WO2020263776A1 - Nucleic acid compositions including a polyalkyleneimine-alkoxylene polymer and methods of making and using the same - Google Patents

Nucleic acid compositions including a polyalkyleneimine-alkoxylene polymer and methods of making and using the same Download PDF

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Publication number
WO2020263776A1
WO2020263776A1 PCT/US2020/039054 US2020039054W WO2020263776A1 WO 2020263776 A1 WO2020263776 A1 WO 2020263776A1 US 2020039054 W US2020039054 W US 2020039054W WO 2020263776 A1 WO2020263776 A1 WO 2020263776A1
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WIPO (PCT)
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polymer
mol
polyalkyleneimine
alkoxylene
nucleic acid
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PCT/US2020/039054
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French (fr)
Inventor
Rodolphe Barrangou
Matthew A. NETHERY
Claudio Hidalgo-Cantabrana
Paul Bernasconi
Marianela RODRIGUEZ
Melissa Ann LAMSON
Frank Reinhold
David DE VLEESSCHAUWER
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North Carolina State University
Basf Plant Science Company Gmbh
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Application filed by North Carolina State University, Basf Plant Science Company Gmbh filed Critical North Carolina State University
Publication of WO2020263776A1 publication Critical patent/WO2020263776A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • NUCLEIC ACID COMPOSITIONS INCLUDING A POLYALKYLENEIMINE- ALKOXYLENE POLYMER AND METHODS OF MAKING AND USING THE SAME
  • the present invention relates generally to nucleic acid compositions including a polyalkyleneimine-alkoxylene polymer such as a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer.
  • a polyalkyleneimine-alkoxylene polymer such as a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer.
  • the present invention also relates to methods of making and using a composition comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) including for use in modifying a genome of an organism and the killing of cells.
  • Embodiments of the invention provide compositions comprising a polyalkyleneimine- alkoxylene polymer (e.g., a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer) and a nucleic acid molecule, optionally wherein the polyalkyleneimine-alkoxylene polymer is complexed with the nucleic acid molecule.
  • a polyalkyleneimine- alkoxylene polymer e.g., a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer
  • PEO polyethyleneimine
  • Embodiments of the invention provide methods of preparing a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)- polyethylene oxide (PEO) polymer) and a nucleic acid molecule, the methods comprising: combining the nucleic acid molecule and the polymer, thereby preparing the composition, optionally wherein the polymer is complexed with the nucleic acid molecule.
  • a polyalkyleneimine-alkoxylene polymer e.g., a polyethyleneimine (PEI)- polyethylene oxide (PEO) polymer
  • PEO polyethylene oxide
  • Embodiments of the invention provide methods of delivering a nucleic acid molecule to (transforming) a cell, comprising contacting the cell with a composition of the invention comprising a polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)- polyethylene oxide (PEO) polymer) and a nucleic acid molecule, thereby delivering the nucleic acid molecule in the composition to the cell.
  • a polyalkyleneimine-alkoxylene polymer e.g., a polyethyleneimine (PEI)- polyethylene oxide (PEO) polymer
  • PEI polyethyleneimine
  • PEO polyethylene oxide
  • Fig. 2 shows chemical-based delivery of DNA using polymers 1, 3, 7, and 9.
  • Plasmid DNA packaged within each polymer, encoding enhanced green fluorescent protein (eGFP) can be delivered by mixing with wheat protoplast cells.
  • Micrographs depicting representative GFP signals were taken two days post transfection.
  • As a positive control cells were transfected with the GFP reporter plasmid in the presence of polyethylene glycol (PEG).
  • Transfection efficiencies were calculated by counting the number of GFP expressing cells and dividing by the number of living cells. Data presented are means of three independent experiments.
  • Fig. 3A-3B shows transfection of the cells using polymers of the invention.
  • Fig. 3A shows transformation efficiency using different amounts of DNA.
  • Fig. 3B shows transformation efficiencies using different amounts of polymer (polymer 9). Transfection efficiencies were calculated by counting the number of GFP expressing cells and dividing by the number of living cells. Data presented are means of two independent experiments. Photographs depicting representative efficiencies were taken two days post transfection. Positive control cells were transfected using polyethylene glycol and negative controls were treated with plasmid DNA only.
  • a measurable value such as an amount or concentration and the like
  • a measurable value such as an amount or concentration and the like
  • about X where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measureable value may include any other range and/or individual value therein.
  • nucleic acid refers to RNA or DNA that is linear, circular or branched, single or double stranded, or a hybrid thereof.
  • the terms also encompass RNA/DNA hybrids.
  • the RNA and/or DNA can include a chemically modified base such as those that are not usually found in nature.
  • dsRNA When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others may be used such as for antisense, dsRNA, and ribozyme pairing.
  • polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression.
  • Other modifications such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.
  • nucleic acid sequence As used herein, the terms“polynucleotide” “nucleotide sequence,”“nucleic acid molecule” and“nucleic acid sequence” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA and/or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded and/or double stranded.
  • nucleotide sequence nucleic acid, nucleic acid molecule, oligonucleotide and polynucleotide may also be used to refer to a heteropolymer of nucleotides.
  • Nucleic acid molecules and/or nucleotide sequences provided herein are presented in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR ⁇ 1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.
  • a 5' region as used herein refers to the region of a polynucleotide that is nearest the 5' end.
  • an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.
  • a 3' region as used herein refers to the region of a polynucleotide that is nearest the 3' end.
  • an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.
  • the term“gene” refers to a nucleic acid molecule capable of being used to produce mRNA, tRNA, rRNA, miRNA, anti-microRNA, regulatory RNA, and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions). A gene may be isolated by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
  • a synthetic nucleic acid or nucleotide sequence refers to a nucleic acid or nucleotide sequence that is not found in nature but is constructed by human intervention and as a consequence is not a product of nature.
  • Alkyl as used herein alone or as part of another group refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms, which can be referred to as a Cl- C20 alkyl.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n- decyl, and the like.
  • Loweralkyl as used herein, is a subset of alkyl, and, in some embodiments, refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.
  • Representative examples of loweralkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like.
  • alkyl or loweralkyl is intended to include both substituted and unsubstituted alkyl or loweralkyl unless otherwise indicated and these groups may be substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(0) m , haloalkyl-S(0)m, alkenyl-S(0) m , alkynyl-S(0) m , cycloalkyl-
  • Alkenyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkenyl 1 to 4 carbon atoms) and includes 1 to 8 double bonds in the normal chain, and can be referred to as a C1-C20 alkenyl.
  • Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4- heptadiene, and the like.
  • alkenyl or loweralkenyl is intended to include both substituted and unsubstituted alkenyl or loweralkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and loweralkyl above.
  • Alkynyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include at least 1 triple bond in the normal chain, and can be referred to as a C1-C20 alkynyl.
  • Representative examples of alkynyl include, but are not limited to, 2- propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like.
  • alkynyl or loweralkynyl is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.
  • Halo as used herein refers to any suitable halogen, including -F, -Cl, -Br, and -I.
  • Mercapto as used herein refers to an -SH group.
  • Cyano as used herein refers to a -CN group.
  • Hydroxyl as used herein refers to an -OH group.
  • Nitro as used herein refers to an -NO2 group.
  • Alkoxy as used herein alone or as part of another group refers to an alkyl or loweralkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -0-.
  • Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
  • Acyl as used herein alone or as part of another group refers to a -C(0)R radical, where R is any suitable substituent such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl or other suitable substituent as described herein.
  • Haloalkyl as used herein alone or as part of another group refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.
  • Aryl as used herein alone or as part of another group refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings.
  • Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
  • the term aryl is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.
  • an aryl is substituted with an alkyl, alkenyl, and/or alkynyl.
  • Arylalkyl as used herein alone or as part of another group refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of arylalkyl include, but are not limited to, benzyl, 2- phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.
  • arylalkenyl and arylalkynyl as used herein alone or as part of another group refer to an aryl group, as defined herein, appended to the parent molecular moiety through an alkenyl group and alkynyl, respectively, each as defined herein.
  • Amino as used herein means the radical -NH2.
  • Alkylamino as used herein alone or as part of another group means the radical -NHR, where R is an alkyl group.
  • Ester as used herein alone or as part of another group refers to a -C(0)OR radical, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • Amide as used herein alone or as part of another group refers to a -C(0)NR a R b radical, where R a and R 3 ⁇ 4 are any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
  • Sulfonamide as used herein alone or as part of another group refers to a -S(0) 2 NR a R b radical, where R a and R b are any suitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.
  • the terms increase, increases, increased, increasing, enhance, and similar terms indicate an elevation in the specified parameter of at least about 2%, 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% or more unless otherwise specifically noted within the text.
  • the terms reduce, reduces, reduced, reduction, and similar terms refer to a decrease in the specified parameter of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% unless otherwise specifically noted within the text.
  • compositions comprising a polyalkyleneimine-alkoxylene polymer and a nucleic acid molecule.
  • a polyalkyleneimine-alkoxylene polymer of the present invention may also be referred to interchangeably herein as an alkoxylated polyethyleneimine.
  • the polyalkyleneimine is a polyethyleneimine and/or a polypropyleneimine.
  • the alkoxylene is one or more ethylene oxide, propylene oxide, and/or butylene oxide unit(s).
  • compositions comprising a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer and a nucleic acid molecule.
  • PEI-PEO may be referenced herein as an example, but other polyalkyleneimine-alkoxylene polymers within the scope of this invention may be used.
  • the polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • the composition may comprise and/or be in an aqueous solution.
  • the composition comprises and/or is a complex of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and at least one nucleic acid molecule.
  • a complex and grammatical variations thereof as used herein refer to a molecular entity comprising at least two components that are associated with one another.
  • the two or more components may be different (e.g., a PEI-PEO polymer and a nucleic acid) and/or one or more of the components may be charged (e.g., ionic) or uncharged.
  • a complex may be formed by covalent, non-covalent, and/or electrostatic interactions between the two or more components.
  • a complex comprises at least two different components that are covalently bonded together.
  • a complex comprises at least two different components that are associated with one another via electrostatic interaction(s).
  • a nucleic acid molecule is electrostatically bound and/or covalently bound to a portion of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
  • a composition of the present invention may comprise one or more (e.g., 1, 2, 5, 10, 50, 100, 1,000, 5,000, 10,000, or more) nucleic acid molecule(s) and/or one or more (e.g., 1, 2, 5, 10, 50, 100, 1,000, 5,000, 10,000, or more) polyalkyleneimine-alkoxylene polymer(s).
  • the composition comprises two or more nucleic acid molecules, which may be complexed with a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
  • a single polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer) is complexed with two or more nucleic acid molecules.
  • a single nucleic acid molecule is complexed with two or more polyalkyleneimine-alkoxylene polymers (e.g., PEI-PEO polymers).
  • a nucleic acid molecule may have any suitable length.
  • a nucleic acid molecule has a length that is not prohibitive of being delivered and/or transformed into a host cell.
  • a nucleic acid molecule has a length of about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 bases to about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilobases in length.
  • a nucleic acid molecule useful with this invention may be any nucleic acid or combination of nucleic acids (e.g, DNA, RNA, or any combination thereof) of interest for transforming the cell of an organism.
  • the nucleic acid molecule may be plasmid DNA, genomic DNA, plastid DNA, mitochondrial DNA, phage DNA, cDNA, mRNA, siRNA, shRNA, miRNA, piRNA, synthetic DNA, PCR amplicons, and/or antisense nucleic acid or any combination thereof.
  • the nucleic acid molecule may be a negatively charged peptide nucleic acid (PNA).
  • the nucleic acid may be any coding or noncoding nucleic acid (e.g., any transcribed and/or non-transcribed nucleic acids).
  • a nucleic acid molecule useful with this invention may be a nucleic acid molecule that is heterologous to the organism into which it is being introduced (e.g., a heterologous nucleic acid molecule or a heterologous polynucleotide) or it may be heterologous with regard to the position in the genome into which it is introduced.
  • heterologous refers to a nucleic acid molecule or nucleotide sequence that either originates from another species or is from the same species or organism but is modified from either its original form or its original position in the genome, or the form primarily expressed in the cell.
  • a polynucleotide derived from an organism or species different from that of the cell into which the polynucleotide is introduced is heterologous with respect to that cell and the cell's descendants.
  • a heterologous polynucleotide includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. present in a different copy number, and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule.
  • a nucleic acid molecule may comprise or be comprised in a vector, a nucleic acid construct, an expression cassette, and/or a plasmid.
  • a vector, a nucleic acid construct, an expression cassette, and/or a plasmid may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • a vector, a nucleic acid construct, an expression cassette, and/or a plasmid may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • Nucleic acid molecules/nucleic acid constructs, expression cassettes, plasmids and/or vectors useful with this invention may be included in a composition with a polymer of the present invention.
  • the nucleic acid molecules/nucleic acid constructs, expression cassettes, plasmids and/or vectors may be complexed with polymers of the present invention.
  • expression cassette means a heterologous nucleic acid construct comprising a nucleotide sequence of interest, wherein said nucleotide sequence of interest is operably associated with at least a control sequence (e.g., a promoter).
  • a control sequence e.g., a promoter
  • nucleic acid molecule, polynucleotide or nucleic acid construct of the present invention is generally free of nucleotide sequences that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid was derived (such as coding sequences present at the 5' or 3' ends).
  • nucleic acid molecule of this invention can include some additional bases or moieties that do not deleteriously or materially affect the basic structural and/or functional characteristics of the nucleic acid molecule.
  • an isolated nucleic acid molecule or isolated nucleotide sequence is a nucleic acid molecule or nucleotide sequence that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Accordingly, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence.
  • 5' non-coding e.g., promoter
  • the term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid nucleic acid molecule encoding an additional polypeptide or peptide sequence.
  • isolated can further refer to a nucleic acid molecule, polynucleotide, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and/or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized).
  • an isolated fragment is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found as such in the natural state. Isolated does not mean that the preparation is technically pure (homogeneous), but rather can be sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • an isolated nucleic acid molecule, nucleotide sequence, and/or polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% pure (w/w) or more.
  • an isolated nucleic acid, nucleotide sequence, and/or polypeptide indicates that at least about a 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or more enrichment of the nucleic acid (w/w) is achieved as compared with the starting material.
  • a nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer may be present in a composition in a weight ratio of about 1 : 10 to about 1 :600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer), or any range or value therein.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1 : 10, 1 :25, 1 :50, 1 :75, 1 : 100, 1 :250, 1 :500, 1 :750, 1 : 1,000, 1 :2,000, 1 :3,000, 1 :4,000, 1 :5,000, 1 :6,000, 1 :7,000, 1 :8,000, 1 :9,000, 1 : 10,000, 1:15,000, 1:20,000, 1:25,000, 1:30,000, 1:35,000, 1:40,000, 1:45,000, 1:50,000, 1:55,000, 1:60,000, 1:70,000, 1:80,000, 1:90,000, 1:100,000, 1:200,000, 1:300,000, 1:400,000, 1:500,000 or 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer), or any
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:10 to about 1:100, about 1:100 to about 1:1,000, about 1:1,000 to about 1:5,000, about 1:5,000 to about 1:10,000, about 1:5,000 to about 1:20,000, about 1:5,000 to about 1:50,000, about 1:5,000 to about 1:100,000, about 1:5,000 to about 1:200,000, about 1:5,000 to about 1:500,000, about 1:5,000 to about 1:600,000, about 1:10,000 to about 1:100,000, about 1:10,000 to about 1:600,000, about 1:30,000 to about 1:50,000, about 1:10,000 to about 1:50,000, about 1:200,000 to about 1:400,000, about 1:50,000 to about 1:500,000 or about 1:50,000 to about 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer).
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:5000 to about 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer).
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:10,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:20,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:100,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:600,000.
  • a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer are present in a composition in a weight ratio of about 1:5000 (nucleic acid molecule polyalkyleneimine-alkoxylene polymer).
  • a nucleic acid molecule is present in a composition of the invention in an amount of about 0.1 nanograms per milligram of the polyalkyleneimine- alkoxylene polymer to about 400 ng/mg of the polyalkyleneimine-alkoxylene polymer or more.
  • a nucleic acid molecule is present in a composition of the invention in an amount of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 nanograms per milligram of the polyalkyleneimine-alkoxylene polymer to about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 or more nanograms per milligram of the polyalkyleneimine- alkoxylene polymer.
  • Example compositions of the invention may comprise a nucleic acid molecule and polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) with the nucleic acid molecule present in an amount relative to 30 mg polymer as set forth in Table 1.
  • polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • about 100 mg PEI-PEO polymer may combined with 100 m ⁇ water to achieve a final volume of about 200 m ⁇ .
  • about 60 m ⁇ of polymer may be combined with DNA.
  • about 60 m ⁇ of this solution comprises about 30 mg of polymer.
  • a ratio of about 10 ng plasmid to 1 mg PEI-PEO polymer is achieved.
  • 1000 ng of plasmid DNA may be added to 30 mg of polymer (60 m ⁇ solution) to achieve ratio of about 33.3 ng plasmid to 1 mg PEI-PEO polymer.
  • 6000 ng of plasmid DNA may be added to 30 mg of polymer (60 m ⁇ solution), then the ratio that is achieved is about 50 ng plasmid in 1 mg PEI-PEO polymer or about 200 ng plasmid in 1 mg PEI-PEO polymer, respectively.
  • a polyalkyleneimine (e.g., PEI) polymer, polyalkoxylene (e.g., PEO) polymer and/or polyalkyleneimine-alkoxylene (e.g., PEI-PEO) polymer of the present invention may be and/or may be prepared as described in U.S. Patent Nos. 7,736,525 and 9,068,147 and/or International Publication No. WO 2017/102556, the contents of each of which are incorporated herein by reference in their entirety.
  • a polyalkyleneimine- alkoxylene polymer may be cationic.
  • a polyalkyleneimine-alkoxylene polymer has an overall cationic charge, but a portion of the polyalkyleneimine-alkoxylene polymer is nonionic and/or anionic, which may reduce the overall charge per weight of the polymer.
  • a portion of the polyalkyleneimine-alkoxylene polymer e.g., an outer portion and/or shell
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may have a zeta potential of at least about +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, or more, optionally when present in a composition having a pH of about 7 and/or measured using methods known to those of skill in the art.
  • a polyalkyleneimine-alkoxylene polymer e.g., a non-quatemized or quatemized PEI-PEO polymer
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a polyalkyleneimine-alkoxylene polymer of the present invention is water soluble, optionally at room temperature.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may have a PEI backbone and one or more alkoxylene unit(s) (e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)) substituted at one or more places (e.g., at one or more nitrogens) along the PEI backbone.
  • alkoxylene unit(s) e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)
  • the polyalkyleneimine-alkoxylene polymer of the present invention may have a polypropyleneimine backbone and one or more alkoxylene unit(s) (e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)) substituted at one or more places (e.g., at one or more nitrogens) along the polypropyleneimine backbone.
  • An alkoxylene unit may replace a hydrogen atom in the PEI or polypropyleneimine backbone (e.g., a hydrogen atom attached to a primary amine, secondary amine, and/or tertiary amine in the PEI backbone).
  • a hydrogen atom in the PEI or polypropyleneimine backbone (e.g., a hydrogen atom attached to a primary amine, secondary amine, and/or tertiary amine in the PEI backbone) is replaced with a single alkoxylene unit (i.e., a monoalkoxylene) or with a polyalkoxylene chain (e.g., a moiety comprising two or more alkoxylene units such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a monoalkoxylene or a polyalkoxylene chain having 2 or 3 to 4, 5, 6, or 8 alkoxylene units (e.g., ethylene oxide units).
  • a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a polyalkoxylene chain having 8, 9, or 10 to 11, 12, or 13 alkoxylene units (e.g., ethylene oxide units).
  • a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a polyalkoxylene chain having 16, 17, or 18 to 19, 20, 21, or 22 alkoxylene units (e.g., ethylene oxide units).
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may be a core-shell polymer.
  • the core comprises an interior portion of the polymer and the shell comprises an outer portion of the polymer.
  • the shell comprises end or terminal portions of a polymer chain.
  • the core comprises a portion or all of the PEI or polypropyleneimine of the polyalkyleneimine-alkoxylene polymer.
  • the core comprises a portion or all of the PEI of a PEI-PEO polymer.
  • the core (e.g., PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer) may have a diameter of about 1, 2, 4, 6, or 8 to about 10, 20, 30, 40, 50, 60, 70, or 80 nm such as about 1 nm to about 10 nm, about 5 nm to about 15 nm, about 20 to about 50 nm, 40 nm to about 80 nm, or 60 nm to about 70 nm.
  • the core has a diameter of about 5, 10, or 65 nm.
  • the shell comprises a portion or all of the alkoxylene (e.g., PEO) of the polyalkyleneimine- alkoxylene polymer.
  • the core comprises a portion or all of the PEI of a PEI-PEO and the shell comprises a portion or all of the PEO of the PEI-PEO polymer.
  • a shell and/or outer surface comprising alkoxylene may aid in controlling and/or reducing the toxicity of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) to a cell (e.g., a host cell and/or the cell in an organism) and/or may block and/or reduce exposure of charged moieties (e.g., cationic moieties) on the polymer.
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a particle e.g., a microparticle or nanoparticle
  • a sphere e.g., a sphere
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may comprise a number of alkoxylene units, such as ethylene oxide units, per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, or 50 alkoxylene units (e.g., ethylene oxide units) per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • alkoxylene units e.g., ethylene oxide units
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • alkoxylene units e.g., ethylene oxide units
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 1, 2, or 3 to about 4, 5, 6, or 8 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, about 8, 9, or 10 to about 11, 12, or 13 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, or about 16, 17, or 18 to about 19, 20, 21, or 22 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, or 2 to about 3, 4, or 5 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, about 5, 6, 7, 8, 9, or 10 to about 11, 12, 13, 14, or 15 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, or about 15 or 20 to about 25 or 30 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer.
  • the number of alkoxylene units may be determined per primary amine of PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
  • a primary amine as used herein in reference to PEI or polypropyleneimine in a polyalkyleneimine-alkoxylene polymer refers to PEI or polypropyleneimine prior to formation of the polyalkyleneimine-alkoxylene polymer or in absence of an alkoxylene unit.
  • a primary amine of PEI or polypropyleneimine may be functionalized with 0, 1 or more alkoxylene unit(s) (e.g., a hydrogen atom of the primary amine may be substituted with an alkoxylene unit).
  • Functionalizing one or more primary amines of PEI or polypropyleneimine with at least one alkoxylene unit may form a polyalkyleneimine- alkoxylene polymer of the present invention.
  • a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, or 50 alkoxylene units (e.g., ethylene oxide units) per primary amine of PEI or polypropyleneimine in the polymer.
  • alkoxylene units e.g., ethylene oxide units
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per primary amine of PEI or polypropyleneimine in the polymer.
  • alkoxylene units e.g., ethylene oxide units
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 1, 2, or 3 to about 4, 5, 6, or 8 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, about 8, 9, or 10 to about 11, 12, or 13 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, or about 16, 17, or 18 to about 19, 20, 21, or 22 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer) comprises about 0, 1, or 2 to about 3, 4, or 5 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, about 5, 6, 7, 8, 9, or 10 to about 11, 12, 13, 14, or 15 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, or about 15 or 20 to about 25 or 30 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer.
  • 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 primary amines of PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer are substituted/functionalized with at least one alkoxylene unit (e.g., at least one ethylene oxide unit).
  • the amount or degree of alkoxylation for a polyalkyleneimine-alkoxylene polymer may be measured using methods known to those of skill in the art such as by determining OH-number and/or by using nuclear magnetic resonance (e.g., 'H NMR).
  • alkoxylation may be quantified by determining the OH-number (also referred to interchangeably as an OH-value) for a polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer), and an alkoxylation degree of 3-5 units may be about 320-213 mg KOH, respectively, to provide an OH-number of about 213 to about 320, an alkoxylation degree of 8-13 alkoxylation may be about 142-91 mg KOH, respectively, to provide an OH-number of about 91 to about 142, and/or an alkoxylation degree of 18-22 may be about 67-55 mg KOH, respectively, to provide an OH-number of about 55-66.
  • an alkoxylation degree of 3-5 units may be about 320-213 mg KOH, respectively, to provide an OH-number of about 213 to about 320
  • an alkoxylation degree of 8-13 alkoxylation may be about 142-91 mg KOH, respectively
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) has an OH-number in a range of about 40 to about 400, about 40 to about 100, about 100 to about 200, or about 200 to about 400.
  • a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine- alkoxylene polymer may be capped with hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, or -C(0)-C1-C22 aryl.
  • a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine-alkoxylene polymer is capped with hydrogen or a -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, or -C(0)-C1-C22 aryl.
  • a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is capped with hydrogen.
  • an internal and/or terminal nitrogen atom e.g., of a primary, secondary, and/or tertiary amine
  • PEI or polypropyleneimine of the polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • one or more nitrogen atom(s) in a polyalkyleneimine- alkoxylene polymer may be substituted with a substituent comprising 1 to 12 carbon atoms (also referred to herein as a C1-C12 substituent).
  • a C1-C12 substituent may be saturated or unsaturated and/or linear or branched.
  • a C1-C12 substituent is a saturated or unsaturated, linear or branched hydrocarbon.
  • a C1-C12 substituent is a substituted or unsubstituted C1-C12 alkyl, alkenyl, alkynyl, aryl, arylalkyl arylalkenyl, or arylalkynyl substituent.
  • one or more nitrogen atom(s) in a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • one or more nitrogen atom(s) in a polyalkyleneimine-alkoxylene polymer may be substituted with a benzyl.
  • one or more nitrogen atom(s) in a polyalkyleneimine- alkoxylene polymer may be substituted with a group selected from a C1-C4 alkyl, a C1-C4 aryl, and/or a C1-C4 arylalkyl.
  • Substitution with a C1-C12 substituent may result in a neutral or cationic charge on a respective nitrogen atom depending on its total number of substituents. In some embodiments, substitution with a C1-C12 substituent may result in permanent quaternization (i.e., a permanent cationic charge) at the substituted position. In some embodiments, substitution with a C1-C12 substituent may result in permanent quaternization of a nitrogen atom in the PEI or polypropyleneimine backbone of a polymer.
  • a polyalkyleneimine-alkoxylene polymer may comprise one or more (e.g., 1, 2, 3, 4, 5, 10, 20, 100, or more) quatemized functional group(s).
  • a nitrogen atom e.g., of a primary, secondary, and/or tertiary amine
  • the polymer may comprise one or more quaternized functional group(s).
  • about 0%, 25%, or 50% to about 60%, 70%, 80%, 90%, or 100% of the nitrogen atoms of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprise a quatemized functional group.
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • about 0%, 5%, 10%, 20%, 40%, or 50% to about 60%, 70%, 80%, 90%, or 100% of the nitrogen atoms in the PEI or polypropyleneimine backbone of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprise a quaternized functional group.
  • a quaternized functional group may comprise a nitrogen atom of PEI or polypropyleneimine bound to a substituent comprising 1 to 12 carbon atoms (also referred to herein as a C1-C12 substituent).
  • exemplary C1-C12 substituents include, but are not limited to, benzyl, a C1-C12 alkyl, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 arylalkyl, a C1-C12 arylalkenyl, or a C1-C12 arylalkynyl, each of which may be substituted or unsubstituted.
  • a quatemized functional group is formed by reacting a C1-C12 substituent with a tertiary amine of PEI or polypropyleneimine.
  • the polyalkyleneimine (e.g., PEI) and/or polyalkoxylene (e.g., PEO) of the polyalkyleneimine-alkoxylene polymer may be linear and/or branched. In some embodiments, PEI and/or PEO of a PEI-PEO polymer are branched.
  • a polyalkyleneimine-alkoxylene polymer may comprise 1, 5, 10, or 20 to 30, 40, or 50 alkoxylene units.
  • PEO of a PEI-PEO polymer may comprise 1, 5, 10, or 20 to 30, 40, or 50 ethylene oxide units.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises a polyalkoxylene in an amount of about 50% to about 99% by weight of the polymer such as about 75% to about 99%, about 80% to about 99%, about 80% to about 95%, or about 90% to about 95% by weight of the polymer.
  • a polyalkyleneimine-alkoxylene polymer may comprise a polyalkoxylene in an amount of about 50%, 51%, 52%, 53%, 54%,
  • a polyalkyleneimine-alkoxylene polymer comprises PEI or polypropyleneimine in an amount of about 0.5% to about 50% by weight of the polymer such as about 1% to about 25%, about 5% to about 20%, about 5% to about 10%, or about 0.5% to about 10% by weight of the polymer.
  • a polyalkyleneimine-alkoxylene polymer comprises PEI or polypropyleneimine in an amount of about 0.5%, 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%, 31%,
  • a PEI-PEO polymer comprises PEO in an amount of about 50% to about 99% by weight of the PEI-PEO polymer such as about 75% to about 99%, about 80% to about 99%, about 80% to about 95%, or about 90% to about 95% by weight of the PEI-PEO polymer.
  • a PEI-PEO polymer may comprise PEO in an amount of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
  • a PEI-PEO polymer comprises PEI in an amount of about 0.5% to about 50% by weight of the PEI-PEO polymer such as about 1% to about 25%, about 5% to about 20%, about 5% to about 10%, or about 0.5% to about 10% by weight of the PEI-PEO polymer.
  • a PEI-PEO polymer comprises PEI in an amount of about 0.5%, 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% by weight of the PEI-PEO polymer.
  • PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer of the present invention may have an amine value in a range of about 5, 10, or 15 to about 20, 25, or 30.
  • PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an amine value of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, or 25.
  • amine value is the number of milligrams of potassium hydroxide (KOH) equivalent to the basicity in 1 gram of sample (e.g., 1 gram of polymer). Amine value may be measured in accordance with ASTM D 2073-92 and optionally calculating the total amine value assuming all amines are primary amines.
  • KOH potassium hydroxide
  • PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has a molecular weight of about 400 g/mol to about 900,000 g/mol such as about 400 g/mol to about 825,000 g/mol, about 400 g/mol to about 750,000 g/mol, about 400 g/mol to about 10,000 g/mol, about 400 g/mol to about 600 g/mol, about 800 g/mol to about 20,000 g/mol, about 600,000 g/mol to about 900,000 g/mol, about 600,000 g/mol to about 825,000 g/mol, about 4,000 g/mol to about 6,000 g/mol, about 1,000 g/mol to about 500,000 g/mol, about 10,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 30,000 g/mol, or about
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an average molecular weight of about 1,000 g/mol to about 30,000,000 g/mol, about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 30,000 g/mol to about 160,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has a molecular weight of about 1,000 g/mol to about 10,000,000 g/mol such as about 1,000 g/mol to about 10,000 g/mol, about 100,000 g/mol to about 1,000,000 g/mol, about 1,000,000 g/mol to about 10,000,000 g/mol, about 1,900 g/mol to about 2,900 g/mol, about 2,100 g/mol to about 2,400 g/mol, about 3,300 g/mol to about 5,000 g/mol, about 3,700 g/mol to about 4,600 g/mol, about 6,100 g/mol to about 9,300 g/mol, about 6,900 g/mol to about 8,500 g/mol, about 100,000 g/mol to about 160,000 g/mol, about 115,000 g/mol to about 141,000 g/mol, about 266,000 g/mol to about 400,000 g/mol, about 299,000
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an EO value (degree of ethoxylation) of about 2 to about 6, about 2 to about 20, about 2 to about 28, about 2 to about 30, about 3 to about 5, about 3 to about 6, about 3 to about 9, about 3 to about 15, about 3 to about 22, about 5 to about 28, about 7 to about 15, about 8 to about 14, or about 17 to about 30 or any range or value therein.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an OH-number of about 70 mg to about 40 mg, about 70 mg to about 50 mg, about 100 mg to about 40 mg, about 100 mg to about 60 mg, about 130 mg to about 40 mg, about 130 mg to about 80 mg, about 200 mg to about 40 mg, about
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 20,000 g/mol to about 200,000 g/mol, about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, 20,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 90,000 g/mol, about 25,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 30,000 g/mol to about 90,000 g/mol, about 30,000 g/mol to about 100,000 g/mol, about
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer had/has an amine value of about 15 to about 20 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 3,000,000 g/mol to about 30,000,000 g/mol, about 4,000,000 g/mol to about 12,000,000 g/mol, about 4,000,000 g/mol to about 15,000,000 g/mol, about 4,000,000 g/mol to about 20,000,000 g/mol, about 4,000,000 g/mol to about 25,000,000 g/mol, about 4,5000,000 g/mol to about 10,000,000 g/mol, about 4,5000,000 g/mol to about 12,000,000 g/mol, about 4,5000,000 g/mol to about 15,000,000 g/mol, about 4,5000,000 g/mol to about 20,000,000 g/mol, about 4,5000,000 g/mol to about 24,000,000 g/mol, about 10,000,000 g/mol to about 24,000,000 g/mol, about 15,000,000 g/mol to about 24,000,000 g/mol, about 20,000,000 g/mol,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 15 to about 20 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 120,000 g/mol to about 1,000,000 g/mol, about 145,000 g/mol to about 450,000 g/mol, about 145,000 g/mol to about 500,000 g/mol, about 145,000 g/mol to about 600,000 g/mol, about 145,000 g/mol to about 800,000 g/mol, about 150,000 g/mol to about 400,000 g/mol, about 150,000 g/mol to about 450,000 g/mol, about 150,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 700,000 g/mol, about 150,000 g/mol to about 800,000 g/mol, about 350,000 g/mol to about 800,000 g/mol, about 400,000 g/mol to about 800,000 g/mol, about 121,000 g/mol to
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 15 to about 20 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 20,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 160,000 g/mol, about 25,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 140,000 g/mol, about 25,000 g/mol to about 150,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 25,000 g/mol to about 180,000 g/mol, about 25,000 g/mol to about 200,000 g/mol, about 25,000 g/mol to about 500,000 g/mol, about 25,000 g/mol to about 1,500,000 g/mol, about 25,000 g/mol to about 3,000,000 g/mol, about 25,000 g/mol to about 5,000,000 g/mol, about 25,000 g
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 75,000 g/mol to about 400,000 g/mol, about 75,000 g/mol to about 500,000 g/mol, about 75,000 g/mol to about 1,000,000 g/mol, about 75,000 g/mol to about 5,000,000 g/mol, about 75,000 g/mol to about 8,000,000 g/mol, about 75,000 g/mol to about 12,000,000 g/mol, about 80,000 g/mol to about 450,000 g/mol, about 80,000 g/mol to about 500,000 g/mol, about 80,000 g/mol to about 750,000 g/mol, about 80,000 g/mol to about 1,000,000 g/mol, about 80,000 g/mol to about 5,000,000 g/mol, about 100,000 g/mol to about 450,000 g/mol, about 100,000 g/mol to about 500,000 g/mol, about 100,000 g
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 150,000 g/mol to about 250,000 g/mol, about 150,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 800,000 g/mol, bout 150,000 g/mol to about 900,000 g/mol, bout 150,000 g/mol to about 1,000,000 g/mol, about 150,000 g/mol to about 5,000,000 g/mol, about 150,000 g/mol to about 12,000,000 g/mol, 150,000 g/mol to about 24,000,000 g/mol, about 150,000 g/mol to about 25,000,000 g/mol, about 500,000 g/mol to about 1,000,000 g/mol, about 500,000 g/mol to about 5,000,000 g/mol, about 500,000 g/mol to about 10,000,000 g/mol, about 500,000 g/mol to about 12,000,000
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 25,000 g/mol to about 40,000 g/mol, about 25,000 g/mol to about 35,000 g/mol or any range or value therein, (e.g., about 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, or 40,000 g/mol), optionally, an average molecular weight of about 30,000 g/mol; a molecular weight of about 1,900 g/mol to about 2,900 g/mol or about 2,100 g/mol to about 2,400 g/mol; about 2 to about 5 alkoxylene units per amine of the polypropy
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 75,000 g/mol to about 90,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 75,000 g/mol to about 85,000 g/mol or any range or value therein, (e.g., about 75,000, 76,000, 77,000, 78,000, 79,000, 80,000, 81,000, 82,000, 83,000, 84,000, 85,000, 86,000, 87,000, 88,000, 89,000, or 90,000 g/mol), optionally, an average molecular weight of about 82,000 g/mol; a molecular weight of about 3,300 g/mol to about 5,000 g/mol or about 3,700 g/mol to about 4,600 g/mol; about 7 to about 10 alkoxylene units per amine of the poly
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 140,000 g/mol to about 180,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, about 140,000 g/mol to about 170,000 g/mol or any range or value therein, (e.g., about 140,000, 141,000, 142,000, 143,000, 144,000, 145,000, 146,000, 147,000, 148,000, 149,000, 150,000, 155,000, 156,000, 157,000, 158,000, 159,000, 160,000, 161,000, 162,000, 163,000, 164,000, 165,000, 166,000, 167,000, 168,000, 169,000, 170,000, 171,000, 172,000, 173,000, 174,000, 175,000, 176,000, 177,000, 178,000, 179,000 or
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 4,000,000 g/mol to about 5,000,000 g/mol, about 3,600,000 g/mol to about 5,400,000 g/mol, about 4,000,000 g/mol to about 4,900,000 g/mol, or any range or value therein, (e.g., about 4,000,000,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 10,000,000 g/mol to about 15,000,000 g/mol, about 9,000,000 g/mol to about 15,000,000 g/mol, about 10,000,000 g/mol to about 13,000,000 g/mol, or any range or value therein, (e.g., about 10,000,000, 10,500,000, 11,000,000, 11,500,000, 12,00,000, 12,500,000, 13,000,000, 13,500,000, 14,000,000, 14,500,000, or 15,000,000 g/mol), optionally, an average molecular weight of about 12,000,000 g/mol; a molecular weight of about 4,000,000 g/mol to about 6,100,000 g/mol or about 4,500,000 g/mol to about 5,600,000 g/mol; about 7 to about 10 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 7,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 22,000,000 g/mol to about 25,000,000 g/mol, about 19,000,000 g/mol to about 29,000,000 g/mol, about 17,000,000 g/mol to about 26,000,000 g/mol, or any range or value therein, (e.g., about
  • alkoxylene e.g., ethylene oxide
  • a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 60 nm to about 70 nm such as about 65 nm
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 140,000 g/mol to about 180,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about 167,000 g/mol, about 140,000 g/mol to about 170,000 g/mol or any range or value therein, (e.g., about 140,000, 141,000, 142,000, 143,000, 144,000, 145,000, 146,000, 147,000, 148,000, 149,000, 150,000, 155,000, 156,000, 157,000, 158,000, 159,000, 160,000, 161,000, 162,000, 163,000, 164,000, 165,000, 166,000, 167,000, 168,000, 169,000, 170,000, 171,000, 172,000, 173,000, 174,000, 175,000, 176,000, 177,000, 178,000, 179,000 or
  • 180,000 g/mol optionally, an average molecular weight of about 152,000 g/mol; a molecular weight of about 100,000 g/mol to about 160,000 g/mol or about 115,000 g/mol to about 141,000 g/mol,; about 2 to about 6 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6), optionally an EO value of 5; alkoxylene (e.g., ethylene oxide) in an amount of about 80% to about 85% by weight of
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 300,000 g/mol to about 500,000 g/mol, about 326,000 g/mol to about 490,000 g/mol, about 367,000 g/mol to about 448,000 g/mol, about 350,000 g/mol to about 450,000 g/mol or any range or value therein, (e.g., about 300,000, 305,000, 310,000, 315,000, 320,000, 325,000, 340,000, 345,000, 350,000, 355,000, 360,000, 365,000, 370,000, 375,000, 380,000, 385,000, 390,000, 395,000, 400,000, 401,000, 402,000, 403,000, 404,000, 405,000, 406,000, 407,000, 408,000, 409,000, 410,000, 415,000, 420,000, 425,000, 430,000, 435,000, 440,000, 450,000, 460,000, 470,000, 480,000, 490,000,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 650,000 g/mol to about 850,000 g/mol, about 633,000 g/mol to about 950,000 g/mol, about 712,000 g/mol to about 871,000 g/mol, about 700,000 g/mol to about 800,000 g/mol or any range or value therein, (e.g., about 650,000, 660,000, 680,000, 690,000, 700,000, 710,000, 720,000, 730,000, 740,000, 750,000, 760,000, 780,000, 781,000, 782,000, 783,000, 784,000, 785,000, 786,000, 787,000, 788,000, 789,000, 790,000, 791,000, 792,000, 793,000, 794,000, 795,000, 796,000, 797,000, 798,000, 799,000, 800,000, 810,000,
  • the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
  • a portion or all of the nitrogen atoms in PEI or polypropyleneimine of a polyalkyleneimine-alkoxylene polymer of the present invention may be substituted with at least one alkoxylene unit (e.g., at least one ethylene oxide unit).
  • a portion or all of the nitrogen atoms in PEI of a PEI-PEO polymer of the present invention may be substituted with at least one alkoxylene unit (e.g., at least one ethylene oxide unit).
  • Scheme 1 Exemplary alkoxylation modification for a terminal nitrogen atom in a polyalkyleneimine.
  • a further exemplary alkoxylation modification is shown in Scheme 2 for an internal nitrogen atom in the polyalkyleneimine backbone of a polyalkyleneimine-alkoxylene polymer where R represents an ethylene spacer and E represents a C1-C12 substituent (e.g., a Cl -Cl 2 alkyl) and X represents a water soluble counterion.
  • R represents an ethylene spacer
  • E represents a C1-C12 substituent (e.g., a Cl -Cl 2 alkyl)
  • X represents a water soluble counterion.
  • Scheme 2 Exemplary alkoxylation modification for an internal nitrogen atom in a polyalkyleneimine.
  • Scheme 3 Another exemplary alkoxylation modification is shown in Scheme 3 for an internal nitrogen atom in the polyalkyleneimine backbone of a polyalkyleneimine-alkoxylene polymer where each R represents an ethylene spacer and E represents a C1-C12 substituent (e.g., a Cl -Cl 2 alkyl) and X represents a water soluble counterion.
  • Scheme 3 Additional exemplary alkoxylation modification for an internal nitrogen atom in a polyalkyleneimine.
  • an alkoxylation modification of a polyalkyleneimine backbone may comprise substitution of a hydrogen atom with a polyalkoxylene chain having an average of about 1 to about 50 alkoxy units such as about 2 to about 40 alkoxy units, about 3 to about 30 units, or about 3 to about 20 alkoxy units.
  • Exemplary alkoxy units include, but are not limited to, ethoxy (EO), 1,2-propoxy (1,2-PO), and/or butoxy (BO).
  • a polyalkoxylene chain comprises, consists essentially of, or consists of ethoxy units.
  • a polyalkoxylene chain comprises, consists essentially of, or consists of ethoxy units and propoxy units.
  • a polyalkoxylene chain comprising, consisting essentially of, or consisting of ethoxy units and propoxy units may comprise, on average, about 1 to about 20 or 30 ethoxy units and, on average, about 0 or 1 to about 10 propoxy units.
  • An exemplary alkoxylated polyethyleneimine polymer of the present invention may have a structure of Formula (I):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl; and
  • each n is an integer of 1 to 50.
  • An exemplary quaternized alkoxylated polyethyleneimine polymer of the present invention may have a structure of formula (II):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
  • each E is independent a Cl -Cl 2 substituent
  • each X is independently a water soluble counterion (e.g., chlorine, bromine, iodine, sulphate, alkyl sulfonate, etc.); and
  • each n is an integer of 1 to 50.
  • a compound of Formula (I) and/or (II) may comprise a polyalkyleneimine (e.g., polyethyleneimine) backbone with a weight average molecular weight of about 600 g/mole to about 500,0000 g/mole.
  • n may be determined based on an average number of units such as, n, on average, may be an integer of 1 to 30 or 50.
  • the degree of quaternization of nitrogen atoms in the polyalkyleneimine backbone of Formula (II) may be at least 5%, 20%, 70% or more.
  • an exemplary alkoxylated polyethyleneimine polymer of the present invention may have a structure of Formula (III):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
  • each n is an integer of 0 to 50;
  • each m is an integer of 1 to 10.
  • structure of the polymer of formula (III) is not limited to block structures.
  • structure of the polymer of formula (III) comprises heteric blocks (e.g., blocks where EO and PO are randomly mixed).
  • Another exemplary quaternized alkoxylated polyethyleneimine polymer of the present invention may have a structure of formula (IV):
  • each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
  • each E is independent a Cl -Cl 2 substituent
  • each X is independently a water soluble counterion (e.g., chlorine, bromine, iodine, sulphate, alkyl sulfonate, etc.);
  • each n is an integer of 1 to 50;
  • each m is an integer of 1 to 10.
  • the structure of the polymer of formula (IV) is not limited to block structures.
  • structure of the polymer of formula (IV) comprises heteric blocks (e.g., blocks where EO and PO are randomly mixed).
  • a compound of Formula (III) and/or (IV) may comprise a polyalkyleneimine (e.g., polyethyleneimine) backbone with a weight average molecular weight of about 600 g/mole to about 500,0000 g/mole.
  • n may be determined based on an average number of units such as, n, on average, may be an integer of 1 to 20, 30, or 50.
  • m may be determined based on an average number of units such as, m, on average, may be an integer of 1 to 10.
  • the degree of quatemization of nitrogen atoms in the polyalkyleneimine backbone of Formula (IV) may be at least 5%, 20%, 70% or more.
  • a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may be prepared in a known manner by reaction of polyalkyleneimines with alkoxy units, an exemplary process is described for the ethoxylation of polyethyleneimine.
  • a method of preparing a polyalkyleneimine-alkoxylene polymer of the present invention comprises, consists essentially of, or consists of alkoxylation (e.g., ethoxylation) of a polyalkyleneimine (e.g., PEI).
  • the polyalkyleneimine may be reacted with all or a portion of the total amount of an alkylene oxide (e.g., ethylene oxide) used in the alkoxylation reaction.
  • a portion or a total amount of alkylene oxide used in the alkoxylation reaction may be about 1 mole of alkylene oxide (e.g., ethylene oxide) per amine (based on total amine value) of polyalkyleneimine.
  • An alkoxylation reaction may be undertaken in the absence of a catalyst and/or in an aqueous solution at a reaction temperature in a range of about 70°C to about 200°C.
  • the alkoxylation reaction is carried out in the presence of water at a reaction temperature in a range of about 80°C to about 160°C and in the absence of a catalyst. In some embodiments, the alkoxylation reaction is carried out at a pressure of up to about 5, 6, 7, 8, 9, or 10 bar.
  • a polyalkyleneimine e.g., PEI
  • a first portion of alkylene oxide e.g., ethylene oxide
  • an at least partially alkoxylated polyalkyleneimine e.g., at least partially alkoxylated PEI
  • the at least partially alkoxylated polyalkyleneimine is reacted with a second portion of an alkylene oxide.
  • PEI polyalkyleneimine
  • ethylene oxide as the exemplary alkylene oxide
  • the at least partially ethoxylated PEI is reacted with a second portion of an ethylene oxide.
  • the second portion of ethylene oxide is the final ethylene oxide addition and/or the second portion in combination with the first portion of ethylene oxide (and/or any other portions) makes up the total amount of ethylene oxide used in the ethoxylation reaction.
  • reaction of the at least partially ethoxylated PEI and the second portion of ethylene oxide is carried out in the presence of a basic catalyst.
  • Examples of basic catalysts include, but are not limited to, alkali metal hydroxides and alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide and/or calcium hydroxide; alkali metal alkoxides such as sodium and potassium Ci-C4-alkoxides (e.g., sodium methoxide, sodium ethoxide and potassium tert-butoxide); alkali metal and alkaline earth metal hydrides such as sodium hydride and calcium hydride; and/or alkali metal carbonates such as sodium carbonate and potassium carbonate.
  • the basic catalyst is an alkali metal hydroxide or alkali metal alkoxide such as potassium hydroxide and/or sodium hydroxide.
  • a basic catalyst may be used and/or present in an amount of about 0.05% to about 10% by weight of polyalkyleneimine and the alkylene oxide.
  • the at least partially alkoxylated polyalkyleneimine e.g., at least partially ethoxylated PEI
  • a second portion of alkylene oxide e.g., ethylene oxide
  • reacting an at least partially alkoxylated polyalkyleneimine e.g., at least partially ethoxylated PEI
  • a second portion of alkylene oxide e.g., ethylene oxide
  • reacting an at least partially alkoxylated polyalkyleneimine e.g., at least partially ethoxylated PEI
  • a second portion of alkylene oxide e.g., ethylene oxide
  • the method will be further described with PEI as the exemplary polyalkyleneimine and ethylene oxide as the exemplary alkylene oxide.
  • the at least partially ethoxylated PEI may be dewatered, optionally in the presence of a basic catalyst.
  • Dewatering may be carried out by heating a composition comprising the at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) and optionally basic catalyst to a temperature of about 80°C or 120°C to about 150°C or 180°C and distilling off the water under a reduced pressure of about 0.01 bar to about 0.5 bar.
  • the dewatering may be carried out and/or supported by a gentle nitrogen stream.
  • a subsequent reaction of the at least partially ethoxylated PEI with a second portion of ethylene oxide in a dewatered composition in the presence of a basic catalyst may be effected at a reaction temperature from about 70°C to about 200°C or about 100°C to about 180°C and/or at a pressure of up to about 8 or 10 bar.
  • reacting an at least partially ethoxylated PEI with a second portion of ethylene oxide in a dewatered composition and in the presence of a basic catalyst is carried out for a period of time of about 30 minutes to about 4 hours.
  • the PEI-PEO polymer may be obtained directly in substance or may be obtained in and/or converted to an aqueous solution.
  • reacting an at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) with a second portion of alkylene oxide (e.g., ethylene oxide) is carried out in an organic solvent such as a nonpolar organic solvent and/or polar aprotic organic solvent.
  • organic solvent such as a nonpolar organic solvent and/or polar aprotic organic solvent.
  • nonpolar aprotic solvents include, but are not limited to, aliphatic and aromatic hydrocarbons such as hexane, cyclohexane, toluene and xylene.
  • Exemplary polar aprotic solvents include, but are not limited to, ethers such as cyclic ethers (e.g., tetrahydrofuran and dioxane), N,N-dialkylamides such as dimethylformamide and dimethylacetamide, and/or N-alkyl lactams such as N-methylpyrrolidone.
  • the organic solvent comprises two or more (e.g., 2, 3, 4 or more) organic solvents.
  • the organic solvent comprises xylene and/or toluene.
  • a composition comprising an at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI), a basic catalyst, and an organic solvent may be dewatered as described herein to provide a dewatered composition.
  • dewatering is carried out by separating out the water from the composition at a temperature of about 120°C to about 180°C, optionally supported by a gentle nitrogen stream.
  • a subsequent reaction of the at least partially alkoxylated polyalkyleneimine with a second portion of alkylene oxide in an organic solvent in the presence of a basic catalyst may be effected at a reaction temperature from about 70°C to about 200°C or about 100°C to about 180°C and/or at a pressure of up to about 8 or 10 bar.
  • the obtained polyalkyleneimine- alkoxylene polymer e.g., PEI-PEO polymer
  • the organic solvent may be removed and optionally replaced with water.
  • the polyalkyleneimine-alkoxylene polymer e.g., PEI-PEO polymer
  • Quaternization may be carried out with methods known to those of skill in the art.
  • quaternization of a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • quaternization of a polyalkyleneimine-alkoxylene polymer is achieved by introducing a Cl- C12 alkyl, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 arylalkyl, a Cl- C12 arylalkenyl, or a Cl -Cl 2 arylalkynyl group such as by reaction of the polymer with the corresponding C1-C12 alkyl- halide or dialkylsulfate, C1-C12 alkenyl- halide or dialkylsulfate, C1-C12 alkynyl- halide or dialkylsulfate, C
  • quaternization of a polyalkyleneimine-alkoxylene polymer is achieved by reacting an amine of the polymer with at least one alkylating compound.
  • the at least one alkylating compound may be represented by the chemical formula EX, wherein E is a C1-C12 alkyl, a C1-C12 alkenyl, a Cl -Cl 2 alkynyl, a Cl -Cl 2 aryl, a Cl -Cl 2 arylalkyl, a Cl -Cl 2 arylalkenyl, or a Cl -Cl 2 arylalkynyl and X is a leaving group that is capable of being replaced by nitrogen or a C2-C6 alkylene oxide (e.g., ethylene oxide or propylene oxide).
  • E is a C1-C12 alkyl, a C1-C12 alkenyl, a Cl -Cl 2 alkynyl, a Cl -Cl 2 aryl, a Cl -Cl 2 arylalkyl, a Cl -Cl 2 arylalkenyl, or a Cl -Cl
  • Exemplary leaving groups include, but are not limited to, halogens (e.g., chlorine, bromine, or iodine), sulphate, alkyl sulfonate (e.g., methyl sulfonate), arylsulfonate (e.g., tolyl sulfonate), and alkyl sulphate (e.g., methosulphate).
  • halogens e.g., chlorine, bromine, or iodine
  • alkyl sulfonate e.g., methyl sulfonate
  • arylsulfonate e.g., tolyl sulfonate
  • alkyl sulphate e.g., methosulphate
  • Exemplary alkylating agents include, but are not limited to, Cl -Cl 2 alkyl halides, bis(Cl-C12-alkyl)sulfates, and benzyl hal
  • the at least one alkylating agent is selected from ethyl chloride, ethyl bromide, methyl chloride, methyl bromide, benzyl chloride, dimethyl sulphate, and/or diethyl sulphate.
  • the method may comprise combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer), thereby preparing the composition.
  • the composition may comprise two or more polyalkyleneimine-alkoxylene polymers and/or two or more nucleic acid molecules.
  • the polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • is complexed with the nucleic acid molecule optionally wherein a single polymer is complexed with 2, 5, 10, 100, or more nucleic acid molecules as described herein.
  • a single nucleic acid molecule is complexed with 2, 5, 10, 25, 50, 75, 100, or more polyalkyleneimine-alkoxylene polymers as described herein.
  • Combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer may be carried out by contacting a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer either of which may be present in the form of a solution or a solid.
  • the nucleic acid molecule and/or the polyalkyleneimine-alkoxylene polymer is/are present in the form of a solid that is added to a solution (e.g., an aqueous solution).
  • combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer comprises mixing a first solution comprising the nucleic acid molecule and a second solution comprising the polymer to form a third solution comprising the nucleic acid molecule and the polymer.
  • combining a nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer comprises providing the nucleic acid molecule and the polymer in contact for a period of time such as about 1, 5, 10, 15, or 30 minutes to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.
  • combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer comprises providing the nucleic acid molecule and the polymer in the same composition (e.g., an aqueous solution) for a period of time, optionally with mixing and/or agitation.
  • the polymer may be dialyzed prior to combining/contact with the nucleic acid (e.g., the polymer may be dialyzed about 1, 5, 10, 15, 20, 30, 40, 50 minutes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours prior to contact with the nucleic acid.
  • compositions comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) that can be used to deliver the nucleic acid molecule to a cell (e.g. host cell) of an organism (e.g., a target organism).
  • a cell e.g. host cell
  • an organism e.g., a target organism.
  • the organism can be any eukaryote (e.g., animal, plant, fungal, protozoa, and the like).
  • An animal useful with this invention may be any animal including but not limited to, a mammal, an insect, a plant, a fungus, a bird, a fish, an amphibian, a reptile, or a cnidarian.
  • a mammal can include, but is not limited to, a rodent, a horse, a dog a cat, a human, a non human primate (e.g., monkeys, baboons, and chimpanzees), a goat, a pig, a cow (e.g., cattle), a sheep, laboratory animals (e.g, rats, mice, gerbils, hamsters, and the like) and the like.
  • compositions of the invention comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) can be used to deliver the nucleic acid molecule to, for example, a cell of a cell line (e.g., a mammalian cell line, an insect cell line).
  • a cell of a cell line e.g., a mammalian cell line, an insect cell line.
  • Non-limiting examples of mammalian and insect cell lines include HEK293 cells, HeLa cells, CHO cells, MEF cells, 3T3 cells, Hi-5 cells, and Sf21 cells.
  • Suitable target organisms (and cells therefrom) can include both males and females and subjects of all ages including embryonic ( e.g ., in utero or in ovo ), infant, juvenile, adolescent, adult and geriatric subjects.
  • the target organism is not a human subject or a human embryonic subject.
  • a plant, plant part and/or plant cell useful with this invention can include, but not is not limited to, Camelina , Glycine , Sorghum , Brassica, Allium , Armoracia , Poa, Solanum, Cucurbita, Musa, Agrostis , Lolium , Festuca, Calamogrostis, Deschampsia , Spinacia , Beta , Pisum, Chenopodium , Helianthus , Pastinaca , Daucus, Petroselium , Populus , Prunus , Castanea, Eucalyptus, Acer , Quercus , LZ///C, Juglans , Picea, Pinus, Malus , Abies, Lemna, Wolffia, Spirodela, Oryza, Zea or Gossypium.
  • the plant and/or plant cell can include, but is not limited to, Camelina alyssum (Mill.) Thell., Camelina microcarpa Andrz. ex DC., Camelina rumelica Velen., Camelina sativa (L.) Crantz, Sorghum bicolor (e.g., Sorghum bicolor L.
  • a plant and/or plant cell can be, but is not limited to, wheat, barley, rye, com, oats, turfgrass (bluegrass, bentgrass, ryegrass, fescue), sugar cane, feather reed grass, tufted hair grass, spinach, cucumber, melon, leak, tomato, potato, beets, chard, quinoa, sugar beets, lettuce, sunflower ( Helianthus annuus), peas (. Pisum sativum), parsnips (.
  • the plant and/or plant cell can be Arabidopsis thaliana.
  • the plant and/or plant cell can be camelina, wheat, rice, com, rape, canola, soybean, sorghum, tomato, bamboo or cotton.
  • a plant, plant part, and/or plant cell can be an algae or algae cell including, but not limited to, a Bacillariophyceae (diatoms), Haptophyceae, Phaeophyceae (brown algae), Rhodophyceae (red algae) or Glaucophyceae (red algae).
  • a Bacillariophyceae diatoms
  • Haptophyceae Haptophyceae
  • Phaeophyceae brown algae
  • Rhodophyceae red algae
  • Glaucophyceae red algae
  • non-limiting examples of an algae or algae cell can be Achnanthidium , Actinella, Nitzschia, Nupela, Geissleria, Gomphonema, Planothidium, Halamphora, Psammothidium, Navicula, Eunotia, Stauroneis, Chlamydomonas, Dunaliella, Nannochloris, Nannochloropsis, Scenedesmus, Chlorella, Cyclotella, Amphora, Thalassiosira , Phaeodactylum, Chrysochromulina, Prymmsium, Thalassiosira, Phaeodactylum, Glaucocystis, Cyanophora, Galdieria, or Porphyridium.
  • plant part includes reproductive tissues (e.g ., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g, phloem and xylem); specialized cells such as epidermal cells, parenchyma cells,
  • plant part also includes plant cells, including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like.
  • plant protoplasts plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like.
  • shoot refers to the above ground parts including the leaves and stems.
  • tissue culture encompasses cultures of tissue, cells, protoplasts and callus.
  • plant cell refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts.
  • a plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ.
  • a plant cell can be an algal cell.
  • the polymers of this invention will be especially useful in transforming intact plant cells (e.g., cells with cell walls, e.g., plants and plant tissues), providing a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium and the like.
  • intact plant cells e.g., cells with cell walls, e.g., plants and plant tissues
  • Non-limiting examples of fungi useful with this invention include Candida spp., Fusarium spp., Aspergillus spp., Cryptococcus spp., Coccidioides spp., Tinea spp., Sporothrix spp., Blastomyces spp., Histoplasma spp., Pneumocystis spp, Saccharomyces spp., Saccharomycodes spp., Hansenula spp., Kluyveromyces spp., Yarrowia spp., Pichia spp., Candida spp., Ashbya spp., Zygosaccharomyces spp.
  • the fungus can include, but is not limited to, Saccharomyces cerevisiae, S. uvarum (carlsbergensis), S. diastaticus, Saccharomycodes ludwigii, Hansenula polymorpha, Kluveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia pastor is, Pichia methanolica, Candida stellata, C.
  • a fungal cell useful with the invention may include its cell wall or may be without a cell wall, e.g., a spheroplast.
  • the polymers of the invention may be used to transfer nucleic acids to organelles, including, but not limited to, chloroplasts or mitochondria.
  • organelles including, but not limited to, chloroplasts or mitochondria.
  • an organelle to be transformed as described herein may be isolated from the natural cellular environment.
  • One or more cells of an organism may be transformed with one or more nucleic acid molecules according to embodiments of the present invention.
  • one or more nucleic acid molecules may be delivered to a host cell or to one or more host cells in a population.
  • a host cell population may comprise cells of at least two different species and/or strains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different species and/or strains).
  • compositions of the invention comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid as described herein are able to transform eukaryotic cells (e.g., generate transformants that have received DNA) and overcome typical problem(s) associated with other transformation protocols
  • a method of delivering a nucleic acid molecule to a host cell may comprise contacting the cell with a composition of the present invention, thereby delivering/introducing the nucleic acid molecule in the composition to/into the cell and/or transforming the cell with the nucleic acid molecule.
  • the composition may be in the form of a solid (e.g., that may be added to a solution comprising a cell to be transformed) or it may be a solution (e.g., an aqueous solution).
  • the composition is and/or comprises a complex comprising the nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer), optionally in an aqueous solution.
  • a polyalkyleneimine- alkoxylene polymer e.g., a PEI-PEO polymer
  • compositions of this invention i.e., a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and a nucleic acid molecule
  • a composition comprising a polyalkyleneimine-alkoxylene polymer e.g., a PEI-PEO polymer
  • nucleic acid molecule e.g., polyalkyleneimine-alkoxylene polymer/nucleic acid molecule
  • “contact” or“contacting” can include contacting target nucleic acid with a composition of this invention in a cell free system, thereby delivering the nucleic acid molecule to the target nucleic acid (for example for modifying the target nucleic acid).
  • nucleic acid molecules may be assembled as part of a single nucleic acid molecule or nucleic acid construct, or as separate nucleic acid molecules or nucleic acid constructs, and can be located on the same or different expression constructs or transformation vectors.
  • a construct and/or vector may be present in a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the invention and/or complexed with the same or different polyalkyleneimine-alkoxylene polymers (e.g., PEI-PEO polymers).
  • one or more polynucleotide(s) can be introduced into cells in a single transformation/transfection event, in separate transformation/transfection events, or any combination thereof.
  • one or more nucleic acid molecules of this invention can be introduced singly or in combination into a cell of a host organism.
  • “contacting” or“introducing” means contacting the population with a composition of the invention under conditions where at least the nucleic acid molecule gains access to the interior of one or more cells of the population, thereby transforming the one or more cells (e.g., stably or transiently).
  • “contacting” or“introducing” means contacting the population with a composition of the invention under conditions where the composition (e.g., polyalkyleneimine-alkoxylene polymer/ nucleic acid molecules) gains access to the interior of one or more cells of the population, thereby transforming the one or more cells of the population (e.g., stably or transiently).
  • a composition of the invention e.g., polyalkyleneimine-alkoxylene polymer/ nucleic acid molecules
  • “contacting” or“introducing” a composition of the invention i.e., a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule
  • a cell may be contacted and/or incubated with a composition of the present invention for about 0.5, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes or about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,
  • 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, or 24 hours, and any value or range therein) e.g., about 5 sec to about 30 min, about 1 min to about 1 hour, about 30 min to about 3 hours, about 1 hour to about 5 hours, about 1 hour to about 10 hours, about 1 hour to about 15 hours, about 1 hour to about 20 hours, about 1 hour to about 24 hours, about 5 hours to 10 hours, about 5 hours to 20 hours, about 10 hours to about 20 hours, about 10 hours to about 24 hours, or longer, or any value or range therein).
  • the incubation time may vary so long as the time is sufficient for the composition (i.e., the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule) to gain access to the interior of the cell.
  • the composition i.e., the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule
  • a cell may be contacted and/or incubated with a composition of the present invention at a pH from about 0 to about 14 (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 and any value or range therein).
  • a cell may be contacted and/or incubated with a composition of the present invention at a pH from about 3 to about 10.
  • a composition including a host cell, nucleic acid molecule, and polyalkyleneimine-alkoxylene polymer may have a pH of about 6, 7, or 8 to about 8.5, 9, or 9.5.
  • a composition including a cell, nucleic acid molecule, and polyalkyleneimine- alkoxylene polymer has a pH of about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.
  • a cell may be contacted and/or incubated with a composition of the present invention at a temperature from about 0°C to about 100°C (e.g., about 1, 2, 3,
  • a cell may be contacted and/or incubated with a composition of the present invention at a temperature from about 4°C to about 50°C or about 15°C to about 37°C.
  • a method of the present invention delivers a nucleic acid molecule to a cell and/or a population of cells without adversely affecting the viability of the cell and/or population.
  • growth of such cells may be delayed or even prevented at an amount above about 5% weight/vol. of the polymer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% wt/vol. of the polymer.
  • a nucleic acid molecule of the invention may be operatively associated with a variety of promoters and other regulatory elements for expression in host cells.
  • a recombinant nucleic acid molecule of this invention may further comprise one or more promoters operably linked to one or more nucleotide sequences.
  • operably linked or“operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related.
  • the term“operably linked” or“operably associated” as used herein refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated.
  • a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence.
  • a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence.
  • control sequences e.g., promoter
  • the control sequences need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof.
  • intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered“operably linked” to the nucleotide sequence.
  • A“promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter.
  • the coding sequence may encode a polypeptide and/or a functional RNA.
  • a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription.
  • promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence.
  • the promoter region may comprise other elements that act as regulators of gene expression.
  • Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e.,“chimeric genes” or“chimeric polynucleotides.” These various types of promoters are known in the art.
  • promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.
  • promoters include, but are not limited to, promoters functional in eukaryotes and prokaryotes including but not limited to, plants, fungi, and animals (e.g., mammals, insects, fish, amphibians, reptiles and the lik).
  • Exemplary promoters useful with yeast can include a promoter from phosphoglycerate kinase ( PGK ), glyceraldehyde-3 -phosphate dehydrogenase (GAP), triose phosphate isomerase (777), galactose-regulon ( GAL1 , GAL10 ), alcohol dehydrogenase (ADH1, ADH2), phosphatase ( PH05 ), copper-activated metallothionine ( CUP1 ), MFal, PGK/a2 operator, TPI/a2 operator, GAP/GAL, PGK/GAL, GAP/ADH2, GAP/PH05, iso-1- cytochrome c/glucocorticoid response element (CYC/GRE), phosphoglycerate kinase/angrogen response element (PGK/ARE), transcription elongation factor EF-la ( TEF1 ), triose phosphate dehydrogenase ( T
  • Non-limiting examples of a promoter functional in a plant include the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007); Li et al. Mol Biol. Rep. 37:1143-1154 (2010)). PrbcSl and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters.
  • PrbcSl and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters.
  • Pnr is induced by nitrate and repressed by ammonium (Li et al. Gene 403: 132-142 (2007)) and Pdcal is induced by salt (Li et al. Mol Biol. Rep. 37: 1143-1154 (2010)).
  • constitutive promoters useful for plants include, but are not limited to, cestrum virus promoter (cmp) (U.S. Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as US Patent No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad.
  • the maize ubiquitin promoter ( UbiP ) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926.
  • the ubiquitin promoter is suitable for the expression of the nucleotide sequences of the invention in transgenic plants, especially monocotyledons.
  • the promoter expression cassettes described by McElroy et al. ⁇ Mol. Gen. Genet. 231 : 150-160 (1991)) can be easily modified for the expression of the nucleotide sequences of the invention and are particularly suitable for use in monocotyledonous hosts.
  • tissue specific/tissue preferred promoters can be used for expression of a heterologous polynucleotide in a plant cell.
  • tissue- specific promoters include those associated with genes encoding the seed storage proteins (such as b-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g. , Kridl et al. (1991) Seed Sci. Res.
  • tissue- specific/tissue preferred promoters include, but are not limited to, the root hair-specific cis- elernents (RFSEs) (Kirn et al. The Plant Cell 18:2958-2970 (2006)), the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153: 185-197 (2010)) and RB7 (U.S. Patent No. 5459252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11 : 160-167; and Vodkin (1983) Prog. Clin. Biol. Res.
  • RFSEs root hair-specific cis- elernents
  • promoters functional in chloroplasts can be used.
  • Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516.
  • Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).
  • inducible promoters can be used.
  • chemical -regulated promoters can be used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences of the invention via promoters that are chemically regulated enables the RNAs and/or the polypeptides of the invention to be synthesized only when, for example, a crop of plants are treated with the inducing chemicals.
  • the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression.
  • a promoter can also include a light- inducible promoter, where application of specific wavelengths of light induce gene expression (Levskaya et al. 2005. Nature 438:441-442).
  • a promoter can include a light-repressible promoter, where application of specific wavelengths of light repress gene expression (Ye et al. 2011. Science 332: 1565-1568).
  • Chemical inducible promoters useful with plants are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid ( e.g ., the PRla system), steroid-responsive promoters (see, e.g, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al.
  • promoters useful with algae include, but are not limited to, the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007); Li et al. Mol Biol. Rep.
  • the promoter of the o 70 -type plastid rRNA gene (Prrn), the promoter of the psbA gene (encoding the photosystem -II reaction center protein Dl) (PpsbA), the promoter of the psbD gene (encoding the photosystem-II reaction center protein D2) (PpsbD), the promoter of the psaA gene (encoding an apoprotein of photosystem I) (PpsaA), the promoter of the ATPase alpha subunit gene (PatpA), and promoter of the RuBisCo large subunit gene (PrbcL), and any combination thereof ⁇ See, e.g., De Cosa et al.
  • a promoter useful with this invention can include, but is not limited to, pol III promoters such as the human U6 small nuclear promoter (U6) and the human HI promoter (HI) (Makinen et al. J Gene Med. 8(4):433-41 (2006)), and pol II promoters such as the CMV (Cytomegalovirus) promoter (Barrow et al. Methods in Mol. Biol.
  • pol III promoters such as the human U6 small nuclear promoter (U6) and the human HI promoter (HI) (Makinen et al. J Gene Med. 8(4):433-41 (2006)
  • pol II promoters such as the CMV (Cytomegalovirus) promoter (Barrow et al. Methods in Mol. Biol.
  • the SV40 Sema virus 40-derived initial promoter
  • the EF-la Elongation Factor-la
  • the Ubc Human Ubiquitin C
  • the PGK Mitine Phosphogly cerate Kinase- 1 promoter and/or constitutive protein gene promoters such as the b-actin gene promoter, the tRNA promoter and the like.
  • tissue-specific regulated nucleic acids and/or promoters as well as tumor- specific regulated nucleic acids and/or promoters have been reported.
  • tissue-specific or tumor-specific promoters can be used.
  • Some reported tissue- specific nucleic acids include, without limitation, B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (hematopoietic cells), CD68 (macrophages), desmin (muscle), elastase-1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin
  • FLT-1 endothelial cells
  • GFAP astrocytes
  • GPIIb megakaryocytes
  • ICAM-2 endothelial cells
  • INF-b hematopoietic cells
  • Mb muscle
  • NPHSI podocytes
  • OG-2 osteoblasts
  • SP-B lungs
  • SYN1 neutral neurons
  • WASP hematopoietic cells
  • tumor-specific nucleic acids and promoters include, without limitation, AFP (hepatocellular carcinoma), CCKAR (pancreatic cancer), CEA (epithelial cancer), c-erbB2 (breast and pancreatic cancer), COX-2, CXCR4, E2F-1, HE4, LP, MUC1 (carcinoma), PRC1 (breast cancer), PSA (prostate cancer), RRM2 (breast cancer), survivin, TRPl (melanoma), and TYR (melanoma).
  • inducible promoters can be used in mammalian cells.
  • inducible promoters include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, copper-inducible system promoters, salicylate- inducible system promoters (e.g., the PRla system), glucocorticoid-inducible promoters, and ecdysone-inducible system promoters.
  • a recombinant nucleic acid molecule of the invention can be an “expression cassette” or can be comprised within an expression cassette.
  • expression cassette means a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the nucleotide sequences of the invention; e.g., a nucleic acid of interest), wherein said nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter).
  • a control sequence e.g., a promoter
  • An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in the selected host cell.
  • a transcriptional and/or translational termination region i.e., termination region
  • a variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation.
  • the termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host cell, or may be derived from another source (i.e., foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).
  • An expression cassette of the invention also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed host cell.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker.
  • Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g, fluorescence).
  • a selective agent e.g., an antibiotic and the like
  • screening e.g, fluorescence
  • vector refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell.
  • a vector comprises a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered or introduced.
  • Vectors for use in transformation of host organisms are well known in the art.
  • Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
  • a vector as defined herein can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic species (e.g. higher plant, mammalian, yeast, insect, fungi, and the like).
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell.
  • the vector may be a bi functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • the nucleic acid molecules of this invention and/or expression cassettes may be comprised in vectors as described herein and as known in the art.
  • the terms“transformed” and“transgenic” refer to any cell that contains all or part of at least one recombinant (e.g., heterologous) polynucleotide even if temporarily (i.e., transient).
  • transformation refers to the introduction of a nucleic acid molecule into a cell. Transformation of a cell or organism may be stable or transient.
  • Transient transformation in the context of a nucleic acid molecule means that a nucleic acid molecule is introduced into the cell and does not integrate into the genome of the cell.
  • nucleic acid molecule introduced into a cell By stably introducing or stably introduced in the context of a nucleic acid molecule introduced into a cell it is intended that the introduced nucleic acid molecule is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the nucleic acid molecule.
  • “Stable transformation” or“stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Genome as used herein also includes the nuclear/chromosomal and the plasmid genome, and therefore includes integration of the nucleic acid molecule into, for example, the plasmid genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome.
  • an“extrachromosomal nucleic acid” refers to select nucleic acids in eukaryotic cells such as in a mitochondrion, a plasmid, a plastid (e.g., chloroplast, amyloplast, leucoplast, proplastid, chromoplast, etioplast, elaiosplast, proteinoplast, tannosome), and/or an extrachromosomal circular DNA (eccDNA)).
  • an extrachromosomal nucleic acid may be referred to as“extranuclear DNA” or“cytoplasmic DNA.”
  • Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism.
  • Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., an animal, a plant, a fungus).
  • Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism.
  • Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and/or hybridization protocols that are well known in the art.
  • PCR polymerase chain reaction
  • “percent sequence identity” or“percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.
  • “sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g.
  • nucleotides or amino acids “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA).
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e ., the entire reference sequence or a smaller defined part of the reference sequence.
  • Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
  • “percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001.
  • the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.
  • the phrase “substantially complementary,” or “substantial complementarity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences refers to two or more sequences or subsequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide or amino acid residue complementary, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantial complementarity can refer to two or more sequences or subsequences that have at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 96, 97, 98, or 99% complementarity (e.g., about 80% to about 90%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99% or more, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99% or more, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99% or more, about 95% to about 97%, about 95% to about 98%, about 95% to about 99% or more).
  • Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridizations
  • Stringent hybridization conditions and“stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra , for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes.
  • An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C.
  • SDS sodium dodecyl sulfate
  • the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.
  • SDS sodium dodecyl sulfate
  • Any nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in any species of interest. Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences.
  • nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in the particular species of interest.
  • Polyethyleneimines such as Lupasol ® P e.g., 50% concentrated in water, 750,000 MW
  • Lupasol ® G100 e.g., 50% in water, 5,000 MW
  • Lupasol ® HF e.g., 56% in water, 25 000 MW
  • PEIs were in water and used as obtained.
  • Lupasol ® P and Lupasol ® G100 were used in 50% water and Lupasol ® HF was used in 56% water.
  • Dimethyl sulfate, n-bromobutane, 1-bromooctane, 1- bromododecane, and 1-bromooctadecane were each obtained from Sigma Aldrich. The purity of each halogenated hydrocarbon was, at minimum, greater than 95%.
  • the molecular weight of the polyethyleneimines were determined by GPC against PEG standards and reported in g/mol.
  • the molecular weight (as reported in g/mol) of the PEI-alkoxylates were determined by hydroxyl value and extrapolated by the average molar equivalent of primary amine per polyethylamine.
  • the acid value was measured by titrating 1 gram of PEI-alkoxylate with a 0.1 N KOH solution in methanol (as reported mg KOH/lg of material)
  • the amine functionality was measured by carbon- 13 NMR; taking the average area of each amine region (i.e., 35 - 45 PPM for primary amines) over the total amine region (35 - 60 PPM). The results for each amine type were then reported as the ratio of primary, secondary and tertiary amines.
  • the charge density of the PEIs (unless otherwise stated) were measured from the dried substance at pH 4.5
  • the hydroxyl number was measured by reacting 1 g of PEI-alkoxylate with acetic anhydride in the presence of DMAP (Dimethyl aminopyridine) and back titrating with 0.5 N KOH solution in Methanol.
  • DMAP Dimethyl aminopyridine
  • PEI polyethyleneimine
  • Agitation was set to 350 rpm.
  • the reactor was sealed and nitrogen purged (3 times at 6.2 bar) before heating. Pressure was adjusted to 1.4 bar with nitrogen and the reactor heated to 120°C.
  • Ethylene oxide (6.84 molar equivalents) was added over 2 hours and reacted to constant pressure.
  • the reactor was cooled to 90°C and an aqueous solution including KOH in an amount of 45 % by weight of the aqueous solution was added to the reactor in an amount of 0.15 wt% of the mixture in the reactor. Water was stripped for 1 hour at 120°C and 0.13 bar.
  • Ethylene oxide (33.86 molar equivalent) was fed into the reactor at a rate of 4 g/min. The rate was increased to 6 g/min as quickly as temperature control and pressure allowed. Total addition time was 5 hrs. After the ethylene oxide addition was completed, reacted to constant pressure for 2 additional hours. The reactor was vented and cooled to 85°C before discharging. By this procedure, an ethoxylated polyethyleneimine was obtained as a brown liquid or as a solid. Degree of ethoxylation was determine by OH#. Average molecular weight was determined by GPC in water/THF. Amine functionality confirmed by 13 C NMR.
  • step 2 Take about 660 m ⁇ of polymer solution from step 1, transfer to 1.5 mL micro-centrifuge tube.
  • the condition of the protoplasts can be checked under a microscope; the healthy cells should appear full and round.
  • the transformation efficiency may be checked two days post transfection by counting the number of GFP-expressing cells using a fluorescence microscope.
  • the parameters of these protocols may be modified to include variations in the ratio of nucleic acid to polymer; the type of nucleic acid used; as well as adjusted for use with cells from different species.
  • Polymers 1, 3, 7, and 9 were evaluated for their effect on plant cell viability, penetration, and DNA delivery efficiency.
  • Polymers 7 an 9 have a larger core than polymers 1 and 3 and a greater cationic charge. Polymers 1 and 7 have a higher cationic charge than polymers 3 and 9.
  • Protoplasts are naked plant cells which have had their cell wall removed by either mechanical or enzymatic means.
  • protoplasts derived from several plant species have been widely used to study cellular processes and elucidate molecular mechanisms underlying pathways involved in plant physiology, immunity, growth and development.
  • the transformation potential of the polymers for plasmid DNA delivery into wheat protoplast cells was assessed.
  • cells were transfected with a GFP-expressing plasmid and assayed for fluorescence 2 days post treatment. Initially, each of the polymers was used at the highest non-lethal concentration as determined in abovementioned experiment and mixed with 10 pg of plasmid DNA.
  • Control cells were transfected using polyethylene glycol (PEG) following our standard transfection protocol. The transfection efficiency was calculated as the number of GFP-positive cells divided by the total number of living cells at the end of the experiments.
  • PEG polyethylene glycol
  • the polymers will be particularly useful in transforming intact plant cells (e.g., cells with cell walls, e.g., plants and plant tissues). This method should provide a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium and the like.

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Abstract

The present invention relates generally to nucleic acid compositions including a polyalkyleneimine-alkoxylene polymer such as a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer. The present invention also relates to methods of making and using a composition comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) including for use in modifying a genome of an organism and the killing of cells.

Description

NUCLEIC ACID COMPOSITIONS INCLUDING A POLYALKYLENEIMINE- ALKOXYLENE POLYMER AND METHODS OF MAKING AND USING THE SAME
Priority
This application claims the benefit, under 35 Ei.S.C. § 119 (e), of U.S. Provisional Application No. 62/865644 filed on June 24, 2019, the entire contents of each of which is incorporated by reference herein.
Field
The present invention relates generally to nucleic acid compositions including a polyalkyleneimine-alkoxylene polymer such as a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer. The present invention also relates to methods of making and using a composition comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) including for use in modifying a genome of an organism and the killing of cells.
Background
Delivery of nucleic acids to living organisms is challenging and currently hinges on limited technologies such as chemical transformation and electroporation. These protocols are difficult to develop and implement and have to be developed and customized to each target organism of interest.
Summary
Embodiments of the invention provide compositions comprising a polyalkyleneimine- alkoxylene polymer (e.g., a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer) and a nucleic acid molecule, optionally wherein the polyalkyleneimine-alkoxylene polymer is complexed with the nucleic acid molecule.
Embodiments of the invention provide methods of preparing a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)- polyethylene oxide (PEO) polymer) and a nucleic acid molecule, the methods comprising: combining the nucleic acid molecule and the polymer, thereby preparing the composition, optionally wherein the polymer is complexed with the nucleic acid molecule.
Embodiments of the invention provide methods of delivering a nucleic acid molecule to (transforming) a cell, comprising contacting the cell with a composition of the invention comprising a polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)- polyethylene oxide (PEO) polymer) and a nucleic acid molecule, thereby delivering the nucleic acid molecule in the composition to the cell.
It is noted that aspects of the invention described with respect to one embodiment, may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination. Applicant reserves the right to change any originally filed claim and/or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and/or incorporate any feature of any other claim or claims although not originally claimed in that manner. These and other objects and/or aspects of the present invention are explained in detail in the specification set forth below. Further features, advantages and details of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments that follow, such description being merely illustrative of the present invention.
Brief Description of the Drawings
Fig. 1 shows the impact of polymers on wheat protoplast survival. Isolated wheat protoplasts (7.5 x 105 cells) were treated with 660 mΐ of an aqueous solution containing increasing concentrations of polymer. Cell viability was assessed two days post treatment. Ctrl = control, non-treated cells.
Fig. 2 shows chemical-based delivery of DNA using polymers 1, 3, 7, and 9. Plasmid DNA packaged within each polymer, encoding enhanced green fluorescent protein (eGFP), can be delivered by mixing with wheat protoplast cells. Micrographs depicting representative GFP signals were taken two days post transfection. As a positive control, cells were transfected with the GFP reporter plasmid in the presence of polyethylene glycol (PEG). Transfection efficiencies were calculated by counting the number of GFP expressing cells and dividing by the number of living cells. Data presented are means of three independent experiments.
Fig. 3A-3B shows transfection of the cells using polymers of the invention. Fig. 3A shows transformation efficiency using different amounts of DNA. Fig. 3B shows transformation efficiencies using different amounts of polymer (polymer 9). Transfection efficiencies were calculated by counting the number of GFP expressing cells and dividing by the number of living cells. Data presented are means of two independent experiments. Photographs depicting representative efficiencies were taken two days post transfection. Positive control cells were transfected using polyethylene glycol and negative controls were treated with plasmid DNA only.
Detailed Description of Example Embodiments
The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.
As used in the description of the invention and the appended claims, the singular forms a, an, and the are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.
Also as used herein, and/or refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed. As used herein, the transitional phrase consisting essentially of (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. See, In re Herz , 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term consisting essentially of as used herein should not be interpreted as equivalent to comprising.
It will also be understood that, as used herein, the terms example, exemplary, and grammatical variations thereof are intended to refer to non-limiting examples and/or variant embodiments discussed herein, and are not intended to indicate preference for one or more embodiments discussed herein compared to one or more other embodiments.
The term about, as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, about X where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measureable value may include any other range and/or individual value therein.
As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,”“nucleic acid sequence,”“oligonucleotide,” and“polynucleotide” refer to RNA or DNA that is linear, circular or branched, single or double stranded, or a hybrid thereof. The terms also encompass RNA/DNA hybrids. In some embodiments, the RNA and/or DNA can include a chemically modified base such as those that are not usually found in nature. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others may be used such as for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.
As used herein, the terms“polynucleotide” “nucleotide sequence,”“nucleic acid molecule” and“nucleic acid sequence” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA and/or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded and/or double stranded. The terms nucleotide sequence, nucleic acid, nucleic acid molecule, oligonucleotide and polynucleotide may also be used to refer to a heteropolymer of nucleotides. Nucleic acid molecules and/or nucleotide sequences provided herein are presented in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25. A 5' region as used herein refers to the region of a polynucleotide that is nearest the 5' end. Thus, for example, an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to the nucleotide located halfway through the polynucleotide. A 3' region as used herein refers to the region of a polynucleotide that is nearest the 3' end. Thus, for example, an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to the nucleotide located halfway through the polynucleotide.
As used herein, the term“gene” refers to a nucleic acid molecule capable of being used to produce mRNA, tRNA, rRNA, miRNA, anti-microRNA, regulatory RNA, and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions). A gene may be isolated by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
A synthetic nucleic acid or nucleotide sequence, as used herein, refers to a nucleic acid or nucleotide sequence that is not found in nature but is constructed by human intervention and as a consequence is not a product of nature.
Alkyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms, which can be referred to as a Cl- C20 alkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n- decyl, and the like. Loweralkyl as used herein, is a subset of alkyl, and, in some embodiments, refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of loweralkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. The term alkyl or loweralkyl is intended to include both substituted and unsubstituted alkyl or loweralkyl unless otherwise indicated and these groups may be substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(0)m, haloalkyl-S(0)m, alkenyl-S(0)m, alkynyl-S(0)m, cycloalkyl-S(0)m, cycloalkylalkyl-S(0)m, aryl-S(0)m, arylalkyl-S(0)m, heterocyclo-S(0)m, heterocycloalkyl-S(0)m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m= 0, 1, 2 or 3.
Alkenyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkenyl 1 to 4 carbon atoms) and includes 1 to 8 double bonds in the normal chain, and can be referred to as a C1-C20 alkenyl. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4- heptadiene, and the like. The term alkenyl or loweralkenyl is intended to include both substituted and unsubstituted alkenyl or loweralkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and loweralkyl above.
Alkynyl as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include at least 1 triple bond in the normal chain, and can be referred to as a C1-C20 alkynyl. Representative examples of alkynyl include, but are not limited to, 2- propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term alkynyl or loweralkynyl is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.
Halo as used herein refers to any suitable halogen, including -F, -Cl, -Br, and -I.
Mercapto as used herein refers to an -SH group.
Cyano as used herein refers to a -CN group.
Hydroxyl as used herein refers to an -OH group.
Nitro as used herein refers to an -NO2 group. Alkoxy as used herein alone or as part of another group, refers to an alkyl or loweralkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -0-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.
Acyl as used herein alone or as part of another group refers to a -C(0)R radical, where R is any suitable substituent such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl or other suitable substituent as described herein.
Haloalkyl as used herein alone or as part of another group, refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.
Aryl as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The term aryl is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above. In some embodiments, an aryl is substituted with an alkyl, alkenyl, and/or alkynyl.
Arylalkyl as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2- phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like. Similarly, arylalkenyl and arylalkynyl as used herein alone or as part of another group, refer to an aryl group, as defined herein, appended to the parent molecular moiety through an alkenyl group and alkynyl, respectively, each as defined herein.
Amino as used herein means the radical -NH2.
Alkylamino as used herein alone or as part of another group means the radical -NHR, where R is an alkyl group.
Ester as used herein alone or as part of another group refers to a -C(0)OR radical, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl. Amide as used herein alone or as part of another group refers to a -C(0)NRaRb radical, where Ra and R¾ are any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.
Sulfonamide as used herein alone or as part of another group refers to a -S(0)2NRaRb radical, where Ra and Rb are any suitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.
As used herein, the terms increase, increases, increased, increasing, enhance, and similar terms indicate an elevation in the specified parameter of at least about 2%, 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% or more unless otherwise specifically noted within the text.
As used herein, the terms reduce, reduces, reduced, reduction, and similar terms refer to a decrease in the specified parameter of at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% unless otherwise specifically noted within the text.
Provided according to embodiments of the present invention are compositions comprising a polyalkyleneimine-alkoxylene polymer and a nucleic acid molecule. A polyalkyleneimine-alkoxylene polymer of the present invention may also be referred to interchangeably herein as an alkoxylated polyethyleneimine. In some embodiments, the polyalkyleneimine is a polyethyleneimine and/or a polypropyleneimine. In some embodiments, the alkoxylene is one or more ethylene oxide, propylene oxide, and/or butylene oxide unit(s). According to some embodiments provided are compositions comprising a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer and a nucleic acid molecule. For convenience PEI-PEO may be referenced herein as an example, but other polyalkyleneimine-alkoxylene polymers within the scope of this invention may be used. In some embodiments, the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is complexed with the nucleic acid molecule. The composition may comprise and/or be in an aqueous solution. In some embodiments, the composition comprises and/or is a complex of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and at least one nucleic acid molecule.
A complex and grammatical variations thereof as used herein refer to a molecular entity comprising at least two components that are associated with one another. The two or more components may be different (e.g., a PEI-PEO polymer and a nucleic acid) and/or one or more of the components may be charged (e.g., ionic) or uncharged. A complex may be formed by covalent, non-covalent, and/or electrostatic interactions between the two or more components. In some embodiments, a complex comprises at least two different components that are covalently bonded together. In some embodiments, a complex comprises at least two different components that are associated with one another via electrostatic interaction(s). In some embodiments, at least two components of a complex are associated with one another through bonding that is weaker than a covalent bond. In some embodiments, a nucleic acid molecule is electrostatically bound and/or covalently bound to a portion of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer).
A composition of the present invention may comprise one or more (e.g., 1, 2, 5, 10, 50, 100, 1,000, 5,000, 10,000, or more) nucleic acid molecule(s) and/or one or more (e.g., 1, 2, 5, 10, 50, 100, 1,000, 5,000, 10,000, or more) polyalkyleneimine-alkoxylene polymer(s). In some embodiments, the composition comprises two or more nucleic acid molecules, which may be complexed with a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer). In some embodiments, a single polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer) is complexed with two or more nucleic acid molecules. In some embodiments, a single nucleic acid molecule is complexed with two or more polyalkyleneimine-alkoxylene polymers (e.g., PEI-PEO polymers).
A nucleic acid molecule may have any suitable length. In some embodiments, a nucleic acid molecule has a length that is not prohibitive of being delivered and/or transformed into a host cell. In some embodiments, a nucleic acid molecule has a length of about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 bases to about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kilobases in length.
A nucleic acid molecule useful with this invention may be any nucleic acid or combination of nucleic acids (e.g, DNA, RNA, or any combination thereof) of interest for transforming the cell of an organism. In some embodiments, the nucleic acid molecule may be plasmid DNA, genomic DNA, plastid DNA, mitochondrial DNA, phage DNA, cDNA, mRNA, siRNA, shRNA, miRNA, piRNA, synthetic DNA, PCR amplicons, and/or antisense nucleic acid or any combination thereof. In some embodiments, the nucleic acid molecule may be a negatively charged peptide nucleic acid (PNA). The nucleic acid may be any coding or noncoding nucleic acid (e.g., any transcribed and/or non-transcribed nucleic acids).
In some embodiments, a nucleic acid molecule useful with this invention may be a nucleic acid molecule that is heterologous to the organism into which it is being introduced (e.g., a heterologous nucleic acid molecule or a heterologous polynucleotide) or it may be heterologous with regard to the position in the genome into which it is introduced. Thus, as used herein, heterologous refers to a nucleic acid molecule or nucleotide sequence that either originates from another species or is from the same species or organism but is modified from either its original form or its original position in the genome, or the form primarily expressed in the cell. Thus, a polynucleotide derived from an organism or species different from that of the cell into which the polynucleotide is introduced, is heterologous with respect to that cell and the cell's descendants. In addition, a heterologous polynucleotide includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. present in a different copy number, and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule.
A nucleic acid molecule may comprise or be comprised in a vector, a nucleic acid construct, an expression cassette, and/or a plasmid. A vector, a nucleic acid construct, an expression cassette, and/or a plasmid may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. A vector, a nucleic acid construct, an expression cassette, and/or a plasmid may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
Nucleic acid molecules/nucleic acid constructs, expression cassettes, plasmids and/or vectors useful with this invention may be included in a composition with a polymer of the present invention. In some embodiments, the nucleic acid molecules/nucleic acid constructs, expression cassettes, plasmids and/or vectors may be complexed with polymers of the present invention.
As used herein,“expression cassette” means a heterologous nucleic acid construct comprising a nucleotide sequence of interest, wherein said nucleotide sequence of interest is operably associated with at least a control sequence (e.g., a promoter). Thus, some aspects of the invention provide expression cassettes designed to express the nucleotides sequences of the invention.
An isolated nucleic acid molecule, polynucleotide or nucleic acid construct of the present invention is generally free of nucleotide sequences that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid was derived (such as coding sequences present at the 5' or 3' ends). However, the nucleic acid molecule of this invention can include some additional bases or moieties that do not deleteriously or materially affect the basic structural and/or functional characteristics of the nucleic acid molecule. Thus, an isolated nucleic acid molecule or isolated nucleotide sequence is a nucleic acid molecule or nucleotide sequence that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Accordingly, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence. The term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid nucleic acid molecule encoding an additional polypeptide or peptide sequence.
The term“isolated” can further refer to a nucleic acid molecule, polynucleotide, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and/or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized). Moreover, an isolated fragment is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found as such in the natural state. Isolated does not mean that the preparation is technically pure (homogeneous), but rather can be sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose. In representative embodiments of the invention, an isolated nucleic acid molecule, nucleotide sequence, and/or polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% pure (w/w) or more. In other embodiments, an isolated nucleic acid, nucleotide sequence, and/or polypeptide indicates that at least about a 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or more enrichment of the nucleic acid (w/w) is achieved as compared with the starting material.
According to some embodiments, a nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) may be present in a composition in a weight ratio of about 1 : 10 to about 1 :600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer), or any range or value therein. In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1 : 10, 1 :25, 1 :50, 1 :75, 1 : 100, 1 :250, 1 :500, 1 :750, 1 : 1,000, 1 :2,000, 1 :3,000, 1 :4,000, 1 :5,000, 1 :6,000, 1 :7,000, 1 :8,000, 1 :9,000, 1 : 10,000, 1:15,000, 1:20,000, 1:25,000, 1:30,000, 1:35,000, 1:40,000, 1:45,000, 1:50,000, 1:55,000, 1:60,000, 1:70,000, 1:80,000, 1:90,000, 1:100,000, 1:200,000, 1:300,000, 1:400,000, 1:500,000 or 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer), or any range or value therein. In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1:10 to about 1:100, about 1:100 to about 1:1,000, about 1:1,000 to about 1:5,000, about 1:5,000 to about 1:10,000, about 1:5,000 to about 1:20,000, about 1:5,000 to about 1:50,000, about 1:5,000 to about 1:100,000, about 1:5,000 to about 1:200,000, about 1:5,000 to about 1:500,000, about 1:5,000 to about 1:600,000, about 1:10,000 to about 1:100,000, about 1:10,000 to about 1:600,000, about 1:30,000 to about 1:50,000, about 1:10,000 to about 1:50,000, about 1:200,000 to about 1:400,000, about 1:50,000 to about 1:500,000 or about 1:50,000 to about 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer). In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1:5000 to about 1:600,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer). In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1:10,000. In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1:20,000. In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1:100,000. In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1:600,000. In some embodiments, a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) are present in a composition in a weight ratio of about 1:5000 (nucleic acid molecule polyalkyleneimine-alkoxylene polymer).
In some embodiments, a nucleic acid molecule is present in a composition of the invention in an amount of about 0.1 nanograms per milligram of the polyalkyleneimine- alkoxylene polymer to about 400 ng/mg of the polyalkyleneimine-alkoxylene polymer or more. In some embodiments, a nucleic acid molecule is present in a composition of the invention in an amount of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 nanograms per milligram of the polyalkyleneimine-alkoxylene polymer to about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 or more nanograms per milligram of the polyalkyleneimine- alkoxylene polymer.
Example compositions of the invention may comprise a nucleic acid molecule and polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) with the nucleic acid molecule present in an amount relative to 30 mg polymer as set forth in Table 1.
Table 1. Example amounts of plasmid (ng) to polymer (mg) useful for transformation.
Figure imgf000015_0001
As an example, about 100 mg PEI-PEO polymer may combined with 100 mΐ water to achieve a final volume of about 200 mΐ. From this solution, about 60 mΐ of polymer may be combined with DNA. Thus, in this example, about 60 mΐ of this solution comprises about 30 mg of polymer. Thus, for 300 ng of plasmid DNA added to 30 mg of polymer (60 mΐ solution), then a ratio of about 10 ng plasmid to 1 mg PEI-PEO polymer is achieved. As a further example, 1000 ng of plasmid DNA may be added to 30 mg of polymer (60 mΐ solution) to achieve ratio of about 33.3 ng plasmid to 1 mg PEI-PEO polymer. A still further example, 1500 ng or
6000 ng of plasmid DNA may be added to 30 mg of polymer (60 mΐ solution), then the ratio that is achieved is about 50 ng plasmid in 1 mg PEI-PEO polymer or about 200 ng plasmid in 1 mg PEI-PEO polymer, respectively. A polyalkyleneimine (e.g., PEI) polymer, polyalkoxylene (e.g., PEO) polymer and/or polyalkyleneimine-alkoxylene (e.g., PEI-PEO) polymer of the present invention may be and/or may be prepared as described in U.S. Patent Nos. 7,736,525 and 9,068,147 and/or International Publication No. WO 2017/102556, the contents of each of which are incorporated herein by reference in their entirety.
According to some embodiments of the present invention, a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) may be cationic. In some embodiments, a polyalkyleneimine-alkoxylene polymer has an overall cationic charge, but a portion of the polyalkyleneimine-alkoxylene polymer is nonionic and/or anionic, which may reduce the overall charge per weight of the polymer. In some embodiments, a portion of the polyalkyleneimine-alkoxylene polymer (e.g., an outer portion and/or shell) is nonionic. A polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may have a zeta potential of at least about +1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, or more, optionally when present in a composition having a pH of about 7 and/or measured using methods known to those of skill in the art. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a non-quatemized or quatemized PEI-PEO polymer) has a zeta potential of greater than 0 and/or a zeta potential in a range of about +1 to about +4, +7, or +15, optionally when present in a composition having a pH of about 7 and/or measured using methods known to those of skill in the art. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention is water soluble, optionally at room temperature.
A polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may have a PEI backbone and one or more alkoxylene unit(s) (e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)) substituted at one or more places (e.g., at one or more nitrogens) along the PEI backbone. In some embodiments, the polyalkyleneimine-alkoxylene polymer of the present invention may have a polypropyleneimine backbone and one or more alkoxylene unit(s) (e.g., ethylene oxide unit(s), propylene oxide unit(s), and/or butylene oxide unit(s)) substituted at one or more places (e.g., at one or more nitrogens) along the polypropyleneimine backbone. An alkoxylene unit may replace a hydrogen atom in the PEI or polypropyleneimine backbone (e.g., a hydrogen atom attached to a primary amine, secondary amine, and/or tertiary amine in the PEI backbone). In some embodiments, a hydrogen atom in the PEI or polypropyleneimine backbone (e.g., a hydrogen atom attached to a primary amine, secondary amine, and/or tertiary amine in the PEI backbone) is replaced with a single alkoxylene unit (i.e., a monoalkoxylene) or with a polyalkoxylene chain (e.g., a moiety comprising two or more alkoxylene units such as 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45
46, 47, 48, 49, or 50 alkoxylene units). In some embodiments, a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a monoalkoxylene or a polyalkoxylene chain having 2 or 3 to 4, 5, 6, or 8 alkoxylene units (e.g., ethylene oxide units). In some embodiments, a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a polyalkoxylene chain having 8, 9, or 10 to 11, 12, or 13 alkoxylene units (e.g., ethylene oxide units). In some embodiments, a hydrogen atom in the PEI or polypropyleneimine backbone is replaced with a polyalkoxylene chain having 16, 17, or 18 to 19, 20, 21, or 22 alkoxylene units (e.g., ethylene oxide units).
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may be a core-shell polymer. The core comprises an interior portion of the polymer and the shell comprises an outer portion of the polymer. In some embodiments, the shell comprises end or terminal portions of a polymer chain. In some embodiments, the core comprises a portion or all of the PEI or polypropyleneimine of the polyalkyleneimine-alkoxylene polymer. In some embodiments, the core comprises a portion or all of the PEI of a PEI-PEO polymer. The core (e.g., PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer) may have a diameter of about 1, 2, 4, 6, or 8 to about 10, 20, 30, 40, 50, 60, 70, or 80 nm such as about 1 nm to about 10 nm, about 5 nm to about 15 nm, about 20 to about 50 nm, 40 nm to about 80 nm, or 60 nm to about 70 nm. In some embodiments, the core has a diameter of about 5, 10, or 65 nm. In some embodiments, the shell comprises a portion or all of the alkoxylene (e.g., PEO) of the polyalkyleneimine- alkoxylene polymer. In some embodiments, the core comprises a portion or all of the PEI of a PEI-PEO and the shell comprises a portion or all of the PEO of the PEI-PEO polymer. A shell and/or outer surface comprising alkoxylene (e.g., PEO) may aid in controlling and/or reducing the toxicity of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) to a cell (e.g., a host cell and/or the cell in an organism) and/or may block and/or reduce exposure of charged moieties (e.g., cationic moieties) on the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is in the form of a particle (e.g., a microparticle or nanoparticle) and/or a sphere.
A polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may comprise a number of alkoxylene units, such as ethylene oxide units, per nitrogen atom of PEI or polypropyleneimine in the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, or 50 alkoxylene units (e.g., ethylene oxide units) per nitrogen atom of PEI or polypropyleneimine in the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per nitrogen atom of PEI or polypropyleneimine in the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 1, 2, or 3 to about 4, 5, 6, or 8 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, about 8, 9, or 10 to about 11, 12, or 13 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, or about 16, 17, or 18 to about 19, 20, 21, or 22 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, or 2 to about 3, 4, or 5 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, about 5, 6, 7, 8, 9, or 10 to about 11, 12, 13, 14, or 15 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer, or about 15 or 20 to about 25 or 30 alkoxylene units per nitrogen atom of PEI or polypropyleneimine in the polymer.
The number of alkoxylene units (e.g., ethylene oxide units) may be determined per primary amine of PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer). A primary amine as used herein in reference to PEI or polypropyleneimine in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) refers to PEI or polypropyleneimine prior to formation of the polyalkyleneimine-alkoxylene polymer or in absence of an alkoxylene unit. As one of skill in the art will understand and as described herein, a primary amine of PEI or polypropyleneimine may be functionalized with 0, 1 or more alkoxylene unit(s) (e.g., a hydrogen atom of the primary amine may be substituted with an alkoxylene unit). Functionalizing one or more primary amines of PEI or polypropyleneimine with at least one alkoxylene unit may form a polyalkyleneimine- alkoxylene polymer of the present invention. In some embodiments, a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, or 50 alkoxylene units (e.g., ethylene oxide units) per primary amine of PEI or polypropyleneimine in the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per primary amine of PEI or polypropyleneimine in the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises about 1, 2, or 3 to about 4, 5, 6, or 8 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, about 8, 9, or 10 to about 11, 12, or 13 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, or about 16, 17, or 18 to about 19, 20, 21, or 22 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer) comprises about 0, 1, or 2 to about 3, 4, or 5 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, about 5, 6, 7, 8, 9, or 10 to about 11, 12, 13, 14, or 15 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer, or about 15 or 20 to about 25 or 30 alkoxylene units per primary amine of PEI or polypropyleneimine in the polymer.
According to some embodiments 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 primary amines of PEI or polypropyleneimine in the polyalkyleneimine-alkoxylene polymer are substituted/functionalized with at least one alkoxylene unit (e.g., at least one ethylene oxide unit). The amount or degree of alkoxylation for a polyalkyleneimine-alkoxylene polymer may be measured using methods known to those of skill in the art such as by determining OH-number and/or by using nuclear magnetic resonance (e.g., 'H NMR). In some embodiments, alkoxylation may be quantified by determining the OH-number (also referred to interchangeably as an OH-value) for a polyalkyleneimine-alkoxylene polymer (e.g., a PEI- PEO polymer), and an alkoxylation degree of 3-5 units may be about 320-213 mg KOH, respectively, to provide an OH-number of about 213 to about 320, an alkoxylation degree of 8-13 alkoxylation may be about 142-91 mg KOH, respectively, to provide an OH-number of about 91 to about 142, and/or an alkoxylation degree of 18-22 may be about 67-55 mg KOH, respectively, to provide an OH-number of about 55-66. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) has an OH-number in a range of about 40 to about 400, about 40 to about 100, about 100 to about 200, or about 200 to about 400.
A terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) may be capped with hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, or -C(0)-C1-C22 aryl. In some embodiments, a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is capped with hydrogen or a -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, or -C(0)-C1-C22 aryl. In some embodiments, a terminal alkoxylene unit (e.g., ethylene oxide unit) of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is capped with hydrogen. In some embodiments, an internal and/or terminal nitrogen atom (e.g., of a primary, secondary, and/or tertiary amine) of PEI or polypropyleneimine of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is bound to a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl arylalkenyl, or arylalkynyl substituent, or any combination thereof.
In some embodiments, one or more nitrogen atom(s) in a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) may be substituted with a substituent comprising 1 to 12 carbon atoms (also referred to herein as a C1-C12 substituent). A C1-C12 substituent may be saturated or unsaturated and/or linear or branched. In some embodiments, a C1-C12 substituent is a saturated or unsaturated, linear or branched hydrocarbon. In some embodiments, a C1-C12 substituent is a substituted or unsubstituted C1-C12 alkyl, alkenyl, alkynyl, aryl, arylalkyl arylalkenyl, or arylalkynyl substituent. In some embodiments, one or more nitrogen atom(s) in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may be substituted with a group selected from a C1-C12 alkyl, a C1-C12 aryl, and/or a Cl- C12 arylalkyl (e.g., benzyl). In some embodiments, one or more nitrogen atom(s) in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may be substituted with a benzyl. In some embodiments, one or more nitrogen atom(s) in a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) may be substituted with a group selected from a C1-C4 alkyl, a C1-C4 aryl, and/or a C1-C4 arylalkyl. Substitution with a C1-C12 substituent may result in a neutral or cationic charge on a respective nitrogen atom depending on its total number of substituents. In some embodiments, substitution with a C1-C12 substituent may result in permanent quaternization (i.e., a permanent cationic charge) at the substituted position. In some embodiments, substitution with a C1-C12 substituent may result in permanent quaternization of a nitrogen atom in the PEI or polypropyleneimine backbone of a polymer.
A polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may comprise one or more (e.g., 1, 2, 3, 4, 5, 10, 20, 100, or more) quatemized functional group(s). A nitrogen atom (e.g., of a primary, secondary, and/or tertiary amine) of the polymer may comprise one or more quaternized functional group(s). In some embodiments, about 0%, 25%, or 50% to about 60%, 70%, 80%, 90%, or 100% of the nitrogen atoms of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprise a quatemized functional group. In some embodiments, about 0%, 0.5%, 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,
40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,
56%, 57%, 58%, 59%, 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nitrogen atoms of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprise a quaternized functional group. In some embodiments, about 0%, 5%, 10%, 20%, 40%, or 50% to about 60%, 70%, 80%, 90%, or 100% of the nitrogen atoms in the PEI or polypropyleneimine backbone of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprise a quaternized functional group.
A quaternized functional group may comprise a nitrogen atom of PEI or polypropyleneimine bound to a substituent comprising 1 to 12 carbon atoms (also referred to herein as a C1-C12 substituent). Exemplary C1-C12 substituents include, but are not limited to, benzyl, a C1-C12 alkyl, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 arylalkyl, a C1-C12 arylalkenyl, or a C1-C12 arylalkynyl, each of which may be substituted or unsubstituted. In some embodiments, a quatemized functional group is formed by reacting a C1-C12 substituent with a tertiary amine of PEI or polypropyleneimine.
The polyalkyleneimine (e.g., PEI) and/or polyalkoxylene (e.g., PEO) of the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may be linear and/or branched. In some embodiments, PEI and/or PEO of a PEI-PEO polymer are branched. A polyalkyleneimine-alkoxylene polymer may comprise 1, 5, 10, or 20 to 30, 40, or 50 alkoxylene units. PEO of a PEI-PEO polymer may comprise 1, 5, 10, or 20 to 30, 40, or 50 ethylene oxide units.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises a polyalkoxylene in an amount of about 50% to about 99% by weight of the polymer such as about 75% to about 99%, about 80% to about 99%, about 80% to about 95%, or about 90% to about 95% by weight of the polymer. A polyalkyleneimine-alkoxylene polymer may comprise a polyalkoxylene in an amount of about 50%, 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or more by weight of the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer comprises PEI or polypropyleneimine in an amount of about 0.5% to about 50% by weight of the polymer such as about 1% to about 25%, about 5% to about 20%, about 5% to about 10%, or about 0.5% to about 10% by weight of the polymer. In some embodiments, a polyalkyleneimine-alkoxylene polymer comprises PEI or polypropyleneimine in an amount of about 0.5%, 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%, 31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,
48%, 49%, or 50% by weight of the polymer.
In some embodiments, a PEI-PEO polymer comprises PEO in an amount of about 50% to about 99% by weight of the PEI-PEO polymer such as about 75% to about 99%, about 80% to about 99%, about 80% to about 95%, or about 90% to about 95% by weight of the PEI-PEO polymer. A PEI-PEO polymer may comprise PEO in an amount of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 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%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
98.5%, 99%, 99.5% or more by weight of the PEI-PEO polymer. In some embodiments, a PEI-PEO polymer comprises PEI in an amount of about 0.5% to about 50% by weight of the PEI-PEO polymer such as about 1% to about 25%, about 5% to about 20%, about 5% to about 10%, or about 0.5% to about 10% by weight of the PEI-PEO polymer. In some embodiments, a PEI-PEO polymer comprises PEI in an amount of about 0.5%, 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%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% by weight of the PEI-PEO polymer.
PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer of the present invention may have an amine value in a range of about 5, 10, or 15 to about 20, 25, or 30. In some embodiments, PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an amine value of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, or 25. As one of skill in the art would recognize, amine value is the number of milligrams of potassium hydroxide (KOH) equivalent to the basicity in 1 gram of sample (e.g., 1 gram of polymer). Amine value may be measured in accordance with ASTM D 2073-92 and optionally calculating the total amine value assuming all amines are primary amines. In some embodiments, PEI or polypropyleneimine used to form and/or in a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has a molecular weight of about 400 g/mol to about 900,000 g/mol such as about 400 g/mol to about 825,000 g/mol, about 400 g/mol to about 750,000 g/mol, about 400 g/mol to about 10,000 g/mol, about 400 g/mol to about 600 g/mol, about 800 g/mol to about 20,000 g/mol, about 600,000 g/mol to about 900,000 g/mol, about 600,000 g/mol to about 825,000 g/mol, about 4,000 g/mol to about 6,000 g/mol, about 1,000 g/mol to about 500,000 g/mol, about 10,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 30,000 g/mol, or about 50,000 g/mol to about 500,000 g/mol. A molecular weight as used herein in reference to a polyalkyleneimine, polyalkoxylene, and polyalkyleneimine-alkoxylene polymer is a number average molecular weight (Mn).
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an average molecular weight of about 1,000 g/mol to about 30,000,000 g/mol, about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 30,000 g/mol to about 160,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about
167,000 g/mol, about 145,000 g/mol to about 450,000 g/mol, about 145,000 g/mol to about
800,000 g/mol, about 150,000 g/mol to about 450,000 g/mol, about 150,000 g/mol to about
800,000 g/mol, about 326,000 g/mol to about 490,000 g/mol, about 367,000 g/mol to about
448,000 g/mol, about 400,000 g/mol to about 800,000 g/mol, about 633,000 g/mol to about
950,000 g/mol, about 712,000 g/mol to about 871,000 g/mol, about 25,000 g/mol to about 4,500,000 g/mol, about 25,000 g/mol to about 5,000,000 g/mol, about 80,000 g/mol to about 12,000,000 g/mol, 20,000 g/mol to about 24,000,000 g/mol, about 150,000 g/mol to about 24,000,000 g/mol, about 4,000,000 g/mol to about 25,000,000 g/mol, about 4,5000,000 g/mol to about 24,000,000 g/mol, about 4,5000,000 g/mol to about 12,000,000 g/mol, about 4,5000,000 g/mol to about 15,000,000 g/mol, about 4,5000,000 g/mol to about 20,000,000 g/mol, about 3,600,000 g/mol to about 5,400,000 g/mol, about 4,000,000 g/mol to about 4,900,000 g/mol, about 4,5000,000 g/mol to about 24,000,000 g/mol, about 9,000,000 g/mol to about 15,000,000 g/mol, about 10,000,000 g/mol to about 13,000,000 g/mol, about 19,000,000 g/mol to about 29,000,000 g/mol, about 17,000,000 g/mol to about 26,000,000 g/mol, or any range or value therein. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has a molecular weight of about 1,000 g/mol to about 10,000,000 g/mol such as about 1,000 g/mol to about 10,000 g/mol, about 100,000 g/mol to about 1,000,000 g/mol, about 1,000,000 g/mol to about 10,000,000 g/mol, about 1,900 g/mol to about 2,900 g/mol, about 2,100 g/mol to about 2,400 g/mol, about 3,300 g/mol to about 5,000 g/mol, about 3,700 g/mol to about 4,600 g/mol, about 6,100 g/mol to about 9,300 g/mol, about 6,900 g/mol to about 8,500 g/mol, about 100,000 g/mol to about 160,000 g/mol, about 115,000 g/mol to about 141,000 g/mol, about 266,000 g/mol to about 400,000 g/mol, about 299,000 g/mol to about 367,000 g/mol, about 520,000 g/mol to about 782,000 g/mol, about 585,000 g/mol to about 717,000 g/mol, about 1,780,000 g/mol to about 2,670,000 g/mol, about 2,000,000 g/mol to about 2,460,000 g/mol, about 4,000,000 g/mol to about 6,100,000 g/mol, about 4,500,000 g/mol to about 5,600,000 g/mol, about 6,900,000 g/mol to about 10,000,000 g/mol, or about 7,700,000 g/mol to about 9,500,000 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an EO value (degree of ethoxylation) of about 2 to about 6, about 2 to about 20, about 2 to about 28, about 2 to about 30, about 3 to about 5, about 3 to about 6, about 3 to about 9, about 3 to about 15, about 3 to about 22, about 5 to about 28, about 7 to about 15, about 8 to about 14, or about 17 to about 30 or any range or value therein.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has an OH-number of about 70 mg to about 40 mg, about 70 mg to about 50 mg, about 100 mg to about 40 mg, about 100 mg to about 60 mg, about 130 mg to about 40 mg, about 130 mg to about 80 mg, about 200 mg to about 40 mg, about
200 mg to about 60 mg, about 200 mg to about 80 mg, about 220 mg to about 40 mg, about
220 mg to about 80 mg, about 250 mg to about 40 mg, about 250 mg to about 70 mg, about
300 mg to about 40 mg, about 300 mg to about 50 mg, about 300 mg to about 60 mg, about
300 mg to about 70 mg, about 300 mg to about 80 mg, about 340 mg to about 60 mg, about
340 mg to about 80 mg, about 350 mg to about 40 mg, about 350 mg to about 50 mg, about
350 mg to about 70 mg, or about 350 mg to about 80 mg, or any range or value therein.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 20,000 g/mol to about 200,000 g/mol, about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, 20,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 90,000 g/mol, about 25,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 30,000 g/mol to about 90,000 g/mol, about 30,000 g/mol to about 100,000 g/mol, about 30,000 g/mol to about 160,000 g/mol, or any range or value therein; a molecular weight of about 1,900 g/mol to about 2,900 g/mol, about 2,100 g/mol to about 2,400 g/mol, about 3,300 g/mol to about 5,000 g/mol, about 3,700 g/mol to about 4,600 g/mol, about 6,100 g/mol to about 9,300 g/mol, or about 6,900 g/mol to about 8,500 g/mol; about 1 to about 25 alkoxylene units per amine of the polypropyleneimine in the polymer such as about 2 to about 4, about 2 to about 9, about 3 to about 4, about 3 to about 9, about 5 to about 10, about 3 to about 15, about 3 to about 20, about 3 to about 22, or about 20 to about 25, or any range or value therein; alkoxylene (e.g., ethylene oxide) in an amount of about 75% to about 95% by weight of the polyalkyleneimine-alkoxylene polymer; a core diameter or diameter of the polyalkyleneimine in the polyalkyleneimine-alkoxylene polymer of about 1 nm to about 10 nm; a zeta potential in a range of about +1, +2, +3, +4, +5, +6, +7, or +8, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 300 mg to about 50 mg, about 300 mg to about 60 mg, about 300 mg to about 70 mg, about 300 mg to about 80 mg, about 300 mg to about 100 mg, about 300 mg to about 120 mg, about 300 mg to about 140 mg, about 300 mg to about 150 mg, about 150 mg to about 50 mg, about 150 mg to about 60 mg, about 150 mg to about 70 mg, or about 150 mg to about 80 mg, or any range or value therein. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer had/has an amine value of about 15 to about 20 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 3,000,000 g/mol to about 30,000,000 g/mol, about 4,000,000 g/mol to about 12,000,000 g/mol, about 4,000,000 g/mol to about 15,000,000 g/mol, about 4,000,000 g/mol to about 20,000,000 g/mol, about 4,000,000 g/mol to about 25,000,000 g/mol, about 4,5000,000 g/mol to about 10,000,000 g/mol, about 4,5000,000 g/mol to about 12,000,000 g/mol, about 4,5000,000 g/mol to about 15,000,000 g/mol, about 4,5000,000 g/mol to about 20,000,000 g/mol, about 4,5000,000 g/mol to about 24,000,000 g/mol, about 10,000,000 g/mol to about 24,000,000 g/mol, about 15,000,000 g/mol to about 24,000,000 g/mol, about 20,000,000 g/mol to about 25,000,000 g/mol, about 3,600,000 g/mol to about 5,400,000 g/mol, about 4,000,000 g/mol to about 4,900,000 g/mol, about 9,000,000 g/mol to about 15,000,000 g/mol, about 10,000,000 g/mol to about 13,000,000 g/mol, about 19,000,000 g/mol to about 29,000,000 g/mol, about 17,000,000 g/mol to about 26,000,000 g/mol, or any range or value therein; a molecular weight of about 1,780,000 g/mol to about 2,670,000 g/mol, about 2,000,000 g/mol to about 2,460,000 g/mol, about 4,000,000 g/mol to about 6,100,000 g/mol, about 4,500,000 g/mol to about 5,600,000 g/mol, about 6,900,000 g/mol to about 10,000,000 g/mol, or about 7,700,000 g/mol to about 9,500,000 g/mol; about 1 to about 20 alkoxylene units per amine of the polypropyleneimine in the polymer such as about 2 to about 4, about 2 to about 9, about 3 to about 4, about 3 to about 9, about 3 to about 10, about 3 to about 15, or about 3 to about 20, or any range or value therein; alkoxylene (e.g., ethylene oxide) in an amount of about 70% to about 95% by weight of the polyalkyleneimine-alkoxylene polymer; a core diameter or diameter of the polyalkyleneimine in the polyalkyleneimine-alkoxylene polymer of about 5 nm to about 15 nm; a zeta potential in a range of about +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, or +14, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 350 mg to about 50 mg, about 350 mg to about 60 mg, about 350 mg to about 70 mg, about 350 mg to about 80 mg, about 350 mg to about 100 mg, about 350 mg to about 120 mg, about 350 mg to about 140 mg, about 350 mg to about 150 mg, about 300 mg to about 60 mg, about 300 mg to about 70 mg, about 300 mg to about 80 mg, about 300 mg to about 100 mg, about 300 mg to about 120 mg, about 300 mg to about 140 mg, about 300 mg to about 150 mg, about 150 mg to about 60 mg, about 150 mg to about 70 mg, or about 150 mg to about 80 mg, or any range or value therein. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 15 to about 20 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 120,000 g/mol to about 1,000,000 g/mol, about 145,000 g/mol to about 450,000 g/mol, about 145,000 g/mol to about 500,000 g/mol, about 145,000 g/mol to about 600,000 g/mol, about 145,000 g/mol to about 800,000 g/mol, about 150,000 g/mol to about 400,000 g/mol, about 150,000 g/mol to about 450,000 g/mol, about 150,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 700,000 g/mol, about 150,000 g/mol to about 800,000 g/mol, about 350,000 g/mol to about 800,000 g/mol, about 400,000 g/mol to about 800,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about 167,000 g/mol, about 326,000 g/mol to about 490,000 g/mol, about 367,000 g/mol to about 448,000 g/mol, about 633,000 g/mol to about 950,000 g/mol, about 712,000 g/mol to about 871,000 g/mol, or any range or value therein; a molecular weight of about 100,000 g/mol to about 160,000 g/mol, about 115,000 g/mol to about 141,000 g/mol, about 266,000 g/mol to about 400,000 g/mol, about 299,000 g/mol to about 367,000 g/mol, about 520,000 g/mol to about 782,000 g/mol, or about 585,000 g/mol to about 717,000 g/mol; about 3 to about 30 alkoxylene units per amine of the polypropyleneimine in the polymer such as about 4 to about 8, about 4 to about 10, about 4 to about 15, about 4 to about 25, about 4 to about 30, about 5 to about 10, about 5 to about 15, about 5 to about 25, about 5 to about 30, about 10 to about 15, about 10 to about 25, about 10 to about 30, or any range or value therein; alkoxylene (e.g., ethylene oxide) in an amount of about 80% to about 98% by weight of the polyalkyleneimine-alkoxylene polymer; a core diameter or diameter of the polyalkyleneimine in the polyalkyleneimine-alkoxylene polymer of about 60 nm to about 70 nm; a zeta potential in a range of about +2, +3, +4, or +5, optionally when present in a composition having a pH of about 7; and/or an OH-Number of about 250 mg to about 50 mg, about 250 mg to about 60 mg, about 250 mg to about 70 mg, about 250 mg to about 80 mg, about 250 mg to about 100 mg, about 350 mg to about 120 mg, about 350 mg to about 140 mg, about 350 mg to about 150 mg, about 200 mg to about 60 mg, about 200 mg to about 70 mg, about 200 mg to about 80 mg, about 200 mg to about 100 mg, about 100 mg to about 40 mg, or about 90 mg to about 40 mg, or any range or value therein. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 15 to about 20 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 20,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 100,000 g/mol, about 20,000 g/mol to about 160,000 g/mol, about 25,000 g/mol to about 100,000 g/mol, about 25,000 g/mol to about 140,000 g/mol, about 25,000 g/mol to about 150,000 g/mol, about 25,000 g/mol to about 160,000 g/mol, about 25,000 g/mol to about 180,000 g/mol, about 25,000 g/mol to about 200,000 g/mol, about 25,000 g/mol to about 500,000 g/mol, about 25,000 g/mol to about 1,500,000 g/mol, about 25,000 g/mol to about 3,000,000 g/mol, about 25,000 g/mol to about 5,000,000 g/mol, about 30,000 g/mol to about 100,000 g/mol, about 30,000 g/mol to about 160,000 g/mol, about 30,000 g/mol to about 180,000 g/mol, about 30,000 g/mol to about 200,000 g/mol, about 30,000 g/mol to about 500,000 g/mol, about 30,000 g/mol to about 1,500,000 g/mol, about 30,000 g/mol to about 3,000,000 g/mol, about 30,000 g/mol to about 5,000,000 g/mol, about 140,000 g/mol to about 160,000 g/mol, about 140,000 g/mol to about 180,000 g/mol, about 140,000 g/mol to about 200,000 g/mol, about 140,000 g/mol to about 500,000 g/mol, about 140,000 g/mol to about 1,500,000 g/mol, about 140,000 g/mol to about 3,000,000 g/mol, about 140,000 g/mol to about 5,000,000 g/mol, 150,000 g/mol to about 180,000 g/mol, about 150,000 g/mol to about 200,000 g/mol, about 150,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 1,500,000 g/mol, about 150,000 g/mol to about 3,000,000 g/mol, about 150,000 g/mol to about 5,000,000 g/mol, about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about 167,000 g/mol, about 3,600,000 g/mol to about 5,400,000 g/mol, about 4,000,000 g/mol to about 4,900,000 g/mol, or any range or value therein; a molecular weight of about 1,900 g/mol to about 2,900 g/mol, about 2,100 g/mol to about 2,400 g/mol, about 100,000 g/mol to about 160,000 g/mol, about 115,000 g/mol to about 141,000 g/mol, about 1,780,000 g/mol to about 2,670,000 g/mol, or about 2,000,000 g/mol to about 2,460,000 g/mol; about 1 to about 8 alkoxylene units per amine of the polypropyleneimine in the polymer such as about 2 to about 4, about 2 to about 5, about 2 to about 6, about 3 to about 4, about 3 to about 5, or about 3 to about 6, or any range or value therein; alkoxylene (e.g., ethylene oxide) in an amount of about 70% to about 85% by weight of the polyalkyleneimine-alkoxylene polymer; and/oran OH-number of about 350 mg to about 200 mg, about 350 mg to about 225 mg, about 350 mg to about 250 mg, about 350 mg to about 275 mg, 330 mg to about 200 mg, about 330 mg to about 225 mg, about 330 mg to about 250 mg, about 330 mg to about 275 mg, 300 mg to about 200 mg, about 300 mg to about 225 mg, about 300 mg to about 250 mg, about 300 mg to about 275 mg, 280 mg to about 200 mg, about 280 mg to about 210 mg, or any range or value therein.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 75,000 g/mol to about 400,000 g/mol, about 75,000 g/mol to about 500,000 g/mol, about 75,000 g/mol to about 1,000,000 g/mol, about 75,000 g/mol to about 5,000,000 g/mol, about 75,000 g/mol to about 8,000,000 g/mol, about 75,000 g/mol to about 12,000,000 g/mol, about 80,000 g/mol to about 450,000 g/mol, about 80,000 g/mol to about 500,000 g/mol, about 80,000 g/mol to about 750,000 g/mol, about 80,000 g/mol to about 1,000,000 g/mol, about 80,000 g/mol to about 5,000,000 g/mol, about 100,000 g/mol to about 450,000 g/mol, about 100,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 450,000 g/mol, about 500,000 g/mol to about 1,000,000 g/mol, about 500,000 g/mol to about 5,000,000 g/mol, about 500,000 g/mol to about 12,000,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 326,000 g/mol to about 490,000 g/mol, about 367,000 g/mol to about 448,000 g/mol, about 9,000,000 g/mol to about 15,000,000 g/mol, about 10,000,000 g/mol to about 13,000,000 g/mol, or any range or value therein; a molecular weight of about 3,300 g/mol to about 5,000 g/mol, about 3,700 g/mol to about 4,600 g/mol, about 266,000 g/mol to about 400,000 g/mol, about 299,000 g/mol to about 367,000 g/mol, about 4,000,000 g/mol to about 6,100,000 g/mol, or about 4,500,000 g/mol to about 5,600,000 g/mol; about 5 to about 15 alkoxylene units per amine of the polypropyleneimine in the polymer such as about 8 to about 9, about 8 to about 10, about 8 to about 12, about 8 to about 13, about 8 to about 14, or about 8 to about 15, or any range or value therein; alkoxylene (e.g., ethylene oxide) in an amount of about 85% to about 95% by weight of the polyalkyleneimine- alkoxylene polymer; and/or an OH-number of about 150 mg to about 70 mg, about 150 mg to about 80 mg, about 150 mg to about 90 mg, about 150 mg to about 100 mg, about 150 mg to about 120 mg, about 140 mg to about 70 mg, about 140 mg to about 80 mg, about 140 mg to about 90 mg, about 140 mg to about 100 mg, about 140 mg to about 120 mg, or about 100 mg to about 80 mg, or any range or value therein.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 150,000 g/mol to about 250,000 g/mol, about 150,000 g/mol to about 500,000 g/mol, about 150,000 g/mol to about 800,000 g/mol, bout 150,000 g/mol to about 900,000 g/mol, bout 150,000 g/mol to about 1,000,000 g/mol, about 150,000 g/mol to about 5,000,000 g/mol, about 150,000 g/mol to about 12,000,000 g/mol, 150,000 g/mol to about 24,000,000 g/mol, about 150,000 g/mol to about 25,000,000 g/mol, about 500,000 g/mol to about 1,000,000 g/mol, about 500,000 g/mol to about 5,000,000 g/mol, about 500,000 g/mol to about 10,000,000 g/mol, about 500,000 g/mol to about 12,000,000 g/mol, about 500,000 g/mol to about 24,000,000 g/mol, about 750,000 g/mol to about 1,000,000 g/mol, about 750,000 g/mol to about 5,000,000 g/mol, about 750,000 g/mol to about 10,000,000 g/mol, about 750,000 g/mol to about 12,000,000 g/mol, about 750,000 g/mol to about 24,000,000 g/mol, about 5,000,000 g/mol to about 25,000,000 g/mol, about 10,000,000 g/mol to about 25,000,000 g/mol, about 12,000,000 g/mol to about 25,000,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, about 633,000 g/mol to about 950,000 g/mol, about 712,000 g/mol to about 871,000 g/mol, about 19,000,000 g/mol to about 29,000,000 g/mol, about 17,000,000 g/mol to about 26,000,000 g/mol, or any range or value therein; a molecular weight of about 6,100 g/mol to about 9,300 g/mol, about 6,900 g/mol to about 8,500 g/mol, about 520,000 g/mol to about 782,000 g/mol, about 585,000 g/mol to about 717,000 g/mol, about 6,900,000 g/mol to about 10,000,000 g/mol, or about 7,700,000 g/mol to about 9,500,000 g/mol; about 15 to about 30 alkoxylene units per amine of the polypropyleneimine in the polymer such as about 15 to about 20, about 15 to about 25, about 15 to about 30, about 18 to about 22, about 18 to about 28, about 20 to about 28, about 20 to about 30, or any range or value therein; alkoxylene (e.g., ethylene oxide) in an amount of about 90% to about 98% by weight of the polyalkyleneimine-alkoxylene polymer; and/or an OH-number of about 80 mg to about 40 mg, about 80 mg to about 50 mg, about 80 mg to about 60 mg, about 70 mg to about 40 mg, about 70 mg to about 50 mg, about 70 mg to about 60 mg, about 60 mg to about 40 mg, about 60 mg to about 50 mg, or about 50 mg to about 40 mg, or any range or value therein.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 24,000 g/mol to about 36,000 g/mol, about 27,000 g/mol to about 33,000 g/mol, about 25,000 g/mol to about 40,000 g/mol, about 25,000 g/mol to about 35,000 g/mol or any range or value therein, (e.g., about 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, or 40,000 g/mol), optionally, an average molecular weight of about 30,000 g/mol; a molecular weight of about 1,900 g/mol to about 2,900 g/mol or about 2,100 g/mol to about 2,400 g/mol; about 2 to about 5 alkoxylene units per amine of the polypropyleneimine in the polymer (i.e., an EO value), or any range or value therein, (e.g., about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5), optionally an EO value of 3.6; alkoxylene (e.g., ethylene oxide) in an amount of about 75% to about 85% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 79%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 1 nm to about 10 nm such as about 5 nm; a zeta potential in a range of about +8, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 300 mg to about 250 mg, about 290 mg to about 260 mg, or about 280 mg to about 260 mg, or any range or value therein (e.g., about 300, 299, 298, 297, 296, 295, 294, 293, 292, 291, 290, 289, 288, 287, 286, 285, 284, 283, 282, 281, 280, 279, 278, 277, 276, 275, 274, 273, 272, 271, 270, 269, 268, 267, 266, 265, 264, 263, 262, 261, 260, 259, 258, 257, 256, 255, 254, 253, 252, 251, or 250 mg), optionally an OH-number of 276 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 75,000 g/mol to about 90,000 g/mol, about 65,000 g/mol to about 99,000 g/mol, about 73,000 g/mol to about 91,000 g/mol, about 75,000 g/mol to about 85,000 g/mol or any range or value therein, (e.g., about 75,000, 76,000, 77,000, 78,000, 79,000, 80,000, 81,000, 82,000, 83,000, 84,000, 85,000, 86,000, 87,000, 88,000, 89,000, or 90,000 g/mol), optionally, an average molecular weight of about 82,000 g/mol; a molecular weight of about 3,300 g/mol to about 5,000 g/mol or about 3,700 g/mol to about 4,600 g/mol; about 7 to about 10 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10), optionally an EO value of 8.4; alkoxylene (e.g., ethylene oxide) in an amount of about 85% to about 90% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 88%; a core diameter or diameter of the polyalkyleneimine- alkoxylene polymer of about 1 nm to about 10 nm such as about 5 nm; a zeta potential in a range of about +2, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 150 mg to about 100 mg, or about 150 mg to about 120 mg, or any range or value therein (e.g., about 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139,
138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120 mg), optionally an OH-number of 136 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 140,000 g/mol to about 180,000 g/mol, about 126,000 g/mol to about 190,000 g/mol, about 142,000 g/mol to about 174,000 g/mol, about 140,000 g/mol to about 170,000 g/mol or any range or value therein, (e.g., about 140,000, 141,000, 142,000, 143,000, 144,000, 145,000, 146,000, 147,000, 148,000, 149,000, 150,000, 155,000, 156,000, 157,000, 158,000, 159,000, 160,000, 161,000, 162,000, 163,000, 164,000, 165,000, 166,000, 167,000, 168,000, 169,000, 170,000, 171,000, 172,000, 173,000, 174,000, 175,000, 176,000, 177,000, 178,000, 179,000 or
180,000 g/mol), optionally, an average molecular weight of about 158,000 g/mol; a molecular weight of about 6,100 g/mol to about 9,300 g/mol or about 6,900 g/mol to about 8,500 g/mol; about 18 to about 23 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, or 23), optionally an EO value of 21.8; alkoxylene (e.g., ethylene oxide) in an amount of about 90% to about 95% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 93% to about 94%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 1 nm to about 10 nm such as about 5 nm; a zeta potential in a range of about +1, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 75 mg to about 45 mg, or about 65 mg to about 45 mg, or any range or value therein (e.g., about 75, 74, 73, 72, 71,
70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, or 45 mg), optionally an OH-number of 56 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 19 and/or a molecular weight of about 400 g/mol to about 600 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 4,000,000 g/mol to about 5,000,000 g/mol, about 3,600,000 g/mol to about 5,400,000 g/mol, about 4,000,000 g/mol to about 4,900,000 g/mol, or any range or value therein, (e.g., about 4,000,000,
4,100,000, 4,200,000, 4,300,000, 4,400,000, 4,500,000, 4,600,000, 4,700,000, 4,700,000,
4,900,000, or 5,000,000 g/mol), optionally, an average molecular weight of about 4,500,000 g/mol; a molecular weight of about 1,780,000 g/mol to about 2,670,000 g/mol or about 2,000,000 g/mol to about 2,460,000 g/mol; about 2 to about 4 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4), optionally an EO value of 2.9; alkoxylene (e.g., ethylene oxide) in an amount of about 70% to about 80% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 74% to about 75%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 60 nm to about 70 nm such as about 65 nm; a zeta potential in a range of about +13, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 350 mg to about 300 mg, or any range or value therein (e.g., about 350, 349, 348, 347, 346, 345, 344,
343, 342, 341, 340, 339, 338, 337, 336, 335, 334, 333, 332, 331, 330, 329, 328, 327, 326,
325, 324, 323, 322, 321, 320, 319, 318, 317, 316, 315, 314, 313, 312, 311, 310, 309, 308,
307, 306, 305, 304, 303, 302, 301, or 300 mg), optionally an OH-number of 331 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 10,000,000 g/mol to about 15,000,000 g/mol, about 9,000,000 g/mol to about 15,000,000 g/mol, about 10,000,000 g/mol to about 13,000,000 g/mol, or any range or value therein, (e.g., about 10,000,000, 10,500,000, 11,000,000, 11,500,000, 12,00,000, 12,500,000, 13,000,000, 13,500,000, 14,000,000, 14,500,000, or 15,000,000 g/mol), optionally, an average molecular weight of about 12,000,000 g/mol; a molecular weight of about 4,000,000 g/mol to about 6,100,000 g/mol or about 4,500,000 g/mol to about 5,600,000 g/mol; about 7 to about 10 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10), optionally an EO value of 8.6; alkoxylene (e.g., ethylene oxide) in an amount of about 85% to about 90% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 87%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 60 nm to about 70 nm such as about 65 nm; a zeta potential in a range of about +4, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 150 mg to about 100 mg, or about 150 mg to about 120 mg, or any range or value therein (e.g., about 150, 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, 127, 126,
125, 124, 123, 122, 121, 120 mg), optionally an OH-number of 133 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 22,000,000 g/mol to about 25,000,000 g/mol, about 19,000,000 g/mol to about 29,000,000 g/mol, about 17,000,000 g/mol to about 26,000,000 g/mol, or any range or value therein, (e.g., about
22,000,000, 22,500,000, 23,000,000, 23,500,000, 24,000,000, 24,500,000, 25,00,000, 25,500,000 g/mol), optionally, an average molecular weight of about 24,000,000 g/mol; a molecular weight of about 6,900,000 g/mol to about 10,000,000 g/mol or about 7,700,000 g/mol to about 9,500,000 g/mol; about 16 to about 21 alkoxylene units per amine of the polypropyleneimine in the polymer , or any range or value therein, (e.g., about 16, 16.1, 16.2,
16.3, 16.4, 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6,
19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, or 21), optionally an EO value of 18.1; alkoxylene (e.g., ethylene oxide) in an amount of about 90% to about 95% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 92%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 60 nm to about 70 nm such as about 65 nm; a zeta potential in a range of about +3, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 80 mg to about 45 mg, or about 75 mg to about 55 mg, or any range or value therein (e.g., about 80, 79, 78, 77,
76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52
51, 50, 49, 48, 47, 46, or 45 mg), optionally an OH-number of 67 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 600,000 g/mol to about 900,000 g/mol or about 600,000 g/mol to about 825,000 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 140,000 g/mol to about 180,000 g/mol, about 121,000 g/mol to about 182,000 g/mol, about 136,000 g/mol to about 167,000 g/mol, about 140,000 g/mol to about 170,000 g/mol or any range or value therein, (e.g., about 140,000, 141,000, 142,000, 143,000, 144,000, 145,000, 146,000, 147,000, 148,000, 149,000, 150,000, 155,000, 156,000, 157,000, 158,000, 159,000, 160,000, 161,000, 162,000, 163,000, 164,000, 165,000, 166,000, 167,000, 168,000, 169,000, 170,000, 171,000, 172,000, 173,000, 174,000, 175,000, 176,000, 177,000, 178,000, 179,000 or
180,000 g/mol), optionally, an average molecular weight of about 152,000 g/mol; a molecular weight of about 100,000 g/mol to about 160,000 g/mol or about 115,000 g/mol to about 141,000 g/mol,; about 2 to about 6 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6), optionally an EO value of 5; alkoxylene (e.g., ethylene oxide) in an amount of about 80% to about 85% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 82% to about 83%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 5 nm to about 15 nm such as about 10 nm; a zeta potential in a range of about +4, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 240 mg to about 190 mg, about 220 mg to about 200 mg, or any range or value therein (e.g., about 240, 239, 238, 237, 236, 235, 234, 233, 232, 231, 230, 229, 228, 227, 226, 225, 224, 223, 222, 221, 220, 219,
218, 217, 216, 215, 214, 213, 212, 211, 210, 209, 208, 207, 206, 205, 204, 203, 202, 201,
200, 199, 198, 197, 196, 195, 194, 193, 192, 191, or 190 mg), optionally an OH-number of 215 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol. In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 300,000 g/mol to about 500,000 g/mol, about 326,000 g/mol to about 490,000 g/mol, about 367,000 g/mol to about 448,000 g/mol, about 350,000 g/mol to about 450,000 g/mol or any range or value therein, (e.g., about 300,000, 305,000, 310,000, 315,000, 320,000, 325,000, 340,000, 345,000, 350,000, 355,000, 360,000, 365,000, 370,000, 375,000, 380,000, 385,000, 390,000, 395,000, 400,000, 401,000, 402,000, 403,000, 404,000, 405,000, 406,000, 407,000, 408,000, 409,000, 410,000, 415,000, 420,000, 425,000, 430,000, 435,000, 440,000, 450,000, 460,000, 470,000, 480,000, 490,000, or 500,000 g/mol), optionally, an average molecular weight of about 408,000 g/mol; a molecular weight of about 266,000 g/mol to about 400,000 g/mol or about 299,000 g/mol to about 367,000 g/mol; about 10 to about 15 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15), optionally an EO value of 13.3; alkoxylene (e.g., ethylene oxide) in an amount of about 90% to about 95% by weight of the polyalkyleneimine-alkoxylene polymer, optionally about 93%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 5 nm to about 15 nm such as about 10 nm; a zeta potential in a range of about +4, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 100 mg to about 70 mg, or about 100 mg to about 80 mg, or any range or value therein (e.g., about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75 74, 73, 72, 71, or 70 mg), optionally an OH-number of 89 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine- alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
In some embodiments, a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention has: an average molecular weight of about 650,000 g/mol to about 850,000 g/mol, about 633,000 g/mol to about 950,000 g/mol, about 712,000 g/mol to about 871,000 g/mol, about 700,000 g/mol to about 800,000 g/mol or any range or value therein, (e.g., about 650,000, 660,000, 680,000, 690,000, 700,000, 710,000, 720,000, 730,000, 740,000, 750,000, 760,000, 780,000, 781,000, 782,000, 783,000, 784,000, 785,000, 786,000, 787,000, 788,000, 789,000, 790,000, 791,000, 792,000, 793,000, 794,000, 795,000, 796,000, 797,000, 798,000, 799,000, 800,000, 810,000, 820,000, 830,000, 840,000, or 850,000 g/mol), optionally, an average molecular weight of about 792,000 g/mol; a molecular weight of about 520,000 g/mol to about 782,000 g/mol or about 585,000 g/mol to about 717,000 g/mol; about 25 to about 30 alkoxylene units per amine of the polypropyleneimine in the polymer, or any range or value therein, (e.g., about 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, or 30), optionally an EO value of 27.4; alkoxylene (e.g., ethylene oxide) in an amount of about 95% to about 98% by weight of the polyalkyleneimine- alkoxylene polymer, optionally about 96% to about 97%; a core diameter or diameter of the polyalkyleneimine-alkoxylene polymer of about 5 nm to about 15 nm such as about 10 nm; a zeta potential in a range of about +1, optionally when present in a composition having a pH of about 7; and/or an OH-number of about 60 mg to about 30 mg, or about 55 mg to about 35 mg, or any range or value therein (e.g., about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 mg), optionally an OH- number of 45 mg. In some embodiments, the PEI or polypropyleneimine used to form and/or in the polyalkyleneimine-alkoxylene polymer has/had an amine value of about 18 and/or a molecular weight of about 20,000 g/mol to about 30,000 g/mol.
According to some embodiments, a portion or all of the nitrogen atoms in PEI or polypropyleneimine of a polyalkyleneimine-alkoxylene polymer of the present invention may be substituted with at least one alkoxylene unit (e.g., at least one ethylene oxide unit). In some embodiments, a portion or all of the nitrogen atoms in PEI of a PEI-PEO polymer of the present invention may be substituted with at least one alkoxylene unit (e.g., at least one ethylene oxide unit). An exemplary alkoxylation modification is shown in Scheme 1 for a terminal nitrogen atom in the polyalkyleneimine backbone of a polyalkyleneimine- alkoxylene polymer where R represents an ethylene spacer and E represents a Cl -Cl 2 substituent (e.g., a C1-C12 alkyl) and X represents a water soluble counterion (e.g., a halo (e.g., chlorine, bromine, iodine); sulphate (-O-SO3H or -O-SO3 ); alkyl sulfonate such as methyl sulfonate, arylsulfonate (e.g., tolyl sulfonate), and alkyl sulphate (e.g., methosulphate (-0-S02-0CH3); etc ).
Scheme 1: Exemplary alkoxylation modification for a terminal nitrogen atom in a polyalkyleneimine.
Figure imgf000037_0001
or hydro en i or hydrogen j
aHiffiK ia&sn mo sfiffc aiion slcoxviaiioi} s diilcaiiois
A further exemplary alkoxylation modification is shown in Scheme 2 for an internal nitrogen atom in the polyalkyleneimine backbone of a polyalkyleneimine-alkoxylene polymer where R represents an ethylene spacer and E represents a C1-C12 substituent (e.g., a Cl -Cl 2 alkyl) and X represents a water soluble counterion.
Scheme 2: Exemplary alkoxylation modification for an internal nitrogen atom in a polyalkyleneimine.
Figure imgf000037_0002
Another exemplary alkoxylation modification is shown in Scheme 3 for an internal nitrogen atom in the polyalkyleneimine backbone of a polyalkyleneimine-alkoxylene polymer where each R represents an ethylene spacer and E represents a C1-C12 substituent (e.g., a Cl -Cl 2 alkyl) and X represents a water soluble counterion. Scheme 3: Additional exemplary alkoxylation modification for an internal nitrogen atom in a polyalkyleneimine.
E X-
R— N— R R— N— R
R R
In some embodiments, an alkoxylation modification of a polyalkyleneimine backbone may comprise substitution of a hydrogen atom with a polyalkoxylene chain having an average of about 1 to about 50 alkoxy units such as about 2 to about 40 alkoxy units, about 3 to about 30 units, or about 3 to about 20 alkoxy units. Exemplary alkoxy units include, but are not limited to, ethoxy (EO), 1,2-propoxy (1,2-PO), and/or butoxy (BO). In some embodiments, a polyalkoxylene chain comprises, consists essentially of, or consists of ethoxy units. In some embodiments, a polyalkoxylene chain comprises, consists essentially of, or consists of ethoxy units and propoxy units. A polyalkoxylene chain comprising, consisting essentially of, or consisting of ethoxy units and propoxy units may comprise, on average, about 1 to about 20 or 30 ethoxy units and, on average, about 0 or 1 to about 10 propoxy units.
An exemplary alkoxylated polyethyleneimine polymer of the present invention (e.g., a PEI-PEO polymer) may have a structure of Formula (I):
Figure imgf000038_0001
wherein:
each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl; and
each n is an integer of 1 to 50.
An exemplary quaternized alkoxylated polyethyleneimine polymer of the present invention (e.g., a quantemized PEI-PEO polymer) may have a structure of formula (II):
Figure imgf000038_0002
wherein: each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
each E is independent a Cl -Cl 2 substituent;
each X is independently a water soluble counterion (e.g., chlorine, bromine, iodine, sulphate, alkyl sulfonate, etc.); and
each n is an integer of 1 to 50.
A compound of Formula (I) and/or (II) may comprise a polyalkyleneimine (e.g., polyethyleneimine) backbone with a weight average molecular weight of about 600 g/mole to about 500,0000 g/mole. In some embodiments, n may be determined based on an average number of units such as, n, on average, may be an integer of 1 to 30 or 50. The degree of quaternization of nitrogen atoms in the polyalkyleneimine backbone of Formula (II) may be at least 5%, 20%, 70% or more.
In some embodiments, an exemplary alkoxylated polyethyleneimine polymer of the present invention (e.g., a PEI-(PEO-propylene oxide (PO)) polymer) may have a structure of Formula (III):
Figure imgf000039_0001
wherein: each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
each n is an integer of 0 to 50; and
each m is an integer of 1 to 10.
The structure of the polymer of formula (III) is not limited to block structures. In some embodiments, structure of the polymer of formula (III) comprises heteric blocks (e.g., blocks where EO and PO are randomly mixed).
Another exemplary quaternized alkoxylated polyethyleneimine polymer of the present invention (e.g., a PEI-(PEO-PO) polymer) may have a structure of formula (IV):
Figure imgf000040_0001
wherein:
each R is independently selected from a hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, -C(0)-C1-C22 aryl;
each E is independent a Cl -Cl 2 substituent;
each X is independently a water soluble counterion (e.g., chlorine, bromine, iodine, sulphate, alkyl sulfonate, etc.);
each n is an integer of 1 to 50; and
each m is an integer of 1 to 10. The structure of the polymer of formula (IV) is not limited to block structures. In some embodiments, structure of the polymer of formula (IV) comprises heteric blocks (e.g., blocks where EO and PO are randomly mixed).
A compound of Formula (III) and/or (IV) may comprise a polyalkyleneimine (e.g., polyethyleneimine) backbone with a weight average molecular weight of about 600 g/mole to about 500,0000 g/mole. In some embodiments, n may be determined based on an average number of units such as, n, on average, may be an integer of 1 to 20, 30, or 50. In some embodiments, m may be determined based on an average number of units such as, m, on average, may be an integer of 1 to 10. The degree of quatemization of nitrogen atoms in the polyalkyleneimine backbone of Formula (IV) may be at least 5%, 20%, 70% or more.
The representations in Formulas (I), (II), (III), and Formula (IV) are to be understood as general structural descriptions of exemplary polyalkyleneimine-alkoxylene polymers of the present invention with the wavy lines ( >LLL/ ) indicating that the polymer backbone may continue in the direction of the wavy line.
A polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention may be prepared in a known manner by reaction of polyalkyleneimines with alkoxy units, an exemplary process is described for the ethoxylation of polyethyleneimine. According to some embodiments a method of preparing a polyalkyleneimine-alkoxylene polymer of the present invention comprises, consists essentially of, or consists of alkoxylation (e.g., ethoxylation) of a polyalkyleneimine (e.g., PEI). The polyalkyleneimine may be reacted with all or a portion of the total amount of an alkylene oxide (e.g., ethylene oxide) used in the alkoxylation reaction. In some embodiments, a portion or a total amount of alkylene oxide used in the alkoxylation reaction may be about 1 mole of alkylene oxide (e.g., ethylene oxide) per amine (based on total amine value) of polyalkyleneimine. An alkoxylation reaction may be undertaken in the absence of a catalyst and/or in an aqueous solution at a reaction temperature in a range of about 70°C to about 200°C. In some embodiments, the alkoxylation reaction is carried out in the presence of water at a reaction temperature in a range of about 80°C to about 160°C and in the absence of a catalyst. In some embodiments, the alkoxylation reaction is carried out at a pressure of up to about 5, 6, 7, 8, 9, or 10 bar.
In some embodiments, following reaction of a polyalkyleneimine (e.g., PEI) with a first portion of alkylene oxide (e.g., ethylene oxide) to provide an at least partially alkoxylated polyalkyleneimine (e.g., at least partially alkoxylated PEI) (e.g., as described above), the at least partially alkoxylated polyalkyleneimine is reacted with a second portion of an alkylene oxide. For simplification, the method will be further described with PEI as the exemplary polyalkyleneimine and ethylene oxide as the exemplary alkylene oxide. Thus, in some embodiments, following reaction of PEI with a first portion of ethylene oxide to provide an at least partially ethoxylated PEI (e.g., as described above), the at least partially ethoxylated PEI is reacted with a second portion of an ethylene oxide. In some embodiments, the second portion of ethylene oxide is the final ethylene oxide addition and/or the second portion in combination with the first portion of ethylene oxide (and/or any other portions) makes up the total amount of ethylene oxide used in the ethoxylation reaction. In some embodiments, reaction of the at least partially ethoxylated PEI and the second portion of ethylene oxide is carried out in the presence of a basic catalyst. Examples of basic catalysts include, but are not limited to, alkali metal hydroxides and alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide and/or calcium hydroxide; alkali metal alkoxides such as sodium and potassium Ci-C4-alkoxides (e.g., sodium methoxide, sodium ethoxide and potassium tert-butoxide); alkali metal and alkaline earth metal hydrides such as sodium hydride and calcium hydride; and/or alkali metal carbonates such as sodium carbonate and potassium carbonate. In some embodiments, the basic catalyst is an alkali metal hydroxide or alkali metal alkoxide such as potassium hydroxide and/or sodium hydroxide. A basic catalyst may be used and/or present in an amount of about 0.05% to about 10% by weight of polyalkyleneimine and the alkylene oxide. In some embodiments, the at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) is reacted with a second portion of alkylene oxide (e.g., ethylene oxide) in the presence of a basic catalyst in an amount of about 0.5% to about 2% by weight of the at least partially alkoxylated polyalkyleneimine and the alkylene oxide.
According to some embodiments, reacting an at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) with a second portion of alkylene oxide (e.g., ethylene oxide) may be carried out in a dewatered composition and/or in an organic solvent. For simplification, the method will be further described with PEI as the exemplary polyalkyleneimine and ethylene oxide as the exemplary alkylene oxide. In some embodiments, the at least partially ethoxylated PEI may be dewatered, optionally in the presence of a basic catalyst. Dewatering may be carried out by heating a composition comprising the at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) and optionally basic catalyst to a temperature of about 80°C or 120°C to about 150°C or 180°C and distilling off the water under a reduced pressure of about 0.01 bar to about 0.5 bar. The dewatering may be carried out and/or supported by a gentle nitrogen stream. A subsequent reaction of the at least partially ethoxylated PEI with a second portion of ethylene oxide in a dewatered composition in the presence of a basic catalyst may be effected at a reaction temperature from about 70°C to about 200°C or about 100°C to about 180°C and/or at a pressure of up to about 8 or 10 bar. In some embodiments, reacting an at least partially ethoxylated PEI with a second portion of ethylene oxide in a dewatered composition and in the presence of a basic catalyst is carried out for a period of time of about 30 minutes to about 4 hours. The PEI-PEO polymer may be obtained directly in substance or may be obtained in and/or converted to an aqueous solution.
In some embodiments, reacting an at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI) with a second portion of alkylene oxide (e.g., ethylene oxide) is carried out in an organic solvent such as a nonpolar organic solvent and/or polar aprotic organic solvent. Exemplary nonpolar aprotic solvents include, but are not limited to, aliphatic and aromatic hydrocarbons such as hexane, cyclohexane, toluene and xylene. Exemplary polar aprotic solvents include, but are not limited to, ethers such as cyclic ethers (e.g., tetrahydrofuran and dioxane), N,N-dialkylamides such as dimethylformamide and dimethylacetamide, and/or N-alkyl lactams such as N-methylpyrrolidone. In some embodiments, the organic solvent comprises two or more (e.g., 2, 3, 4 or more) organic solvents. In some embodiments, the organic solvent comprises xylene and/or toluene.
A composition comprising an at least partially alkoxylated polyalkyleneimine (e.g., at least partially ethoxylated PEI), a basic catalyst, and an organic solvent may be dewatered as described herein to provide a dewatered composition. In some embodiments, dewatering is carried out by separating out the water from the composition at a temperature of about 120°C to about 180°C, optionally supported by a gentle nitrogen stream. A subsequent reaction of the at least partially alkoxylated polyalkyleneimine with a second portion of alkylene oxide in an organic solvent in the presence of a basic catalyst may be effected at a reaction temperature from about 70°C to about 200°C or about 100°C to about 180°C and/or at a pressure of up to about 8 or 10 bar. In some embodiments, the obtained polyalkyleneimine- alkoxylene polymer (e.g., PEI-PEO polymer) may be removed from the organic solvent and/or the organic solvent may be removed and optionally replaced with water. In some embodiments, the polyalkyleneimine-alkoxylene polymer (e.g., PEI-PEO polymer) may be isolated in substance and/or may be provided in an aqueous solution.
Quaternization may be carried out with methods known to those of skill in the art. For example, in some embodiments, quaternization of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention is achieved by introducing a Cl- C12 alkyl, a C1-C12 alkenyl, a C1-C12 alkynyl, a C1-C12 aryl, a C1-C12 arylalkyl, a Cl- C12 arylalkenyl, or a Cl -Cl 2 arylalkynyl group such as by reaction of the polymer with the corresponding C1-C12 alkyl- halide or dialkylsulfate, C1-C12 alkenyl- halide or dialkylsulfate, C1-C12 alkynyl- halide or dialkylsulfate, C1-C12 aryl- halide or dialkylsulfate, C1-C12 arylalkyl- halide or dialkylsulfate, C1-C12 arylalkenyl- halide or dialkylsulfate, or C1-C12 arylalkynyl- halide or dialkylsulfate. A quaternization reaction may be carried out as described in International Publication No. WO 2009/060059, the contents of which are incorporated herein by reference in their entirety.
In some embodiments, quaternization of a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the present invention is achieved by reacting an amine of the polymer with at least one alkylating compound. The at least one alkylating compound may be represented by the chemical formula EX, wherein E is a C1-C12 alkyl, a C1-C12 alkenyl, a Cl -Cl 2 alkynyl, a Cl -Cl 2 aryl, a Cl -Cl 2 arylalkyl, a Cl -Cl 2 arylalkenyl, or a Cl -Cl 2 arylalkynyl and X is a leaving group that is capable of being replaced by nitrogen or a C2-C6 alkylene oxide (e.g., ethylene oxide or propylene oxide). Exemplary leaving groups include, but are not limited to, halogens (e.g., chlorine, bromine, or iodine), sulphate, alkyl sulfonate (e.g., methyl sulfonate), arylsulfonate (e.g., tolyl sulfonate), and alkyl sulphate (e.g., methosulphate). Exemplary alkylating agents include, but are not limited to, Cl -Cl 2 alkyl halides, bis(Cl-C12-alkyl)sulfates, and benzyl halides. In some embodiments, the at least one alkylating agent is selected from ethyl chloride, ethyl bromide, methyl chloride, methyl bromide, benzyl chloride, dimethyl sulphate, and/or diethyl sulphate.
Provided according to some embodiments of the present invention is a method of preparing a composition of the present invention. The method may comprise combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer), thereby preparing the composition. The composition may comprise two or more polyalkyleneimine-alkoxylene polymers and/or two or more nucleic acid molecules. In some embodiments, the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) is complexed with the nucleic acid molecule, optionally wherein a single polymer is complexed with 2, 5, 10, 100, or more nucleic acid molecules as described herein. In some embodiments, a single nucleic acid molecule is complexed with 2, 5, 10, 25, 50, 75, 100, or more polyalkyleneimine-alkoxylene polymers as described herein.
Combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may be carried out by contacting a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer either of which may be present in the form of a solution or a solid. In some embodiments, the nucleic acid molecule and/or the polyalkyleneimine-alkoxylene polymer is/are present in the form of a solid that is added to a solution (e.g., an aqueous solution). In some embodiments, combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises mixing a first solution comprising the nucleic acid molecule and a second solution comprising the polymer to form a third solution comprising the nucleic acid molecule and the polymer.
In some embodiments, combining a nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer) comprises providing the nucleic acid molecule and the polymer in contact for a period of time such as about 1, 5, 10, 15, or 30 minutes to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, combining a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) comprises providing the nucleic acid molecule and the polymer in the same composition (e.g., an aqueous solution) for a period of time, optionally with mixing and/or agitation. In some embodiments, the polymer may be dialyzed prior to combining/contact with the nucleic acid (e.g., the polymer may be dialyzed about 1, 5, 10, 15, 20, 30, 40, 50 minutes about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours prior to contact with the nucleic acid.
Described herein are compositions comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) that can be used to deliver the nucleic acid molecule to a cell (e.g. host cell) of an organism (e.g., a target organism). The organism can be any eukaryote (e.g., animal, plant, fungal, protozoa, and the like).
An animal useful with this invention may be any animal including but not limited to, a mammal, an insect, a plant, a fungus, a bird, a fish, an amphibian, a reptile, or a cnidarian. A mammal can include, but is not limited to, a rodent, a horse, a dog a cat, a human, a non human primate (e.g., monkeys, baboons, and chimpanzees), a goat, a pig, a cow (e.g., cattle), a sheep, laboratory animals (e.g, rats, mice, gerbils, hamsters, and the like) and the like. Non-limiting examples of birds useful with this invention include chickens, ducks, turkeys, geese, quails and birds kept as pets (e.g, parakeets, parrots, macaws, and the like). In some embodiments, compositions of the invention comprising a nucleic acid molecule and a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) can be used to deliver the nucleic acid molecule to, for example, a cell of a cell line (e.g., a mammalian cell line, an insect cell line). Non-limiting examples of mammalian and insect cell lines include HEK293 cells, HeLa cells, CHO cells, MEF cells, 3T3 cells, Hi-5 cells, and Sf21 cells. Suitable target organisms (and cells therefrom) can include both males and females and subjects of all ages including embryonic ( e.g ., in utero or in ovo ), infant, juvenile, adolescent, adult and geriatric subjects. In embodiments of the invention, the target organism is not a human subject or a human embryonic subject.
Any plant or fungus, or part thereof, can be employed in practicing the present invention.
In some embodiments of this invention, a plant, plant part and/or plant cell useful with this invention can include, but not is not limited to, Camelina , Glycine , Sorghum , Brassica, Allium , Armoracia , Poa, Solanum, Cucurbita, Musa, Agrostis , Lolium , Festuca, Calamogrostis, Deschampsia , Spinacia , Beta , Pisum, Chenopodium , Helianthus , Pastinaca , Daucus, Petroselium , Populus , Prunus , Castanea, Eucalyptus, Acer , Quercus , LZ///C, Juglans , Picea, Pinus, Malus , Abies, Lemna, Wolffia, Spirodela, Oryza, Zea or Gossypium. In other embodiments, the plant and/or plant cell can include, but is not limited to, Camelina alyssum (Mill.) Thell., Camelina microcarpa Andrz. ex DC., Camelina rumelica Velen., Camelina sativa (L.) Crantz, Sorghum bicolor (e.g., Sorghum bicolor L. Moench), Gossypium hirsutum, Glycine max, Zea mays, Brassica oleracea, Brassica rapa, Brassica napus, Solanum tuberosum, Solanum lycopersicum, Raphanus sativus, Armoracia rusticana, Allium sative, Allium cepa, Allium ampeloprasum, Malus domestica, Populus grandidentata, Populus tremula, Populus tremuloides, Prunus serotina, Prunus pensylvanica, Castanea dentate, Populus balsamifer, Populus deltoids, Acer Saccharum, Acer nigrum, Acer negundo, Acer rubrum, Acer saccharinum, Acer pseudoplatanus, Oryza sativa, Saccharum offwinarum, Triticum aestivum, Triticum turgidum, Secale cereal, Hordeum vulgar e or Avena sativa. In additional embodiments, a plant and/or plant cell can be, but is not limited to, wheat, barley, rye, com, oats, turfgrass (bluegrass, bentgrass, ryegrass, fescue), sugar cane, feather reed grass, tufted hair grass, spinach, cucumber, melon, leak, tomato, potato, beets, chard, quinoa, sugar beets, lettuce, sunflower ( Helianthus annuus), peas (. Pisum sativum), parsnips (. Pastinaca sativa), carrots ( Daucus carota), parsley ( Petroselinum crispum), duckweed, pine, spruce, fir, eucalyptus, oak, walnut, or willow. In particular embodiments, the plant and/or plant cell can be Arabidopsis thaliana. In some representative embodiments, the plant and/or plant cell can be camelina, wheat, rice, com, rape, canola, soybean, sorghum, tomato, bamboo or cotton.
In further embodiments, a plant, plant part, and/or plant cell can be an algae or algae cell including, but not limited to, a Bacillariophyceae (diatoms), Haptophyceae, Phaeophyceae (brown algae), Rhodophyceae (red algae) or Glaucophyceae (red algae). In still other embodiments, non-limiting examples of an algae or algae cell can be Achnanthidium , Actinella, Nitzschia, Nupela, Geissleria, Gomphonema, Planothidium, Halamphora, Psammothidium, Navicula, Eunotia, Stauroneis, Chlamydomonas, Dunaliella, Nannochloris, Nannochloropsis, Scenedesmus, Chlorella, Cyclotella, Amphora, Thalassiosira , Phaeodactylum, Chrysochromulina, Prymmsium, Thalassiosira, Phaeodactylum, Glaucocystis, Cyanophora, Galdieria, or Porphyridium.
The term“plant part,” as used herein, includes but is not limited to reproductive tissues ( e.g ., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g, phloem and xylem); specialized cells such as epidermal cells, parenchyma cells,
chollenchyma cells, schlerenchyma cells, stomata, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term“plant part” also includes plant cells, including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like. As used herein,“shoot” refers to the above ground parts including the leaves and stems. As used herein, the term“tissue culture” encompasses cultures of tissue, cells, protoplasts and callus.
As used herein,“plant cell” refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts. A plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ. In some embodiments, a plant cell can be an algal cell.
The polymers of this invention will be especially useful in transforming intact plant cells (e.g., cells with cell walls, e.g., plants and plant tissues), providing a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium and the like.
Non-limiting examples of fungi useful with this invention include Candida spp., Fusarium spp., Aspergillus spp., Cryptococcus spp., Coccidioides spp., Tinea spp., Sporothrix spp., Blastomyces spp., Histoplasma spp., Pneumocystis spp, Saccharomyces spp., Saccharomycodes spp., Hansenula spp., Kluyveromyces spp., Yarrowia spp., Pichia spp., Candida spp., Ashbya spp., Zygosaccharomyces spp. or Hanseniaspora spp. In representative embodiments, the fungus can include, but is not limited to, Saccharomyces cerevisiae, S. uvarum (carlsbergensis), S. diastaticus, Saccharomycodes ludwigii, Hansenula polymorpha, Kluveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia pastor is, Pichia methanolica, Candida stellata, C. pulcherrima , Ashbya gossypii , Zygosaccharomyces fermentati , Zygosaccharomyces ruxii, Zygosaccharomyces bailii , Hanseniaspora uvarum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, Candida albicans, Candida boidinii, Candida utilis, Candida freyschussii, Candida glabrata, Candida sonorensis, Coccidioides immitis, Cryptococcus neoformans, Fusarium solani, Fusarium culmorum, Tinea unguium, Tinea corporis, Tinea cruris, Sporothrix schenckii, Blastomyces dermatitidis, Histoplasma capsulatum, Pneumocystis carinii, or Histoplasma duboisii.
In some embodiments, a fungal cell useful with the invention may include its cell wall or may be without a cell wall, e.g., a spheroplast.
In some embodiments, the polymers of the invention may be used to transfer nucleic acids to organelles, including, but not limited to, chloroplasts or mitochondria. In some embodiments, an organelle to be transformed as described herein may be isolated from the natural cellular environment.
One or more cells of an organism (e.g., host cells) may be transformed with one or more nucleic acid molecules according to embodiments of the present invention. In some embodiments, one or more nucleic acid molecules may be delivered to a host cell or to one or more host cells in a population. In some embodiments, a host cell population may comprise cells of at least two different species and/or strains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different species and/or strains).
The inventors have surprisingly discovered that compositions of the invention comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid as described herein are able to transform eukaryotic cells (e.g., generate transformants that have received DNA) and overcome typical problem(s) associated with other transformation protocols
In some embodiments, a method of delivering a nucleic acid molecule to a host cell may comprise contacting the cell with a composition of the present invention, thereby delivering/introducing the nucleic acid molecule in the composition to/into the cell and/or transforming the cell with the nucleic acid molecule. The composition may be in the form of a solid (e.g., that may be added to a solution comprising a cell to be transformed) or it may be a solution (e.g., an aqueous solution). In some embodiments, the composition is and/or comprises a complex comprising the nucleic acid molecule and a polyalkyleneimine- alkoxylene polymer (e.g., a PEI-PEO polymer), optionally in an aqueous solution. “Contact,” “contacting,” “introduce,” and/or “introducing” (and grammatical variations thereof) in the context of a composition of this invention (i.e., a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and a nucleic acid molecule) means presenting the composition to a host organism or cell of said organism (e.g., host cell; e.g., animal, plant, and/or fungal) in such a manner that the nucleic acid molecule (e.g., polyalkyleneimine-alkoxylene polymer/nucleic acid molecule) gains access to the interior of the cell. In some embodiments, “contact” or“contacting” can include contacting target nucleic acid with a composition of this invention in a cell free system, thereby delivering the nucleic acid molecule to the target nucleic acid (for example for modifying the target nucleic acid).
Where more than one nucleic acid molecule is to be introduced , these nucleic acid molecules may be assembled as part of a single nucleic acid molecule or nucleic acid construct, or as separate nucleic acid molecules or nucleic acid constructs, and can be located on the same or different expression constructs or transformation vectors. A construct and/or vector may be present in a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) of the invention and/or complexed with the same or different polyalkyleneimine-alkoxylene polymers (e.g., PEI-PEO polymers). Accordingly, one or more polynucleotide(s) can be introduced into cells in a single transformation/transfection event, in separate transformation/transfection events, or any combination thereof. Thus, in some aspects, one or more nucleic acid molecules of this invention can be introduced singly or in combination into a cell of a host organism. In the context of a population of cells, “contacting” or“introducing” means contacting the population with a composition of the invention under conditions where at least the nucleic acid molecule gains access to the interior of one or more cells of the population, thereby transforming the one or more cells (e.g., stably or transiently). In some embodiments, in the context of a population of cells, “contacting” or“introducing” means contacting the population with a composition of the invention under conditions where the composition (e.g., polyalkyleneimine-alkoxylene polymer/ nucleic acid molecules) gains access to the interior of one or more cells of the population, thereby transforming the one or more cells of the population (e.g., stably or transiently).
In some embodiments,“contacting” or“introducing” a composition of the invention (i.e., a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule) with/into a cell comprises“incubating” the cell, or a population of cells, with the composition of the present invention for about 0 minutes (e.g., 1, 2, 3, 4, 5 sec to about 54, 55, 56, 57, 58, 59 sec) to about 24 hours. For example, a cell may be contacted and/or incubated with a composition of the present invention for about 0.5, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes or about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,
19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, or 24 hours, and any value or range therein) (e.g., about 5 sec to about 30 min, about 1 min to about 1 hour, about 30 min to about 3 hours, about 1 hour to about 5 hours, about 1 hour to about 10 hours, about 1 hour to about 15 hours, about 1 hour to about 20 hours, about 1 hour to about 24 hours, about 5 hours to 10 hours, about 5 hours to 20 hours, about 10 hours to about 20 hours, about 10 hours to about 24 hours, or longer, or any value or range therein). The incubation time may vary so long as the time is sufficient for the composition (i.e., the polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) and nucleic acid molecule) to gain access to the interior of the cell.
In some embodiments, a cell may be contacted and/or incubated with a composition of the present invention at a pH from about 0 to about 14 (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 and any value or range therein). In some embodiments, a cell may be contacted and/or incubated with a composition of the present invention at a pH from about 3 to about 10. A composition including a host cell, nucleic acid molecule, and polyalkyleneimine-alkoxylene polymer (e.g., a PEI-PEO polymer) may have a pH of about 6, 7, or 8 to about 8.5, 9, or 9.5. In some embodiments, a composition including a cell, nucleic acid molecule, and polyalkyleneimine- alkoxylene polymer has a pH of about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.
In some embodiments, a cell may be contacted and/or incubated with a composition of the present invention at a temperature from about 0°C to about 100°C (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,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100°C and any value or range therein). In some embodiments, a cell may be contacted and/or incubated with a composition of the present invention at a temperature from about 4°C to about 50°C or about 15°C to about 37°C.
In some embodiments, a method of the present invention delivers a nucleic acid molecule to a cell and/or a population of cells without adversely affecting the viability of the cell and/or population. Thus, for example, growth of such cells may be delayed or even prevented at an amount above about 5% weight/vol. of the polymer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% wt/vol. of the polymer.
A nucleic acid molecule of the invention may be operatively associated with a variety of promoters and other regulatory elements for expression in host cells. Thus, for example, a recombinant nucleic acid molecule of this invention may further comprise one or more promoters operably linked to one or more nucleotide sequences.
By“operably linked” or“operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term“operably linked” or“operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Thus, a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For instance, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered“operably linked” to the nucleotide sequence.
A“promoter” is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (i.e., a coding sequence) that is operably associated with the promoter. The coding sequence may encode a polypeptide and/or a functional RNA. Typically, a “promoter” refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. In general, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence. The promoter region may comprise other elements that act as regulators of gene expression. These include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants, for example, the CAAT box may be substituted by the AGGA box (Messing et al, (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).
Promoters can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and/or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, i.e.,“chimeric genes” or“chimeric polynucleotides.” These various types of promoters are known in the art.
The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.
Exemplary promoters include, but are not limited to, promoters functional in eukaryotes and prokaryotes including but not limited to, plants, fungi, and animals (e.g., mammals, insects, fish, amphibians, reptiles and the lik).
Exemplary promoters useful with yeast can include a promoter from phosphoglycerate kinase ( PGK ), glyceraldehyde-3 -phosphate dehydrogenase (GAP), triose phosphate isomerase (777), galactose-regulon ( GAL1 , GAL10 ), alcohol dehydrogenase (ADH1, ADH2), phosphatase ( PH05 ), copper-activated metallothionine ( CUP1 ), MFal, PGK/a2 operator, TPI/a2 operator, GAP/GAL, PGK/GAL, GAP/ADH2, GAP/PH05, iso-1- cytochrome c/glucocorticoid response element (CYC/GRE), phosphoglycerate kinase/angrogen response element (PGK/ARE), transcription elongation factor EF-la ( TEF1 ), triose phosphate dehydrogenase ( TDH3 ), phosphoglycerate kinase 1 ( PGK1), pyruvate kinase 1 ( PYK1 ), and/or hexose transporter ( ΉCT7 ) (See, Romanos et al. Yeast 8:423-488 (1992); and Partow et al. Yeast 27:955-964 (2010)).
Non-limiting examples of a promoter functional in a plant include the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007); Li et al. Mol Biol. Rep. 37:1143-1154 (2010)). PrbcSl and Pactin are constitutive promoters and Pnr and Pdcal are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al. Gene 403: 132-142 (2007)) and Pdcal is induced by salt (Li et al. Mol Biol. Rep. 37: 1143-1154 (2010)).
Examples of constitutive promoters useful for plants include, but are not limited to, cestrum virus promoter (cmp) (U.S. Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; as well as US Patent No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter. The constitutive promoter derived from ubiquitin accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al ., 1991. Plant Science 79: 87-94), maize (Christensen et al ., 1989. Plant Molec. Biol. 12: 619-632), and arabidopsis (Norris et al. 1993. Plant Molec. Biol. 21 :895-906). The maize ubiquitin promoter ( UbiP ) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926. The ubiquitin promoter is suitable for the expression of the nucleotide sequences of the invention in transgenic plants, especially monocotyledons. Further, the promoter expression cassettes described by McElroy et al. {Mol. Gen. Genet. 231 : 150-160 (1991)) can be easily modified for the expression of the nucleotide sequences of the invention and are particularly suitable for use in monocotyledonous hosts.
In some embodiments, tissue specific/tissue preferred promoters can be used for expression of a heterologous polynucleotide in a plant cell. Non-limiting examples of tissue- specific promoters include those associated with genes encoding the seed storage proteins (such as b-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g. , Kridl et al. (1991) Seed Sci. Res. 1 :209-219; as well as EP Patent No. 255378). Additional examples of plant tissue- specific/tissue preferred promoters include, but are not limited to, the root hair-specific cis- elernents (RFSEs) (Kirn et al. The Plant Cell 18:2958-2970 (2006)), the root-specific promoters RCc3 (Jeong et al. Plant Physiol. 153: 185-197 (2010)) and RB7 (U.S. Patent No. 5459252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11 : 160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), and/or S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology , 37(8): 1108-1115).
In addition, promoters functional in chloroplasts can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).
In some embodiments of the invention, inducible promoters can be used. Thus, for example, chemical -regulated promoters can be used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator. Regulation of the expression of nucleotide sequences of the invention via promoters that are chemically regulated enables the RNAs and/or the polypeptides of the invention to be synthesized only when, for example, a crop of plants are treated with the inducing chemicals. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of a chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. In some aspects, a promoter can also include a light- inducible promoter, where application of specific wavelengths of light induce gene expression (Levskaya et al. 2005. Nature 438:441-442). In other aspects, a promoter can include a light-repressible promoter, where application of specific wavelengths of light repress gene expression (Ye et al. 2011. Science 332: 1565-1568).
Chemical inducible promoters useful with plants are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid ( e.g ., the PRla system), steroid-responsive promoters (see, e.g, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, e.g, Gatz et al. (1991) Mol. Gen. Genet. 227, 229- 237, and U.S. Patent Numbers 5,814,618 and 5,789,156, Lac repressor system promoters, copper-inducible system promoters, salicylate-inducible system promoters (e.g, the PRla system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. 11 :605-612), and ecdysone-inducible system promoters.
In some particular embodiments, promoters useful with algae include, but are not limited to, the promoter of the RubisCo small subunit gene 1 (PrbcSl), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdcal) (See, Walker et al. Plant Cell Rep. 23:727-735 (2005); Li et al. Gene 403: 132-142 (2007); Li et al. Mol Biol. Rep. 37: 1143-1154 (2010)), the promoter of the o70-type plastid rRNA gene (Prrn), the promoter of the psbA gene (encoding the photosystem -II reaction center protein Dl) (PpsbA), the promoter of the psbD gene (encoding the photosystem-II reaction center protein D2) (PpsbD), the promoter of the psaA gene (encoding an apoprotein of photosystem I) (PpsaA), the promoter of the ATPase alpha subunit gene (PatpA), and promoter of the RuBisCo large subunit gene (PrbcL), and any combination thereof {See, e.g., De Cosa et al. Nat. Biotechnol. 19:71-74 (2001); Daniell et al. BMC Biotechnol. 9:33 (2009); Muto et al. BMC Biotechnol. 9:26 (2009); Surzycki et al. Biologicals 37: 133-138 (2009)).
In further embodiments, a promoter useful with this invention can include, but is not limited to, pol III promoters such as the human U6 small nuclear promoter (U6) and the human HI promoter (HI) (Makinen et al. J Gene Med. 8(4):433-41 (2006)), and pol II promoters such as the CMV (Cytomegalovirus) promoter (Barrow et al. Methods in Mol. Biol. 329:283-294 (2006)), the SV40 (Simian Virus 40)-derived initial promoter, the EF-la (Elongation Factor-la) promoter, the Ubc (Human Ubiquitin C) promoter, the PGK (Murine Phosphogly cerate Kinase- 1) promoter and/or constitutive protein gene promoters such as the b-actin gene promoter, the tRNA promoter and the like.
Moreover, tissue-specific regulated nucleic acids and/or promoters as well as tumor- specific regulated nucleic acids and/or promoters have been reported. Thus, in some embodiments, tissue-specific or tumor-specific promoters can be used. Some reported tissue- specific nucleic acids include, without limitation, B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (hematopoietic cells), CD68 (macrophages), desmin (muscle), elastase-1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin
(differentiating cells and healing tissues), FLT-1 (endothelial cells), GFAP (astrocytes),
GPIIb (megakaryocytes), ICAM-2 (endothelial cells), INF-b (hematopoietic cells), Mb (muscle), NPHSI (podocytes), OG-2 (osteoblasts, SP-B (lungs), SYN1 (neurons), and WASP (hematopoietic cells). Some reported tumor-specific nucleic acids and promoters include, without limitation, AFP (hepatocellular carcinoma), CCKAR (pancreatic cancer), CEA (epithelial cancer), c-erbB2 (breast and pancreatic cancer), COX-2, CXCR4, E2F-1, HE4, LP, MUC1 (carcinoma), PRC1 (breast cancer), PSA (prostate cancer), RRM2 (breast cancer), survivin, TRPl (melanoma), and TYR (melanoma).
In some embodiments, inducible promoters can be used in mammalian cells. Examples of inducible promoters include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, copper-inducible system promoters, salicylate- inducible system promoters (e.g., the PRla system), glucocorticoid-inducible promoters, and ecdysone-inducible system promoters. In some embodiments, a recombinant nucleic acid molecule of the invention can be an “expression cassette” or can be comprised within an expression cassette. As used herein, “expression cassette” means a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the nucleotide sequences of the invention; e.g., a nucleic acid of interest), wherein said nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter). Thus, some embodiments of the invention provide expression cassettes designed to express the nucleotides sequences of the invention.
An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
An expression cassette also can optionally include a transcriptional and/or translational termination region (i.e., termination region) that is functional in the selected host cell. A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host cell, or may be derived from another source (i.e., foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).
An expression cassette of the invention also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed host cell. As used herein, “selectable marker” means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g, fluorescence). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.
In addition to expression cassettes, the nucleic acid molecules and nucleotide sequences described herein can be used in connection with vectors. The term“vector” refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell. A vector comprises a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered or introduced. Vectors for use in transformation of host organisms are well known in the art. Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable. A vector as defined herein can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Additionally included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic species (e.g. higher plant, mammalian, yeast, insect, fungi, and the like). In some representative embodiments, the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell. Accordingly, the nucleic acid molecules of this invention and/or expression cassettes may be comprised in vectors as described herein and as known in the art.
As used herein, the terms“transformed” and“transgenic” refer to any cell that contains all or part of at least one recombinant (e.g., heterologous) polynucleotide even if temporarily (i.e., transient).
The term“transformation” as used herein refers to the introduction of a nucleic acid molecule into a cell. Transformation of a cell or organism may be stable or transient.
Transient transformation in the context of a nucleic acid molecule means that a nucleic acid molecule is introduced into the cell and does not integrate into the genome of the cell.
By stably introducing or stably introduced in the context of a nucleic acid molecule introduced into a cell it is intended that the introduced nucleic acid molecule is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the nucleic acid molecule.
“Stable transformation” or“stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Genome as used herein also includes the nuclear/chromosomal and the plasmid genome, and therefore includes integration of the nucleic acid molecule into, for example, the plasmid genome. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome.
As used herein, an“extrachromosomal nucleic acid” refers to select nucleic acids in eukaryotic cells such as in a mitochondrion, a plasmid, a plastid (e.g., chloroplast, amyloplast, leucoplast, proplastid, chromoplast, etioplast, elaiosplast, proteinoplast, tannosome), and/or an extrachromosomal circular DNA (eccDNA)). In some embodiments, an extrachromosomal nucleic acid may be referred to as“extranuclear DNA” or“cytoplasmic DNA.”
Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., an animal, a plant, a fungus). Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and/or hybridization protocols that are well known in the art.
As used herein, the term“percent sequence identity” or“percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments,“percent identity” can refer to the percentage of identical amino acids in an amino acid sequence. As used herein“sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g. , nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e ., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention“percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al ., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.
As used herein, the phrase “substantially complementary,” or “substantial complementarity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, refers to two or more sequences or subsequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide or amino acid residue complementary, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments, substantial complementarity can refer to two or more sequences or subsequences that have at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 96, 97, 98, or 99% complementarity (e.g., about 80% to about 90%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99% or more, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99% or more, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99% or more, about 95% to about 97%, about 95% to about 98%, about 95% to about 99% or more). Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. In some representative embodiments, two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions.
“Stringent hybridization conditions” and“stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra , for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g, about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.
The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the invention. In one embodiment, a reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C. In another embodiment, the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C. In still further embodiments, the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP04, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.
Any nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in any species of interest. Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences. As is understood in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original, native nucleotide sequence. Thus, in representative embodiments of the invention, the nucleotide sequence and/or heterologous nucleic acid construct of this invention can be codon optimized for expression in the particular species of interest.
The present invention is explained in greater detail in the following non-limiting examples.
EXAMPLES
Example 1:
Polyethyleneimines such as Lupasol® P (e.g., 50% concentrated in water, 750,000 MW), Lupasol® G100 (e.g., 50% in water, 5,000 MW), and Lupasol® HF (e.g., 56% in water, 25 000 MW), were obtained from BASF Corporation. PEIs were in water and used as obtained. Specifically, Lupasol® P and Lupasol® G100 were used in 50% water and Lupasol® HF was used in 56% water. Dimethyl sulfate, n-bromobutane, 1-bromooctane, 1- bromododecane, and 1-bromooctadecane were each obtained from Sigma Aldrich. The purity of each halogenated hydrocarbon was, at minimum, greater than 95%.
Molecular weight determination
The molecular weight of the polyethyleneimines were determined by GPC against PEG standards and reported in g/mol. The molecular weight (as reported in g/mol) of the PEI-alkoxylates were determined by hydroxyl value and extrapolated by the average molar equivalent of primary amine per polyethylamine.
Thin layer chromatography
Thin layer chromatography was used to monitor reaction completeness. Silica plates were spotted with a 1 pL aliquot of diluted material: product and starting materials (1 part sample to 100 parts chloroform). The plate was then eluded with a solution of 3 : 1 ethyl acetate to hexanes. The plate was stained and the reaction was halted once the starting material bands were no longer visible.
Determination of acid value The acid value was measured by titrating 1 gram of PEI-alkoxylate with a 0.1 N KOH solution in methanol (as reported mg KOH/lg of material)
Determination of amine functionality and polymer charge density by 13C-NMR
The amine functionality was measured by carbon- 13 NMR; taking the average area of each amine region (i.e., 35 - 45 PPM for primary amines) over the total amine region (35 - 60 PPM). The results for each amine type were then reported as the ratio of primary, secondary and tertiary amines. The charge density of the PEIs (unless otherwise stated) were measured from the dried substance at pH 4.5
Determination of hydroxy number (# OH)
The hydroxyl number was measured by reacting 1 g of PEI-alkoxylate with acetic anhydride in the presence of DMAP (Dimethyl aminopyridine) and back titrating with 0.5 N KOH solution in Methanol.
Table 2: Summary of synthesized PEI particles
Figure imgf000064_0001
Synthesis
Ethoxylation of polyethyleneimines:
To a clean, dry, leak checked 7.57 1 autoclave, 8.14 molar equivalents of polyethyleneimine (PEI) in 50% or 56% water were charged. Agitation was set to 350 rpm. The reactor was sealed and nitrogen purged (3 times at 6.2 bar) before heating. Pressure was adjusted to 1.4 bar with nitrogen and the reactor heated to 120°C. Ethylene oxide (6.84 molar equivalents) was added over 2 hours and reacted to constant pressure. The reactor was cooled to 90°C and an aqueous solution including KOH in an amount of 45 % by weight of the aqueous solution was added to the reactor in an amount of 0.15 wt% of the mixture in the reactor. Water was stripped for 1 hour at 120°C and 0.13 bar. Vacuum was relieved with nitrogen to 1.4 bar and the vessel heated to 165°C. Ethylene oxide (33.86 molar equivalent) was fed into the reactor at a rate of 4 g/min. The rate was increased to 6 g/min as quickly as temperature control and pressure allowed. Total addition time was 5 hrs. After the ethylene oxide addition was completed, reacted to constant pressure for 2 additional hours. The reactor was vented and cooled to 85°C before discharging. By this procedure, an ethoxylated polyethyleneimine was obtained as a brown liquid or as a solid. Degree of ethoxylation was determine by OH#. Average molecular weight was determined by GPC in water/THF. Amine functionality confirmed by 13C NMR.
Methylation of tertiary amines:
In a 2 L vessel fitted with a stirrer, 500 mmol of PEI ethoxylate and 3.69 mol of sodium bicarbonate were dispersed in 250 mL of water. The flask was cooled to a temperature of 18-20°C with an ice bath as 3.37 moles of dimethyl sulfate were added dropwise. The addition occurred over 50 minutes and resulted in a maximum exotherm of 25°C with active cooling. The reaction persisted at room temperature for 20 minutes before it was heated to 65°C and reacted for an additional 30 minutes. The completeness of the reaction was verified by thin layer chromatography (TLC) using a solvent composition of 3 to 1 ethyl acetate to hexanes. Excess water was decanted from the resultant brown slurry. The reaction product was extracted with ether. Dried with sodium sulfate. Ether was removed by rotary evaporation. By this procedure, the quaternary polyethyleneimine ethoxylate was obtained. Amine functionality and polymer charge density was confirmed by C NMR.
Alkylation of PEI particles by esterification of OH groups with fatty acid:
In a 250 mL flask fitted with a stirrer and dean-stark distillation column, 200 mmol of PEI ethoxylate, 800 mmol of lauric acid, and methanesulfonic acid (at 2 mol % to initiator) were charged. The reaction contents were heated to 140 °C. The reaction continued at this temperature for 5 hours under reduced pressure (<0.12 bar) as water was collected. The reaction was completed when the measured acid value was <10 mgKOH. Table 3: Summary of PEI polymers used and PEI-PEO polymers.
Figure imgf000066_0001
Example 2: Example transformation protocol
Example transformation protocols for transforming plant cells using the compositions of the invention is described below.
1. Add about 400 mg polymer to about 400 mΐ ultra-pure water. Mix by pipetting. If still highly viscous, heat in water bath (37°C) until viscosity decreases.
2. Take about 660 mΐ of polymer solution from step 1, transfer to 1.5 mL micro-centrifuge tube.
3. Add about 120 ug plasmid DNA to 1.5 mL micro-centrifuge tube.
4. Mix for about 30 seconds (e.g., vortex)
5. Add about 220 mΐ of polymer-DNA solution to about 200 mΐ protoplast cells (about 2.5 x 105 cells) and immediately mix (e.g., by slowly inverting or tapping the tube).
6. Incubate the mixture for about 15-20 min in the dark. Add about 880 mΐ of W5 solution to the tube and mix well (e.g., invert the tube) to stop the transformation process. 7. Centrifuge the protoplasts at 80 g for 3 min at RT and remove the supernatant carefully by pipetting.
8. Resuspend the protoplasts gently in 2 ml of solution (e.g., W5 solution).
9. Transfer the protoplasts into plates (e.g., 6-well, 12-well or 24-well plates or tissue culture plates). Wrap the plates in aluminum foil and incubate them overnight at about 23 °C
The condition of the protoplasts can be checked under a microscope; the healthy cells should appear full and round. The transformation efficiency may be checked two days post transfection by counting the number of GFP-expressing cells using a fluorescence microscope.
The parameters of these protocols may be modified to include variations in the ratio of nucleic acid to polymer; the type of nucleic acid used; as well as adjusted for use with cells from different species.
Example 3: DNA delivery to plants
Polymers 1, 3, 7, and 9 were evaluated for their effect on plant cell viability, penetration, and DNA delivery efficiency.
Impact on cell viability
Four polymers were selected for evaluation in plant cells, i.e. 1, 3, 7 and 9. Polymers 7 an 9 have a larger core than polymers 1 and 3 and a greater cationic charge. Polymers 1 and 7 have a higher cationic charge than polymers 3 and 9.
The testing was performed in wheat protoplast cells. Protoplasts are naked plant cells which have had their cell wall removed by either mechanical or enzymatic means. Over the past decades, protoplasts derived from several plant species have been widely used to study cellular processes and elucidate molecular mechanisms underlying pathways involved in plant physiology, immunity, growth and development.
The impact of the selected polymers on protoplast survival was tested first. To this end, isolated protoplasts were mixed with increasing amounts of the polymers and incubated in six-well culture plates. Two days post treatment, cell viability was assessed by checking the appearance of the cells under a fluorescence microscope. Healthy cells appear full and round containing visually intact membranes and show strong autofluorescence. As illustrated in Fig. 1, incubating protoplasts in the presence of increasing doses of polymer 1 and polymer 7 caused strong cytotoxic effects, with both polymers killing off more than 90% of the treated cells at a concentration of 400 mg. Higher survival rates were found for polymer 7 and especially polymer 9, in which case we failed to observe any significant differences in the number of living cells as compared to non-treated controls.
Transformation
The transformation potential of the polymers for plasmid DNA delivery into wheat protoplast cells was assessed. For this purpose, cells were transfected with a GFP-expressing plasmid and assayed for fluorescence 2 days post treatment. Initially, each of the polymers was used at the highest non-lethal concentration as determined in abovementioned experiment and mixed with 10 pg of plasmid DNA. Control cells were transfected using polyethylene glycol (PEG) following our standard transfection protocol. The transfection efficiency was calculated as the number of GFP-positive cells divided by the total number of living cells at the end of the experiments.
As shown in Fig. 2, no GFP-expressing cells could be detected upon treatment with 100 mg of either polymer 1 or polymer 3. We also failed to observe any GFP-positive cells at higher concentrations, suggesting that these polymers are not suited for transfection of plasmid DNA in wheat cells. In contrast, mixing cells with 400 mg of polymer 7 or polymer 9 did yield a significant, albeit rather low, number of green fluorescent cells, indicating successful polymer-mediated DNA uptake. Polymer 9 consistently outperformed polymer 7 over three independent experiments, showing a mean transfection efficiency of almost 2%.
It is believed that the polymers will be particularly useful in transforming intact plant cells (e.g., cells with cell walls, e.g., plants and plant tissues). This method should provide a rapid and direct means for transforming intact plant cells and tissues that eliminates the need for production of protoplasts or infection by Agrobacterium and the like.
To further improve the transformation efficiency, a series of experiments were set up to evaluate and assess the impact of protoplast density, polymer concentration and plasmid amount on the number of GFP-positive cells. Based on the previous results, these experiments were performed with polymer 9 only. We found that the transfection efficiency was linearly correlated with the amount of plasmid DNA, reaching a maximum of 3.6% when mixing 2.5 x 105 protoplast cells with 220 mΐ of a 400 mg polymer solution containing 40 pg DNA (Fig. 3A). Enhancing the amount of polymer had a negative impact on the number of GFP-expressing cells, with concentrations higher than 600 mg reducing efficiencies to almost zero (Fig. 3B).
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:
1. A composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-polyethylene oxide (PEO) polymer) and a nucleic acid molecule, optionally wherein the polyalkyleneimine-alkoxylene polymer is complexed with the nucleic acid molecule.
2. The composition of claim 1, wherein the composition comprises two or more nucleic acid molecules, optionally wherein the polyalkyleneimine-alkoxylene polymer is complexed with two or more nucleic acid molecules.
3. The composition of claim 1 or claim 2, wherein the nucleic acid molecule is about 20 bases to about 100 kilobases in length.
4. The composition of any of the preceding claims, wherein the nucleic acid molecule is DNA, RNA, or any combination thereof.
5. The composition of any of the preceding claims, wherein the nucleic acid molecule and the polyalkyleneimine-alkoxylene polymer are present in the composition in a weight ratio of about 1 : 10 to about 1 : 10,000 (nucleic acid molecule : polyalkyleneimine-alkoxylene polymer).
6. The composition of any of the preceding claims, wherein the nucleic acid molecule is present in the composition in an amount of about 0.1, 0.5, 1, 5, 10, 15, 20, or 25 nanograms per microgram of the polyalkyleneimine-alkoxylene polymer to about 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nanograms per microgram of the polyalkyleneimine-alkoxylene polymer.
7. The composition of any one of the preceding claims, wherein the polyalkyleneimine- alkoxylene polymer is cationic.
8. The composition of any one of the preceding claims, wherein the nucleic acid molecule is electrostatically and/or covalently bound to a portion of the polyalkyleneimine- alkoxylene polymer
9. The composition of any one of the preceding claims, wherein the polyalkyleneimine- alkoxylene polymer has a zeta potential of at least about +1, +2, +3, +4, +5, or +6 when the composition has a pH of about 7.
10. The composition of any one of the preceding claims, wherein the polyalkyleneimine- alkoxylene polymer comprises 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per nitrogen atom of the polyalkyleneimine (e.g., PEI or polypropyleneimine) in the polyalkyleneimine-alkoxylene polymer,
optionally wherein the polyalkyleneimine-alkoxylene polymer comprises 0, 1, 2, or 5 to 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 alkoxylene units (e.g., ethylene oxide units) per primary amine of the polyalkyleneimine (e.g., PEI or polypropyleneimine) in the polyalkyleneimine-alkoxylene polymer.
11. The composition of any one of the preceding claims, wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the primary amines of the polyalkyleneimine (e.g., PEI or polypropyleneimine) of the polyalkyleneimine-alkoxylene polymer are functionalized with at least one alkoxylene unit (e.g., ethylene oxide unit), optionally wherein one or more primary amines of the polyalkyleneimine are functionalized with at least one alkoxylene unit to form the polyalkyleneimine-alkoxylene.
12. The composition of any one of the preceding claims, wherein a terminal alkoxylene unit (e.g., ethylene oxide unit) of the polyalkyleneimine-alkoxylene polymer is capped with hydrogen, C1-C22 alkyl, C1-C22 alkenyl, C1-C22 alkynyl, C1-C22 aryl, -C(0)-C1-C22 alkyl, -C(0)-C1-C22 alkenyl, -C(0)-C1-C22 alkynyl, or -C(0)-C1-C22 aryl.
13. The composition of any one of the preceding claims, wherein an internal and/or terminal nitrogen atom of the polyalkyleneimine (e.g., PEI or polypropyleneimine) of the polyalkyleneimine-alkoxylene polymer is bound to an alkyl, alkenyl, alkynyl, aryl, arylalkyl arylalkenyl, and/or arylalkynyl substituent, which optionally is substituted or unsubstituted.
14. The composition of any one of the preceding claims, wherein the polyalkyleneimine- alkoxylene polymer comprises one or more quaternized functional group(s), optionally wherein the polyalkyleneimine-alkoxylene polymer comprises at least 5% of the nitrogen atoms in the polyalkyleneimine (e.g., PEI or polypropyleneimine) backbone are quaternized based on the total number of nitrogen atoms in the polyalkyleneimine backbone.
15. The composition of claim 14, wherein the one or more quaternized functional group(s) include a nitrogen atom of the polyalkyleneimine (e.g., PEI) bound to a C1-C12 substituent (e.g., a substituted or unsubstituted C1-C12 alkyl, alkenyl, alkynyl, aryl, arylalkyl arylalkenyl, or arylalkynyl substituent), optionally wherein the one or more quaternized group(s) are formed by reacting a C1-C12 substituent with a tertiary amine of the polyalkyleneimine.
16. The composition of any one of the preceding claims, wherein the polyalkyleneimine and/or alkoxylene of the polyalkyleneimine-alkoxylene polymer is/are branched.
17. The composition of any one of the preceding claims, wherein the polyalkyleneimine- alkoxylene polymer is a core-shell polymer, optionally wherein the core of the polyalkyleneimine-alkoxylene polymer comprises the polyalkyleneimine (e.g., PEI or polypropyleneimine) and the shell of the polymer comprises alkoxylene (e.g., PEO).
18. The composition of any one of the preceding claims, wherein the alkoxylene of the polymer comprises 1 to 30 alkoxylene units (e.g., PEO units).
19. The composition of any one of the preceding claims, wherein the polyalkyleneimine- alkoxylene polymer comprises PEI in an amount of about 1 to about 40 % by weight of the polymer and comprises PEO in an amount of about 60 to about 99% by weight of the polymer.
20. The composition of any one of the preceding claims, wherein the polyalkyleneimine- alkoxylenepolymer is water soluble, optionally at room temperature.
21. The composition of any one of the preceding claims, wherein the polyalkyleneimine of the polyalkyleneimine-alkoxylenepolymer has a molecular weight of about 800 g/mol to about 750,000 g/mol.
22. The composition of any one of the preceding claims, wherein the polyalkyleneimine of the polyalkyleneimine-alkoxylenepolymer has an amine value of about 16, 17, 18, 19, 20, or 21.
23. A method of preparing a composition comprising a polyalkyleneimine-alkoxylene polymer (e.g., a polyethyleneimine (PEI)-poly ethylene oxide (PEO) polymer) and a nucleic acid molecule, comprising:
combining the nucleic acid molecule and the polymer, thereby preparing the composition, optionally wherein the polymer is complexed with the nucleic acid molecule.
24. The method of claim 23, wherein the composition comprises two or more nucleic acid molecules, optionally wherein the polymer is complexed with two or more nucleic acid molecules.
25. The method of claim 23 or 24, wherein combining the nucleic acid molecule and the polymer comprises contacting and/or mixing a first solution comprising the nucleic acid molecule and a second solution comprising the polymer to form a third solution comprising the nucleic acid molecule and the polymer.
26. The method of any one of claims 23 to 25, wherein combining the nucleic acid molecule and the polymer comprises providing the nucleic acid molecule and the polymer in contact (e.g., in the same composition, optionally with agitation) for about 1 min to about 24 hours.
27. A method of delivering a nucleic acid molecule to (transforming) a cell, comprising contacting the cell with the composition of any one of claims 1 to 22, thereby delivering the nucleic acid molecule in the composition to the cell.
28. The method of claim 27, wherein the cell is from or in an animal, a plant, and/or a fungus.
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US20120021006A1 (en) * 2008-12-22 2012-01-26 Alexander Levitzki Egfr-homing double-stranded rna vector for systemic cancer treatment
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