dendritic polyelectrolyte zwitterions, Janus-type conjugate comprising same and uses thereof

Abstract

The present invention relates to a polyelectrolyte-zwitterion-based dendrimer and a Janus-shaped polyelectrolyte-zwitterion-based dendrimer as a carrier that is non-contaminating, has high intracellular delivery efficiency, and is non-toxic. In the case of the Janus-shaped polyelectrolyte-zwitterion-based dendrimer, it exists as a self-assembled supramolecular structure in solution without a separate process. Since the polyelectrolyte-zwitterion-based dendrimer and the Janus-shaped polyelectrolyte-zwitterion-based dendrimer can efficiently deliver loaded drugs or target genes into the cell without cytotoxicity, they can be usefully employed in various therapeutic drugs or nucleic acid delivery systems, drug treatment technologies, cell labeling systems, as well as gene therapy.

Description

Technology Field

[0001] The present invention relates to a polyelectrolyte-zwitterion-based dendrimer, a Janus-type polyelectrolyte-zwitterion-based dendrimer, and the use thereof, as a carrier that is non-contaminating, has high intracellular delivery efficiency, and is non-toxic. Background Technology

[0002] Ionic macromolecules include polyelectrolytes and zwitterions. These exhibit opposite solution behaviors. Polyelectrolytes are polymers composed of repeating units carrying positive or negative charges; in deionized water (DW), they exist in an expanded structure due to the repulsion between like charges, but as the salt concentration increases, they clump together and consequently become poorly soluble in water. This is called the polyelectrolyte effect.

[0003] Zygotic ions have equal numbers of positive and negative charges within a single molecule, resulting in a net charge of zero. Due to the attraction between the positive and negative charges within the molecule, they exist in a clustered form in deionized water (DW). However, as the salt concentration increases, the molecules spread out and move further apart due to a wrapping effect that counteracts the attractive forces, causing them to dissolve well in water. This is called the anti-polyelextrolyte effect.

[0004] Due to the fact that cell membranes carry a negative charge, most drug delivery systems are being developed to possess positively charged properties. While these positively charged polymers or nanoparticles have the advantage of effectively delivering into cells by forming strong binding forces with negatively charged cell membranes, they have high toxicity and can induce immune responses. Furthermore, there is a problem of polymers or nanoparticles aggregating due to the polyelectrolyte effect under ionic strength conditions similar to those inside the body. Additionally, they have the disadvantage of low non-contamination properties, which can lead to serum or cellular proteins adsorbing onto the surface of the polymers or nanoparticles, causing further clumping and potentially impairing biological functions.

[0005] Therefore, there is an urgent need for a new structure as a drug delivery system that possesses high non-contamination, high intracellular delivery efficiency, low toxicity, and high structural stability at physiological salt concentrations. Prior art literature

[0006] Patent Document 1. Korean Published Patent Application No. 10-2022-0086875 The problem to be solved

[0007] Accordingly, the present invention has been devised in consideration of the above-mentioned problems, and the objective of the present invention is to provide a polyelectrolyte-zwitterion-based dendrimer for producing dendritic compounds and nanostructures having high non-contamination characteristics, high intracellular delivery efficiency, low toxicity, and high structural stability at physiological salt concentrations.

[0008] Another objective of the present invention is to provide a Janus-type dendritic polyelectrolyte zwitterion (JDPZ) comprising the above-mentioned polyelectrolyte zwitterion-based hydrophilic dendrimer (dendritic polyelectrolyte zwitterion; DPZ) and a hydrophobic compound.

[0009] Another objective of the present invention is to provide a composition capable of safely storing and retaining hydrophobic drugs and genetic material while delivering them into a cell. means of solving the problem

[0010] Provides a polyelectrolyte-zwitterion-based dendritic compound represented by the following general formula 1 or 2.

[0011] [General Formula 1]

[0012]

[0013] [General Formula 2]

[0014]

[0015] In the above formula,

[0016] The above A and C may each be independently selected from the group consisting of arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), and guanidine.

[0017] The above B and D may each be independently selected from the group consisting of glutamic acid (Glu, E) and aspartic acid (Asp, D).

[0018] The above n, m, o, and p are each independently selected from any one integer between 3 and 16.

[0019] The above arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), guanidine, glutamic acid (Glu, E) and aspartic acid (Asp, D) may each be L-type or D-type amino acids independently of each other.

[0020] The above dendritic compound is characterized in that, under conditions of pH 2 to pH 13, n, m, o, and p are all the same.

[0021] To achieve the other objectives mentioned above, the present invention provides a polyelectrolyte-zwitterionic dendrimer characterized by a core based on a dendrimer of the first to fifth generation; and a positively charged peptide represented by SEQ ID NO. 1 and / or a negatively charged peptide represented by SEQ ID NO. 2 being bonded to the terminal functional group of the dendrimer core.

[0022] [Sequence No. 1]

[0023] [X1] n

[0024] [Sequence No. 2]

[0025] [X2] n

[0026] In the above sequence, X1 is any one selected from arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn) and guanidine, and X2 is glutamic acid (Glu, E) or aspartic acid (Asp, D).

[0027] Above, n is 3 It is an integer of up to 16.

[0028] The above arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), guanidine, glutamic acid (Glu, E) and aspartic acid (Asp, D) may each be L-type or D-type amino acids independently of each other.

[0029] The above-mentioned dendrimer-based core is not particularly limited thereto as long as it includes a terminal functional group capable of binding to a positively charged peptide and / or a negatively charged peptide, but, for example, it may be any one selected from the group consisting of polyamidoamine (PAMAM) dendrimer, polylysine dendrimer, polyimine (PI) dendrimer, polypropyleneimine (PPI) dendrimer, polyester dendrimer, polyamide dendrimer, polyurethane dendrimer, polyornithine dendrimer, carbosilane dendrimer, polyether dendrimer, polyglutamic acid dendrimer, polyaspartic acid dendrimer, polyglycerol dendrimer, and polymelamine dendrimer, and more preferably, it may be a polylysine dendrimer.

[0030] The above dendrimer-based core may be of the first or second generation.

[0031] The positively charged peptide represented by SEQ ID NO. 1 and the negatively charged peptide represented by SEQ ID NO. 2 may be bonded to the ends of the dendrimer-based core in equal numbers.

[0032] When the above-mentioned dendrimer-based core is of the first generation, one strand of the positively charged peptide chain and one strand of the negatively charged peptide chain are combined, and when the above-mentioned dendrimer-based core is of the second generation, two strands of the positively charged peptide chain and two strands of the negatively charged peptide chain may be combined.

[0033] The above positively charged peptide may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 86.

[0034] The above negatively charged peptide may be represented by any one selected from the group consisting of SEQ ID NOs 87 to 142.

[0035] To achieve the other objectives mentioned above, the present invention provides a Janus-type polyelectrolyte-zwitterion-based dendrimer characterized by a core based on a dendrimer of the first to fifth generation; wherein a plurality of positively charged peptides represented by SEQ ID NO. 1 and negatively charged peptides represented by SEQ ID NO. 2 are alternately bonded to one end functional group based on the dendrimer-based core, and a hydrophobic compound or amino acid derivative is independently bonded to the other end functional group based on the core of the dendrimer.

[0036] [Sequence No. 1]

[0037] [X1]n

[0038] [Sequence No. 2]

[0039] [X2]n

[0040] In the above sequence, X1 is any one selected from arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), and guanidine, and X2 is glutamic acid (Glu, E) or aspartic acid (Asp, D). The above, n is 3 It is an integer of up to 16.

[0041] The above dendrimer-based core may be of the 2nd or 3rd generation.

[0042] The positively charged peptide represented by SEQ ID NO. 1 and the negatively charged peptide represented by SEQ ID NO. 2 may have an equal number of dendrimer-based cores (one end functional group of the core) attached to each other.

[0043] The above-mentioned dendrimer-based core has 2 on the surface depending on the number of generations xIt may have terminal functional groups (where x is the number of generations), and among the total terminal functional groups present in the dendrimer-based core, half may have a peptide represented by SEQ ID NO. 1 or 2 attached, and the other half may each have a hydrophobic compound or an amino acid derivative attached independently.

[0044] The above positively charged peptide may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 86.

[0045] The above negatively charged peptide may be represented by any one selected from the group consisting of SEQ ID NOs 87 to 142.

[0046] The above dendrimer is characterized by having the same number of positively charged peptides and negatively charged peptides under conditions of pH 2 to pH 13.

[0047] The above hydrophobic compound may be any one selected from the group consisting of fatty acids having 3 to 30 carbon atoms, hydrophobic drugs, and fluorescent substances.

[0048] To achieve the other objectives mentioned above, the present invention provides a nanostructure comprising a Janus-type multivalent electrolyte-bio-ion-based dendrimer.

[0049] The above nanostructure may be an elongated fiber structure in the form of nanofibers.

[0050] The average diameter of the above nanostructure may be 8 to 30 nm.

[0051] To achieve the other objectives mentioned above, the present invention provides a gene or drug delivery system comprising: a Janus-shaped polyelectrolyte-bio-ion-based dendrimer; and a target gene or drug supported on the dendrimer.

[0052] The above gene may be a plasmid, mRNA, RNA, DNA, or a combination thereof.

[0053] The above drug may be a low molecular weight drug, a gene drug, a protein drug, an antibody drug, a synthetic compound drug, or a combination thereof. Effects of the invention

[0054] The present invention provides a novel molecule in the form of a dendrimer composed of a positively charged peptide and a negatively charged peptide that simultaneously possess the characteristics of a polyelectrolyte and a binary ion, thereby having no cytotoxicity and non-contaminating properties, and minimizing interactions with nucleic acids, proteins, etc. in vivo, so that it can maintain its structure stably in vivo.

[0055] Furthermore, the Janus-type polyelectrolyte-bio-ion-based dendrimer according to the present invention can provide a delivery vehicle with a stable structure and excellent intracellular delivery efficiency by forming a complex with a target gene or drug.

[0056] The Janus-shaped polyelectrolyte-zwitterion-based dendrimer according to the present invention is completely protected from the external environment, so it exists as a self-assembled supramolecular structure in solution without a separate process, and can efficiently deliver loaded drugs or target genes into the cell without cytotoxicity, so it can be usefully used in various therapeutic drugs or nucleic acid delivery systems, drug treatment technologies, cell labeling systems, as well as gene therapy. Brief explanation of the drawing

[0057] Figure 1 is a conceptual diagram showing the basic structure of a polyelectrolyte-bio-ion based dendrimer. FIG. 2a is a MALDI-TOF MS graph of (+1)2(-1)2 prepared from Example 1-1, FIG. 2b is a MALDI-TOF MS graph of (+2)2(-2)2 prepared from Example 1-2, FIG. 2c is a MALDI-TOF MS graph of (+3)2(-3)2 prepared from Example 1-3, FIG. 2d is a MALDI-TOF MS graph of (+4)2(-4)2 prepared from Example 1-4, FIG. 2e is a MALDI-TOF MS graph of (+5)2(-5)2 prepared from Example 1-5, and FIG. 2f is a MALDI-TOF MS graph of (+6)2(-6)2 prepared from Example 1-6. FIG. 3 shows polyelectrolyte-zwitterion-based dendrimers (DPZ(+M)) prepared from Examples 1-1 to 1-6 according to concentrations (0, 2, 4, 8, 16, 32, 64, 128 μM) n (-M) n This is a graph showing the cytotoxicity of peptides prepared from ) and Comparative Examples 3-1 and 3-2. Figure 4a is the EMSA result analyzing the protein non-contamination of the polyelectrolyte-zwitterion-based dendrimers ((+1)2(-1)2 and (+2)2(-2)2) of Examples 1-1 and 1-2. Figure 4b is the EMSA result analyzing the protein non-contamination of the polyelectrolyte-zwitterion-based dendrimers ((+3)2(-3)2 and (+4)2(-4)2) of Examples 1-3 and 1-4. Figure 4c is the EMSA result analyzing the protein non-contamination of the polyelectrolyte-zwitterion-based dendrimers ((+5)2(-5)2 and (+6)2(-6)2) of Examples 1-5 and 1-6. Figure 5 is the result of an EMSA analysis of the protein non-contamination of linear peptide R12 (sequence number 145). Figure 6 is the result of EMSA analyzing the interaction patterns between the multivalent electrolyte-bio-ion-based dendrimer ((+6)2(-6)2) of Examples 1-6 and various genetic materials (single-stranded DNA, mRNA, and plasmid DNA). Figure 7 is the result of an EMSA experiment analyzing the complex formation between mRNA and the polyelectrolyte-bioelectrolyte-zwitterion-based dendrimers ((+3)2(-3)2, (+4)2(-4)2, (+5)2(-5)2 and (+6)2(-6)2) of Examples 1-3 to 1-6 according to the charge ratio. Figure 8 is a MALDI-TOF graph for DPZ introduced with FAM, a fluorescent material prepared from Examples 2-1 to 2-6. Figure 8a is for (+1)2(-1)2_FAM of Example 2-1, Figure 8b is for (+2)2(-2)2_FAM of Example 2-2, Figure 8c is for (+3)2(-3)2_FAM of Example 2-3, Figure 8d is for (+4)2(-4)2_FAM of Example 2-4, Figure 8e is for (+5)2(-5)2_FAM of Example 2-5, and Figure 8f is for (+6)2(-6)2_FAM of Example 2-6. FIG. 9 shows the (+M) of Examples 2-1 to 2-6 according to concentration (16 μM (top), 32 μM (bottom)). n (-M) n This is a graph showing the intracellular delivery efficiency of FAM in HeLa cell lines analyzed by flow cytometry. Figure 10 is a MALDI-TOF graph for G1 Janus-type polyelectrolyte-bio-ion-based dendrimers (JDPZ, (+M)1(-M)1conjugate_1) prepared from Examples 3-1 to 3-6. FIG. 10a is for (+1)1(-1)1conjugate_1 of Example 3-1, FIG. 10b is for (+2)1(-2)1conjugate_1 of Example 3-2, FIG. 10c is for (+3)1(-3)1conjugate_1 of Example 3-3, FIG. 10d is for (+4)1(-4)1conjugate_1 of Example 3-4, FIG. 10e is for (+5)1(-5)1conjugate_1 of Example 3-5, and FIG. 10f is for (+6)1(-6)1conjugate_1 of Example 3-6. FIG. 11 is an AFM image of Janus-type polyelectrolyte-bio-ion-based dendrimers ((+1)1(-1)1-conjugate_1, (+2)1(-2)1-conjugate_1, (+3)1(-3)1-conjugate_1, (+4)1(-4)1-conjugate_1, (+5)1(-5)1-conjugate_1, and (+6)1(-6)1-conjugate_1) of Examples 3-1 to 3-6 in DW solution. Figure 12 is a graph showing the cytotoxicity of (+M)1(-M)1conjugate_1 of Examples 3-1 to 3-6 according to concentrations (0, 4, 8, 16, 32, 64 μM). Figure 13 is a MALDI-TOF graph for a G1 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ, (+M)1(-M)1conjugate_FAM) with a fluorescent material introduced from Examples 4-1 to 4-12. FIG. 13a is for the (+1)1(-1)1conjugate_FAM of Example 4-1, FIG. 13b is for the (+2)1(-2)1conjugate_FAM of Example 4-2, FIG. 13c is for the (+3)1(-3)1conjugate_FAM of Example 4-3, FIG. 13d is for the (+4)1(-4)1conjugate_FAM of Example 4-4, FIG. 13e is for the (+5)1(-5)1conjugate_FAM of Example 4-5, FIG. 13f is for the (+6)1(-6)1conjugate_FAM of Example 4-6, FIG. 13g is for the (+7)1(-7)1conjugate_FAM of Example 4-7, and FIG. 13h is for Example 4-8 Figure 13i is for (+8)1(-8)1conjugate_FAM, Figure 13i is for (+5)1(-5)1conjugate (RD)_FAM of Example 4-9, Figure 13j is for (+6)1(-6)1conjugate (RD)_FAM of Example 4-10, Figure 13k is for (+5)1(-5)1conjugate (KE)_FAM of Example 4-11, and Figure 13l is for (+6)1(-6)1conjugate (KE)_FAM of Example 4-12. Figure 14 is a graph showing the intracellular delivery efficiency (degree of internalization) of JDPZ((+M)1(-M)1conjugate_FAM) prepared from Examples 4-1 to 4-6, analyzed by flow cytometry. Figure 15 is a graph showing the intracellular delivery efficiency of JDPZ((+M)1(-M)1conjugate_FAM) prepared from Examples 4-6 to 4-12, analyzed by flow cytometry. FIG. 16 is a MALDI-TOF graph for G2 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ((+M)2(-M)2conjugate_1)) prepared from Examples 5-1 to 5-4. FIG. 16a is for (+3)2(-3)2conjugate_1 prepared from Example 5-1, FIG. 16b is for (+4)2(-4)2conjugate_1 prepared from Example 5-2, FIG. 16c is for (+5)2(-5)2conjugate_1 prepared from Example 5-3, and FIG. 16d is for (+6)2(-6)2conjugate_1 prepared from Example 5-4. FIG. 17a is for a (+3)4 conjugate prepared from Comparative Example 1-1, FIG. 17b is for a (+3)2(Ø)2 conjugate prepared from Comparative Example 1-2, and FIG. 17c is for a (Ø)2(-3)2 conjugate prepared from Comparative Example 1-3. FIG. 18 is an AFM image of the G2 Janus-type polyelectrolyte-bio-ion based dendrimers (JDPZ, (+M)2(-M)2conjugate_1) of Examples 5-1 to 5-4. FIG. 18a is for (+3)2(-3)2conjugate_1 prepared from Example 5-1, FIG. 18b is for (+4)2(-4)2conjugate_1 prepared from Example 5-2, FIG. 18c is for (+5)2(-5)2conjugate_1 prepared from Example 5-3, and FIG. 18d is for (+6)2(-6)2conjugate_1 prepared from Example 5-4. FIG. 19 is a graph analyzing the cytotoxicity of a G2 Janus-type polyelectrolyte-bio-ion-based dendrimer (JDPZ, (+M)2(-M)2conjugate_1) prepared from Example 5-1 and G2 Janus-type dendrimers prepared from Comparative Examples 1-1 to 1-3. FIG. 20 is the DLS spectrum of the Janus-shaped polyelectrolyte-zwitterion-based dendrimer ((+6)2(-6)2conjugate_1)(a) of Example 5-4 and the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate)(b) of Comparative Example 1-2 under different solution conditions (DW or PBS). FIG. 21a is the EMSA result analyzing the protein non-contamination of the Janus-shaped polyelectrolyte-bio-ion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) structure of Example 5-4 and the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) of Comparative Example 1-2. FIG. 21b is the EMSA result analyzing the non-contamination of genetic material by the Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) of Example 5-4 and the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) of Comparative Example 1-2. Here, 'L' is an RNA marker. FIG. 22 is a MALDI-TOF graph for G2 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ((+M)2(-M)2conjugate_FAM)) introduced with fluorescent materials prepared from Examples 6-1, 6-2 and Comparative Examples 2-1 to 2-2. FIG. 22a is for (+3)2(-3)2conjugate_FAM prepared from Example 6-1, FIG. 22b is for (+6)2(-6)2conjugate_FAM prepared from Example 6-2. FIG. 22c is for (+3)2(Ø3)2conjugate_FAM of Comparative Example 2-1, FIG. 22d is for (Ø3)2(-3)2conjugate_FAM of Comparative Example 2-2. Figure 23 is a graph showing the intracellular delivery efficiency of JDPZ ((+6)2(-6)2conjugate_FAM) prepared from Example 6-2, analyzed by flow cytometry in the HCT116 cell line. Figure 24 is an image analyzed by confocal microscopy (LSM 980, Carl Zeiss, Germany) after treating HeLa cells with a G2 Janus-type multivalent electrolyte-bio-ion-based dendrimer (Example 6-2) into which a fluorescent substance was introduced. FIG. 25 is a graph showing the cytotoxicity of G2 Janus-type polyelectrolyte-zwitterion-based dendrimers ((+M)2(-M)2conjugate_FAM) with fluorescent substances introduced from Examples 6-1 and 6-2 and G2 Janus-type dendrimers with fluorescent substances introduced from Comparative Examples 2-1 and 2-2, measured by flow cytometry (FACS). Figure 26 is a MALDI-TOF graph for a G2 Janus-type polyelectrolyte-bio-ion-based dendrimer ((+6)2(-6)2conjugate_2) prepared from Example 7. Figure 27 is a MALDI-TOF graph for a G2 Janus-type polyelectrolyte-bio-ion-based dendrimer ((+6)2(-6)2conjugate_3) prepared from Example 8. Figure 28 shows the results of quantifying the expressed GFP protein by flow cytometry after treating HeLa cell lines with a complex of Janus-shaped multi-electrolyte-zwitterion-based dendrimer (+6)2(-6)2conjugate_2 prepared from Example 7 and EGFP (enhanced green fluorescent protein) mRNA. Figure 29 is a graph showing the quantification of GFP protein expression by flow cytometry after treating HeLa cell lines with a complex of Janus-shaped multi-electrolyte-zwitterion-based dendrimer (+6)2(-6)2conjugate 3 prepared from Example 8 and EGFP (enhanced green fluorescent protein) mRNA. Fig. 30 is in vivo ( in vivo This is a graph showing the confirmation and quantification of the expression of a complex ((+6)2(-6)2_conjugate 3_complex) loaded with EGFP mRNA in ). Specific details for implementing the invention

[0058] Before disclosing and describing the compounds, compositions, and / or methods of the present invention, it should be understood that the aspects described below are not limited to specific compounds, synthesis methods, or uses, which may vary. Furthermore, it should be understood that the terms used herein are merely for describing specific aspects and are not intended to limit the present invention.

[0059] In the present specification and the claims below, reference will be made to many terms defined as having the following meanings.

[0060] Unless specifically stated otherwise, the weight percentage of the component is based on the total weight of the formulation or composition containing the component.

[0061] In the present invention, "A and / or B" means "A and B, or A or B".

[0062] In the present invention, a "dendrimer" is a molecule having a regular branched structure, generally synthesized by connecting constituent units one by one. Depending on its structure and properties, such a dendrimer can be utilized for various purposes, such as drug delivery systems, therapeutic agents, bioimaging, and contrast agents.

[0063] In the present invention, "Janus" is named after the two-faced god of Roman mythology and refers to a molecule containing two structures. In a narrow sense, it refers to a case where a spherical particle is divided in half, with each having a separate structure; however, in the present invention, it is used in a general sense to include a case where a dendrimer molecule is divided into two parts based on its center, with materials of different characteristics bonded to each part.

[0064] In the present invention, "amino acid" is used in the broadest sense and is intended to include naturally occurring L-amino acids or residues. One-letter and / or three-letter abbreviations commonly used for naturally occurring amino acids may be used herein. Amino acids include not only D-amino acids but also chemically modified amino acids, e.g., amino acid analogs; naturally occurring amino acids not typically incorporated into proteins, e.g., norleucine; and chemically synthesized compounds having properties known in the art to which the present invention belongs, characterized by including carboxy- and / or amino-terminal amino acids. For example, analogs or mimics of phenylalanine or proline that allow stereotype restriction of peptide compounds identical to natural Phe or Pro are included within the definition of amino acid. Such analogs and mimics are referred to herein as "functional equivalents" of amino acids. Other examples of amino acids are [Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Eds. Gross and Meiehofer, Vol. It is listed in 5, p. 341 (Academic Press, Inc.: NY 1983).

[0065] In the present invention, "peptide" includes all proteins, protein fragments, and peptides isolated from naturally occurring sources, synthesized by recombinant techniques, or chemically synthesized.

[0066] The present invention may include a substituent having at least 70%, preferably at least 90%, and more preferably at least 95% identity with the original "peptide".

[0067] In the present invention, a "substitution having identity" means that when one amino acid is substituted with another amino acid, there is no significant change in properties such as the secondary structure and hydropathic nature of the polypeptide. Amino acid variation can be obtained based on the relative similarity of amino acid side chain substituents, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature.

[0068] For example, amino acids can be classified according to common side chain characteristics into i) hydrophobic (norleucine, methionine, alanine, valine, leucine, isoleucine), ii) neutral hydrophilic (cysteine, serine, threonine, asparagine, glutamine), iii) acidic (aspartic acid, glutamic acid), iv) basic (histidine, lysine, arginine), v) residues affecting chain orientation (glycine, proline), and vi) aromatic (tryptophan, tyrosine, phenylalanine). Conservative substitution will involve exchanging a member of one of these classes for another member of the same class.

[0069] Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; aspartic acid and glutamic acid are both negatively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; aspartic acid and glutamic acid; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0070] The present invention will be described in detail below.

[0071] One aspect of the present invention relates to a polyelectrolyte-zwitterion-based dendritic compound represented by the following general formula 1 or 2.

[0072] [General Formula 1]

[0073]

[0074] [General Formula 2]

[0075]

[0076] In the above formula, A and C may each be independently selected from the group consisting of arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), and guanidine.

[0077] The above B and D may each be independently selected from one or more of the group consisting of glutamic acid (Glu, E) and aspartic acid (Asp, D). The above n, m, o, and p are each independently 3 It may be any one integer selected from to 16, preferably 3 To 12, more preferably 3 It is an integer of up to 6.

[0078] The above arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), guanidine, glutamic acid (Glu, E) and aspartic acid (Asp, D) may each be L-type or D-type amino acids independently of each other.

[0079] It is preferable that the above dendritic compound has n, m, o, and p all the same under pH 2 to pH 13 conditions.

[0080] In the above Equation 1, the repeating unit indicated by A and the repeating unit indicated by B are connected to each other through a linker, and in the above Equation 2, the repeating unit indicated by A, the repeating unit indicated by B, the repeating unit indicated by C, and the repeating unit indicated by D may be connected to each other through a linker.

[0081] The above linker may be at least one or more lysine residues (Lys or K). The lysine may have a side chain reactive group to provide a reactive group for the polymerization of the repeating units represented by A, B, C, and D.

[0082] The polyelectrolyte-zwitterion-based dendritic compound represented by the above general formula 1 or 2 may preferably be any one selected from those represented by formulas 1-1 to 1-6.

[0083] Another aspect of the present invention relates to a polyelectrolyte-zwitterionic dendrimer characterized by a core based on a dendrimer of the 1st to 5th generation; and a positively charged peptide represented by SEQ ID NO. 1 and / or a negatively charged peptide represented by SEQ ID NO. 2 being bonded to the terminal functional group of the core.

[0084] [Sequence No. 1]

[0085] [X1] n

[0086] [Sequence No. 2]

[0087] [X2] n

[0088] In the above sequence, X1 is any one selected from arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn) and guanidine, and X2 is glutamic acid (Glu, E) or aspartic acid (Asp, D).

[0089] Above, n is 3 It may be an integer from 1 to 16, preferably 3 To 12, more preferably 3 It is an integer of up to 6.

[0090] In the present invention, "core" refers to a material that acts as a support by binding to a positively charged peptide and a negatively charged peptide, thereby enabling the positively charged peptide and the negatively charged peptide to be arranged at regular intervals from each other.

[0091] The above-mentioned dendrimer-based core is not particularly limited thereto as long as it includes a terminal functional group capable of binding to a positively charged peptide and / or a negatively charged peptide, but, for example, it may be any one selected from the group consisting of polyamidoamine (PAMAM) dendrimer, polylysine dendrimer, polyimine (PI) dendrimer, polypropyleneimine (PPI) dendrimer, polyester dendrimer, polyamide dendrimer, polyurethane dendrimer, polyornithine dendrimer, carbosilane dendrimer, polyether dendrimer, polyglutamic acid dendrimer, polyaspartic acid dendrimer, polyglycerol dendrimer, and polymelamine dendrimer, and more preferably, it may be a polylysine dendrimer.

[0092] The above-mentioned dendrimer-based core is preferably of the 1st to 5th generation, and more preferably may be of the 1st or 2nd generation.

[0093] In the present invention, the "dendrimer" can specify the generation of the dendrimer according to the overlap level of the base compounds forming the dendrimer, and can be expressed as a 1st generation dendrimer (G1), a 2nd generation dendrimer (G2), a 3rd generation dendrimer (G3), a 4th generation dendrimer (G4), and a 5th generation dendrimer (G5). That is, as one additional base compound is bonded, the generation number increases by one generation.

[0094] When the core based on the above dendrimer is a polylysine dendrimer, the polylysine dendrimer is composed of at least one or more "lysine (K)", and a first-generation dendrimer is formed by adding one additional lysine to the lysine core. The first-generation dendrimer has one or more "branches" attached to the core. The second-generation dendrimer can be formed by reacting lysine with the first-generation dendrimer. Third, fourth, fifth generations, etc., can be produced by performing a similar reaction.

[0095] The polylysine dendrimer formed by repeating the above lysine is not particularly limited as long as it is at least one generation, but preferably it is one to five generations, and more preferably it may be one or two generations. The technique for producing the above polylysine dendrimer can be produced through subsequent coupling / deprotection using lysine, and since lysine has three reactive groups, it can produce branched branches.

[0096] Specifically, the lysine has an amino group, a carboxyl group, and a side chain of the amino group, and possesses an α-hydrogen levorotatory stereostructure. That is, a core composed of one or more lysine residues is bonded to one or more lysine residues, and said bonding may be due to a peptide bond or a peptide bond with the side chain of the lysine residue.

[0097] In the present invention, the polylysine has a plurality of terminal amino groups on its surface, and a plurality of positively charged peptides represented by SEQ ID NO. 1 and / or negatively charged peptides represented by SEQ ID NO. 2 are bonded thereto and arranged spaced apart from each other.

[0098] Specifically, a positively charged peptide represented by SEQ ID NO. 1 and a negatively charged peptide represented by SEQ ID NO. 2 may each be bonded to the terminal amino groups of the polylysine dendrimer. That is, the polyelectrolyte-zwitterionic dendrimer according to the present invention can simultaneously form polyelectrolyte properties and zwitterionic properties by alternately bonding peptides with different charges to the branched terminal amino groups of the dendrimer (Fig. 1).

[0099] The polyelectrolyte-zwitterionic dendrimer according to the present invention is characterized by having a positively charged peptide represented by SEQ ID NO. 1 and a negatively charged peptide represented by SEQ ID NO. 2 bonded to each other in equal numbers. If either of the peptides becomes more numerous or fewer than the other, the net charge deviates from zero, making it impossible to properly exhibit zwitterionic properties, which may cause cytotoxicity. For example, if the positively charged peptide chain strands become more numerous than the negatively charged peptide chain strands, the dendrimer molecule becomes positively charged, causing cytotoxicity; if the negatively charged peptide chain strands become more numerous than the positively charged peptide chain strands, there is no cytotoxicity, but a problem may arise in which the intracellular delivery efficiency is significantly reduced.

[0100] The number of positively charged peptides and negatively charged peptides per molecule of the polylysine dendrimer is not particularly limited, but the optimal number may be selected depending on the terminal amino groups present on the surface of the polylysine dendrimer. For example, if the polylysine dendrimer is a first-generation polylysine dendrimer, one strand of a positively charged peptide chain represented by SEQ ID NO. 1 and one strand of a negatively charged peptide chain represented by SEQ ID NO. 2 may be combined. Additionally, if the polylysine dendrimer is a second-generation polylysine dendrimer, two strands of a positively charged peptide chain represented by SEQ ID NO. 1 and two strands of a negatively charged peptide chain represented by SEQ ID NO. 2 may be combined.

[0101] In the above sequence, arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), guanidine, glutamic acid (Glu, E), and aspartic acid (Asp, D) may each be L-type or D-type amino acids independently of each other.

[0102] The above positively charged peptide is not particularly limited as long as it consists of a positively charged amino acid residue, but may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 98, preferably may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 50, and may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 8.

[0103] The above negatively charged peptide may be composed of negatively charged amino acid residues, but is not particularly limited thereto; it may be represented by any one selected from the group consisting of SEQ ID NOs 99 to 162, and preferably may be represented by any one selected from the group consisting of 99 to 130.

[0104] The polyelectrolyte-zwitterion dendrimer according to the present invention may be represented by any one of the following chemical formulas 1-1 to 1-6. The weight-average molecular weight (MW) of the polyelectrolyte-zwitterion dendrimer according to the present invention is It can be 2,200 to 4,000 Da, and is a safe substance that does not exhibit toxicity to cells even at high concentrations.

[0105] The polyelectrolyte-zwitterion dendrimer according to the present invention is capable of drug delivery and fluorescent labeling / delivery, and can be manufactured as a polyelectrolyte-zwitterion-based dendrimer capable of delivering the drug or contrast agent to a target through binding with the drug or contrast agent, and may be represented, for example, by any one of the following chemical formulas 2-1 to 2-6.

[0106] In the present invention, "drug" may be a synthetic compound drug for alleviating, preventing, treating, or diagnosing a disease, injury, or specific symptoms, and specifically, doxorubicin, daunorubicin, epirubicin, idarubicin, valubicin, mitoxantrone, dexamethasone, triamcinolone, beclomethasone diproprionate, triamcinolone acetonide, triamcinolone diacetate, bethamethasone diproprionate, testosterone, budesonide, 17α-ethinylestradiol, levonorgestrel, fluticasone proprionate, sorafenib, It may be one or more selected from paclitaxel, docetaxel, doxorubicin, cyclosporine A, amphothericin B, indinavir, rapamycin, doxorubicin, coenzyme Q10, ursodeoxycholic acid, ilaprazole, imatinib mesilate, and tanespimycin.

[0107] In the present invention, "contrast material" refers to any material used for imaging structures or fluids within the body in medical imaging. The contrast material may include, but is not limited to, a radiopaque contrast agent, a paramagnetic contrast agent, a superparamagnetic contrast agent, a CT (computed tomography) contrast agent, or other contrast agents. For example, a radiopaque contrast agent (for X-ray imaging) may include inorganic iodine compounds and organic iodine compounds (e.g., diatrisoates), radiopaque metals and their salts (e.g., silver, gold, platinum, etc.), and other radiopaque compounds (e.g., calcium salts, barium salts such as barium sulfate, tantalum, and tantalum oxide). Paramagnetic contrast agents (for MR imaging) include gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and its derivatives, and other gadolinium, manganese, iron, dysprosium, copper, europium, erbium, chromium, nickel, and cobalt complexes, for example, 1,4,7,10-tetraazcyclododecane-N,N',N",N"-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazcyclododecane-N,-N',N"-triacetic acid (D03A), 1,4,7-triazcyclononan-N,N',N"-triacetic acid (NOTA), It may include 1,4,8,10-tetraazcyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA) and hydroxybenzylethylene-diaminediacetic acid (HBED).Superparamagnetic contrast materials (for MR imaging) may include magnetite, superparamagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO), and monocrystalline iron oxide. Other suitable contrast materials may include iodinated and non-iodinated, ionic and non-ionic CT contrast materials, contrast materials such as spin-labels, or other diagnostically effective agents. Additionally, the contrast materials may include β-galactosidase, green fluorescent protein, blue fluorescent protein, or luciferase. They may include marker genes encoding proteins that are easily detectable when expressed in cells. Various markers such as radionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, etc., may be used.

[0108] The above fluorescent substances are fluorescein, FAM (carboxyfluorescein), rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, phycoerythrin, PerCP (peridinin chlorophyll-α protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, and Oregon Green. 488, Oregon Green 500, Oregon Green 514, Alexa Fluor (trademark) 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, 7-Amino-4-Methylcomarin-3-Acetic Acid, BODIPY (trademark) FL, BODIPY FL-Br2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY It may be one or more selected from the group consisting of 630 / 650, Bodipi 650 / 665, Bodipi R6G, Bodipi TMR, and Bodipi TR, but is not limited thereto.

[0109] The above drugs and contrast agents may additionally bind to the functional groups at the other end of the dendrimer that are not bound to the peptide, based on the core of the dendrimer.

[0110] According to a specific embodiment of the present invention, a composition comprising a polyelectrolyte-bio-ionic bonded dendrimer can be utilized as a composition with excellent effect of delivering contrast agents or drugs into cells.

[0111] Another aspect of the present invention is A dendrimer-based core of the 1st to 5th generation; a positively charged peptide represented by SEQ ID NO. 1 and / or a negatively charged peptide represented by SEQ ID NO. 2 is bonded to one end functional group centered on the dendrimer core, and a hydrophobic compound is bonded to the other end functional group centered on the dendrimer core.

[0112] [Sequence No. 1]

[0113] [X1] n

[0114] [Sequence No. 2]

[0115] [X2] n

[0116] In the above sequence, X1 is any one selected from arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), and guanidine, and X2 is glutamic acid (Glu, E) or aspartic acid (Asp, D). The above, n is 3 It is an integer of up to 16.

[0117] As described above, the dendrimer-based core is identical to the dendrimer-based core in 1. a polyelectrolyte-zwitterion-based dendrimer, so it is to be referenced thereto. In the present invention, the dendrimer-based core is characterized by having a peptide or a hydrophobic compound attached to the terminal functional group of each end.

[0118] The above dendrimer-based core has terminal functional groups on its surface depending on the number of generations, specifically the number of terminal functional groups is (2 xIt is equivalent to ), where x is the number of generations of the dendrimer. For example, in the case of a 1st generation dendrimer-based core, it has 2 surface-terminal functional groups, in the case of the 2nd generation, it has 4 surface-terminal functional groups, in the case of the 3rd generation, it has 8 surface-terminal functional groups, in the case of the 4th generation, it has 16 surface-terminal functional groups, and in the case of the 5th generation, it has 32 surface-terminal functional groups.

[0119] Here, the total number of terminal functional groups (2) present in the dendrimer-based core. x Half (50%) of the peptide may have a site (one side) where the peptide is bound, and the other half (50%) of the terminal functional groups (the other side) where the peptide is not bound may each have a hydrophobic compound or an amino acid derivative independently bound to them.

[0120] When the above-mentioned dendrimer-based core is lysine, it may specifically be represented by the following [Chemical Formula A] or [Chemical Formula B], and the terminal functional group may be the N-terminal or side chain terminal of lysine or both.

[0121] [Chemical Formula A] G1

[0122] Lys(α,ε)

[0123] [Chemical Formula B] G2

[0124] Lys(α,ε)-Lys

[0125] ┃

[0126] Lys(α,ε)

[0127] The Janus-type polyelectrolyte-zwitterion-based dendrimer of the present invention is characterized in that substances with different characteristics are bonded to one side and the other side among the entire terminal functional groups based on the core molecule of the dendrimer, wherein a hydrophilic positively charged peptide and a negatively charged peptide are bonded to the amino group at the α position of lysine and the amino group of the side chain at the ε position, respectively, to form a polyelectrolyte-zwitterion-based dendrimer, and a hydrophobic compound is bonded to the other side.

[0128] The positively charged peptide represented by SEQ ID NO. 1 and the negatively charged peptide represented by SEQ ID NO. 2 may be bound to the core end of a dendrimer-based peptide in equal numbers. For example, if the positively charged peptide chain strands are greater than the negatively charged peptide chain strands, the dendrimer molecules become positively charged, causing cytotoxicity; and if the negatively charged peptide chain strands are greater than the positively charged peptide chain strands, there is no cytotoxicity, but a problem may arise in which the intracellular delivery efficiency is significantly reduced.

[0129] The number of positively charged peptides and negatively charged peptides per molecule of the dendrimer-based core is not particularly limited, but the optimal number may be selected depending on the terminal functional groups present on the surface of the dendrimer-based core. For example, the dendrimer-based core is 2 In the case of the generation, one strand of a positively charged peptide chain represented by SEQ ID NO. 1 and one strand of a negatively charged peptide chain represented by SEQ ID NO. 2 may be combined. In addition, the dendrimer-based core is 3 In the case of a generation, two strands of a positively charged peptide chain represented by SEQ ID NO. 1 and two strands of a negatively charged peptide chain represented by SEQ ID NO. 2 may be combined.

[0130] The above positively charged peptide is not particularly limited as long as it consists of a positively charged amino acid residue, but may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 98, preferably may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 50, and may be represented by any one selected from the group consisting of SEQ ID NOs 3 to 8.

[0131] The above negatively charged peptide may be composed of negatively charged amino acid residues, and is not particularly limited thereto, but may be represented by any one selected from the group consisting of SEQ ID NOs 99 to 162, and most preferably may be represented by SEQ ID NOs 99 to 130.

[0132] Due to the Janus shape described above, the polyelectrolyte-zwitterion-based dendrimer exists as a self-assembled nanostructure having an elongated fiber-shaped nanofiber structure in solution. This is formed because the Janus-shaped polyelectrolyte-zwitterion-based dendrimer possesses amphiphilicity, having both hydrophilic and hydrophobic groups simultaneously with the polyelectrolyte-zwitterion molecular structure, and is a thermodynamically stable and uniform structure formed by amphiphilic materials.

[0133] Due to the amphiphilicity of this nanofiber form and the Janus form, the polyelectrolyte-zwitterion-based dendrimer is protected from various mechanisms within the human body and can efficiently move to the target site.

[0134] Furthermore, Janus-shaped polyelectrolyte-zwitterion-based dendrimers can stably deliver hydrophobic compounds into cells without toxicity by configuring them with hydrophobic compounds bound within the dendrimer molecules. Additionally, since they exhibit non-contaminating properties toward biomaterials (nucleic acids, proteins, etc.) present in the body when administered, Janus-shaped polyelectrolyte-zwitterion-based dendrimers can resolve side effects such as additional deterioration of biological function. Therefore, the Janus-shaped polyelectrolyte-zwitterion-based dendrimers can be excellently utilized for various applications, such as drug delivery systems, therapeutic agents, bioimaging, and contrast agents.

[0135] The above-mentioned hydrophobic compound is not particularly limited to any substance that exhibits hydrophobicity. For example, the above-mentioned hydrophobic compound may be one or more selected from the group consisting of fatty acids, bile acids, hydrophobic drugs, contrast agents, and fluorescent substances.

[0136] The above fatty acid passes through the N-terminus and carboxyl group of the polylysine dendrimer female Hydrophobic fatty acids capable of forming amide bonds include fatty acids having 3 to 30 carbon atoms, aliphatic carboxylic acids, for example, aliphatic carboxylic acids having 4 to 20 carbon atoms. For example, aliphatic carboxylic acids include saturated aliphatic carboxylic acids and unsaturated aliphatic carboxylic acids, and are aliphatic carboxylic acids in a straight-chain or branch-chain form.

[0137] The above saturated aliphatic carboxylic acids are butanoic acid / butyric acid, pentanic acid / valeric acid, hexanoic acid / caproic acid, heptanoic acid / enanthic acid, octanoic acid / caprylic acid, nonanoic acid / pelargonic acid, decanoic acid / capric acid, undecylic acid / undecanoic acid, lauric acid / dodecanoic acid, tridecyl acid / tridecanoic acid, and myristic acid / tetradecanoic acid. Pentadecylic acid / pentadecanoic acid, palmitic acid / hexadecanoic acid, margaric acid / heptadecanoic acid, stearic acid / octadecanoic acid, nonadecylic acid / nonodecanoic acid, arachidic acid / eicosanoic acid, heniicosylic acid / heneicosanoic acid, behenic acid / docosanoic acid, tricosylic acid / tricosanoic acid, lignoceric acid / tetracosanoic acid, Pentacosylic acid / pentocosanoic acidIt may include, but is not limited to, one or more saturated aliphatic monocarboxylic acids selected from the group consisting of cerotic acid / hexacosanoic acid, heptacosylic acid / heptocosanoic acid, montanic acid / octacosanoic acid, nonocosylic acid / nonocosanoic acid, and melisic acid / triacontanoic acid.

[0138] In addition, it may include aliphatic dicarboxylic acids such as succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, and sebacic acid; and aliphatic tricarboxylic acids such as citric acid, isocitric acid, and propane-1,2,3-tricarboxylic acid.

[0139] In addition, the above unsaturated aliphatic carboxylic acid is butenoic acid, pentenic acid, hexenoic acid, heptenoic acid, such as crotonic acid.Decenoic acids such as octenoic acid, nonenoic acid, cis-4-decenoic acid / obtusilic acid (10:1(n-6)), and cis-9-decenoic acid / caproleic acid (10:(n-1)), aconitic acid, myristoleic acid (14:1), alpha-linolenic acid (18:3), stearidonic acid (18:4), eiosapentadienoic acid (EPA; 20:5), docosahexaenoic acid (DHA; 22:6), and linoleic acid aicd; 18:2), gamma-linolenic acid (γ-linolenic acid; 18:3), dihomo-γ-linolenic acid (dihomo-γ-linolenic acid; 20:3), arachidonic acid (20:4), adrenic acid (22:4), palmitoleic acid (16:1), vaccenic acid (18:1), paullinic acid (20:1), oleic acid (18:1), elaidic acid (trans-18:1), gondoic acid / 11-eicosenoic acid (20:1), erucic acid (22:1), nervonic acid (24:1), It may be one or more selected from the group consisting of mead acid (20:3) and xymenic acid (26:1).

[0140] For example, the above hydrophobic fatty acids are lauric acid / dodecanoic acid, tridecyl acid / tridecanoic acid, myristic acid / tetradecanoic acid, pentadecylic acid / pentadecanoic acid, palmitic acid / hexadecanoic acid, margaric acid / heptadecanoic acid, stearic acid / octadecanoic acid, nonadecylic acid / nonodecanoic acid, arachidic acid / eicosanoic acid, and heniicosylic acid / heneicosanoic acid. Behenic acid / docosanoic acid, myristoleic acid (14:1), alpha-linolenic acid (18:3), stearidonic acid (18:4), eiosapentadienoic acid (EPA; 20:5), docosahexaenoic acid (DHA; 22:6), linoleic acid (18:2), gamma-linolenic acid (18:3), dihomo-gamma-linolenic acid (20:3), arachidonic acid (20:4), adrenic acid (22:4), palmitoleic acid; 16:1), vaccenic acid (18:1), paullinic acid (20:1), oleic acid (It may be one or more selected from the group consisting of elaidic acid (trans-18:1), gondoic acid / 11-eicosenoic acid (20:1), and erucic acid (22:1), and more preferably, it may be one or more selected from the group consisting of dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, palmitoleic acid, and oleic acid.;

[0141] The above hydrophobic drugs are not particularly limited thereto, but preferably may include chemical substances or biodrugs having a water solubility of about 10 mg / ml or less. For example, the hydrophobic drugs may be anthracycline-based substances, hydrophobic glucocorticoids, steroid-based substances, taxane-based drugs, cyclic peptide-based drugs, or combinations thereof. The anthracycline-based substances may be doxorubicin, daunorubicin, epirubicin, idarubicin, valubicin, mitoxantrone, or combinations thereof. Hydrophobic glucocorticoids may be, for example, dexamethasone, triamcinolone, beclomethasone diproprionate, triamcinolone acetonide, triamcinolone diacetate, bethamethasone diproprionate, testosterone, budesonide, 17α-ethinylestradiol, levonorgestrel, fluticasone proprionate, or combinations thereof. For example, hydrophobic drugs may be sorafenib, paclitaxel, docetaxel, doxorubicin, cyclosporine A, amphothericin B, indinavir, rapamycin, doxorubicin, coenzyme Q10, ursodeoxycholic acid, ilaprazole, imatinib mesilate, tanespimycin, or combinations thereof.

[0142] The above-mentioned contrast agent (imaging agent or contrast media) refers to a substance that increases the contrast of an image by artificially increasing the difference in X-ray absorption of each tissue so that tissues or blood vessels can be clearly seen during examinations such as magnetic resonance imaging or computed tomography. The contrast agent may be, for example, a transition element or a chelate complex of a transition element.

[0143] The above fluorescent material is not particularly limited as long as it is hydrophobic, but preferably fluorescein, FAM (carboxyfluorescein), rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrotamine 6G, carboxyrodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, phycoerythrin, PerCP (peridinin chlorophyll-α protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue Blue), Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor (trademark) 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, 7-Amino-4-Methylcomarin-3-Acetic Acid, BODIPY (trademark) FL, BODIPY FL-Br2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY It may be one or more selected from the group consisting of 581 / 591, Bodipi 630 / 650, Bodipi 650 / 665, Bodipi R6G, Bodipi TMR, and Bodipi TR, but is not limited thereto.

[0144] The above amino acid derivative may be one or more selected from the group consisting of allantoin, N,N-dimethylglycine, homoserine, hypoxanthine, lactate, malic acid, and glycerol 3-phosphate.

[0145] The Janus-type polyelectrolyte-zwitterion-based dendrimer according to the present invention may be represented by any one of the following chemical formulas 3-1 to 3-6, 4-1 to 4-12, 5-1 to 5-4, 6-1 to 6-2, 7, and 8. In the above chemical formulas, the hydrophobic compound or amino acid derivative is R1 to It can be indicated with one or more of R4, and R1 to R4 may be any one selected from the hydrophobic compounds or amino acid derivatives described above, each independently.

[0146] The structure of the Janus-shaped polyelectrolyte-zwitterion-based dendrimer according to the present invention, which possesses polyelectrolyte and zwitterion properties along with hydrophilic and hydrophobic properties simultaneously, is a newly designed structure derived from nowhere. Conventional drug delivery systems mostly possess polyelectrolyte properties. It is known that drug delivery systems with polyelectrolyte properties can effectively deliver drugs into cells by forming strong binding forces with negatively charged cell membranes. However, positively charged particles induce high toxicity and immune responses in vivo, and the problem of polyelectrolyte effects arises, where nanoparticles aggregate at ion concentrations similar to biological conditions. In fact, various positively charged nanoparticles are designed to inhibit aggregation through surface modification; however, in this case, intracellular delivery efficiency is reduced, or even if delivered, they fail to bind and maintain with other substances (nucleic acids, proteins, etc.) in vivo and remain in an aggregated state, which can lead to side effects that impair biological functions.

[0147] However, the present invention provides a dendrimer core (preferably polylysine) based on which a positively charged peptide represented by SEQ ID NO. 1 and / or a negatively charged peptide represented by SEQ ID NO. 2 are alternately bound to one end functional group, thereby providing stable polyelectrolyte and zwitterionic properties while having appropriate hydrophilicity for self-assembly, and can achieve high intracellular delivery efficiency, low toxicity, and high structural stability at physiological salt concentrations even when a hydrophobic compound is bound to the other end functional group.

[0148] According to one embodiment of the present invention, the Janus-shaped multivalent electrolyte-bio-ion based dendrimer of the present invention has the advantage of not aggregating with each other, self-assembling into a nanofiber-shaped structure to exist stably in a solution, and having non-contaminating properties for biomaterials (nucleic acids, proteins, etc.).

[0149] In addition, the Janus-shaped polyelectrolyte-zwitterion-based dendrimer according to the present invention has the advantage of being usable immediately without separate surface modification or the addition of a carrier, as it does not form mutual bonds or aggregate even when mixed with external materials such as nucleic acid materials or proteins. Therefore, it can be utilized in various fields such as pharmaceuticals, stimulus-responsive molecular machines, and cosmetics.

[0150] Another aspect of the present invention relates to a nanostructure comprising a Janus-shaped polyelectrolyte-bio-ion-based dendrimer. This can have a wide range of medical applications. Specifically, it can be used to deliver drugs, fluorescent substances, etc., to a target requiring treatment, or in therapies to deliver target genes or drugs to cells and tissues.

[0151] Another aspect of the present invention relates to a gene or drug delivery system comprising: a Janus-shaped polyelectrolyte-bio-ion-based dendrimer; and a target gene or drug supported on said dendrimer.

[0152] As described above, the Janus-shaped polyelectrolyte-zwitterion-based dendrimer according to the present invention is an amphiphilic molecule having both hydrophilic and hydrophobic properties simultaneously, and thus can form a nanostructure of a specific structure through self-assembly in solution.

[0153] The Janus-shaped polyelectrolyte-zwitterion-based dendrimer of the present invention is characterized by being formed as a supramolecular nanostructure having an elongated fiber structure in the form of nanofibers in solution. The nanofiber structure is formed by amphiphilic molecules forming aggregated nanostructures of uniform size and shape, and the present invention may be an elongated fiber, that is, a thread structure, having a nanofiber structure.

[0154] The above nanostructure may typically have an average diameter of 10 to 400 nm, and preferably 8 to 30 nm.

[0155] The above Janus-shaped polyelectrolyte-zwitterion-based dendrimer may carry a target gene or drug. Specifically, when the Janus-shaped polyelectrolyte-zwitterion-based dendrimer is mixed with the target gene or drug in a solution, the target gene or drug is encapsulated in a nanostructure formed by the self-assembly of the Janus-shaped polyelectrolyte-zwitterion-based dendrimer. This exists very stably in solution and possesses high non-contamination properties, allowing for long-term protection from substances present both inside and outside the body, as well as efficient delivery into cells. Furthermore, since the above-described process proceeds automatically through self-assembly in solution, it has the advantage of being easy and rapid to perform. Additionally, the nanostructure present in the composition of the present invention has non-contamination properties against external substances present outside the body, so it has the advantage of being able to be stored and used without separate surface modification or a carrier.

[0156] The nanostructure of the present invention improves upon the problems associated with conventional drug delivery systems, namely the inability of the delivery system's membrane structure to maintain its shape for extended periods or the rapid drop in in vivo concentration caused by binding with in vivo or external substances. Furthermore, the nanostructure according to the present invention can be stored in various solvents such as deionized water (DW) and PBS, and it has been confirmed that stability is maintained during storage since the average diameter and size distribution do not change during storage, thereby resolving the instability inherent in conventional drug delivery systems. Moreover, it has been confirmed that the nanostructure of the present invention can be effectively delivered into cells (the nucleus and cytoplasm) and, in particular, can efficiently deliver the loaded target gene or drug to be expressed as a protein.

[0157] The nanostructures according to the present invention may be administered to a subject using techniques known in the art. It will be understood that the actual preferred amount of nanostructures in a particular patient will vary depending on the specific compound used, the specific composition being formulated, the method of application, and the specific site and target being treated.

[0158] The nanostructure according to the present invention is effective for delivering target genes or drugs into cells and can ultimately be used for the treatment or prevention of many diseases, including genetic disorders or cancer. As used herein, the term "treat" is defined as reducing the symptoms of a disease or maintaining the symptoms so as not to worsen them. Additionally, the term "treat" is also defined as the prevention of any symptoms associated with a specific disease.

[0159] The above-mentioned target gene or drug is a substance to be delivered into a cell and is not particularly limited as long as it does not hinder the purpose of the present invention; the gene may be a plasmid, mRNA, RNA, DNA, or a combination thereof, and the drug may be a low molecular weight drug, a gene drug, a protein drug, an antibody drug, a synthetic compound drug, or a combination thereof.

[0160] The above-mentioned gene may be a normal gene of a target gene related to a disease, or a gene that suppresses the expression of a target protein. For example, as a cancer therapeutic gene that induces the death of cancer cells and ultimately causes tumor regression, it may be a tumor suppressor gene, an immune regulatory gene, a cytokine gene, a chemokine gene, an antigenic gene, a suicide gene, a cytotoxic gene, a cell proliferation inhibitory gene, a pro-apoptotic gene, and an anti-angiogenic gene, but is not limited thereto. In addition, many therapeutic genes that can be usefully utilized to treat various diseases may also be delivered by the complex according to one embodiment. For example, genes encoding cytokines, interleukins, chemokines, or colony-stimulating factors, genes expressing tissue plasminogen activator (tPA) or urokinase, and genes producing LAL that prevent hypercholesterolemia by providing a sustained thrombotic effect may be included, and various polynucleotides for treating viral, malignant, and inflammatory diseases and conditions such as cystic fibrosis, adenosine deaminase deficiency, and AIDS may also be included. The nucleotide sequences of the said genes or polynucleotides may be obtained from nucleotide sequence databases such as GenBank or EMBL.

[0161] The above cancers may be, but are not limited to, ovarian cancer, colorectal cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, bile duct cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain cancer, pro-anal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine gland cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renopelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma.

[0162] The composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, intranasally, by inhalation, or topically) depending on the intended method, and the dosage may be appropriately selected by those skilled in the art, although it depends on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0163] The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat or diagnose a disease with a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level may be determined based on factors including the patient's disease type and severity, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. Other pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above-mentioned factors, and this can be easily determined by a person skilled in the art.

[0164] Specifically, the effective amount of the composition of the present invention may vary depending on the patient's age, gender, condition, body weight, absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and concomitant drugs. Additionally, the dosage may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may increase or decrease depending on the route of administration, severity of obesity, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0165] When the composition of the present invention is administered, it may be formulated with a suitable amount of pharmaceutically acceptable vehicle or carrier to provide an appropriate form of administration.

[0166] Meanwhile, the above composition may further include a carrier, an excipient, and a diluent used in the manufacture of a pharmaceutical composition.

[0167] The above carrier is commonly used and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may additionally include other common additives such as antioxidants and buffers as needed.

[0168] In addition, excipients, diluents, dispersants, surfactants, binders, lubricants, etc. can be added to formulate it into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0169] Examples of the above excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0170] Regarding suitable pharmaceutically acceptable carriers and formulations, each component may be preferably formulated using the methods disclosed in Remington’s literature. Although there are no particular restrictions on the formulation of the pharmaceutical composition of the present invention, it may be formulated into an injectable, infusion, spray, inhalation, or topical application.

[0171] In addition, the above composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions.

[0172] Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc., and the solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, with the nanostructures according to the present invention. In addition, lubricants such as magnesium styrate and talc may be used in addition to the excipients. Liquid dosage forms for oral administration may include suspensions, liquid formulations, emulsions, syrups, etc., and various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be used in addition to simple diluents such as water and liquid paraffin.

[0173] Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. For the non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. Witepsol, macrogol, tween 61, cacao oil, laurin oil, and glycerogelatin may be used as bases for the suppositories.

[0174] Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these embodiments are intended to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0175] <Examples and Experimental Examples>

[0176] Examples 1-1 to 1-6. Synthesis of Polyelectrolyte-Bitionic-Based Dendrimers (DPZ)

[0177] [Reaction Equation 1]

[0178]

[0179] Although the Solid Phase Peptide Synthesis Reaction (SPSS) allows for the convenient synthesis of peptides, it has limitations in that hydrophobic sequences with a tendency to aggregate are difficult to synthesize because the aggregation reaction is not controlled by their own hydrophobicity and hydrogen bonding. To overcome these limitations, a dendritic polyelectrolyte zwitterion (DPZ) was synthesized using a bifurcation synthesis method utilizing the Solid Phase Peptide Synthesis Reaction (SPPS) from lysine protected by 9-fluorenylmethyloxycarbonyl (Fmoc) and Dde(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl groups according to Reaction Scheme 1.

[0180] All Fmoc-amino acids were purchased from AAPPTec (USA). Polylysine dendrimers were prepared stepwise from two newly generated amines by selective deprotection of two Fmoc groups with piperidine and SPPS, as shown in Reaction Scheme 1.

[0181] Specifically, a polyelectrolyte-zwitterion-based dendrimer (DPZ) was synthesized on a link amide MBHA (4-methylbenzhydrylamine) LL (low loading) resin. A second-generation (G2) polylysine dendrimer was synthesized by sequentially coupling Fmoc-Lys(Fmoc)-OH and Fmoc-Lys(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Dde)-OH onto the resin. To attach a positively charged peptide and a negatively charged peptide to the terminal amino groups of the polylysine dendrimer, the Fmoc group was removed with 20% piperidine, and two positively charged peptide strands were sequentially synthesized using SPPS. Then, the Dde group was removed with a 2% hydrazine monohydrate solution in DMF, and the other two negatively charged peptide strands were extended using SPPS.

[0182] The positively charged peptide chains included in the above DPZ synthesis were synthesized as SEQ ID NOs 3 to 8 according to the length of the positively charged arginine residue (Arg, R), and the negatively charged peptide chains were synthesized as SEQ ID NOs 99 to 104 according to the length of the negatively charged glutamic acid (Glu, E) residue.

[0183] Polyelectrolyte binary ion-based dendrimer (DPZ(+M)) manufactured through the above-described process n (-M) n (wherein M is the number of amino acid residues constituting the positively charged peptide and the negatively charged peptide, and n is the number of peptide strands. M is an integer from 1 to 16, preferably from 1 to 12, more preferably from 1 to 6. n is an integer from 1 to 10, preferably from 1 to 4, more preferably from 1 to 2) is characterized by having both the characteristics of a polyelectrolyte and a binary ion while exhibiting hydrophilicity, because the positively charged peptide and the negatively charged peptide chain are each connected through a polylysine dendrimer and bonded in a dendrimer form.

[0184] According to the experimental method described above, (+1)2(-1)2 (Example 1-1, Chemical Formula 1-1), (+2)2(-2)2 (Example 1-2, Chemical Formula 1-2), (+3)2(-3)2 (Example 1-3, Chemical Formula 1-3), (+4)2(-4)2 (Example 1-4, Chemical Formula 1-4), (+5)2(-5)2 (Example 1-5, Chemical Formula 1-5), and (+6)2(-6)2 (Example 1-6, Chemical Formula 1-6) were synthesized.

[0185] The synthesized molecules were purified at room temperature using high-performance liquid chromatography (HPLC) with a C4 reversed-phase column (Waters, USA). Distilled water (0.1% TFA) and acetonitrile (ACN 0-100%) were used as the eluent. The molecular weight of the molecules was measured using a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometer (Fig. 2). The purity was confirmed to be > 95% as a result of analytical HPLC analysis.

[0186] [Chemical Formula 1-1]

[0187]

[0188] [Chemical Formula 1-2]

[0189]

[0190] [Chemical Formula 1-3]

[0191]

[0192] [Chemical Formula 1-4]

[0193]

[0194] [Chemical Formula 1-5]

[0195]

[0196] [Chemical Formula 1-6]

[0197]

[0198] Examples 2-1 to 2-6. Synthesis of fluorescently introduced polyelectrolyte-zwitterion-based dendrimers (DPZ)

[0199] Synthesis was performed in the same manner as in Examples 1-1 to 1-6, except that after completing the growth of the peptide chain as shown in Reaction Scheme 1, the Mmt group was deprotected and coupling with 5(6)-FAM (FAM=carboxyfluorescein) was carried out. Through the above-described process, a polyelectrolyte-zwitterion-based dendrimer (DPZ((+M)2(-M)2_FAM)) with the fluorescent substance FAM introduced was synthesized (in order, Chemical Formulas 2-1 to 2-6). Since deprotection using the Mmt group allows for the formation of bonds to various compounds, it was confirmed that the compounds applicable to the present invention are not particularly limited as long as they can form bonds with amino groups.

[0200] [Chemical Formula 2-1]

[0201]

[0202] [Chemical Formula 2-2]

[0203]

[0204] [Chemical Formula 2-3]

[0205]

[0206] [Chemical Formula 2-4]

[0207]

[0208] [Chemical Formula 2-5]

[0209]

[0210] [Chemical Formula 2-6]

[0211]

[0212] Examples 3-1 to 3-6. G1 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M) 1 (-M) 1 -(C o ) p ) = (+M) 1 (-M) 1 conjugate_1)

[0213] After synthesizing a dendritic polyelectrolyte zwitterion (DPZ) using the bifurcation synthesis method, a Janus-type polyelectrolyte zwitterion-based dendrimer (JDPZ((+M))) consisting of two strands of peptide and two strands of hydrophobic compound was formed by sequentially coupling Fmoc-Lys(Fmoc)-OH and fatty acid alkyl chains to the amine generated from Mmt deprotection. n (-M) n -(C o ) p )) was synthesized (chemical formulas 3-1 to 3-6 in order).

[0214] [Chemical Formula 3-1] JDPZ((+1)1(-1)1-(C 12 )2) = (+1)1(-1)1conjugate_1

[0215]

[0216] [Chemical Formula 3-2] JDPZ((+2)1(-2)1-(C 12 )2) = (+2)1(-2)1conjugate_1

[0217]

[0218] [Chemical Formula 3-3] JDPZ((+3)1(-3)1-(C 12 )2) = (+3)1(-3)1conjugate_1

[0219]

[0220] [Chemical Formula 3-4] JDPZ((+4)1(-4)1-(C 12 )2) = (+4)1(-4)1conjugate_1

[0221]

[0222] [Chemical Formula 3-5] JDPZ((+5)1(-5)1-(C 12 )2) = (+5)1(-5)1conjugate_1

[0223]

[0224] [Chemical Formula 3-6] JDPZ((+6)1(-6)1-(C 12 )2) = (+6)1(-6)1conjugate_1

[0225]

[0226] First, a link amide MBHA (4-Methylbenzhydrylamine) LL (low loading) resin was synthesized, and a first-generation (G1) polylysine dendrimer was synthesized by sequentially coupling Fmoc-Lys(Mmt)-OH and Fmoc-Lys(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Dde)-OH onto the resin.

[0227] The deprotection process of the Mmt group was performed by coupling Fmoc-Lys(Fmoc)-OH to the primary amine produced after treating the resin with 1% trifluoroacetic acid in dichloromethane several times (1 min × up to 8 times). After deprotecting two Fmoc groups with piperidine, 12-aminododecanoic acid was synthesized by coupling it to the primary amine using O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HCTU), Hydroxybenzotriazole (HOBt), and N,N-diisopropylethylamine (DIPEA) dissolved in NMP.

[0228] JDPZ((+M)1(-M)1-(C) manufactured through the above-described process o ) p(wherein M is the number of amino acid residues constituting the positively charged peptide and the negatively charged peptide, n=1 is the number of peptide strands, o is the number of carbon atoms, and p is the number of fatty acid alkyl chains. M is an integer from 1 to 30, o is an integer from 10 to 30, and p is an integer from 1 to 10) was purified at room temperature using HPLC (high-performance liquid chromatography) with a C4 reversed-phase column (Waters, USA). Distilled water (0.1% TFA) and acetonitrile (ACN 0-100%) were used as the eluent. The molecular weight of the molecule was measured using a MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometer and is shown in Fig. 10. The purity was confirmed to be > 95% as a result of analytical HPLC analysis.

[0229] Examples 4-1 to 4-12. Fluorescent-introduced G1 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ((+M) 1 (-M) 1 -(C o ) p _FAM) = (+M) 1 (-M) 1 conjugate_FAM)

[0230] After synthesizing a dendritic polyelectrolyte zwitterion (DPZ) using the bifurcation synthesis method, Dde-Lys(Fmoc)-OH was coupled to the amine generated from Mmt deprotection. First, the Fmoc group was selectively deprotected, followed by coupling of a fatty acid alkyl chain. Subsequently, by deprotecting the Dde group and sequentially coupling the fatty acid alkyl chain and the fluorescent agent 5(6)-FAM, a Janus-shaped polyelectrolyte zwitterion-based dendrimer (JDPZ((+M)1(-M)1-(C)) composed of two peptide strands, two hydrophobic strands, and FAM attached to the terminals of the hydrophobic compounds was formed. o ) p _FAM)) was synthesized (chemical formulas 4-1 to 4-12 in order).

[0231] [Chemical Formula 4-1]

[0232]

[0233] [Chemical Formula 4-2]

[0234]

[0235] [Chemical Formula 4-3]

[0236]

[0237] [Chemical Formula 4-4]

[0238]

[0239] [Chemical Formula 4-5]

[0240]

[0241] [Chemical Formula 4-6]

[0242]

[0243] [Chemical Formula 4-7]

[0244]

[0245] [Chemical Formula 4-8]

[0246]

[0247] [Chemical Formula 4-9]

[0248]

[0249] [Chemical Formula 4-10]

[0250]

[0251] [Chemical Formula 4-11]

[0252]

[0253] [Chemical Formula 4-12]

[0254]

[0255] Examples 5-1 to 5-4. G2 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M) 2 (-M) 2 -(C o ) p ) = (+M) 2 (-M) 2 conjugate_1)

[0256] [Reaction Equation 2]

[0257]

[0258] A dendritic polyelectrolyte zwitterion (DPZ) was synthesized using a bifurcation synthesis method. It was synthesized on a link amide MBHA (4-Methylbenzhydrylamine) LL (low loading) resin, and a second-generation (G2) polylysine dendrimer was synthesized by sequentially coupling Fmoc-Lys(Mmt)-OH, Fmoc-Lys(Fmoc)-OH, and Fmoc-Lys(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, Dde)-OH onto the resin. To attach a positively charged peptide and a negatively charged peptide to the terminal amino groups of the polylysine dendrimer, the Fmoc group was removed with 20% piperidine, and two positively charged peptide strands were sequentially synthesized using SPPS. Then, the Dde group was removed with a 2% hydrazine monohydrate solution in DMF, and the other two negatively charged peptide strands were extended into SPPS.

[0259] Subsequently, Fmoc-Lys(Fmoc)-OH was coupled to the amine generated from Mmt deprotection. After deprotecting two Fmoc groups with piperidine, Fmoc-Lys(Dde)-OH was coupled to each amine group. By using a method in which two fatty acid alkyl chains were coupled after removing the Fmoc group with 20% piperidine, and then two fatty acid alkyl chains were coupled after removing the Dde group, a Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M)2(-M)2-(C)) was formed consisting of four strands of peptide and four strands of hydrophobic compound. o ) p )) was synthesized (chemical formulas 5-1 to 5-4 in order).

[0260] [Chemical Formula 5-1] JDPZ((+3)2(-3)2-(C 12)4) = (+3)2(-3)2conjugate_1

[0261]

[0262] [Chemical Formula 5-2] JDPZ((+4)2(-4)2-(C 12 )4) = (+4)2(-4)2conjugate_1

[0263]

[0264] [Chemical Formula 5-3] JDPZ((+5)2(-5)2-(C 12 )4) = (+5)2(-5)2conjugate_1

[0265]

[0266] [Chemical Formula 5-4] JDPZ((+6)2(-6)2-(C 12 )4) = (+6)2(-6)2conjugate_1

[0267]

[0268] JDPZ((+M)2(-M)2-(C) manufactured through the above-described process o ) p(wherein M is the number of amino acid residues constituting the positively charged peptide and the negatively charged peptide, n=2 is the number of peptide strands, o is the number of carbon atoms, and p is the number of fatty acid alkyl chains. M is an integer from 1 to 30, o is an integer from 10 to 30, and p is an integer from 1 to 10) was purified at room temperature using HPLC (high-performance liquid chromatography) with a C4 reversed-phase column (Waters, USA). Distilled water (0.1% TFA) and acetonitrile (ACN 0-100%) were used as the eluent. The molecular weight of the molecule was measured using a MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometer and is shown in Fig. 16. The purity was confirmed to be > 95% as a result of analytical HPLC analysis.

[0269] Examples 6-1 to 6-2. Fluorescent-introduced G2 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M) 2 (-M) 2 -(C o ) p _FAM) = (+M) 2 (-M) 2 conjugate_FAM)

[0270] [Reaction Equation 3]

[0271]

[0272] After completing the growth of the peptide chain as shown in Reaction Scheme 3, Dde-Lys(Fmoc)-OH was coupled to the amine produced from Mmt deprotection. Subsequently, Fmoc was selectively deprotected, and Fmoc-Lys(Fmoc)-OH was coupled to the amine produced. After deprotecting two Fmoc groups with piperidine, two fatty acid alkyl chains were coupled to each amine group. After deprotecting the Dde group, Dde-Lys(Fmoc)-OH was coupled, and the synthesis was completed by sequentially coupling one fatty acid alkyl chain after deprotecting the Fmoc group and coupling 5(6)-FAM after deprotecting the Dde group. Through the above-described process, a Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M)2(-M)2-(C)) composed of four strands of peptide, three strands of hydrophobic compound, and one hydrophobic fluorescent agent o ) p _FAM) was synthesized (chemical formulas 6-1 to 6-2 in order). Since bonds can be formed to various hydrophobic compounds through deprotection using the Mmt group, it can be seen that hydrophobic compounds applicable to the present invention are not particularly limited as long as they can form bonds with amino groups.

[0273] [Chemical Formula 6-1] JDPZ((+3)2(-3)2-(C 12 )3_FAM) = (+3)2(-3)2conjugate_FAM

[0274]

[0275] [Chemical Formula 6-2] JDPZ((+6)2(-6)2-(C 12 )3_FAM) = (+6)2(-6)2conjugate_FAM

[0276]

[0277] Example 7. G2 Janus-type polyelectrolyte-bioelectrolyte-based dendrimer (JDPZ((+6)) 2 (-6) 2 -(C o ) p (C q ) r ) = (+6) 2 (-6) 2 conjugate_2)

[0278] Second-generation (G2) polylysine dendrimers were synthesized using the bifurcation synthesis method. Subsequently, two positively charged peptide strands and two negatively charged peptide strands were synthesized sequentially. Then, Fmoc-Lys(Fmoc)-OH groups were coupled to the amines generated from Mmt deprotection. After removing the Fmoc groups with 20% piperidine, two fatty acid alkyl chains were coupled to form a G2 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M)2(-M)2-(C o ) p ) = (+M)2(-M)2conjugate_2) was synthesized.

[0279] [Chemical Formula 7] JDPZ((+6)2(-6)2-(C 16 )1(C 18 )1) = (+6)2(-6)2conjugate_2

[0280]

[0281] Example 8. G2 Janus-type polyelectrolyte-bio-ion based dendrimer (JDPZ((+6)) 2 (-6) 2 -(C o ) p (DMG) q ) = (+6) 2 (-6) 2 conjugate_3)

[0282] A second-generation (G2) polylysine dendrimer was synthesized using the bifurcation synthesis method. Subsequently, two positively charged peptide strands and two negatively charged peptide strands were synthesized sequentially. Then, Fmoc-Lys(Fmoc)-OH was coupled to the amine generated from Mmt deprotection. After removing the Fmoc group with 20% piperidine, two Dde-Lys(Fmoc)-OH groups were coupled. After deprotecting the Fmoc group, two DMG(N,N-dimethylglycine) groups were coupled, and subsequently, after deprotecting the Dde group, two Dde-Lys(Fmoc)-OH groups were coupled. Next, the Fmoc group was deprotected and two fatty acid alkyl chains were coupled, and subsequently, the Dde group was deprotected and two DMG groups were coupled to form a G2 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2-(C o ) p (DMG) q ) = (+6)2(-6)2conjugate_3) was synthesized.

[0283] [Chemical Formula 8] JDPZ((+6)2(-6)2-(C 18 )1(C 18 )1(DMG)4) = (+6)2(-6)2conjugate_3

[0284]

[0285] Comparative Examples 1-1 to 1-3. G2 Janus-shaped dendrimer

[0286] A G2 Janus-type dendrimer was synthesized in the same manner as in Example 5-1, except that arginine was used as the positively charged peptide, glutamic acid as the negatively charged peptide, and serine (Ser, S) as the neutral charge (Ø) (in order, Comparative Examples 1-1 to 1-3).

[0287] [Chemical Formula 5-5] (+3)4-(C 12 )4 = (+3)4conjugate

[0288]

[0289] [Chemical Formula 5-6] (+3)2(Ø3)2-(C 12 )4= (+3)2(Ø3)2conjugate

[0290]

[0291] [Chemical Formula 5-7] (Ø3)2(-3)2-(C 12 )4= (Ø3)2(-3)2conjugate

[0292]

[0293] Comparative Examples 2-1 to 2-2. G2 Janus-shaped dendrimers with introduced fluorescent material

[0294] A G2 Janus-type dendrimer was synthesized in the same manner as in Example 5-1, except that arginine was used as the positively charged peptide, glutamic acid as the negatively charged peptide, and serine (Ser, S) as the neutral charge (Ø) (in order, Comparative Examples 2-1 to 2-2).

[0295] [Chemical Formula 6-3] (+3)2(Ø3)2-(C 12 )3_FAM = (+3)2(Ø3)2conjugate_FAM

[0296]

[0297] [Chemical Formula 6-4] (Ø3)2(-3)2-(C 12 )3_FAM = (Ø3)2(-3)2conjugate_FAM

[0298]

[0299] Comparative Examples 3-1, 3-2. Zigionic peptide complex (Ac(ER)12, AcE12R12)

[0300] Using a solid-state peptide synthesis reaction (SPSS), a peptide (3-1) represented by SEQ ID NO. 163, in which a cationic amino acid and an anionic amino acid are alternately combined, and a peptide (3-2) represented by SEQ ID NO. 164, in which a zwitterionic peptide and an anionic peptide are combined, were prepared.

[0301] Experimental Example 1. Characterization of Polyelectrolyte-Bitcoion-Based Dendrimers (DPZ)

[0302] 1) MALDI-TOF Mass Spectrometry

[0303] To confirm whether polyelectrolyte-zwitterion-based dendrimers (DPZ) prepared from Examples 1-1 to 1-6 were synthesized, they were analyzed by MALDI-TOF.

[0304] FIG. 2a is the MALDI-TOF graph of (+1)2(-1)2 prepared from Example 1-1, FIG. 2b is the MALDI-TOF graph of (+2)2(-2)2 prepared from Example 1-2, FIG. 2c is the MALDI-TOF graph of (+3)2(-3)2 prepared from Example 1-3, FIG. 2d is the MALDI-TOF graph of (+4)2(-4)2 prepared from Example 1-4, FIG. 2e is the MALDI-TOF graph of (+5)2(-5)2 prepared from Example 1-5, and FIG. 2f is the MALDI-TOF graph of (+6)2(-6)2 prepared from Example 1-6. According to this, it can be seen that the polyelectrolyte-zwitterion-based dendrimers (DPZ) prepared from Examples 1-1 to 1-6 were successfully synthesized.

[0305] 2) Cytotoxicity

[0306] Since DPZ contains both polyvalent electrolytes and binary ions within a single molecule, it was expected to possess non-contamination and low toxicity, while simultaneously exhibiting favorable intracellular delivery efficiency due to its interaction with the cell membrane. First, cytotoxicity was analyzed. HeLa cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 1 × 10⁶ cells were placed in each well. 4 After dispensing cells / wells, they were cultured at 37°C for 24 hours, and samples of various concentrations (Examples 1-1 to 1-6 or Comparative Examples 3-1, 3-2) were each treated and cultured at 37°C for 4 hours. WST-8 solution was added to the cells after culture was complete, and after 4 hours, the absorbance was measured at 450 nm using a microplate reader (Perkin-Elmer, USA).

[0307] FIG. 3 shows polyelectrolyte-zwitterion-based dendrimers (DPZ(+M)) prepared from Examples 1-1 to 1-6 according to concentrations (0, 2, 4, 8, 16, 32, 64, 128 μM) n (-M) n This is a graph showing the cytotoxicity of peptides prepared from ) and Comparative Examples 3-1 and 3-2 analyzed in HeLa cell lines. As shown in Fig. 3, the zwitterion of Comparative Example 3-1, the polyelectrolyte-zwitterion of Comparative Example 3-2, and the polyelectrolyte-zwitterion-based dendrimers (DPZ(+M)) of Examples 1-1 to 1-6 n (-M) n It can be seen that ) is a stable substance that does not cause toxicity to cells. In the case of the polyelectrolyte-zwitterion of Comparative Example 3-2, it can be confirmed that it promotes cell growth.

[0308] 3) Analysis of nonfouling characteristics

[0309] Various proteins and genetic materials exist within the body. Consequently, it has been confirmed that conventional cell-penetrating peptides or carriers form interactions with various molecules within the body, leading to problems such as insufficient delivery of drug efficacy or the occurrence of side effects. In this invention, it is believed that non-contamination of molecules within the body has been secured by presenting a new structure, and this was verified through an Electrophoretic Mobility Shift Assay (EMSA).

[0310] First, 0.5 μg of BSA, a sticky protein present in high proportions in blood, was prepared as a model protein, and the polyelectrolyte-zwitterion-based dendrimers (DPZ(+M)) of Examples 1-1 to 1-6 were prepared at various concentration ratios. n (-M) nIt was mixed with ). The mixing ratio used was the R / B ratio, which is the arginine / BSA ratio. After reacting the mixture at room temperature for 12 hours, electrophoresis was performed on a 12% native polyacrylamide gel electrophoresis (PAGE) gel at 22 mA for 90 minutes. To visualize the bands, they were stained with Coomassie Brilliant Blue R-250.

[0311] Figure 4a shows the EMSA results analyzing the protein non-contamination of the polyelectrolyte-zwitterion-based dendrimers ((+1)2(-1)2 and (+2)2(-2)2) of Examples 1-1 and 1-2. Figure 4b shows the EMSA results analyzing the protein non-contamination of the polyelectrolyte-zwitterion-based dendrimers ((+3)2(-3)2 and (+4)2(-4)2) of Examples 1-3 and 1-4. Figure 4c shows the EMSA results analyzing the protein non-contamination of the polyelectrolyte-zwitterion-based dendrimers ((+5)2(-5)2 and (+6)2(-6)2) of Examples 1-5 and 1-6. In Figure 4, 'L' is a protein marker, and the R / B ratio is the mixing ratio of arginine and BSA present in DPZ.

[0312] As shown in Fig. 4, it was confirmed that the polyelectrolyte-zwitterion-based dendrimers of Examples 1-1 to 1-6 do not bind to BSA even in a mixed solution with excess BSA (R / B ratio > 1). In other words, it can be seen that the polyelectrolyte-zwitterion-based dendrimers of Examples 1-1 to 1-6 have significantly higher non-contamination properties for proteins present in vivo.

[0313] 4) Comparative analysis of nonfouling for linear R12 having the same number of cationic residues as in Examples 1-6

[0314] A linear peptide R12 having the same number of cationic residues (12 arginine groups) as the polyelectrolyte-zwitterion-based dendrimer ((+6)2(-6)2) of Examples 1-6 was prepared using solid-phase peptide synthesis reaction (SPSS), and '3) Analysis of nonfouling characteristics It was analyzed using EMSA in the same way as.

[0315] Figure 5 shows the EMSA results analyzing the protein-non-contamination properties of linear peptide R12 (SEQ No. 165). According to the results, it was confirmed that linear peptide R12 (SEQ No. 165) interacts with BSA to induce an electrophoretic mobility shift (EMS) and weakening of the band intensity of the BSA band. Therefore, linear peptide R12 (SEQ No. 165), which possesses only cationic properties, did not exhibit non-contamination characteristics.

[0316] Experimental Example 3. mRNA complex formation, cell delivery, and expression

[0317] It was confirmed that the polyelectrolyte-zwitterion-based dendrimer (DPZ) of the present invention exists stably inside cells without toxicity. Next, we sought to confirm whether the polyelectrolyte-zwitterion-based dendrimer according to the present invention acts as a genetic material carrier and performs a role as a therapeutic agent through fusion with genetic material.

[0318] 1) Analysis of the presence or absence of complex formation between polyelectrolyte-zwitterion-based dendrimers and dielectric material

[0319] Single-stranded DNA (ssDNA) (Sequence No. 166) and mRNA (Sequence No. 167) were purchased from Bioneer (Korea) and TriLink BioTechnologies (USA), respectively, and used as genetic materials. Double-helix plasmid DNA (Sequence No. 168) was used and prepared by in-house cloning. In the case of mRNA, the total sequence length is 997 nt, but since the selling company (Trilink BioTechnologies) has disclosed only the open reading frame (ORF) of 720 nt, only that sequence is described.

[0320] Complexes were prepared by varying the mixing ratio of each genetic material with the polyelectrolyte-bio-ion-based dendrimer ((+6)2(-6)2) of Examples 1-6. The mixing ratio is denoted as the Charge ratio (+ / -), which is calculated as (total positive charge of DPZ) / (total negative charge of genetic material). The DPZ of Examples 1-6 was calculated and mixed based on the weight of the genetic material (ssDNA 200 nt, mRNA 997 nt, plasmid DNA 4,848 bp). Distilled water (DW) was used as the mixing solvent. The mixture was subjected to electrophoresis on a 0.8% agarose gel at 100 V for 30 minutes to confirm the formation of the complex.

[0321] Figure 6 is the result of EMSA analyzing the interaction patterns between the multivalent electrolyte-bio-ion-based dendrimer ((+6)2(-6)2) of Examples 1-6 and various genetic materials (single-stranded DNA, mRNA, and plasmid DNA).

[0322] As shown in Fig. 6, it was confirmed that the multivalent electrolyte-bio-ion based dendrimers ((+6)2(-6)2) of Examples 1-6 form a complex with various forms of genetic materials, such as single-stranded DNA, mRNA, and plasmid DNA, and induce an electrophoretic mobility shift (EMS) of these genetic materials.

[0323] 2) Presence or absence of complex formation between polyelectrolyte-bioelectrolyte-based dendrimers and mRNA

[0324] mRNA and polyelectrolyte-bioelectrolyte-difficion-based dendrimers according to charge ratio (DPZ(+M) of Examples 1-3 to 1-6) n (-M) n A complex was prepared. Specifically, 997 nt of EGFP mRNA was immobilized, and the polyelectrolyte-bioelectrolyte-based dendrimers ((+3)2(-3)2, (+4)2(-4)2, (+5)2(-5)2, and (+6)2(-6)2) of Examples 1-3 to 1-6 were each mixed to achieve a charge ratio of 0 to 4. The mixing ratio is denoted as Charge ratio(+ / -), which is calculated as (total positive charge of DPZ) / (total negative charge of the genetic material). The mixture was subjected to electrophoresis on a 0.8% agarose gel at 100 V for 30 minutes to confirm the formation of the complex.

[0325] Figure 7 is the result of an EMSA experiment analyzing the complex formation between mRNA and the polyelectrolyte-bioelectrolyte-zwitterion-based dendrimers ((+3)2(-3)2, (+4)2(-4)2, (+5)2(-5)2 and (+6)2(-6)2) of Examples 1-3 to 1-6 according to the charge ratio.

[0326] As shown in Fig. 7, the polyelectrolyte-zwitterion-based dendrimers of Examples 1-3 to 1-6 induced a stronger EMS as the charge ratio (+ / -) increased. In addition, it was confirmed that the polyelectrolyte-zwitterion-based dendrimers of Examples 1-3 to 1-6 induced a stronger EMS as the lengths of the positively charged peptide and the negatively charged peptide increased.

[0327] Experimental Example 4. Characterization of Fluorescently Introduced Polyelectrolyte-Bitcoion-Based Dendrimers (DPZ)

[0328] 1) MALDI-TOF Mass Spectrometry

[0329] DPZ ((+M) introduced with FAM, a fluorescent material prepared from Examples 2-1 to 2-6 n (-M) n To confirm the synthesis of _FAM), it was analyzed using MALDI-TOF.

[0330] FIG. 8 shows DPZ(+M) introduced with FAM, a fluorescent material prepared from Examples 2-1 to 2-6 n (-M) n This is a MALDI-TOF graph for _FAM). FIG. 8a is for (+1)2(-1)2_FAM of Example 2-1, FIG. 8b is for (+2)2(-2)2_FAM of Example 2-2, FIG. 8c is for (+3)2(-3)2_FAM of Example 2-3, FIG. 8d is for (+4)2(-4)2_FAM of Example 2-4, FIG. 8e is for (+5)2(-5)2_FAM of Example 2-5, and FIG. 8f is for (+6)2(-6)2_FAM of Example 2-6.

[0331] As shown in Fig. 8, a fluorescently introduced polyelectrolyte-bio-ion-based dendrimer ((+M) n (-M) n It was confirmed that _FAM) (Examples 2-1 to 2-6) was also successfully synthesized.

[0332] 2) Intracellular delivery efficiency

[0333] DPZ ((+M) introduced with FAM, a fluorescent material prepared from Examples 2-1 to 2-6 n (-M) n We aimed to verify whether _FAM) is effectively delivered into cells. To this end, intracellular delivery efficiency was analyzed using HeLa cells. First, HeLa cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 1–3 × 10⁶ cells were placed in each well. 4 After dispensing cells / wells, incubate at 37°C for 24 hours, then apply 16 μM or 32 μM (+M) n (-M) n Each cell was treated with FAM (Examples 2-1 to 2-6) and cultured at 37°C for 4 hours. Afterward, the cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), treated with trypsin, and then resuspended in DPBS supplemented with 10% cell dissociation buffer (Gibco) and 1% FBS. Subsequently, intracellular fluorescence was analyzed using a flow cytometer (BD LSR II SORT flow cytometer, BD Bioscience, USA).

[0334] FIG. 9 shows the (+M) of Examples 2-1 to 2-6 according to concentration (16 μM (top), 32 μM (bottom)). n (-M) n This is a graph showing the intracellular delivery efficiency of FAM in HeLa cell lines analyzed by flow cytometry (FACS). As shown in Figure 9, it was confirmed that all of the fluorescently introduced polyelectrolyte-zwitterion-based dendrimers of Examples 2-1 to 2-6 had a high intracellular delivery efficiency of over 95%.

[0335] Experimental Example 5. Characterization of G1 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ)

[0336] 1) MALDI-TOF Mass Spectrometry

[0337] To confirm the synthesis of the G1 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ, (+M)1(-M)1conjugate_1) prepared from Examples 3-1 to 3-6, it was analyzed by MALDI-TOF.

[0338] Figure 10 is a MALDI-TOF graph for G1 Janus-type polyelectrolyte-bio-ion-based dendrimers (JDPZ, (+M)1(-M)1conjugate_1) prepared from Examples 3-1 to 3-6. FIG. 10a is for (+1)1(-1)1conjugate_1 of Example 3-1, FIG. 10b is for (+2)1(-2)1conjugate_1 of Example 3-2, FIG. 10c is for (+3)1(-3)1conjugate_1 of Example 3-3, FIG. 10d is for (+4)1(-4)1conjugate_1 of Example 3-4, FIG. 10e is for (+5)1(-5)1conjugate_1 of Example 3-5, and FIG. 10f is for (+6)1(-6)1conjugate_1 of Example 3-6.

[0339] As shown in Fig. 10, it was confirmed that the G1 Janus-type polyelectrolyte-zwitterion-based dendrimers prepared from Examples 3-1 to 3-6 were also successfully synthesized.

[0340] 2) Structural Analysis

[0341] As it has been confirmed that the Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ) according to the present invention forms a supramolecular structure through self-assembly in solution, we intended to determine the morphology and structure of the Janus-shaped polyelectrolyte-zwitterion-based dendrimer supramolecular structure according to the present invention.

[0342] The G1 Janus-type polyelectrolyte-bio-ion based dendrimers (JDPZ, (+M)1(-M)1conjugate_1)((+1)1(-1)1-conjugate_1, (+2)1(-2)1-conjugate_1, (+3)1(-3)1-conjugate_1, (+4)1(-4)1-conjugate_1, (+5)1(-5)1-conjugate_1, and (+6)1(-6)1-conjugate_1) of Examples 3-1 to 3-6 were each mixed in DW and analyzed by AFM as follows. AFM was performed using an NX10 system (Park Systems, Korea) in non-contact mode. After dissolving each sample in DW, 1 µl was cast onto a freshly exfoliated mica surface and dried. The data were analyzed using XEN software (Park Systems, Korea).

[0343] FIG. 11 is an AFM image of Janus-type polyelectrolyte-bio-ion-based dendrimers ((+1)1(-1)1-conjugate_1, (+2)1(-2)1-conjugate_1, (+3)1(-3)1-conjugate_1, (+4)1(-4)1-conjugate_1, (+5)1(-5)1-conjugate_1, and (+6)1(-6)1-conjugate_1) of Examples 3-1 to 3-6 in DW solution.

[0344] According to this, the Janus-shaped multi-electrolyte-zwitterion-based dendrimers of Examples 3-1, 3-2, 3-4, and 3-6 formed elongated nanofiber-shaped supramolecular nanostructures, and the Janus-shaped multi-electrolyte-zwitterion-based dendrimers of Examples 3-3 and 3-5 formed round supramolecular nanostructures.

[0345] 3) Cytotoxicity analysis

[0346] We intended to analyze the cytotoxicity of the Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ) according to the present invention. Since JDPZ contains both a polyelectrolyte and a zwitterion within a single molecule, it was expected to have non-contamination and low toxicity, while also having favorable intracellular delivery efficiency due to its interaction with the cell membrane.

[0347] Cytotoxicity was performed using HeLa cells. Specifically, HeLa cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 1 × 10⁶ cells were placed in each well. 4 After dispensing cells / wells, they were cultured at 37°C for 24 hours, and samples of various concentrations (Examples 3-1 to 3-6) were each treated and cultured at 37°C for 4 hours. WST-8 solution was added to the cells after culture was complete, and after 4 hours, the absorbance was measured at 450 nm using a microplate reader (Perkin-Elmer, USA).

[0348] Figure 12 is a graph showing the cytotoxicity of (+M)1(-M)1conjugate_1 of Examples 3-1 to 3-6 according to concentrations (0, 4, 8, 16, 32, 64 μM). According to this, it can be seen that the Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ) ((+1)1(-1)1-conjugate_1, (+2)1(-2)1-conjugate_1, (+3)1(-3)1-conjugate_1, (+4)1(-4)1-conjugate_1, (+5)1(-5)1-conjugate_1, and (+6)1(-6)1-conjugate_1) of Examples 3-1 to 3-6 are all stable substances that do not cause toxicity to cells.

[0349] Experimental Example 6. Characterization of G1 Janus-shaped polyelectrolyte-zwitterion-based dendrimers (JDPZ_FAM) with introduced fluorescent material

[0350] 1) MALDI-TOF Mass Spectrometry

[0351] To confirm the synthesis of the fluorescently introduced G1 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ, (+M)1(-M)1conjugate_FAM) prepared from Examples 4-1 to 4-12, analysis was performed using MALDI-TOF.

[0352] Figure 13 is a MALDI-TOF graph for a G1 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ, (+M)1(-M)1conjugate_FAM) with a fluorescent material introduced from Examples 4-1 to 4-12. FIG. 13a is for the (+1)1(-1)1conjugate_FAM of Example 4-1, FIG. 13b is for the (+2)1(-2)1conjugate_FAM of Example 4-2, FIG. 13c is for the (+3)1(-3)1conjugate_FAM of Example 4-3, FIG. 13d is for the (+4)1(-4)1conjugate_FAM of Example 4-4, FIG. 13e is for the (+5)1(-5)1conjugate_FAM of Example 4-5, FIG. 13f is for the (+6)1(-6)1conjugate_FAM of Example 4-6, FIG. 13g is for the (+7)1(-7)1conjugate_FAM of Example 4-7, and FIG. 13h is for Example 4-8 Figure 13i is for (+8)1(-8)1conjugate_FAM, Figure 13i is for (+5)1(-5)1conjugate (RD)_FAM of Example 4-9, Figure 13j is for (+6)1(-6)1conjugate (RD)_FAM of Example 4-10, Figure 13k is for (+5)1(-5)1conjugate (KE)_FAM of Example 4-11, and Figure 13l is for (+6)1(-6)1conjugate (KE)_FAM of Example 4-12.

[0353] As shown in Fig. 13, it was confirmed that the fluorescent material introduced in the G1 Janus-type multi-electrolyte-zwitterion-based dendrimer prepared from Examples 4-1 to 4-12 was also successfully synthesized.

[0354] 2) Intracellular delivery efficiency

[0355] We aimed to verify the intracellular delivery efficiency of the fluorescently introduced G1 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ, (+M)1(-M)1conjugate_FAM) prepared from Examples 4-1 to 4-12. Intracellular delivery efficiency was analyzed using HeLa cells. First, HeLa cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 1–3 × 10⁶ cells were placed in each well. 4 After dispensing cells / wells, incubated at 37°C for 24 hours, then treated with JDPZ((+M)1(-M)1conjugate_FAM) (Examples 4-1 to 4-12) at concentrations of 4 μM, 8 μM, or 32 μM, respectively, and incubated at 37°C for 4 hours. Subsequently, the cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), treated with trypsin, resuspended in DPBS supplemented with 10% cell dissociation buffer (Gibco) and 1% FBS, and then intracellular fluorescence was analyzed using a flow cytometer (BD LSR II SORT flow cytometer, BD Bioscience, USA).

[0356] FIG. 14 is a graph showing the intracellular delivery efficiency (degree of internalization) of JDPZ((+M)1(-M)1conjugate_FAM) prepared from Examples 4-1 to 4-6, analyzed by flow cytometry (FACS). FIG. 14a shows the treatment with 4 μM of JDPZ((+M)1(-M)1conjugate_FAM) prepared from Examples 4-1 to 4-6, FIG. 14b shows the treatment with 8 μM of JDPZ((+M)1(-M)1conjugate_FAM) prepared from Examples 4-1 to 4-6, and FIG. 14c shows the treatment with 16 μM of JDPZ((+M)1(-M)1conjugate_FAM) prepared from Examples 4-1 to 4-6.

[0357] Figure 15 is a graph showing the intracellular delivery efficiency of JDPZ((+M)1(-M)1conjugate_FAM) prepared from Examples 4-6 to 4-12, analyzed by flow cytometry (FACS).

[0358] As shown in Fig. 14, the G1 Janus-type multi-electrolyte-zwitterion-based dendrimers with fluorescent substances introduced in Examples 4-1 to 4-6 were all efficiently delivered into cells, and it was confirmed that there was a dose-response tendency in which the delivery efficiency into cells increased in proportion to the concentration.

[0359] As shown in Fig. 15, the G1 Janus-type multi-electrolyte-zwitterion-based dendrimers with fluorescent materials introduced in Examples 4-6 to 4-12 showed a high internalization efficiency of 94.4% to 100%, and it was confirmed that the internalization efficiency tended to increase in proportion to the concentration.

[0360] Experimental Example 7. Characterization of G2 Janus-type polyelectrolyte-zwitterion-based dendrimers

[0361] 1) MALDI-TOF Mass Spectrometry

[0362] MALDI-TOF analysis was performed to confirm the synthesis of the G2 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ, (+M)2(-M)2conjugate_1) prepared from Examples 5-1 to 5-4 and the G2 Janus-type polyelectrolyte-zwitterion-based dendrimers prepared from Comparative Examples 1-1 to 1-3.

[0363] FIG. 16 is a MALDI-TOF graph for G2 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ((+M)2(-M)2conjugate_1)) prepared from Examples 5-1 to 5-4. FIG. 16a is for (+3)2(-3)2conjugate_1 prepared from Example 5-1, FIG. 16b is for (+4)2(-4)2conjugate_1 prepared from Example 5-2, FIG. 16c is for (+5)2(-5)2conjugate_1 prepared from Example 5-3, and FIG. 16d is for (+6)2(-6)2conjugate_1 prepared from Example 5-4.

[0364] FIG. 17a is for a (+3)4 conjugate prepared from Comparative Example 1-1, FIG. 17b is for a (+3)2(Ø)2 conjugate prepared from Comparative Example 1-2, and FIG. 17c is for a (Ø)2(-3)2 conjugate prepared from Comparative Example 1-3.

[0365] As shown in FIGS. 16 and 17, it was confirmed that not only the G2 Janus-type polyelectrolyte-zwitterion-based dendrimers prepared from Examples 5-1 to 5-4 but also the G2 Janus-type dendrimers prepared from Comparative Examples 1-1 to 1-3 were successfully synthesized.

[0366] 2) Structural Analysis

[0367] The G2 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ, (+M)2(-M)2conjugate_1) of Examples 5-1 to 5-4 were added to each DW to prepare aqueous solutions, and AFM analysis was performed. AFM was performed using an NX10 system (Park Systems, Korea) in non-contact mode. 1 µl of the sample was cast onto a freshly exfoliated mica surface and dried. The data were analyzed using XEN software (Park Systems, Korea).

[0368] FIG. 18 is an AFM image of the G2 Janus-type polyelectrolyte-bio-ion based dendrimers (JDPZ, (+M)2(-M)2conjugate_1) of Examples 5-1 to 5-4. FIG. 18a is for (+3)2(-3)2conjugate_1 prepared from Example 5-1, FIG. 18b is for (+4)2(-4)2conjugate_1 prepared from Example 5-2, FIG. 18c is for (+5)2(-5)2conjugate_1 prepared from Example 5-3, and FIG. 18d is for (+6)2(-6)2conjugate_1 prepared from Example 5-4.

[0369] According to this, it was confirmed that the G2 Janus-type polyelectrolyte-bio-ion-based dendrimer (JDPZ, (+M)2(-M)2conjugate_1) of Examples 5-1 to 5-4 is a supramolecular structure in the form of an elongated nanofiber.

[0370] 3) Cytotoxicity analysis

[0371] To analyze the cytotoxicity of the G2 Janus-type polyelectrolyte-bio-ion-based dendrimer (JDPZ, (+M)2(-M)2conjugate_1) according to the present invention, the study was conducted using HCT116 cells (human colorectal cancer cell line). Specifically, HCT116 cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 1 × 10⁶ cells were placed in each well.4 After dispensing cells / wells, they were cultured at 37°C for 24 hours, and samples of various concentrations (0, 1.25, 2.5, 5, 10 μM) (Examples 5-1 and Comparative Examples 1-1 to 1-3) were treated, respectively, and cultured at 37°C for 4 hours. WST-8 solution was added to the cells after culture was complete, and after 4 hours, the absorbance was measured at 450 nm using a microplate reader (Perkin-Elmer, USA).

[0372] FIG. 19 is a graph analyzing the cytotoxicity of a G2 Janus-type polyelectrolyte-bio-ion-based dendrimer (JDPZ, (+M)2(-M)2conjugate_1) prepared from Example 5-1 and G2 Janus-type dendrimers prepared from Comparative Examples 1-1 to 1-3.

[0373] As shown in Fig. 19, Example 5-1 (JDPZ, (+3)2(-3)2conjugate_1) was confirmed to be a safe substance that does not exhibit cytotoxicity. On the other hand, Comparative Example 1-1 ((+3)4conjugate) or Comparative Example 1-2 ((+3)2(Ø3)2conjugate), in which one of the peptides is present in excess, were confirmed to exhibit very high toxicity to cells. Comparative Example 1-2 was confirmed to exhibit cytotoxicity, although lower than that of Comparative Example 1-1.

[0374] 4) DLS Analysis

[0375] Solutions were prepared by adding the G2 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ, (+M)2(-M)2conjugate_1) prepared from Examples 5-1, 5-4 and Comparative Examples 1-2 to DW and PBS, respectively, and analyzed by DLS. Dynamic light scattering (DLS) was performed to confirm the size and size distribution using an ELS-Z1000 particle size analyzer (Otsuka, Japan), and the results are shown in Table 1 and Figure 20.

[0376] FIG. 20 shows the DLS spectra of the Janus-shaped polyelectrolyte-zwitterion-based dendrimer ((+6)2(-6)2conjugate_1) of Example 5-4 (a) and the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) of Comparative Example 1-2 (b) under different solution conditions (DW or PBS). The DLS results of the Janus-shaped polyelectrolyte-zwitterion-based dendrimers ((+3)2(-3)2conjugate_1, (+6)2(-6)2conjugate_1) of Example 5-1 and Example 5-4 and the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) of Comparative Example 1-2 under different solution conditions (DW or PBS) are shown in Table 1.

[0377] division Net charge Diameter (nm) in DW Diameter (nm) in PBS Zeta potential (mV) Example 5-1 0 109.8 75.4 +42.3 Examples 5-4 0 649.0 128.9 +35.2 Comparative Example 1-2 +6 742.3 5,688.2 +52.8

[0378] According to Table 1 and Figure 20, it was confirmed that the Janus-shaped polyelectrolyte-bio-ion-based dendrimer ((+6)2(-6)2conjugate_1) of Example 5-4 had a significantly smaller size or diameter under PBS conditions than under DW conditions, and the size distribution was also narrower.

[0379] That is, in the Janus-shaped polyelectrolyte-zwitterion-based dendrimers of Examples 5-1 and 5-4 according to the present invention, due to the antipolyelectrolyte effect, at high salt concentrations such as PBS, the volume fraction of the water-soluble polyelectrolyte-zwitterion-based dendrimer portion increases, and the packing parameter of the amphiphilic molecule decreases, and as a result, it can be seen that the size of the self-assembled supramolecular structure decreases under PBS conditions compared to DW conditions.

[0380] In contrast, it was confirmed that the Janus-shaped dendrimer ((+3)2(Ø3)2 conjugate) prepared from Comparative Example 1-2, which was positively charged, increased in size in PBS compared to DW. That is, it was confirmed that the size or diameter of the positively charged dendrimer of Comparative Example 1-2 increased significantly under PBS conditions. This is attributed to the polyelectrolyte effect, which is a general characteristic of polyelectrolytes.

[0381] Synthesizing the DLS results, it can be seen that Janus-type polyelectrolyte-zwitterion-based dendrimers and Janus-type dendrimers consisting only of positive charges have completely different solution behaviors. Therefore, the Janus-type polyelectrolyte-zwitterion-based dendrimers of Examples 5-1 and 5-4 can maintain a more stable structure than the dendrimers of Comparative Examples 1-1 to 1-2 under in vivo conditions, indicating that they are highly suitable for in vivo applications.

[0382] 5) Analysis of Nonfouling Characteristics - BSA

[0383] It was confirmed through EMSA (Electrophoretic Mobility Shift Assay) that when the dendrimer structure of the present invention is administered into the body, it is non-contaminating to various proteins present in the body.

[0384] First, 1 μg of BSA, a sticky protein present in high proportions in blood, was prepared as a model protein and mixed with the Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ, (+6)2(-6)2conjugate_1) of Example 5-4 at various concentration ratios. The mixing ratio used was the peptide / BSA ratio, which is peptide / BSA (w / w). After reacting the mixture at room temperature for 1 hour, electrophoresis was performed on a 12% native polyacrylamide gel electrophoresis (PAGE) gel at 20 mA for 90 minutes. To visualize the bands, they were stained with Coomassie Brilliant Blue R-250.

[0385] At this time, the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) structure of Comparative Example 1-2 was used as a comparison group instead of the JDPZ ((+6)2(-6)2conjugate_1) of Example 5-4.

[0386] FIG. 21a is the EMSA result analyzing the protein non-contamination of the Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) structure of Example 5-4 and the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) of Comparative Example 1-2. Here, 'L' is a protein marker.

[0387] As shown in FIG. 21a, it was confirmed that the Janus-shaped dendrimer ((+3)2(Ø3)2-conjugate) of Comparative Example 1-2 readily combines with BSA. That is, the Janus-shaped dendrimer ((+3)2(Ø3)2-(C 12 )4) interacts with BSA to induce an electrophoretic mobility shift (EMS) in the BSA band, indicating that it is non-contaminating.

[0388] On the other hand, it was confirmed that the Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) of Example 5-4 hardly binds to BSA even under high concentration BSA conditions. In other words, it was confirmed that the Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) of Example 5-4 has high non-contamination properties, maintaining its structure and function stably without being hindered by BSA even at high concentrations of BSA.

[0389] 6) Analysis of Nonfouling Characteristics - rRNA

[0390] It was confirmed through an Electrophoretic Mobility Shift Assay (EMSA) that the dendrimer structure of the present invention is non-contaminating to various genetic materials present in vivo when administered in vivo. Meanwhile, 200 ng of yeast rRNA (yeast ribosomal RNA) was prepared as a model genetic material, and the Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) of Example 5-4 was mixed at various concentration ratios. The mixing ratio used was the peptide / RNA ratio, which is peptide / RNA (w / w). After reacting the mixture at room temperature for 1 hour, electrophoresis was performed on a 2% agarose gel at 90 V for 100 minutes. To visualize the bands, the gel was stained with SYBR Gold Nucleic Acid Gel Stain (Invitrogen, USA).

[0391] At this time, as a comparison group, the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) structure of Comparative Example 1-2 was used instead of the JDPZ ((+6)2(-6)2conjugate_1) of Example 5-4.

[0392] FIG. 21b is the EMSA result analyzing the non-contamination of genetic material by the Janus-shaped polyelectrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) of Example 5-4 and the Janus-shaped dendrimer ((+3)2(Ø3)2conjugate) of Comparative Example 1-2. Here, 'L' is an RNA marker.

[0393] Looking at Fig. 21b, it was confirmed that the Janus-shaped dendrimer ((+3)2(Ø3)2 conjugate) of Comparative Example 1-2 forms non-specific interactions with rRNA even under low rRNA concentration conditions. This is consistent with the known problem that among cell-penetrating peptides known to deliver genetic material, arginine-rich CPPs also exhibit strong non-specific interactions with genetic material present in vivo / in cells.

[0394] On the other hand, the Janus-shaped multi-electrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) of Example 5-4 according to the present invention forms interactions only under high concentration conditions where rRNA is present in greater than 10 times the amount, and thus the Janus-shaped multi-electrolyte-zwitterion-based dendrimer (JDPZ((+6)2(-6)2conjugate_1)) of Example 5-4 has high non-contamination properties for genetic materials such as nucleic acids.

[0395] Experimental Example 8. Fluorescently introduced G2 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M) 2 (-M) 2 Analysis of the characteristics of conjugate_FAM

[0396] 1) MALDI-TOF Mass Spectrometry

[0397] To confirm the synthesis of the fluorescently introduced G2 Janus-type polyelectrolyte-zwitterion-based dendrimer (JDPZ((+M)2(-M)2conjugate_FAM) prepared from Examples 6-1 to 6-2, it was analyzed by MALDI-TOF.

[0398] FIG. 22 is a MALDI-TOF graph for G2 Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ((+M)2(-M)2conjugate_FAM)) introduced with fluorescent materials prepared from Examples 6-1, 6-2 and Comparative Examples 2-1 to 2-2. FIG. 22a is for (+3)2(-3)2conjugate_FAM prepared from Example 6-1, FIG. 22b is for (+6)2(-6)2conjugate_FAM prepared from Example 6-2. FIG. 22c is for (+3)2(Ø3)2conjugate_FAM of Comparative Example 2-1, FIG. 22d is for (Ø3)2(-3)2conjugate_FAM of Comparative Example 2-2.

[0399] As shown in FIG. 22, it was confirmed that G2 Janus-type multi-electrolyte-zwitterion-based dendrimers with introduced fluorescent materials prepared from Examples 6-1, 6-2 and Comparative Examples 2-1 to 2-2 were also successfully synthesized.

[0400] 2) Intracellular delivery efficiency

[0401] To confirm intracellular delivery efficiency, HCT116 cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 1–3 × 10⁶ cells were placed in each well. 4 After dispensing cells / wells, incubated at 37°C for 24 hours, each was treated with (+6)2(-6)2conjugate_FAM prepared from Example 6-2 at a concentration of 10 μM and incubated at 37°C for 4 hours. Subsequently, the cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), triedpsinated, and then resuspended in DPBS supplemented with 10% cell dissociation buffer (Gibco) and 1% FBS. Intracellular fluorescence was then analyzed using a flow cytometer (BD LSR II SORT flow cytometer, BD Bioscience, USA).

[0402] Figure 23 is a graph showing the intracellular delivery efficiency of JDPZ((+6)2(-6)2conjugate_FAM) prepared from Example 6-2, analyzed by flow cytometry in the HCT116 cell line. As shown in Figure 23, it was confirmed that the G2 Janus-type polyelectrolyte-zwitterion-based dendrimer with the fluorescent substance introduced in Example 6-2 had a high internalization efficiency of about 100%.

[0403] The polyelectrolyte-zwitterion-based dendrimers (DPZ) and Janus-type polyelectrolyte-zwitterion-based dendrimers (JDPZ) according to the present invention were found to be non-toxic and have high intracellular delivery efficiency. In contrast, Janus-type dendrimers in which positively charged peptides form strong supramolecular nanostructures (Comparative Examples 1-1, 1-2, 2-1) have high cell delivery efficiency but exhibit very high toxicity.

[0404] 3) Intracellular drug delivery efficiency

[0405] We confirmed that Janus-shaped multi-electrolyte-bio-ion-based dendrimers (JDPZ) were stably internalized inside the cell, and then analyzed whether the delivered drug was delivered into the cell using a confocal laser scanning microscope (CLSM).

[0406] The fluorescently introduced G2 Janus-type polyelectrolyte-bio-ion-based dendrimer (JDPZ((+6)2(-6)2conjugate_FAM)) prepared from Example 6-2 was treated to HeLa cells, and their intracellular locations were measured using a confocal laser scanning microscope (CLSM). Specifically, HCT116 cells were placed in 2 × 10⁶ 8-well LabTek II chamber cover glass systems (Nunc, USA) in DMEM containing 10% FBS (Fetal Bovine Serum) and 1% Pen-Strep. 4After inoculating HeLa cells / well, the cells were cultured at 37°C for 24 hours. Subsequently, the cells were washed with DPBS, and 10 μM of the sample (Example 6-2) was treated in Opti-MEM for 1 hour. The cells were then visualized using a confocal microscope (LSM 980, Carl Zeiss, Germany).

[0407] Figure 24 is an image analyzed by confocal microscopy (LSM 980, Carl Zeiss, Germany) after treating HeLa cells with a G2 Janus-type multivalent electrolyte-bio-ion-based dendrimer (Example 6-2) into which a fluorescent substance was introduced.

[0408] As shown in Fig. 24, it was confirmed that the G2 Janus-type polyelectrolyte-zwitterion-based dendrimer with the fluorescent substance introduced in Example 6-2 efficiently penetrates into the cell and successfully delivers the drug into the cell nucleus and cytoplasm.

[0409] 4) Cytotoxicity

[0410] After treating HCT116 human colorectal cancer cell lines with Examples 6-1 and 6-2 and Comparative Examples 2-1 and 2-2, respectively, cytotoxicity was measured by determining the percentage of live cells using flow cytometry (FACS).

[0411] FIG. 25 is a graph showing the cytotoxicity of G2 Janus-type polyelectrolyte-zwitterion-based dendrimers ((+M)2(-M)2conjugate_FAM) with fluorescent substances introduced from Examples 6-1 and 6-2 and G2 Janus-type dendrimers with fluorescent substances introduced from Comparative Examples 2-1 and 2-2, measured by flow cytometry (FACS).

[0412] According to this, Example 6-1 ((+3)2(-3)2conjugate_FAM) and Example 6-2 ((+6)2(-6)2conjugate_FAM) showed no cytotoxicity. In contrast, Comparative Example 2-1 ((+3)2(Ø3)2conjugate_FAM) was found to exhibit very high toxicity, with 90% of HCT116 cells dying.

[0413] That is, it was confirmed that the polyelectrolyte-zwitterion-based dendrimers according to the present invention (Examples 1 to 6) are in a very stable state that does not exhibit any cytotoxicity in the fluid phase, even though they contain a number of positively charged peptides, because they have a special structure called a polyelectrolyte-zwitterion-based dendrimer.

[0414] Experimental Example 9. G2 Janus-type polyelectrolyte-zwitterion-based dendrimer ((+6)) 2 (-6) 2 conjugate_2)((+6) 2 (-6) 2 Analysis of the characteristics of conjugate_3)

[0415] 1) MALDI-TOF Mass Spectrometry

[0416] The G2 Janus-type polyelectrolyte-bio-ion-based dendrimers ((+6)2(-6)2conjugate_2)((+6)2(-6)2conjugate_3) prepared from Examples 7 and 8 were purified and analyzed by MALDI-TOF.

[0417] FIG. 26 is a MALDI-TOF graph for a G2 Janus-type polyelectrolyte-zwitterion-based dendrimer ((+6)2(-6)2conjugate_2) prepared from Example 7, and FIG. 27 is a MALDI-TOF graph for a G2 Janus-type polyelectrolyte-zwitterion-based dendrimer ((+6)2(-6)2conjugate_3) prepared from Example 8.

[0418] According to this, it was confirmed that the G2 Janus-type polyelectrolyte-zwitterion-based dendrimers prepared from Examples 7 and 8 were also successfully synthesized regardless of the number and type of hydrophobic compounds. It was also confirmed that they are successfully synthesized even when hydrophilic amino acid derivatives are included instead of hydrophobic compounds.

[0419] 2) Preparation of Janus-shaped multi-electrolyte-bio-ion-based dendrimer complexes loaded with EGFP mRNA and analysis of intracellular delivery efficiency

[0420] A Janus-type polyelectrolyte-bioelectrolyte-bioelectrolyte-based dendrimer complex loaded with GFP mRNA was prepared by mixing 1 μg of EGFP mRNA (SEQ ID NO. 167) with the G2 Janus-type polyelectrolyte-bioelectrolyte-based dendrimer ((+6)2(-6)2_conjugate 2) prepared from Example 7 or the G2 Janus-type polyelectrolyte-bioelectrolyte-based dendrimer ((+6)2(-6)2_conjugate 3) prepared from Example 8.

[0421] HeLa cells were subcultured in DMEM supplemented with 10% FBS (Fetal Bovine Serum), and 1–3 × 10⁶ cells were placed in each well. 4 After dispensing cells / wells, incubated at 37°C for 24 hours, then treated with complexes ((+6)2(-6)2_conjugate 2_complex) or ((+6)2(-6)2_conjugate 3_complex) at concentrations of 16 μM or 32 μM, respectively, and incubated at 37°C for 4 hours. Subsequently, the cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), triedpsinated, and then resuspended in DPBS supplemented with 10% cell dissociation buffer (Gibco) and 1% FBS. The expressed eGFP protein (Enhanced Green Fluorescence Protein) was then analyzed using a flow cytometer (BD LSR II SORT flow cytometer, BD Bioscience, USA).

[0422] Figure 28 shows the results of quantifying the expressed GFP protein by flow cytometry after treating HeLa cell lines with a complex of Janus-shaped multi-electrolyte-zwitterion-based dendrimer (+6)2(-6)2conjugate_2 prepared from Example 7 and EGFP (enhanced green fluorescent protein) mRNA.

[0423] Figure 29 is a graph showing the quantification of GFP protein expression by flow cytometry after treating HeLa cell lines with a complex of Janus-shaped multi-electrolyte-zwitterion-based dendrimer (+6)2(-6)2conjugate 3 prepared from Example 8 and EGFP (enhanced green fluorescent protein) mRNA.

[0424] As shown in FIGS. 28 and 29, the Janus-type multi-electrolyte-zwitterion-based dendrimer ((+6)2(-6)2conjugate_2 / 3) of Examples 7 and 8 not only effectively carried EGFP mRNA but also successfully delivered the mRNA to the nucleus and cytoplasm of the cell, and it was confirmed that eGFP protein was expressed from the internalized EGFP mRNA with a high efficiency of 82.9%.

[0425] 3) Preparation of Janus-shaped multi-electrolyte-bio-ion-based dendrimer complexes loaded with EGFP mRNA and analysis of in vivo delivery efficiency

[0426] A Janus-shaped multielectrolyte-bioelectrolyte-bioelectrolyte-based dendrimer complex ((+6)2(-6)2_conjugate 2_complex) loaded with GFP mRNA was prepared by mixing 5 μg of EGFP mRNA (SEQ No. 167) with the G2 Janus-shaped multielectrolyte-bioelectrolyte-based dendrimer ((+6)2(-6)2_conjugate 2_complex) prepared from Example 7. The complex ((+6)2(-6)2_conjugate 2_complex) was administered to BALB / c nude mice. At 0.1 mg / kg After subcutaneous injection, the expressed eGFP (Enhanced Green Fluorescence Protein) was imaged and quantified using IVIS (In Vivo Imaging System). In this case, only EGFP mRNA was used as a control instead of the complex.

[0427] Fig. 30 is in vivo ( in vivo This is the result of confirming whether the complex ((+6)2(-6)2_conjugate 2_complex) loaded with EGFP mRNA was expressed in ).

[0428] As shown in Fig. 30, the Janus-type polyelectrolyte-bio-ion-based dendrimer ((+6)2(-6)2conjugate_2) of Example 7 not only effectively carried EGFP mRNA but also successfully delivered the mRNA in vivo, and the EGFP mRNA delivered in vivo was expressed as EGFP protein. In contrast, when only EGFP mRNA was administered, significant expression of eGFP protein was not observed.

[0429] In summary, the present invention fabricates a polyelectrolyte-zwitterion-based dendrimer by attaching positively and negatively charged peptides to the ends of a polylysine dendrimer, and fabricates a Janus-shaped nanostructure by attaching a hydrophobic compound thereto. The Janus-shaped polyelectrolyte-zwitterion-based dendrimer does not exhibit toxicity even when containing a large amount of positively charged peptides, exists stably by forming a supramolecular nanostructure in a solution such as body fluid, and possesses high non-contamination properties regarding interactions with proteins and genetic material present in the body. Therefore, when genetic material is loaded onto the Janus-shaped polyelectrolyte-zwitterion-based dendrimer according to the present invention, excellent intracellular delivery and protein expression effects can be achieved when administered to cells or the body. Thus, the present invention can be utilized not only as a delivery carrier for the safe delivery of genetic material within the body, but also as a therapeutic agent or diagnostic agent based on genetic material.

Claims

Claim 1 Polyelectrolyte-zwitterion-based dendritic compounds represented by the following general formula 1 or 2.[General Formula 1] [General Formula 2] In the above formula, A and C may each be independently selected from the group consisting of arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn), and guanidine. B and D may each be independently selected from the group consisting of glutamic acid (Glu, E) and aspartic acid (Asp, D). n, m, o, and p are each independently selected from any one integer between 3 and 16. Claim 2 A polyelectrolyte-zwitterion-based dendritic compound according to claim 1, characterized in that n, m, o, and p are all the same under pH 2 to pH 13 conditions. Claim 3 A polyelectrolyte-zwitterion-based dendritic compound according to claim 1, characterized in that A and C are each independently represented by a positively charged peptide selected from the group consisting of SEQ ID NOs 3 to 98. Claim 4 A polyelectrolyte-zwitterion-based dendritic compound according to claim 1, characterized in that B and D are each independently represented by a negatively charged peptide selected from the group consisting of SEQ ID NOs 99 to 162. Claim 5 A polyelectrolyte-zwitterionic dendrimer characterized by a dendrimer-based core of the 1st to 5th generation; and a positively charged peptide represented by SEQ ID NO. 1 and / or a negatively charged peptide represented by SEQ ID NO. 2 being bonded to the terminal functional group of the core. [SEQ ID NO. 1][X1] n [Sequence No. 2][X2] n In the above sequence, X1 is any one selected from arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn) and guanidine, and X2 is glutamic acid (Glu, E) or aspartic acid (Asp, D). In the above, n is an integer from 3 to 16. Claim 6 A polyelectrolyte-zwitterionic dendrimer according to claim 5, wherein the dendrimer-based core is selected from the group consisting of polyamidoamine (PAMAM) dendrimer, polylysine dendrimer, polyimine (PI) dendrimer, polypropyleneimine (PPI) dendrimer, polyester dendrimer, polyamide dendrimer, polyurethane dendrimer, polyornithine dendrimer, carbosilane dendrimer, polyether dendrimer, polyglutamic acid dendrimer, polyaspartic acid dendrimer, polyglycerol dendrimer, and polymelamine dendrimer. Claim 7 A polyelectrolyte-bio-ionic dendrimer according to claim 5, characterized in that the dendrimer-based core is of the 1st or 2nd generation. Claim 8 A polyelectrolyte-zwitterionic bonded dendrimer according to claim 5, characterized in that the positively charged peptide represented by SEQ ID NO. 1 and the negatively charged peptide represented by SEQ ID NO. 2 are bonded to the ends of a dendrimer-based core in equal numbers. Claim 9 A polyelectrolyte-zwitterionic bonded dendrimer according to claim 5, characterized in that when the dendrimer-based core is of the first generation, one strand of a positively charged peptide chain represented by SEQ ID NO. 1 and one strand of a negatively charged peptide chain represented by SEQ ID NO. 2 are bonded, and when the dendrimer-based core is of the second generation, two strands of a positively charged peptide chain represented by SEQ ID NO. 1 and two strands of a negatively charged peptide chain represented by SEQ ID NO. 2 are bonded. Claim 10 A polyelectrolyte-zwitterionic bonded dendrimer according to claim 5, characterized in that the positively charged peptide is represented by any one selected from the group consisting of SEQ ID NOs 3 to 98. Claim 11 A polyelectrolyte-zwitterionic bonded dendrimer according to claim 5, characterized in that the negatively charged peptide is represented by any one selected from the group consisting of SEQ ID NOs 99 to 162. Claim 12 A Janus-type polyelectrolyte-zwitterion-based dendrimer characterized by a 1st to 5th generation dendrimer-based core; wherein a positively charged peptide represented by SEQ ID NO. 1 and / or a negatively charged peptide represented by SEQ ID NO. 2 is bonded to a functional group at one end of the dendrimer core, and a hydrophobic compound or amino acid derivative is bonded to a functional group at the other end of the dendrimer core. [SEQ ID NO. 1][X1] n [Sequence No. 2][X2] n In the above sequence, X1 is any one selected from arginine (Arg, R), histidine (His, H), lysine (Lys, K), ornithine (Orn) and guanidine, and X2 is glutamic acid (Glu, E) or aspartic acid (Asp, D). In the above, n is an integer from 3 to 16. Claim 13 A Janus-type multi-electrolyte-zwitterion-based dendrimer according to claim 12, wherein the dendrimer-based core is selected from the group consisting of polyamidoamine (PAMAM) dendrimer, polylysine dendrimer, polyimine (PI) dendrimer, polypropyleneimine (PPI) dendrimer, polyester dendrimer, polyamide dendrimer, polyurethane dendrimer, polyornithine dendrimer, carbosilane dendrimer, polyether dendrimer, polyglutamic acid dendrimer, polyaspartic acid dendrimer, polyglycerol dendrimer, and polymelamine dendrimer. Claim 14 A Janus-type multivalent electrolyte-bio-ion based dendrimer according to claim 12, characterized in that the dendrimer-based core is of the 2nd or 3rd generation. Claim 15 A Janus-type polyelectrolyte-zwitterion-based dendrimer according to claim 12, characterized in that the number of positively charged peptides represented by SEQ ID NO. 1 and negatively charged peptides represented by SEQ ID NO. 2 bonded to the ends of the dendrimer-based core are equal to each other. Claim 16 In Clause 12, the above-mentioned dendrimer-based core has 2 on the surface depending on the number of generations x A Janus-type polyelectrolyte-zwitterion-based dendrimer characterized by having terminal functional groups (where x is the number of generations), wherein half of the total terminal functional groups present in the dendrimer-based core are bound to a peptide represented by SEQ ID NO. 1 or 2, and the other half of the terminal functional groups are each independently bound to a hydrophobic compound or an amino acid derivative. Claim 17 A Janus-type polyelectrolyte-zwitterion-based dendrimer according to claim 12, characterized in that the positively charged peptide is represented by any one selected from the group consisting of SEQ ID NOs 3 to 98. Claim 18 A Janus-type polyelectrolyte-zwitterion-based dendrimer according to claim 12, characterized in that the negatively charged peptide is represented by any one selected from the group consisting of SEQ ID NOs 99 to 162. Claim 19 A Janus-type polyelectrolyte-zwitterion-based dendrimer according to claim 12, characterized in that the hydrophobic compound is any one selected from the group consisting of fatty acids having 3 to 30 carbon atoms, hydrophobic drugs, and fluorescent substances. Claim 20 Nanostructure comprising a Janus-type multivalent electrolyte-bio-ion based dendrimer according to claim 12. Claim 21 In claim 20, the nanostructure is characterized in that the nanostructure is in the form of a nanofiber. Claim 22 In claim 20, the nanostructure is characterized by having an average diameter of 8 to 30 nm. Claim 23 A delivery vehicle comprising: a nanostructure of claim 20; a target gene or drug carried on said nanostructure. Claim 24 A carrier according to claim 23, characterized in that the gene is a plasmid, mRNA, RNA, DNA, or a combination thereof. Claim 25 A delivery vehicle characterized in that, in paragraph 23, the drug is a low molecular weight drug, a gene drug, a protein drug, an antibody drug, a synthetic compound drug, or a combination thereof.

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