阅读说明：本技术 用于核酸递送的叔氨基脂化的阳离子肽 (Tertiary aminolipidated cationic peptides for nucleic acid delivery ) 是由 C·J·麦金利 于 2019-09-27 设计创作，主要内容包括：本公开涉及用于细胞内递送的叔氨基脂化和/或聚乙二醇化的阳离子肽化合物及其与核酸的复合物,用于制备该化合物和复合物的方法,以及用于将聚阴离子化合物递送至细胞的方法。(The present disclosure relates to tertiary aminolipidated and/or pegylated cationic peptide compounds and complexes thereof with nucleic acids for intracellular delivery, methods for making the compounds and complexes, and methods for delivering polyanionic compounds to cells.)

1. A tertiary amino lipidated and/or pegylated cationic peptide compound of formula (I) or a salt thereof:

wherein:

m is an integer of 0 to 10;

n is an integer of 0 to 5;

s is an integer from 0 to 5;

t is an integer from 0 to 10;

wherein at least one of m, n, s and t is non-zero;

r is an integer from 1 to 20;

each o is independently an integer of 0, 1 or 2;

each q is independently an integer of 0, 1 or 2;

each p is independently an integer of 1 or 2;

R¹is-H, alkyl, alkylaryl, -COR^1aOr a lipid moiety, in the form of a lipid,

wherein R is^1ais-H, -OH, alkyl, aryl, alkylaryl, -O-alkyl or-O-alkylaryl;

each R²Independently is of formula-CH₂CH₂O(CH₂CH₂O)_uCH₃And wherein each u is independently an integer from 2 to 200;

each R³Independently a lipid moiety;

each R⁴Independently a neutral spacer moiety or a lipid moiety;

each R⁵Independently a cationic moiety;

each R⁶Independently is of formula-CH₂CH₂O(CH₂CH₂O)_vCH₃And wherein each v is independently an integer from 2 to 200;

R⁷is-H, alkyl, acyl, -OH, -OR^7a、-NH₂、-NHR^7aOr a lipid moiety, wherein R^7aIs an alkyl, acyl or lipid moiety; and

R^aand R^bEach independently is-H, C₁-C₄Alkyl groups or side chain moieties on naturally or non-naturally occurring amino acids.

2. Tertiary aminolipidated and/or pegylated cationic peptide compound according to claim 1, wherein at least one of n or s is non-zero.

3. The tertiary amino lipidated and/or pegylated cationic peptide compound according to claim 1 or claim 2, wherein said tertiary amino lipidated and/or pegylated cationic peptide compound comprises a block of N-lipidated amino acid residues, wherein at least one of N and s is at least 2, at least 3 or at least 4, optionally 2,3 or 4.

4. Tertiary aminolipidated and/or pegylated cationic peptide compound according to any of claims 1 to 3, wherein each R³Independently is C₄-C₂₂Alkyl or C₄-C₂₂Alkenyl, and wherein said C₄-C₂₂The alkenyl group is optionally mono-or polyunsaturated.

5. Tertiary aminolipidated and/or pegylated cationic peptide compound according to any of claims 1 to 4, wherein each R³Independently is C₈-C₁₂An alkyl group.

6. Tertiary aminolipidated and/or pegylated cationic peptide compound according to any of claims 1 to 4, wherein each R³Independently selected from 2-ethylhexyl-1-yl, hexanoyl, oleyl, stearyl, linoleyl, myristyl and lauryl.

7. Tertiary aminolipidated and/or pegylated cationic peptide compound according to any of claims 1 to 6, wherein said tertiary amino lipidated and/or pegylated cationic peptide compoundThe compound comprises a polymer containing at least two moieties R having a cation⁵A cationic domain of a cationic amino acid residue of (a).

8. Tertiary aminolipidated and/or pegylated cationic peptide compound according to any of claims 1 to 7, wherein each R⁵Independently an aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, N-heterocyclylalkyl, or N-heteroaryl group.

9. The tertiary amino lipidated and/or pegylated cationic peptide compound according to any of claims 1-8, wherein each R⁵Independently selected from:

10. the tertiary amino lipidated and/or pegylated cationic peptide compound according to any of claims 1 to 9, wherein each R⁵Is composed of

11. Tertiary amino lipidated and/or pegylated cationic peptide compound according to any of claims 1 to 10, wherein each neutral spacer moiety R⁴Independently C substituted by cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl or hydroxyalkyl₁-C₄Alkyl, wherein each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl group is optionally substituted with one or more substituents-OH, halo, or alkoxy.

12. Tertiary amino lipidated and/or pegylated cationic peptide compound according to any of claims 1 to 11, wherein each neutral spacer moiety R⁴Independently selected from:

13. tertiary amino lipidated and/or pegylated cationic peptide compound according to any of claims 1 to 12, wherein each neutral spacer moiety R⁴Is that

14. The tertiary amino lipidated and/or pegylated cationic peptide compound of any of claims 1-13, wherein said tertiary amino lipidated and/or pegylated cationic peptide compound comprises a mixture comprising at least two cationic peptide compounds having R⁵A cationic domain of amino acid residues of a cationic moiety, and wherein each of said at least two cationic amino acid residues within said cationic domain is at least one R having a neutral spacer or a lipid moiety⁴Are separated.

15. The tertiary amino lipidated and/or pegylated cationic peptide compound according to any of claims 1 to 14, wherein said tertiary amino lipidated and/or pegylated cationic peptide compound comprises at least one trimeric subunit-R^cation-R^neutral-R^neutralAnd wherein R is^cationIs a salt comprising a cationic moiety R⁵And each R is^neutralIs a polymer containing a neutral spacer moiety R⁴The amino acid residue of (1).

16. Tertiary aminolipidated and/or pegylated cationic peptidation according to any of claims 1 to 15A compound in which each cationic moiety R⁵Is composed ofAnd each neutral spacer moiety R⁴Is composed of

17. Tertiary aminolipidated and/or pegylated cationic peptide compound according to any of claims 1 to 16, wherein

When m is an integer from 0 to 3, each u is independently an integer from 20 to 200, optionally from 30 to 50; and

when m is an integer from 4 to 10, each u is independently an integer from 2 to 10.

18. The tertiary aminolipidated and/or pegylated cationic peptide compound of any of claims 1-17, wherein

When t is an integer from 0 to 3, each v is independently an integer from 30 to 50;

when t is an integer from 4 to 10, each v is independently an integer from 2 to 10.

19. Tertiary aminolipidated and/or pegylated cationic peptide compound according to any of claims 1 to 18, wherein

m is 1, and u is an integer of 40 to 45; or

t is 1, and v is an integer of 40 to 45.

20. The tertiary aminolipidated and/or pegylated cationic peptide compound of any of claims 1-19, wherein R^aAnd R^bIndependently selected from-H and-CH₃。

21. The tertiary aminolipidated and/or pegylated cationic peptide compound of any of claims 1-20, wherein R^aAnd R^bis-H.

22. A tertiary amino lipidated and pegylated cationic peptide compound of formula (I) or a salt thereof:

wherein:

m is an integer of 0 to 10;

n is an integer of 0 to 5;

s is an integer from 0 to 10;

t is an integer from 0 to 5;

wherein at least one of m and t is non-zero; and wherein at least one of n and s is non-zero

r is an integer from 1 to 20;

each o is independently an integer of 0, 1 or 2;

each q is independently an integer of 0, 1 or 2;

each p is independently an integer of 1 or 2;

R¹is-H, alkyl, alkylaryl, -COR^1aOr a lipid moiety, in the form of a lipid,

wherein R is^1ais-H, -OH, alkyl, aryl, alkylaryl, -O-alkyl or-O-alkylaryl;

each R²Independently is of formula-CH₂CH₂O(CH₂CH₂O)_uCH₃And wherein each u is independently an integer from 2 to 200;

each R³Independently a lipid moiety;

each R⁴Independently a neutral spacer moiety or a lipid moiety;

each R⁵Independently a cationic moiety;

each R⁶Independently is of formula-CH₂CH₂O(CH₂CH₂O)_vCH₃And wherein each v is independently an integer from 2 to 200;

R⁷is-H, alkyl, acyl, -OH, -OR^7a、-NH₂、-NHR^7aOr a lipid moiety, wherein R^7aIs an alkyl, acyl or lipid moiety; and

R^aand R^bEach independently is-H, C₁-C₄Alkyl groups or side chain moieties on naturally or non-naturally occurring amino acids.

23. A complex comprising one or more tertiary amino lipidated and/or pegylated cationic peptide compounds according to any one of claims 1-22 complexed with a polyanionic compound.

24. The complex of claim 23, wherein the polyanionic compound comprises a nucleic acid.

25. The complex of claim 23 or claim 24, wherein the polyanionic compound comprises a nucleic acid, and wherein the nucleic acid is an mRNA encoding a polypeptide.

26. The complex of any one of claims 23-25, wherein the polyanionic compound comprises a nucleic acid, and wherein the nucleic acid is an mRNA encoding a protein.

27. The complex of any one of claims 23-26, wherein said polyanionic compound comprises a nucleic acid, and wherein said complex comprises at least one said tertiary amino lipidated and/or pegylated cationic peptide and said nucleic acid in a mass ratio of from 0.5:1 to 50: 1.

28. The complex of any one of claims 23-27, wherein the complex comprises: the one or more tertiary amino lipidated and/or pegylated cationic peptide compounds, nucleic acids, phospholipids, optionally structural lipids, and PEG lipids.

29. The complex of any one of claims 23 to 28, further comprising one or more small molecule active agents or drug substances.

30. A method of delivering a polyanionic compound to a cell, comprising contacting the cell with the complex of any one of claims 23 to 29.

31. The method of claim 30, wherein said contacting is by endocytosis.

32. The method according to claim 30 or 31, wherein the cells are contacted in vivo or in vitro.

33. The method of any one of claims 30 to 32, wherein said polyanionic compound comprises a nucleic acid that is an mRNA encoding a polypeptide, and said cell expresses said polypeptide upon contact with said complex.

34. The method of any one of claims 30 to 33, wherein said polyanionic compound comprises nucleic acid that is mRNA encoding a protein, and said cell expresses said protein upon contact with said complex.

35. A method of forming a complex of any one of claims 23 to 29, comprising contacting the tertiary amino lipidated and/or pegylated cationic peptide compound with the polyanionic compound.

36. The method of claim 35, comprising contacting a solution comprising the tertiary amino lipidated and/or pegylated cationic peptide compound with a solution comprising the polyanionic compound.

Technical Field

The present disclosure relates to tertiary amino lipidated and/or pegylated cationic peptide compounds, and more particularly, to tertiary amino lipidated and/or pegylated peptoids for nucleic acid delivery and complexes thereof. The present disclosure also provides methods for preparing complexes thereof comprising one or more tertiary amino lipidated and/or pegylated cationic peptides and polyanionic compounds (e.g., nucleic acids), and methods for delivering the polyanionic compounds to cells.

Background

It is generally believed that a consistent, efficient method for delivering and transferring nucleic acids into cells is of critical importance for any practical application of gene therapy, siRNA, miRNA, antisense or mRNA based therapies. Cationic peptoid-phospholipid conjugate constructs, also known as lipidoids (lipids), are a class of agents developed to encapsulate polyanionic nucleic acids and stabilize nucleic acids against nucleases in vivo, while also facilitating uptake and subsequent release of polynucleotides into the cell cytosol. The lipid, such as lipid 1 (shown in fig. 1), has proven itself to be an efficient and non-immunogenic vesicle for complexing and intracellular delivery with nucleic acids (e.g., plasmid dna (pdna) and short interfering rna (sirna)).

However, because of the many types of nucleic acids-with different base pair lengths, chain numbers, conformations, and the like, each particular type of nucleic acid presents different challenges when attempting to optimize its insertion into a cell. Although lipidoids have shown success in improving the uptake of a variety of nucleic acids, existing lipidoid constructs focus primarily on the delivery of certain types of polynucleotides (e.g., pDNA and siRNA), and may not necessarily be as effective in promoting intracellular uptake of other polynucleotides (e.g., messenger rna (mrna)). In addition, even for lipid-like constructs optimized for a particular nucleic acid type, lipid-nucleic acid complexes may still suffer from further complications in vivo, including, for example, particle stability and aggregation. Thus, to date, no lipid-like drugs have been approved for use in humans.

Thus, there remains a need for alternative agents for delivering nucleic acids (particularly mRNA) into cells with less in vivo complexity such as particle aggregation.

Disclosure of Invention

The present disclosure provides lipid based alternative conjugation methods for lipids and even for pegylation with greater flexibility to overcome difficulties encountered with other forms of lipids.

In a first aspect, there is provided a tertiary amino lipidated and/or pegylated cationic peptide compound of formula (I) or a salt thereof:

wherein:

m is an integer of 0 to 10;

n is an integer of 0 to 5;

s is an integer from 0 to 5;

t is an integer from 0 to 10;

wherein at least one of m, n, s and t is non-zero;

r is an integer from 1 to 20;

each o is independently an integer of 0, 1 or 2;

each q is independently an integer of 0, 1 or 2;

each p is independently an integer of 1 or 2;

R¹is-H, alkyl, alkylaryl, COR^1aOr a lipid moiety, in the form of a lipid,

wherein R is^1ais-H, -OH, alkyl, aryl, alkylaryl, -O-alkyl or-O-alkylaryl;

each R²Independently is of formula-CH₂CH₂O(CH₂CH₂O)_uCH₃And wherein each u is independently an integer from 2 to 200;

each R³Independently is a lipidA moiety;

each R⁴Independently a neutral spacer moiety or a lipid moiety;

each R⁵Independently a cationic moiety;

each R⁶Independently is of formula-CH₂CH₂O(CH₂CH₂O)_vCH₃And wherein each v is independently an integer from 2 to 200;

R⁷is-H, alkyl, acyl, -OH, -OR^7a、-NH₂、-NHR^7aOr a lipid moiety, wherein R^7aIs an alkyl, acyl or lipid moiety; and

R^aand R^bEach independently is-H, C₁-C₄Alkyl groups or side chain moieties on naturally or non-naturally occurring amino acids.

In some embodiments of this aspect, the tertiary amino lipidated and/or pegylated cationic peptide compound has at least one of n or s is non-zero. In certain embodiments of this aspect, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a block of N-lipidated amino acid residues, wherein at least one of N and s is at least 2, at least 3, or at least 4, optionally 2,3, or 4. In some embodiments of this aspect, each R is³Independently is C₄-C₂₂Alkyl or C₄-C₂₂Alkenyl radical, and wherein C₄-C₂₂The alkenyl group is optionally mono-or polyunsaturated. Further embodiments of this aspect include tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure, wherein each R is³Independently is C₈-C₁₂An alkyl group. In other embodiments of this aspect which may be combined with any one or both of the preceding embodiments, each R is³Independently selected from 2-ethylhexyl-1-yl, hexanoyl, oleyl, stearyl, linoleyl, myristyl and lauryl.

In still further embodiments of this aspect which may be combined with any one or more of the preceding embodiments, the tertiary amino group is lipidated and/or pegylatedComprising a peptide containing at least two moieties R having a cation⁵A cationic domain of a cationic amino acid residue of (a). In still other embodiments that may be combined with any one or more of the preceding embodiments, each R is a hydrogen atom⁵Independently an aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoalkyl, N-heterocyclylalkyl, or N-heteroaryl group. In certain other embodiments that may be combined with any one or more of the preceding embodiments, each R is a hydrogen atom⁵Is independently selected fromAndin certain embodiments, each R is⁵Is that

In some embodiments of this aspect which may be combined with any one or more of the preceding embodiments, each neutral spacer moiety R is a cyclic alkyl moiety⁴Independently C substituted by cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl or hydroxyalkyl₁-C₄Alkyl, wherein each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl group is optionally substituted with one or more substituents-OH, halogen, or alkoxy. In still other embodiments that may be combined with any one or more of the preceding embodiments, each neutral spacer moiety R⁴Is independently selected from In certain embodiments of any of the preceding embodiments, each neutral spacer moiety R⁴Is thatIn some embodiments of this aspect which may be combined with any one or more of the preceding embodiments, each lipid moiety R is a lipid moiety R⁴Independently is C₄-C₂₂Alkyl or C₄-C₂₂Alkenyl radical and wherein C₄-C₂₂The alkenyl group is optionally mono-or polyunsaturated. In some embodiments, each lipid moiety R is a pharmaceutically acceptable salt, solvate, or solvate of the compound⁴Is C₆-C₁₈Alkyl or C₆-C₁₈An alkenyl group. In certain embodiments, each lipid moiety R is a pharmaceutically acceptable salt, solvate, or solvate of the compound⁴Is C₈-C₁₂An alkyl group. In still other embodiments, each lipid moiety R⁴Is C₁₀Alkyl groups, such as n-decyl. In other embodiments of this aspect which may be combined with any one or both of the preceding embodiments, each lipid moiety R is a lipid moiety R⁴Independently selected from 2-ethylhexyl-1-yl, hexanoyl, oleyl, stearyl, linoleyl, myristyl and lauryl.

In still further embodiments of this aspect, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a mixture comprising at least two cationic peptide compounds having R⁵A cationic domain of amino acid residues of a cationic moiety, and wherein each of at least two cationic amino acid residues within the cationic domain is substituted with at least one neutral spacer or lipid moiety R⁴Are separated. In still other embodiments of this aspect which may be combined with one or more of any of the preceding embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises at least one trimeric subunit-R^cation-R^neutral-R^neutralAnd wherein R is^cationIs a salt comprising a cationic moiety R⁵And each R is^neutralIs a polymer containing a neutral spacer moiety R⁴The amino acid residue of (1). In still other embodiments of this aspect that may be combined with one or more of any of the preceding embodiments, each cationic moiety R⁵Is composed ofAnd each neutral spacer moiety R⁴Is composed of

In still other embodiments of this aspect that may be combined with any one or more of the preceding embodiments, when m is an integer from 0 to 3, each u is independently an integer from 20 to 200, optionally from 30 to 50; and when m is an integer from 4 to 10, each u is independently an integer from 2 to 10.

In some embodiments of this aspect that may be combined with any one or more of the preceding embodiments, when t is an integer from 0 to 3, each v is independently an integer from 30 to 50; and when t is an integer from 4 to 10, each v is independently an integer from 2 to 10. In other embodiments of this aspect that may be combined with any one or more of the preceding embodiments, m is 1, and u is an integer from 40 to 45; and/or t is 1, and v is an integer from 40 to 45. In other embodiments of this aspect that may be combined with any one or more of the preceding embodiments, R^aAnd R^bIndependently selected from-H and-CH₃. In certain other embodiments of this aspect that may be combined with any one or more of the preceding embodiments, R^aAnd R^bis-H.

Another aspect of the present disclosure provides a tertiary amino lipidated and pegylated cationic peptide compound of formula (I) or a salt thereof:

wherein:

m is an integer of 0 to 10;

n is an integer of 0 to 5;

s is an integer from 0 to 10;

t is an integer from 0 to 5;

wherein at least one of m and t is non-zero, and at least one of n and s is non-zero;

r is an integer from 1 to 20;

each o is independently an integer of 0, 1 or 2;

each q is independently an integer of 0, 1 or 2;

each p is independently an integer of 1 or 2;

R¹is-H, alkyl, alkylaryl, COR^1aOr a lipid moiety, in the form of a lipid,

wherein R is^1ais-H, -OH, alkyl, aryl, alkylaryl, -O-alkyl or-O-alkylaryl;

each R²Independently is of formula-CH₂CH₂O(CH₂CH₂O)_uCH₃And wherein each u is independently an integer from 2 to 200;

each R³Independently a lipid moiety;

each R⁴Independently a neutral spacer moiety or a lipid moiety;

each R⁵Independently a cationic moiety;

each R⁶Independently is of formula-CH₂CH₂O(CH₂CH₂O)_vCH₃And wherein each v is independently an integer from 2 to 200;

R⁷is-H, alkyl, acyl, -OH, -OR^7a、-NH₂、-NHR^7aOr a lipid moiety, wherein R^7aIs an alkyl, acyl or lipid moiety; and

R^aand R^bEach independently is-H, C₁-C₄Alkyl groups or side chain moieties on naturally or non-naturally occurring amino acids. Where applicable, this aspect of the disclosure also includes various embodiments of the first aspect.

A further aspect of the present disclosure provides a complex comprising one or more tertiary amino lipidated and/or pegylated cationic peptide compounds of any or both of the preceding aspects in any and all various embodiments thereof complexed with a polyanionic compound. In certain embodiments of this aspect, the polyanionic compound comprises a nucleic acid. In particular embodiments of this aspect that include nucleic acids, the nucleic acid is mRNA encoding a polypeptide. In a still further embodiment of this aspect, the nucleic acid is mRNA encoding a protein. In certain embodiments of this aspect that may be combined with the preceding embodiments, including nucleic acids, the complex comprises at least one tertiary amino lipidated and/or pegylated cationic peptide and nucleic acid in a mass ratio of from 0.5:1 to 50: 1. In certain embodiments of this aspect that may be combined with any one or both of the preceding embodiments, the complex comprises: one or more tertiary amino lipidated and/or pegylated cationic peptide compounds, nucleic acids, phospholipids, structural lipids, and PEG lipids. In yet a further embodiment that may be combined with any of the preceding embodiments, the complex further comprises one or more small molecule active agents or drug substances.

A further aspect of the present disclosure provides a method of delivering a polyanionic compound to a cell comprising contacting the cell with the complex of the preceding aspect of any and all of the various embodiments thereof. In certain embodiments of this aspect, the contacting is by endocytosis. In other embodiments of this aspect which may be combined with the preceding aspects, the cell is contacted in vivo or in vitro. In certain embodiments of this aspect that may be combined with any one or both of the preceding embodiments, the cell expresses the polypeptide upon contact with the complex. In certain embodiments of this aspect that may be combined with one or more of any of the preceding embodiments, the cell expresses the protein upon contact with the complex.

Further aspects of the present disclosure provide methods of forming complexes of aspects related to the complexes and any and all various embodiments thereof by contacting a tertiary amino lipidated and/or pegylated cationic peptide compound with a polyanionic compound. In one embodiment of this aspect, the complex is formed by contacting a solution comprising a tertiary amino lipidated and/or pegylated cationic peptide compound with a solution comprising a polyanionic compound.

Drawings

Figure 1 shows an exemplary secondary amine phospholipid-modified cationic peptide "lipid class 1".

Figures 2A-2F generally show tertiary amino lipidated cationic peptoids. Fig. 2A shows the generalized structure of a tertiary amino lipidated peptoid. Figure 2B shows exemplary tertiary amino lipidated cationic peptoids. Fig. 2C shows exemplary lipid monomers. Fig. 2D shows an exemplary cationic monomer. Figure 2E shows exemplary spacer monomers and polyethylene glycol monomers. Figure 2F shows an exemplary tertiary amino lipidated cationic peptoid compound 8.

FIGS. 3A-3E show an exemplary process for preparing tertiary amino lipidated and/or pegylated cationic peptide compounds by sequential addition of amino acid residues in a solid phase. Fig. 3A shows a generalized acylation reaction of a resin-bound secondary amine with an acylating agent, bromoacetic acid. FIG. 3B shows the reaction of the resulting acylation product with a suitable amine via nucleophilic substitution to yield the corresponding N-substituted amino acid residue. Fig. 3C and 3D show successive iterations of the acylation and nucleophilic substitution reactions to produce the desired resin-bound cationic peptide compound. FIG. 3E shows cleavage of the cationic peptide compound from the solid resin support to provide free peptide.

Figure 4 depicts a graph of mean bioluminescence (RLU) results as measured for HeLa cells treated with tertiary amino lipidated peptoid compounds 1-18 complexed with firefly luciferase (Fluc) mRNA alone at peptoid to mRNA mass ratios of 2:1, 3.5:1, 5:1, and 7.5:1 in vitro.

Figure 5 shows a graph of the mean bioluminescence (RLU) observed in HeLa cells treated in vitro with one of the selected multicomponent lipid formulations containing the lipid 1 or the aminolipidated peptoid compounds of the present disclosure. Lipid 1 or selected aminolipidated peptoid compounds were combined with cholesterol, 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) in three mass ratios (50:16:32:2, 63:0:35:2, and 42:23:33:2) and further mixed with firefly luciferase (Fluc) mRNA in either a peptoid to mRNA mass ratio of 7:1 or 10:1 to provide the test formulation.

FIGS. 6A-6B show in vitro expression of Fluc mRNA in HeLa cells treated with multicomponent lipid formulations of depsipeptide-cholesterol: DOPE: DMG-PEG2000 mass ratio of 41:23:33:3 and depsipeptide-mRNA mass ratio of amino-lipidated peptoids to cholesterol, DOPE and DMG-PEG2000 of 10: 1. FIGS. 6A and 6B show the mean bioluminescence observed in HeLa cells treated with formulations 1-36 (FIG. 6A) and 37-72 (FIG. 6B).

FIGS. 7A-7B show in vivo expression of Fluc mRNA in Balb/c mice treated with subcutaneously administered multicomponent lipid formulations of an aminolipidated peptoid with a mass ratio of 41:23:33:3, cholesterol: DOPE: DMG-PEG2000 and a mass ratio of 10:1, peptoid: mRNA. FIGS. 7A and 7B show the mean bioluminescence observed 6 hours after administration of formulations 1-36 (FIG. 7A) and 37-72 (FIG. 7B).

Detailed Description

The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

The present disclosure provides N-substituted cationic peptide compounds, and compositions and complexes thereof, for delivering nucleic acids and other polyanionic materials into cells. More specifically, the present disclosure relates to cationic peptide compounds comprising one or more amino acid residues N-substituted with a lipid moiety and/or (oligo and/or poly) ethylene glycol moiety at or near the N-terminus and/or C-terminus to facilitate efficient complexing and intracellular delivery of messenger rna (mrna) and other polynucleotides.

In contrast to previous lipid-like structures (e.g., lipid 1), the tertiary aminolipidated and/or pegylated cationic peptide compounds of the present disclosure not only replace the phospholipid component of the lipid conjugate with N-lipidated amino acid residues at or near the N-and C-termini of the peptide, but also allow for the presence of multiple lipid moieties along the backbone of the peptide and the incorporation of oligomeric and/or polyethylene glycol moieties. The inclusion of multiple lipid moieties and/or oligo-and polyethylene glycol moieties along the oligopeptide backbone of the compounds described herein results in improved complexation with nucleic acids, and thus also in cellular uptake of nucleic acids, compared to previous lipid-like constructs comprising a single terminal phospholipid or other lipid moiety.

As used herein, "alkenyl" refers to an unsaturated, straight or branched, monovalent hydrocarbon chain, or combination thereof, having at least one site of ethylenic 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)₂-C₁₀Meaning 2 to 10 carbon atoms). The alkenyl group may be in the "cis" or "trans" configuration, or in the "E" or "Z" configuration. Particular alkenyl radicals are those having from 2 to 20 carbon atoms ("C₂-C₂₀Alkenyl ") having 2 to 8 carbon atoms (" C)₂-C₈Alkenyl ") having 2 to 6 carbon atoms (" C)₂-C₆Alkenyl) or having 2 to 4 carbon atoms ("C)₂-C₄Alkenyl ") groups. Examples of alkenyl groups include, but are not limited to, groups such as vinyl (or ethylene), prop-1-enyl, prop-2-enyl (or allyl), 2-methylprop-1-enyl, but-2-enyl, but-3-enyl, but-1, 3-dienyl, 2-methylbut-1, 3-dienyl, homologs and isomers thereof, and the like.

The term "alkyl" refers to and includes saturated straight and branched chain monovalent hydrocarbon structures and combinations thereof having the indicated number of carbon atoms (i.e., C)₁-C₁₀Meaning 1 to 10 carbons). Particular alkyl groups are those having from 1 to 20 carbon atoms ("C)₁-C₂₀Alkyl groups "). More particularly alkyl radicals are those having from 1 to 8 carbon atoms ("C)₁-C₈Alkyl group "), 3 to 8 carbon atoms (" C₃-C₈Alkyl "), 1-6 carbon atoms (" C₁-C₆Alkyl "), 1-5 carbon atoms (" C₁-C₅Alkyl group ") or 1 to 4 carbon atoms (" C)₁-C₄Alkyl groups "). Examples of alkyl groups include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, e.g., n-pentyl, n-hexyl, n-heptyl, homologs and isomers of n-octyl, and the like.

As used herein, "alkylene" refers to the same residue as alkyl, but is divalent. Particular alkylene radicals are those having from 1 to 6 carbon atoms ("C)₁-C₆Alkylene "), 1 to 5 carbon atoms (" C₁-C₅Alkylene group ") 1-4 carbon atoms (' C)₁-C₄Alkylene ") or 1 to 3 carbon atoms (" C)₁-C₃Alkylene ") groups. Examples of alkylene groups include, but are not limited to, groups such as methylene (-CH)₂-) ethylene (-CH₂CH₂-) propylene (-CH)₂CH₂CH₂-) butylene (-CH)₂CH₂CH₂CH₂-) and the like.

As used herein, "alkynyl" refers to an unsaturated, straight or branched, monovalent hydrocarbon chain, or combination 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)₂-C₁₀Meaning 2 to 10 carbon atoms). Particular alkynyl groups are those having 2 to 20 carbon atoms ("C)₂-C₂₀Alkynyl ") having 2 to 8 carbon atoms (" C₂-C₈Alkynyl ") having 2 to 6 carbon atoms (" C₂-C₆Alkynyl ") or having 2 to 4 carbon atoms (" C)₂-C₄Alkynyl ") of a cyclic alkyl group. Examples of alkynyl groups include, but are not limited to, groups such as ethynyl (or ethynyl), prop-1-ynyl, prop-2-ynyl (or propynyl), but-1-ynyl, but-2-ynyl, but-3-ynyl, homologs and isomers thereof, and the like.

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

"carbonyl" refers to a C ═ O group.

As used herein, "complex" includes any chemical association between two or more molecules that may be mediated by ionic interactions, hydrogen bonding, van der waals interactions, metal-ligand coordination, other chemical forces, and combinations of one or more of the foregoing. Complexes may form higher order structures including, for example, polyplexes, coacervate complexes, nanocomposites, nanoparticles, and microparticles.

The term "cycloalkyl" refers to and includes cyclic monovalent hydrocarbon structures that may be fully saturated, monounsaturated, or polyunsaturated, but which are non-aromatic, having the indicated number of carbon atoms (e.g., C)₁-C₁₀Meaning 1 to 10 carbons). Cycloalkyl groups may consist of one ring (e.g., cyclohexyl) or multiple rings (e.g., adamantyl), but do not include aryl groups. Cycloalkyl groups containing more than one ring may be fused, spiro, or bridged, or a combination thereof. Preferred cycloalkyl groups are cyclic hydrocarbons having from 3 to 13 ring carbon atoms. More preferred cycloalkyl groups are cyclic hydrocarbons having from 3 to 8 ring carbon atoms ("C)₃-C₈Cycloalkyl "). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, norbornyl, and the like.

"halo" or "halogen" refers to an element of the group 17 series of atomic numbers 9 to 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, but are not necessarily, the same halo group; thus, 4-chloro-3-fluorophenyl falls within the scope of dihaloaryl groups. Alkyl groups in which each hydrogen is substituted with a halogen group are referred to as "perhaloalkyl groups". Preferred perhaloalkyl groups are trifluoroalkyl (-CF)₃). Similarly, "perhaloalkoxy" refers to an alkoxy group in which a halogen is substituted for each hydrogen in the hydrocarbon that makes up the alkyl portion of the alkoxy group. An example of perhaloalkoxy is trifluoromethoxy (-OCF)₃)。

The term "heteroaryl" refers to and includes unsaturated aromatic cyclic groups having from 1 to 10 ring carbon atoms and at least one ring heteroatom (including but not limited to heteroatoms such as nitrogen, oxygen, and sulfur), wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atoms are optionally quaternized. Heteroaryl groups may be attached to the rest of the molecule at a ring carbon or a ring heteroatom. Heteroaryl groups can contain additional fused rings (e.g., 1 to 3 rings), including additional fused aryl, heteroaryl, cycloalkyl, and/or heterocyclyl rings. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, thienyl, furyl, thiazolyl, and the like.

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

"oxo" refers to an ═ O moiety.

"thiocarbonyl" refers to a C ═ S group.

Unless otherwise indicated, "optionally substituted" means that the group may be unsubstituted or substituted with one or more (e.g., 1,2, 3, 4, or 5) of the substituents listed for the group, wherein the substituents may be the same or different. In one embodiment, the optionally substituted group has one substituent. In another embodiment, the optionally substituted group has two substituents. In another embodiment, the optionally substituted group has three substituents. In another embodiment, the optionally substituted group has four substituents. In some embodiments, an optionally substituted group has 1-2, 2-5, 3-5, 2-3, 2-4, 3-4, 1-3, 1-4, or 1-5 substituents.

The term "substituted" means that one or more hydrogen atoms of a moiety is replaced by a monovalent or divalent group. "optionally substituted" means that the moiety may be substituted or unsubstituted. Suitable substituents include, for example, hydroxy, nitro, amino (e.g., -NH)₂Or dialkylamino), imino, cyano, halogen (e.g. F)Cl, Br, I), haloalkyl (e.g. CCl)₃Or CF₃) Thio, sulfonyl, thioamido, amidino (imidino), oxo, oxoamidino (oxamidino), methoxyamidino (methoxamidino), amidino, guanidino, sulfonamido, carboxyl, formyl, alkyl, alkoxy-alkyl, alkylcarbonyl, alkylcarbonyloxy (-OCOR), aminocarbonyl, arylcarbonyl, aralkylcarbonyl, carbonylamino, heteroarylcarbonyl, heteroaralkyl-carbonyl, alkylthio, aminoalkyl, cyanoalkyl, carbamoyl (-NHCOOR-or-OCONHR-), urea (-NHCONHR-), aryl, and the like, wherein R is any suitable group, e.g., alkyl or alkenyl. In some embodiments, as described, the optionally substituted moiety is optionally substituted with only selected groups. In some embodiments, the aforementioned groups (e.g., alkyl) are optionally substituted with, for example, alkyl (e.g., methyl or ethyl), haloalkyl (e.g., -CCl)₃、-CH₂CHCl₂or-CF₃) Cycloalkyl (e.g., -C)₃H₅、-C₄H₇、-C₅H₉) Amino (e.g., -NH)₂Or dialkylamino), alkoxy (e.g., methoxy), heterocycloalkyl (e.g., as morpholine, piperazine, piperidine, azetidine), hydroxy, and/or heteroaryl (e.g., oxazolyl). In some embodiments, the substituents themselves are optionally substituted. In some embodiments, the substituent is itself unsubstituted. The group substituted on the substituent may be, for example, carboxyl, halo, nitro, amino, cyano, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, aminocarbonyl, -SR, thioamido, -SO₃H、-SO₂R or cycloalkyl, wherein R is any suitable group, such as hydrogen or alkyl.

Tertiary amino lipidated and/or pegylated cationic peptide compounds

The tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure are peptide chains comprising N-substituted amino acid residues. The tertiary aminolipidated and/or pegylated cationic peptide compounds of the present disclosure comprise an oligopeptide backbone, wherein the oligopeptide backbone comprises repeating subunits of N-substituted cationic amino acid residues optionally interleaved with N-substituted neutral ("spacer") and/or lipid amino acid residues. The oligopeptide backbone is further terminated at the N-and/or C-terminus by amino acid residues N-substituted with lipid moieties ("N-lipidation") and/or N-substituted with oligo-and/or poly-ethylene glycols ("N-pegylation").

In one aspect, provided herein is a tertiary amino lipidated and/or pegylated cationic peptide compound of formula (I):

in some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds can be characterized by the total number of amino acid residues present in the peptide compound, wherein each amino acid residue is represented by the general structure- (NR-CR)^aR^b-C (O) -represents. 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 to 20 amino acid residues. In certain embodiments, the total number of amino acid residues is m, n, s, t, and [ r × (o + p + q)]And (4) summing.

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 wherein 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 wherein 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., is electrically neutral) or a net positive charge. In some embodiments wherein 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) +.

In certain embodiments, the amino acid residue of the cationic peptide compounds described herein is an N-substituted variant of a naturally occurring amino acid or a non-naturally occurring amino acid, wherein the carbon side chain is formed by R^aAnd R^bAnd (4) showing. The amino acid residues may be present in the D-or L-configuration. Additionally, it is recognized that substitution at the N-position of an amino acid residue may limit the free rotation of the amide bond. Thus, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure may also exist in various rotameric conformations (rotamers).

In some embodiments, each R is^aAnd each R^bIndependently a side chain moiety on a naturally or non-naturally occurring amino acid. The term "naturally occurring amino acid" as used herein 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 an amino acid that does not normally occur in nature, and includes, for example, the D-isomer of a naturally occurring amino acid, 2-aminoadipic acid, 2-aminobutyric acid, norvaline, norleucine, and ornithine. In certain embodiments, R^aAnd R^bEach independently is-H, -CH₃Or

In other embodiments, each R is^aAnd each R^bIndependently is-H or C₁-C₄An alkyl group. In some casesIn embodiments, each R is^aAnd each R^bIndependently is-H or CH₃. In other embodiments, R^aAnd R^bis-H. In which R is^aAnd R^bIn embodiments where the tertiary amino group is lipidated and/or pegylated cationic peptide compound may also be referred to as an N-lipidated and/or pegylated polyglycine compound or an N-lipidated and/or pegylated peptoid compound. In certain embodiments, each R is^aAnd each R^bIndependently is-H, C₁-C₄Alkyl groups or side chain moieties on naturally or non-naturally occurring amino acids.

Oligopeptide backbone or repeat subunits and structural motifs

As described herein, tertiary aminolipidated and/or pegylated cationic peptide compounds are useful for complexation with polyanionic compounds (e.g., nucleic acids), as well as for delivery of such polyanionic compounds into 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 represented by fragments of the following formula (I):

cationic amino acid residues in the repeating subunits of the oligopeptide backbone impart a positive charge to the compounds of the present disclosure, which allows for favorable electrostatic interaction and charge neutralization with polyanionic materials such as nucleic acids. The staggering of neutral or lipidated amino acid residues between cationic residues allows for better control of the spatial distribution of positive charges throughout the tertiary amino lipidated and/or pegylated cationic peptide compounds, which enables improved complexation of the cationic peptide compounds with polyanionic substances having a specific length, charge distribution and/or conformation.

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.

It will be appreciated that each subunit r may or may not be strictly identical to other subunits within the overall tertiary amino lipidated and/or pegylated cationic peptide compound. For example, in tertiary amino lipidated and/or pegylated cationic peptide compounds, each subunit r may comprise a cationic amino acid residue, and optionally also a neutral and/or lipidated amino acid residue, independently selected with respect to the cationic and neutral and/or lipidated amino acid residues present in the other subunits.

Cationic moiety

Each repeating subunit of the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure comprises at least one cationic amino acid residue. The cationic amino acid residue provides a positive charge that enables the peptide compounds described herein to form electrostatic complexes with nucleic acids or other polyanionic compounds by interacting with a negative charge on the nucleic acids or polyanionic compounds. Complexing of nucleic acids partially or completely masks the negative charge of the nucleic acids and facilitates transport through the lipid membrane of the cell and into the interior of the cell.

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

Each cationic amino acid residue comprising a cationic moiety R in the N-position⁵. It will be appreciated that each cationic moiety R⁵It may be independently selected not only within the repeating subunits of the cationic peptide compound, but also throughout the entire oligopeptide backbone.

The cationic moieties described herein can be substituents having a stable net positive charge over 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. Typically, the physiological pH is less than about 8.5, typically less than about 8.0. More typically, the physiological pH is less than about 7.5. The cationic moiety can be characterized, for example, by the threshold pKa value of the functional groups present in the moiety (which is sufficient to generate a positive charge at physiological pH). In certain embodiments, the cationic moiety has a pKa value of at least 7.5. In other embodiments, the cationic moiety has a pKa value between physiological pH and more acidic pH (e.g., pH 4.5-5.5). In further embodiments, substituents having multiple independent pKa values are used.

It will be appreciated that the tertiary amino lipidated and/or pegylated cationic peptide compounds described herein may comprise a plurality of cationic moieties in close proximity to each other along the oligopeptide backbone. When multiple cationic moieties are present along the oligopeptide backbone, the protonated or deprotonated state of certain cationic moieties may affect the pKa values of other cationic moieties in close proximity. By this mechanism, the pKa of a particular cationic moiety in the cationic peptide compounds described herein can be altered relative to its pKa value measured in isolation. For example, protonation of one amine in the oligopeptide backbone (having a pKa of about 8) can reduce the pKa of the nearby cationic moiety from a normal value of about 7.5 to a pKa of about 5-6.

The ability of the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure to accommodate multiple cationic moieties that are in varying proximity to each other enables these cationic peptide compounds and any complexes thereof to react with high sensitivity to changes in physiological pH. The sensitivity of these compounds and their complexes to physiological pH changes may be important to facilitate delivery of nucleic acids and release into the cytosol following endosomal transport (endosomal compartment pH of about 4.5 to 5.5).

The cationic or positively charged moiety may include, for example, nitrogen-based substituents such as those comprising the following functional groups: amino, guanidino, hydrazino, and amidino groups. These functional groups may be aromatic, saturated cyclic or aliphatic. In some embodiments, each R is⁵Independently aminoalkyl, alkylaminoalkyl, aminoalkylaminoalkyl, guanidinoAlkyl, N-heterocyclylalkyl or N-heteroaryl. In other embodiments, each R is⁵Independently is

In certain embodiments, each R is⁵Independently selected from: in still other embodiments, each R is⁵Is that

In still further embodiments in which the cationic residue is a terminal residue of the entire peptide compound, additional cationic moieties R that are not compatible with synthetic or deprotection conditions (e.g., acid-labile linkers) or for which no suitable protecting group strategy exists (e.g., polyamines) may be used⁵. For example, in some embodiments, the cationic moiety R of the terminal cationic residue⁵Is a polyamine. R at the terminal residue therein⁵In some embodiments that are polyamines, the polyamine is In certain embodiments, the polyamine is selected from

In other embodimentsCationic moiety R of the terminal cationic residue⁵Is hydroxyalkyl, hydroxyether, alkoxyalkyl or hydroxyheteroalkyl. In certain embodiments, the cationic moiety R of the terminal cationic residue⁵Is composed of

In a still further embodiment, the cationic moiety R of the terminal cationic residue⁵Is composed of

It will be further appreciated that the nitrogen atom in the peptide chain is unsubstituted (i.e. wherein R is⁵Is hydrogen) can also be used as ionizable cationic moiety under physiological conditions. In some embodiments, the cationic moiety R⁵Is a hydrogen atom.

Neutral spacer moiety

Within the oligopeptide backbone of the tertiary amino lipidated and/or pegylated cationic peptide compounds, the cationic amino acid residues may optionally be interleaved with neutral spacer amino acid residues, thereby having a neutral spacer moiety at the N-position. Neutral amino acid residues may be used to modulate the spatial distribution of positive charges in tertiary amino lipidated and/or pegylated cationic peptide compounds to improve electrostatic interactions with polyanionic compounds (including polynucleotides) to be complexed with the cationic peptide compounds.

In each repeating subunit of the tertiary amino lipidated and/or pegylated cationic peptide compound, a neutral amino acid residue may be taken as one or more R⁴The group is present at the N-terminus or C-terminus of the cationic amino acid residue. In which the subunit R comprises a neutral spacer moiety R⁴In some embodiments, the corresponding o and/or q for each neutral spacer moiety present represents the respective number of neutral spacer residues bonded to the N-and C-termini of the cationic amino acid residues within subunit r. In some embodimentsIn the case, each o is independently an integer of 0, 1 or 2. In other embodiments, each q is independently an integer of 0, 1, or 2.

Each neutral spacer amino acid residue comprising a neutral spacer moiety R at the N-position⁴. As with the cationic moieties described herein, it will be recognized that each neutral spacer moiety R⁴Independently selected within the repeating subunits of the cationic peptide compound and between the repeating subunits r of the oligopeptide backbone.

It will also be appreciated that a neutral spacer moiety may include any substituent that is neutral or has a zero net charge over a physiologically relevant pH range. In some embodiments, each neutral moiety R⁴Independently C substituted by cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl or hydroxyalkyl₁-C₄Alkyl, wherein each cycloalkyl, heterocyclylalkyl, alkylaryl, arylalkyl, alkylheteroaryl, heteroarylalkyl, alkoxy, alkoxyalkyl, or hydroxyalkyl group is optionally substituted with one or more substituents-OH, halogen, or alkoxy. In some embodiments, each neutral spacer moiety R is a cyclic alkyl moiety⁴Is independently selected from In certain embodiments, each neutral spacer moiety R⁴Is that

Lipid fraction

In addition to the optional interleaving 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, N-lipidated amino acid residues (having a lipid moiety at the N-position) may also optionally be interleaved with cationic (and optionally neutral spacer) amino acid residues. In some embodiments, wherein the tertiary amino lipidated and/or pegylated cationic peptide compound comprises N-lipidated amino acid residues, the tertiary amino lipidated and/or pegylated cationic peptide compound is N-lipidated. Like the neutral amino acid residues, N-lipidated amino acid residues within the oligopeptide backbone may be used to modulate the spatial distribution of positive charges in tertiary amino lipidated and/or pegylated cationic peptide compounds, as well as enhance their lipophilicity for improved encapsulation and intracellular delivery of polyanionic materials. The spacing of lipids along the peptoid backbone may also affect lipid mobility/crystallinity, which is known to affect cellular uptake and endosomal release.

As with the neutral spacer residues, in each repeating subunit of the tertiary amino lipidated and/or pegylated cationic peptide compound, the N-lipidated amino acid residue may serve as one or more R⁴The group is present at the N-terminus or C-terminus or both of the cationic amino acid residue. In which the subunit R comprises a lipid moiety R⁴In some embodiments, the respective o and/or q of each lipid moiety present may also represent the respective number of lipidated residues bonded to the N-and C-termini of the cationic amino acid residues within subunit r. In some embodiments, each o is independently an integer of 0, 1, or 2. In other embodiments, each q is independently an integer of 0, 1, or 2.

Each N-lipidated amino acid residue comprising a lipid moiety R in the N-position⁴. As with the cationic and neutral moieties described herein, it will be appreciated that each lipid moiety R⁴Independently selected within the repeating subunits of the cationic peptide compound and between the repeating subunits r of the oligopeptide backbone.

Suitable lipid moieties may 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.

In some embodiments, the lipid moiety may comprise a branched or straight chain aliphatic moiety having from about 6 to about 50 carbon atoms or from about 10 to about 50 carbon atoms, optionally comprising one or more heteroatoms, and optionally comprising one or more double or triple bonds (i.e., saturated or otherwise)Mono-or poly-unsaturated). In certain embodiments, the lipid moiety may comprise an optionally substituted aliphatic, straight or branched chain moiety, 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 include, for example, an aliphatic carbon chain derived from a fatty acid and a fatty alcohol. In some embodiments, each lipid moiety R is a pharmaceutically acceptable salt, solvate, or solvate of the compound⁴Independently is C₈-C₂₄Alkyl or C₈-C₂₄Alkenyl radical, wherein C₈-C₂₄The alkenyl group is optionally mono-or polyunsaturated. In some embodiments, each lipid moiety R is a pharmaceutically acceptable salt, solvate, or solvate of the compound⁴Is C₆-C₁₈Alkyl or C₆-C₁₈An alkenyl group. In certain embodiments, each lipid moiety R is a pharmaceutically acceptable salt, solvate, or solvate of the compound⁴Is C₈-C₁₂An alkyl group. In still other embodiments, each lipid moiety R⁴Is C₁₀Alkyl groups, such as n-decyl. In other embodiments, each lipid moiety R⁴Independently selected from 2-ethylhexyl-1-yl, hexenyl, oleyl, stearyl, linoleyl, myristyl and lauryl.

In still other embodiments that may be combined with any of the preceding embodiments, each lipid moiety R⁴Independently is In still other embodiments that may be combined with any of the preceding embodiments, each lipid moiety R⁴Independently of formula (II) Wherein R is⁸Is of from about 6 to about 50 carbon atomsOr a branched or straight chain aliphatic moiety of from about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds. In certain embodiments, each lipid moiety R is a pharmaceutically acceptable salt, solvate, or solvate of the compound⁴Independently is

The natural lipid fraction used in the practice of the present invention may be derived from, for example, phospholipids, glycerides (e.g., di-or tri-glycerides), glycoglycerides, sphingolipids, ceramides, and saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

Other suitable lipid moieties may include lipophilic carbocyclic or aromatic groups, such as optionally substituted aryl, cycloalkyl, cycloalkylalkyl or arylalkyl moieties, including for example naphthyl or ethylbenzyl, or lipids containing an ester functional group, including for example sterol esters and wax esters. In still other embodiments, the lipid moiety R⁴Is that

Structural motifs

In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure can comprise a specific sequence or arrangement of cationic, neutral spacer, and lipidated amino acid residues relative to each other, which can be described similarly to the various classifications of linear copolymers (random, block, alternating, periodic, stereoblock, etc.).

For example, cationic peptide compounds in which the tertiary amino group is lipidated and/or pegylated comprise a generic moiety R^cation、R^neutralAnd R^lipidIn some embodiments, the amino acid residues may be arranged in a random sequence, an alternating sequence, or a block sequence. It is to be understood that R^cationAnd R^neutralAre respectively equivalent to containing a cationic moiety R⁵And a neutral moiety R⁴The amino acid residue of (1). Cationic peptidation depending on the lipidation of N-lipidated residues at tertiary amino groups and/or pegylationPosition in the compounds, generic part R^lipidCan be understood to include having a lipid moiety R⁴And a lipid moiety R³Both amino acid residues of (1).

An example of an alternating sequence of cationic and neutral spacer amino acid residues can be represented by R^cation-R^neutral-R^cation-R^neutralOr R^neutral-R^cation-R^neutral-R^cationAnd (4) showing. Another example of an alternating sequence of cationic and lipidated amino acid residues may be represented by R^cation-R^lipid-R^cation-R^lipidOr R^neutral-R^cation-R^neutral-R^cationicAnd (4) showing. Some examples of block sequences may be represented by R^cation-R^cation-R^cation-R^neutral-R^neutral-R^neutral、R^neutral-R^neutral-R^neutral-R^cation-R^cation-R^cation、R^cation-R^cation-R^cation-R^lipid-R^lipid-R^lipidOr R^lipid-R^lipid-R^lipid-R^cation-R^cation-R^cationAnd (4) showing. In still other embodiments, the cationic moiety, neutral spacer moiety, and lipidating moiety may be arranged in block sequences and alternating sequences of different repeating subunit fragments throughout the oligopeptidic compound. For example, in some embodiments, repeating motifs of larger oligomeric units, such as R, may be present within the cationic peptide compound^cation-R^neutral-R^neutral-R^cation-R^neutral-R^neutral. In other embodiments, the cationic peptide compound may comprise blocks of N-lipidated residues in combination with blocks of alternating cationic and neutral residues.

As described herein, tertiary amino lipidated and/or pegylated cationic peptides of the present disclosure comprise at least one peptide having a cationic moiety R⁵A cationic amino acid residue of (a). In some embodiments, the backbone of the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a "cationSubdomains "or" cationic blocks ". A cationic domain is to be understood broadly as having multiple cationic moieties R within the oligopeptide chain⁵(e.g., at least two cationic residues) of a sequence of amino acid residues. In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a cationic domain, wherein the cationic domain comprises at least two, at least three, or at least four cationic amino acid residues.

The cationic domain may include, for example, a plurality of R's in a continuous linear sequence⁵Cationic moiety (e.g., R)⁵R⁵R⁵Or R^cationR^cationR^cation) The amino acid residue block of (a). In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a "cationic domain", wherein the tertiary amino lipidated and/or pegylated cationic peptide compound comprises at least two, at least three, or at least four consecutive cationic peptide compounds having R⁵Amino acid residues of the cationic moiety.

In other embodiments, a "cationic domain" can include a block of adjacent amino acid residues, wherein a plurality of cationic residues have an additional non-cationic (i.e., neutral or lipidated) residue spaced between two cationic residues, such that no one cationic residue within the block is directly bonded to another cationic residue. For example, in some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a "cationic domain", wherein the cationic domain comprises at least two moieties R having a cation⁵And wherein each of at least two cationic amino acid residues within the cationic domain are substituted with at least one or at least two neutral spacer or lipid moieties R⁴Are separated. In some embodiments, the non-cationic residues are staggered between two cationic residues within a cationic domain at regularly spaced intervals of uniform length. In some embodiments in which the cationic domain comprises non-cationic residues that are staggered between cationic residues, the cationic domain can be described as a submonous comprising repeats (dimers, trimers, tetramers, etc.)A meta domain. These subunits may include, but are not limited to-R^cationR^neutral-、-R^cationR^neutralR^neutral-、-R^cationR^lipid-、-R^cationR^lipidR^lipid-、-R^cationR^neutralR^lipid-or-R^cationR^lipidR^neutral-. In other embodiments, the non-cationic residues are interspersed between two cationic residues within a cationic domain, with a different number of non-cationic residues 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, wherein the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a cationic domain, each R is a hydroxyl group⁵Is composed ofIn other embodiments in which the tertiary amino lipidated and/or pegylated cationic peptide comprises a cationic domain, each cationic moiety R⁵Is composed ofAnd each neutral spacer base portion isIn certain embodiments in which the tertiary amino lipidated and/or pegylated cationic peptide comprises a cationic domain comprising one or more dimer or trimer subunits, each cationic moiety R is a cationic moiety⁵Is composed ofAnd each neutral spacer base portion isIn still other embodiments, the tertiary amino lipidated and/or pegylated cationic peptide packageComprising a monomer containing one or more trimeric subunits-R^cationR^neutralR^neutralA cationic domain of (A), each cationic moiety R⁵Is composed ofAnd each neutral spacer base part

As described herein, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure can comprise one or more distinct cationic moieties (R)^cation1、R^cation2、R^cation3Etc.), one or more different neutral spacer moieties (R)^neutral1、R^neutral2、R^neutral3Etc.) and/or one or more different lipid moieties (R)^lipid1、R^lipid2、R^lipid3Etc.). It will be appreciated that the above examples of sequences or arrangements of generic cations and neutral spacer amino acid residues are not intended to be limiting to the peptide compounds of the present disclosure. The cationic, neutral spacer and lipid moieties (which may be present as random, alternating or block sequences as generally described above) may also be present as specific sequences of individual cationic, neutral and lipid moieties within a larger block or alternating structural motif. For example, some exemplary residue permutations may include, but are not limited to, the following: r^cation1R^{neut ral1}R^neutral1R^lipid1R^cation1R^neutral1R^neutral1R^lipid1R^cation1R^neutral1R^neutral1、R^cation1R^neutral1R^neutral1R^{c ation1}R^neutral1R^neutral1R^cation1R^neutral1R^neutral1R^lipid1R^lipid1、R^lipid1R^cation1R^neutral1R^lipid1R^cation1R^{neu tral1}-R^lipid2R^cation2R^neutral2R^lipid2R^cation2R^neutral2、R^lipid1R^lipid2R^lipid3R^lipid4R^neutral1R^neutral2R^{catio n1}R^neutral1R^neutral2R^lipid1R^lipid2R^lipid3R^lipid4 or R^lipid1R^lipid2R^lipid1R^lipid2R^cation1R^neutral1R^cation1R^{ne utral1}R^lipid1R^lipid2R^lipid1R^lipid2。

Side chain functionalized and peptide conjugated compounds

As described above, the oligopeptide backbone comprises predominantly a moiety having side chains (R) present on naturally or non-naturally occurring amino acids^aAnd R^b) And a cationic, neutral spacer or lipid moiety (R) at the N-position⁴And R⁵) The amino acid residue of (1).

In one aspect, R of the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure^a、R^b、R⁴And R⁵The group may further comprise a reactive linking group that may be used to covalently bond to a therapeutic agent and/or a targeting element (such as a small molecule or antibody). These additional functional groups may also comprise cationic groups that are incompatible with synthesis or deprotection conditions (e.g., acid-labile linkers) or for which no suitable protecting group strategy exists (e.g., polyamines). For example, the reactive group may be attached to an existing side chain R by chemical methods known in the art^aAnd/or R^bOr is attached to R⁵Cation moiety, R at N-position⁴Neutral spacer or lipid moiety. Or, if R has the desired reactive moiety^a、R^b、R⁴Or R⁵Groups are commercially available as the corresponding free amines, which can then be incorporated during general synthesis of the peptide chain (e.g., during the subunit synthesis).

In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide comprises one or more amino acid residues comprising a reactive group or a linking group. Suitable reactive groups may include, but are not limited to, esters, amides, isocyanates, thiols, or "click" chemically compatible moieties (e.g., azido, alkynyl).

In another aspect, the present disclosure further provides tertiary aminolipidated and/or pegylated cationic peptide compounds, wherein the peptide compounds are covalently bound or conjugated to a therapeutic agent and/or targeting element. In certain embodiments, the therapeutic agent and/or targeting element is a small molecule, peptide sequence, antibody or antibody fragment, aptamer, monosaccharide or oligosaccharide (e.g., galactose), or glycan.

Linking groups or linkages that are sensitive to or labile under particular physiological conditions may help achieve targeted delivery to a particular organ or cell, followed by selective release of the therapeutic agent and/or polyanionic material into the cell (release in the cell). In addition, labile linkages may also facilitate the elimination and clearance of tertiary amino lipidated and/or pegylated cationic compounds from cells (or organs or the whole body) following polyanionic material delivery. In certain embodiments, the linking 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 labile, chemically labile, pH labile, photolabile, thermally labile, or enzymatically cleavable.

Terminal (capping) residue and protecting end group

As described herein, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure comprise an oligopeptide backbone that is terminated at the N-terminus and/or C-terminus by an amino acid residue that is N-substituted with a lipid moiety ("N-lipidated") and/or N-substituted with an oligo-and/or polyethylene glycol ("N-pegylated"). N-lipidation of the cationic peptide compound confers advantageous lipophilicity to the compound and to any complexes formed between the compound and the polyanionic material. N-pegylation provides better control of particle formation and aggregation in vivo. The terminal amino and carboxylic acid moieties at the N-and C-termini may be further capped with protecting end groups.

As described herein, 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, 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 compounds of the present disclosure comprise 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.

Lipid fraction

As mentioned above, the tertiary amino lipidated and/or pegylated cationic peptide compounds may comprise amino acid residues at the N-terminus and/or C-terminus, wherein the amino acid residue is N-substituted with a lipid moiety, or "N-lipidated". 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 increased lipophilicity of the cationic peptide compound enhances its affinity for hydrophobic environments, such as the lipid bilayer of a cell membrane, thus increasing the propensity of the tertiary amino lipidated and/or pegylated cationic peptide compound and any complex thereof with the polyanionic compound to be transported into the cell.

In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound is N-lipidated. In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure comprise an N-lipidated amino acid residue at the N-terminus. In other embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure comprise an N-lipidated amino acid residue at the C-terminus. In certain embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure comprise N-lipidated amino acid residues at the N-terminus and the C-terminus.

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.

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 of 0, 1,2, 3, 4, 5, 6, or 7. In still other embodiments, n is an integer of 1,2, 3, or 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 of 0, 1,2, 3, 4, 5, 6, or 7. In still other embodiments, s is an integer of 1,2, 3, or 4. In some embodiments, wherein the tertiary amino lipidated and/or pegylated cationic peptide compound is N-lipidated, at least one of N or s is non-zero. In some embodiments, n is non-zero. In other embodiments, s is non-zero. In certain embodiments, both n and s are non-zero.

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, optionally 2,3, or 4.

In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a block of N-lipidated residues, or "N-lipid block", wherein 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 each other (e.g., R)^lipidR^lipidR^lipid). In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a block of N-lipidated residues, wherein N is at least 2, at least 3, or at least 4. In other embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a block of N-lipidated residues, wherein s is at least 2, at least 3, or at least 4.

The N-lipidated amino acid residues in the tertiary amino lipidated and/or pegylated cationic peptides of the present disclosure are covered by a lipid moiety R³N-substitution. The lipid moieties of the present disclosure may include hydrophobic or lipophilic moieties that are neutral (i.e., have no charge or have a net charge of zero). The lipid moiety of the tertiary amino lipidated and/or pegylated cationic compounds described herein may be naturally or synthetically derived. Each R³Independently a lipid moiety, which may be the same or different. In some embodiments, each R is³Are the same. In other embodiments, each R is³Is different.

It will be appreciated that particular sequences or arrangements of N-lipidated amino acid residues may be particularly useful for improving complexing with and delivery of nucleic acids. For example, in which the tertiary amino lipidated and/or pegylated cationic compound comprises a group of two different lipid moieties R^3aAnd R^3bOne of the embodiments of mixed N-lipidated amino acid residues, the N-lipidated amino acid residues may be arranged in an alternating or block sequence at the N-terminus or C-terminus. An example of an alternating sequence of N-lipidated amino acid residues can be represented by R^3a-R^3b-R^3a-R^3bOr R^3b-R^3a-R^3b-R^3aAnd (4) showing. Examples of block sequences may be represented by R^3a-R^3a-R^3b-R^3bOr R^3b-R^3b-R^3a-R^3aAnd (4) showing. In other embodiments, the sequence of N-lipidated amino acid residues may be randomly ordered. It will be appreciated that the above examples of sequences or arrangements of two N-lipidated amino acid residues are not intended to be limiting. In addition, the tertiary aminolipidated and/or pegylated cationic peptide compounds of the present disclosure can comprise two or more different R' s³A lipid moiety, which may be present in a random, alternating or block sequence as generally described above.

Suitable lipid moieties may 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.

In some embodiments, the lipid moiety may comprise a branched or straight chain aliphatic moiety having from about 6 to about 50 carbon atoms or from about 10 to about 50 carbon atoms, optionally comprising one or more heteroatoms, and optionally comprising 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 or branched chain moiety, 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 include, for example, an aliphatic carbon chain derived from a fatty acid and a fatty alcohol. In some embodiments, each R is³Independently is C₈-C₂₄Alkyl or C₈-C₂₄Alkenyl radical, wherein C₈-C₂₄The alkenyl group is optionally mono-or polyunsaturated. In some embodiments, each R is³Is C₆-C₁₈Alkyl or C₆-C₁₈An alkenyl group. In certain embodiments, each R is³Is C₈-C₁₂An alkyl group. In still other embodiments, each R is³Is C₁₀Alkyl groups, such as n-decyl. In some embodiments, each R is³Independently selected from 3-ethylhexyl-1-yl, octanoyl, hexanoyl, oleyl, stearyl, linoleyl, myristyl and lauryl. In other embodiments, each R is³Independently selected from oleyl, stearyl, linoleyl, myristyl and lauryl.

In still other embodiments that may be combined with any of the preceding embodiments, each R³Independently is In still other embodiments that may be combined with any of the preceding embodiments, each R³Independently of formula (II) Wherein R is⁸Is a branched or straight chain aliphatic moiety having from about 6 to about 50 carbon atoms or from about 10 to about 50 carbon atoms, optionally containing one or more heteroatoms, and optionally containing one or more double or triple bonds. In certain embodiments, each R is³Independently is

The natural lipid fraction used in the practice of the present invention may be derived from, for example, phospholipids, glycerides (e.g., di-or tri-glycerides), glycoglycerides, sphingolipids, ceramides, and saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

Other suitable lipid moieties may include lipophilic carbocyclic or aromatic groups such as optionally substituted aryl, cycloalkyl, cycloalkylalkyl or arylalkyl moieties including, for example, naphthyl or ethylbenzyl, or lipids containing an ester functional group including, for example, sterol esters and wax esters. In still other embodiments, the lipid moiety R⁴Is that

Ethylene glycol moiety

The tertiary aminolipidated and/or pegylated cationic peptides of the present disclosure may comprise a capped amino acid residue N-substituted with an oligomer or polymer of ethylene glycol (i.e., N-substituted with an oligomeric ethylene glycol and/or polyethylene glycol) at the N-terminus and/or C-terminus. Incorporation of oligo-and/or polyethylene glycol moieties into the tertiary amino lipidated and/or pegylated cationic peptide compounds described herein can promote particle stability of the complex formed with the nucleic acid and prevent particle aggregation in vivo.

It will be appreciated that the term "pegylated" is used herein to describe cationic peptide compounds comprising terminal amino acid residues that can be N-substituted with an oligoethylene glycol, a polyethylene glycol, or a combination thereof. In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds provided herein are N-pegylated.

In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises N-pegylated amino acid residues at the N-terminus, as shown in the following fragment of formula (I):

in certain embodiments wherein the tertiary amino lipidated and/or pegylated cationic peptide compound comprises N-pegylated amino acid residues at the N-terminus, m represents the number of N-pegylated amino acid residues at the N-terminus, and each R represents²Independently is of formula-CH₂CH₂O(CH₂CH₂O)_uR^2aWherein R is^2ais-H or C₁-C₄An alkyl group. In some embodiments, R^2ais-H, -CH₃Or CH₂CH₃. In certain embodiments, R^2ais-H. In other embodiments, R^2ais-CH₃. In still other embodiments, R^2ais-CH₂CH₃。

In some embodiments, m is an integer from 0 to 10, an integer from 0 to 3, or an integer from 4 to 10. In some embodiments, each u is independently 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.

In certain embodiments, m is an integer from 0 to 3, and each u is an integer from 20 to 200, or optionally an integer 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 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:

in certain embodiments wherein 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 represents⁶Independently is of formula-CH₂CH₂O(CH₂CH₂O)_vR^6aWherein R is^6ais-H or C₁-C₄An alkyl group. In some embodiments, R^6ais-H, -CH₃Or CH₂CH₃. In certain embodiments, R^6ais-H. In other embodiments, R^6ais-CH₃. In still other embodiments, R^6ais-CH₂CH₃。

In some embodiments, t is an integer from 0 to 10, an integer from 0 to 3, or an integer from 4 to 10. In some embodiments, each u is independently 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.

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 some embodiments, wherein the tertiary amino lipidated and/or pegylated cationic peptide compound is N-pegylated, at least one of m or t is non-zero. In some embodiments, m is non-zero. In other embodiments, t is non-zero. In certain embodiments, m and t are both non-zero.

N-lipidated and/or N-pegylated

As described herein, tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure can comprise amino acid residues that are N-lipidated and/or N-pegylated. The cationic peptide compounds provided herein comprise at least one amino acid residue that is N-lipidated or N-pegylated. In some embodiments of the tertiary amino lipidated and/or pegylated cationic peptide compounds, at least one of m, n, s, or t is non-zero.

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 non-zero. In certain embodiments, both n and s are non-zero. In still other embodiments, the tertiary aminolipidated and/or pegylated cationic peptide compound of formula (I) is a tertiary aminolipidated cationic peptide compound of formula (Ia):

wherein at least one of n and s is non-zero, and wherein R¹、R³、R⁴、R⁵、R⁷、R^a、R^bO, p, q and r are as defined for formula (I). Certain embodiments of the compounds of formula (Ia)In embodiments, n and s are both non-zero.

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 non-zero. In certain embodiments, m and t are both non-zero. 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):

wherein at least one of m and t is non-zero, and wherein R¹、R²、R⁴、R⁵、R⁶、R⁷、R^a、R^bO, p, q and r are as defined for formula (I). In certain embodiments of the compounds of formula (Ib), m and t are both non-zero.

In still other embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure comprise 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 wherein 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 non-zero, and at least one of n and s is non-zero.

In a still 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):

wherein at least one of m and t is non-zeroAnd at least one of n and s is non-zero, and wherein R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R^a、R^bO, p, q and r are as defined for formula (I).

In other embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds of the present disclosure may also comprise N-substituted amino acid residues with other neutral shielding polymers or moieties at the N-terminus or C-terminus that provide similar shielding effects as the N-pegylated residues. These may include, for example, hydroxyalkyl groups, hyaluronic acid, polysaccharides, polyphosphates and polyphosphoesters, poly (vinylpyrrolidone), polyols, hydrophilic polypeptides, or other examples of synthetic hydrophilic polymers.

N-and C-terminal end groups

The tertiary aminolipidated and/or pegylated cationic peptide compounds of the present disclosure may have an N-terminus and a C-terminus in their free amine and free acid forms or in protected form, respectively. In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound further comprises a protecting or terminal capping group at the N-terminal and/or C-terminal residue.

In some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compounds comprise a protecting or terminal end-capping group R at the N-terminal residue¹. In some embodiments, R¹is-H, alkyl, alkylaryl, -COR^1a、-CH₂-COR^1aA cationic moiety, a lipid moiety or an oligoethylene glycol or polyethylene glycol moiety, wherein R^1ais-H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, -O-alkylaryl or a lipid moiety. In certain embodiments, R¹is-H, alkyl, alkylaryl, -COR^1aOr a lipid moiety, wherein R^1ais-H, -OH, alkyl, aryl, alkylaryl, -O-alkyl, or-O-alkylaryl. In which R is¹In certain embodiments that are lipid moieties, the lipid moiety is not a phospholipid. In which R is¹Is alkyl or-COR^1aIn still other embodiments, wherein R is^1aIs alkyl, optionally substituted with-OH or halogen.

In other embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound comprises a protecting or terminal end-capping group R at the C-terminal residue⁷. In some embodiments, R⁷is-H, alkyl, acyl, -OH, -OR^7a、-NH₂、-NHR^7aA cationic moiety, a lipid moiety or an oligoethylene glycol or polyethylene glycol moiety, wherein R^7aIs an alkyl, acyl or lipid moiety. In some embodiments, R⁷is-H, alkyl, acyl, -OH, -OR^7a、-NH₂、-NHR^7aOr a lipid moiety, wherein R^7aIs an alkyl, acyl or lipid moiety. In which R is⁷In certain embodiments that are lipid moieties, the lipid moiety is not a phospholipid.

In still further embodiments, the N-and C-termini of tertiary amino lipidated and/or pegylated cationic peptide compounds may be selected or further modified such that R¹And R⁷With additional functional groups, such as reactive linking groups.

In some embodiments, R of the tertiary amino lipidated and/or pegylated cationic peptide¹And/or R⁷Independently comprise one or more reactive linking groups. Suitable reactive groups may include, but are not limited to, esters, amides, isocyanates, thiols, or "click" chemically compatible moieties (e.g., azido, alkynyl).

The reactive linking group can then be used to covalently bind other useful compounds (including targeting elements or therapeutic agents) to the peptide compound. In some embodiments, the tertiary aminolipidated and/or pegylated cationic peptide compound is covalently bound or conjugated to a therapeutic agent and/or targeting element. In certain embodiments, the therapeutic agent and/or targeting element is a small molecule, antibody or antibody fragment.

In addition, for R¹And R⁷May also contain cationic groups that are incompatible with the synthesis or deprotection conditions (e.g. acid-labile linkers) or for which no access is availableSuitable protecting group strategies are cationic groups (e.g., polyamines). In still further embodiments, R¹Is a polyamine. In which R is¹In some embodiments that are polyamines, the polyamine is In certain embodiments, the polyamine is selected from

As described above, a linker or linkage that is sensitive to or labile under a particular physiological condition environment may facilitate targeted delivery of the polyanionic material to a particular cell and improve a particular pharmacokinetic property such as elimination. In certain embodiments, the linking 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 labile, chemically labile, pH labile, photolabile, thermally labile, or enzymatically cleavable.

Synthesis of tertiary amino lipidated and/or pegylated cationic peptide compounds

In another aspect, provided herein are methods of making the tertiary amino lipidated and/or pegylated cationic peptide compounds described herein.

Existing lipid-like constructs are typically limited to direct lipid conjugation at the N-terminus, or the use of reactive linking groups on the side chains of the residues to indirectly conjugate the lipid moiety to the oligopeptidic compound. Furthermore, for lipidoids, modifications are only made to conjugate lipids to oligopeptides when the oligopeptides are fully synthesized. The tertiary aminolipidated and/or pegylated cationic peptide compounds of the present disclosure can be synthesized without the need for additional linker species to conjugate the lipid moiety to the oligopeptide core. For the tertiary aminolipidated and/or pegylated cationic peptide compounds described herein, the lipid and ethylene glycol moieties are covalently and directly bound to a nitrogen atom within the peptide itself, that is, at the amide nitrogen or N-position in the amino acid residue. Thus, tertiary amino lipidated and/or pegylated cationic peptides can be synthesized entirely by methods known in the art for producing N-substituted residues in the peptide chain.

Tertiary amino lipidated and/or pegylated cationic peptide compounds can be prepared by a series of acylation (amidation) and nucleophilic substitution (amination) reactions for each amino acid residue to be added, whether or not 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 a peptide chain. The sequential addition of residues may be repeated until the desired amino acid sequence and length is achieved.

The compounds of the invention can be synthesized by solid and liquid phase methods. Exemplary methods for preparing N-substituted peptides (including tertiary amino lipidated and/or pegylated cationic peptide compounds described herein) by solid phase synthesis are discussed below and shown in FIGS. 3A-3E.

As shown in fig. 3A, a solid resin support with a terminal secondary amine is provided at the beginning of the synthesis. The acylating agent is added to the terminal secondary amine 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 to facilitate amidation and subsequent amination at the alpha-carbon. In certain embodiments, the acylating agent is a haloacetic acid. In other embodiments, the acylating agent is bromoacetic acid.

In FIG. 3B, the acylation product produced in FIG. 3A is reacted with a desired substituted primary or secondary amine to provide the corresponding N-substituted terminal amino acid residue. The primary or secondary amine selected replaces the leaving group on the alpha-carbon, such as bromine in bromoacetic acid, to produce the corresponding aminated product. In some embodiments, the primary or secondary amine is selected from NHR^pR²、NHR^pR³、NHR^pR⁴、NHR^pR⁵And NHR^pR⁶Wherein R is^pis-H or a protecting group, and wherein R²、R³、R⁴、R⁵And R⁶As defined for the tertiary amino lipidated and/or pegylated cationic peptide compounds described herein.

The amidation and amination reactions are repeated in series (fig. 3C) until the desired peptide sequence is obtained (fig. 3D). It will be appreciated that the methods of preparing tertiary amino lipidated and/or pegylated cationic peptide compounds described herein may further comprise protection and deprotection steps to prevent any undesired reaction with reactive moieties in the peptide chain during the sequential amidation/amination reactions. For example, a protecting group R^pMay be added during synthesis to the side chain of the alpha-carbon and/or N-substituent at any position along the peptide compound described herein. Suitable protecting groups R^pAny protecting group known in the art may be included, particularly those suitable for peptide synthesis in orthogonal protection schemes, such as Boc/Bzl or Fmoc/tBu.

N-and C-terminal (R)¹And R⁷) Incorporating terminal groups and/or amino acid residues (R) along the oligopeptide backbone^a、R^b、R⁴、R⁵) The functionalization of the side chain of (a) can be performed by methods known in the art. One of ordinary skill in the art will recognize that the timing of selecting an appropriate method and additional functionalization steps relative to the overall solid phase synthesis depends on the end group and/or linking group to be added, as well as the compatibility of the method with other moieties and/or protecting groups present on the peptide compound.

Depending on any protection scheme used in the above synthesis, 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 (fig. 3E). Cleavage of the tertiary amino lipidated and/or pegylated cationic peptide compound from the solid resin support yields the corresponding free cationic peptide compound in solution.

Further steps may be taken to isolate and purify the cationic peptide compound from the solution, including, for example, filtering the peptide-containing solution from a solid resin support and freeze drying the isolated filtrate to provide a solid product.

Due to the acidic conditions used to effect resin cleavage, it will be appreciated that tertiary amino lipidated and/or pegylated cationic peptide compounds may exist in the form of the corresponding acidic salts. For example, in some embodiments, the tertiary amino lipidated and/or pegylated cationic peptide compound is in the form of a salt thereof. 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 salt (hydrochloric acid addition salt), a hydrobromide salt (hydrobromic acid addition salt), or a trifluoroacetate salt (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 trifluoroacetate salt of the tertiary amino lipidated and/or pegylated cationic peptide compound.

The acid addition salt forms of the tertiary amino lipidated and/or pegylated cationic peptide compounds can be further modified by methods in the art (e.g., ion exchange) to obtain one or more pharmaceutically acceptable salt forms. The phrase "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the subject to which the formulation is administered. The term "pharmaceutically acceptable acid addition salts" can include, but is not limited to, those pharmaceutically acceptable salts formed with inorganic acids (e.g., hydrochloric, hydrobromic, hydroiodic, sulfuric, nitric, carbonic, phosphoric) and organic acids selected from the aliphatic, cycloaliphatic, aromatic, aryl-aliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids (e.g., formic, acetic, propionic, gluconic, lactic, pyruvic, oxalic, maleic, malonic, succinic, fumaric, tartaric, citric, aspartic, ascorbic, glutamic, benzoic, phenylacetic, methanesulfonic "methanesulfonate", ethanesulfonic, p-toluenesulfonic and salicylic).

In some embodiments, the salt of the tertiary amino lipidated and/or pegylated cationic peptide compound is a pharmaceutically acceptable salt. In certain embodiments, the salt is a 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.

Complexes of tertiary amino lipidated and/or pegylated cationic peptides with polyanionic compounds, formulations thereof and methods of preparing complexes and formulations thereof

Complexes of tertiary amino lipidated and/or pegylated cationic peptides with polyanionic compounds

As described herein, tertiary aminolipidated and/or pegylated cationic peptide compounds of the present disclosure can be used for complexation with one or more polyanionic compounds (e.g., nucleic acids). Complexes of tertiary amino lipidated and/or pegylated cationic peptide compounds with polyanionic compounds can be used to deliver polyanionic compounds to the interior of cells with improved efficiency compared to complexes formed with other polycationic constructs (e.g., lipid 1).

In one aspect, the present disclosure provides a complex comprising one or more tertiary aminolipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof, as described herein, complexed with one or more polyanionic compounds. In some embodiments, the one or more polyanionic compounds comprise a nucleic acid. Nucleic acids as used herein include naturally occurring nucleic acids, such as DNA, RNA, and/or hybrids thereof, as well as non-naturally occurring variants having non-natural backbones and modified backbone linkages, such as phosphorothioates, non-natural and modified bases, and non-natural and modified termini. Exemplary nucleic acids include genomic DNA, cDNA, mRNA, miRNA, and siRNA.

The nucleic acid may be a recombinantly produced or chemically synthesized molecule. Nucleic acids can be single-stranded, double-stranded, triple-stranded, and quadruple-stranded, as well as more complex three-dimensional forms (including single-stranded and double-stranded regions).

The length of a nucleic acid (suitably defined in nucleotide units or base pairs (bp)) may vary depending on the type of nucleic acid. In some embodiments, wherein the nucleic acid is mRNA, the mRNA can have 100 to 10,000 nucleotide units or 1,000 to 3,000 nucleotide units. In other embodiments where the nucleic acid is DNA, the DNA may have between 5,000bp and 20,000bp, or about 10,000 bp.

In some embodiments where the nucleic acid is mRNA, the mRNA is mRNA encoding a protein or peptide. In some embodiments wherein the nucleic acid is mRNA, the mRNA is mRNA encoding a peptide (including an oligopeptide or polypeptide). In certain embodiments, the mRNA is an mRNA encoding a polypeptide. In yet a further embodiment, the mRNA is an mRNA encoding a protein. In other embodiments, the mRNA is an mRNA encoding a peptide. As described above, mRNA can be naturally occurring (e.g., isolated tumor RNA), or can be synthetic (e.g., produced by in vitro transcription). For synthetic or non-naturally occurring mRNA variants, the mRNA may comprise a non-natural backbone with modified backbone linkages 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 may comprise a specific sequence, such as a self-amplifying sequence or an internal ribosome entry site.

In some embodiments, the combined delivery of two or more specific nucleic acids together may be particularly useful for therapeutic applications. For example, in some embodiments, the one or more polyanionic compounds include a combination of sgrnas (single guide RNAs) as CRISPR sequences and mrnas encoding Cas 9. In yet further embodiments, the nucleic acid may also be complexed with a protein, for example with a CRISPR/Cas9 ribonucleoprotein complex.

In some embodiments, the one or more polyanionic compounds may include non-nucleic acid anions or polyanionic compounds. 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 a Cas9 protein. In other embodiments, the one or more polyanionic compounds include a polyphosphate. In still other embodiments, the one or more polyanionic compounds include heparin or other glycosaminoglycan derivatives.

Although tertiary amino lipidated and/or pegylated cationic peptides are particularly suitable for carrying and delivering polyanionic loads into cells, it is recognized that tertiary amino lipidated and/or pegylated cationic peptide compounds described herein can also be used to form complexes with other non-anionic agents or loads (including hydrophobic compounds) for delivery into cells. Such other loads may include, but are not limited to, for example, one or more small molecule active agents or drug substances (as a sole therapeutic agent or in combination with another agent (e.g., a nucleic acid)) and/or an immunological adjuvant. It should be further appreciated that these other load molecules may or may not be combined with any of the polyanionic compounds described herein for complexing. In some embodiments, a complex described herein comprises an endosomal escape modulator, a TLR agonist, and a chemotherapeutic agent. In other embodiments, the complexes of the present disclosure comprise an adjuvant or immune co-stimulant. For example, in some embodiments, the complex comprises an immunological adjuvant selected from the group consisting of CpG Oligodeoxynucleotides (ODNs), Lipopolysaccharides (LPS), and any combination thereof.

The complexes described herein can be characterized by the ratio of the number of cationic groups on the tertiary amino lipidated and/or pegylated cationic peptide to the number of anionic phosphate groups on the nucleic acid. In some embodiments, the complexes comprise cationic peptides and nucleic acids that are tertiary amino lipidated and/or pegylated at a cationic to 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, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5: 1. In certain embodiments, the complexes comprise a tertiary amino lipidated and/or pegylated cationic peptide and a nucleic acid in a cationic to anionic charge ratio of 2:1 to 5: 1. In still other embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide and nucleic acid having a cationic to anionic charge ratio of 3: 1.

Alternatively, the complexes described herein can be characterized by the relative mass ratio of the tertiary amino lipidated and/or pegylated cationic peptide compound to the polyanionic compound and/or other loading in the complex. The mass ratios of the components in the composite can be readily calculated based on the known concentrations and volumes of the component stock solutions used to prepare the composite. Furthermore, if a non-anionic load is present in the complex, the mass ratio can provide a more accurate representation of the relative amount of tertiary amino lipidated and/or pegylated cationic peptide compound to total load compared to the cationic to anionic charge ratio, which does not take into account the non-anionic material.

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

In certain embodiments wherein the complex comprises a nucleic acid, the complex comprises a tertiary aminolipidated and/or pegylated cationic peptide and nucleic acid 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 complex comprises a tertiary amino lipidated and/or pegylated cationic peptide and a nucleic acid in a mass ratio of 2:1 to 5: 1. In still other embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide and nucleic acid in a mass ratio of 3: 1.

The complexes of the present disclosure comprise at least one tertiary amino lipidated and/or pegylated cationic peptide compound complexed with at least one polyanionic compound or other suitable carrier compound. In some embodiments, the complexes described herein may comprise 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 enable 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, percent encapsulation, transfection efficiency, plasma half-life, and the like. Different applications may require different combinations of these properties.

For example, in certain embodiments, the complex may comprise a combination of one or more cationic cation-rich tertiary amino lipidated and/or pegylated cationic peptide compounds and one or more highly lipidated tertiary amino lipidated and/or pegylated cationic peptide compounds. Theoretically, this combination could confer improved delivery of polyanionic compounds by providing greater charge stability (by the cation rich peptide compound) and greater lipophilic shielding (by the lipidated peptide compound). It will further be appreciated that the individual amounts of each of the individual tertiary amino lipidated and/or pegylated cationic peptide compounds can be adjusted to achieve the desired properties. In some embodiments, the complex may comprise a further component in addition to the tertiary amino lipidated and/or pegylated cationic peptide compound and the polyanionic compound.

Complexes of tertiary amino lipidated and/or pegylated cationic peptides and other lipid components with polyanionic compounds and compositions thereof

The present disclosure further provides compositions comprising complexes, wherein the complexes comprise one or more polyanionic compounds and three or more lipid components. In some embodiments of the foregoing, the complex comprises one or more polyanionic compounds, one or more tertiary aminolipidated and/or cationic peptide compounds, and two or more other lipid components. In some embodiments, compositions and complexes comprising one or more tertiary amino lipidated and/or cationic peptide compounds with one or more polyanionic compounds and/or non-anionic compounds further comprise two or more lipid components, such as phospholipids, structural lipids, and/or shielding lipids (e.g., PEG lipids) as described herein, the compositions can be described as lipid formulations. In certain embodiments, where the composition comprises two or more lipid components, the lipid composition may be described as a multi-component lipid formulation or composition. In certain embodiments, wherein the complex comprises a tertiary amino lipidated and/or cationic peptide compound, a polyanionic compound, a phospholipid, a structural lipid, and a shielding lipid, the composition comprises a lipid nanoparticle. In still other embodiments, where the composition comprises a lipid nanoparticle complex, the composition can be characterized as a Lipid Nanoparticle (LNP) composition.

In addition to tertiary amino lipidation and/or cationic peptide compounds for the main lipid component used as charge neutralization of the loaded polyanionic compound, the multi-component lipid composition and complexes therein may comprise other lipid components, including structural lipids, phospholipids and shielding lipids. The additional lipid components described herein provide physical structure and stability to the complexes of cationic peptide compounds and polyanionic materials, which facilitates their administration in solution and facilitates uptake of the complexes into cells.

For example, in some embodiments, the compositions of the present disclosure comprise a complex of one or more polyanionic compounds and a lipid component, wherein the lipid component comprises optionally one or more structural lipids; one or more phospholipids, one or more shielding lipids and one or more tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof. In some embodiments, the compositions of the present disclosure comprise a complex of one or more polyanionic compounds and a lipid component, wherein the lipid component comprises one or more phospholipids, one or more shielding lipids, and one or more tertiary aminolipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof. In other embodiments, the composition comprises a complex of one or more polyanionic compounds and a lipid component, wherein the lipid component comprises one or more structural lipids; one or more phospholipids, one or more shielding lipids and one or more tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof.

In some embodiments, the complex comprises: tertiary amino lipidated and/or pegylated cationic peptides, nucleic acids, phospholipids, optional structural lipids, and PEG lipids. In certain embodiments wherein the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide, a nucleic acid, a phospholipid, an optional structured lipid, and a PEG lipid, the complex is a Lipid Nanoparticle (LNP) or an Amphiphilic Nanoparticle (ANP). In some embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide, a nucleic acid, a phospholipid, a structural lipid, and a PEG lipid. In other embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide, a nucleic acid, a phospholipid, and a PEG lipid.

Phospholipids may be incorporated into the complexes and compositions of the present disclosure. Through its amphiphilic properties and ability to disrupt cell membranes, phospholipids provide further stabilization of complexes in solution, as well as promote endocytosis. In some embodiments, the compositions provided herein comprise a complex comprising one or more phospholipids as a lipid component. In certain embodiments, the one or more phospholipids comprise one or more zwitterionic phospholipids.

In some embodiments, the phospholipid is selected from 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-di-undecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-cholestanyl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1, 2-dilinolyl-sn-glycero-3-phosphocholine, 1, 2-dineoyl-sn-glycero-3-phosphocholine, 1, 2-docosahexenoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dineotetraenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-docosahexenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (1-Glycerol) sodium salt (DOPG), sphingomyelin, and any mixtures thereof. In certain embodiments, the phospholipid is DOPE.

It will be appreciated that the amphiphilic properties provided by phospholipids to assist in disrupting cell membranes for endocytosis may alternatively be provided by non-phospholipid zwitterionic lipids. In some embodiments, the composition comprises a complex comprising one or more non-phospholipid zwitterionic lipids. In yet a further embodiment, the composition comprises a complex comprising one or more phospholipids, one or more zwitterionic lipids, or any combination thereof. As described herein, structured lipids can be used to impart physical stability to complexes of polyanionic compounds in multicomponent compositions and enhance the lipophilic character of the complexes to facilitate binding to and endocytosis of target cells. In some embodiments, the compositions of the present disclosure comprise a complex comprising, as a lipid component, optionally one or more structural lipids. In some variations, the composition comprises a complex comprising one or more structural lipids.

Structural lipids suitable for use in the compositions and complexes of the present disclosure may include, but are not limited to, sterols. In some embodiments, the structural lipid is selected from the group consisting of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol.

It should be noted, however, that in some embodiments of the present disclosure, the compositions and complexes provided herein are free of structural lipids, yet still exhibit very good delivery efficiency. In certain other embodiments, the compositions and complexes provided herein are free of structural lipids. As described herein, the complexes and compositions of the present disclosure may further comprise one or more shielding lipids. The shielding lipid (e.g., a pegylated lipid or PEG lipid) can provide an additional charge neutralizing layer to the one or more tertiary amino lipidated and/or pegylated cationic peptide compounds as counter charges to the one or more polyanionic compounds and prevent clearance by the process of phagocytosis of cells. In some embodiments, the compositions provided herein comprise a complex comprising one or more shielding lipids as the lipid component.

In some embodiments, the shielding lipid is a PEG lipid. In other embodiments, the PEG lipid is selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG lipid is PEG-modified phosphatidylethanol selected from PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), and PEG-modified DOPE (DOPE-PEG). In certain embodiments, the PEG lipid is selected from the group consisting of dimyristoyl glycerol-polyethylene glycol (DMG-PEG), distearoyl glycerol-polyethylene glycol (DSG-PEG), dipalmitoyl glycerol-polyethylene glycol (DPG-PEG), and dioleoyl glycerol-polyethylene glycol (DOG-PEG). In certain embodiments, the PEG lipid is DMG-PEG.

It should be further recognized that the particular molecular weight of the PEG chains in the aforementioned PEG lipids may be particularly advantageous for incorporation into the complexes of the present disclosure. For example, in some embodiments, the molecular weight of the PEG chain is 350-5,000 g/mole, 1,000-5,000 g/mole, or 2,000-5,000 g/mole. In certain embodiments, the PEG chain of the PEG lipid has a molecular weight of about 350 g/mole, 500 g/mole, 600 g/mole, 750 g/mole, 1,000 g/mole, 2,000 g/mole, 3,000 g/mole, 5,000 g/mole, or 10,000 g/mole. In certain other embodiments, the PEG chain of the PEG lipid has a molecular weight of about 500 g/mole, 750 g/mole, 1,000 g/mole, 2,000 g/mole, or 5,000 g/mole. For example, in certain embodiments, the pegylated lipid is dimyristoyl glycerol-polyethylene glycol 2000(DMG-PEG 2000).

In yet a further embodiment, the one or more PEG lipids comprise a tertiary amino pegylated cationic peptide compound of formula (I) comprising at least one oligomeric or polyethylene glycol moiety. The tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) of the present disclosure, by virtue of their flexibility in accommodating both lipid and polyethylene glycol moieties along the backbone of the peptide chain and depending on the nature of their particular substituents, can be used not only as lipidated cationic peptide compounds for charge neutralization, but also as suitable shielding lipids to stabilize the cationic compound-polyanionic compound complex. It will be appreciated that the tertiary amino pegylated cationic peptide compounds of formula (I) may be combined with other tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I).

In some embodiments, the PEG lipid is a tertiary amino lipidated and/or pegylated cationic peptide compound of formula (I) as described herein, wherein at least one of m and t is non-zero (i.e., N-pegylated). In a still further embodiment, the PEG lipid is a tertiary amino lipidated and/or pegylated cationic peptide compound of formula (I), wherein m and t are independently integers from 0 to 10, and wherein at least one of m and t is non-zero. In some embodiments, the PEG lipid is a tertiary amino pegylated cationic peptide compound of formula (Ib). In still other embodiments, the PEG lipid is a tertiary amino lipidated and pegylated cationic compound of formula (Ic).

As with the PEG lipids described above, the particular molecular weight of the PEG chain in the aforementioned tertiary amino lipidated and pegylated cationic peptide compounds can be particularly advantageous for incorporation into the complexes of the present disclosure. For example, in some embodiments, the molecular weight of the PEG chain is 350-5,000 g/mole, 1,000-5,000 g/mole, or 2,000-5,000 g/mole. In certain embodiments, the PEG chain of the tertiary aminolipidated and pegylated cationic peptide compound has a molecular weight of about 350 g/mole, 500 g/mole, 600 g/mole, 750 g/mole, 1,000 g/mole, 2,000 g/mole, 3,000 g/mole, or 5,000 g/mole. In certain other embodiments, the PEG chain of the PEG lipid has a molecular weight of about 500 g/mole, 750 g/mole, 1,000 g/mole, 2,000 g/mole, or 5,000 g/mole.

However, it will be appreciated that the tertiary aminolipidated and/or pegylated cationic peptide compounds of formula (I) may comprise several short oligoethylene glycol moieties, rather than fewer longer polyethylene glycol moieties, and provide similar particle stability to the complex. In some embodiments wherein the PEG lipid is an N-pegylated cationic peptide compound of formula (I), the tertiary amino lipidated and/or pegylated cationic peptide compound comprises at least one moiety of formula-CH₂CH₂O(CH₂CH₂O)_uR^2aOf ethylene glycol moiety R²Wherein each R is^2aIndependently is-H or C₁-C₄An alkyl group. In other embodiments, at least one is of the formula-CH₂CH₂O(CH₂CH₂O)_vR^6aOf ethylene glycol moiety R⁶Wherein each R is^6aIndependently is-H or C₁-C₄An alkyl group. In some embodiments that may be combined with any of the preceding embodiments (wherein m is non-zero), m is an integer from 0 to 10, wherein each u is independently an integer from 2 to 200. In still other embodiments that may be combined with any of the preceding embodiments wherein t is non-zero, t is an integer from 0 to 10, wherein each v is independently an integer from 2 to 200.

In some embodiments, the PEG lipid is a tertiary amino lipidated and/or pegylated cationic peptide compound of formula (I) as described herein, wherein at least one of m and t is non-zero and at least one of N and s is non-zero (i.e., N-lipidated). In certain embodiments wherein the N-pegylated cationic peptide compound of formula (I) is also N-lipidated, at least one of N and s is non-zero. 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 a further embodiment, n is 4.

As described above, the compositions of the present disclosure comprise a complex comprising one or more polyanionic compounds and a lipid component, wherein the lipid component comprises one or more tertiary amino lipidated and/or pegylated cationic peptide compounds, one or more phospholipids, one or more shielding lipids, and optionally one or more structural lipids. The physical properties of the complexes and compositions described herein can be influenced by the particular choice of lipid component for a given polyanionic compound and the amounts of each component in the complex and composition. In some embodiments, the lipid components in the complexes and compositions thereof may be characterized by the mass percentage of the lipid components (alone or in combination) relative to the total lipid components present and/or the mass ratio of the individual lipid components relative to each other.

In some embodiments, the compositions of the present disclosure are characterized by the mass percentage of lipid components present. As described herein, the total mass or weight of the lipid component is the sum of the individual masses of any tertiary amino lipidated and/or pegylated cationic peptide compound, any structural lipid, any phospholipid and any shielding lipid present.

In some embodiments, the compositions and complexes therein comprise 40-80% w/w of the total weight of the lipid component of one or more tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof. In certain variations, the compositions and complexes therein comprise 40-70% w/w of the total weight of the lipid component of one or more tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof.

As described herein, a composition comprising a complex may optionally comprise one or more structural lipids. In some embodiments that may be combined with any of the preceding embodiments, the compositions and complexes therein comprise 0-25% w/w of one or more structured lipids, based on the total weight of the lipid component. In certain variations, the compositions and complexes therein comprise 0-25% w/w cholesterol, based on the total weight of the lipid component.

The compositions and complexes described herein also comprise one or more phospholipids. In still other embodiments, the compositions and complexes therein comprise 10-60% w/w of one or more phospholipids, based on the total weight of the lipid component. In certain variations, the compositions and complexes therein comprise 20-40% w/w of one or more phospholipids, based on the total weight of the lipid component. In some embodiments, the compositions and complexes therein comprise 10-60% w/w 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by total weight of lipid components. In certain embodiments, the compositions and complexes therein comprise 20-40% w/w 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by total weight of lipid components.

In further embodiments, the compositions and complexes therein comprise one or more shielding lipids. In some embodiments, the compositions and complexes therein comprise 1-5% of one or more shielding lipids, based on the total weight of the lipid component. In certain embodiments, the compositions and complexes therein comprise 1-5% of one or more PEG lipids, based on the total weight of the lipid component. In still other embodiments, the compositions and complexes therein comprise 1-5% 1, 2-dimyristyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) by total weight of the lipid component.

In other embodiments, the compositions of the present disclosure and complexes therein comprise 40-80% w/w of the total weight of the lipid component of one or more tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof; 0-25% w/w cholesterol; 10-60% w/w DOPE; and 1-5% DMG-PEG 2000. In yet a further embodiment, the compositions of the present disclosure and complexes therein comprise 40-70% w/w of the total weight of the lipid component of one or more tertiary aminolipidated and/or pegylated cationic peptide compounds of formula (I) or salts thereof; 0-25% w/w cholesterol; 20-40% w/w DOPE; and 1-5% DMG-PEG 2000.

In some embodiments, wherein the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide compound, a nucleic acid, a phospholipid, a PEG lipid, and optionally a structural lipid, the complex can be characterized by the mass ratio of the individual components to each other, or the mass ratio of one or more components to one or more other components. For example, in some embodiments, the compositions and complexes of the present disclosure can be described by a first mass ratio of tertiary amino lipidated and/or pegylated cationic peptide compound to phospholipid to structural lipid (if present) to shielding lipid and a second mass ratio of tertiary amino lipidated and/or pegylated cationic peptide compound to nucleic acid. Alternatively, in other embodiments, the compositions and complexes provided herein can be described by the mass ratio of the total lipid component to the nucleic acid, wherein the total lipid component includes any tertiary amino lipidated and/or pegylated cationic peptide compounds present, structural lipids, phospholipids, and shielding lipids.

In another aspect, the composites 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, complexes of the present disclosure comprise one or more phospholipids and optionally one or more structural lipids. In some embodiments, wherein the composition comprises a complex comprising one or more structural lipids and one or more phospholipids, the composition can be characterized by the mass ratio of the one or more structural lipids to the one or more phospholipids. In some embodiments, the mass ratio of the one or more structural lipids (when present) to the one or more phospholipids is between 0.5:1 and 2: 1. In certain embodiments wherein the composition comprises cholesterol and DOPE, the mass ratio of cholesterol to DOPE is between 0.5:1 and 2: 1.

As described above, the complex comprising the tertiary amino lipidated and/or pegylated cationic peptide, nucleic acid, phospholipid, optional structural lipid and PEG lipid is characterized by mass percent and/or mass ratio. In some embodiments, wherein the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide, a nucleic acid, a phospholipid, an optional structural lipid, and a PEG lipid, the complex can be characterized by the mass ratio of the individual components to each other or the mass ratio of one or more components to one or more other components. For example, in some embodiments, complexes of the present disclosure can be described by a first mass ratio of tertiary amino lipidated and/or pegylated cationic peptide to phospholipid to structural lipid to PEG lipid and a second mass ratio of tertiary amino lipidated and/or pegylated cationic peptide to nucleic acid.

With respect to the polyanionic compounds present in the complexes and compositions of the present disclosure, the amount of the polyanionic compound in the complex, and thus the composition, can be characterized in a variety of ways. In some embodiments, the compositions and complexes described herein can be characterized by the ratio of the number of cationic groups on the tertiary amino lipidated and/or pegylated cationic peptide compound to the number of anionic phosphate groups on the nucleic acid. In some embodiments, the complexes comprise cationic peptide compounds and nucleic acids that are tertiary amino lipidated and/or pegylated having a cationic to 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, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5: 1. In certain embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide compound having a cationic to anionic charge ratio of 2:1 to 5:1 and a nucleic acid. In still other embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide compound having a cationic to anionic charge ratio of 3:1 and a nucleic acid.

Alternatively, the complexes described herein and compositions comprising the complexes can be characterized by the relative mass ratio of the tertiary amino lipidated and/or pegylated cationic peptide compound to the polyanionic compound and/or other loading in the complex. The mass ratios of the components in the composite can be readily calculated based on the known concentrations and volumes of the component stock solutions used to prepare the composite. Furthermore, if a non-anionic load is present in the complex, the mass ratio can provide a more accurate representation of the relative amount of tertiary amino lipidated and/or pegylated cationic peptide compound to total load compared to the cationic to anionic charge ratio, which does not take into account the non-anionic material.

In some embodiments, the complex comprises one or more tertiary aminolipidated and/or pegylated cationic peptide compounds and one or more polyanionic compounds 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, 1:1 to 20:1, 1:1 to 5:1, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5. 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 still further other 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 certain embodiments wherein the complex comprises a nucleic acid, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide compound and a nucleic acid 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, 2:1 to 20:1, 2:1 to 10:1, or 2:1 to 5: 1. In certain embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide compound and a nucleic acid in a mass ratio of 2:1 to 5: 1. In still other embodiments, the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide compound and a nucleic acid in a mass ratio of 3: 1.

In still other embodiments, the amount of polyanionic compound present in the complexes and compositions thereof can be characterized by the mass ratio of the lipid component (tertiary amino lipidated and/or pegylated cationic peptide compound, phospholipid, shielding lipid, and structural lipid, if present) to the polyanionic compound(s). In some embodiments, the mass ratio of lipid component to polyanion compound(s) is from 0.5:1 to 50:1, from 0.5:1 to 20:1, from 0.5:1 to 10:1, from 0.5:1 to 5:1, from 1:1 to 20:1, from 1:1 to 10:1, from 1:1 to 5:1, from 2:1 to 25:1, from 2:1 to 20:1, from 2:1 to 10:1, or from 2:1 to 5: 1. In certain embodiments, the mass ratio of the lipid component to the one or more polyanionic compounds is from 5:1 to 10:1 or from 6:1 to 7: 1.

Additional Components

Additional components may also be added to the complex to facilitate high encapsulation of the polyanion load and/or targeted controlled release thereof. These additional components may include, for example, polymers and surface active components.

Incorporation of polymers into the complexes described herein may stabilize complexes comprising tertiary amino lipidated and/or pegylated cationic peptides and polyanionic compounds by forming polymer nanoparticle vesicles or forming mixed lipid-polymer nanoparticles in the presence of additional lipid components. In some embodiments, the composites of the present disclosure comprise a polymer. Suitable polymers may include neutral polymers (e.g., poly (lactic-co-glycolic acid) (PLGA) or polyglycolic acid (PGA)), anionic polymers (including poly (aspartic acid), poly (glutamic acid), and heparin), and/or cationic polymers (e.g., polyethyleneimine, protamine).

In other embodiments, the complexes described herein comprise a surface active component, such as a targeting ligand, a stabilizer, and/or a surfactant.

In another aspect, the present disclosure provides a formulation comprising a complex described herein. The formulations described herein include mixtures of stock solutions for preparing the complexes herein as well as purified pharmaceutical formulations for administration. It is understood that a formulation comprising a complex of the present disclosure will necessarily also comprise the components present in the complex described herein.

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, parenteral, or 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, or an intrathecal 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 intra-anal formulation, an oral formulation, or an intravaginal formulation, and the like.

As described above for the complexes of the present disclosure, the formulations provided herein may comprise one or more tertiary aminolipidated and/or pegylated cationic peptide compounds of formula (I) with one or more polyanionic compounds and/or non-anionic compounds. In addition to the tertiary amino lipidated and/or pegylated cationic peptide and the polyanionic compound(s) and/or non-anionic compound(s), it will be appreciated that other components may be included in the formulation to modulate its pharmacokinetic and pharmacodynamic properties.

In some embodiments, the complex may include further components that may be used to deliver nucleic acid loads and other compounds to the cell. These components may include, but are not limited to, those components that together form, for example, a solid Lipid Nanoparticle (LNP) or other comparable super-complex or delivery system (e.g., a mixed lipid-polymer nanoparticle). In some embodiments, the formulation comprises a tertiary amino lipidated and/or pegylated cationic peptide, a nucleic acid, a phospholipid, a structural lipid, and a PEG lipid. In certain embodiments, wherein the complex comprises a tertiary amino lipidated and/or pegylated cationic peptide, a nucleic acid, a phospholipid, a structured lipid, and a PEG lipid, the formulation comprises a lipid nanoparticle.

In some embodiments, where a formulation comprising one or more tertiary aminolipidated and/or pegylated cationic peptide compounds of formula (I) and one or more polyanionic compounds and/or non-anionic compounds further comprises one or more lipid components (e.g., phospholipids, structural lipids, and/or PEG lipids as described herein), the formulation may be described as a lipid formulation. In certain embodiments, where the lipid formulation comprises two or more lipid components, the lipid formulation may be described as a multi-component lipid formulation. In still other embodiments, where the lipid formulation comprises a lipid nanoparticle complex, the lipid formulation may be characterized as a lipid nanoparticle formulation.

In other embodiments, the formulation comprises a tertiary amino lipidated and/or pegylated cationic peptide, a nucleic acid, and a polymer (e.g., PLGA).

It will further be appreciated that the components used to prepare the complexes and formulations of the present disclosure, as well as the process parameters used to prepare the complexes or formulations, may be adapted depending on whether the complex and/or formulation is intended for immediate use ("on-the-fly") or storage for future use. In particular, considerations for storage may include the temperature at which the complex and/or formulation is maintained (e.g., at room temperature, 4 ℃, -20 ℃, -78 ℃) and the duration of storage.

Suitable excipients may include, but are not limited to, those excipients that facilitate administration or enhance storage stability (e.g., cryoprotectants). For example, the formulations described herein may contain, in addition to the above components, pharmaceutically acceptable excipients such as carriers, solvents, dispersants, diluents, fillers, stabilizers, preservatives and the like.

Method for preparing compound and preparation thereof

Complexes and formulations of tertiary amino lipidated and/or pegylated cationic peptide compounds and polyanionic compounds and complexes thereof can be prepared by a variety of physical and/or chemical methods to modulate their physical, chemical and biological properties. These typically include a tertiary aminolipidated and/or pegylated cationic peptide (e.g., an aminolipidated peptoid) in water or a water-miscible organic solvent in rapid combination with a desired polyanionic compound (e.g., an oligonucleotide) in water or an aqueous buffer. These methods may include simple mixing of components by pipetting or microfluidic mixing processes, such as those involving T-type mixers, vortex mixers, or other chaotic mixing structures.

In one aspect, the present disclosure provides a method of forming a complex described herein, comprising contacting a tertiary amino lipidated and/or pegylated cationic peptide compound with a polyanionic compound.

In some embodiments, a method of forming a cationic peptide compound comprising tertiary amino lipidation and/or pegylation and a polyanion compound comprises contacting a solution comprising a cationic peptide compound comprising tertiary amino lipidation and/or pegylation with a solution comprising a polyanion compound. In certain embodiments, the polyanionic compound comprises a nucleic acid.

In yet another aspect, the present disclosure provides a method of preparing a formulation comprising a tertiary amino lipidated and/or pegylated cationic peptide compound and a polyanionic compound, wherein the polyanionic compound is a nucleic acid, the method comprising contacting a solution comprising the tertiary amino lipidated and/or pegylated cationic peptide compound with a solution comprising the polyanionic compound to provide the formulation.

The complex and further components of the formulation, such as the lipid component forming the lipid nanoparticle, the polymer, the surfactant or the excipient may be mixed and combined with the tertiary amino lipidated and/or pegylated cationic peptide compound in water or a water-miscible organic solvent before, during or after mixing the tertiary amino lipidated and/or pegylated cationic peptide compound with the polyanionic compound.

The particular process conditions for preparing the complexes and formulations described herein can be adjusted or selected accordingly to provide the desired physical properties of the complexes and formulations. For example, parameters for mixing the components of the complex and formulation that may affect the final complex and formulation may include, but are not limited to, the order of mixing, the temperature of mixing, the mixing speed/rate, the flow rate, the stock solution concentration, the component mass ratio (e.g., peptide: loading), and the solvent.

Methods of using complexes and formulations thereof

As mentioned above, tertiary aminolipidated and/or pegylated cationic peptide compounds, complexes thereof with polyanionic compounds, and formulations thereof facilitate the delivery of polyanionic compounds to cells, particularly the intracellular environment. Thus, peptide compounds, their complexes with polyanionic compounds, and their formulations are useful in many clinical and research applications. The delivery of polyanionic compounds to cells may be used for clinical applications, such as those associated with prophylactic, 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 in research or non-clinical applications, including bioassays and reagents.

In another aspect, provided herein are methods of delivering a polyanionic compound to a cell. In some embodiments, the method of delivering a polyanionic compound to a cell comprises contacting the cell with a complex comprising a tertiary amino lipidated and/or pegylated cationic peptide compound and a polyanionic compound. In other embodiments, the method of delivering a polyanionic compound to a cell comprises contacting the cell with a formulation comprising a tertiary amino lipidated and/or pegylated cationic peptide compound and a polyanionic compound. In some embodiments of the foregoing methods, the contacting is by endocytosis.

In some embodiments, the method of delivering a polyanionic compound to a cell comprises contacting the cell with a complex comprising a tertiary amino lipidated and/or pegylated cationic peptide compound and a polyanionic compound, wherein the cell is contacted in vitro, ex vivo, or in vivo. In some embodiments, the method of delivering a polyanionic compound to a cell comprises contacting the cell with a formulation comprising a tertiary amino lipidated and/or pegylated cationic peptide compound and a polyanionic compound, wherein the cell is contacted in vitro, ex vivo, or in vivo.

In some embodiments, wherein the cell is contacted in vitro, the cell is a HeLa cell. In other embodiments, in which the cells are contacted in vivo, the complexes or formulations of the present disclosure are administered to a mammalian subject. Mammalian subjects may include, but are not limited to, human or mouse subjects. In still other embodiments in which the cells are contacted ex vivo, the cells are obtained from a human or mouse subject.

In some embodiments of the foregoing methods in which cells are contacted in vivo, the complexes and formulations described herein can be administered by injection. In certain embodiments, the complexes and formulations described herein can be administered by injection (intravenous (IV), Subcutaneous (SC), Intramuscular (IM), intrathecal). In some embodiments, the complexes and formulations are administered by Intravenous (IV), Subcutaneous (SC), Intramuscular (IM), or intrathecal injection. In other embodiments, the complexes and formulations described herein are administered by bolus injection or intravenous infusion. In other embodiments, where cells are contacted in vivo, the complexes and formulations of the present disclosure are administered by nasal or oral inhalation. In some embodiments, wherein the cells are contacted in vivo, the complexes and formulations described herein are administered orally. In still other embodiments where the cells are contacted in vivo, the complexes and formulations are administered by absorption into the mucosa (including topically, intra-anal, intra-oral, intra-vaginal, etc.).

It is to be understood that clinical applications, such as the diagnostic, prophylactic and therapeutic examples disclosed above, can involve dosing regimens (e.g., dosage levels and time courses of administration) which can be varied as appropriate to the particular complex and/or formulation used, the route of administration, the subject to whom the complex and/or formulation is administered, and/or the physiological effect desired. For example, in some embodiments, the methods of the present disclosure comprise administering the complex or formulation at a dose of 0.0001mg/kg to about 10mg/kg body weight.

Having generally described this invention, the invention will be better understood by reference to the specific examples which are included herein to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

Examples

The present disclosure is further described in detail in the following examples, which are not intended to limit the scope of the present disclosure as claimed in any way. The drawings are to be regarded as forming a part of the specification and description of the present disclosure. The following examples are provided for the purpose of illustration and are not intended to limit the claimed disclosure.

Example 1 Synthesis of exemplary Tertiary amino lipidated cationic peptides for nucleic acid delivery

The following examples describe general schemes for the synthesis of tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) as described herein.

In the description provided below, all R^aAnd R^bAre all-H. All polymers were synthesized using bromoacetic acid and a primary amine. FIGS. 2B-2E provide R for the preparation of tertiary aminolipidated and/or pegylated cationic peptide compounds of the present disclosure²、R³、R⁴、R⁵And R⁶Some exemplary removal of primary aminesAnd (4) generation of base.

Fmoc-Rink amide resin was used as a solid support. The Fmoc group on the resin was deprotected with 20% (v/v) piperidine-Dimethylformamide (DMF). The amino resin was then amidated with bromoacetic acid. Followed by amination of the alpha-carbon by nucleophilic substitution of bromine with a primary amine. These two steps are repeated in succession to produce the desired cationic peptide sequence.

All reactions and washes were performed at room temperature unless otherwise indicated. The washing of the resin is directed to the addition of a washing solvent (usually DMF or dimethyl sulfoxide (DMSO)) to the resin, stirring the resin to obtain a homogeneous slurry, and then draining the solvent completely from the resin. The solvent was removed by vacuum filtration through the sintered bottom of the reaction vessel until the resin appeared dry. In all syntheses, the resin slurry was stirred by bubbling argon through the bottom of the sintering vessel.

The initial resin is deprotected. Loading Fmoc-Rink amide resin into the sintered reaction vessel. DMF was added to the resin and the solution was stirred to swell the resin. Then, the DMF was drained. The Fmoc group was removed by adding 20% piperidine in DMF to the resin, stirring the resin and draining the resin. To the resin was added 20% piperidine in DMF and stirred for 15 min, then drained. The resin was then washed six times with DMF.

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

Nucleophilic substitution/amination. The acylated resin was treated with the desired primary or secondary amine to undergo nucleophilic substitution of the bromo leaving group on the α -carbon (fig. 3B). This acylation/displacement cycle (fig. 3C) is repeated until the desired peptide sequence is obtained (fig. 3D).

Peptide cleavage from resin. The dried resin was placed in a glass scintillation vial containing a teflon-coated microscler stirring bar and 95% aqueous trifluoroacetic acid (TFA) was added. The solution was stirred for 20 minutes and then filtered through a Solid Phase Extraction (SPE) cartridge fitted with a polyethylene frit into a polypropylene conical centrifuge tube.

The resin was washed with 1mL 95% TFA. The combined filtrates were then lyophilized three times from 1:1 acetonitrile to water. The lyophilized peptide (FIG. 3E) was re-dissolved in absolute ethanol at a concentration of 5mg/mL or dissolved in DMSO at a concentration of 10 mg/mL.

And (5) purifying and characterizing. The re-dissolved crude peptide was purified by preparative HPLC. The purified peptide was characterized by LC-MS analysis.

Example 2 Synthesis and characterization of representative amino lipidated peptoids

Aminolipidated polyglycine compounds ("peptoids") were synthesized by the sub-monomer method described in example 1 above using bromoacetic acid and N, N' -Diisopropylcarbodiimide (DIC). Polystyrene-supported MBHA Fmoc-protected Rink amide (200 mg representative scale, 0.64 mmol/g loading, Protein Technologies) resin was used as solid support. For bromoacetylation, the resin was combined with a 1:1 mixture of 2M bromoacetic acid and 2M N, N' -Diisopropylcarbodiimide (DIC) for 5 minutes. Amine replacement was performed using a 1M amine solution in DMF for 1 hour. After synthesis, the crude peptoid was cleaved from the resin using 5ml of a mixture of 95:2.5:2.5 trifluoroacetic acid (TFA), water, triisopropylsilane at room temperature for 40 minutes. The resin was removed by filtration and the filtrate was concentrated using a Biotage V10 evaporator. The crude peptoid was further concentrated by lyophilization from a 25% aqueous solution of MeCN. Purity and identity were analyzed on a Waters Acquity UPLC Peptide BEH C4 column using a Waters Acquity UPLC system (with Acquity Diode Array UV detector and Waters SQD2 mass spectrometer) in a gradient of 5-95%. Selected crude peptoids were purified by a preparative Waters Prep150LC system with Waters 2489 UV/visible light detector on a Waters XBridge BEH300 Prep C4 column over 30 minutes using a 5-40% acetonitrile gradient in water with 0.1% TFA.

Table 1A shows representative amino lipidated peptoid compounds 1-72 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 weight, retention time (in minutes, determined by UPLC-UV measurement at λ ═ 218 nm), and major observed mass-to-charge ratios (m/z, MH) in terms of minutes⁺By electrospray ionization-mass spectrometry). For each of the aminolipidated and pegylated peptoid compounds 48, 56, 64, and 72, the mass spectrum contains several peak distributions due to the polydispersity (average molecular weight 2000g/mol) of the PEG moiety attached to the peptoid. The peak mass to charge ratio reported for these aminolipidated and pegylated peptoid compounds is MH2²⁺The central value of the peak distribution has effective monomer separation delta-22 m/z (-OCH)₂CH₂-, ethylene glycol molecular weight 44 g/mol).

TABLE 1A

TABLE 1B

Example 3 formulation of representative amino lipidated peptoids with oligonucleotides to form nanoparticle compositions

The following examples describe general schemes for the formulation of tertiary amino lipidated and/or pegylated cationic peptide compounds of formula (I) with oligonucleotides as described herein.

In standard formulations, the tertiary amino lipidated and/or pegylated cationic peptide compounds are dissolved in anhydrous ethanol at a concentration of 0.5mg/mL (for in vitro experiments) or 5mg/mL (for in vivo experiments). The resulting solution is stable at room temperature, but should be stored at-20 ℃. The nucleic acid load was dissolved in water without DNAse or RNAse at a final concentration of 0.2mg/mL (for in vitro experiments) or 1mg/mL (for in vivo experiments). These solutions should be stored at-20 ℃ or at-78 ℃ (for longer periods of time).

To prepare the nanoparticle formulation, tertiary amino lipidated and/or pegylated cationic peptide compounds are mixed with nucleic acids by pipetting in a mass ratio of about 1:1 (peptide compound: loading) to 20: 1. Before formulation, the tertiary amino lipidated and/or pegylated cationic peptide compound and the load (e.g., nucleic acid) are diluted in ethanol and acidic buffer (PBS, adjusted to ph5.5 with 0.1M HCl) to a volume ratio of 1:3, respectively, and targeted at a final load concentration of about 0.05mg/mL to 0.2 mg/mL.

Example 4 characterization of physical Properties of representative mRNA/peptoid formulations

The exemplary aminolipidated cationic peptoids 1-36 described in example 2 were combined with firefly luciferase (Fluc) mRNA to form nanoparticle compositions for evaluation for in vitro or in vivo therapeutic and/or prophylactic purposes. The formulations were prepared according to the protocol of example 3 and mixed by simple pipetting.

The mRNA/peptoid formulation at a 5:1 mass ratio of loading was evaluated by Dynamic Light Scattering (DLS) to determine the volume average particle size/diameter (nm) of the mRNA/peptoid complex and the size polydispersity index (PDI) within the formulation. The percent mRNA encapsulation of each preparation of exemplary compounds was determined by fluorescence of the Qubit RNA HS (Invitrogen) dye by Triton X-100 before and after particle lysis. The results are shown in table 2 below.

TABLE 2

NA ═ sample is not suitable for DLS measurement

Example 5 in vitro expression of firefly luciferase (Fluc) following treatment with a representative Fluc mRNA/amino-lipidated cationic peptoid formulation

The efficacy of mRNA/aminolipidated peptoid formulations was evaluated in vitro based on their ability to deliver a firefly luciferase (Fluc) reporter gene to cultured cells. The aminolipidated cationic peptoids 1-36 of example 2 were combined with Fluc mRNA alone at a ratio of 5:1w/w and the resulting particles were added to cultured HeLa cells at a dose of 100 ng/well (total volume 100 μ L). The resulting luciferase expression (RLU) was measured by a luminescence plate reader after 6 hours and 24 hours of treatment. Table 3 below shows the luciferase expression observed for the Fluc mRNA/aminolipidated peptoid formulation at two time points.

TABLE 3 in vitro expression of firefly luciferase (Fluc) following treatment with representative Fluc mRNA/amino lipidated peptoid formulations

In vitro luciferase expression measurements were also performed on the exemplary aminolipidated cationic peptoid compounds 1-18 of example 2 at peptoid to mRNA mass ratios of 2:1, 3.5:1, 5:1, and 7.5: 1. The observed luciferase expression was measured as mean bioluminescence (RLU) by a luminescence plate reader, as shown in figure 4.

Example 6-in vitro expression of firefly luciferase (Fluc) following treatment with a representative Fluc mRNA/amino-lipidated peptoid Complex in a Multi-component lipid formulation

The efficacy of mRNA/aminolipidated peptoids in multi-component lipid formulations was evaluated based on their ability to deliver firefly luciferase (Fluc) reporter genes to cultured HeLa cells in vitro. Three lipid formulations with different mass percentages of lipid components were prepared using cholesterol, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) versus amino-lipidated peptoids (peptoids: cholesterol: DOPE: DMG-PEG2000, 50:165:32:2, 63:0:35:2 and 42:23:33: 2). Each base preparation of aminolipidated peptoids was then combined with mRNA in a mass ratio of 7:1 or 10:1 (peptoid: loading). The compositions of the six lipid formulations evaluated in this example are shown in table 4 below. The formulation using the lipidoid 1 instead of the amino lipidated cationic peptoid compound was prepared for comparison at the same mass percentages in table 4.

TABLE 4

Cell culture: HeLa cells were seeded at 10,000 wells in 100 μ L DMEM containing 10% FBS and 1% penicillin/streptomycin 18 hours prior to treatment and allowed to adhere. The medium was replaced immediately before transfection with 100 μ L of fresh serum-containing DMEM.

mRNA preparation: fluc mRNA was prepared internally for transfection experiments using standard In Vitro Transcription (IVT) methods.

mRNA preparation: lipid mixtures were prepared according to the above ratios using a master stock of aminolipidated peptoids (compounds 2 and 6-18) or lipidoids 1 (5mg/mL in ethanol), cholesterol (5mg/mL), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, 5mg/mL) and 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000, 0.5 mg/mL). Considering the remaining volume (and adjusting the effective ratio of total lipid to mRNA), 100% ethanol was added. Then 8. mu.L of this lipid mixture was added to 24. mu.L of mRNA dissolved in acidified PBS (pH 5.5) at a concentration of 15 ng/. mu.L. After mixing, 10. mu.L of the resulting solution was added to the corresponding well of a 96-well plate. All treatments were performed in triplicate and values are expressed as mean values. HeLa cells were treated with the resulting solution for 6 hours, after which the medium was replaced with fresh DMEM. As a negative control, HeLa cells were treated with a solution of mRNA alone. For Lipo conditions, mRNA was formulated with Lipofectamine 2000(Thermo Fisher) according to the manufacturer's instructions.

In vitro imaging: before imaging, 10. mu.l of a 30mg/mL solution of D-fluorescein was added to each well, followed by incubation at 37 ℃ for 10 minutes. After this time, luminescence of the whole plate was measured using a SpectraMax iD3 plate reader (Molecular Devices). Fig. 5 depicts a histogram of the observed mean bioluminescence (RLU) for each lipid formulation.

Example 7 in vitro expression of firefly luciferase (Fluc) following treatment with representative Fluc mRNA/amino lipidated peptoid complexes in a Multi-component lipid formulation

Additional experiments were performed to evaluate the in vitro delivery efficiency of formulations of aminolipidated peptoids 1-72 with cholesterol, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) to HeLa cells, where the mass ratio of peptoid: cholesterol: DOPE: DMG-PEG2000 was 41:23:33:3 and the peptoid: Fluc mRNA ratio was 10: 1.

The protocol as described in example 6 was used for the full panel evaluation of the amino lipidated peptoids 1 to 72 of the multicomponent lipid formulation. Briefly, HeLa cells were treated with the formulation for 6 hours (50 ng Fluc mRNA per well) after which the medium was replaced with fresh DMEM. Before imaging, 10 liters of 30mg/mL D-fluorescein solution was added to each well followed by incubation at 37 ℃ for 10 minutes. Thereafter, luminescence of the entire plate was measured using a SpectraMax iD3 plate reader (Molecular Devices). The average luminescence (RLU) observed for the amino lipidated peptoid compounds 1-72 is shown in FIG. 6A (Compounds 1-36) and FIG. 6B (Compounds 37-72). All treatments were performed in triplicate and expressed as mean values.

Example 8-in vivo systemic expression of firefly luciferase (Fluc) following subcutaneous administration of a representative Fluc mRNA/aminolipidated multi-component lipid formulation.

The in vivo delivery efficiency of aminolipidated peptoid compounds to Fluc mRNA of Balb/c mice (n ═ 3) was further evaluated by subcutaneous injection administration. A multi-component lipid formulation comprising aminolipidated lipidoids 1-72 with Fluc mRNA (2g), cholesterol, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) was prepared, wherein the mass ratio of peptoid: cholesterol: DOPE: DMG-PEG2000 was 41:23:33:3 and the ratio of peptoid: Fluc mRNA was 10: 1. The formulation was administered by tail vein injection at a dose of 0.1 mg/kg (about 2g Fluc mRNA/dose) and the resulting bioluminescence was quantified after 6 hours.

Figures 7A and 7B show the in vivo systemic expression of Fluc in test mice quantified as mean bioluminescence (RLU).