Transdermal drug delivery composition comprising gh625-h2a histone or its derivative and nucleic acid

Abstract

The intracellular or transdermal drug delivery composition of the present invention comprising a gH625-H2A histone or a derivative thereof and a nucleic acid comprises a gH625-H2A histone or a derivative thereof in which a peptide having the amino acid sequence of General Formula 1 is fused to the N-terminus and a nucleic acid: R1-His-Gly-Leu-X1-X2-X3-Leu-X4-Arg-Trp-X5-His-X6-X7-X8-Leu-Ile-X9-X10-Phe -R2(General Formula 1): in the above General Formula 1, R1 is H (free amino group), R2 is OH (free carboxyl group), X1 is A (alanine) or S (serine), X2, X3 or X4 is S (serine) or T (threonine), and X5, X8 or X10 is A is A (alanine) or G (glycine), X6 is Y (tyrosine) or F (phenylalanine), X7 is N (asparagine) or Q (glutamine), and X9 is R (arginine) or K (lysine).

Description

Technology Field

[0001] The present invention relates to a chromatin delivery system using a cell-permeable peptide, and more specifically, to a composition for intracellular or transdermal drug delivery comprising a gH625-H2A histone or its derivative and a nucleic acid. Background Technology

[0003] DNA is considered a highly negatively charged polyanionic polymer and does not easily bind to or penetrate cell membranes without specialized absorption mechanisms. Commercial transfection reagents are commonly used to introduce nucleic acids into mammalian cells. These reagents typically use cationic lipids to encapsulate DNA or condense nucleic acids through the formation of polycationic polymers. Membrane fusion and endocytosis are the primary mechanisms of intracellular DNA delivery using these reagents. However, commercial transfection reagents in vitro Despite its usefulness, there are limitations. Specifically, the transfection efficiency is lower compared to viral vectors, and due to the instability of the transfection reaction complex, it aggregates, precipitates, or decomposes over time, hindering long-term storage and in vivo It is unsuitable for use.

[0004] Eukaryotic DNA is packaged within the nucleus through a multi-step chromatin compression process. This compression process begins with the assembly of 10 nm nucleosomes, where DNA is wrapped 1.65 times around standard histone cores (two each of H3, H4, H2A, and H2B). A single nucleosome consists of approximately 146 base pairs of DNA and eight histone proteins, with a total molecular weight of approximately 179 kDa.

[0005] The chromatin structure can be distinguished as a condensed polyanionic polymer resulting from the compression of DNA surrounding the positively charged N-terminal tails of histones and core histones. Meanwhile, the four core histones possess protein transduction domains that facilitate membrane penetration. These core histones can condense DNA individually or together to facilitate DNA delivery into mammalian cells. More importantly, the reconstituted chromatin enables DNA delivery via endocytosis-independent pathways.

[0006] As the largest superficial organ in the human body, the skin can serve as a non-invasive and convenient route for intracellular or transdermal drug delivery. Transdermal routes are also highly advantageous for both biopharmaceutical and skincare applications by minimizing non-specific systemic side effects. However, the epidermis, particularly the stratum corneum, acts as a powerful impermeable barrier, making the delivery of large biomolecules via passive topical application difficult. These large-molecular-weight biomolecules generally require mildly invasive delivery enhancers, such as microneedles and electroporation. Despite these various physical and biochemical approaches, efficient transdermal drug delivery remains a significant challenge. The problem to be solved

[0008] The present invention aims to provide a composition for intracellular or transdermal drug delivery comprising gH625-H2A histones with enhanced cell membrane and / or skin permeability for in vivo gene delivery.

[0009] The present invention aims to provide a composition for intracellular or transdermal drug delivery capable of effectively delivering biomolecules such as DNA, RNA, and proteins into cells.

[0010] The present invention aims to provide a composition for intracellular or transdermal drug delivery capable of delivering various biomolecules through the skin in a non-invasive and convenient manner.

[0011] The purpose is to provide a platform technology applicable to various fields, such as gene therapy, cosmetic formulations, and biopharmaceutical delivery, through applications according to embodiments of the present invention.

[0013] The objectives of the present invention are not limited to those mentioned above, and other unmentioned objectives will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0015] A transdermal drug delivery composition according to one aspect of the present invention for solving the above technical problem comprises a gH625-H2A histone or a derivative thereof in which a peptide having the amino acid sequence of General Formula 1 is fused to the N-terminus, and a nucleic acid:

[0016] R1-His-Gly-Leu-X1-X2-X3-Leu-X4-Arg-Trp-X5-His-X6-X7-X8-Leu-Ile-X9-X10-Phe-R2 (Formula 1):

[0017] In the above general formula 1,

[0018] R1 is H (free amino group), R2 is OH (free carboxyl group), and

[0019] X1 is A (alanine) or S (serine), and

[0020] X2, X3, or X4 is S (serine) or T (threonine), and

[0021] X5, X8, or X10 is A (alanine) or G (glycine), and

[0022] X6 is Y (tyrosine) or F (phenylalanine), and

[0023] X7 is N (asparagine) or Q (glutamine), and

[0024] X9 is R (arginine) or K (lysine).

[0026] The amino acid substitution of the above general formula 1 can maintain the major physicochemical properties of the peptide of SEQ ID NO. 1 based on similarity of amino acid properties, charge distribution, hydrophobic / hydrophilic pattern, and structural properties.

[0027] Here, the gH625 peptide can improve stability while maintaining cell membrane permeability and amphiphilicity. His(H), Gly(G), and Leu(L) located at the N-terminus of the peptide play important roles in the function of the peptide, and Trp(W) plays an important role in hydrophobicity and aromatic properties. In this case, as for substitutable amino acids, hydrophobicity can be increased by substituting Ala(A) with Val(V) or Ile(I), and similar polarity can be maintained by substituting Ser(S) with Thr(T) and Thr(T) with Ser(S). Additionally, positive charge can be maintained by substituting Arg(R) with Lys(K), and aromatic properties can be maintained by substituting Tyr(Y) with Phe(F).

[0028] His(H), Gly(G), and Leu(L), located at the N-terminus of the peptide, play important roles in the function of the peptide, and Trp(W) plays an important role in hydrophobicity and aromatic properties, so they need to be preserved. Meanwhile, in some amino acid sequences, hydrophobicity can be increased by substituting Ala(A) with Val(V) or Ile(I), similar polarity can be maintained by substituting Ser(S) with Thr(T) and Thr(T) with Ser(S), positive charge can be maintained by substituting Arg(R) with Lys(K), and aromatic properties can be maintained by substituting Tyr(Y) with Phe(F).

[0029] In the substitution of alanine (A) and serine (S) at the X1 position, alanine is a nonpolar amino acid with a -CH3 side chain, and serine is a polar amino acid with a -CH2OH side chain. Both amino acids have small side chains (89 Da and 105 Da, respectively), so structural changes are minimized. The hydroxyl group (-OH) of serine can form hydrogen bonds, but the effect on the protein structure is limited due to the short side chain.

[0030] In the substitution of serine (S) and threonine (T) at the X2, X3, and X4 positions, serine (-CH2OH) and threonine (-CH(CH3)OH) are both polar amino acids containing a hydroxyl group. Although threonine has one more methyl group than serine (105Da and 119Da, respectively), both amino acids can act as hydrogen bond donors and acceptors. Due to these characteristics, they can form similar hydrogen bond networks in protein structures.

[0031] In the substitution of alanine (A) and glycine (G) at the X5, X8, and X10 positions, alanine (-CH3) and glycine (-H) are the simplest amino acids (89Da and 75Da, respectively). Glycine has the greatest structural flexibility as it lacks a side chain, while alanine exhibits similar characteristics by having only a small methyl group. These positions do not impose any special functional constraints on protein structure.

[0032] In the substitution of tyrosine (Y) and phenylalanine (F) at the X6 position, tyrosine (-CH2-C6H4OH) and phenylalanine (-CH2-C6H5) are aromatic amino acids containing a benzene ring (181Da and 165Da, respectively). Tyrosine has a structure in which a hydroxyl group is added to phenylalanine, and both amino acids can participate in hydrophobic interactions and π-π stacking.

[0033] In the substitution of asparagine (N) and glutamine (Q) at the X7 position, asparagine (-CH2-CONH2) and glutamine (-CH2-CH2-CONH2) are polar amino acids with amide groups at the ends (132 Da and 146 Da, respectively). With only a difference of one methylene group (-CH2-) in side chain length, their hydrogen bonding ability and polarity can be very similar.

[0034] In the substitution of arginine (R) and lysine (K) at the X9 position, arginine (-CH2-CH2-CH2-NH-C(=NH)-NH2) and lysine (-CH2-CH2-CH2-CH2-NH2) are basic amino acids that carry a positive charge at physiological pH (174 Da and 146 Da, respectively). Both amino acids carry a positive charge at the end of their long aliphatic side chains and can participate in ionic bonding and electrostatic interactions.

[0035] These substitutions provide structural flexibility while preserving the physicochemical properties of amino acids, such as size, charge, polarity, and hydrophobicity, to the greatest extent possible at each position. As a substitution pattern that is evolutionarily conserved, this allows for structural stability while maintaining protein function.

[0036] Here, the gH625-H2A histone may be one in which the gH625 peptide of SEQ ID NO. 1-16 is fused to the N-terminus of H2A. Here, the histone included in the intracellular or transdermal drug delivery composition may be gH625-H2A alone or in combination with other canonical histones (H3, H4, H2B) or derivatives thereof.

[0037] Here, the nucleic acid may be DNA or RNA.

[0038] Here, the intracellular or transdermal drug delivery composition may include a nucleosome structure.

[0039] Here, the above histone may be chemically synthesized.

[0040] Here, the histone may be produced by the recombinant expression of histone-encoding DNA.

[0041] Here, the nucleic acid may include a sequence encoding at least one protein.

[0042] Here, the nucleic acid may include a sequence expressing at least one noncoding RNA.

[0043] Here, the nucleic acid may include a sequence encoding a promoter for the controlled expression of an encoded protein and a non-coding RNA.

[0044] Here, the intracellular or transdermal drug delivery composition may be in the form of a synthetic chromatin delivery system (SCDS) containing nucleosomes.

[0045] Here, the nucleic acid may be plasmid DNA containing a 601 Widom repeat sequence.

[0046] The term "intracellular or transdermal drug delivery" of the present invention may refer to a non-invasive method of delivering a drug to a limited local site or to the systemic circulation directly into a cell or through the skin.

[0047] The term “Synthetic Chromatin” in the present invention may refer to chromatin comprising proteins and DNA that is synthetically produced through a self-assembly process in vitro from biological components that have been isolated, purified, or chemically synthesized, rather than chromatin fibers obtained from nature.

[0048] The term “Chromatin” of the present invention may refer to a complex of DNA and related proteins including histones containing repeating units of a nucleosome structure.

[0049] The term “nucleosome” in the present invention refers to a structure formed by the in vitro assembly of DNA and four types of histone proteins [H3, H4, H2A, H2B], which may represent a “bead-thread” structure.

[0050] The term "Synthetic Chromatin Delivery System (SCDS)" in this invention refers to a gene delivery system with enhanced cell membrane and skin permeability, wherein SCDS is a complex comprising a gH625-H2A histone or its derivative and a nucleic acid.

[0051] The term "gH625-H2A histone" of the present invention may refer to a recombinant protein in which a gH625 peptide (sequence number 1-16) is fused to the N-terminus of an H2A histone.

[0052] Here, the amino acid sequence of the gH625-H2A histone may be sequence number 21.

[0053] The intracellular or transdermal drug delivery composition of the present invention may be provided in a patch form. For example, in the case of a patch form, it may be provided as a matrix-type transdermal patch containing 1-5 mg of drug per patch with a size of about 10-30 cm². Alternatively, it may be provided in a gel form of a hydrogel or hydrocolloid formulation. For example, it may be provided containing an active ingredient at a concentration of about 1-5% (w / w) and at a concentration of about 0.5-2.0 mg / kg per day based on an adult weighing about 70 kg, such that the maximum daily dose does not exceed 100 mg.

[0054] When provided in patch form, the replacement cycle may be approximately 24 to 72 hours.

[0055] The gH625 peptide is a peptide composed of the amino acid sequence in Table 1 below.

[0056] Sequence number amino acid sequence 1 HGLASTLTRWAHYNALIRAF 2 HGLASTLTRWAHYNALIRGF 3 HGLASTLTRWAHYQALIKAF 4 HGLASSLSRWGHFNGLIRAF 5 HGLSTTLTRWAHYNALIRAF 6 HGLSTSLSRWGHFQGLIKGF 7 HGLASTLTRWGHFNALIKAF 8 HGLSTSLSRWAHYQGLIRGF 9 HGLASSLTRWGHFQALIKGF 10 HGLSTTLSRWAHYNGLIRAF 11 HGLASSLSRWAHFQGLIKAF 12 HGLSTTLTRWGHYNALIRGF 13 HGLASTLSRWGHFQALIKGF 14 HGLSTSLTRWAHYNGLIRAF 15 HGLASSLTRWAHFNGLIKAF 16 HGLSTTLSRWGHYQALIRGF Effects of the invention

[0057] A composition for intracellular or transdermal drug delivery (SCDS) comprising gH625-H2A histone according to an embodiment of the present invention can provide enhanced cell membrane and skin permeability.

[0058] The SCDS according to an embodiment of the present invention provides an efficient gene and protein delivery system, which can facilitate the intracellular delivery of various biomolecules.

[0059] The SCDS according to an embodiment of the present invention provides a composition for non-invasive intracellular or transdermal drug delivery, which can enable the effective delivery of various biomolecules through the skin.

[0060] The SCDS according to an embodiment of the present invention provides a platform technology capable of multi-purpose applications, enabling utilization in various fields such as gene therapy, cosmetics, and biopharmaceuticals.

[0062] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below. Brief explanation of the drawing

[0064] Figure 1 shows the results of the cell membrane permeability analysis of biotinylated gH625-H2A and wild-type H2A (individual histone proteins were purified from AE coli BL21 and confirmed by SDS-PAGE and Coomassie Brilliant Blue G250 staining; B. Cell membrane permeability analysis of gH625-H2A was performed, and 5 μg of biotinylated wild-type H2A and gH625-H2A were directly added to the culture medium of a 96-well plate and incubated at 37°C for 1 hour. Subsequently, the cells were washed and permeated, stained with DAPI and Avidin Flur 488, and representative images taken with a 1-second exposure at 100x magnification are shown. Fluorescence intensity was analyzed using ImageJ software to analyze relative intensity; C. The time-dependent cell membrane permeability pattern of gH625-H2A was analyzed, and 5 μg of biotinylated gH625-H2A or in HeLa cells Directly treat with bovine serum albumin and incubate at 37°C for a specified time. Measure intracellular residual fluorescence with a microplate reader and plot it as a function of time. Figure 2 shows the results of the cell membrane permeability analysis of biotinylated chromatin using gH625-H2A histone octamer (A. Wild-type core histones and histone octamers containing gH625-H2A were prepared and verified by SDS-PAGE and Coomassie Brilliant Blue G250 staining; B. MNase analysis of assembled chromatin was performed. 500 ng of pUC19 / 16x601 plasmid and an equal amount of assembled chromatin were digested with 2 and 8 units of MNase for 10 minutes, and the reaction products were analyzed by 1% agarose gel electrophoresis and Gel red staining; C. Cell membrane permeability analysis of chromatin was performed. 100 ng of biotinylated pUC19 / 16x601 plasmid and chromatin assembled with wild-type octamer or gH625-H2A octamer were cultured in 96-well plates Added directly to the medium and incubated at 37°C for 1 hour. After washing and permeating the cells, stained with DAPI and Avidin Flur 488, and a representative image was taken at 100x magnification with a 1-second exposure; relative intensity obtained by analyzing the fluorescence intensity of D.C. using ImageJ software). Figure 3. Results observing that chromatin permeation into cell membranes occurs effectively regardless of endocytosis inhibitors (A. FACS analysis of Cy5-positive cells was performed, and synthetic chromatin assembled with Cy5-labeled pUC19 / 16x601 plasmid was directly treated to HeLa cells for 3 hours. After washing the cells, excess DNA and chromatin were digested with 5 U / ml DNaseI in DMEM containing 15 mM MgCl2 for 1 hour, and the Cy5-labeled cell population was analyzed using a flow cytometer; B. The location of Cy5-labeled pUC19 / 16x601 within cells was analyzed using a confocal microscope. HeLa cells were directly treated with wild-type chromatin or gH chromatin for 3 hours, and Cy5 and DAPI staining were examined to track intracellular DNA locations; C. Results investigating the effect of endocytosis inhibitors on gH chromatin-mediated intracellular delivery. HeLa cells Pre-treat cells with methyl-β-cyclodextrin (MCD, 1.5 mM) and amiloride (AMI, 10 μM) for 1 hour, and directly treat the cells with Cy5-labeled pUC19 / 16x601 or an equal amount of gH625-H2A chromatin as in A. Analyze the Cy5-labeled cell population using a flow cytometer. Fig. 4. Results of skin penetration analysis using a porcine skin-based Franz cell system (A. Porcine skin penetration analysis was performed. 1 μg of Cy5-labeled pUC19 / 16x601 DNA and an equal amount of wild-type or gH chromatin were independently applied to porcine skin three times at 24-hour intervals using Franz diffuse cells, and histological sections were photographed using a fluorescence microscope at each time interval. Representative images are shown. SC: Stratum corneum; E: Epidermis; D: Dermis; B. Fluorescence intensity of histological sections was measured at each time interval and relative intensities were plotted). Figure 5. Results of analyzing the assembly and cell growth / collagen secretion stimulating effects of EGF surface-expressing chromatin (A. Wild-type core histones and histone octamers including gH625-H2A and H2B-EGF were prepared and verified by SDS-PAGE and Coomassie Brilliant Blue G250 staining; B. MNase analysis of the assembled chromatin was performed. 500 ng of pUC19 / 16x601 plasmid and an equal amount of assembled chromatin were digested with 2 and 8 units of MNase for 10 minutes, and the reaction products were analyzed by 1% agarose gel electrophoresis and Gel red staining; C. The effect of EGF on HaCa-T cell growth was analyzed. EGF (12.5 ng / ml), wild-type chromatin (250 ng / ml), and various concentrations of EGF surface-expressing chromatin (125, 250 Cell growth was evaluated by CCK-8 assay after treatment with ng / ml for 3 days; D. Showing the effect of EGF on collagen type Iα1 secretion. After treating HaCa-T cells with EGF (12.5 ng / ml) and various concentrations of EGF surface expression chromatin (125, 250 ng / ml) for 2 days, the amount of collagen type Iα1 in the culture medium was measured three times repeatedly by ELISA). Specific details for implementing the invention

[0065] The objectives and effects of the present invention, and the technical configurations for achieving them, will become clear by referring to the embodiments described in detail below in conjunction with the accompanying drawings. In describing the present invention, if it is determined that a detailed description of known functions or configurations may unnecessarily obscure the essence of the invention, such detailed description will be omitted. Furthermore, the terms described below are defined with consideration of their function in the present invention, and these may vary depending on the intentions or practices of the user or operator.

[0066] However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Therefore, such definition should be based on the content throughout this specification.

[0067] Table 2 below summarizes the biophysical properties of the histone variants used in the study and shows the characteristics of six histone proteins.

[0068] Protein name Protein ID Number of constituent amino acids (Length) Molecular weight (MW, KDa) isoelectric point (PI) Extinction coefficient Instability Index H2A Human 2-A (NP_003507.1) 132 14.1 10.9 4470 53.96 H2B Human H2-E (NP_003519.1) 128 13.9 10.3 7450 46.08 H3 Human H3.2 (NP_066403.2) 138 15.3 11.3 4470 47.55 H4 Human H4 (NP_003539.1) 105 11.3 11.4 5960 43.5 gH625-H2A HSV_gH625-H2A 150 16.3 11.1 11460 53.24 H2B-EGF HSV_gH625-H2A 185 20.7 9.6 25900 48.26

[0070] The biophysical characteristics of the histone variants in Table 2 above are as follows.

[0071] First, the human H2A (Human-2-A) histone has the protein ID NP_003507.1 and consists of 132 amino acids. Its molecular weight is 14.1 kDa, isoelectric point is 10.9, quenching coefficient is 4470, and instability index is 53.96, and it possesses the highest cell membrane movement ability among core histones.

[0072] Next, human H2B (Human H2-E) histone has the protein ID NP_003519.1 and consists of 128 amino acids. Its molecular weight is 13.9 kDa, isoelectric point is 10.3, quenching factor is 7450, and instability index is 46.08, and stable protein expression is possible when EGF is fused to the C-terminus.

[0073] Next, human H3 (Human H3.2) histone has the protein ID NP_066403.2 and consists of 138 amino acids. Its molecular weight is 15.3 kDa, isoelectric point is 11.3, quenching coefficient is 4470, and instability index is 47.55, and it plays an important role in the formation of the histone core structure.

[0074] Next, human H4 histone has the protein ID NP_003539.1 and consists of 105 amino acids. Its molecular weight is 11.3 kDa, isoelectric point is 11.4, quenching coefficient is 5960, and instability index is 43.5, and it contributes to the stabilization of the nucleosome structure.

[0075] Next, gH625-H2A (HSV_gH625-H2A) histone is composed of 150 amino acids, with a molecular weight of 16.3 kDa, an isoelectric point of 11.1, a quenching coefficient of 11460, and an instability index of 53.24. It exhibits a cell permeability approximately 6 times higher than that of wild-type H2A and can penetrate the cell membrane within 30 minutes.

[0076] Next, H2B-EGF histones consist of 185 amino acids, with a molecular weight of 20.7 kDa, an isoelectric point of 9.6, a quenching coefficient of 25900, and an instability index of 48.26. 6.8 kDa of EGF is added to the C-terminus, allowing for the display of two EGF peptides per nucleosome, which stimulates the growth of keratinocytes and collagen secretion.

[0077] These histones are key proteins for DNA packaging, with two of each of the four core histones gathering to form a disc-shaped protein core, around which DNA is wound to form a nucleosome. In particular, gH625-H2A chromatin exhibits enhanced cell membrane and skin permeability, high DNA delivery efficiency, and is unaffected by endocytosis inhibitors, giving it high potential as a non-invasive intracellular or transdermal drug delivery system.

[0079] The amino acid sequences of the histone variants in Table 2 above are as shown in Table 3 below.

[0080] Sequence number Protein name (Protein name) Protein reference number (Protein ID) 17 H2A MAMSGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVYMAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK 18 H2B MAMPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK 19 H3 MAMARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA 20 H4 MAMSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG 21 gH-H2A MHGLASTLTRWAHYNALIRAFSGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVYMAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK 22 H2B-EGF MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSKSRPVATNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

[0082] In the present invention, DNA containing the Widom 601 sequence (pUC19 / 16x601, sequence number 23) repeated 16 times was used for gene transfer detection, and pEGFP-C1 / Mlu16x601 (sequence number 24) superhelical plasmid DNA was constructed.

[0084] The sequence of sequence number 23 is as follows:

[0085]

[0087] The sequence of sequence number 24 is as follows:

[0088]

[0090] The present invention will be described in detail below through embodiments.

[0092] Example 1: Purification of Histone Octamers and Salt-Gradation Chromatin Assembly

[0094] The pET3 expression vector was extensively used for wild-type histone expression and purification. The addition of peptides gH625 and EGF to histone genes was performed via DNA synthesis (GenScript Biotech Corp) using the sequences shown in the appendix. The preparation of histone octamers containing wild-type or hybrid wild-type and modified histones was performed as described in the prior literature. For physiologically isolated nucleosome arrays, DNA containing 16 repeats of the wild 601 sequence on plasmid DNA (pUC19 / 16x601) was used. Large-scale assembly of up to 60 μg of chromatin was performed using stepwise gradient dialysis on a dialysis membrane, and the quality of the synthetic nucleosome arrays was evaluated via Micrococcal Nuclease (MNase) analysis.

[0096] Example 2: Analysis of Cell Permeation of Biotinylated Histones and Chromatin

[0098] Partially purified individual histones, H2A and gH625-H2A, were conjugated with biotin molecules using the commercial kit EZ-Link Pentylamine-Biotin (Thermo Scientific). The biotinylated products were dialyzed in phosphate-buffered saline (PBS), and the biotinylated histones were further purified via a Superdex S200 gel filtration column. For cell permeation analysis, HeLa cells were seeded at 10,000 per well in 96-well plates and cultured in growth medium for 24 hours. 5 μg of biotinylated wild-type H2A or gH625-H2A was added directly to the growth medium and incubated at 37°C for 1 hour. Cells were thoroughly washed with PBS containing 0.5 mM MgCl2, fixed with 2% paraformaldehyde, and permeated with PBS containing 0.1% Triton X-100 for 15 minutes.

[0099] After blocking with 10% BSA in PBS containing 0.5% Tween20, Avidin Alexa Fluor™ 488 (Thermo Scientific, product number A21370) was added to each well (0.5 μg per 100 μl of PBS), and DAPI staining was performed. Avidin Fluor 488 without added histones was used as a negative control. Quantitative analysis of histone permeability was performed by measuring fluorescence intensity three times using a plate reader. For cell permeability analysis using biotin-labeled gH625-chromatin, HeLa cells were treated directly with chromatin, similar to the biotinized histone treatment described above. Cell fluorescence was visualized quantitatively or qualitatively using a fluorescence microscope (Nikon Inverted Microscope Ti-S), a confocal fluorescence microscope (LSM 880, Zeiss), or a plate reader (Varioskan Lux, Thermo Scientific). Cell imaging was analyzed using ImageJ. The DAPI signal particle selection threshold was adjusted to 850 infinity (ImageJ parameter), and valid cell nuclei were selected in the blue channel. The effective intensity at the same location in the green channel was measured and statistically analyzed. The measurements were normalized to the mean.

[0101] Example 3: Analysis of Cell Permeation of Cy5-Labeled gH625-Chromatin

[0102] pUC19 / 16x601 plasmid DNA was labeled with the Label IT Tracker Intracellular Nucleic Acid Localization Kit (Mirus Bio, MIR7021), and chromatins were prepared from wild-type or modified histone-containing histone octamers. The synthetic chromatins were added directly to adherent cells in serum-free DMEM. After incubation at 37°C for 3 hours, the cells were washed once with PBS and incubated for 1 hour with 1 mL of 5 U DNase in DMEM containing 15 mM MgCl2. Cells were collected, washed with PBS, and fixed with 4% paraformaldehyde. The collected cells were analyzed by FACS.

[0104] Example 4: Skin permeability investigation using a porcine skin-based Franz cell system

[0106] Dorsal skin stored at -20°C was thawed using deionized water and washed repeatedly with PBS buffer. The porcine skin was fixed between the donor and receiver chambers of the Franz cell so that the stratum corneum faced the donor chamber. 7.0 ml of aqueous solution was added to the receiver chamber, followed by 1.0 ml of PBS through a sampler to ensure close contact with the dermis. A sample of 0.5 μg of DNA equivalent was applied to the skin surface of the donor chamber, which had an effective area of ​​approximately 3.14 cm², and spread radially from the center. To promote absorption, the chamber solution was agitated at 300 rpm in a water bath maintained at 32°C. Skin samples were collected at specified time points (24, 48, and 72 hours) and washed five times with PBS. The samples were fixed in 4% paraformaldehyde for at least 24 hours, after which frozen section analysis was performed.

[0108] Example 5: Analysis of Cell Growth and Collagenase Stimulation in Human Keratinocyte HaCa-T Cell Lines

[0110] 10,000 HaCa-T cells were seeded into 96-well plates and treated with various reagents on the same day. These treatments included various concentrations of EGF peptide (12.5 ng / mL) and EGF-labeled chromatin (125, 250, 500 ng / mL). After a 2-day culture period, cell growth was evaluated using the Cell Counting Kit (CCK-8) (Beyotime, China) as described in the product manual. To analyze collagen production in HaCa-T cell cultures over 48 hours, cells were treated with EGF peptide and EGF-labeled chromatin in a manner similar to the cell growth assay. Culture medium from the 96-well plates was collected in three replicates at 48 hours. For collagen quantification, the Human COL1α1 ELISA Kit (Elabscience, China) was used according to the product manual.

[0112] Through the above examples, the following results can be confirmed.

[0114] First, H2A fused with the gH625 peptide at the N-terminus enhances the cellular permeability of histones.

[0115] The gH625 peptide was fused to the N-terminus of the human H2A protein via gene synthesis, and recombinant gH625-H2A (sequence number 21) and wild-type standard human histones were purified from an E. coli expression system (Fig. 1A). Both gH625-H2A and wild-type H2A were biotinized by NHS (N-hydroxysuccinimide)-mediated biotin binding, and the labeled histones were further purified by gel filtration chromatography.

[0116] Previous studies have shown that H2A possesses the highest mobility across the cell membrane independently of the endocytosis pathway among the four core histones. To compare the cellular permeability of gH625-H2A and wild-type H2A in HeLa cells, equal amounts of each histone were added directly to the cell culture medium, and relative permeation efficiency was visualized using Avidin-Flur 488 (Fig. 1B). While treatment with wild-type H2A exhibited moderate visual fluorescence intensity, treatment with gH625-H2A significantly increased the cellular fluorescence signal, suggesting that more gH625-H2A histones can permeate across the membrane into the cell (Fig. 1B, image from Avidin-Flur 488, comparison of gH-H2A and WT H2A). Microplate readings of the fluorescence signal show that gH625-H2A exhibits a fluorescence signal approximately six times higher than that of wild-type H2A (Fig. 1B, graph in the right panel).

[0117] To investigate the permeation kinetics of gH625-H2A, cells were treated with gH625-H2A in full growth medium containing 5% FBS for varying durations up to 60 minutes (Fig. 1C). The results indicate that gH625-H2A histones can permeate into the cell to saturation levels within 30 minutes, suggesting that translocation across the membrane occurs relatively faster than with most endocytosis-dependent commercial transformation reagents. Similar results were obtained when using growth medium containing 10% FBS. All these results demonstrate that gH625 significantly enhances the intrinsic cell membrane permeability of H2A, which occurs in less than one hour regardless of the presence of FBS in the culture medium.

[0118] Chromatin labeled with the gH625 peptide enhances the movement of DNA across the membrane.

[0119] Previous studies have shown that chromatins assembled in vitro using histones derived from frog or calf thymus enhance intracellular plasmid DNA delivery. To investigate whether the gH625 peptide can enhance intracellular DNA delivery when labeled on chromatin, reconstituted octamers were prepared for in vitro chromatin assembly (Fig. 2A). Reconstituted octamers containing additional gH625 modifications to other human core histones precipitated severely during the purification process and were not further attempted.

[0120] Chromatin was assembled using a biotinylated pUC19 plasmid containing 16 repeats of the 601-Widom nucleosome position sequence. The quality of the assembled chromatin was investigated by MNase analysis (Fig. 2B). While the synthesized DNA produced mainly low molecular weight DNA fragments of less than 100 bp, the chromatin assembled using wild-type or gH625-H2A-containing octamers produced 200 bp-DNA ladders, suggesting that the chromatin consists mainly of physiologically spaced nucleosomes (Fig. 2B, compare lanes 2 with lanes 4 and 6). These results demonstrate that biotinylation of the histone H2A N-terminus gH625 peptide or the plasmid DNA main chain does not affect the quality of chromatin assembly.

[0121] Cell permeability analysis similar to that in Fig. 1B was performed using wild-type and gH625 peptide-labeled chromatins, respectively (Fig. 2C). The results show that gH625 peptide-labeled chromatin permeates into cells with higher efficiency than wild-type chromatin (Fig. 2C, comparison of WT H2A chromatin and gH625-H2A chromatin in Avidin-Fluor 488). Considering the native nuclear localization signals of the four human core histones, gH625-labeled chromatin appears to be primarily located in the nucleus (Fig. 2C, synthetic image of Avidin-Fluor 488 and DAPI). Quantification of the images in Fig. 2C using Image J software confirms that wild-type chromatin possesses native membrane permeability and that additional gH625 peptide labeling on the chromatin significantly increases plasmid DNA delivery across the cell membrane toward the nucleus (Fig. 2D). However, observation of the migration of biotin-labeled DNA through chromatin assembly has the disadvantage that membrane permeability treatment is required to visualize secondary Avidin Flur 488 binding, which can lead to endogenous cellular biotin-mediated signaling and artifacts.

[0122] To visualize the transferred plasmid DNA more directly, the DNA was pre-labeled with Cy5 using the Label IT nucleic acid labeling kit prior to chromatin assembly. HeLa cells were treated with Cy5-labeled chromatin in growth medium for 3 hours, and Cy5-positive cells were analyzed by cytometry (Fig. 3A). The results show that over 90% of cells exhibited a Cy5-positive signal upon treatment with wild-type or gH625 chromatin during the 3-hour incubation period, which is significantly higher than the control Effectene transformation method.

[0123] To further confirm plasmid delivery across the membrane, HeLa cells grown on coverslips were treated under conditions similar to those of the cell permeability assay, and confocal fluorescence microscopy images were analyzed (Fig. 3B). Consistent with the FACS analysis, Cy5-labeled DNA derived from wild-type or gH625-labeled chromatin was found intracellularly in both the cytoplasm and the nucleus.

[0124] Chromatin and gH625 peptides are known to enter cells primarily through a direct transport mechanism. To analyze the effects of endocytosis inhibitors, HeLa cells were pretreated with methyl-beta-cyclodextrin (MCD) and amiloride (AMI), followed by transformation with Cy5-labeled DNA or direct addition of gH625-H2A-containing chromatin (Fig. 3C). While effectene-mediated transformation was significantly affected by both inhibitors, cell entry of gH625-H2A-containing chromatin was relatively insensitive to the macrophage inhibitor amiloride and the broad-spectrum endocytosis inhibitor methyl-beta-cyclodextrin.

[0125] Although not confirmed by an MNase-based multiple nucleosome ladder, reconstituted chromatin has previously been reported to exhibit reporter gene expression upon extracellular application. We explored the potential for ectopic reporter gene expression using gH625-H2A-containing chromatin, but no detectable gene expression of EGFP-encoding plasmid DNA was observed. It was not determined whether gene expression of the transformed DNA requires appropriate downstream processes, such as the release of endosomes from the chromatin or the dissociation of a possible repressive chromatin structure.

[0126] Chromatin labeled with gH peptides improves the skin permeability of DNA.

[0127] Transdermal delivery through the skin is an attractive pathway for delivering bioactive macromolecules, including DNA and proteins. A small number of cell-permeable peptides (CPPs) are known to facilitate direct skin penetration through the stratum corneum and epidermis, which are major barriers. Considering the direct transport mechanisms across histone and chromatin membranes, the direct skin penetration ability of the derived DNA and chromatin was investigated using a suckling pig skin-Franz cell system.

[0128] Cy5-labeled plasmid DNA and the chromatin derived therefrom were applied to porcine skin three times in three repetitions at 24-hour intervals, and frozen sections were analyzed using a fluorescence microscope (Figs. 4A and 4B). Significantly high fluorescence was observed in the stratum corneum after 24 hours of exposure to gH625-H2A chromatin (Fig. 4A, 24-hour column), and deeper penetration into the epidermal and dermal regions was observed after 48 and 72 hours of exposure. Wild-type chromatin also showed better permeability than the labeled DNA, but its efficiency was significantly lower than that of gH625-H2A chromatin (Fig. 4B). These results demonstrate that the gH625 peptide labeling on the chromatin enhances the skin penetration of chromatin through the skin barrier.

[0129] EGF-labeled gH625-chromatin stimulates keratinocyte growth and collagen 1α secretion.

[0130] The outermost skin region of the epidermis consists of up to 90% keratinocytes. Skin penetration testing of gH625-H2A chromatin was necessary to investigate whether gH625-H2A chromatin could reach the epidermis and serve as a delivery vehicle for bioactive peptides capable of promoting keratinocyte cell growth and collagen stimulation. Human EGF peptides were fused to the C-terminus of human H2B histones via gene synthesis, and histone octamers containing modified histones of gH625-H2A and H2B-EGF were purified from an E. coli expression system (Fig. 5A). The modified H2B-EGF possesses an additional 6.8 kDa EGF at the C-terminus and, once incorporated into a nucleosome, enables the display of two EGF peptides per nucleosome on the chromatin structure (Table 2).

[0131] In vitro chromatin assembly using octamers of modified histones was investigated by MNase analysis (Fig. 5B). Chromatin assembly was successfully performed with gH625-H2A / H2B-EGF octamers, but the quality of assembly based on multiple DNA ladders appears to be reduced (Fig. 5B, lanes 1 and 2 versus lanes 3 and 4).

[0132] EGF is a growth factor that promotes cell division and stimulates the biosynthesis of collagen, elastin, and extracellular matrix, and is known to have a short half-life due to its low thermal stability. Since H2B-EGF underwent a denaturation-regeneration cycle during the purification process, the biological activity of EGF (EGF-chromatin) labeled on the chromatin was evaluated by comparison with natural EGF peptides (Figs. 5C and 5D).

[0133] Recommended doses of EGF peptide and EGF-labeled chromatin stimulated the growth of immortalized human keratinocytes, HaCa-T cells, in minimal growth medium by up to 50% compared to the control group (Fig. 5C). Assuming that up to 5% of the chromatin mass is derived from the labeled EGF, the EGF-labeled chromatins at 125 and 250 ng / ml are within a comparable range to the known active concentration of EGF peptide in tissue culture cells, which is 10–100 ng / ml.

[0134] EGF is known to slightly enhance the synthesis of type I collagen upon acute exposure. EGF peptides appear to increase the production of collagen type I, but the increase is not statistically significant compared to the control group (Fig. 5D). However, treatment of HaCa-T cells with EGF-labeled chromatin induces higher concentrations of collagen type I secretion into the medium (Fig. 5D). Taken together, these results demonstrate that EGF-labeled chromatin maintains growth factor activity in cell proliferation and collagen synthesis.

[0136] In summary, the gH625 peptide significantly enhances the cell permeability of histone H2A and chromatin. Specifically, gH625-H2A exhibited a cell permeability approximately six times higher than wild-type H2A, indicating that gH625-labeled chromatin passes through the cell membrane and nuclear membrane more effectively; in particular, gH625-chromatin demonstrated enhanced transdermal permeability in a porcine skin model.

[0137] In addition, since gH625-chromatin labeled with EGF stimulates the growth of keratinocytes and collagen secretion, these results indicate that chromatin modified with gH625 can be used as an effective carrier for the transdermal delivery of various biomolecules, such as DNA, peptides, and proteins. The present invention is expected to present new possibilities in the fields of non-invasive gene therapy and intracellular or transdermal drug delivery.

[0139] The present specification and drawings disclose preferred embodiments of the present invention. Although specific terms have been used, they are used merely in a general sense to facilitate the explanation of the technical content of the present invention and to aid in understanding the invention, and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that, in addition to the embodiments disclosed herein, other variations based on the technical concept of the present invention are possible.

Claims

Claim 1 A composition for intracellular or transdermal drug delivery comprising a gH625-H2A histone or its derivative and a nucleic acid, wherein a peptide having the amino acid sequence of General Formula 1 is fused to the N-terminus of the gH625-H2A histone or its derivative and a nucleic acid: R1-His-Gly-Leu-X1-X2-X3-Leu-X4-Arg-Trp-X5-His-X6-X7-X8-Leu-Ile-X9-X10-Phe -R2(General Formula 1): In the above General Formula 1, R1 is H (free amino group), R2 is OH (free carboxyl group), X1 is A (alanine) or S (serine), X2, X3, or X4 is S (serine) or T (threonine), and X5, X8 or X10 is A (alanine) or G (glycine), X6 is Y (tyrosine) or F (phenylalanine), X7 is N (asparagine) or Q (glutamine), and X9 is R (arginine) or K (lysine). Claim 2 A composition for intracellular or transdermal drug delivery according to claim 1, characterized in that the peptide is an amino acid sequence of any one of SEQ ID NOs 1 to 16. Claim 3 A composition for intracellular or transdermal drug delivery according to claim 1, characterized in that the nucleic acid is DNA or RNA. Claim 4 A composition for intracellular or transdermal drug delivery according to claim 1, characterized in that the histone included in the composition for intracellular or transdermal drug delivery is gH625-H2A alone or in combination with other canonical histones (H3, H4, H2B) or derivatives thereof. Claim 5 A composition for intracellular or transdermal drug delivery according to claim 1, characterized in that the histone is produced by recombinant expression of histone-encoding DNA. Claim 6 A composition for intracellular or transdermal drug delivery according to claim 1, characterized in that the nucleic acid comprises a sequence encoding at least one protein. Claim 7 A composition for intracellular or transdermal drug delivery according to claim 1, characterized in that the nucleic acid comprises a sequence expressing at least one noncoding RNA. Claim 8 A composition for intracellular or transdermal drug delivery according to claim 1, characterized in that it comprises a sequence encoding a promoter for the controlled expression of a protein encoded by the nucleic acid and a non-coding RNA. Claim 9 An intracellular or transdermal drug delivery composition according to any one of claims 1 to 8, characterized in that the intracellular or transdermal drug delivery composition is in the form of a synthetic chromatin delivery system (SCDS) comprising a nucleosome. Claim 10 A composition for intracellular or transdermal drug delivery, characterized in that, in any one of claims 1 to 9, the nucleic acid is plasmid DNA containing a 601 Widom repeat sequence.

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