Lipidated Cationic Peptide-PEG Compositions for Nucleic Acid Delivery

Abstract

The present disclosure relates to complexes and compositions of cationic compounds combined with a low mass percent of PEGylated compounds, such as PEGylated lipids, for delivery of nucleic acids and other polyanionic cargoes to cells, methods for preparing complexes and compositions comprising one or more cationic compounds and a low mass percent of PEGylated compounds with polyanionic compounds, and methods for delivering polyanionic compounds to cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 885,022, filed August 9, 2019, and U.S. Provisional Application No. 62 / 907,470, filed September 27, 2019, which are incorporated by reference in their entireties.

[0002] Technical Field The present disclosure relates to complexes and compositions of cationic compounds combined with low mass percent PEGylated compounds, such as PEGylated lipids or PEGylated diacylglycerol derivatives, for delivery of nucleic acids and other polyanionic cargoes.More specifically, the present disclosure relates to tertiary amino lipidated and / or PEGylated peptoids, and their complexes and compositions combined with small amounts of PEGylated lipids for nucleic acid delivery.The present disclosure also provides methods for preparing complexes and compositions comprising one or more cationic compounds and low mass percent PEGylated compounds with polyanionic compounds, such as nucleic acids, and methods for delivering polyanionic compounds to cells. [Background technology]

[0003] The ability to consistently and effectively deliver nucleic acids to cells is a key requirement for the practical implementation of gene therapy, siRNA-, miRNA-, antisense-, or mRNA-based therapy in clinical settings. A variety of cationic compounds have been explored as delivery vehicles, which can encapsulate polyanionic nucleic acids and stabilize the nucleic acid against nucleases in vivo. Lipitoid 1 (shown in Figure 1) is an example of a cationic peptoid-phospholipid conjugate construct that has been widely evaluated as a stand-alone cationic delivery vehicle. However, nucleic acid and lipidoid complexes can still be subject to additional complications in vivo, including, for example, particle instability and aggregation.

[0004] Multicomponent lipid mixtures, such as lipid nanoparticle formulations, have become an area of ​​great focus to achieve the necessary pharmacokinetic properties to support in vivo nucleic acid delivery, including improved formulation (particle) stability. Lipid nanoparticles incorporate cationic compounds along with a number of compatible lipid co-components (e.g., shielding lipids, structural lipids, and phospholipids) that provide charge insulation and confer structural and chemical stability to the delivery vehicle-nucleic acid complex under physiological conditions.

[0005] Despite the promise of lipid nanoparticle formulations to achieve the properties required for in vivo nucleic acid transfer, optimizing lipid formulations is often difficult due to the interaction of many variables, including identifying compatible co-components for the nucleic acid to be delivered and carefully adjusting the relative amounts of each component to achieve the desired pharmacokinetic properties. Furthermore, most lipid-based formulations usually contain four or more separate components, and the majority of existing formulations consist primarily of delivery components rather than the nucleic acid material itself. As a result, the active therapeutic agent constitutes only a small portion of the overall formulation by weight.

[0006] Thus, there remains a need for formulations for gene therapy that achieve effective and efficient delivery of higher mass percentages of active nucleic acid material without compromising the pharmacokinetic properties required for in vivo applications. Summary of the Invention

[0007] The present disclosure provides complexes and compositions comprising cationic compounds, such as lipidoids or tertiary amino lipidated and / or PEGylated cationic peptide compounds, in combination with PEGylated compounds, such as PEG lipids, as delivery systems for polyanionic compounds. The complexes and compositions described herein utilize a minimal amount of PEG compound, on a weight basis, relative to the content of cationic and polyanionic compounds, to stabilize against particle aggregation, thus providing easily optimizable and stable formulations with a high mass percent of active polyanionic material.

[0008] In one aspect, the present disclosure provides: (i) one or more polyanionic compounds; (ii) one or more PEG compounds; and (iii) A composition comprising a complex of one or more cationic compounds, wherein the one or more cationic compounds are (a) one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) or salts thereof, [ka] During the ceremony, m is 0, n is an integer from 0 to 5; s is an integer from 0 to 5; t is 0, At least one of n and s is other than 0; r is an integer from 1 to 20; each o is independently an integer 0, 1, 2, 3, or 4; each q is independently an integer 0, 1, 2, 3, or 4; each p is independently an integer 1 or 2; R 1 is -H, alkyl, alkylaryl, -COR 1a or a lipid moiety, R 1a is -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl; Each R 2 is, independently, Formula -CH2CH2O(CH2CH2O) u an ethylene glycol portion of CH3, and each u is independently an integer from 1 to 200; Each R 3 is independently a lipid moiety, Each R 4 is independently a neutral spacer moiety or a lipid moiety, Each R 5 is independently a cationic moiety; Each R 6are independently represented by the formula -CH2CH2O(CH2CH2O) v an ethylene glycol portion of CH3, and each v is independently an integer from 1 to 200; R 7 is -H, alkyl, acyl, -OH, -OR 7a , -NH2, -NHR 7a , or a lipid moiety, R 7a is an alkyl, acyl, or lipid moiety; Each R a and R bは、独立して、-H、C one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) or salts thereof, wherein the tertiary amino acid is 1-C4-alkyl or a side chain moiety found in naturally or non-naturally occurring amino acids; (b) one or more lipoids of formula (II), [ka] During the ceremony, x is an integer from 1 to 100; Each R 9 is independently a lipid moiety, Each R 10 is independently a cationic or neutral spacer moiety, (c) one or more lipid-like compounds of formula (III), [ka] During the ceremony, Each R 11 is independently substituted or unsubstituted alkyl; Each R 12 is independently substituted or unsubstituted alkyl; Each R 13 are independently hydrogen or substituted or unsubstituted alkyl; one or more lipid-like compounds of formula (III), wherein each y is independently an integer from 1 to 8; or (d) any combination thereof; The composition includes one or more cationic compounds, one or more PEG compounds, and one or more polyanionic compounds, having a combined weight percent of at least 90% w / w of the complex.

[0009] In some embodiments of this aspect, the one or more cationic compounds are one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds. In certain embodiments, the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprise a block of N-lipidated amino acid residues, and either n or s is at least 2. In certain embodiments, the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprise a block of N-lipidated amino acid residues, and either n or s is 4. In other embodiments of this aspect, each R 3 is independently 22 -Alkyl or C4-C 22 -alkenyl, C4-C 22 -alkenyl is optionally mono- or polyunsaturated. In certain embodiments, which may be combined with one or more of the preceding embodiments, each R 3 is independently C8-C 12 In still other embodiments, which may be combined with one or more of the preceding embodiments, each R 3 are independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl. In some embodiments, which may be combined with one or more of the preceding embodiments, one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds are provided with two different lipid moieties R 3a and R 3b In yet another embodiment, which may be combined with one or more of the preceding embodiments, at least one lipid moiety R 3 (or R3a Or R 3b ) is a branched aliphatic moiety. In certain embodiments, which may be combined with one or more of the preceding embodiments, at least one lipid moiety R 3 (or R 3a Or R 3b ) is 2-ethylhex-1-yl.

[0010] In some embodiments of this aspect, at least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprises a cationic moiety R 5 In other embodiments, which may be combined with one or more of any of the preceding embodiments, each R 5 is independently aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, N-heterocyclylalkyl, or N-heteroaryl. In certain embodiments, which may be combined with any of the preceding embodiments, each R 5 is, independently, [ka] In another embodiment, each R 5 is -(C1-C4 alkylene)-NR 5a R 5b and R 5a and R 5b are independently H, C1-C3 alkyl, or C6-C 10 aryl or R 5a and R 5b are taken together with the nitrogen atom to which they are attached to form a 4- to 7-membered N-heterocycloalkyl ring, which may optionally contain one additional heteroatom selected from the group consisting of N and O. In yet other embodiments, each R 5 teeth, [ka] In some embodiments, each R 5 is independently [ka] and R 5a and R 5b are independently H, C1-C3 alkyl, or C6-C 10 aryl or R 5a and R 5b together with the nitrogen atom to which they are attached form a 4- to 7-membered N-heterocycloalkyl ring, which may optionally contain one additional heteroatom selected from the group consisting of N and O. In certain embodiments, each R 5 teeth, [ka] It is.

[0011] In yet another embodiment of this aspect, each neutral spacer moiety R 4 is independently C1-C4-alkyl, C1-C5 straight chain alkyl substituted with cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, optionally substituted with one or more substituents -OH, halo, or alkoxy. In some embodiments, which may be combined with one or more of any of the preceding embodiments, each neutral spacer moiety R 4 are independently -CH3, [ka] In certain embodiments, each neutral spacer moiety R 4 teeth, [ka] It is.

[0012] In yet a further embodiment, which may be combined with one or more of any of the preceding embodiments, at least one of the tertiary amino lipidated and / or PEGylated cationic peptide compounds is R 5 The cationic domain comprises at least two amino acid residues having a cationic moiety, and each of the at least two cationic amino acid residues in the cationic domain is linked to a neutral spacer or a lipid moiety R 4 In some embodiments of this aspect, which may be combined with one or more of any of the preceding embodiments, at least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds is separated by at least one amino acid residue having at least one trimeric subunit -R カチオン -R 中性 -R 中性 Including R カチオン is a cationic moiety R 5 Each R 中性 However, the neutral spacer portion R 4 In another embodiment of this aspect, at least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprises at least one trimeric subunit -R カチオン -R 中性 -R 脂質 Including R カチオン is a cationic moiety R 5 Each R 中性 However, the neutral spacer portion R 4 Each R 脂質 But the lipid portion R 4 In certain embodiments of any of the foregoing embodiments, each cationic moiety R 5 teeth, [ka] and each neutral spacer moiety R 4 teeth, [ka] It is.

[0013] In other embodiments of this aspect, which may be combined with any one or more of the preceding embodiments, R a and R b is independently selected from the group consisting of -H and -CH3. In other particular embodiments of this aspect, which may be combined with any one or more of the preceding embodiments, R a and R b is -H.

[0014] In yet other embodiments, which may be combined with any one or more of the preceding embodiments, the one or more cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of peptide compound to PEG compound of 90:10 to 99:1. In yet further embodiments of this aspect, which may be combined with any one or more of the preceding embodiments, the one or more cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of peptide compound to PEG compound of 95:5 to 98:2. In certain embodiments of this aspect, which may be combined with any one or more of the preceding embodiments, the one or more PEG compounds comprise DMG-PEG2000. In still further embodiments of this aspect, which may be combined with any one or more of the preceding embodiments, the one or more PEG compounds comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) or salts thereof, [ka] During the ceremony, m is an integer from 0 to 15; n is an integer from 0 to 5; s is an integer from 0 to 5; t is an integer from 0 to 15; At least one of m and t is other than 0; r is an integer from 1 to 20; each o is independently an integer 0, 1, 2, 3, or 4; each q is independently an integer 0, 1, 2, 3, or 4; each p is independently an integer 1 or 2; R 1 is -H, alkyl, alkylaryl, -COR 1a or a lipid moiety, R 1a is -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl; Each R 2 are independently represented by the formula -CH2CH2O(CH2CH2O) u an ethylene glycol portion of CH3, and each u is independently an integer from 1 to 200; Each R 3 is independently a lipid moiety, Each R 4 is independently a neutral spacer moiety or a lipid moiety; Each R 5 is independently a cationic moiety; Each R 6 are independently represented by the formula -CH2CH2O(CH2CH2O) v an ethylene glycol portion of CH3, and each v is independently an integer from 1 to 200; R 7 is -H, alkyl, acyl, -OH, -OR 7a , -NH2, -NHR 7a , or a lipid moiety, R 7a is an alkyl, acyl, or lipid moiety; Each R a and R bは、独立して、-H、C 1-C4-alkyl or a side chain moiety found in natural or non-naturally occurring amino acids.

[0015] In still other embodiments of this aspect, where the one or more PEG compounds comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, when m is an integer between 0 and 3, each u is independently an integer between 30 and 50, and when m is an integer between 4 and 15, each u is independently an integer between 1 and 10. In still other embodiments, where the one or more PEG compounds comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, which may be combined with any one or more of the preceding embodiments, the composition comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, when t is an integer between 0 and 3, each v is independently an integer between 30 and 50, and when t is an integer between 4 and 15, each v is independently an integer between 1 and 10. In certain embodiments, which may be combined with any of the preceding embodiments, m is 1 and u is an integer between 40 and 45, or t is 1 and v is an integer between 40 and 45.

[0016] In yet another embodiment of this aspect, which may be combined with any one or more of the preceding embodiments, the one or more polyanionic compounds include nucleic acids. In yet another embodiment of this aspect, which may be combined with any one or more of the preceding embodiments, the one or more polyanionic compounds include nucleic acids, and the mass ratio of the one or more cationic peptide compounds to the nucleic acids is 0.5:1 to 20:1. In some embodiments of this aspect, which may be combined with any one or more of the preceding embodiments, the one or more polyanionic compounds include nucleic acids, and the nucleic acids are mRNAs encoding polypeptides. In certain embodiments of this aspect, which may be combined with any one or more of the preceding embodiments, the one or more polyanionic compounds include nucleic acids, and the nucleic acids are mRNAs encoding proteins.

[0017] In yet another embodiment of this aspect, which may be combined with any one or more of the above-mentioned embodiments, the complex further comprises one or more second agents.In yet another embodiment, which may be combined with any one of the above-mentioned embodiments, the one or more second agents are selected from the group consisting of polyethylene glycol, targeting elements, and combinations thereof.In yet another embodiment, which may be combined with any one of the above-mentioned embodiments, the complex further comprises one or more small molecule active agents or drug substances.

[0018] In yet another aspect of the present disclosure, there is provided herein a method for delivering a polyanionic compound to a cell, comprising contacting the cell with the composition of the above aspect in any and all of its various embodiments.In certain embodiments of this aspect, the contacting is by endocytosis.In other embodiments of this aspect, it may be combined with the above aspect, and the cell is contacted in vivo or in vitro.In certain embodiments of this aspect, it may be combined with either or both of the above embodiments, and the cell expresses the polypeptide after contacting with the composition.In still further embodiments, it may be combined with one or more of the above embodiments, and the cell expresses the protein after contacting with the composition.

[0019] An additional aspect of the present disclosure provides a method for preparing any and all of the compositions of the aforementioned aspects and various embodiments thereof related to compositions by contacting one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds with one or more PEG compounds and one or more polyanionic compounds. In an embodiment of this aspect, the composition is formed by contacting a solution containing one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more PEG compounds with a solution containing one or more polyanionic compounds.

[0020] In yet another aspect of the disclosure, provided herein is a method of delivering a polyanionic compound to the lungs of a subject, comprising administering to the subject a composition of the preceding aspect, wherein the one or more cationic compounds are one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I), and the mass ratio of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) to the polyanionic compound is 1:1 to 10:1. In some embodiments of this aspect, the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) include Compound 8, Compound 24, Compound 28, Compound 73, Compound 79, Compound 93, Compound 98, or Compound 112, or any combination thereof. In some embodiments of this aspect, the one or more cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of peptide compounds to PEG compounds of 90:10 to 99:1. In other embodiments, which may be combined with the preceding embodiments, administration is intravenous (IV) (e.g., bolus injection or intravenous infusion), subcutaneous (SC), intramuscular (IM), intrathecal, or intratumoral injection. In other embodiments, which may be combined with any of the preceding embodiments, the polyanionic compound comprises a nucleic acid that is an mRNA encoding a polypeptide, and the cells express the polypeptide after contacting with the composition. In certain embodiments, which may be combined with any of the preceding embodiments, the polyanionic compound comprises a nucleic acid that is an mRNA encoding a protein, and the cells express the protein after contacting with the composition.

[0021] In another aspect of the disclosure, provided herein is a method of delivering a polyanionic compound to the lung and / or spleen of a subject, comprising administering to the subject the composition of claim 1, wherein the one or more cationic compounds are one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I), and the mass ratio of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) to the polyanionic compound is 1:1 to 10:1. In some embodiments of this aspect, the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) include Compound 8, Compound 19, Compound 24, Compound 28, Compound 73, Compound 79, Compound 93, Compound 98, or Compound 112, or any combination thereof. In some embodiments of this aspect, the one or more cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of peptide compounds to PEG compounds of 90:10 to 99:1. In other embodiments, which may be combined with the preceding embodiments, administration is intravenous (IV) (e.g., bolus injection or intravenous infusion), subcutaneous (SC), intramuscular (IM), intrathecal, or intratumoral injection. In other embodiments, which may be combined with any of the preceding embodiments, the polyanionic compound comprises a nucleic acid that is an mRNA encoding a polypeptide, and the cells express the polypeptide after contacting with the composition. In certain embodiments, which may be combined with any of the preceding embodiments, the polyanionic compound comprises a nucleic acid that is an mRNA encoding a protein, and the cells express the protein after contacting with the composition. [Brief description of the drawings]

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] [Figure 1] FIG. 1 shows an exemplary secondary amine phospholipidated cationic peptide, "Lipitoid 1." [Diagram 2] Figures 2A-2F generally depict tertiary amino lipidated cationic peptoids. Figure 2A depicts a generalized structure of a tertiary amino lipidated peptoid. Figure 2B depicts an exemplary tertiary amino lipidated cationic peptoid. Figure 2C depicts an exemplary lipid monomer. Figure 2D depicts an exemplary cationic monomer. Figure 2E depicts an exemplary spacer monomer and polyethylene glycol monomer. Figure 2F depicts an exemplary tertiary amino lipidated cationic peptoid compound 8. [Diagram 3] Figures 3A-3E show an exemplary process for preparing tertiary amino lipidated and / or PEGylated cationic peptide compounds via sequential addition of amino acid residues on the solid phase. Figure 3A shows a generalized acylation reaction of a resin-bound secondary amine with the acylating agent bromoacetic acid. Figure 3B shows the reaction of the resulting acylated product with an appropriate amine via nucleophilic displacement to generate the corresponding N-substituted amino acid residue. Figures 3C and 3D show successive iterations of acylation and nucleophilic displacement reactions to generate the desired resin-bound cationic peptide compound. Figure 3E shows the cleavage of the cationic peptide compound from the solid resin support to provide the free peptide. [Figure 4] Figures 4A and 4B show the variation in particle size stability over time and transfection efficiency in formulations of an exemplary aminolipidated peptoid compound 8 formulated with Fluc mRNA alone (no PEG compound) or with either 2% w / w or 5% w / w DMG-PEG2000 incorporation. Figure 4A shows a graph of the number average particle size observed for each of the three formulations immediately after formulation (30 minutes after formulation) and 8 hours after formulation. Figure 4B shows a bar graph of the average bioluminescence recorded 8 hours after treatment for HeLa cells treated with the three formulations. [Diagram 5]Figures 5A-5D show the in vivo systemic distribution of a formulation of aminolipidated peptoid compound 28 with Fluc mRNA and 2% w / w DMG-PEG2000 as a function of storage time at 4°C prior to injection. Figures 5A-5C show the systemic luminescence after 1 hour (Figure 5A), 3 days (Figure 5B), and 7 days (Figure 5C) of storage. Figure 5D shows plots of the total photon flux observed at each time point. [Figure 6] Figures 6A-6E show the bioluminescence (RLU) recorded in HeLa cells 24 hours after treatment with formulations of aminolipidated peptoids and Fluc mRNA with or without PEG incorporation (DMG-PEG2000). Figure 6A shows the bioluminescence observed in cells treated with formulations of aminolipidated peptoid compounds (1-18) and Fluc mRNA only (no DMG-PEG2000). Figures 6B and 6C show the bioluminescence observed in cells treated with formulations of aminolipidated peptoid compounds (1-36) and Fluc mRNA with 2% w / w DMG-PEG2000 added. Figures 6D and 6E show the average bioluminescence observed in HeLa cells treated with formulations of aminolipidated peptoid compounds 1-72 and Fluc mRNA with 2% w / w DMG-PEG2000 added at a peptoid:mRNA ratio of 5:1. [Figure 7] Figures 7A-7C show the in vivo systemic and organ-specific distribution of a formulation of aminolipidated peptoid with Fluc mRNA and 2% w / w DMG-PEG2000, as represented by bioluminescence intensity and total photon flux. Figure 7A shows the systemic distribution of the formulation in Balb / c mice. Figure 7B shows the corresponding organ-specific biodistribution of the formulation (lung, kidney, liver, spleen, inguinal lymph node, and mesenteric lymph node). Figure 7C shows a bar graph quantifying the organ-specific biodistribution of the formulation as a function of total photon flux. [Figure 8]Figures 8A-8C show the total photon flux observed in Balb / c mice treated with subcutaneous and intramuscular injections of formulations of aminolipidated peptoids with 2% w / w DMG-PEG2000 and Fluc mRNA. Figure 8A shows plots of the total photon flux in a formulation of aminolipidated peptoid compound 28 with 2% w / w DMG-PEG2000 and Fluc mRNA at different peptoid:mRNA ratios (1:1, 2:1, 5:1, 7.5:1 and 10:1) administered either subcutaneously (SC) or intramuscularly (IM) to Balb / c mice. Figures 8B and 8C show plots of the total photon flux observed in mice treated with formulations of aminolipidated peptoid compounds 1-72 with 2% DMG-PEG2000 and Fluc mRNA at a peptoid:mRNA ratio of 5:1. [Figure 9] 9A-9C show normalized total photon flux for in vitro assessment of transfection efficiency in various cell types (HeLa in FIG. 9A, HepG2 in FIG. 9B, and JAWSII in FIG. 9C) of Fluc mRNA delivered in different formulations of aminolipidated peptoids (compounds 24 and 79), linear aminoPEGylated peptoids (compounds 48, 56, 64, and 72), and branched aminoPEGylated peptoids (compounds 106, 107, 108, and 109) in combination with commercially available PEG lipids (DMG-PEG2000, 18:0 PEG1000PE, 14:0 PEG2000PE, 14:0 PEG5000PE). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The following description sets forth example methods, parameters, etc., however, it should be recognized that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0025] The present disclosure relates to complexes and compositions of cationic compounds, such as tertiary amino lipidated and / or PEGylated cationic peptide compounds, with low mass percent of PEGylated compounds, such as PEGylated lipids, for delivery of nucleic acids and other polyanionic cargoes.More specifically, the present disclosure relates to tertiary amino lipidated and / or PEGylated peptoids, and their complexes and compositions with low concentrations of PEGylated lipids, for delivery of nucleic acids.The present disclosure also provides methods for preparing complexes and compositions that include one or more tertiary amino lipidated and / or PEGylated cationic peptides and low mass percent of PEGylated compounds with polyanionic compounds, such as nucleic acids, and methods for delivering polyanionic compounds to cells.In one aspect, the present disclosure provides complexes and compositions that include one or more polyanionic compounds, one or more PEGylated compounds, and one or more cationic compounds.

[0026] It should be recognized that one or more cationic compounds can form complexes with one or more polyanionic compounds in the composition due to their favorable charge-charge interactions.It should be further recognized that one or more PEGylated compounds can further bind to the charge complex of cationic compound-polyanionic compound to form complexes.It should be understood that the compositions provided herein include all components present in the complex that includes polyanionic compound with cationic compound and PEGylated compound, in addition to any additional components that do not form complexes with cationic compound and polyanionic compound.

[0027] Surprisingly, it has been observed that the addition of PEGylated compounds (such as the PEG lipid DMG-PEG2000) to complexes and compositions containing cationic delivery vehicle compounds and polyanionic compounds can provide improved particle stability over time without the need for further incorporation of structural lipids and phospholipids used as co-components in standard lipid nanoparticle formulations. Furthermore, it has also been observed that long-term stabilizing effects can be achieved by incorporating only a small mass percentage of PEGylated compounds relative to the total complex, thus providing a stable, easily optimizable formulation that can support the delivery of a much larger weight fraction of active polyanionic substances in the complex to cells. It has been shown that complexes containing a small mass percentage of PEGylated compounds, and therefore compositions containing the complexes, have transfection efficiencies of their polyanionic cargo comparable to complexes of cationic compounds and polyanionic compounds alone, but with significantly improved formulation stability.

[0028] As used herein, "alkenyl" refers to an unsaturated linear or branched monovalent hydrocarbon chain, or combinations thereof, having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C=C) and having the specified number of carbon atoms (i.e., C-C 10 means 2 to 10 carbon atoms). Alkenyl groups may be in the "cis" or "trans" configuration, or alternatively in the "E" or "Z" configuration. Particular alkenyl groups are those having 2 to 20 carbon atoms ("C2-C 20 alkenyl"), those having 2 to 8 carbon atoms ("C2-C8 alkenyl"), those having 2 to 6 carbon atoms ("C2-C6 alkenyl"), or those having 2 to 4 carbon atoms ("C2-C4 alkenyl"). Examples of alkenyl include, but are not limited to, groups such as ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (or allyl), 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, homologs and isomers thereof.

[0029] The term "alkyl" refers to saturated linear and branched monovalent hydrocarbon structures and combinations thereof having the specified number of carbon atoms (i.e., C1-C2). 10 means 1 to 10 carbons). Particular alkyl groups are those having 1 to 20 carbon atoms ("C1-C 20 More specific alkyl groups are those having 1 to 8 carbon atoms ("C1-C8 alkyl"), 3 to 8 carbon atoms ("C3-C8 alkyl"), 1 to 6 carbon atoms ("C1-C6 alkyl"), 1 to 5 carbon atoms ("C1-C5 alkyl"), or 1 to 4 carbon atoms ("C1-C4 alkyl"). Examples of alkyl include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, e.g., homologs and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0030] As used herein, "alkylene" refers to the same residues as alkyl, but with divalency. Particular alkylene groups are those having 1 to 6 carbon atoms ("C1-C6 alkylene"), 1 to 5 carbon atoms ("C1-C5 alkylene"), 1 to 4 carbon atoms ("C1-C4 alkylene"), or 1 to 3 carbon atoms ("C1-C3 alkylene"). Examples of alkylene include, but are not limited to, groups such as methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), and the like.

[0031] As used herein, "alkynyl" refers to an unsaturated linear or branched monovalent hydrocarbon chain, or combinations thereof, having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C) and having the specified number of carbon atoms (i.e., C-C 10 means 2 to 10 carbon atoms). Particular alkynyl groups are those having 2 to 20 carbon atoms ("C 20alkynyl"), those having 2 to 8 carbon atoms ("C2-C8 alkynyl"), those having 2 to 6 carbon atoms ("C2-C6 alkynyl"), or those having 2 to 4 carbon atoms ("C2-C4 alkynyl"). Examples of alkynyl include, but are not limited to, groups such as ethynyl (or acetylenyl), prop-1-ynyl, prop-2-ynyl (or propargyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs and isomers thereof.

[0032] The term "aryl" refers to and includes polyunsaturated aromatic hydrocarbon groups. Aryl may contain additional fused rings (e.g., 1-3 rings), including additional fused aryl, heteroaryl, cycloalkyl, and / or heterocyclyl rings. In one variation, the aryl group contains 6-14 ring carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, and the like.

[0033] "Carbonyl" refers to the group C=O.

[0034] As used herein, a "complex" includes any chemical association between two or more molecules, which may be mediated by ionic interactions, hydrogen bonds, van der Waals interactions, metal-ligand coordination, other chemical forces, and combinations of one or more of the above. Complexes may form higher-order structures, including, for example, polyplexes, coacervate complexes, nanocomplexes, nanoparticles, and microparticles.

[0035] The term "cycloalkyl" refers to and includes cyclic monovalent hydrocarbon structures, which may be fully saturated, monounsaturated, or polyunsaturated, but are non-aromatic, having a specified number of carbon atoms (e.g., C-C 10means 1-10 carbons). Cycloalkyls can consist of one ring, such as cyclohexyl, or multiple rings, such as adamantyl, but aryl groups are excluded. Cycloalkyls containing more than one ring can be fused, spiro, or bridged, or combinations thereof. Preferred cycloalkyls are cyclic hydrocarbons having 3-13 annular carbon atoms. More preferred cycloalkyls are cyclic hydrocarbons having 3-8 annular carbon atoms ("C3-C8 cycloalkyls"). Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, and the like.

[0036] "Halo" or "halogen" refers to elements of group 17 having atomic numbers 9-85. Preferred halo groups include fluoro, chloro, bromo, and iodo. When a residue is substituted with more than one halogen, it may be referred to by using a prefix corresponding to the number of halogen moieties attached, e.g., dihaloaryl, dihaloalkyl, trihaloaryl, etc. refer to aryl and alkyl substituted with two ("di") or three ("tri") halo groups, which may be the same halo, but are not necessarily the same, so that 4-chloro-3-fluorophenyl is within the scope of dihaloaryl. An alkyl group in which each hydrogen is replaced with a halo group is referred to as a "perhaloalkyl." A preferred perhaloalkyl group is trifluoroalkyl (-CF3). Similarly, "perhaloalkoxy" refers to an alkoxy group in which a halogen replaces each H in the hydrocarbon that makes up the alkyl portion of the alkoxy group. An example of a perhaloalkoxy group is trifluoromethoxy (-OCF3).

[0037] The term "heteroaryl" refers to an unsaturated aromatic cyclic group having 1-10 cyclic carbon atoms and at least one cyclic heteroatom, including but not limited to nitrogen, oxygen, and sulfur, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group can be attached to the remainder of the molecule at a cyclic carbon or cyclic heteroatom. The heteroaryl can contain additional fused rings (e.g., 1-3 rings), including additional fused aryl, heteroaryl, cycloalkyl, and / or heterocyclyl rings. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidyl, thiophenyl, furanyl, thiazole, and the like.

[0038] The term "heterocycle" or "heterocyclyl" refers to a saturated or unsaturated non-aromatic group having 1-10 ring carbon atoms and 1-4 ring heteroatoms, such as nitrogen, sulfur, or oxygen, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Heterocyclyl groups can have a single ring or multiple fused rings, but exclude heteroaryl groups. Heterocycles containing two or more rings can be fused, spiro, or bridged, or any combination thereof. In fused ring systems, one or more of the fused rings can be aryl or heteroaryl. Examples of heterocyclyl groups include, but are not limited to, tetrahydropyranyl, dihydropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, thiazolinyl, thiazolidinyl, tetrahydrofuran, tetrahydrothiophenyl, 2,3-dihydrobenzo[b]thiophen-2-yl, 4-amino-2-oxopyrimidin-1(2H)-yl, and the like.

[0039] "Oxo" refers to the moiety =O.

[0040] "Thiocarbonyl" refers to the group C=S.

[0041] "Optionally substituted" means that, unless otherwise specified, the group is unsubstituted or can be substituted with one or more (e.g., 1, 2, 3, 4, or 5) of the substituents listed for that group, where the substituents can be the same and different. In one embodiment, an optionally substituted group has one substituent. In another embodiment, an optionally substituted group has two substituents. In another embodiment, an optionally substituted group has three substituents. In another embodiment, an optionally substituted group has four substituents. In some embodiments, an optionally substituted group has 1 to 2, 2 to 5, 3 to 5, 2 to 3, 2 to 4, 3 to 4, 1 to 3, 1 to 4, or 1 to 5 substituents.

[0042] The term "substituted" refers to the replacement of one or more hydrogen atoms of a moiety with a monovalent or divalent radical. "Optionally substituted" indicates that the moiety can be substituted or unsubstituted. Suitable substituents include, for example, hydroxyl, nitro, amino (e.g., -NH2 or dialkylamino), imino, cyano, halo (F, Cl, Br, I, etc.), haloalkyl (such as -CC13 or -CF3), thio, sulfonyl, thioamido, amidino, imidino, oxo, oxamidino, methoxamidino, imidino, guanidino, sulfonamido, carboxyl, formyl, alkyl, alkoxy, alkoxy-alkyl, alkylcarbonyl, alkylcarbonyloxy (-OCOR), aminocarbonyl, arylcarbonyl, aralkylcarbonyl, carbonylamino, heteroarylcarbonyl, heteroaralkyl-carbonyl, alkylthio, aminoalkyl, cyanoalkyl, carbamoyl (-NHCOOR- or -OCONHR-), urea (-NHCONHR-), aryl, etc., where R is any suitable group, e.g., alkyl or alkylene. In some embodiments, the optionally substituted moieties are only optionally substituted by selected radicals as described. In some embodiments, the above groups (e.g., alkyl groups) are optionally substituted by, for example, alkyl (e.g., methyl or ethyl), haloalkyl (e.g., -CC13, -CH2CHCl2, or -CF3), cycloalkyl (e.g., -C3H5, -C4H7, -C5H9), amino (e.g., -NH2 or dialkylamino), alkoxy (e.g., methoxy), heterocycloalkyl (e.g., morpholine, piperazine, piperidine, azetidine), hydroxyl, and / or heteroaryl (e.g., oxazolyl). In some embodiments, the substituents are themselves optionally substituted. In some embodiments, the substituents are not themselves substituted.The group substituted by the substituent can be, for example, carboxyl, halo, nitro, amino, cyano, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, aminocarbonyl, -SR, thioamide, -SO3H, -SO2R, or cycloalkyl, where R is any suitable group, for example, hydrogen or alkyl.

[0043] Cationic Compounds as Delivery Vehicles As detailed above, the complexes and compositions provided herein comprise one or more polyanionic compounds, one or more PEGylated compounds, and one or more cationic compounds. Due to their positive charges, the cationic compounds in the complexes and compositions can counterbalance the negative charges of the polyanionic cargoes and form charge-neutral complexes (also called cationic compound-polyanionic compound complexes), thus facilitating the transport of anionic substances through lipophilic cell membranes to target cells.

[0044] The cationic compounds described herein have a net zero charge or a net positive charge. In some embodiments where the complex or composition comprises one or more cationic compounds, the one or more cationic compounds independently have a net zero charge or a net positive charge. In certain embodiments, the one or more cationic compounds independently have a net positive charge of at least +1. In other embodiments, the one or more cationic compounds independently have a net positive charge of at least +2. In still other embodiments, the one or more cationic compounds independently have a net positive charge of +4 or less or +3 or less. It should be appreciated that the net charge present on the one or more cationic compounds may vary depending on environmental conditions. For example, in some embodiments, the one or more cationic compounds independently have a stable net positive charge in a physiologically relevant pH range. For example, the physiological pH is at least about 5.5, typically at least about 6.0. More typically, the physiological pH is at least about 6.5. Usually, the physiological pH is less than about 8.5, usually less than about 8.0. More typically, the physiological pH is less than about 7.5.

[0045] It should be appreciated that there are various cationic compounds that can serve as a delivery vehicle for nucleic acids and are suitable for the complexes and compositions described herein. The cationic compounds in the complexes and compositions described herein can include individual small molecules as well as larger polymeric constructs. Suitable cationic compounds can include, but are not limited to, cationic lipids, cationic lipid-peptide conjugates (e.g., lipitoids), cationic peptides, cationic polymers, and lipid-like (lipophilic) cationic compounds. In some embodiments, the complexes and compositions of the present disclosure include one or more cationic compounds, and the one or more cationic compounds are selected from the group consisting of cationic lipids, cationic lipid-peptide conjugates (e.g., lipitoids), cationic peptides, cationic polymers, and lipid-like (lipophilic) cationic compounds, and any combination thereof.

[0046] Tertiary Amino Lipidated and / or PEGylated Cationic Peptide Compounds In some embodiments, the complexes and compositions of the present disclosure comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds. The tertiary amino lipidated and / or PEGylated cationic peptide compounds are peptide chains comprising N-substituted amino acid residues. The tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure comprise an oligopeptide backbone, which comprises repeating subunits of N-substituted cationic amino acid residues, optionally alternating with N-substituted neutral ("spacer") and / or lipid amino acid residues. The oligopeptide backbone is further capped at the N- and / or C-terminus by amino acid residues that are N-substituted with lipid moieties ("N-lipidation") and / or N-substituted with oligoethylene glycol and / or polyethylene glycol ("N-PEGylation").

[0047] In one aspect, provided herein is a tertiary amino lipidated and / or PEGylated cationic peptide compound of formula (I) or a salt thereof: [ka]

[0048] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptidic compounds may be characterized by the total number of amino acid residues present in the peptidic compound, each amino acid residue having the general structure -(NR-CR a R) b -C(O))-. In some embodiments, the total number of amino acid residues is 2-40 amino acid residues, 2-30 amino acid residues, 3-25 amino acid residues, 5-20 amino acid residues, or 7-15 amino acid residues. In certain embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises 5-20 amino acid residues. In certain embodiments, the total number of amino acid residues is the sum of m, n, s, t, and [r×(o+p+q)].

[0049] In other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound has a net zero charge or a net positive charge. In certain embodiments where the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino lipidated cationic peptide compound, the tertiary amino lipidated cationic peptide compound has a net positive charge of at least +1. In other embodiments where the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino PEGylated cationic peptide compound or a tertiary amino lipidated and PEGylated cationic peptide compound, the cationic peptide compound has a net zero charge (i.e., charge neutral) or a net positive charge. In some embodiments where the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino PEGylated cationic peptide compound or a tertiary amino lipidated and PEGylated cationic peptide compound, the cationic peptide compound has a net positive charge of +1. In certain embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound has a net positive charge of (r×p)+.

[0050] In certain embodiments, the amino acid residues of the cationic peptide compounds described herein are N-substituted variants of naturally occurring or non-naturally occurring amino acids, and the carbon side chains are selected from the group consisting of R a and R b The amino acid residue can be in either D- or L-configuration. It should be further recognized that substitution at the N-position of an amino acid residue can restrict the free rotation of an amide bond. Thus, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure can also exist in various rotamer conformations (rotamers).

[0051] In some embodiments, each R a and each R bare independently side chain moieties found in natural or non-naturally occurring amino acids. As used herein, the term "naturally occurring amino acid" refers to Gly, Ala, Val, Leu, Ile, Ser, Thr, Cys, Met, Asp, Glu, Asn, Gln, Lys, Arg, Phe, Tyr, His, or Trp. The term "non-naturally occurring amino acid" refers to amino acids that are not normally found in nature, including, for example, the D isomers of the naturally occurring amino acids, 2-aminoadipic acid, 2-aminobutyric acid, norvaline, norleucine, and ornithine. In certain embodiments, each R a and R b are independently -H, -CH3, or [ka] It is.

[0052] In other embodiments, each R a and each R b are independently -H or C1-C 4- In certain embodiments, each R a and each R b is independently -H or CH3. In other embodiments, R a and R b is -H. a and R b In embodiments where R is -H, the tertiary amino lipidated and / or PEGylated cationic peptide compounds may also be referred to as N-lipidated and / or PEGylated polyglycine compounds or N-lipidated and / or PEGylated peptoid compounds. a and each R b are independently -H, C1-C4-alkyl, or a side chain moiety found in natural or non-naturally occurring amino acids.

[0053] Oligopeptide backbones, or repeating subunits and structural motifs As described herein, tertiary amino lipidated and / or PEGylated cationic peptide compounds can be useful for complexing with polyanionic compounds such as nucleic acids and for delivering such polyanionic compounds to cells. The tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure comprise an oligopeptide backbone of repeating subunits of N-substituted cationic amino acid residues, optionally interleaved with N-substituted neutral spacer amino acid residues and / or N-lipidated amino acid residues, as shown in the following fragment of formula (I): [ka]

[0054] The cationic amino acid residues in the repeating subunits of the oligopeptide backbone impart a positive charge to the compounds of the present disclosure, allowing for favorable electrostatic interactions with and charge neutralization of polyanionic species such as nucleic acids. The alternation of neutral or lipidated amino acid residues between the cationic residues allows for greater control over the spatial distribution of positive charges throughout the tertiary amino lipidated and / or PEGylated cationic peptide compounds, allowing for improved complexation of the cationic compounds with polyanionic species of specific length, charge distribution, and / or conformation.

[0055] In the oligopeptide backbone, r represents the number of repeating subunits of cationic and neutral and / or lipidated amino acid residues in the tertiary amino lipidated and / or PEGylated cationic peptide compound. In some embodiments, r is an integer from 1 to 25. In certain embodiments, r is an integer from 1 to 20. In other embodiments, r is an integer from 1 to 15. In some embodiments, r is an integer from 1 to 5. In certain embodiments, r is an integer from 2 to 4.

[0056] It should be appreciated that each subunit r may or may not be exactly the same as the other subunits within the overall tertiary amino lipidated and / or PEGylated cationic peptide compound. For example, within a tertiary amino lipidated and / or PEGylated cationic peptide compound, each subunit r contains cationic amino acid residues, and optionally also neutral and / or lipidated amino acid residues, and is independently selected relative to the cationic and neutral and / or lipidated amino acid residues present in the other subunits.

[0057] Cationic Moiety Each repeating subunit of the tertiary amino lipidated and / or PEGylated cationic peptide compound of the present disclosure comprises at least one cationic amino acid residue.The cationic amino acid residue provides a positive charge that allows the peptide compound described herein to form an electrostatic complex with nucleic acid or other polyanionic compound by interacting with the negative charge of nucleic acid or polyanionic compound.The complexation of nucleic acid partially or completely shields the negative charge of nucleic acid and facilitates its transport through the lipid membrane of the cell to the inside of the cell.

[0058] Within each subunit r, p represents the number of cationic amino acid residues present in that subunit. In some embodiments, each p is independently the integer 1 or 2. In certain embodiments, p is 1.

[0059] Each cationic amino acid residue contains a cationic moiety R at the N-position. 5 Each cationic moiety R 5 It will be appreciated that may be independently selected not only within the repeating subunits of the cationic peptide compound, but throughout the oligopeptide backbone.

[0060] The cationic moiety described herein can be a substituent that has a stable net positive charge in a physiologically relevant pH range. For example, physiological pH is at least about 5.5, typically at least about 6.0. More typically, physiological pH is at least about 6.5. Usually, physiological pH is less than about 8.5, typically less than about 8.0. More typically, physiological pH is less than about 7.5. A cationic moiety can be, for example, a functional group present in the moiety that is sufficient to generate a positive charge at physiological pH. a In certain embodiments, the cationic moiety may be characterized by a pK value of at least 7.5. a In other embodiments, the cationic moiety has a pK value between physiological pH and a more acidic pH, e.g., pH 4.5 to 5.5. a In additional embodiments, multiple independent pK a A substituent having a value is used.

[0061] It will be appreciated that the tertiary amino lipidated and / or PEGylated cationic peptide compounds described herein may contain multiple cationic moieties in close proximity to one another along the oligopeptide backbone. When multiple cationic moieties are present along the oligopeptide backbone, the protonation or deprotonation state of a particular cationic moiety will be related to the pK of other cationic moieties in close proximity. a This mechanism can affect the pK value of a particular cationic moiety in the cationic peptide compounds described herein. a is isolated and measured its pK a For example, the pK a Protonation of one amine in an oligopeptide backbone with a pK of approximately 8 increases the pK of a nearby cationic moiety. a value from the usual pK value of about 7.5 a It can be reduced to about 5-6.

[0062] The ability of the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure to accommodate multiple cationic moieties in varying proximity to one another allows these cationic peptide compounds, and any complexes thereof, to respond with high sensitivity to variations in physiological pH. The sensitivity of these compounds and their complexes to changes in physiological pH may be important for facilitating the delivery and release of nucleic acids into the cytosol following endosomal trafficking (endosomal compartment pH ∼4.5-5.5).

[0063] Cationic, or positively charged, moieties may include nitrogen-based substituents, such as those containing functional groups that are amino, guanidino, hydrazide, and amidino. These functional groups can be either aromatic, saturated cyclic, or aliphatic. In some embodiments, each R 5 is independently aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, N-heterocyclylalkyl, or N-heteroaryl. 5 is, independently, [ka] It is.

[0064] In certain embodiments, each R 5 is, independently, [ka] In still other embodiments, each R 5 teeth, [ka] It is.

[0065] In certain embodiments, each R 5 is -(C1-C4 alkylene)-NR 5a R5b and R 5a and R 5b are independently H, C1-C3 alkyl, or C6-C 10 aryl or R 5a and R 5b together with the nitrogen atom to which they are attached form a 4- to 7-membered N-heterocycloalkyl ring, which may optionally contain one additional heteroatom selected from the group consisting of N and O. In certain embodiments, each R 5 teeth, [ka] In some embodiments, each R 5 is independently [ka] and R 5a and R 5b are independently H, C1-C3 alkyl, or C6-C 10 aryl or R 5a and R 5b together with the nitrogen atom to which they are attached form a 4- to 7-membered N-heterocycloalkyl ring, which may optionally contain one additional heteroatom selected from the group consisting of N and O. In certain embodiments, each R 5 is, independently, [ka] In still other particular embodiments, each R 5 teeth, [ka] It is.

[0066] In still further embodiments where the cationic residue is the terminal residue of the overall peptide compound, additional cationic moieties R may be added that are not compatible with the synthesis or deprotection conditions (such as acid labile linkers) or for which suitable protecting group strategies are not available (e.g., polyamines). 5 For example, in some embodiments, the cationic moiety R of the terminal cationic residue may be 5 is a polyamine. The terminal residue R 5 In some embodiments where [ka] In certain embodiments, the polyamine is [ka] is selected from the group consisting of:

[0067] In other embodiments, the cationic moiety R of the terminal cationic residue 5 is hydroxyalkyl, hydroxyether, alkoxyalkyl, or hydroxylheteroalkyl. In certain embodiments, the cationic moiety R of the terminal cationic residue 5 teeth, [ka] It is.

[0068] In yet a further embodiment, the cationic moiety R of the terminal cationic residue 5 teeth, [ka] It is.

[0069] The unsubstituted nitrogen atoms in the peptide chain are, i.e., R 5It should be further recognized that when R is hydrogen, it can also function as a cationic moiety that is ionizable under physiological conditions. In some embodiments, the cationic moiety R 5 is a hydrogen atom.

[0070] Neutral spacer part Within the oligopeptide backbone of the tertiary amino lipidated and / or PEGylated cationic peptide compounds, the cationic amino acid residues may be optionally interleaved with neutral spacer amino acid residues having neutral spacer moieties at the N-positions. The neutral amino acid residues may be useful for modulating the spatial distribution of positive charges in the tertiary amino lipidated and / or PEGylated cationic peptide compounds for improved electrostatic interactions with polyanionic compounds (including polynucleotides) that are complexed with the cationic peptide compounds.

[0071] In each repeating subunit of a tertiary amino lipidated and / or PEGylated cationic peptide compound, the neutral amino acid residue may be attached to either the N-terminus or C-terminus of a cationic amino acid residue by one or more R 4 The subunit r can be present as a neutral spacer moiety R 4 In some embodiments, including, o and / or q, corresponding to each neutral spacer moiety present, represent the respective number of neutral spacer residues attached to the N-terminus and C-terminus of the cationic amino residue in subunit r. In some embodiments, each o is independently an integer 0, 1, 2, 3, or 4. In other embodiments, each q is independently an integer 0, 1, 2, 3, or 4.

[0072] Each neutral spacer amino acid residue has at its N-position a neutral spacer moiety R 4 As with the cationic moieties described herein, each neutral spacer moiety R 4 It will be appreciated that is independently selected within the repeating subunits of the cationic peptide compound, as well as among the repeating subunits r of the oligopeptide backbone.

[0073] It should also be appreciated that the neutral spacer moiety may include any substituent that is neutral or has a net charge of zero in the physiologically relevant pH range. In some embodiments, each neutral moiety R 4 is independently a C1-C4-alkyl, C1-C5 straight chain alkyl substituted with cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, optionally substituted with one or more substituents -OH, halo, or alkoxy. In further embodiments, each neutral spacer moiety R4 is independently -CH3, [ka] In certain embodiments, each neutral spacer moiety R 4 teeth, [ka] It is.

[0074] In yet other embodiments, a neutral spacer moiety can include a substituent that can become anionic (i.e., negatively charged) in the physiologically relevant pH range. For example, in some embodiments, the spacer moiety R 4 teeth, [ka] It is.

[0075] lipid part In addition to the optional alternation of neutral amino acid residues with cationic amino acid residues within the oligopeptide backbone of the tertiary amino lipidated and / or PEGylated cationic peptide compounds, the N-lipidated amino acid residues having lipid moieties at the N-position may also be optionally alternating with cationic (and optional neutral spacer) amino acid residues. In some embodiments in which the tertiary amino lipidated and / or PEGylated cationic peptide compounds contain N-lipidated amino acid residues, the tertiary amino lipidated and / or PEGylated cationic peptide compounds are N-lipidated. As with the neutral amino acid residues, the N-lipidated amino acid residues within the oligopeptide backbone may be useful for modulating the spatial distribution of positive charges in the tertiary amino lipidated and / or PEGylated cationic peptide compounds, as well as for increasing their lipophilicity for improved encapsulation and intracellular delivery of polyanionic substances. The spacing of lipids along the peptoid backbone may also affect the fluidity / crystallinity of the lipids, which are known to affect cellular uptake and endosomal release.

[0076] Similar to the neutral spacer residue, in each repeating subunit of the tertiary amino lipidated and / or PEGylated cationic peptide compound, the N-lipidated amino acid residue may be provided with one or more R 4 The subunit r can be present as a lipid moiety R 4 In some embodiments, including those described above, o and / or q corresponding to each lipid moiety present may also represent the respective number of lipidated residues attached to the N-terminus and C-terminus of the cationic amino acid residue in unit r. In some embodiments, each o is independently an integer 0, 1, 2, 3, or 4. In other embodiments, each q is independently an integer 0, 1, 2, 3, or 4.

[0077] Each N-lipidated amino acid residue contains a lipid moiety R at the N-position. 4 Each lipid moiety R, as well as the cationic and neutral moieties described herein, 4It will be appreciated that is independently selected within the repeating subunits of the cationic peptide compound, as well as among the repeating subunits r of the oligopeptide backbone.

[0078] Suitable lipid moieties can include, for example, optionally substituted branched or straight chain aliphatic moieties, or optionally substituted moieties derived from natural lipid compounds, including fatty acids, sterols, and isoprenoids.

[0079] In some embodiments, the lipid moiety may comprise a branched or straight chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds (i.e., saturated or mono- or polyunsaturated). In certain embodiments, the lipid moiety may comprise an optionally substituted aliphatic, straight chain or branched moiety, each hydrophobic tail independently having about 8 to about 30 carbon atoms or about 6 to about 30 carbon atoms. In certain embodiments, the lipid moiety may comprise an aliphatic carbon chain derived, for example, from fatty acids and fatty alcohols. In some embodiments, each lipid moiety R 4 Independently, C8-C 24 -Alkyl or C8-C 24 -alkenyl, C8-C 24 -alkenyl is optionally mono- or polyunsaturated. In some embodiments, each lipid moiety R 4 is C6-C 18 Alkyl or C6-C 18 alkenyl. In certain embodiments, each lipid moiety R 4 is C6-C 18 Alkyl or C6-C 18 Alkenyl, C6-C 18 In certain other embodiments, each lipid moiety R 4 is C6-C 17 Alkyl or C6-C 17 alkenyl. In certain embodiments, each lipid moiety R 4 is C8-C12 In yet another embodiment, each lipid moiety R 4 C such as n-decyl 10 -alkyl. In some embodiments, each R 4 is independently selected from the group consisting of 2-ethylhex-1-yl, caprylyl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl. 4 is independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl. 4 is independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, myristyl, and lauryl.

[0080] In yet another embodiment, which may be combined with any of the other embodiments, each lipid moiety R 4 is, independently, [ka] In still other embodiments which may be combined with any of the preceding embodiments, each lipid moiety R 4 are independently expressed as [ka] and R 8 is a branched or straight chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds. 4 is, independently, [ka] It is.

[0081] The natural lipid moieties used in the practice of the present invention can be derived, for example, from phospholipids, glycerides (such as diglycerides or triglycerides), glycosylglycerides, sphingolipids, ceramides, as well as saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

[0082] Other suitable lipid moieties may include lipids that include a lipophilic carbocyclic or aromatic group, such as an optionally substituted aryl, cycloalkyl, cycloalkylalkyl, or arylalkyl moiety, including, for example, naphthalenyl or ethylbenzyl, or an ester functional group, including, for example, sterol esters and wax esters. 4 teeth, [ka] It is.

[0083] Structural motif In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure may contain specific sequences or arrangements of cationic, neutral spacer, and lipidated amino acid residues relative to one another, which can be described similarly to the various classifications of linear copolymers (random, block, alternating, periodic, stereoblock, etc.).

[0084] For example, tertiary amino lipidated and / or PEGylated cationic compounds are common moieties R カチオン , R 中性 , and R 脂質 In some embodiments, including the above, the amino acid residues may be arranged in a random, alternating, or block sequence. カチオン and R 中性 The expressions for 、 Cationic moiety R 5 and the neutral part R 4 It should be understood that the general moiety R is equivalent to an amino acid residue containing 脂質Depending on the position of the N-lipidated residue in the tertiary amino lipidated and / or PEGylated cationic peptide compound, the lipid moiety R 4 and lipid moiety R 3 It can be understood to include both amino acid residues having the following structure:

[0085] An example of an alternating sequence of cationic and neutral spacer amino acid residues is R カチオン -R 中性 -R カチオン -R 中性 , or R 中性 -R カチオン -R 中性 -R カチオン Another example of an alternating sequence of cationic and lipidated amino acid residues can be represented by R カチオン -R 脂質 -R カチオン -R 脂質 , or R 中性 -R カチオン -R 中性 -R カチオン Some examples of block sequences are カチオン -R カチオン -R カチオン -R 中性 -R 中性 -R 中性 , R 中性 -R 中性 -R 中性 -R カチオン -R カチオン -R カチオン , R カチオン -R カチオン -R カチオン -R 脂質 -R 脂質 -R 脂質 , or R 脂質 -R 脂質 -R 脂質 -R カチオン -R カチオン -R カチオン Further, in some embodiments, the cationic moieties, neutral spacer moieties, and lipidated moieties may be arranged in block and alternating sequences in different repeating subunit segments within the overall oligopeptidic compound. For example, in some embodiments, a cationic peptidic compound may have a structure such as Rカチオン -R 中性 -R 中性 -R カチオン -R 中性 -R 中性 There may be repeating motifs of larger oligomeric units such as, In other embodiments, the cationic peptide compounds may contain blocks of N-lipidated residues combined with blocks of alternating cationic and neutral residues.

[0086] As described herein, the tertiary amino lipidated and / or PEGylated cationic peptides of the present disclosure include a cationic moiety R 5 In some embodiments, the backbone of the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises a "cationic domain" or "cationic block." A cationic domain comprises a plurality of cationic moieties R 5 A cationic domain can be broadly understood as, for example, a segment of consecutive amino acid residues in an oligopeptide chain having at least two cationic residues. In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises a cationic domain, which comprises at least two, at least three, or at least four cationic amino acid residues.

[0087] The cationic domain may, for example, be a continuous linear sequence of multiple R 5 A block of amino acid residues having a cationic moiety (e.g., R 5 R 5 R 5 , or R カチオン R カチオン R カチオン In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises a "cationic domain", where the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises R 5 It comprises at least two, at least three, or at least four contiguous amino acid residues having a cationic moiety.

[0088] In other embodiments, a "cationic domain" may comprise a block of contiguous amino acid residues, the plurality of cationic residues being characterized by an additional non-cationic (i.e., neutral or lipidated) residue disposed between two cationic residues, such that a cationic residue within the block is not directly bonded to another cationic residue. For example, in some embodiments, a tertiary amino lipidated and / or PEGylated cationic peptide compound comprises a "cationic domain", the cationic domain being characterized by a cationic moiety R 5 and each of the at least two cationic amino acid residues in the cationic domain is linked to a neutral spacer or lipid moiety R 4 In some embodiments, the non-cationic residues are interleaved between two cationic residues in the cationic domain at regularly spaced intervals. In some embodiments, where the cationic domain comprises non-cationic residues interleaved between cationic residues, the cationic domain may be described as a domain comprising repeating (dimeric, trimeric, tetrameric, etc.) subunits. These subunits include -R カチオン R 中性 -, -R カチオン R 中性 R 中性 -, -R カチオン R 脂質 -, -R カチオン R 脂質 R 脂質 -, -R 脂質 R カチオン R 脂質 -, -R 脂質 R 脂質 R カチオン -, -R カチオン R 中性 R 脂質 -, or -R カチオン R 脂質 R 中性In other embodiments, the non-cationic residues are interspersed between two cationic residues in the cationic domain, and the number of non-cationic residues varies between each pair of cationic residues. In certain embodiments in which the cationic domain comprises non-cationic residues interspersed between cationic residues, each pair of cationic residues is independently separated by at least one non-cationic residue. In some embodiments, at least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprises at least one trimeric subunit -R カチオン -R 中性 -R 中性 In another embodiment, at least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprises at least one trimeric subunit -R カチオン -R 中性 -R 脂質 Includes.

[0089] In some embodiments, where the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises a cationic domain, each R 5 teeth, [ka] In other embodiments in which the tertiary amino lipidated and / or PEGylated cationic peptide comprises a cationic domain, each cationic moiety R 5 teeth, [ka] and each neutral spacer moiety is [ka] In certain embodiments in which the tertiary amino lipidated and / or PEGylated cationic peptide comprises a cationic domain comprising one or more dimeric or trimeric subunits, each cationic moiety R 5teeth, [ka] and each neutral spacer moiety is [ka] In yet another embodiment, the tertiary amino lipidated and / or PEGylated cationic peptide comprises one or more trimeric subunits -R カチオン R 中性 R 中性 -, each cationic moiety R 5 teeth, [ka] and each neutral spacer moiety is [ka] In yet another embodiment, the tertiary amino lipidated and / or PEGylated cationic peptide comprises at least one trimeric subunit -R カチオン R 中性 R 脂質 -, each cationic moiety R 5 teeth, [ka] and each neutral spacer moiety is [ka] It is.

[0090] As described herein, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure may comprise one or more distinct cationic moieties (R カチオン1 , R カチオン2 , R カチオン3 ), one or more distinct neutral spacer moieties (R中性1 , R 中性2 , R 中性 3), and / or one or more different lipid moieties (R 脂質1 , R 脂質2 , R 脂質 3, etc. It should be appreciated that the above examples of typical cationic and neutral spacer amino acid residue sequences or arrangements are not intended to be limiting to the peptide compounds of the present disclosure. The cationic, neutral spacer, and lipid moieties may generally be present in random, alternating, or block arrangements as previously described, and may be present in specific arrangements of individual cationic, neutral, and lipid moieties within larger blocks or alternating structural motifs. For example, some exemplary arrangements of residues include those having R カチオン1 R 中性1 R 中性1 R 脂質1 R カチオン1 R 中性1 R 中性1 R 脂質1 R カチオン1 R 中性1 R 中性1 , R カチオン1 R 中性1 R 中性1 R カチオン1 R 中性1 R 中性1 R カチオン1 R 中性1 R 中性1 R 脂質1 R 脂質1 , R 脂質1 R カチオン1 R 中性1 R 脂質1 R カチオン1 R 中性1 -R 脂質2 R カチオン2 R 中性2 R 脂質2 R カチオン2 R 中性2 , R 脂質1 R 脂質2 R 脂質3 R 脂質4 R 中性1 R 中性2 R カチオン1 R 中性1 R 中性2 R 脂質1 R 脂質2 R 脂質3 R脂質4 , or R 脂質1 R 脂質2 R 脂質1 R 脂質2 R カチオン1 R 中性1 R カチオン1 R 中性1 R 脂質1 R 脂質2 R 脂質1 R 脂質2 These may include, but are not limited to:

[0091] Side Chain Functionalization and Peptide Conjugate Compounds As described above, the oligopeptide backbone is primarily composed of natural or unnatural amino acids (R a and R b ) 4 and R 5 ).

[0092] In one embodiment, the R of the tertiary amino lipidated and / or PEGylated cationic peptide compound of the present disclosure is a , R b , R 4 , and R 5 The groups may further include reactive linker groups that can be used to covalently attach to therapeutic and / or targeting elements, such as small molecules or antibodies. These additional functionalities may also include cationic groups that are not compatible with synthesis or deprotection conditions (such as acid-labile linkers) or for which suitable protecting group strategies are not available (e.g., polyamines). Reactive groups may, for example, be attached to existing side chains R a and / or R b , or R 5 cationic moiety, R at N-position 4 Alternatively, R with the desired reactive moiety can be added to the neutral spacer or lipid moiety of R by chemical methods known in the art. a , R b , R 4 , or R 5If the group is commercially available as the corresponding free amine, the corresponding free amine can be incorporated during the general synthesis of the peptide chain (eg, during submonomer synthesis).

[0093] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptides contain one or more amino acid residues that contain a reactive group or linker group. Suitable reactive groups can include, but are not limited to, esters, amides, isocyanates, thiols, or "click" chemistry compatible moieties (e.g., azides, alkynyls).

[0094] In another aspect, the present disclosure further provides a tertiary amino lipidated and / or PEGylated cationic peptide compound as the cationic compound, wherein the peptide compound is covalently linked or conjugated to a therapeutic agent and / or a targeting element. In certain embodiments, the therapeutic agent and / or the targeting element is a small molecule, a peptide sequence, an antibody or antibody fragment, an aptamer, a mono- or oligosaccharide (e.g., galactose), or a glycan.

[0095] Linker groups or bonds that are sensitive or unstable under certain physiological conditions can be useful to allow targeted delivery to specific organs or cells, followed by selective release of therapeutic agents and / or polyanionic substances in or to cells.In addition, labile bonds can also facilitate the clearance and disappearance of tertiary amino lipidated and / or PEGylated cationic compounds from cells (or organs, or the whole body) after delivery of polyanionic substances.In certain embodiments, linker groups are selected so that the covalent bond between tertiary amino lipidated and / or PEGylated cationic peptide compounds and therapeutic agents or targeting elements is hydrolytically unstable, chemically unstable, pH labile, photolabile, heat labile, or enzymatically cleavable.

[0096] Terminal (capping) residues and end-group protection As described herein, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure comprise an oligopeptide backbone whose N-terminus and / or C-terminus are capped by amino acid residues that are N-substituted with lipid moieties ("N-lipidation") and / or N-substituted with oligoethylene glycol and / or polyethylene glycol ("N-PEGylation"). The N-lipidation of cationic peptide compounds confers favorable lipophilicity to the compounds and any complexes formed between the compounds and polyanionic species. N-PEGylation provides better control over particle formation and aggregation in vivo. The terminal amino acids and carboxylic acid moieties of the N-terminus and C-terminus can be further capped with protective end groups.

[0097] As described herein, the tertiary amino lipidated and / or PEGylated cationic peptide compound may comprise an amino acid residue that is N-lipidated and / or N-PEGylated. The cationic peptide compound provided herein comprises at least one amino acid residue that is N-lipidated or N-PEGylated. In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino lipidated cationic peptide compound. In other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino PEGylated cationic peptide compound. In still other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound of the present disclosure comprises both N-lipidated and N-PEGylated amino acid residues. In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino lipidated and PEGylated cationic peptide compound.

[0098] lipid part As described above, the tertiary amino lipidated and / or PEGylated cationic peptide compounds may contain amino acid residues at the N-terminus and / or C-terminus, which are N-substituted with lipid moieties or "N-lipidated". The incorporation of N-lipidated amino acid residues at the N-terminus and / or C-terminus of the cationic peptide compounds described herein increases the lipophilicity of the compounds. The increase in lipophilicity of the cationic peptide compounds increases their affinity for hydrophobic environments, such as the lipid bilayer of cell membranes, and thus increases the propensity of the tertiary amino lipidated and / or PEGylated cationic peptide compounds, and their complexes with polyanionic compounds, to be transported into cells.

[0099] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds are N-lipidated. In some embodiments, the tertiary amine lipidated and / or PEGylated cationic peptide compounds of the present disclosure comprise N-lipidated amino acid residues at the N-terminus. In other embodiments, the tertiary amine lipidated and / or PEGylated cationic peptide compounds of the present disclosure comprise N-lipidated amino acid residues at the C-terminus. In certain embodiments, the tertiary amine lipidated and / or PEGylated cationic peptide compounds of the present disclosure comprise N-lipidated amino acid residues at the N-terminus and C-terminus.

[0100] In some embodiments, the number of N-lipidated amino acid residues at the N-terminus of the cationic peptide compounds described herein is represented by n. In other embodiments, the number of N-lipidated amino acid residues at the N-terminus of the cationic peptide compounds described herein is represented by s.

[0101] In some embodiments, n is an integer from 0 to 8. In certain embodiments, n is an integer from 0 to 5. In other embodiments, n is an integer from 0, 1, 2, 3, 4, 5, 6, or 7. In some embodiments, n is an integer from 4 or less. In still other embodiments, n is an integer from 1, 2, 3, or 4. In certain embodiments, n is 4. In some embodiments, s is an integer from 0 to 8. In certain embodiments, s is an integer from 0 to 5. In other embodiments, s is an integer from 0, 1, 2, 3, 4, 5, 6, or 7. In some embodiments, s is an integer from 4 or less. In still other embodiments, s is an integer from 1, 2, 3, or 4. In certain embodiments, s is 4. In some embodiments where the tertiary amino lipidated and / or PEGylated cationic peptide compound is N-lipidated, at least one of n or s is other than 0. In some embodiments, n is other than 0. In certain embodiments, n is other than 0 and s is 0. In other embodiments, s is other than 0. In certain embodiments, s is other than 0 and n is 0. In certain embodiments, both n and s are other than 0.

[0102] In some embodiments, the sum of n and s is an integer from 1 to 8, from 2 to 7, or from 4 to 6. In other embodiments, the sum of n and s is at least 2, at least 3, or at least 4.

[0103] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds comprise a block of N-lipidated residues, or an "N-lipid block," where the tertiary amino lipidated and / or PEGylated cationic peptide comprises at least two, at least three, or at least four N-lipidated residues adjacent to one another (e.g., R 脂質 R 脂質 R 脂質In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds comprise a block of N-lipidated residues and n is at least 2, at least 3, or at least 4. In other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds comprise a block of N-lipidated residues and s is at least 2, at least 3, or at least 4.

[0104] The N-lipidated amino acid residues in the tertiary amino lipidated and / or PEGylated cationic peptides of the present disclosure are represented by the lipid moiety R 3 N-substituted by 。 The lipid moieties of the present disclosure may comprise hydrophobic or lipophilic moieties that are neutral (i.e., uncharged or have a net charge of zero). The lipid moieties of the tertiary amino lipidated and / or PEGylated cationic compounds described herein may be of either natural or synthetic origin. Each R 3 are independently lipid moieties and can be the same or different. In some embodiments, each R 3 are the same. In another embodiment, each R 3 In yet other embodiments where the sum of n and s is at least 2 (i.e., n+s≧2), the lipid moiety R 3 At least two of the are different from each other.

[0105] It will be appreciated that certain sequences or arrangements of N-lipidated amino acid residues may be particularly useful for improving complexation with and delivery of nucleic acids. For example, a tertiary amino lipidated and / or PEGylated cationic compound may be coupled to two different lipid moieties R 3a and R 3b In some embodiments, including a set of mixed N-lipidated amino acid residues having one of the following structures: 3a -R 3b -R 3a -R 3b or R 3b -R3a -R 3b -R 3a An example of a block array is R 3a -R 3a -R 3b -R 3b or R 3b -R 3b -R 3a -R 3a In other embodiments, the sequence of the N-lipidated amino acid residues may be randomly ordered. It should be appreciated that the above examples of sequences or arrangements of two N-lipidated amino acid residues are not intended to be limiting. Additionally, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure may be represented by R 3 The lipid moieties may comprise two or more different lipid moieties, which may be present in a random, alternating, or block arrangement, generally as described above.

[0106] Suitable lipid moieties can include, for example, optionally substituted branched or straight chain aliphatic moieties, or optionally substituted moieties derived from natural lipid compounds, including fatty acids, sterols, and isoprenoids.

[0107] In some embodiments, the lipid moiety may comprise a branched or straight chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds (i.e., saturated or mono- or polyunsaturated). In certain embodiments, the lipid moiety may comprise an optionally substituted aliphatic, straight chain or branched moiety, each hydrophobic tail independently having about 8 to about 30 carbon atoms or about 6 to about 30 carbon atoms. In certain embodiments, the lipid moiety may comprise an aliphatic carbon chain derived, for example, from fatty acids and fatty alcohols. In some embodiments, each R 3 Independently, C8-C 24 -Alkyl or C8-C 24 -alkenyl, C8-C 24-alkenyl is optionally mono- or polyunsaturated. In some embodiments, each R 3 is C6-C 18 Alkyl or C6-C 18 alkenyl. In certain embodiments, each lipid moiety R 3 is C6-C 18 Alkyl or C6-C 18 Alkenyl, C6-C 18 In certain other embodiments, each lipid moiety R 3 is C6-C 17 Alkyl or C6-C 17 alkenyl. In certain embodiments, each R 3 is C8-C 12 In yet other embodiments, each R 3 C such as n-decyl 10 -alkyl. In some embodiments, each R 3 is independently selected from the group consisting of 2-ethylhex-1-yl, caprylyl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl. 3 is independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl. 3 is independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, myristyl, and lauryl. 3 is independently selected from the group consisting of oleyl, stearyl, linoleyl, myristyl, and lauryl.

[0108] In still other embodiments which may be combined with any of the other embodiments, each R 3 is, independently, [ka] It is.

[0109] In some embodiments, each R 3 is independently a branched or straight chain aliphatic moiety having about 6 to 50 carbon atoms. 3 is independently a straight chain aliphatic moiety. In certain other embodiments, each R 3 is independently a branched aliphatic moiety. In still other embodiments where the sum of n and s is at least 2 (i.e., n+s≧2), each R 3 are independently branched or straight chain aliphatic moieties, and the lipid moiety R 3 At least two of them are different from each other (R 3a and R 3b In some embodiments, the lipid moiety R 3 is composed of two different lipid moieties R 3a and R 3b In other embodiments where the sum of n and s is at least 2 (i.e., n+s≧2), at least one R 3 is or contains a branched aliphatic moiety and at least one R 3 is or comprises a straight chain aliphatic moiety. In some embodiments, at least one lipid moiety R 3 (or R 3a Or R 3b In certain embodiments that may be combined with any of the preceding embodiments, at least one lipid moiety R 3 (or R 3a Or R 3b ) is 2-ethylhex-1-yl.

[0110] In still yet other embodiments which may be combined with any of the still further embodiments, each R 3 are independently expressed as [ka] and R 8is a branched or straight chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds. 3 is, independently, [ka] It is.

[0111] The natural lipid moieties used in the practice of the present invention can be derived, for example, from phospholipids, glycerides (such as diglycerides or triglycerides), glycosylglycerides, sphingolipids, ceramides, as well as saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

[0112] Other suitable lipid moieties may include lipids that include a lipophilic carbon ring or aromatic group, such as an optionally substituted aryl, cycloalkyl, cycloalkylalkyl, or arylalkyl moiety, including, for example, naphthalenyl or ethylbenzyl, or an ester functional group, including, for example, sterol esters and wax esters. 4 teeth, [ka] It is.

[0113] Ethylene glycol portion The tertiary amino lipidated and / or PEGylated cationic peptides of the present disclosure may include capping amino acid residues at the N-terminus and / or C-terminus that are N-substituted with an oligomer or polymer of ethylene glycol, i.e., N-substituted with oligoethylene glycol and / or polyethylene glycol. Incorporation of oligoethylene glycol and / or polyethylene glycol moieties into the tertiary amino lipidated and / or PEGylated cationic peptide compounds described herein may promote particle stability of complexes formed with nucleic acids and may prevent particle aggregation in vivo.

[0114] It should be appreciated that the term "PEGylated" is used herein to describe cationic peptide compounds that include a terminal amino acid residue that may be N-substituted with an oligoethylene glycol, or polyethylene glycol, or a combination thereof. In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds provided herein are N-PEGylated.

[0115] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises an N-PEGylated amino acid residue at the N-terminus, as shown in the fragment of formula (I) below: [ka]

[0116] In certain embodiments where the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises an N-PEGylated amino acid residue at the N-terminus, m represents the number of N-PEGylated amino acid residues at the N-terminus, and each R 2 are independently represented by the formula -CH2CH2O(CH2CH2O) u R 2a is the ethylene glycol portion of R 2a is -H or C1-C4-alkyl. In some embodiments, R 2a is -H, -CH, or CHCH. In certain embodiments, R2a is -H. In other embodiments, R 2a -CH 3である。 In still yet other embodiments, R 2a is -CH2CH3.

[0117] In some embodiments, m is an integer from 0 to 15, an integer from 0 to 10, an integer from 0 to 3, an integer from 4 to 10, or an integer from 4 to 15. In some embodiments, each u is independently an integer from 1 to 200, an integer from 2 to 200, an integer from 2 to 100, an integer from 2 to 50, an integer from 50 to 200, an integer from 50 to 100, an integer from 100 to 200, or an integer from 150 to 200.

[0118] In certain embodiments, m is an integer from 0 to 3, and each u is an integer from 20 to 200, or optionally from 30 to 50. In certain embodiments, m is an integer from 0 to 3, and u is an integer from 40 to 45. In still other embodiments, m is 1, and u is an integer from 40 to 45. In other embodiments, m is an integer from 4 to 10, and each u is an integer from 2 to 10. In certain embodiments, m is an integer from 4 to 10, and u is an integer from 2 to 5. In still other embodiments, m is an integer from 7 to 10, and u is 3. In other embodiments, m is an integer from 4 to 15, and each u is an integer from 2 to 10. In certain embodiments, m is an integer from 4 to 15, and u is an integer from 2 to 5. In some embodiments, m is an integer from 10 to 15, and u is an integer from 1 to 10. In certain embodiments, m is an integer from 10 to 15, and u is an integer from 1 to 5. In other embodiments, m is an integer from 10 to 15, and u is an integer from 2 to 10. In certain embodiments, m is an integer from 10 to 15, and u is an integer from 2 to 5.

[0119] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises an N-PEGylated amino acid residue at the C-terminus, as shown in the fragment of formula (I) below: [ka]

[0120] In certain embodiments where the tertiary amino lipidated and / or PEGylated cationic peptide compound comprises an N-PEGylated amino acid residue at the C-terminus, t represents the number of N-PEGylated amino acid residues at the N-terminus, and each R 6 are independently represented by the formula -CH2CH2O(CH2CH2O) v R 6a is the ethylene glycol portion of R 6a is -H or C1-C4-alkyl. In some embodiments, R 6a is -H, -CH, or CHCH. In certain embodiments, R 6a is -H. In other embodiments, R 6a is -CH3. In still yet other embodiments, R 6a is -CH2CH3.

[0121] In some embodiments, t is an integer from 0 to 15, an integer from 0 to 10, an integer from 0 to 3, an integer from 4 to 10, or an integer from 4 to 15. In some embodiments, each v is independently an integer from 1 to 200, an integer from 2 to 200, an integer from 2 to 100, an integer from 2 to 50, an integer from 50 to 200, an integer from 50 to 100, an integer from 100 to 200, or an integer from 150 to 200.

[0122] In some embodiments, t is an integer from 0 to 3, and each v is an integer from 30 to 50. In certain embodiments, t is an integer from 0 to 3, and v is an integer from 40 to 45. In still other embodiments, t is 1, and v is an integer from 40 to 45. In other embodiments, t is an integer from 4 to 10, and each v is an integer from 2 to 10. In certain embodiments, t is an integer from 4 to 10, and v is an integer from 2 to 5. In still other embodiments, t is an integer from 7 to 10, and v is 3. In other embodiments, t is an integer from 4 to 15, and each v is an integer from 2 to 10. In certain embodiments, t is an integer from 4 to 15, and v is an integer from 2 to 5. In some embodiments, t is an integer from 10 to 15, and v is an integer from 1 to 10. In certain embodiments, t is an integer from 10 to 15, and v is an integer from 1 to 5. In other embodiments, t is an integer from 10 to 15, and v is an integer from 2 to 10. In certain embodiments, t is an integer from 10 to 15, and v is an integer from 2 to 5.

[0123] In some embodiments where the tertiary amino lipidated and / or PEGylated cationic peptide compound is N-PEGylated, at least one of m or t is other than 0. In some embodiments, m is other than 0. In other embodiments, t is other than 0. In certain embodiments, both m and t are other than 0.

[0124] N-Lipidation and / or N-PEGylation As described herein, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure can comprise N-lipidated and / or N-PEGylated amino acid residues.The cationic peptide compounds provided herein comprise at least one N-lipidated or N-PEGylated amino acid residue.In some embodiments of the tertiary amino lipidated and / or PEGylated cationic peptide compounds, at least one of m, n, s, or t is other than 0.

[0125] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino lipidated cationic peptide compound, wherein at least one of n and s is other than 0. In certain embodiments, both n and s are other than 0. In further particular embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound of formula (I) is a tertiary amino lipidated cationic peptide compound of formula (Ia): [ka] wherein at least one of n and s is other than 0; 1 , R 3 , R 4 , R 5 , R 7 , R a , R b , o, p, q, and r are as defined for formula (I). In certain embodiments of compounds of formula (Ia), both n and s are other than 0.

[0126] In other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino PEGylated cationic peptide compound, wherein at least one of m and t is other than 0. In certain embodiments, both m and t are other than 0. In certain embodiments, both m and t are other than 0. In other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound of formula (I) is a tertiary amino PEGylated cationic peptide compound of formula (Ib): [ka] wherein at least one of m and t is other than 0; 1 , R 2 , R 4 , R 5 , R 6 , R 7 , R a , R b, o, p, q, and r are as defined for formula (I). In certain embodiments of compounds of formula (Ib), both m and t are other than 0.

[0127] In still other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound of the present disclosure comprises both N-lipidated and N-PEGylated amino acid residues.In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino lipidated and PEGylated cationic peptide compound.In certain embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is a tertiary amino lipidated and PEGylated cationic peptide compound, at least one of m and t is other than 0, and at least one of n and s is other than 0.

[0128] In yet a further embodiment, the tertiary amino lipidated and / or PEGylated cationic peptide compound of formula (I) is a tertiary amino lipidated and PEGylated cationic peptide compound of formula (Ic), [ka] wherein at least one of m and t is other than 0, at least one of n and s is other than 0, and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R a , R b , o, p, q, and r are as defined for formula (I).

[0129] In other embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure may also contain N-substituted amino acid residues bearing other neutral shielding polymers or moieties at the N- or C-terminus that provide similar shielding effects as the N-PEGylated residues. These may include examples such as hydroxyl alkyls, hyaluronic acid, polysaccharides, polyphosphates and polyphosphoesters, poly(vinylpyrrolidone), polyols, hydrophilic polypeptides, or other synthetic hydrophilic polymers.

[0130] N- and C-Terminal Groups The tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure may be provided in their free amine and free acid forms, or in protected forms, at the N-terminus and C-terminus, respectively. In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds further comprise protecting or terminal capping groups at the N-terminus and / or C-terminus residues.

[0131] In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compounds have a protecting group or end-capping group R at the N-terminal residue. 1 In some embodiments, R 1 is -H, alkyl, alkylaryl, -COR 1a , -CH2-COR 1a , a cationic moiety, a lipid moiety, or an oligoethylene glycol or polyethylene glycol moiety; R 1a is -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, -O-alkylaryl, or a lipid moiety. 1 is -H, alkyl, alkylaryl, -COR 1a , or a lipid moiety, R 1a is -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl. 1 In certain embodiments where R is a lipid moiety, the lipid moiety is not a phospholipid. 1is alkyl or -COR 1a and R 1a In still other embodiments where is alkyl, the alkyl is optionally substituted with --OH or halo.

[0132] In another embodiment, the tertiary amino lipidated and / or PEGylated cationic peptide compound is provided with a protecting group or end-capping group R 7 In some embodiments, R 7 is -H, alkyl, acyl, -OH, -OR 7a , -NH2, -NHR 7a , a cationic moiety, a lipid moiety, or an oligoethylene glycol or polyethylene glycol moiety; R 7a is an alkyl, acyl, or lipid moiety. In some embodiments, R 7 is -H, alkyl, acyl, -OH, -OR 7a , -NH2, -NHR 7a , or a lipid moiety, R 7a is an alkyl, acyl, or lipid moiety. 7 In certain embodiments where is a lipid moiety, the lipid moiety is not a phospholipid.

[0133] In yet a further embodiment, the N-terminus and C-terminus of the tertiary amino lipidated and / or PEGylated cationic peptide compound are 1 and R 7 may be selected or further modified to carry additional functional groups, such as reactive linker groups.

[0134] In some embodiments, the R and / or R of the tertiary amino lipidated and / or PEGylated cationic peptide 7 independently comprise one or more reactive linker groups. Suitable reactive groups can include, but are not limited to, esters, amides, isocyanates, thiols, or "click" chemistry compatible moieties (e.g., azides, alkynyls).

[0135] The reactive linker group can then be used to covalently link other useful compounds, including targeting elements or therapeutic agents, to the peptide compound. In some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is covalently linked or conjugated to a therapeutic agent and / or targeting element. In certain embodiments, the therapeutic agent and / or targeting element is a small molecule, an antibody, or an antibody fragment.

[0136] In addition, R 1 and R 7 Additional functional groups in R may also include cationic groups that are incompatible with synthesis or deprotection conditions (such as acid labile linkers) or for which suitable protecting group strategies are not available (e.g., polyamines). 1 is a polyamine. 1 In some embodiments where [ka] In certain embodiments, the polyamine is [ka] is selected from the group consisting of:

[0137] As mentioned above, linker groups or bonds that are sensitive or unstable under certain physiological conditions can facilitate targeted delivery of polyanionic substances to certain cells and improve certain pharmacokinetic properties, such as clearance. In certain embodiments, the linker group is selected such that the covalent bond between the tertiary amino lipidated and / or PEGylated cationic peptide compound and the therapeutic agent or targeting element is hydrolytically unstable, chemically unstable, pH labile, photolabile, heat labile, or enzymatically cleavable.

[0138] Combinations of tertiary amino lipidated and / or PEGylated cationic peptide compounds The complexes and compositions of the present disclosure may include one or more cationic compounds. In some embodiments, the complexes described herein may include one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I). The use of mixtures and combinations of multiple cationic peptide compounds of the present disclosure in a single complex may allow the preparation of formulations tailored to specific pharmacokinetic and pharmacodynamic properties. Relevant pharmacokinetic and pharmacodynamic properties may include, but are not limited to, biodistribution, immunogenicity, formulation stability, encapsulation rate, transfection efficiency, plasma half-life, etc. Different combinations of these properties may be required for different applications.

[0139] For example, in certain embodiments, a complex or composition may comprise a combination of one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds that are highly lipidated and / or rich in cations with one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds that contain at least one PEG moiety.Theoretically, such a combination may confer improved delivery of polyanionic compounds by providing greater charge stabilization (via the cation-rich peptide compounds) along with higher lipophilic shielding (by the lipidated peptide compounds).It should further be appreciated that the amount of each of the individual tertiary amino lipidated and / or PEGylated cationic peptide compounds may be adjusted to achieve desired properties.

[0140] Synthesis of tertiary amino lipidated and / or PEGylated cationic peptide compounds In another aspect, provided herein are methods for preparing the tertiary amino lipidated and / or PEGylated cationic peptide compounds described herein.

[0141] The tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure can be synthesized without the need for additional linker species to conjugate lipid moiety to oligopeptide core.In the tertiary amino lipidated and / or PEGylated cationic peptide compounds described herein, lipid and ethylene glycol moiety are covalently and directly bonded to nitrogen atom in peptide itself, i.e., amide nitrogen or N-position in amino acid residue.Therefore, the tertiary amino lipidated and / or PEGylated cationic peptide can be fully synthesized by the method known in the art for generating N-substituted residue in peptide chain.

[0142] Tertiary amino lipidated and / or PEGylated cationic peptide compounds can be prepared by a series of acylation (amidation) and nucleophilic substitution (amination) reactions of each added amino acid residue, regardless of whether the residue has a cationic, neutral, lipid, or oligo / polyethylene glycol moiety at the N-position. The cationic peptide compounds described herein are synthesized by the sequential addition of individual residues to the peptide chain. The sequential addition of residues can be carried out iteratively until the desired sequence and length of amino acids is achieved.

[0143] The compounds of the present invention can be synthesized by both solid-phase and solution-phase methods. An exemplary method for preparing N-substituted peptides, including the tertiary amino lipidated and / or PEGylated cationic peptide compounds described herein, is discussed below by solid-phase synthesis and illustrated in Figures 3A-3E.

[0144] As shown in Figure 3A, a solid resin support having a terminal secondary amine is provided at the start of the synthesis. An acylating agent is added to the terminal secondary amine along with a suitable peptide coupling reagent and solvent to form an amide bond between the terminal secondary amine and the acylating agent. The acylating agent is preferably an acetylating agent. The acylating agent contains at least two suitable leaving groups that facilitate amidation at the alpha carbon and subsequent amination. In certain embodiments, the acylating agent is a haloacetic acid. In other embodiments, the acylating agent is bromoacetic acid.

[0145] In Figure 3B, the acylation product produced in Figure 3A is reacted with a desired substituted primary or secondary amine to provide the corresponding N-substituted terminal amino acid residue. The selected primary or secondary amine displaces a leaving group, such as the bromine in bromoacetic acid, on the α-carbon to produce the corresponding amination product. In some embodiments, the primary or secondary amine is NHR p R 2 , N.H.R. p R 3 , N.H.R. p R 4 , N.H.R. p R 5 , and N.H.R. p R 6 and R is an amine selected from the group consisting of p is -H or a protecting group, R 2 , R 3 , R 4 , R 5 , and R 6 is as defined for the tertiary amino lipidated and / or PEGylated cationic peptide compounds described herein.

[0146] The amidation and amination reactions are successively repeated (FIG. 3C) until the desired peptide sequence is achieved (FIG. 3D). It will be appreciated that the methods for preparing tertiary amino lipidated and / or PEGylated cationic peptide compounds described herein may further include protection and deprotection steps to prevent any undesired reactions with reactive moieties of the peptide chain during the successive amidation / amination reactions. For example, protecting groups R p can be added to the side chain at the α-carbon and / or N-substituent anywhere along the peptide compounds described herein during synthesis. p may include any protecting group known in the art, particularly those suitable for peptide synthesis in orthogonal protection schemes such as Boc / Bzl or Fmoc / tBu.

[0147] N-terminus and C-terminus (R 1 and R 7 ) as well as the incorporation of terminal groups at the end groups and / or side chains (R a , R b , R 4 , R 5 Functionalization of ) can be carried out by methods known in the art. One skilled in the art will recognize that the selection of a suitable method and the timing of additional functionalization steps relative to the overall solid phase synthesis will depend on the compatibility of the method with the terminal and / or linker groups being added, as well as other moieties and / or protecting groups present in the peptide compound.

[0148] The desired peptide compound is cleaved from the solid resin support under suitable reaction conditions, such as acidic conditions, including hydrochloric acid, hydrobromic acid, or trifluoroacetic acid, depending on any protection scheme utilized in the synthesis above (Figure 3E). Cleavage of the tertiary amino lipidated and / or PEGylated cationic peptide compound from the solid resin support generates the corresponding free cationic peptide compound in solution.

[0149] Further steps may be carried out to isolate and purify the cationic peptide compound from the solution, including, for example, filtration of the peptide-containing solution from the solid resin support and lyophilization of the isolated filtrate to provide a solid product.

[0150] It should be appreciated that depending on the acidic conditions used to effect resin cleavage, the tertiary amino lipidated and / or PEGylated cationic peptide compound may exist in the form of the corresponding acid salt. For example, in some embodiments, the tertiary amino lipidated and / or PEGylated cationic peptide compound is in its salt form. In certain embodiments, the salt form is an acid addition salt. In some embodiments, the salt of the tertiary amino lipidated and / or PEGylated cationic peptide compound is a hydrochloride (hydrochloric acid addition salt), a hydrobromide (hydrobromic acid addition salt), or a trifluoroacetate (trifluoroacetic acid addition salt). In certain embodiments, the salt of the tertiary amino lipidated and / or PEGylated cationic peptide compound is a trifluoroacetic acid addition salt, or a trifluoroacetic acid salt, of the tertiary amino lipidated and / or PEGylated cationic peptide compound.

[0151] The acid addition salt forms of the tertiary amino lipidated and / or PEGylated cationic peptide compounds can be further modified via methods known in the art (e.g., ion exchange) to obtain one or more pharma- ceutically acceptable salt forms. The phrase "pharmaceutically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other components that make up the formulation and / or with the subject to which the formulation is administered. The term "pharmaceutically acceptable acid addition salts" may include, but are not limited to, pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the like, as well as organic acids selected from the aliphatic, alicyclic, aromatic, arylaliphatic, heterocyclic, carboxylic, and sulfonic acid classes of organic acids such as formic acid, acetic acid, propionic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, benzoic acid, phenylacetic acid, methanesulfonic acid "mesylate", ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid.

[0152] In some embodiments, the salt of tertiary amino lipidated and / or PEGylated cationic peptide compound is a pharmaceutically acceptable salt.In certain embodiments, the salt is hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, formate, acetate, propionate, gluconate, lactate, pyruvate, oxalate, maleate, malonate, succinate, fumarate, tartrate, bitartrate, citrate, aspartate, ascorbate, glutamate, benzoate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, or salicylate.

[0153] Lipitoid In some embodiments, one or more cationic compounds of the complexes and compositions described herein comprise one or more cationic peptoid-phospholipid conjugate constructs, also known as lipitoids. Lipitoids are N-substituted polyglycine compounds (also known as "peptoids") having a combination of cationic and / or neutral side chains at the N-positions of the glycine residues along the peptoid backbone, which are further conjugated to a single terminal phospholipid group of the peptoid chain. Lipitoids and their synthesis are generally described in Simon RJ et al., Proc. Natl. Acad. Sci. USA 89, 9367-9371 (1992), Lobo, BA; Vetro, JA; Suich, DM; Zuckermann, RN; Middaugh, CR Structure / Function Analysis of Peptoid / Lipitoid: DNA Complexes. Journal of Pharmaceutical Sciences 2003, 92(9), 1905-1918, and Utku, Y.; Dehan, E.; Ouerfelli, O.; Piano, F.; Zuckermann, RN; Pagano, M.; Kirshenbaum, KA Peptidomimetic SiRNA Transfection Reagent for Highly Effective Gene Silencing. Molecular BioSystems 2006, 2(6-7), 312. An exemplary lipitoid is the nonamer "lipitoid 1" as shown in Figure 1. The general structure of a lipitoid compound is shown in formula (II) below, where x is an integer between 1 and 100, and each R 9 is independently a lipid moiety, and each R 10 is independently a cationic or neutral moiety. [ka]

[0154] In the above formula (II), x is an integer from 1 to 100. Certain numbers of residues may be particularly suitable for use in the conjugates and compositions described herein. For example, in some embodiments, x is an integer from 1 to 50, 2 to 25, or 3 to 15.

[0155] As shown in formula (II) above, R 9 represents a lipid moiety, including but not limited to, a branched or straight chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds (i.e., saturated or mono- or polyunsaturated). In certain embodiments, the lipid moiety R 9 may comprise an optionally substituted aliphatic, straight chain, or branched moiety, with each hydrophobic tail independently having from about 8 to about 30 carbon atoms or from about 6 to about 30 carbon atoms. In certain embodiments, the lipid moiety may comprise an aliphatic carbon chain derived, for example, from fatty acids and fatty alcohols. In some embodiments, each lipid moiety R 9 Independently, C8-C 24 -Alkyl or C8-C 24 -alkenyl, C8-C 24 -alkenyl is optionally mono- or polyunsaturated. In another embodiment, each lipid moiety R 9 is independently selected from the group consisting of oleyl, stearyl, linoleyl, myristyl, and lauryl. In still other embodiments that may be combined with any of the other embodiments, each lipid moiety R 9 is, independently, [ka] It is.

[0156] Group R at the N-position of the aminoglycine residue 10 contains cationic and neutral moieties similar to those described above for the tertiary amino lipidated and / or PEGylated cationic peptide compounds.

[0157] The cationic, or positively charged, moieties may include nitrogen-based substituents, such as those containing functional groups that are amino, guanidino, hydrazide, and amidino. These functional groups can be either aromatic, saturated cyclic, or aliphatic. In some embodiments of the lipitoids, each cationic moiety is independently an aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, or N-heterocyclylalkyl. In other embodiments, each cationic moiety is independently an aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, or N-heterocyclylalkyl. [ka] In certain embodiments, each cationic moiety is independently: [ka] In yet other embodiments, each cationic moiety is selected from the group consisting of: [ka] It is.

[0158] The neutral moiety may be, but is not limited to, a C1-C4-alkyl substituted with cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, each of which is optionally substituted with one or more substituents -OH, halo, or alkoxy. In some embodiments of the lipitoid, each neutral moiety is [ka] In certain embodiments, each neutral spacer moiety is selected from the group consisting of: [ka] In yet other embodiments, each neutral spacer moiety is: [ka] It is.

[0159] In yet other embodiments, each cationic moiety is: [ka] and each neutral spacer moiety is [ka] In still further embodiments, the one or more lipitoids comprises lipitoid 1.

[0160] Similar to the tertiary amino lipidated and / or PEGylated cationic peptide compounds described above, the lipidoids described herein may contain amino acid residues that may be arranged in random sequences or repeating motifs in alternating or block sequences. In some embodiments, the lipidoids of formula (II) may contain repeating (dimeric, trimeric, tetrameric, etc.) subunits within a chain of x residues. These subunits may include -R カチオン R 中性 -or-R カチオン R 中性 R 中性 - may include, but is not limited to, R カチオン is an amino acid residue containing a cationic moiety, and each R 中性 is an amino acid residue comprising a neutral spacer moiety. In another embodiment, the lipitoid of formula (II) comprises at least one trimeric subunit -R カチオン -R 中性 -R 中性 Including Rカチオン is an amino acid residue containing a cationic moiety, and each R 中性 are the amino acid residues which comprise the neutral spacer moiety.

[0161] In some embodiments in which the lipitoid of formula (II) comprises a cationic domain, each cationic moiety R 10 teeth, [ka] In other embodiments of the above embodiments, each cationic moiety R 10 teeth, [ka] and each neutral spacer moiety R 10 teeth, [ka] In certain embodiments, the lipitoid of formula (II) comprises one or more dimeric or trimeric subunits, each cationic moiety being: [ka] and each neutral spacer moiety is [ka] In yet another embodiment, the lipitoid of formula (II) comprises one or more trimeric subunits -R カチオン R 中性 R 中性 -, a cationic domain comprising R カチオン is an amino acid residue containing a cationic moiety, and each R 中性 are amino acid residues comprising a neutral spacer moiety, and each cationic moiety is [ka] and each neutral spacer moiety is [ka] It is.

[0162] As well as the tertiary amino lipidated and / or PEGylated cationic peptide compounds, the complexes and compositions described herein may contain one or more lipitoids.In certain embodiments, the one or more cationic compounds contain one or more lipitoids.In yet other embodiments, the one or more cationic compounds contain lipitoid 1.

[0163] Lipid-like cationic compounds In still other embodiments, the one or more cationic compounds of the complexes and compositions described herein may include one or more lipid-like cationic compounds.

[0164] Lipid-like cationic compounds are amphipathic compounds that contain one or more ionizable (cationic) nitrogen-containing moieties and one or more lipophilic tails. Lipid-like cationic compounds are alternatively referred to as "cationic lipids" or "ionizable lipids." The ionizable (cationic) moieties in lipid-like cationic compounds may include neutral tertiary amines that ionize to positively charged moieties at suitable physiological pH levels. Alternatively, the ionizable (cationic) atoms may be quaternary ammonium groups provided as positively charged groups with suitable counterions. The lipophilic tail may include, for example, a branched or straight chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds (i.e., saturated or mono- or polyunsaturated).

[0165] Examples of lipid-like cationic compounds are described in U.S. Patent Publication No. 2018 / 0147166A1 and are shown as formula (III) below.

[0166] In some embodiments, the one or more cationic compounds comprise one or more lipid-like cationic compounds of formula (III): [ka] In the formula, each R 11 is independently substituted or unsubstituted alkyl, and each R 12 is independently substituted or unsubstituted alkyl, and each R 13 is independently hydrogen or substituted or unsubstituted alkyl, and each y is independently an integer from 1 to 8.

[0167] PEGylated Compounds As described herein, the complexes and compositions of the present disclosure include one or more PEGylated compounds, such as PEGylated lipids or compounds with PEG (and optionally lipid) functionality. The PEGylated compounds described herein may alternatively be referred to as "PEG compounds." The incorporation of PEGylated compounds into complexes that include one or more cationic compounds and one or more polyanionic cargos promotes intracellular uptake of the cationic compound-cargo by preventing particle aggregation, shielding the surface charge of the particles, and providing protection against endogenous degradation pathways, including, for example, attack by nucleases.

[0168] PEGylated lipids In some embodiments, the conjugates and compositions described herein comprise one or more PEGylated compounds, and the one or more PEGylated compounds comprise one or more PEGylated lipids. It should be appreciated that the term "PEGylated lipid" can be interchangeably referred to as "PEG lipid" or "PEG-modified lipid". As described herein, PEGylated lipid can be understood to include any lipid or lipid-like compound covalently attached to a polyethylene glycol moiety.

[0169] Suitable lipid moieties for PEGylated lipids can include, for example, optionally substituted branched or straight chain aliphatic moieties, or optionally substituted moieties derived from natural lipid compounds, including fatty acids, sterols, and isoprenoids.

[0170] In some embodiments, the lipid moieties may include branched or straight chain aliphatic moieties having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds (i.e., saturated or mono- or polyunsaturated). In certain embodiments, the lipid moieties may include optionally substituted aliphatic, straight chain or branched moieties, each hydrophobic tail independently having about 8 to about 30 carbon atoms or about 6 to about 30 carbon atoms. In certain embodiments, the lipid moieties may include aliphatic carbon chains derived, for example, from fatty acids and fatty alcohols. In some embodiments, each lipid moiety may independently include a C8-C 24 -Alkyl or C8-C 24 -alkenyl, C8-C 24 -Alkenyl is optionally mono- or polyunsaturated.

[0171] The natural lipid moieties used in the practice of the present invention can be derived, for example, from phospholipids, glycerides (such as diglycerides or triglycerides), glycosylglycerides, sphingolipids, ceramides, as well as saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

[0172] Other suitable lipid moieties may include lipids containing a lipophilic aromatic group, such as an optionally substituted aryl or arylalkyl moiety, including, for example, naphthalenyl or ethylbenzyl, or an ester functional group, including, for example, sterol esters and wax esters.

[0173] In other embodiments, the one or more PEG lipids are selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and any combination thereof.

[0174] In some embodiments, the PEG lipid comprises a PEG-modified sterol. In certain embodiments, the PEG lipid comprises a PEG-modified cholesterol.

[0175] In some embodiments, the PEG lipid is a PEG-modified ceramide. In certain embodiments, the PEG-modified ceramine is selected from the group consisting of N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]} and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]}, and any combination thereof.

[0176] In some embodiments, the phospholipid of the PEG-modified phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). ), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (ChemsPC), 16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In certain embodiments, the phospholipid is DOPE.

[0177] In some embodiments, one or more of the PEG lipids comprises a PEG-modified phosphatidylethanol. In some embodiments, the PEG lipid is a PEG-modified phosphatidylethanol selected from the group consisting of PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), and PEG-modified DOPE (DOPE-PEG).

[0178] In certain embodiments, the PEG lipid is selected from the group consisting of dimyristoylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerol-polyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), and dioleoylglycerol-polyethylene glycol (DOG-PEG). In certain embodiments, the PEG lipid is DMG-PEG.

[0179] It should further be appreciated that certain molecular weights of the PEG chains in the aforementioned PEG lipids may be particularly advantageous for incorporation into the conjugates of the present disclosure. For example, in some embodiments, the PEG chains have a molecular weight of 350-6,000 g / mol, 1,000-5,000 g / mol, or 2000-5,000 g / mol. In certain embodiments, the PEG chains of the PEG lipids have a molecular weight of about 350 g / mol, 500 g / mol, 600 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, 3,000 g / mol, 5,000 g / mol, or 10,000 g / mol. In certain other embodiments, the PEG chains of the PEG lipids have a molecular weight of about 500 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, or 5,000 g / mol. For example, in certain embodiments, the PEGylated lipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000).

[0180] Tertiary Aminolipidated and PEGylated Cationic Peptides In yet another embodiment, one or more PEGylated compounds comprise tertiary amino PEGylated cationic peptide compounds of formula (I) that contain at least one oligo or polyethylene glycol moiety.By virtue of their flexibility to accommodate both lipid and PEG moieties along the backbone of the peptide chain, and depending on the nature of their specific substituents, the tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) of the present disclosure function not only as cationic delivery vehicles, but also as suitable PEGylated compounds that stabilize cationic compound-polyanionic compound complexes.It should be recognized that the tertiary amino PEGylated cationic peptide compounds of formula (I) can be combined with other classes of cationic compounds, such as lipoids or lipid-like compounds, as well as other tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I).

[0181] In some embodiments, the PEGylated compound is a tertiary amino lipidated and / or PEGylated cationic peptide compound of formula (I) described herein, where at least one of m and t is other than 0 (i.e., N-PEGylated). In still further embodiments, the PEGylated compound is a tertiary amino lipidated and / or PEGylated cationic peptide compound of formula (I), where m and t are independently integers 0-15, and at least one of m and t is other than 0. In some embodiments, the PEGylated compound is a tertiary amino lipidated and PEGylated cationic peptide compound of formula (Ib). In yet other embodiments, the PEGylated compound is a tertiary amino lipidated and PEGylated cationic compound of formula (Ic).

[0182] As with the PEGylated lipids described above, certain molecular weights of the PEG chains in the aforementioned tertiary amino lipidated and PEGylated cationic peptide compounds may be particularly advantageous for incorporation into the conjugates of the present disclosure. For example, in some embodiments, the PEG chains have a molecular weight of 350-6,000 g / mol, 1,000-5,000 g / mol, or 2000-5,000 g / mol. In certain embodiments, the PEG chains of the tertiary amino lipidated and PEGylated cationic peptide compounds have a molecular weight of about 350 g / mol, 500 g / mol, 600 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, 3,000 g / mol, or 5,000 g / mol. In certain other embodiments, the PEG chains of the PEG lipids have a molecular weight of about 500 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, or 5,000 g / mol.

[0183] However, it should be appreciated that the tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) may contain several short oligoethylene glycol moieties in place of fewer and longer polyethylene glycol moieties to provide similar particle stabilization to the complex. In some embodiments where the PEGylated compound is an N-PEGylated cationic peptide compound of formula (I), the tertiary amino lipidated and / or PEGylated cationic peptide compound has the formula -CH2CHO(CH2CHO) u R 2a At least one ethylene glycol moiety R 2 wherein each R 2a is independently -H or C1-C4-alkyl. In another embodiment, the formula -CH2CH2O(CH2CH2O) v R 6 At least one ethylene glycol moiety R 6a where each R 6a are independently -H or C1-C 4-In some embodiments that may be combined with any of the preceding embodiments where m is other than 0, m is an integer from 0 to 15, and each u is independently an integer from 1 to 200. In still further embodiments that may be combined with any of the preceding embodiments where t is other than 0, t is an integer from 0 to 15, and each v is independently an integer from 1 to 200.

[0184] In some embodiments, the PEGylated compound is a tertiary amino lipidated and / or PEGylated cationic peptide compound of formula (I) described herein, where at least one of m and t is other than 0 and at least one of n and s is other than 0 (i.e., N-lipidated). In certain embodiments, where the N-PEGylated cationic peptide compound of formula (I) is also N-lipidated, at least one of n and s is other than 0. In some embodiments, the sum of n and s is at least 1, 2, 3, or 4. In certain embodiments, s is 4. In yet another embodiment, n is 4.

[0185] Polyanionic Compounds and Other Delivery Cargoes As described herein, the complexes and compositions of the present disclosure may include one or more polyanionic compounds, such as nucleic acids, complexed with cationic compounds.The complexes and compositions of the present disclosure, which include one or more cationic compounds, such as tertiary amino lipidated and / or PEGylated cationic peptide compounds, and one or more PEGylated lipids, together with polyanionic compounds, may be used to deliver polyanionic compounds, such as nucleic acids, to the inside of cells.In addition to polyanionic cargoes, the complexes and compositions provided herein may also be utilized for intracellular delivery of a wide range of non-anionic cargoes.Depending on the polyanionic and non-anionic compounds or cargoes delivered to cells, the complexes and compositions provided herein may be useful in many different applications, including, for example, vaccination and gene therapy.

[0186] In some embodiments, one or more polyanionic compounds comprise nucleic acid.Nucleic acid as used herein includes naturally occurring nucleic acid such as DNA, RNA, and / or their hybrids, as well as non-naturally occurring variants with non-natural backbone and modified backbone linkages, such as phosphorothioate, non-natural and modified bases, and non-natural and modified termini.Exemplary nucleic acids include genomic DNA, cDNA, mRNA, miRNA, and siRNA.

[0187] Nucleic acids can be recombinantly produced or chemically synthesized molecules. Nucleic acids can be single-stranded, double-stranded, triple-stranded, and quadruple-stranded, as well as more complex three-dimensional forms that contain single- and double-stranded regions.

[0188] Depending on the type of nucleic acid, the length of the nucleic acid (defined in nucleotide units or base pairs (bp) as appropriate) may vary. In some embodiments where the nucleic acid is mRNA, the mRNA may have 100-10,000 nucleotide units, or 1,000-3,000 nucleotide units. In other embodiments where the nucleic acid is DNA, the DNA may have 5,000 bp-20,000 bp, or about 10,000 bp.

[0189] In some embodiments where the nucleic acid is an mRNA, the mRNA is an mRNA that codes for a protein or peptide. In some embodiments where the nucleic acid is an mRNA, the mRNA is an mRNA that codes for a peptide, including an oligopeptide or a polypeptide. In certain embodiments, the mRNA is an mRNA that codes for a polypeptide. In yet further embodiments, the mRNA is an mRNA that codes for a protein. In other embodiments, the mRNA is an mRNA that codes for a peptide. As described above, the mRNA can be naturally occurring (e.g., isolated tumor RNA) or synthetic (e.g., produced by in vitro transcription). In synthetic or non-naturally occurring variants of mRNA, the mRNA can include non-natural backbones with modified backbone bonds, such as phosphorothioates, non-natural and modified bases, and / or non-natural and modified termini. In certain embodiments where the nucleic acid is an mRNA, the mRNA can include special sequences, such as self-amplifying sequences or internal ribosome entry sites.

[0190] In some embodiments, the combined delivery of two or more specific nucleic acids may be particularly useful for therapeutic applications.For example, in some embodiments, one or more polyanionic compounds include a combination of sgRNA (single guide RNA) as a CRISPR sequence and mRNA encoding Cas9.In yet further embodiments, the nucleic acid may also be complexed with a protein, such as a CRISPR / Cas9 ribonucleoprotein complex.

[0191] In some embodiments, the one or more polyanionic compounds may include anionic or polyanionic compounds that are not nucleic acids. Suitable anionic compounds may include, but are not limited to, proteins, polyphosphates, or heparin. In some embodiments, the one or more polyanionic compounds include one or more proteins. In one embodiment, the one or more polyanionic compounds include Cas9 proteins. In other embodiments, the one or more polyanionic compounds include polyphosphates. In yet other embodiments, the one or more polyanionic compounds include heparin or other glycosaminoglycan derivatives.

[0192] In addition to the suitability of the complexes and compositions provided herein to deliver polyanionic cargo to cells, it should be appreciated that the complexes and compositions described herein may also be utilized to deliver other non-anionic agents or cargos (including hydrophobic compounds) to cells. These other cargoes may include, but are not limited to, small molecule active agents (either as independent therapeutic agents or in combination with another agent, such as a nucleic acid) and / or immunological adjuvants. It should be further appreciated that these other cargo molecules may or may not be combined with any of the polyanionic compounds described herein for complex formation. In some embodiments, the complexes described herein include an endosomal escape modulating agent, a TLR agonist, and a chemotherapeutic agent. In other embodiments, the complexes of the present disclosure include an adjuvant or immune co-stimulatory agent. For example, in some embodiments, the complexes or compositions include an immunological adjuvant selected from the group consisting of CpG oligodeoxynucleotides (ODN), lipopolysaccharide (LPS), and any combination thereof.

[0193] Additional Ingredients The conjugates and compositions of the present disclosure may include one or more additional components, including polyethylene glycol and / or a targeting agent.

[0194] In some embodiments, the conjugates and compositions include one or more additional components, and the one or more additional components include polyethylene glycol (PEG). It is further recognized that certain molecular weights of polyethylene glycol may be particularly suitable for incorporation into the conjugates and compositions of the present disclosure. For example, in some embodiments, the polyethylene glycol has a molecular weight of 350-6,000 g / mol, 1,000-5,000 g / mol, or 2000-5,000 g / mol. In certain embodiments, the polyethylene glycol has a molecular weight of about 350 g / mol, 500 g / mol, 600 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, 3,000 g / mol, 5,000 g / mol, or 10,000 g / mol. In certain other embodiments, the polyethylene glycol has a molecular weight of about 500 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, or 5,000 g / mol. It will be appreciated that in some embodiments in which the composition includes polyethylene glycol as an additional component, one or more polyethylene glycol polymers of different molecular weights may be included in the conjugates and compositions.

[0195] In other embodiments, the complex or composition comprises one or more additional components, wherein the one or more additional components comprises a targeting agent, hi certain embodiments, the targeting agent is a small molecule, an antibody, or an antibody fragment.

[0196] It should be appreciated that the compositions described herein may be prepared as a formulation for administration. In some embodiments, the formulation is a solution, suspension, colloidal suspension, spray, or aerosol. In other embodiments, the formulation is a lipid complex formulation. In some embodiments, the formulation is an enteral formulation, a parenteral formulation, or a topical formulation. In some embodiments, the formulation is an injectable formulation, such as an intravenous formulation, a subcutaneous formulation, an intramuscular formulation, an intradermal formulation, an intraocular formulation, an intrathecal formulation, or an intratumoral formulation. In other embodiments, the formulation is an oral formulation. In still other embodiments, the formulation is a mucosal formulation, including, for example, a nasal formulation, an anal formulation, an oral formulation, or an intravaginal formulation.

[0197] It will be appreciated that the components used in preparing the conjugates and formulations of the present disclosure, as well as the process parameters for making said conjugates or formulations, may be adapted depending on whether the conjugate and / or formulation is intended for immediate use ("just-in-time") or will be placed in storage for future use. In particular, storage considerations may include the temperature at which the conjugate and / or formulation will be maintained (e.g., room temperature, 4°C, -20°C, -78°C) and the storage period.

[0198] Thus, suitable excipients may include, but are not limited to, those that facilitate administration or improve storage stability (e.g., cryoprotectants).For example, in addition to the above components, the formulations described herein may include pharma- ceutically acceptable excipients such as carriers, solvents, dispersants, diluents, fillers, stabilizers, preservatives, etc.In still other embodiments, the compositions may include pharma-ceutically acceptable excipients or carriers, including, for example, solvents, diluents, buffers, and viscosity modifiers.

[0199] However, in contrast to standard lipid nanoparticle formulations, the conjugates and compositions of the present disclosure do not contain standalone structural lipids (such as cholesterol or related sterols) or standalone phospholipids (e.g., DOPE) unless conjugated to a polyethylene glycol moiety (PEG modification) as described above.

[0200] Properties of the Complex and Composition As described herein, the complexes and compositions of the present disclosure are primarily composed of one or more cationic compounds, one or more PEG compounds, and one or more polyanionic compounds, such as nucleic acids for intracellular delivery. In contrast to many other existing lipid-based formulations, such as standard lipid nanoparticle formulations, the complexes and compositions of the present disclosure do not contain structural lipids (such as cholesterol) or phospholipids (such as DOPE). These additional co-ingredients are typically used to provide improved structural stability and cell membrane permeability and constitute a significant proportion of the overall formulation. Traditional lipid nanoparticle formulations contain large amounts of inactive ingredients and therefore require larger amounts of the formulation to be administered to deliver a specific amount of nucleic acid, e.g., to elicit a desired therapeutic or prophylactic effect. In contrast, formulations that contain only cationic compounds complexed with polyanionic cargo to neutralize charge contain a large proportion of polyanionic material by weight, but these cationic compound-polyanionic compound complexes suffer from particle instability and aggregation without the benefit of any additional co-ingredients.

[0201] The complexes and compositions of the present disclosure overcome the shortcomings of conventional lipid formulations by combining cationic and polyanionic compounds with small proportions of PEGylated lipids, thereby stabilizing the cationic compound-polyanionic compound complex, protecting it from in vivo degradation, and promoting endocytosis, without detrimentally reducing the weight percentage of active polyanionic material in the complex, and therefore the overall composition.

[0202] In one aspect, the complexes and compositions of the present disclosure may be characterized by the mass percentage (or weight percentage) of each of the individual components present in the complex. Alternatively, the complexes and compositions may be characterized by the combined mass percentage of two or more components relative to the total mass of the complex. For example, in some embodiments, the one or more polyanionic compounds, the one or more PEG compounds, and the one or more cationic compounds have a combined mass percentage of at least 90% w / w, at least 95% w / w, or at least 99% w / w of the complex. In certain embodiments, the one or more polyanionic compounds, the one or more PEG compounds, and the one or more cationic compounds have a combined mass percentage of at least 90% w / w of the complex. In embodiments in which the complex comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds or salts thereof, the one or more polyanionic compounds, the one or more PEG compounds, and the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds or salts thereof have a combined mass percentage of at least 90% w / w, at least 95% w / w, or at least 99% w / w of the complex. In certain embodiments, the one or more polyanionic compounds, the one or more PEG compounds, and the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds or salts thereof comprise a total weight percentage of at least 90% w / w of the complex.In other embodiments, the complex comprises a complex containing one or more lipitoids, the one or more polyanionic compounds, the one or more PEG compounds, and the one or more lipitoids comprise a total weight percentage of at least 90% w / w, at least 95% w / w, or at least 99% w / w of the complex.In certain embodiments, the one or more polyanionic compounds, the one or more PEG compounds, and the one or more lipitoids comprise a total weight percentage of at least 90% w / w of the complex.

[0203] In some embodiments where the conjugate or composition includes one or more additional components, such as a polyethylene glycol polymer or a targeting agent, the one or more additional components are present in a weight percent of the conjugate of 10% or less.

[0204] In another aspect, the complexes and compositions of the present disclosure can be characterized by the mass ratio of the individual components to each other. For example, as described herein, the complexes of the present disclosure include a low mass percentage of the PEGylated compound compared to the mass percentage of the cationic delivery vehicle. In some embodiments where the complex or composition includes one or more cationic compounds and one or more PEG compounds, the one or more cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of 90:10 to 99:1, 92:8 to 98:2, 95:5 to 99:1, or 95:5 to 98:2. In certain embodiments where the complex or composition includes one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of 90:10 to 99:1, 92:8 to 98:2, 95:5 to 99:1, or 95:5 to 98:2. In still other embodiments where the complex or composition comprises one or more lipitoids, the one or more lipitoids and the one or more PEG compounds are present in a weight ratio of 90:10 to 99:1, 92:8 to 98:2, 95:5 to 99:1, or 95:5 to 98:2.

[0205] In some embodiments where the complex or composition comprises one or more cationic compounds and one or more PEG lipids, the one or more cationic peptide compounds and the one or more PEG lipids are present in a mass ratio of 90:10 to 99:1, 92:8 to 98:2, 95:5 to 99:1, or 95:5 to 98:2. In certain embodiments where the complex or composition comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and the one or more PEG lipids are present in a mass ratio of 90:10 to 99:1, 92:8 to 98:2, 95:5 to 99:1, or 95:5 to 98:2. In still other embodiments where the complex or composition comprises one or more lipitoids, the one or more lipitoids and the one or more PEG lipids are present in a mass ratio of 90:10 to 99:1, 92:8 to 98:2, 95:5 to 99:1, or 95:5 to 98:2.

[0206] The complexes and compositions comprising the complexes described herein may be characterized by the total cationic charge in one or more cationic compounds, for example, the ratio of the number of cationic groups in the tertiary amino lipidated and / or PEGylated cationic peptides to the number of anionic phosphate groups in the nucleic acid. In some embodiments, the complexes comprise one or more cationic compounds and one or more polyanionic compounds in a cationic:anionic charge ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1. In certain embodiments, the complexes comprise one or more tertiary amino lipidated and / or PEGylated cationic peptides and one or more polyanionic compounds, such as nucleic acids, in a cation:anion charge ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1. In certain other embodiments, the complex comprises one or more lipitoids and one or more polyanionic compounds in a cation:anion charge ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1.

[0207] In other embodiments, the complex comprises one or more cationic compounds and one or more nucleic acids at a cation:anion charge ratio of 2:1 to 5:1. In some embodiments, the complex comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more nucleic acids at a cation:anion charge ratio of 2:1 to 5:1. In still other embodiments, the complex comprises one or more lipitoids and one or more nucleic acids at a cation:anion charge ratio of 2:1 to 5:1. In some embodiments, the complex comprises one or more cationic compounds and one or more nucleic acids at a cation:anion charge ratio of 3:1. In other embodiments, the complex comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more nucleic acids at a cation:anion charge ratio of 3:1. In still other embodiments, the complex comprises one or more lipitoids and one or more nucleic acids at a cation:anion charge ratio of 3:1.

[0208] Alternatively, the complexes and compositions may be characterized by the relative mass ratio of the cationic compound to the polyanionic compound and / or other cargo in the complex. The mass ratio of components in a complex can be easily calculated based on the known concentrations and volumes of stock solutions of each component used in preparing the complex. Furthermore, when non-anionic cargo is present in the complex, the mass ratio may provide a more accurate indication of the relative amount of cationic compound to the total cargo than the cationic:anionic charge ratio, which does not take into account the non-anionic material.

[0209] In some embodiments, the complex comprises one or more cationic compounds and one or more polyanionic and / or non-anionic compounds in a mass ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1. In certain embodiments, the complexes comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more polyanionic and / or non-anionic compounds in a mass ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1. In other embodiments, the complex comprises one or more lipitoids and one or more polyanionic and / or non-anionic compounds in a mass ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1.

[0210] In other embodiments, the complex comprises one or more cationic compounds and one or more polyanionic compounds and / or non-anionic compounds in a mass ratio of 2:1 to 5:1. In certain embodiments, the complex comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more polyanionic compounds and / or non-anionic compounds in a mass ratio of 2:1 to 5:1. In some embodiments, the complex comprises one or more lipitoids and one or more polyanionic compounds and / or non-anionic compounds in a mass ratio of 2:1 to 5:1. In still other embodiments, the complex comprises one or more cationic compounds and one or more polyanionic compounds and / or non-anionic compounds in a mass ratio of 3:1. In certain embodiments, the complex comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more polyanionic compounds and / or non-anionic compounds in a mass ratio of 3:1. In yet a further embodiment, the complex comprises one or more lipitoids and one or more polyanionic and / or non-anionic compounds in a mass ratio of 3:1.

[0211] In certain embodiments in which a complex comprises one or more nucleic acids as part of one or more polyanionic compounds, the complex comprises one or more cationic compounds and one or more nucleic acids in a mass ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1. In some embodiments, the complexes comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more nucleic acids in a mass ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1. In other embodiments, the complex comprises one or more lipidoids and one or more nucleic acids in a mass ratio of 0.5:1 to 50:1, 0.5:1 to 20:1, 0.5:1 to 10:1, 0.5:1 to 5:1, 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5:1. In some embodiments, the complex comprises one or more cationic compounds and one or more nucleic acids in a mass ratio of 2:1 to 5:1. In certain embodiments, the complex comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more nucleic acids in a mass ratio of 2:1 to 5:1. In certain other embodiments, the complex comprises one or more lipidoids and one or more nucleic acids in a mass ratio of 2:1 to 5:1. In still other embodiments, the complex comprises one or more cationic compounds and one or more nucleic acids in a mass ratio of 3:1. In certain embodiments, the complex comprises one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds and one or more nucleic acids in a mass ratio of 3:1. In certain other embodiments, the complex comprises one or more lipidoids and one or more nucleic acids in a mass ratio of 3:1.

[0212] Methods for Preparing Complexes and Compositions Cationic and polyanionic compound complexes and their compositions can be prepared by various physical and / or chemical methods to adjust their physical, chemical, and biological properties. These usually include rapid mixing of cationic compounds (e.g., tertiary amino lipidated and / or PEGylated cationic peptides) in water or in a water-miscible organic solvent with the desired polyanionic compounds (e.g., oligonucleotides) in water or in an aqueous buffer solution. These methods include simple mixing of the components by pipetting, or microfluidic mixing processes such as those involving T-mixers, vortex mixers, or other chaotic mixing structures. The compositions of the present disclosure can be prepared in a similar manner.

[0213] In one aspect, the present disclosure provides a method for preparing the complexes and compositions described herein, comprising contacting one or more cationic compounds with one or more polyanionic compounds and one or more PEG compounds, such as PEG lipids.In some embodiments, the present disclosure provides a method for preparing a composition, comprising contacting one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds with one or more polyanionic compounds and one or more PEG compounds.In other embodiments, the present disclosure provides a method for preparing a composition, comprising contacting one or more lipoids with one or more polyanionic compounds and one or more PEG compounds.

[0214] In some embodiments, the method for preparing a complex or composition comprising one or more cationic compounds, one or more PEG compounds, and one or more polyanionic compounds includes contacting a solution comprising one or more cationic compounds with a solution comprising one or more polyanionic compounds and a solution comprising one or more PEG compounds. In certain embodiments, the polyanionic compound comprises a nucleic acid. In some embodiments, the one or more cationic compounds comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds. In other embodiments, the one or more cationic compounds comprise one or more lipoids.

[0215] Additional components in the complexes and compositions, such as polyethylene glycol and / or targeting agent or excipient additional components, may be mixed or admixed with the composition before, during, or after the main components of the cationic compound, polyanionic compound, and PEG compound are mixed.

[0216] The specific process conditions for preparing the conjugates and compositions described herein can be adjusted or selected accordingly to provide the desired physical properties of the composition. For example, parameters for mixing the components of the composition that can affect the final composition can include, but are not limited to, the order of mixing, the temperature of mixing, the speed / rate of mixing, the flow rate, the concentration of the stock solution, the mass ratio of the components (e.g., peptide:cargo), and the solvent.

[0217] Methods of using the conjugates and compositions thereof As described above, the compositions described herein facilitate the delivery of polyanionic compounds to cells, particularly to the intracellular environment. As such, the compositions may find use in many clinical applications as well as research applications. Delivery of polyanionic compounds to cells may be used for clinical applications, such as those related to preventive, diagnostic, and / or therapeutic methods. For example, in some embodiments, suitable clinical applications may include vaccination, cancer immunotherapy, protein replacement therapy, and / or in vivo gene editing, ex vivo cell therapy transfection, ex vivo stem cell induction. Methods of delivering polyanionic compounds to cells may also be useful for research or non-clinical applications, including for biological assays and reagents.

[0218] In another aspect, provided herein is a method for delivering a polyanionic compound to a cell. In some embodiments, the method for delivering a polyanionic compound to a cell comprises contacting the cell with a complex comprising one or more cationic compounds, one or more PEGylated compounds, and a polyanionic compound. In other embodiments, the method for delivering a polyanionic compound to a cell comprises contacting the cell with a complex comprising one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, one or more PEGylated compounds, and a polyanionic compound. In some embodiments of the aforementioned method, the contacting is by endocytosis. In yet other embodiments, provided herein is a method for delivering a polyanionic compound to a cell, comprising contacting the cell with a composition comprising a complex comprising one or more cationic compounds, one or more PEGylated compounds, and a polyanionic compound. In other embodiments, provided herein is a method comprising contacting the cell with a composition comprising a complex comprising one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, one or more PEGylated compounds, and a polyanionic compound.

[0219] In some embodiments, the method of delivering a polyanionic compound to a cell comprises contacting the cell with a complex comprising one or more cationic compounds, one or more PEGylated compounds, and a polyanionic compound, and the cell is contacted in vitro, ex vivo, or in vivo. In certain embodiments, the method of delivering a polyanionic compound to a cell comprises contacting the cell with a complex comprising one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, one or more PEGylated compounds, and a polyanionic compound, and the cell is contacted in vitro, ex vivo, or in vivo. In other embodiments, the method of delivering a polyanionic compound to a cell comprises contacting the cell with a composition comprising a complex comprising one or more cationic compounds, one or more PEGylated compounds, and a polyanionic compound, and the cell is contacted in vitro, ex vivo, or in vivo. In certain embodiments, a method of delivering a polyanionic compound to a cell comprises contacting the cell with a composition comprising a complex comprising one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds, one or more PEGylated compounds, and a polyanionic compound, wherein the cell is contacted in vitro, ex vivo, or in vivo.

[0220] In some embodiments where the cells are contacted in vitro, the cells are HeLa, HepG2, or JAWSII cells. In other embodiments where the cells are contacted in vivo, the complex of the present disclosure, or its composition, or its formulation, is administered to a mammalian subject. The mammalian subject may include, but is not limited to, a human or mouse subject. In yet other embodiments where the cells are contacted ex vivo, the cells are obtained from a human or mouse subject.

[0221] In some embodiments of the aforementioned methods of contacting cells in vivo, the complexes and compositions described herein may be administered by injection. In certain embodiments, the complexes and compositions described herein may be administered by injection (intravenous (IV), subcutaneous (SC), intramuscular (IM), intrathecal, or intratumoral). In some embodiments, the complexes and compositions are administered by intravenous (IV), subcutaneous (SC), intramuscular (IM), intrathecal, or intratumoral injection. In other embodiments, the complexes and compositions described herein are administered by bolus injection or intravenous infusion. In other embodiments of contacting cells in vivo, the complexes and compositions of the present disclosure are administered by nasal or oral inhalation. In some embodiments of contacting cells in vivo, the complexes and compositions described herein are administered orally. In yet other embodiments of contacting cells in vivo, the complexes and compositions are administered via absorption into mucosa (including topical, anal, buccal, vaginal, etc.).

[0222] In another aspect, provided herein is a method for delivering a polyanionic compound to one or more target organs of a subject (e.g., lung, spleen, liver, kidney, inguinal lymph node, and / or mesenteric lymph node). Organ specificity (e.g., targeting to the lung) can be achieved by delivering one or more cationic compounds (e.g., having at least a 2+ charge and a pK within a particular range). a The peptoid:cargo ratio can be influenced by the choice of the tertiary amino lipidated and / or PEGylated compounds of formula (I), such as having

[0223] It should be understood that clinical applications, such as the diagnostic, prophylactic, and therapeutic examples disclosed above, may involve dosing regimens (e.g., dosage levels and time course of administration) that may vary depending on the particular conjugates and compositions used, the route of administration, the subject to which the conjugates and compositions are administered, and / or the physiological effect desired. For example, in some embodiments, the methods of the present disclosure include administering the conjugate or composition at a dose of 0.001 mg / kg to about 1 mg / kg body weight.

[0224] Enumeration of embodiments The following enumerated embodiments are representative of some aspects of the present invention. 1. (i) one or more polyanionic compounds; (ii) one or more PEG compounds; and (iii) A composition comprising a complex of one or more cationic compounds, wherein the one or more cationic compounds are (a) one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I), or salts thereof, [ka] During the ceremony, m is 0, n is an integer from 0 to 5; s is an integer from 0 to 5; t is 0, At least one of n and s is other than 0; r is an integer from 1 to 20; each o is independently an integer 0, 1, or 2; each q is independently an integer 0, 1, or 2; each p is independently an integer 1 or 2; R 1 is -H, alkyl, alkylaryl, -COR 1a or a lipid moiety, R 1ais -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl; Each R 2 is, independently, Formula -CH2CH2O(CH2CH2O) u an ethylene glycol portion of CH3, and each u is independently an integer from 2 to 200; Each R 3 is independently a lipid moiety, Each R 4 is independently a neutral spacer moiety or a lipid moiety; Each R 5 is independently a cationic moiety; Each R 6 are independently represented by the formula -CH2CH2O(CH2CH2O) v the ethylene glycol portion of CH3, and each v is independently an integer from 2 to 200; R 7 is -H, alkyl, acyl, -OH, -OR 7a , -NH2, -NHR 7a , or a lipid moiety, R 7a is an alkyl, acyl, or lipid moiety; Each R a and R bは、独立して、-H、C one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) or salts thereof, wherein the tertiary amino acid is 1-C4-alkyl or a side chain moiety found in naturally or non-naturally occurring amino acids; (b) one or more lipoids of formula (II), [ka] During the ceremony, x is an integer from 1 to 100; Each R 9 is independently a lipid moiety, Each R 10 is independently a cationic or neutral spacer moiety, (c) one or more lipid-like compounds of formula (III), [ka] During the ceremony, Each R 11 is independently substituted or unsubstituted alkyl; Each R 12 is independently substituted or unsubstituted alkyl; Each R 13 are independently hydrogen or substituted or unsubstituted alkyl; one or more lipid-like compounds of formula (III), wherein each y is independently an integer from 1 to 8; And (d) any combination thereof; A composition, wherein the one or more cationic compounds, the one or more PEG compounds, and the one or more polyanionic compounds have a combined weight percent of at least 90% w / w of the complex. 2. The composition of embodiment 1, wherein the one or more cationic compounds are one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I). 3. The composition of embodiment 1 or embodiment 2, wherein the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprise a block of N-lipidated amino acid residues, and either n or s is at least 2. 4.Each R 3 But independently, C4-C 22 -Alkyl or C4-C 22 -alkenyl, C4-C 22 The composition of any one of embodiments 1-3, wherein -alkenyl is optionally mono- or polyunsaturated. 5.Each R 3 But independently, C8-C 12 The composition of any one of embodiments 1-4, wherein is alkyl. 6.Each R 3 is independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl. 7. At least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprises a cationic moiety R 5 7. The composition of any one of embodiments 1-6, comprising a cationic domain comprising at least two cationic amino acid residues having the formula: 8.Each R 5 The composition of any one of embodiments 1-7, wherein is independently aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, N-heterocyclylalkyl, or N-heteroaryl. 9.Each R 5 But independently, [ka] The composition of any one of embodiments 1-8, selected from the group consisting of: 10.Each R 5 but, [ka] 10. The composition of any one of embodiments 1 to 9, wherein 11. Each neutral spacer portion R 4 is independently C1-C4-alkyl substituted by cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl, and each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl is optionally substituted by one or more substituents -OH, halo, or alkoxy. 12. Each neutral spacer portion R 4 But independently, [ka] 12. The composition of any one of embodiments 1-11, selected from the group consisting of: 13. Each neutral spacer portion R 4 but, [ka] 13. The composition of any one of embodiments 1-12, wherein 14. At least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds is selected from the group consisting of R 5 The cationic domain comprises at least two amino acid residues having a cationic moiety, and each of the at least two cationic amino acid residues in the cationic domain is linked to a neutral spacer or a lipid moiety R 4 14. The composition of any one of embodiments 1-13, wherein the amino acids are separated by at least one amino acid residue having the formula: 15. At least one of the one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds comprises at least one trimeric subunit -R カチオン -R 中性 -R 中性 Including R カチオン is a cationic moiety R 5 Each R 中性 is a neutral spacer moiety R 4 The composition of any one of embodiments 1-14, wherein the amino acid residue comprises: 16. Each cationic moiety R 5 but, [ka] and each neutral spacer moiety R 4 but, [ka] 16. The composition of any one of embodiments 1-15, wherein 17.R aand Rb is independently selected from the group consisting of -H and -CH3. 18.R a and Rb is -H. 19. The composition of any one of embodiments 1-18, wherein the one or more cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of peptide compounds to PEG compounds of 90:10 to 99:1. 20. The composition of any one of embodiments 1-19, wherein the one or more cationic peptide compounds and the one or more PEG compounds are present in a mass ratio of peptide compounds to PEG compounds of from 95:5 to 98:2. 21. The composition of any one of embodiments 1-20, wherein the one or more PEG compounds comprises DMG-PEG2000. 22. The one or more PEG compounds comprise one or more tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) or salts thereof; [ka] During the ceremony, m is an integer from 0 to 10; n is an integer from 0 to 5; s is an integer from 0 to 5; t is an integer from 0 to 10; At least one of m and t is other than 0; r is an integer from 1 to 20; each o is independently an integer 0, 1, or 2; each q is independently an integer 0, 1, or 2; each p is independently an integer 1 or 2; R 1 is -H, alkyl, alkylaryl, -COR 1a or a lipid moiety, R 1a is -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl; Each R2 are independently represented by the formula -CH2CH2O(CH2CH2O) u an ethylene glycol portion of CH3, and each u is independently an integer from 2 to 200; Each R 3 is independently a lipid moiety, Each R 4 is independently a neutral spacer moiety or a lipid moiety; Each R 5 is independently a cationic moiety; Each R 6 are independently represented by the formula -CH2CH2O(CH2CH2O) v the ethylene glycol portion of CH3, and each v is independently an integer from 2 to 200; R 7 is -H, alkyl, acyl, -OH, -OR 7a , -NH2, -NHR 7a , or a lipid moiety, R 7a is an alkyl, acyl, or lipid moiety; Each R a and R bは、独立して、-H、C The composition of any one of embodiments 1-21, which is 1-C4-alkyl or a side chain moiety found in natural or non-naturally occurring amino acids. twenty three. when m is an integer from 0 to 3, each u is independently an integer from 20 to 200, optionally from 30 to 50; 23. The composition of embodiment 22, wherein when m is an integer from 4 to 10, each u is independently an integer from 2 to 10. twenty four. when t is an integer from 0 to 3, each v is independently an integer from 30 to 50; 24. The composition of embodiment 22 or embodiment 23, wherein when t is an integer from 4 to 10, each v is independently an integer from 2 to 10. twenty five. m is 1 and u is an integer between 40 and 45; or The composition of any one of embodiments 22-24, wherein t is 1 and v is an integer from 40 to 45. 26. The composition of any one of embodiments 1-25, wherein the one or more polyanionic compounds comprises a nucleic acid. 27. The composition of any one of embodiments 1-26, wherein the one or more polyanionic compounds comprise a nucleic acid and the mass ratio of the one or more cationic peptide compounds to nucleic acid is from 0.5:1 to 20:1. 28. The composition of any one of embodiments 1-27, wherein the one or more polyanionic compounds comprise a nucleic acid, and the nucleic acid is an mRNA encoding a polypeptide. 29. The composition of any one of embodiments 1-28, wherein the one or more polyanionic compounds comprises a nucleic acid, and the nucleic acid is an mRNA encoding a protein. 30. The composition of any one of embodiments 1-29, wherein the conjugate further comprises one or more second agents. 31. The composition of any one of embodiments 1-30, wherein the one or more second agents are selected from the group consisting of polyethylene glycol, targeting elements, and combinations thereof. 32. The composition of any one of embodiments 1-31, wherein the conjugate further comprises one or more small molecule active agents or drug substances. 33. The composition of any one of embodiments 1-31, wherein the complex is suspended in an aqueous solution. 34. A method for delivering a polyanionic compound to a cell, comprising contacting the cell with the composition of any one of embodiments 1 to 33. 35. The method of embodiment 34, wherein the contacting is by endocytosis. 36. The method of embodiment 34 or 35, wherein the cell is contacted in vivo or in vitro. 37. The method of any one of embodiments 35-36, wherein the polyanionic compound comprises a nucleic acid that is an mRNA encoding a polypeptide, and the cell expresses the polypeptide after contacting with the composition. 38. The method of any one of embodiments 34-37, wherein the polyanionic compound comprises a nucleic acid that is an mRNA encoding a protein, and the cell expresses the protein after contacting with the composition. 39. A method for preparing the composition of any one of embodiments 1 to 33, comprising contacting one or more cationic peptide compounds with one or more PEG compounds and one or more polyanionic compounds. 40. The method of embodiment 39, comprising contacting a solution comprising one or more cationic peptide compounds and one or more PEG compounds with a solution comprising one or more polyanionic compounds. EXAMPLES

[0225] The present disclosure is described in further detail in the following examples, which are in no way intended to limit the scope of the disclosure claimed.The attached figures are intended to be considered as an integral part of the details and description of the present disclosure.The following examples are provided for illustration, but are not intended to limit the disclosure claimed.

[0226] Example 1 - Synthesis of exemplary tertiary amino lipidated cationic peptides for nucleic acid delivery The following examples illustrate general protocols for the synthesis of the tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) described herein. [ka]

[0227] In the explanation provided below, R a and R b are all -H. All polymers are synthesized using bromoacetic acid and primary amines. Figures 2B-2E show the synthesis of tertiary amino lipidated and / or PEGylated cationic peptide compounds of the present disclosure, R 2 , R 3 , R 4 , R 5 , and R 6 Some exemplary substituents for primary amines of the formula are provided below.

[0228] Fmoc-Rink amide resin is used as the solid support. The Fmoc group of the resin is deprotected with 20% (v / v) piperidine-dimethylformamide (DMF). The amino resin is then amidated with bromoacetic acid. This is followed by amination of the α-carbon by nucleophilic displacement of the bromide with a primary amine. The two steps are repeated consecutively to generate the desired cationic peptide sequence.

[0229] All reactions and washes are carried out at room temperature unless otherwise stated. Washing the resin refers to adding the wash solvent (usually DMF or dimethylsulfoxide (DMSO)) to the resin, agitating the resin to obtain a homogenous slurry, followed by complete draining of the solvent from the resin. The solvent is removed by vacuum filtration through the fritted bottom of the reaction vessel until the resin appears dry. In all syntheses, the resin slurry is agitated by bubbling argon through the bottom of the fritted vessel.

[0230] Initial resin deprotection. Add Fmoc-Rink amide resin to a fritted reaction vessel. Add DMF to the resin and agitate the solution to swell the resin. Then drain the DMF. The Fmoc group is removed by adding 20% ​​piperidine in DMF to the resin, agitating the resin, and draining the resin. Add 20% piperidine in DMF to the resin, agitate for 15 minutes, then drain. Next, wash the resin six times with DMF.

[0231] Acylation / amidation. The deblocked amine is then acylated by adding bromoacetic acid in DMF to the resin followed by N,N-diisopropylcarbodiimide (DIC) in DMF (Figure 3A). The solution is stirred at room temperature for 30 minutes and then drained. This step is repeated twice. The resin is then washed twice with DMF and once with DMSO. This is one complete reaction cycle.

[0232] Nucleophilic Substitution / Amination. The acylation resin is treated with the desired primary or secondary amine to effect nucleophilic substitution at the α-carbon bromine leaving group (Figure 3B). This acylation / substitution cycle is repeated (Figure 3D) until the desired peptide sequence is obtained (Figure 3C).

[0233] Peptide cleavage from the resin. Place the dried resin into a glass scintillation vial containing a Teflon-coated micro stir bar and add 95% trifluoroacetic acid (TFA) in water. Stir the solution for 20 min and then filter through a solid-phase extraction (SPE) column fitted with a polyethylene frit into a polypropylene conical centrifuge tube.

[0234] The resin is washed with 1 mL of 95% TFA. The combined filtrates are then lyophilized three times from 1:1 acetonitrile:water. The lyophilized peptide (Figure 3E) is redissolved in absolute ethanol at a concentration of 5 mg / mL or in DMSO at a concentration of 10 mg / mL.

[0235] Purification and characterization. The redissolved crude peptides are purified by preparative HPLC. The purified peptides are characterized by LC-MS analysis.

[0236] Example 2 - Synthesis and characterization of representative aminolipidated peptoids Aminolipidated peptoids were synthesized by the submonomer method described above in Example 1 using bromoacetic acid and N,N'-diisopropylcarbodiimide (DIC). Polystyrene-supported MBHA Fmoc-protected Rink amide (representative scale 200 mg, 0.64 mmol / g loading, Protein Technologies) resin was used as the solid support. For bromoacetylation, the resin was mixed with a 1:1 mixture of 2 M bromoacetic acid and 2 M N,N'-diisopropylcarbodiimide (DIC) for 5 min. Amine displacement was carried out using a 1 M solution of the amine in DMF for 1 h. After synthesis, the crude peptoids were cleaved from the resin using 5 mL of a 95:2.5:2.5 mixture of trifluoroacetic acid (TFA):water:triisopropylsilane for 40 min at room temperature. The resin was removed by filtration and the filtrate was concentrated using a Biotage V10 evaporator. The crude peptoids were further concentrated by lyophilization from a 25% aqueous solution of MeCN. Purity and identity were analyzed using a Waters Acquity UPLC Peptide BEH C18 column over a 5-95% gradient using a Waters Acquity UPLC system equipped with an Acquity Diode Array UV detector and a Waters SQD2 mass spectrometer. Selected crude peptoids were purified by a preparative Waters Prep 150LC system equipped with a Waters 2489 UV / Visible detector on a Waters Xbridge BEH300 Prep C4 column using a 5-40% acetonitrile gradient in water with 0.1% TFA over 30 min.

[0237] Table 1A shows representative aminolipidated peptoids prepared by the method described in Example 2. Table 1B provides characterization data for the aminolipidated peptoid compounds 1-72 prepared in Table 1A, including predicted molecular weights, retention times (in minutes, as determined by UPLC-UV measurements at λ=218 nm), and primary observed mass-to-charge ratios (m / z, M-H). +, by electrospray ionization-mass spectrometry). For each of the aminolipidated and PEGylated peptoid compounds 48, 56, 64, and 72, the mass spectrum contained several distributions of peaks due to the polydispersity of the PEG moieties (average molecular weight 2000 g / mol) attached to the peptoids. The mass-to-charge ratio peaks reported for these aminolipidated and PEGylated peptoid compounds were the MH2 peak. 2+ This is the center of the distribution and is the effective monomer separation δ about 22 m / z (-OCH2CH 2- , ethylene glycol molecular weight 44 g / mol).

[0238] [Table 1A] JPEG0007657773000084.jpg228170JPEG0007657773000085.jpg210170JPEG0007657773000086.jpg229170JPEG0007657773000087.jpg220170JPEG0007657773000088.jpg228170JPEG0007657773000089.jpg252170JPEG0007657773000090.jpg237170JPEG0007657773000091.jpg255170JPEG0007657773000092.jpg198170JPEG0007657773000093.jpg197170JPEG0007657773000094.jpg241170JPEG0007657773000095.jpg231170JPEG0007657773000096.jpg251170JPEG0007657773000097.jpg238170JPEG0007657773000098.jpg246170JPEG0007657773000099.jpg242170JPEG0007657773000100.jpg249170JPEG0007657773000101.jpg218170JPEG0007657773000102.jpg199170JPEG0007657773000103.jpg206170JPEG0007657773000104.jpg228170JPEG0007657773000105.jpg173170JPEG0007657773000106.jpg199170JPEG0007657773000107.jpg196170JPEG0007657773000108.jpg150170JPEG0007657773000109.jpg206170JPEG0007657773000110.jpg213170JPEG0007657773000111.jpg196170JPEG0007657773000112.jpg196170JPEG0007657773000113.jpg228170JPEG0007657773000114.jpg216170JPEG0007657773000115.jpg185170JPEG0007657773000116.jpg228170JPEG0007657773000117.jpg202170JPEG0007657773000118.jpg202170JPEG0007657773000119.jpg205170JPEG0007657773000120.jpg2 13170JPEG0007657773000121.jpg210170JPEG0007657773000122.jpg249170JPEG0007657773000123.jpg244170JPEG0007657773000124.jpg88170.

[0239] [Table 1B] JPEG0007657773000126.jpg203170

[0240] Example 3 - Formulation of representative aminolipidated peptoids with oligonucleotides to form nanoparticle compositions The following examples describe general protocols for the formulation of tertiary amino lipidated and / or PEGylated cationic peptide compounds of formula (I) described herein with oligonucleotides.

[0241] In a standard formulation, the aminolipidated peptoid is dissolved in absolute ethanol at a concentration of 0.5 mg / mL (for in vitro experiments) or 5 mg / mL (for in vivo experiments). The resulting solution is stable at room temperature but should be stored at -20°C. The nucleic acid cargo is dissolved in DNAse- or RNAse-free water at a final concentration of 0.2 mg / mL (for in vitro experiments) or 1 mg / mL (for in vivo experiments). For longer periods, these solutions should be stored at -20°C or -78°C.

[0242] To prepare nanoparticle formulations, aminolipidated peptoids are mixed with nucleic acid by pipetting in a mass ratio of approximately 1:1 (peptoid:cargo) to 20:1. Prior to formulation, aminolipidated peptoids and nucleic acid cargo are diluted in a volume ratio of 1:3 with ethanol and acidic buffer (PBS adjusted to pH 5.5 with 0.1 M HCl), respectively, targeting a final cargo concentration of approximately 0.05 mg / mL to 0.2 mg / mL.

[0243] Example 4 - Preparation and characterization of physical properties of representative mRNA / peptoid formulations Exemplary aminolipidated cationic peptoids described in Example 2 were combined with firefly luciferase (Fluc) mRNA to form nanoparticle compositions and evaluated for therapeutic and / or prophylactic purposes in vitro or in vivo. Formulations were prepared according to the protocol in Example 3 and mixed by simple pipetting.

[0244] The mRNA / peptoid formulations with a mass ratio of peptoid:cargo of 5:1 were evaluated by dynamic light scattering (DLS) to determine the volume average particle size / diameter (nm) and size polydispersity index (PDI) of the mRNA / peptoid complexes within the formulation. The percentage of mRNA encapsulation in each formulation of the exemplary compounds was determined by fluorescence of Qubit RNA HS (Invitrogen) dye before and after dissolution of the particles with Triton X-100. The results are shown in Table 2 below.

[0245] [Table 2]

[0246] Example 5 - Effect of PEG on formulation stability and transfection efficiency To evaluate the effect of variable amounts of PEG incorporation on the formulation stability (i.e., particle size) and transfection efficiency of peptoid formulations, three formulations were prepared using compound 8 alone or in combination with DMG-PEG2000 at 2% w / w or 5% w / w. Particles were formulated using 5 μg of mRNA at a concentration of 0.2 μg / μL. Immediately after formulation, the particles were added to PBS (pH 7.4).

[0247] The volume average particle size (nm) of the three formulations was evaluated immediately after formulation, after addition to PBS buffer (0.5 h), and 8 h after formulation (8 h) by the same method as in Example 4 above. The measured number average particle size is shown in Figure 4A.

[0248] Transfection efficiency was also evaluated for three formulations of compound 8 alone, with 2% DMG-PEG2000, or with 5% DMG-PEG2000. Figure 4B shows the mean bioluminescence (RLU) of HeLa cells observed 8 hours after treatment with 100 ng mRNA / well, approximately 30 minutes after formulation. Untreated HeLa cells were used as a control for bioluminescence measurements.

[0249] Example 6 - Storage stability To evaluate the storage stability of formulations containing aminolipidated peptoid compound with 2% w / w PEG incorporation, formulations of Fluc mRNA and aminolipidated peptoid compound 28 with 2% w / w DMG-PEG2000 were prepared 7 days, 3 days, or 1 hour prior to injection. During that time, the formulations were stored at 4° C. Each of these three formulations was injected into three mice via tail vein injection.

[0250] The storage stability of the formulation was assessed by recording the systemic distributed luminescence (or photon flux) 8 h after injection. Figures 5A-5C show the systemic luminescence after 1 h (Figure 5A), 3 days (Figure 5B), and 7 days (Figure 5C) of storage. Figure 5D shows a plot of the total photon flux observed at each time point.

[0251] Example 7 - In vitro expression of firefly luciferase (Fluc) following treatment with a representative Fluc mRNA / aminolipidated peptoid formulation alone (no PEG) or containing 2% (w / w) DMG-PEG2000 (2% PEG) The efficacy of the mRNA / aminolipidated peptoid formulations was evaluated in vitro based on their ability to deliver the firefly luciferase (Fluc) reporter gene to cultured cells. The aminolipidated peptoids were combined with Fluc mRNA in a 5:1 w / w ratio, and the resulting particles were added to cultured HeLa cells at a dose of 100 ng / well (in a total volume of 100 μL). The resulting luciferase expression (RLU) was measured 6 and 24 hours after treatment by a luminescence plate reader. Table 3 below shows the luciferase expression of the mRNA / aminolipidated peptoid formulations observed at the two time points.

[0252] [Table 3]

[0253] In addition to evaluating nanoparticle complexes formed between mRNA cargo and aminolipidated peptoids alone, multicomponent formulations containing small amounts of the PEG-lipid DMG-PEG2000 were also tested for Fluc mRNA delivery in vitro. In these formulations, DMG-PEG2000 was premixed with the candidate peptoids at an overall 2% incorporation (w / w) while maintaining the same 5:1 mass ratio of peptoid:mRNA used previously. Table 4 below shows the luciferase expression observed at two time points (6 and 24 hours after treatment) for mRNA / aminolipidated peptoid formulations with 2% DMG-PEG2000 incorporation.

[0254] [Table 4]

[0255] Example 8 - In vitro expression of firefly luciferase (Fluc) following treatment with a representative Fluc mRNA / aminolipidated peptoid formulation containing no or 2% (w / w) DMG-PEG2000 at varying peptoid:cargo mass ratios The charge ratio dependence of the aminolipidated peptoid delivery vehicle was assessed by formulating particles at various mass ratios between Fluc mRNA and delivery vehicle. Formulations varied peptoid:mRNA (w / w) ratios from 2:1 to 7.5:1 (2:1, 3.5:1, 5:1, 7.5:1). Additionally, the role of DMG-PEG2000 was also explored by either excluding DMG-PEG2000 or incorporating DMG-PEG2000 at 2% (w / w) relative to the aminolipidated peptoid. Fluc expression from these formulations was measured in HeLa cells after 24 h of treatment.

[0256] Figure 6A shows the results of in vitro luciferase expression measurements performed for exemplary amino lipidated cationic peptoid compounds 1-18 from Example 2 that do not have PEG incorporation. Figures 6B and 6C show plots of the mean bioluminescence (RLU) in HeLa cells treated with formulations of exemplary amino lipidated cationic peptoid compounds 1-36, all formulated with 2% w / w DMG-PEG2000. Table 5 shows the Fluc expression observed for HeLa cells treated with formulations containing 2% w / w DMG-PEG2000.

[0257] [Table 5]

[0258] Formulations of aminolipidated peptoid compounds 37-72 with 2% DMG-PEG2000 were also evaluated for their ability to deliver Fluc mRNA to HeLa cells. Figures 6D and 6E show plots of the mean bioluminescence (RLU) observed for HeLa cells treated with various formulations of aminolipidated peptoid compounds 1-72 in combination with Fluc mRNA (50 ng / well, peptoid:mRNA ratio of 5:1) and 2% DMG-PEG2000.

[0259] Example 9 - In vitro cell viability following treatment with a representative Fluc mRNA / aminolipidated peptoid formulation alone (no PEG) or containing 2% (w / w) DMG-PEG2000 (2% PEG) Cell viability was measured by a conventional MTT viability assay after treatment with Fluc mRNA / aminolipidated peptoid formulations. All peptoid / mRNA formulations were prepared at a mass ratio of 5:1 with the addition of 2% (w / w) DMG-PEG2000. In this assay, cells were first treated with peptoid formulations at a final mRNA concentration of 100 ng / well for 8 h. Cells were then grown for 48 h before being exposed to 1.5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 2 h and then lysed using a solubilizing solution of 10% Triton-X 100 in acidified isopropanol (0.1 N HCl). The signal corresponding to reduced formazan was normalized to untreated cells to obtain the relative viability of each treatment condition.

[0260] Only minor changes in viability were observed in HeLa cells treated with mRNA / aminolipidated peptoid formulations. In general, at least 80% viability was maintained in almost all conditions, with the exception of compound 5, 34, and 35 formulations only. On average, formulations containing 2% DMG-PEG2000 showed slightly higher values ​​(average 97% viability vs. 89% for formulations without PEG). Table 6 shows the relative viability of cells treated with PEG-free and 2%-PEG formulations.

[0261] [Table 6]

[0262] Example 10 - In vitro systemic expression of firefly luciferase (Fluc) following IV administration of a representative Fluc mRNA / aminolipidated peptoid formulation. The aminolipidated peptoid delivery vehicle is effective for in vivo administration of Fluc mRNA to Balb / c mice (n=3) via multiple routes of administration. Briefly, a representative aminolipidated peptoid / Fluc formulation (prepared at a mass ratio of 5:1 aminolipidated peptoid:mRNA with 2% w / w DMG-PEG2000 unless otherwise noted) was administered via tail vein injection at a dose of 0.5 mg / kg and the resulting bioluminescence was quantified 8 hours later. Figure 7A shows the systemic distribution of the formulation in Balb / c mice. Table 7 shows the in vivo systemic expression of Fluc in the test mice quantified as total flux.

[0263] [Table 7]

[0264] Organ-specific bioluminescence was quantified by sacrificing treated animals, removing the organs of interest, and quantifying the resulting bioluminescence separately. The relative expression of each of the highest performing aminolipidated peptoids varied significantly. For example, compound 8 showed the highest expression overall, but nearly all of its expression was localized to the lung. However, other compounds showed preferential localization in other organs, such as the spleen in the case of compound 28, and a mixture of liver, spleen, and lung in the case of compound 24.

[0265] Figure 7B shows the corresponding organ-specific biodistribution of the formulation (lung, kidney, liver, spleen, inguinal lymph node, and mesenteric lymph node). Figure 7C shows a bar graph quantifying the organ-specific biodistribution of the formulation as a function of total photon flux. Table 8 shows the total flux measured for liver, lung, and spleen samples obtained from treated mice.

[0266] [Table 8]

[0267] In addition to IV administration, aminolipidated peptoids are also effective in local administration of mRNA via subcutaneous (SC) or intramuscular (IM) routes of administration. Table 9 shows the total flux observed with selected aminolipidated peptoid formulations administered subcutaneously or intramuscularly in Balb / c mice.

[0268] [Table 9]

[0269] In these examples, mRNA was administered at a dose of 0.1 milligrams per kilogram (mpk) subcutaneously and 0.05 mpk intramuscularly. In these cases, overall expression was higher after intramuscular injection. The highest expression was observed with compound 8 for IV administration, but compounds 24 and 28 surpassed that example with IM administration, suggesting the need to tailor these structures for different routes of administration.

[0270] Additional experiments were performed to evaluate any variability in biodistribution as a function of the loading mass ratio of aminolipidated peptoid to Fluc mRNA. Figure 8A shows plots of total photon flux in formulations of aminolipidated peptoid compound 28 with 2% DMG-PEG2000 and Fluc mRNA at different peptoid:mRNA ratios (1:1, 2:1, 5:1, 7.5:1, and 10:1). As shown in Figure 8A, the maximum total photon flux in formulations of compound 28 was observed at 5:1 for subcutaneous injection and 7.5:1 for intramuscular injection.

[0271] The evaluation of the biodistribution profile upon subcutaneous administration was extended to aminolipidated peptoid compounds 1-72. Formulations of aminolipidated peptoid compounds 1-72 containing 2% w / w DMG-PEG2000 and Fluc mRNA at a peptoid:mRNA ratio of 5:1 were administered subcutaneously to Balb / c mice. Measurements of the total photon flux observed for each of the formulations are shown in Figures 8B and 8C.

[0272] Example 11 - In vitro cell type transfection efficiency of firefly luciferase (Fluc) in aminolipidated peptoid formulations with PEG Cell culture: HeLa cells were seeded at 10,000 cells / well in 100 μL of DMEM containing 10% FBS and 1% penicillin / streptomycin and allowed to attach 18 hours before treatment. The medium was replaced with 100 μL of fresh serum-containing DMEM immediately before transfection. HepG2 cells were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin. They were seeded at 10,000 cells / well and allowed to attach 18 hours before treatment. JAWSII cells were cultured in RPMI medium containing 10% FBS, 1% penicillin / streptomycin, 1 mM sodium pyruvate, and 5 ng / mL mouse GM-CSF.

[0273] mRNA preparation: Fluc mRNA for transfection experiments was prepared in-house using standard in vitro transcription (IVT) methods.

[0274] Representative aminolipidated peptoid / Fluc formulations for mRNA formulations: Compounds 24 and 79 (prepared at a 5:1 mass ratio of aminolipidated peptoid:mRNA unless otherwise noted). The aminolipidated peptoid:mRNA mixtures were combined with commercially available PEG lipids (DMG-PEG2000, 18:0 PEG1000PE, 14:0 PEG2000 PE, 14:0 PEG5000 PE), linear aminoPEGylated peptoids (compounds 48, 56, 64, and 72), or branched aminoPEGylated peptoids (compounds 106, 107, 108, and 109) at weight percentages of 0.01%, 0.02%, 0.05%, and 0.1% of the PEGylated compounds.

[0275] Fluc expression from these formulations was measured in cells after 24 hours of treatment based on observed bioluminescence (total photon flux). For ease of comparison, observed total photon flux was normalized to the formulation containing DMG-PEG2000 as the standard condition.

[0276] 9A-9C show the normalized total photon flux (cps) for in vitro assessment of transfection efficiency of Fluc mRNA in various cell types (HeLa in FIG. 9, HepG2 in FIG. 9B, and JAWSII in FIG. 9C).

[0277] It was observed that linear PEGylated peptoids exhibited high transfection efficiency at lower weight percent incorporation, whereas branched PEGylated peptoids exhibited better transfection when incorporated at higher than lower weight percents.

Claims

1. (i) one or more polyanionic compounds; (ii) one or more PEG compounds; and (iii) one or more cationic compounds, wherein the one or more cationic compounds are having the structure of formula (Ia) or a salt thereof: 【Chemistry 49】 During the ceremony, n is an integer from 0 to 5; s is an integer from 0 to 5; At least one of n and s is other than 0, r is an integer from 1 to 20; each o is independently an integer 0, 1, 2, 3, or 4; each q is independently an integer 0, 1, 2, 3, or 4; each p is independently an integer 1 or 2; R 1 is -H, alkyl, alkylaryl, -COR 1a , or a lipid moiety, where R 1a is -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl; each R 3 is independently a lipid moiety; each R 4 is independently a neutral spacer moiety or a lipid moiety; Each R 5 is independently a cationic moiety; R 7 is -H, alkyl, acyl, -OH, -OR 7a , -NH 2 , -NHR 7a , or a lipid moiety, where R 7a is an alkyl, acyl, or a lipid moiety; each R a and R b is independently -H, C 1 -C 4 -alkyl, or a side chain moiety found in a natural or non-naturally occurring amino acid; A composition, wherein said one or more cationic compounds, said one or more PEG compounds, and said one or more polyanionic compounds comprise a combined weight percent of said composition of at least 90% w / w.

2. The composition of claim 1 , wherein either n or s is at least 2.

3. Each R ３ But independently, C ４ -C ２２ -Alkyl or C ４ -C ２２ -alkenyl, wherein the C ４ -C ２２ The composition of claim 1 or 2, wherein the alkenyl is optionally mono- or polyunsaturated.

4. Each R ３ But independently, C ８ -C １２ The composition of any one of claims 1 to 3, wherein the alkyl group is alkyl.

5. Each R ３ The composition of any one of claims 1 to 3, wherein is independently selected from the group consisting of 2-ethylhex-1-yl, caproyl, oleyl, stearyl, linoleyl, myristyl, and lauryl.

6. The cationic compound comprises a cationic moiety R ５ The composition according to any one of claims 1 to 5, comprising at least two cationic amino acid residues having the formula:

7. Each R ５ The composition of any one of claims 1 to 6, wherein is independently aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, N-heterocyclylalkyl, or N-heteroaryl.

8. Each R ５ But independently, 【Chemistry 4】 The composition according to any one of claims 1 to 7, selected from the group consisting of:

9. R ４ is each neutral spacer moiety, independently １ -C ５ C substituted with linear alkyl, cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, and hydroxyalkyl １ -C ４ -alkyl, wherein each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl is optionally substituted by one or more substituents -OH, halo, or alkoxy.

10. Each neutral spacer moiety R ４ But independently, 【Chemistry 8】 The composition according to any one of claims 1 to 9, selected from the group consisting of:

11. Each neutral spacer moiety R ４ but, 【Chemistry 9】 The composition according to any one of claims 1 to 10,

12. At least one of the cationic compounds is R ５ a cationic domain comprising at least two amino acid residues having a cationic moiety, each of the at least two cationic amino acid residues in the cationic domain being one R ４ The composition according to any one of claims 1 to 11, wherein said amino acid residues are separated by at least one amino acid residue having the formula:

13. R ａ and R ｂ The composition of any one of claims 1 to 12, wherein is -H.

14. 14. The composition of any one of claims 1 to 13, wherein the one or more cationic compounds and one or more PEG compounds are present in a mass ratio of cationic compound to PEG compound of from 90:10 to 99:

1.

15. The composition of any one of claims 1 to 14, wherein the one or more PEG compounds comprises DMG-PEG2000.

16. The one or more PEG compounds include one or more compounds of formula (I) or salts thereof: 【Chemistry 12】 During the ceremony, m is an integer from 0 to 15; n is an integer from 0 to 5; s is an integer from 0 to 5; t is an integer from 0 to 15; At least one of m and t is other than 0; r is an integer from 1 to 20; each o is independently an integer 0, 1, 2, 3, or 4; each q is independently an integer 0, 1, 2, 3, or 4; each p is independently an integer 1 or 2; R １ is -H, alkyl, alkylaryl, -COR １ａ or a lipid moiety, R １ａ is -H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or -O-alkylaryl; Each R ２ are independently a group of the formula -CH ２ CH ２ O (CH ２ CH ２ O) ｕ CH ３ where each u is independently an integer from 1 to 200; Each R ３ is independently a lipid moiety, Each R ４ is independently a neutral spacer moiety or a lipid moiety; Each R ５ is independently a cationic moiety; Each R ６ are independently a group of the formula -CH ２ CH ２ O (CH ２ CH ２ O) ｖ CH ３ where each v is independently an integer from 1 to 200; R ７ is -H, alkyl, acyl, -OH, -OR ７ａ , -NH ２ , -NHR ７ａ or a lipid moiety, R ７ａ is an alkyl, acyl, or lipid moiety; Each R ａ and R ｂ are independently -H, C １ -C ４ -alkyl, or a side chain moiety found in natural or non-naturally occurring amino acids.

17. 17. The composition of claim 16, wherein when m is an integer from 0 to 3, each u is independently an integer from 30 to 50, or when m is an integer from 4 to 15, each u is independently an integer from 1 to 10.

18. 18. The composition of claim 16 or 17, wherein when t is an integer from 0 to 3, each v is independently an integer from 30 to 50, or when t is an integer from 4 to 15, each v is independently an integer from 1 to 10.

19. 19. The composition of any one of claims 16 to 18, wherein m is 1 and u is an integer from 40 to 45, or t is 1 and v is an integer from 40 to 45.

20. The composition of any one of claims 1 to 19, wherein the one or more polyanionic compounds comprises a nucleic acid.

21. 21. The composition of any one of claims 1 to 20, wherein the one or more polyanionic compounds comprise a nucleic acid and the mass ratio of the one or more cationic compounds to the nucleic acid is from 0.5:1 to 20:

1.

22. 22. The composition of any one of claims 1 to 21, wherein the one or more polyanionic compounds comprises a nucleic acid, the nucleic acid being an mRNA encoding a polypeptide.

23. 23. The composition of any one of claims 1 to 22, wherein the one or more polyanionic compounds comprises a nucleic acid, the nucleic acid being an mRNA encoding a protein.

24. The composition of any one of claims 1 to 23, wherein the composition further comprises one or more second pharmaceutical agents.

25. The composition of any one of claims 1 to 24, wherein the one or more second agents are selected from the group consisting of polyethylene glycol, targeting elements, and combinations thereof.

26. The composition of any one of claims 1 to 25, wherein the composition further comprises one or more small molecule active agents or drug substances.

27. A pharmaceutical composition for delivering a polyanionic compound to a subject in need thereof, comprising a composition according to any one of claims 1 to 26.

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