Cell-penetrating peptide for delivery biomaterials

Abstract

The present invention relates to a cell-permeable peptide comprising the amino acid sequence of SEQ ID NO. 1.

Description

Technology Field

[0001] The present invention relates to a novel cell-permeable peptide, wherein a biomaterial-binding peptide comprising one or more amino acids at at least one end of the cell-permeable peptide binds to a biomaterial and can deliver the biomaterial into a cell. Background Technology

[0002] Cells consist of cytoplasm surrounded by a cell membrane and contain many biomolecules such as proteins and nucleic acids.

[0003] Eukaryotic cells contain a nucleus, which is involved in heredity by regulating cell activity and division through maintaining genes without modification and controlling gene expression.

[0004] Gene expression occurs based on genetic information stored in DNA, by using DNA to create RNA in the cell nucleus, and then synthesizing proteins from the RNA. Various proteins obtained through the genetic information expressed in the cell nucleus play a role in performing cell activity and function, and thus have a significant impact on the maintenance of life.

[0005] As such, to regulate the important role of the cell nucleus and gene expression, it is crucial to block the influx of substances into the cytoplasm where the nucleus is located. The cell membrane is composed of a phospholipid bilayer, which separates the cytoplasm from the outside of the cell and prevents external substances from entering the cell.

[0006] Utilizing the intracellular action of DNA as described above, various therapeutic methods are being developed to treat human diseases, such as introducing DNA into cells via drugs and enabling the DNA that has finally entered the nucleus to synthesize proteins capable of treating the disease.

[0007] However, as mentioned above, the cell membrane, which exists to prevent the influx of external substances from outside the cell and to protect internal organelles in order to maintain cell function, is configured to allow limited permeability of substances that need to be delivered into or out of the cell through special channels or passage mechanisms; therefore, it is not easy to introduce substances into the cell from the outside.

[0008] After conducting repeated research to solve the aforementioned problem, the inventors of the present invention developed a novel peptide having a novel amino acid sequence that has cell permeability and the ability to deliver biomaterials into cells by directly or indirectly connecting biomaterials to the ends of the peptide. The problem to be solved

[0009] The present invention aims to provide a peptide having a novel amino acid sequence having cell permeability.

[0010] The present invention aims to provide a novel cell-permeable peptide capable of delivering a biomaterial into a cell by directly or indirectly connecting the biomaterial to the cell-permeable peptide.

[0011] The present invention aims to provide a novel cell-permeable peptide with good cytotoxicity.

[0012] The present invention also provides a method for delivering biomaterials into a cell using a cell-permeable peptide according to the present invention. means of solving the problem

[0013] The cell-permeable peptide according to the present invention for solving the above-mentioned problems comprises the amino acid sequence of SEQ ID NO. 1.

[0014] In the present invention, the cell-permeable peptide may have a biomaterial-binding peptide comprising one or more amino acids attached to its N-terminus and / or C-terminus.

[0015] In the present invention, the cell-permeable peptide may be one in which the biomaterial-binding peptide binds to a biomaterial and delivers the biomaterial into the cell.

[0016] In the present invention, the biomaterial may be one or more selected from the group consisting of DNA, RNA, and protein.

[0017] In the present invention, the cell-permeable peptide may be bound to a DNA-binding domain (DNA-Binding Domain, DBD). Effects of the invention

[0018] The cell-permeable peptide according to the present invention has the effect of being able to penetrate into a cell without relying on a separate channel or mechanism.

[0019] The cell-permeable peptide according to the present invention has the effect of not reducing its cell permeability even when additional proteins or functional groups are connected to its terminals.

[0020] The cell-permeable peptide according to the present invention has the effect of being able to deliver biomaterials into the cell by directly or indirectly attaching biomaterials to its terminals and permeating into the cell.

[0021] The cell-permeable peptide according to the present invention has the effect of delivering biomaterials into cells for the treatment of diseases, improvement of constitution, etc. Brief explanation of the drawing

[0022] Figure 1 is a cleavage map of the plasmid DNA pEGFP-N1 (Clontech); FIG. 2 is a graph showing the results of measuring the delivery efficiency of plasmid DNA to HEK293 cells using the cell-permeable peptide and lipofectamine according to the present invention, respectively; Figure 3 is a graph showing the results of measuring the cytotoxicity of the SRE-CPP peptide of the example; Figure 4 is a graph showing the results of measuring the cytotoxicity of the SRE-DBD peptide of the example. Specific details for implementing the invention

[0023] Hereinafter, each component of the present invention is described in more detail so that a person skilled in the art to which the present invention pertains can easily implement it; however, this is merely an example, and the scope of the rights of the present invention is not limited by the following.

[0024] The cell-permeable peptide according to the present invention comprises the amino acid sequence of SEQ ID NO. 1.

[0025] The cell-permeable peptide described above may include the amino acid sequence of SEQ ID NO. 1, even if additional amino acids are connected.

[0026] For example, the amino acid sequence of the above sequence number 1 is the same as the following sequence number 1.

[0027] Sequence No. 1: SREFHRKYRIPA

[0028] The amino acid sequence of SEQ ID NO. 1 may further include an additional peptide at the end, and preferably, it may be a peptide having the amino acid sequence of SEQ ID NO. 1. That is, it may be a peptide composed of 12 amino acids as in SEQ ID NO. 1.

[0029] The cell-permeable peptide according to the present invention may have additional amino acids or peptides attached to its N-terminus and / or C-terminus.

[0030] More specifically, the additional amino acid or peptide may be a biomaterial binding peptide comprising one or more amino acids.

[0031] The cell-permeable peptide according to the present invention plays a role in delivering a biomaterial into a cell by penetrating the cell while being directly or indirectly bound to the biomaterial. The biomaterial may be directly connected to the end of the cell-permeable peptide, such as additional amino acids or peptides, or may be a biomaterial that can be bound to the peptide connected to the end.

[0032] The above-mentioned biomaterial may be, for example, a substance that has a function within the body such as DNA, RNA, or protein, and any other substance that needs to be delivered into a cell may be applied without limitation.

[0033] Specifically, the cell-permeable peptide may be bound to a DNA-binding domain (DBD), and accordingly, after the cell-permeable peptide bound to the DBD is introduced into the cell with DNA bound to the DBD, the DNA is released from the cell-permeable peptide, thereby having the effect of delivering DNA into the cell.

[0034] Specifically, the amino acid sequence of DBD, one of the above biomaterial-binding peptides, is as shown in SEQ ID NO. 2 below. However, this is a peptide corresponding to one example, and is the DBD peptide disclosed in Korean Patent Application No. 10-2015-0131427. This is merely one possible example, and it is also possible to bind any other peptide, amino acid, functional group, etc., that has the function of binding to biomaterials.

[0035] Sequence No. 2: KSPKKAKKKSPKKAKK

[0036] The cell-permeable peptide according to the present invention combined with the above DBD may, for example, have the amino acid sequence of SEQ ID NO. 3 below. Likewise, this is merely one exemplary peptide form, and the cell-permeable peptide according to the present invention can be implemented in various forms as long as it includes the amino acid sequence of SEQ ID NO. 1 above.

[0037] Sequence No. 3: SREFHRKYRIPAKSPKKAKKKSPKKAKK

[0038] Hereinafter, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the present invention; however, this is merely an example, and the scope of the present invention is not limited by the following.

[0039] [Example: Synthesis of Cell-Permeable Peptides]

[0040] The peptide of SEQ ID NO. 1 (12-mer, named 'SRE-CPP'), the peptide of SEQ ID NO. 2 (16-mer, named 'DBD'), and the fusion peptide of SEQ ID NO. 3 (28-mer, named 'SRE-DBD', a fusion peptide of SEQ ID NO. 1 and SEQ ID NO. 2) were synthesized by Peptron (Daejeon, South Korea) using the FMOC solid-phase method with an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, South Korea). The synthesized peptides were purified and analyzed using reverse-phase high-speed liquid chromatography (reverse-phase HPLC) with a C18 analysis RP column (Shiseido capcell pak) (Prominence LC-20AB, Shimadzu, Japan) and identified using a mass spectrometer (HP 1100 Series LC / MSD, Hewlett-Packard, Roseville, USA).

[0041] [Experimental Example 1: Experiment to Confirm Plasmid DNA Delivery Efficiency Using SRE-DBD Fusion Peptide]

[0042] The transfection efficiency of the fusion peptide of the present invention was evaluated using HEK293 cells (ATCC), and for comparison, the transfection efficiency of lipofectamine, a reagent currently used for plasmid DNA delivery, was evaluated.

[0043] The plasmid DNA used to measure DNA transfer efficiency was pEGFP-N1 (Clontech), which contains the Enhanced Green Fluorescent Protein (EGFP) gene. The cleavage map of this plasmid DNA is shown in Figure 1.

[0044] 1 x 10 in a 6-well plate 6 HEK293 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) for 12 hours, then 4 µg of pEGFP-N1 and 1 µg of the fusion peptide SRE-DBD were mixed and reacted for 30 minutes before being treated with the cells. The control group consisted of an experimental group treated with a mixture of 4 µg of pEGFP-N1 and 1 µg of DBD, and an experimental group treated with 4 µg of pEGFP-N1 and 10 µl of Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. After 24 hours following the treatment, each experimental group was washed with PBS, the cells were detached, transferred to a 96-well plate, and fluorescence values ​​were measured using a Fluorescence Spectrometer (excitation 488 nm, emission 509 nm). From the results in Fig. 2, it can be seen that the DNA delivery activity of the test group treated with the fusion peptide SRE-DBD is about 5 times higher than that of the control group treated with Lipofectamine 2000 (Fig. 2).

[0045] [Experimental Example 2: Cytotoxicity Confirmation Experiment Using SRE-CCP and SRE-DBD Fusion Peptides]

[0046] The cytotoxicity of the SRE-CPP peptide and the fusion peptide SRE-DBD of the present invention was evaluated. 1 x 10⁶ in a 24-well plate 5 HEK293 cells were cultured with RPMI 1640 medium (Thermo Fisher Scientific) and treated with SRE-CPP peptide and SRE-DBD peptide at different concentrations. After 48 hours, an MTT assay was performed to measure cell viability, and the results are shown in Figures 3 and 4.

[0047] Referring to Fig. 3, it can be seen that the cell-permeable peptide of the present invention does not cause significant cytotoxicity regardless of the treatment concentration (Fig. 3). As a result of measuring the cell viability of the fusion peptide SRE-DBD using the same method as in the experiment above, no cytotoxicity was detected (Fig. 4).

Claims

Claim 1 A cell-permeable peptide comprising the amino acid sequence of SEQ ID NO. 1, wherein the cell-permeable peptide has a biomaterial-binding peptide attached to its C-terminus, and the biomaterial-binding peptide has the amino acid sequence of SEQ ID NO.

2. Claim 2 delete Claim 3 In claim 1, the cell-permeable peptide is characterized in that the biomaterial-binding peptide binds to a biomaterial and delivers the biomaterial into the cell. Claim 4 A cell-permeable peptide according to claim 3, characterized in that the biomaterial is one or more selected from the group consisting of DNA, RNA, and protein. Claim 5 A cell-permeable peptide according to claim 1, characterized in that the cell-permeable peptide is bound to a DNA-binding domain (DNA-Binding Domain, DBD).

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