Collapsible cell-penetrating complexes for nucleic acid delivery

Abstract

To provide complexes, compositions and methods for the delivery of therapeutic, diagnostic and imaging agents, including nucleic acids, into cells.SOLUTION: This invention may facilitate complexation, protection, delivery and release of oligonucleotides and polyanionic cargos into target cells, tissues, and organs both in vitro and in vivo.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 367,555, filed July 27, 2016, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] STATEMENT OF RIGHTS TO DISCLOSURES MADE IN FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Contract No. DE-SC0005430 awarded by the U.S. Department of Energy, Contract No. 1306730 awarded by the National Science Foundation, and Contract Nos. CA031841 and CA031845 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] References to "Sequence Listings," tables, or computer programs listed in an attachment submitted as an ASCII file Not applicable. [Background technology]

[0004] There is a need for new materials and strategies that enable or facilitate the delivery of therapeutic agents, diagnostic probes, and / or research tools across cell membranes and other biological barriers as required for a wide range of clinical, diagnostic, and / or research applications. Delivery of such cargoes, e.g., nucleic acids, has considerable clinical potential in the context of vaccination strategies against infectious diseases, cancer immunotherapy, protein therapy, and gene editing. Provided herein are solutions to these and other problems in the art. Summary of the Invention

[0005] In a first aspect, a cell-penetrating complex is provided that comprises a nucleic acid non-covalently bound to a cationic amphiphilic polymer, wherein the cationic amphiphilic polymer comprises a pH-sensitive disintegration domain.

[0006] In another aspect, there is provided a nanoparticle composition comprising a plurality of cell-penetrating complexes as disclosed herein.

[0007] In another embodiment, there is provided a cationic amphiphilic polymer of the formula H-L1-[(LP1)-(IM)-(LP2)]-L2-H(I), wherein L1 and L2 are independently a bond, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—, —NHC(O)—, —S(O)—, —S(O)NH—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkenyl, or substituted or unsubstituted heterocycloalkenyl.

[0013] Provided is a cationic amphiphilic polymer wherein L1 is alkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; L1 and L12 are independently a bond or a lipophilic polymer domain, at least one of L1 or L12 being a lipophilic polymer domain; I M is a pH-sensitive disintegratable domain; z1, z3, and z4 are independently integers of 0 to 100, at least one of z1 or z3 being not 0, and z2 being an integer of 2 to 100. In embodiments, L1 is a substituted or unsubstituted alkylene. In embodiments, L1 is a peptide (e.g., an amino acid sequence).

[0008] In another embodiment, there is provided a cationic amphiphilic polymer of the formula H-L1-[(LP1)z1-(LP3)z1a-(IM)z2-(LP2)z3-(LP4)z3b]z4-L2-H(I), wherein L1 and L2 are independently a bond, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—, —NHC(O)—, —S(O)2-, —S(O)NH—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, or LP1, LP2, LP3, and LP4 are independently a bond or a lipophilic polymer domain, at least one of LP1, LP2, LP3, and LP4 is a lipophilic polymer domain, IM is a pH-sensitive disintegratable domain, z1, z1a, z3, z3b, and z4 are independently an integer of 0 to 100, at least one of z1 or z3 is not 0, and z2 is an integer of 2 to 100. In embodiments, L1 is a substituted or unsubstituted alkylene. In embodiments, L1 is a peptide (e.g., an amino acid sequence).

[0009] In another aspect, there is provided a method of transfecting a nucleic acid into a cell, the method comprising contacting the cell with a complex as disclosed herein.

[0010] In another embodiment, there is provided a cationic amphiphilic polymer of the formula R1A-[L1-[(LP1)z1-(IM)z2-(LP2)z3]z4-L2-R2A]z5, wherein R1A is hydrogen, halogen, —CCl3, —CBr3, —CF3, —CI3, CHCl2, —CHBr2, —CHF2, —CHI2, —CH2Cl, —CH2Br, —CH2F, —CH2I, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —S03H, —S04H, —SON2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, —NHC (O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R2A is independently hydrogen, Halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H , -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCH I2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; L1 and L2 are independently a bond, -C(O)O-, -O-, -S-, -NH-, -C(O)NH-, -NHC(O)-, -S(O)2-, -S(O)NH-, -NHC(O)NH-, substituted or unsubstituted alkylene,

[0023] Provided is a cationic amphiphilic polymer in which R1A is a substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; LP1 and LP2 are independently a bond or a lipophilic polymer domain; at least one of LP1 or LP2 is a lipophilic polymer domain; IM is independently a pH-sensitive disintegration domain; z5 is an integer of 1 to 10; z1, z3, and z4 are independently integers of 0 to 100; at least one of z1 or z3 is not 0; and z2 is independently an integer of 2 to 100. In an embodiment, R1A is hydrogen. When R1A is hydrogen, z5 is 1.

[0011] In another aspect, there is provided a method for inducing an immune response in a subject in need thereof, the method comprising administering an effective amount of a cell-permeable complex disclosed herein. [Brief explanation of the drawings]

[0012] [Figure 1] Data showing the average brightness (p / s / cm2 / sr) of cells after stable transfection with the CART system are shown. [Figure 2] 1 presents data demonstrating the antitumor immunogenicity of CART and CART in combination with adjuvants. [Figure 3] Figure 3A shows data demonstrating the efficacy of the prophylactic vaccination strategy, showing a graph of tumor size. Figure 3B shows data demonstrating the efficacy of the prophylactic vaccination strategy, showing a graph of survival rate. Figure 3C shows data demonstrating the efficacy of the prophylactic vaccination strategy, showing a graph of tumor size in mice re-challenged with TSA-expressing A20 lymphoma (Note: overlapping lines are shown separately, and all black lines have values ​​of zero). [Figure 4]Figure 4A shows the size of pre-established tumors (prime vaccination: day 7, boost: days 10 and 13), which is tumor size data. Figure 4B shows the survival rate data (prime vaccination: day 7, boost: days 10 and 13), which is survival curve data. [Figure 5] Figure 5A shows data demonstrating in situ vaccination with CART and mRNA encoding an immunostimulatory protein. Figure 5B shows data demonstrating in situ vaccination with CART and mRNA encoding an immunostimulatory protein. [Figure 6] FIG. 1 shows a diagram depicting a charge-variable efflux transporter (CART) for delivering mRNA. [Figure 7] Figure 7A shows an oligo(carbonate-b-α-amino ester) CART synthesized for mRNA delivery, demonstrating the mechanism of oligo(α-amino ester) rearrangement via tandem five- and six-membered transition states to form an amide. Figure 7B shows an oligo(carbonate-b-α-amino ester) CART synthesized for mRNA delivery, demonstrating the synthesis of an oligo(carbonate-b-α-amino ester) CART for mRNA delivery by OROP of cyclic carbonate and ester monomers. Figure 7C shows an oligo(carbonate-b-α-amino ester) CART synthesized for mRNA delivery, using an amphiphilic oligo(carbonate) synthesized by the OROP methodology described above. [Figure 8] Figure 8A shows the results of the CART rearrangement mechanism of oligo(α-amino ester), demonstrating that the self-immolative rearrangement of the α-amino ester moiety of the block co-oligomer yields an intact lipophilic oligocarbonate block along with the attached initiator and small molecule HEGD2. Figure 8B shows the results of the CART rearrangement mechanism of oligo(α-amino ester), comparing the GPC traces of the protected block co-oligomer Pyr-D15:A12 (10) (red) and its derivative after deprotection and base-catalyzed rearrangement (blue) with the independently synthesized D15 homo-oligomer 11. [Figure 9] Figure 9A shows data related to the evaluation of CART for eGFP mRNA delivery, where the mean eGFP fluorescence values ​​obtained from HeLa cells treated with various transporters were determined by flow cytometry. Figure 9B shows data related to the evaluation of CART for eGFP mRNA delivery, where a representative flow cytometry histogram of eGFP fluorescence values ​​indicates the percent transfection rate in HeLa cells treated with eGFP mRNA transporters. Figure 9C shows data related to the evaluation of CART for eGFP mRNA delivery, where the effect of charge ratio on eGFP expression resulting from mRNA delivery with D13:A11 7 is shown. Figure 9D shows data related to the evaluation of CART for eGFP mRNA delivery, where epifluorescence microscopy images of HeLa cells treated with naked mRNA, mRNA complexed with Lipofectamine, or mRNA complexed with D13:A11 7 are shown. All data shown are from HeLa cells treated with an mRNA concentration of 0.125 μg / well in a 24-well plate for 8 hours. [Figure 10]Figure 10A shows the uptake of Cy5-mRNA / D13:A117 complexes at 4°C (conditions that inhibit endocytosis), demonstrating that mRNA expression is due to a charge-fluctuation self-immolative mechanism driven by CART oligomers, which releases mRNA and induces endosomal escape. Figure 10B shows the relative uptake and expression of eGFP mRNA after treatment with complexes formed with degradable and nondegradable transporter systems, demonstrating that mRNA expression is due to a charge-fluctuation self-immolative mechanism driven by CART oligomers, which releases mRNA and induces endosomal escape. The colored bars represent GFP expression, and the white bars represent Cy5-mRNA fluorescence. Figure 10C shows eGFP expression after co-treatment of mRNA / D13:A11 7 complexes with concanamycin A (ConA), a compound known to inhibit endosomal acidification, and chloroquine (ChL), an endosome-disrupting agent, demonstrating that mRNA expression is due to the charge-fluctuation self-immolative mechanism driving mRNA release and endosomal escape by CART oligomers. Figure 10D shows confocal microscopy images of cells treated with CART D13:A11 7 or non-releasable oligomer 7 for 4 hours, demonstrating that mRNA expression is due to the charge-fluctuation self-immolative mechanism driving mRNA release and endosomal escape by CART oligomers. Cells were co-treated with transporter / Cy5-mRNA complexes and TRITC-dextran 4400. [Figure 11]Figure 11A shows the application of CART-mediated mRNA delivery in multiple cell lines. The transfection efficiency of eGFP mRNA delivery by D13:A11## is compared with that of Lipofectamine in HeLa (blue), J774 (red), HEK293 (gray), CHO (yellow), and HepG2 (green) cell lines. Figure 11B shows the application of CART-mediated mRNA delivery in multiple cell lines. CART7-mediated delivery of Fluc mRNA follows the same trend in charge ratio as eGFP. Charges are reported as theoretical (+ / -) ratios. Figure 11C shows the application of CART-mediated mRNA delivery in mice. In vivo BLI after intramuscular injection of naked Fluc mRNA (open circles) and mRNA / D13:A11## complexes (filled circles). Bars represent the average across all animals (n = 3 for 1 h, 4 h, and 7 h, n = 5 for 24 h and 48 h). Figure 11D shows the application of CART-based mRNA delivery in mice. Representative bioluminescence images are shown after intramuscular injection of naked mRNA (left flank) or mRNA / CART complexes (right flank). Figure 11E shows the application of CART-based mRNA delivery in mice. In vivo BLI is shown after intravenous tail vein injection of naked mRNA (open circles) and mRNA / CART complexes (filled circles). Bars represent the average across all animals (n = 2 for 1 h and 7 h, n = 4 for 4 h, 24 h, and 48 h). The dotted line represents the background bioluminescence signal from animals not injected with D-luciferin. Figure 11F shows the application of CART-based mRNA delivery in multiple cell lines and mice. Representative bioluminescence images are shown from mice treated with mRNA / D13:A11 complexes via intravenous tail vein injection. DETAILED DESCRIPTION OF THE INVENTION

[0013] While various embodiments and aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only, and that numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It will be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure.

[0014] Unless the context requires otherwise, it is expressly contemplated that the various features of the present disclosure described herein can be used in any combination. Furthermore, the present disclosure contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if a composite is described herein as having components A, B, and C, it is expressly contemplated that A, B, or C, or any combination thereof, can be omitted or eliminated, individually or in any combination.

[0015] As used in this specification and the appended claims, please note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "cancer cell" includes a plurality of cancer cells. As another example, a "nucleic acid" or a "nucleic acid" includes a plurality of nucleic acid molecules, i.e., a plurality of nucleic acids.

[0016] The term "about" refers to a range of values ​​that includes the specified value and that one of ordinary skill in the art would reasonably consider to be comparable to the specified value. In embodiments, about refers to within a standard deviation using measurements generally accepted in the art. In embodiments, about refers to a range of up to ±10% of the specified value. In embodiments, about refers to the specified value.

[0017] Also, as used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, as well as exclusive combinations when interpreted as an alternative ("or").

[0018] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. As used herein, the transitional phrase "consisting essentially of" (and grammatical equivalents) is intended to be interpreted as including the recited materials or steps and materials or steps that do not materially affect the basic and novel feature(s) of the recited embodiment. Thus, the term "consisting essentially of," as used herein, should not be interpreted as an equivalent term to "comprising." "Consisting of" is intended to mean excluding more than trace amounts of other component elements and other substantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transitional phrases are within the scope of the present disclosure.

[0019] definition The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae depicted herein should be construed in accordance with standard rules of chemical valency known in the chemical arts.

[0020] As used herein, the terms "oligomer" and "polymer" refer to a compound having multiple repeating subunits (e.g., polymerized monomers). The terms "co-oligomer" or "copolymer" refer to an oligomer or polymer containing two or more types of residues (monomer units or monomers, which are used interchangeably herein). The number of monomers in an oligomer is generally less than the number of monomers in a polymer. Thus, in some examples, an oligomer can be from 1 to about 10 monomers, from 1 to about 20 monomers, from 1 to about 30 monomers, from 1 to about 40 monomers, from 1 to about 50 monomers, from 1 to about 100 monomers, from 1 to about 150 monomers, from 1 to about 200 monomers, from 1 to about 250 monomers, from 1 to about 300 monomers, from 1 to about 350 monomers, from 1 to about 400 monomers, from 1 to about 450 monomers, or from 1 to about 500 monomers in length. In some examples, an oligomer can be less than about 500 monomers, less than about 450 monomers, less than about 400 monomers, less than about 350 monomers, less than about 300 monomers, less than about 250 monomers, less than about 200 monomers, less than about 150 monomers, less than about 100 monomers, less than about 50 monomers, less than about 40 monomers, less than about 30 monomers, less than about 20 monomers, or less than about 10 monomers in length. With respect to polymers, the number of monomers in a polymer generally exceeds the number of monomers in an oligomer. Thus, in some examples, the polymer can be from about 500 to about 1000 monomers, from about 500 to about 2000 monomers, from about 500 to about 3000 monomers, from about 500 to about 4000 monomers, from about 500 to about 5000 monomers, from about 500 to about 6000 monomers, from about 500 to about 7000 monomers, from about 500 to about 8000 monomers, from about 500 to about 9000 monomers, from about 500 to about 10,000 monomers, or greater than 10,000 monomers in length.

[0021] The term "polymerizable monomer" is used according to its meaning in the art of polymer chemistry to refer to a compound that can be chemically covalently bonded to other monomer molecules (such as other polymerizable monomers, whether the same or different) to form a polymer.

[0022] The term "block copolymer" is used according to its ordinary meaning to refer to two or more portions (e.g., blocks) of polymerized monomers linked by covalent bonds. In embodiments, a block copolymer is a repeating pattern of polymer. In embodiments, a block copolymer includes two or more monomers in a periodic (e.g., repeating) arrangement. For example, a diblock copolymer has the formula -BBBBBBAAAAA-, where "B" is the first subunit and "A" is the second subunit, covalently bonded together. Thus, a triblock copolymer is a copolymer having three distinct blocks, two of which may be the same (e.g., -AAAAABBBBBBAAAAA-) or all three of which are different (e.g., -AAAAABBBBBBCCCCC-), where "A" is the first subunit, "B" is the second subunit, and "C" is the third subunit, covalently bonded together.

[0023] The term "alkyl," by itself or as part of another substituent, unless otherwise specified, means straight (i.e., unbranched) or branched chain, or combinations thereof, which may be fully saturated, monounsaturated, or polyunsaturated, and may include divalent and polyvalent groups. Alkyl may have the number of carbon atoms indicated (i.e., C1-C10 means 1 to 10 carbons). Alkyl is a non-cyclized chain. Examples of saturated hydrocarbon groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, homologs and isomers thereof, e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are groups that have one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologs and isomers. Alkoxy is an alkyl group attached to the remainder of the molecule via an oxygen linker (-O-).

[0024] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from alkyl, exemplified, but not limited to, by -CHCHCHCH-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred. A "lower alkyl" or "lower alkylene" is a C1-C8 alkyl or alkylene group.

[0025] The term "heteroalkyl," unless otherwise specified, alone or in combination with another term, means a stable linear or branched chain consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, or any combination thereof, wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N, P, S, and Si may be located at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. A heteroalkyl is a non-cyclizing chain. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3.

[0026] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, and is exemplified, but not limited to, by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. In heteroalkylene groups, heteroatoms can occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). As used herein, heteroalkyl groups include groups attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SOR'. When "heteroalkyl" is referenced followed by a list of specific heteroalkyl groups, such as -NR'R'', it will be understood that the terms heteroalkyl and -NR'R'' are not redundant or mutually exclusive. To the contrary, specific heteroalkyl groups are provided for added clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyl groups such as -NR'R'', etc.

[0027] The terms "cycloalkyl" and "heterocycloalkyl," alone or in combination with other terms, refer to cyclic forms of "alkyl" and "heteroalkyl," respectively, unless otherwise specified. Cycloalkyls and heteroalkyls are not aromatic. Additionally, in a heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyls include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0028] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise specified, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0029] The term "acyl," unless otherwise specified, means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0030] The term "aryl," unless otherwise specified, refers to a polyunsaturated aromatic hydrocarbon substituent, which may be a single ring, fused (i.e., fused-ring aryl), or covalently linked multiple rings (preferably 1 to 3 rings). Fused-ring aryl refers to a fused multiple ring group in which at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 4 heteroatoms selected from N, O, and S, the nitrogen and sulfur atoms of which are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., fused multiple rings in which at least one of the fused rings is a heteroaromatic ring). 5,6-fused-ring heteroarylene refers to two fused rings, one of which is 5-membered and the other is 6-membered, and at least one of the rings is a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two fused rings, one of which is 6-membered and the other of which is 6-membered, and at least one of which is a heteroaryl ring. A 6,5-fused ring heteroarylene refers to two fused rings, one of which is 6-membered and the other of which is 5-membered, and at least one of which is a heteroaryl ring. A heteroaryl group can be attached to the rest of the molecule through a carbon or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, Examples include 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above-listed aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "Arylene" and "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl or heteroaryl, respectively.

[0031] For brevity, when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl), the term "aryl" includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is intended to include groups in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced with, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, etc.).

[0032] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0033] The term "alkylsulfonyl," as used herein, refers to a moiety having the formula -S(O)-R', where R' is an alkyl group as defined above. R' can have a specified number of carbons (e.g., "C-C alkylsulfonyl").

[0034] Each of the above terms (eg, "alkyl," "heteroalkyl," "aryl" and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical.

[0035] Substituents on alkyl and heteroalkyl groups (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) range in number from 0 to (2m'+1) (m' is the total number of carbon atoms in the group) and include -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', and -NOR. R', R'', R''', and R'' each independently represent a hydrogen atom, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group (e.g., an aryl group substituted with 1 to 3 halogens), a substituted or unsubstituted alkyl group, an alkoxy group, a thioalkoxy group, or an arylalkyl group. When a compound disclosed herein includes more than one R group, for example, each of the R groups is independently selected; when more than one of R', R", R'", and R"" groups is present, each of those groups is also independently selected. When R' and R" are attached to the same nitrogen atom, R' and R" can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will recognize that the term "alkyl" is intended to include groups that include carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0036] Similar to the substituents described for the alkyl group, substituents on the aryl and heteroaryl groups can vary and include, for example, —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′′, —OC(O)R′, —C(O)R′, —COR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′-C(O)NR″R′′, —NR″C(O)R′, —NR-C(NR′R″R′′)═NR′″, and —NR-C(NR′R″)═NR′ ", -S(O)R', -S(O)R', -S(O)NR'R'', -NRSO2R', -CN, -NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, where R', R'', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When the compounds disclosed herein include more than one R group, for example, each R group is independently selected, and when more than one of R', R'', R''', and R'''' groups is present, each such group is independently selected.

[0037] Two or more substituents may optionally be linked to form an aryl group, a heteroaryl group, a cycloalkyl group, or a heterocycloalkyl group. It is understood that these so-called ring-forming substituents are typically, but not necessarily, attached to a cyclic parent structure. In an embodiment, the ring-forming substituents are attached to adjacent ring members of the parent structure. For example, two ring-forming substituents attached to adjacent ring members of the cyclic parent structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to one ring member of the parent structure. For example, two ring-forming substituents attached to one ring member of the cyclic parent structure create a spiro ring structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent ring members of the parent structure.

[0038] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -TC(O)-(CRR')qU-, where T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)rB-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 4. One of the single bonds in the new ring thus formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'-(C''R''')d-, where s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The R, R', R'' and R''' substituents are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0039] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0040] "Substituent," as used herein, means a group selected from the moieties described below. (A) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroaryl. alkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). (B) Alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl substituted with at least one substituent selected from the following: (i) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl or C1-C4 alkyl), unsubstituted heteroalkyl ... For example, 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl), and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl substituted with at least one substituent selected from the following: (a) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl or C1-C4 alkyl), unsubstituted heteroalkyl unsubstituted cycloalkyl (e.g., a 2- to 8-membered heteroalkyl, a 2- to 6-membered heteroalkyl, or a 2- to 4-membered heteroalkyl), unsubstituted cycloalkyl (e.g., a C3-C8 cycloalkyl, a C3-C6 cycloalkyl, or a C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., a 3- to 8-membered heterocycloalkyl, a 3- to 6-membered heterocycloalkyl, or a 5- to 6-membered heterocycloalkyl), unsubstituted aryl (e.g., a C6-C10 aryl, a C10 aryl, or a phenyl), or unsubstituted heteroaryl (e.g., a 5- to 10-membered heteroaryl, a 5- to 9-membered heteroaryl, or a 5- to 6-membered heteroaryl); (b) oxo, halogen, -CCl3, -CBr3, -CF3, -CI3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with at least one substituent selected from: unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3-8 membered heterocycloalkyl, 3-6 membered heterocycloalkyl, or 5-6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5-10 membered heteroaryl, 5-9 membered heteroaryl, or 5-6 membered heteroaryl).

[0041] As used herein, the term "size-limited substituent" or "size-limited substituent group" refers to a group selected from all of the substituents described above for "substituents," wherein the substituted or unsubstituted alkyl is, respectively, a substituted or unsubstituted C1-C20 alkyl, the substituted or unsubstituted heteroalkyl is, respectively, a substituted or unsubstituted 2- to 20-membered heteroalkyl, the substituted or unsubstituted cycloalkyl is, respectively, a substituted or unsubstituted C4-C8 cycloalkyl, and the substituted or unsubstituted heterocycloalkyl is, respectively, a substituted or unsubstituted 4- to 8-membered heterocycloalkyl.

[0042] As used herein, the term "lower substituent" or "lower substituent group" refers to a group selected from all of the substituents described above for "substituents," wherein the substituted or unsubstituted alkyl is, respectively, substituted or unsubstituted C1-C8 alkyl, the substituted or unsubstituted heteroalkyl is, respectively, substituted or unsubstituted 2- to 8-membered heteroalkyl, the substituted or unsubstituted cycloalkyl is, respectively, substituted or unsubstituted C5-C7 cycloalkyl, and the substituted or unsubstituted heterocycloalkyl is, respectively, substituted or unsubstituted 5- to 7-membered heterocycloalkyl.

[0043] In embodiments, each of the substituted groups described in the compounds of the present invention is substituted with at least one substituent. More specifically, in embodiments, each of the substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds of the present invention is substituted with at least one substituent. In embodiments, at least one or all of these groups are substituted with at least one size-limited substituent. In embodiments, at least one or all of these groups are substituted with at least one lower-rank substituent.

[0044] In embodiments of the compounds of the present invention, each substituted or unsubstituted alkyl can be a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 20-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 10-membered heteroaryl. In embodiments of the compounds of the invention, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2- to 20-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and / or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5- to 10-membered heteroarylene.

[0045] In embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and / or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5- to 9-membered heteroaryl. In embodiments, each substituted or unsubstituted alkylene is substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is substituted or unsubstituted 2- to 8-membered heteroalkylene, each substituted or unsubstituted cycloalkylene is substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is substituted or unsubstituted 3- to 7-membered heterocycloalkylene, each substituted or unsubstituted arylene is substituted or unsubstituted C6-C10 arylene, and / or each substituted or unsubstituted heteroarylene is substituted or unsubstituted 5- to 9-membered heteroarylene. In embodiments, the compound is a species depicted herein.

[0046] The terms "a" or "an," as used herein, mean one or more. Additionally, the phrase "substituted with a + [noun]," as used herein, means that the specified group may be substituted with one or more of any or all of the specified substituents. For example, if a group, such as an alkyl group or heteroaryl group, is "substituted with unsubstituted C1-C20 alkyl or unsubstituted 2-20 membered heteroalkyl," the group may contain one or more unsubstituted C1-C20 alkyls and / or one or more unsubstituted 2-20 membered heteroalkyls. Additionally, if a moiety is substituted with an R substituent, the group may be referred to as "substituted with R." If a moiety is substituted with R, the moiety is substituted with at least one R substituent, and each R substituent is optionally a different group.

[0047] The term "nucleophilic moiety" refers to a chemical species or functional group that can donate one or more (e.g., two) electrons to an electrophile. In embodiments, a nucleophilic moiety refers to a chemical species or functional group that can donate electrons to an electrophile in a chemical reaction to form a bond.

[0048] The term "electrophilic moiety" refers to a chemical species or functional group that can accept one or more (e.g., two) electrons. In embodiments, an electrophilic moiety refers to a chemical species or functional group that has an empty orbital and can therefore accept electrons to form a bond in a chemical reaction.

[0049] The term "oligoglycol moiety" means [ka] where R400 is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and n300 is an integer greater than or equal to 1. In some examples, R400 is H or alkyl.

[0050] The description of the compounds of the present disclosure is limited by the principles of chemical bonding known to those skilled in the art.Therefore, when a group can be substituted with one or more of many substituents, the substitution is selected in accordance with the principles of chemical bonding and to produce a compound that is not inherently unstable and / or is known to those skilled in the art to be likely to be unstable under ambient conditions (such as aqueous conditions, neutral conditions, and some known physiological conditions).For example, heterocycloalkyl or heteroaryl is bonded to the rest of the molecule through a ring heteroatom according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0051] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). In practicing the present disclosure, any methods, instruments, and materials similar or equivalent to those described herein can be used. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0052] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, or their complements, in either single-, double-, or multi-stranded form. The terms "polynucleotide," "oligonucleotide," "oligo," and the like, in their ordinary and customary sense, refer to a linear sequence of nucleotides. The term "nucleotide," in its ordinary and customary sense, refers to a unit, i.e., a monomer, of a polynucleotide. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or modified forms thereof. Examples of polynucleotides contemplated by the present invention include single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid molecules comprising a mixture of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. Examples of nucleic acids, e.g., polynucleotides, contemplated by the present invention include any type of RNA, such as messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), plasmid DNA (pDNA), minicircle DNA, genomic DNA (gDNA), and fragments of any of these. In the context of polynucleotides, the term "duplex" refers to a double-stranded state in its usual and customary sense. Nucleic acids can be linear or branched. For example, nucleic acids can be linear nucleotides, or they can be branched, e.g., having one or more nucleotide arms or nucleotide branches. Optionally, branched nucleic acids are repeatedly branched to form higher-order structures, such as dendrimers.

[0053] Nucleic acids (including, for example, nucleic acids having a phosphothioate backbone) can contain one or more reactive moieties. As used herein, the term reactive moiety includes any group that can react with another molecule, e.g., a nucleic acid or a polypeptide, through a covalent, non-covalent, or other interaction. By way of example, a nucleic acid can contain an amino acid reactive moiety that reacts with an amino acid of a protein or polypeptide through a covalent, non-covalent, or other interaction.

[0054] The term also includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, both synthetic, natural, and non-natural, that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphothioates, which have a double-bonded sulfur replacing the oxygen in the phosphate), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acids, phosphonoformic acids, methylphosphonates, boronphosphonates, or O-methylphosphoramidite linkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford University Press), modifications to the nucleotide base, such as in 5-methylcytidine or pseudouridine, peptide nucleic acid backbones, and phosphodiester derivatives containing peptide nucleic acid linkages. Other analog nucleic acids include those with cationic backbones, non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNAs), as known in the art), including those described in U.S. Pat. Nos. 5,235,033, 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acid. Modifications to the ribose-phosphate backbone may be made for various reasons, such as to improve the stability and half-life of the molecule in physiological environments or as a probe on a biochip. Mixtures of natural nucleic acids and analogs can be made, or mixtures of different nucleic acid analogs and mixtures of natural nucleic acids and analogs can be made. In embodiments, the internucleotide linkages in the DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0055] A nucleic acid can include a non-specific sequence. As used herein, the term "non-specific sequence" refers to a nucleic acid sequence that includes a series of residues that are designed to be non-complementary or only partially complementary to any other nucleic acid sequence. For example, a non-specific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism. An "inhibitory nucleic acid" is a nucleic acid (e.g., a polymer of DNA, RNA, or nucleotide analog) that can bind to a target nucleic acid (e.g., an mRNA translatable into a protein) and reduce transcription of the target nucleic acid (e.g., transcription from DNA to mRNA), reduce translation of the target nucleic acid (e.g., mRNA), or alter splicing of the transcript (e.g., a single-stranded morpholino oligo). In an embodiment, the nucleic acid is RNA (e.g., mRNA). In an embodiment, the nucleic acid is 10 to 100,000 bases in length. In an embodiment, the nucleic acid is 50 to 10,000 bases in length. In an embodiment, the nucleic acid is 50 to 5,000 bases in length. In an embodiment, the nucleic acid is 50 to 1,000 bases in length.

[0056] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which may be conjugated to a non-amino acid moiety. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, to naturally occurring amino acid polymers, and to unnatural amino acid polymers. These terms also apply to macrocyclic peptides, peptides modified with non-peptide functional groups, peptidomimetics, polyamides, and macrolactams. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as one part.

[0057] The terms "peptidyl" and "peptidyl moiety" refer to a monovalent peptide.

[0058] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature.

[0059] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly accepted one-letter abbreviations.

[0060] "Contacting" is used according to its plain and simple meaning to refer to the process of bringing at least two distinct species (e.g., chemical compounds, including biological molecules or cells) into sufficient proximity so as to react, interact, or physically touch. However, it will be understood that the resulting reaction product may result directly from the reaction between the added reagents or from an intermediate derived from one or more of the added reagents that may be produced in the reaction mixture. In embodiments, contacting includes, for example, allowing a nucleic acid to interact with an endonuclease.

[0061] A "control" sample or "control" value refers to a sample that serves as a control, usually a known control, for comparison with a test sample. For example, a test sample can be obtained under test conditions, e.g., the presence of a test compound, and compared to a sample obtained under known conditions, e.g., the absence of the test compound (negative control) or the presence of a known compound (positive control). A control can also be an average value obtained from numerous tests or results. Those skilled in the art will recognize that controls can be designed for evaluation of any number of parameters. For example, controls can be established for the purpose of comparing therapeutic effects based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Those skilled in the art will know which standard control is most appropriate in a given situation and will be able to analyze data based on comparison of the standard control value. Standard controls are also useful for determining the significance (e.g., statistical significance) of data. For example, if the standard control shows a high variability in the value of a given parameter, the variation in the test sample will not be considered significant.

[0062] A "label" or "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens, proteins or other substances that can be made detectable by incorporating a radioactive label into, for example, a peptide or antibody specifically reactive with a target peptide. Any suitable method known in the art for conjugating an antibody to a label may be used (e.g., using the methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego).

[0063] "Biological sample" or "sample" refers to material obtained from or derived from a subject or patient. Biological samples include tissue sections such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include body fluids such as blood, blood fractions, blood products (e.g., serum, plasma, platelets, red blood cells, etc.), saliva, tissues, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, synovial fluid, articular tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, and the like. Biological samples are typically obtained from eukaryotic organisms such as mammals (primates, e.g., chimpanzees or humans, cows, dogs, cats, rodents (e.g., guinea pigs, rats, mice, etc.), rabbits, or birds), reptiles, or fish.

[0064] "Cell," as used herein, refers to a cell that performs metabolic or other functions sufficient to maintain or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane (stained with certain dyes), the ability to produce offspring, or, in the case of gametes, the ability to combine with a second gamete to produce a viable fetus. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammalian cells, insect (e.g., Spodoptera) cells, and human cells.

[0065] The term "stem cell" or "stem cells" refers to a clonal, self-renewing cell population that is pluripotent and can thereby generate several types of differentiated cells.

[0066] The term "gene" refers to a DNA segment involved in producing a protein, including regions preceding and following the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). The leader, trailer, and introns contain regulatory elements required for the transcription and translation of a gene. Furthermore, a "protein gene product" is a protein expressed from a particular gene.

[0067] The terms "expression" or "expressed," as used herein with respect to a gene, refer to the transcription and / or translation products of that gene. The level of expression of a DNA molecule in a cell may be determined based on either the amount of corresponding mRNA that is present in the cell or the amount of protein encoded by the DNA that is produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0068] Expression of a transfected gene can be transient or stable in a cell. In "transient expression," the transfected gene is not passed to daughter cells during cell division. Because expression of the gene is limited to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of the transfected gene can be achieved by co-transfecting the transfected gene with another gene that confers a selective advantage on the transfected cell. Such a selective advantage can be resistance to a particular toxin found in the cell.

[0069] The term "plasmid" refers to a nucleic acid molecule that encodes a gene and / or regulatory elements necessary for expression of the gene. Expression of a gene from a plasmid can be in cis or trans. When a gene is expressed in cis, the gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to when the gene and regulatory elements are encoded by separate plasmids.

[0070] The term "exogenous" refers to a molecule or substance (e.g., a nucleic acid or protein) that originates outside a given cell or organism, whereas the term "endogenous" refers to a molecule or substance that is naturally present in or originates within a given cell or organism.

[0071] A "vector" is a nucleic acid that can transport another nucleic acid into a cell. When in the appropriate environment, a vector can direct the expression of one or more proteins encoded by one or more genes carried by the vector.

[0072] The term "codon optimization," when referring to genes or coding regions of nucleic acid molecules intended for transformation into various hosts, refers to modifying the codons in the genes or coding regions of the nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization can involve replacing at least one, two, or more, or a significant number of codons with one or more codons that are more frequently used in the genes of the host organism. Given the large number of gene sequences available for a wide range of animal, plant, and microbial species, relative codon usage can be calculated. Codon usage tables are readily available, for example, at the "Codon Usage Database," available at www.kazusa.or.jp / codon / . Using knowledge of codon usage or codon preference in each organism, one skilled in the art can apply these frequencies to any given polypeptide sequence to create a nucleic acid fragment with a codon-optimized coding region that encodes that polypeptide but uses the codons optimal for that species. Codon-optimized coding regions can be designed by a variety of methods known to those skilled in the art.

[0073] A "cell culture" is an in vitro population of cells outside of a living organism. Cell cultures can be established from primary cells isolated from a cell bank or animal, or from secondary cells derived from one of these sources and immortalized for long-term in vitro culture.

[0074] The terms "transfection," "transduction," "transfecting," or "transducing" can be used interchangeably and are defined as the process of introducing nucleic acid molecules and / or proteins into cells. Nucleic acids may be introduced into cells using non-viral or viral-based methods. The nucleic acid molecule can be a sequence encoding an entire protein or a functional portion thereof. Typically, a nucleic acid vector contains elements necessary for protein expression (e.g., a promoter, a transcription initiation site, etc.). Non-viral transfection methods include any suitable method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, transfection via heat shock, magnetofection, and electroporation. In the methods described herein, for viral-based methods, any useful viral vector can be used. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In some embodiments, the nucleic acid molecule is introduced into cells using a retroviral vector, according to standard procedures well known in the art. The terms "transfection" or "transduction" also refer to the introduction of proteins into cells from the external environment. Typically, protein transduction or transfection relies on the binding of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

[0075] As used herein, the terms "specific binding" or "specifically binds" refer to two molecules forming a complex (e.g., a ribonucleoprotein and a transfection peptide) that is relatively stable under physiological conditions.

[0076] Methods for determining whether a ligand binds to another species (e.g., a protein or nucleic acid) and / or determining the affinity of the interaction between such a ligand and a species are known in the art. For example, binding of a ligand to a protein can be detected and / or quantified using a variety of techniques, including, but not limited to, Western blot, dot blot, surface plasmon resonance (e.g., BIAcore system, Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), isothermal titration calorimetry (ITC), or enzyme-linked immunosorbent assay (ELISA).

[0077] Immunoassays that can be used to analyze immunospecific binding and cross-reactivity of ligands include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIAs, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, and fluorescent immunoassays. Such assays are routine and well known in the art.

[0078] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or functional fragment thereof that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively.

[0079] The terms "antigen" and "epitope" refer interchangeably to that portion of a molecule (e.g., a polypeptide) that is specifically recognized by a component of the immune system, e.g., an antibody, T cell receptor, or other immune receptor, such as a receptor on a natural killer (NK) cell. As used herein, the term "antigen" includes antigenic epitopes and antigenic fragments thereof.

[0080] An exemplary immunoglobulin (antibody) structural unit can be a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms "variable heavy chain," "VH," or "VH" refer to the variable region of an immunoglobulin heavy chain, including Fv, scFv, dsFv, or Fab, and the terms "variable light chain," "VL," or "VL" refer to the variable region of an immunoglobulin light chain, including Fv, scFv, dsFv, or Fab.

[0081] Examples of antibody functional fragments include, but are not limited to, a complete antibody molecule, an antibody fragment (such as Fv, single-chain Fv (scFv), complementarity-determining region (CDR), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2', and any combination thereof), or any other functional portion of an immunoglobulin peptide that is capable of binding to a target antigen (see, for example, FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th ed. 2001). As will be appreciated by those skilled in the art, various antibody fragments can be obtained by a variety of methods, for example, digestion of intact antibodies with enzymes such as pepsin, or de novo synthesis. Antibody fragments are often synthesized de novo, either chemically or by using recombinant DNA techniques. Thus, the term "antibody," as used herein, includes antibody fragments produced by the modification of whole antibodies, antibody fragments synthesized de novo using recombinant DNA techniques (e.g., single-chain Fvs), or antibody fragments identified using phage display libraries (see, e.g., McCafferty et al., (1990) Nature 348:552). The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described, for example, in Kostelny et al. (1992) J. Immunol. 148:1547; Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al. (1993), PNAS.USA 90:6444, Gruber et al. (1994) J Immunol. 152:5368, Zhu et al. (1997) Protein Sci. 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026 and McCartney, et al. (1995) Protein Eng. 8:301.

[0082] As used herein, terms such as "disintegration," "self-immolation," "self-immolation mechanism," "disintegrating moiety," "disintegrating domain," and the like refer to a chemical group that undergoes an intramolecular reaction that allows the chemical group to undergo a chemical rearrangement and be released from the remainder of the compound to which it is attached. A "pH-sensitive" disintegrating domain refers to a chemical group that undergoes a disintegrating reaction at a lower pH range but does not substantially undergo a disintegrating reaction outside of that lower pH range (e.g., about pH 1-5, about pH 5-7, or about pH 7-10). In embodiments, the lower pH range is pH 1-3, pH 2-4, pH 3-5, pH 4-6, pH 5-7, pH 6-8, pH 7-9, or pH 8-10. In embodiments, the pH-sensitive disintegrating region comprises a cationic α-amino ester (oligo(α-amino ester)). In embodiments, the cationic component of the cationic α-amino ester is a positively charged nitrogen atom (e.g., a cationic amine). In embodiments, the cationic component of the cationic alpha amino ester is not a guanidinium group. In embodiments, the cationic component of the cationic alpha amino ester is not a piperidinium group.

[0083] Terms such as "cell-permeable complex" refer in their usual and customary sense to chemical complexes (e.g., complexes or compositions disclosed herein and embodiments thereof) that can permeate cells (biological cells, such as eukaryotic or prokaryotic cells). In embodiments, the cell-permeable complex comprises a nucleic acid ionically bound to a cationic amphiphilic polymer. In embodiments, the nucleic acid is substantially unable to permeate cells in the absence of the cationic amphiphilic polymer. Thus, in embodiments, the cationic amphiphilic polymer facilitates the transport of the nucleic acid into the cell. As used herein, terms such as "cationic charge-variable releasable transporter," "CART," and the like refer to the cell-permeable complexes disclosed herein. The CART compounds of the present invention can release the nucleic acid component within the cell through the action of a pH-sensitive disintegration domain within the cationic amphiphilic polymer component (which reacts in response to intracellular pH, thereby releasing the nucleic acid within the cell). In embodiments, the cationic amphiphilic polymer rapidly degrades within the cell (e.g., T1 / 2 of less than 6 hours at pH 7.4). In at least some embodiments, polyplex, complex, electrostatic complex, CART / mRNA complex, CART / oligonucleotide complex, and nanoparticle can be used interchangeably to refer to a cell-penetrating complex.

[0084] The term "amphiphilic polymer," as used herein, refers to a polymer that includes both hydrophilic and hydrophobic portions. In embodiments, the hydrophilic and hydrophobic portions are present in a 1:1 weight ratio. In embodiments, the hydrophilic and hydrophobic portions are present in a 1:2 weight ratio. In embodiments, the hydrophilic and hydrophobic portions are present in a 1:5 weight ratio. In embodiments, the hydrophilic and hydrophobic portions are present in a 2:1 weight ratio. In embodiments, the hydrophilic and hydrophobic portions are present in a 5:1 weight ratio. An amphiphilic polymer may be a diblock copolymer or a triblock copolymer. In embodiments, an amphiphilic polymer may include two hydrophilic portions (e.g., blocks) and one hydrophobic portion (e.g., block).

[0085] Terms such as "lipophilic polymer domain" (often referred to as "lipid block") refer to cationic amphiphilic polymer regions that are not hydrophilic (e.g., that are insoluble in water by themselves). In embodiments, the lipophilic polymer domain has low water solubility. For example, low water solubility refers to a solubility of the lipophilic polymer domain of about 0.0005 mg / mL to about 10 mg / mL dissolved in water.

[0086] The term "initiator" refers to a compound involved in the reaction to synthesize a cationic amphiphilic polymer, whose purpose is to initiate the polymerization reaction. Thus, the initiator is typically incorporated at the end of the polymer being synthesized. For example, to produce a cationic amphiphilic polymer, multiple molecules of one type (formula) of monomer or two or more types of monomers (e.g., two different types of monomers) can be reacted with the initiator. The initiator can be present at at least one end of the resulting polymer, but cannot constitute the repeat (or polymerized) unit(s) present in the polymer.

[0087] The term "disease" or "condition" refers to a state of survival or health of a subject that can be treated with the compounds, pharmaceutical compositions, or methods provided herein. The disease can be an autoimmune disease, inflammatory disease, cancer disease, infectious disease, metabolic disease, developmental disease, cardiovascular disease, liver disease, intestinal disease, endocrine disease, neurological disease, or other disease. In some examples, the disease is cancer (e.g., breast cancer, ovarian cancer, sarcoma, osteosarcoma, lung cancer, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, neuroblastoma).

[0088] The terms "infection" or "infectious disease" refer to a disease or condition that can be caused by an organism such as a bacterium, virus, fungus, or any other pathogenic microbial agent.

[0089] As used herein, the term "cancer" refers to any type of cancer, neoplasm, or malignant tumor found in mammals, including leukemia, lymphoma, melanoma, neuroendocrine tumors, carcinoma, and sarcoma. Exemplary cancers that may be treated with the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, and pancreatic cancer. cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, gliosarcoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., of the head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include thyroid cancer, cancer of the endocrine system, brain tumors, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma. tumor, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, uterine cancer, adrenocortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

[0090] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like, with respect to the activity and / or functionality of a molecule (e.g., a polynucleotide or protein), mean to negatively affect the activity or function of the molecule (e.g., decrease or reduce the activity or function of the molecule) compared to the activity or function of the protein in the absence of the inhibition. Thus, inhibition includes at least partially, partially or totally blocking a stimulus, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signal transduction, or enzymatic activity, or the amount of a protein or polynucleotide. Similarly, an "inhibitor" is a compound that inhibits a target biomolecule (i.e., a nucleic acid, peptide, carbohydrate, lipid, or any other molecule found in nature), for example, by binding to the target biomolecule or partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or downregulating the activity of the target biomolecule. In the context of prophylactic treatment of a disease, inhibition refers to a reduction in the disease or symptoms of the disease.

[0091] "Treatment," "treating," and "treat" are defined as acting on a disease, disorder, or condition with an agent to reduce or ameliorate the harmful or any other undesirable effects of the disease, disorder, or condition and / or its symptoms. "Treating" a condition or subject in need of treatment, or "treatment" of a condition or subject in need of treatment, refers to (1) taking steps to obtain a beneficial or desired result, including a clinical result such as a reduction in symptoms; (2) inhibiting the disease, e.g., arresting or reducing the onset of the disease or its clinical symptoms; (3) relieving the disease, e.g., causing regression of the disease or its clinical symptoms; or (4) delaying the disease. For example, beneficial or desired clinical results include, but are not limited to, the reduction and / or elimination of cancer cells and the prevention and / or reduction of cancer cell metastasis.

[0092] The terms "prevent," "preventing," or "prevention," in the context of disease, refer to the non-development of clinical symptoms of a disease in a subject who has not yet experienced or exhibited symptoms of the disease. In some instances, such prevention can occur in subjects who may be considered to have a predisposition to the disease, while in some other instances, the subject may not necessarily be considered to have a predisposition to the disease.

[0093] As used herein, "administering" refers to the physical introduction of a composition into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the compositions described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes, for example, by injection or infusion. The phrase "parenteral administration," as used herein, refers to a method of administration other than enteral administration or topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, intrasternal, and in vivo electroporation. Alternatively, the compositions described herein can be administered by a parenteral route, such as a topical, epidermal, or mucosal route, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. Also, administration can be, for example, once, multiple times, and / or over one or more extended periods of time.

[0094] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" are used interchangeably and refer to the spread of a proliferative disease or disorder, such as cancer, from one organ or another non-adjacent organ or body part. When cancer begins at a site of origin, such as the breast, that site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or site of origin gain the ability to penetrate and invade surrounding normal tissue in that local area and / or penetrate the walls of the lymphatic or vascular system circulating therethrough to reach other sites and tissues in the body. A clinically detectable secondary tumor formed from cancer cells of the primary tumor is referred to as a metastatic tumor or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor in the breast site is composed of abnormal lung cells, not abnormal breast cells. The secondary tumor in the breast is referred to as metastatic lung cancer. Therefore, the term metastatic cancer refers to the disease that the subject has or has had primary tumor, and has one or more secondary tumors.The term non-metastatic cancer or the subject with non-metastatic cancer refers to the disease that the subject has primary tumor, but does not have one or more secondary tumors.For example, metastatic lung cancer refers to the disease that the subject has primary lung tumor or has a history of primary lung tumor, and has one or more secondary tumors in one second location or multiple second locations, such as breast.

[0095] An "anti-cancer agent" is a therapeutic agent that has anti-cancer activity and can be used to treat or prevent cancer. Anti-cancer agents can be large or small molecules. Examples of anti-cancer agents include antibodies, small and large molecules, or combinations thereof. Examples of "anti-cancer activity" include, but are not limited to, reducing the number of cancer cells, reducing the size of the cancer, killing cancer cells, reducing and / or inhibiting metastasis, and reducing the growth and / or proliferation of cancer cells.

[0096] The term "associated with" or "associated with," in the context of a substance or the activity or function of a substance associated with a disease, means that the disease can be caused (in whole or in part) by the substance or the activity or function of the substance, or that the symptoms of the disease can be caused (in whole or in part) by the substance or the activity or function of the substance. When the term is used in the context of a symptom, e.g., a symptom associated with a disease or condition, it means that the symptom can be indicative of the disease or condition present in a subject exhibiting the symptom.

[0097] The terms "subject," "individual," "host," or "subject in need thereof" refer to an organism that is suffering from a disease or condition or that may suffer from a disease or condition in the future. The term "patient" refers to an organism that already suffers from a disease or condition, e.g., a patient that has been diagnosed with a disease or condition or has one or more symptoms associated with a disease or condition. Non-limiting examples include humans, other mammals, bovine, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In some embodiments, the patient is a human.

[0098] The term "vaccine" refers to a composition capable of providing active adaptive immunity and / or a therapeutic effect (e.g., treatment) against a particular disease or pathogen. Vaccines typically contain one or more agents capable of inducing an immune response in a subject against a pathogen or disease, i.e., a target pathogen or disease. The immunogenic agent stimulates the body's immune system to recognize the agent as a threat or indicator of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any pathogen upon subsequent exposure. Vaccines can be prophylactic (e.g., preventing or ameliorating the effects of future infection, either natural or pathogenic, or the anticipated development of cancer in a predisposed subject) or therapeutic (e.g., treating cancer in a subject diagnosed with cancer). Administration of a vaccine is referred to as vaccination. In some examples, the vaccine composition can provide a subject with a nucleic acid, e.g., mRNA encoding an antigenic molecule (e.g., a peptide). In a subject, the nucleic acid delivered by the vaccine composition can be expressed into an antigen molecule, allowing the subject to acquire immunity against the antigen molecule. In the context of vaccination against infectious diseases, the vaccine composition can provide mRNA encoding an antigen molecule associated with a specific pathogen, for example, one or more peptides known to be expressed in the pathogen (e.g., pathogenic bacteria or pathogenic viruses). In the context of cancer vaccines, the vaccine composition can provide mRNA encoding a specific peptide associated with cancer, for example, a peptide that is expressed substantially only in cancer cells or is more highly expressed in cancer cells than normal cells. After vaccination with the cancer vaccine composition, the subject will have immunity against the cancer-associated peptide and can specifically kill cancer cells.

[0099] As used herein, the term "immune response" includes, but is not limited to, an "adaptive immune response" (also known as an "acquired immune response"), which elicits an initial response to a specific pathogen or type of cell that is targeted by the immune response, followed by an enhanced response against the target upon subsequent encounter. Induction of immune memory can provide the basis for vaccination.

[0100] The terms "immunogenic" or "antigenic" refer to a compound or composition that, when administered to an immunocompetent subject, induces an immune response, such as a cytotoxic T lymphocyte (CTL) response, a B cell response (e.g., the production of antibodies that specifically bind to an epitope), an NK cell response, or any combination thereof. Thus, an immunogenic or antigenic composition is a composition capable of eliciting an immune response in an immunocompetent subject. For example, an immunogenic or antigenic composition can include one or more immunogenic epitopes associated with a pathogen or specific cell type that is the target of the immune response. In addition, an immunogenic composition can include an isolated nucleic acid construct (DNA or RNA) encoding one or more immunogenic epitopes of an antigenic polypeptide that can be used to express the epitope(s) (and thus be used to elicit an immune response against the polypeptide or related polypeptides associated with the target pathogen or cell type).

[0101] According to the methods provided herein, a subject can be administered an effective amount of one or more of the agents, compositions, or complexes (all of which are used interchangeably herein) (e.g., cell-penetrating complexes or vaccine compositions) provided herein. The terms "effective amount" and "effective dose" are used interchangeably. The term "effective amount" is defined as any amount necessary to produce a desired effect (e.g., transfecting a nucleic acid into a cell and causing the transfected nucleic acid to exhibit its intended outcome). One of ordinary skill in the art can experimentally determine the effective amount and schedule for administering the agent. The dosage range is large enough to achieve the desired effect, such as transfection of a nucleic acid, modulation of gene expression, gene editing, induction of stem cells, induction of an immune response, etc. The dosage should not be so large as to cause significant adverse side effects (e.g., undesirable cross-reactions, anaphylactic reactions, etc.). Generally, dosages can vary depending on age, condition, sex, type of disease, severity of disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. Dosage can be adjusted by the individual physician if any contraindications exist. Dosage can vary, and one or more doses can be administered daily for one or several days. Guidance can be found in the literature regarding appropriate dosages for a given class of pharmaceuticals. For example, an effective amount can result in at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least a 100% increase or decrease in a given parameter. Efficacy can also be expressed as a "-fold" increase or decrease. For example, a therapeutically effective amount can result in at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or greater increase or decrease in an effect compared to a control.The exact dosage and formulation can depend on the therapeutic purpose, and can be ascertained by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003); and Pickar, Dosage Calculations (1999)).

[0102] The term "killing," in the context of cancer therapy, is intended to include any type of manipulation that results in the death of at least a portion of the cancer cells or population of cancer cells.

[0103] cell permeable complex In a first aspect, a cell-penetrating complex is provided, comprising a nucleic acid non-covalently bound to a cationic amphiphilic polymer, wherein the cationic amphiphilic polymer comprises a pH-sensitive disintegration domain. In embodiments, one or more counterions (e.g., anions) may be present as countercharges to the positive charges in the cationic amphiphilic polymer. In embodiments, the nucleic acid is non-covalently bound to the cationic amphiphilic polymer. In embodiments, the nucleic acid is ionically bound to the cationic amphiphilic polymer. In embodiments, the cell-penetrating complex comprises a plurality of, optionally different, nucleic acids (e.g., 1 to 10 additional nucleic acids, 1 to 5 additional nucleic acids, 1 to 5 additional nucleic acids, 2 additional nucleic acids, or 1 additional nucleic acid). In embodiments, the nucleic acid is RNA. In embodiments, the nucleic acid is mRNA.

[0104] In embodiments, the ratio of the number of cations in the cationic amphiphilic polymer molecule to the number of anions in the nucleic acid molecule present in the cell-penetrating complex can be about 1:1, about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, or greater, or any range therebetween. In another embodiment, the ratio of the number of anions on the nucleic acid molecule to the number of cations on the cationic amphiphilic polymer molecule present in the cell-penetrating complex can be about 1:1, about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, or greater, or any range therebetween. In some preferred embodiments, this ratio is about 10 cationic charges on the amphiphilic polymer molecule for every 1 negative charge on the nucleic acid. Another embodiment may have 5 cationic charges on the amphiphilic polymer molecule for every 1 negative charge on the nucleic acid, or 20 cationic charges on the amphiphilic polymer molecule for every 1 negative charge on the nucleic acid.

[0105] In embodiments, the ratio of the number of nucleic acid molecules to the number of cationic amphiphilic polymer molecules present in the cell-penetrating complex can be about 1:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, or greater, or any range therebetween. In another embodiment, the ratio of the number of cations in the cationic amphiphilic polymer molecules to the number of nucleic acid molecules present in the cell-penetrating complex can be about 1:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, about 10:1, or greater, or any range therebetween.

[0106] In embodiments, the cationic amphiphilic polymer may be a cationic charge-variable release transporter (CART), which may comprise oligomeric chains containing a range of cationic sequences that exhibit a pH-sensitive change in charge from cationic to neutral or from cationic to anionic.

[0107] In embodiments, the cationic amphiphilic polymer has a pH-sensitive disintegration domain and a lipophilic polymer domain. In embodiments, the lipophilic polymer domain may facilitate cell penetration, cellular delivery, and / or transport across a cell membrane. In embodiments, the lipophilic polymer domain may be substantially water-insoluble (e.g., soluble in water at about 0.0005 mg / mL to less than about 10 mg / mL). In embodiments, the lipophilic polymer domain may facilitate aggregation of the cationic amphiphilic polymer into nanoparticles. In embodiments, such nanoparticles may have an average longest dimension of about 50 nm to about 500 nm. In embodiments, the lipophilic polymer domain may facilitate entry into an endosome, disintegration, and subsequent fusion of the remainder of the cationic amphiphilic polymer with the endosome. In embodiments, the cell-penetrating complexes of the present disclosure protect nucleic acid cargo from degradation. Terms such as "nucleic acid cargo" refer in their ordinary and customary sense to a species desired for transport into cells by the cell-penetrating complexes and embodiments disclosed herein.

[0108] In embodiments, the cationic amphiphilic polymer has the formula (I): H-L1-[(LP1)-(IM)-(LP2)]-L2-H(I), wherein L1 and L2 are independently a bond, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—, —NHC(O)—, —S(O)—, —S(O)NH—, —NHC(O)NH—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted aryl ... or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; LP1 and LP2 are independently a bond or a lipophilic polymer domain, at least one of LP1 or LP2 is a lipophilic polymer domain; IM is a pH-sensitive disintegratable domain; z1, z3, and z4 are independently integers of 0 to 100, at least one of z1 or z3 is not 0, and z2 is an integer of 2 to 100.

[0109] In embodiments, L1 is substituted or unsubstituted C1-C3 alkylene. In embodiments, L1 is substituted or unsubstituted methylene. In embodiments, L1 is substituted or unsubstituted C1-C6 alkylene or substituted or unsubstituted 2- to 6-membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted C1-C3 alkylene or substituted or unsubstituted 2- to 3-membered heteroalkylene.

[0110] In embodiments, L1 is substituted or unsubstituted alkylene (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkylene (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkylene (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted arylene (e.g., C6-C10 or phenylene), or substituted or unsubstituted heteroarylene (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, L is a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkylene or unsubstituted alkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkylene or unsubstituted heteroalkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkylene or unsubstituted cycloalkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkylene or unsubstituted heterocycloalkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) arylene or unsubstituted arylene, or a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroarylene or unsubstituted heteroarylene. In embodiments, L is an unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, or unsubstituted heteroarylene. In embodiments, L1 is unsubstituted alkylene (e.g., C1-C6 alkylene). In embodiments, L1 is a bond.

[0111] In embodiments, L2 is substituted or unsubstituted C1-C3 alkylene. In embodiments, L2 is substituted or unsubstituted methylene. In embodiments, L2 is substituted or unsubstituted C1-C6 alkylene or substituted or unsubstituted 2- to 6-membered heteroalkylene. In embodiments, L2 is substituted or unsubstituted C1-C3 alkylene or substituted or unsubstituted 2- to 3-membered heteroalkylene.

[0112] In embodiments, L2 is substituted or unsubstituted alkylene (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkylene (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkylene (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted arylene (e.g., C6-C10 or phenylene), or substituted or unsubstituted heteroarylene (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, L2 is a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkylene or unsubstituted alkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkylene or unsubstituted heteroalkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkylene or unsubstituted cycloalkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkylene or unsubstituted heterocycloalkylene, a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) arylene or unsubstituted arylene, or a substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroarylene or unsubstituted heteroarylene. In embodiments, L2 is an unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, or unsubstituted heteroarylene. In embodiments, L2 is unsubstituted alkylene (e.g., C1-C6 alkylene). In embodiments, L2 is a bond.

[0113] In an embodiment, z2 is an integer from 2 to 90 (e.g., 5 to 90, 10 to 90, or 20 to 90), 2 to 80 (e.g., 5 to 80, 10 to 80, or 20 to 80), 2 to 70 (e.g., 5 to 70, 10 to 70, or 20 to 70), 2 to 50 (e.g., 5 to 50, 10 to 50, or 20 to 50), or 2 to 25. In an embodiment, z1, z3, and z4 are independently an integer from 0 to 90 (e.g., 5 to 90, 10 to 90, or 20 to 90), 0 to 80 (e.g., 5 to 80, 10 to 80, or 20 to 80), 0 to 70 (e.g., 5 to 70, 10 to 70, or 20 to 70), 0 to 50 (e.g., 5 to 50, 10 to 50, or 20 to 50), or 2 to 25. In an embodiment, z1, z3, and z4 are independently an integer from 2 to 90 (e.g., 5 to 90, 10 to 90, or 20 to 90), 2 to 80 (e.g., 5 to 80, 10 to 80, or 20 to 80), 2 to 70 (e.g., 5 to 70, 10 to 70, or 20 to 70), 2 to 50 (e.g., 5 to 50, 10 to 50, or 20 to 50), or 2 to 25.

[0114] In embodiments of the cell-penetrating complex, the pH-sensitive disruptive domain comprises a first nucleophilic moiety and a first electrophilic moiety, where the first nucleophilic moiety is reactive with the first electrophilic moiety over a pH range and is substantially unreactive with the electrophilic moiety outside of that pH range (e.g., about pH 1-5, about pH 5-7, or about pH 7-10). In embodiments, the pH ranges at which the first nucleophilic moiety is most reactive with the first electrophilic moiety are pH 1-3, pH 2-4, pH 3-5, pH 4-6, pH 5-7, pH 6-8, pH 7-9, or pH 8-10. The term "nucleophilic moiety" is used according to its simple and clear meaning in chemistry to refer to a moiety (e.g., a functional group) that can donate electrons.

[0115] In embodiments, the pH range in which the reactivity of the first nucleophilic moiety with the first electrophilic moiety is greatest is pH 1-3. In embodiments, the pH range in which the reactivity of the first nucleophilic moiety with the first electrophilic moiety is greatest is pH 2-4. In embodiments, the pH range in which the reactivity of the first nucleophilic moiety with the first electrophilic moiety is greatest is pH 3-5. In embodiments, the pH range in which the reactivity of the first nucleophilic moiety with the first electrophilic moiety is greatest is pH 4-6. In embodiments, the pH range in which the reactivity of the first nucleophilic moiety with the first electrophilic moiety is greatest is pH 5-7. In embodiments, the pH range in which the reactivity of the first nucleophilic moiety with the first electrophilic moiety is greatest is pH 6-8. In embodiments, the pH range in which the reactivity of the first nucleophilic moiety with the first electrophilic moiety is greatest is pH 7-9. In embodiments, the pH range in which the first nucleophilic moiety and the first electrophilic moiety are most reactive is pH 8-10. In embodiments, the pH is 1. In embodiments, the pH is 2. In embodiments, the pH is 3. In embodiments, the pH is 4. In embodiments, the pH is 5. In embodiments, the pH is 6. In embodiments, the pH is 7. In embodiments, the pH is 8. In embodiments, the pH is 9. In embodiments, the pH is 10. In embodiments, the pH is about 1. In embodiments, the pH is about 2. In embodiments, the pH is about 3. In embodiments, the pH is about 4. In embodiments, the pH is about 5. In embodiments, the pH is about 6. In embodiments, the pH is about 7. In embodiments, the pH is about 8. In embodiments, the pH is about 9. In embodiments, the pH is about 10.

[0116] In embodiments, the first nucleophilic moiety substantially protonates at low pH (e.g., pH about 1 to about 5). In embodiments, the first nucleophilic moiety substantially protonates in the pH range of 5 to 7. In embodiments, the first nucleophilic moiety is cationic. In embodiments, the first nucleophilic moiety includes a cationic nitrogen (e.g., a cationic amine).

[0117] In embodiments, the first nucleophilic moiety can be attached to a pH-labile protecting group. Terms such as "pH-labile protecting group" refer in their ordinary and customary sense to a chemical moiety capable of protecting another functional group attached to it, where the protecting group can be cleaved or otherwise inactivated as a protecting group under certain pH conditions (e.g., conditions that lower the pH). In one embodiment, the pH-labile protecting group is -CO2-t-Bu, a group that is removed under acidic conditions (e.g., below pH 7). Additional nucleophilic protecting groups can also include protecting groups that are cleaved by light, heat, nucleophiles, and bases.

[0118] In embodiments of the cell-penetrating complex disclosed above, the pH-sensitive disruption domain may have the structure of formula (II): [ka] In the formula, n is an integer of 2 or greater, n1 is an integer of 0 to 50, Z is a nucleophilic moiety, X1 is a bond, -C(R5)(R6)-, -C(R5)(R6)-C(R7)(R8)-, -OC(R5)(R6)-, or -OC(R5)(R6)-C(R7)(R8)-, X2 is -O- or -S-, and R1, R2, R3, R4, R5, R6, R7, and R8 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In an embodiment, n is an integer ranging from 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10. In an embodiment, n1 is an integer ranging from 0 to 25, 0 to 10, or 0 to 5. In an embodiment, n1 is 0, 1, 2, 3, 4, or 5. In an embodiment, n1 is 1 or 2.

[0119] In embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R, R, R, R, R, R, R, R, and R are independently substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R, R, R, R, R, R, R, R, and R are independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are independently hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl). In embodiments, R1, R2, R3, R4, R5, R6, R7, and R8 are hydrogen.

[0120] In the cell-penetrating complex embodiments disclosed above, the pH-sensitive disruption domain is [ka] wherein R1, R2, R3, R4, X1, Z, and X2 are as defined herein, and n1 and n2 are integers greater than or equal to 2.

[0121] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure of formula (III): [ka] wherein n is an integer greater than or equal to 2, Z is a nucleophilic moiety, X1 is a bond, -C(R5)(R6)-, -C(R5)(R6)-C(R7)(R8)-, -OC(R5)(R6)-, or -OC(R5)(R6)-C(R7)(R8)-, X2 is -O- or -S-, and R1.1, R1.2, R2.1, R2.2, R3, R4, R5, R6, R7, and R8 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In an embodiment, n is an integer in the range of 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10. In an embodiment, n is an integer in the range of 2 to 100 or 2 to 50.

[0122] In embodiments, R1.1, R1.2, R2.1, R2.2, R3, R4, R5, R6, R7, and R8 are independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R1.1, R1.2, R2.1, R2.2, R3, R4, R5, R6, R7, and R8 are independently substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R1.1, R1.2, R2.1, R2.2, R3, R4, R5, R6, R7, and R8 are independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R1.1, R1.2, R2.1, R2.2, R3, R4, R5, R6, R7, and R8 are independently hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl).In an embodiment, R1.1, R1.2, R2.1, R2.2, R3, R4, R5, R6, R7 and R8 are hydrogen.

[0123] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure of formula (IV): [ka] In the formula, n is an integer of 2 or greater. In an embodiment, n is an integer in the range of 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10. In an embodiment, n is an integer in the range of 2 to 100 or 2 to 50. In an embodiment, n is 2 to 15.

[0124] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure of formula (IV): [ka] In the formula, n is an integer of 2 or greater. In an embodiment, n is an integer in the range of 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10. In an embodiment, n is an integer in the range of 2 to 100 or 2 to 50.

[0125] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure of formula (V): [ka] In the formula, n is an integer of 2 or greater. In an embodiment, n is an integer in the range of 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10. In an embodiment, n is an integer in the range of 2 to 100 or 2 to 50.

[0126] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure of formula (Va): [ka] In the formula, n is an integer of 2 or greater. In an embodiment, n is an integer in the range of 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10. In an embodiment, n is an integer in the range of 2 to 100 or 2 to 50.

[0127] In embodiments, the pH sensitive disruption domain has the structure: [ka] wherein X6 is -O-, -NH-, -CONH-, -COO-, -OCO-, -NHCO-, R20 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and R21 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In embodiments, R20 is an oligoglycol moiety.

[0128] In embodiments, R20 is substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R20 is substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R20 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R20 is hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl). In embodiments, R20 is hydrogen.

[0129] In embodiments, R21 is substituted or substituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R21 is substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R21 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R21 is hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl). In embodiments, R21 is hydrogen.

[0130] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure: [ka] In the formula, R24, R25, and R26 are hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and n3 is an integer of 0 to 50.

[0131] In embodiments, R24, R25, and R26 are independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R24, R25, and R26 are independently substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R1.1, R24, R25, and R26 are independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R24, R25, and R26 are independently hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl). In embodiments, R24, R25, and R26 are independently hydrogen.

[0132] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure of formula (VI): [ka] In the formula, n is an integer of 2 or more, n1 is an integer of 0 to 50, X1 is a bond, -O-, -NR5-, -C(R5)(R6)-, or -C(R5)(R6)-C(R7)(R8)-, X2 is a bond, -O-, -C(R9)(R10)-, or -C(R9)(R10)-C(R11)(R12)-, and X4 is a bond, -NR16-, -O-, -C(R16)(R17)-, or -C(R16)(R17)-C(R18) )(R19)-, wherein X5 is a nucleophilic moiety and R1, R2, R5, R6, R7, R8, R9, R10, R11, R12, R15, R16, R17, R18 and R19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0133] In embodiments, R1, R2, R5, R6, R7, R8, R9, R10, R11, R12, R15, R16, R17, R18, and R19 are independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., cycloalkyl, cyclohexyl ... cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R, R, R, R, R, R, R, R, R, R, R, R, R, R, and R are independently substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R1, R2, R5, R6, R7, R8, R9, R10, R11, R12, R15, R16, R17, R18 and R19 are independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl.In embodiments, R1, R2, R5, R6, R7, R8, R9, R10, R11, R12, R15, R16, R17, R18, and R19 are independently hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl). In embodiments, R1, R2, R5, R6, R7, R8, R9, R10, R11, R12, R15, R16, R17, R18, and R19 are hydrogen.

[0134] In embodiments, Z is a nucleophilic moiety. In embodiments, Z is -S-, -OR13-, -S+R13-, -NR13-, or -N+(R13)(H)-, where R13 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In embodiments, Z is -S-. In embodiments, Z is -S+R13-. In embodiments, Z is -NR13-. In embodiments, Z is -N+(R13)(H)-. In embodiments, Z is -S+H-. In embodiments, Z is -NH-. In embodiments, Z is -N+H2-. In embodiments, Z is -OH-.

[0135] In embodiments, R13 is independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R is independently substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R is independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R is independently hydrogen or unsubstituted alkyl (e.g., C-C alkyl). In embodiments, R is hydrogen.

[0136] In an embodiment, Z is [ka] wherein X3 is -C(R15)- or -N-; X4 is a bond, -C(O)-, -P(O)(OR16)2-, -S(O)(OR17)2-, -C(R16)(R17)-, or -C(R16)(R17)-C(R18)(R19)-; X5 is a nucleophilic moiety; and R13, R14, R15, R16, R17, R18, and R19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In embodiments, X3 is -CH.

[0137] In embodiments, R13, R14, R15, R16, R17, R18, and R19 are independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R, R, R, R, R, R, R, and R are independently substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, a size-limiting substituent, or a lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R13, R14, R15, R16, R17, R18, and R19 are independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R13, R14, R15, R16, R17, R18, and R19 are independently hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl).

[0138] In embodiments, X5 is -N+(R13)(H), where R13 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0139] With respect to the cell-penetrating complexes and embodiments thereof disclosed herein, in embodiments, the lipophilic polymer domain has the formula: [ka] wherein n2 is an integer from 1 to 100, and R20 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0140] In embodiments, R20 is substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R20 is substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R20 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R20 is hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl).

[0141] In embodiments, R20 is unsubstituted C1-C30 alkyl. In embodiments, R20 is unsubstituted C1-C20 alkyl. In embodiments, R20 is unsubstituted C8-C30 alkyl. In embodiments, R20 is unsubstituted C8-C20 alkyl. In embodiments, R20 is unsubstituted C9-C20 alkyl. In embodiments, R20 is unsubstituted C9-C18 alkyl. In embodiments, R20 is unsubstituted C18 alkyl. In embodiments, R20 is unsubstituted C17 alkyl. In embodiments, R20 is unsubstituted C16 alkyl. In embodiments, R20 is unsubstituted C15 alkyl. In embodiments, R20 is unsubstituted C14 alkyl. In embodiments, R20 is unsubstituted C13 alkyl. In embodiments, R20 is unsubstituted C12 alkyl. In embodiments, R20 is an unsubstituted C11 alkyl. In embodiments, R20 is an unsubstituted C10 alkyl. In embodiments, R20 is an unsubstituted C9 alkyl. In embodiments, R20 is an unsubstituted C8 alkyl. In embodiments, R20 is an unsubstituted C7 alkyl. In embodiments, R20 is an unsubstituted C6 alkyl. In embodiments, R20 is an unsubstituted C5 alkyl. In embodiments, R20 is an unsubstituted C4 alkyl. In embodiments, R20 is an unsubstituted C3 alkyl. In embodiments, R20 is an unsubstituted C2 alkyl.

[0142] In embodiments, R20 is an unsubstituted C1-C30 alkenyl. In embodiments, R20 is an unsubstituted C1-C20 alkenyl. In embodiments, R20 is an unsubstituted C8-C30 alkenyl. In embodiments, R20 is an unsubstituted C8-C20 alkenyl. In embodiments, R20 is an unsubstituted C9-C20 alkenyl. In embodiments, R20 is an unsubstituted C9-C18 alkenyl. In embodiments, R20 is an unsubstituted C18 alkenyl. In embodiments, R20 is an unsubstituted C17 alkenyl. In embodiments, R20 is an unsubstituted C16 alkenyl. In embodiments, R20 is an unsubstituted C15 alkenyl. In embodiments, R20 is an unsubstituted C14 alkenyl. In embodiments, R20 is an unsubstituted C13 alkenyl. In embodiments, R20 is an unsubstituted C12 alkenyl. In embodiments, R20 is an unsubstituted C11 alkenyl. In embodiments, R20 is an unsubstituted C10 alkenyl. In embodiments, R20 is an unsubstituted C9 alkenyl. In embodiments, R20 is an unsubstituted C8 alkenyl. In embodiments, R20 is an unsubstituted C7 alkenyl. In embodiments, R20 is an unsubstituted C6 alkenyl. In embodiments, R20 is an unsubstituted C5 alkenyl. In embodiments, R20 is an unsubstituted C4 alkenyl. In embodiments, R20 is an unsubstituted C3 alkenyl. In embodiments, R20 is an unsubstituted C2 alkenyl.

[0143] In embodiments, R20 is a stearyl moiety (e.g., unsubstituted C18 alkyl). In embodiments, R20 is an oleyl moiety (e.g., unsubstituted C18 alkenyl). In embodiments, R20 is a linoleyl moiety (e.g., unsubstituted C18 alkenyl). In embodiments, R20 is a dodecyl moiety (e.g., unsubstituted C12 alkyl). In embodiments, R20 is a nonenyl moiety (e.g., unsubstituted C9 alkenyl). In embodiments, R20 is [ka] is.

[0144] In embodiments, the lipophilic polymer domain is a compound of formula (Ia): [ka] wherein X6 can be -O-, -NH-, -CO2-, -CONH-, -OC-, or -NHCO-, R20 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, R21 is hydrogen, substituted or unsubstituted alkyl, and n is as defined herein. In embodiments, R20 is an oligoglycol moiety.

[0145] In embodiments, R20 is substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R20 is substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R20 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R20 is hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl).

[0146] In embodiments, R21 is substituted or substituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R21 is substituted (e.g., substituted with a substituent, size-limited substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limited substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limited substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limited substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limited substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limited substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R21 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R21 is hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl).

[0147] In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure: [ka] In the formula, n4 is an integer of 0 to 50. In an embodiment, n4 is an integer of 0 to 10. In an embodiment, n4 is an integer of 1 to 15.

[0148] In embodiments of the cell-penetrating complexes disclosed herein, the lipophilic polymer has the structure: [ka] wherein X7 is -O-, -NH-, -CO2-, -CONH-, -OC-, or -NHCO-; R22 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and R23 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In embodiments, R22 is an oligoglycol moiety.

[0149] In embodiments, R22 is substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R22 is substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R22 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R22 is hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl).

[0150] In embodiments, the lipophilic polymer domain may be a compound of formula (Ib): [ka] wherein R100 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, R1, R2, R3, R4 are hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and n100 is an integer equal to or greater than 1 and is as defined herein.

[0151] In embodiments, R1, R2, R3, and R4 are independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R, R, R, and R are independently substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R, R, R, and R are independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R1, R2, R3, and R4 are independently hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl). In embodiments, R1, R2, R3, and R4 are hydrogen.

[0152] In embodiments, the lipophilic polymer domain may be a compound of formula (Ic): [ka] wherein R200 is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, and n200 is an integer equal to or greater than 2. In embodiments, R200 is an oligoglycol moiety. In embodiments, R200 is an amine-terminated oligoglycol moiety. The term "oligoglycol moiety" refers to [ka] The term "amine-terminated oligoglycol moiety" refers to [ka] In the formula, n200 is an integer of 2 or more.

[0153] In embodiments, R200 is substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4 or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6 or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered or 5-6 membered). In embodiments, R200 is substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R200 is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R200 is hydrogen or unsubstituted alkyl (e.g., C1-C6 alkyl). In embodiments, R200 is hydrogen.

[0154] In embodiments, the lipophilic polymer domain may be a compound of formula (Id): [ka] wherein R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R and R are independently hydrogen or substituted or unsubstituted alkyl; and n is as defined herein. In embodiments, R is an oligoglycol moiety. In embodiments, R is an amine-terminated oligoglycol moiety. In embodiments, R, R, and R are hydrogen.

[0155] In embodiments, R300, R301, and R302 are independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R, R, and R are independently substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R, R, and R are independently unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In embodiments, R300, R301, and R302 are independently hydrogen or unsubstituted alkyl (eg, C1-C6 alkyl).

[0156] In one aspect, the present disclosure provides a cell-penetrating complex having a nucleic acid non-covalently bound to a cationic amphiphilic polymer. The cationic amphiphilic polymer can have a pH-sensitive disintegration domain. In some embodiments, the cationic amphiphilic polymer has a pH-sensitive disintegration domain and a lipophilic polymer domain. In some embodiments, the cell-penetrating complex has a cationic amphiphilic polymer of formula (VII): R1A-[L1-[(LP1)z1-(IM)z2-(LP2)z3]z4-L2-R2A]z5 During the ceremony, R1A is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH , -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R2A is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH , -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; L1 and L2 are independently a bond, -C(O)O-, -O-, -S-, -NH-, -C(O)NH-, -NHC(O)-, -S(O)2-, -S(O)NH-, -NHC(O)NH-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; LP1 and LP2 are independently a bond or a lipophilic polymer domain, and at least one of LP1 or LP2 is a lipophilic polymer domain; IM is a pH-sensitive disintegration domain, z5 is an integer from 1 to 10, z1, z3 and z4 are independently an integer of 0 to 100, provided that at least one of z1 or z3 is not 0, and z2 is an integer of 2 to 100.

[0157] In embodiments, R 1A is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0158] In embodiments, R1A is independently substituted or unsubstituted alkyl (e.g., C1-C8, C1-C6, C1-C4, or C1-C2), substituted or unsubstituted heteroalkyl (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered), substituted or unsubstituted aryl (e.g., C6-C10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10 membered, 5-9 membered, or 5-6 membered). In embodiments, R is substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) alkyl or unsubstituted alkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroalkyl or unsubstituted heteroalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) cycloalkyl or unsubstituted cycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heterocycloalkyl or unsubstituted heterocycloalkyl, substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) aryl or unsubstituted aryl, or substituted (e.g., substituted with a substituent, size-limiting substituent, or lower substituent) heteroaryl or unsubstituted heteroaryl. In embodiments, R is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

[0159] In embodiments, R1A is hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 2- to 6-membered heteroalkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 3- to 6-membered heterocycloalkyl, substituted or unsubstituted phenyl, or substituted or unsubstituted 5- to 6-membered heteroaryl.

[0160] In embodiments, R1A is substituted or unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R1A is substituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R1A is unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl). In embodiments, R1A is substituted or unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R1A is substituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R1A is unsubstituted heteroalkyl (e.g., 2- to 8-membered heteroalkyl, 2- to 6-membered heteroalkyl, or 2- to 4-membered heteroalkyl). In embodiments, R1A is substituted or unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R1A is substituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R1A is unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl). In embodiments, R1A is substituted or unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl). In embodiments, R1A is substituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl). In embodiments, R1A is unsubstituted heterocycloalkyl (e.g., 3- to 8-membered heterocycloalkyl, 3- to 6-membered heterocycloalkyl, or 5- to 6-membered heterocycloalkyl). In embodiments, R1A is substituted or unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl). In embodiments, R1A is substituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl).In embodiments, R1A is unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl). In embodiments, R1A is substituted or unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, R1A is substituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl). In embodiments, R1A is unsubstituted heteroaryl (e.g., 5- to 10-membered heteroaryl, 5- to 9-membered heteroaryl, or 5- to 6-membered heteroaryl).

[0161] In embodiments, R is substituted or unsubstituted aryl. In some other embodiments, R is substituted or unsubstituted phenyl. In yet some other embodiments, R is substituted or unsubstituted aryl. In yet some other embodiments, R is substituted or unsubstituted phenyl or naphthalenyl.

[0162] In some embodiments, the cell-penetrating complex can have a cationic amphiphilic polymer having the following formula (VIII): [ka] During the ceremony, Ring A is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; The CART can have the formula -L1-[(LP1)z1-(IM)z2-(LP2)z3]z4-L2-R2A; During the ceremony, R2A is hydrogen, halogen, -CCl3, -CBr3, -CF3, -CI3, CHCl2, -CHBr2, -CHF2, -CHI2, -CH2Cl, -CH2Br, -CH2F, -CH2I, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O) H, -NHC(O)OH, -NHOH, -OCCl3, -OCF3, -OCBr3, -OCI3, -OCHCl2, -OCHBr2, -OCHI2, -OCHF2, -OCH2Cl, -OCH2Br, -OCH2I, -OCH2F, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl, wherein L1 and L2 independently represent a bond, -C(O)O-, -O-, -S-, -NH-, -C(O)NH-, -NHC(O)-, -S(O)2-, -S(O)NH-, -NHC(O)NH-, a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, or a substituted or unsubstituted arylene; or a substituted or unsubstituted heteroarylene, LP1 and LP2 are independently a bond or a lipophilic polymer domain, at least one of LP1 or LP2 is a lipophilic polymer domain, IM is a pH-sensitive disintegratable domain, z5 is an integer of 1 to 10, z1, z3, and z4 are independently integers of 0 to 100, at least one of z1 or z3 is not 0, and z2 is an integer of 2 to 100.

[0163] In some embodiments, in the above formula (VIII), ring A is substituted or unsubstituted aryl. In some other embodiments, ring A is substituted or unsubstituted phenyl. In still some other embodiments, ring A is substituted or unsubstituted aryl. In still some other embodiments, ring A is substituted or unsubstituted phenyl or naphthalenyl.

[0164] In embodiments, ring A is unsubstituted aryl (i.e., unsubstituted except for the CART portion). In embodiments, ring A is unsubstituted phenyl (i.e., unsubstituted except for the CART portion). In embodiments, ring A is unsubstituted phenyl or naphthalenyl (i.e., unsubstituted except for the CART portion). In embodiments, ring A is substituted aryl (i.e., substituted in addition to the CART portion). In embodiments, ring A is substituted phenyl (i.e., substituted in addition to the CART portion). In embodiments, ring A is substituted phenyl or naphthalenyl (i.e., substituted in addition to the CART portion).

[0165] In embodiments, the cell-permeable complex is a detectable agent (eg, a fluorophore).

[0166] In embodiments, R1A is aryl substituted with a methoxy linker. In embodiments, R1A is aryl substituted with a linker (e.g., -CH2-O-). A non-limiting example where R1A is aryl substituted with a methoxy linker has the formula: [ka]

[0167] In some embodiments, the cationic amphiphilic polymer has formula (IX): [ka]

[0168] In some embodiments, the cationic amphiphilic polymer has the following formula (X): [ka]

[0169] In some embodiments, the cationic amphiphilic polymer can have the following formula (XI): [ka] wherein CART1, CART2, and CART3 are independently CART moieties as defined in formula (VIII) (e.g., -L1-[(LP1)z1-(IM)z2-(LP2)z3]z4-L2-R2A). In embodiments, each CART moiety is optionally different.

[0170] In embodiments, the cationic amphiphilic polymer is [ka] It has the formula:

[0171] In embodiments, the cationic amphiphilic polymer is [ka] In embodiments, the cationic amphiphilic polymer has the formula: [ka] In embodiments, the cationic amphiphilic polymer has the formula: [ka] In embodiments, the cationic amphiphilic polymer has the formula: [ka] In embodiments, the cationic amphiphilic polymer has the formula: [ka]

[0172] In embodiments, ring A is substituted with a detectable agent via a linker (e.g., a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene).

[0173] In some embodiments, the cell-penetrating complex comprises a cationic amphiphilic polymer having any of the formulas above, wherein L is -CH2-O-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. In embodiments, L is -CH2-O-.

[0174] In some embodiments, the cationic amphiphilic polymer has any of the formulas above, wherein L1 is -CH2-O-, [ka] In an embodiment, L1 is -CH2-O-. In an embodiment, L1 is [ka] In an embodiment, L1 is [ka] In an embodiment, L1 is [ka] is.

[0175] In some embodiments, the cationic amphiphilic polymer can have a formula of any of the above formulas, wherein z1, z3, and z4 can independently be integers from 0 to 100, and at least one of z1 or z3 is not 0. In some embodiments, z1, z3, and z4 can independently be integers ranging from 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10, and at least one of z1 or z3 is not 0. In embodiments, z1, z3, and z4 can independently be integers ranging from 2 to 100 or 2 to 50, and at least one of z1 or z3 is not 0.

[0176] In embodiments, z1 is 0. In embodiments, z1 is 1. In embodiments, z1 is 2. In embodiments, z1 is 3. In embodiments, z1 is 4. In embodiments, z1 is 5. In embodiments, z1 is 6. In embodiments, z1 is 7. In embodiments, z1 is 8. In embodiments, z1 is 9. In embodiments, z1 is 10. In embodiments, z1 is 11. In embodiments, z1 is 12. In embodiments, z1 is 13. In embodiments, z1 is 14. In embodiments, z1 is 15. In embodiments, z1 is 16. In embodiments, z1 is 17. In embodiments, z1 is 18. In embodiments, z1 is 19. In embodiments, z1 is 20. In embodiments, z1 is 21. In embodiments, z1 is 22. In embodiments, z1 is 23. In embodiments, z1 is 24. In embodiments, z1 is 25. In embodiments, z1 is 26. In embodiments, z1 is 27. In embodiments, z1 is 28. In embodiments, z1 is 29. In embodiments, z1 is 30. In embodiments, z1 is 31. In embodiments, z1 is 32. In embodiments, z1 is 33. In embodiments, z1 is 34. In embodiments, z1 is 35. In embodiments, z1 is 36. In embodiments, z1 is 37. In embodiments, z1 is 38. In embodiments, z1 is 39. In embodiments, z1 is 40. In embodiments, z1 is 41. In embodiments, z1 is 42. In embodiments, z1 is 43. In embodiments, z1 is 44. In embodiments, z1 is 45. In embodiments, z1 is 46. In embodiments, z1 is 47. In embodiments, z1 is 48. In embodiments, z1 is 49. In embodiments, z1 is 50. In embodiments, z1 is 51. In embodiments, z1 is 52. In embodiments, z1 is 53. In embodiments, z1 is 54. In embodiments, z1 is 55. In embodiments, z1 is 56. In embodiments, z1 is 57. In embodiments, z1 is 58. In embodiments, z1 is 59. In embodiments, z1 is 60. In embodiments, z1 is 61. In embodiments, z1 is 62.In embodiments, z1 is 63. In embodiments, z1 is 64. In embodiments, z1 is 65. In embodiments, z1 is 66. In embodiments, z1 is 67. In embodiments, z1 is 68. In embodiments, z1 is 69. In embodiments, z1 is 70. In embodiments, z1 is 71. In embodiments, z1 is 72. In embodiments, z1 is 73. In embodiments, z1 is 74. In embodiments, z1 is 75. In embodiments, z1 is 76. In embodiments, z1 is 77. In embodiments, z1 is 78. In embodiments, z1 is 79. In embodiments, z1 is 80. In embodiments, z1 is 81. In embodiments, z1 is 82. In embodiments, z1 is 83. In embodiments, z1 is 84. In embodiments, z1 is 85. In embodiments, z1 is 86. In embodiments, z1 is 87. In embodiments, z1 is 88. In embodiments, z1 is 89. In embodiments, z1 is 90. In embodiments, z1 is 91. In embodiments, z1 is 92. In embodiments, z1 is 93. In embodiments, z1 is 94. In embodiments, z1 is 95. In embodiments, z1 is 96. In embodiments, z1 is 97. In embodiments, z1 is 98. In embodiments, z1 is 99. In embodiments, z1 is 100.

[0177] In embodiments, z3 is 0. In embodiments, z3 is 1. In embodiments, z3 is 2. In embodiments, z3 is 3. In embodiments, z3 is 4. In embodiments, z3 is 5. In embodiments, z3 is 6. In embodiments, z3 is 7. In embodiments, z3 is 8. In embodiments, z3 is 9. In embodiments, z3 is 10. In embodiments, z3 is 11. In embodiments, z3 is 12. In embodiments, z3 is 13. In embodiments, z3 is 14. In embodiments, z3 is 15. In embodiments, z3 is 16. In embodiments, z3 is 17. In embodiments, z3 is 18. In embodiments, z3 is 19. In embodiments, z3 is 20. In embodiments, z3 is 21. In embodiments, z3 is 22. In embodiments, z3 is 23. In embodiments, z3 is 24. In embodiments, z3 is 25. In embodiments, z3 is 26. In embodiments, z3 is 27. In embodiments, z3 is 28. In embodiments, z3 is 29. In embodiments, z3 is 30. In embodiments, z3 is 31. In embodiments, z3 is 32. In embodiments, z3 is 33. In embodiments, z3 is 34. In embodiments, z3 is 35. In embodiments, z3 is 36. In embodiments, z3 is 37. In embodiments, z3 is 38. In embodiments, z3 is 39. In embodiments, z3 is 40. In embodiments, z3 is 41. In embodiments, z3 is 42. In embodiments, z3 is 43. In embodiments, z3 is 44. In embodiments, z3 is 45. In embodiments, z3 is 46. In embodiments, z3 is 47. In embodiments, z3 is 48. In embodiments, z3 is 49. In embodiments, z3 is 50. In embodiments, z3 is 51. In embodiments, z3 is 52. In embodiments, z3 is 53. In embodiments, z3 is 54. In embodiments, z3 is 55. In embodiments, z3 is 56. In embodiments, z3 is 57. In embodiments, z3 is 58. In embodiments, z3 is 59. In embodiments, z3 is 60. In embodiments, z3 is 61. In embodiments, z3 is 62.In embodiments, z3 is 63. In embodiments, z3 is 64. In embodiments, z3 is 65. In embodiments, z3 is 66. In embodiments, z3 is 67. In embodiments, z3 is 68. In embodiments, z3 is 69. In embodiments, z3 is 70. In embodiments, z3 is 71. In embodiments, z3 is 72. In embodiments, z3 is 73. In embodiments, z3 is 74. In embodiments, z3 is 75. In embodiments, z3 is 76. In embodiments, z3 is 77. In embodiments, z3 is 78. In embodiments, z3 is 79. In embodiments, z3 is 80. In embodiments, z3 is 81. In embodiments, z3 is 82. In embodiments, z3 is 83. In embodiments, z3 is 84. In embodiments, z3 is 85. In embodiments, z3 is 86. In embodiments, z3 is 87. In embodiments, z3 is 88. In embodiments, z3 is 89. In embodiments, z3 is 90. In embodiments, z3 is 91. In embodiments, z3 is 92. In embodiments, z3 is 93. In embodiments, z3 is 94. In embodiments, z3 is 95. In embodiments, z3 is 96. In embodiments, z3 is 97. In embodiments, z3 is 98. In embodiments, z3 is 99. In embodiments, z3 is 100.

[0178] In embodiments, z4 is 0. In embodiments, z4 is 1. In embodiments, z4 is 2. In embodiments, z4 is 3. In embodiments, z4 is 4. In embodiments, z4 is 5. In embodiments, z4 is 6. In embodiments, z4 is 7. In embodiments, z4 is 8. In embodiments, z4 is 9. In embodiments, z4 is 10. In embodiments, z4 is 11. In embodiments, z4 is 12. In embodiments, z4 is 13. In embodiments, z4 is 14. In embodiments, z4 is 15. In embodiments, z4 is 16. In embodiments, z4 is 17. In embodiments, z4 is 18. In embodiments, z4 is 19. In embodiments, z4 is 20. In embodiments, z4 is 21. In embodiments, z4 is 22. In embodiments, z4 is 23. In embodiments, z4 is 24. In embodiments, z4 is 25. In embodiments, z4 is 26. In embodiments, z4 is 27. In embodiments, z4 is 28. In embodiments, z4 is 29. In embodiments, z4 is 30. In embodiments, z4 is 31. In embodiments, z4 is 32. In embodiments, z4 is 33. In embodiments, z4 is 34. In embodiments, z4 is 35. In embodiments, z4 is 36. In embodiments, z4 is 37. In embodiments, z4 is 38. In embodiments, z4 is 39. In embodiments, z4 is 40. In embodiments, z4 is 41. In embodiments, z4 is 42. In embodiments, z4 is 43. In embodiments, z4 is 44. In embodiments, z4 is 45. In embodiments, z4 is 46. In embodiments, z4 is 47. In embodiments, z4 is 48. In embodiments, z4 is 49. In embodiments, z4 is 50. In embodiments, z4 is 51. In embodiments, z4 is 52. In embodiments, z4 is 53. In embodiments, z4 is 54. In embodiments, z4 is 55. In embodiments, z4 is 56. In embodiments, z4 is 57. In embodiments, z4 is 58. In embodiments, z4 is 59. In embodiments, z4 is 60. In embodiments, z4 is 61. In embodiments, z4 is 62.In embodiments, z4 is 63. In embodiments, z4 is 64. In embodiments, z4 is 65. In embodiments, z4 is 66. In embodiments, z4 is 67. In embodiments, z4 is 68. In embodiments, z4 is 69. In embodiments, z4 is 70. In embodiments, z4 is 71. In embodiments, z4 is 72. In embodiments, z4 is 73. In embodiments, z4 is 74. In embodiments, z4 is 75. In embodiments, z4 is 76. In embodiments, z4 is 77. In embodiments, z4 is 78. In embodiments, z4 is 79. In embodiments, z4 is 80. In embodiments, z4 is 81. In embodiments, z4 is 82. In embodiments, z4 is 83. In embodiments, z4 is 84. In embodiments, z4 is 85. In embodiments, z4 is 86. In embodiments, z4 is 87. In embodiments, z4 is 88. In embodiments, z4 is 89. In embodiments, z4 is 90. In embodiments, z4 is 91. In embodiments, z4 is 92. In embodiments, z4 is 93. In embodiments, z4 is 94. In embodiments, z4 is 95. In embodiments, z4 is 96. In embodiments, z4 is 97. In embodiments, z4 is 98. In embodiments, z4 is 99. In embodiments, z4 is 100.

[0179] In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7. In embodiments, n is 8. In embodiments, n is 9. In embodiments, n is 10. In embodiments, n is 11. In embodiments, n is 12. In embodiments, n is 13. In embodiments, n is 14. In embodiments, n is 15. In embodiments, n is 16. In embodiments, n is 17. In embodiments, n is 18. In embodiments, n is 19. In embodiments, n is 20. In embodiments, n is 21. In embodiments, n is 22. In embodiments, n is 23. In embodiments, n is 24. In embodiments, n is 25. In embodiments, n is 26. In embodiments, n is 27. In embodiments, n is 28. In embodiments, n is 29. In embodiments, n is 30. In embodiments, n is 31. In embodiments, n is 32. In embodiments, n is 33. In embodiments, n is 34. In embodiments, n is 35. In embodiments, n is 36. In embodiments, n is 37. In embodiments, n is 38. In embodiments, n is 39. In embodiments, n is 40. In embodiments, n is 41. In embodiments, n is 42. In embodiments, n is 43. In embodiments, n is 44. In embodiments, n is 45. In embodiments, n is 46. In embodiments, n is 47. In embodiments, n is 48. In embodiments, n is 49. In embodiments, n is 50. In embodiments, n is 51. In embodiments, n is 52. In embodiments, n is 53. In embodiments, n is 54. In embodiments, n is 55. In embodiments, n is 56. In embodiments, n is 57. In embodiments, n is 58. In embodiments, n is 59. In embodiments, n is 60. In embodiments, n is 61. In embodiments, n is 62. In embodiments, n is 63. In embodiments, n is 64. In embodiments, n is 65. In embodiments, n is 66. In embodiments, n is 67. In embodiments, n is 68.In embodiments, n is 69. In embodiments, n is 70. In embodiments, n is 71. In embodiments, n is 72. In embodiments, n is 73. In embodiments, n is 74. In embodiments, n is 75. In embodiments, n is 76. In embodiments, n is 77. In embodiments, n is 78. In embodiments, n is 79. In embodiments, n is 80. In embodiments, n is 81. In embodiments, n is 82. In embodiments, n is 83. In embodiments, n is 84. In embodiments, n is 85. In embodiments, n is 86. In embodiments, n is 87. In embodiments, n is 88. In embodiments, n is 89. In embodiments, n is 90. In embodiments, n is 91. In embodiments, n is 92. In embodiments, n is 93. In embodiments, n is 94. In embodiments, n is 95. In embodiments, n is 96. In embodiments, n is 97. In embodiments, n is 98. In embodiments, n is 99. In embodiments, n is 100.

[0180] In embodiments, n1 is 0. In embodiments, n1 is 1. In embodiments, n1 is 2. In embodiments, n1 is 3. In embodiments, n1 is 4. In embodiments, n1 is 5. In embodiments, n1 is 6. In embodiments, n1 is 7. In embodiments, n1 is 8. In embodiments, n1 is 9. In embodiments, n1 is 10. In embodiments, n1 is 11. In embodiments, n1 is 12. In embodiments, n1 is 13. In embodiments, n1 is 14. In embodiments, n1 is 15. In embodiments, n1 is 16. In embodiments, n1 is 17. In embodiments, n1 is 18. In embodiments, n1 is 19. In embodiments, n1 is 20. In embodiments, n1 is 21. In embodiments, n1 is 22. In embodiments, n1 is 23. In embodiments, n1 is 24. In embodiments, n1 is 25. In embodiments, n1 is 26. In embodiments, n1 is 27. In embodiments, n1 is 28. In embodiments, n1 is 29. In embodiments, n1 is 30. In embodiments, n1 is 31. In embodiments, n1 is 32. In embodiments, n1 is 33. In embodiments, n1 is 34. In embodiments, n1 is 35. In embodiments, n1 is 36. In embodiments, n1 is 37. In embodiments, n1 is 38. In embodiments, n1 is 39. In embodiments, n1 is 40. In embodiments, n1 is 41. In embodiments, n1 is 42. In embodiments, n1 is 43. In embodiments, n1 is 44. In embodiments, n1 is 45. In embodiments, n1 is 46. In embodiments, n1 is 47. In embodiments, n1 is 48. In an embodiment, n1 is 49. In an embodiment, n1 is 50.

[0181] In embodiments, n2 is 1. In embodiments, n2 is 2. In embodiments, n2 is 3. In embodiments, n2 is 4. In embodiments, n2 is 5. In embodiments, n2 is 6. In embodiments, n2 is 7. In embodiments, n2 is 8. In embodiments, n2 is 9. In embodiments, n2 is 10. In embodiments, n2 is 11. In embodiments, n2 is 12. In embodiments, n2 is 13. In embodiments, n2 is 14. In embodiments, n2 is 15. In embodiments, n2 is 16. In embodiments, n2 is 17. In embodiments, n2 is 18. In embodiments, n2 is 19. In embodiments, n2 is 20. In embodiments, n2 is 21. In embodiments, n2 is 22. In embodiments, n2 is 23. In embodiments, n2 is 24. In embodiments, n2 is 25. In embodiments, n2 is 26. In embodiments, n2 is 27. In embodiments, n2 is 28. In embodiments, n2 is 29. In embodiments, n2 is 30. In embodiments, n2 is 31. In embodiments, n2 is 32. In embodiments, n2 is 33. In embodiments, n2 is 34. In embodiments, n2 is 35. In embodiments, n2 is 36. In embodiments, n2 is 37. In embodiments, n2 is 38. In embodiments, n2 is 39. In embodiments, n2 is 40. In embodiments, n2 is 41. In embodiments, n2 is 42. In embodiments, n2 is 43. In embodiments, n2 is 44. In embodiments, n2 is 45. In embodiments, n2 is 46. In embodiments, n2 is 47. In embodiments, n2 is 48. In embodiments, n2 is 49. In embodiments, n2 is 50. In embodiments, n2 is 51. In embodiments, n2 is 52. In embodiments, n2 is 53. In embodiments, n2 is 54. In embodiments, n2 is 55. In embodiments, n2 is 56. In embodiments, n2 is 57. In embodiments, n2 is 58. In embodiments, n2 is 59. In embodiments, n2 is 60. In embodiments, n2 is 61. In embodiments, n2 is 62. In embodiments, n2 is 63.In embodiments, n2 is 64. In embodiments, n2 is 65. In embodiments, n2 is 66. In embodiments, n2 is 67. In embodiments, n2 is 68. In embodiments, n2 is 69. In embodiments, n2 is 70. In embodiments, n2 is 71. In embodiments, n2 is 72. In embodiments, n2 is 73. In embodiments, n2 is 74. In embodiments, n2 is 75. In embodiments, n2 is 76. In embodiments, n2 is 77. In embodiments, n2 is 78. In embodiments, n2 is 79. In embodiments, n2 is 80. In embodiments, n2 is 81. In embodiments, n2 is 82. In embodiments, n2 is 83. In embodiments, n2 is 84. In embodiments, n2 is 85. In embodiments, n2 is 86. In embodiments, n2 is 87. In embodiments, n2 is 88. In embodiments, n2 is 89. In embodiments, n2 is 90. In embodiments, n2 is 91. In embodiments, n2 is 92. In embodiments, n2 is 93. In embodiments, n2 is 94. In embodiments, n2 is 95. In embodiments, n2 is 96. In embodiments, n2 is 97. In embodiments, n2 is 98. In embodiments, n2 is 99. In embodiments, n2 is 100.

[0182] In embodiments, z2 is 2. In embodiments, z2 is 3. In embodiments, z2 is 4. In embodiments, z2 is 5. In embodiments, z2 is 6. In embodiments, z2 is 7. In embodiments, z2 is 8. In embodiments, z2 is 9. In embodiments, z2 is 10. In embodiments, z2 is 11. In embodiments, z2 is 12. In embodiments, z2 is 13. In embodiments, z2 is 14. In embodiments, z2 is 15. In embodiments, z2 is 16. In embodiments, z2 is 17. In embodiments, z2 is 18. In embodiments, z2 is 19. In embodiments, z2 is 20. In embodiments, z2 is 21. In embodiments, z2 is 22. In embodiments, z2 is 23. In embodiments, z2 is 24. In embodiments, z2 is 25. In embodiments, z2 is 26. In embodiments, z2 is 27. In embodiments, z2 is 28. In embodiments, z2 is 29. In embodiments, z2 is 30. In embodiments, z2 is 31. In embodiments, z2 is 32. In embodiments, z2 is 33. In embodiments, z2 is 34. In embodiments, z2 is 35. In embodiments, z2 is 36. In embodiments, z2 is 37. In embodiments, z2 is 38. In embodiments, z2 is 39. In embodiments, z2 is 40. In embodiments, z2 is 41. In embodiments, z2 is 42. In embodiments, z2 is 43. In embodiments, z2 is 44. In embodiments, z2 is 45. In embodiments, z2 is 46. In embodiments, z2 is 47. In embodiments, z2 is 48. In embodiments, z2 is 49. In embodiments, z2 is 50. In embodiments, z2 is 51. In embodiments, z2 is 52. In embodiments, z2 is 53. In embodiments, z2 is 54. In embodiments, z2 is 55. In embodiments, z2 is 56. In embodiments, z2 is 57. In embodiments, z2 is 58. In embodiments, z2 is 59. In embodiments, z2 is 60. In embodiments, z2 is 61. In embodiments, z2 is 62. In embodiments, z2 is 63. In embodiments, z2 is 64.In embodiments, z2 is 65. In embodiments, z2 is 66. In embodiments, z2 is 67. In embodiments, z2 is 68. In embodiments, z2 is 69. In embodiments, z2 is 70. In embodiments, z2 is 71. In embodiments, z2 is 72. In embodiments, z2 is 73. In embodiments, z2 is 74. In embodiments, z2 is 75. In embodiments, z2 is 76. In embodiments, z2 is 77. In embodiments, z2 is 78. In embodiments, z2 is 79. In embodiments, z2 is 80. In embodiments, z2 is 81. In embodiments, z2 is 82. In embodiments, z2 is 83. In embodiments, z2 is 84. In embodiments, z2 is 85. In embodiments, z2 is 86. In embodiments, z2 is 87. In embodiments, z2 is 88. In embodiments, z2 is 89. In embodiments, z2 is 90. In embodiments, z2 is 91. In embodiments, z2 is 92. In embodiments, z2 is 93. In embodiments, z2 is 94. In embodiments, z2 is 95. In embodiments, z2 is 96. In embodiments, z2 is 97. In embodiments, z2 is 98. In embodiments, z2 is 99. In embodiments, z2 is 100.

[0183] In embodiments, z5 is 1. In embodiments, z5 is 2. In embodiments, z5 is 3. In embodiments, z5 is 4. In embodiments, z5 is 5. In embodiments, z5 is 6. In embodiments, z5 is 7. In embodiments, z5 is 8. In embodiments, z5 is 9. In embodiments, z5 is 10.

[0184] In some embodiments, the cationic amphiphilic polymer can have any of the above formulas, where z2 is an integer from 2 to 100. In some embodiments, z2 can be an integer ranging from 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 2, or 2 to 10. In embodiments, z2 can be an integer ranging from 2 to 100 or 2 to 50.

[0185] In some embodiments, the cationic amphiphilic polymer can have any of the above formulas, wherein z5 is an integer from 1 to 3. In some other embodiments, z5 is 1 or 3. In yet some other embodiments, z5 is 1. In yet some other embodiments, z5 is 3.

[0186] In some embodiments, the cationic amphiphilic polymer can have any of the above formulas, where R2 is hydrogen.

[0187] In some embodiments, the cationic amphiphilic polymer can have any of the formulas above, where L2 is a bond.

[0188] In embodiments, the CART is [ka] It has the formula TIFF0007738099000039.tif231167TIFF0007738099000040.tif74167.

[0189] In some embodiments, the pH-sensitive disruption domain is [ka] where n is an integer greater than or equal to 2. In embodiments, n is an integer ranging from 2 to 100, from 2 to 90, from 2 to 80, from 2 to 70, from 2 to 60, from 2 to 50, from 2 to 40, from 2 to 30, from 2 to 2, or from 2 to 10. In embodiments, n is an integer ranging from 2 to 100 or from 2 to 50.

[0190] In some embodiments, in the above formula (IV), n is an integer ranging from 2 to 50.

[0191] In some embodiments, the pH-sensitive disruption domain is [ka] wherein: n is an integer of 2 or greater, n1 is an integer from 0 to 50, Z is a nucleophilic moiety, X1 is a bond, -C(R5)(R6)-, -C(R5)(R6)-C(R7)(R8)-, -OC(R5)(R6)- or -OC(R5)(R6)-C(R7)(R8)-; X2 is -O- or -S-; R1, R2, R3, R4, R5, R6, R7, and R8 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0192] In some embodiments, the pH-sensitive disruption domain is [ka] wherein: n is an integer of 2 or greater, Z is a nucleophilic moiety, X1 is a bond, -C(R5)(R6)-, -C(R5)(R6)-C(R7)(R8)-, -OC(R5)(R6)- or -OC(R5)(R6)-C(R7)(R8)-; X2 is -O- or -S-; R1.1, R1.2, R2.1, R2.2, R3, R4, R5, R6, R7 and R8 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0193] In some embodiments, the pH-sensitive disruption domain is [ka] where n is an integer greater than or equal to 2. In embodiments, n is an integer ranging from 2 to 100, from 2 to 90, from 2 to 80, from 2 to 70, from 2 to 60, from 2 to 50, from 2 to 40, from 2 to 30, from 2 to 2, or from 2 to 10. In embodiments, n is an integer ranging from 2 to 100 or from 2 to 50.

[0194] In some embodiments, the pH-sensitive disruption domain is [ka] where n is an integer greater than or equal to 2. In embodiments, n is an integer ranging from 2 to 100, from 2 to 90, from 2 to 80, from 2 to 70, from 2 to 60, from 2 to 50, from 2 to 40, from 2 to 30, from 2 to 2, or from 2 to 10. In embodiments, n is an integer ranging from 2 to 100 or from 2 to 50.

[0195] In some embodiments, the pH-sensitive disruption domain is [ka] wherein n is an integer of 2 or more, n1 is an integer of 0 to 50, X1 is a bond, -O-, -NR5-, -C(R5)(R6)-, or -C(R5)(R6)-C(R7)(R8)-, X2 is a bond, -O-, -C(R9)(R10)-, or -C(R9)(R10)-C(R11)(R12)-, X4 is a bond, -C(O)-, -P(O)(OR16)2-, -S(O)(OR17)2-, -C(R16)(R17)-, or is -C(R16)(R17)-C(R18)(R19)-, X5 is a nucleophilic moiety, and R1, R2, R5, R6, R7, R8, R9, R10, R11, R12, R15, R16, R17, R18, and R19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0196] In some embodiments, in a pH-sensitive disruptive domain having any of the above defined formulas, Z is -S-, -S+R13-, -NR13-, or -N+(R13)(H)-, where R13 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0197] In some embodiments, in a pH-sensitive disruptive domain having any of the above defined formulas, Z is -S-, -S+R13-, -NR13-, or -N+(R13)(H)-, where R13 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0198] In some embodiments, in the pH-sensitive disruptive domain having any of the above defined formulas, Z is [ka] wherein: X3 is C(R15) or N; X4 is a bond, -C(O)-, -P(O)(OR16)2-, -S(O)(OR17)2-, -C(R16)(R17)- or -C(R16)(R17)-C(R18)(R19)-; X5 is a nucleophilic moiety, R13, R14, R15, R16, R17, R18, and R19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0199] In some embodiments, in the pH-sensitive disruptive domain having any of the above defined formulas, Z is [ka] and where: X3 is C(R15) or N; X4 is a bond, -C(O)-, -P(O)(OR16)2-, -S(O)(OR17)2-, -C(R16)(R17)- or -C(R16)(R17)-C(R18)(R19)-; X5 is a nucleophilic moiety, R13, R14, R15, R16, R17, R18, and R19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0200] In some embodiments, in a pH-sensitive disruptive domain having any of the above defined formulas, X5 is -N+(R13)(H)-, where R13 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0201] In some embodiments, the pH-sensitive disruption domain can have one of the following formulas: In embodiments of the cell-penetrating complexes disclosed herein, the pH-sensitive disruption domain has the structure of formula (IV): [ka]

[0202] In some embodiments, the lipophilic polymer domain comprises: [ka] wherein n2 is an integer from 1 to 100; R20 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0203] In some embodiments, lipophilic polymer domains, or what are synonymously referred to as lipophilic polymer domains, can have one of the following variations of the R group: [ka]

[0204] With respect to the cell-penetrating complexes and embodiments thereof disclosed herein, in embodiments, the nucleic acid may be DNA or RNA, such as messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), plasmid DNA (pDNA), minicircle DNA, or genomic DNA (gDNA). The cell-penetrating complexes of the present invention may further comprise a protein or peptide.

[0205] With respect to the cell-penetrating complexes and embodiments thereof disclosed herein, in embodiments, the cell-penetrating complexes further comprise a plurality of lipophilic moieties.

[0206] With respect to the cell-penetrating complexes and embodiments thereof disclosed herein, in embodiments, the cell-penetrating complexes further comprise a plurality of disruptive domains.

[0207] With respect to the cell-penetrating complexes and embodiments thereof disclosed herein, in embodiments, the counter anion for the cationic sequence may include common counter ions known in the art (e.g., acetate, trifluoroacetate, triflate, chloride, bromide, sulfate, phosphate, succinate, citrate, etc.). In embodiments, the counter anion is acetate, trifluoroacetate, triflate, chloride, bromide, sulfate, phosphate, succinate, or citrate.

[0208] Transfection In another aspect, there is provided a method of transfecting a nucleic acid into a cell, the method comprising contacting the cell with a cell-penetrating complex or an embodiment thereof as disclosed herein.

[0209] In embodiments, the method further comprises degrading the cationic amphiphilic polymer within the cell, thereby forming a degradation product, which in embodiments is a substituted or unsubstituted diketopiperazine.

[0210] With respect to any of the embodiments of the method for transfecting a nucleic acid into a cell, in embodiments, the nucleic acid is mRNA. In embodiments, the method further comprises expressing the mRNA in the cell. In embodiments, the cell forms part of an organism. In embodiments, the organism is a human.

[0211] Provided herein are, inter alia, novel materials and strategies that enable or facilitate the complexation, protection, delivery and release of oligonucleotides and polyanionic cargo, such as messenger RNA (mRNA), to target cells, tissues and organs, both in vitro and in vivo.

[0212] For example, one mRNA delivery strategy disclosed herein utilizes biodegradable poly(carbonate-co-amino ester) oligomers and variations thereof, which have been found to electrostatically complex with polyanions (such as mRNA), creating non-covalently linked macromolecular particles that protect the mRNA cargo and readily enter cells to independently release the oligonucleotide cargo, where it is converted by cellular processes into peptides and proteins whose sequences, and therefore activities, are determined by the sequence of the mRNA.

[0213] This results in significantly improved cell transfection efficiency compared to, for example, the use of nucleic acids themselves and known gene delivery vectors. The materials and strategies used for mRNA delivery can also be used to deliver other oligonucleotides, such as siRNA, pDNA, shRNA, and gDNA. Furthermore, the materials and strategies can also be used to deliver other anionic biomolecules, such as heparin, inorganic polyphosphates, and inositol polyphosphates (e.g., IP3, IP7, and IP8). This delivery can be achieved in vivo using a variety of human and non-human cell lines and through multiple administration methods, including, but not limited to, intramuscular, intravenous, intraperitoneal, intraocular, intranasal, subcutaneous, buccal, and topical administration. The poly(carbonate-co-amino ester)s disclosed herein can be used, for example, as customizable, biodegradable, and biocompatible materials in biomedical therapy, imaging, and device applications. Copolymerization with non-toxic, biodegradable compounds such as valerolactone, caprolactone, lactide, and cyclic carbonates allows for tailoring of physical and biological properties, including cargo release rate, hydrophobicity, incorporation of targeting ligands, biodistribution, and toxicity.

[0214] Thus, in some embodiments, agents provided herein include oligomers, polymers, cooligomers and copolymers that may be derived from cyclic amino ester monomers and cyclic methyl trimethylene carbonate (MTC) monomers, where the cyclic amino esters have the parent structure of morpholin-2-one and its homologs, with multiple possible substitution patterns, including: (1) N-acylation with various hydrophobic groups (e.g., R = alkyl, alkenyl, aryl, polycyclic (including steroids), heterocyclic), cationic groups (e.g., ammonium, phosphonium, sulfonium, guanidinium, including acylation with amino acids such as glycine, lysine, ornithine, and arginine), anionic groups (e.g., carboxylate, sulfate, phosphate), or hydrophilic (e.g., PEG) carbamates. Protection of the morpholine nitrogen with an N-Boc or N-Cbz group followed by organocatalytic ring-opening oligomerization or polymerization can afford cationic polymer or oligomer backbones after deprotection. (2) α-Alkylation or functionalization next to the ester carbonyl with any of the possible functional groups listed above, selected to allow for the formation of a cargo conjugate and subsequent release of the cargo by biodegradation. (3) Alkylation in the vicinity of the morpholine nitrogen with the above functional groups. (4) A combination of the above modifications.

[0215] Additionally, copolymers or cooligomers (block or statistical copolymers or cooligomers) can be made by blending two or more morpholin-2-one monomers or by copolymerizing (or cooligomerizing) one or more morpholin-2-one monomers with one or more cyclic carbonate monomers described herein. These carbonate monomers can incorporate a similar variety of side chain functional groups, highly lipophilic groups, or cationic groups to modulate the stability, delivery, and release properties of oligonucleotides. Furthermore, various other commercially available cyclic ester monomers (including, but not limited to, lactide, glycolide, valerolactone, and / or caprolactone) can be used to incorporate lipophilic functional groups. Polyaminoesters and poly(carbonate-co-aminoesters) are synthesized through ring-opening polymerization and / or copolymerization of morpholin-2-one monomers and cyclic carbonate monomers. N-Boc-protected morpholinone (MBoc) was polymerized using an organocatalytic system to high conversions (>85%), tunable Mn (1 kDa–20 kDa), and low molecular weight distributions (Mw / Mn: 1.1–1.3). Deprotection of the Boc group after polymerization afforded cationic (diprotic, secondary amine) water-soluble polymers (stable for >3 days at 0.5 M in D20). Furthermore, copolymerization of MBoc with MTC-dodecyl carbonate monomer followed by deprotection afforded moderately charged cationic materials in high yields (>60%), with narrow polydispersities (<1.4 PDI), and tunable block lengths. Block lengths were controlled by the initiator to monomer ratio.

[0216] Polyaminoesters and poly(carbonate-co-aminoesters) are biocompatible and biodegradable. The cationic polyaminoesters rapidly degrade through a novel pH- and buffer-dependent degradation mechanism, producing, in one embodiment, bis-N-hydroxyethyl-2,5-piperidinedione and bis-hydroxyethylglycine. This unexpected degradation yields products that are nontoxic at therapeutic concentrations, and their monomeric forms (the predicted products of further hydrolysis) are natural biomarkers for phospholipid modification in the Maillard reaction. The carbonate segments of these aminoester / carbonate copolymers degrade through hydrolysis and decarboxylation, and the by-products have previously been shown to be nontoxic. The novel poly(carbonate-co-aminoesters) and oligo(carbonate-co-aminoesters) exhibit unexpected performance as gene delivery agents due to their unique degradation mechanism. These novel materials noncovalently complex with mRNA at a moderate theoretical charge ratio (e.g., approximately 10:1) to protect, deliver, and release the mRNA, resulting in excellent transfection efficiencies (greater than 99% in some cases) and robust induction of gene expression in vitro and in vivo. This strategy is effective for the delivery of mRNA molecules of various lengths (test transcripts of 1000 and 2000 nucleotides). In one embodiment, gene delivery is achieved through the formulation of cationic poly(carbonate-co-amino ester) with anionic cargo to form self-assembled particles sized between 200 and 400 nm. These particles are stable over the timescale required for intracellular gene delivery and subsequently release the oligonucleotide cargo upon cell entry. Without being bound by any particular theory, these materials degrade to the bis-N-hydroxyethyl-2,5-piperidinedione product, bis-hydroxyethylglycine. Treatment of various human and non-human cell lines (e.g., HeLa, HaCaT, J774, HEK293) with this mRNA / amphiphile complex induces protein expression (e.g., GFP, luciferase) in vitro and in vivo through multiple administration methods (intramuscular and intravenous administration tested).

[0217] Protein expression has been measured by flow cytometry and fluorescence microscopy using mRNA encoding a fluorescent reporter gene (GFP) and bioluminescence (firefly luciferase). This poly(carbonate-co-aminoester) has been shown to be a more efficient transfection agent than the commercially available standard Lipofectamine 2000 and many other lead compounds previously described for the delivery of siRNA.

[0218] In embodiments, gene delivery is achieved by combining mixed amphiphilic oligomers with mRNA cargo in the presence of a third component selected to modulate the stability and size of the resulting complex, increase cellular uptake, modulate the release rate of the mRNA from the complex, and increase expression of the cargo mRNA. Third components include, but are not limited to, coordinating metals (e.g., Zn+2, Mg+2, Ca+2), dynamic non-covalent crosslinkers (e.g., carbohydrates), counterions (e.g., Cl-, AcO-, succinate, and citrate), and solubility modifiers (e.g., lipids and PEG).

[0219] Applications of this technology may include the following: Clinical Applications: (1a) Nucleic Acid Transfection Vectors: The use of DNA and RNA has long been proposed to treat genetic diseases, but the greatest barrier to the clinical use of gene therapy remains the effective delivery of oligonucleotide cargo. (1b) RNA Vaccination to Prevent Infectious Diseases: mRNA-based vaccines offer significant safety advantages over DNA vaccines, but are currently limited in the clinic by the delivery of mRNA to cells. This application is currently undergoing clinical trials, but current technology requires the removal of primary cells from patients for in vitro transfection by electroporation, followed by subsequent reintroduction of the transformed cells into the patient. This method can be significantly improved using the delivery technology of the present invention to directly induce mRNA expression in vivo. (1c) Stem Cell Induction: Pluripotency can be induced in undifferentiated stem cells by using the technology of the present invention to induce the expression of four known transcription factors. The modular nature of the poly(carbonate-co-amino ester) delivery vehicle allows for the facile delivery of all four desired mRNA transcripts simultaneously: (1) basic research applications (including, but not limited to, in vitro transfection of cultured cells, gene editing using CRISPR / Cas9, pathway validation using combinatorial gene expression (mRNA translation) and gene knockdown (RNAi), cancer immunotherapy, allergy tolerance, protein replacement therapy, gene editing, and diagnostics);

[0220] Advantages of the conjugate, composition and method disclosed in the present invention may include, for example, the following: (1) Its in vitro mRNA transfection efficiency is higher than that of commercially available transfection agents (e.g., Lipofectamine 2000), even in difficult-to-transfect cell lines (e.g., J774 macrophages), thereby improving efficacy and at the same time increasing tolerability. (2) Robust gene expression in vivo (BALB / c mice) demonstrates the clinical applicability of this technology, which avoids the toxicity of cationic carriers (e.g., lipofectamine) and provides a clinical alternative to ex vivo methods of gene delivery and expression. (3) Different routes of administration can achieve different in vivo gene expression: intravenous injection results in predominant expression in the liver and spleen, while (for example) intramuscular delivery maintains local expression at the administration site. Nasal delivery provides routes of uptake to the mucosa and / or lungs. (4) It is rapidly degraded to a known metabolite (bis-hydroxyethylglycine), which allows efficient gene expression. (5) For example, oligonucleotide-loaded particles are stable in low pH environments (such as the skin or intestinal tract) and decompose in high pH environments, releasing mRNA in a pH-dependent manner. (6) Materials are readily available through metal-free synthesis to create oligomers, polymers, or block / statistical copolymers or cooligomers with targeted molecular weights and a high degree of control over dispersity. (7) The material is amenable to targeting by adding targeting ligands (such as folate or biotin) to the surface of the particles formed or by binding to monoclonal antibodies. (8) A unique mechanism for the collapse of cationic polyaminoester domains into isolable neutral small molecules leads to the formation of the biocompatible / biodegradable product bis- N -hydroxyethyl-2,5-piperidinedione (a cyclic dimer of hydroxyethylglycine).

[0221] Features of the conjugates, compositions, and methods of the present invention include the following: In embodiments, poly(carbonate-co-amino ester) poly(amino ester)s and cationic materials derived therefrom can exhibit at least one of the following properties and functionalities: (1) The unique pH-responsive collapse mechanism of cationic polyaminoesters, leading to biocompatible / biodegradable hydroxyethylglycine dimer domains, which lead to the release of oligonucleotide cargo, is unique among other responsive biomaterials in that it occurs via an unexpected intramolecular bond formation event that irreversibly neutralizes the cationic ammonium, rapidly triggering the release of the anionic cargo. (2) isolatable products of intramolecular degradation (such as bis-N-hydroxyethyl-2,5-piperidinedione, which further decomposes to hydroxyethylglycine). (3) Electrostatically packaging anionic cargo into particles for delivery and then providing a time window of activity for rapid release after cellular internalization. (4) It allows for the copolymerization of multiple lactone monomers while providing control over macromolecular architecture. Functionalized monomers can be polymerized in block or statistical structures, which further allows for the combination of multiple monomer types, such as cyclic carbonates or phosphates. (5) In vivo use of these substances can be performed without acute toxicity when administered locally or systemically, and they have been shown to be well tolerated at the concentrations required for a therapeutic response. (6) The use of poly(carbonate-co-amino ester)s as gene delivery vehicles enables efficient delivery and release of oligonucleotides, including messenger RNA. Amphiphilic block co-oligomers of MTC-dodecyl carbonate and N-Boc morpholin-2-one monomers can be formulated with large anionic cargoes (e.g., mRNA) to form stable sub-400 nm particles. These resulting particles can be efficiently internalized by cells and release their mRNA cargo, resulting in robust gene expression. This concept has been demonstrated in vitro in multiple cell lines and in vivo in mouse studies. The effectiveness of these materials has been shown to be due to the pH-responsive rearrangement of the cationic amino ester block to form the neutral small molecule bis-N-hydroxyethyl-2,5-piperidinedione, bis-hydroxyethylglycine. Although other cationic gene delivery vehicles have been reported in the past, the oligo(carbonate-co-amino esters) described above are unique and represent the highest level of performance due to their unique ability to release mRNA (or other oligonucleotide) cargo on a timescale suitable for cellular uptake and their tolerability. (7) The experimentally determined optimal length for mRNA delivery, in which an average DP12 diblock in both the MTC-dodecyl carbonate domain and the N-Boc morpholin-2-one domain works optimally, is a departure from the prior art teachings of the present inventors. This length is significantly shorter than commercially available cationic polyamine vectors (e.g., PEI) and longer than the siRNA delivery vectors previously discovered by the present inventors (see WO2013036532A1 and PNAS2012,109(33),13171-13176).

[0222] In one aspect, the present invention provides a method for transfecting a nucleic acid into a cell. The method can include contacting the cell with a cell-permeable complex described elsewhere herein. In some embodiments, the method can perform gene editing in the cell. In embodiments, the gene editing can include genome editing, which is a type of genetic manipulation that uses an isolated or engineered nuclease system to insert, delete, or replace DNA in the genome of an organism. In certain embodiments, the methods disclosed herein can be used to deliver genetic tools or systems capable of performing gene editing in transfected cells. Some non-limiting examples of genetic tools or systems for gene editing include CRISPR-Cas systems and transposon systems.

[0223] In one aspect, the nucleic acid (i.e., cargo nucleic acid) transfected by the transfection methods according to some embodiments can comprise one or more vectors having a first nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes to a target sequence in the genome of a cell and a second nucleotide sequence encoding a Cas9 protein. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence can be located on the same vector or on different vectors.

[0224] In general, CRISPR / Cas9 systems offer high fidelity and relatively simple gene editing. For specificity, the system relies on two factors: the target sequence and the protospacer adjacent motif (PAM). The target sequence can be, for example, 20 bases long, as part of each CRISPR locus in the crRNA array. The crRNA array can have multiple unique target sequences. The Cas9 protein can select a precise location in the host genome by using sequences that bind to base pairs in the host DNA. The PAM sequence in the host genome can be recognized by Cas9. Once the elements are assembled, e.g., into one or more plasmids, and transfected into cells, the Cas9 protein, with the help of the crRNA, finds the correct sequence in the host cell's DNA and creates a single- or double-strand break in the DNA, depending on the Cas9 variant. Homologous-directed repair can be induced by appropriately spaced cuts in the host DNA. Provision of a DNA repair template allows specific DNA sequences to be inserted at the desired location in the genome. Once introduced, the new sequence becomes part of the cell's genetic material and can be inherited by its daughter cells. Many online tools are available in the art to assist in the design of effective sgRNA sequences. In some embodiments, certain embodiments of the methods and compositions of the present invention can deliver or transfect a nucleotide sequence encoding a CRISPR-Cas system guide RNA and a nucleotide sequence encoding a Cas9 protein to induce gene editing in transfected cells.

[0225] In some embodiments, the cargo nucleic acid transfected by the transfection methods according to certain embodiments can comprise a CRISPR RNA (crRNA), which can be in the same vector as the first nucleotide sequence encoding the CRISPR-Cas system guide RNA.

[0226] In some embodiments, the cargo nucleic acid transfected by the transfection method according to certain embodiments can comprise a transactivating RNA (tracrRNA). In some embodiments, the tracrRNA can be in the same vector as the second nucleotide sequence encoding the Cas9 protein.

[0227] In some embodiments, the Cas9 protein used in the transfection methods according to some embodiments can be codon-optimized for expression in the transfected cells.

[0228] In another aspect, the nucleic acid (i.e., cargo nucleic acid) to be transfected by the transfection method according to some embodiments can comprise one or more vectors having a first nucleotide sequence encoding a transposase and a second nucleotide sequence having the nucleic acid sequence of a gene of interest flanked by transposase recognition sites. In some embodiments, the first nucleotide sequence and the second nucleotide sequence can be located on the same vector or on different vectors.

[0229] A transposable element (or transposon) generally refers to a DNA sequence that can change its position within a genome, potentially causing mutations or inversions, as well as altering the genetic composition and genome size of a cell. A transposase generally refers to an enzyme that can bind to a transposon and catalyze the movement of the transposon to another part of the genome, for example, by a cut-and-paste mechanism or a replicative transposition mechanism. Introducing a transposase and a gene of interest flanked by transposase recognition sites in a cell can induce insertion of the gene of interest into the cell's genome. According to some embodiments, methods and compositions according to certain embodiments of the present invention can deliver or transfect a nucleic acid encoding a transposase and a gene of interest to induce gene editing in transfected cells.

[0230] In some embodiments, the transposase used in the transfection methods of some embodiments is capable of recognizing and excising a genomic sequence, hi some other embodiments, the nucleic acid sequence of the gene of interest transfected by the transfection method is capable of being integrated into the genome of the transfected cell.

[0231] In some embodiments, gene editing performed by the transfection methods according to some embodiments can perform one or more of DNA deletions, gene disruptions, DNA insertions, DNA inversions, point mutations, DNA substitutions, knock-ins, and knock-downs.

[0232] Method for inducing an immune response In another aspect, the present invention provides methods for inducing an immune response in a subject. In some embodiments, the methods can use a cell-permeable complex to treat and / or prevent a disease or condition. The methods generally involve administering to a subject in need thereof a therapeutically effective amount of a cell-permeable complex or a pharmaceutical composition comprising a cell-permeable complex described herein, alone (e.g., in monotherapy) or in combination with one or more additional components, such as a pharmaceutically acceptable excipient and / or additional therapeutic agent (e.g., in combination therapy).

[0233] In some embodiments, the cell-penetrating complex or a pharmaceutical composition comprising the cell-penetrating complex can be used as a vaccine capable of inducing an immune response in a subject administered the cell-penetrating complex or pharmaceutical composition thereof.

[0234] In some embodiments, a vaccine can have prophylactic activity, such that it can prevent or reduce the likelihood of a disease or condition occurring in a subject. In some instances where a vaccine is used for prophylactic purposes, the subject can be an animal that does not exhibit the disease or condition, such as a human who has not been diagnosed with the disease or condition or who does not exhibit significant symptoms associated with the disease or condition. In some other embodiments, the vaccine has a therapeutic effect, such that it can be used to treat a disease or condition. Some examples of therapeutic vaccines can include, but are not limited to, cancer vaccines that can be administered to patients who already have cancer. The cancer vaccine can exhibit one or more anti-cancer activities, such as reducing the number of cancer cells, reducing the size of the cancer, killing cancer cells, reducing and / or inhibiting metastasis, and reducing the growth and / or proliferation of cancer cells. In some other embodiments, a cancer vaccine can also be used for prophylactic purposes, particularly in subjects who are considered to be predisposed to cancer but who do not currently have cancer. A prophylactic vaccine can be administered to a subject who is predisposed to a particular cancer to prevent that cancer or reduce the likelihood of that cancer occurring in that subject.

[0235] In one aspect, the present disclosure provides a method of inducing an immune response against a disease in a subject in need thereof, which method can include administering to the subject an effective amount of a cell-permeable complex.

[0236] In some embodiments, the cell-penetrating complex can be used as a vaccine capable of inducing an immune response in a subject administered the complex. The complex can include a nucleic acid non-covalently bound to a cationic amphiphilic polymer, and the cationic amphiphilic polymer can have a pH-sensitive disintegration domain.

[0237] In some embodiments, the disease or condition targeted by the vaccine or vaccine composition can include, but is not limited to, an autoimmune disease, an inflammatory disease, a cancer disease, an infectious disease, a metabolic disease, a developmental disease, a cardiovascular disease, a liver disease, an intestinal disease, an endocrine disease, a neurological disease, or other disease.

[0238] In some embodiments, the nucleic acid included in the vaccine or composition thereof can be a nucleic acid sequence encoding an antigen or immunogenic epitope. For example, when an infectious disease is of concern, the nucleic acid included in the vaccine can encode one or more peptides known to be expressed in the pathogen of that infectious disease (e.g., a pathogenic bacterium or virus) and can induce an immune response when administered to a subject. In another example, when the disease is a specific type of cancer, the nucleic acid administered to the subject using the vaccine composition can encode one or more peptides associated with that cancer, such as peptides expressed substantially only in that type of cancer or peptides whose expression levels are significantly higher in cancer cells than in non-cancer cells. When nucleic acids encoding antigenic peptide(s) or immunogenic peptide(s) are administered to a subject and delivered (i.e., transfected) to specific cells of the subject, the transfected nucleic acid can ultimately be translated to express the antigenic peptide(s). The expressed peptide(s) are antigenic or immunogenic, and can therefore induce an immune response in the subject against the expressed peptide(s). The induced immune response can function to treat the targeted disease, for example, by reducing specific diseased cell populations and exerting a therapeutic effect if the subject already has the disease. Alternatively, the subject can have an adaptive immune response, where after an initial response to an immunogenic peptide that is the target of the adaptive immune response, the subject has this vaccination, which can elicit immunological memory, and then when the target is encountered, an enhanced response is exerted against the target, exerting a prophylactic effect.

[0239] In some embodiments, vaccination can provide dual therapeutic and prophylactic activity by delivering two separate types (or sequences) of nucleic acids in a single vaccine composition. The two separate nucleic acids can encode two different immunogenic peptides. Thus, in some embodiments, a vaccine composition can transfect (1) a first nucleic acid encoding a first immunogenic peptide that can induce a more rapid therapeutic effect against an existing disease or condition, and (2) a second nucleic acid encoding a different second immunogenic peptide intended to induce adaptive immunity in a subject against the future onset of a different disease or condition. In some embodiments, a vaccine can deliver two or more different nucleic acids to a subject, each independently exerting a therapeutic or prophylactic effect.

[0240] In embodiments, a vaccine composition can have two or more different types (or different formulas) of cationic amphiphilic polymers. Alternatively, a vaccine composition can have only one type (or one formula) of cationic amphiphilic polymer. In some embodiments, one type of cationic amphiphilic polymer can be non-covalently bound to one type (sequence) of nucleic acid. Alternatively, one type of cationic amphiphilic polymer can be non-covalently bound to two or more types (sequences) of nucleic acid. Thus, in some examples, to deliver two or more sequences (or types) of nucleic acid, a mixture of different types of cationic amphiphilic polymers, each bound to a different sequence of nucleic acid, can be administered to a subject. Alternatively, to deliver two or more sequences (or types) of nucleic acid, a single type (or formula) of cationic amphiphilic polymer bound to multiple types (or sequences) of nucleic acid can be administered to a subject. Alternatively, a single type (or formula) of cationic amphiphilic polymer bound to a single sequence (or type) of nucleic acid can be administered to a subject.

[0241] In some embodiments, the nucleic acid included in the vaccine or composition thereof can be messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), plasmid DNA (pDNA), minicircle DNA, or genomic DNA (gDNA). In alternative embodiments, the nucleic acid included in the vaccine or composition thereof can be mRNA. In some embodiments, the nucleic acid is transfected into one or more cells in a subject by vaccination. In some embodiments, one or more nucleic acid sequences can be transfected by the vaccine composition. Thus, in some embodiments, the vaccine composition comprises two different nucleic acids, each encoding a different antigenic peptide. Thus, when the vaccine is administered to a subject in need of vaccination, two or more types of antigenic epitopes can be expressed in the subject to induce an immune response. In alternative embodiments, one type of nucleic acid can be transfected by vaccination to express one type of epitope in the subject to induce an immune response.

[0242] In some embodiments, a method for inducing an immune response in a subject in need thereof can include administering to the subject an effective amount of a cell-permeable complex and, optionally, administering to the subject an effective amount of one or more additional pharmaceutical compositions. In some embodiments, the additional pharmaceutical compositions can include an anti-cancer agent and, optionally, a pharmaceutically acceptable carrier. The additional anti-cancer agent can be, for example, an antibody, a small molecule, a large molecule, or a combination thereof. Examples of anti-cancer activity include, but are not limited to, reducing the number of cancer cells, reducing the size of the cancer, killing cancer cells, reducing and / or inhibiting metastasis, and reducing the growth and / or proliferation of cancer cells. In some examples, the administration of the cell-permeable complex and the additional pharmaceutical composition can produce a synergistic effect that is greater than the combined effect of the two agents administered individually.

[0243] composition In one aspect, the present disclosure provides a cationic amphiphilic polymer as described herein. In some embodiments, the cationic amphiphilic polymer can be non-covalently bound to nucleic acids. In some embodiments, the cationic amphiphilic polymer can have one or more lipophilic polymer domains and one or more pH-sensitive disintegration domains. In some embodiments, the cationic amphiphilic polymer can be formulated into a composition together with one or more optional ingredients. The cationic amphiphilic polymer or composition thereof can be formulated into a cell-penetrating complex by non-covalently binding one or more nucleic acids.

[0244] In another aspect, the present disclosure provides a cell-penetrating complex comprising a nucleic acid and a cationic amphiphilic polymer. In some embodiments, the cationic amphiphilic polymer can be non-covalently bound to the nucleic acid. In some embodiments, the cationic amphiphilic polymer can have one or more lipophilic polymer domains and one or more pH-sensitive disintegration domains. In some embodiments, the cell-penetrating complex can be formulated into a composition together with one or more optional components.

[0245] In some embodiments, the cationic amphiphilic polymer or cell-penetrating complex can be formulated into a composition that can be used to transfect a nucleic acid into a cell. These compositions that can transfect a nucleic acid into a cell are, at least in some embodiments, referred to as transfection compositions. In some embodiments, the cargo nucleic acid that can be bound to the cationic amphiphilic polymer to form the cell-penetrating complex can be messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), transactivating RNA (tracrRNA), plasmid DNA (pDNA), minicircle DNA, or genomic DNA (gDNA). A transfection composition containing a cationic amphiphilic polymer but no cargo nucleic acid can be formulated with the cargo nucleic acid, for example, by contacting (or mixing) the cationic amphiphilic polymer with the nucleic acid prior to transfection.

[0246] In embodiments, transfection using the compositions and methods disclosed herein can alter one or more cellular characteristics. In some examples, transfection can alter the gene expression profile in the transfected cell (e.g., increase or decrease expression of one or more gene products (e.g., RNA or peptides)). In some other examples, transfection can alter genome structure (e.g., gene editing can be achieved by transfecting components of CRISPR / Cas9 or transposon systems). In some other examples, transfection can modulate the activity of a cellular pathway. Thus, in some such examples, transfection can induce stem cell development. In some other examples, the composition can deliver (or transfect) a therapeutic cargo nucleic acid, such that the transfection can treat and / or prevent a disease or condition. In some embodiments, a composition delivering (or transfecting) a therapeutic cargo nucleic acid can induce an immune response in a subject to which the composition is administered. As is apparent from the above, compositions according to the present disclosure (including compositions having cationic amphiphilic polymers or cell-penetrating complexes) can be used to achieve a variety of outcomes in transfected cells or administered subjects, depending on the cargo nucleic acid and its function.

[0247] Pharmaceutical Composition In some embodiments, compositions comprising cationic amphiphilic polymers or cell-penetrating complexes can be used for therapeutic purposes. In some embodiments, therapeutic purposes include prophylactic purposes (to prevent the onset of a disease or condition) and therapeutic purposes (to treat an existing disease or condition). When a composition comprises a cationic amphiphilic polymer but no cargo nucleic acid, a cargo nucleic acid capable of exerting a therapeutic effect can be non-covalently attached to the cationic amphiphilic polymer before administration to a subject.

[0248] In some embodiments, the composition can be a vaccine or composition thereof, i.e., a composition comprising a vaccine and, optionally, a pharmaceutically acceptable carrier. The vaccine or vaccine composition can be used to prevent and / or treat a disease or condition, or a pathogen associated with a disease or condition. In some embodiments, the vaccine or vaccine composition comprises a cell-permeable complex having a cationic amphiphilic polymer and a cargo nucleic acid. In some embodiments, the cell-permeable complex can induce an immune response, i.e., immunogenicity, when administered to a subject. This immunogenicity can be induced, at least in part, upon expression in the transfected cell of one or more antigenic peptides encoded by the cargo nucleic acid.

[0249] In one aspect, the cationic amphiphilic polymer or cell-penetrating complex disclosed herein can be incorporated into a pharmaceutical composition. The cationic amphiphilic polymer can have a pH-sensitive disintegration domain. In one embodiment, the pharmaceutical composition can further comprise a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable carrier.

[0250] In some embodiments, the pharmaceutical composition comprises a cell-penetrating complex having a nucleic acid non-covalently bound to a cationic amphiphilic polymer as an active ingredient, and may further comprise a pharmaceutically acceptable excipient or additive depending on the route of administration. Examples of such excipients or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and pharmaceutically acceptable surfactants. The additives used may be selected from, but are not limited to, the above additives or combinations thereof, depending on the dosage form of the present disclosure.

[0251] In some embodiments, the pharmaceutically acceptable carrier is an immunoadjuvant. In some examples, the immunoadjuvant may include, but is not limited to, a Toll-like receptor (TLR) agonist, a STING pathway agonist, an agonist antibody against CD40, OX40, CTLA4, PD1 or PD1-L, Freund's adjuvant, bryostatin, and a ligand for CD40, OX40, CD137, PD1, CTLA4, and any combination thereof. In some embodiments, the adjuvant, when co-administered with the cell-permeable complex to a subject, can enhance the immunogenicity induced by the cell-permeable complex.

[0252] The formulation of the pharmaceutical composition of the present disclosure can vary (e.g., solution, emulsion) depending on the selected route of administration, which can be, for example, intramuscular, subcutaneous, intravenous, intralymphatic, subcutaneous, intramuscular, intraocular, topical cutaneous, topical conjunctival, oral, intravesical (bladder), intraarterial, and intravaginal.

[0253] In some embodiments, the composition can include a cryoprotectant, non-limiting examples of which include glycols (e.g., ethylene glycol, propylene glycol, and glycerol), dimethyl sulfoxide (DMSO), formamide, sucrose, trehalose, dextrose, and any combination thereof.

[0254] In some embodiments, the formulation is a controlled-release formulation. The term "controlled-release formulation" includes sustained-release and extended-release formulations. Controlled-release formulations are well known in the art. These formulations contain excipients that allow for sustained, periodic, pulsed, or delayed release of the composition. Controlled-release formulations include, but are not limited to, formulations in which the composition is embedded in a matrix, enteric coatings, microencapsulation, gels and hydrogels, implants, and any other formulation that allows for controlled release of the composition.

[0255] In one aspect, a kit of moieties comprising a cell-permeable complex or composition thereof is provided. In another aspect, a kit of moieties comprising a cationic amphiphilic polymer or composition thereof that is not bound to a nucleic acid is provided. The kit can further include documentation or instructions describing a protocol for creating a cell-permeable complex using the cationic amphiphilic polymer and a cargo nucleic acid. The documentation or instructions for the kit can also describe a protocol for administering the composition to a subject in need thereof.

[0256] Therapeutic formulations described herein can be prepared for storage by mixing the active ingredient, i.e., immunogenic substance(s), at the desired degree of purity with any physiologically acceptable carrier, excipient, or stabilizer. Acceptable carriers, excipients, or stabilizers can be non-toxic to recipients at the dosages and concentrations employed, and can include buffers (such as phosphate, citrate, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, tannins, and the like. Proteins (such as serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants (such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG)).

[0257] The formulations of the invention may also contain more than one active compound (e.g., a second active agent in addition to the immunogenic agent(s) having a cell-permeable complex), which may be selected to have complementary activities that do not adversely affect each other. Such molecules may suitably be present together in amounts that may be effective for the intended purpose.

[0258] Administration In some aspects, provided are methods for delivering a composition to a cell or a subject to produce a desired activity in the cell or subject. In some embodiments, the composition can comprise a cell-penetrating complex having a cargo nucleic acid non-covalently attached to a cationic amphiphilic polymer. When transfected into a cell or administered to a subject, the cargo nucleic acid can produce a variety of intended effects depending on the nature of the nucleic acid sequence. Some non-limiting examples of intended effects include modulation of gene expression, modulation of cellular pathways, genome editing, and induction of an immune response. In some embodiments, the composition can be administered to a subject in an effective amount sufficient to produce at least a portion of the intended effect in the subject.

[0259] "Administration," "administering," and the like, when used in the context of a composition, refer to both direct administration (which may be in vitro administration to a cell, in vivo administration to a cell, or administration to a subject by a medical professional or self-administration by the subject) and / or indirect administration (which may be the act of formulating a composition of the present disclosure). As used herein with respect to a cell, the term refers to introducing a composition into the cell. Typically, an effective amount is administered, which amount can be determined by one of skill in the art. Any method of administration may be used. Compounds (e.g., drugs and antibodies) may be administered to cells, for example, by adding the compound to cell culture medium or by in vivo injection. Administration to a subject can be by, for example, intravascular injection, direct intratumoral delivery, etc.

[0260] Administration may refer to oral administration, administration as a suppository, topical contact, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration, or subcutaneous administration to a subject, or implantation of a sustained-release device, such as a mini-osmotic pump. Administration may be by any route, including parenteral and transmucosal routes (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarticular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. "Concomitantly administered" refers to administration of a composition described herein simultaneously with, immediately before, or immediately after the administration of one or more additional therapies, e.g., cancer therapies such as chemotherapy, hormonal therapy, radiation therapy, or immunotherapy. The compounds of the present disclosure can be administered to a patient singly or in combination, which is intended to include simultaneous or sequential administration of the compounds individually or in combination (two or more compounds).

[0261] The amount and frequency of administration (single or multiple administrations) to a subject can vary depending on various factors, such as whether the subject is suffering from another disease, the route of administration, the recipient's size, age, sex, health, weight, body mass index, and diet, the nature and extent of symptoms of the disease being treated, the type of concurrent treatment, complications resulting from the disease being treated, or other health-related issues. Other treatment regimens or therapeutic agents can be used in conjunction with the methods and compositions described herein (including embodiments thereof). Adjustment and manipulation of established administration methods (e.g., frequency and duration) are well within the capabilities of one of ordinary skill in the art.

[0262] Using the teachings provided herein, effective prophylactic or therapeutic regimens can be designed that are both less toxic and effective in treating the clinical symptoms seen in a particular patient. This design must involve careful selection of active compounds by considering factors such as the compound's potency, relative bioavailability, the patient's weight, the presence and severity of adverse side effects, the preferred method of administration, and the toxicity profile of the selected agent.

[0263] In some embodiments, the subject is a mammal, such as a human, a non-human primate, a murine (i.e., mouse and rat), a dog, a cat, or a horse. In one embodiment, the subject is a human.

[0264] In some embodiments, the composition contains about 1 ng / kg of subject body weight, about 10 ng / kg of subject body weight, about 50 ng / kg of subject body weight, about 100 ng / kg of subject body weight, about 500 ng / kg of subject body weight, about 1 ug / kg of subject body weight, about 10 μg / kg of subject body weight, about 50 ug / kg of subject body weight, about 100 μg / kg of subject body weight, about 150 μg / kg of subject body weight, about 200 μg / kg of subject body weight, about 250 μg / kg of subject body weight, about 300 μg / kg of subject body weight, about 350 μg / kg of subject body weight, about 375 μg / kg of subject body weight, about 400 μg / kg of subject body weight, about 450 μg / kg of subject body weight. μg, about 500 μg / kg of subject body weight, about 550 μg / kg of subject body weight, about 600 μg / kg of subject body weight, about 650 μg / kg of subject body weight, about 700 μg / kg of subject body weight, about 750 μg / kg of subject body weight, about 800 μg / kg of subject body weight, about 850 μg / kg of subject body weight, about 900 μg / kg of subject body weight, about 1 mg / kg of subject body weight, about 10 mg / kg of subject body weight, about 50 mg / kg of subject body weight, about 100 mg / kg of subject body weight, about 500 mg / kg of subject body weight, about 1 g / kg of subject body weight, or greater, or any range therebetween. In some embodiments, the composition contains about 0.5 μg, about 1.0 μg, about 1.5 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about 3.5 μg, about 4.0 μg, about 4.5 μg, about 5.0 μg, about 5.5 μg, about 6.0 μg, about 6.5 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg, about 8.5 μg, about 9.0 μg, about 9.5 μg, about 1.0 mg, about 1.5 mg g, about 2.0 mg, about 2.5 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, about 5.0 mg, about 5.5 mg, about 6.0 mg, about 6.5 mg, about 7.0 mg, about 7.5 mg, about 8.0 mg, about 8.5 mg, about 9.0 mg, about 9.5 mg, about 1 g, or more, or any range of doses (or amounts) between the above values.In some embodiments, the composition can be administered at a dose (or amount) of about 7.5 μg or about 0.375 mg per kg of subject body weight. Administration can be repeated for a desired period, for example, over a period of about 1 to about 5 days, or once every few days (e.g., about 5 days), for about 1 month, for about 2 months, etc. The weight can be the weight of the cell-permeable complex, or the weight of the composition or pharmaceutical formulation thereof. In some embodiments,

[0265] In one embodiment, the composition can be administered systemically or locally (e.g., intratumoral injection, intravenous injection) at intervals of 6 hours, 12 hours, once a day, every other day, or on a weekly or monthly basis to induce a desired effect or otherwise provide a therapeutic effect.

[0266] In one embodiment, the response rate to the composition, particularly a cancer vaccine, can be reduced compared to a baseline or control control. The term "response rate" is used herein in its conventional sense to refer to the percentage of patients who respond by cancer regression after treatment. Response rates include, for example, partial or complete regression. A partial response can include regression of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% of cancer cells. In some embodiments, the control control is obtained from healthy subjects, cancer subjects (e.g., cancer subjects being treated or subjects with a different cancer), or any of these populations.

[0267] The following examples and detailed protocols further illustrate embodiments of the present invention. However, these examples are intended to illustrate embodiments only and are not to be construed as limiting the scope of the present invention. The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference. [Example]

[0268] General methods and experiments material Unless otherwise specified, reagents were purchased from Sigma-Aldrich and used as received. 1-(3,5-bis-trifluoromethyl-phenyl)-3-cyclohexyl-thiourea (Macromolecules 39(23):7863-7871), MTC-guanidine monomer (J Am Chem Soc 131(45):16401-16403), MTC-dodecyl monomer (Proc Natl Acad Sci 109(33):13171-13176), MTC-piperidine monomer (Chem Commun(1):114-116), N-Boc morpholinone monomer (J Am Chem Soc 136(26):9252-9255), and dansyl alcohol (J Am Chem Soc 131(45):16401-16403) were all prepared according to literature procedures. Unless otherwise noted, all commercially available solvents and reagents were used without further purification. Methylene chloride (CHCl) and tetrahydrofuran (THF) were passed through an alumina drying column (Solv-tek Inc.) under nitrogen pressure. Petroleum ether, pentane, hexane, ethyl acetate (EtOAc), and methanol (MeOH) were obtained from Fisher Scientific. Deuterated solvents were purchased from Cambridge Isotope Laboratories. Regenerated cellulose dialysis membrane (Spectra / Por® 6 Standard RC, MWCO 1000) was purchased from Spectrum Laboratories, Inc.

[0269] mRNA In all examples below, eGFP mRNA (5meC, ψ, L-6101), Fluc mRNA (5meC, ψ, L-6107), OVA mRNA (5meC, ψ, L-7210), and Cy5-eGFP mRNA (5meC, ψ, L-6402) were purchased from TriLink BioTechnologies Inc.

[0270] Instrument formation Particle size was measured by dynamic light scattering using a Malvern Zetasizer Nano ZS90. Flow cytometry analysis was performed using a BD LSRII FACS Analyzer (Stanford University Shared FACS Facility). Laser scanning confocal microscopy was performed using a Leica SP8 White Light Confocal microscope equipped with a 40x HC PL APO CS2 oil immersion objective (Stanford University Cell Sciences Imaging Facility). Bioluminescence was measured using a charge-coupled device (CCD) camera (IVIS100, Xenogen Corp., Alameda, CA) and analyzed using Living Image Software (Perkin-Elmer). Epifluorescence microscopy was performed using a Zeiss Axio Observer.Z1 equipped with an X-Cite 120Q wide-field excitation light source and a GFP filter set. Images were acquired with a CoolSNAP HQ2 camera and transferred to a computer for image analysis.

[0271] cell line HeLa, J774, HepG2, and HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. CHO cells were maintained in F12 medium supplemented with 10% FBS and 1% penicillin / streptomycin. All cells were grown at 37°C in a 5% CO2 atmosphere. Cells were passaged when approximately 80% confluent.

[0272] Mesenchymal stem cells (MSCs) were prepared according to the method of Huang et al. (J. Orthopaedic Trans. 3(1):26-33). Briefly, femurs were removed from two 8-week-old female CD1 mice, and tissue was removed from the outside of the bones. The ends of the bones were cut with sterile scissors. Bone marrow was flushed from four bones in a 10-cm tissue-culture-treated Petri dish using a 3 mL syringe and a 25 g needle with DMEM 10% fetal bovine serum containing penicillin / streptomycin. The marrow was disrupted and dispersed by pipetting, but was not filtered or otherwise manipulated. The Petri dish was incubated for 6 days, at which time a characteristic monolayer developed. The culture medium was then washed twice with PBS and trypsinized with 0.25% trypsin (Gibco) for 5 minutes at 37°C. The cells were then harvested, transferred to a 75 cm2 cell culture flask, and incubated for 3 days until they reached 90% confluence. The cultures could be maintained for more than two passages, but proliferation significantly decreased at passage 4. For transfection, cells were seeded at 1.2 × 104 cells per well in a 24-well plate.

[0273] Example 1. Monomer synthesis In some examples, novel lactone or carbonate monomers are used to generate CARTs with diverse cationic disruptive domains. Examples of precursor and monomer synthesis are described below.

[0274] Example 1.1. Synthesis of tert-butyl 2-oxomorpholine-4-carboxylate (N-Boc morpholinone): In these examples, the synthesis of N-Boc morpholinone monomer was adapted from Chung, et al. J. Am. Chem. Soc., 2013, 135(20), 7593-7602. [ka]

[0275] Synthesis of tert-butyl 2-oxomorpholine-4-carboxylate: 2.086 g (10.2 mmol) of tert-butyl bis(2-hydroxyethyl)carbamate was dissolved in 75 mL of CH3CN and bubbled with oxygen for 5 minutes. 398 mg (0.38 mmol) of [(neocuproine)Pd(OAc)]2(OTf)2 was added, resulting in the formation of a dark red solution. The reaction was placed in a 60 °C oil bath, and oxygen was bubbled through the reaction mixture, which was monitored by TLC (1:1 hexane:EtOAc). After 24 h, 263 mg (0.25 mmol) of [(neo)Pd(OTf)(Ac)]2 was added to the reaction. After 48 h, an additional 426 mg (0.41 mmol) of [(neocuproine)Pd(OAc)]2(OTf)2 was added to the reaction. After a total of 72 h, the oxygen flow was stopped and the reaction was allowed to cool to room temperature. The reaction mixture was concentrated to 5–10 mL to give a black, non-viscous mixture that was loaded onto a SiO plug and eluted with 1:1 hexanes:EtOAc. Concentration afforded 1.766 g of a yellow oil (8.78 mmol, 86.4%), which solidified upon standing. This crude product was dissolved in 10 mL of EtO, followed by the addition of 20 mL of hexanes and storage at −30° C. overnight. The precipitate was collected by filtration and washed with pentane to give 1.54 g of a white powder (7.67 mmol, 76% yield). 1H NMR (500 MHz, CDCl3): δ 4.41 (br, 2 H), 4.26 (s, 2 H), 3.66 (t, 2 H), 1.47 (s, 9H). 13C NMR (125 MHz, CD3Cl): δ 28.24, 40.63, 45.49, 67.19, 81.33, 153.49, 166.63. HRMS (m / z): M calculated for C9H15NO4Na: 224.0893; found: 224.0896.

[0276] Example 1.2. Synthesis of tert-butyl 7-oxo-1,4-oxazepane-4-carboxylate (7-lacto): [ka] A flame-dried flask was charged with tert-butyl 4-oxopiperidine-1-carboxylate (205.8 mg, 1.03 mmol) and DCM (10 mL) and stirred at 0 °C for 15 min. mCPBA (600.1 mg, 2.9 mmol) in DCM (5 mL) was added in two portions and the mixture was allowed to warm to room temperature. After 18 h, the reaction mixture was transferred to a separatory funnel, washed with saturated NaCHO (20 mL x 3), dried over NaSO, and filtered. The solvent was removed under reduced pressure to give 317.0 mg of a white solid. Silica gel chromatography was performed eluting with 9:1 DCM:EtOAc. The relevant fractions were concentrated to give 151.0 mg of a white solid (0.71 mmol, 68.9% yield). 1H NMR (500 MHz, CD3Cl): δ 4.21 (s, 2 H), 3.82 (s, 2 H), 3.65 (s, 2 H), 2.81 (s, 2 H), 1.46 (s, 9H).13C NMR (101 MHz, CD3Cl): δ 173.9, 154.4, 81.1, 69.5, 47.3, 41. 3,37. 6,28.4

[0277] Example 1.3. Synthesis of tert-butyl (2-(bis(2-hydroxyethyl)amino)-2-oxoethyl)carbamate: [ka] A flame-dried flask was charged with diethanolamine (225 mg, 2.15 mmol) and methyl (tert-butoxycarbonyl)glycinate (370 mg, 1.95 mmol) and stirred in a 75°C heating bath. After 18 h, the reaction was passed through a silica plug and eluted with acetone. The solvent was removed under reduced pressure to give 505.1 mg of a pale yellow oil (1.93 mmol, 99% yield). 1H NMR (500 MHz, CD3Cl): δ 5.7-5.6 (br, 1H), 3.99(d, 2 H), 3.78-3.65 (m, 4H), 3.53-3.35 (m, 4H), 3.05 (br, 1H). 13C NMR (101 MHz, CD3Cl): δ 170.4, 156.3, 79.9, 60.0, 51.1, 42.4, 28.3

[0278] Example 1.4. Synthesis of tert-butyl (2-oxo-2-(2-oxomorpholino)ethyl)carbamate (glycine monomer): [ka] A flame-dried flask was charged with the diol (450 mg, 1.72 mmol) and acetonitrile (12 mL), followed by air bubbling for 5 minutes. Pd(neo)OAc (84.1 mg, 0.16 mmol, 9.4 mol%) was added to the reaction and allowed to stir at 50°C under a constant stream of air. After 18 hours, the reaction was concentrated, triturated with EtOAc (30 mL), and filtered to give 220 mg of a foamy orange residue. Silica gel chromatography was performed, eluting with 100% EtOAc. The relevant fractions were concentrated to give 53.1 mg of a clear oil (0.205 mmol, 12% yield), which solidified upon standing. 1H NMR (500 MHz, CD3Cl): δ 5.45-5.3 (br, 1H), 4.5-4.45(q,2 H), 4.28-4.4(d, 2 H), 4.0-3.93(dd,2 H), 3.85-3.7(dt,2 H), 1.45 (s, 9H)13C NMR (125 MHz, CD3Cl): δ 167.7, 166.1, 164.8, 155.8, 80.2, 66.9, 65.9, 45.8, 44.1, 42.4, 41.2, 39.3, 28.3, 15.316

[0279] Example 1. Synthesis of 5.7-Gly (ketone): [ka] To a flask containing hydroxybenzotriazole (600 mg, 4.4 mmol), EDC-HCl (453 mg, 2.9 mmol), and (tert-butoxycarbonyl)glycine (520 mg) in 10 mL of THF, TEA (500 μL) was added and stirred for 20 min. A solution of 4-piperidinone-HCl-monohydrate (462 mg, 3.0 mmol) in a 10:1 mixture of THF:HO (11 mL total) was added in one portion. The reaction was allowed to stir overnight. The reaction was concentrated under reduced pressure, taken up in 15 mL of EtOAc, washed with AcOH (500 μL) in HO (10 mL), and the aqueous phase extracted with 15 mL of EtOAc. The combined organic layers were subsequently washed with NaHCO (2 × 20 mL) and brine (1 × 20 mL) and then dried over MgSO. Concentration under reduced pressure gave 375 mg (50% yield) of a white crystalline powder which was used without further purification.

[0280] Example 1.6.7-Gly Monomer Synthesis: [ka] To a flask containing the 7-Gly monomer: N-glycinyl(Boc)4-piperidinone (28 mg, 0.11 mmol) was added mCPBA (60 mg, 0.35 mmol) in 5 mL of DCM. The reaction was allowed to stir at 0°C for 2 hours, after which an additional portion of mCPBA (20 mg, 0.12 mmol) in 2 mL of DCM was added to the reaction mixture and allowed to stir overnight. The reaction was concentrated under reduced pressure, taken up in 10 mL of EtOAc, washed with NaHCO (4 x 10 mL), and dried over MgSO. The crude product was purified by SiO chromatography using a 9:1 EtOAc:DCM solvent system. The relevant fractions were concentrated under reduced pressure to give 21 mg of pdt (71% yield).

[0281] Example 1.7. Synthesis of 8-membered carbonate-based monomers: [ka] 8-membered carbonate-based monomers: These examples were prepared by literature procedures reported in J. Am. Chem. Soc. 2015, 137, 13851-13860.

[0282] Example 1.8. Synthesis of tert-butyl (2-oxotetrahydro-2H-pyran-3-yl)carbamate (Mglut) [ka] Synthesis of tert-butyl (2-oxotetrahydro-2H-pyran-3-yl)carbamate (Mglut): 1.078 g (4.9 mmol) of (S)-(-)-2-(Boc-amino)-1,5-pentanediol was dissolved in 10 mL of CH3CN (0.49 M) and air was bubbled through for 5 minutes. 0.233 g (0.45 mmol) of [(neo)Pd(OTf)(OAc)]2 was added, causing the clear orange solution to darken to black. The reaction was placed in a 45 °C oil bath and air was bubbled through the reaction mixture. The reaction was monitored by H NMR to confirm the disappearance of the lactol peak at 5.1 ppm. Over a 44-hour period, based on the progress of the reaction, a total of 0.310 g (0.6 mmol, 12 mol%) of [(neo)Pd(OTf)(OAc)]2 was added in small increments. At 44 hours, 1H NMR indicated the reaction was complete. The airflow was stopped and the reaction was allowed to cool to room temperature. The reaction mixture was concentrated, and the residue was taken up in 50 mL of EtOAc, sonicated, and then filtered through a plug of Celite and concentrated to give 0.940 g of a pink-orange oil, which solidified upon standing. Silica gel chromatography was performed eluting with 4:1 DCM:EtOAc. The relevant fractions were concentrated to give 0.480 g (2.23 mmol) of a white solid (46% yield). Spectroscopic data were consistent with previous reports. Xie, X. and Stahl, SSJAm. Chem. Soc., 2015, 137(11):3767-3770

[0283] Example 1.9. Synthesis of phosphoester-based monomers [ka] Phosphoester-Based Monomers: Some examples include the synthesis of phosphate- or phosphoester-based cationic disruptive domains. In this example, the required monomers were synthesized according to previous literature procedures (e.g., McKinlay, CJ et. al. J. Am. Chem. Soc. 2016, 138(10), 3510-3517, WO2017083637A1). Briefly (e.g., 2-(6-bis-boc guanidinohexyloxy)-1,3,2-dioxaphospholane-2-oxide): 2-chloro-1,3,2-dioxaphospholane-2-oxide (797 mg, 5.59 mmol, 1.25 equiv.) was weighed into a Schlenk flask under an inert N2 atmosphere (glove box). This was placed on ice under nitrogen, and THF (75 mL) was added. In a separate vial, guanhexanol (7.16 g, 20.0 mmol, 1 equiv.) was dissolved in THF (10 mL) and triethylamine (2.8 mL, 20 mmol, 1 equiv.) was added. The contents of the vial were added dropwise to a Schlenk flask via syringe over 10 minutes and allowed to react for 20 hours. After the reaction, the product was filtered through a Celite® pad. The crude product was dissolved in a small amount (5 mL) of THF and triturated with 20 mL of dry pentane. The product was allowed to oil out in a -55°C freezer overnight. The pentane layer was removed and dried under vacuum for 10 hours to afford the pure product as a slightly yellow oil (79% yield). This monomer was synthesized and deprotected according to the standard procedure described below.

[0284] Example 2. Polymerization of CART General Procedure A solution of the DBU:TU cocatalyst system is added to a toluene solution of the monomer and initiator. The reaction is stirred for 2 hours, followed by the addition of the monomer with the disruptive domain as a solid and stirring for 2 hours. The reaction is quenched with acetic acid (or benzoic acid) and then dialyzed against MeOH in DCM. After dialysis, the solvent is removed under reduced pressure to yield the pure oligomer. The oligomer is then deprotected using TFA / DCM or HCl / Et2O to yield the amphiphilic CART. The CART is diluted to 2.0 mM in DMSO and used in transfection studies without further purification. [ka]

[0285] Deprotection involves the use of TFA / DCM or HCl Et2O Example of TFA deprotection: To 10.5 mg of oligomer dissolved in 1.0 mL of dry DCM, add 100 uL of TFA. The reaction is stirred for 8 hours. The solvent is removed under reduced pressure to give 10.3 mg of CART (TFA salt) as a residue.

[0286] HCl deprotection example: To 15 mg of protected CART was added two portions of 2.0 M HCl in EtO (total 2 mL). The reaction was stirred for 18 h and then concentrated under reduced pressure to give 12.1 mg of CART (HCl salt) as a residue.

[0287] Examples of initiators: monofunctional alcohols (i.e., benzyl alcohol, 1-pyrenebutanol), PEG, fluorescent dyes (dansyl, BHQ, BDK), multifunctional alcohols (trisbenzyl alcohol, PEG diol), targeting ligands (biotin, folate), and conjugated ligands (biotin).

[0288] The parent morpholinone-based CART was adapted from that described in the literature report McKinlay, et al. PNAS, 2017, E448-E456.

[0289] [ka] Preparation of Co-oligomers Dn:Am: Representative synthesis of D13:A11: A flame-dried vial was charged with MTC-dodecyl monomer 5 (33.2 mg, 0.1 mmol), initiator dansyl 3 (3.9 mg, 0.013 mmol), and 50 μL of CHCl. ​​The catalyst diazabicycloundecene (DBU) (0.8 mg, 0.005 mmol) and thiourea (TU) (2.0 mg, 0.005 mmol) in 50 μL of CHCl were added to the reaction vial and stirred. After 2 h, N-Boc monomer (22.3 mg, 0.11 mmol) was added as a solid to the vial and the reaction was stirred for 3 h. After a total of 5 h, the reaction was quenched with 5 drops of AcOH and then concentrated under reduced pressure. The crude material was dialyzed (1.0 kDa dialysis bag) against MeOH in CHCl. ​​Concentration yielded 37.9 mg of a pale green residue. Analysis of each group by 1H NMR (2.8 ppm) indicates a DP of 13:11.

[0290] To 23.5 mg of oligomer dissolved in 0.8 mL of dry DCM was added 20 uL of TFA. The reaction was stirred for 18 h. The solvent was removed under reduced pressure to give 23.1 mg of CART as a residue.

[0291] Cl-based CART Examples of CARTs with various cationic disruptive domains are as follows: [ka] A flame-dried vial was charged with dodecyl-MTC (33.0 mg, 0.1 mmol), 1-pyrenebutanol (2.3 mg, 0.0083 mmol), and 50 μL of toluene. DBU (0.76 mg, 0.005 mmol) and TU (1.85 mg, 0.005 mmol) in 50 μL of toluene were added to the reaction vial and stirred. After 2 h, 7-lacto (Example 1.2) (21.5 mg, 0.1 mmol) was added as a solid and the reaction was stirred. After stirring for an additional 2 h, the reaction was quenched with one drop of acetic acid. The crude material was dialyzed (1.0 kDa dialysis bag) against MeOH in DCM. Concentration under reduced pressure gave 41 mg (76% yield).

[0292] To 10.5 mg of oligomer dissolved in 1.0 mL of dry DCM was added 100 uL of TFA. The reaction was stirred for 8 h. The solvent was removed under reduced pressure to give 10.3 mg of CART as a residue.

[0293] 6-Gly-based CART [ka] A flame-dried vial was charged with dodecyl-MTC (32.8 mg, 0.1 mmol), 1-pyrenebutanol (2.3 mg, 0.0083 mmol), and 50 μL of DCM. DBU (0.76 mg, 0.005 mmol) and TU (1.85 mg, 0.005 mmol) in 50 μL of DCM were added to the reaction vial and stirred. After 2 h, tert-butyl (2-oxo-2-(2-oxomorpholino)ethyl)carbamate (36 mg, 0.14 mmol) was added as a solid and the reaction was stirred. After stirring for an additional 2 h, the reaction was quenched with one drop of acetic acid. The crude material in DCM was dialyzed against MeOH (1.0 kDa dialysis bag). Concentration under reduced pressure gave 47 mg (68% yield).

[0294] To 11.1 mg of oligomer dissolved in 1.0 mL of dry DCM was added 100 uL of TFA. The reaction was stirred for 8 h. The solvent was removed under reduced pressure to give 11 mg of CART as a residue.

[0295] 7-Gly-based CART [ka] A flame-dried vial was charged with dodecyl-MTC (32.2 mg, 0.1 mmol), benzyl alcohol (0.9 mg, 0.0083 mmol), and 50 μL of toluene. DBU (0.76 mg, 0.005 mmol) and TU (1.85 mg, 0.005 mmol) in 50 μL of toluene were added to the reaction vial and stirred. After 2 h, tert-butyl (2-oxo-2-(7-oxo-1,4-oxazepan-4-yl)ethyl)carbamate (32.1 mg, 0.1 mmol) was added as a solid and the reaction was stirred. After stirring for an additional 2 h, the reaction was quenched with one drop of acetic acid. The crude material was dialyzed (1.0 kDa dialysis bag) against MeOH in DCM. Concentration under reduced pressure gave 49 mg (77% yield).

[0296] To 21 mg of oligomer dissolved in 2.0 mL of dry DCM was added 200 μL of TFA. The reaction was stirred for 8 h. The solvent was removed under reduced pressure to give 21.3 mg of CART as a clear yellow residue.

[0297] Carb-based CART [ka] A flame-dried vial was charged with dodecyl-MTC (33.1 mg, 0.1 mmol), 5-(dimethylamino)-N-(2-hydroxyethyl)naphthalene-1-sulfonamide (dansyl alcohol) (2.8 mg, 0.0083 mmol), and 50 mL of DCM. DBU (0.76 mg, 0.005 mmol) and TU (1.85 mg, 0.005 mmol) in 50 μL of DCM were added to the reaction vial and stirred. After 2 h, 8-Carb (23.2 mg, 0.1 mmol) was added as a solid. After stirring for an additional 2 h, the reaction was quenched with one drop of acetic acid. The crude material in DCM was dialyzed against MeOH (1.0 kDa dialysis bag). Concentration under reduced pressure gave 54.1 mg (96% yield).

[0298] To 12 mg of oligomer dissolved in 1.0 mL of dry DCM was added 100 μL of TFA. The reaction was stirred for 9 h. The solvent was removed under reduced pressure to give 12 mg of CART as a residue.

[0299] Glu-based CART [ka] A flame-dried vial was charged with dodecyl-MTC (15.5 mg, 0.05 mmol), benzyl alcohol (0.5 mg, 0.004 mmol), and 50 μL of DCM. DBU (0.4 mg, 0.0025 mmol) and TU (0.9 mg, 0.0025 mmol) in 50 μL of DCM were added to the reaction vial and stirred. After 2 h, tert-butyl (2-oxotetrahydro-2H-pyran-3-yl)carbamate (MGlut) (18.1 mg, 0.084 mmol) was added as a solid. After stirring for an additional 2 h, the reaction was quenched with one drop of acetic acid. The crude material in DCM was dialyzed (1.0 kDa dialysis bag) against MeOH. Concentration under reduced pressure gave 17.9 mg (51% yield).

[0300] To 9.0 mg of oligomer dissolved in 1.0 mL of dry DCM was added 100 uL of TFA. The reaction was stirred for 9 h. The solvent was removed under reduced pressure to give 9.0 mg of CART as a residue.

[0301] Additional examples of CARTs synthesized through these methods are detailed in the table below. [Table 1] TIFF0007738099000069.tif119165

[0302] Example 3. CART / Nucleic Acid Complex In some cases, CART / mRNA polyplexes were prepared by mixing 5.71 μL of RNAse-free PBS (adjusted to pH 5.5 with 1N HCl) and eGFP mRNA (from a 0.2 μg / μL stock in PBS, pH 7.4) with 0.59 μL of CART (from a 2 mM stock in DMSO) to achieve a 10:1 + / - CART / mRNA ratio. These CART / mRNA complexes were incubated for 20 s at room temperature before treatment or analysis.

[0303] In comparative examples, other CARTs were used in corresponding amounts to give the same + / - charge ratio of 10:1.

[0304] DLS characterization of particle size and zeta potential In a specific example, dynamic light scattering (DLS) was used to analyze the polyelectrolyte complexes formed between CART D13:A11 and the CART and eGFP mRNA. At pH 5.5, the resulting polyplexes had a hydrodynamic diameter of 254 nm ± 10 nm. When these polyplexes were added to cell culture media, the hydrodynamic diameter changed from 254 nm to 512 nm over a 2-hour period.

[0305] When this CART / mRNA complex is added to unbuffered water, the size remains at 257 nm ± 24 nm throughout the 2-hour experiment.

[0306] Zeta potential measurements are consistent with the particle size data, with the surface charge starting at +33 ± 7 mV and changing to -30 ± 3 mV over 2 hours. This is consistent with the cationic ammonium being rearranged to a neutral amide, leaving the surface primarily anionic with the accompanying oligonucleotides. Without being bound by theory, the difference between the rearrangement rates of the homo-oligomers (minutes) and the mRNA polyplexes (hours) likely reflects the complexation-dependent enhanced stability of the α-amino ester material in a buffered aqueous environment. This allows the CART / mRNA complexes to remain stable at pH 7.4 over therapeutically relevant timescales until intracellular degradation.

[0307] As a comparison, when complexed with the non-degradable transporter D13:G12 (Figure 7B), particles of approximately 250 nm were also formed, but the size and zeta potential of these particles did not change with time, indicating that degradation was not occurring.

[0308] As a comparative example, the size of the formulated polyplexes was not cargo-dependent: When polyplexes were formed with luciferase (Fluc) mRNA, which is approximately twice as long as eGFP (eGFP = 996 nt vs. Fluc = 1929 nt), and added to cell culture medium at pH 7.4, the polyplexes exhibited the same behavior as those formed with eGFP mRNA, suggesting that CART-enabled delivery may be versatile and work with mRNAs of various sizes.

[0309] In these specific examples, mRNA / co-oligomer complexes were prepared as described above using 500 ng of eGFP mRNA at a 10:1 (cation:anion) charge ratio and added to 120 μL of RNase-free PBS, pH 5.5, pH 7.4, or neutral RNase-free water. The solution was immediately transferred to a disposable clear plastic cuvette and measured for size. Size measurements were taken at the initial time point (1 min) and at 15-min intervals over a 2-h period. The reported size is the z-average. Zeta potential measurements were obtained by diluting the mRNA:co-oligomer complexes formulated for DLS with 800 μL of water, transferring them to a ZetaCell (DTS1060), and measuring the zeta potential. All reported values ​​are the average of a minimum of three test runs. Errors are expressed as ±SD. In some other examples, the ratio of cations to anions can be about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:30 or greater. In some other examples, the ratio of anions to cations can be about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:30 or greater.

[0310] Example 4. Delivery of mRNA by CART Delivery and expression of eGFP mRNA in multiple cell lines In some cases, CART D13:A11 conferred high levels of eGFP expression in HeLa cells, with transfection efficiencies exceeding 99% and high mean fluorescence intensities (Figure 9A, B, D).

[0311] For example, for transfection with CART D13:A11, HeLa cells were seeded at 40,000 cells / well in 24-well plates and allowed to adhere overnight. Oligomer / mRNA polyplexes were prepared by mixing RNAse-free PBS (pH 5.5) and eGFP mRNA with varying amounts of oligomer from a DMSO stock solution to achieve specific co-oligomer / mRNA ratios (optimized for a theoretical cation:anion ratio of 10:1, total volume 8.4 μL). The complexes were incubated for 20 seconds at room temperature before treatment. A control Lipofectamine™ 2000 solution was prepared in OptiMEM according to the manufacturer's instructions. Cells were washed with serum-free DMEM, and the mRNA / Lipo solution was added to a final volume of 200 μL / well and 125 ng mRNA / well. After washing with serum-free DMEM, 2.5 μL of mRNA / co-oligomer complexes were added to a total volume of 200 μL. All conditions were performed in triplicate, resulting in a final mRNA concentration of 125 ng / well. Cells were incubated for 8 hours at 37°C and then trypsinized with trypsin-EDTA (0.05%) for 10 minutes at 37°C. Serum-containing DMEM was added, and the contents of each were thoroughly centrifuged. The supernatant was removed, and the pelleted cells were resuspended in PBS (125 μL), transferred to FACS tubes, and read on a flow cytometry analyzer (LSR-II.UV, Stanford University). Data shown (Figure 9) are the geometric mean fluorescence signals from 10,000 analyzed cells. For transfection efficiency, untreated cells were gated for lack of eGFP expression, and the data shown are the percentage of cells expressing eGFP higher than untreated cells out of 10,000 analyzed cells. Error bars are expressed as ±SD. NOTE: All other cell lines were used in their respective media as described above. For HepG2 cells, 5 mM EDTA was added to the PBS used to resuspend the cell pellet for flow cytometry.

[0312] A second CART with a longer block length (D18:A17) resulted in high transfection efficiency (>90%) but lower mean transfection values ​​(Figure 9A). In a comparative example, complexes formed with the α-aminoester homo-oligomer A13 failed to induce eGFP expression, indicating that the hydrophobic domain is essential for oligonucleotide delivery. As a further comparison, when HeLa cells were treated with mRNA formulated with Lipofectamine, only moderate levels of eGFP expression were observed; only approximately 50% of the cells were fluorescent.

[0313] In several other examples, to optimize delivery parameters for CART D13:A11, the charge ratio between the cationic oligomer and the anionic mRNA was varied from 1:1 to 50:1 (+ / -), and the resulting eGFP fluorescence was determined (Figure 9C). Values ​​are reported as the theoretical (+ / -) charge molar ratio of ammonium cations to phosphate anions, assuming complete amine protonation and phosphate deprotonation. eGFP expression showed a roughly parabolic dependence on charge ratio, with maximum eGFP fluorescence observed for complexes formed at a (+ / -) charge ratio of 10:1. This value is higher than that observed for guanidinium-rich oligocarbonate complexes (used in siRNA delivery, which showed optimal performance at a charge ratio of 4.8:1). Furthermore, epifluorescence microscopy was used to confirm the flow cytometry results (Figure 9D). HeLa cells treated with CART / mRNA complexes exhibited significant fluorescence in nearly all visible cells. In contrast, cells treated with Lipofectamine showed partial expression of eGFP, while other cells remained untransfected.

[0314] In a further example, we assayed eGFP mRNA expression after delivery with CART D13:A11 in a panel of cell lines, including some typically considered difficult to transfect. In addition to HeLa cells, we also compared mRNA expression in mouse macrophage cells (J774), human embryonic kidney cells (HEK-293), Chinese hamster ovary cells (CHO), and human hepatocellular carcinoma cells (HepG2) treated with CART complexes formed with eGFP mRNA to that observed with Lipofectamine (Figure 6A). In all cell lines tested, eGFP expression was observed in over 90% of cells treated with CART D13:A11, whereas Lipofectamine treatment induced expression in only 22–55% of cells (Figure 11A). This suggests that this delivery system is versatile for a wide variety of human and nonhuman cell types. In addition to immortalized cell lines, mRNA expression was also observed in primary mesenchymal stem cells (MSCs) derived from CD1 mice, with high transfection efficiency (>85%).

[0315] In other examples, CART activity has been demonstrated in other difficult-to-transfect cell lines, including Jurkat T cells (11% transfection rate with CART vs. 7% with Lipofectamine), primary T cells (6% transfection rate vs. 0% with Lipo), 3T3 fibroblasts (70% with CART vs. 59% with Lipo), and trophoblast stem cells (19% with CART vs. 69% with Lipo).

[0316] In some instances, consistent transfection of mRNA with CART D13:A11 was also observed using mRNAs of different lengths (such as the even larger firefly luciferase (Fluc) mRNA), substantially exceeding the performance of Lipofectamine by more than three-fold (Figure 11B). Similar to the trend observed with eGFP mRNA, a 10:1 (cation:anion) ratio yielded the highest levels of Fluc bioluminescence, regardless of mRNA length, indicating that delivery efficiency is largely independent of cargo size.

[0317] In these examples, HeLa cells were seeded at 10,000 cells / well in black 96-well plates and allowed to adhere overnight. mRNA polyplexes and control Lipofectamine™ 2000 were prepared as described above using Fluc mRNA (final concentration of 50 ng mRNA / well in a total volume of 50 μL). All conditions were performed in sextuplicate. Cells were incubated for 8 hours at 37°C under treatment, after which the medium was removed and 100 μL of a solution of D-luciferin (300 μg / mL) in DMEM was added to the cells. The resulting luminescence was measured using an IVIS50 or IVIS200 (Perkin-Elmer's Xenogen Product line) charge-coupled device camera and Living Image Software. Data represent the mean of three experiments, with errors expressed as ±SD.

[0318] Charge-fluctuating collapsible domains By delivering a mixture of eGFP mRNA and Cy5-labeled eGFP mRNA, we were able to simultaneously quantify and separate mRNA internalization and expression. Cy5 fluorescence indicates internalized mRNA, regardless of localization, while eGFP fluorescence indicates cytoplasmic release and subsequent mRNA expression. This method revealed the effects of backbone structure and cation type by comparing the cellular uptake and mRNA expression of two oligomers: CART D13:A11, the non-degradable guanidinium-containing D13:G12, and the non-degradable ammonium-containing D13:Pip13.

[0319] In these examples, when Cy-5 mRNA was formulated with CART D13:A11, high levels of both intracellular Cy5 and eGFP fluorescence were observed (Figure 10B, D). As a comparison, only Cy5 fluorescence was observed when non-disintegrating guanidinium-containing D13:G12 or non-disintegrating ammonium-containing D13:Pip13 were used. This example demonstrates that all three mRNA complexes were efficiently internalized by cells, but because they lack a backbone to disassemble, non-disintegrating polyplexes derived from D13:G12 and D13:Pip13 did not release mRNA on the timescale required to enable detectable levels of translation. Furthermore, the lack of eGFP expression observed with complexes formed with ammonium-containing D13:Pip13 suggests that the effectiveness of CART D13:A11 is not simply due to differences in the electrostatic binding affinity of the ammonium and guanidinium cations. Rather, specific, controlled loss of cationic charge through disruptive rearrangements is essential for efficacy.

[0320] Specifically, in these examples, to measure cellular uptake and release of oligomer / mRNA polyplexes, HeLa cells were treated with polyplexes prepared as described above using Cy5-labeled eGFP mRNA at a final concentration of 62.5 ng mRNA / well. Cells were prepared and analyzed by flow cytometry for both eGFP and Cy5 fluorescence as described above.

[0321] In additional examples, confocal microscopy was used to confirm the need for disruption in this embodiment. CART-mediated mRNA release and endosomal escape by CART D13:A11 compared to an ineffective transporter (D13:G12) was further confirmed by confocal microscopy, where simultaneous detection of the dansylated transporter, Cy5-mRNA, and tetramethylrhodamine (TRITC)-dextran 4400 was performed to stain for the endosomal compartment. Four hours after treatment with the CART D13:A11 / Cy5-mRNA complex, cells were imaged, revealing scattered fluorescence for both the Cy5 and dansyl fluorophores, indicating that these entities had successfully escaped from the endosome and dissociated from the polyplex (Figure 10D). Scattered fluorescence from (TRITC)-dextran 4400 was also observed, which may be due to endosomal rupture and release of the trapped dextran. As a comparison, when cells were treated with the non-disruptive D13:G12 / Cy5-mRNA complex, both Cy5 and dansyl fluorescence remained punctate and colocalized. These signals largely overlapped with punctate TRITC-dextran 4400, indicating endosomal uptake (Figure 10D). Taken together, these data strongly suggest that the charge-variable behavior of CART D13:A11 allows endosomal rupture and mRNA release, contributing to the high mRNA delivery efficiency of these agents.

[0322] Specifically, in the confocal microscopy example, HeLa cells were seeded at 10,000 cells / well in eight-chamber glass-bottom dishes (Nunc Lab-Tek II, Thermo Scientific) and allowed to adhere overnight. Prior to treatment, cells were washed with serum-free DMEM, and 200 μL of serum-free DMEM was added to each well along with 100 μM TRITC-dextran (Sigma, average molecular weight = 4,400). Cy5-eGFP mRNA polyplexes were prepared as described above (final concentration: 125 ng mRNA / well) and added to corresponding wells. Cells were incubated for 4 hours at 37°C, after which the medium was removed and 500 μL of 10 mM HEPES buffer solution in PBS was added. Cells were imaged using a Leica SP8 White Light Confocal microscope tuned for DAPI (dansyl), GFP, DsRed (TRITC-dextran), and Cy5.

[0323] In vivo delivery of mRNA by CART In certain examples, CARTs such as D13:A11 demonstrated superior gene expression in vivo. In one example, CART D13:A11 complexed with luciferase mRNA and administered intramuscularly to Balb / c mice produced high levels of bioluminescence. In comparison, mRNA not complexed with a delivery agent did not produce bioluminescence. This expression peaked at 4 hours and was still observable at 48 hours. In these examples, 7.5 μg of luciferase mRNA was complexed with D13:A11 at a 10:1 charge ratio in a total volume of 75 μL of PBS (pH 5.5) and injected into the right thigh muscle of Balb / c mice (Figures 11C and 11D).

[0324] In an additional example, high levels of bioluminescence were observed when D13:A11 was complexed with luciferase mRNA and administered via intravenous tail vein injection. High levels of expression persisted for 24 hours, with detectable bioluminescence even after 48 hours. Bioluminescence was primarily localized to the spleen and liver in these images (Figure 11E, F). No bioluminescent signal was observed when naked mRNA was administered IV. No toxicity was observed immediately after injection or for several weeks following treatment in any of the mice tested.

[0325] As an additional example, IV administration of Fluc mRNA complexed with CART O11:A9, D12:Gly10.5, and D14:α-methyl14.5 all resulted in significant luciferase expression in the liver and / or spleen. In these examples, 7.5 μg of luciferase mRNA complexed with CART at a 10:1 charge ratio was injected into the tail vein of Balb / c mice in a total volume of 75 μL of PBS (pH 5.5).

[0326] In an additional example, high levels of bioluminescence were observed when D13:A11 was complexed with luciferase mRNA and administered via intratumoral injection. In this example, 4 μg of luciferase mRNA was complexed with D13:A11 at a 10:1 charge ratio in a total volume of 75 μL of PBS (pH 5.5) and injected into the center of a solid subcutaneous A20 lymphoma tumor.

[0327] In a further example, CART O11:A9, D12:Gly10.5, and D14:α-methyl14.5 complexed with luciferase mRNA and administered by subcutaneous injection produced high levels of bioluminescence. In this example, 5 μg of luciferase mRNA was complexed with D13:A11 at a 10:1 charge ratio in a total volume of 75 μL of PBS (pH 5.5) and injected subcutaneously into the backs of Balb / C mice.

[0328] As a comparative example, when D12:G12 was complexed with Fluc mRNA according to the prior art, no bioluminescence was observed. In this example, 5 μg of luciferase mRNA was complexed with D13:A11 at a charge ratio of 10:1 in a total volume of 75 μL of PBS (pH 5.5) and injected subcutaneously into the backs of Balb / C mice.

[0329] mRNA delivery and expression via novel charged domains Comparative Example: Using the charge-variable motifs demonstrated by the original embodiment, new monomers and new CART oligomers of diverse chemical structures were synthesized. These CARTs were shown to effectively package and transfect fluorescently labeled (Cy5) mRNA, with several variants (e.g., D9:(7-lacto)11 and D12:(7-glycine)11) resulting in Cy5 intracellular fluorescence that was 1.1- to 1.2-fold higher than the parent D13:A11 system. Some species of these CARTs (a-Me, a-Me-co-A) showed some mRNA expression.

[0330] Lipophilic Polymer Domains Some examples include CARTs in which the lipophilic polymer domain (or, equivalently, the lipid block) of the material has been modified. Applicants have demonstrated the synthesis of CARTs containing cholesterol, nonenyl, stearyl, oleyl, and linoleyl side chains incorporated as the lipophilic polymer domain. These variants exhibit different performance when complexed with eGFP mRNA, with oleyl- and linoleyl-functionalized materials achieving transfection efficiencies similar to those of dodecyl-based systems.

[0331] Some efforts have suggested that altering the lipid blocks or lipophilic polymer domains affects particle size and zeta potential, with linoleyl and oleyl materials retaining a positive zeta potential longer than stearyl and dodecyl-based CARTs, as measured by dynamic light scattering.

[0332] New initiator In some examples, variations in the initiator alcohol for ring-opening polymerization have been explored to incorporate additional functionality into CARTs. Initiators that have been explored include fluorophores (dansyl and BDK), branched (3-arm) moieties, fluorescence quenchers (BHQ), and biotinylated derivatives. CARTs of equivalent monomer block length containing different initiator functional groups generally exhibit eGFP mRNA transfection efficiencies similar to the original embodiment.

[0333] In some embodiments, the initiator can have the formula: [ka]

[0334] Fluorescent initiators have been used to independently track the uptake of cargo (mRNA) and transporter (dansyl or BDK) by flow cytometry and confocal microscopy. Quenching initiators have been used to monitor the release of fluorescently labeled mRNA or miRNA cargo by monitoring the re-emission of fluorescence upon release from the quenched particle.

[0335] Initial results suggest that three-arm initiators can introduce structural changes (e.g., size and zeta potential) into CART / mRNA particles while maintaining mRNA transfection efficiency.

[0336] Early results suggest that biotin-derived initiators can be used either to target cancer cells or to attach additional cargo through biotin / streptavidin complementarity.

[0337] Example 5. Counterions A conventional example involves Boc deprotection using trifluoroacetic acid, resulting in a trifluoroacetate counteranion to the ammonium cation. Applicants have also demonstrated similar efficacy for CARTs deprotected under different conditions, including hydrochloric acid (HCl) to yield a chloride counterion. The resulting CART salts exhibit transfection efficiency / expression similar to the original embodiment.

[0338] Example 6. mRNA combinations In some embodiments, multiple mRNAs can be combined in a single CART complex. One example of this is the co-mixing of CART D13:A11 with a binary mixture of eGFP mRNA and mCherry mRNA. Under these conditions, the resulting ratio of eGFP to mCherry reflects the loading rate of the mRNA transcripts into the complex. Additional flow cytometry analysis showed that all cells were either double-positive or double-negative, suggesting that both transcripts were introduced into the same CART / mRNA particle, rather than each transcript being contained in a separate particle. This co-mixing was achieved by simply mixing the two transcripts prior to processing with CART, and no additional optimization was required.

[0339] In an additional example, we demonstrated the simultaneous expression of multiple mRNA transcripts by co-formulating CART D13:A11 / mRNA complexes with a binary mixture of eGFP mRNA and Fluc mRNA. These polyplexes induce the expression of two unique proteins at levels proportional to the mass % of that transcript in the formulation.

[0340] Example 7. Other oligonucleotide cargoes In addition to the delivery of mRNA, the present applicant has also explored various CART systems of the present invention to deliver other nucleic acid cargoes. The uptake and activity of siRNA, miRNA, minicircle DNA, and pDNA have been shown to be amenable to CART technology. For many of these cargoes, CART optimization (e.g., charge ratio, lipid domain length / degree of unsaturation) has been experimentally determined.

[0341] siRNA Several CARTs, including an unsubstituted morpholinone (D13:A11) and a seven-membered glycine-functionalized lactone (D10:(7-gly)11), demonstrated high levels of efficacy in siRNA-induced gene knockdown. Unexpectedly, delivery of low siRNA doses (e.g., 5 pmol / well) resulted in >85% knockdown of protein expression, even in systems such as D10:(7-gly)11, which demonstrated fluorescent mRNA uptake but no mRNA translation.

[0342] Comparative Example: The performance of these two CART delivery agents far exceeded that of the prior art, which consisted of a non-releasable transporter (D:G 4:4 (see WO2013036532A1 and PNAS 2012, 109(33), 13171-13176)), which only showed 50% knockdown under the above conditions.

[0343] Specifically, siRNA knockdown experiments were performed according to a modified procedure (Geihe, et. al. PNAS 2012, 109(33), 13171-13176). HaCaT cells expressing TdTomato / EGFP were seeded at 10,000 cells / well in a 96-well plate and incubated at 37°C for 18-24 hours. To prepare the siRNA / CART complex, 2 μL of a 25 μM stock of CBL3 siRNA was premixed with 17.60 μL of PBS (pH 5.5). CART was added to the above mixture from a 2 mM stock in DMSO to achieve a net cation:anion charge ratio of 10:1. The solution was mixed for 20 seconds and then 5 μL was added to each of three wells of a 96-well plate containing 100 μL of serum-free DMEM, resulting in a net siRNA concentration of 5 pmol / well. Cells were incubated with the complex for 4 hours, then the medium was replaced with serum-containing DMEM and incubated for 48 hours. After incubation, cells were washed with PBS, trypsinized, and analyzed by flow cytometry. The normalized expression rate of tdTOM was calculated by the formula: (mean fluorescence value of tdTOM-treated cells / mean fluorescence value of EGFP-treated cells) / (mean fluorescence value of tdTOM-untreated cells / mean fluorescence value of EGFP-untreated cells) × 100.

[0344] miRNA In some instances, the nucleic acid cargo is a fluorescently labeled miRNA. Flow cytometry analysis demonstrates robust uptake of labeled miRNA using CART D13:A11 compared with other transfection methods, such as Lipofectamine and poly(lactic-co-glycolic acid) nanoparticles. In these instances, the optimal charge ratio for miRNA uptake is 20:1 (cation:anion), which is higher than that observed with mRNA (10:1).

[0345] Specifically, miRNA uptake was measured using Cy5-labeled miRNA. Briefly, HeLa cells were seeded at 40,000 cells / well in 24-well plates and allowed to adhere overnight at 37°C. To prepare CART / miRNA complexes, 2.1 μL of a 0.2 μg / μL stock of Cy5-miRNA (sequence: Cy5-UpCpApACAUCAGUCUGAUAApGpCpUpA) was premixed with 5.72 μL of PBS, pH 5.5. Immediately prior to transfection, CART was added to this mixture from a 2 mM stock in DMSO to achieve a net cation:anion charge ratio of 10:1. This mixture was mixed for 20 seconds and then 5 μL was added to each of three wells of a 24-well plate containing 200 μL of serum-free DMEM, resulting in a final miRNA concentration of 125 ng / well. This mixture was then incubated for 8 hours at 37°C. After this, the cells were washed with PBS, trypsinized, and the intracellular fluorescence of Cy5 was determined by flow cytometry.

[0346] pDNA In some instances, the nucleic acid cargo was double-stranded plasmid DNA (pDNA) rather than the RNA cargo described above. In these cases, CARTs demonstrated improved efficacy (approximately 2.5-fold) over commercially available agents (e.g., Lipofectamine), with higher transfection efficiencies and median fluorescence intensities. Specifically, a plasmid encoding pPKCδ-GFP was used as the fluorescent reporter. In some cases, CARTs containing different lipid blocks or lipophilic polymer domains were more effective at delivering the plasmid than CART D13:A11, with oleyl- and linoleyl-functionalized materials showing the highest transfection efficiencies.

[0347] The particle size of CART / pDNA complexes was generally smaller than that of similar mRNA-containing particles, with hydrodynamic diameters ranging from 90 to 110 nm for most compounds and 390 nm for linoleyl-functionalized CARTs. The zeta potential followed the same progression as mRNA particles, starting at approximately +40 mV and decreasing to -40 mV as charge-variable rearrangements occurred.

[0348] Specific experimental details for transfection: For example, for transfection with CART O11:A9, CHO cells were seeded at 40,000 cells / well in 24-well plates and allowed to adhere overnight. pDNA:CART complexes were prepared by mixing PBS (pH 5.5) and pPKCδ-GFP with various amounts of oligomers from DMSO stock solutions to yield specific pDNA / CART ratios (optimized for a theoretical cation:anion ratio of 25:1; total volume 115 μL). The complexes were incubated for 20 seconds at room temperature before treatment. A control Lipofectamine 2000 solution was prepared in OptiMEM according to the manufacturer's instructions. Cells were washed with serum-free F-12 medium, and the pDNA / Lipofectamine solution was added. The final volume was 500 μL / well and 679 ng pDNA / well. After washing with serum-free F-12 medium, 37.5 μL of pDNA / CART complexes were added to each of three wells, for a total volume of 500 μL and a final pDNA concentration of 674 ng / well. Cells were incubated for 24 hours at 37°C, after which the medium was replaced with serum-containing F-12 medium. After an additional 24 hours, cells were trypsinized with trypsin-EDTA (0.25%) for 5 minutes at 37°C. Serum-containing F-12 medium was added, and the contents of each well were thoroughly centrifuged. The supernatant was removed, and the pelleted cells were resuspended in PBS (200 μL), transferred to FACS tubes, and read on a flow cytometry analyzer (LSR-II.UV, Stanford University). Data shown are the geometric mean fluorescence signals from 5,000–10,000 cells analyzed. For transfection efficiency, untreated cells were gated for lack of eGFP expression, and the data shown are the percentage of analyzed cells with higher eGFP expression than untreated cells. Error bars are expressed ±SD.

[0349] Stable transfection by delivery of CART / p transposase In some cases, multiple plasmids, such as a combination of pLuciferase (containing transposase recognition sites) and a plasmid encoding pTransposase separately, could be delivered simultaneously, resulting in stable expression of the target luciferase gene. After 6 days, the performance of CART O11:A9 significantly outperformed that of Lipofectamine, with bioluminescence levels over 10-fold higher.

[0350] In these examples, CHO cells were seeded at 10,000 cells per well in black 96-well plates and allowed to adhere overnight at 37°C. To prepare pDNA:CART complexes, PBS (pH 5.5) was first premixed with either 470 ng of pDNA encoding both firefly luciferase and TdTomato, or 585 ng of the same plasmid mixed with pTransposase (plasmid and pTransposase mixed at a 2:1 ratio). A Lipofectamine control was prepared in serum-free OptiMEM medium according to the manufacturer's specifications. Immediately prior to transfection, CART was added to the plasmid / PBS solution from a 2 mM stock solution in DMSO to achieve a net charge ratio of 5:1. The resulting complexes were incubated at room temperature for 20 seconds before being added to the cells. After rinsing the cells with serum-free F-12 medium, 7.3 μL of the complex was added to each of three wells of a 96-well plate containing serum-free medium, resulting in a final volume of 50 μL per well and a pDNA concentration of 137 or 171 ng / well. The cells were incubated with the treatment for 24 hours at 37°C, after which the medium was replaced with 100 μL of serum-containing F-12 medium containing 0.3 mg / mL luciferin. The resulting bioluminescence was measured using an IVIS50 or IVIS200 (Perkin-Elmer's Xenogen Product line) charge-coupled device camera and Living Image Software. Errors are expressed as ±SD. Once confluent, the cells were passaged into new wells using 0.25% trypsin.

[0351] In further examples, effective delivery and stable transfection by CART was confirmed with multiple plasmids. In these cases, the combination of plasmids encoding pLuciferase (both of which confer puromycin resistance and contain transposase recognition sites) and pTransposase separately conferred stable expression of the target luciferase gene along with puromycin resistance. Puromycin selection over several generations verified stable transfection and produced a twofold improvement in bioluminescence over Lipofectamine.

[0352] In these examples, CHO cells were seeded at 10,000 cells per well in a black 96-well plate and allowed to adhere overnight at 37°C. To prepare pDNA:CART complexes, 585 ng of pLuc-tdTom / pTransposase mix, encoding firefly luciferase and tdTomato, was mixed with PBS at pH 5.5. A control Lipofectamine 2000 solution was prepared in serum-free OptiMEM medium according to the manufacturer's specifications. Immediately prior to transfection, CART was added to a pDNA / PBS solution derived from a 2 mM stock solution in DMSO to achieve a net + / - charge ratio of 5:1. The complexes were incubated at room temperature for 20 seconds before being added to the cells. The cells were then rinsed with serum-free F-12 medium, and 7.3 μL of the complex was added to each of three wells of a 96-well plate containing serum-free medium, resulting in a final volume of 50 μL per well and a pDNA concentration of 171 ng / well. After 24 hours, the medium was replaced with 100 μL of serum-containing F-12 medium (containing 0.3 mg / mL luciferin and 6 μg / mL puromycin). Cells were imaged daily using an IVIS50 or IVIS200 (Perkin-Elmer's Xenogen Product line) charge-coupled device camera and Living Image Software. Errors are expressed as ±SD. After treatment, cells were passaged for 72 hours using 20 μL of 0.25% trypsin per well. Cells were incubated for 5 minutes and then diluted with 80 μL of serum-containing F-12 medium to a final volume of 100 μL per well. 30 μL of cell solution from each well was added to each of three new wells and diluted with 70 μL of serum-containing F-12 medium containing 0.3 mg / mL luciferin and 6 μg / mL puromycin. The 10 μL of cell solution remaining in the original well was diluted with 90 μL of serum-containing F-12 medium (containing 0.3 mg / mL luciferin and 6 μg / mL puromycin) to a final volume of 100 μL in each well.

[0353] Mini Circle DNA Further examples demonstrate the effective delivery of minicircle DNA (mcDNA) using CART. In these cases, both CART D13:A11 and oleyl CART O11:A9 delivered luciferase-encoding minicircle DNA into HeLa cells. This resulted in higher levels of luciferase expression (approximately 10-fold higher) than both Lipofectamine and polyethyleneimine (PEI) formulations.

[0354] Specifically, we used a luciferase reporter system to measure the uptake and expression of minicircle DNA. Briefly, HeLa cells were seeded at 15,000 cells / well in 96-well plates and allowed to adhere overnight at 37°C. To prepare CART / minicircle DNA complexes, 2.1 μL of a 0.2 μg / μL stock of minicircle DNA containing the firefly luciferase gene and constitutive promoter was premixed with 5.71 μL of PBS, pH 5.5. Control Lipofectamine and PEI were prepared according to the manufacturer's specifications. Then, just prior to transfection, CART was added to the above mixture from a 2 mM stock in DMSO to achieve a net cation:anion charge ratio of 10:1 (or any other charge ratio to be tested). This mixture was mixed for 20 seconds and then 2.5 μL was added to each of six wells of a 96-well plate containing 100 μL of serum-free DMEM, resulting in a final minicircle DNA concentration of 62.5 ng / well. This was incubated for 8 hours at 37°C, after which the medium was replaced with serum-containing DMEM containing 0.3 mg / mL luciferin, and the cells were imaged for bioluminescence using the IVIS camera system.

[0355] Example 8. Editing with CRISPR / Cas9 Several examples of CART-mediated transfection have demonstrated successful knock-in of targeted genes through specific CRISPR / Cas9 gene editing. In this case, mRNA encoding Cas9 was co-expressed with D13:A11 along with an sgRNA targeting the untranslated region of the mouse β-actin gene. A promoterless minicircle DNA containing a luciferase or mCherry gene and the same CRISPR cut site from the β-actin gene was also co-expressed. Using this system, Cas9 cleaved both the mouse genome and the minicircle, resulting in reporter gene expression only when the minicircle vector was inserted into the β-actin untranslated region and under the control of the β-actin promoter.

[0356] The co-formulation of Cas9 mRNA and sgRNA constructs with fluorescent reporter genes was performed to ensure that the presence of these cargoes did not interfere with CART-mediated nucleic acid delivery. In this case, eGFP mRNA was formulated as a 50% w / w mixture with either Cas9 mRNA or sgRNA, or in a formulation consisting of 50% eGFP mRNA, 25% Cas9 mRNA, and 25% sgRNA. In both of these examples, intracellular eGFP fluorescence was approximately 50% of that observed in a formulation consisting of eGFP mRNA alone. This same result was obtained with both CART D13:A11 and CART O11:A9, confirming the successful co-formulation of CRISPR components with the CART described above. Further characterization by DLS (DLS by standard procedures) showed that particles formed with mixtures of either D13:A11 or O11:A9 with Cas9 mRNA and sgRNA were approximately 173 nm in size, consistent with sizes observed for other mRNA transcripts.

[0357] When 3T3 mouse fibroblasts were treated with complexes formulated as described above using the luc minicircle construct, significant bioluminescence was observed. This signal was not observed when a scrambled sgRNA sequence was used, confirming that the signal was due to specific insertion of the luciferase gene into the 3T3 genome. The higher the proportion of minicircle DNA in the formulation, the greater the bioluminescence obtained. Additionally, when complexes were formed with mCherry-containing minicircles and exposed to 3T3 fibroblasts, flow cytometry analysis showed that approximately 2% of the target population exhibited strong mCherry fluorescence, indicating efficient genome integration.

[0358] Specifically, CRISPR / Cas9 gene editing was performed in 3T3 fibroblasts according to the following general procedure. Briefly, HeLa cells were seeded at 40,000 cells / well in a 24-well plate and allowed to adhere overnight at 37°C. To prepare the CART / oligonucleotide complex, 134 ng of Cas9 mRNA, 134 ng of sgRNA, and 134 ng of minicircle DNA containing the luciferase gene were mixed with 22.8 μL of PBS, pH 5.5. Immediately prior to transfection, CART was added to this mixture from a 2 mM stock in DMSO to achieve a net cation:anion charge ratio of 10:1. This mixture was mixed for 20 seconds and then 7.5 μL was added to each of three wells of a 24-well plate containing 400 μL of serum-free DMEM, resulting in a final concentration of 40 ng of each component. This mixture was then incubated for 8 hours at 37°C. After 8 hours, the medium was replaced with serum-containing DMEM and allowed to stand for 24–48 hours. After incubation, the medium was replaced with serum-containing DMEM containing 0.3 mg / mL luciferin, and the cells were imaged for bioluminescence using an IVIS camera system.

[0359] Example 9. Vaccination with mRNA vaccine In a vaccination example, neither CART alone nor CART combined with an adjuvant (no mRNA) induced protective antitumor immunity in a surrogate antigen model (Figure 2). Specifically, in this example, 6- to 8-week-old mice were co-inoculated with 107 tumor-specific antigen (TSA)-expressing A20 cells and then treated with 206 μg of CART alone or 206 μg of CART with 50 μg of adjuvant via subcutaneous injection at a different anatomical site from the tumor. Tumor size was measured daily, and mice with tumor diameters greater than 15 mm were euthanized according to the animal protocol. No extension of lifespan was observed in the inoculated mice in either of these control groups. CART does not elicit an immune response.

[0360] Prophylactic vaccination with concurrent exposure to tumor Vaccination either prior to or at the time of tumor inoculation results in antigen-specific protective responses. These studies demonstrate that vaccination with CART containing TSA-mRNA plus adjuvant induces protective immunity in mouse models bearing TSA-expressing tumor cells, either in a co-formulation embodiment or when administered separately at the time of vaccination. Treatment with CART / TSA-mRNA provides protection, and the use of an immunostimulatory adjuvant enhances efficacy. Mice treated with adjuvant alone rapidly developed large tumors and were sacrificed according to the animal protocol. A single dose produced a robust response, eliminating the need for a vaccine boost, and recall was possible upon a second tumor challenge (Figure 3).

[0361] Specifically, in this example, 6- to 8-week-old mice were co-inoculated with 107 tumor-specific antigen (TSA)-expressing A20 cells via subcutaneous injection and treated with either CART+TSA-mRNA+adjuvant (prepared as described above) or adjuvant alone via subcutaneous injection at a different anatomical site from the tumor. Tumor size was measured every other day for the first 30 days. Mice with tumor diameters greater than 15 mm were euthanized. In recall response experiments, surviving mice from the CART+TSA-mRNA+adjuvant-treated group were re-challenged with 107 TSA-expressing A20 cells.

[0362] Treatment of established tumors After the establishment of large tumors, vaccination with a CART / mRNA plus adjuvant vaccine induces antigen-specific therapeutic responses. Seven days after vaccination of large tumors, treatment with CART / mRNA plus adjuvant (3 x 3 μg of TSA-mRNA / CART complex) resulted in both tumor regression and remission. In this study (a total of five mice), tumor progression was significantly delayed, and even after approximately 60-90 days, 40% of mice (2 of 5 mice) were completely cured of their tumors. The performance of vaccination with mRNA / CART plus adjuvant surpasses that of established protein vaccination strategies, which have repeatedly demonstrated protective effects in animal studies.

[0363] Specifically, in this example, 6- to 8-week-old mice were subcutaneously inoculated with 107 tumor-specific antigen (TSA)-expressing A20 lymphoma cells. When tumor size reached 150 mm3, mice were treated three times with 3 μg of TSA-mRNA plus CART complexed with adjuvant, adjuvant alone, or saline by subcutaneous injection at different anatomical sites from the tumor, with a 4-day gap between each treatment. Tumor size was measured every other day for the first 40 days. Mice with tumor diameters greater than 15 mm were euthanized.

[0364] Intratumoral vaccination Intratumoral injection of CART / mRNA complexes results in tumor regression. Treatment of established A20 lymphoma tumors with mRNA complexed with CART results in protein expression in four distinct cell population subsets in the tumor. A protein candidate thought to modulate antitumor immune responses by enhancing T cell function demonstrated a significant effect on the treated tumors. Notably, growth rate retardation was also observed in distant, untreated tumors in the same animals (Figure 5). These findings suggest that in situ vaccination / boosting strategies have potential for treating metastatic disease.

[0365] Specifically, in this example, 6- to 8-week-old mice were inoculated subcutaneously with 107 A20 cells simultaneously at two different anatomical sites. Once tumor size reached 150 mm3, one tumor in each mouse was treated with three intratumoral injections of either CART + 10 µg of immune-modulating protein mRNA or saline. The other tumor was left untreated. Both tumors were measured every other day for the first 20 days or until the tumors reached a size that required euthanasia of the mice.

[0366] Example 10. Charge-variable release transporters (CARTs) for delivery and release of messenger RNA The present invention provides a novel class of synthetic biodegradable materials, charge-stable release transporters (CARTs), that are tunable and highly effective, synthesized in a few steps and characterized. The CARTs provided herein, in some embodiments, are structurally unique and operate via a previously unseen mechanism, initially complexing with mRNA and functioning as α-aminoester cations to protect and deliver the mRNA for efficient protein translation in both cell culture and animals. They then undergo a degradable, charge-neutralizing intramolecular rearrangement, changing their physical properties and releasing the mRNA. This novel mRNA delivery technology may be broadly applicable to numerous research and therapeutic applications.

[0367] Messenger RNA (mRNA) allows for the in vivo synthesis of the protein it encodes, providing the platform for a rapidly evolving class of gene therapy drugs that have the potential to transform the treatment of a variety of diseases, including cancer, genetic disorders, and infectious diseases. The use of methods and compositions according to certain embodiments of the invention to specifically express proteins encoded by mRNA can be utilized in research applications, imaging applications, therapeutic applications requiring protein supplementation or enhancement, and novel vaccination strategies in both prophylactic and immunotherapeutic applications.

[0368] Charge-variable release transporters (CARTs) according to some embodiments provided herein initially function as polycations that transiently complex with and protect polyanionic mRNAs, and then rapidly change their cationic charge, i.e., anion-binding capacity, through a controlled self-destruction mechanism.

[0369] As an example of a specific embodiment, we demonstrate the development of CARTs, a novel class of materials desi...

Claims

1. The following formula: 【Chemical 1】 A cationic amphiphilic polymer having the formula: During the ceremony, Ring A is a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl; z5 is 1 to 10, CART has the following formula: -L １ -[(LP １ ) ｚ１ -(LP ３ ) ｚ１ａ -(IM) ｚ２ -(LP ２ ) ｚ３ -(LP ４ ) ｚ３ｂ ] ｚ４ -L ２ -H wherein L １ and L ２ are independently a bond, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—, —NHC(O)—, or S(O) ２ -, -S(O)NH-, -NHC(O)NH-, substituted or unsubstituted C １ ~C ２０ Alkylene or heteroalkylene, substituted or unsubstituted C ３ ~C ８ Cycloalkylene or heterocycloalkylene, or substituted or unsubstituted C ６ ~C １０ arylene or heteroarylene, LP １ , LP ２ , LP ３ , and L.P. ４ are independently a bond or a lipophilic polymer domain, and LP １ , LP ２ , LP ３ , and L.P. ４ at least two of which are lipophilic polymer domains; z1, z1a, z3, and z3b are independently 0 to 100, and at least two of z1, z1a, z3, and z3b are not 0; z4 is 2 to 100, z2 is 2 to 100; Each lipophilic polymer domain independently has the formula: 【Chemistry 2】 and where R is an unbranched C alkyl group which may be fully saturated, monounsaturated, or polyunsaturated. １ ~C ３０ alkyl, or cholesterol, IM is represented by the following formula (1), (2), or (3): 【Chemistry 3】 A cationic amphiphilic polymer, which is a pH-sensitive disintegration domain of

2. 2. The cationic amphiphilic polymer according to claim 1, wherein z3 and z3b are each 0.

3. LP １ and L.P. ３ A cationic amphiphilic polymer according to claim 2, wherein each of

4. 4. The cationic amphiphilic polymer of claim 3, wherein each R is independently stearyl, oleyl, linoleyl, dodecyl, or nonenyl.

5. 5. The cationic amphiphilic polymer of claim 4, wherein ring A is a substituted or unsubstituted aryl, optionally wherein the aryl is benzyl, phenyl, or naphthalenyl.

6. L １ 6. The cationic amphiphilic polymer of claim 5, wherein is a substituted or unsubstituted alkylene, a substituted or unsubstituted heteroalkylene, a substituted or unsubstituted cycloalkylene, a substituted or unsubstituted heterocycloalkylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene.

7. L １ but, 【Chemistry 4】 7. The cationic amphiphilic polymer according to claim 6, wherein

8. A nanoparticle composition comprising a plurality of complexes of the cationic amphiphilic polymer of any one of claims 1 to 7 with nucleic acids.

9. The nanoparticle composition of claim 8 , wherein the nucleic acid is RNA or DNA.

10. 9. The nanoparticle composition of claim 8, wherein the nucleic acid is messenger RNA, small interfering RNA, short hairpin RNA, microRNA, guide RNA, CRISPR RNA, transactivating RNA, plasmid DNA, minicircle DNA, or genomic DNA.

11. A pharmaceutical composition comprising the nanoparticle composition of claim 8 and a pharmaceutically acceptable excipient.

12. A vaccine composition comprising the nanoparticle composition of claim 8 and optionally an immunoadjuvant.

13. A method for transfecting a nucleic acid into a cell in vitro, comprising contacting the cell with the nanoparticle composition of claim 8.

14. 10. A method for in vitro gene editing comprising contacting a cell with the nanoparticle composition of claim 8, wherein the nucleic acid comprises a first nucleotide encoding a CRISPR-Cas system guide RNA that hybridizes to a target sequence in the genome of the cell, and a second nucleotide encoding a Cas9 protein, wherein the first and second nucleotides are in the same or different vectors.

15. 13. The pharmaceutical composition of claim 11 or the vaccine composition of claim 12 for inducing an immune response in a subject in need thereof.

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