WO2024196082A1 - Micelle containing novel peptide, and use thereof - Google Patents

Abstract

The present invention pertains to a novel peptide, a micelle containing the peptide, a drug delivery complex, and a pharmaceutical composition for preventing or treating disease. Micelles containing the novel peptide according to embodiments of the present invention are formed through the self-assembly of peptides that are easy to synthesize, isolate, and purify, and thus the micelles have a simple manufacturing process and a stable structure. In addition, the micelles have low cytotoxicity and high cell permeability, can efficiently deliver siRNA into the body through the interaction of a positively charged portion and the negatively charged siRNA, and can target various mRNAs. Moreover, the micelles can effectively deliver two or more types of nucleic acids (including, but not limited to, DNA, mRNA, siRNA, microRNA, ASO, gapmer, aptamer, antagomir, agomir, and oligonucleotide) having specific sequences into the body to target various types of nucleic acids complementary thereto.

Description

Micelles containing novel peptides and their uses

The present invention relates to a micelle comprising a novel peptide and its use as a drug delivery complex or a pharmaceutical composition for preventing or treating proliferative diseases.

Existing new drug development technologies have mainly used small molecule compounds or antibodies, which are applied to produced proteins, and thus have the disadvantages of requiring a long period of time to derive new drug candidates and having limited target proteins.

To overcome the above shortcomings, nucleic acid therapeutics using RNA interference technology are attracting attention in the development of new drugs. Nucleic acid therapeutics using RNA interference technology act on mRNA to suppress the expression of specific proteins, and have the advantage of requiring a short period of time to derive new drug candidates and enabling multi-targeting.

However, siRNA, which is mainly used as a nucleic acid therapeutic using RNA interference technology, has a disadvantage in that it has a negative charge and thus has very low cell membrane permeability, so it must be delivered into cells using a specific carrier.

Recently, N-Acetylgalactosamine (GalNAc) has been utilized as a carrier for siRNA, but it is mostly used as a treatment for liver-related diseases because it has high affinity for ASGPR, a specific cell surface protein expressed in hepatocytes.

Accordingly, the inventors of the present invention synthesized a novel peptide that exhibits high cell permeability without exhibiting cytotoxicity, and confirmed that micelles are formed by self-assembly thereof, and that the micelles form a stable complex with siRNA and exhibit excellent cell internalization and endosomal escape capabilities. In addition, it was confirmed that the micelle-siRNA complex according to the present invention can be used to silence the corresponding gene.

The present invention provides a novel peptide, a micelle comprising the same, a drug delivery complex thereof, or a pharmaceutical composition for preventing or treating proliferative diseases.

However, the problems that the present invention seeks to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The first aspect of the present invention provides a peptide comprising an amino acid sequence of structural formula 1.

The second aspect of the present invention provides a peptide comprising an amino acid sequence of structural formula 4.

The third aspect of the present invention provides a micelle comprising the peptide.

The fourth aspect of the present invention provides a drug delivery complex comprising the micelle; and a desired drug.

The fifth aspect of the present invention provides a composition for drug delivery, comprising the drug delivery complex.

The sixth aspect of the present invention provides a pharmaceutical composition for preventing or treating a proliferative disease, comprising the micelle and siRNA.

The micelles containing the novel peptides according to the embodiments of the present invention are formed by self-assembly of peptides that are easy to synthesize and purify, and thus have a simple manufacturing process and a stable structure. In addition, they have low cytotoxicity and high cell permeability, can efficiently deliver siRNA into the body through the interaction of a positively charged portion and a negatively charged siRNA, and can target various mRNAs. In addition, they can effectively deliver two or more kinds of nucleic acids (non-limiting examples include DNA, mRNA, siRNA, microRNA, ASO, gapmer, aptamer, antagomir, agomir, and oligonucleotides) having a specific sequence into the body, and target various kinds of complementary nucleic acids thereto.

Figure 1 shows the results of confirming the critical micelle concentration (CMC), which is the concentration at which micelles are formed by self-assembly of an amphipathic peptide according to one embodiment of the present invention.

Figure 2 shows the results of confirming the critical micelle concentration (CMC), which is the concentration at which micelles are formed by self-assembly of a cationic peptide according to one embodiment of the present invention.

Figure 3 shows the results of confirming the size of micelles containing amphipathic peptides according to one embodiment of the present invention.

Figure 4 shows the results of confirming the size of micelles containing cationic peptides according to one embodiment of the present invention.

Figure 5 shows the results of confirming whether a micelle containing a cationic peptide and siRNA form a complex according to one embodiment of the present invention.

Figure 6 shows the results of confirming whether a micelle containing a cationic peptide and siRNA form a complex according to one embodiment of the present invention.

Figure 7 shows the results of confirming the size of a micelle-siRNA complex containing an amphipathic peptide according to one embodiment of the present invention.

Figure 8 shows the results of confirming the size of a micelle-siRNA complex containing a cationic peptide according to one embodiment of the present invention.

Figure 9 shows the results of confirming the form of a micelle-siRNA complex containing an amphipathic peptide according to one embodiment of the present invention.

FIG. 10 is a graph comparing cell proliferation rates after treating human pancreatic adenocarcinoma cell lines (Capan-1) with various concentrations of Lipofectamin ^TM 2000, micelles containing SCL1001, an amphipathic peptide (SCM100), or micelles containing SCL2001 (SCM2001), according to one embodiment of the present invention.

Figure 11 is a photograph of a cell morphology observed under a microscope after treating a human pancreatic ductal adenocarcinoma cell line (Capan-1) with Lipofectamin ^TM 2000 at a concentration of 40 μg/㎖ and micelles (SCM1001 or SCM2001) containing an amphipathic peptide, SCL1001 or SCL2001, respectively, according to one embodiment of the present invention.

FIG. 12 is a graph comparing cell proliferation rates after treating a micelle-siRNA complex formed by mixing a specific concentration of siRNA, various concentrations of Lipofectamin ^TM 2000, and micelles (SCM1001 or SCM2001) containing an amphipathic peptide, SCL1001 or SCL2001, to a human pancreatic adenocarcinoma cell line (Capan-1) according to one embodiment of the present invention.

FIG. 13 is a photograph showing a microscopic observation of changes in cell appearance after treating a micelle-siRNA complex formed by mixing 200 nM siRNA and 40 μg/㎖ of Lipofectamin ^TM 2000 and micelles containing the amphipathic peptide SCL1001 or SCL2001 (SCM1001 or SCM2001) into a human pancreatic adenocarcinoma cell line (Capan-1) according to one embodiment of the present invention.

Figure 14 shows the results of confirming the cell internalization and endosomal escape ability of a micelle-siRNA complex prepared by mixing siRNA and micelles (SCM1001 or SCM2001) containing an amphipathic peptide, SCL1001 or SCL2001, according to one embodiment of the present invention.

Figure 15 shows the results of confirming the stability in serum of a micelle-siRNA complex prepared by mixing siRNA and micelle (SCM100) containing an amphipathic peptide, SCL1001, according to one embodiment of the present invention.

Figure 16 shows the results of confirming the KRAS protein expression inhibitory effect of a micelle-siRNA complex prepared by mixing siRNA and micelles (SCM1001 or SCM2001) containing an amphipathic peptide, SCL1001 or SCL2001, according to one embodiment of the present invention.

Figure 17 is a graph quantifying the efficiency of inhibiting KRAS protein expression of a micelle-siRNA complex prepared by mixing siRNA and micelles (SCM1001 or SCM2001) containing an amphipathic peptide, SCL1001 or SCL2001, according to one embodiment of the present invention.

Figure 18 shows the results of confirming the KRAS protein expression inhibitory effect of a micelle-siRNA complex prepared by mixing siRNA and micelles (SCM1016 or SCM1020) containing cationic peptide SCL1016 or SCL1020 according to one embodiment of the present invention.

FIG. 19 is a graph quantifying the efficiency of inhibiting KRAS protein expression of a micelle-siRNA complex prepared by mixing siRNA and micelles (SCM1016 or SCM1020) containing cationic peptide SCL1016 or SCL1020 according to one embodiment of the present invention.

Figure 20 shows the results of confirming the KRAS protein expression inhibitory effect of a micelle-siRNA complex prepared by mixing micelles (SCM1023) containing cationic peptide SCL1023 and siRNA according to one embodiment of the present invention.

Figure 21 is a graph quantifying the efficiency of inhibiting KRAS protein expression of a micelle-siRNA complex prepared by mixing siRNA and micelle (SCM1023) containing cationic peptide SCL1023 according to one embodiment of the present invention.

Figure 22 is a schematic diagram showing the binding of micelles to siRNA conjugated with Cy5 and/or FAM according to one embodiment of the present invention.

Figure 23 shows the results of confirming the intracellular delivery effects of various types of siRNA using micelle-siRNA complexes prepared by mixing two types of siRNAs and micelles (SCM1001 or SCM2001) containing amphipathic peptides SCL1001 or SCL2001 according to one embodiment of the present invention.

Figure 24 is a schematic diagram showing a peptide constituting the micelle of the present invention, showing a peptide in which a hydrophobic moiety is bound to the amino terminal of the peptide, and a targeting/labeling moiety or a linker for connecting them is bound to the carboxyl terminal.

Figure 25 is a schematic drawing showing the structure of a micelle containing the peptide of the present invention.

Hereinafter, with reference to the attached drawings, implementation examples and embodiments of the present invention will be described in detail so that those with ordinary skill in the art can easily practice the present invention. However, the present invention may be implemented in various different forms and is not limited to the implementation examples and embodiments described herein. In addition, in order to clearly describe the present invention in the drawings, parts that are not related to the description are omitted, and similar parts are assigned similar drawing reference numerals throughout the specification.

Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather includes other components, unless otherwise specifically stated.

The terms “about,” “substantially,” and the like, as used herein, are used in a meaning that is at or close to the numerical value when manufacturing and material tolerances inherent in the meanings stated are presented, and are used to prevent unscrupulous infringers from unfairly utilizing the disclosure in which exact or absolute numerical values are stated to aid the understanding of the present application.

Throughout this specification, the term "combination(s) thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the Makushi format, and means including one or more selected from the group consisting of said components.

Throughout this specification, references to “A and/or B” mean “A or B, or A and B.”

A first aspect of the present invention provides a peptide comprising an amino acid sequence of structural formula 1:

[Structural formula 1]

(X ¹ L) _a X ² Q(X ³ ) _b

In the above structural formula 1,

The above X ¹ , X ² and X ³ are each independently R (Arginine), H (Histidine), or K (Lysine),

The above L stands for Leucine,

The above Q stands for Glutamine,

The above a is an integer from 2 to 5,

The above b is an integer from 1 to 13.

The above peptide may be a cationic peptide or a hydrophilic peptide. Additionally, the peptide may be a cell-penetrating peptide.

The above peptide may be for forming micelles and/or drug delivery vehicles. Specifically, the peptide may be for forming micelles and/or drug delivery vehicles by self-assembly. Accordingly, the present invention may provide micelles and/or drug delivery vehicles comprising the peptide.

In one embodiment, X ¹ , X ² and X ³ of the structural formula 1 can each independently be R (Arginine), H (Histidine), or K (Lysine), specifically, X ¹ , X ² and X ³ can each independently be R (Arginine) or K (Lysine), and more specifically, X ¹ , X ² and X ³ can all be R (Arginine) or all can be K (Lysine).

In one embodiment, a in the structural formula 1 may be an integer from 2 to 5, specifically an integer from 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4 or 4 to 5, and more specifically, a may be 2, 3, 4 or 5.

In one embodiment, b of the structural formula 1 may be an integer from 1 to 13, and specifically, may be an integer from 1 to 13, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 2, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 13, 4 to 10, 4 to 8, or 4 to 6, and more specifically, b may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

In one embodiment, in the amino acid sequence having the structure of the structural formula 1, the terminal of X ³ may be the amino terminal (N`-terminal) or the carboxyl terminal (C`-terminal). In the case of a peptide in which the terminal of X ³ is the carboxyl terminal, it may include an amino acid sequence having a structure of the structural formula 2 below, and in the case of a peptide in which the terminal of X ³ is the amino terminal, it may include an amino acid sequence having a structure of the structural formula 3 below.

[Structural formula 2]

N`-(X <sup>1</sup> L) <sub>a</sub> X <sup>2</sup> Q(X <sup>3</sup> ) <sub>b</sub> -C`

[Structural formula 3]

N`-(X <sup>3</sup> ) <sub>b</sub> QX <sup>2</sup> (LX <sup>1</sup> ) <sub>a</sub> -C`

In the above structural formula 2 or 3,

The above X ¹ , X ² and X ³ are each independently R (Arginine), H (Histidine), or K (Lysine),

The above L stands for Leucine,

The above Q stands for Glutamine,

The above a is an integer from 2 to 5,

The above b is an integer from 1 to 13,

N' represents the amino terminus of the peptide, and C' represents the carboxyl terminus of the peptide.

In one embodiment, the peptide may include an amino acid sequence having at least one H (Histidine) additionally linked to at least one of the amino terminus and the carboxyl terminus of an amino acid sequence having the structure of the structural formula 1 (specifically, an amino acid sequence having the structure of the structural formula 2 or 3). The above H may further include or be connected to 1 to 21, specifically 1 to 21, 1 to 18, 1 to 15, 1 to 12, 1 to 9, 1 to 6, 1 to 3, 3 to 21, 3 to 18, 3 to 15, 3 to 12, 3 to 9, 3 to 6, 6 to 21, 6 to 18, 6 to 15, 6 to 12, 6 to 9, 9 to 21, 9 to 18, 9 to 15, 9 to 12, 12 to 21, 12 to 18, or 12 to 15 Hs.

In one embodiment of the present invention, the peptide may include at least one selected from the group consisting of amino acid sequences described in Table 1 below, and specifically may include an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 31.

Sequence number Amino acid sequence 1 RLRLRQRRRR 2 KLKLKQKKKK 3 RLRLRQRR 4 RLRLRQRRR 5 RLRLRQRRRRR 6 RLRLRLRQRR 7 RLRLRLRQRRR 8 RLRLRLRQRRRRR 9 RLRLRLRLRQRR 10 RLRLRLRLRQRRR 11 RLRLRLRLRQRRRRR 12 RLRLRLRLRLRQRR 13 RLRLRLRLRLRQRRR 14 RLRLRLRLRLRQRRRRR 15 KLKLKQKK 16 KLKLKQKKK 17 KLKLKQKKKKK 18 KLKLKLKQKK 19 KLKLKLKQKKK 20 KLKLKLKQKKKKK 21 KLKLKLKLKQKK 22 KLKLKLKLKQKKK 23 KLKLKLKLKQKKKKK 24 KLKLKLKLKLKQKK 25 KLKLKLKLKLKQKKK 26 KLKLKLKLKLKQKKKKK 27 HHHRLRLRQRRRR 28 RLRLRQRRRRHHH 29 HHHRLRLRQRRRRHHH 30 HHHHHHRLRLRQRRRR 31 RLRLRQRRRRHHHHHH 32 HHHHHHRLRLRQRRRRHHHHH 33 HHHKLKLKQKKKK 34 KLKLKQKKKKHHH 35 HHHKLKLKQKKKKHHH 36 HHHHHHHKLKLKQKKKK 37 KLKLKQKKKKHHHHHH 38 HHHHHHHKLKLKQKKKKHHHHH 39 KLKLKQRRRR 40 RLRLRQKKKK

In one embodiment of the present invention, the peptide may additionally comprise one or more hydrophobic amino acids at its N-terminus or C-terminus, and specifically, may additionally comprise one or more amino acids selected from the group consisting of A (Alanine), V (Valine), I (Isoleucine), and L (Leucine) at the N-terminus. The peptide may additionally comprise 1 to 20 hydrophobic amino acids.

In one embodiment, a hydrophobic peptide comprising the amino acid sequence of VLVALAIV (SEQ ID NO: 41) may be additionally linked to the N-terminus of the peptide, and the hydrophobic peptide may be directly linked or indirectly linked via a linker comprising one or more amino acid residues (e.g., P (Proline)).

When the linker is a proline residue, it can form a folded structure in the three-dimensional structure of the peptide. When the linker is a proline residue, it can include one or more prolines. In addition, the linker can be a glycine residue, and any amino acid residue that can form a folded structure in the three-dimensional structure of the peptide can be used without limitation.

In one embodiment of the present invention, the peptide may comprise or consist of one or more sequences selected from the group consisting of amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 31, SEQ ID NO: 42 (VLVALAIVRLRLRQRRRR), and SEQ ID NO: 43 (VLVALAIVPRLRLRQRRRR). In addition, the peptide may comprise each selected amino acid sequence repeated two or more times.

In one embodiment of the present invention, when one or more hydrophobic amino acids are linked to the N-terminus or C-terminus of the peptide, or a hydrophobic peptide is linked, the peptide may be an amphipathic peptide.

As used throughout this specification, the term "amphipathic peptide" means a peptide that simultaneously contains regions having different physical properties, for example, different solubility parameters, and may mean, for example, a peptide that simultaneously contains a hydrophilic region and a hydrophobic region.

In one embodiment of the present invention, the peptide may be manufactured by a chemical peptide synthesis method known in the art, for example, a SPSS (Solid Phase Peptide Synthesis) method, or a process in which a gene encoding the peptide is amplified by PCR (polymerase chain reaction) or synthesized by a known method and then cloned into an expression vector for expression, but is not limited thereto. In one embodiment of the present invention, the peptide may refer to a peptide manufactured using a cell, or an artificially synthesized peptide, but is not limited thereto. For example, the peptide may be obtained as a recombinant by inserting DNA encoding the peptide into an appropriate expression system, or may be artificially synthesized, but is not limited thereto.

In one embodiment of the present invention, the peptide may be prepared using, but is not limited to, a human-derived peptide, a non-human-derived peptide, or a viral peptide.

As used throughout this specification, the terms “hydrophilic” and “hydrophobic” mean a region contained within a peptide while forming micelles, for example, in a state where each region can be confirmed to be phase separated, and the degree of each hydrophilicity or hydrophobicity may be relative.

In one embodiment of the present invention, the amino acid comprising and/or consisting of the amino acid sequence of SEQ ID NO: 1 may comprise and/or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with the amino acid sequence of SEQ ID NO: 1 of the present invention, but is not limited thereto.

In one embodiment of the present invention, the amino acid comprising and/or consisting of the amino acid sequence of SEQ ID NO: 2 may comprise and/or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with the amino acid sequence of SEQ ID NO: 2 of the present invention, but is not limited thereto.

In one embodiment of the present invention, the amino acid comprising and/or consisting of the amino acid sequence of SEQ ID NO: 31 may comprise and/or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with the amino acid sequence of SEQ ID NO: 31 of the present invention, but is not limited thereto.

In one embodiment of the present invention, the amino acid comprising and/or consisting of the amino acid sequence of SEQ ID NO: 42 may comprise and/or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with the amino acid sequence of SEQ ID NO: 42 of the present invention, but is not limited thereto.

In one embodiment of the present invention, the amino acid comprising and/or consisting of the amino acid sequence of SEQ ID NO: 43 may comprise and/or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology or identity with the amino acid sequence of SEQ ID NO: 43 of the present invention, but is not limited thereto.

As used throughout this specification, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences are related, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

In one embodiment of the present invention, the peptide may further comprise a hydrophobic substance at the amino terminus or the carboxyl terminus to form a micelle and/or a drug delivery vehicle, and specifically, may further comprise the hydrophobic substance at the amino terminus. Specifically, the hydrophobic substance may comprise one or more of hydrophobic moieties such as sterol, cholesterol, and fatty acid.

A second aspect of the present invention provides a peptide comprising an amino acid sequence of structural formula 4:

[Structural formula 4]

(X ³ ) _b X ² Q(X ¹ L) _a

In the above structural formula 4,

The above X ¹ , X ² and X ³ are each independently R (Arginine), H (Histidine), or K (Lysine),

The above L stands for Leucine,

The above Q stands for Glutamine,

The above a is an integer from 2 to 5,

The above b is an integer from 1 to 13.

The same parts as described above also apply to the above peptides.

The above structural formula 4 is obtained by changing the order of the domains in the amino acid sequence of structural formula 1 by setting '(X ¹ L) _a ', 'X ² Q', and '(X ³ ) _b ' as the first domain, the second domain, and the third domain, respectively. If structural formula 1 is arranged in the order of 'first domain-second domain-third domain', structural formula 4 is the reverse order of structural formula 1, and is arranged in the order of 'third domain-second domain-first domain'.

The above peptide may be a cationic peptide or a hydrophilic peptide. Additionally, the peptide may be a cell-penetrating peptide.

The above peptide may be for forming micelles and/or drug delivery vehicles. Specifically, the peptide may be for forming micelles and/or drug delivery vehicles by self-assembly. Accordingly, the present invention may provide micelles and/or drug delivery vehicles comprising the peptide.

In one embodiment, X ¹ , X ² and X ³ of the structural formula 4 can each independently be R (Arginine), H (Histidine), or K (Lysine), specifically, X ¹ , X ² and X ³ can each independently be R (Arginine) or K (Lysine), and more specifically, X ¹ , X ² and X ³ can all be R (Arginine) or all can be K (Lysine).

In one embodiment, a in the structural formula 4 may be an integer from 2 to 5, specifically an integer from 2 to 5, 2 to 4, 2 to 3, 3 to 5, 3 to 4 or 4 to 5, and more specifically, a may be 2, 3, 4 or 5.

In one embodiment, b in the structural formula 4 may be an integer from 1 to 13, and specifically, may be an integer from 1 to 13, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 2, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 13, 4 to 10, 4 to 8, or 4 to 6, and more specifically, b may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

In one embodiment, in the amino acid sequence having the structure of the structural formula 4, the terminal of X ³ may be the amino terminal (N`-terminal) or the carboxyl terminal (C`-terminal). In the case of a peptide in which the terminal of X ³ is the amino terminal, it may include an amino acid sequence having a structure of the structural formula 5 below, and in the case of a peptide in which the terminal of X ³ is the carboxyl terminal, it may include an amino acid sequence having a structure of the structural formula 6 below.

[Structural formula 5]

N`-(X <sup>3</sup> ) <sub>b</sub> X <sup>2</sup> Q(X <sup>1</sup> L) <sub>a</sub> -C`

[Structural formula 6]

N`-(LX <sup>1</sup> ) <sub>a</sub> QX <sup>2</sup> (X <sup>3</sup> ) <sub>b</sub> -C`

In the above structural formula 5 or 6,

The above X ¹ , X ² and X ³ are each independently R (Arginine), H (Histidine), or K (Lysine),

The above L stands for Leucine,

The above Q stands for Glutamine,

The above a is an integer from 2 to 5,

The above b is an integer from 1 to 13,

N' represents the amino terminus of the peptide, and C' represents the carboxyl terminus of the peptide.

In one embodiment, the peptide may include an amino acid sequence having at least one H (Histidine) additionally linked to at least one of the amino terminus and the carboxyl terminus of the amino acid sequence having the structure of the structural formula 4. The above H may further include or be connected to 1 to 21, specifically 1 to 21, 1 to 18, 1 to 15, 1 to 12, 1 to 9, 1 to 6, 1 to 3, 3 to 21, 3 to 18, 3 to 15, 3 to 12, 3 to 9, 3 to 6, 6 to 21, 6 to 18, 6 to 15, 6 to 12, 6 to 9, 9 to 21, 9 to 18, 9 to 15, 9 to 12, 12 to 21, 12 to 18, or 12 to 15 Hs.

In one embodiment of the present invention, the peptide may additionally comprise one or more hydrophobic amino acids at its N-terminus or C-terminus, and specifically, may additionally comprise one or more amino acids selected from the group consisting of A (Alanine), V (Valine), I (Isoleucine), and L (Leucine) at the N-terminus. The peptide may additionally comprise 1 to 20 hydrophobic amino acids.

In one embodiment, a hydrophobic peptide comprising the amino acid sequence of VLVALAIV (SEQ ID NO: 41) may be additionally linked to the N-terminus of the peptide, and the hydrophobic peptide may be directly linked or indirectly linked via a linker comprising one or more amino acid residues (e.g., P (Proline)).

When the linker is a proline residue, it can form a folded structure in the three-dimensional structure of the peptide. When the linker is a proline residue, it can include one or more prolines. In addition, the linker can be a glycine residue, and any amino acid residue that can form a folded structure in the three-dimensional structure of the peptide can be used without limitation.

In one embodiment of the present invention, the peptide may further comprise a hydrophobic substance at the amino terminus or the carboxyl terminus to form a micelle and/or a drug delivery vehicle, and specifically, may further comprise the hydrophobic substance at the amino terminus. Specifically, the hydrophobic substance may comprise one or more of hydrophobic moieties such as sterol, cholesterol, and fatty acid.

The third aspect of the present invention provides a micelle comprising a peptide comprising the amino acid sequence of the structural formula 1 or a peptide comprising the amino acid sequence of the structural formula 4. The same parts as described above are also applied to the micelle.

As used throughout this specification, “micelle” may mean a nano-sized particle having a core/shell structure due to the self-assembly properties of peptides.

In one embodiment of the present invention, the micelle may be cell permeable.

The term "cell permeability" as used throughout this specification means the ability or property of a peptide, a micelle comprising the same, or a complex or composition comprising the micelle, to penetrate a cell membrane and enter the interior of a cell.

In one embodiment of the present invention, the micelle may be for drug delivery, and specifically for delivering any drug to a specific targeting region (cell, tissue and/or organ).

In one embodiment of the present invention, the peptide may further include a hydrophobic substance at the amino terminus or the carboxyl terminus, and specifically, may further include one at the amino terminus. Specifically, the hydrophobic substance may include at least one of hydrophobic moieties such as sterol, cholesterol, and fatty acid. The fatty acid is a carboxylic acid including a saturated or unsaturated aliphatic chain having about 4 or more carbon atoms, and may be, for example, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, and the like. The above sterol may include animal sterols and derivatives such as cholesterol, cholesteryl chloride, cholesteryl octanoate, cholesteryl nonanoate, cholesteryl oleyl carbonate, and cholesteryl isostearyl carbonate; plant sterols and derivatives such as phytosterol, campesterol, sitosterol, and stigmasterol; or a combination thereof. For example, the additionally included hydrophobic substance may be included as a core of the micelle to further increase the stability of the micelle.

In one embodiment of the present invention, the peptide may further comprise a linker for linking a targeting agent, a labeling agent or a targeting/labeling agent to the amino terminal or the carboxyl terminal, and specifically, may further comprise a linker at the carboxyl terminal.

The above targeting agent refers to a agent capable of targeting a region (cell, tissue and/or organ) to which the micelle is to be delivered, and the targeting agent may include a ligand capable of binding to a receptor present in the targeting region or a receptor capable of binding to a ligand present in the targeting agent.

The targeting agent may be directly bound to the peptide, or may be linked via a linker for linking the targeting/labeling agent. The linker may refer to a material used to effectively link (bind) the targeting/labeling agent to the peptide.

Therefore, micelles containing the above peptides can exhibit high targeting efficiency and can be easily tracked in vitro and in vivo .

In one embodiment of the present invention, the micelle comprising the peptide can be easily tracked in vitro and in vivo by binding to a fluorescent substance such as a fluorescent functional group, an isotope substance, or a targeting ligand. In one embodiment of the present invention, the micelle comprising the peptide can be tracked by binding to a fluorescent substance such as a fluorescent functional group, an isotope substance, and can also target a specific cell or organ by binding to a targeting ligand.

The fourth aspect of the present invention provides a drug delivery complex comprising the micelle; and a desired drug. The same parts as described above also apply to the drug delivery complex.

In one embodiment of the present invention, the drug may be a nucleic acid, a protein, or a compound.

In one embodiment of the present invention, the drug may be any one selected from the group consisting of DNA, mRNA, siRNA, microRNA, ASO, gapmer, aptamer, antagomir, agomir, and oligonucleotide, but is not limited thereto. Specifically, the drug may be siRNA.

The compound may be any one selected from the group consisting of, but is not limited to, fats, carbohydrates, dyes, photosensitizers, anticancer agents, antibiotics, and low molecular weight compounds. The protein may be any one selected from the group consisting of, but is not limited to, enzymes, ligands, hormones, carriers, immunoglobulins, antibodies, structural proteins, motor peptides, receptors, signaling peptides, storage peptides, membrane peptides, transmembrane peptides, internal peptides, external peptides, secreted peptides, viral peptides, native peptides, glycosylated proteins, fragmented proteins, disulfide bond proteins, recombinant proteins, and chemically modified proteins.

In one embodiment of the present invention, the drug can electrostatically bind to the peptide. In one embodiment of the present invention, since the peptide has a positive charge, a drug having a negative charge can be utilized by electrostatically binding to the peptide without limitation, as long as the present invention can be applied. In one embodiment of the present invention, the drug can electrostatically bind to the hydrophilic amino acid residue. In one embodiment of the present invention, since the hydrophilic amino acid residue has a positive charge, a drug having a negative charge can be utilized by electrostatically binding to the hydrophilic amino acid residue without limitation, as long as the present invention can be applied.

In one embodiment of the present invention, the drug may be mutually combined with the peptide within the micelle to form a complex or a complex, or may not be mutually combined but may be mixed with each other to form a non-covalent complex, and may be applied without limitation as long as it can improve cell permeability without inhibiting the pharmacological activity of the drug.

In one embodiment of the present invention, the conjugate may be one in which the peptide and the drug are chemically bonded, or physically bonded, for example, covalently bonded or non-covalently bonded, and rapidly and safely penetrate into cells through in vivo or in vitro processing. For example, the composition in which the peptide and the drug are bound may be introduced into cells directly through the endocytosis process, which is a conventional cellular absorption method, or without such a process, but is not limited thereto.

Specifically, since the peptide has a positive charge, it can electrostatically bind to siRNA that has a negative charge.

In one embodiment of the present invention, the micelle and the target drug (specifically, nucleic acid, etc.) may be included in the drug delivery vehicle at a mass ratio (w/w) of 0.1:1 to 10:1, and specifically, 0.1:1 to 10:1 (w/w), 0.1:1 to 8:1 (w/w), 0.1:1 to 6:1 (w/w), 0.1:1 to 5:1 (w/w), 0.1:1 to 4:1 (w/w), 0.1:1 to 3:1 (w/w), 0.1:1 to 2:1 (w/w), 0.1:1 to 1:1 (w/w), 0.5:1 to 10:1 (w/w), 0.5:1 to 8:1 (w/w), 0.5:1 to 6:1 (w/w), 0.5:1 to 5:1 (w/w), 0.5:1 to 4:1 (w/w), 0.5:1 to 3:1 (w/w), 0.5:1 to 2:1 (w/w), 0.5:1 to 1:1 (w/w), 1:1 to 10:1 (w/w), 1:1 to 8:1 (w/w), 1:1 to 6:1 (w/w), 1:1 to 5:1 (w/w), 1:1 to 4:1 (w/w), 1:1 to 3:1 (w/w), 1:1 to 2:1 (w/w), 2:1 to 10:1 (w/w), 2:1 to 8:1 (w/w), 2:1 to 6:1 (w/w), 2:1 to 5:1 (w/w), or It may be included in a mass ratio of 2:1 to 3:1 (w/w).

In one embodiment of the present invention, the drug delivery complex may be capable of simultaneously delivering two or more different types of drugs into cells. For example, the drug delivery complex may be capable of simultaneously delivering two or more different types of siRNA into cells.

In one embodiment of the present invention, the drug delivery complex can be easily tracked in vitro and in vivo by a fluorescent material such as a fluorescent functional group, an isotope material, etc. In addition, the drug delivery complex to which a targeting ligand is bound can be easily and effectively targeted. For example, the drug delivery complex to which folate is bound can be easily and effectively targeted to a specific cell or organ.

The fifth aspect of the present invention provides a drug delivery composition comprising the peptide, micelle or drug delivery complex. The same parts as described above are also applicable to the composition.

The sixth aspect of the present invention provides a pharmaceutical composition for preventing or treating a proliferative disease, comprising the micelle; and siRNA. The same parts as described above are also applied to the composition.

In one embodiment of the present invention, the proliferative disease may include, but is not limited to, one or more selected from the group consisting of neoplasms (including intraepithelial neoplasms), tumors, cancers, leukemias, psoriasis, bone diseases, fibroproliferative disorders, and atherosclerosis.

In one embodiment of the present invention, the cancer may be a solid tumor or a blood cancer. The above cancers are basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, peritoneal cancer, choriocarcinoma, connective tissue cancer, digestive organ cancer, endometrial cancer, esophageal cancer, eye cancer, colon cancer, rectal cancer, kidney cancer, melanoma, stomach cancer, liver cancer, lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma), colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, respiratory cancer, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, urinary system cancer, vulvar cancer, oral cancer, leukemia, myeloma, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, brain tumor, glioblastoma, neuroblastoma, rhabdomyosarcoma, retinoblastoma, head and neck cancer, salivary gland cancer, Hodgkin's lymphoma, non-Hodgkin's It may include, but is not limited to, one or more selected from the group consisting of lymphoma, mantle cell lymphoma, AIDS-associated lymphoma, Waldenstrom macroglobulinemia, post-transplant lymphoproliferative disorder, nevus, Meigs syndrome, and lymphoma.

In one embodiment of the present invention, the pharmaceutical composition can be used for the prevention or treatment of age-related macular degeneration (wet or dry macular degeneration), a connective tissue growth factor-related disease or disorder (including but not limited to keloids, hypertrophic scars, fibrosis, etc.), a hair loss disease or a pigmentation-related disorder. Here, the keloid can be a burn keloid, a posttraumatic keloid, and an abnormal proliferation of other types of scar tissue. Non-limiting examples of fibrosis include renal fibrosis, retinal fibrosis, pulmonary fibrosis, liver fibrosis, systemic sclerosis, pachydermatosis, or dermal fibrosis.

The term "cancer" as used throughout this specification is a general term for a disease caused by cells that have aggressive characteristics in which cells divide and proliferate while ignoring normal growth limits, invasive characteristics in which cells infiltrate surrounding tissues, and metastatic characteristics in which cells spread to other parts of the body, and can be used with the same meaning as a malignant tumor.

In addition, "treatment of cancer" means inhibiting or preventing the growth of cancer cells or tissues, and this also includes reducing the growth and metastasis of cancer and reducing resistance to anticancer drugs to enhance the treatment effect compared to when no treatment or processing is performed. The cancer metastasis refers to the process in which tumor (cancer) cells spread to distant parts of the body, and "resistance to anticancer drugs" or "anticancer drug resistance" refers to the absence of a therapeutic effect from the beginning of treatment when treating a cancer patient with an anticancer drug, or the initial cancer treatment effect is lost over the course of continued treatment. "Prevention" may refer to any act of inhibiting the occurrence of cancer or delaying its onset by administering the pharmaceutical composition.

The pharmaceutical composition of the present invention may include a conventional, non-toxic, pharmaceutically acceptable carrier that is formulated into a formulation according to a conventional method.

The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

The term "pharmaceutically acceptable carrier" as used throughout the present specification means a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers include those that are sterile and biocompatible, and include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as sweeteners, solubilizers, wetting agents, emulsifiers, isotonic agents, absorbents, antioxidants, preservatives, lubricants, fillers, buffers, and bacteriostatic agents may be added.

The composition of the present invention can be prepared in various dosage forms for parenteral administration (e.g., intramuscular, intravenous or subcutaneous injection). When the pharmaceutical composition of the present invention is prepared in a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation and suppositories according to methods known in the art together with a suitable carrier.

Injectable preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, and the like. Meanwhile, injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

Formulation of pharmaceutical compositions is well known in the art, and reference can be made to references, such as Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

The composition of the present invention may be administered to a patient in a therapeutically effective amount or a pharmaceutically effective amount.

The term "administration" as used throughout this specification means introducing a given substance into a subject in an appropriate manner, and the route of administration of the composition may be administered through any common route as long as it can reach the target tissue. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, topically, intranasally, or rectally, but is not limited thereto.

Here, the term "therapeutically effective amount" or "pharmaceutically effective amount" means an amount of the composition that is effective in preventing or treating the target disease, and is sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

Specifically, the therapeutically effective amount refers to an amount of a drug that is effective in treating a proliferative disease (a non-limiting example, cancer). The scope of the present invention is not limited thereto, as it may increase or decrease depending on the route of administration, severity of the disease, gender, weight, age, etc.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. At this time, the other therapeutic agent may additionally include any compound or natural extract that has already been verified as safe and known to have anticancer activity in order to increase or enhance anticancer activity. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount of side effects or without side effects, and this can be easily determined by those skilled in the art.

The seventh aspect of the present invention provides a method for preventing or treating a proliferative disease, comprising the step of administering to a subject a pharmaceutical composition for preventing or treating the proliferative disease. The same parts as described above are also applicable to the method.

The term "subject" as used herein may include, without limitation, mammals, birds, reptiles, farmed fish, etc., including dogs, cats, rats, livestock, humans, etc., that are afflicted with or at risk of developing cancer, and the subject may exclude humans.

The pharmaceutical composition above can be administered in single or multiple doses in a pharmaceutically effective amount. At this time, the composition can be formulated and administered in the form of a solution, powder, aerosol, injection, infusion (Ringel), capsule, pill, tablet, suppository or patch. The route of administration of the pharmaceutical composition for preventing or treating cancer can be administered through any general route as long as it can reach the target tissue.

The pharmaceutical composition above is not particularly limited thereto, but may be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, and rectal administration depending on the intended purpose. However, when administered orally, it may be administered in an unformulated form, and since the active ingredient of the pharmaceutical composition may be denatured or decomposed by gastric acid, the oral composition may be administered orally in a form that coats the active agent or is formulated to protect it from decomposition in the stomach, or in the form of an oral patch. In addition, the composition may be administered by any device that allows the active ingredient to move to the target cell.

The eighth aspect of the present invention provides a use of a micelle comprising said peptide for use in delivering a drug, and a drug delivery complex comprising said micelle. The same as described above applies to said use as well.

The ninth aspect of the present invention provides a use of the pharmaceutical composition for preventing or treating a proliferative disease, for use in preventing or treating a proliferative disease. The same parts as described above apply to the above use.

The tenth aspect of the present invention provides a use of a pharmaceutical composition for preventing or treating a proliferative disease in the manufacture of a medicament for preventing or treating a proliferative disease. The same parts as described above apply to the above use.

The eleventh aspect of the present invention provides a polynucleotide comprising a sequence encoding a peptide comprising an amino acid sequence of the structural formula 1 or a peptide comprising an amino acid sequence of the structural formula 4. The same parts as described above are also applied to the polynucleotide.

The polynucleotide may undergo various modifications to the coding region within a range that does not change the amino acid sequence of the peptide expressed from the coding region due to the degeneracy of the codon or in consideration of the codon preferred in an organism that is to express the peptide, and various modifications or alterations may also be made in a portion excluding the coding region within a range that does not affect the expression of the coding sequence, and it will be readily understood by those skilled in the art that such modified coding sequences are also included in the scope of the present invention. That is, a polynucleotide according to one aspect may undergo mutations by substitution, deletion, insertion or a combination of these of one or more nucleic acid bases, as long as it encodes a peptide or protein having an activity equivalent thereto, and these are also included in the scope of the present invention.

Hereinafter, the present invention will be described in more detail using examples. However, the following examples are provided only to help understand the present invention, and the contents of the present invention are not limited to the following examples.

[Example]

Manufacturing Example 1. Manufacturing of amphipathic peptide

To prepare an amphipathic peptide, a hydrophilic cell-penetrating peptide sequence RLRLRQRRRR (SEQ ID NO: 1) was combined with a peptide sequence VLVALAIV (SEQ ID NO: 41), which is known as a hydrophobic moiety derived from the MUC1 transmembrane domain. In addition, an amphipathic peptide was prepared by adding a proline residue between the hydrophilic peptide and the hydrophobic peptide. The amphipathic peptide was prepared using a solid-phase synthesis (SPPS) method by requesting Wellpep Co., Ltd. (Korea), and the specific sequence is as described in Table 2 below.

name Amino acid sequence SCL0003 VLVALAIVRLRLRQRRRR (SEQ ID NO: 42) SCL0008 VLVALAIV P RLRLRQRRRR (SEQ ID NO: 43)

Manufacturing Example 2. Manufacturing of an amphipathic peptide bound to a hydrophobic substance

A peptide was prepared by binding cholesterol, as an example of a hydrophobic substance, to the amino terminal of the amphipathic peptide prepared in Manufacturing Example 1. The amphipathic peptide bound to cholesterol was manufactured by request to Wellpep Co., Ltd. (Korea), and its specific composition is as described in Table 3 below.

name structure SCL1001 cholesterol -VLVALAIVRLRLRQRRRR (SEQ ID NO: 42) SCL2001 cholesterol -VLVALAIV P RLRLRQRRRR (SEQ ID NO: 43)

Manufacturing Example 3. Manufacturing of micelles containing amphipathic peptides

The 10 mg/mL amphiphilic peptides (SCL0003 and SCL0008) manufactured in Manufacturing Example 1 and the 10 mg/mL hydrophobic substance-conjugated amphiphilic peptides (SCL1001 and SCL2001) manufactured in Manufacturing Example 2 were each diluted in PBS (Gibco, 10010023) to a final concentration of 25 μg/mL to manufacture the respective amphiphilic peptide-based micelles, SCM0003 (manufactured using SCL0003), SCM0008 (manufactured using SCL0008), SCM1001 (manufactured using SCL1001), and SCM2001 (manufactured using SCL2001).

Manufacturing Example 4. Manufacturing of cationic cell-penetrating peptide combined with hydrophobic substance

A peptide was prepared by conjugating stearic acid (represented as C18 in this specification) as an example of a hydrophobic substance to the amino terminus of the hydrophilic cell-penetrating peptide sequence RLRLRQRRRR (SEQ ID NO: 1) or a variant thereof [sequence in which R is replaced with K, KLKLKQKKKK (SEQ ID NO: 2)] included in the amphipathic peptide of Manufacturing Example 1. The cationic cell-penetrating peptide conjugated with stearic acid was manufactured by request to Wellpep Co., Ltd. (Korea), and its specific composition is as described in Table 4 below.

name structure SCL1016 C18 -KLKLKQKKKK (SEQ ID NO: 2) SCL1020 C18 -RLRLRQRRRR (SEQ ID NO: 1)

Manufacturing Example 5. Manufacturing of micelles containing PEGylated lipids and cationic cell-penetrating peptides

In Manufacturing Example 4, cationic peptides (SCL1016 and SCL1020) conjugated with hydrophobic substances were mixed with 50 mg/mL of PEGylated lipids in ethanol, then dropped into 1 mL of PBS (Gibco, 10010023), shaken well, and diluted to a final concentration of 1 mg/mL to prepare SCM1016 micelles (manufactured using SCL1016) and SCM1020 micelles (manufactured using SCL1020).

Example 1. Formation and size confirmation of micelles containing amphipathic peptides

Example 1.1 Confirmation of formation of micelles containing amphipathic peptides

To confirm whether micelles containing the amphipathic peptide manufactured in Manufacturing Example 3 could be formed, the following experiment was performed.

To determine the critical micelle concentration (CMC) of the micelle containing the amphiphilic peptide prepared in Manufacturing Example 3, fluorescence spectroscopy using pyrene (Sigma, 48570) as a probe was used. Specifically, the amphiphilic peptide prepared in acetonitrile and pyrene were mixed, and then acetonitrile was removed through air drying. The sample was reconstituted by adding distilled water, and the fluorescence intensity was measured using a spectrofluorometer (varioskan lux, Thermo-fisher) at wavelengths of Ex = 330 nm, Em(I1) = 372 nm, and Em(I2) = 392 nm. The intensity ratio (I392 nm/I372 nm) was expressed as a logarithmic function of the peptide concentration.

As shown in Fig. 1, an experiment was conducted using pyrene as a probe to confirm the critical micelle concentration, and it was confirmed that micelles were formed at a concentration of 25 μg/mL or higher for SCL1001 and SCL2001.

Example 1.2 Confirmation of formation of micelles containing cationic cell-penetrating peptides

To confirm whether micelles containing the cationic cell-penetrating peptide manufactured in Manufacturing Example 4 could be formed, the following experiments were performed.

The CMC of micelles containing SCL1020, one of the cationic cell-penetrating peptides manufactured in Manufacturing Example 4, was confirmed by absorbance spectroscopy using pyrene (Sigma, 48570) as a probe. Specifically, as in Manufacturing Example 5, the peptide was manufactured to a concentration of 50 mg/mL in ethanol, mixed with a PEGylated lipid, and then dropped into 1 mL of PBS (Gibco, 10010023) to manufacture SCM1020 at each concentration.

After adding pyrene (0.01 mg/mL) and micelles at various concentrations to the microplate, the absorbance was measured at wavelengths of 323 nm and 339 nm using a microplate reader (MOLECULAR DEVICES, Spectra Max iD3) when the reaction was completed for 1 to 3 hours. The absorbance was expressed as a logarithmic function of the peptide concentration, and Boltzmann regression was performed using the Prism program (GraphPad). After confirming the four parameters (Bottom, Top, V50, Slope) derived through Boltzmann regression, if V50/Slope < 10, CMC was equal to V50, and if V50/Slope > 10, CMC was calculated as V50 + 2xSlope.

As shown in Fig. 2, an experiment was conducted using pyrene as a probe to confirm the CMC, and it was confirmed that micelles were formed at a concentration of 149 μg/mL or higher.

Example 1.3. Determination of the size of micelles containing amphipathic peptides

Each micelle liquid phase prepared in Manufacturing Example 3 at a final concentration of 25 ㎍/mL in 1 mL of PBS was shaken well to prepare a dispersion, and then the particle size was measured. Specifically, the particle size was measured by the photon correlation spectroscopy (QELS method) using a Zetasizer (ZS90, Malvern, UK), and the same sample was measured at least five times to obtain the average value, and the results are shown in Table 5 below.

Particle Size (nm) Standard Deviation SCM0003 342.6 23.2 SCM0008 265.7 15.1 SCM1001 20.1 3.8 SCM2001 20.5 10.6

As shown in Table 4 and Fig. 3 above, it was confirmed that micelles containing amphipathic peptides at a concentration of 25 μg/mL exhibited a particle size of about 20 nm.

Example 1.4. Size determination of micelles containing PEGylated lipids and cationic cell-penetrating peptides

In Manufacturing Example 5, each micelle liquid phase manufactured to a final concentration of 1 mg/mL was shaken well to disperse, and then the particle size was measured. Specifically, the particle size was measured by photon correlation spectroscopy (PCS method) using a Zetasizer (Zetasizer Pro Blue Label, Malvern, UK), and the same sample was measured more than 5 times to obtain the average value, and the results are shown in Table 6 below.

Particle Size (nm) Standard Deviation SCM1016 7.9 0.2 SCM1020 9.8 0.6

As shown in Table 5 and Figure 4 above, it was confirmed that micelles containing PEGylated lipids and cationic peptides at a concentration of 1 mg/mL exhibited a particle size of about 10 nm.

Example 2. Formation and size confirmation of micelle-siRNA complexes

Example 2.1 Confirmation of formation of micelle-siRNA complex

In order to determine the optimal mixing ratio for micelles containing amphipathic peptides (SCL0008, SCL0003), micelles containing amphipathic peptides bound to hydrophobic substances (SCL1001, SCL2001), and micelles containing cationic cell-penetrating peptides bound to hydrophobic substances (SCL1016, SCL1020) to form complexes with siRNA, the degree of complex formation according to each ratio (micelle:siRNA) was determined.

Analysis was performed using a gel retardation assay, and siRNA was stained with GelRed Nucleic Acid stain and visualized by GelDoc Go imaging System (BMS, BR12009077). siRNA When mixed (complexed or conjugated) with micelles, siRNA is not observed when stained with GelRed Nucleic Acid due to the interference effect of the peptide.

As a result, as shown in Figures 5 and 6, it was confirmed that in the case of low concentrations of peptide, binding with siRNA was not complete, and that the complex of siRNA and peptide was completely formed when a certain concentration of peptide was included.

Example 2.2 Size determination of amphipathic peptide-based micelle-siRNA complexes

Each micelle prepared in Preparation Example 3 at a final concentration of 25 μg/mL and 200 nM siRNA liquid were mixed well in 1 mL of PBS to prepare a dispersion, and then the particle size was measured. Specifically, the particle size was measured by the photon correlation spectroscopy (QELS) method using a Zetasizer (ZS90, Malvern, UK), and the same sample was measured at least five times to obtain the average value, and the results are shown in Table 7 below.

Particle Size (nm) Standard Deviation SCM0003/siRNA 1046.6 83.2 SCM0008/siRNA 563.3 44.1 SCM1001/siRNA 60.9 2.4 SCM2001/siRNA 72.4 7.2

As shown in Table 7 and Figure 7 above, it was confirmed that the micelle-siRNA complex containing the amphipathic peptide at a concentration of 25 μg/mL exhibited a particle size of about 60 to 75 nm.

Example 2.3. Size determination of cationic peptide-based micelle-siRNA complexes

In Manufacturing Example 5, each micelle and siRNA prepared at a final concentration of 1 mg/mL were mixed well in a 3:1 (w/w) ratio by shaking to prepare a dispersion, and then the particle size was measured. Specifically, the particle size was measured by photon correlation spectroscopy (PCS method) using a Zetasizer (Zetasizer Pro Blue Label, Malvern, UK), and the same sample was measured at least five times to obtain the average value, and the results are shown in Table 8 below.

Particle Size (nm) Standard Deviation SCM1016/siRNA 22.7 1.9 SCM1020/siRNA 35.2 2.4

As shown in Table 8 and Figure 8, the micelle-siRNA complex containing the PEGylated lipid and the cationic cell-penetrating peptide prepared at a ratio of 3:1 (w/w) was confirmed to exhibit a particle size of about 23 to 35 nm.

Example 2.4 Morphological analysis of micelle-siRNA complexes

The morphology of micelles containing an amphipathic peptide (SCM2001) bound to a hydrophobic substance and siRNA complexes was observed using a transmission electron microscope (H-7600 (Hitachi)). Specifically, 1 mL each of 25 ㎍/mL SCM2001 and 25 ㎍/mL SCM2001/400 nM siRNA complexes were prepared and incubated at room temperature for 30 minutes. Subsequently, the samples were loaded onto a carbon grid, stained with 1% uranyl acetate for 10 seconds, and washed with distilled water. The samples were dried for 5 minutes and then observed under an electron microscope.

As shown in Fig. 9, the micelle-siRNA complex containing the amphiphilic peptide bound to the hydrophobic substance was confirmed to exhibit a uniform size distribution (less than 100 nm). Through this, it was found that the micelle-siRNA complex containing the amphiphilic peptide had a relatively well-distinguished spherical shape and did not aggregation, which facilitated its absorption into cells.

Example 3. Measurement of zeta potential changes of amphipathic peptide micelle-siRNA complexes

Each micelle (not including siRNA) manufactured in Preparation Example 3 at a final concentration of 25 μg/mL and each micelle liquid phase containing 200 nM siRNA manufactured in Example 2.1 were mixed well by shaking in 1 mL of PBS to prepare a dispersion, and then the zeta potential was measured. Specifically, the zeta potential of the particles was measured using a Zetasizer (ZS90, Malvern, UK), and the same sample was measured at least five times to obtain the average value, and the results are shown in Table 9 below.

without siRNA with siRNA Zeta potential
(mV) SD Zeta potential
(mV) SD SCM0003 15.3 2.2 -5.5 4.8 SCM0008 11.9 4.5 -10.5 0.7 SCM1001 14.5 4.5 17.8 1.3 SCM2001 12.6 2.7 19.7 2.5

As shown in Table 9 above, the micelles containing SCM1001 and SCM2001 were confirmed to be structurally stable as evidenced by a further increase in the zeta potential value after forming a complex with siRNA.

Example 4. Confirmation of cytotoxicity of micelles containing amphipathic peptides

To determine the cytotoxicity of micelles containing amphipathic peptides, Capan-1 cells (KCLB, NO. 30079) were treated with peptides and Lipofectamine ^TM 2000 (Invitrogen ^TM , 11668027) as a control at concentrations of 0, 5, 10, 20, and 40 ㎍/mL, respectively, and cultured in a CO ₂ incubator for 24 h.

At the point when the culture was completed, WST-1 (Water Soluble Tetrazolium salt-1, EZ-assay kit, Duzen Bio) analysis was performed. Specifically, 0.1 mL of WST-1 solution was added to the wells of cells where additional culture was completed, and after culturing for 3 hours, the absorbance was measured at 450 nm using a Microplate Spectrophotometer (Multiskan™ GO, thermo).

As shown in Fig. 10, it was confirmed that the cell viability was significantly higher when treated with SCM1001 micelle or SCM2001 micelle in a concentration-dependent manner compared to Lipofectamine ^TM 2000 transfection reagent.

In addition, as shown in Fig. 11, when the Lipofectamine ^TM 2000 transfection reagent was treated at a concentration of 40 ㎍/mL, a change in the cell morphology occurred, but when the micelle containing SCM1001 or SCM2001 was treated at the same concentration, it was confirmed that there was no change in the cell morphology, respectively.

Through this, it was found that SCM1001 micelle or SCM2001 micelle did not exhibit cytotoxicity.

Example 5. Confirmation of cytotoxicity of amphipathic peptide-based micelle-siRNA complexes

To examine the cytotoxicity of micelle-siRNA complexes containing amphipathic peptides, Capan-1 cell line (KCLB, NO. 30079) was treated with SCM1001/siRNA, SCM2001/siRNA complexes, and Lipofectamine ^TM 2000 (Invitrogen ^TM , 11668027)/siRNA complexes as a control at concentrations of 0, 5, 10, 20, and 40 μg/mL, respectively, and cultured in a CO ₂ incubator for 24 h.

At the point when the culture was completed, WST-1 (Water Soluble Tetrazolium salt-1, EZ-assay kit, Duzen Bio) analysis was performed. Specifically, 0.1 mL of WST-1 solution was added to the well of cells where additional culture was completed, and after culturing for 3 hours, the absorbance was measured at 450 nm using a Microplate Spectrophotometer (Multiskan™ GO, thermo).

As shown in Fig. 12, it was confirmed that the cell viability was significantly higher when micelles containing SCM1001 or SCM2001 were treated in a concentration-dependent manner compared to the Lipofectamine ^TM 2000 transfection reagent.

In addition, as shown in Fig. 13, when the cells were treated with 200 nM siRNA and 40 ㎍/mL of Lipofectamine ^TM 2000 transfection reagent, a change in the cell morphology occurred, but when the micelle-siRNA complex containing SCM1001 or SCM2001 was treated at the same concentration, it was confirmed that there was no change in the cell morphology, respectively.

Through this, it was found that micelle-siRNA complexes containing SCM1001 or SCM2001 did not exhibit cytotoxicity.

Example 6. Confirmation of cell internalization and endosomal escape ability of micelle-siRNA complexes

The cellular internalization and endosomal escape ability of micelle-siRNA complexes containing amphipathic peptides were confirmed. Specifically, 1.5 × 10 ⁴ Capan-1 (KCLB, NO. 30079) cells were dispensed into each well of an optical 96-well plate (M0562, Greiner) and cultured for 24 h.

After incubating the SCM1001/FAM-siRNA complex, SCM2001/FAM-siRNA complex, and the control Lipofectamine ^TM 2000 (Invitrogen ^TM , 11668027)/FAM-siRNA complex for 6 hours, 1 uM LysoTracker ^TM Deep Red (L12492, Invitrogen ^TM ) was added, and the cell penetration rate and distribution were confirmed using a super resolution confocal laser microscope (LSM800, super resolution confocal laser microscope).

As shown in Fig. 14, when treated with Lipofectamine ^TM 2000, it was confirmed that most of it was entrapped in lysosomes, which are red in color, and that a relatively small amount existed in the cytoplasm. On the other hand, when treated with micelle-siRNA complexes containing SCM1001 or SCM2001, it was confirmed that FAM-siRNA, which is green in color, was distributed throughout the cytoplasm.

Through this, it was found that micelle-siRNA complexes containing SCM1001 or SCM2001 had excellent cell internalization and endosomal escape capabilities.

Example 7. Measurement of serum stability of micelle-siRNA complexes

To confirm the serum stability of the micelle-siRNA complex containing the amphipathic peptide, the stability was measured using Fetal Bovine Serum (16000044, gibco). Specifically, SCM1001 at a concentration of 25 ㎍/mL or 50 ㎍/mL was mixed with 400 nM siRNA and PBS (0.5 mL) to form micelle-siRNA complexes, which were then mixed with heat-inactivated FBS and incubated at 37°C. Samples were collected at the desired time points (24 h, 48 h, and 72 h), 10 uL per sample was collected, mixed with 2 uL 6X Loading dye, and electrophoresis was performed on a 1.5% agarose gel at 100 V for 30 minutes.

As shown in Fig. 15, the micelle-siRNA complex containing SCM1001 was confirmed to be stable for 72 hours in 50% FBS, indicating that the micelle-siRNA complex produced in the present invention has excellent serum stability.

Example 8. Confirmation of the target protein expression inhibition effect of amphipathic peptide-based micelle-siRNA complex

To evaluate the target protein expression inhibition efficacy of the amphiphilic peptide-based micelle-siRNA complex of the present invention, the following experiments were performed.

Specifically, HeLa cells were transfected with R6 siRNA (AM51334, ambion) or R5 siRNA (#4390826, ambion), which are known to target KRAS protein, using micelles containing Lipofectamin2000 (LIPO2K), SCM1001, or SCM2001, and 48 hours later, Western blot was performed to confirm the efficiency of siRNA-mediated inhibition of KRAS protein expression (#sc-30, santa cruz). KRAS protein expression was induced by sequential treatment with the following agents: control, 30 nM of R6 siRNA, 5 nM of R5 siRNA, Lipo2K and 30 nM of R6 siRNA, SCM1001 and 30 nM of R6 siRNA, SCM2001 and 30 nM of R6 siRNA, LIPO2k and 5 nM of R5 siRNA, SCM1001 and 5 nM of R5 siNRA, and SCM2001 and 5 nM of R5 siRNA. KRAS and β-Actin (#4967S, CST) were used as a loading control. The analysis equipment used was the iBright750 Imaging System (invitrogen).

As a result, as shown in Fig. 16, it was confirmed that the expression of KRAS protein was effectively suppressed when treated with micelle-siRNA complexes containing SCM1001 or SCM2001.

In addition, as shown in Fig. 17, when the micelle-siRNA complex containing SCM1001 or SCM2001 was treated, the results of the Western blot normalization showed that both types of siRNA showed about 80% or about 50% inhibition efficiency of KRAS expression compared to the control group.

Example 9. Confirmation of the target protein expression inhibition effect of cationic peptide-based micelle-siRNA complex

To evaluate the target protein expression inhibition efficacy of the cationic peptide-based micelle-siRNA complex of the present invention, the following experiments were performed.

Specifically, transfection was performed using a micelle complex containing R5 siRNA (#4390824, ambion) known to target KRAS protein as an example in HeLa cells, and SCM1016, SCM1020, and SCM1023, and the mass ratios of micelles and siRNA in the complexes were 5:1 and 3:1, respectively. After 48 hours, Western blot was performed to confirm the efficiency of siRNA-mediated inhibition of KRAS protein expression (415700, Invitrogen). Meanwhile, the SCM1023 is a micelle manufactured using SCL1023 (RLRLRQRRRRHHHHHH, SEQ ID NO: 31), a cationic peptide, as an example of a peptide having one or more histidines added to the amino terminus and/or carboxyl terminus, through the method described in Manufacturing Example 5.

First, KRAS protein expression was suppressed by treating with Control, siRNA 40 ng and Lipofectamin2000 (LIPO2K) complex, siRNA 40 ng and SCM1016 micelle-complex, or siRNA 40 ng and SCM1020 micelle-complex. To confirm the expression of KRAS protein, KRAS antibody (415700, Invitrogen) and β-Actin (#) as a loading control were used.　MA5-15739, Invitrogen) were used at ratios of 1:500 and 1:1000, respectively, and HRP-conjugated anti-mouse secondary antibody (# 31430, Invitrogen) was used at a ratio of 1:1000 for detection. Detection reagent (34075, thermo fisher) was used for detection of each protein band, and the iBright750 Imaging System (Invitrogen) was used as the analysis equipment.

As shown in Figure 18, it was confirmed that the expression of KRAS protein was effectively inhibited when treated with micelle-siRNA complexes containing SCM1016 or SCM1020.

In addition, as shown in Fig. 19, when the micelle-siRNA complex containing SCM1016 or SCM1020 was treated, compared to the control group, SCM1016 inhibited KRAS expression by about 80% at 3:1, and SCM1020 inhibited KRAS expression by about 90% at 3:1.

Next, the expression of KRAS protein was confirmed after inducing inhibition of KRAS protein expression by treating Control, siRNA 40 ng and Lipofectamin2000 (LIPO2K) complex, and siRNA 40 ng and SCM1023 micelle complex in the same manner as above.

As shown in Figures 20 and 21, it was confirmed that the expression of KRAS protein was effectively suppressed even when treated with micelle-siRNA complexes containing SCM1023.

Example 10. Confirmation of multi-siRNA intracellular delivery effect of micelle-siRNA complex

To confirm whether the micelle of the present invention can effectively deliver multiple siRNAs into cells by forming a complex with two or more siRNAs, the following experiments were performed.

Specifically, siRNA (Bioneer, Korea) conjugated with two types of fluorescent materials (cy5, FAM) respectively was produced and transfected into Capan-1 (KCLB, NO. 30079) cells using Lipofectamin2000 (LIPO2K), SCM1001, or micelles containing SCM2001. Thereafter, the cells were observed using a confocal microscope (LS800) (Fig. 22).

As a result, as shown in Fig. 22, Lipo2K tended to deliver more FAM-siRNA than Cy5-siRNA into cells, but it was confirmed that micelles containing SCM1001 or SCM2001 delivered FAM-siRNA and Cy5-siRNA into cells at the same ratio. Based on this, it can be seen that the micelle of the present invention can effectively deliver two or more types of multiple siRNAs into cells simultaneously.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single component may be implemented in a distributed manner, and likewise, components described as distributed may be implemented in a combined manner.

The scope of the present application is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present application.

<Task information>

[National Research and Development Project 1 that supported this invention]

[Task ID] 1425174451

[Task Number] S3314019

[Ministry Name] Ministry of SMEs and Startups

[Name of the task management (specialized) organization] Small and Medium Business Technology Information Promotion Agency

[Research Project Name] Startup Growth Technology Development

[Research Project Name] Development of a Low-Temperature Process-Based Sphere Microneedle Patch for High-Active Vaccine Delivery

[Name of the organization performing the task] Eskin

[Research Period] 2023.01.01 ~ 2023.12.31

[National Research and Development Project 2 that supported this invention]

[Task ID] 1425178753

[Task Number] 00261713

[Ministry Name] Ministry of SMEs and Startups

[Name of the task management (specialized) organization] Small and Medium Business Technology Information Promotion Agency

[Research Project Name] Startup Growth Technology Development

[Research Project Name] Cell-Permeable Peptide Micelles-Based Target-Directed Multiplexed RNAi New Drug Platform

[Name of the organization performing the task] Eskin

[Research Period] 2023.06.01 ~ 2024.05.31

[National Research and Development Project 3 that supported this invention]

[Task ID] 1425172554

[Task Number] S3312967

[Ministry Name] Ministry of SMEs and Startups

[Name of the task management (specialized) organization] Small and Medium Business Technology Information Promotion Agency

[Research Project Name] Development of Commercialization Technology for Small and Medium Enterprises

[Research Project Name] Development of Micro Needle Patch for Acne Skin Care Using 3-Structure HA Nanogel Combined with Vitamin C

[Name of the organization performing the task] Eskin

[Research Period] 2023.01.01 ~ 2023.12.31

Claims (23)

A peptide comprising an amino acid sequence of the following structural formula 1: [Structural formula 1] (X ¹ L) _a X ² Q(X ³ ) _b In the above structural formula 1, The above X ¹ , X ² and X ³ are each independently R (Arginine), H (Histidine), or K (Lysine), The above a is an integer from 2 to 5, The above b is an integer from 1 to 13. A peptide according to claim 1, wherein in the amino acid sequence having the structure of structural formula 1, the terminal of X ³ is the amino terminal (N`-terminal) or the carboxyl terminal (C`-terminal). A peptide according to claim 1, wherein the peptide comprises an amino acid sequence in which at least one H (Histidine) is additionally linked to at least one of the amino terminus and the carboxyl terminus of the amino acid sequence having the structure of the structural formula 1. A peptide according to claim 1, wherein the peptide comprises at least one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 40. A peptide according to claim 1, further comprising at least one hydrophobic amino acid at the amino terminal or carboxyl terminal of the peptide. A peptide comprising an amino acid sequence of the following structural formula 4: [Structural formula 4] (X ³ ) _b X ² Q(X ¹ L) _a In the above structural formula 4, The above X ¹ , X ² and X ³ are each independently R (Arginine), H (Histidine), or K (Lysine), The above a is an integer from 2 to 5, The above b is an integer from 1 to 13, N' represents the amino terminus of the peptide, and C' represents the carboxyl terminus of the peptide. A peptide according to claim 6, wherein in the amino acid sequence having the structure of structural formula 4, the terminal of X ³ is the amino terminal (N`-terminal) or the carboxyl terminal (C`-terminal). A peptide according to claim 6, wherein the peptide comprises an amino acid sequence in which at least one H (Histidine) is additionally linked to at least one of the amino terminus and the carboxyl terminus of the amino acid sequence having the structure of the structural formula 4. A peptide according to claim 6, further comprising at least one hydrophobic amino acid at the amino terminal or carboxyl terminal of the peptide. A micelle comprising a peptide according to any one of claims 1 to 9. A micelle according to claim 10, wherein the micelle is cell permeable. A micelle according to claim 10, wherein the micelle is formed by self-assembly of the peptide. A micelle according to claim 10, further comprising a hydrophobic substance at the amino terminal or carboxyl terminal of the peptide. A micelle according to claim 13, wherein the hydrophobic substance is at least one hydrophobic moiety selected from the group consisting of cholesterol and fatty acids. A micelle according to claim 10, further comprising a targeting agent, a labeling agent, or a linker for linking the targeting agent or the labeling agent to the amino terminal or the carboxyl terminal of the peptide. Micelles of claim 10; and A drug delivery complex comprising a drug of interest. A drug delivery complex according to claim 16, wherein the drug is a nucleic acid, a protein or a compound. A drug delivery complex according to claim 16, wherein the drug is at least one selected from the group consisting of DNA, mRNA, siRNA, microRNA, ASO, gapmer, aptamer, antagomir, agomir, and oligonucleotide. A drug delivery complex according to claim 16, wherein the drug is electrostatically bound to the peptide. A drug delivery complex according to claim 16, wherein the drug electrostatically binds to a hydrophilic amino acid residue of the peptide. Micelles of claim 10; and A pharmaceutical composition for preventing or treating a proliferative disease comprising siRNA. A pharmaceutical composition for preventing or treating a proliferative disease according to claim 21, wherein the proliferative disease is at least one selected from the group consisting of a neoplasm, a tumor, cancer, leukemia, psoriasis, a bone disease, a fibroproliferative disorder, and atherosclerosis. A pharmaceutical composition for preventing or treating a proliferative disease according to claim 22, wherein the cancer is at least one selected from the group consisting of melanoma, gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

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