WO2017205566A1 - Oxidized aminolipidoids and uses thereof - Google Patents

Oxidized aminolipidoids and uses thereof Download PDF

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Publication number
WO2017205566A1
WO2017205566A1 PCT/US2017/034376 US2017034376W WO2017205566A1 WO 2017205566 A1 WO2017205566 A1 WO 2017205566A1 US 2017034376 W US2017034376 W US 2017034376W WO 2017205566 A1 WO2017205566 A1 WO 2017205566A1
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group
optionally substituted
formulation
plant
compound
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PCT/US2017/034376
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French (fr)
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Richard W. Heidebrecht
Jennifer BEAUDOIN
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Preceres Inc.
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Publication of WO2017205566A1 publication Critical patent/WO2017205566A1/en

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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
    • A01N33/02—Amines; Quaternary ammonium compounds
    • A01N33/08—Amines; Quaternary ammonium compounds containing oxygen or sulfur
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/24—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with two or more hetero atoms
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    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/24—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with two or more hetero atoms
    • A01N43/26—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with two or more hetero atoms five-membered rings
    • A01N43/28—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with two or more hetero atoms five-membered rings with two hetero atoms in positions 1,3
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    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/24—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with two or more hetero atoms
    • A01N43/32—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with two or more hetero atoms six-membered rings
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    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/74—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,3
    • A01N43/76—1,3-Oxazoles; Hydrogenated 1,3-oxazoles
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    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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    • A01N55/00—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C211/14—Amines containing amino groups bound to at least two aminoalkyl groups, e.g. diethylenetriamines
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/10—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic with one amino group and at least two hydroxy groups bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/12—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic the nitrogen atom of the amino group being further bound to hydrocarbon groups substituted by hydroxy groups
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
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    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/26—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by nitrogen atoms
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    • C07D263/04—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D263/06—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by oxygen atoms, attached to ring carbon atoms
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    • C07D273/08—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00 having two nitrogen atoms and more than one oxygen atom
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    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
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    • C07D317/10—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings
    • C07D317/14—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 not condensed with other rings with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D317/28—Radicals substituted by nitrogen atoms
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    • C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
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    • C07D498/04—Ortho-condensed systems
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    • A01N2300/00—Combinations or mixtures of active ingredients covered by classes A01N27/00 - A01N65/48 with other active or formulation relevant ingredients, e.g. specific carrier materials or surfactants, covered by classes A01N25/00 - A01N65/48

Definitions

  • aminolipidoids containing oxygen-based functionalities, such as hydroxyl and ether groups
  • aminolipidoids oxygen-based functionalities, such as hydroxyl and ether groups
  • the present disclosure also relates generally to methods of preparing such aminolipidoids, their formulation with one or more active agents, and the delivery of such formulations to target organisms.
  • BACKGROUND OF THE INVENTION [0003] A wide range of molecules have been employed for delivering polynucleotides and other active agents to cells.
  • polymers such as polyethylenimine or poly(beta-aminoesters) have been used to effectively complex DNA for delivery into cells.
  • Polymers in these classes of delivery agent typically contain amine functionalities that serve to electrostatically bind to DNA to form nanoparticles that are then taken up by the cell via endocytosis. Once in the cell, it is hypothesized that these amine groups serve to buffer the endosome and cause an influx of ions due to the proton-sponge mechanism. The resulting burst of the endocytic vesicle leads to the release of the payload of the particle, which is then free to travel to the nucleus where the DNA is expressed.
  • RNA Ribonucleic acid
  • RNA Ribonucleic acid
  • the delivery system needs to protect RNA from nuclease degradation, allow for the proper concentration and distribution profile in the target tissues, facilitate efficient uptake of RNA into target cells, and release RNA into cytoplasm to knockout expression of the target gene.
  • Promising results have been achieved with materials that form liposomes or lipoplexes that entrap the RNA or form nanoparticles, which can then be internalized by a cell.
  • the materials utilized to form a lipid-based delivery system generally consist of a positively charged headgroup and a hydrophobic tail. The charged portion serves to electrostatically bind the negatively charged RNA, while the hydrophobic tail leads to self- assembly into lipophilic particles.
  • One embodiment of the present invention is a compound of formula (I):
  • X is an oxygen-containing core derived from an oxidized amine comprising (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups;
  • R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
  • R' is identically or different in each instance hydrogen, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or
  • heterocycloaliphatic group an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
  • n is an integer in the range of from 2 to 10;
  • Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least two hydroxyl groups. [0009] Another embodiment of the present invention is the above compound, wherein the oxidized amine comprises three hydroxyl groups. [0010] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least two ether groups. [0011] Another embodiment of the present invention is the above compound, wherein the oxidized amine comprises three, four, or five ether groups. [0012] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least one hydroxyl group and at least one ether group.
  • Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least two primary amino groups.
  • Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least one primary amino group and at least one secondary amino group.
  • Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine selected from the group consisting of compounds of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), and (22):
  • R is an optionally substituted aliphatic or heteroaliphatic group.
  • R' is an optionally substituted aliphatic or heteroaliphatic group.
  • R' is an optionally substituted aliphatic or heteroaliphatic group.
  • R' is an optionally substituted aliphatic or heteroaliphatic group.
  • the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond.
  • the optionally substituted heteroaliphatic group comprises at least one ether group.
  • Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic group is a C 6 to C 20 aliphatic group.
  • R and R' are independently selected from the group consisting of moieties of formulae 23-41:
  • Another embodiment of the present invention is the above compound, wherein R is a group of formula (II), wherein R 5 is H and R 6 is an optionally substituted aliphatic or heteroaliphatic group.
  • R' is a group of formula (II), wherein R 5 is H and R 6 is an optionally substituted aliphatic or heteroaliphatic group.
  • R 5 is H and R 6 is an optionally substituted aliphatic or heteroaliphatic group.
  • Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond.
  • Another embodiment of the present invention is the above compound, wherein the optionally substituted heteroaliphatic group comprises at least one ether group.
  • Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one fluorine atom.
  • Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one trialkoxysilane group.
  • R and R' are independently selected from the group consisting of moieties of formulae 42-60:
  • Another embodiment of the present invention is the above compound, wherein the compound is selected from the group consisting of compounds of formulae 61 through 186:
  • Yet another embodiment of the present invention is a formulation comprising a compound of formula (I):
  • X is an oxygen-containing core derived from an oxidized amine comprising (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups;
  • R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
  • R' is identically or different in each instance hydrogen, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
  • R 5 and R 6 are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
  • n is an integer in the range of from 2 to 10;
  • Another embodiment of the present invention is the above formulation, further comprising at least one additional active agent to be delivered.
  • Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent to be delivered is contained within or on the surface of the non-covalent complex.
  • Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent to be delivered is not contained within or on the surface of the non-covalent complex.
  • Another embodiment of the present invention is the above formulation, further comprising one or more excipients.
  • excipients is selected from the group consisting of fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, coating
  • permeability adjusters and combinations thereof.
  • the one or more excipients is selected from the group consisting of carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof.
  • the one or more water soluble polymers comprises a polyethylene glycol, a polypropylene oxide, a polyvinylpyrrolidone, a polyvinyl alcohol, polylactic acid, poly(lactic-co-glycolic acid), or any combination thereof.
  • Another embodiment of the present invention is the above formulation, further comprising an adjuvant selected from the group consisting of chloroquine, chlorpromazine, amodiaquine, perphenazine, coronatine, tolbutamide, glyburide, glybenclamide, arginine, lysine, and histidine.
  • an adjuvant selected from the group consisting of chloroquine, chlorpromazine, amodiaquine, perphenazine, coronatine, tolbutamide, glyburide, glybenclamide, arginine, lysine, and histidine.
  • the first active agent to be delivered is an oligonucleotide or a polynucleotide.
  • Another embodiment of the present invention is the above formulation, further comprising an agriculturally acceptable carrier.
  • Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in a plant.
  • Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in an insect.
  • Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in a plant pathogen.
  • Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent is selected from the group consisting of an herbicide, an insecticide, a fungicide, a nematicide, a bactericide, a viricide, and any combination thereof.
  • the formulation is in the form of a microparticle or nanoparticle.
  • the active agent to be delivered is selected from the group consisting of
  • polynucleotides, oligonucleotides, proteins, peptides, and small molecules are also embodiments.
  • the active agent to be delivered is an oligonucleotide or a polynucleotide.
  • Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide is modified.
  • Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide is unmodified.
  • Another embodiment of the present invention is the above formulation, wherein the active agent to be delivered is an RNA.
  • RNA is a single-stranded RNA.
  • RNA is a double-stranded RNA.
  • siRNA siRNA
  • RNA is an siRNA.
  • RNA is an mRNA.
  • Yet another embodiment of the present invention is a method of regulating expression of a gene in a target organism, comprising applying the above formulation to the target organism.
  • Yet another embodiment of the present invention is a method of modulating a trait of a plant, comprising delivering to the plant an effective amount of the above formulation that modulates the expression of a gene in a plant.
  • the trait is selected from the group consisting of total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, plant sensitivity to herbicide, and any combination thereof.
  • Another embodiment of the present invention is the above method, wherein one or more of the traits is improved relative to a plant not treated with the formulation.
  • Another embodiment of the present invention is the above method, wherein at least one trait selected from the group consisting of plant growth, plant lifespan, plant size, fruit size, fruit yield, total yield, fruit freshness, fruit ripening time, plant nutritional content, and fruit nutritional content, is improved relative to a plant not treated with the formulation.
  • Another embodiment of the present invention is the above method, wherein one or more of the traits is decreased relative to a plant not treated with the formulation.
  • Another embodiment of the present invention is the above method, wherein the plant growth and/or the plant lifespan is decreased relative to a plant not treated with the formulation.
  • Another embodiment of the present invention is the above method, wherein the fruit ripening time is decreased relative to a plant not treated with the formulation.
  • Another embodiment of the present invention is the above method, wherein the plant sensitivity to herbicide is increased relative to a plant not treated with the formulation.
  • Yet another embodiment of the present invention is a method of modulating a trait of an insect, comprising delivering an effective amount of the above formulation that modulates the expression of a gene in an insect to the insect, to a plant infested with the insect, or to a plant prior to infestation with the insect.
  • Another embodiment of the present invention is the above method, wherein the trait modulated is insect growth, development,and/or lifespan.
  • Yet another embodiment of the present invention is a method of modulating the pathogenicity of a plant pathogen, comprising applying the above formulation that modulates the expression of a gene in a plant pathogen to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen.
  • Yet another embodiment of the present invention is a plant cell, insect cell, fungal cell, nematodic cell, or bacterial cell comprising the above formulation.
  • Another embodiment of the present invention is the above formulation, wherein X is derived from 1,10-diaza-18-crown-6.
  • Another embodiment of the present invention is the above formulation, wherein R and R' are each, independently, an optionally substituted aliphatic or
  • R 1 and R 2 are independently selected from the group consisting of hydrogen or a moiety of formula 26 through 29, 32, 34, 35, 37, 40, 41, 46, 47, 50 through 52, 60, and 187 through 191:
  • novel aminolipidoids of the present disclosure provide for several different advantages in the delivery of active agents to target organisms.
  • the aminolipidoids may be used to complex oligonucleotides and polynucleotides, thereby enhancing their delivery to the target organism and preventing their degradation.
  • These aminolipidoids may also be used generate formulations, such as microparticles, nanoparticles, picoparticles, liposomes, and micelles, containing the active agent to be delivered.
  • These aminolipidoids, as well as the formulations thereof may be biocompatible and biodegradable and may be used to provide controlled, sustained release of the active agent to be delivered.
  • aminolipidoids and their corresponding formulations may also be responsive to pH changes given that these aminolipidoids are protonated at lower pH. These aminolipidoids may also act as proton sponges in the delivery of an active agent to a cell to cause endosome lysis.
  • the aminolipidoids of the present disclosure possess oxygen enriched cores (e.g., a higher density of hydroxyl and ether groups) compared to conventional aminolipidoids. The presence of the oxygen-based functional groups in the presently disclosed aminolipidoids results in a very polar core, which, when coupled with their highly lipophilic tails, results in agents well suited for the delivery of polynucleotides.
  • the presently disclosed aminolipidoids form well-defined nanoparticles with polynucleotides and conserve the desired pKa during their delivery to the target organism.
  • the oxygen-rich cores of the presently disclosed aminolipidoids mimic natural products (e.g., antibiotics) and, thus, exhibit enhanced uptake and cell recognition for delivery of
  • polynucleotides as well as superior biocompatibility.
  • present disclosure provides for novel aminolipidoids of formula (I):
  • X is an oxygen-containing core.
  • the term“oxygen-containing core” is defined as core derived from an oxidized amine.
  • the term“oxidized amine” is defined as an amine that comprises (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups.
  • the term“oxidized amine” does not encompass aminoglycosides.
  • the compounds of formula (I) do not encompass the following compounds:
  • X is derived from an oxidized amine comprising at least two hydroxyl groups.
  • the oxidized amine comprises three hydroxyl groups.
  • X is derived from an oxidized amine comprising at least two ether groups.
  • the oxidized amine comprises three, four, or five ether groups.
  • X is derived from an oxidized amine comprising at least one hydroxyl group and at least one ether group.
  • X is derived from an oxidized amine comprising at least two primary amino groups.
  • X is derived from an oxidized amine comprising at least one primary amino group and at least one secondary amino group. In certain embodiments, X is derived from an oxidized amine having any combination of the foregoing embodiments. In certain embodiments, X is derived from an oxidized amine selected from the group consisting of compounds of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), and (22):
  • X is not derived from 1,10-diaza-18-crown-6.
  • R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
  • R 5 and R 6 are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group.
  • the aliphatic or cycloaliphatic group can optionally further comprise an ester or amide linkage in the aliphatic or cycloaliphatic chain.
  • R 5 and R 6 cannot both be hydrogen.
  • R' is identically or different in each instance, hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or
  • heterocycloaliphatic group an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
  • R 5 and R 6 are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group.
  • the aliphatic or cycloaliphatic group can optionally further comprise an ester or amide linkage in the aliphatic or cycloaliphatic chain.
  • R 5 and R 6 cannot both be hydrogen.
  • the aliphatic groups of the presently disclosed aminolipidoids refers to both saturated and unsaturated aliphatic hydrocarbyl groups, which can be straight chain (i.e., unbranched), branched, or cyclic (including polycyclic) and are optionally substituted with one or more functional groups.
  • aliphatic groups include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl groups, each of which are optionally substituted with one or more functional groups.
  • alkyl refers to saturated hydrocarbyl groups, which can be unbranched, branched, or cyclic (i.e., alicyclic) alkyl groups.
  • alkenyl and“alkylene” refers to unsaturated hydrocarbyl groups having at least one carbon-carbon double bond.
  • alkynyl refers to unsaturated hydrocarbyl groups having at least one carbon-carbon triple bond.
  • Examples of such aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH 2 -cyclopropyl, n-butyl, sec-butyl, isobutyl, tert- butyl, cyclobutyl, -CH 2 -cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, neopentyl, cyclopentyl, -CH 2 -cyclopentyl, hexyl, cyclohexyl, -CH 2 -cyclohexyl, heptyl, cycloheptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl,
  • heteroaliphatic groups of the presently disclosed aminolipidoids refers to aliphatic groups, as described above, that independently in one or more instances contain an oxygen, sulfur, nitrogen, phosphorus, or silicon atom between two carbon atoms of the aliphatic group.
  • Such heteroaliphatic groups include saturated and unsaturated heterocycles.
  • heterocycles refers to a non-aromatic partially unsaturated or fully saturated 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms in size and bi- and tri-cyclic ring systems, which may include aromatic six-membered aryl or aromatic heterocyclic groups fused to a non-aromatic heterocyclic ring.
  • heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, 2,5-dihydro-1H-imidazole, and tetrahydrofuryl.
  • the aliphatic and/or heteroaliphatic groups of the aminolipidoids of the present disclosure independently contain from 1 to 20, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 5, or from 1 to 4, or from 4 to 5, or from 4 to 8, or from 4 to 10, or from 4 to 12, or from 4 to 20, or from 5 to 20, or from 5 to 12, or from 5 to 10, or from 5 to 8, or from 8 to 10, or from 8 to 12, or from 8 to 20, or from 10 to 12, or from 10 to 20, or from 12 to 20 carbon atoms.
  • the aryl and heteroaryl groups of the presently disclosed aminolipidoids refer to mono- or polycyclic aromatic carbocyclic groups and mono- or polycyclic aromatic heterocyclic groups.
  • these groups have 3-14 carbon atoms, each of which is optionally substituted.
  • the aryl group is a mono- or bicyclic carbocyclic ring system having one or two aromatic rings.
  • the heteroaryl group is a mono- or bicyclic heterocyclic ring system having one or two aromatic rings.
  • the heteroaryl group (1) has from five to ten ring atoms of which one ring atom is selected from S, O, and N, (2) has zero, one, or two ring atoms that are additional heteroatoms independently selected from S, O, and N, and (3) the remaining ring atoms are carbon.
  • heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, and isoquinolinyl.
  • the term“substituted,” whether preceded by the term“optionally” or not, and substituents contained in formulas of the present disclosure, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • the term“substituted” is contemplated to include all permissible substituents of organic compounds. Broadly, permissible substituents include all acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • heteroatoms such as nitrogen
  • heteroatoms such as nitrogen
  • the presently disclosed aminolipidoids are not intended to be limited in any manner by the permissible substituents of organic compounds.
  • Any substituents disclosed herein may also be further substituted (e.g., an aryl substituent may itself be substituted, such as with another aryl group, which, in turn, is further substituted with fluorine at one or more positions).
  • Examples of optional substituents of the aminolipidoids of the present disclosure include, but are not limited to, aliphatic groups, heteroaliphatic groups, aryl groups, heteroaryl groups, arylalkyl groups, heteroarylalkyl groups, alkoxy groups, aryloxy groups, heteroalkoxy groups, heteroaryloxy groups, alkylthio groups, arylthio groups, heteroalkylthio groups, heteroarylthio groups, F, Cl, Br, I, -OH, -NO 2 , -CN, -CF 3 , -CH 2 CF 3 , - CHCl2, -CH2OH, -CH2CH2OH, -CH2NH2, -CH2SO2R, -C(O)R, -CO2(R), -CON(R)2, -OC(O)R, -OCO 2 R, -OCON(R) 2 , -N(R) 2 , -S(O) 2 R, and
  • R is an optionally substituted aliphatic or heteroaliphatic group.
  • R' is an optionally substituted aliphatic or heteroaliphatic group.
  • R is a group of formula (II), R 5 is H, and R 6 is an optionally substituted aliphatic or
  • R' is a group of formula (II), R 5 is H, and R 6 is an optionally substituted aliphatic or heteroaliphatic group.
  • the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond.
  • the optionally substituted heteroaliphatic group comprises at least one ether group.
  • the optionally substituted aliphatic group is a C 6 to C 20 aliphatic group.
  • the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond.
  • the optionally substituted heteroaliphatic group comprises at least one ether group.
  • the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one fluorine atom. In certain embodiments, the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one trialkoxysilane group. [0088] In certain embodiments of the presently disclosed aminolipidoids of formula (I), R and R' are independently selected from the group consisting of moieties of formulae 23- 60:
  • n is an integer in the range of from 1 to 10.
  • the compound of formula (I) is selected from the group consisting of compounds of formulae 61 through 186:
  • the presently disclosed aminolipidoids can be a salt, either from protonation by a mineral or organic acid or by quaternization of one more available tertiary nitrogens.
  • counterions for such salts include, but are not limited to, halides, such as fluoride, chloride, bromide, or iodide, nitrate, hydrogen sulfate, dihydrogen phosphate, bicarbonate, nitrite, perchlorate, iodate, chlorate, bromate, chlorite, hypochlorite, hypobromite, cyanide, amide, cyanate, hydroxide, permanganate, an acid anion such as acetate or formate, or anions with negative charges greater than -1 (e.g., having in some embodiments one or more than one adsorbent functional group as counterion), such as oxide, sulfide, nitride, arsenate, phosphate, arsenite, hydrogen
  • the presently disclosed aminolipidoids, and optional substitutents thereon can contain isotopes of various common atoms. Examples of such isotopes include, but are not limited to, deuterium, C 13 , N 15 , O 18 , and F 18 .
  • R, R 1 , and R 2 can, independently, be perfluorinated C 1-20 alkyl groups.certain embodiments, the presently disclosed aminolipidoids are relatively non-cytotoxic.
  • the presently disclosed aminolipidoids are
  • the presently disclosed aminolipidoids have a pKa in the range of from about 3.0 to about 9.0, or in the range of from about 5.0 to about 8.0, or in the range of from about 5.5 to about 7.5, or in the range of from about 6.0 to about 7.0.
  • the presently disclosed aminolipidoids may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such forms, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present disclosure. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present disclosure. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.
  • a particular enantiomer of the presently disclosed aminolipidoids may be prepared by asymmetric synthesis, for example, by reacting an optionally substituted compound comprising (1) at least one primary amino group, (2) at least one primary amino group and at least one secondary amino group, or (3) at least two secondary amino groups with a chiral epoxide, acrylate, acrylamide, or aldehyde. It may also be prepared by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • diastereomeric salts can be formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • the aminolipidoid can contain at least one stereocenter. Therefore, in certain embodiments of the presently disclosed lipidoids, each group of formula (II):
  • The“enantiomeric excess” of a substance is a measure of how pure a desired enantiomer is relative to the undesired enantiomer. Enantiomeric excess is defined as the absolute difference between the mole fraction of each enantiomer which is most often expressed as a percent enantiomeric excess. For mixtures of diastereomers, there are analogous definitions and uses for“diastereomeric excess” and percent diastereomeric excess. For example, a sample with 70% of R isomer and 30% of S will have an
  • the aminolipidoids of the present disclosure can have an enantiomeric excess or a diastereomeric excess up to and including 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.
  • amino lipidoids may be prepared by any method known in the art.
  • the aminolipidoids of the present disclosure are synthesized by reacting the oxidized amine with one or more terminal epoxides, interior epoxides, acrylates, acrylamides, aldehydes, or any combination thereof.
  • these starting materials are commercially available and/or are easily and/or inexpensively prepared.
  • the presently disclosed aminolipidoids are synthesized by reacting the oxidized amine with a stoichiometric amount or stoichiometric excess of a terminal epoxide, interior epoxide, acrylate, acrylamide, aldehyde, or any combination thereof.
  • the synthesis of the presently disclosed aminolipidoids is performed without solvent (i.e., neat).
  • the synthesis of the presently disclosed lipidoids is performed in a suitable solvent, such as a lower alcohol (e.g., isopropanol) or DMF.
  • these syntheses are performed at a temperature in the range of about 90 to about 120 °C for about 2 to about 96 hours.
  • conventional heating sources can be employed.
  • non-conventional heating sources such as microwave radiation, can be employed.
  • the reaction mixture is cooled, any solvent used is removed in vacuo, and the crude product is purified or used“as is.”
  • the synthesized aminolipidoids may be purified by any technique known in the art including, but not limited to, precipitation, crystallization, chromatography (e.g., silica gel chromatography, size exclusion chromatography, ion-exchange chromatography, and HPLC), and distillation.
  • the crude product is purified by silica gel chromatography.
  • the aminolipidoids of the present disclosure can be synthesized such that the aminolipidoid contains two or more different R and R' groups.
  • this can be achieved by reacting the oxidized amine with a less-than- stoichiometric amount of a terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde followed by reacting the now-partially substituted compound with a further, different terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde and so on.
  • this can be achived by reacting the oxidized amine with a mixture of different terminal epoxides, interior epoxides, acrylates, acrylamides, and /or aldehydes.
  • this can be achieved through substituting one or more of the amino groups of the the oxidized amine with a protecting group and then reacting the remaining unprotected amino groups of the partially protected comppund with a terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde.
  • the protecting groups can then be removed and the now unprotected amino groups of the partially modified compound can then be further reacted with a different terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde.
  • amino-protecting groups examples include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl- [9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1- methylethyl carbamate (Adpoc), 1,1-dimethyl
  • TBOC 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1- methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N- dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N- hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cb
  • tungsten)carbonyl]amine N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzene sulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4- methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- methoxybenzenes
  • the present disclosure provides for formulations comprising the presently disclosed aminolipidoids that may be used to deliver one or more active agents to a target organism. Therefore, in certain embodiments, the presently disclosed formulations can comprise at least one aminolipidoid of the present disclosure and at least one active agent to be delivered. In certain embodiments, the at least one aminolipidoid of the present disclosure and a first active agent to be delivered are non-covalently associated to one another to form a non-covalent complex.
  • non-covalently associated encompasses any kind of intermolecular interaction between the at least one aminolipidoid of the present disclosure and the first active agent to be delivered other than covalent interactions (i.e., interactions that involve the sharing of electrons).
  • non- covalent interactions include, but are not limited to, electrostatic interactions, such as ionic interactions, hydrogen bonding, and halogen bonding, Van der Waals forces, such as the Keesom force, the Debye force, and London dispersion forces, ⁇ -effects, such as ⁇ - ⁇ interactions, cation- ⁇ interactions, anion- ⁇ interactions, and polar ⁇ interactions, and hydrophobic interactions.
  • non-covalent complex encompasses a complex of at least one aminolipidoid of the present disclosure and a first active agent to be delivered wherein the least one aminolipidoid and the first active agent are associated to each other via non-covalent interactions, as defined above.
  • oxygen- containing core X in the compound of formula (I) is derived from 1,10-diaza-18-crown-6.
  • R and R' can each, independently, be an optionally substituted aliphatic or heteroaliphatic or group.
  • compound of formula (I) is a compound of formula (III):
  • R 1 and R 2 are independently selected from the group consisting of hydrogen or a moiety of formula 26 through 29, 32, 34, 35, 37, 40, 41, 46, 47, 50 through 52, 60, and 187 through 191:
  • the presently disclosed formulations further comprise at least one additional active agent to be delivered.
  • this at least one additional active agent is part of the non-covalent complex of the least one aminolipidoid and the first active agent.
  • the at least one additional active agent can be contained within the non-covalent complex or adhered to the surface of the non-covalent complex via non-covalent interactions, as defined above.
  • the at least one additional active agent is not contained within the non-covalent complex or adhered to the surface of the non-covalent complex, e.g., the at least one additional active agent is simply in a physical mixture with the non-covalent complex.
  • the first active agent is an oligonucleotide or a polynucleotide
  • the at least one additional active agent is an herbicide, an insecticide, a fungicide, a bactericide, and/or a viricide.
  • the first active agent is used to increase the sensitivity of the target organism to the additional active agent, for example, to increase the sensitivity of a plant to an herbicide, or to increase the sensitivity of an insect to an insecticide.
  • the presently disclosed formulations may also comprise one or more excipients.
  • Suitable excipients include, but are not limited to, fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters.
  • the one or more excipients may be selected from the group consisting of sterols, carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof.
  • the one or more excipients is a phytosterol. In certain other embodiments, the one or more excipients is cholesterol. In certain other embodiments, the one or more excipients comprises a water-soluble polymer such as polyethylene glycol (PEG), a polypropylene oxide (PPO), a polyvinylpyrrolidone (PVP), a polyvinyl alcohol (PVA), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), or any combination thereof. In certain embodiments, the water-soluble polymer can be contained within or adhered to the surface of the non-covalent complexes of the present disclosure via non- covalent interactions, as defined above.
  • the water-soluble polymer can be tethered to the surface of the non-covalent complexes of the present disclosure via a lipid tail that is covalently bound on one end to the water-soluble polymer and which is entrained within the surface and/or interior of the non-covalent complex.
  • the presently disclosed formulations are combined with an agriculturally acceptable carrier.
  • the agriculturally acceptable carrier can be solid or liquid and is a substance useful in formulation of agricultural products. Examples of such agricultural products include, but are not limited to, fertilizers, herbicides, insecticides, fungicides, bactericides, viricides, and nematicides.
  • Such agriculturally acceptable carriers for use in the presently disclosed formulations include, but are not limited to, surface active agents, stickers, spreader stickers, inert carriers, preservatives, humectants, dyes, UV (ultra-violet) protectants, buffers, flow agents, antifoams (e.g.,
  • polydimethylsiloxane sodium aluminosilicate, or other components which facilitate product handling and application of the compositions.
  • agriculturally acceptable inert carriers include inorganic minerals, such as kaolin, mica, gypsum, fertilizer, carbonates, sulfates, and phosphates, organic materials, such as sugar, starches, and cyclodextrins, and botanical materials, such as wood products, cork, powdered corn cobs, rice hulls, peanut hulls, and walnut shells.
  • Agriculturally acceptable carriers are described, for example, in U.S. Patent No. 6,984,609.
  • the agriculturally acceptable carriers include, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders, or fertilizers. Such carriers are described, for example, in WO 97/33890.
  • the presently disclosed formulations may further comprise one or more additional compounds to facilitate passage of the active agent(s) through the plant cell wall.
  • additional compounds to facilitate passage of the active agent(s) through the plant cell wall.
  • 8,609,420 describes conjugation of the active agent to a semi-conductor nanoparticle within the size range of 3-5 nm (e.g., a“quantum dot”) and one or more cell penetrating peptides to improve penetration of the plant cell and intracellular delivery of the active agent.
  • U.S. Patent No.8,686,222 describes interacting a polyamidoamine dendrimer and one or more cell penetrating peptides with the active agent to improve cell penetration.
  • U.S. Patent No.8,653,327 describes delivery of active agents through plant cell walls by coating a PEGylated semiconductor nanoparticle with the active agent.
  • 8,722,410 describes transferring active agents into plant cells by applying the active agent to a nanoparticle coated with a subcellular compartment targeting protein.
  • U.S. Patent Nos.8,609,420, 8,686,222, 8,653,327, and 8,722,410 are incorporated by reference herein in their entireties.
  • the formulations of the present disclosure can take any form. Examples of such forms include, but are not limited to, complexes, particles (e.g., microparticles, nanoparticles, and picoparticles), micelles, and liposomes.
  • two or more active agents can be formulated with the presently disclosed aminolipidoids to form a single complex, particle, micelle or liposome containing the two or more active agents.
  • the two or more active agents can be separately formulated to form a single complex, particle, micelle or liposome, each containing a single active agent, and are then combined to form a mixture prior to delivery to a target organism.
  • the aminolipidoids of the present disclosure possess one or more primary, secondary, and/or tertiary amino groups that, although hindered, are available to interact with an active agent (e.g., a polynucleotide). As such, a non-covalent complex is formed when an active agent is contacted with the presently disclosed aminolipidoids under conditions suitable to form an active agent/aminolipidoid non-covalent complex.
  • an active agent e.g., a polynucleotide
  • multiple aminolipidoid molecules may non-covalently complex with an active agent molecule.
  • the non-covalent complex may include 1-100 aminolipidoid molecules, 1- 1000 aminolipidoid molecules, 10-1000 aminolipidoid molecules, or 100-10,000
  • the aminolipidoids of the present disclosure may be used to encapsulate active agents.
  • the aminolipidoids of the present disclosure have several properties that make them particularly suitable in the preparation of drug delivery devices. These include, but are not limited to: (1) the ability of the lipidoid to complex and“protect” labile agents, (2) the ability to buffer the pH in the endosome (3) the ability to act as a“proton sponge” and cause endosomolysis, and (4) the ability to neutralize the charge on negatively charged agents.
  • the aminolipidoids of the present disclosure are used to form particles containing at least one active agent to be delivered.
  • these particles may include other materials, such as proteins, carbohydrates, synthetic polymers (e.g., PEG, PLGA), and natural polymers.
  • the presently disclosed aminolipidoids are combined with an active agent to be delivered to a cell or a subject to form microparticles,
  • the agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a
  • the diameter of the particles of the present disclosure range from 1 to 1,000 micrometers. In certain embodiments, the diameter of the particles range from 1 to 100 micrometers. In certain embodiments, the diameter of the particles range from 1 to 10 micrometers. In certain embodiments, the diameter of the particles range from 10 to 100 micrometers. In certain embodiments, the diameter of the particles range from 100 to 1,000 micrometers. In certain embodiments, the diameter of the particles range from 1 to 5 micrometers.
  • the diameter of the particles range from 1 to 1,000 nm. In certain embodiments, the diameter of the particles range from 1 to 100 nm. In certain embodiments, the diameter of the particles range from 1 to 10 nm. In certain embodiments, the diameter of the particles range from 10 nm to 100 nm. In certain embodiments, the diameter of the particles range from 100 nm to 1,000 nm. In certain embodiments, the diameters of the particles range from 1 to 5 nm. In certain embodiments, the diameter of the particles range from 1 to 1,000 pm. In certain embodiments, the diameter of the particles range from 1 to 100 pm. In certain embodiments, the diameter of the particles range from 1 to 10 pm. In certain embodiments, the diameter of the particles range from 10 to 100 pm.
  • the diameter of the particles range from 100 to 1,000 pm. In certain embodiments, the diameter of the particles range from 1 to 5 pm.
  • the particles of the present disclosure may be prepared using any method known in the art. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. In certain embodiments, methods of preparing the particles are the double emulsion process and spray drying. In other embodiments, methods of preparing the particles are nanoprecipitation or flash precipitation, for example, as disclosed in U.S. Patent Nos.
  • the conditions used in preparing the particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology,“stickiness”, shape, etc.).
  • the method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may also depend on the agent being encapsulated and/or the composition of the matrix.
  • the particles are coated with a targeting agent. In other embodiments, the particles are coated to achieve desirable surface properties (e.g., a particular charge).
  • the aminolipidoids of the invention may be used to prepare micelles or liposomes containing an active agent to be delivered. Many techniques for preparing micelles and liposomes are known in the art, and any method may be used with the aminolipidoids of the present disclosure to make micelles and liposomes. Micelles and liposomes are particularly useful in delivering hydrophobic agents, such as hydrophobic small molecules.
  • liposomes containing aminolipidoids of the present disclosure are formed through spontaneous assembly.
  • these liposomes are formed when thin lipid films or lipid cakes are hydrated and stacks of lipid crystalline bilayers become fluid and swell.
  • the hydrated lipid sheets detach during agitation and self- close to form large, multilamellar vesicles (LMV). This prevents interaction of water with the hydrocarbon core of the bilayers at the edges.
  • LMV multilamellar vesicles
  • reducing the size of the particle can be modified through input of sonic energy (sonication) or mechanical energy (extrusion). See Walde, P.“Preparation of Vesicles (Liposomes)” In Encyclopedia of Nanoscience and Nanotechnology; Nalwa, H. S. Ed. American Scientific Publishers: Los Angeles, 2004; Vol. 9, pp.43-79 and Szoka et al.“Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes)” Ann. Rev. Biophys.
  • the preparation of liposomes of the present disclosure involves preparing the aminolipidoids for hydration, hydrating the aminolipidoids with agitation, and sizing the vesicles to achieve a homogenous distribution of liposomes.
  • Aminolipidoids are first dissolved in an organic solvent to assure a homogeneous mixture. The solvent is then removed to form a lipidoid film. This film is thoroughly dried to remove residual organic solvent by placing the vial or flask on a vacuum pump overnight. Hydration of the lipidoid film/cake is accomplished by adding an aqueous medium to the container of dry lipidoid and agitating the mixture.
  • LMV small unilamellar vesicles
  • LUV unilamellar vesicles
  • Certain aminolipidoids of the present disclosure can spontaneously self- assemble around certain molecules, such as DNA and RNA, to form liposomes.
  • the application is the delivery of polynucleotides to a target cell.
  • use of the aminolipidoids of the present disclosure allows for simple assembly of liposomes without the need for additional steps or devices such as an extruder.
  • the complexes, microparticles, nanoparticles, picoparticles, liposomes, and micelles of the present disclosure may be modified to include targeting agents since it is often desirable to target a particular cell, collection of cells, or tissue.
  • targeting agents that direct pharmaceutical compositions to particular cells are known in the art (e.g., Cotten et al. Methods Enzym. 217:618, 1993; which is incorporated herein by reference in its entirety).
  • the targeting agents may be included throughout the particle or may be only on the surface.
  • the targeting agent may be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, and/or nucleic acid.
  • the targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the particle.
  • Examples of targeting agents include, but are not limited to, antibodies, fragments of antibodies, low- density lipoproteins (LDLs), transferrin, asialycoproteins, gp120 envelope protein of the human immunodeficiency virus (HIV), carbohydrates, receptor ligands, sialic acid, and aptamers.
  • the targeting agent may be included in the mixture that is used to form the particles. If the targeting agent is only on the surface on the particle, the targeting agent may be associated with (i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals, or other interactions) the formed particles using standard chemical techniques.
  • the formulations of the present disclosure can be formulated as a bait, a food substance, or an attractant.
  • the formulations of the present disclosure can be incorporated into an insect bait suitable for oral administration of the formulation to the target insect.
  • the bait may comprise a formulation comprising an active agent and an aminolipidoid of the present disclosure dispersed in a carrier and an edible insect attractant.
  • the bait comprises an edible insect attractant and a nanoparticle or microparticle comprising at least one active agent and at least one aminolipidoid of the present disclosure, wherein the nanoparticle or microparticle is dispersed in a carrier.
  • the formulation of the present disclosure and attractant can be mixed together before being dispersed in the desired carrier.
  • Suitable attractants include any type of insect food and/or attractant which will lure the insect to the bait to ingest the bait.
  • Exemplary insect foods or attractants include, but are not limited to, any type of insect food, including various sugars, proteins, carbohydrates, yeast, fats, and/or oils.
  • the bait can be in any form suitable for delivery and ingestion of the composition, depending on the habitat and target insect, but will typically be a liquid, gel, self-sustaining gel-matrix, or solid bait (e.g., tablets, granules, etc.).
  • Exemplary carriers include, without limitation, agarose gel, gelatin gel, and/or pectin gel. In certain embodiments, the carrier is agarose gel, which is especially suited for aquatic habitats and breeding grounds. Insect baits are known in the art and are described, for example, in U.S. Pat.
  • the presently disclosed formulations can be present in the bait in an effective amount (i.e., concentration) for the activity of the active agent, such as gene silencing.
  • concentration of the active agent in the bait may be about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% by weight of the bait. Any of these values may be used to define a range for the concentration of the active agent in the bait.
  • the concentration of the active agent in the bait may range from about 0.1 to about 1%, or from about 1 to about 5% by weight of the bait.
  • the weight ratio of active agent to insect attractant (food) in the bait may be about 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150 or 1:200. Any of these values may be used to define a range for the weight ratio of the active agent to the insect attractant in the bait.
  • the weight ratio of the active agent to the insect attractant in the bait may be from about 1:20 to about 1:200, or from about 1:50 to about 1:100.
  • the concentration of a microparticle or nanoparticle comprising the at least one active agent and at least one aminolipidoid of the present disclosure in the bait may be about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% by weight of the bait. Any of these values may be used to define a range for the concentration of the microparticle or nanoparticle in the bait. For example, the concentration of the microparticle or nanoparticle in the bait may range from about 0.1 to about 1%, or from about 1 to about 5% by weight of the bait.
  • the weight ratio of the microparticle or nanoparticle to insect attractant (food) in the bait may be about 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150 or 1:200. Any of these values may be used to define a range for the weight ratio of the microparticle or nanoparticle to the insect attractant in the bait.
  • the weight ratio of the microparticle or nanoparticle to the insect attractant in the bait may be from about 1:20 to about 1:200, or from about 1:50 to about 1:100.
  • the presently disclosed aminolipidoid-based formulations can be used to deliver an active agent to target organisms for the purpose of killing and/or controlling the proliferation of the target organisms, such as insects, plant pathogens (e.g., fungi, bacteria, viruses, and nematodes), and weeds.
  • the presently disclosed aminolipidoid-based formulations comprise an insecticidal, nematidicidal, fungicidal, bacteriocidal, viricidal, or herbicidal active agent, or combinations thereof.
  • these formulations are combined with an agriculturally acceptable carrier to form a insecticidal, nematodicidal, fungicidal, bacteriocidal, viricidal, or herbicidal formulation.
  • a target organism is an organism in which the presently disclosed herbicidal, insecticidal, or fungicidal formulations are intended to be functional, for example, to mediate gene silencing or suppression.
  • a target organism is also a host organism, as described herein below.
  • a target organism is separate and distinct from a host organism that serves as a source of the active agent to be functional in the target organism.
  • the insecticidal, nematidicidal, fungicidal, bacteriocidal, viricidal, or herbicidal formulation may further be combined with an agriculturally acceptable carrier.
  • the agriculturally acceptale carrier can be solid or liquid and is a substance useful in formulation of agricultural products, for example, fertilizers, herbicides, insecticides, fungicides, bactericides, viricides, and nematicides.
  • Agriculturally acceptable carriers include, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders or fertilizers. Such carriers are described for example, in WO 97/33890, which is incorporated herein by reference.
  • the presently disclosed formulations can be applied to the crop area or plant to be treated, simultaneously or in succession with further compounds.
  • further compounds can be, for example, fertilizers or micronutrient donors or other preparations, which influence the growth of plants. They can also be selective herbicides or non-selective herbicides as well as insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation.
  • Active agents that can be delivered to a target organism using the presently disclosed formulations include, but are not limited to, any type of molecule or compound including, but not limited to, nucleic acids, peptides, polypeptides, small molecules, and mixtures thereof.
  • nucleic acids include, but are not limited to, interfering RNA molecules (e.g., siRNA, aiRNA, miRNA), antisense oligonucleotides, plasmids, ribozymes, immunostimulatory oligonucleotides, and mixtures thereof.
  • the active agent comprises a nucleic acid.
  • the nucleic acid comprises an interfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, or mixtures thereof.
  • the nucleic acid comprises single-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid such as, e.g., an antisense oligonucleotide, a ribozyme, a plasmid, an immunostimulatory
  • oligonucleotide or mixtures thereof.
  • presently disclosed particles are associated with a nucleic acid.
  • the nucleic acid is fully encapsulated in a lipidoid particle.
  • the term“nucleic acid” includes any oligonucleotide or
  • polynucleotide with fragments containing up to 60 nucleotides generally termed
  • oligonucleotides, and longer fragments termed polynucleotides.
  • oligonucleotides of the invention are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 nucleotides in length. Any of these values may be used to define a range for the size of the oligonucleotide. For example, the size of the oligonucleotide may range from 15-60, 20-60 or 25-60 nucleotides in length.
  • the polynucleotide is 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more nucleotides in length.
  • the polynucleotide is at least 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nucleotides in length. Any of these values may be used to define a range for the size of the polynucleotide.
  • the polynucleotide may range from 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900- 950, or 950-1000 nucleotides in length.
  • the nucleic acid may be administered alone in the particles of the present disclosure, or in combination (e.g., co-administered) with particles of the present disclosure comprising peptides, polypeptides, or small molecules, such as conventional drugs.
  • oligonucleotide refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally-occurring bases, sugars, and intersugar (backbone) linkages.
  • the terms“polynucleotide” and“oligonucleotide” also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases.
  • Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides.
  • a deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5′ and 3′ carbons of this sugar to form an alternating, unbranched polymer.
  • a ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.
  • Nucleic acids that can be used in the presently disclosed formulations includes any form of nucleic acid that is known. The nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids.
  • double-stranded DNA examples include, e.g., structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA.
  • double-stranded RNA examples include, e.g., siRNA and other RNAi agents such as aiRNA and pre-miRNA.
  • Single-stranded nucleic acids include, e.g., antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides.
  • Nucleic acids that can be used in the formulations of the present disclosure may be of various lengths, which is generally dependent upon the particular form of nucleic acid.
  • plasmids or genes may be from about 1,000 to about 100,000 nucleotide residues in length.
  • oligonucleotides may range from about 10 to about 100 nucleotides in length. In certain embodiments,
  • oligonucleotides both single-stranded, double-stranded, and triple-stranded, may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides in length.
  • an oligonucleotide (or a strand thereof) that can be used in the presently disclosed formulations specifically hybridizes to or is complementary to a target polynucleotide sequence.
  • “complementary” as used herein indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In certain embodiments, an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired.
  • the oligonucleotide may include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence that it is targeting or to which it specifically hybridizes.
  • the oligo- or polynucleotide is optionally purified and substantially pure.
  • the polynucleotide is greater than 50% pure.
  • the oligo- or polynucleotide is greater than 75% pure.
  • the oligo- or polynucleotide is greater than 95% pure.
  • the oligo- or polynucleotide may be provided by any means known in the art.
  • the oligo- or polynucleotide has been engineered using recombinant techniques.
  • the oligo- or polynucleotide may also be obtained from natural sources and purified from contaminating components found normally in nature.
  • the oligo- or polynucleotide may also be chemically synthesized in a laboratory. In certain embodiments, the oligo- or polynucleotide is synthesized using standard solid phase chemistry.
  • the oligo- or polynucleotide may be modified by chemical or biological means. In certain embodiments, these modifications lead to increased stability of the oligo- or polynucleotide.
  • the oligo- or polynucleotide to be delivered may be in any form. Examples of such forms include, but are not limited to, a circular plasmid, a linearized plasmid, a cosmid, a viral genome, a modified viral genome, an artificial chromosome, dsRNA, ssRNA, dsDNA, ssDNA, RNA/DNA hybrids, dsRNA hairpins, siRNA, aiRNA, and miRNA.
  • the oligo- or polynucleotide may be of any sequence. In certain
  • the oligo- or polynucleotide encodes a protein or peptide.
  • the encoded proteins may be enzymes, structural proteins, receptors, soluble receptors, ion channels, or cytokines.
  • the oligo- or polynucleotide may also comprise regulatory regions to control the expression of a gene. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA box, ribosomal binding sites, and stop site for transcription.
  • the polynucleotide is not intended to encode a protein.
  • the polynucleotide may be used to fix an error in the genome of the cell being transfected.
  • the nucleic acid is modified.
  • the term "modified" in reference to a nucleic acid is defined as a nucleic acid that contains variations of the standard bases, sugars and/or phosphate backbone chemical structures occurring in ribonucleic (i.e., A, C, G and U) and deoxyribonucleic (i.e., A, C, G and T) acids. Particular modifications of nucleic acids are further described below.
  • the oligo- or polynucleotide is an RNA that carries out RNA interference (RNAi).
  • interfering RNA or“RNAi” or“interfering RNA sequence” refers to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (e.g., duplex RNA, such as siRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence.
  • RNAi double-stranded RNA
  • duplex RNA such as siRNA, aiRNA, or pre-miRNA
  • Interfering RNA thus refers to the single-stranded RNA that is complementary to a target mRNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand.
  • Interfering RNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif).
  • the sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof.
  • the active agent comprises an siRNA.
  • the siRNA molecule can comprise a double-stranded region of about 15 to about 60 nucleotides in length (e.g., about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).
  • the siRNA molecules used in the presently disclosed formulations are capable of silencing the expression of a target sequence in vitro and/or in vivo.
  • the siRNA molecule comprises modified nucleotides including, but not limited to, 2′-O-methyl (2′OMe) nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof.
  • 2′-O-methyl (2′OMe) nucleotides 2′-deoxy-2′-fluoro(2′F) nucleotides
  • MOE 2-methoxyethyl
  • LNA locked nucleic acid
  • the siRNA comprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidine nucleotides) such as, for example, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, and mixtures thereof.
  • the siRNA does not comprise 2′OMe-cytosine nucleotides.
  • the siRNA comprises a hairpin loop structure.
  • the siRNA may comprise modified nucleotides in one strand (i.e., sense or antisense) or both strands of the double-stranded region of the siRNA molecule.
  • uridine and/or guanosine nucleotides are modified at selective positions in the double-stranded region of the siRNA duplex.
  • at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and/or antisense strand can be a modified uridine nucleotide such as a 2′OMe-uridine nucleotide.
  • every uridine nucleotide in the sense and/or antisense strand is a 2′OMe-uridine nucleotide.
  • at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense and/or antisense strand can be a modified guanosine nucleotide such as a 2′OMe- guanosine nucleotide.
  • every guanosine nucleotide in the sense and/or antisense strand is a 2′OMe-guanosine nucleotide.
  • At least one, two, three, four, five, six, seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified, e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/or by introducing modified nucleotides such as 2′OMe nucleotides.
  • the 5′-GU-3′ motif can be in the sense strand, the antisense strand, or both strands of the siRNA sequence.
  • the 5′-GU-3′ motifs may be adjacent to each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.
  • a modified siRNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the target sequence relative to the corresponding unmodified siRNA sequence.
  • the siRNA molecule does not comprise phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double-stranded region.
  • the siRNA comprises one, two, three, four, or more phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double- stranded region. In certain embodiments, the siRNA does not comprise phosphate backbone modifications. [0146] In certain embodiments, the siRNA does not comprise 2′-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region. In certain
  • the siRNA comprises one, two, three, four, or more 2′-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region. In certain embodiments, the siRNA does not comprise 2′-deoxy nucleotides. [0147] In certain embodiments, the nucleotide at the 3′-end of the double-stranded region in the sense and/or antisense strand is not a modified nucleotide. In certain
  • the nucleotides near the 3′-end (e.g., within one, two, three, or four nucleotides of the 3′-end) of the double-stranded region in the sense and/or antisense strand are not modified nucleotides.
  • the siRNA molecules described herein may have 3′ overhangs of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or may lack overhangs (i.e., have blunt ends) on one or both sides of the double-stranded region.
  • the siRNA has 3′ overhangs of two nucleotides on each side of the double-stranded region.
  • the 3′ overhang on the antisense strand has complementarity to the target sequence and the 3′ overhang on the sense strand has complementarity to a complementary strand of the target sequence.
  • the 3′ overhangs do not have complementarity to the target sequence or the complementary strand thereof.
  • the 3′ overhangs comprise one, two, three, four, or more nucleotides such as 2′-deoxy(2′H) nucleotides.
  • the 3′ overhangs comprise deoxythymidine (dT) and/or uridine nucleotides.
  • one or more of the nucleotides in the 3′ overhangs on one or both sides of the double-stranded region comprise modified nucleotides.
  • modified nucleotides include, but are not limited to, 2′OMe nucleotides, 2′-deoxy-2′F nucleotides, 2′-deoxy nucleotides, 2′-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof.
  • one, two, three, four, or more nucleotides in the 3′ overhangs present on the sense and/or antisense strand of the siRNA comprise 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidine nucleotides) such as, for example, 2′OMe-guanosine nucleotides, 2′OMe- uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, and mixtures thereof.
  • 2′OMe nucleotides e.g., 2′OMe purine and/or pyrimidine nucleotides
  • 2′OMe-guanosine nucleotides e.g., 2′OMe- uridine nucleotides
  • 2′OMe-adenosine nucleotides e-cytosine nucleotides
  • the siRNA may comprise at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and/or modified siRNA sequences that silence target gene expression.
  • the cocktail of siRNA may comprise sequences, which are directed to the same region or domain (e.g., a“hot spot”) and/or to different regions or domains of one or more target genes.
  • one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) modified siRNA that silence target gene expression are present in a cocktail.
  • one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence target gene expression are present in a cocktail.
  • the antisense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof.
  • the antisense strand of the siRNA molecule comprises or consists of a sequence that is 100% complementary to the target sequence or a portion thereof.
  • the antisense strand of the siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.
  • the sense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof.
  • the sense strand of the siRNA molecule comprises or consists of a sequence that is 100% identical to the target sequence or a portion thereof.
  • the siRNA that can be used in the presently disclosed formulations are capable of silencing the expression of a target gene of interest.
  • each strand of the siRNA duplex can be about 15 to about 60 nucleotides in length, or about 15 to about 30 nucleotides in length.
  • the siRNA comprises at least one modified nucleotide.
  • the modified siRNA contains at least one 2′OMe purine or pyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine, 2′OMe-adenosine, and/or 2′OMe- cytosine nucleotide.
  • one or more of the uridine and/or guanosine nucleotides are modified.
  • the modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the siRNA.
  • the siRNA sequences may have overhangs or may lack overhangs (i.e., have blunt ends).
  • the modified siRNA generally comprises from about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA duplex.
  • one, two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the double-stranded region of the siRNA comprise modified nucleotides.
  • less than about 25% e.g., less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the nucleotides in the double-stranded region of the siRNA comprise modified nucleotides.
  • from about 1% to about 25% e.g., from about 1%- 25%, 2%-25%, 3%-25%, 4%-25%, 5%-25%, 6%-25%, 7%-25%, 8%-25%, 9%-25%, 10%- 25%, 11%-25%, 12%-25%, 13%-25%, 14%-25%, 15%-25%, 16%-25%, 17%-25%, 18%- 25%, 19%-25%, 20%-25%, 21%-25%, 22%-25%, 23%-25%, 24%-25%, etc.) or from about 1% to about 20% (e.g., from about 1%-20%, 2%-20%, 3%-20%, 4%-20%, 5%-20%, 6%- 20%, 7%-20%, 8%-20%, 9%-20%, 10%-20%, 11%-20%, 12%-20%, 13%-20%, 14%-20%, 15%-20%, 16%-20%, 17%-20%, 18%-20%, 19%-20%, 1%
  • the resulting modified siRNA can comprise less than about 30% modified nucleotides (e.g., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% modified nucleotides) or from about 1% to about 30% modified nucleotides (e.g., from about 1%-30%, 2%-30%, 3%-30%, 4%-30%, 5%-30%, 6%-30%, 7%-30%, 8%-30%, 9%-30%, 10%-30%, 11%-30%, 12%-30%, 13%-30%, 14%
  • modified nucleotides suitable for use in the presently disclosed formulations include, but are not limited to, ribonucleotides having a 2′-O-methyl (2′OMe), 2′-deoxy-2′-fluoro(2′F), 2′-deoxy, 5-C-methyl, 2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′- amino, or 2′-C-allyl group.
  • Modified nucleotides having a Northern conformation are also suitable for use in siRNA molecules.
  • Such modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl) (MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′- deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro(2′Cl) nucleotides, and 2′-azido nucleotides.
  • LNA locked nucleic acid
  • MOE 2-methoxyethyl
  • siRNA molecules described herein include one or more G-clamp nucleotides.
  • a G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex.
  • nucleotides having a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4- nitroindole, 5-nitroindole, and 6-nitroindole can be incorporated into siRNA molecules.
  • the siRNA molecules may further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone
  • terminal cap moieties include, but are not limited to, inverted deoxy abasic residues, glyceryl modifications, 4′,5′-methylene nucleotides, 1-( ⁇ - D-erythrofuranosyl) nucleotides, 4′-thio nucleotides, carbocyclic nucleotides, 1,5- anhydrohexitol nucleotides, L-nucleotides, ⁇ -nucleotides, modified base nucleotides, threo- pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties, 3′- 3′-inverted abasic moieties, 3′-2′-inverted nucleotide
  • phosphorodithioate methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions.
  • Such chemical modifications can occur at the 5′- end and/or 3′-end of the sense strand, antisense strand, or both strands of the siRNA.
  • the sense and/or antisense strand of the siRNA molecule can further comprise a 3′-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides and/or any combination of modified and unmodified nucleotides.
  • the siRNA molecules can optionally comprise one or more non-nucleotides in one or both strands of the siRNA.
  • non-nucleotide refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore lacks a base at the 1′-position.
  • chemical modification of the siRNA comprises attaching a conjugate to the siRNA molecule.
  • the conjugate can be attached at the 5′ and/or 3′-end of the sense and/or antisense strand of the siRNA via a covalent attachment such as, e.g., a biodegradable linker.
  • the conjugate can also be attached to the siRNA, e.g., through a carbamate group or other linking group.
  • the conjugate is a molecule that facilitates the delivery of the siRNA into a cell.
  • the active agent comprises an asymmetrical interfering RNA (aiRNA).
  • aiRNA duplexes of various lengths may be designed with overhangs at the 3′ and 5′ ends of the antisense strand to target an mRNA of interest.
  • the sense strand of the aiRNA molecule is about 10-25, 12- 20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, or about 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length.
  • the 5′ antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g.,“AA”,“UU”,“dTdT”, etc.). In other
  • the 3′ antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g.,“AA”,“UU”,“dTdT”, etc.).
  • the aiRNA molecules described herein may comprise one or more modified nucleotides, e.g., in the double-stranded (duplex) region and/or in the antisense overhangs.
  • aiRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences.
  • the aiRNA molecule comprises 2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, or mixtures thereof.
  • aiRNA molecules may comprise an antisense strand which corresponds to the antisense strand of an siRNA molecule, e.g., one of the siRNA molecules described herein.
  • aiRNA molecules may be used to silence the expression of any of a target gene.
  • the aiRNA molecule comprises a double-stranded (duplex) region of about 10 to about 25 (base paired) nucleotides in length, wherein the aiRNA molecule comprises an antisense strand comprising 5′ and 3′ overhangs, and wherein the aiRNA molecule is capable of silencing target gene expression.
  • each of the 5′ and 3′ overhangs on the antisense strand comprises or consists of one, two, three, four, five, six, seven, or more nucleotides.
  • the aiRNA molecule comprises modified nucleotides selected from the group consisting of 2′OMe nucleotides, 2′F nucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNA nucleotides, and mixtures thereof.
  • miRNA the active agent comprises a microRNAs (miRNA). Generally, miRNA are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression.
  • the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, or about 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length.
  • the miRNA molecule comprises about 15 to about 60 nucleotides in length, wherein the miRNA molecule is capable of silencing target gene expression.
  • miRNA molecules may comprise one or more modified nucleotides.
  • miRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences.
  • the miRNA molecule comprises 2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, or mixtures thereof.
  • the miRNA molecule comprises modified nucleotides selected from the group consisting of 2′F nucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNA nucleotides, and mixtures thereof.
  • dsRNA [0173]
  • the active agent is a dsRNA (double-stranded RNA).
  • the active agent is an shRNA (short hairpin RNA).
  • the active agent is an antisense oligonucleotide.
  • the terms“antisense polynucleotide” or“antisense” include polynucleotides that are
  • Antisense polynucleotides are single strands of DNA or RNA that are complementary to a chosen sequence.
  • the polynucleotide is an antisense RNA.
  • Antisense RNA polynucleotides prevent the translation of complementary RNA strands by binding to the RNA.
  • Antisense DNA polynucleotides can be used to target a specific, complementary (coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid can be degraded by the enzyme RNase H.
  • antisense polynucleotides comprise from about 10 to about 60 nucleotides, or from about 15 to about 30 nucleotides.
  • antisense polynucleotides that may not be exactly complementary to the desired target gene.
  • the invention can be utilized in instances where non-target specific- activities are found with antisense, or where an antisense sequence containing one or more mismatches with the target sequence is the most preferred for a particular use.
  • Methods of producing antisense polynucleotides are known in the art and can be readily adapted to produce an antisense polynucleotides that targets any polynucleotide sequence. Selection of antisense polynucleotide sequences specific for a given target sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, and relative stability.
  • Antisense polynucleotides may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
  • Highly preferred target regions of the mRNA include those regions at or near the AUG translation initiation codon and those sequences that are substantially complementary to 5′ regions of the mRNA.
  • Ribozymes are RNA- protein complexes having specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • the enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, hepatitis ⁇ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence), or Neurospora VS RNA motif, for example.
  • Important characteristics of enzymatic nucleic acid molecules used according to the invention are that they have a specific substrate binding site which is complementary to one or more of the target gene DNA or RNA regions, and that they have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
  • Methods of producing a ribozyme targeted to any polynucleotide sequence are known in the art.
  • Ribozyme activity can be optimized by altering the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases, modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.
  • Insecticides [0183] In certain embodiments, an insecticide for killing or controlling the proliferation of an insect is combined with the active agent described above. Examples of suitable insecticides include, but are not limited to, those provided in Table 1. [0184] Table 1.
  • suitable insecticides include biologics, hormones or pheromones such as azadirachtin, Bacillus species, Beauveria species, codlemone, Metarrhizium species, Paecilomyces species, thuringiensis and Verticillium species, and active compounds having unknown or non-specified mechanisms of action such as fumigants (such as aluminium phosphide, methyl bromide and sulphuryl fluoride) and selective feeding inhibitors (such as cryolite, flonicamid and pymetrozine).
  • biologics such as azadirachtin, Bacillus species, Beauveria species, codlemone, Metarrhizium species, Paecilomyces species, thuringiensis and Verticillium species
  • active compounds having unknown or non-specified mechanisms of action such as fumigants (such as aluminium phosphide, methyl bromide and sulphuryl fluoride) and selective feeding inhibitors (such as cryolite,
  • mite growth inhibitors include, but are not limited to, clofentezine, etoxazole and hexythiazox, amidoflumet, benclothiaz, benzoximate, bifenazate, bromopropylate, buprofezin,
  • chinomethioat chlordimeform, chlorobenzilate, chloropicrin, clothiazoben, cycloprene, cyflumetofen, dicyclanil, fenoxacrim, fentrifanil, flubenzimine, flufenerim, flutenzin, gossyplure, hydramethylnone, japonilure, metoxadiazone, petroleum, piperonyl butoxide, potassium oleate, pyrafluprole, pyridalyl, pyriprole, sulfluramid, tetradifon, tetrasul, triarathene, verbutin, furthermore the compound 3-methylphenyl propylcarbamate
  • herbicides include, but are not limited to, benzoic acid herbicides such as dicamba esters, phenoxyalkanoic acid herbicides such as 2,4-D, MCPA and 2,4-DB esters, aryloxyphenoxypropionic acid herbicides such as clodinafop, cyhalofop, fenoxaprop, fluazifop, haloxyfop and quizalofop esters, pyridinecarboxylic acid herbicides such as aminopyralid, picloram and clopyralid esters, pyrimidinecarboxylic acid herbicides such as aminocyclopyrachlor esters, pyridyloxyalkanoic acid herbicides such as fluoroxypyr and triclopyr esters, and hydroxybenzonitrile herbicides such as bromoxynil and ioxynil esters, esters of the arylpyridine carboxylic acids and arylpyrimidine carboxylic acids of the following generic structures as disclosed
  • the herbicide is selected from the group consisting of 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, amitrole, asulam, atrazine, azafenidin, benefin, bensulfuron, bensulide, bentazon, bromacil,
  • fungicides include, but are not limited to, strobilurins, azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl, metominostrobin, picoxystrobin,pyraclostrobin,
  • trifloxystrobin, orysastrobin carboxamides, carboxanilides, benalaxyl, benalaxyl-M, benodanil, carboxin, mebenil, mepronil, fenfuram, fenhexamid, flutolanil, furalaxyl, furcarbanil, furametpyr, metalaxyl, metalaxyl-M (mefenoxam), methfuroxam, metsulfovax, ofurace, oxadixyl, oxycarboxin, penthiopyrad, pyracarbolid, salicylanilide, tecloftalam, thifluzamide, tiadinil, N-biphenylamides, bixafen, boscalid, carboxylic acid morpholides, dimethomorph, flumorph, benzamides, flumetover, fluopicolid (picobenzamid), zo
  • procymidone vinclozolin, acibenzolar-S-methyl, anilazine, captan, captafol, dazomet, diclomezin, fenoxanil, folpet, fenpropidin, famoxadon, fenamidon, octhilinone, probenazole, proquinazid, pyroquilon, quinoxyfen, tricyclazole, carbamates, dithiocarbamates, ferbam, mancozeb, maneb, metiram, metam, propineb, thiram, zineb, ziram, diethofencarb, flubenthiavalicarb, iprovalicarb, propamocarb, guanidines, dodine, iminoctadine, guazatine, kasugamycin, polyoxins, streptomycin, validamycin A, organometallic compounds, fentin salts, sulfur-containing
  • organophosphorous compounds edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, pyrazophos, tolclofos-methyl, Organochlorine compounds, thiophanate-methyl,
  • chlorothalonil dichlofluanid, tolylfluanid, flusulfamide, phthalide, hexachlorobenzene, pencycuron, quintozene, nitrophenyl derivatives, binapacryl, dinocap, dinobuton,
  • the present disclosure provides for a method of modulating a trait of a plant, comprising delivering to the plant an effective amount of a modified polyethyleneimine-based formulation comprising an oligonucleotide or polynucleotide that modulates the expression of a gene in the plant.
  • Oligonucleotides or polynucleotides that modulate the expression of a gene in a plant include, but are not limited to, RNA molecules (e.g., siRNA, aiRNA, miRNA, dsRNA, and shRNA) and DNA molecules (e.g., antisense polynucleotides) that decrease expression of the gene in the plant, and RNA molecules (e.g., mRNA) and DNA molecules (e.g., expression cassettes and plasmids) that increase expression of the gene in the plant.
  • the oligonucleotide or polynucleotide modulates the expression of a gene that is endogenous to the plant.
  • the oligonucleotide or polynucleotide modulates the expression of a gene that is heterologous to the plant, e.g., a transgene that does not naturally occur within the plant.
  • the oligonucleotide or polynucleotide that modulates the expression of a gene in the plant hybridizes to a gene or gene product that is endogenous to the plant.
  • Traits that may be modulated in a plant include, but are not limited to, total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, and any combination thereof.
  • the presently disclosed modified polyethylenimine-based formulations can be used to deliver an active agent to a plant (e.g., a weed), for the purpose of killing and/or controlling the proliferation of the plant.
  • a plant e.g., a weed
  • one or more of the above-mentioned traits in a plant is increased or improved relative to a plant that is not treated with the modified
  • polyethyleneimine-based formulation The trait in the plant as described herein may be increased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% by delivery of the modified polyethyleneimine-based formulation to the plant relative to a plant that is not treated with the formulation. In other embodiments, one or more of the above mentioned traits is decreased relative to a plant that is not treated with the modified polyethyleneimine-based formulation.
  • the trait in the plant as described herein may be decreased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% by delivery of the modified polyethyleneimine-based formulation to the plant relative to a plant that is not treated with the formulation.
  • the present disclosure provides for a method of modulating a trait of an insect, comprising delivering to the insect, to a plant infested with the insect, or to a plant prior to infestation with the insect an effective amount of a modified
  • RNA molecules e.g., siRNA, aiRNA, miRNA, dsRNA, and shRNA
  • DNA molecules e.g., antisense polynucleotides
  • RNA molecules e.g., mRNA
  • DNA molecules e.g., expression cassettes and plasmids
  • the oligonucleotide or polynucleotide that modulates the expression of a gene in the insect hybridizes to a gene or gene product that is endogenous to the insect.
  • Traits that may be modulated in the insect include, but are not limited to, insect growth, development, activity,and/or lifespan.
  • delivery of the formulation to the insect kills the insect.
  • delivery of the formulation to the insect reduces its growth and/or lifespan, thereby reducing the damage done by the insect to a plant.
  • delivery of the formulation to the insect causes the insect to remain in a young or immature stage, thus preventing the insect from completing its lifecycle.
  • delivery of the formulation to the insect interferes with enzymes involved in the molting process that stimulate the synthesis and formation of chitin, which is an essential component of an insect’s exoskeleton. As a result, the insect fails to reach adulthood because it dies in an immature stage.
  • delivery of the formulation to the insect disrupts the feeding activity of the insect. As a result, insects starve to death because they are unable to obtain nutrients.
  • the delivery of the formulation to the insect decreases its growth, activity or lifespan by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% relative to an insect that is not treated with the
  • the delivery of the formulation to the insect increases its growth, activity or lifespan by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% relative to an insect that is not treated with the formulation.
  • Plant Pathogens [0200] In another aspect, the present disclosure provides a method of modulating the pathogenicity of a plant pathogen, comprising applying to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen a modified polyethyleneimine-based formulation comprising an oligonucleotide or
  • the pathogenicity of the plant pathogen is decreased, for example by decreasing the growth, activity, or lifespan of the plant pathogen, or delaying the
  • the formulation is used to kill the plant pathogen and/or control its proliferation.
  • the pathogenicity of the plant pathogen is increased, for example, by increasing the growth, activity or lifespan of the plant pathogen, or accelerating its development.
  • Increasing pathogenicity of a plant pathogen may be used, for example, to kill or reduce the growth of a plant such as a weed.
  • the growth, activity or lifespan of the plant pathogen may be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% relative to a plant pathogen that is not treated with the modified polyethyleneimine-based formulation.
  • the growth, activity or lifespan of the plant pathogen may be increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% relative to a plant pathogen that is not treated with the modified polyethyleneimine-based formulation.
  • Target Organisms [0202] In certain embodiments, the target organism is any organism in which one or more traits is modulated by the active agent. In certain embodiments, a target organism is also a host organism, as described herein below.
  • the target organism is an organism comprising one or more genes that is targeted by an oligonucleotide or polynucleotide active agent.
  • the target organism is a plant in which one or more yield-related traits is improved by the active agent.
  • the target organism is a beneficial insect whose growth, fecundity, or disease resistance is improved by the active agent.
  • the target organisms are plant pests or pathogens whose damage to the plant can be reduced or eliminated by active agents according to the invention.
  • plant pests and pathogens include, but are not limited to, insects, nematodes, fungi, bacteria, viruses, and parasitic plants such as striga, dodder, and mistletoe.
  • Insect pests that may be targeted according to the invention include, but are not limited to, chewing, sucking, and boring insects that belong, for example, to the non-limiting Orders Coleoptera, Diptera, Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera, and Orthoptera.
  • the composition may be taken up by an insect by direct contact with the composition, for example, by topical adsorption or inhalation of the composition or by direct feeding on a bait comprising the composition, as described below.
  • the compositions may also be taken up by the insect by direct feeding on a plant that has been treated with the composition. Examples of insect pests that may be targeted by the invention include, but are not limited to, those provided in Table 2.
  • nematodes examples include, but are not limited to, those provided in Table 3.
  • fungi examples include, but are not limited to, those provided in Table 4.
  • bacteria examples include, but are not limited to, those shown in Table 5.
  • plant viruses that may be targeted include, but are not limited to, those shown in the Table 6.
  • the target organism is a weed.
  • the term“weed” refers to any unwanted plant.
  • the weed to be controlled may include monocotyledonous species, such as species of the genus Agrostis, Alopecurus, Avena, Bromus, Cyperus, Digitaria, Echinochloa, Lolium, Monochoria, Rottboellia, Sagittaria, Scirpus, Setaria, Sida or Sorghum, and dicotyledonous species, for example species of the genus Abutilon, Amaranthus, Chenopodium, Chrysanthemum, Conyza, Galium, Ipomoea, Nasturtium, Sinapis, Solanum, Stellaria, Veronica, Viola or Xanthium.
  • Weeds can also include plants which may be considered crop plants but which are growing outside a crop area (escapes), or which grow from seed left over from a previous planting of a different crop (volunteers). Such volunteers or escapes may be tolerant to certain other herbicides.
  • dsRNAs targeting specific genes in specific species can be applied topically to alter plant traits as well, and in some cases, offers the farmer more flexibility with regard to timing and endurance of application.
  • the compositions of the present invention may be used to enhance a yield-related trait in a plant.
  • Yield-related traits that may be enhanced by the compositions of the present invention include, but are not limited to, total seed germination, rate of seed germination, plant biomass, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, root growth, early vigor, plant biomass, plant size, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, and leaf number.
  • Crop Plants [0222] Representative crop plants that may be target organisms include
  • monocotyledonous and dicotyledonous plants including but not limited to fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from Acer spp., Allium spp., Amaranthus spp., Ananas comosus, Apium graveolens, Arachis spp, Asparagus officinalis, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp.
  • Camellia sinensis Canna indica, Cannabis saliva, Capsicum spp., Castanea spp., Cichorium endivia, Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucumis spp., Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Ginkgo biloba, Glycine spp.
  • Lycopersicon pyriforme Malus spp., Medicago sativa, Mentha spp., Miscanthus sinensis, Morus nigra, Musa spp., Nicotiana spp., Olea spp., Oryza spp.
  • Solanum lycopersicum Solanum lycopersicum
  • Sorghum bicolor Sorghum halepense
  • Spinacia spp. Tamarindus indica, Theobroma cacao
  • Trifolium spp. Triticosecale rimpaui
  • Triticum spp. e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare
  • Vaccinium spp. Vicia spp., Vigna spp., Viola odorata, Vitis spp., and Zea mays.
  • a target gene of interest my also include a gene that is essential to the survival of an organism, such as a weed, insect, or plant pathogen, and can serve as a target for controlling growth and proliferation of the organism.
  • dsRNA-mediated silencing of an essential gene in an insect pest can induce cessation of feeding and ultimately growth inhibition, morbidity, or mortality.
  • RNAi provides a unique mode of action for the control of insect pests that could complement the current strategy of expressing Bacillus thuringiensis insecticidal proteins in plants of agricultural importance.
  • dsRNAs targeting essential insect genes can be delivered via topical sprays for RNAi-mediated insect control.
  • the compositions of the invention may be applied to an organism that is different from the target organism.
  • the target organism is an insect
  • the composition is applied to a non-target organism, such as a plant, that is a host for the insect.
  • a non-target organism is any organism other than the target organism.
  • a non-target organism can comprise a host organism and organisms that consume the host organism or otherwise contact polynucleotides (e.g., siRNAs or antisense polynucleotides) or proteins expressed in a host organism.
  • Non-target organisms include crop plants that may be infected with a target organism, such as a plant pathogen or insect. Representative crop plants include
  • monocotyledonous and dicotyledonous plants including but not limited to fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from Acer spp., Allium spp., Amaranthus spp., Ananas comosus, Apium graveolens, Arachis spp, Asparagus officinalis, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp.
  • Camellia sinensis Canna indica, Cannabis saliva, Capsicum spp., Castanea spp., Cichorium endivia, Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucumis spp., Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Ginkgo biloba, Glycine spp.
  • Lycopersicon esculenturn e.g., Lycopersicon esculenturn, Lycopersicon lycopersicum, Lycopersicon pyriforme
  • Malus spp. Medicago sativa, Mentha spp., Miscanthus sinensis, Morus nigra, Musa spp., Nicotiana spp., Olea spp., Oryza spp.
  • Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare
  • Vaccinium spp. Vicia spp., Vigna spp., Viola odorata, Vitis spp., and Zea mays.
  • rice, oilseed rape, canola, soybean, corn (maize), cotton, sugarcane, alfalfa, sorghum, and wheat e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgar
  • the presently disclosed formulations can be applied as a spray or powder to the plant, plant part, seed, a pest, or an area of cultivation.
  • the presently disclosed formulations may also be applied as concentrated emulsions, dusts, emulsifiable concentrates, fumigants, gels, granules, seed treatments, suspension concentrates, suspoemulsions, tablets, water soluble liquids, water dispersible granules or dry flowables, wettable powders, and ultra-low volume solutions.
  • formulations For further information on formulation types see“Catalogue of Pesticide Formulation Types and International Coding System” Technical Monograph No.2, 5th Edition by CropLife International (2002), which is incorporated herein by reference in its entirety. Agricultural formulations are also described, for example, in U.S. Pat. No. 8,815,27, which is incorporated herein by reference in its entirety. . [0229] For example, the presently disclosed formulations may be applied as aqueous suspensions or emulsions prepared from concentrated formulations.
  • Such water-soluble, water-suspendable, or emulsifiable formulations can either be solids, usually known as wettable powders, or water dispersible granules, or liquids usually known as emulsifiable concentrates, or aqueous suspensions.
  • Wettable powders which may be compacted to form water dispersible granules, comprise an intimate mixture of the composition, a carrier, and surfactants.
  • the carrier may be selected from attapulgite clays, montmorillonite clays, diatomaceous earths, and purified silicates.
  • Effective surfactants comprising from about 0.5% to about 10% of the wettable powder, include sulfonated lignins, condensed
  • Emulsifiable concentrates can comprise a suitable concentration of the formulation, such as from about 50 to about 500 grams per liter of liquid dissolved in a carrier that is either a water-miscible solvent or a mixture of water-immiscible organic solvent and emulsifiers.
  • Suitable organic solvents include aromatics, especially xylenes and petroleum fractions, especially the high-boiling naphthalenic and olefinic portions of petroleum, such as heavy aromatic naphtha.
  • Other organic solvents may also be used, such as the terpenic solvents, including rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols such as 2-ethoxyethanol.
  • Suitable emulsifiers for emulsifiable concentrates can be selected from conventional anionic and non-ionic surfactants.
  • Aqueous suspensions comprise suspensions of water-insoluble forms of the presently disclosed formulations dispersed in an aqueous carrier at a concentration in the range from about 5% to about 50% by weight. Ingredients, such as inorganic salts and synthetic or natural gums, may also be added to increase the density and viscosity of the aqueous carrier.
  • the presently disclosed formulations may also be applied as granular formulations, for example, for applications to the soil. Granular formulations may contain from about 0.5% to about 10% by weight of the composition, dispersed in a carrier that comprises clay or a similar substance.
  • Such formulations may be prepared by dissolving the formulation in a suitable solvent and applying it to a granular carrier which has been pre- formed to a suitable particle size, for example, in the range of from about 0.5 to about 3 mm. Such formulations may also be prepared by making a dough or paste of the carrier and compound and crushing and drying to obtain the desired granular particle size.
  • Dusts comprising the presently disclosed formulations may be prepared by intimately mixing the formulation in powdered form with a suitable dusty agricultural carrier, such as kaolin clay, ground volcanic rock, and the like. Dusts may contain from about 1% to about 10% by weight of the composition. They may be applied as a seed dressing or as a foliage application with a dust blower machine.
  • the presently disclosed formulations may also be applied in the form of a solution in an appropriate organic solvent (e.g., petroleum oil), such as the spray oils, which are widely used in agricultural chemistry.
  • an appropriate organic solvent e.g., petroleum oil
  • the presently disclosed formulations may also be applied in the form of an aerosol composition.
  • the formulation can be dissolved or dispersed in a carrier, which is a pressure-generating propellant mixture.
  • the aerosol composition is packaged in a container from which the mixture is dispensed through an atomizing valve.
  • the presently disclosed formulations may be applied to the crop area or plant to be treated, simultaneously or in succession, with further compounds. These further compounds can be, for example, fertilizers or micronutrient donors or other preparations, which influence the growth of plants.
  • the presently disclosed aminolipidoid formulations can be used to deliver a therapeutic agent to a target in a subject for the purpose of treating or preventing a disease or disorder.
  • the presently disclosed aminolipidoid formulations can be used to deliver a therapeutic agent to a target in a subject for the purpose of treating or preventing a disease or disorder.
  • aminolipidoid formulations are combined with a pharmaceutically acceptable excipient and/or carrier to form a pharmaceutical formulation.
  • the disease or disorder is treated or prevented by administering a therapeutically effective amount of the pharmaceutical formulation to a subject in need thereof.
  • the subject is a mammal.
  • the subject is a human.
  • the presently disclosed aminolipidoid formulations i.e., complexes, microparticles, nanoparticles, picoparticles, liposomes, and micelles, are combined with one or more pharmaceutically acceptable excipients and/or carriers to form pharmaceutical formulations suitable to administer to mammals, including humans.
  • excipients and carriers examples include, but are not limited to, fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters.
  • Excipients and/or carriers may comprise about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or higher percentage of the presently disclosed pharmaceutical formulations.
  • compositions can be administered to mammals, including humans, by any conventional route.
  • routes include, but are not limited to, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically, bucally, or as an oral or nasal spray.
  • Dosage forms for oral administration include, but are not limited to, solid and liquid dosage forms.
  • Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules.
  • Liquid dosage forms for oral administration include, but are not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • parenteral administration and “administered parenterally” means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.
  • the dosage will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage of from 0.01 to 2000 mg of the therapeutic agent is recommended for an adult human patient, and this may be administered in a single dose or in divided doses.
  • the term "therapeutic agent” includes any synthetic or naturally occurring biologically active compound or composition which, when administered to subject, induces a desired pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action.
  • the term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, and gene constructs. More particularly, the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas.
  • therapeutic agents include, but are not limited to, nucleic acids, adjuvants, anti-infectives such as antibiotics and antiviral agents, analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics,
  • nucleic acids such as antibiotics and antiviral agents, analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and chol
  • antiarrhythmics antihypertensive agents, hormones, and nutrients, antiarthritics,
  • antiasthmatic agents anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators, central nervous system stimulants, cough and cold preparations, decongestants, diagnostics, hormones, bone growth stimulants and bone resorption inhibitors, oncology drugs (e.g., chemotherapy drugs, hormonal therapeutic agents, immunotherapeutic agents, radiotherapeutic agents), lipid-lowering agents, antidepressants, stimulants, antibiotics, birth control medication, anti-angiogenics, cytovascular agents, signal
  • transduction inhibitors hormones, vasoconstrictors, and steroids.
  • immunosuppressives muscle relaxants, psychostimulants, sedatives, tranquilizers, proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced), small molecules and other biologically active macromolecules such as, for example, proteins and enzymes.
  • the agent may be a biologically active agent used in medical, including veterinary, applications.
  • therapeutic agent also includes without limitation, medicaments, vitamins; mineral supplements, substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, substances which affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • nucleic acids include, but are not limited to, interfering RNA molecules (e.g., siRNA, aiRNA, miRNA), antisense oligonucleotides, plasmids, ribozymes, immunostimulatory oligonucleotides, and mixtures thereof.
  • Nucleic acids delivered as the therapeutic agent in the presently disclosed pharmaceutical formulations can be used to downregulate or silence the translation (i.e., expression) of a target gene of interest in the target organism.
  • genes associated with viral infection and survival include, but are not limited to, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.
  • metabolic diseases and disorders e.g., liver diseases and disorders
  • tumorigenesis and cell transformation e.g., cancer
  • angiogenic genes include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.
  • viral sequences that can be downregulated or silenced include filoviruses such as Ebola virus and Marburg virus, arenaviruses such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus, influenza viruses such as Influenza A, B, and C viruses, hepatitis viruses, Human Immunodeficiency Virus (HIV), herpes viruses, and Human Papilloma Viruses (HPV).
  • filoviruses such as Ebola virus and Marburg virus
  • arenaviruses such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus
  • influenza viruses such as Influenza A, B, and C viruses
  • hepatitis viruses such as Human Immunodeficiency Virus (HIV), herpes viruses, and Human Papilloma Viruses (HPV).
  • Examples of peptides or polypeptide that may be used as therapeutic agents include, but are not limited to, an antibodies such as a polyclonal antibodies, a monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, PrimatizedTM antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell-surface receptors, ligands, hormones, or small molecules.
  • an antibodies such as a polyclonal antibodies, a monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, PrimatizedTM antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell-surface receptors, ligands, hormones, or small molecules.
  • oncology drugs that may be used as therapeutic agents include, but are not limited to, alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosine arabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin, interferon, letrozole, Leustatin, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone
  • anti-viral drugs examples include, but are not limited to, abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III (e.g., IFN- ⁇ molecules such as IFN- ⁇ 1,
  • the therapeutic agent to be delivered may be a prophylactic agent.
  • Prophylactic agents include, but are not limited to, antibiotics, nutritional supplements, and vaccines.
  • Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts.
  • Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, and Freund's adjuvant.
  • prophylactic agents include, but are not limited to, (1) antigens of the following bacterial organisms: Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,
  • Example 17 General Procedure for Nanoparticle Formulation of Active Agents with Aminolipidoids
  • Stock ethanolic solutions of one or more aminolipidoids of the present disclosure, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG) are prepared and combined to yield a molar ratio of 50:10:38.5:1.5 with the organic solution of 10% 10 mM citrate buffer at pH 5.
  • An siRNA is dissolved in 10 mM citrate at pH5 buffer at a concentration of 0.1 mg/mL.
  • Example 18– General Procedure for Evaluating Lipidoid Formulations is Insect Feeding Assays [0256] Lipidoid formulations of the present disclosure can be evaluated in insect feeding assays to determine their efficacy in RNA delivery to an insect cell.
  • WTPB western tarnished plant bug
  • TPB tarnished plant bug
  • TPB tarnished plant bug
  • Each aminolipidoid of the present disclosure to be evaluated is separately formulated with an siRNA that targets an essential gene in TPB and an siRNA that targets an essential gene in WTPB according to the general procedure described above in Example 4.
  • the feeding assay employed is based on a 96 well format and a sachet system as described by Habibi et al. (2002, Archives of Insect Biochem. and Phys. 50: 62-74) and U.S. Patent No. 8,609,936, each of which is incorporated herein by reference in their entireties.
  • the insect artificial diet is commercially available from Bio-Serv TM (Bio-Serv TM Diet F9644B, Frenchtown, N.J.). [0257] Autoclaved boiling water is combined with Bio-Serv ® Diet F9644B in a surface sterilized blender. Four surface sterilized chicken eggs are broken and the contents are added to the blender containing the diet mix. The mixture is blended until smooth and adjusted to one liter of volume and allowed to cool. Feeding samples are prepared by mixing the siRNA formulations described above in the desired concentration with an equivalent volume of the blended diet.
  • a sheet of Parafilm ® (Pechiney Plastic Packing, Chicago, Ill.) is placed over a 96-well format vacuum manifold with a vacuum of approximately -20 millimeters mercury, which is sufficient to cause extrusion of the Parafilm ® into the wells. Forty microliters of test sample are added to the Parafilm ® wells.
  • a sheet of Mylar film (Clear Lam Packaging, Inc., Elk Grove Village, Ill.) is then placed over the Parafilm ® and sealed gently with a tacking iron (Bienfang Sealector II, Hunt Corporation, Philadelphia, Pa.).
  • the Parafilm ® sachets are then placed over a flat-bottom 96-well plate containing the Lygus eggs suspended in agarose.

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Abstract

The present disclosure is directed to novel oxidized aminolipidoids, formulations thereof further comprising at least one active agent, as well as methods of delivering the at least one active agent to a target organism.

Description

OXIDIZED AMINOLIPIDOIDS AND USES THEREOF RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No.
62/342,443, filed May 27, 2016, which is hereby incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] The present disclosure relates generally to novel aminolipidoids containing oxygen-based functionalities, such as hydroxyl and ether groups (hereinafter referred to as “aminolipidoids”). The present disclosure also relates generally to methods of preparing such aminolipidoids, their formulation with one or more active agents, and the delivery of such formulations to target organisms. BACKGROUND OF THE INVENTION [0003] A wide range of molecules have been employed for delivering polynucleotides and other active agents to cells. For example, polymers such as polyethylenimine or poly(beta-aminoesters) have been used to effectively complex DNA for delivery into cells. Polymers in these classes of delivery agent typically contain amine functionalities that serve to electrostatically bind to DNA to form nanoparticles that are then taken up by the cell via endocytosis. Once in the cell, it is hypothesized that these amine groups serve to buffer the endosome and cause an influx of ions due to the proton-sponge mechanism. The resulting burst of the endocytic vesicle leads to the release of the payload of the particle, which is then free to travel to the nucleus where the DNA is expressed. [0004] While such polymer based systems have been used extensively for DNA delivery, the delivery of other molecules, such as RNA, presents distinct challenges. In many cases, polymeric materials do not work as effectively for RNA delivery. This is likely due to differences in the chemical structure of the RNA being delivered compared to DNA. RNA are generally short, linear fragments containing additional hydroxyl moieties on each ribose ring. These differences necessitate an alternative approach that is suited for complexation with short RNA strands. In particular, an improved delivery system is required for the use of RNA for agricultural and pharmaceutical applications. The delivery system needs to protect RNA from nuclease degradation, allow for the proper concentration and distribution profile in the target tissues, facilitate efficient uptake of RNA into target cells, and release RNA into cytoplasm to knockout expression of the target gene. [0005] Promising results have been achieved with materials that form liposomes or lipoplexes that entrap the RNA or form nanoparticles, which can then be internalized by a cell. The materials utilized to form a lipid-based delivery system generally consist of a positively charged headgroup and a hydrophobic tail. The charged portion serves to electrostatically bind the negatively charged RNA, while the hydrophobic tail leads to self- assembly into lipophilic particles. Such cationic lipids are promising but still fall short of the transfection efficiency achieved by viral vectors. Few advances have been made in the field, in part due to the limited structural diversity of these lipid-like molecules, which is a result of the difficult synthetic procedures oftentimes required to access these structures. Furthermore, many lipid-based nucleotide formulations are toxic and/or require the action of endosome formation to deliver their payloads. [0006] Thus, there exists a continuing need for novel amine-based lipidoid molecules that possess an improved toxicity profile and ability to deliver RNA and DNA, as well as other active agents, to cells over existing amine-based lipidoid materials. EMBODIMENTS OF THE INVENTION [0007] One embodiment of the present invention is a compound of formula (I):
Figure imgf000003_0001
wherein
X is an oxygen-containing core derived from an oxidized amine comprising (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups;
R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000004_0001
wherein
R5 and R6
are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic
group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
with the proviso that R5 and R6 cannot both be hydrogen;
R' is identically or different in each instance hydrogen, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or
heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000004_0002
wherein
R5 and R6
are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic
group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
with the proviso that R5 and R6 cannot both be hydrogen;
or a covalent bond to X; and
n is an integer in the range of from 2 to 10;
with the proviso that X is not derived from 1,10-diaza-18-crown-6, and
with the proviso that the compound of formula (I) is not one of the following compounds:
Figure imgf000005_0001
[0008] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least two hydroxyl groups. [0009] Another embodiment of the present invention is the above compound, wherein the oxidized amine comprises three hydroxyl groups. [0010] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least two ether groups. [0011] Another embodiment of the present invention is the above compound, wherein the oxidized amine comprises three, four, or five ether groups. [0012] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least one hydroxyl group and at least one ether group. [0013] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least two primary amino groups. [0014] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine comprising at least one primary amino group and at least one secondary amino group. [0015] Another embodiment of the present invention is the above compound, wherein X is derived from an oxidized amine selected from the group consisting of compounds of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), and (22):
Figure imgf000006_0001
Figure imgf000007_0001
[0016] Another embodiment of the present invention is the above compound, wherein R is an optionally substituted aliphatic or heteroaliphatic group. [0017] Another embodiment of the present invention is the above compound, wherein R' is an optionally substituted aliphatic or heteroaliphatic group. [0018] Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond. [0019] Another embodiment of the present invention is the above compound, wherein the optionally substituted heteroaliphatic group comprises at least one ether group. [0020] Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic group is a C6 to C20 aliphatic group.
[0021] Another embodiment of the present invention is the above compound, wherein R and R' are independently selected from the group consisting of moieties of formulae 23-41:
Figure imgf000008_0001
Figure imgf000009_0001
. [0022] Another embodiment of the present invention is the above compound, wherein R is a group of formula (II), wherein R5 is H and R6 is an optionally substituted aliphatic or heteroaliphatic group. [0023] Another embodiment of the present invention is the above compound, wherein R' is a group of formula (II), wherein R5 is H and R6 is an optionally substituted aliphatic or heteroaliphatic group. [0024] Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond. [0025] Another embodiment of the present invention is the above compound, wherein the optionally substituted heteroaliphatic group comprises at least one ether group. [0026] Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one fluorine atom. [0027] Another embodiment of the present invention is the above compound, wherein the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one trialkoxysilane group. [0028] Another embodiment of the present invention is the above compound, wherein R and R' are independently selected from the group consisting of moieties of formulae 42-60:
Figure imgf000009_0002
Figure imgf000010_0001
Figure imgf000011_0001
. [0029] Another embodiment of the present invention is the above compound, wherein the compound is selected from the group consisting of compounds of formulae 61 through 186:
Figure imgf000011_0002
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
. [0030] Yet another embodiment of the present invention is a formulation comprising a compound of formula (I):
Figure imgf000019_0002
wherein
X is an oxygen-containing core derived from an oxidized amine comprising (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups; R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000020_0001
wherein
R5 and R6
are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
with the proviso that R5 and R6 cannot both be hydrogen;
R' is identically or different in each instance hydrogen, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000020_0002
wherein
R5 and R6 are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
with the proviso that R5 and R6 cannot both be hydrogen;
or a covalent bond to X; and
n is an integer in the range of from 2 to 10;
and a first active agent to be delivered, and
with the proviso that the compound of formula (I) is not one of the following compounds:
Figure imgf000021_0001
[0031] Another embodiment of the present invention is the above formulation, further comprising at least one additional active agent to be delivered. [0032] Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent to be delivered is contained within or on the surface of the non-covalent complex. [0033] Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent to be delivered is not contained within or on the surface of the non-covalent complex. [0034] Another embodiment of the present invention is the above formulation, further comprising one or more excipients. [0035] Another embodiment of the present invention is the above formulation, wherein the one or more excipients is selected from the group consisting of fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, coating
permeability adjusters, and combinations thereof. [0036] Another embodiment of the present invention is the above formulation, wherein the one or more excipients is selected from the group consisting of carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof. [0037] Another embodiment of the present invention is the above formulation, wherein the one or more water soluble polymers comprises a polyethylene glycol, a polypropylene oxide, a polyvinylpyrrolidone, a polyvinyl alcohol, polylactic acid, poly(lactic-co-glycolic acid), or any combination thereof. [0038] Another embodiment of the present invention is the above formulation, further comprising an adjuvant selected from the group consisting of chloroquine, chlorpromazine, amodiaquine, perphenazine, coronatine, tolbutamide, glyburide, glybenclamide, arginine, lysine, and histidine. [0039] Another embodiment of the present invention is the above formulation, wherein the first active agent to be delivered is an oligonucleotide or a polynucleotide. [0040] Another embodiment of the present invention is the above formulation, further comprising an agriculturally acceptable carrier. [0041] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in a plant. [0042] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in an insect. [0043] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide modulates the expression of a gene in a plant pathogen. [0044] Another embodiment of the present invention is the above formulation, wherein the at least one additional active agent is selected from the group consisting of an herbicide, an insecticide, a fungicide, a nematicide, a bactericide, a viricide, and any combination thereof. [0045] Another embodiment of the present invention is the above formulation, wherein the formulation is in the form of a microparticle or nanoparticle. [0046] Another embodiment of the present invention is the above formulation, wherein the active agent to be delivered is selected from the group consisting of
polynucleotides, oligonucleotides, proteins, peptides, and small molecules. [0047] Another embodiment of the present invention is the above formulation, wherein the active agent to be delivered is an oligonucleotide or a polynucleotide. [0048] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide is modified. [0049] Another embodiment of the present invention is the above formulation, wherein the oligonucleotide or polynucleotide is unmodified. [0050] Another embodiment of the present invention is the above formulation, wherein the active agent to be delivered is an RNA. [0051] Another embodiment of the present invention is the above formulation, wherein the RNA is a single-stranded RNA. [0052] Another embodiment of the present invention is the above formulation, wherein the RNA is a double-stranded RNA. [0053] Another embodiment of the present invention is the above formulation, wherein the RNA is an siRNA. [0054] Another embodiment of the present invention is the above formulation, wherein the RNA is an mRNA. [0055] Yet another embodiment of the present invention is a method of regulating expression of a gene in a target organism, comprising applying the above formulation to the target organism. [0056] Yet another embodiment of the present invention is a method of modulating a trait of a plant, comprising delivering to the plant an effective amount of the above formulation that modulates the expression of a gene in a plant. [0057] Another embodiment of the present invention is the above method, wherein the trait is selected from the group consisting of total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, plant sensitivity to herbicide, and any combination thereof. [0058] Another embodiment of the present invention is the above method, wherein one or more of the traits is improved relative to a plant not treated with the formulation. [0059] Another embodiment of the present invention is the above method, wherein at least one trait selected from the group consisting of plant growth, plant lifespan, plant size, fruit size, fruit yield, total yield, fruit freshness, fruit ripening time, plant nutritional content, and fruit nutritional content, is improved relative to a plant not treated with the formulation. [0060] Another embodiment of the present invention is the above method, wherein one or more of the traits is decreased relative to a plant not treated with the formulation. [0061] Another embodiment of the present invention is the above method, wherein the plant growth and/or the plant lifespan is decreased relative to a plant not treated with the formulation. [0062] Another embodiment of the present invention is the above method, wherein the fruit ripening time is decreased relative to a plant not treated with the formulation. [0063] Another embodiment of the present invention is the above method, wherein the plant sensitivity to herbicide is increased relative to a plant not treated with the formulation. [0064] Yet another embodiment of the present invention is a method of modulating a trait of an insect, comprising delivering an effective amount of the above formulation that modulates the expression of a gene in an insect to the insect, to a plant infested with the insect, or to a plant prior to infestation with the insect. [0065] Another embodiment of the present invention is the above method, wherein the trait modulated is insect growth, development,and/or lifespan. [0066] Yet another embodiment of the present invention is a method of modulating the pathogenicity of a plant pathogen, comprising applying the above formulation that modulates the expression of a gene in a plant pathogen to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen. [0067] Yet another embodiment of the present invention is a plant cell, insect cell, fungal cell, nematodic cell, or bacterial cell comprising the above formulation. [0068] Another embodiment of the present invention is the above formulation, wherein X is derived from 1,10-diaza-18-crown-6. [0069] Another embodiment of the present invention is the above formulation, wherein R and R' are each, independently, an optionally substituted aliphatic or
heteroaliphatic or group. [0070] Another embodiment of the present invention is the above formulation, wherein the compound of formula (I) is a compound of formula (III):
Figure imgf000026_0001
wherein R1 and R2 are independently selected from the group consisting of hydrogen or a moiety of formula 26 through 29, 32, 34, 35, 37, 40, 41, 46, 47, 50 through 52, 60, and 187 through 191:
Figure imgf000026_0002
Figure imgf000027_0001
.
DETAILED DESCRIPTION OF THE INVENTION [0071] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. [0072] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of“or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and“included”, is not limiting. Any ranges described herein will be understood to include the endpoints and all values between the endpoints. [0073] The novel aminolipidoids of the present disclosure provide for several different advantages in the delivery of active agents to target organisms. For example, the aminolipidoids may be used to complex oligonucleotides and polynucleotides, thereby enhancing their delivery to the target organism and preventing their degradation. These aminolipidoids may also be used generate formulations, such as microparticles, nanoparticles, picoparticles, liposomes, and micelles, containing the active agent to be delivered. These aminolipidoids, as well as the formulations thereof, may be biocompatible and biodegradable and may be used to provide controlled, sustained release of the active agent to be delivered. These aminolipidoids and their corresponding formulations may also be responsive to pH changes given that these aminolipidoids are protonated at lower pH. These aminolipidoids may also act as proton sponges in the delivery of an active agent to a cell to cause endosome lysis. [0074] In particular, the aminolipidoids of the present disclosure possess oxygen enriched cores (e.g., a higher density of hydroxyl and ether groups) compared to conventional aminolipidoids. The presence of the oxygen-based functional groups in the presently disclosed aminolipidoids results in a very polar core, which, when coupled with their highly lipophilic tails, results in agents well suited for the delivery of polynucleotides. For example, the presently disclosed aminolipidoids form well-defined nanoparticles with polynucleotides and conserve the desired pKa during their delivery to the target organism. Furthermore, the oxygen-rich cores of the presently disclosed aminolipidoids mimic natural products (e.g., antibiotics) and, thus, exhibit enhanced uptake and cell recognition for delivery of
polynucleotides, as well as superior biocompatibility. [0075] In one aspect, the present disclosure provides for novel aminolipidoids of formula (I):
Figure imgf000028_0001
In the presently disclosed aminolipidoids of formula (I), X is an oxygen-containing core. As used herein, the term“oxygen-containing core” is defined as core derived from an oxidized amine. As used herein, the term“oxidized amine” is defined as an amine that comprises (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups. Furthermore, as used herein, the term“oxidized amine” does not encompass aminoglycosides. Moreover, the compounds of formula (I) do not encompass the following compounds:
Figure imgf000029_0001
[0076] In certain embodiments of the presently disclosed aminolipidoids of formula (I), X is derived from an oxidized amine comprising at least two hydroxyl groups. In certain embodiments, the oxidized amine comprises three hydroxyl groups. In certain embodiments, X is derived from an oxidized amine comprising at least two ether groups. In certain embodiments, the oxidized amine comprises three, four, or five ether groups. In certain embodiments, X is derived from an oxidized amine comprising at least one hydroxyl group and at least one ether group. In certain embodiments, X is derived from an oxidized amine comprising at least two primary amino groups. In certain embodiments, X is derived from an oxidized amine comprising at least one primary amino group and at least one secondary amino group. In certain embodiments, X is derived from an oxidized amine having any combination of the foregoing embodiments. In certain embodiments, X is derived from an oxidized amine selected from the group consisting of compounds of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), and (22):
Figure imgf000030_0001
Figure imgf000031_0001
[0077] In certain embodiments of the presently disclosed aminolipidoids of formula (I), X is not derived from 1,10-diaza-18-crown-6. [0078] In certain embodiments of the presently disclosed aminolipidoids of formula (I), R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000031_0002
wherein R5 and R6 are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group. In certain embodiments, the aliphatic or cycloaliphatic group can optionally further comprise an ester or amide linkage in the aliphatic or cycloaliphatic chain. R5 and R6 cannot both be hydrogen. [0079] In certain embodiments of the presently disclosed aminolipidoids of formula (I), R', is identically or different in each instance, hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or
heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000032_0001
wherein R5 and R6 are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group. In certain embodiments, the aliphatic or cycloaliphatic group can optionally further comprise an ester or amide linkage in the aliphatic or cycloaliphatic chain. R5 and R6 cannot both be hydrogen. [0080] The aliphatic groups of the presently disclosed aminolipidoids refers to both saturated and unsaturated aliphatic hydrocarbyl groups, which can be straight chain (i.e., unbranched), branched, or cyclic (including polycyclic) and are optionally substituted with one or more functional groups. Examples of aliphatic groups include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl groups, each of which are optionally substituted with one or more functional groups. As used herein, the term“alkyl” refers to saturated hydrocarbyl groups, which can be unbranched, branched, or cyclic (i.e., alicyclic) alkyl groups. As used herein, the terms“alkenyl” and“alkylene” refers to unsaturated hydrocarbyl groups having at least one carbon-carbon double bond. As used herein, the term“alkynyl” refers to unsaturated hydrocarbyl groups having at least one carbon-carbon triple bond. [0081] Examples of such aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH2-cyclopropyl, n-butyl, sec-butyl, isobutyl, tert- butyl, cyclobutyl, -CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, neopentyl, cyclopentyl, -CH2-cyclopentyl, hexyl, cyclohexyl, -CH2-cyclohexyl, heptyl, cycloheptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tridecynyl,
tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, nonadecynyl, icosynyl, and all isomers thereof. [0082] The heteroaliphatic groups of the presently disclosed aminolipidoids refers to aliphatic groups, as described above, that independently in one or more instances contain an oxygen, sulfur, nitrogen, phosphorus, or silicon atom between two carbon atoms of the aliphatic group. Such heteroaliphatic groups include saturated and unsaturated heterocycles. As used herein, the term“heterocycles,” refers to a non-aromatic partially unsaturated or fully saturated 3- to 10-membered ring system, which includes single rings of 3 to 8 atoms in size and bi- and tri-cyclic ring systems, which may include aromatic six-membered aryl or aromatic heterocyclic groups fused to a non-aromatic heterocyclic ring. Such heterocyclic rings include those having from one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, in which the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Examples of such heterocycles include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, 2,5-dihydro-1H-imidazole, and tetrahydrofuryl. [0083] In certain embodiments, the aliphatic and/or heteroaliphatic groups of the aminolipidoids of the present disclosure independently contain from 1 to 20, or from 1 to 12, or from 1 to 10, or from 1 to 8, or from 1 to 5, or from 1 to 4, or from 4 to 5, or from 4 to 8, or from 4 to 10, or from 4 to 12, or from 4 to 20, or from 5 to 20, or from 5 to 12, or from 5 to 10, or from 5 to 8, or from 8 to 10, or from 8 to 12, or from 8 to 20, or from 10 to 12, or from 10 to 20, or from 12 to 20 carbon atoms. [0084] The aryl and heteroaryl groups of the presently disclosed aminolipidoids refer to mono- or polycyclic aromatic carbocyclic groups and mono- or polycyclic aromatic heterocyclic groups. In certain embodiments, these groups have 3-14 carbon atoms, each of which is optionally substituted. In certain embodiments, the aryl group is a mono- or bicyclic carbocyclic ring system having one or two aromatic rings. The term“carbocyclic” as used herein, refers to an aromatic or non-aromatic ring system in which each atom of the ring is a carbon atom. Examples of such aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and indenyl. In certain embodiments, the heteroaryl group is a mono- or bicyclic heterocyclic ring system having one or two aromatic rings. In certain embodiments, the heteroaryl group (1) has from five to ten ring atoms of which one ring atom is selected from S, O, and N, (2) has zero, one, or two ring atoms that are additional heteroatoms independently selected from S, O, and N, and (3) the remaining ring atoms are carbon. Examples of such heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, and isoquinolinyl. [0085] It will be appreciated that the aminolipidoids of the presented disclosure may be substituted with any number of substituents. In general, the term“substituted,” whether preceded by the term“optionally” or not, and substituents contained in formulas of the present disclosure, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents of organic compounds. Broadly, permissible substituents include all acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this disclosure, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, the presently disclosed aminolipidoids are not intended to be limited in any manner by the permissible substituents of organic compounds. Any substituents disclosed herein may also be further substituted (e.g., an aryl substituent may itself be substituted, such as with another aryl group, which, in turn, is further substituted with fluorine at one or more positions). [0086] Examples of optional substituents of the aminolipidoids of the present disclosure include, but are not limited to, aliphatic groups, heteroaliphatic groups, aryl groups, heteroaryl groups, arylalkyl groups, heteroarylalkyl groups, alkoxy groups, aryloxy groups, heteroalkoxy groups, heteroaryloxy groups, alkylthio groups, arylthio groups, heteroalkylthio groups, heteroarylthio groups, F, Cl, Br, I, -OH, -NO2, -CN, -CF3, -CH2CF3, - CHCl2, -CH2OH, -CH2CH2OH, -CH2NH2, -CH2SO2R, -C(O)R, -CO2(R), -CON(R)2, -OC(O)R, -OCO2R, -OCON(R)2, -N(R)2, -S(O)2R, and -NR(CO)R, wherein each R is, independently, hydrogen, an aliphatic group, a heteroaliphatic group, an aryl group, a heteroaryl group, an arylalkyl group, or a heteroarylalkyl group, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be optionally substituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be optionally substituted. [0087] In certain embodiments of the presently disclosed aminolipidoids of formula (I), R is an optionally substituted aliphatic or heteroaliphatic group. In certain embodiments, R' is an optionally substituted aliphatic or heteroaliphatic group. In certain embodiments, R is a group of formula (II), R5 is H, and R6 is an optionally substituted aliphatic or
heteroaliphatic group. In certain embodiments, R' is a group of formula (II), R5 is H, and R6 is an optionally substituted aliphatic or heteroaliphatic group. In certain embodiments, the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond. In certain embodiments the optionally substituted heteroaliphatic group comprises at least one ether group. In certain embodiments, the optionally substituted aliphatic group is a C6 to C20 aliphatic group. In certain embodiments, the optionally substituted aliphatic or heteroaliphatic group comprises at least one double bond. In certain embodiments, the optionally substituted heteroaliphatic group comprises at least one ether group. In certain embodiments, the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one fluorine atom. In certain embodiments, the optionally substituted aliphatic or heteroaliphatic group is substituted with at least one trialkoxysilane group. [0088] In certain embodiments of the presently disclosed aminolipidoids of formula (I), R and R' are independently selected from the group consisting of moieties of formulae 23- 60:
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
. [0089] In certain embodiments of the presently disclosed aminolipidoids of formula (I), n is an integer in the range of from 1 to 10. [0090] In certain embodiments of the presently disclosed aminolipidoids of formula (I), the compound of formula (I) is selected from the group consisting of compounds of formulae 61 through 186:
Figure imgf000038_0002
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
. [0091] In certain embodiments, the presently disclosed aminolipidoids can be a salt, either from protonation by a mineral or organic acid or by quaternization of one more available tertiary nitrogens. Examples of counterions for such salts include, but are not limited to, halides, such as fluoride, chloride, bromide, or iodide, nitrate, hydrogen sulfate, dihydrogen phosphate, bicarbonate, nitrite, perchlorate, iodate, chlorate, bromate, chlorite, hypochlorite, hypobromite, cyanide, amide, cyanate, hydroxide, permanganate, an acid anion such as acetate or formate, or anions with negative charges greater than -1 (e.g., having in some embodiments one or more than one adsorbent functional group as counterion), such as oxide, sulfide, nitride, arsenate, phosphate, arsenite, hydrogen phosphate, sulfate, thio sulfate, sulfite, carbonate, chromate, dichromate, peroxide, or oxalate. [0092] In certain embodiments, the presently disclosed aminolipidoids, and optional substitutents thereon, can contain isotopes of various common atoms. Examples of such isotopes include, but are not limited to, deuterium, C13, N15, O18, and F18. In certain embodiments, R, R1, and R2 can, independently, be perfluorinated C1-20 alkyl groups.certain embodiments, the presently disclosed aminolipidoids are relatively non-cytotoxic. [0093] In certain embodiments, the presently disclosed aminolipidoids are
biocompatible and biodegradable. In certain embodiments, the presently disclosed aminolipidoids have a pKa in the range of from about 3.0 to about 9.0, or in the range of from about 5.0 to about 8.0, or in the range of from about 5.5 to about 7.5, or in the range of from about 6.0 to about 7.0. [0094] The presently disclosed aminolipidoids may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such forms, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this disclosure. [0095] Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present disclosure. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present disclosure. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures. [0096] If a particular enantiomer of the presently disclosed aminolipidoids is desired, it may be prepared by asymmetric synthesis, for example, by reacting an optionally substituted compound comprising (1) at least one primary amino group, (2) at least one primary amino group and at least one secondary amino group, or (3) at least two secondary amino groups with a chiral epoxide, acrylate, acrylamide, or aldehyde. It may also be prepared by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts can be formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. [0097] In certain embodiments, where the presently disclosed aminolipidoids contain at least one group of formula (II), the aminolipidoid can contain at least one stereocenter. Therefore, in certain embodiments of the presently disclosed lipidoids, each group of formula (II):
Figure imgf000046_0001
is, independently, a group of formulae (IIa) or (IIb):
Figure imgf000047_0001
[0098] The“enantiomeric excess” of a substance is a measure of how pure a desired enantiomer is relative to the undesired enantiomer. Enantiomeric excess is defined as the absolute difference between the mole fraction of each enantiomer which is most often expressed as a percent enantiomeric excess. For mixtures of diastereomers, there are analogous definitions and uses for“diastereomeric excess” and percent diastereomeric excess. For example, a sample with 70% of R isomer and 30% of S will have an
enantiomeric excess of 40%. This can also be thought of as a mixture of 40% pure R with 60% of a racemic mixture (which contributes 30% R and 30% S to the overall composition). [0099] The aminolipidoids of the present disclosure can have an enantiomeric excess or a diastereomeric excess up to and including 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%. [0100] The presently disclosed amino lipidoids may be prepared by any method known in the art. In certain embodiments, the aminolipidoids of the present disclosure are synthesized by reacting the oxidized amine with one or more terminal epoxides, interior epoxides, acrylates, acrylamides, aldehydes, or any combination thereof. In certain embodiments, these starting materials are commercially available and/or are easily and/or inexpensively prepared. [0101] In certain embodiments, the presently disclosed aminolipidoids are synthesized by reacting the oxidized amine with a stoichiometric amount or stoichiometric excess of a terminal epoxide, interior epoxide, acrylate, acrylamide, aldehyde, or any combination thereof. In certain embodiments, the synthesis of the presently disclosed aminolipidoids is performed without solvent (i.e., neat). In certain other embodiments, the synthesis of the presently disclosed lipidoids is performed in a suitable solvent, such as a lower alcohol (e.g., isopropanol) or DMF. In certain embodiments, these syntheses are performed at a temperature in the range of about 90 to about 120 °C for about 2 to about 96 hours. In certain embodiments, conventional heating sources can be employed. In certain other embodiments, non-conventional heating sources, such as microwave radiation, can be employed. After the reaction is complete, the reaction mixture is cooled, any solvent used is removed in vacuo, and the crude product is purified or used“as is.” [0102] Reaction Scheme (A) exemplifies conditions that may be employed to react an oxidized amine with a terminal epoxide or an aldehyde to synthesize compounds according to the present invention: Scheme A
Figure imgf000048_0001
[0103] The synthesized aminolipidoids may be purified by any technique known in the art including, but not limited to, precipitation, crystallization, chromatography (e.g., silica gel chromatography, size exclusion chromatography, ion-exchange chromatography, and HPLC), and distillation. In certain embodiments, the crude product is purified by silica gel chromatography. [0104] In certain embodiments, the aminolipidoids of the present disclosure can be synthesized such that the aminolipidoid contains two or more different R and R' groups. In certain embodiments, this can be achieved by reacting the oxidized amine with a less-than- stoichiometric amount of a terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde followed by reacting the now-partially substituted compound with a further, different terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde and so on. In certain other embodiments, this can be achived by reacting the oxidized amine with a mixture of different terminal epoxides, interior epoxides, acrylates, acrylamides, and /or aldehydes. In certain embodiments this can be achieved through substituting one or more of the amino groups of the the oxidized amine with a protecting group and then reacting the remaining unprotected amino groups of the partially protected comppund with a terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde. The protecting groups can then be removed and the now unprotected amino groups of the partially modified compound can then be further reacted with a different terminal epoxide, interior epoxide, acrylate, acrylamide, or aldehyde. [0105] Examples of amino-protecting groups that may be used include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl- [9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1- methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2- dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1- methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N- dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N- hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p- methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p- chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3- dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4- dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m- chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5- benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4- dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl- (10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′- phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p- cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2- pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1- cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl- 1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4- pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N- benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide, 3-(p- hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2- one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5- dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5- substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5- triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl- 4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4- methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N- [(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2- picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2- pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′- isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or
tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzene sulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4- methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β- trimethylsilylethane sulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′- dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide,
trifluoromethylsulfonamide, and phenacylsulfonamide. [0106] Formulations of Aminolipidoids [0107] In another aspect, the present disclosure provides for formulations comprising the presently disclosed aminolipidoids that may be used to deliver one or more active agents to a target organism. Therefore, in certain embodiments, the presently disclosed formulations can comprise at least one aminolipidoid of the present disclosure and at least one active agent to be delivered. In certain embodiments, the at least one aminolipidoid of the present disclosure and a first active agent to be delivered are non-covalently associated to one another to form a non-covalent complex. As used herein, the term“non-covalently associated” encompasses any kind of intermolecular interaction between the at least one aminolipidoid of the present disclosure and the first active agent to be delivered other than covalent interactions (i.e., interactions that involve the sharing of electrons). Examples of such non- covalent interactions include, but are not limited to, electrostatic interactions, such as ionic interactions, hydrogen bonding, and halogen bonding, Van der Waals forces, such as the Keesom force, the Debye force, and London dispersion forces, π-effects, such as π-π interactions, cation-π interactions, anion-π interactions, and polar π interactions, and hydrophobic interactions. As such, the term“non-covalent complex,” as used herein, encompasses a complex of at least one aminolipidoid of the present disclosure and a first active agent to be delivered wherein the least one aminolipidoid and the first active agent are associated to each other via non-covalent interactions, as defined above. [0108] In certain embodiments of the presently disclosed formulations, oxygen- containing core X in the compound of formula (I) is derived from 1,10-diaza-18-crown-6. In those embodiments, R and R' can each, independently, be an optionally substituted aliphatic or heteroaliphatic or group. In certain embodiments of the presently disclosed formulations, compound of formula (I) is a compound of formula (III):
Figure imgf000052_0001
wherein R1 and R2 are independently selected from the group consisting of hydrogen or a moiety of formula 26 through 29, 32, 34, 35, 37, 40, 41, 46, 47, 50 through 52, 60, and 187 through 191:
Figure imgf000052_0002
Figure imgf000053_0001
. [0100] In certain embodiments, the presently disclosed formulations further comprise at least one additional active agent to be delivered. In certain embodiments, this at least one additional active agent is part of the non-covalent complex of the least one aminolipidoid and the first active agent. In other words, the at least one additional active agent can be contained within the non-covalent complex or adhered to the surface of the non-covalent complex via non-covalent interactions, as defined above. In certain other embodiments, the at least one additional active agent is not contained within the non-covalent complex or adhered to the surface of the non-covalent complex, e.g., the at least one additional active agent is simply in a physical mixture with the non-covalent complex. In certain embodiments, the first active agent is an oligonucleotide or a polynucleotide, and the at least one additional active agent is an herbicide, an insecticide, a fungicide, a bactericide, and/or a viricide. In certain embodiments, the first active agent is used to increase the sensitivity of the target organism to the additional active agent, for example, to increase the sensitivity of a plant to an herbicide, or to increase the sensitivity of an insect to an insecticide. [0101] The presently disclosed formulations may also comprise one or more excipients. Suitable excipients include, but are not limited to, fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters. The one or more excipients may be selected from the group consisting of sterols, carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof. In certain embodiments, the one or more excipients is a phytosterol. In certain other embodiments, the one or more excipients is cholesterol. In certain other embodiments, the one or more excipients comprises a water-soluble polymer such as polyethylene glycol (PEG), a polypropylene oxide (PPO), a polyvinylpyrrolidone (PVP), a polyvinyl alcohol (PVA), a polylactic acid (PLA), a poly(lactic-co-glycolic acid) (PLGA), or any combination thereof. In certain embodiments, the water-soluble polymer can be contained within or adhered to the surface of the non-covalent complexes of the present disclosure via non- covalent interactions, as defined above. In certain other embodiments, the water-soluble polymer can be tethered to the surface of the non-covalent complexes of the present disclosure via a lipid tail that is covalently bound on one end to the water-soluble polymer and which is entrained within the surface and/or interior of the non-covalent complex. [0102] In certain embodiments, the presently disclosed formulations are combined with an agriculturally acceptable carrier. The agriculturally acceptable carrier can be solid or liquid and is a substance useful in formulation of agricultural products. Examples of such agricultural products include, but are not limited to, fertilizers, herbicides, insecticides, fungicides, bactericides, viricides, and nematicides. Examples of such agriculturally acceptable carriers for use in the presently disclosed formulations include, but are not limited to, surface active agents, stickers, spreader stickers, inert carriers, preservatives, humectants, dyes, UV (ultra-violet) protectants, buffers, flow agents, antifoams (e.g.,
polydimethylsiloxane), sodium aluminosilicate, or other components which facilitate product handling and application of the compositions. Examples of agriculturally acceptable inert carriers include inorganic minerals, such as kaolin, mica, gypsum, fertilizer, carbonates, sulfates, and phosphates, organic materials, such as sugar, starches, and cyclodextrins, and botanical materials, such as wood products, cork, powdered corn cobs, rice hulls, peanut hulls, and walnut shells. Agriculturally acceptable carriers are described, for example, in U.S. Patent No. 6,984,609. In certain embodiments, the agriculturally acceptable carriers include, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders, or fertilizers. Such carriers are described, for example, in WO 97/33890. [0103] The presently disclosed formulations may further comprise one or more additional compounds to facilitate passage of the active agent(s) through the plant cell wall. Several technologies for facilitating passage of compounds through the plant cell wall are known in the art. For example, U.S. Patent No. 8,609,420 describes conjugation of the active agent to a semi-conductor nanoparticle within the size range of 3-5 nm (e.g., a“quantum dot”) and one or more cell penetrating peptides to improve penetration of the plant cell and intracellular delivery of the active agent. U.S. Patent No.8,686,222 describes interacting a polyamidoamine dendrimer and one or more cell penetrating peptides with the active agent to improve cell penetration. U.S. Patent No.8,653,327 describes delivery of active agents through plant cell walls by coating a PEGylated semiconductor nanoparticle with the active agent. U.S. Patent No. 8,722,410 describes transferring active agents into plant cells by applying the active agent to a nanoparticle coated with a subcellular compartment targeting protein. U.S. Patent Nos.8,609,420, 8,686,222, 8,653,327, and 8,722,410 are incorporated by reference herein in their entireties. [0104] The formulations of the present disclosure can take any form. Examples of such forms include, but are not limited to, complexes, particles (e.g., microparticles, nanoparticles, and picoparticles), micelles, and liposomes. In certain embodiments, two or more active agents (e.g., two or more siRNA) can be formulated with the presently disclosed aminolipidoids to form a single complex, particle, micelle or liposome containing the two or more active agents. Alternatively, in certain embodiments, the two or more active agents can be separately formulated to form a single complex, particle, micelle or liposome, each containing a single active agent, and are then combined to form a mixture prior to delivery to a target organism. [0105] The aminolipidoids of the present disclosure possess one or more primary, secondary, and/or tertiary amino groups that, although hindered, are available to interact with an active agent (e.g., a polynucleotide). As such, a non-covalent complex is formed when an active agent is contacted with the presently disclosed aminolipidoids under conditions suitable to form an active agent/aminolipidoid non-covalent complex. In certain
embodiments, multiple aminolipidoid molecules may non-covalently complex with an active agent molecule. The non-covalent complex may include 1-100 aminolipidoid molecules, 1- 1000 aminolipidoid molecules, 10-1000 aminolipidoid molecules, or 100-10,000
aminolipidoid molecules. [0106] The aminolipidoids of the present disclosure may be used to encapsulate active agents. The aminolipidoids of the present disclosure have several properties that make them particularly suitable in the preparation of drug delivery devices. These include, but are not limited to: (1) the ability of the lipidoid to complex and“protect” labile agents, (2) the ability to buffer the pH in the endosome (3) the ability to act as a“proton sponge” and cause endosomolysis, and (4) the ability to neutralize the charge on negatively charged agents. Thus, in certain embodiments, the aminolipidoids of the present disclosure are used to form particles containing at least one active agent to be delivered. These particles may include other materials, such as proteins, carbohydrates, synthetic polymers (e.g., PEG, PLGA), and natural polymers. [0107] In certain embodiments, the presently disclosed aminolipidoids are combined with an active agent to be delivered to a cell or a subject to form microparticles,
nanoparticles, liposomes, or micelles. The agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a
polynucleotide, protein, peptide, or small molecule. The presently disclosed aminolipidoids may be combined with other aminolipidoids of the present disclosure, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, and lipids, to form the particles. [0108] In certain embodiments, the diameter of the particles of the present disclosure range from 1 to 1,000 micrometers. In certain embodiments, the diameter of the particles range from 1 to 100 micrometers. In certain embodiments, the diameter of the particles range from 1 to 10 micrometers. In certain embodiments, the diameter of the particles range from 10 to 100 micrometers. In certain embodiments, the diameter of the particles range from 100 to 1,000 micrometers. In certain embodiments, the diameter of the particles range from 1 to 5 micrometers. In certain embodiments, the diameter of the particles range from 1 to 1,000 nm. In certain embodiments, the diameter of the particles range from 1 to 100 nm. In certain embodiments, the diameter of the particles range from 1 to 10 nm. In certain embodiments, the diameter of the particles range from 10 nm to 100 nm. In certain embodiments, the diameter of the particles range from 100 nm to 1,000 nm. In certain embodiments, the diameters of the particles range from 1 to 5 nm. In certain embodiments, the diameter of the particles range from 1 to 1,000 pm. In certain embodiments, the diameter of the particles range from 1 to 100 pm. In certain embodiments, the diameter of the particles range from 1 to 10 pm. In certain embodiments, the diameter of the particles range from 10 to 100 pm. In certain embodiments, the diameter of the particles range from 100 to 1,000 pm. In certain embodiments, the diameter of the particles range from 1 to 5 pm. [0109] The particles of the present disclosure may be prepared using any method known in the art. These include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. In certain embodiments, methods of preparing the particles are the double emulsion process and spray drying. In other embodiments, methods of preparing the particles are nanoprecipitation or flash precipitation, for example, as disclosed in U.S. Patent Nos. 8,207,290, 8,404,799, 8,546,521, 8,618,240, and 8,809,492, each of which are incorporated herein in its entirety. The conditions used in preparing the particles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology,“stickiness”, shape, etc.). The method of preparing the particle and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may also depend on the agent being encapsulated and/or the composition of the matrix. Methods developed for making particles for delivery of encapsulated agents are described in the literature (e.g., Doubrow, M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release 5:13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283, 1987; Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774, 1988; each of which is incorporated herein by reference in their entirety). [0110] If the particles prepared by any of the above methods have a size range outside of the desired range, the particles can be sized, for example, using a sieve. The particle may also be coated. In certain embodiments, the particles are coated with a targeting agent. In other embodiments, the particles are coated to achieve desirable surface properties (e.g., a particular charge). [0111] The aminolipidoids of the invention may be used to prepare micelles or liposomes containing an active agent to be delivered. Many techniques for preparing micelles and liposomes are known in the art, and any method may be used with the aminolipidoids of the present disclosure to make micelles and liposomes. Micelles and liposomes are particularly useful in delivering hydrophobic agents, such as hydrophobic small molecules. [0112] In certain embodiments, liposomes containing aminolipidoids of the present disclosure are formed through spontaneous assembly. In other embodiments, these liposomes are formed when thin lipid films or lipid cakes are hydrated and stacks of lipid crystalline bilayers become fluid and swell. The hydrated lipid sheets detach during agitation and self- close to form large, multilamellar vesicles (LMV). This prevents interaction of water with the hydrocarbon core of the bilayers at the edges. Once these particles have formed, reducing the size of the particle can be modified through input of sonic energy (sonication) or mechanical energy (extrusion). See Walde, P.“Preparation of Vesicles (Liposomes)” In Encyclopedia of Nanoscience and Nanotechnology; Nalwa, H. S. Ed. American Scientific Publishers: Los Angeles, 2004; Vol. 9, pp.43-79 and Szoka et al.“Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes)” Ann. Rev. Biophys.
Bioeng. 9:467-508, 1980, each of which is incorporated herein in its entirety. [0113] The preparation of liposomes of the present disclosure involves preparing the aminolipidoids for hydration, hydrating the aminolipidoids with agitation, and sizing the vesicles to achieve a homogenous distribution of liposomes. Aminolipidoids are first dissolved in an organic solvent to assure a homogeneous mixture. The solvent is then removed to form a lipidoid film. This film is thoroughly dried to remove residual organic solvent by placing the vial or flask on a vacuum pump overnight. Hydration of the lipidoid film/cake is accomplished by adding an aqueous medium to the container of dry lipidoid and agitating the mixture. Disruption of LMV suspensions using sonic energy typically produces small unilamellar vesicles (SUV) with diameters in the range of from 15 to 50 nm. Lipid extrusion is a technique in which a lipid suspension is forced through a polycarbonate filter with a defined pore size to yield particles having a diameter near the pore size of the filter used. Extrusion through filters with 100 nm pores typically yields large, unilamellar vesicles (LUV) with a mean diameter of from 120 to 140 nm. [0114] Certain aminolipidoids of the present disclosure can spontaneously self- assemble around certain molecules, such as DNA and RNA, to form liposomes. In some embodiments, the application is the delivery of polynucleotides to a target cell. Thus, use of the aminolipidoids of the present disclosure allows for simple assembly of liposomes without the need for additional steps or devices such as an extruder. [0115] The complexes, microparticles, nanoparticles, picoparticles, liposomes, and micelles of the present disclosure may be modified to include targeting agents since it is often desirable to target a particular cell, collection of cells, or tissue. A variety of targeting agents that direct pharmaceutical compositions to particular cells are known in the art (e.g., Cotten et al. Methods Enzym. 217:618, 1993; which is incorporated herein by reference in its entirety). The targeting agents may be included throughout the particle or may be only on the surface. The targeting agent may be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, and/or nucleic acid. The targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the particle. Examples of targeting agents include, but are not limited to, antibodies, fragments of antibodies, low- density lipoproteins (LDLs), transferrin, asialycoproteins, gp120 envelope protein of the human immunodeficiency virus (HIV), carbohydrates, receptor ligands, sialic acid, and aptamers. If the targeting agent is included throughout the particle, the targeting agent may be included in the mixture that is used to form the particles. If the targeting agent is only on the surface on the particle, the targeting agent may be associated with (i.e., by covalent, hydrophobic, hydrogen bonding, van der Waals, or other interactions) the formed particles using standard chemical techniques. [0116] In certain embodiments, the formulations of the present disclosure can be formulated as a bait, a food substance, or an attractant. For example, the formulations of the present disclosure can be incorporated into an insect bait suitable for oral administration of the formulation to the target insect. The bait may comprise a formulation comprising an active agent and an aminolipidoid of the present disclosure dispersed in a carrier and an edible insect attractant. In certain embodiments, the bait comprises an edible insect attractant and a nanoparticle or microparticle comprising at least one active agent and at least one aminolipidoid of the present disclosure, wherein the nanoparticle or microparticle is dispersed in a carrier. The formulation of the present disclosure and attractant can be mixed together before being dispersed in the desired carrier. Suitable attractants include any type of insect food and/or attractant which will lure the insect to the bait to ingest the bait.
Exemplary insect foods or attractants include, but are not limited to, any type of insect food, including various sugars, proteins, carbohydrates, yeast, fats, and/or oils. The bait can be in any form suitable for delivery and ingestion of the composition, depending on the habitat and target insect, but will typically be a liquid, gel, self-sustaining gel-matrix, or solid bait (e.g., tablets, granules, etc.). Exemplary carriers include, without limitation, agarose gel, gelatin gel, and/or pectin gel. In certain embodiments, the carrier is agarose gel, which is especially suited for aquatic habitats and breeding grounds. Insect baits are known in the art and are described, for example, in U.S. Pat. No.8,841,272, which is incorporated herein by reference in its entirety. [0117] The presently disclosed formulations can be present in the bait in an effective amount (i.e., concentration) for the activity of the active agent, such as gene silencing. The concentration of the active agent in the bait may be about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% by weight of the bait. Any of these values may be used to define a range for the concentration of the active agent in the bait. For example, the concentration of the active agent in the bait may range from about 0.1 to about 1%, or from about 1 to about 5% by weight of the bait. The weight ratio of active agent to insect attractant (food) in the bait may be about 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150 or 1:200. Any of these values may be used to define a range for the weight ratio of the active agent to the insect attractant in the bait. For example, the weight ratio of the active agent to the insect attractant in the bait may be from about 1:20 to about 1:200, or from about 1:50 to about 1:100. [0118] In certain embodiments, the concentration of a microparticle or nanoparticle comprising the at least one active agent and at least one aminolipidoid of the present disclosure in the bait may be about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% by weight of the bait. Any of these values may be used to define a range for the concentration of the microparticle or nanoparticle in the bait. For example, the concentration of the microparticle or nanoparticle in the bait may range from about 0.1 to about 1%, or from about 1 to about 5% by weight of the bait. The weight ratio of the microparticle or nanoparticle to insect attractant (food) in the bait may be about 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150 or 1:200. Any of these values may be used to define a range for the weight ratio of the microparticle or nanoparticle to the insect attractant in the bait. For example, the weight ratio of the microparticle or nanoparticle to the insect attractant in the bait may be from about 1:20 to about 1:200, or from about 1:50 to about 1:100. [0119] Herbicidal and Pesticidal Applications [0120] In another aspect, the presently disclosed aminolipidoid-based formulations can be used to deliver an active agent to target organisms for the purpose of killing and/or controlling the proliferation of the target organisms, such as insects, plant pathogens (e.g., fungi, bacteria, viruses, and nematodes), and weeds. In certain embodiments, the presently disclosed aminolipidoid-based formulations comprise an insecticidal, nematidicidal, fungicidal, bacteriocidal, viricidal, or herbicidal active agent, or combinations thereof. In certain embodiments, these formulations are combined with an agriculturally acceptable carrier to form a insecticidal, nematodicidal, fungicidal, bacteriocidal, viricidal, or herbicidal formulation. [0121] A target organism is an organism in which the presently disclosed herbicidal, insecticidal, or fungicidal formulations are intended to be functional, for example, to mediate gene silencing or suppression. In certain embodiments, a target organism is also a host organism, as described herein below. In other embodiments, a target organism is separate and distinct from a host organism that serves as a source of the active agent to be functional in the target organism. [0122] The insecticidal, nematidicidal, fungicidal, bacteriocidal, viricidal, or herbicidal formulation may further be combined with an agriculturally acceptable carrier. The agriculturally acceptale carrier can be solid or liquid and is a substance useful in formulation of agricultural products, for example, fertilizers, herbicides, insecticides, fungicides, bactericides, viricides, and nematicides. Agriculturally acceptable carriers include, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, thickeners, binders or fertilizers. Such carriers are described for example, in WO 97/33890, which is incorporated herein by reference. [0123] The presently disclosed formulations can be applied to the crop area or plant to be treated, simultaneously or in succession with further compounds. These further compounds can be, for example, fertilizers or micronutrient donors or other preparations, which influence the growth of plants. They can also be selective herbicides or non-selective herbicides as well as insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application promoting adjuvants customarily employed in the art of formulation. [0124] Active Agents [0125] Active agents that can be delivered to a target organism using the presently disclosed formulations include, but are not limited to, any type of molecule or compound including, but not limited to, nucleic acids, peptides, polypeptides, small molecules, and mixtures thereof. Examples of nucleic acids include, but are not limited to, interfering RNA molecules (e.g., siRNA, aiRNA, miRNA), antisense oligonucleotides, plasmids, ribozymes, immunostimulatory oligonucleotides, and mixtures thereof. [0126] In certain embodiments, the active agent comprises a nucleic acid. In certain embodiments, the nucleic acid comprises an interfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, or mixtures thereof. In certain embodiments, the nucleic acid comprises single-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid such as, e.g., an antisense oligonucleotide, a ribozyme, a plasmid, an immunostimulatory
oligonucleotide, or mixtures thereof. [0127] In certain embodiments, presently disclosed particles are associated with a nucleic acid. In some embodiments, the nucleic acid is fully encapsulated in a lipidoid particle. As used herein, the term“nucleic acid” includes any oligonucleotide or
polynucleotide, with fragments containing up to 60 nucleotides generally termed
oligonucleotides, and longer fragments termed polynucleotides. In particular embodiments, oligonucleotides of the invention are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 nucleotides in length. Any of these values may be used to define a range for the size of the oligonucleotide. For example, the size of the oligonucleotide may range from 15-60, 20-60 or 25-60 nucleotides in length. In particular embodiments, the polynucleotide is 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more nucleotides in length. In particular embodiments, the polynucleotide is at least 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nucleotides in length. Any of these values may be used to define a range for the size of the polynucleotide. For example, the polynucleotide may range from 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900- 950, or 950-1000 nucleotides in length. The nucleic acid may be administered alone in the particles of the present disclosure, or in combination (e.g., co-administered) with particles of the present disclosure comprising peptides, polypeptides, or small molecules, such as conventional drugs. [0128] In the context of this invention, the terms“polynucleotide” and
“oligonucleotide” refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally-occurring bases, sugars, and intersugar (backbone) linkages. The terms“polynucleotide” and“oligonucleotide” also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. [0129] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5′ and 3′ carbons of this sugar to form an alternating, unbranched polymer. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose. [0130] Nucleic acids that can be used in the presently disclosed formulations includes any form of nucleic acid that is known. The nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA are described herein and include, e.g., structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA are described herein and include, e.g., siRNA and other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic acids include, e.g., antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides. [0131] Nucleic acids that can be used in the formulations of the present disclosure may be of various lengths, which is generally dependent upon the particular form of nucleic acid. For example, in certain embodiments, plasmids or genes may be from about 1,000 to about 100,000 nucleotide residues in length. In certain embodiments, oligonucleotides may range from about 10 to about 100 nucleotides in length. In certain embodiments,
oligonucleotides, both single-stranded, double-stranded, and triple-stranded, may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides in length. [0132] In certain embodiments, an oligonucleotide (or a strand thereof) that can be used in the presently disclosed formulations specifically hybridizes to or is complementary to a target polynucleotide sequence. The terms“specifically hybridizable” and
“complementary” as used herein indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In certain embodiments, an oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence to cause a loss of utility or expression therefrom, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired. Thus, the oligonucleotide may include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence that it is targeting or to which it specifically hybridizes. [0133] In certain embodiments, the oligo- or polynucleotide is optionally purified and substantially pure. In some embodiments, the polynucleotide is greater than 50% pure. In some embodiments, the oligo- or polynucleotide is greater than 75% pure. In some embodiments, the oligo- or polynucleotide is greater than 95% pure. The oligo- or polynucleotide may be provided by any means known in the art. In certain embodiments, the oligo- or polynucleotide has been engineered using recombinant techniques. The oligo- or polynucleotide may also be obtained from natural sources and purified from contaminating components found normally in nature. The oligo- or polynucleotide may also be chemically synthesized in a laboratory. In certain embodiments, the oligo- or polynucleotide is synthesized using standard solid phase chemistry. [0134] The oligo- or polynucleotide may be modified by chemical or biological means. In certain embodiments, these modifications lead to increased stability of the oligo- or polynucleotide. Examples of such modifications include, but are not limited to, methylation, phosphorylation, and end-capping. [0135] The oligo- or polynucleotide to be delivered may be in any form. Examples of such forms include, but are not limited to, a circular plasmid, a linearized plasmid, a cosmid, a viral genome, a modified viral genome, an artificial chromosome, dsRNA, ssRNA, dsDNA, ssDNA, RNA/DNA hybrids, dsRNA hairpins, siRNA, aiRNA, and miRNA. [0136] The oligo- or polynucleotide may be of any sequence. In certain
embodiments, the oligo- or polynucleotide encodes a protein or peptide. The encoded proteins may be enzymes, structural proteins, receptors, soluble receptors, ion channels, or cytokines. The oligo- or polynucleotide may also comprise regulatory regions to control the expression of a gene. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA box, ribosomal binding sites, and stop site for transcription. In certain embodiments, the polynucleotide is not intended to encode a protein. For example, the polynucleotide may be used to fix an error in the genome of the cell being transfected. [0137] In certain embodiments, the nucleic acid is modified. As used herein, the term "modified" in reference to a nucleic acid (e.g., an oligonucleotide or polynucleotide) is defined as a nucleic acid that contains variations of the standard bases, sugars and/or phosphate backbone chemical structures occurring in ribonucleic (i.e., A, C, G and U) and deoxyribonucleic (i.e., A, C, G and T) acids. Particular modifications of nucleic acids are further described below. [0138] In certain embodiments, the oligo- or polynucleotide is an RNA that carries out RNA interference (RNAi). The term“interfering RNA” or“RNAi” or“interfering RNA sequence” refers to single-stranded RNA (e.g., mature miRNA) or double-stranded RNA (e.g., duplex RNA, such as siRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence.
Interfering RNA thus refers to the single-stranded RNA that is complementary to a target mRNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering RNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof. [0139] siRNA [0140] In certain embodiments, the active agent comprises an siRNA. The siRNA molecule can comprise a double-stranded region of about 15 to about 60 nucleotides in length (e.g., about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The siRNA molecules used in the presently disclosed formulations are capable of silencing the expression of a target sequence in vitro and/or in vivo. [0141] In certain embodiments, the siRNA molecule comprises modified nucleotides including, but not limited to, 2′-O-methyl (2′OMe) nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In other embodiments, the siRNA comprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidine nucleotides) such as, for example, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, and mixtures thereof. In certain embodiments, the siRNA does not comprise 2′OMe-cytosine nucleotides. In certain embodiments, the siRNA comprises a hairpin loop structure. [0142] In certain embodiments, the siRNA may comprise modified nucleotides in one strand (i.e., sense or antisense) or both strands of the double-stranded region of the siRNA molecule. In certain embodiments, uridine and/or guanosine nucleotides are modified at selective positions in the double-stranded region of the siRNA duplex. With regard to uridine nucleotide modifications, at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and/or antisense strand can be a modified uridine nucleotide such as a 2′OMe-uridine nucleotide. In certain embodiments, every uridine nucleotide in the sense and/or antisense strand is a 2′OMe-uridine nucleotide. With regard to guanosine nucleotide modifications, at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense and/or antisense strand can be a modified guanosine nucleotide such as a 2′OMe- guanosine nucleotide. In certain embodiments, every guanosine nucleotide in the sense and/or antisense strand is a 2′OMe-guanosine nucleotide. [0143] In certain embodiments, at least one, two, three, four, five, six, seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified, e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/or by introducing modified nucleotides such as 2′OMe nucleotides. The 5′-GU-3′ motif can be in the sense strand, the antisense strand, or both strands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent to each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. [0144] In certain embodiments, a modified siRNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the target sequence relative to the corresponding unmodified siRNA sequence. [0145] In certain embodiments, the siRNA molecule does not comprise phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double-stranded region. In certain embodiments, the siRNA comprises one, two, three, four, or more phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double- stranded region. In certain embodiments, the siRNA does not comprise phosphate backbone modifications. [0146] In certain embodiments, the siRNA does not comprise 2′-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region. In certain
embodiments, the siRNA comprises one, two, three, four, or more 2′-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region. In certain embodiments, the siRNA does not comprise 2′-deoxy nucleotides. [0147] In certain embodiments, the nucleotide at the 3′-end of the double-stranded region in the sense and/or antisense strand is not a modified nucleotide. In certain
embodiments, the nucleotides near the 3′-end (e.g., within one, two, three, or four nucleotides of the 3′-end) of the double-stranded region in the sense and/or antisense strand are not modified nucleotides. [0148] The siRNA molecules described herein may have 3′ overhangs of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or may lack overhangs (i.e., have blunt ends) on one or both sides of the double-stranded region. In certain embodiments, the siRNA has 3′ overhangs of two nucleotides on each side of the double-stranded region. In certain embodiments, the 3′ overhang on the antisense strand has complementarity to the target sequence and the 3′ overhang on the sense strand has complementarity to a complementary strand of the target sequence. Alternatively, the 3′ overhangs do not have complementarity to the target sequence or the complementary strand thereof. In certain embodiments, the 3′ overhangs comprise one, two, three, four, or more nucleotides such as 2′-deoxy(2′H) nucleotides. In certain embodiments, the 3′ overhangs comprise deoxythymidine (dT) and/or uridine nucleotides. In certain embodiments, one or more of the nucleotides in the 3′ overhangs on one or both sides of the double-stranded region comprise modified nucleotides. Examples of modified nucleotides are described above and include, but are not limited to, 2′OMe nucleotides, 2′-deoxy-2′F nucleotides, 2′-deoxy nucleotides, 2′-O-2-MOE nucleotides, LNA nucleotides, and mixtures thereof. In certain embodiments, one, two, three, four, or more nucleotides in the 3′ overhangs present on the sense and/or antisense strand of the siRNA comprise 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidine nucleotides) such as, for example, 2′OMe-guanosine nucleotides, 2′OMe- uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, and mixtures thereof. [0149] The siRNA may comprise at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and/or modified siRNA sequences that silence target gene expression. The cocktail of siRNA may comprise sequences, which are directed to the same region or domain (e.g., a“hot spot”) and/or to different regions or domains of one or more target genes. In certain embodiments, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) modified siRNA that silence target gene expression are present in a cocktail. In certain embodiments, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence target gene expression are present in a cocktail. [0150] In certain embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence or a portion thereof. In certain embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In certain embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof. [0151] In certain embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof. In certain embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is 100% identical to the target sequence or a portion thereof. [0152] The siRNA that can be used in the presently disclosed formulations are capable of silencing the expression of a target gene of interest. Each strand of the siRNA duplex can be about 15 to about 60 nucleotides in length, or about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA comprises at least one modified nucleotide. In some embodiments, the modified siRNA contains at least one 2′OMe purine or pyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine, 2′OMe-adenosine, and/or 2′OMe- cytosine nucleotide. In certain embodiments, one or more of the uridine and/or guanosine nucleotides are modified. The modified nucleotides can be present in one strand (i.e., sense or antisense) or both strands of the siRNA. The siRNA sequences may have overhangs or may lack overhangs (i.e., have blunt ends). [0153] The modified siRNA generally comprises from about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA duplex. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the double-stranded region of the siRNA comprise modified nucleotides. [0154] In certain embodiments, less than about 25% (e.g., less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the nucleotides in the double-stranded region of the siRNA comprise modified nucleotides. [0155] In certain embodiments, from about 1% to about 25% (e.g., from about 1%- 25%, 2%-25%, 3%-25%, 4%-25%, 5%-25%, 6%-25%, 7%-25%, 8%-25%, 9%-25%, 10%- 25%, 11%-25%, 12%-25%, 13%-25%, 14%-25%, 15%-25%, 16%-25%, 17%-25%, 18%- 25%, 19%-25%, 20%-25%, 21%-25%, 22%-25%, 23%-25%, 24%-25%, etc.) or from about 1% to about 20% (e.g., from about 1%-20%, 2%-20%, 3%-20%, 4%-20%, 5%-20%, 6%- 20%, 7%-20%, 8%-20%, 9%-20%, 10%-20%, 11%-20%, 12%-20%, 13%-20%, 14%-20%, 15%-20%, 16%-20%, 17%-20%, 18%-20%, 19%-20%, 1%-19%, 2%-19%, 3%-19%, 4%- 19%, 5%-19%, 6%-19%, 7%-19%, 8%-19%, 9%-19%, 10%-19%, 11%-19%, 12%-19%, 13%-19%, 14%-19%, 15%-19%, 16%-19%, 17%-19%, 18%-19%, 1%-18%, 2%-18%, 3%- 18%, 4%-18%, 5%-18%, 6%-18%, 7%-18%, 8%-18%, 9%-18%, 10%-18%, 11%-18%, 12%- 18%, 13%-18%, 14%-18%, 15%-18%, 16%-18%, 17%-18%, 1%-17%, 2%-17%, 3%-17%, 4%-17%, 5%-17%, 6%-17%, 7%-17%, 8%-17%, 9%-17%, 10%-17%, 11%-17%, 12%-17%, 13%-17%, 14%-17%, 15%-17%, 16%-17%, 1%-16%, 2%-16%, 3%-16%, 4%-16%, 5%- 16%, 6%-16%, 7%-16%, 8%-16%, 9%-16%, 10%-16%, 11%-16%, 12%-16%, 13%-16%, 14%-16%, 15%-16%, 1%-15%, 2%-15%, 3%-15%, 4%-15%, 5%-15%, 6%-15%, 7%-15%, 8%-15%, 9%-15%, 10%-15%, 11%-15%, 12%-15%, 13%-15%, 14%-15%, etc.) of the nucleotides in the double-stranded region of the siRNA comprise modified nucleotides. [0156] In certain embodiments, e.g., when one or both strands of the siRNA are selectively modified at uridine and/or guanosine nucleotides, the resulting modified siRNA can comprise less than about 30% modified nucleotides (e.g., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% modified nucleotides) or from about 1% to about 30% modified nucleotides (e.g., from about 1%-30%, 2%-30%, 3%-30%, 4%-30%, 5%-30%, 6%-30%, 7%-30%, 8%-30%, 9%-30%, 10%-30%, 11%-30%, 12%-30%, 13%-30%, 14%-30%, 15%-30%, 16%-30%, 17%-30%, 18%-30%, 19%-30%, 20%-30%, 21%-30%, 22%-30%, 23%-30%, 24%-30%, 25%-30%, 26%-30%, 27%-30%, 28%-30%, or 29%-30% modified nucleotides). [0157] Examples of modified nucleotides suitable for use in the presently disclosed formulations include, but are not limited to, ribonucleotides having a 2′-O-methyl (2′OMe), 2′-deoxy-2′-fluoro(2′F), 2′-deoxy, 5-C-methyl, 2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′- amino, or 2′-C-allyl group. Modified nucleotides having a Northern conformation are also suitable for use in siRNA molecules. Such modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl) (MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′- deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro(2′Cl) nucleotides, and 2′-azido nucleotides. In certain instances, the siRNA molecules described herein include one or more G-clamp nucleotides. A G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex. In addition, nucleotides having a nucleotide base analog such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4- nitroindole, 5-nitroindole, and 6-nitroindole can be incorporated into siRNA molecules. [0158] In certain embodiments, the siRNA molecules may further comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone
modifications, and the like. Examples of terminal cap moieties include, but are not limited to, inverted deoxy abasic residues, glyceryl modifications, 4′,5′-methylene nucleotides, 1-(β- D-erythrofuranosyl) nucleotides, 4′-thio nucleotides, carbocyclic nucleotides, 1,5- anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo- pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties, 3′- 3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties, 3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties, 5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties, 5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate, 5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate, 5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate, and bridging or non-bridging methylphosphonate or 5′-mercapto moieties. Examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include, but are not limited to, phosphorothioate,
phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions. Such chemical modifications can occur at the 5′- end and/or 3′-end of the sense strand, antisense strand, or both strands of the siRNA. [0159] In certain embodiments, the sense and/or antisense strand of the siRNA molecule can further comprise a 3′-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides and/or any combination of modified and unmodified nucleotides. [0160] The siRNA molecules can optionally comprise one or more non-nucleotides in one or both strands of the siRNA. As used herein, the term“non-nucleotide” refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their activity. The group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore lacks a base at the 1′-position. [0161] In certain embodiments, chemical modification of the siRNA comprises attaching a conjugate to the siRNA molecule. The conjugate can be attached at the 5′ and/or 3′-end of the sense and/or antisense strand of the siRNA via a covalent attachment such as, e.g., a biodegradable linker. The conjugate can also be attached to the siRNA, e.g., through a carbamate group or other linking group. In certain instances, the conjugate is a molecule that facilitates the delivery of the siRNA into a cell. [0162] aiRNA [0163] In certain embodiments, the active agent comprises an asymmetrical interfering RNA (aiRNA). In certain embodiments, aiRNA duplexes of various lengths may be designed with overhangs at the 3′ and 5′ ends of the antisense strand to target an mRNA of interest. In certain embodiments, the sense strand of the aiRNA molecule is about 10-25, 12- 20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, or about 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length. [0164] In certain embodiments, the 5′ antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g.,“AA”,“UU”,“dTdT”, etc.). In other
embodiments, the 3′ antisense overhang contains one, two, three, four, or more nontargeting nucleotides (e.g.,“AA”,“UU”,“dTdT”, etc.). In certain embodiments, the aiRNA molecules described herein may comprise one or more modified nucleotides, e.g., in the double-stranded (duplex) region and/or in the antisense overhangs. As a non-limiting example, aiRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences. In certain embodiments, the aiRNA molecule comprises 2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, or mixtures thereof. [0165] In certain embodiments, aiRNA molecules may comprise an antisense strand which corresponds to the antisense strand of an siRNA molecule, e.g., one of the siRNA molecules described herein. In certain embodiments, aiRNA molecules may be used to silence the expression of any of a target gene. [0166] In certain embodiments, the aiRNA molecule comprises a double-stranded (duplex) region of about 10 to about 25 (base paired) nucleotides in length, wherein the aiRNA molecule comprises an antisense strand comprising 5′ and 3′ overhangs, and wherein the aiRNA molecule is capable of silencing target gene expression. [0167] In certain embodiments, each of the 5′ and 3′ overhangs on the antisense strand comprises or consists of one, two, three, four, five, six, seven, or more nucleotides. [0168] In certain embodiments, the aiRNA molecule comprises modified nucleotides selected from the group consisting of 2′OMe nucleotides, 2′F nucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. [0169] miRNA [0170] In certain embodiments, the active agent comprises a microRNAs (miRNA). Generally, miRNA are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. In certain embodiments, the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, or about 15-30, 15-25, or 19-25 nucleotides in length, or about 20-24, 21-22, or 21-23 nucleotides in length. In certain embodiments, the miRNA molecule comprises about 15 to about 60 nucleotides in length, wherein the miRNA molecule is capable of silencing target gene expression. [0171] In certain embodiments, miRNA molecules may comprise one or more modified nucleotides. As a non-limiting example, miRNA sequences may comprise one or more of the modified nucleotides described above for siRNA sequences. In certain embodiments, the miRNA molecule comprises 2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, or mixtures thereof. In certain embodiments, the miRNA molecule comprises modified nucleotides selected from the group consisting of 2′F nucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNA nucleotides, and mixtures thereof. [0172] dsRNA [0173] In certain embodiments, the active agent is a dsRNA (double-stranded RNA). In certain embodiments, the active agent is an shRNA (short hairpin RNA). [0174] Antisense Polynucleotide [0175] In certain embodiments, the active agent is an antisense oligonucleotide. The terms“antisense polynucleotide” or“antisense” include polynucleotides that are
complementary to a targeted polynucleotide sequence. Antisense polynucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. [0176] In certain embodiments, the polynucleotide is an antisense RNA. Antisense RNA polynucleotides prevent the translation of complementary RNA strands by binding to the RNA. Antisense DNA polynucleotides can be used to target a specific, complementary (coding or non-coding) RNA. If binding occurs, this DNA/RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, antisense polynucleotides comprise from about 10 to about 60 nucleotides, or from about 15 to about 30 nucleotides. The term also encompasses antisense polynucleotides that may not be exactly complementary to the desired target gene. Thus, the invention can be utilized in instances where non-target specific- activities are found with antisense, or where an antisense sequence containing one or more mismatches with the target sequence is the most preferred for a particular use. [0177] Methods of producing antisense polynucleotides are known in the art and can be readily adapted to produce an antisense polynucleotides that targets any polynucleotide sequence. Selection of antisense polynucleotide sequences specific for a given target sequence is based upon analysis of the chosen target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense polynucleotides may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell. Highly preferred target regions of the mRNA include those regions at or near the AUG translation initiation codon and those sequences that are substantially complementary to 5′ regions of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithm software
(Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)). [0178] Ribozymes [0179] In certain embodiments, the active agent is a ribozyme. Ribozymes are RNA- protein complexes having specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction. [0180] The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, hepatitis δ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence), or Neurospora VS RNA motif, for example. Important characteristics of enzymatic nucleic acid molecules used according to the invention are that they have a specific substrate binding site which is complementary to one or more of the target gene DNA or RNA regions, and that they have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. [0181] Methods of producing a ribozyme targeted to any polynucleotide sequence are known in the art. Ribozyme activity can be optimized by altering the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases, modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements. [0182] Insecticides [0183] In certain embodiments, an insecticide for killing or controlling the proliferation of an insect is combined with the active agent described above. Examples of suitable insecticides include, but are not limited to, those provided in Table 1. [0184] Table 1.
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
[0185] Additional non-limiting examples of suitable insecticides include biologics, hormones or pheromones such as azadirachtin, Bacillus species, Beauveria species, codlemone, Metarrhizium species, Paecilomyces species, thuringiensis and Verticillium species, and active compounds having unknown or non-specified mechanisms of action such as fumigants (such as aluminium phosphide, methyl bromide and sulphuryl fluoride) and selective feeding inhibitors (such as cryolite, flonicamid and pymetrozine). Examples of mite growth inhibitors include, but are not limited to, clofentezine, etoxazole and hexythiazox, amidoflumet, benclothiaz, benzoximate, bifenazate, bromopropylate, buprofezin,
chinomethioat, chlordimeform, chlorobenzilate, chloropicrin, clothiazoben, cycloprene, cyflumetofen, dicyclanil, fenoxacrim, fentrifanil, flubenzimine, flufenerim, flutenzin, gossyplure, hydramethylnone, japonilure, metoxadiazone, petroleum, piperonyl butoxide, potassium oleate, pyrafluprole, pyridalyl, pyriprole, sulfluramid, tetradifon, tetrasul, triarathene, verbutin, furthermore the compound 3-methylphenyl propylcarbamate
(Tsumacide Z), the compound 3-(5-chloro-3-pyridinyl)-8-(2,2,2-trifluoroethyl)-8- azabicyclo[3.2.1]octa- ne-3-carbonitrile (CAS reg. No.185982-80-3) and the corresponding 3-endo isomer (CAS reg. No.185984-60-5) (cf. WO 96/37494, WO 98/25923), and also preparations comprising insecticidally effective plant extracts, nematodes, fungi, or viruses. [0186] Herbicides [0187] In certain embodiments, an herbicide for killing or controlling the proliferation of weeds and other unwanted plants is combined with one of the active agents described above. Examples of herbicides include, but are not limited to, benzoic acid herbicides such as dicamba esters, phenoxyalkanoic acid herbicides such as 2,4-D, MCPA and 2,4-DB esters, aryloxyphenoxypropionic acid herbicides such as clodinafop, cyhalofop, fenoxaprop, fluazifop, haloxyfop and quizalofop esters, pyridinecarboxylic acid herbicides such as aminopyralid, picloram and clopyralid esters, pyrimidinecarboxylic acid herbicides such as aminocyclopyrachlor esters, pyridyloxyalkanoic acid herbicides such as fluoroxypyr and triclopyr esters, and hydroxybenzonitrile herbicides such as bromoxynil and ioxynil esters, esters of the arylpyridine carboxylic acids and arylpyrimidine carboxylic acids of the following generic structures as disclosed in U.S. Pat. No.7,314,849, U.S. Pat. No. 7,300,907 and U.S. Pat. No.7,642,220. In certain embodiments, the herbicide is selected from the group consisting of 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, amitrole, asulam, atrazine, azafenidin, benefin, bensulfuron, bensulide, bentazon, bromacil,
bromoxynil, butylate, carfentrazone, chloramben, chlorimuron, chlorproham, chlorsulfuron, clethodim, clomazone, clopyralid, cloransulam, cyanazine, cycloate, DCPA, desmedipham, dichlobenil, diclofop, diclosulam, diethatyl, difenzoquat, diflufenzopyr, dimethenamid-p, diquat, diuron, DSMA, endothall, EPTC, ethalfluralin, ethametsulfuron, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fluthiacet, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, haloxyfop, hexazinone, imazamethabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione, methazole, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxasulfuron, oxyfluorfen, paraquat, pebulate, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, prodiamine, prometryn, pronamide, propachlor, propanil, prosulfuron, pyrazon, pyridate, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine,
sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin,
triflusulfuron, vernolate, and any combination thereof. [0188] Fungicides [0189] In certain embodiments, a fungicide for killing or controlling the proliferation of a fungus is combined with the active agent described above. Exemplary fungicides include, but are not limited to, strobilurins, azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl, metominostrobin, picoxystrobin,pyraclostrobin,
trifloxystrobin, orysastrobin, carboxamides, carboxanilides, benalaxyl, benalaxyl-M, benodanil, carboxin, mebenil, mepronil, fenfuram, fenhexamid, flutolanil, furalaxyl, furcarbanil, furametpyr, metalaxyl, metalaxyl-M (mefenoxam), methfuroxam, metsulfovax, ofurace, oxadixyl, oxycarboxin, penthiopyrad, pyracarbolid, salicylanilide, tecloftalam, thifluzamide, tiadinil, N-biphenylamides, bixafen, boscalid, carboxylic acid morpholides, dimethomorph, flumorph, benzamides, flumetover, fluopicolid (picobenzamid), zoxamid, carboxamides, carpropamid, diclocymet, mandipropamid, silthiofam, azoles, triazoles, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, enilconazole, epoxiconazole, fenbuconazole, flusilazol, fluquinconazole, flutriafol, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimenol, triadimefon, triticonazole, Imidazoles, cyazofamid, imazalil, pefurazoate, prochloraz, triflumizole, benzimidazoles, benomyl, carbendazim, fuberidazole, thiabendazole, ethaboxam, etridiazole, hymexazol, nitrogen-containing heterocyclyl compounds, pyridines, fuazinam, pyrifenox, pyrimidines, bupirimate, cyprodinil, ferimzone, fenarimol, mepanipyrim, nuarimol, pyrimethanil, piperazines, triforine, pyrroles, fludioxonil, fenpiclonil, morpholines, aldimorph, dodemorph, fenpropimorph, tridemorph, dicarboximides, iprodione,
procymidone, vinclozolin, acibenzolar-S-methyl, anilazine, captan, captafol, dazomet, diclomezin, fenoxanil, folpet, fenpropidin, famoxadon, fenamidon, octhilinone, probenazole, proquinazid, pyroquilon, quinoxyfen, tricyclazole, carbamates, dithiocarbamates, ferbam, mancozeb, maneb, metiram, metam, propineb, thiram, zineb, ziram, diethofencarb, flubenthiavalicarb, iprovalicarb, propamocarb, guanidines, dodine, iminoctadine, guazatine, kasugamycin, polyoxins, streptomycin, validamycin A, organometallic compounds, fentin salts, sulfur-containing heterocyclyl compounds, isoprothiolane, dithianone,
organophosphorous compounds, edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, pyrazophos, tolclofos-methyl, Organochlorine compounds, thiophanate-methyl,
chlorothalonil, dichlofluanid, tolylfluanid, flusulfamide, phthalide, hexachlorobenzene, pencycuron, quintozene, nitrophenyl derivatives, binapacryl, dinocap, dinobuton,
spiroxamine, cyflufenamid, cymoxanil, metrafenon, N-2-cyanophenyl-3,4-dichloroisothiazol- 5-carboxamide (isotianil), N-(3',4',5'-trifluorobiphenyl-2-yl)-3-difluoromethyl-1- methylpyrazole-4-carboxamide, 3-[5-(4-chlorophenyl)-2,3-dimethylisoxazolidin-3-yl]- pyridine, N-(3',4'-dichloro-4-fluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazol- e-4- carboxamide, 5-chloro-7-(4-methylpiperidin-1-yl)-6-(2,4,6-trifluorophenyl)-[1,2,4]tria- zolo[1,5-a]pyrimidine, 2-butoxy-6-iodo-3-propylchromen-4-one, N,N-dimethyl-3-(3-bromo- 6-fluoro-2-methylindole-1-sulfonyl)-[1,2,4]triazo- le-1-sulfonamide, methyl-(2-chloro-5-[1- (3-methylbenzyloxyimino)-ethyl]benzyl)carbamate, methyl-(2-chloro-5-[1-(6-methylpyridin- 2-ylmethoxy-imino)ethyl]benzyl)carbamate, methyl 3-(4-chlorophenyl)-3-(2- isopropoxycarbonylamino-3-methylbutyryl-amino)propionate, 4-fluorophenyl N-(1-(1-(4- cyanophenyl)ethanesulfonyl)but-2-yl)carbamate , N-(2-(4-[3-(4-chlorophenyl)prop-2- ynyloxy]-3-methoxyphenyl)ethyl)-2-metha- nesulfonylamino-3-methylbutyramide, N-(2-(4- [3-(4-chlorophenyl)prop-2-ynyloxy]-3-methoxyphenyl)ethyl)-2-ethan- esulfonylamino-3- methylbutyramide, N-(4'-bromobiphenyl-2-yl)-4-difluoromethyl-2-methylthiazol-5- carboxamide, N-(4'-trifluoromethylbiphenyl-2-yl)-4-difluoromethyl-2-methylthiazol-5- carboxamide, N-(4'-chloro-3'-fluorobiphenyl-2-yl)-4-difluoromethyl-2-methylt- hiazol-5- carboxamide, and methyl 2-(ortho-((2,5-dimethylphenyloxy-methylene)phenyl)-3- methoxyacrylate. [0190] Modulation of Traits in Plants, Insects and Plant Pathogens [0191] Plants [0192] In another aspect, the present disclosure provides for a method of modulating a trait of a plant, comprising delivering to the plant an effective amount of a modified polyethyleneimine-based formulation comprising an oligonucleotide or polynucleotide that modulates the expression of a gene in the plant. Oligonucleotides or polynucleotides that modulate the expression of a gene in a plant include, but are not limited to, RNA molecules (e.g., siRNA, aiRNA, miRNA, dsRNA, and shRNA) and DNA molecules (e.g., antisense polynucleotides) that decrease expression of the gene in the plant, and RNA molecules (e.g., mRNA) and DNA molecules (e.g., expression cassettes and plasmids) that increase expression of the gene in the plant. In certain embodiments, the oligonucleotide or polynucleotide modulates the expression of a gene that is endogenous to the plant. In other embodiments, the oligonucleotide or polynucleotide modulates the expression of a gene that is heterologous to the plant, e.g., a transgene that does not naturally occur within the plant. In certain embodiments, the oligonucleotide or polynucleotide that modulates the expression of a gene in the plant hybridizes to a gene or gene product that is endogenous to the plant. [0193] Traits that may be modulated in a plant include, but are not limited to, total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, and any combination thereof. In a particular embodiment, the presently disclosed modified polyethylenimine-based formulations can be used to deliver an active agent to a plant (e.g., a weed), for the purpose of killing and/or controlling the proliferation of the plant. [0194] In certain embodiments, one or more of the above-mentioned traits in a plant is increased or improved relative to a plant that is not treated with the modified
polyethyleneimine-based formulation. The trait in the plant as described herein may be increased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% by delivery of the modified polyethyleneimine-based formulation to the plant relative to a plant that is not treated with the formulation. In other embodiments, one or more of the above mentioned traits is decreased relative to a plant that is not treated with the modified polyethyleneimine-based formulation. The trait in the plant as described herein may be decreased by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% by delivery of the modified polyethyleneimine-based formulation to the plant relative to a plant that is not treated with the formulation. [0195] Insects [0196] In another aspect, the present disclosure provides for a method of modulating a trait of an insect, comprising delivering to the insect, to a plant infested with the insect, or to a plant prior to infestation with the insect an effective amount of a modified
polyethyleneimine-based formulation comprising an oligonucleotide or polynucleotide that modulates expression of a gene in the insect. Oligonucleotides or polynucleotides that modulate the expression of a gene in the insect include, but are not limited to, RNA molecules (e.g., siRNA, aiRNA, miRNA, dsRNA, and shRNA) and DNA molecules (e.g., antisense polynucleotides) that decrease expression of the gene in the insect, and RNA molecules (e.g., mRNA) and DNA molecules (e.g., expression cassettes and plasmids) that increase expression of the gene in the insect. In certain embodiments, the oligonucleotide or polynucleotide that modulates the expression of a gene in the insect hybridizes to a gene or gene product that is endogenous to the insect. [0197] Traits that may be modulated in the insect include, but are not limited to, insect growth, development, activity,and/or lifespan. For example, in certain embodiments, delivery of the formulation to the insect kills the insect. In certain embodiments, delivery of the formulation to the insect reduces its growth and/or lifespan, thereby reducing the damage done by the insect to a plant. In certain embodiments, delivery of the formulation to the insect causes the insect to remain in a young or immature stage, thus preventing the insect from completing its lifecycle. For example, in certain embodiments, delivery of the formulation to the insect interferes with enzymes involved in the molting process that stimulate the synthesis and formation of chitin, which is an essential component of an insect’s exoskeleton. As a result, the insect fails to reach adulthood because it dies in an immature stage. In certain embodiments, delivery of the formulation to the insect disrupts the feeding activity of the insect. As a result, insects starve to death because they are unable to obtain nutrients. [0198] In certain embodiments, the delivery of the formulation to the insect decreases its growth, activity or lifespan by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% relative to an insect that is not treated with the
formulation. In certain embodiments, the delivery of the formulation to the insect increases its growth, activity or lifespan by at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% relative to an insect that is not treated with the formulation. [0199] Plant Pathogens [0200] In another aspect, the present disclosure provides a method of modulating the pathogenicity of a plant pathogen, comprising applying to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen a modified polyethyleneimine-based formulation comprising an oligonucleotide or
polynucleotide that modulates expression of a gene in the plant pathogen. For example, in certain embodiments, the pathogenicity of the plant pathogen is decreased, for example by decreasing the growth, activity, or lifespan of the plant pathogen, or delaying the
development of the plant pathogen. In a particular embodiment, the formulation is used to kill the plant pathogen and/or control its proliferation. In certain other embodiments, the pathogenicity of the plant pathogen is increased, for example, by increasing the growth, activity or lifespan of the plant pathogen, or accelerating its development. Increasing pathogenicity of a plant pathogen may be used, for example, to kill or reduce the growth of a plant such as a weed. In certain embodiments, the growth, activity or lifespan of the plant pathogen may be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% relative to a plant pathogen that is not treated with the modified polyethyleneimine-based formulation. In certain embodiments, the growth, activity or lifespan of the plant pathogen may be increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000% relative to a plant pathogen that is not treated with the modified polyethyleneimine-based formulation. [0201] Target Organisms [0202] In certain embodiments, the target organism is any organism in which one or more traits is modulated by the active agent. In certain embodiments, a target organism is also a host organism, as described herein below. For example, in some embodiments, the target organism is an organism comprising one or more genes that is targeted by an oligonucleotide or polynucleotide active agent. In some embodiments, the target organism is a plant in which one or more yield-related traits is improved by the active agent. In some embodiments, the target organism is a beneficial insect whose growth, fecundity, or disease resistance is improved by the active agent. In certain embodiments, the target organisms are plant pests or pathogens whose damage to the plant can be reduced or eliminated by active agents according to the invention. Examples of plant pests and pathogens include, but are not limited to, insects, nematodes, fungi, bacteria, viruses, and parasitic plants such as striga, dodder, and mistletoe. Insect pests that may be targeted according to the invention include, but are not limited to, chewing, sucking, and boring insects that belong, for example, to the non-limiting Orders Coleoptera, Diptera, Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera, and Orthoptera. [0203] Insects [0204] In some embodiments the composition may be taken up by an insect by direct contact with the composition, for example, by topical adsorption or inhalation of the composition or by direct feeding on a bait comprising the composition, as described below. The compositions may also be taken up by the insect by direct feeding on a plant that has been treated with the composition. Examples of insect pests that may be targeted by the invention include, but are not limited to, those provided in Table 2.
[0205] Table 2.
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
[0206] Nematodes
[0207] Examples of nematodes that may be targeted include, but are not limited to, those provided in Table 3.
[0208] Table 3.
Figure imgf000089_0001
Figure imgf000090_0002
[0209] Fungi
[0210] Examples of fungi that may be targeted include, but are not limited to, those provided in Table 4.
[0211] Table 4.
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0002
[0212] Bacteria
[0213] Examples of bacteria that may be targeted include, but are not limited to, those shown in Table 5.
[0214] Table 5.
Figure imgf000095_0001
Figure imgf000096_0002
[0215] Viruses
[0216] Examples of plant viruses that may be targeted include, but are not limited to, those shown in the Table 6.
[0217] Table 6.
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
[0218] Weeds [0219] In certain embodiments, the target organism is a weed. As used herein, the term“weed” refers to any unwanted plant. The weed to be controlled may include monocotyledonous species, such as species of the genus Agrostis, Alopecurus, Avena, Bromus, Cyperus, Digitaria, Echinochloa, Lolium, Monochoria, Rottboellia, Sagittaria, Scirpus, Setaria, Sida or Sorghum, and dicotyledonous species, for example species of the genus Abutilon, Amaranthus, Chenopodium, Chrysanthemum, Conyza, Galium, Ipomoea, Nasturtium, Sinapis, Solanum, Stellaria, Veronica, Viola or Xanthium. Weeds can also include plants which may be considered crop plants but which are growing outside a crop area (escapes), or which grow from seed left over from a previous planting of a different crop (volunteers). Such volunteers or escapes may be tolerant to certain other herbicides. [0220] It has been demonstrated that several agriculturally relevant traits in plants can be modified via the introduction of transgenes that target the silencing of specific genes, including soybean oil composition and corn kernel protein composition. dsRNAs targeting specific genes in specific species can be applied topically to alter plant traits as well, and in some cases, offers the farmer more flexibility with regard to timing and endurance of application. In certain embodiments the compositions of the present invention may be used to enhance a yield-related trait in a plant. Yield-related traits that may be enhanced by the compositions of the present invention include, but are not limited to, total seed germination, rate of seed germination, plant biomass, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, root growth, early vigor, plant biomass, plant size, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, and leaf number. [0221] Crop Plants [0222] Representative crop plants that may be target organisms include
monocotyledonous and dicotyledonous plants including but not limited to fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from Acer spp., Allium spp., Amaranthus spp., Ananas comosus, Apium graveolens, Arachis spp, Asparagus officinalis, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Camellia sinensis, Canna indica, Cannabis saliva, Capsicum spp., Castanea spp., Cichorium endivia, Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucumis spp., Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Lycopersicon spp. (e.g., Lycopersicon esculenturn, Lycopersicon lycopersicum,
Lycopersicon pyriforme), Malus spp., Medicago sativa, Mentha spp., Miscanthus sinensis, Morus nigra, Musa spp., Nicotiana spp., Olea spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Petroselinum crispum, Phaseolus spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prunus spp., Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp. (e.g., Solanum tuberosum, Solanum integrifolium or
Solanum lycopersicum), Sorghum bicolor, Sorghum halepense, Spinacia spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., and Zea mays. Especially preferred are rice, oilseed rape, canola, soybean, corn (maize), cotton, sugarcane, alfalfa, sorghum, and wheat. [0223] In certain embodiments, a target gene of interest my also include a gene that is essential to the survival of an organism, such as a weed, insect, or plant pathogen, and can serve as a target for controlling growth and proliferation of the organism. For example, dsRNA-mediated silencing of an essential gene in an insect pest can induce cessation of feeding and ultimately growth inhibition, morbidity, or mortality. Recent studies have shown that certain coleopteran insect species, most notably the western corn rootworm, Diabrotica virgifera virgifera, are exquisitely sensitive to ingested dsRNAs. Highly efficacious dsRNAs yield LC50 values in the parts-per-billion (ppb or ng/ml) range with this species. RNAi provides a unique mode of action for the control of insect pests that could complement the current strategy of expressing Bacillus thuringiensis insecticidal proteins in plants of agricultural importance. In certain embodimetnts, dsRNAs targeting essential insect genes can be delivered via topical sprays for RNAi-mediated insect control. [0224] Non-Target Organisms [0225] In some embodiments, the compositions of the invention may be applied to an organism that is different from the target organism. For example, in some embodiments the target organism is an insect, and the composition is applied to a non-target organism, such as a plant, that is a host for the insect. As used herein, a "non-target organism” is any organism other than the target organism. Where the target organism and host organism differ, a non- target organism can comprise a host organism and organisms that consume the host organism or otherwise contact polynucleotides (e.g., siRNAs or antisense polynucleotides) or proteins expressed in a host organism. The target-specific design of polynucleotides such as RNAi and antisense polynucleotides, as described herein, provides that such polynucleotides have little or no gene silencing activity in non-target organisms. [0226] Non-target organisms include crop plants that may be infected with a target organism, such as a plant pathogen or insect. Representative crop plants include
monocotyledonous and dicotyledonous plants including but not limited to fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from Acer spp., Allium spp., Amaranthus spp., Ananas comosus, Apium graveolens, Arachis spp, Asparagus officinalis, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Camellia sinensis, Canna indica, Cannabis saliva, Capsicum spp., Castanea spp., Cichorium endivia, Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucumis spp., Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Lycopersicon spp. (e.g., Lycopersicon esculenturn, Lycopersicon lycopersicum, Lycopersicon pyriforme), Malus spp., Medicago sativa, Mentha spp., Miscanthus sinensis, Morus nigra, Musa spp., Nicotiana spp., Olea spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Petroselinum crispum, Phaseolus spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prunus spp., Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp. (e.g., Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Sorghum halepense, Spinacia spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., and Zea mays. Especially preferred are rice, oilseed rape, canola, soybean, corn (maize), cotton, sugarcane, alfalfa, sorghum, and wheat. [0227] Application of the Formulations [0228] In certain embodiments, the presently disclosed formulations can be applied as a spray or powder to the plant, plant part, seed, a pest, or an area of cultivation. The presently disclosed formulations may also be applied as concentrated emulsions, dusts, emulsifiable concentrates, fumigants, gels, granules, seed treatments, suspension concentrates, suspoemulsions, tablets, water soluble liquids, water dispersible granules or dry flowables, wettable powders, and ultra-low volume solutions. For further information on formulation types see“Catalogue of Pesticide Formulation Types and International Coding System” Technical Monograph No.2, 5th Edition by CropLife International (2002), which is incorporated herein by reference in its entirety. Agricultural formulations are also described, for example, in U.S. Pat. No. 8,815,27, which is incorporated herein by reference in its entirety. . [0229] For example, the presently disclosed formulations may be applied as aqueous suspensions or emulsions prepared from concentrated formulations. Such water-soluble, water-suspendable, or emulsifiable formulations can either be solids, usually known as wettable powders, or water dispersible granules, or liquids usually known as emulsifiable concentrates, or aqueous suspensions. Wettable powders, which may be compacted to form water dispersible granules, comprise an intimate mixture of the composition, a carrier, and surfactants. The carrier may be selected from attapulgite clays, montmorillonite clays, diatomaceous earths, and purified silicates. Effective surfactants, comprising from about 0.5% to about 10% of the wettable powder, include sulfonated lignins, condensed
naphthalenesulfonates, naphthalenesulfonates, alkylbenzenesulfonates, alkyl sulfates, and non-ionic surfactants such as ethylene oxide adducts of alkyl phenols. [0230] Emulsifiable concentrates can comprise a suitable concentration of the formulation, such as from about 50 to about 500 grams per liter of liquid dissolved in a carrier that is either a water-miscible solvent or a mixture of water-immiscible organic solvent and emulsifiers. Suitable organic solvents include aromatics, especially xylenes and petroleum fractions, especially the high-boiling naphthalenic and olefinic portions of petroleum, such as heavy aromatic naphtha. Other organic solvents may also be used, such as the terpenic solvents, including rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols such as 2-ethoxyethanol. Suitable emulsifiers for emulsifiable concentrates can be selected from conventional anionic and non-ionic surfactants. [0231] Aqueous suspensions comprise suspensions of water-insoluble forms of the presently disclosed formulations dispersed in an aqueous carrier at a concentration in the range from about 5% to about 50% by weight. Ingredients, such as inorganic salts and synthetic or natural gums, may also be added to increase the density and viscosity of the aqueous carrier. [0232] The presently disclosed formulations may also be applied as granular formulations, for example, for applications to the soil. Granular formulations may contain from about 0.5% to about 10% by weight of the composition, dispersed in a carrier that comprises clay or a similar substance. Such formulations may be prepared by dissolving the formulation in a suitable solvent and applying it to a granular carrier which has been pre- formed to a suitable particle size, for example, in the range of from about 0.5 to about 3 mm. Such formulations may also be prepared by making a dough or paste of the carrier and compound and crushing and drying to obtain the desired granular particle size. [0233] Dusts comprising the presently disclosed formulations may be prepared by intimately mixing the formulation in powdered form with a suitable dusty agricultural carrier, such as kaolin clay, ground volcanic rock, and the like. Dusts may contain from about 1% to about 10% by weight of the composition. They may be applied as a seed dressing or as a foliage application with a dust blower machine. [0234] The presently disclosed formulations may also be applied in the form of a solution in an appropriate organic solvent (e.g., petroleum oil), such as the spray oils, which are widely used in agricultural chemistry. [0235] The presently disclosed formulations may also be applied in the form of an aerosol composition. The formulation can be dissolved or dispersed in a carrier, which is a pressure-generating propellant mixture. The aerosol composition is packaged in a container from which the mixture is dispensed through an atomizing valve. [0236] The presently disclosed formulations may be applied to the crop area or plant to be treated, simultaneously or in succession, with further compounds. These further compounds can be, for example, fertilizers or micronutrient donors or other preparations, which influence the growth of plants. They can also be selective herbicides or non-selective herbicides as well as insecticides, fungicides, bactericides, nematicides, or mixtures of several of these preparations, if desired together with further carriers, surfactants, or application promoting adjuvants customarily employed in the art of formulation. [0237] Therapeutic Applications [0238] In another aspect, the presently disclosed aminolipidoid formulations can be used to deliver a therapeutic agent to a target in a subject for the purpose of treating or preventing a disease or disorder. In certain embodiments, the presently disclosed
aminolipidoid formulations are combined with a pharmaceutically acceptable excipient and/or carrier to form a pharmaceutical formulation. In certain embodiments, the disease or disorder is treated or prevented by administering a therapeutically effective amount of the pharmaceutical formulation to a subject in need thereof. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. [0239] In certain embodiments, the presently disclosed aminolipidoid formulations, i.e., complexes, microparticles, nanoparticles, picoparticles, liposomes, and micelles, are combined with one or more pharmaceutically acceptable excipients and/or carriers to form pharmaceutical formulations suitable to administer to mammals, including humans.
Examples of classes of such excipients and carriers include, but are not limited to, fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, and coating permeability adjusters. Excipients and/or carriers may comprise about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or higher percentage of the presently disclosed pharmaceutical formulations. [0240] The presently disclosed pharmaceutical compositions can be administered to mammals, including humans, by any conventional route. Examples of such routes include, but are not limited to, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically, bucally, or as an oral or nasal spray. Dosage forms for oral administration include, but are not limited to, solid and liquid dosage forms. Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. Liquid dosage forms for oral administration include, but are not limited to, emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Dosage forms for topical administration include, but are not limited to, ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and transdermal patches. [0241] As used herein, "parenteral administration" and "administered parenterally” means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. [0242] Although the dosage will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage of from 0.01 to 2000 mg of the therapeutic agent is recommended for an adult human patient, and this may be administered in a single dose or in divided doses. [0243] As used herein, the term "therapeutic agent" includes any synthetic or naturally occurring biologically active compound or composition which, when administered to subject, induces a desired pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, and gene constructs. More particularly, the term "therapeutic agent" includes compounds or compositions for use in all of the major therapeutic areas. [0244] Examples of such therapeutic agents include, but are not limited to, nucleic acids, adjuvants, anti-infectives such as antibiotics and antiviral agents, analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics,
antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics,
antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators, central nervous system stimulants, cough and cold preparations, decongestants, diagnostics, hormones, bone growth stimulants and bone resorption inhibitors, oncology drugs (e.g., chemotherapy drugs, hormonal therapeutic agents, immunotherapeutic agents, radiotherapeutic agents), lipid-lowering agents, antidepressants, stimulants, antibiotics, birth control medication, anti-angiogenics, cytovascular agents, signal
transduction inhibitors, hormones, vasoconstrictors, and steroids. immunosuppressives, muscle relaxants, psychostimulants, sedatives, tranquilizers, proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced), small molecules and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications. The term therapeutic agent also includes without limitation, medicaments, vitamins; mineral supplements, substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, substances which affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment. These therapeutic agents may be administered alone with pharmaceutical formulations or in combination (e.g., co-administered) with pharmaceutical formulations comprising nucleic acid, such as interfering RNA. [0245] Examples of nucleic acids include, but are not limited to, interfering RNA molecules (e.g., siRNA, aiRNA, miRNA), antisense oligonucleotides, plasmids, ribozymes, immunostimulatory oligonucleotides, and mixtures thereof. [0246] Nucleic acids delivered as the therapeutic agent in the presently disclosed pharmaceutical formulations can be used to downregulate or silence the translation (i.e., expression) of a target gene of interest in the target organism. Examples of classes of genes that can be downregulated or silenced include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. Examples viral sequences that can be downregulated or silenced include filoviruses such as Ebola virus and Marburg virus, arenaviruses such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus, influenza viruses such as Influenza A, B, and C viruses, hepatitis viruses, Human Immunodeficiency Virus (HIV), herpes viruses, and Human Papilloma Viruses (HPV). [0247] Examples of peptides or polypeptide that may be used as therapeutic agents include, but are not limited to, an antibodies such as a polyclonal antibodies, a monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, Primatized™ antibodies, cytokines, growth factors, apoptotic factors, differentiation-inducing factors, cell-surface receptors, ligands, hormones, or small molecules. [0248] Examples of oncology drugs that may be used as therapeutic agents include, but are not limited to, alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosine arabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin, interferon, letrozole, Leustatin, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone, rituxan, streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene, tretinoin, ATRA, valrubicin, velban, ellipticin and ellipticin analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecins. [0249] Examples of anti-viral drugs that may be used as therapeutic agents include, but are not limited to, abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III (e.g., IFN-λ molecules such as IFN-λ1, IFN-λ2, and IFN-λ3), interferon type II (e.g., IFN-γ), interferon type I (e.g., IFN-α such as PEGylated IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ, interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine. [0250] In certain embodiments, the therapeutic agent to be delivered may be a prophylactic agent. Prophylactic agents include, but are not limited to, antibiotics, nutritional supplements, and vaccines. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, and cell extracts. Prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, and Freund's adjuvant. [0251] Examples of prophylactic agents include, but are not limited to, (1) antigens of the following bacterial organisms: Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,
Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, and Camphylobacter jejuni, (2) antigens of the following viruses: smallpox, influenza A and B, respiratory syncytial virus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and hepatitis A, B, C, D, and E virus, and (3) antigens of the following fungal, protozoan, and parasitic organisms: Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma
pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,
Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni. These antigens may be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof. [0252] The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions. EXAMPLES [0253] Example 17– General Procedure for Nanoparticle Formulation of Active Agents with Aminolipidoids [0254] Stock ethanolic solutions of one or more aminolipidoids of the present disclosure, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG) are prepared and combined to yield a molar ratio of 50:10:38.5:1.5 with the organic solution of 10% 10 mM citrate buffer at pH 5. An siRNA is dissolved in 10 mM citrate at pH5 buffer at a concentration of 0.1 mg/mL. The ethanolic solution is then added to the aqueous siRNA solution while stirring at 700 rpm to resultin the precipitation of aminolipidoid nanoparticles. The aminolipidoid nanoparticles are separated from the supernatant. Particle size of the aminolipidoid nanoparticles is determined by Dynamic Light Scattering. [0255] Example 18– General Procedure for Evaluating Lipidoid Formulations is Insect Feeding Assays [0256] Lipidoid formulations of the present disclosure can be evaluated in insect feeding assays to determine their efficacy in RNA delivery to an insect cell. Two model insects are used: western tarnished plant bug (WTPB, Lygus hesperus) and tarnished plant bug (TPB, Lygus lineolaris). Each aminolipidoid of the present disclosure to be evaluated is separately formulated with an siRNA that targets an essential gene in TPB and an siRNA that targets an essential gene in WTPB according to the general procedure described above in Example 4. The feeding assay employed is based on a 96 well format and a sachet system as described by Habibi et al. (2002, Archives of Insect Biochem. and Phys. 50: 62-74) and U.S. Patent No. 8,609,936, each of which is incorporated herein by reference in their entireties. The insect artificial diet is commercially available from Bio-ServTM (Bio-ServTM Diet F9644B, Frenchtown, N.J.). [0257] Autoclaved boiling water is combined with Bio-Serv® Diet F9644B in a surface sterilized blender. Four surface sterilized chicken eggs are broken and the contents are added to the blender containing the diet mix. The mixture is blended until smooth and adjusted to one liter of volume and allowed to cool. Feeding samples are prepared by mixing the siRNA formulations described above in the desired concentration with an equivalent volume of the blended diet. [0258] A sheet of Parafilm® (Pechiney Plastic Packing, Chicago, Ill.) is placed over a 96-well format vacuum manifold with a vacuum of approximately -20 millimeters mercury, which is sufficient to cause extrusion of the Parafilm® into the wells. Forty microliters of test sample are added to the Parafilm® wells. A sheet of Mylar film (Clear Lam Packaging, Inc., Elk Grove Village, Ill.) is then placed over the Parafilm® and sealed gently with a tacking iron (Bienfang Sealector II, Hunt Corporation, Philadelphia, Pa.). The Parafilm® sachets are then placed over a flat-bottom 96-well plate containing the Lygus eggs suspended in agarose. Upon hatching, Lygus nymphs will feed by piercing the sachet that is presented above them. Insect diet sachets are replaced on days two and four. Stunting and mortality scores are determined on day 5 and compared to the untreated controls. Those siRNA formulations that significantly increase stunting and mortality relative to the untreated controls demonstrate that the formulations are effective in delivering the siRNAs to the insect cells.

Claims

CLAIMS 1. A compound of formula (I):
Figure imgf000118_0001
wherein
X is an oxygen-containing core derived from an oxidized amine comprising (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups;
R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000118_0002
wherein
R5 and R6
are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
with the proviso that R5 and R6 cannot both be hydrogen; R' is identically or different in each instance hydrogen, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or
heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000119_0001
wherein
R5 and R6
are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
with the proviso that R5 and R6 cannot both be hydrogen; or a covalent bond to X; and
n is an integer in the range of from 2 to 10;
with the proviso that X is not derived from 1,10-diaza-18-crown-6, and
with the proviso that the compound of formula (I) is not one of the following compounds:
Figure imgf000120_0001
2. The compound of claim 1, wherein X is derived from an oxidized amine comprising at least two hydroxyl groups.
3. The compound of claim 2, wherein the oxidized amine comprises three hydroxyl groups.
4. The compound of claim 1, wherein X is derived from an oxidized amine comprising at least two ether groups.
5. The compound of claim 4, wherein the oxidized amine comprises three, four, or five ether groups.
6. The compound of claim 1, wherein X is derived from an oxidized amine comprising at least one hydroxyl group and at least one ether group.
7. The compound of claim 1, wherein X is derived from an oxidized amine comprising at least two primary amino groups. 8. The compound of claim 1, wherein X is derived from an oxidized amine comprising at least one primary amino group and at least one secondary amino group. 9. The compound of claim 1, wherein X is derived from an oxidized amine selected from the group consisting of compounds of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21), and (22):
Figure imgf000121_0001
Figure imgf000122_0001
10. The compound of claim 1, wherein R is an optionally substituted aliphatic or
heteroaliphatic group.
11. The compound of claim 10, wherein R' is an optionally substituted aliphatic or
heteroaliphatic group.
12. The compound of claim 11, wherein the optionally substituted aliphatic or
heteroaliphatic group comprises at least one double bond.
13. The compound of claim 11, wherein the optionally substituted heteroaliphatic group comprises at least one ether group.
14. The compound of claim 11, wherein the optionally substituted aliphatic group is a C6 to C20 aliphatic group.
15. The compound of claim 1, wherein R and R' are independently selected from the group consisting of moieties of formulae 23-41:
Figure imgf000122_0002
Figure imgf000123_0001
.
16. The compound of claim 1, wherein R is a group of formula (II), wherein R5 is H and R6 is an optionally substituted aliphatic or heteroaliphatic group.
17. The compound of claim 16, wherein R' is a group of formula (II), wherein R5 is H and R6 is an optionally substituted aliphatic or heteroaliphatic group.
18. The compound of claim 17, wherein the optionally substituted aliphatic or
heteroaliphatic group comprises at least one double bond.
19. The compound of claim 17, wherein the optionally substituted heteroaliphatic group comprises at least one ether group.
20. The compound of claim 17, wherein the optionally substituted aliphatic or
heteroaliphatic group is substituted with at least one fluorine atom.
21. The compound of claim 17, wherein the optionally substituted aliphatic or
heteroaliphatic group is substituted with at least one trialkoxysilane group.
22. The compound of claim 1, wherein R and R' are independently selected from the group consisting of moieties of formulae 42-60:
Figure imgf000124_0001
Figure imgf000124_0002
Figure imgf000125_0001
.
23. The compound of claim 1, wherein the compound is selected from the group
consisting of compounds of formulae 61 through 186:
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
wherein
Figure imgf000132_0002
Figure imgf000133_0001
Figure imgf000134_0001
.
24. A formulation comprising a compound of formula (I):
Figure imgf000134_0002
wherein
X is an oxygen-containing core derived from an oxidized amine comprising (a) at least one hydroxyl group, at least one ether group, or at least one hydroxyl group and at least one ether group, and (b) at least one primary amino group or at least two secondary amino groups;
R is, identically or differently in each instance, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000134_0003
wherein
R5 and R6
are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group; with the proviso that R5 and R6 cannot both be hydrogen;
R' is identically or different in each instance hydrogen, an optionally substituted aliphatic or cycloaliphatic group, wherein the aliphatic or cycloaliphatic group optionally further comprises an ester or amide linkage in the aliphatic or cycloaliphatic chain, an optionally substituted heteroaliphatic or
heterocycloaliphatic group, an optionally substituted aralkyl group, an optionally substituted aryl group, or a group of formula (II):
Figure imgf000135_0001
wherein
R5 and R6
are each selected from the group consisting of hydrogen, an optionally substituted aliphatic or cycloaliphatic group, an optionally substituted heteroaliphatic or heterocycloaliphatic group, an optionally substituted aralkyl group, or an optionally substituted aryl group;
with the proviso that R5 and R6 cannot both be hydrogen; or a covalent bond to X; and
n is an integer in the range of from 2 to 10;
and a first active agent to be delivered, and
with the proviso that the compound of formula (I) is not one of the following compounds:
Figure imgf000135_0002
,
Figure imgf000136_0001
25. The formulation of claim 24, further comprising at least one additional active agent to be delivered.
26. The formulation of claim 25, wherein the at least one additional active agent to be delivered is contained within or on the surface of the non-covalent complex.
27. The formulation of claim 25, wherein the at least one additional active agent to be delivered is not contained within or on the surface of the non-covalent complex.
28. The formulation of claim 24, further comprising one or more excipients.
29. The formulation of claim 28, wherein the one or more excipients is selected from the group consisting of fillers, extenders, binders, humectants, disintegrants, plasticizers, stabilizers, solution retarding agents, wetting agents, suspending agents, thickening agents, absorbents, lubricants, surfactants, buffering agents, diluents, solvents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, opacifying agents, separating agents, coating permeability adjusters, and combinations thereof.
30. The formulation of claim 28, wherein the one or more excipients is selected from the group consisting of carbohydrates, proteins, lipids, water-soluble polymers, and any combination thereof.
31. The formulation of claim 30, wherein the one or more water soluble polymers
comprises a polyethylene glycol, a polypropylene oxide, a polyvinylpyrrolidone, a polyvinyl alcohol, polylactic acid, poly(lactic-co-glycolic acid), or any combination thereof.
32. The formulation of claim 24, further comprising an adjuvant selected from the group consisting of chloroquine, chlorpromazine, amodiaquine, perphenazine, coronatine, tolbutamide, glyburide, glybenclamide, arginine, lysine, and histidine.
33. The formulation of claim 24, wherein the first active agent to be delivered is an
oligonucleotide or a polynucleotide.
34. The formulation of claim 33, further comprising an agriculturally acceptable carrier.
35. The formulation of claim 34, wherein the oligonucleotide or polynucleotide
modulates the expression of a gene in a plant.
36. The formulation of claim 34, wherein the oligonucleotide or polynucleotide
modulates the expression of a gene in an insect.
37. The formulation of claim 34, wherein the oligonucleotide or polynucleotide
modulates the expression of a gene in a plant pathogen.
38. The formulation of claim 25, wherein the at least one additional active agent is
selected from the group consisting of an herbicide, an insecticide, a fungicide, a nematicide, a bactericide, a viricide, and any combination thereof.
39. The formulation of claim 24, wherein the formulation is in the form of a microparticle or nanoparticle.
40. The formulation of claim 39, wherein the active agent to be delivered is selected from the group consisting of polynucleotides, oligonucleotides, proteins, peptides, and small molecules.
41. The formulation of claim 40, wherein the active agent to be delivered is an oligonucleotide or a polynucleotide.
42. The formulation of claim 41, wherein the oligonucleotide or polynucleotide is
modified.
43. The formulation of claim 41, wherein the oligonucleotide or polynucleotide is
unmodified.
44. The formulation of claim 41, wherein the active agent to be delivered is an RNA.
45. The formulation of claim 44, wherein the RNA is a single-stranded RNA.
46. The formulation of claim 44, wherein the RNA is a double-stranded RNA.
47. The formulation of claim 44, wherein the RNA is an siRNA.
48. The formulation of claim 44, wherein the RNA is an mRNA.
49. A method of regulating expression of a gene in a target organism, comprising
applying the formulation of any one of claims 24-48 to the target organism.
50. A method of modulating a trait of a plant, comprising delivering to the plant an
effective amount of the formulation of claim 35.
51. The method of claim 50, wherein the trait is selected from the group consisting of total seed germination, rate of seed germination, disease tolerance, insect tolerance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, tolerance to heavy metals, total yield, seed yield, fruit yield, root growth, early vigor, plant growth, plant biomass, plant size, plant lifespan, total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number, leaf number, fruit size, fruit freshness, fruit ripening time, fruit nutritional content, plant nutritional content, plant sensitivity to herbicide, and any combination thereof.
52. The method of claim 51, wherein one or more of the traits is improved relative to a plant not treated with the formulation.
53. The method of claim 52, wherein at least one trait selected from the group consisting of plant growth, plant lifespan, plant size, fruit size, fruit yield, total yield, fruit freshness, fruit ripening time, plant nutritional content, and fruit nutritional content, is improved relative to a plant not treated with the formulation.
54. The method of claim 51, wherein one or more of the traits is decreased relative to a plant not treated with the formulation.
55. The method of claim 54, wherein the plant growth and/or the plant lifespan is
decreased relative to a plant not treated with the formulation.
56. The method of claim 54, wherein the fruit ripening time is decreased relative to a plant not treated with the formulation.
57. The method of claim 51, wherein the plant sensitivity to herbicide is increased relative to a plant not treated with the formulation.
58. A method of modulating a trait of an insect, comprising delivering an effective
amount of the formulation of claim 36 to the insect, to a plant infested with the insect, or to a plant prior to infestation with the insect.
59. The method of claim 58, wherein the trait modulated is insect growth, development, and/or lifespan.
60. A method of modulating the pathogenicity of a plant pathogen, comprising applying the formulation of claim 37 to the plant pathogen, to a plant infected with the plant pathogen, or to a plant prior to infection with the plant pathogen.
61. A plant cell, insect cell, fungal cell, nematodic cell, or bacterial cell comprising the formulation of claim 24.
62. The formulation of claim 24, wherein X is derived from 1,10-diaza-18-crown-6.
63. The formulation of claim 62, wherein R and R' are each, independently, an optionally substituted aliphatic or heteroaliphatic or group.
64. The formulation of claim 63, wherein the compound of formula (I) is a compound of formula (III):
Figure imgf000140_0001
wherein R1 and R2 are independently selected from the group consisting of hydrogen or a moiety of formula 26 through 29, 32, 34, 35, 37, 40, 41, 46, 47, 50 through 52, 60, and 187 through 191:
Figure imgf000140_0002
Figure imgf000141_0001
.
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