Surfactant peptide nanostructures and uses thereof in drug delivery

Abstract

To provide surfactant peptide nanostructures and uses thereof in drug delivery.SOLUTION: The present disclosure provides a surfactant peptide nanostructure, wherein the peptide has repeating hydrophobic amino acids, the number of which is an integer equal to 4 or less than 4. The peptide nanostructure is useful for therapeutic uses including the delivery of drugs and siRNA. In some embodiments, the peptide has a nanosphere structure. In some embodiments, the peptide forms a nanosphere. In some embodiments, the peptide forms a nanospheric structure with a center that is substantially free of self-assembling surfactant peptide (e.g., a spherical nanovesicle).SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 599,566, filed December 15, 2017, the contents of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated by reference in its entirety. The ASCII copy (created on November 30, 2018) has filename: 2004837-0223_SL.txt and is 29,249 bytes in size. [Background technology]

[0003] background Surfactant peptides typically have a hydrophilic head group and a lipophilic tail with hydrophobic amino acids. Certain surfactant peptides have been previously disclosed (e.g., PCT Publication Nos. WO2003006043, WO2013181511, and WO2009018467). Some of the disclosed surfactant peptides are oligopeptides and diblock and triblock peptide copolymers with structures that include a hydrophilic head group containing charged amino acids and a lipophilic tail containing hydrophobic amino acids in a repeat unit of 5 or more amino acids. Other surfactant peptides with one repeating unit of four alanines have been disclosed for use in stabilizing membrane proteins (eg, US Patent Application Publication No. US20090069547). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2003 / 006043 [Patent Document 2] International Publication No. 2013 / 181511 [Patent Document 3] International Publication No. 2009 / 018467 [Patent Document 4] US Patent Application Publication No. 2009 / 0069547 Summary of the Invention [Means for solving the problem]

[0005] Abstract Previously disclosed surfactant peptides have been reported to self-assemble to form nanotubes with average diameters of about 50 nM.

[0006] The present disclosure provides short surfactant peptides with repeating hydrophobic units of four or fewer amino acids that form nanospherical structures that are particularly useful for the formulation and delivery of therapeutic agents.

[0007] In some embodiments, the present disclosure provides repeating hydrophobic amino acids according to the following formula: Formula I(N→C): (X)a(Y)m Formula II(N→C): (Y)m(X)a Formula III(N→C): (X)a(Y)m(X)b Formula IV(N→C): (Y)m(X)a(Y)n Formula V(N→C): (X)a(Z)m Formula VI(N→C): (Z)m(X)a Formula VII(N→C): (X)a(Z)m(X)b; or Formula VIII(N→C): (Z)m(X)a(Z)n; The present invention provides a composition comprising a peptide having (X) is an amino acid having a side chain that is nonpolar and uncharged at physiological pH; (Y) is an amino acid having a cationic side chain at physiological pH; (Z) is an amino acid having an anionic side chain at physiological pH; And here a is an integer equal to or less than 4; b is an integer equal to or less than 4; m is an integer equal to or greater than 1; and n is an integer equal to or greater than 1.

[0008] In some embodiments, the peptides have a nanospherical structure. In some embodiments, the peptides form nanospheres. In some embodiments, the peptides form nanospherical structures with centers that are substantially free of self-assembled surfactant peptides (e.g., spherical nanovesicles).

[0009] In some embodiments, the (X), (Y), and / or (Z) amino acids are natural amino acids. In some embodiments, the (X), (Y), and / or (Z) amino acids are unnatural amino acids.

[0010] In some embodiments, the integer a or b is 4. In some embodiments, the integer a or b is 3.

[0011] In some embodiments, (X) is alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or glycine. In some embodiments, (X) is alanine. In some embodiments, (Y) is arginine, lysine, histidine, or ornithine. In some embodiments, (Y) is lysine. In some embodiments, (X) is alanine and (Y) is lysine. In some embodiments, (Z) is aspartic acid or glutamic acid.

[0012] In some embodiments, one or more amino acids are L-amino acids. In some embodiments, each amino acid is an L-amino acid. In some embodiments, one or more amino acids are D-amino acids. In some embodiments, each amino acid is a D-amino acid.

[0013] In some embodiments, the peptide is 4-10 amino acids in length. In some embodiments, the peptide is 5 amino acids in length. In some embodiments, the peptide is 6 amino acids in length. In some embodiments, the peptide is 7 amino acids in length.

[0014] In some embodiments, the peptide comprises a modified N-terminus and / or a modified C-terminus, hi some embodiments, the peptide has an acetylated N-terminus and / or an aminated C-terminus.

[0015] In some embodiments, the peptide has a nanosphere structure and an amino acid sequence according to any one of SEQ ID NOs: 1-50. In some embodiments, the peptide has a nanosphere structure and an amino acid sequence according to any one of SEQ ID NOs: 51-100.

[0016] In some embodiments, the peptide has a nanosphere structure and the amino acid sequence AAAK (SEQ ID NO: 1). In some embodiments, the peptide has a nanosphere structure and the amino acid sequence AAAAK (SEQ ID NO: 5). In some embodiments, the peptide has a nanosphere structure and the amino acid sequence AAAKAAA (SEQ ID NO: 15).

[0017] In some embodiments, the present disclosure provides a composition comprising a nanosphere peptide in an aqueous solution. In some embodiments, the peptide is at a concentration of at least 0.01% (w / v). In some embodiments, the aqueous solution has a pH of about 6 to about 8. In some embodiments, the aqueous solution has a pH of about 7. In some embodiments, the aqueous solution is at an ionic strength of about 0M to about 0.3M. In some embodiments, the aqueous solution is at an ionic strength of about 0.15M. In some embodiments, the aqueous solution is isotonic.

[0018] In some embodiments, the disclosure provides a composition for use in drug delivery. In some embodiments, the composition includes an agent for delivery to a subject. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a drug (e.g., a small molecule). In some embodiments, the agent is a biologic. In some embodiments, the agent is an oligonucleotide. In some embodiments, the agent is an inhibitor of RNA. In some embodiments, the agent is an siRNA. In some embodiments, the agent is for delivery to a cancer cell.

[0019] In some embodiments, the present disclosure provides a method of formulating an agent for delivery to a subject, the method comprising contacting the agent with a nanosphere peptide surfactant as described herein.

[0020] In some embodiments, the disclosure provides a method of delivering an agent to a subject, the method comprising administering to the subject a compound of the following formula: Formula I(N→C): (X)a(Y)m Formula II(N→C): (Y)m(X)a Formula III(N→C): (X)a(Y)m(X)b Formula IV(N→C): (Y)m(X)a(Y)n Formula V(N→C): (X)a(Z)m Formula VI(N→C): (Z)m(X)a Formula VII(N→C): (X)a(Z)m(X)b; or Formula VIII(N→C): (Z)m(X)a(Z)n; The method includes administering a composition formulated with a peptide having repeating hydrophobic amino acids according to (X) is an amino acid having a side chain that is nonpolar and uncharged at physiological pH; (Y) is an amino acid having a cationic side chain at physiological pH; (Z) is an amino acid having an anionic side chain at physiological pH; And here a is an integer equal to or less than 4; b is an integer equal to or less than 4; m is an integer equal to or greater than 1; and n is an integer equal to or greater than 1.

[0021] In some embodiments, the peptides form nanospherical structures, In some embodiments, the peptides form nanospheres, In some embodiments, the peptides form spherical nanovesicles.

[0022] In some embodiments, the (X), (Y), and / or (Z) amino acids are natural amino acids. In some embodiments, the (X), (Y), and / or (Z) amino acids are unnatural amino acids.

[0023] In some embodiments, the integer a or b is 4. In some embodiments, the integer a or b is 3.

[0024] In some embodiments, (X) is alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or glycine. In some embodiments, (X) is alanine. In some embodiments, (Y) is arginine, lysine, histidine, or ornithine. In some embodiments, (Y) is lysine. In some embodiments, (X) is alanine and (Y) is lysine. In some embodiments, (Z) is aspartic acid or glutamic acid.

[0025] In some embodiments, one or more amino acids are L-amino acids. In some embodiments, each amino acid is an L-amino acid. In some embodiments, one or more amino acids are D-amino acids. In some embodiments, each amino acid is a D-amino acid.

[0026] In some embodiments, the peptide is 4-10 amino acids in length. In some embodiments, the peptide is 5 amino acids in length. In some embodiments, the peptide is 6 amino acids in length. In some embodiments, the peptide is 7 amino acids in length.

[0027] In some embodiments, the peptide comprises a modified N-terminus and / or a modified C-terminus, hi some embodiments, the peptide has an acetylated N-terminus and / or an aminated C-terminus.

[0028] In some embodiments, the peptide has a nanosphere structure and an amino acid sequence according to any one of SEQ ID NOs: 1-50. In some embodiments, the peptide has a nanosphere structure and an amino acid sequence according to any one of SEQ ID NOs: 51-100.

[0029] In some embodiments, the peptide has a nanosphere structure and the amino acid sequence AAAK (SEQ ID NO: 1). In some embodiments, the peptide has a nanosphere structure and the amino acid sequence AAAAK (SEQ ID NO: 5). In some embodiments, the peptide has a nanosphere structure and the amino acid sequence AAAKAAA (SEQ ID NO: 15).

[0030] In some embodiments, the present disclosure provides a composition comprising a nanosphere peptide in an aqueous solution. In some embodiments, the peptide is at a concentration of at least 0.01% (w / v). In some embodiments, the aqueous solution has a pH of about 6 to about 8. In some embodiments, the aqueous solution has a pH of about 7. In some embodiments, the aqueous solution is at an ionic strength of about 0M to about 0.3M. In some embodiments, the aqueous solution is at an ionic strength of about 0.15M. In some embodiments, the aqueous solution is isotonic.

[0031] definition In order that this disclosure may be more readily understood, certain terms are first described below. Further definitions of the following terms, as well as other terms, are set forth throughout the specification.

[0032] The term "agent" as used herein refers to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. In some embodiments, the agent is or includes a product of nature, in that it is found in nature and / or derived from nature. In some embodiments, the agent is or includes one or more artificial entities, in that it is designed, engineered, and / or generated through human activity and / or is not found in nature. In some embodiments, the agent may be utilized in isolated or pure form; in some embodiments, the agent may be utilized in crude form. Some specific agents that may be utilized in accordance with the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, and ribozymes), peptides, peptidomimetics, and the like. In some embodiments, the agent is or includes a polymer. In some embodiments, the agent is not a polymer and / or is substantially free of any polymers. In some embodiments, the agent comprises at least one polymer moiety. In some embodiments, the agent lacks any polymer moieties or is substantially free of any polymer moieties. In some embodiments, the agent is or comprises a cell lysate.

[0033] As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H 2The amino acid has a NC(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "nonstandard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically prepared or obtained from a natural source. In some embodiments, the amino acid may include structural modifications compared to the general structure above, including the carboxy- and / or amino-terminal amino acids in a polypeptide. For example, in some embodiments, the amino acid may be modified by methylation, amidation, acetylation, and / or substitution compared to its general structure. As will be clear from the context, in some embodiments, the term "amino acid" is used to refer to a free amino acid; in some embodiments, it is used to refer to an amino acid residue of a polypeptide.

[0034] As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value that is similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except when such number exceeds 100% of the possible value).

[0035] Two events or entities, as the term "associated" with each other is used herein, are associated with each other if the presence, level and / or form of one correlates with the presence, level and / or form of the other. In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly such that they are in and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0036] The term "comparable" is used herein to describe two (or more) sets of conditions, environments, individuals, or populations that are sufficiently similar to each other to allow a comparison of results to be obtained or a phenomenon to be observed. In some embodiments, comparable sets of conditions, environments, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few varied characteristics. Those skilled in the art will recognize that sets of environments, individuals, or populations are comparable to each other when they are characterized by a sufficient number and type of substantially identical characteristics to justify a reasonable conclusion that the differences in results obtained under the different sets of environments, individuals, or populations, or the phenomena observed in the different sets of environments, individuals, or populations, are caused by or indicate variations in those varied characteristics. Those skilled in the art will recognize that the relative language used herein (e.g., enhanced, activated, reduced, inhibited, etc.) typically refers to a comparison made under comparable conditions.

[0037] "Complementary" means capable of forming ionic or hydrogen bonding interactions between hydrophilic residues from adjacent peptides, e.g., in a sheet or scaffold, each hydrophilic residue in a peptide either hydrogen bonds or ionically pairs with a hydrophilic residue on an adjacent peptide or is exposed to solvent.

[0038] Certain methodologies may include a "determining step." Upon reading and understanding this specification, one of skill in the art will recognize that such a "determining step" may utilize or be accomplished through the use of any of a variety of techniques available to one of skill in the art (e.g., including the specific techniques explicitly mentioned herein). In some embodiments, the determining step involves the manipulation of a physical sample. In some embodiments, the determining step involves the consideration and / or manipulation of data or information, e.g., utilizing a computer or other processing device adapted to perform the relevant analysis. In some embodiments, the determining step involves receiving relevant information and / or materials from a source. In some embodiments, the determining step involves comparing one or more characteristics of the sample or entity to a comparable reference.

[0039] The term "in vitro" as used herein refers to events that take place not within a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0040] The term "in vivo" as used herein refers to events that occur within a multicellular organism (e.g., humans and non-human animals). In the context of cell-based systems, the term can be used to refer to events that occur within a living cell (as opposed to, e.g., in vitro systems).

[0041] As used herein, the term "nanospheric" refers to a spherical structure having a diameter in the nanometer range. As used herein, a "nanospheric structure" includes nanospheres and / or spherical nanovesicles. Spherical nanovesicles are similar to nanospheres, but have a center that is substantially free of self-assembling surfactant peptides.

[0042] The term "peptide" as used herein refers to any polymeric chain of amino acids. In some embodiments, a peptide has an amino acid sequence that occurs naturally. In some embodiments, a peptide has an amino acid sequence that does not occur naturally. In some embodiments, a peptide has an amino acid sequence that is engineered in that it is designed and / or generated through human activity. In some embodiments, a peptide includes or consists of natural amino acids, non-natural amino acids, or both. In some embodiments, a peptide includes or consists of only natural amino acids or only non-natural amino acids. In some embodiments, a peptide includes D-amino acids, L-amino acids, or both. In some embodiments, a peptide includes only D-amino acids. In some embodiments, a peptide includes only L-amino acids. In some embodiments, a peptide includes one or more pendant groups or other modifications, such as modifications or linkages to one or more amino acid side chains at the N-terminus of the peptide, at the C-terminus of the peptide, or any combination thereof. In some embodiments, such pendant groups or modifications are selected from acetylation, amidation, lipidation, methylation, pegylation, and the like (including combinations thereof). In some embodiments, the term "peptide" may be added to the name of a reference peptide, activity, or structure; in such cases, the term is used herein to refer to peptides that share a related activity or structure and thus may be considered members of the same class or family of peptides. For each such class, the present specification provides, and / or one of skill in the art will know, exemplary peptides within that class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary peptides are the reference peptides of that peptide class or family.In some embodiments, members of a peptide class or family exhibit significant sequence homology or identity with, share common sequence motifs (e.g., characteristic sequence elements) with, and / or share common activities (in some embodiments, at comparable levels or within specified ranges) with reference peptides of that class; in some embodiments, with all peptides within that class.

[0043] The term "pure" is used to indicate the extent to which the peptides described herein are free of deletion adducts of the peptide and other species, including peptides of different lengths.

[0044] The term "reference" as used herein describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to an agent, animal, individual, population, sample, sequence, or value of a reference or control. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, and is embodied in a tangible medium, if necessary. Typically, as will be understood by those skilled in the art, the reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation. Those skilled in the art will recognize when sufficient similarity exists to justify reliance on and / or comparison to a particular possible reference or control.

[0045] The term "self-assembling" is used herein to refer to certain peptides that can spontaneously self-associate into structures (e.g., nanospherical structures, including nanospheres and spherical nanoparticles) in solution (e.g., aqueous solution) under appropriate conditions. In some embodiments, the self-assembly (and / or dis-assembly) into nanospherical structures is responsive to one or more environmental triggers (e.g., changes in one or more of pH, temperature, ionic strength, osmolality, osmolality, applied pressure, applied shear stress, etc.). In some embodiments, compositions of self-assembling polypeptides are characterized by a detectable nanospherical structure when the polypeptide is in an assembled state.

[0046] As used herein, the term "substantially" refers to the qualitative condition of indicating the total or nearly total extent or degree of a desired property or characteristic. Those skilled in the art of biology understand that biological and chemical phenomena rarely, if ever, proceed to and / or completeness, or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0047] As used herein, the phrase "therapeutic agent" generally refers to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it exhibits a statistically significant effect on an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population may be defined by various criteria (e.g., a certain age group, sex, genetic background, pre-existing clinical conditions, etc.). In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is an agent that has been or needs to be approved by a government agency before it can be commercially available for administration to humans. In some embodiments, a "therapeutic agent" is a drug that requires a prescription for administration to a human.

[0048] As used herein, a "therapeutically effective amount" is an amount that produces a desired effect for which it is administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to the disease, disorder, and / or condition according to a therapeutic administration regimen. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of one or more symptoms of the disease, disorder, and / or condition and / or delays its onset. Those skilled in the art will recognize that the term "therapeutically effective amount" does not require that a successful treatment is actually achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to patients in need of such treatment, provides a specific desired pharmacological response in a significant number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to the amount as measured in one or more particular tissues (e.g., tissues affected by the disease, disorder, or condition) or bodily fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of skill in the art will recognize that in some embodiments, a therapeutically effective amount of a particular agent or treatment may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0049] Unless otherwise defined, technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] The following drawings are for illustration purposes only and not by way of limitation. [Brief description of the drawings]

[0051] [Figure 1]Figure 1 shows molecular models of exemplary surfactant peptides; (A) Ac-A3K-NH2 (SEQ ID NO: 51), (B) Ac-A4K-NH2 (SEQ ID NO: 55), (C) Ac-A3KA3-NH2 (SEQ ID NO: 65). Ac-A3K-NH2 (SEQ ID NO: 51) and Ac-A4K-NH2 (SEQ ID NO: 55) have one short hydrophobic leg. Ac-A3KA3-NH2 (SEQ ID NO: 55) has two short hydrophobic legs.

[0052] [Diagram 2] FIG. 2 shows an AFM image of Ac-A6K-NH2 (SEQ ID NO: 105), which has a nanotube structure representative of conventional surfactant peptides.

[0053] [Diagram 3] Figure 3 shows an AFM image of the bilayer structure of Ac-A6K-NH2 (SEQ ID NO: 105) at 0.1% (w / v). The thickness of each layer is 0.8 nm. Ac-A6K-NH2 (SEQ ID NO: 105) formed a bilayer structure at 0.1% (w / v).

[0054] [Figure 4] Figure 4 shows an AFM image of Ac-A3K-NH2 (SEQ ID NO: 51). Ac-A3K-NH2 (SEQ ID NO: 51) forms nanospheres at 0.1% (w / v). The diameter of the nanospheres is 28 nm.

[0055] [Diagram 5] Figure 5 shows an AFM image of Ac-A4K-NH2 (SEQ ID NO: 55). Ac-A4K-NH2 (SEQ ID NO: 55) forms nanospheres (i.e., spherical nanovesicles). The diameter of the nanospheres is 85 nm. The height profile shows that the edges of the nanospheres dried on the mica surface have a higher height than their central parts. This indicates that the interior of the A4K (SEQ ID NO: 55) nanospheres is empty (i.e., forms spherical nanovesicles).

[0056] [Figure 6]Figure 6 shows an AFM image of Ac-A3KA3-NH2 (SEQ ID NO: 65). Ac-A3KA3-NH2 (SEQ ID NO: 65) forms nanospheres (i.e., spherical nanovesicles). The diameter of the nanospheres is 30 nm. The height profile shows that the edges of the nanospheres dried on the mica surface have a higher height than their central parts. This indicates that the interior of the Ac-A3KA3-NH2 (SEQ ID NO: 65) nanospheres is empty (i.e., forms spherical nanovesicles).

[0057] [Figure 7] Figures 7A and 8B illustrate nanostructures of surfactant peptides. In Figure 7A, nanotubes are formed by representative surfactant peptides with a number of repeating hydrophobic amino acids equal to or greater than 5 (e.g., Ac-A6K-NH2 (SEQ ID NO: 105)). In Figure 7B, nanosphere structures of surfactant peptides disclosed herein with a number of repeating hydrophobic amino acids equal to or greater than 3 or 4 (e.g., Ac-A3KA3-NH2 (SEQ ID NO: 65) and Ac-A4K-NH2 (SEQ ID NO: 55)).

[0058] [Figure 8] Figure 8 shows an AFM image of the A6K / siRNA complex (0.1% / 0.01%), which aggregates.

[0059] [Figure 9] 9 shows an AFM image of Ac-A4K-NH2 (SEQ ID NO: 55) / siRNA complex (0.1% / 0.01%), which forms a nanosphere structure.

[0060] [Figure 10] 10 shows an AFM image of Ac-A3KA3-NH2 (SEQ ID NO: 65) / siRNA complex (0.5% / 0.05%), which forms a nanosphere structure.

[0061] [Figure 11A] Figures 11A and 11B show cytotoxicity data as described in Example 1. Luciferase luminescence data from the peptide alone or the peptide / negative control siRNA are shown. [I] or [H] indicates the experimental conditions specified in Table 4. Figures 11A and 11B show data from Method 1 and Method 2, respectively. [Figure 11B] Same as above.

[0062] [Figure 12A] Figures 12A-12E show transfection efficacy data as described in Example 1. Figures 12A-C show luciferase luminescence from control samples (e.g., mock, Dhrama-siRNA, Dhrama-NC siRNA, peptide / NC-siRNA complex samples), peptide / siRNA complex samples. Figures 12A and 12B show results from method 1. Figure 12C shows results from method 2. Figures 12D (method 1) and 12E (method 2) show the percentage inhibition of luciferase luminescence. Each luciferase luminescence data from its peptide / siRNA complex is normalized by the corresponding luciferase luminescence data from the peptide / NC siRNA complex. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above.

[0063] [Figure 13] Figure 13 shows the zeta potential of Ac-A4K-NH2 (SEQ ID NO: 55) / siRIN complexes at various charge ratios, with a sample size of 3 and error bars representing standard deviation (SD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Detailed Description The present disclosure relates to surfactant peptide nanostructures and their use in the delivery of drugs to a subject. The present disclosure encompasses the discovery that certain surfactant peptides having hydrophobic amino acid repeats of four or fewer amino acids form nanosphere structures that are well suited for the delivery of drugs.

[0065] In particular, the present disclosure identifies the shortcomings of surfactant peptides with five or more hydrophobic amino acid repeats, particularly those that tend to form nanotube structures rather than nanospherical structures (nanospheres or spherical nanovesicles). Moreover, they tend to aggregate when complexed with agents for delivery (e.g., siRNA). The present disclosure provides nanospherical structures that are better suited for delivery of agents, particularly siRNA or other therapeutic agents, to a subject.

[0066] peptide The present disclosure provides surfactant peptide nanostructures having a small number (i.e., an integer equal to or less than 4) of repeating hydrophobic amino acids. In some embodiments, the peptide has the following formula I, II, III, IV, V, VI, VII, or VIII: Formula I(N→C): (X)a(Y)m Formula II(N→C): (Y)m(X)a Formula III(N→C): (X)a(Y)m(X)b Formula IV(N→C): (Y)m(X)a(Y)n Formula V(N→C): (X)a(Z)m Formula VI(N→C): (Z)m(X)a Formula VII(N→C): (X)a(Z)m(X)b Formula VIII(N→C): (Z)m(X)a(Z)n; The following is provided, where (X) is an amino acid having a side chain that is nonpolar and uncharged at physiological pH; (Y) is an amino acid having a cationic side chain at physiological pH; (Z) is an amino acid having an anionic side chain at physiological pH; And here a is an integer equal to or less than 4; b is an integer equal to or less than 4; m is an integer equal to or greater than 1; and n is an integer equal to or greater than 1.

[0067] In some embodiments, the (X), (Y), and / or (Z) amino acids are natural amino acids. In some embodiments, the (X), (Y), and / or (Z) amino acids are unnatural amino acids. In some embodiments, (X) is alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or glycine. In some embodiments, (Y) is arginine, lysine, histidine, or ornithine. In some embodiments, (X) is arginine and (Y) is lysine. In some embodiments, (Z) is aspartic acid or glutamic acid. The (X), (Y), and / or (Z) amino acids can be L-amino acids, D-amino acids, or combinations thereof.

[0068] In some embodiments, the number of hydrophobic repeat amino acids is 4. In some embodiments, the number of hydrophobic repeat amino acids is 3. In some embodiments, the number of hydrophobic repeat amino acids is 2.

[0069] In some embodiments, the total number of amino acids in the peptide is about 4 to about 10. In some embodiments, the total number of amino acids in the peptide is 4, 5, 6, 7, 8, 9, or 10.

[0070] Amino acid sequences of exemplary peptide structures are provided in Table 1. Table 1. Exemplary surfactant peptides [Table 1-1] [Table 1-2]

[0071] In some embodiments, the peptide has an acetylated N-terminus and / or an aminated C-terminus. Exemplary peptides with an acetylated N-terminus and / or an aminated C-terminus are shown in Table 2. Table 2. Exemplary surfactant peptides with aminated C-terminus and acetylated N-terminus [Table 2-1] [Table 2-2]

[0072] Nanostructure In some embodiments, the surfactant peptides assemble to form nanospherical structures. As used herein, nanospherical structures include structures including nanospheres and / or spherical nanovesicles. Spherical nanovesicles are similar to nanospheres, but have a center that is substantially free of surfactant peptides that self-assemble.

[0073] Nanostructures play an important role in the formulation of drugs (e.g., therapeutic agents such as siRNA). Atomic force microscopy (AFM) studies show that surface-active peptides with five or more repeating hydrophobic amino acids typically form nanotubes or bilayers, as opposed to the more desirable nanosphere structures. In contrast, surface-active peptides with four or fewer repeating hydrophobic amino acids have been found to form nanosphere structures (e.g., spherical nanovesicles).

[0074] It has been previously reported that the nanostructure of A3K (SEQ ID NO: 1) is a bilayered membrane (J. Phys. Chem. B, 2014, 118 (42), pp 12215-12222). A3K (SEQ ID NO: 1) has also been reported to have a nanostructure as loose peptide stacks. It has been investigated for its potential antibacterial ability (Biomacromolecules, 2010, 11 (2), pp 402-411), and in another report, A3K (SEQ ID NO: 1), A6K (SEQ ID NO: 101), and A9K (SEQ ID NO: 102) were tested for their interaction with lipid membranes (RSC Adv., 2017, 7, 35973). However, unlike those reported in the above references, under appropriate conditions, A3K (SEQ ID NO: 1) can form a nanosphere structure as shown in Figure 4.

[0075] A4K (SEQ ID NO:5) has previously been reported to be highly water soluble and to show no relevant self-assembly in water, unlike A6K, which has been shown to self-assemble to form nanotubes (Langmuir, 2014, 30 (33), pp 10072-10079). However, in contrast to what has been previously reported, it is shown that under appropriate conditions, A4K (SEQ ID NO:5) can form nanosphere structures (spherical nanovesicles) in water, as shown in FIG. 5.

[0076] A3KA3 (SEQ ID NO: 15) has been previously used as a test compound for optimizing electrospray ionization conditions (Rapid Commun Mass Spectrom. 2017, 31(13):1129-1136). However, the nanostructure of A3KA3 (SEQ ID NO: 15) has not been described in the above references. It is shown herein that under appropriate conditions, A3KA3 (SEQ ID NO: 15) can undergo self-assembly to form nanosphere structures (spherical nanovesicles) as shown in FIG. 6.

[0077] This disclosure shows the excellent properties of the surface active peptides disclosed herein. The results of the experiment of peptide complexed with siRNA in aqueous solution at pH 7.5 are shown in Table 3. The peptides AAAK (SEQ ID NO: 1), AAAAK (SEQ ID NO: 5) and AAAKAAA (SEQ ID NO: 15) were found to be transparent and virtually free of phase separation in transmittance studies when complexed with siRNA at concentrations of 0.01% (w / v) and 0.05% (w / v) at peptide concentrations of 0.1% (w / v) and 0.5% (w / v). In contrast, under similar conditions, AAAAAK (SEQ ID NO: 101) and AAAAAAK (SEQ ID NO: 102) became turbid and phase separated (see results in Table 3). Table 3. Permeability studies of surfactant peptides and surfactant peptide / siRNA complexes in aqueous solutions at pH 7.5. [Table 3] #: Exemplary conventional self-assembling peptides for comparison *: Significant turbidity and phase separation occurred.

[0078] composition Surfactant peptides having less than or equal to four repeating hydrophobic amino acids can be formulated with various agents for delivery. In some embodiments, the agent is delivered to a cell (in vitro delivery). In some embodiments, the agent is delivered to a subject (in vivo delivery). In some embodiments, compositions are provided that include the surfactant peptide in an aqueous solution.

[0079] In some embodiments, the peptide is at a concentration of at least 0.01% (w / v). In some embodiments, the peptide is at a concentration of about 0.01% (w / v) to about 0.5% (w / v). In some embodiments, the aqueous solution has a pH of about 6 to about 8. In some embodiments, the aqueous solution has a pH of about 7.5. In some embodiments, the aqueous solution is at an ionic strength of about 0M to about 0.3M. In some embodiments, the aqueous solution is at an ionic strength of about 0.15M. In some embodiments, the aqueous solution is isotonic.

[0080] Payload Drugs In some embodiments, the peptides of the present invention include one or more payload agents, such as therapeutic or detection agents. Such agents include, for example, any chemical class of compounds or entities, including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. In some embodiments, the agent is or includes a natural product, in that it is found in nature and / or derived from nature. In some embodiments, the agent is or includes one or more entities that are artificial, in that it is designed, engineered, and / or generated through human activity, or is not found in nature. In some embodiments, the agent may be utilized in isolated or pure form; in some embodiments, the agent may be utilized in crude form. Some specific embodiments of agents that may be utilized according to the present invention include small molecules, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptidomimetics, and the like.

[0081] A detection agent may refer to any element, molecule, functional group, compound, fragment, or moiety that is detectable. In some embodiments, a detection entity is provided or utilized alone. In some embodiments, a detection entity is provided and / or utilized in association with (e.g., bound to) another agent. Examples of detection entities include, but are not limited to, various ligands, radionuclides (e.g., 3 H, 14 C. 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes (see below for certain exemplary fluorescent dyes), chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (such as, for example, gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (see below for specific examples of enzymes), colorimetric labels (such as, for example, dyes, colloidal gold, etc.), biotin, dioxygenin, haptens, and proteins for which antisera or monoclonal antibodies are available. EXAMPLES

[0082] Example Example 1: Use of surfactant peptides to deliver siRNA to tumor cells in vitro This example describes, inter alia, an exemplary use of surfactant peptides to deliver siRNA to tumor cells in vitro, as well as their cytotoxicity and transfection efficacy.

[0083] siRNA (luciferase GL3 duplex) was synthesized using Ac-A4K-NH 2(SEQ ID NO:55), Ac-A3KA3-NH 2 (SEQ ID NO:55), Ac-A6K-NH 2 (SEQ ID NO: 105), and a control drug (DharmaFECT 1; a commercially available lipid transfection drug). The siRNA and peptide complexes were placed into nanosphere structures. The compositions containing the nanosphere structures were provided to tumor cells (MCF-7 cells; a breast cancer cell line) in vitro to reach preliminary transfection efficacy and cytotoxicity.

[0084] Two methods were utilized for this example. For method 1, MCF-7 cells were cultured on plates. The siRNA and peptide complexes were added to the culture medium covering the cells. The culture dish (96 wells) containing the siRNA and peptide complexes and the cells was incubated for 48 hours. For method 2, a concentrated culture (5×10 6 A total of 1000 cells / ml were prepared in a tube. The siRNA and peptide complexes were added to the suspension. The cell mixture was incubated in the tube for 1 hour. The cell mixture was plated in the culture dish and incubated for 48 hours. Luciferase luminescence from each sample was observed.

[0085] Table 4 summarizes the experimental conditions, such as the concentrations of siRNA and peptide detergent, charge ratios, and the type of siRNA (e.g., luciferase GL3 duplex or its negative control). The buffer used in this example was Opti-MEM1 (serum-free). As control samples, buffer alone, negative control (NC) siRNA with or without peptide detergent, DharmaFECT 1 with or without siRNA (or NC siRNA) were tested on the cells. Table 4. Experimental conditions [Table 4]

[0086] Cytotoxicity. For this test, the samples contained either NC siRNA or no siRNA. As shown in Figures 11A and 11B, the peptide (Ac-A6K-NH 2 (SEQ ID NO: 105), Ac-A4K-NH 2 (SEQ ID NO: 55) and Ac-A3KA3-NH 2 (SEQ ID NO:55)) showed a smaller decrease in luciferase luminescence than DharmaFECT, indicating that the peptide is less toxic than DharmaFECT.

[0087] Efficacy. As shown in Figures 12D and 12E, luciferase luminescence was reduced for all detergents at the charge ratio of 4.5 compared to the siRNA-only sample. The reduction in luciferase luminescence from the siRNA and peptide complex was similar to that from DharmaFECT (e.g., about 20%). An increase in luciferase luminescence was observed for A6K / siRNA at high concentration mixtures (siRNA 250 nM, A6K 51.75 μM). The large reduction in luciferase luminescence from DharmaFECT may be due to cytotoxicity.

[0088] Thus, the data in this example demonstrate that the peptide or its siRNA and peptide complexes are less toxic than known transfection reagents, and that the siRNA and peptide complexes deliver and effectively silence their target genes.

[0089] Example 2: Use of surfactant peptides to deliver siRNA to tumor cells in vivo This example describes, inter alia, an exemplary use of surfactant peptides to deliver siRNA to tumor cells in vivo. siRNA was delivered as Ac-A4K-NH 2 (SEQ ID NO:55), Ac-A3KA3-NH 2 (SEQ ID NO:55), Ac-A6K-NH 2(SEQ ID NO: 105), or a control peptide. The siRNA and peptide complex is placed into a nanosphere structure. A composition comprising the nanosphere structure is administered to a mouse tumor model. One or more cancer-related genes are suppressed, and the size of the tumor is reduced.

[0090] Example 3: Characterization of surfactant peptide / siRNA complexes This embodiment, inter alia, Surfactant peptide / siRNA complexes An exemplary characterization of is described below.

[0091] The ζ potential was measured by Zetasizer Nano ZS (Malvern) to characterize the electrokinetic potential of the complex. Ac-A4K-NH 2 (SEQ ID NO:55) concentration was 0.5 w / v% in aqueous solution. siRNA (ribophorin II, RPN2) concentration was controlled for those different charge ratios of A4K / siRNA. Ac-A4K-NH 2 The zeta potential of (SEQ ID NO: 55) alone was +21.9. This is because the surface of the A4K nanosphere is 2 13. The ζ potential of the A4K / siRNA mixture becomes more negative with more siRNA, as shown in FIG. 13. The instrument only detects dispersed phases larger than about 3.8 nm, and since the size of siRNA in water is about 2 nm in diameter, the measurement only confirms that Ac-A4K-NH siRNA is positively charged, not unassociated siRNA. 2 (SEQ ID NO:55) or Ac-A4K-NH 2 The electrokinetic potential of the (SEQ ID NO: 55) / siRNA complex is read. Thus, the results indicate that the negatively charged siRNA molecules are bound onto the positively charged surface of the A4K nanospheres. Thus, the data indicates that the siRNA is bound to Ac-A4K-NH 2 (SEQ ID NO:55) was successfully conjugated. The present invention provides, for example, the following items. (Item 1) A composition comprising a peptide having a nanosphere structure, wherein the peptide has the following formula I, II, III, IV, V, VI, VII, or VIII: Formula I(N→C): (X)a(Y)m Formula II(N→C): (Y)m(X)a Formula III(N→C): (X)a(Y)m(X)b Formula IV(N→C): (Y)m(X)a(Y)n Formula V(N→C): (X)a(Z)m Formula VI(N→C): (Z)m(X)a Formula VII(N→C): (X)a(Z)m(X)b Formula VIII(N→C): (Z)m(X)a(Z)n; wherein the repeating hydrophobic amino acids conform to (X) is an amino acid having a side chain that is nonpolar and uncharged at physiological pH; (Y) is an amino acid having a cationic side chain at physiological pH; (Z) is an amino acid having an anionic side chain at physiological pH; And here a is an integer equal to or less than 4; b is an integer equal to or less than 4; m is an integer equal to or greater than 1; and n is an integer equal to or greater than 1; composition. (Item 2) 2. The composition according to claim 1, wherein the amino acids (X), (Y) and / or (Z) are natural amino acids. (Item 3) 2. The composition of claim 1, wherein the (X), (Y), and / or (Z) amino acids are unnatural amino acids. (Item 4) 4. The composition according to any one of items 1 to 3, wherein the integer a or b is 4. (Item 5) 5. The composition according to any one of items 1 to 4, wherein the integer of a or b is 3. (Item 6) 6. The composition according to any one of items 1 to 5, wherein (X) is alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or glycine. (Item 7) 7. The composition according to any one of items 1 to 6, wherein (Y) is arginine, lysine, histidine, or ornithine. (Item 8) 8. The composition according to any one of items 1 to 7, wherein (Z) is aspartic acid or glutamic acid. (Item 9) 9. The composition according to any one of items 1 to 8, wherein each amino acid is an L-amino acid. (Item 10) 9. The composition according to any of items 1 to 8, wherein each amino acid is a D-amino acid. (Item 11) 11. The composition according to any one of items 1 to 10, wherein the peptide is 4 to 10 amino acids in length. (Item 12) The composition according to any one of items 1 to 10, wherein the peptide is 5 amino acids in length. The composition according to any one of items 1 to 10, wherein the peptide is 7 amino acids in length. 14. The composition according to any of items 1 to 13, wherein the peptide comprises a modified N-terminus and / or a modified C-terminus. (Item 15) 15. The composition according to any of items 1 to 14, wherein the peptide has an acetylated N-terminus and / or an aminated C-terminus. (Item 16) A composition comprising a peptide having a nanosphere structure, wherein the peptide has an amino acid sequence according to any one of SEQ ID NOs: 1-50. (Item 17) A composition comprising a peptide having a nanosphere structure, wherein the peptide has an amino acid sequence according to any one of SEQ ID NOs: 51-100. (Item 18) A composition comprising a peptide having a nanosphere structure, wherein the peptide has the amino acid sequence AAAK (SEQ ID NO:1). (Item 19) A composition comprising a peptide having a nanosphere structure, wherein the peptide has the amino acid sequence AAAAK (SEQ ID NO:5). (Item 20) A composition comprising a peptide having a nanosphere structure, wherein the peptide has the amino acid sequence AAAKAAA (SEQ ID NO: 15). (Item 21) 22. The composition according to any of items 1 to 21, wherein the peptide is in an aqueous solution. (Item 22) 22. The composition according to item 21, wherein the peptide is at a concentration of at least 0.01% (w / v). (Item 23) 23. The composition according to any one of items 21 to 22, wherein the aqueous solution has a pH of about 6 to about 8. (Item 24) 24. The composition according to any one of items 21 to 23, wherein the aqueous solution has an ionic strength of about 0M to about 0.3M. (Item 25) 24. The composition according to any of items 21 to 23, wherein the aqueous solution is at an ionic strength of about 0.15 M. (Item 26) 26. The composition according to any of items 21 to 25, wherein the aqueous solution is isotonic. (Item 27) 27. The composition according to any of items 1 to 26 for use in drug delivery. (Item 28) The composition according to any one of items 1 to 26, further comprising an agent for delivery to a subject. (Item 29) 29. The composition of claim 28, wherein the agent is a therapeutic agent. (Item 30) 29. The composition of claim 28, wherein the agent is a drug. (Item 31) 29. The composition of claim 28, wherein the agent is a siRNA. (Item 32) 32. The composition of claim 31, wherein the siRNA is for delivery to a cancer cell. (Item 33) 27. A method of formulating an agent for delivery to a subject, the method comprising the step of contacting the agent with a composition according to any one of items 1 to 26. (Item 34) 1. A method of delivering a drug to a subject, the method comprising administering to the subject a composition formulated with a peptide, wherein the peptide has the following formula I, II, III, IV, V, VI, VII, or VIII: Formula I(N→C): (X)a(Y)m Formula II(N→C): (Y)m(X)a Formula III(N→C): (X)a(Y)m(X)b Formula IV(N→C): (Y)m(X)a(Y)n Formula V(N→C): (X)a(Z)m Formula VI(N→C): (Z)m(X)a Formula VII(N→C): (X)a(Z)m(X)b Formula VIII(N→C): (Z)m(X)a(Z)n; wherein the repeating hydrophobic amino acids conform to (X) is an amino acid having a side chain that is nonpolar and uncharged at physiological pH; (Y) is an amino acid having a cationic side chain at physiological pH; (Z) is an amino acid having an anionic side chain at physiological pH; And here a is an integer equal to or less than 4; b is an integer equal to or less than 4; m is an integer equal to or greater than 1; and n is an integer equal to or greater than 1; method. (Item 35) 35. The method of claim 34, wherein the (X), (Y) and / or (Z) amino acids are natural amino acids. (Item 36) 35. The method of claim 34, wherein the (X), (Y) and / or (Z) amino acids are unnatural amino acids. (Item 37) 37. The method according to any one of Items 34 to 36, wherein the integer a or b is 4. (Item 38) 38. The method according to any one of Items 34 to 37, wherein the integer a or b is 3. (Item 39) 39. The method according to any one of items 34 to 38, wherein (X) is alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or glycine. (Item 40) 40. The method according to any one of items 34 to 39, wherein (Y) is arginine, lysine, histidine, or ornithine. (Item 41) 41. The method according to any one of items 34 to 40, wherein (Z) is aspartic acid or glutamic acid. (Item 42) 42. The method according to any one of items 34 to 41, wherein each amino acid is an L-amino acid. (Item 43) 42. The method according to any one of items 34 to 41, wherein each amino acid is a D-amino acid. (Item 44) 43. The method according to any one of items 34 to 42, wherein the peptide is 4 to 10 amino acids in length. (Item 45) The method according to any one of Items 34 to 42, wherein the peptide is 5 amino acids in length. Item 47. The method according to any one of Items 34 to 42, wherein the peptide is 7 amino acids in length. 47. The method according to any of items 34 to 46, wherein the peptide comprises a modified N-terminus and / or a modified C-terminus. (Item 48) 48. The method according to any of items 34 to 47, wherein the peptide has an acetylated N-terminus and / or an aminated C-terminus. (Item 49) 1. A method of delivering a drug to a subject, the method comprising administering to the subject a composition formulated with a peptide, wherein the peptide has an amino acid sequence according to any of SEQ ID NOs: 1-50. (Item 50) 1. A method of delivering a drug to a subject, the method comprising administering to the subject a composition formulated with a peptide, wherein the peptide has an amino acid sequence according to any of SEQ ID NOs: 51-100. (Item 51) 1. A method of delivering a drug to a subject, the method comprising administering to the subject a composition formulated with a peptide, wherein the peptide has the amino acid sequence AAAK (SEQ ID NO:1). (Item 52) 1. A method of delivering a drug to a subject, the method comprising administering to the subject a composition formulated with a peptide, wherein the peptide has the amino acid sequence AAAAK (SEQ ID NO:5). (Item 53) 1. A method of delivering a drug to a subject, the method comprising administering to the subject a composition formulated with a peptide, wherein the peptide has the amino acid sequence AAAKAAA (SEQ ID NO: 15). (Item 54) 54. The method according to any of items 34 to 53, wherein the peptide is in an aqueous solution. (Item 55) 55. The method of claim 54, wherein the peptide is at a concentration of at least 0.01% (w / v). (Item 56) 56. The method according to any one of items 54 to 55, wherein the aqueous solution has a pH of about 6 to about 8. (Item 57) 57. The method according to any one of items 54 to 56, wherein the aqueous solution has an ionic strength of about 0M to about 0.3M. (Item 58) 57. The method according to any of items 54 to 56, wherein the aqueous solution is at an ionic strength of about 0.15 M. (Item 59) 59. The method according to any one of items 54 to 58, wherein the aqueous solution is isotonic. (Item 60) 59. The method according to any one of items 35 to 58, wherein the agent is a therapeutic agent. (Item 61) 59. The method according to any one of items 35 to 58, wherein the agent is a drug. (Item 62) 59. The method according to any of items 35 to 58, wherein the agent is an siRNA. (Item 63) 63. The method of claim 62, wherein the agent is delivered to a cancer cell.

Claims

[Claim 1] The invention as depicted in the drawings.

Images