) a selective organ targeting (SORT) lipid separate from said ionizable cationic lipid, wherein said SORT lipid effects delivery of said therapeutic agent to said non-liver basal cell of said subject characterized by a greater amount or activity of said therapeutic agent in said non-liver basal cell compared to that achieved with a reference lipid composition. WO 2022/216619 discloses a method for enhancing an expression or activity of cystic fibrosis transmembrane conductance regulator (CFTR) protein in a cell, the method comprising: (a) contacting said cell with a nucleic acid editing system assembled with a lipid composition, which nucleic acid editing system comprises (i) a guide nucleic acid, (ii) a heterologous polypeptide comprising an endonuclease or a heterologous polynucleotide encoding said heterologous polypeptide, and (iii) a donor template nucleic acid, to yield a complex of said heterologous endonuclease with said guide nucleic acid in said cell; (b) cleaving a CFTR gene or transcript in said cell with said complex at a cleavage site to yield a cleaved CFTR gene or transcript; and (c) using said donor template nucleic acid to repair said cleaved CFTR gene or transcript to yield a repaired CFTR gene or transcript encoding a functional CFTR protein in said cell, thereby enhancing said expression or activity of CFTR protein in said cell. WO 2022/201167 discloses lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications. Sun et al. (AAPS PharmSciTech. 2022 May 9;23(5):135) describe optimization of 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP)/cholesterol cationic lipid nanoparticles for mRNA, pDNA, and oligonucleotide delivery. Nevertheless, there remains a need in the art for suitable and efficient delivery platforms for targeted delivery of nucleic acid and therapeutic agents to specifically the lungs. SUMMARY OF THE INVENTION The present invention relates to novel lipid nanoparticle (LNP) formulations. These lipid nanoparticles protect nucleic acids from degradation, clearance from circulation and intracellular release. In addition, the nucleic acid encapsulated lipid nanoparticles advantageously are well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient. The present LNP formulations were surprisingly found to specifically target the lung with significantly higher lung expression compared to other organs, including the heart, liver, spleen and kidney. The results provided herein indicate that given similar systemic exposure, the formulations in accordance with the principles of the invention is unexpectedly more effective in delivering biological agents specifically to the lungs than hitherto known formulations. The high specificity of the present lipid nanoparticle formulations in lung targeting is highly advantageous, as it reduces the side effects that accompany the delivery of active agents to undesired sites and organs. Advantageously, this high specificity also enables use of lower dosages, as less active agent is lost, which, in its turn, also contributes to side effect reduction. The present lipid nanoparticle formulations comprise at least one ionizable lipid which is represented by the structure of Formula (IA), Formula (IB) and/or Formula (II) and at least one permanently charged lipid. According to some embodiments, the permanently charged lipid includes a quaternary ammonium moiety. According to some embodiments, the quaternary ammonium moiety comprises a tetra-alkyl ammonium group. According to some embodiments, the permanently charged lipid is selected from the group consisting of:1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DORI), 1,2-dimyristoyl- 3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), Dimethyldioctadecylammonium (DDAB) salts and combinations thereof. According to some embodiments, the permanently charged lipid is DOTAP. Specifically, DOTAP is used in the formulations described herein as a permanently charged lipid. Surprisingly, in contrast with previous neutral lipids, permanently charged lipids were found to exhibit remarkable properties that enable the present formulations to target the lungs. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride salt is represented by the chemical structure drawn below: As specified herein, DOTAP is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof. The terms “DOTAP” and “DOTAP salt” as used herein are intended to include any DOTAP salts, including any counter anion. Suitable counter anions for DOTAP include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like. 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), chloride salt is represented by the chemical structure drawn below: DOTMA-chloride As specified herein, DOTMA is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof. The terms “DOTMA” and “DOTMA salt” as used herein are intended to include any DOTMA salts, including any counter anion. Suitable counter anions for DOTMA include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like. N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DORI), bromide salt is represented by the chemical structure drawn below: DORI-bromide As specified herein, DORI is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof. The terms “DORI” and “DORI salt” as used herein are intended to include any DORI salts, including any counter anion. Suitable counter anions for DORI include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like. 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane chloride salt is represented by the chemical structure drawn below: DMTAP -chloride As specified herein, DMTAP is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof. The terms “DMTAP” and “DMTAP salt” as used herein are intended to include any DMTAP salts, including any counter anion. Suitable counter anions for DMTAP include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like. 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) chloride salt is represented by the chemical structure drawn below: DSTAP-chloride As specified herein, DSTAP is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof. The terms “DSTAP” and “DSTAP salt” as used herein are intended to include any DSTAP salts, including any counter anion. Suitable counter anions for DSTAP include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like. Dimethyldioctadecylammonium (DDAB) bromide salt is represented by the chemical structure drawn below: DDAB As specified herein, DDAB is an ammonium compound and such is permanently charged at the quaternary nitrogen atom thereof. Although the letter B in DDAB stands for bromide, the terms “DDAB” and “DDAB salt” refer to any dimethyldioctadecylammonium salt, which may be bromide or other as described herein. Specifically, the terms “DDAB” and “DDAB salt” as used herein are intended to include any dimethyldioctadecylammonium salts, including any counter anion. Suitable counter anions for dimethyldioctadecylammonium include, but are not limited to, halides (e.g., chloride, bromide, etc.), sulfate derivatives (e.g., triflate, etc.), borate derivatives (e.g., tetrafluoroborate, etc.), phosphate derivatives (e.g. H2PO4-, etc.) and the like. Thus, according to some embodiments, the present invention provides a lipid nanoparticle formulation comprising: at least one ionizable lipid which is represented by the structure of Formula (IA), Formula (IB) or Formula (II), or salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof, wherein the structures of Formula (IA), Formula (IB) and Formula (II) are represented below; and at least one permanently charged lipid having a quaternary ammonium moiety or a salt thereof; Formula (II): , wherein R1C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; R2C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; Y1C is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; W1C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; R5C is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NR NIIRNII’, wherein each one of RNII and RNII’ is individually C1-4 alkyl or R NII and RNII’ together with the nitrogen to which they are bound, form a ring; W2C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; Y2C is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; R3C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; and R4C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl; Formula (IA):
, wherein R1A is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNARNA’, wherein each one of RNA and RNA’ is individually C1-4 alkyl or RNA and RNA’ together with the nitrogen to which they are bound, form a ring; R2A is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; nA is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; XA is selected from the group consisting of:-COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, - OCONH- and -NHCONH-; jA is selected from the group consisting of: 0, 1, 2, 3 and 4; YA is selected from the group consisting of: absent, -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-; mA is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; R3A is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNA’’RNA’’’, wherein each one of RNA’’ and RNA’’’ is individually C1-4 alkyl or RNA’’ and RNA’’’ together with the nitrogen to which they are bound, form a ring; and R4A is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; Formula (IB): wherein R1B is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNBRNB’, wherein each one of RNA and RNA’ is individually C1-4 alkyl or RNA and RNA’ together with the nitrogen to which they are bound, form a ring; R2B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; WB is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; YB is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; R3B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; and R4B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. According to some embodiments, the lipid nanoparticle formulation comprises one ionizable lipid represented be Formula (IA), Formula (IB) or Formula (II). According to some embodiments, the According to some embodiments, the lipid nanoparticle formulation comprises a plurality of ionizable lipids represented be Formula (IA), Formula (IB) and/or Formula (II). The term “at least one” means a single element or object or a plurality of elements and/or objects. The term “plurality” means two or more elements or objects. It is to be understood that “one ionizable lipid” relates to a single species of and ionizable lipid, which may include a plurality of molecules of the same kind. According to some embodiments, the ionizable lipid is represented by the structure of Formula (IA). It is to be understood that specifying that the lipid nanoparticle formulation comprises one ionizable lipid represented be Formula (IA), Formula (IB) or Formula (II) and that the ionizable lipid is represented by the structure of Formula (IA), broadly cover both the option that the lipid nanoparticle formulation comprises only ionizable lipid(s) of Formula (IA) (i.e., not of Formulae (IB) and/or (II)), and the option that the lipid nanoparticle formulation comprises ionizable lipids of Formulae (IB)/(II), as long as the lipid nanoparticle formulation comprises at least one ionizable lipid of Formula (IA). Also, this embodiment comprises lipid nanoparticle formulations that comprise ionizable lipid(s) that are not covered in any of Formulae (IA)/(IB)/(II), as long as the lipid nanoparticle formulation comprises at least one ionizable lipid of Formula (IA). Similar embodiments, which state that the ionizable lipid is represented by the structure of Formula (IB) or (II) have similar breadth. According to some embodiments, the ionizable lipid is represented by the structure of Formula (IA) or Formula (IB). According to some embodiments, the ionizable lipid is represented by the structure of Formula (IA) or Formula (II). According to some embodiments, the ionizable lipid is represented by the structure of Formula (IB) or Formula (II). According to some embodiments, R1A is selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2A is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25, -(CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17 and -(CH2)7CH=CHCH2CH=CHC4H9. Each possibility represents a separate embodiment of the invention. According to some embodiments, nA is selected from the group consisting of: 8, 9 and 10. Each possibility represents a separate embodiment of the invention. According to some embodiments, nA is selected from the group consisting of: 8, 9 and 10, and jA is 0 or 2, or a combination thereof. According to some embodiments, XA is selected from the group consisting of: -COO-, -OOC-, -NHCO- and -CONH. According to some embodiments, XA is selected from the group consisting of: -COO- and -OOC-. According to some embodiments, XA is -COO- and the lipid is represented by Formula (IA1) . According to some embodiments, jA is 0 or 2. Each possibility represents a separate embodiment of the invention. According to some embodiments, YA is selected from the group consisting of: absent, -COO-, -OOC-, - NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-. Each possibility represents a separate embodiment of the invention. According to some embodiments, YA is selected from the group consisting of: absent, -COO- and -OOC-. According to some embodiments, YA is absent or -OOC-. Each possibility represents a separate embodiment of the invention. According to some embodiments, YA is absent, jA is 0 and the lipid of Formula (IA) is represented by Formula (IA2): . According to some embodiments, YA is absent, jA is 0, XA is -COO- and the lipid of Formula (IA) is represented by Formula (IA3): . According to some embodiments, mA is selected from the group consisting of: 8, 9, and 10. Each possibility represents a separate embodiment of the invention. According to some embodiments, R3A is selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH. Each possibility represents a separate embodiment of the invention. According to some embodiments, R4A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25, -(CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17 and -(CH2)7CH=CHCH2CH=CHC4H9. Each possibility represents a separate embodiment of the invention. According to some embodiments, the ionizable lipid is selected from the group consisting of: lipid IA-4, lipid IA-5, lipid IA-8, lipid IA-10, lipid IA-11, lipid IA-12, lipid IA-13, lipid IA-14, and lipid IA-15 and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the ionizable lipid is IA-10 or IA-12. According to some embodiments, the ionizable lipid is IA-10. According to some embodiments, the ionizable lipid is IA-12. According to some embodiments, the ionizable lipid is represented by the structure of Formula (IB). According to some embodiments, R1B is selected from the group consisting of: -CH2CH2OCH2CH2OH, -CH2CH2Cl, -CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2CH2OH and -CH2CH2NMe2. Each possibility represents a separate embodiment of the invention. According to some embodiments, R1B is selected from the group consisting of: CH2CH2OCH2CH2OH, -CH2CH2Cl or -CH2CH2OH. According to some embodiments, R2B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2B is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, - (CH2)7CH=CHCH2CH=CHC6H13, -(CH2)8CH=CHC8H17 and -C12H25. Each possibility represents a separate embodiment of the invention. According to some embodiments, WB is a C5-9 unsubstituted alkylene. According to some embodiments, WB selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, YB is selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-. Each possibility represents a separate embodiment of the invention. According to some embodiments, YB is absent and the lipid of Formula (IB) is represented by Formula (IB1): . According to some embodiments, R3B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R3B is a C4-16 alkyl. According to some embodiments, R4B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R4B is a C4-16 alkyl. According to some embodiments, the ionizable lipid is selected from the group consisting of: lipid IB-4, lipid IB-6, lipid IB-9, lipid IB-10, lipid IB-13, lipid IB-24 and a combination thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the ionizable lipid is selected from the group consisting of: IB-4, IB-6, IB-10 and a combination thereof. According to some embodiments, the ionizable lipid is IB-4. According to some embodiments, the ionizable lipid is represented by the structure of Formula (II). According to some embodiments, R1C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R1C is a C4-10 alkyl. According to some embodiments, R1C is C6H13. According to some embodiments, R1C is n- C6H13. According to some embodiments, R2C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2C is a C4-10 alkyl. According to some embodiments, R2C is C6H13. According to some embodiments, R2C is n- C6H13. According to some embodiments, Y1C is selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of Y1C and Y2C is absent and the lipid of Formula (II) is represented by Formula (II1): . According to some embodiments, W1C is a C5-9 unsubstituted alkylene. According to some embodiments, W1C is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of W1C and W2C is a C4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II2): . According to some embodiments, each one of Y1C and Y2C is absent, each one of W1C and W2C is a C4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II3): . According to some embodiments, R5C is selected from the group consisting of: OH, -(CH2CH2O)2-3H, C1-4 hydroxyalkyl and C1-4 haloalkyl and C1-3 alkylene-NR NIIRNII’, wherein each one of RNII and RNII’ is individually C1-4 alkyl or R NII and RNII’ together with the nitrogen to which they are bound, form a 5-6 membered ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, R5C is selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, - CH2CH2CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2N(CH2)5, -CH2CH2N(CH2)5, -CH2CH2Cl, -OH and -CH2CH2NMe2. Each possibility represents a separate embodiment of the invention. According to some embodiments, R5C is -CH2CH2OH, or -CH2CH2OCH2CH2OH. According to some embodiments, W2C is a C5-9 unsubstituted alkylene. According to some embodiments, W2C is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y2C is selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-. Each possibility represents a separate embodiment of the invention. According to some embodiments, R3C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R3C is a C4-10 alkyl. According to some embodiments, R3C is C6H13. According to some embodiments, R4C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R4C is a C4-10 alkyl. According to some embodiments, R4C is C6H13. According to some embodiments, the ionizable lipid is lipid II-1 or II-5. According to some embodiments, the ionizable lipid is lipid II-. According to some embodiments, the ionizable lipid is lipid II-5. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB-22, IB-23, IB-24, II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II- 15, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-23 and II-24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment of the invention. The chemical structures of each of the specific lipids are detailed below in the “Exemplary Ionizable Lipids” Section and in the claims. According to some embodiments, the permanently charged lipid is selected from the group consisting of:1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DORI), 1,2-dimyristoyl- 3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), dimethyldioctadecylammonium (DDAB), salts and combinations thereof. According to some embodiments, the permanently charged lipid is DOTAP or DOTMA. According to some embodiments, the permanently charged lipid is DOTAP. According to some embodiments, the permanently charged lipid is DOTMA. According to some embodiments, the lipid nanoparticle formulation comprises 10 to 50 mol% of the ionizable lipid, including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 40 mol% of the ionizable lipid. According to some embodiments, the lipid nanoparticle formulation comprises 15 to 30 mol% of the ionizable lipid. According to some embodiments, the lipid nanoparticle formulation comprises 10 to 40 mol% permanently charged lipid(s), including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation comprises 15 to 35 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises about 20 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation further comprises at least one neutral lipid. According to some embodiments, the neutral lipid comprises a sterol, a phospholipid or both. According to some embodiments, the sterol comprises cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 40 mol% cholesterol, including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation comprises 30 to 40 mol% cholesterol. According to some embodiments, the phospholipid comprises a phosphatidylethanolamine. According to some embodiments, the phosphatidylethanolamine is selected from the group consisting of: 1,2-dilauroyl- L-phosphatidyl-ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) 1,3-Dipalmitoyl-sn-glycero-2- phosphoethanolamine (1,3-DPPE), 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), biotin-phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or combinations thereof. According to some embodiments, the phosphatidylethanolamine comprises 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). According to some embodiments, the phospholipid comprises 1,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), Distearoylphosphatidylcholine (DSPC) or both. Each possibility represents a separate embodiment of the invention. According to some embodiments, the phospholipid comprises DOPE. According to some embodiments, the phospholipid comprises DSPC. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 15 mol% DSPC. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE, DSPC or both. It is to be understood that the phrase “the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE, DSPC or both” is intended to mean that the lipid nanoparticle formulation comprises one of the following: 5 to 15 mol% DOPE; 5 to 15 mol% DSPC or 5 to 15 mol% of DOPE and DSPC combined. According to some embodiments, the lipid nanoparticle formulation further comprises a PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises 1 to 5 mol% DMG-PEG 2000. According to some embodiments, the lipid nanoparticle formulation is selected from the group consisting of Formulation 1 to 5 and 11-26, wherein the compositions of Formulations 1 to 5 and 11-26 are described below: Formulation 1: lipid IA-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 2: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 3: lipid IB-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 4: lipid IB-6: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 5: lipid II-5: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 11: lipid II-1: 25 mol% to 35 mol%; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 12: lipid II-1: 25 mol% to 35 mol%; DOTAP: 5 mol% to 15 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 50 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 13: lipid II-1: 20 mol% to 30 mol%; DOTAP: 25 mol% to 35 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 14: lipid II-1: 10 mol% to 20 mol%; DOTAP: 30 mol% to 50 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 15: lipid II-1: 15 mol% to 25 mol%; DOTAP: 15 mol% to 25 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 45 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 16: lipid II-1: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; Distearoylphosphatidylcholine (DSPC): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 17: lipid II-1: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 18: lipid IA-11: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 19: lipid IA-12: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 20: lipid IA-8: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 21: lipid IA-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 22: lipid IB-24: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 23: lipid II-5: 15 mol% to 25 mol%; DOTAP: 15 mol% to 25 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 55 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 24: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; Distearoylphosphatidylcholine (DSPC): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 25: lipid IB-4: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 26: lipid IA-11: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%. According to some embodiments, the lipid nanoparticle formulation is Formulation 15 or Formulation 23. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation is Formulation 1 or Formulation 2. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation has average nanoparticle size (Z average) in the range of 50 to 200 nanometers, including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation has average nanoparticle size (Z average) in the range of 50 to 150 nanometers. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 25 mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential in the range of 28 to 40 mV, including each value and sub–range within the specified range. According to some embodiments, the lipid nanoparticle formulation has polydispersity index (PDI) of no more than 0.25. According to some embodiments, the lipid nanoparticle formulation is devoid of a lung targeting moiety. According to some embodiments, the lipid nanoparticle formulation further comprises a nucleic acid encapsulated within at least one particle thereof. According to some embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation further comprises a therapeutic agent encapsulated within at least one particle thereof. According to some embodiments, the lipid nanoparticle formulation further comprises a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the lipid nanoparticle formulation is liposomal composition. According to some embodiments, the lipid nanoparticle formulation is a liver bypass formulation. According to some embodiments, the lipid nanoparticle formulation is formulated for intravenous (IV) administration. According to some embodiments, the lipid nanoparticle formulation is selectively targeting the lung upon systemic administration. According to some embodiments, the lipid nanoparticle formulation is selectively targeting the lung upon intravenous (IV) administration. According to some embodiments, the lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration in mammals. According to some embodiments, the lipid nanoparticle formulation is for use in the treatment of a lung and/or respiratory disease or disorder. According to some embodiments, the lipid nanoparticle formulation is use in the treatment of a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection. According to some embodiments, the lipid nanoparticle formulation is for use in the treatment of a hereditary lung disorder or a genetic lung disease. According to some embodiments, there is provided a method of treating lung and/or respiratory disease or disorder, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method is for treating a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection. According to some embodiments, the method is for the treatment of a hereditary lung disorder or a genetic lung disease. According to some embodiments, the method comprises a step of systemically administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method comprises a step of step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, there is provided a method of delivery of a therapeutic agent or a nucleic acid to the lung, the method comprising the step of systemically administering to a subject in need thereof the lipid nanoparticle formulation disclosed herein and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method comprises the step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, there is provided a lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB-24. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid is lipid IA-4. According to some embodiments, the lipid is lipid IA-13. According to some embodiments, the lipid is lipid IA-14. According to some embodiments, the lipid is lipid IA-15. According to some embodiments, the lipid is lipid IB-4. According to some embodiments, the lipid is lipid IB-9. According to some embodiments, the lipid is lipid IB-24. According to some embodiments, there is provided a lipid nanoparticle formulation comprising the lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB-24. Each possibility represents a separate embodiment of the invention. According to some embodiments, there is provided a pharmaceutical composition comprising at least one lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB- 24 or the lipid nanoparticle formulation comprising one or more of said lipid. According to some embodiments, there is provided a pharmaceutical composition comprising at least one lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB- 24. According to some embodiments, there is provided a pharmaceutical composition comprising a lipid nanoparticle formulation comprising at least one lipid selected from the group consisting of: lipid IA-4, lipid IA-13, lipid IA-14, lipid IA-15, lipid IB-4, lipid IB-9 and lipid IB-24 . Each possibility represents a separate embodiment of the invention. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a 1H NMR spectrum of lipid IA-12 (NV3-006). Figure 1B is an ESI-MS spectrum of lipid IA-12 (NV3-006). Figure 1C is a 1H NMR spectrum of lipid IA-11 (NV3-004). Figure 1D is an ESI-MS spectrum of lipid IA-11 (NV3-004). Figure 1E is a 1H NMR spectrum of lipid IA-10 (NV3-002). Figure 1F is an ESI-MS spectrum of lipid IA-10 (NV3-002). Figures 2A-E are IVIS imaging analyses for Luciferase expression for Formulation 1 (Figure 2A), Formulation 2 (Figure 2B), Formulation 3 (Figure 2C), Formulation 4 (Figure 2D) and Formulation 5 (Figure 2E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figures 3A-E are IVIS imaging analyses for Luciferase expression for Formulation 6 (Figure 3A), Formulation 7 (Figure 3B), Formulation 8 (Figure 3C), Formulation 9 (Figure 3D) and Formulation 10 (Figure 3E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). In Figures 3A-E, the lung is not targeted and the white appearance of the lungs does not indicate fluorescence, but rather is a result of the B&W limitation. Figure 4A is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 5 (left group), Formulation 1 (second from left group), Formulation 2 (middle group), Formulation 3 (second from right group) and Formulation 4 (right group). Figure 4B is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 10 (left group), Formulation 6 (second from left group), Formulation 7 (middle group), Formulation 8 (second from right group) and Formulation 9 (right group). Figure 5 is a histogram analysis of Luciferase expression comparing between Lung (circles), liver (squares) and spleen (triangles) for Formulation 5 (left group), Formulation 2 (second from left group), Formulation 4 (middle group), Formulation 3 (second from right group) and Formulation 1 (right group). Figures 6A-F are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 6A), Formulation 22 (Figure 6B), Formulation 18 (Figure 6C), Formulation 19 (Figure 6D), Formulation 20 (Figure 6E) and Formulation 21 (Figure 6F) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 7 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group). Figure 8 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group). Figure 9 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group). Figures 10A-E are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 10A), Formulation 17 (Figure 10B), Formulation 18 (Figure 10C), Formulation 26 (Figure 10D), and Formulation 25 (Figure 10E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 11 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group), and Formulation 25 (right group). Figure 12 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group). Figure 13 is bar chart showing luciferase expression in the lungs were compared between DOTAP/DOTMA formulations; Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group). Figures 14A-C are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 14A), Formulation 16 (Figure 14B), and Formulation 24 (Figure 10C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 15 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 11 (left group), Formulation 16 (middle group) and Formulation 24 (right group). Figure 16 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group). Figure 17 is bar chart showing luciferase expression in the lungs were compared between DSPC vs DOPE formulations; Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group). Figures 18A-E are IVIS imaging analysis for Luciferase expression of Formulation 12 (Figure 18A), Formulation 15 (Figure 18B), Formulation 11 (Figure 18C), Formulation 13 (Figure 18D), and Formulation 14 (Figure 18C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 19 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 12 (left group), Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group). Figure 20 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group). Figure 21 is bar chart showing luciferase expression in the lungs were compared between different formulations with increasing DOTAP amount; Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group). Figures 22A-B are IVIS imaging analysis for Luciferase expression of Formulation 15 (Figure 22A), and Formulation 23 (Figure 22B) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 23 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 15 (left group), and Formulation 23 (right group). Figure 24 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (left group), and Formulation 23 (right group). Figure 25 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 15 (left group), and Formulation 23 (right group). Figure 26A is a 1H NMR spectrum of lipid IB-4 (NV2-004). Figure 26B is an ESI-MS spectrum of lipid IB-4 (NV2-004). Figure 27A is a 1H NMR spectrum of lipid IB-13 (NV2-015). Figure 27B is an ESI-MS spectrum of lipid IB-13 (NV2-015). Figure 28A is a 1H NMR spectrum of lipid IA-8 (NV3-009). Figure 28B is an ESI-MS spectrum of lipid IA-8 (NV3-009). Figure 29A is a 1H NMR spectrum of lipid IB-24 (NV2-027). Figure 29B is an ESI-MS spectrum of lipid IB-24 (NV2-027). Figure 30A is a 1H NMR spectrum of lipid IA-13 (NV3-013). Figure 30B is an ESI-MS spectrum of lipid IA-13 (NV3-013). Figure 31A is a 1H NMR spectrum of lipid IA-14 (NV3-014). Figure 31B is an ESI-MS spectrum of lipid IA-14 (NV3-014). Figure 32A is a 1H NMR spectrum of lipid IA-15 (NV3-016). Figure 32B is an ESI-MS spectrum of lipid IA-15 (NV3-016). DETAILED DESCRIPTION OF THE PRESENT INVENTION The present invention is based on the discovery of lipid nanoparticle compositions useful in selectively delivering active agents to the lungs. The lipid nanoparticle compositions of the present invention are useful comprises ionizable lipid(s) represented by Formula (IA), Formula (IB) and/or Formula (II), and a permanently charged lipid. Ionizable lipids - Formula (IA) As contemplated herein, the present invention relates to an ionizable lipid(s) represented by the structure of Formula (IA): including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. According to some embodiments, R1A is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNARNA’, wherein each one of RNA and RNA’ is individually C1-4 alkyl or RNA and RNA’ together with the nitrogen to which they are bound, form a ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. According to some embodiments, R1A is selected from the group consisting of: OH, -(CH2CH2O)2-6H and C1-6 hydroxyalkyl. According to some embodiments, R1A is selected from the group consisting of: OH, - (CH2CH2O)2-4H and C1-4 hydroxyalkyl. According to some embodiments, R1A is -(CH2CH2O)1-4H or OH. According to some embodiments, R1A is selected from the group consisting of: -CH2CH2OH, - CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH. According to some embodiments, R1A is OH. According to some embodiments, R1A is -CH2CH2OH. According to some embodiments, R1A is, - CH2CH2OCH2CH2OH. According to some embodiments, R1A is the same as R3A. It is the be understood that the term “the same” in the previous paragraph means that the two specified R substituents have the same chemical definition. For example, lipid 1A-2 as shown below has the same R1A and R3A, each of which is-CH2CH2OH. This lipid also has the same R2A and R4A, each of which is - (CH2)8CH=CHCH2CH=CHC5H11. According to some embodiments, R2A is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2A is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R2A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25, - (CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17 and -(CH2)7CH=CHCH2CH=CHC4H9. According to some embodiments, R2A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11 and -C12H25. According to some embodiments, R2A is -(CH2)8CH=CHCH2CH=CHC5H11. According to some embodiments, R2A is -C12H25. According to some embodiments, R2A is the same as R4A. According to some embodiments, nA is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. According to some embodiments, nA is selected from the group consisting of: 8, 9 and 10. According to some embodiments, nA is 9 or 10. According to some embodiments, nA is 9. According to some embodiments, nA is 10. According to some embodiments, XA is selected from the group consisting of:-COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-.According to some embodiments, XA is selected from the group consisting of: -COO-, -OOC-, -NHCO- and -CONH. According to some embodiments, XA is -COO- or -OOC-. According to some embodiments, XA is -COO- and the lipid is represented by Formula (IA1) . According to some embodiments, jA is selected from the group consisting of: 0, 1, 2, 3 and 4. Each possibility represents a separate embodiment of the invention. According to some embodiments, jA is selected from the group consisting of: 0, 1 and 2. According to some embodiments, jA is 0 or 2. According to some embodiments, jA is 0. According to some embodiments, YA is selected from the group consisting of: absent, -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-. Each possibility represents a separate embodiment of the invention. According to some embodiments, YA is absent, -COO- or -OOC-. Each possibility represents a separate embodiment of the invention. According to some embodiments, YA is absent or -OOC-. According to some embodiments, YA is absent. According to some embodiments, YA is absent, jA is 0 and the lipid of Formula (IA) is represented by Formula (IA2): . According to some embodiments, YA is absent, jA is 0, XA is -COO- and the lipid of Formula (IA) is represented by Formula (IA3) . According to some embodiments, mA is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15. Each possibility represents a separate embodiment of the invention. According to some embodiments, mA is selected from the group consisting of: 6, 7, 8, 9, 10, 11, 12 and 13. According to some embodiments, mA is selected from the group consisting of: 8, 9, and 10. According to some embodiments, mA is 9 or 10. According to some embodiments, mA is 9. According to some embodiments, mA is 10. According to some embodiments, R3A is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNA’’RNA’’’, wherein each one of RNA’’ and RNA’’’ is individually C1-4 alkyl or RNA’’ and RNA’’’ together with the nitrogen to which they are bound, form a ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. According to some embodiments, R3A is selected from the group consisting of: OH, -(CH2CH2O)2-6H and C1-6 hydroxyalkyl. According to some embodiments, R3A is selected from the group consisting of: OH, - (CH2CH2O)2-4H and C1-4 hydroxyalkyl. According to some embodiments, R3A is -(CH2CH2O)1-4H or OH. According to some embodiments, R3A is selected from the group consisting of: -CH2CH2OH, - CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH. According to some embodiments, R3A is OH. According to some embodiments, R3A is -CH2CH2OH. According to some embodiments, R1A is, -CH2CH2OCH2CH2OH. According to some embodiments, R4A is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R4A is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R4A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25, -(CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17 and -(CH2)7CH=CHCH2CH=CHC4H9. According to some embodiments, R4A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11 and -C12H25. According to some embodiments, R4A is -(CH2)8CH=CHCH2CH=CHC5H11. According to some embodiments, R4A is -C12H25. According to some embodiments, the ionizable lipid of Formula (IA) is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14 and IA-15 including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid of Formula (IA) is selected from the group consisting of: lipid IA-4, lipid IA-5, lipid IA-8, lipid IA-10, lipid IA-11, lipid IA-12, lipid IA-13, lipid IA-14, and lipid IA-15. According to some embodiments, the lipid of Formula (IA) is IA-5. According to some embodiments, the lipid of Formula (IA) is IA-10. According to some embodiments, the lipid of Formula (IA) is IA-11. According to some embodiments, the lipid of Formula (IA) is IA-12. Formula (IB) As contemplated herein, the present invention relates to an ionizable lipid(s) represented by the structure of Formula (IB):
including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. According to some embodiments, R1B is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNBRNB’, wherein each one of RNA and RNA’ is individually C1-4 alkyl or RNA and RNA’ together with the nitrogen to which they are bound, form a ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. According to some embodiments, R1B is selected from the group consisting of: -CH2CH2OCH2CH2OH, -CH2CH2Cl, -CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2CH2OH and -CH2CH2NMe2. According to some embodiments, R1B is selected from the group consisting of: CH2CH2OCH2CH2OH, -CH2CH2Cl or -CH2CH2OH. According to some embodiments, R1B is -CH2CH2OCH2CH2OH or -CH2CH2Cl. According to some embodiments, R1B is -CH2CH2OCH2CH2OH. According to some embodiments, R1B is -CH2CH2Cl. It is to be understood that the group -CH2CH2CH2N(CH2)4, which is specified herein refers to propyl pyrrolidine, i.e., the group drawn below:
According to some embodiments, R1B is the same as R3B. According to some embodiments, R2B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R2B is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, - (CH2)7CH=CHCH2CH=CHC6H13, -(CH2)8CH=CHC8H17 and -C12H25. According to some embodiments, R2B is -(CH2)8CH=CHCH2CH=CHC5H11 or -C12H25. According to some embodiments, R2B is - (CH2)8CH=CHCH2CH=CHC5H11. According to some embodiments, R2B is -C12H25. According to some embodiments, R2A is the same as R4A. According to some embodiments, R2A is the same as R3A. According to some embodiments, WB is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According to some embodiments, WB is a C4-12 alkylene. According to some embodiments, WB is a straight chain alkylene. According to some embodiments, WB is a C5-9 alkylene. According to some embodiments, WB is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, WB is -(CH2)9-. According to some embodiments, YB is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene. According to some embodiments, YB is selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-. It is to be understood by the person having ordinary skill in the art that when a variable is said to be “absent”, no chemical group will appear in the specified place, and the atoms drawn as bonded to the variable, will be bonded to each other. For example, Formula (IB1) is an embodiment of Formula (IB), wherein the variable YB is absent. In Formula (IB), YB is drawn as bonded to an oxygen atom and to a nitrogen atom. Thus, according to some embodiments, if YB is absent the oxygen will be directly bonded to the nitrogen via a single (signa) bond. According to some embodiments, YB is absent and the lipid of Formula (IB) is represented by Formula (IB1): . According to some embodiments, R3B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R3B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R3B is a C4-16 alkyl. According to some embodiments, R3B is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R3B is -C6H13 or -C8H17. According to some embodiments, R3B is -C6H13. According to some embodiments, R3B is -C8H17. According to some embodiments, R3B is the same as R4B. According to some embodiments, R4B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R4B is selected from the group consisting of: C6-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R4B is a C4-16 alkyl. According to some embodiments, R4B is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R4B is -C6H13 or -C8H17. According to some embodiments, R4B is -C6H13. According to some embodiments, R4B is -C8H17. According to some embodiments, the ionizable lipid of Formula (IB) is selected from the group consisting of: IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB-22, IB-23 and IB-24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the ionizable lipid of Formula (IB) is selected from the group consisting of: lipid IB-4, lipid IB-6, lipid IB-9, lipid IB-10, lipid IB- 13, lipid IB-24 and a combination thereof. According to some embodiments, the lipid of Formula (IB) is selected from the group consisting of: IB-4, IB-6 and IB-10. According to some embodiments, the lipid of Formula (IB) is IB-4. According to some embodiments, the lipid of Formula (IB) is IB-6. According to some embodiments, the lipid of Formula (IB) is IB-10. Formula (II) As contemplated herein, the present invention relates to an ionizable lipid(s) represented by the structure of Formula (II):
including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. According to some embodiments, R1C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R1C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R1C is a C4-16 alkyl. According to some embodiments, R1C is a C4-10 alkyl. According to some embodiments, R1C is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R1C is -C6H13 or -C8H17. According to some embodiments, R1C is -C6H13. According to some embodiments, R1C is -C8H17. According to some embodiments, R1C is the same as R2C. According to some embodiments, R1C is the same as R3C. According to some embodiments, R1C is the same as R4C. According to some embodiments, R1C, R2C, R3C, and R4C are the same. According to some embodiments, R2C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R2C is a C4-16 alkyl. According to some embodiments, R2C is a C4-10 alkyl. According to some embodiments, R2C is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R2C is -C6H13 or -C8H17. According to some embodiments, R2C is -C6H13. According to some embodiments, R2C is -C8H17. According to some embodiments, Y1C is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y1C is selected from the groups consisting of: absent, - CH2CH2OCH2CH2- and -CH2CH2-. According to some embodiments, Y1C is absent. According to some embodiments, Y1C is the same as Y2C. According to some embodiments, each one of Y1C and Y2C is absent and the lipid of Formula (II) is represented by Formula (II1): . According to some embodiments, W1C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According to some embodiments, W1C is a C4- 12 alkylene. According to some embodiments, W1C is a straight chain alkylene. According to some embodiments, W1C is a C5-9 alkylene. According to some embodiments, W1C is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, W1C is -(CH2)9-. According to some embodiments, W1C and W2C are the same. According to some embodiments, each one of W1C and W2C is a C4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II2): . According to some embodiments, each one of Y1C and Y2C is absent, each one of W1C and W2C is a C4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II3): . According to some embodiments, R5C is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NR NIIRNII’, wherein each one of RNII and RNII’ is individually C1-4 alkyl or R NII and RNII’ together with the nitrogen to which they are bound, form a ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, the haloalkyl is selected from the group consisting of: chloroalkyl, fluoroalkyl and bromoalkyl. According to some embodiments, the haloalkyl is chloroalkyl. According to some embodiments, R5C is selected from the group consisting of: OH, -(CH2CH2O)2-3H, C1-4 hydroxyalkyl and C1-4 haloalkyl and C1-3 alkylene-NR NIIRNII’, wherein each one of RNII and RNII’ is individually C1-4 alkyl or R NII and RNII’ together with the nitrogen to which they are bound, form a 5-6 membered ring. Each possibility represents a separate embodiment of the invention. According to some embodiments, R5C is selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, - CH2CH2CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2N(CH2)5, -CH2CH2N(CH2)5, -CH2CH2Cl, -OH and -CH2CH2NMe2. According to some embodiments, R5C is -CH2CH2OH or -CH2CH2OCH2CH2OH. According to some embodiments, W2C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen. According to some embodiments, W2C is a C4- 12 alkylene. According to some embodiments, W2C is a straight chain alkylene. According to some embodiments, W2C is a C5-9 alkylene. According to some embodiments, W2C is selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-. Each possibility represents a separate embodiment of the invention. According to some embodiments, W2C is -(CH2)9-. According to some embodiments, Y2C is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y2C is selected from the groups consisting of: absent, - CH2CH2OCH2CH2- and -CH2CH2-. According to some embodiments, Y2C is absent. According to some embodiments, R3C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene-O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R3C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R3C is a C4-16 alkyl. According to some embodiments, R3C is a C4-10 alkyl. According to some embodiments, R3C is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R3C is -C6H13 or -C8H17. According to some embodiments, R3C is -C6H13. According to some embodiments, R3B is -C8H17. According to some embodiments, R4C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, the alkyl is a straight chain alkyl. According to some embodiments, the alkenyl is a straight chain alkenyl. According to some embodiments, the alkyl is an unsubstituted alkyl. According to some embodiments, the alkenyl is an unsubstituted alkenyl. According to some embodiments, R4C is a C4-16 alkyl. According to some embodiments, R4C is a C4-10 alkyl. According to some embodiments, R4C is selected from the group consisting of: -C12H25, -C6H13 and -C8H17. According to some embodiments, R4C is -C6H13 or -C8H17. According to some embodiments, R4C is -C6H13. According to some embodiments, R4C is -C8H17. According to some embodiments, the ionizable lipid of Formula (II) is selected from the group consisting of: II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II-15, II-16, II-17, II-18, II- 19, II-20, II-21, II-22, II-23 and II-24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: II-1, and II-5. According to some embodiments, the lipid of Formula (II) is II- 1. According to some embodiments, the lipid of Formula (II) is II-5. The chemical structures of each of the specific lipids are detailed below in the “Exemplary Ionizable Lipids” Section and in the claims. Exemplary Ionizable Lipids Exemplary lipids according to Formula (IA), Formula (IB) and Formula (II) of the present invention are shown below. It is to be understood that, according to some embodiments, the invention is not limited to any one or more of the following exemplary lipids. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB-22, IB-23, IB-24, II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II- 15, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-23 and II-24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment of the invention. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14 and IA-15. According to some embodiments, the ionizable lipid is selected from the group consisting of: IB-1, IB-2, IB-3, IB-4, IB-5, IB- 6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB-18, IB-19, IB-20, IB-21, IB- 22, IB-23 and IB-24. According to some embodiments, the ionizable lipid is selected from the group consisting of: II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II-12, II-13, II-14, II-15, II-16, II- 17, II-18, II-19, II-20, II-21, II-22, II-23 and II-24. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-4, IA-5, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-4, IB-9, IB-10, IB-13, II-1 and II-5. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-10, IA-11, IA-12, IB-4, IB-10, II-1 and II-5. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-110 IA-12, IB-4 and IB-5. According to some embodiments, the lipid of is selected from the group consisting of: IA-5, IA-10, IA-11 and IA-12. According to some embodiments, the lipid is IA-10. According to some embodiments, the lipid is selected from the group consisting of: IB-4, IB-6 and IB-10. According to some embodiments, the lipid is IB-4. According to some embodiments, the lipid is IB-6. According to some embodiments, the lipid is IB-10. According to some embodiments, the lipid is selected from the group consisting of: II-1 and II-5. According to some embodiments, the lipid is II-5. According to some embodiments, the present invention provides an ionizable lipid represented by the structure of Formula (IA), Formula (IB) or Formula (II). Each possibility represents a separate embodiment of the invention. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-1, IA-2, IA-3, IA-4, IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11, IA-12, IA-13, IA-14, IA-15, IB-1, IB-2, IB-3, IB-4, IB-5, IB-6, IB-7, IB-8, IB-9, IB-10, IB-11, IB-12, IB-13, IB-14, IB-15, IB-16, IB-17, IB- 18, IB-19, IB-20, IB-21, IB-22, IB-23, IB-24, II-1, II-2, II-3, II-4, II-5, II-6, II-7, II-8, II-9, II-10, II-11, II- 12, II-13, II-14, II-15, II-16, II-17, II-18, II-19, II-20, II-21, II-22, II-23 and II-24, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. Each possibility represents a separate embodiment of the invention. According to some embodiments, the ionizable lipid is selected from the group consisting of: IA-4, IA-13, IA-14, IA-15, IB-4, IB-9 and IB-24. The following exemplary lipids are portrayed as non-limiting examples of the ionizable lipids of the present invention (designation below the chemical structure).
Lipid IA-4 is also referred herein as NV3-015. IA-5 Lipid IA-5 is also referred herein as NV3-005.
IA-8 Lipid IA-8 is also referred herein as NV3-009. IA-10 Lipid IA-10 is also referred herein as NV3-002. IA-11 Lipid IA-11 is also referred herein as NV3-004. IA-12 Lipid IA-12 is also referred herein as NV3-006. IA-13 Lipid IA-13 is also referred herein as NV3-013. IA-14 Lipid IA-14 is also referred herein as NV3-014. Lipid IB-4 is also referred herein as NV2-004. IB-8 IB-9 Lipid IB-9 is also referred herein as NV2-009. IB-10 Lipid IB-10 is also referred herein as NV2-011. IB-13 Lipid IB-13 is also referred herein as NV2-015. IB-17
IB-24 Lipid IB-24 is also referred herein as NV2-027. II-1 Lipid II-1 is also referred herein as NV1-001.
II-5 Lipid II-5 is also referred herein as NV1-005.
11-14
-18
11-22
II-24. Chemical Definitions The term “ionizable lipid”, as used herein refers to lipid species that carries a charge at a selected pH. Selected pH values include, but not limited to physiological pH, pH=7 and the like. It is to be understood by the person having ordinary skill in the art that the lipids of Formulae (IA), (IB) and (II) may be considered as ionizable lipids. The term “cationic lipid”, as used herein refers to lipid species that carries a net positive charge at a selected pH. Selected pH values include, but not limited to physiological pH, pH=7 and the like. It is to be understood by the person having ordinary skill in the art that the lipids of Formulae (IA), (IB) and (II) may be considered as cationic lipids, since they bear 2 or more nitrogen atoms, where these atoms are typically basic and protonizable at the selected pH, so that the lipids may carry a net positive charge. According to some embodiments, the lipid of the present invention is a cationic lipid. The term “permanently charged lipid”, as used herein refers to lipid species that carries a charge at on one or more of its atoms and that has a total net (positive or negative) charge. The charged atom may not be de- charged in the permanently charged lipid. Specifically, the charged atom may not be de-charged at different pH environments. According to some embodiments, the charge of the charged atom may not be altered at different pH environments. The term “permanently charged lipid” includes both charged lipids coupled to a counterion and zwitterionic lipids. Zwitterionic permanently charged lipids may require unequal number of positively charged atoms and negatively charged atom in the lipid, and also include a counterion not bonded to the lipid. For example distearoylphosphatidylcholine (DSPC) has permanently charged phosphate and ammonium groups, and has a net zero charge so it in not permanently charged. Charged atoms of the permanently charged lipid include, but are not limited to nitrogen (e.g., ammonium groups) and/or oxygen (e.g., sulfate groups, carboxylate groups, phosphate groups, etc.). According to some embodiments, the permanently charged lipid comprises a quaternary ammonium group. Specifically, quaternary ammonium groups are considered to have permanently charged nitrogen atom, due to the three-valent nitrogen bonded to 4 groups (e.g., alkyl groups). According to some embodiments, the permanently charged lipid have a net charge. It is to be understood that a permanently charged lipid which has a net charge has a permanently charged atom as part of or chemically bonded to the lipid backbone, and a counterion, which is not covalently bonded to the lipid backbone. The term “quaternary ammonium group” as used herein refers to a chemical functional group containing at least one quaternized nitrogen wherein the nitrogen atom is attached to four organic groups. A permanently charged lipid according to the present invention may comprise one or more quaternized nitrogen atoms. According to some embodiments, the quaternary ammonium group is a tetra-alkyl ammonium. The term “tetra-alkyl ammonium” refers to a group or compound (e.g., lipid) which contain such group, that has a nitrogen atom bonded to four alkyl groups. The term “alkyl” is defined below. An “alkyl” group refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain alkyl groups. The alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl. The term "Cn-m alkyl", refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2-methyl-1-butyl, 3-pentyl, hexyl, 1,2,2-trimethylpropyl and the like. The term "alkylene," employed alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkylene group to the remainder of the compound. The term "Cn-m alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethan-l,2-diyl, ethan-l,l-diyl, propan-l,3-diyl, propan-l,2-diyl, propan-l,l-diyl, butan-l,4-diyl, butan-l,3-diyl, butan-1,2- diyl, 2-methyl-propan-l,3-diyl, -(CHMe)-(CH2)4-, -(CHMe)-(CH2)4- and the like. It is to be understood that for branched alkylene groups the total number of carbon atoms is counted. For example, the substituent -(CHMe)-(CH2)4-, is a C6 alkylene. It is to be understood that C0-alkylene means that the specified substituent is absent. In various section of the present application ranges of alkyl chains are presented, e.g., C4-16 alkyl, C6-18 alkyl etc. It is to be understood that such ranges include any sub range thereof. for example, C6-18 alkyl may include and/or be directed to: C6-12 alkyl, C8-14 alkyl, C12-18 alkyl, C8 alkyl etc. An "alkenyl" group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond including straight-chain, branched-chain and cyclic alkenyl groups. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl- butenyl and decenyl. The alkenyl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl. Alkenyls according to the present invention may include more than one carbon-carbon double bond. Thus, dienes (see e.g., lipid IA-11, NV3-004, substituent R4A) and trienes are within the definition of alkenyl. According to some embodiments, the alkenyl is a dienyl. The term "Cn-m alkenyl", refers to an alkyl group having n to m carbon atoms. An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the compound. Examples of alkenyl moieties include, but are not limited to, chemical groups such as ethenyl, propenyl, isopropenyl, n- butenyl, sec-butenyl the like. The term "alkenylene," employed alone or in combination with other terms, refers to a divalent alkenyl linking group. An alkenylene group formally corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkenylene group to the remainder of the compound. The term "Cn-m alkenylene" refers to an alkenylene group having n to m carbon atoms. In various section of the present application ranges of alkenyl chains are presented, e.g., C2-8 alkenyl, C4-20 alkenyl etc. It is to be understood that such ranges include any sub range thereof. for example, C4-14 alkenyl may include and/or be directed to: C4-8 alkenyl, C8-14 alkenyl, C6-12 alkenyl, C9 alkenyl etc. According to some embodiments, each one of the alkenyl double bond has a cis configuration. One or more of the lipids of the invention, may be present as a salt. The term "salt" encompasses both basic and acid addition salts, including but not limited to, carboxylate salts or salts with amine nitrogen atoms, and include salts formed with the organic and inorganic anions and cations discussed below. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and organic or inorganic acids. Such acids include hydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D- camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids. Each possibility represents a separate embodiment of the invention. The term "organic or inorganic cation" refers to counter-ions for the anion of a salt. The counter-ions include, but are not limited to, alkali and alkaline earth metals (such as lithium, sodium, potassium, barium, aluminum and calcium); ammonium and mono-, di- and tri-alkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2- hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations. See, for example, Berge et al., J. Pharm. Sci. (1977), 66:1-19, which is incorporated herein by reference. Particles, Formulations Compositions and Uses According to some embodiments, the present invention provides a particle comprising the present ionizable lipid and the permanently charged lipid. According to some embodiments, there is provided a composition comprising a plurality of particles as discloses herein and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the composition is a liposomal composition. According to some embodiments, the particles of the present invention are in the form of liposomes. According to some embodiments, the composition further comprises one or more components selected from the group consisting of a neutral lipid, a charged lipid, a steroid, and a polymer-conjugated lipid. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid nanoparticle formulation of the present invention comprises 10 to 50 mol% of the ionizable lipid(s) according to Formula (IA), Formula (IB) and or Formula (II). It is to be understood that according to embodiments wherein the lipid nanoparticle formulation comprises a combination of different ionizable lipids according to Formula (IA), Formula (IB) and or Formula (II), the mol% refers to the total mol% of all said different ionizable lipids. According to some embodiments, the lipid nanoparticle formulation of the present invention comprises 15 to 45 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises 20 to 40 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 15 to 30 mol% of the ionizable lipid. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 30 mol% of the ionizable lipid. According to some embodiments, the lipid nanoparticle formulation of the present invention comprises 25 to 35 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises about 30 mol% of the ionizable lipid(s). The term “about” means ±20%, ±15%, ±10% or ±5% or a specified value. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulation of the present invention comprises at least 10 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises at least 15 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises at least 20 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises at least 25 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 60 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 50 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 40 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 35 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 30 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation of the present invention comprises no more than 25 mol% of the ionizable lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 10 to 40 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 15 to 35 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 15 to 30 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises 20 to 30 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises about 25 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises about 20 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 5 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 10 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 15 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 60 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 45 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 40 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 35 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 30 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation comprises no more than 25 mol% permanently charged lipid(s). According to some embodiments, the lipid nanoparticle formulation further comprises at least one neutral lipid. According to some embodiments, “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn- glyccro-3- phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glyccro-3-phosphocholine (DMPC), 1-Palmitoyl- 2-olcoyl-sn-glyccro-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidyl ethanolamines such as 1,2-Diolcoyl-sn-glyccro-3-phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived. According to some embodiments, the lipid nanoparticle formulation further comprises at least one sterol, at least one phospholipid or both. According to some embodiments, the neutral lipid comprises a sterol, a phospholipid or both. Each possibility represents a separate embodiment of the invention. According to some embodiments, the sterol comprises cholesterol. According to some embodiments, the lipid nanoparticle formulation further comprises cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 15 to 45 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 40 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 25 to 37 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 30 to 40 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 30 to 35 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises about 32.5 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 25 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 30 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 40 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 35 mol% of the sterol. According to some embodiments, the lipid nanoparticle formulation comprises 15 to 45 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 20 to 40 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 25 to 37 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises 30 to 35 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises about 32.5 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 20 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 25 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises at least 30 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 50 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 40 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation comprises no more than 35 mol% cholesterol. According to some embodiments, the lipid nanoparticle formulation further comprises at least one phosphatidylethanolamine. According to some embodiments, the phospholipid comprises a phosphatidylethanolamine. According to some embodiments, the phosphatidylethanolamine is selected from the group consisting of: 1,2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) 1,3-Dipalmitoyl-sn-glycero-2- phosphoethanolamine (1,3-DPPE), 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), biotin-phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or combinations thereof. According to some embodiments, the phosphatidylethanolamine comprises DOPE. According to some embodiments, the phospholipid comprises 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Distearoylphosphatidylcholine (DSPC) or both. According to some embodiments, the nanoparticle formulation further comprises DOPE. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is the neutral phospholipid shown below: DOPE. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 20 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 15 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises about 10 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 3 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 5 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 7 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 25 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 20 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 15 mol% of the phospholipid. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 20 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises 5 to 15 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises about 10 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises at least 3 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises at least 5 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises at least 7 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises no more than 25 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises no more than 20 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation comprises no more than 15 mol% DOPE. According to some embodiments, the lipid nanoparticle formulation further comprises a PEGylated lipid. The term “PEG” refers to polyethylene glycol. The term “PEGylated lipid” means a lipid that is bonded to PEG. According to some embodiments, the PEGylated lipid comprises a PEG moiety having a molecular weight in the range of 1000 gr/mol to 3000 gr/mol, including each value and sub-range within the specified range. According to some embodiments, the PEGylated lipid comprises a PEG moiety having a molecular weight in the range of 1000 gr/mol to 2000 gr/mol. According to some embodiments, the PEG moiety has a molecular weight of about 2000 gr/mol. According to some embodiments, the PEGylated lipid comprises DMG-PEG. According to some embodiments, the PEGylated lipid comprises DMG-PEG-2000. The term “DMG-PEG” means 1,2-Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol, or ,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol. The term “DMG-PEG-2000” means 1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol, or ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol, wherein the polyethylene glycol has a molecular weight of about 2000 gr/mol. According to some embodiments, the lipid nanoparticle formulation comprises 1 to 5 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises 1.5 to 4 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises about 2.5 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 1 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises at least 2 mol% PEG. According to some embodiments, the lipid nanoparticle formulation comprises no more than 10 mol% PEGylated lipid. According to some embodiments, the lipid nanoparticle formulation comprises no more than 5 mol% PEGylated lipid. It is to be understood that by the phrase “the molar percentage of the component is at least x mol% of the particle” it is meant that at least x% of the particle molecules are of the component. Similarly, the phrase “the molar percentage of the component is no more than x mol% of the particle” it is meant that no more than x% of the particle molecules are of the component. The unit “mol%” is also sometimes referred as “mol:mol” or “% mol:mol”. According to some embodiments, the particle comprises the membrane stabilizing lipid and a lipid membrane comprising the lipid. According to some embodiments, the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids. According to some embodiments, the membrane stabilizing lipid comprises cholesterol. According to some embodiments, the membrane stabilizing lipids may be selected from, but not limited to: cholesterol, phospholipids (such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols), cephalins, sphingolipids (sphingomyelins and glycosphingolipids), glycoglycerolipids, and combinations thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the phosphatidylethanolamines may be selected from, but not limited to: 1,2-dilauroyl-L-phosphatidyl- ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn- glycero-3-phosphoethanolamine (DPhPE) 1,3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3- DPPE), 1-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), Biotin- Phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or combinations thereof. In some embodiments, the Phosphatidylethanolamines may be conjugated to a PEG-Amine derivative. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid nanoparticle formulation is selected from the group consisting of Formulation 1 to 5 and 11-26, wherein the compositions of Formulations 1 to 5 and 11-26 are described below. According to some embodiments, Formulation 1 comprises: lipid IA-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%. According to some embodiments, Formulation 2 comprises: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%. According to some embodiments, Formulation 3 comprises: lipid IB-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and polyethylene glycol (PEG): 1.5 mol% to 4 mol%. According to some embodiments, Formulation 4 comprises: lipid IB-6: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%. According to some embodiments, Formulation 5 comprises: lipid II-5: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%. According to some embodiments, Formulation 11 comprises: lipid II-1: 25 mol% to 35 mol%; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 12 comprises: lipid II-1: 25 mol% to 35 mol%; DOTAP: 5 mol% to 15 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 50 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 13 comprises: lipid II-1: 20 mol% to 30 mol%; DOTAP: 25 mol% to 35 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 14 comprises: lipid II-1: 10 mol% to 20 mol%; DOTAP: 30 mol% to 50 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 15 comprises: lipid II-1: 15 mol% to 25 mol%; DOTAP: 15 mol% to 25 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 55 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 16 comprises: lipid II-1: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; Distearoylphosphatidylcholine (DSPC): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 17 comprises: lipid II-1: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 18 comprises: lipid IA-11: 25 mol% to 35 mol%;
DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 19 comprises: lipid IA-12: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 20 comprises: lipid IA-8: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 21 comprises: lipid IA-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 22 comprises: lipid IB-24: 25 mol% to 35 mol%;
73 DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 23 comprises: lipid II-5: 15 mol% to 25 mol%; DOTAP: 15 mol% to 25 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 55 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 24 comprises: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; Distearoylphosphatidylcholine (DSPC): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 25 comprises: lipid IB-4: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; According to some embodiments, Formulation 26 comprises: lipid IA-11: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%. According to some embodiments, the lipid nanoparticle formulation is Formulation 1. According to some embodiments, the lipid nanoparticle formulation is Formulation 2. According to some embodiments, the lipid nanoparticle formulation is Formulation 3. According to some embodiments, the lipid nanoparticle formulation is Formulation 4. According to some embodiments, the lipid nanoparticle formulation is Formulation 5. According to some embodiments, the lipid nanoparticle formulation is Formulation 6. According to some embodiments, the lipid nanoparticle formulation is Formulation 7. According to some embodiments, the lipid nanoparticle formulation is Formulation 8. According to some embodiments, the lipid nanoparticle formulation is Formulation 9. According to some embodiments, the lipid nanoparticle formulation is Formulation 10. According to some embodiments, the lipid nanoparticle formulation is Formulation 11. According to some embodiments, the lipid nanoparticle formulation is Formulation 12. According to some embodiments, the lipid nanoparticle formulation is Formulation 13. According to some embodiments, the lipid nanoparticle formulation is Formulation 14. According to some embodiments, the lipid nanoparticle formulation is Formulation 15. According to some embodiments, the lipid nanoparticle formulation is Formulation 16. According to some embodiments, the lipid nanoparticle formulation is Formulation 17. According to some embodiments, the lipid nanoparticle formulation is Formulation 18. According to some embodiments, the lipid nanoparticle formulation is Formulation 19. According to some embodiments, the lipid nanoparticle formulation is Formulation 20. According to some embodiments, the lipid nanoparticle formulation is Formulation 21. According to some embodiments, the lipid nanoparticle formulation is Formulation 22. According to some embodiments, the lipid nanoparticle formulation is Formulation 23. According to some embodiments, the lipid nanoparticle formulation is Formulation 25. According to some embodiments, the lipid nanoparticle formulation is Formulation 25. According to some embodiments, the lipid nanoparticle formulation is Formulation 26. According to some embodiments, the lipid nanoparticle formulation is Formulation 1 or Formulation 2. According to some embodiments, the lipid nanoparticle formulation is Formulation 15 or Formulation 23. According to some embodiments, the lipid nanoparticle formulation comprises a targeting moiety. According to some embodiments, the lipid nanoparticle formulation comprises at least one nanoparticle, which is conjugated to a targeting moiety. According to some embodiments, the targeting moiety is a lung targeting moiety. According to some embodiments, the lipid nanoparticle formulation is devoid of lung targeting moieties. Specifically, it was found that the present lipid nanoparticles are highly effective in targeting the lungs, even in the absence of lung targeting moieties. Accordingly, the incorporation of such moieties, which are typically employed for lung-targeting purposes, may be avoided. This enables a simplified formulation process at significantly reduced costs. According to some embodiments, the lipid nanoparticle formulation further comprises a nucleic acid encapsulated within at least one particle thereof. According to some embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. According to some embodiments, the particle further comprises a nucleic acid. According to some embodiments, the nucleic acid is encapsulated within a particle of the lipid nanoparticle formulation. According to some embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. In some embodiments, the composition may further comprise a nucleic acid. Examples of nucleic acids include small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the weight ratio between the nucleic acid and the lipid mixture may be adjusted so as to achieve maximal biological effect by the nucleic acid on the target site. In some embodiments, the ratio between the nucleic acid and the lipid phase may be 1:1. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:2. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:5. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:10. For example, the weight ratio between the nucleic acid and the lipids phase may be 1:16. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:20. In some embodiments, the weight ratio between the nucleic acid and the lipid phase is about 1:1 to 1:20 (w:w). According to some embodiments, the particle further comprises a therapeutic agent. According to some embodiments, the therapeutic agent is encapsulated within a particle comprising the lipid. According to some embodiments, the lipid nanoparticle formulation comprises a therapeutic agent encapsulated within at least one particle thereof. As detailed herein the present lipid nanoparticle formulation may include a plurality of nanoparticle. The following section related to the diameter of the nanoparticles of the present lipid nanoparticle formulation. According to some embodiments, the lipid nanoparticle formulation has average nanoparticle size (Z average) in the range of 10 to 500 nanometers. According to some embodiments, the lipid nanoparticle formulation has average nanoparticle size in the range of 25 to 400 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Z average) in the range of 50 to 200 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Z average) in the range of 50 to 150 nanometers. According to some embodiments, the lipid nanoparticle formulation has nanoparticle size (Z average) in the range of 60 to 120 nanometers. In some embodiments, the particles (including any nucleic acid, therapeutic agent and the like encapsulated within and any targeting moiety conjugated thereto) have a particle size (diameter) in the range of about 10 to about 500 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 10 to about 350 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 40 to about 270 nm. In some embodiments, the particles have a particle size (diameter) in the range of over about 10 nm. In some embodiments, the particles have a particle size (diameter) of over about 20 nm. In some embodiments, the particles have a particle size (diameter) of over about 30 nm. In some embodiments, the particles have a particle size (diameter) of over about 40 nm. In some embodiments, the particles have a particle size (diameter) of over about 45 nm. In some embodiments, the particles have a particle size (diameter) of over about 50 nm. In some embodiments, the particles have a particle size (diameter) of over about 60 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 500 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 400 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 300 nm. In some embodiments, the size is a hydrodynamic diameter. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 10mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 15mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 20mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 25mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 27mV.According to some embodiments, the lipid nanoparticle formulation has Zeta potential of at least 29mV. According to some embodiments, the lipid nanoparticle formulation has Zeta potential in the range of 28 to 40 mV. The term “zeta potential” refers to a physical measurement of a colloidal system by electrophoresis. It gives the value of the potential (in mV) of a colloid in a suspension at the boundary between the Stern layer and the diffuse layer. In other words, the zeta potential in a colloidal system is the difference in potential between the immovable layer attached to the surface of the dispersed phase and the dispersion medium. The zeta potential is related to stability of suspensions of particles. Zeta potential may be adjusted, in part, for example, by adjusting the concentration of an electrolyte in the buffer system. According to some embodiments, the lipid nanoparticle formulation has polydispersity index (PDI) of no more than 0.75. According to some embodiments, the lipid nanoparticle formulation has PDI of no more than 0.5. According to some embodiments, the lipid nanoparticle formulation has PDI of no more than 0.25. According to some embodiments, the lipid nanoparticle formulation has PDI of no more than 0.2. According to some embodiments, there is provided a composition suitable for administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation. Each possibility represents a separate embodiment of the invention. According to some embodiments, there is provided a liquid composition suitable for IV administration wherein the liquid composition comprises the present lipid nanoparticle formulation. It was found that the present lipid nanoparticle formulations are suitable for IV delivery. According to some embodiments, the composition is a liquid composition. According to some embodiments, there is provided a liquid composition suitable for IV administration, wherein the liquid composition comprises the present lipid nanoparticle formulation. According to some embodiments, the liquid composition is in the form of an aqueous solution, emulsion or suspension. According to some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like. Aqueous injection suspensions may also contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. The parenteral formulations can be present in unit dose or multiple dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as, for example, water for injection, immediately prior to use. In some embodiments, parenteral administration includes intravenous administration. As used herein, intravenous administration refers to one or more boluses injections as well as a continuous intravenous infusion. Each possibility represents a separate embodiment. The term “intravenous bolus” refers to administration into a vein of an animal or human in a duration of several minutes or less. The term “intravenous infusion” refers to administration into the vein of an animal or human patient over a period of time greater than 5 minutes, for example between about 30 to about 120 minutes, including each value within the specified range. According to some embodiments, the present lipid nanoparticle formulation is selectively targeting the lung upon systemic administration. According to some embodiments, the present lipid nanoparticle formulation is selectively targeting the lung upon administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation. According to some embodiments, the present lipid nanoparticle formulation is selectively targeting the lung upon administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation. Each possibility represents a separate embodiment of the invention. According to some embodiments, the present lipid nanoparticle formulation is selectively targeting the lung upon intravenous (IV) administration. According to some embodiments, the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney upon administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation. Each possibility represents a separate embodiment of the invention. According to some embodiments, the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney upon intravenous (IV) administration. Each possibility represents a separate embodiment of the invention. According to some embodiments, the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration. According to some embodiments, the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration in mammals. According to some embodiments, the present lipid nanoparticle formulation exhibits higher targeting to the lung compared to the heart, liver, spleen and kidney upon intravenous (IV) administration in humans. According to some embodiments, the present lipid nanoparticle formulation comprises a nucleic acid and/or protein encapsulated within at least one particle thereof, wherein administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation, in mammals of the formulation results in higher expression in the lungs of the mammal compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney. Each possibility represents a separate embodiment of the invention. According to some embodiments, the present lipid nanoparticle formulation comprises a nucleic acid and/or protein encapsulated within at least one particle thereof, wherein intravenous (IV) administration in mammals of the formulation results in higher expression in the lungs of the mammal compared to one or more organs selected from the group consisting of: heart, liver, spleen and kidney. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lung expression is higher than each one of the heart, liver, spleen and kidney. According to some embodiments, the compositions of the present invention may be used as a delivery system to administer a therapeutic agent to its target location in the body, wherein the target location is the lungs. Thus, according to some embodiments, the present invention relates to a method for administering a therapeutic agent, by preparing a composition comprising a lipid as described herein and a biologically active agent (e.g., a therapeutic agent, a protein, a nucleic acid and the like), and administering the composition to a subject in need thereof. According to some embodiments, the present invention relates to a method for administering a therapeutic agent, by preparing a particle as described herein comprising an active agent, and administering the composition to a subject in need thereof. According to some embodiments, the method further comprises encapsulating the active agent within a particle comprising the lipid. According to some embodiments, these lipid nanoparticle formulations are useful for expression of protein encoded by mRNA. According to some embodiments, these improved lipid nanoparticles formulations are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA. According to some embodiments, these improved lipid nanoparticle formulations are useful for down- regulating (e.g., silencing) the protein levels and/or mRNA levels of target genes. According to some embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes. According to some embodiments, the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antibody. According to some embodiments, there is provided a method of delivery of an active agent to the lung, the method comprising the step of systemically administering to a subject in need thereof the lipid nanoparticle formulation of the present invention and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the active agent is selected from the group consisting of: a protein, a therapeutic agent and a nucleic acid. Each possibility represents a separate embodiment of the invention. According to some embodiments, the active agent is a therapeutic agent. According to some embodiments, the active agent is a nucleic acid. Exemplary nucleic acids are discussed above. According to some embodiments, the method comprises the step of administering intravenously (IV), intranasally or via inhalation to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method comprises the step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the is provided a method for lung targeting, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation of the present invention. According to some embodiments, the administration is systemic. According to some embodiments, the administration is selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation. According to some embodiments, the administration is IV. According to some embodiments, the lipid nanoparticle formulation is for use in the treatment of a lung disease or disorder. According to some embodiments, the lipid nanoparticle formulation is for use in the treatment of a respiratory disease or disorder. According to some embodiments, the lipid nanoparticle formulation is for use in the treatment of a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection. Each possibility represents a separate embodiment of the invention. According to some embodiments, the respiratory infection is caused by a genetic disease or disorder. According to some embodiments, the lipid nanoparticle formulation is for use in the treatment of a hereditary lung disorder or a genetic lung disease. According to some embodiments, the present invention provides a method of treating a lung disease or disorder, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the present invention provides a method of treating a respiratory disease or disorder, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the present invention provides a method of treating a disease or disorder selected from the group consisting of: cystic fibrosis, lung cancer and a respiratory infection, the method comprising the step of administering to a subject in need thereof the lipid nanoparticle formulation and a pharmaceutically acceptable carrier, diluent or excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the respiratory infection is caused by a genetic disease or disorder. According to some embodiments, the method is for the treatment of a hereditary lung disorder or a genetic lung disease. According to some embodiments, the method comprises the step of systemically administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method comprises the step of administering intravenously (IV), intranasally or via inhalation to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the method comprises the step of intravenously (IV) administering to a subject in need thereof the lipid nanoparticle formulation and the pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the administration in any one of the methods disclosed herein is in the liquid phase. According to some embodiments, the lipid nanoparticle formulations are administered IV as solutions, emulsions or suspensions. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid nanoparticle formulations are administered IV as solutions. According to some embodiments, the lipid nanoparticle formulations are administered via inhalation as aerosols. According to some embodiments, the present invention provides a dosage form suitable for administration selected from the group consisting of: intravenous (IV) administration, internasal administration or administration via inhalation, wherein the dosage form comprises at least one container, which contains the present lipid nanoparticle formulation. Each possibility represents a separate embodiment of the invention. According to some embodiments, the present invention provides a dosage form suitable for IV administration, wherein the dosage form comprises at least one container, which contains the present lipid nanoparticle formulation. According to some embodiments, the dosage form comprises at least two containers, wherein the first container comprises the present lipid nanoparticle formulation in solid form and the second container comprises a biocompatible solvent suitable for reconstitution prior to IV administration. Typically, the biocompatible solvent is an aqueous medium, for example water or saline to maintain electrolyte balance. Intravenous (IV) administration can be performed by bolus injections, for example using a hypodermic syringe. Alternatively, IV administration can be performed by continuous infusion using a needle or a plastic or silicone catheter. Definitions To facilitate an understanding of the present invention, a number of terms and phrases are defined below. It is to be understood that these terms and phrases are for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art. As used herein, the term “nanoparticle” refers to a nanostructure that is generally or substantially spherical or spheroidal. Typically, each dimension of a nanoparticle is in a range of about 1 nm to about 1000 nm, As referred to herein, the terms "nucleic acid", "nucleic acid molecules" “oligonucleotide”, "polynucleotide", and "nucleotide" may interchangeably be used herein. The terms are directed to polymers of deoxy ribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof. The term also encompasses RNA/DNA hybrids. The polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA. The DNA or RNA molecules may be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, Antisense RNA, CRISPR/Cas and the like. Each possibility represents a separate embodiment of the present invention. The terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent inter nucleoside linkages, as well as oligonucleotides having non- naturally occurring portions, which function similarly to respective naturally occurring portions. The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term "construct", as used herein, refers to an artificially assembled or isolated nucleic acid molecule which may include one or more nucleic acid sequences, wherein the nucleic acid sequences may include coding sequences (that is, sequence which encodes an end product), regulatory sequences, non-coding sequences, or any combination thereof. The term construct includes, for example, vector but should not be seen as being limited thereto. "Expression vector" refers to constructs that have the ability to incorporate and express heterologous nucleic acid fragments (such as, for example, DNA), in a foreign cell. In other words, an expression vector comprises nucleic acid sequences/fragments (such as DNA, mRNA, tRNA, rRNA), capable of being transcribed. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art. In some representative embodiments, the expression vector may encode for a double stranded RNA molecule in the target site. The term "expression", as used herein, refers to the production of a desired end-product molecule in a target cell. The end-product molecule may include, for example an RNA molecule; a peptide or a protein; and the like; or combinations thereof. As used herein, the terms "introducing" and "transfection" may interchangeably be used and refer to the transfer of molecules, such as, for example, nucleic acids, polynucleotide molecules, vectors, and the like into a target cell(s), and more specifically into the interior of a membrane-enclosed space of a target cell(s). The molecules can be "introduced" into the target cell(s) by any means known to those of skill in the art, for example as taught by Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001), the contents of which are incorporated by reference herein. Means of "introducing" molecules into a cell include, for example, but are not limited to: heat shock, calcium phosphate transfection, PEI transfection, electroporation, lipofection, transfection reagent(s), viral-mediated transfer, and the like, or combinations thereof. The transfection of the cell may be performed on any type of cell, of any origin, such as, for example, human cells, animal cells, plant cells, virus cell, and the like. The cells may be selected from isolated cells, tissue cultured cells, cell lines, cells present within an organism body, and the like. The term “treating” and "treatment" as used herein refers to abrogating, inhibiting, slowing or reversing the progression of a disease or condition, ameliorating clinical symptoms of a disease or condition or preventing the appearance of clinical symptoms of a disease or condition. The term “preventing” is defined herein as barring a subject from acquiring a disorder or disease or condition. The term "treatment of cancer" is directed to include one or more of the following: a decrease in the rate of growth of the cancer (i.e. the cancer still grows but at a slower rate); cessation of growth of the cancerous growth, i.e., stasis of the tumor growth, and, the tumor diminishes or is reduced in size. The term also includes reduction in the number of metastases, reduction in the number of new metastases formed, slowing of the progression of cancer from one stage to the other and a decrease in the angiogenesis induced by the cancer. In most preferred cases, the tumor is totally eliminated. Additionally included in this term is lengthening of the survival period of the subject undergoing treatment, lengthening the time of diseases progression, tumor regression, and the like. In some embodiments, the cancer is a blood cancer. As used herein, the term “liver bypass” refers to a property of a lipid nanoparticle (LNP) formulation wherein the accumulation of the LNPs and/or the expression of a delivered payload (e.g., mRNA) in the liver is substantially reduced relative to other organs, such as the lungs or spleen. In the context of the present disclosure, “liver bypass” characterizes formulations designed to avoid or minimize hepatic uptake following systemic administration, thereby favoring delivery to non-hepatic target organs. For example, liver bypass may be reflected by a liver-to-target organ (e.g., lung or spleen) expression ratio of less than about 30%, 20%, 10%, 5%, or even less than about 1%, as determined by normalized luciferase activity or other quantitative expression assays. According to some embodiments, the liver-to-lung expression ratio is less than about 30%, 20%, 10%, 5%, or 1%, indicating preferential accumulation or expression in the lungs relative to the liver. The extent of liver bypass may be determined experimentally, for example by administering an LNP formulation comprising an mRNA encoding a reporter protein such as luciferase, and subsequently quantifying organ-specific expression using bioluminescent imaging or tissue-based luciferase assays. A formulation is considered to exhibit liver bypass when the liver shows minimal or significantly lower expression levels compared to one or more target organs, such as the lungs. Liver bypass may result from various formulation parameters, including but not limited to lipid composition, charge ratio, helper lipids, PEG-lipid content, and overall particle size or surface properties. As used herein, the term “lung targeting formulation” refers to a lipid nanoparticle (LNP) composition that, upon systemic administration, preferentially delivers its payload (e.g., mRNA) to the lung tissue relative to other organs. Such formulations result in enhanced accumulation of the LNPs and/or increased expression of the delivered nucleic acid in the lungs, as determined by appropriate in vivo or ex vivo assays, including but not limited to luciferase-based bioluminescence imaging or tissue lysate quantification. Lung targeting may be achieved by modifying one or more characteristics of the LNP formulation, such as lipid composition, molar ratios, helper lipid identity, PEG-lipid content, particle size, surface charge, or other physicochemical properties. According to some embodiments, lung targeting formulations also exhibit liver bypass properties, such that the liver receives minimal expression relative to the lung. Examples EXAMPLE 1: Synthesis of ionizable lipids Example 1A: Synthesis of lipid IA-12:
To a solution of linoleic alcohol (4.0 g, 15.0 mmol, 1 equiv.) in dry CH2Cl2 (80 mL), molecular sieves (4Å MS) were added under argon atmosphere. Then PCC (6.4 g, 30.0 mmol, 2 equiv.) was added portion wise over a period of 10 min and stirred for 2 hr at room temperature. After completing the reaction filtered it through a silica gel pad using CH2Cl2 to remove PCC. The solvent was evaporated under reduced pressure to obtain the linoleic aldehyde 3.9 g (99%) as a colorless liquid. Linoleic aldehyde (3.20 g, 12.12 mmol, 1 equiv.) and ethanolamine (0.89 ml, 14.54 mmol, 1.2 equiv.) were dissolved in dry CH2Cl2 (60 mL) under nitrogen atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (5.10 g, 24.24 mmol, 2 equiv.) was added portion wise over a period of 15 min, and stirred it for 16 hr at the same temperature. Later, the reaction mixture was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column chromatography using 0-10% MeOH in CHCl3 to get 1 (2.0 g, 54%) as a pale yellowish liquid. 1H NMR (400 MHz, CDCl3): δ 5.40-5.30 (4 H, m), 3.64 (2 H, t, J = 5.2 Hz), 2.83-2.73 (4 H, m), 2.62 (2 H, t, J = 7.2 Hz), 2.04 (4 H, q, J = 6.8 Hz), 1.54-1.43 (2 H, m), 1.41-1.21 (16 H, m), 0.88 (3 H, t, J = 6.8 HZ). ESI-MS: m/z 310.5 [M+1]+ (9Z,12Z)-N-(2-((Tert-butyldiphenylsilyl)oxy)ethyl)octadeca-9,12-dien-1-amine To a stirred solution of 1 (1.30 g, 4.2 mmol, 1 equiv.) and imidazole (628 mg, 9.24 mmol, 2.2 equiv.) in dry CH2Cl2 (40 mL), TBDPS-Cl (1.31 mL, 5.04 mmol, 1.2 equiv.) was added drop-wise under argon atmosphere and stirred it for overnight at room temperature. The reaction mixture was then poured in brain solution and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-3% MeOH in CHCl3 to afford TBDPS protected compound 2 in quantitative yield as a yellow color liquid. 1H NMR (400 MHz, CDCl3): δ 7.69-7.63 (4 H, m), 7.45-7.33 (6 H, m), 5.44-5.28 (4 H, m), 3.78 (2 H, t, J = 5.6 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.74 (2 H, t, J = 5.2 Hz), 2.59 (2 H, t, J = 7.2 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.53-1.42 (2 H, m), 1.41-1.21 (16 H, m), 1.05 (9 H, s), 0.89 (3 H, t, J = 6.8 HZ) ESI-MS: m/z 548.7 [M+1]+ 10-Oxodecanoic acid To a solution of IBX (11.9 g, 45 wt.%, 19.15 mmol, 1.2 equiv.) in DMSO (40 mL), a solution of 10- Hydroxydecanoic acid (3.0 g, 15.96 mmol, 1.0 equiv.) in THF (20 mL) was added and stirred for 6 hr at room temperature. After that, the reaction was quenched with water (20 mL) and the precipitated solid was removed by filtration. The filtrate was diluted with water and extracted with diethyl ether (4 x 100 mL). The organic layer dried over anhydrous Na2SO4 and the solvent was removed on rotary evaporator. The crude product was purified by column chromatography using 0-20% ethyl acetate in hexane to obtain 10- oxodecanioc acid 3 (2.60 g, 87%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.75 (1 H, t, J = 2.0 Hz), 2.41 (2 H, dt, J = 7.2, 2.0 Hz), 2.33 (2 H, t, J = 7.6 Hz), 1.61 (4 H, quint, J = 7.2 Hz), 1.39-1.23 (8 H, m). 10-((2-((tert-Butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decanoic acid The compound 2 (700 mg, 1.27 mmol, 1.0 equiv.) and 10-oxodecanioc acid 3 (285 mg, 1.53 mmol, 1.2 equiv.) were dissolved in dry CH2Cl2 (30 mL) under argon atmosphere and stirred for 1 hr at room temperature. Then sodium triacetoxyborohydride (404 mg, 1.91 mmol, 1.5 equiv.) was added and stirred it for 24 hr at the same temperature. Later, the reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated rotary evaporator and the residue was purified by column chromatography using 0-3% MeOH in CHCl3 to provide 5 (778 mg, 85%) as a pale yellowish viscus liquid. 1H NMR (400 MHz, CDCl3): δ 7.67-7.61 (4 H, m), 7.46-7.34 (6 H, m), 5.43-5.27 (4 H, m), 3.86 (2 H, t, J = 5.6 Hz), 2.96 (2 H, t, J = 5.6 Hz), 2.85-2.66 (6 H, m), 2.21 (2 H, t, J = 7.2 Hz), 2.11-1.96 (4 H, m), 1.65-1.44 (6 H, m), 1.42-1.13 (26 H, m), 1.04 (9 H, s), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m/z 718.9 [M+1]+; 716.9 [M- 1]- 10-Hydroxydecanal 1, 10-Decanediol (1.0 g, 5.7 mmol, 1 equiv.) was dissolved in dry THF (40 mL) under argon atmosphere and molecular sieves (4Å MS) were added. Then PCC (1.5 g, 6.9 mmol, 1.2 equiv.) was added portion wise to the reaction mixture over a period of 5 min and stirred for 2 hr at room temperature. After that, the reaction mixture was filtered through silica gel pad to remove PCC followed by wash with 30% ethyl acetate in hexane (2 × 100 mL). The solvent was evaporated under reduced pressure and the residue was purified by column chromatography using 5-15% ethyl acetate in hexane to obtain the 10-hydroxydecanal 4 (0.49 g, 49%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.77 (1 H, t, J = 2.0 Hz), 3.64 (2 H, t, J = 6.8 Hz), 2.43 (2 H, dt, J = 7.6, 2.0 Hz), 1.69-1.52 (4 H, m), 1.41-1.25 (10 H, m). 10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decan-1-ol The compound 2 (1.8 g, 3.28 mmol, 1 equiv.) and 10-hydroxydecanal 4 (678 mg, 3.94 mmol, 1.2 equiv.) were dissolved in dry CH2Cl2 (50 mL) under argon atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (1.38 g, 6.56 mmol, 2 equiv.) was added and stirred it for 24 hr at the same temperature. After that, the reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 × 30 mL). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated on rotary evaporator and the residue was purified by column chromatography using 0-2% MeOH in CHCl3 to obtain 6 (1.9 g, 82%) as a yellowish viscus liquid. 1H NMR (400 MHz, CDCl3): δ 7.70-7.63 (4 H, m), 7.45-7.33 (6 H, m), 5.43-5.27 (4 H, m), 3.85-3.68 (2 H, br), 3.63 (2 H, dt, J = 6.8, 2.0 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.72-2.57 (2 H, br), 2.53-2.32 (4 H, br), 2.04 (4 H, q, J= 8.0 Hz) 1.65-1.47 (4 H, m), 1.45-1.12 (30 H, m), 1.05 (9 H, s), 0.89 (3 H, t, J = 7.2 Hz). ESI-MS: m/z 704.9 [M+1]+ 10-((2-Hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10-((2-hydroxyethyl)((9Z,12Z)- octadeca-9,12-dien-1-yl)amino)decanoate The alcohol 6 (303 mg, 0.43 mmol, 1.0 equiv.), acid 5 (370 mg, 0.51 mmol, 1.2 equiv.), EDC.HCl (165 mg, 0.86 mmol, 2.0 equiv.) and DMAP (11 mg, 0.09 mmol, 0.2 equiv.) were dissolved in dry CH2Cl2 (15 mL) under nitrogen atmosphere and stirred for 24 hr at room temperature. Later, the reaction was quenched with sat. NaHCO3 and extracted with CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue purified with a short silica gel column (2% IPA/CHCl3) to get the desired product (417 mg, 69%). The obtained product (417 mg, 0.3 mmol, 1 equiv.) was dissolved in THF (5 mL), and TBAF (1.2 mL, 1.0 M in THF, 1.19 mmol, 4.0 equiv.) was added. The reaction was stirred for 3 hr at room temperature and quenched with sat. NH4Cl, and extracted with 20% ethyl acetate in diethyl ether. Then the combined organic portion was washed with sat. NH4Cl solution (3 times) to remove TBAF completely. The solvent was evaporated under reduced pressure and the residue purified by column chromatography using 0-15% isopropanol in chloroform to obtain the IA-12 (NV3-006) (220 mg, 80%) as a yellow color viscus liquid. 1H NMR (400 MHz, CDCl3): δ 5.45-5.26 (8 H, m), 4.05 (2 H, t, J = 6.8 Hz), 3.60 (4 H, t, J = 4.8 Hz), 2.77 (4 H, t, J = 6.4 Hz), 2.71-2.62 (4 H, br), 2.59-2.47 (8 H, br), 2.28 (4 H, t, J = 7.6), 2.05 (8 H, q, J = 6.8 Hz), 1.60 (6 H, quint, J = 6.8 Hz), 1.55-1.42 (8 H, m), 1.40-1.20 (50 H, m), 0.89 (6 H, t, J = 7.2 Hz). ESI-MS: m/z 928.3 [M+1]+; 464.7 [M/2+1]+ Figure 1A is a 1H NMR spectrum of lipid IA-12 (NV3-006). Figure 1B is an ESI-MS spectrum of lipid IA- 12 (NV3-006). Example 1B: Synthesis of lipid IA-11:
2-(Dodecylamino)ethan-1-ol To a solution of dodecanal (2.4 mL, 10.87 mmol, 1.0 equiv.) in dry CH2Cl2 (60 mL), ethanolamine (0.79 ml, 13.04 mmol, 1.2 equiv.) was added under nitrogen atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (4.6 g, 21.74 mmol, 2.0 equiv.) was added portion wise over a period of 15 min and stirred for 24 hr at the same temperature. The reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column chromatography using 0-10% MeOH in CHCl3 to obtain 7 (0.97 g, 40%) and 8 (0.45 g, 19%) as white solid and pale yellowish liquid respectively. 1H NMR (400 MHz, CDCl3): δ 3.64 (2 H, t, J = 5.2 Hz), 3.08 (2 H, br), 2.75 (2 H, t, J = 5.2 Hz), 2.60 (2 H, t, J = 7.2 Hz), 1.48 (2 H, quint, J = 7.2 Hz), 1.35-1.17 (18 H, m), 0.86 (3 H, t, J = 7.2 Hz). ESI-MS: m/z 230.4 [M+1]+ 2-(Didodecylamino)ethan-1-ol 1H NMR (400 MHz, CDCl3): δ 3.54 (2 H, t, J = 5.6 Hz), 2.60 (2 H, t, J = 5.2 Hz), 2.46 (4 H, t, J = 7.6 Hz), 1.44 (4 H, quint, J = 6.8 Hz), 1.34-1.19 (36 H, m), 0.88 (6 H, t, J = 7.2 Hz). ESI-MS: m/z 398.7 [M+1]+ N-(2-((Tert-butyldiphenylsilyl)oxy)ethyl)dodecan-1-amine To a solution of 7 (4.0 g, 17.47 mmol, 1.0 equiv.) and imidazole (2.38 g, 34.94 mmol, 2.0 equiv.) in dry CH2Cl2 (100 mL), TBDPS-Cl (5.0 mL, 19.22 mmol, 1.1 equiv.) was added drop-wise over a period of 5 min under argon atmosphere and stirred for overnight at room temperature. Then the reaction mixture was poured in brain solution and extracted with CH2Cl2 (3 times). The organic portion was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-2% MeOH in CHCl3 to afford TBDPS protected compound 9 (7.73 g, 95%) as a pale yellowish liquid. 1H NMR (400 MHz, CDCl3): δ 7.69-7.63 (4 H, m), 7.45-7.34 (6 H, m), 3.78 (2 H, t, J = 5.2 Hz), 2.74 (2 H, t, J = 5.2 Hz), 2.60 (2 H, t, J = 7.2 Hz), 1.49 (2 H, quint, J = 6.8 Hz), 1.36-1.18 (18 H, m), 1.05 (9 H, s), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m/z 468.7 [M+1]+ 10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)(dodecyl)amino)decanoic acid The compound 9 (5.03 g, 10.75 mmol, 1.0 equiv.) and 10-oxodecanioc acid 3 (2.4 g, 12.90 mmol, 1.2 equiv.) were dissolved in dry CH2Cl2 (180 mL) under argon atmosphere and stirred for 1 hr. at room temperature. Then sodium triacetoxyborohydride (3.4 g, 16.12 mmol, 1.5 equiv.) was added, and the reaction mixture was stirred for 24 hr at the same temperature. Later, the reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated rotary evaporator and the residue was purified by column chromatography using 0-5% IPA in CHCl3 to provide 10 (6.6 g, 97%) as a pale yellowish viscus liquid. 1H NMR (400 MHz, CDCl3): δ 7.69-7.60 (4 H, m), 7.46-7.33 (6 H, m), 3.86 (2 H, t, J = 5.6 Hz), 2.95 (2 H, t, J = 5.6 Hz), 2.81-2.66 (4 H, m), 2.21 (2 H, t, J = 7.2 Hz), 1.64-1.43 (6 H, m), 1.36-1.13 (28 H, m), 1.04 (9 H, s), 0.87 (3 H, t, J = 6.8 Hz). ESI-MS: m/z 638.9 [M+1]+; 636.8 [M-1]- 10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10-((2-((tert- butyldiphenylsilyl)oxy)ethyl)(dodecyl)amino)decanoate The alcohol 6 (338 mg, 0.48 mmol, 1.0 equiv.), acid 10 (460 mg, 0.72 mmol, 1.5 equiv.), EDC.HCl (183 mg, 0.96 mmol, 2.0 equiv.) and DMAP (12 mg, 0.01 mmol, 0.2 equiv.) were dissolved in dry CH2Cl2 (10 mL) under argon atmosphere and stirred for 24 hr at room temperature. Then the reaction was quenched with sat. NaHCO3 and extracted with CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue purified by column chromatography using 0-2% IPA/CHCl3 to obtain the desired product 11 (590 mg, 93%) as pale yellowish liquid. 1H NMR (400 MHz, CDCl3): δ 7.71-7.64 (8 H, m), 7.45-7.33 (12 H, m), 5.44-5.23 (4 H, m), 4.05 (2 H, t, J = 6.8 Hz), 3.70 (4 H, t, J = 6.8 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.61 (4 H, t, J = 6.8 Hz), 2.37 (8 H, t, J = 7.2 Hz), 2.28 (2 H, t, J = 7.6 Hz), 2.05 (4 H, q, J = 7.2 Hz), 1.72-1.52 (6 H, m), 1.44-1.11 (62 H, m), 1.05 (18 H, s), 0.93-0.84 (6 H, m). ESI-MS: m/z 663.3 [M/2+1]+ 10-((2-Hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10-(dodecyl(2- hydroxyethyl)amino)decanoate To a solution of 11 (520 mg, 0.39 mmol, 1 equiv.) in dry THF (10 mL), TBAF (1.6 mL, 1.57 mmol, 4.0 equiv.) was added. The reaction was stirred for 3 hrs at room temperature and quenched with sat. NH4Cl and extracted with the mixture of ethyl acetate and diethyl ether (20:80). Then the combined organic portion was washed with sat. NH4Cl solution (3 times) to remove TBAF completely. The solvent was evaporated, and the residue purified by column chromatography using 0-15% isopropanol in chloroform to obtain the IA-11 (NV3-004; 244 mg, 74%) as a yellow color viscus liquid. 1H NMR (400 MHz, CDCl3): δ 5.44-5.26 (4 H, m), 4.05 (2 H, t, J = 6.4 Hz), 3.55 (4 H, t, J = 5.2 Hz), 2.77 (2 H, t, J = 6.4 Hz), 2.60 (4 H, t, J = 5.2 Hz), 2.47 (8 H, t, J = 7.2 Hz), 2.28 (2 H, t, J = 7.2 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.61 (6 H, quint, J = 6.8 Hz),1.50-1.39 (8 H, m), 1.38-1.13 (54 H, m), 0.93-0.82 (6 H, m). ESI-MS: m/z 848.3 [M+1]+; 424.7 [M/2+1]+ Figure 1C is a 1H NMR spectrum of lipid IA-11 (NV3-004). Figure 1D is an ESI-MS spectrum of lipid IA- 11 (NV3-004). Example 1C: Synthesis of lipid IA-10:
10-((2-((Tert-butyldiphenylsilyl)oxy)ethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10- oxodecanoate The alcohol 6 (512 mg, 0.73 mmol, 1.0 equiv.), 10-oxodecanioc acid 3 (202 mg, 1.09 mmol, 1.5 equiv.), EDC.HCl (277 mg, 1.45 mmol, 2.0 equiv.) and DMAP (18 mg, 0.14 mmol, 0.2 equiv.) were dissolved in dry CH2Cl2 (10 mL) under argon atmosphere and stirred for 24 hr at room temperature. The reaction mixture was quenched with sat. NaHCO3 and extracted with CH2Cl2 (3 times). The organic portion was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated on rotary evaporator and the crude product was purified by column chromatography using 0-5% isopropanol in chloroform to obtain the 13 (564 mg, 89%) as a pale yellow color viscus liquid. 10-((2-Hydroxyethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)decyl 10- (dodecyl(hydroxy)amino)decanoate To a suspension of hydroxylamine hydrochloride (34 mg, 0.48 mmol, 1.0 equiv.) in dry CH2Cl2 (5 mL), trimethylamine (67 µL, 0.48 mmol, 1.0 equiv.) was added under argon atmosphere and stirred for 5 min at room temperature. Later, a solution of aldehyde 13 (420 mg, 0.48 mmol, 1.0 equiv.) in dry CH2Cl2 (10 mL) was added drop wisely and stirred it for 2 hr. After that, the reaction mixture was diluted with another 10 mL of dry CH2Cl2, and sodium triacetoxyborohydride (150 mg, 0.72 mmol, 1.5 equiv.) was added portion wise over a period of 10 min and stirred for another 10 min. Then a solution of dodecanal (160 µl, 0.721 mmol, 1.5 equiv.) in dry CH2Cl2 (10 mL) was added drop wisely to the reaction mixture and stirred for another 10 min. Later the remaining amount of sodium triacetoxyborohydride (150 mg, 0.72 mmol, 1.5 equiv.) was added portion wise over a period of 15 min and left for the overnight stirring at room temperature under argon atmosphere. The reaction was quenched with sat.NaHCO3 solution and extracted with CH2Cl2 (3 times). The solvent was evaporated on rotary evaporator and the crude product was dissolved in THF (10 mL) and TBAF (1.0 mL, 1.0 M in THF, 0.96 mmol, 2.0 equiv.) was added. The reaction was stirred for 3 hr at room temperature and quenched with sat. NH4Cl, and extracted with 20% ethyl acetate in diethyl ether (3 times). The combined organic portion was washed with sat. NH4Cl solution (3 times) to remove TBAF completely. The solvent was evaporated under reduced pressure and the residue purified by column chromatography using 0-5% isopropanol in chloroform to bestow the IA-10 (NV3-002; 208 mg, 53%) as a pale yellowish gum. 1H NMR (400 MHz, CDCl3): δ 5.44-5.27 (4 H, m), 4.06 (2 H, t, J = 6.8 Hz), 3.81-3.58 (2 H, m), 2.77 (4 H, t, J = 6.4 Hz), 2.64 (8 H, t, J = 7.6 Hz), 2.29 (2 H, t, J = 7.6 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.69-1.48 (14 H, m), 1.38-1.13 (54 H, m), 0.89 (3 H, t, J = 6.8 Hz), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m/z 820.3 [M+1]+; 410.7 [M/2+1]+1H NMR (400 MHz, CDCl3): δ 5.44-5.27 (4 H, m), 4.06 (2 H, t, J = 6.8 Hz), 3.69-3.59 (2 H, br), 2.77 (2 H, t, J = 6.4 Hz), 2.74-2.67 (2 H, br), 2.63 (4 H, t, J = 7.6 Hz), 2.67-2.51 (4 H, br), 2.28 (2 H, t, J = 7.6 Hz), 2.05 (4 H, q, J = 6.8 Hz), 1.66-1.45 (14 H, m), 1.38-1.13 (54 H, m), 0.89 (3 H, t, J = 6.8 Hz), 0.88 (3 H, t, J = 6.8 Hz). ESI-MS: m/z 820.28 [M+1]+; 410.68 [M/2+1]+ Figure 1E is a 1H NMR spectrum of lipid IA-10 (NV3-002). Figure 1F is an ESI-MS spectrum of lipid IA- 10 (NV3-002). Examples 1D and 1E: Syntheses of lipids II-1 and II-5: To a solution of hexanol (4.0 g, 39.2 mmol, 1 equiv.) in dry CH2Cl2 (100 mL), molecular sieves (4Å MS) were added under argon atmosphere. Then PCC (12.64 g, 58.8 mmol, 1.5 equiv.) was added portion wise over a period of 10 min and stirred for 2 hr at room temperature. After completing the reaction filtered it through a silica gel pad using CH2Cl2 to remove PCC. The solvent was evaporated on rotary evaporator with low vacuum to get a solution of hexanal (20 mL). The aldehyde solution was dried over anhydrous sodium sulfate and subjected to the next step. To a suspension of hydroxylamine hydrochloride (871 mg, 11.76 mmol, 0.3 equiv.) in dry CH2Cl2 (20 mL), dry trimethylamine (1.6 mL, 11.76 mmol, 0.3 equiv.) was added under argon atmosphere and stirred for 10 min at room temperature. Then, a solution of hexanal in dry CH2Cl2 (30 mL) was added drop wisely and stirred for 2 hr. After that, the reaction mixture was diluted with another 50 mL of dry CH2Cl2 and sodium triacetoxyborohydride (7.4 g, 35.3 mmol, 0.9 equiv.) was added portion wise and left for the overnight stirring at room temperature. Later, the reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column chromatography using 0-10% EtOAc in Hexane to obtain N,N-dihexylhydroxylamine 18 (2.2 g, 91%). 1H NMR (400 MHz, CDCl3): δ 2.64 (4 H, t, J = 7.6 Hz), 1.58 (4 H, quint, J = 7.2 Hz), 1.40-1.18 (12 H, m), 0.87 (6 H, t, J = 6.8 Hz). ESI-MS: m/z 202.4 [M+1]+ 10-((Dihexylamino)oxy)-10-oxodecan-1-ol The above hydroxylamine 18 (500 mg, 2.49 mmol, 1.0 equiv.), 10-((tert-butyldiphenylsilyl)oxy)decanoic acid 19 (1.27 g, 2.98 mmol, 1.2 equiv.), EDC.HCl (950 mg, 4.97 mmol, 2.0 equiv.) and DMAP (60 mg, 0.50 mmol, 0.2 equiv.) were dissolved in dry CH2Cl2 (20 mL) under argon atmosphere and left for the overnight stirring at room temperature. Then the reaction was quenched with sat. NaHCO3 followed by extract with CH2Cl2 (3 times) and washed with brine solution and dried over with anhydrous Na2SO4. The solvent was evaporated on rotary evaporator and the crude product was dissolved in THF (10 mL) and TBAF (5.0 mL, 1.0 M in THF, 4.97 mmol, 2.0 equiv.) was added. The reaction was stirred for 3 hr at room temperature and quenched with sat. NH4Cl, and extracted with ethyl acetate (3 times). The solvent was evaporated and the residue purified by column chromatography using 0-5% EtOAc in hexane to obtain the desired alcohol 20 (721 mg, 78%) as a colorless liquid. 1H NMR (400 MHz, CDCl3): δ 3.63 (2 H, q, J = 4.4 Hz), 2.80 (4 H, t, J = 7.6 Hz), 2.27 (2 H, t, J = 7.6 Hz), 1.71-1.44 (8 H, m), 1.41-1.17 (22 H, m), 0.87 (6 H, t, J = 6.8 Hz). ESI-MS: m/z 394.5 [M+Na]+ 2-(Bis(10-((dihexylamino)oxy)-10-oxodecyl)amino)ethanol To a solution of IBX (704 mg, 45 wt.%, 1.13 mmol, 1.2 equiv.) in DMSO (6 mL), a solution of a solution of alcohol 20 (350 mg, 0.94 mmol, 1.0 equiv.) in THF (6 mL) was added and stirred for 5 hr at room temperature. After that, the reaction was quenched with water (10 mL) and the precipitated solid was removed by filtration. The filtrate was diluted with water and extracted with diethyl ether (4 x 10 mL). The organic layer dried over anhydrous Na2SO4 and the solvent was removed on rotary evaporator. The crude product was dried and subject to the next step. The crude product was dissolved in dry CH2Cl2 (20 mL) and ethanolamine (27 µL, 0.45 mmol, 0.475 equiv.) was added under argon atmosphere and stirred for 2 hr. at room temperature. Then sodium triacetoxyborohydride (298 mg, 1.41 mmol, 1.5 equiv.) was added portion wise and stirred for 24 hr at the same temperature. The reaction was quenched with sat.NaHCO3 solution followed by extract with CH2Cl2 (3 times). The organic layer was washed with brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and the residue was purified by column chromatography using 0-6% IPA in CHCl3 to obtain the lipid II-1 (NV1-001) (195 mg, 57%) as colorless liquid. 1H NMR (400 MHz, CDCl3): δ 3.54 (2 H, t, J = 5.2 Hz), 2.80 (8 H, t, J = 7.6 Hz), 2.60 (2 H, t, J = 5.2 Hz), 2.46 (4 H, t, J = 7.2 Hz), 2.27 (4 H, t, J = 7.7.2 Hz), 1.64 (6 H, quint, J = 7.6 Hz), 1.56-1.38 (10 H, m), 1.41- 1.17 (44 H, m), 0.87 (12 H, t, J = 6.8 Hz). ESI-MS: m/z 769.01 [M+1]+ 6-(10-((dihexylamino)oxy)-10-oxodecyl)-18-hexyl-16-oxo-3,17-dioxa-6,18-diazatetracosan-1-ol: The crude product of 20 (460.0 mg, 1.00 wt) was dried and subject to the next step. The crude product (1.00 wt, 1.00 equiv) was dissolved in dry DCM (20 mL, 40 vol) and 2-(aminoethoxy)ethanol (56.2 mg, 0.12 wt, 0.45 equiv.) was added under a nitrogen atmosphere. The reaction was stirred for 2 h at room temperature then sodium triacetoxyborohydride (754.6 mg, 3.0 equiv.) was added portion wise and stirred for at least 5 h at the same temperature. The reaction was quenched with saturated NaHCO3 solution (40 vol) followed by extract with DCM (3 x 10 vol). The organic layer was washed with brine solution (10 vol) and dried over anhydrous Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography using 0-1% (v/v) MeOH in CHCl3 to obtain II-5 (NV1-005) as colorless oil in 3% th yield. 1H NMR (400 MHz, CDCl3): δ 3.71-3.63 (3 H, m), 3.61 (2 H, t), 2.80 (7 H, t), 2.74 (2 H, t), 2.57 (5 H, t), 2.27 (6 H, t), 1.69-1.59 (9 H, m), 1.54-1.43 (10 H, m), 1.41-1.22 (48 H, m), 0.87 (12 H, t). ESI-MS: m/z 812.0 [M+H]+ Example 1F: Synthesis of lipid IB-10:
4-((10-((dihexylamino)oxy)-10-oxodecyl)(dodecyl)amino)butan-1-ol To a clean flask were charged 10-((dihexylamino)oxy)-10-oxodecan-1-ol (1 wt, 1.0 equiv) and PCC, 98% (2 wt, 2.0 equiv). The reagents were suspended in anhydrous DCM (5 vol) and celite (3 wt) was added to the mixture. The reaction mixture was stirred at 18 to 25 °C for at least 3 h or until complete by TLC analysis in 20% (v/v) EtOAc in hexane. On reaction completion, the crude reaction mixture was filtered over silica gel in hexane. The silica was washed with 20% (v/v) EtOAc in hexane (2 x 25 vol). The filtrates were combined, and solvents were removed using rotavapor to yield orange colored oil. The crude intermediate aldehyde (0.34g, 0.9mol) was dissolved in dry CH2Cl2 (20 mL) and 4-aminobutanol (0.08g, 0.9mol) was added under a nitrogen atmosphere. The reaction was stirred for 2 h at room temperature then sodium triacetoxyborohydride (0.3g, 1.35mol) was added portion-wise and stirred for 5 h at the same temperature. On detection of the single chain intermediate charged dodecanal (1.10 equiv). The reaction was quenched with saturated NaHCO3 solution followed by extraction with CH2Cl2 (3 x 10 vol). The organic layer was washed with brine solution (10 vol) and dried over anhydrous Na2SO4. The solvent was evaporated under vacuum at 40 °C and the residue was purified by column chromatography. The chromatography was performed in 0 - 3% v/v MeOH in chloroform to remove the impurities and the lipid desired lipid IB-10 (NV2-011) was isolated as a pale-yellow oil (0.05g). 1H NMR (400 MHz, CDCl3): δ 3.6 (2 H, s), 2.77 (3 H, t), 2.56 (5 H, s), 2.45 (3 H, t), 2.11 (2 H, 2), 1.66- 1.60 (8 H, m), 1.55-1.37 (8 H, m), 1.36-1.17 (42 H, m), 0.88 (9 H, t). ESI-MS: m/z 612.0 [M+H]+ Example 1G: Synthesis of lipid IB-4 (NV2-004): To a clean flask were charged 10-((dihexylamino)oxy)-10-oxodecan-1-ol (1 wt, 1.0 equiv) and PCC, 98% (2 wt, 2.0 equiv). The reagents were suspended in anhydrous DCM (5 vol) and celite (3 wt) was added to the mixture. The reaction mixture was stirred at 18 to 25 °C for at least 3 h or until complete by TLC analysis in 20% (v/v) EtOAc in hexane. On reaction completion, the crude reaction mixture was filtered over silica gel in hexane. The silica was washed with 20% (v/v) EtOAc in hexane (2 x 25 vol). The filtrates were combined, and solvents were removed using rotavapor to yield orange colored oil 1H NMR (400 MHz, CDCl3): δ 9.76 (t, J = 1.8 Hz, 1H), 2.84 – 2.76 (m, 4H), 2.42 (td, J = 7.3, 1.8 Hz, 2H), 2.27 (t, J = 7.5 Hz, 2H), 1.68 – 1.56 (m, 4H), 1.55 (s, 8H), 1.37 – 1.19 (m, 23H), 0.87 (t, J = 6.7 Hz, 9H). ESI-MS: m/z 370.8 [M+H] + Synthesis of (6Z,9Z)-18-bromooctadeca-6,9-diene To a stirred solution of linoleyl alcohol (3.0g, 11.300 mmol, 1.0 equiv.) in DCM (100 mL) was added triphenyl phosphine (3.56g, 13.600mmol, 1.2 equiv.), stirred for 10 min then carbon tetra bromide (4.510g, 13.600 mmol, 1.2 equiv.) added in one portion. Then the reaction mixture stirred at room temperature for overnight. The progress of the reaction monitored by TLC analysis (5% EtOAc in Hexane). After completion of the reaction, the solvent was removed and purified by column chromatography using 0-5% EtOAc in Hexane to afford 3.7g of (6Z,9Z)-18-bromooctadeca-6,9-diene (2) as a colourless liquid. SM = starting material; co= SM and RM and RM= reaction mixture; Blue spot indicates product (It applies where these terms appear). 1H NMR (400 MHz, CDCl3): δ 5.44 – 5.28 (m, 4H), 3.41 (t, J = 6.9 Hz, 2H), 2.78 (t, J = 6.5 Hz, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.91 – 1.80 (m, 2H), 1.46 – 1.35 (m, 4H), 1.35 – 1.22 (m, 12H), 0.93 – 0.85 (m, 3H). Synthesis of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) ethoxy) ethan-1-ol (8) To a stirred solution of (6Z,9Z)-18-bromooctadeca-6,9-diene (2) (4.0 g, 12.144 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 2-(2-aminoethoxy) ethan-1-ol (12.16mL, 121.43 mmol, 10.0 equiv.) at room temperature and then stirred for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 3.2 g of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) ethoxy) ethan-1-ol (8) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 5.44 – 5.27 (m, 4H), 3.76 – 3.69 (m, 2H),3.67-3.57 (m,4H), 2.85 – 2.73 (m, 4H), 2.66 – 2.58 (m, 2H), 2.10-2.00 (m,4H), 1.50 (q, J = 7.2 Hz, 2H), 1.42 – 1.28 (m, 18H), 0.94-0.84(m, 3H). ESI-MS: m/z 354.7 [M+H] +. Synthesis of 18-hexyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)-16-oxo-3,17-dioxa-6,18-diazatetracosan-1- ol (NV2-004) To a stirred solution of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) ethoxy) ethan-1-ol (8) (0.2g, 0.565 mmol, 1.0 equiv.) in DCM (15 mL) was added 10-((dihexylamino)oxy)-10-oxodecanal (0.25 g, 0.678 mmol, 1.2 equiv.) at room temperature, stirred for 1 h. Then STAB (0.239 g, 1.131 mmol, 2.0 equiv.) added at room temperature, stirred for 3h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of aldehyde, the reaction mixture was quenched with sat. NaHCO3 solution (pH=7-8) and extracted with DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-12% IPA in CHCl3 to give the 270mg g of 18-hexyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)-16-oxo-3,17-dioxa-6,18- diazatetracosan-1-ol (NV2-004; lipid IB-4) as yellow colour liquid. 1H NMR (400 MHz, CDCl3): δ 5.44 – 5.27 (m, 4H), 3.75 – 3.69 (m, 3H), 3.62 (dd, J = 5.3, 3.3 Hz, 2H), 2.84 – 2.73 (m, 8H), 2.27 (t, J = 7.5 Hz, 2H), 2.05 (q, J = 6.9 Hz, 5H), 1.63 (p, J = 7.5 Hz, 5H), 1.51 (s, 6H), 1.42 – 1.34 (m, 3H), 1.34 – 1.22 (m, 36H), 0.88 (t, J = 6.6 Hz, 9H). ESI-MS: m/z 708 [M+H] + Figure 26A is a 1H NMR spectrum of lipid IB-4 (NV2-004). Figure 26B is an ESI-MS spectrum of lipid IB- 4 (NV2-004). Example 1H: Synthesis of lipid IB-13 (NV2-015):
Synthesis of 6-oxohexanoic acid To a stirred solution of 6-hydroxyhexanoic acid (1.5g, 11.349 mmol, 1.0 equiv.) in DMSO (12 mL) was added IBX (4.76g, 17.024mmol, 1.5equiv.) at rt, stirred for overnight. The progress of the reaction monitored by TLC analysis (60% EtOAc in Hexane). After completion of the reaction, the reaction mixture quenched with the addition of H2O (20ml), solids were precipitated out which were collected by filtration. Collect the filtrate and extracted into diethyl ether (2x50mL). Then combined organic layers were dried over anhydrous Na2SO4, filtered and distilled off to give the crude material. The crude was purified by Buchi flash using0- 30% EtOAc in hexane to afford 608 mg of 6-oxohexanoic acid as a semi solid. 1H NMR (400 MHz, CDCl3): δ 9.78 (t, J = 1.6 Hz, 1H), 2.55- 2.27 (m, 4H), 1.77-1.60 (m, 4H). ESI-MS: N/A Synthesis of 2-(didodecylamino) ethan-1-ol : To a stirred solution of dodecanal (3.0 g, 16.276 mmol, 1.0 equiv.) in dry DCM (30 mL) was added ethanol amine (0.447g, 7.324 mmol, 0.45 equiv.) at rt, stirred for 2h. Then reaction mixture was further diluted with dry DCM (30 mL). Then STAB (6.9 g, 32.552 mmol, 2.0 equiv.) was charged to the reaction mixture at rt, stirred for 18 h. The progress of the reaction was monitored by TLC (5% MeOH in CHCl3). The reaction mixture was quenched with sat. NaHCO3 solution and extracted with DCM (2x100 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash pure system using 0-5% MeOH in CHCl3 to afford 1.75 g of 2- (didodecylamino) ethan-1-ol (2) as yellow liquid. 1H NMR (400 MHz, CDCl3): δ 3.53 (t, J = 5.4 Hz, 2H), 2.58 (t, J = 5.4 Hz, 2H), 2.49 – 2.41 (m, 4H), 1.43 (q, J = 6.6 Hz, 4H), 1.26 (d, J = 3.6 Hz, 40H), 0.92 – 0.85 (m, 7H). ESI-MS: N/A Synthesis of 2-(didodecylamino) ethyl 6-oxohexanoate : To a stirred solution of 6-oxohexanoic acid (3) (0.5 g, 3.842 mmol, 1.0 equiv.) and of 2-(didodecylamino) ethan-1-ol (2) (1.22 g, 3.073 mmol, 0.80 equiv.) in dry DCM (25 mL) were added EDC.HCl (1.32 g, 6.915 mmol, 1.8 equiv.) and DMAP (0.093 g, 0.768 mmol, 0.2 equiv. ) at room temperature, stirred for 3-4 h.The progress of the reaction was monitored by TLC (5% MeOH in CHCl3). After completion of acid, the reaction mixture was quenched with water and extracted into 100%EtOAc (3x50 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% IPA in CHCl3 to give 0.495 g of 2-(didodecylamino) ethyl 6- oxohexanoate (4) as yellow liquid. 1H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.6 Hz, 1H), 4.12 (td, J = 6.3, 2.5 Hz, 2H), 2.67 (t, J = 6.3 Hz, 2H), 2.45 (ddd, J = 9.5, 7.2, 5.3 Hz, 5H), 2.33 (td, J = 5.8, 2.5 Hz, 2H), 1.66 (h, J = 3.2 Hz, 4H), 1.40 (q, J = 6.8 Hz, 4H), 1.26 (s, 35H), 0.92 – 0.84 (m, 6H). ESI-MS: m/z 542.8 [M+MeOH] + and 510.8 [M+H] + Synthesis of 2-(2-(dodecylamino) ethoxy) ethan-1-ol (6) To a stirred solution of 1-Bromo dodecane (4.0 g, 16.048 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 2-(2-aminoethoxy) ethan-1-ol (16 mL, 160.48 mmol, 10.0 equiv.) at room temperature and then stirred for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 3.1 g of 2-(2- (dodecylamino) ethoxy) ethan-1-ol (6) as an off-white solid. 1H NMR (400 MHz, CDCl3): δ 3.88 (t, J = 4.9 Hz, 2H), 3.80 – 3.73 (m, 2H), 3.65 (dd, J = 5.0, 3.3 Hz, 2H), 3.19 (t, J = 4.9 Hz, 2H), 3.05 – 2.96 (m, 2H), 1.95 – 1.83 (m, 2H), 1.31-1.25 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H). ESI-MS: m/z 274.8 [M+H] + Synthesis of 2-(didodecylamino) ethyl 6-(dodecyl(2-(2-hydroxyethoxy) ethyl) amino) hexanoate (NV2- 015; IB-13): To a stirred solution of 2-(2-(dodecylamino) ethoxy) ethan-1-ol (0.2 g, 0.731 mmol, 1.0 equiv.) in dry DCM (15 mL) was added 2-(didodecylamino) ethyl 6-oxohexanoate (4) (0.447 g, 0.877 mmol, 1.2 equiv.) at room temperature, stirred for 1 h. Then STAB (0.310 g, 1.462 mmol, 2.0 equiv.) added at room temperature, stirred for 3h at rt. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of aldehyde, the reaction mixture was quenched with saturated Sat.NaHCO3 solution and extracted with DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% IPA in CHCl3 to give the 0.280 g of 2-(didodecylamino) ethyl 6-(dodecyl(2-(2-hydroxyethoxy) ethyl) amino)hexanoate (NV2-015) as a pale-yellow liquid in pure and 0.20g of NV2-015 as yellow liquid in less pure. 1H NMR (400 MHz, CDCl3): δ 4.11 (t, J = 6.3 Hz, 2H), 3.72 – 3.64 (m, 2H), 3.64 – 3.57 (m, 4H), 2.64 (dt, J = 20.5, 5.9 Hz, 4H), 2.44 (s, 8H), 2.30 (t, J = 7.5 Hz, 3H), 1.64 (p, J = 7.5 Hz, 2H), 1.55 – 1.42 (m, 4H), 1.42 – 1.38 (m, 2H), 1.26 (s, 57H), 0.92 – 0.84 (m, 9H). ESI-MS: m/z 768.2 ; 384.8 [M+H] + Figure 27A is a 1H NMR spectrum of lipid IB-13 (NV2-015). Figure 27B is an ESI-MS spectrum of lipid IB-13 (NV2-015). Example 1I: Synthesis of lipid IB-9 (NV2-009): Experimental procedure for NV2-009 Synthesis of 10-oxodecanoic acid (2) To a stirred solution of IBX (2.602g, 9.295mmol, 1.75equiv.) in DMSO (13 mL) was added a solution of 10-hydroxydecanoic acid (1.0g, 5.311 mmol, 1.0 equiv.) in THF (10 mL) at rt, stirred for 4-5h. The progress of the reaction monitored by TLC analysis (60% EtOAc in Hexane). After completion of the reaction, the reaction mixture quenched with the addition of H2O (20ml), solids were precipitated out which were collected by filtration. Collect the filtrate and extracted into diethyl ether (2x50mL). Then combined organic layers were dried over anhydrous Na2SO4, filtered and distilled off to give the crude material. The crude was purified by Buchi flash using0-30% EtOAc in hexane to afford 620mg of 10-oxodecanoic acid as a white solid. 1H NMR (400 MHz, CDCl3): δ 9.77 (t, J = 1.8 Hz, 1H), 2.42 (td, J = 7.3, 1.8 Hz, 2H), 2.35 (t, J = 7.5 Hz, 2H), 1.63 – 1.59(m, 4H), 1.35 - 1.32 (m, 8H). ESI-MS: m/z 186.6 [M+H] + Synthesis of N, N-dioctylhydroxylamine (4): To a stirred solution of Octanal (3.03g, 23.632 mmol, 1.0 equiv.) in dry DCM (30 mL) was added NH2OH.HCl (0.821g, 11.8160mmol, 0.5 equiv.) and TEA (1.19g, 11.816 mmol, 0.5 equiv.) at rt, stirred for 2h. Then reaction mixture was further diluted with dry DCM (20 mL). Then STAB (15.03g, 70.899mmol, 3.0 equiv.) was charged to the reaction mixture at rt, stirred for 4h. The progress of the reaction was monitored by TLC (20% EtOAc in hexane). The reaction mixture was quenched with sat. NaHCO3 solution and extracted with DCM (2x100 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was triturated with acetonitrile and collected the solids by filtration to afford 2.78 g of N, N-dioctylhydroxylamine (4) as an off-white solid. 1H NMR (400 MHz, CDCl3): δ 2.68 – 2.60 (m, 4H), 1.58 (p, J = 7.8 Hz, 4H), 1.35 – 1.25 (m, 20H), 0.92 – 0.84 (m, 6H). ESI-MS: m/z 258.8 [M+H] + Synthesis of 10-((dioctylamino)oxy)-10-oxodecanal (5): To a stirred solution of 10-oxodecanoic acid (2) (1.22 g, 6.583 mmol, 1.13 equiv.) and N, N- dioctylhydroxylamine (4) (1.5 g, 5.826 mmol, 1.0 equiv.) in dry DCM (40 mL) was added EDC.HCl (2.23 g, 11.65 mmol, 2.0 equiv.) and DMAP (0.142 g, 1.165 mmol, 0.2 equiv. ) at room temperature, stirred for 3-4 h.The progress of the reaction was monitored by TLC (30% EtOAc in hexane). After completion of acid, the reaction mixture was quenched with water and extracted into 100%EtOAc (3x50 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-20% EtOAc in hexane to give 0.66 g of 10- ((dioctylamino)oxy)-10-oxodecanal (5) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 9.76 (t, J = 1.8 Hz, 1H), 2.84 – 2.76 (m, 4H), 2.42 (td, J = 7.3, 1.8 Hz, 2H), 2.28 (t, J = 7.5 Hz, 2H), 1.63 (h, J = 6.9 Hz, 4H), 1.50 (s, 4H), 1.37 – 1.12 (m, 31H), 0.88 (q, J = 8.1 Hz, 6H). ESI-MS: m/z 426.6 [M+H] + Synthesis of 2-(dodecylamino) ethan-1-ol (7) To a stirred solution of 1-Bromo dodecane (4.0 g, 16.049 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added ethanolamine (9.7 mL, 160.494 mmol, 10.0 equiv.) at room temperature and then stirred for 24h- 48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 3.1 g of 2-(dodecylamino) ethan-1- ol (7) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 3.67 – 3.60 (m, 2H), 2.81 – 2.74 (m, 2H), 2.65 – 2.57 (m, 2H), 1.47 (q, J = 7.0 Hz, 2H), 1.32-1.26 (m, 18H), 0.88 (t, J = 8.0 Hz, 3H). ESI-MS: m/z 230.5 [M+H] +. Synthesis 2-((10-((dioctylamino)oxy)-10-oxodecyl) (dodecyl)amino) ethan-1-ol (NV2-009):
To a stirred solution of 2-(dodecylamino) ethan-1-ol (7) (0.2 g, 0.871 mmol, 1.0 equiv.) in dry DCM (10 mL) was added 10-((dioctylamino)oxy)-10-oxodecanal (5) (0.408 g, 0.958 mmol, 1.1 equiv.) at room temperature, stirred for 1 h. Then STAB (0.369 g, 1.743 mmol, 2.0 equiv.) added at room temperature, stirred for 3h at rt. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of aldehyde, the reaction mixture was quenched with saturated Sat.NaHCO3 solution and extracted with DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% IPA in DCM to give the 0.383 g of 2-((10-((dioctylamino)oxy)-10-oxodecyl) (dodecyl)amino) ethan-1-ol (NV2-009; lipid IB-9) as a yellow gummy liquid. 1H NMR (400 MHz, CDCl3): δ 3.52 (t, J = 5.4 Hz, 2H), 2.84 – 2.76 (m, 4H), 2.57 (t, J = 5.4 Hz, 2H), 2.44 (t, J = 7.4 Hz, 4H), 2.27 (t, J = 7.5 Hz, 2H), 1.64 (s, 3H), 1.46 (dp, J = 29.3, 6.8 Hz, 9H), 1.27 (d, J = 6.3 Hz, 47H), 0.88 (td, J = 6.9, 2.9 Hz, 9H). ESI-MS: m/z 640.0 [M+H] + Figure 28A is a 1H NMR spectrum of lipid IB-9 (NV2-009). Figure 28B is an ESI-MS spectrum of lipid IB- 9 (NV2-009). Example 1J: Synthesis of lipid IB-24 (NV2-027):
Experimental procedure for NV2-027 Synthesis of 2-(dioctyl amino) ethan-1-ol (2) To a stirred solution of Octanal (3.0 g, 23.397 mmol, 1.0 equiv.) in dry DCM (50 mL) was added ethanolamine (0.7 mL, 11.698mmol, 0.5 equiv.) at room temperature and then stirred for 2h. Then STAB (9.920g, 46.794mmol, 2.0 equiv.) was charged at room temperature, stirred for 16h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the reaction was quenched with sat. NaHCO3 solution and extracted into DCM (2x60ml). The combined organic layers washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 1.797 g of 2-(dioctyl amino) ethan-1-ol (2) as a pale- yellow liquid. 1H NMR (400 MHz, CDCl3) δ 3.76 (t, J = 5.0 Hz, 2H), 2.86 (t, J = 5.0 Hz, 2H), 2.79 – 2.71 (m, 4H), 1.62 (dq, J = 13.1, 6.5 Hz, 4H), 1.29 (dt, J = 10.6, 4.5 Hz, 20H), 0.92 – 0.83 (m, 6H). ESI-MS: m/z 286.8 [M+H] +. Synthesis of 2-(dioctyl amino) ethyl 6-oxoheptanoate (4) To a stirred solution of 2-(dioctyl amino) ethan-1-ol (2) (1.73 g, 6.059 mmol, 1.0 equiv.) and 6-oxoheptanoic acid (1.048g, 7.270 mmol, 1.2 equiv.) in DCM (40 mL) was added DMAP (0.148g, 1.218 mmol, 0.2 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl (2.32 g, 12.118 mmol, 2.0 equiv.) was added at room temperature, stirred for 16 h. The progress of the reaction was monitored by TLC (60% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x25 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by column using 0-30% EtOAc in hexane to give the 1.98 g of 2-(dioctyl amino) ethyl 6-oxoheptanoate (4) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 4.11 (t, J = 6.3 Hz, 2H), 2.67 (t, J = 6.3 Hz, 2H), 2.49 – 2.39 (m, 6H), 2.36 – 2.28 (m, 2H), 2.14 (s, 3H), 1.56 (s, 7H), 1.41 (h, J = 6.7 Hz, 4H), 1.27 (d, J = 2.3 Hz, 20H), 0.88 (t, J = 6.7 Hz, 6H). ESI-MS: m/z 412.38 [M+H] +. Synthesis of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) amino) heptanoate (5): To a stirred solution of 2-(dioctyl amino) ethyl 6-oxoheptanoate (4) (1.97g, 4.785 mmol, 1.0 equiv.) in dry DCM (30 mL) was added ethanolamine (2.3 mL, 38.283 mmol, 8.0 equiv.) at room temperature and then stirred for 2h. Then STAB (5.07 g, 23.926 mmol, 5.0 equiv.) was charged at room temperature, stirred for 16h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the reaction was quenched with sat. NaHCO3 solution and extracted into DCM (2x60ml). The combined organic layers washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 1.90 g of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) amino) heptanoate (5) as a pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 4.12 (t, J = 6.3 Hz, 2H), 3.71 (td, J = 4.5, 2.3 Hz, 2H), 2.95 – 2.73 (m, 3H), 2.68 (t, J = 6.3 Hz, 2H), 2.49 – 2.40 (m, 4H), 2.32 (t, J = 7.4 Hz, 2H), 1.64 (p, J = 7.5 Hz, 3H), 1.41 (dq, J = 11.8, 7.5 Hz, 7H), 1.35 – 1.20 (m, 23H), 1.15 (d, J = 6.3 Hz, 3H), 0.92 – 0.84 (m, 7H). ESI-MS: m/z 458.0 [M+H] + Synthesis of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) ((9Z,12Z)-octadeca-9,12-dien-1-yl) amino) heptanoate (NV2-027): To a stirred solution of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) amino) heptanoate (5) (0.442 g, 0.968 mmol, 0.8 equiv.) in dry DCM (10 mL) was added linoleyl aldehyde (0.32 g, 1.210 mmol, 1.0 equiv.) at room temperature, stirred for 1 h. Then STAB (0.513 g, 2.420 mmol, 2.0 equiv.) added at room temperature, stirred for 24h at rt. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of aldehyde, the reaction mixture was quenched with saturated Sat.NaHCO3 solution and extracted with DCM (3x20 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% IPA in CHCl3 to give the 0.405 g of 2-(dioctyl amino) ethyl 6-((2-hydroxyethyl) ((9Z,12Z)-octadeca-9,12-dien- 1-yl) amino) heptanoate (NV2-027; IB-24) as a yellow liquid. 1H NMR (400 MHz, CDCl3): δ 5.44 – 5.27 (m, 4H), 4.12 (t, J = 6.3 Hz, 2H), 3.48 (tt, J = 10.6, 4.7 Hz, 2H), 2.81 – 2.62 (m, 5H), 2.53 – 2.42 (m, 4H), 2.42 – 2.26 (m, 5H), 2.05 (q, J = 6.8 Hz, 4H), 1.62 (p, J = 7.5 Hz, 4H), 1.50 – 1.21 (m, 47H), 0.94 (d, J = 6.5 Hz, 3H), 0.89 (td, J = 6.8, 4.1 Hz, 9H). ESI-MS: m/z 706.0; 353.8 [M+H] + Figure 29A is a 1H NMR spectrum of lipid IB-24 (NV2-027). Figure 29B is an ESI-MS spectrum of lipid IB-24 (NV2-027). Example 1K: Synthesis of lipid IA-13 (NV3-013):
Experimental procedure for NV3-013 Synthesis of 4-(dodecylamino) butan-1-ol (2) To a stirred solution of 1-Bromo dodecane (4.0 g, 16.049 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 4-amino butanol (9.7 mL, 160.494 mmol, 10.0 equiv.) at room temperature and then stirred for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was triturated with acetonitrile to give the 3.1 g of 4-(dodecylamino) butan-1-ol (2) as an white solid. 1H NMR (400 MHz, CDCl3) δ 3.60 – 3.53 (m, 2H), 2.68 – 2.56 (m, 4H), 1.63 (s, 5H), 1.49 (p, J = 7.3 Hz, 2H), 1.27 (d, J = 9.3 Hz, 18H), 0.88 (t, J = 6.8 Hz, 3H). ESI-MS: m/z 258.28 [M+H] +. Synthesis of 10-bromodecyl 10-bromodecanoate (5) To a stirred solution of 10-bromodecan-1-ol (0.5 g, 2.100 mmol, 1.0 equiv.) and 10-bromodecanoic acid (0.627g, 2.500mmol,1.2 equiv.) in DCM (20 mL) was added DMAP (0.04g, 0.400 mmol, 0.2 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl (0.805 g, 4.200 mmol, 2.0 equiv.) was added at room temperature, stirred for 2-3 h. The progress of the reaction was monitored by TLC (30% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x15 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by column using 0-5% EtOAc in hexane to give the 0.87 g of 10-bromodecyl 10- bromodecanoate (3) as color-less liquid. 1H NMR (400 MHz, CDCl3): δ 4.06 (t, J = 6.7 Hz, 2H), 3.41 (td, J = 6.8, 1.3 Hz, 4H), 2.29 (t, J = 7.5 Hz, 2H), 1.91 – 1.79 (m, 4H), 1.61 (q, J = 7.1 Hz, 4H), 1.42 (t, J = 7.2 Hz, 4H), 1.30-1.25 (m, 18H). Synthesis of 10-(dodecyl(4-hydroxybutyl) amino) decyl 10-(dodecyl(4-hydroxybutyl) amino) decanoate (NV3-013, IA-13): To a stirred solution of 4-(dodecylamino) butan-1-ol (2) (0.421g, 1.637mmol, 2.2 equiv.) and 10-bromodecyl 10-bromodecanoate (0.350 g, 0.744 mmol, 1.0 equiv.) in DMF (12 mL) was added KI (0.296 g, 1.785 mmol, 2.4 equiv.) and K2CO3 (0.514g, 3.720 mmol, 5.0 equiv. ) at room temperature, stirred at 70°C for 36h.The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of amino alcohol, the reaction mixture was quenched with water and extracted into EtOAc (3x15 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-22% IPA in CHCl3 to give the 0.302 g of 10-(dodecyl(4- hydroxybutyl) amino) decyl 10-(dodecyl(4-hydroxybutyl) amino) decanoate (NV3-013) as pale-yellow liquid. 1H NMR (400 MHz, CDCl3): δ 4.05 (t, J = 6.8 Hz, 2H), 3.55 (t, J = 4.6 Hz, 4H), 2.49 – 2.37 (m, 11H), 2.28 (t, J = 7.6 Hz, 2H), 1.71 – 1.55 (m, 12H), 1.53 – 1.41 (m, 8H), 1.27 (d, J = 7.0 Hz, 57H), 0.88 (t, J = 6.8 Hz, 6H). ESI-MS: m/z 823.71; 412.41 [M+H] + Figure 30A is a 1H NMR spectrum of lipid IA-13 (NV3-013). Figure 30B is an ESI-MS spectrum of lipid IA-13 (NV3-013). Example 1L: Synthesis of lipid IA-14 (NV3-014): Experimental procedure for NV3-014 Synthesis of ethane-1,2-diyl bis(10-bromodecanoate) (2): To a stirred solution of ethylene glycol (0.3 g, 4.8 mmol, 1.0 equiv.) and 10-bromodecanoic acid (3.61 g, 14.4 mmol,1.2 equiv.) in DCM (60 mL) was added DMAP (0.238g, 1.900 mmol, 0.4 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl (3.68 g, 19.200 mmol, 2.0 equiv.) was added at room temperature, stirred for 16 h. The progress of the reaction was monitored by TLC (20% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x50 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by column using 0-10% EtOAc in hexane to give the 2.3 g of ethane-1,2-diyl bis(10- bromodecanoate) (2) as color-less liquid. 1H NMR (400 MHz, CDCl3): δ 4.27 (s, 4H), 3.41 (t, J = 6.8 Hz, 4H), 2.32 (t, J = 7.5 Hz, 4H), 1.85 (dq, J = 8.7, 6.9 Hz, 4H), 1.64 (s, 4H), 1.55 (s, 2H), 1.41 (q, J = 7.0 Hz, 4H), 1.36 – 1.26 (m, 16H). Synthesis of 4-(dodecylamino) butan-1-ol (4): To a stirred solution of 1-Bromo dodecane (3.0 g, 12.036 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added butanol amine (10.71 g, 120.365 mmol, 10.0 equiv.) at room temperature and then stirred for 24h- 48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was triturated with acetonitrile to afford 2.36g of 4-(dodecylamino) butan-1-ol (4) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 3.60 – 3.53 (m, 2H), 2.68 – 2.56 (m, 4H), 1.63 (s, 5H), 1.49 (p, J = 7.3 Hz, 2H), 1.27 (d, J = 9.3 Hz, 18H), 0.88 (t, J = 6.8 Hz, 3H). ESI-MS: m/z 258.28 [M+H] +. Synthesis of ethane-1,2-diyl bis(10-(dodecyl(4-hydroxybutyl) amino) decanoate (NV3-014. Lipid IA- 14): To a stirred solution of 4-(dodecylamino) butan-1-ol (4) (0.428 g, 1.665 mmol, 2.2 equiv.) and ethane-1,2- diyl bis(10-bromodecanoate) (2) (0.40 g, 0.757 mmol, 1.0 equiv.) in DMF (12 mL) was added KI (0.289 g, 1.741 mmol, 2.3 equiv.) and K2CO3 (0.522 g, 3.785 mmol, 5.0 equiv. ) at room temperature, stirred at 70°C for 48 h.The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of amino alcohol, the reaction mixture was quenched with water and extracted into EtOAc (3x20 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 0.35 g of ethane-1,2-diyl bis(10-(dodecyl(4-hydroxybutyl) amino) decanoate (NV3-014) as yellow gummy liquid. 1H NMR (400 MHz, CDCl3): δ 4.27 (s, 4H), 3.59 – 3.52 (m, 4H), 2.47 (t, J = 7.5 Hz, 10H), 2.32 (t, J = 7.6 Hz, 4H), 1.70 – 1.62 (m, 10H), 1.59 (d, J = 7.3 Hz, 2H), 1.49 (s, 8H), 1.27 (d, J = 10.9 Hz, 60H), 0.88 (t, J = 6.8 Hz, 6H). ESI-MS: m/z 882.4 [M+H] +; 441.9 [M+H] +. Figure 31A is a 1H NMR spectrum of lipid IA-14 (NV3-014). Figure 31B is an ESI-MS spectrum of lipid IA-14 (NV3-014). Example 1M: Synthesis of lipid IA-15 (NV3-016): Experimental procedure for NV3-016 Synthesis of (Z)-1-bromooctadec-9-ene (1) To a stirred solution of Oleyl alcohol (5.0g, 18.622 mmol, 1.0 equiv.) in DCM (100 mL) was added triphenyl phosphine (5.85g, 22.347 mmol, 1.2 equiv.), stirred for 10 min then carbon tetra bromide (7.41g, 22.347 mmol, 1.2 equiv.) added in one portion. Then the reaction mixture stirred at room temperature for overnight. The progress of the reaction monitored by TLC analysis (5% EtOAc in Hexane). After completion of the reaction, the solvent was removed and purified by column chromatography using 0-5% EtOAc in Hexane to afford 6.14g of (Z)-1-bromooctadec-9-ene (1) as a pale-brown liquid. 1H NMR (400 MHz, CDCl3): δ 5.35 (td, J = 7.0, 4.0 Hz, 2H), 3.40 (t, J = 6.9 Hz, 2H), 2.01 (q, J = 6.3 Hz, 4H), 1.85 (p, J = 7.0 Hz, 2H), 1.48 – 1.37 (m, 2H), 1.30 (t, J = 9.8 Hz, 20H), 0.88 (t, J = 6.7 Hz, 3H). Synthesis of (Z)-2-(octadec-9-en-1-ylamino) ethan-1-ol (2) To a stirred solution of (Z)-1-bromooctadec-9-ene (4.0 g, 12.070 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added ethanolamine (14.74g, 241.41 mmol, 20.0 equiv.) at room temperature and then stirred for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL), washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 2.08 g of (Z)-2-(octadec-9-en-1- ylamino) ethan-1-ol (2) as pale-brown liquid. 1H NMR (400 MHz, CDCl3): δ 5.41 – 5.28 (m, 2H), 4.12 (q, J = 7.2 Hz, 1H), 3.66 (dd, J = 6.3, 4.0 Hz, 2H), 2.85 – 2.74 (m, 3H), 2.62 (q, J = 5.7 Hz, 2H), 2.07 – 1.92 (m, 4H), 1.50 (q, J = 7.4 Hz, 2H), 1.35 – 1.22 (m, 22H), 0.92 – 0.83 (m, 3H). ESI-MS: m/z 312.9 [M+H] +. Synthesis of 10-bromodecyl 10-bromodecanoate (5) To a stirred solution of 10-bromodecan-1-ol (0.5 g, 2.100 mmol, 1.0 equiv.) and 10-bromodecanoic acid (0.627g, 2.500mmol,1.2 equiv.) in DCM (20 mL) was added DMAP (0.04g, 0.400 mmol, 0.2 equiv.) at room temperature, stirred for 10 min. Then EDC.HCl (0.805 g, 4.200 mmol, 2.0 equiv.) was added at room temperature, stirred for 2-3 h. The progress of the reaction was monitored by TLC (30% EtOAc in hexane). The reaction mixture was quenched with water and extracted with DCM (3x15 mL). The combined DCM layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by column using 0-5% EtOAc in hexane to give the 0.87 g of 10-bromodecyl 10- bromodecanoate (5) as color-less liquid. 1H NMR (400 MHz, CDCl3): δ 5.40 – 5.30 (m, 2H), 3.68 – 3.61 (m, 2H), 2.82 – 2.75 (m, 2H), 2.67 – 2.58 (m, 2H), 2.07 – 1.94 (m, 7H), 1.49 (s, 2H), 1.37 – 1.25 (m, 22H), 0.92 – 0.84 (m, 3H). Synthesis of 10-((2-hydroxyethyl) ((Z)-octadec-9-en-1-yl) amino) decyl 10-((2-hydroxyethyl) ((Z)-octadec-9-en-1-yl) amino) decanoate (NV3-016, lipid IA-15): To a stirred solution of (Z)-2-(octadec-9-en-1-ylamino) ethan-1-ol (2) (0.582g, 1.871mmol, 2.2equiv.) and 10-bromodecyl 10-bromodecanoate (0.400 g, 0.850 mmol, 1.0 equiv.) in DMF(12 mL) was added KI (0.324 g, 1.956 mmol, 2.3 equiv.) and K2CO3 (0.586g, 4.252 mmol, 5.0 equiv. )at room temperature, stirred at 65- 70°C for 32 h.The progress of the reaction was monitored by TLC (10% MeOH in CHCl3). After completion of amino alcohol, the reaction mixture was quenched with water and extracted into EtOAc (3x15 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and evaporated the solvent. The crude was purified by Buchi flash using 0-10% MeOH in CHCl3 to give the 0.498 and 0.14g of 10-((2-hydroxyethyl) ((Z)-octadec-9-en-1-yl) amino) decyl 10-((2-hydroxyethyl) ((Z)-octadec-9- en-1-yl) amino) decanoate (NV3-016) as yellow liquid. 1H NMR (400 MHz, CDCl3) δ 5.40 – 5.28 (m, 4H), 4.05 (t, J = 6.8 Hz, 2H), 3.53 (t, J = 5.4 Hz, 4H), 2.58 (t, J = 5.4 Hz, 4H), 2.45 (dd, J = 8.5, 6.4 Hz, 8H), 2.29 (t, J = 7.5 Hz, 2H), 2.06 – 1.97 (m, 8H), 1.61 (s, 4H), 1.44 (p, J = 6.8 Hz, 9H), 1.37 – 1.24 (m, 65H), 0.92 – 0.82 (m, 6H). ESI-MS: m/z 932.4 [M+H] +;466.9[M+H] + Figure 32A is a 1H NMR spectrum of lipid IA-15 (NV3-016). Figure 32B is an ESI-MS spectrum of lipid IA-15 (NV3-016). EXAMPLE 2: Preparation and characterization of LNPs comprising ionizable lipids Ionizable lipids were synthesized as detailed in Example 1. Other lipids DSPC, DOPE, DOTAP, Cholesterol and DMG-PEG were purchased from Avanti polar lipids. Example 2A: LNPs comprising ionizable lipids, including a permanently charge lipid: Lipid nanoparticles were synthesized by microfluidic mixing device. Briefly, one volume of lipid mix (Ionizable lipid, DOTAP, DOPE, DMG-PEG, Cholesterol at 30:25:10:32.5:2.5 mol ratio) in ethanol solution and three volumes of mRNA (lipid to mRNA ratio at 40:1w/w) in citrate buffer (pH 4.5) were mixed through a microfluidic mixing device Ignite (Precision Nanosystems Inc) at total flow rate of 12 ml/min. The resultant mRNA-LNPs were dialyzed against phosphate-buffered saline (PBS, pH 7.4) for 24 hours. The details of Formulations 1-5 are presented in Table 1: Table 1: Formulation details (with permanently charge lipid) Example 2B: LNPs comprising ionizable lipids, without a permanently charge lipid: Lipid nanoparticles were synthesized by microfluidic mixing device. Briefly, one volume of lipid mix (Ionizable lipid, DOPE or DSPC, Cholesterol and DMG-PEG, at mol ratios as reflected in Table 2) in ethanol solution and three volumes of mRNA (lipid to mRNA ratio at 40:1w/w) in citrate buffer (pH 4.5) were mixed through a microfluidic mixing device Ignite (Precision Nanosystems Inc) at total flow rate of 12 ml/min. The resultant mRNA-LNPs were dialyzed against phosphate-buffered saline (PBS, pH 7.4) for 24 hours. The details of Formulations 6-10 are presented in Table 2: Table 2: Formulation details (without permanently charge lipid)
EXAMPLE 3: RNA encapsulation and quantification The Quant-iT RiboGreen RNA assay kit (Life Technologies) was used to measure the mRNA encapsulation in LNPs. In brief, 0.5 µL of LNP was diluted in a final volume of 100 µL of TE buffer (20 mм EDTA, 10 mм Tris-HCL) with or without Triton X-100 (1%, Sigma-Aldrich). Samples were loaded in a 96-well black plate (Costar, Corning). The plate was incubated for 5 min at 37 °C before adding 100 µL of RiboGreen in TE buffer (1:200 v/v) to each well. The fluorescence was detected using GloMax plate reader (Promega) according to the manufacturer’s protocol. EXAMPLE 4: Size and ζ-potential analysis of LNPs Nano size and ζ-potential of mRNA-LNPs were analyzed by dynamic light scattering (DLS) using a Malvern nano ZS ζ-sizer (Malvern Instruments). Briefly, mRNA-LNPs were diluted in double-distilled water (1:50, volume ratio) and PBS (1:50, volume ratio) for ζ potential and size measurements, respectively. The physico-chemical properties - particle size, polydispersity index (PDI), Zeta potential and encapsulation efficiency (EE%) - of Formulations 1, 2, 3, 4 and 5 are presented in Table 3. The physico-chemical properties of Formulations 6, 7, 8, 9 and 10 are presented in Table 4 Table 3: Physico-chemical properties of formulations with permanently charge lipid Table 4: Physico-chemical properties of formulations without permanently charge lipid EXAMPLE 5: In vivo luciferase assay & organ distribution study Female 8-10 weeks-old C57BL/6 mice (Envigo, Rehovot, Israel) were intravenously injected with mLuc- LNPs at 10ug/mouse mRNA dose. After 6 hours, the mice were intraperitoneally injected with D-Luciferin (150 mg kg−1), and major organs were harvested for imaging using IVIS-spectrum-CT (Perkin Elmer Inc). All the animal experiments were performed at Tel Aviv university, Israel. All the animal protocols were approved by the Tel Aviv University, Institutional Animal Care and Usage Committee (IACUC) in accordance with current regulations and standards of the Israel Ministry of Health. Pre-established criteria for removing animals from the experiments were based on animal health, behavior, and well-being as required by ethical guidelines. IVIS imaging analysis for Luciferase expression is presented for Formulation 1 (Figure 2A), Formulation 2 (Figure 2B), Formulation 3 (Figure 2C), Formulation 4 (Figure 2D), Formulation 5 (Figure 2E), Formulation 6 (Figure 3A), Formulation 7 (Figure 3B), Formulation 8 (Figure 3C), Formulation 9 (Figure 3D) and Formulation 10 (Figure 3E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). In Figures 3A-E, the lung is not targeted and the white appearance of the lungs does not indicate fluorescence, but rather is a result of the B&W limitation. As shown in Figures 2A-E, Luciferase expression was observed mostly in lungs for all the liquid nanoparticles (LNPs) comprising permanently charged lipids. In some mice treated with mRNA-LNPs (Formulation 3, Formulation 4 and Formulation 5), luciferase expression was also observed in spleen. However, total expression levels varied depending on the type of ionizable lipid. LNPs composed of ionizable lipid NV3-002 (IA-10) exhibited higher protein expression in lungs followed by NV2-004 (IB-4) and NV2-011 (IB-10). In contrast, as shown in Figures 3A-E, Luciferase expression was not observed mostly in lungs for all the liquid nanoparticles (LNPs) which are devoid of permanently charged lipids. Figure 4A is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 5 (left group), Formulation 1 (second from left group), Formulation 2 (middle group), Formulation 3 (second from right group) and Formulation 4 (right group). Figure 4B is a histogram analysis of Luciferase expression in the heart (dotted, dark), lung (dotted, bright), liver (black), spleen (dark) and kidney (squares) for Formulation 10 (left group), Formulation 6 (second from left group), Formulation 7 (middle group), Formulation 8 (second from right group) and Formulation 9 (right group). Figure 5 is a histogram analysis of Luciferase expression comparing between Lung (circles), liver (squares) and spleen (triangles) for Formulation 5 (left group), Formulation 2 (second from left group), Formulation 4 (middle group), Formulation 3 (second from right group) and Formulation 1 (right group). Thus, the quantification of luciferase expression in different organs, as shown in Figure 4A and Figure 5 clearly demonstrates the preferential lung luciferase expression enabled by the present Formulations 1-5. The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the figures. While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow. EXAMPLE 6: Preparation and characterization of LNPs comprising ionizable lipids Ionizable lipids were synthesized as detailed in Example 1. Other lipids DSPC, DOPE, DOTAP, Cholesterol and DMG-PEG were purchased from Avanti polar lipids. Lipid nanoparticles were synthesized as described in Example 2A. The details of Formulations 11-26 are presented in Table 6: Table 6: Formulation details (with permanently charge lipid)
EXAMPLE 7: RNA encapsulation and quantification and Size and ζ-potential analysis of LNPs RNA encapsulation and quantification of Formulations 11-26 were performed as described in Example 3. Size and ζ-potential analysis of LNPs were performed as described in Example 4. The physico-chemical properties - particle size, polydispersity index (PDI), Zeta potential and encapsulation efficiency (EE%) - of Formulations 1126 are presented in Table 7. Table 7: Physico-chemical properties of formulations with permanently charge lipid
EXAMPLE 8: In vivo luciferase assay & organ distribution study - Formulations 11 and 18-22 LNPs composed of iL, DOTAP, DOPE, Cholesterol & DMG-PEG at 30:25:10:32.5:2.5 mole ratios were formulated with Luc-mRNA respectively. Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analyzed for Luciferase expression by IVIS imaging system. IVIS imaging analysis for Luciferase expression is presented for Formulation 11 (Figure 6A), Formulation 22 (Figure 6B), Formulation 18 (Figure 6C), Formulation 19 (Figure 6D), Formulation 20 (Figure 6E) and Formulation 21 (Figure 6F) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 7 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21(right group). Figure 8 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21 (right group). Figure 9 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 11 (left group), Formulation 22 (second from left group), Formulation 18 (third from left group), Formulation 19 (third from right group), Formulation 20 (second from right group) and Formulation 21 (right group). All the formulations are distributed to the lungs and observed significant luciferase expression in the lung tissue. However, there was a significant difference in the protein expression in the lungs between different ionizable lipids. Additionally, lung distribution over the liver depends on the type of ionizable lipid. Luciferase expressions of NV1-001 & NV2-027 lipid LNPs were similar in both lung and liver. EXAMPLE 9: In vivo luciferase assay & organ distribution study – Formulations 11, 17-18 and 25-26 LNPs composed of iL, DOTAP or DOTMA, DOPE, Cholesterol & DMG-PEG at 30:25:10:32.5:2.5 mole ratios were formulated with Luc-mRNA respectively (F3.1 or F3.7). Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analyzed for Luciferase expression by IVIS imaging system. Figures 10A-E are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 10A), Formulation 17 (Figure 10B), Formulation 18 (Figure 10C), Formulation 26 (Figure 10D), and Formulation 25 (Figure 10E) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 11 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group), and Formulation 25 (right group). Figure 12 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group). Figure 13 is bar chart showing luciferase expression in the lungs were compared between DOTAP/DOTMA formulations; Formulation 11 (left group), Formulation 17 (second from left group), Formulation 18 (middle group), Formulation 26 (second from right group) and Formulation 25 (right group). The effect of cationic lipid DOTAP or DOTMA in the formulation has no significant effect on lung distribution and expression. However, for the lipid NV1-001 composed of DOTMA has lowered liver distribution, whereas for the lipid NV3-004 has no difference. EXAMPLE 10: In vivo luciferase assay & organ distribution study – Formulations 11, 16 and 24 LNPs composed of iL, DOTAP, DOPE or DSPC, Cholesterol & DMG-PEG at 30:25:10:32.5:2.5 mole ratios were formulated with Luc-mRNA respectively (F3.1 or F3.6). Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analysed for Luciferase expression by IVIS imaging system. Figures 14A-C are IVIS imaging analysis for Luciferase expression of Formulation 11 (Figure 14A), Formulation 16 (Figure 14B), and Formulation 24 (Figure 10C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 15 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 11 (left group), Formulation 16 (middle group) and Formulation 24 (right group). Figure 16 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group). Figure 17 is bar chart showing luciferase expression in the lungs were compared between DSPC vs DOPE formulations; Formulation 11 (left group), Formulation 16 (middle group), and Formulation 24 (right group). Formulations composed of either DOPE or DSPC helper lipids have no significant effect on lung distribution. The protein expression was similar in the lungs and other organs as well. EXAMPLE 11: In vivo luciferase assay & organ distribution study – Formulations 11-15 LNPs composed of iL (NV1-001), DOTAP, DOPE, Cholesterol & DMG-PEG at various DOTAP mole ratios were formulated with Luc-mRNA. Mice were administered Luc-mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analysed for Luciferase expression by IVIS imaging system. Figures 18A-E are IVIS imaging analysis for Luciferase expression of Formulation 12 (Figure 18A), Formulation 15 (Figure 18B), Formulation 11 (Figure 18C), Formulation 13 (Figure 18D), and Formulation 14 (Figure 18C) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 19 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 12 (left group), Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group). Figure 20 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group). Figure 21 is bar chart showing luciferase expression in the lungs were compared between different formulations with increasing DOTAP amount; Formulation 15 (second from left group), Formulation 11 (middle group), Formulation 13 (second from right group), and Formulation 14 (right group). The bio-distribution of NV1-001 LNPs varied on DOTAP amount. Significant protein expression in the lungs compared to liver was observed with 20% DOTAP. EXAMPLE 12: In vivo luciferase assay & organ distribution study – Formulations 15 and 23 LNPs composed of iL (1-001 or 1-005), DOTAP, DOPE, Cholesterol & DMG-PEG at 20:20:10:38.5:2.5 mole ratios were formulated with Luc-mRNA respectively (F3.1 or F3.6). Mice were administered Luc- mRNA encapsulated LNPs intravenously at 0.5mg/kg mRNA dose. Six hours post administration organs were isolated and analysed for Luciferase expression by IVIS imaging system. Figures 22A-B are IVIS imaging analysis for Luciferase expression of Formulation 15 (Figure 22A), and Formulation 23 (Figure 22B) in the heart (top row), lung (second row), liver (third row), spleen (fourth row) and kidney (bottom row). Figure 23 is a histogram analysis of Luciferase expression in the heart (clear), lung (dotted, bright), liver (right leaning “/” diagonal lines), spleen (dotted, dark) and kidney (left leaning “\” diagonal lines) for Formulation 15 (left group), and Formulation 23 (right group). Figure 24 is a lung-to-liver (clear/ left vertical axis) and lung-to-spleen (grey/ right vertical axis) measured ratio for Formulation 15 (left group), and Formulation 23 (right group). Figure 25 is bar chart showing luciferase expression in the lungs were compared between different ionizable lipid LNPs of the same composition; Formulation 15 (left group), and Formulation 23 (right group). The lung distribution between the two different ionizable lipids formulations each containing equal amount of DOTAP amount were similar. The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the figures. While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow

Claims

CLAIMS What is claimed is: 1. A lipid nanoparticle formulation comprising: at least one ionizable lipid which is represented by the structure of Formula (II), Formula (IA) or Formula (IB), or salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof, wherein the structures of Formula (II), Formula (IA) and Formula (IB) are represented below; and at least one permanently charged lipid having a quaternary ammonium moiety or a salt thereof; Formula (II): , wherein R1C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; R2C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; Y1C is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; W1C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; R5C is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NR NIIRNII’, wherein each one of RNII and RNII’ is individually C1-4 alkyl or R NII and RNII’ together with the nitrogen to which they are bound, form a ring; W2C is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; Y2C is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; R3C is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; and R4C is selected from the group consisting of: C4-18 alkyl and C12-24 alkenyl; Formula (IA): , wherein R1A is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNARNA’, wherein each one of RNA and RNA’ is individually C1-4 alkyl or RNA and RNA’ together with the nitrogen to which they are bound, form a ring; R2A is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; nA is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; XA is selected from the group consisting of: -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, - OCONH- and -NHCONH-; jA is selected from the group consisting of: 0, 1, 2, 3 and 4; YA is selected from the group consisting of: absent, -COO-, -OOC-, -NHCO-, -CONH-, -NHCOO-, -OCONH- and -NHCONH-; mA is selected from the group consisting of: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15; R3A is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNA’’RNA’’’, wherein each one of RNA’’ and RNA’’’ is individually C1-4 alkyl or RNA’’ and RNA’’’ together with the nitrogen to which they are bound, form a ring; and R4A is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; Formula (IB): , wherein R1B is selected from the group consisting of: OH, -(CH2CH2O)2-6H, C1-6 hydroxyalkyl and C1-6 haloalkyl and C1-3 alkylene-NRNBRNB’, wherein each one of RNA and RNA’ is individually C1-4 alkyl or RNA and RNA’ together with the nitrogen to which they are bound, form a ring; R2B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; WB is a C4-12 alkylene, optionally substituted with at least one substituent selected from the group consisting of hydroxy and halogen; YB is selected from the groups consisting of: absent, -(CH2CH2O)1-5CH2CH2- and C1-6 alkylene; R3B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl; and R4B is selected from the group consisting of: C5-25 alkyl, C5-25 alkenyl, C5-15 alkylene-CO2-C5-15 alkyl, C5-15 alkylene-CO2-C5-15 alkenyl, C5-15 alkylene-O2C-C5-15 alkyl, C5-15 alkylene-O2C-C5-15 alkenyl, C5-15 alkenylene-CO2-C5-15 alkyl, C5-15 alkenylene-CO2-C5-15 alkenyl, C5-15 alkenylene- O2C-C5-15 alkyl, C5-15 alkenylene-O2C-C5-15 alkenyl.
2. The lipid nanoparticle formulation of claim 1, wherein the ionizable lipid is represented by the structure of Formula (IA).
3. The lipid nanoparticle formulation of any one of claims 1 to 2, wherein R1A is selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH.
4. The lipid nanoparticle of formulation any one of claims 1 to 3, wherein R2A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25, -(CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17 and -(CH2)7CH=CHCH2CH=CHC4H9.
5. The lipid nanoparticle formulation of any one of claims 1 to 4, wherein nA is selected from the group consisting of: 8, 9 and 10, and jA is 0 or 2, or a combination thereof.
6. The lipid nanoparticle formulation of any one of claims 1 to 5, wherein XA is -COO- and the lipid is represented by Formula (IA1)
.
7. The lipid nanoparticle formulation of any one of claims 1 to 6, wherein YA is absent or -OOC-.
8. The lipid nanoparticle formulation of any one of claims 1 to 7, wherein mA is selected from the group consisting of: 8, 9, and 10.
9. The lipid nanoparticle formulation of any one of claims 1 to 8, wherein R3A is selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH and OH.
10. The lipid nanoparticle formulation of any one of claims 1 to 9, wherein R4A is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -C12H25, -(CH2)5CH=CHCH2CH=CHC8H17, -(CH2)8CH=CHC8H17 and -(CH2)7CH=CHCH2CH=CHC4H9.
11. The lipid nanoparticle formulation of claim 2, wherein the ionizable lipid is selected from the group consisting of: IA-4
IA-15 and a combination thereof, preferably, wherein the lipid is lipid IA-10 or lipid IA12.
12. The lipid nanoparticle formulation of claim 1, wherein the ionizable lipid is represented by the structure of Formula (IB).
13. The lipid nanoparticle formulation of any one of claims 1, 3 to 10 or 12, wherein R1B is selected from the group consisting of: -CH2CH2OCH2CH2OH, -CH2CH2Cl, -CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2CH2OH and -CH2CH2NMe2, preferably, wherein R1B is CH2CH2OCH2CH2OH, -CH2CH2Cl or -CH2CH2OH.
14. The lipid nanoparticle formulation of any one of claims 1, 3 to 10 or 12 to 13, wherein R2B is selected from the group consisting of: -(CH2)8CH=CHCH2CH=CHC5H11, -(CH2)7CH=CHCH2CH=CHC6H13, -(CH2)8CH=CHC8H17 and -C12H25, preferably, wherein R2B is -(CH2)8CH=CHCH2CH=CHC5H11 or -C12H25.
15. The lipid nanoparticle formulation of any one of claims 1, 3 to 10 or 12 to 14, wherein WB selected from the group consisting of: -(CH2)9-, -(CHMe)-(CH2)4- and -(CH2)5-.
16. The lipid nanoparticle formulation of any one of claims 1, 3 to 10 or 12 to 25, wherein YB is selected from the groups consisting of: absent, -CH2CH2OCH2CH2- and -CH2CH2-, preferably, wherein YB is absent or -CH2CH2-.
17. The lipid nanoparticle formulation of any one of claims 1, 3 to 10 or 12 to 16, wherein R3B is a C4-16 alkyl.
18. The lipid nanoparticle formulation of any one of claims 1, 3 to 10 or 12 to 17, wherein R4B is a C4-16 alkyl.
19. The lipid nanoparticle formulation of claim 12, wherein the ionizable lipid is selected from the group consisting of: IB-10 IB-24 and a combination thereof, preferably wherein the lipid is IB-4.
20. The lipid nanoparticle formulation of claim 1, wherein the ionizable lipid is represented by the structure of Formula (II).
21. The lipid nanoparticle formulation of any one of claims 1, 3 to 10, 12 to 18 or 20, wherein R1C is a C4-10 alkyl, preferably, wherein R1C is C6H13.
22. The lipid nanoparticle formulation of any one of claims 1, 3 to 10, 12 to 18 or 20 to 31, wherein R2C is a C4-10 alkyl, preferably, wherein R2C is C6H13.
23. The lipid nanoparticle formulation of any one of claims 1, 3 to 10, 12 to 18 or 20 to 22, wherein each one of Y1C and Y2C is absent and the lipid of Formula (II) is represented by Formula (II1): .
24. The lipid nanoparticle formulation of any one of claims 1, 3 to 10, 12 to 18 or 20 to 23, wherein each one of Y1C and Y2C is absent, each one of W1C and W2C is a C4-12 straight chain alkylene and the lipid of Formula (II) is represented by Formula (II3):
.
25. The lipid nanoparticle formulation of any one of claims 1, 3 to 10, 12 to 18 or 20 to 24, wherein R5C is selected from the group consisting of: -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2CH2N(CH2)4, -CH2CH2CH2N(CH2)5, -CH2CH2N(CH2)5, -CH2CH2Cl, -OH and -CH2CH2NMe2, preferably, wherein R5C is -CH2CH2OH, or -CH2CH2OCH2CH2OH.
26. The lipid nanoparticle formulation of any one of claims 1, 3 to 10, 12 to 18 or 20 to 25, wherein R3C is a C4-10 alkyl, preferably, wherein R3C is C6H13.
27. The lipid nanoparticle formulation of any one of claims 1, 3 to 10, 12 to 18 or 20 to 26, wherein R4C is a C4-10 alkyl, preferably, wherein R4C is C6H13.
28. The lipid nanoparticle of claim 20, wherein the ionizable lipid is, II-1.
29. The lipid nanoparticle of claim 1, wherein the ionizable lipid is selected from the group consisting of: IB-1 IB- 13
IB- 18
-15 53
and a combination thereof.
30. The lipid nanoparticle formulation of any one of claims 1 to 29, wherein the permanently charged lipid is selected from the group consisting of:1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), N-(2-hydroxyethyl)-N,N-dimethyl- 2,3-bis(oleoyloxy)propan-1-aminium (DORI), 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), dimethyldioctadecylammonium (DDAB), salts and combinations thereof.
31. The lipid nanoparticle formulation of claim 30, wherein the permanently charged lipid is DOTAP or DOTMA.
32. The lipid nanoparticle formulation of any one of claims 1 to 31, comprising 15 to 30 mol% of the ionizable lipid.
33. The lipid nanoparticle formulation of any one of claims 1 to 32, comprising 10 to 40 mol% permanently charged lipid(s).
34. The lipid nanoparticle formulation of any one of claims 1 to 33, comprising 15 to 35 mol% permanently charged lipid(s).
35. The lipid nanoparticle formulation of any one of claims 1 to 34, comprising about 20 mol% permanently charged lipid(s).
36. The lipid nanoparticle formulation of any one of claims 1 to 35, further comprising at least one neutral lipid, preferably, wherein the neutral lipid comprises a sterol, a phospholipid or both, more preferably wherein the sterol comprises cholesterol.
37. The lipid nanoparticle formulation of claim 36, comprising 30 to 40 mol% cholesterol.
38. The lipid nanoparticle formulation of claim 36, wherein the phospholipid comprises 1,2-Dioleoyl- sn-glycero-3-phosphoethanolamine (DOPE), Distearoylphosphatidylcholine (DSPC), or both, preferably 5 to 15 mol% DOPE, DSPC or both.
39. The lipid nanoparticle formulation of any one of claims 1 to 38, further comprising a PEGylated lipid, preferably, comprising 1 to 5 mol% DMG-PEG 2000.
40. The lipid nanoparticle formulation of claim 29, which is selected from the group consisting of Formulation 1 to 5 and 11-26, wherein the compositions of Formulations 1 to 5 and 11-26 are described below: Formulation 1: lipid IA-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 2: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 3: lipid IB-10: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 4: lipid IB-6: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 5: lipid II-5: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 11: lipid II-1: 25 mol% to 35 mol%; 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 12: lipid II-1: 25 mol% to 35 mol%; DOTAP: 5 mol% to 15 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 50 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 13: lipid II-1: 20 mol% to 30 mol%; DOTAP: 25 mol% to 35 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 14: lipid II-1: 10 mol% to 20 mol%; DOTAP: 30 mol% to 50 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 15: lipid II-1: 15 mol% to 25 mol%; DOTAP: 15 mol% to 25 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 55 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 16: lipid II-1: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; Distearoylphosphatidylcholine (DSPC): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 17: lipid II-1: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 18: lipid IA-11: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 19: lipid IA-12: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 20: lipid IA-8: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 21: lipid IA-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 22: lipid IB-24: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 23: lipid II-5: 15 mol% to 25 mol%; DOTAP: 15 mol% to 25 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 40 mol% to 55 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 24: lipid IB-4: 25 mol% to 35 mol%; DOTAP: 20 mol% to 30 mol%; Distearoylphosphatidylcholine (DSPC): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 25: lipid IB-4: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%; Formulation 26: lipid IA-11: 25 mol% to 35 mol%; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA): 20 mol% to 30 mol%; 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 7 mol% to 13 mol%; cholesterol: 27 mol% to 38 mol%; and DMG-PEG 2000: 1.5 mol% to 4 mol%.
41. The lipid nanoparticle formulation of claim 40, which is Formulation 15 or Formulation 23.
42. The lipid nanoparticle formulation of any one of claims 1 to 41, further comprising a nucleic acid encapsulated within at least one particle thereof, preferably, wherein the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), micro RNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids.
43. The lipid nanoparticle formulation of any one of claims 1 to 42, formulated for intravenous (IV) administration.
44. The lipid nanoparticle formulation of any one of claims 1 to 43, which is a liver bypass formulation.
45. The lipid nanoparticle formulation of any one of claims 1 to 44, which is selectively targeting the lung upon intravenous (IV) administration.
46. A lipid selected from the group consisting of:
IB-24.
47. A lipid nanoparticle formulation comprising at least one lipid according to claim 46.
48. A pharmaceutical composition comprising at least one lipid according to claim 46 or the lipid nanoparticle formulation of claim 47, and a pharmaceutically acceptable carrier, diluent or excipient.
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