CN106795205B - Cell membrane penetrating peptides and their use as bioactive substance carriers - Google Patents
Cell membrane penetrating peptides and their use as bioactive substance carriers Download PDFInfo
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
Abstract
The present invention relates to a novel peptide that is capable of delivering a substance that is difficult to penetrate a cell membrane of a eukaryotic cell and is delivered into the interior of the eukaryotic cell through various binding, and relates to a peptide transporter that is capable of efficiently delivering a variety of bioactive substances into a cell by penetrating a cell membrane as a peptide that is searched for in an amino acid sequence derived from a human. The cell membrane-penetrating peptide found by the research was bound to a protein and a nucleic acid, and the efficacy of the peptide was confirmed to be delivered into the cell, and the peptide was confirmed to have superior efficacy compared to the conventional cell-penetrating peptide R9. The present invention is expected to contribute to the development and utilization of pharmaceuticals, cosmetics, functional products, and the like, which utilize a variety of bioactive substances that are difficult to transfer into the interior of cells and thus cannot be utilized.
Description
Technical Field
The present invention relates to a novel peptide (peptide) that penetrates cell membranes, which enables a substance that is difficult to penetrate cell membranes of eukaryotic cells and is delivered into cells to the inside of the cells through various binding, and from the C-terminal amino acid sequence of melittin peptide that plays a central role in cell membrane lysis of melittin peptide, a similar amino acid sequence is searched and developed from the amino acid sequence of human protein. The peptides developed are expected to be effective in delivering various bioactive substances into the body, which are difficult to deliver into the cell and thus cannot be used as substances beneficial to human beings, and which are difficult to develop, as the peptides that function as a transporter that penetrates the cell membrane and efficiently delivers various bioactive substances into the body, and are expected to be greatly beneficial for the development and utilization of products beneficial to human beings, such as pharmaceuticals, cosmetics, and functional products.
Background
The cell membrane of a eukaryotic cell is a monomer, which is formed of a double lipid membrane, protects the substances inside the cell, and controls the inflow of substances from the outside or the movement of the substances inside. In particular, it is almost impossible to widely know a mechanism of organizing and organizing a hydrophilic substance like nucleic acid which is an active substance in a living body, or a molecule having a large size and a surface charge like a protein or a peptide, but not a specific substance like a receptor on a cell membrane surface. Therefore, since most of the drugs developed so far are developed using cell surface substances other than intracellular substances, if a mediator capable of efficiently transferring such hydrophilic substances or large-sized substances into cells is developed, it is possible to develop a new drug or product useful for countless intracellular substances that have not been developed.
The innumerable substances that act inside the cell cannot function properly due to lack or deformation, and the simplest approach to treat such diseases is to replenish the insufficient and deformed biological substances. However, since the barrier of the cell membrane restricts the transfer of large-sized biomolecules into the cell, there has been a demand for the development of a carrier capable of transferring a substance into the cell. Cell membrane-penetrating peptides developed on the basis of this background are Protein Transduction Domains (PTDs) or Cell-penetrating peptides (CPPs).
In 1988, there was a first report that the Tat protein of HIV-1(Human immunodeficiency virus, type 1; Human immunodeficiency virus type 1) can be introduced into mammalian cells, and studies on a carrier that transfers a substance into the inside of a cell have been actively conducted (document 1). Peptides, which consist of about 20 shorter amino acids and are capable of penetrating cell membranes to deliver substances to the interior of cells, were then named PTDs or CPPs, thereby developing countless new cell membrane penetrating peptides. Representative PTDs include Tat-PTD (references 2 and 3) (Tat-49-57, sequence: RKKKRRQRRR) found in HIV-1Tat protein, Antp (reference 4) (Antennapedia gene (Antennapedia) _43-58, sequence: RQIKIWFQNRRMKWKK) derived from the homeodomain of drosophila antenna gene, VP22 (reference 5) (VP22_267-300, sequence: DAATATRGRSAASRPTERPRAPARSASRPRRPVE) derived from HSV-1 (Herpessimixvirrutype 1, herpes simplex virus type 1) VP22 protein, Poly-arginine (R9, RRRRRRRRR) developed from artificial sequences, and the like, and these peptides are commonly characterized by constituting cationic amino acids at a high ratio.
In contrast, a cell Membrane-penetrating peptide mainly composed of a hydrophobic peptide has also been developed, and representative is MTS (document 6) (Membrane-displacing Sequence). MTS consists of 16 amino acids derived from the human FGF-4(fibroblast growth factor) protein, and is composed mainly of hydrophobic amino acids (sequence: AAVALLPAVLLALLAP). It is widely known that hydrophobic peptides have a mechanism of passing through a hydrophobic region inside a double lipid membrane constituting a cell membrane to directly transfer a substance to cytoplasm. In addition to this, cell-penetrating peptides were developed from various proteins of various biological species, and artificial sequences were also designed (design) for the development of cell membrane-penetrating peptides. Research for the development of useful substances has been continuously conducted by utilizing the effects of countless cell membrane-penetrating peptides developed in this way to deliver various substances such as proteins, nucleic acids, compounds, metals, and the like to the interior of cells. However, the mechanism of action is still unclear, and the fate of a mediator and a mediator (cargo) after delivery into the cell has been studied more, and the development of pharmaceuticals using cell membrane-penetrating peptides has not been successfully achieved.
PTDs have been developed in view of the functional side of penetrating cell membranes, mostly from substances that must penetrate cell membranes to function, like viruses (viruses) or toxins (toxins). However, PTDs derived from other biological species than humans may also cause safety problems such as immune response and toxicity when useful substances similar to pharmaceuticals applied in vivo are developed for humans in the future.
Melittin (Gene ID: 406130) peptide, which is a major toxic peptide, has more than 50% of the composition of Melittin (Apitoxin), and it is widely known that toxicity is expressed by strong hemolytic action. It is more known that a more specific mechanism of action is that, after first binding to a polysaccharide or a phosphorylated site on the surface of a cell membrane, an N-terminal hydrophobic peptide portion is allowed to permeate the double lipid membrane of the cell membrane to form pores, and the cell is lysed. In addition, binding to the Phospholipase A2(Phospholipase A2) protein, thereby blocking the transport pumps similar to Na + -K + -ATPase and H + -K + -ATPase, and further leading to increased intracellular permeation of ions similar to Na + and Ca2 +.
The research using melittin mainly studies the function of targeting and killing specific cells using the toxic effect of melittin. Soman n n.r. et al performed the following studies: perfluorocarbon (perfluorcarbon) nanoparticles capable of targeting cancer cells are conjugated with melittin to target cancer cells and thereby cause them to die (document 7).
The research of using melittin's cell membrane lytic function as a carrier of genetic genes has also been actively carried out in recent years. The Schellinger j.g. et al performed the following studies: poly-lysine and melittin were bound to HPMA (N- (2-hydroxypropyl) methacrylamide) to be used as a genetic gene transporter (reference 8). In this study, melittin was used with the aim of perforating the cell membrane, thereby increasing the efficiency of gene delivery. US patent application US20120165393 a1 is the following invention: ligands against Asialoglycoprotein (Asialoglycoprotein) receptors and melittin-bound fusion proteins are used as siRNA transporters, in this invention melittin is used for the following purposes: binds to siRNA, thereby penetrating cell membranes (document 9).
The present inventors searched for similar sequences in the amino acid sequence of human protein from 12 amino acid sequences (SEQ ID NO: 3) including the hydrophilic amino acid sequence at the C-terminal end of melittin peptide, and finally developed the most ideal LayN peptide. The amino acid sequence of the LayN peptide is derived from the amino acid sequence of a human protein named Layilin.
The Layilin protein functions as a hyaluronic acid receptor and is composed of an extracellular domain (transmembrane domain) (SEQ ID NO: 5) from the N-terminus to the cytoplasmic domain. The LayN peptide was found to be the most preferable sequence (SEQ ID NO: 1) in which cysteine (cysteine) at position 259 was substituted with serine (serine) from 10 amino acids (SEQ ID NO: 2) from amino acid sequence 257 to 266 of the Layilin protein.
Reference to the literature
1.Frankel A.D.,Pabo C.O.Cellular uptake of the tat protein from humanimmunodeficiency virus.Cell,1988 23；55(6):1189–93.
2.Fawell S.,Seery J.,Daikh Y.,Moore C.,Chen L.L.,Pepinsky B.,BarsoumJ.Tat-mediated delivery of heterologous proteins into cells.Proc Natl AcadSci USA.1994 18；91(2):664-8.
3.US 5804604 A(Biogen Inc.)1995.5.25
4.Derossi D.,JoliotA.H.,Chassaing G.,Prochiantz A.The third helix ofthe Antennapedia homeodomain translocates through biologicalmembranes.J.Biol.Chem.1994 8；269(14):10444-50.
5.Elliott G.,O’Hare P.Intercellular trafficking and protein deliveryby a herpesvirus structural protein.Cell.1997 24；88(2):223-33.
6.US 5807746 A(Vanderbilt University)1994.6.13
7.Soman N.R.,Baldwin S.L.,Hu G.,Marsh J.N.,Lanza G.M.,Heuser J.E.,Arbeit J.M.,Wickline S.A.,Schlesinger P.H.Molecularly targeted nanocarriersdeliver the cytolytic peptide melittin specifically to tumor cells in mice,reducing tumor growth.J.Clin.Invest.2009 119(9):2830-42.
8.Schellinger J.G.,Pahang J.A.,Johnson R.N.,Chu D.S.,Sellers D.L.,Maris D.O.,Convertine A.J.,Stayton P.S.,Horner P.J.,Pun S.H.Melittin-graftedHPMA-oligolysine based copolymers for gene delivery.Biomaterials 2013 34(9):2318-26.
9.US 20120165393 A1(Arrowhead Madison Inc.)2011.12.15
Disclosure of Invention
The present invention addresses the problem of providing a novel cell membrane-penetrating peptide that enables the delivery of a biologically active substance into the interior of a cell, in order to solve the following problems: various bioactive substances, which are capable of functioning and functioning inside cells, are difficult to penetrate cell membranes and are thus delivered inside cells, and thus, the use rate is reduced and development is not easy. The cell membrane-penetrating peptide developed by the present invention is derived from an amino acid sequence constituting a human protein, and is expected to ensure higher safety in the future development of useful substances such as pharmaceuticals and cosmetics for humans.
The present inventors explored the development of a novel cell-penetrating peptide sequence having the efficiency of penetrating cell membranes by the amino acid sequence of the peptide. The problem solving method of the invention is as follows.
Melittin peptide is the major peptide, which has more than 50% of the toxic components. Melittin is a peptide consisting of 26 amino acids, 20 amino acids from the N-terminus are mainly composed of hydrophobic amino acids, and 6 amino acids from the C-terminus are composed of hydrophilic amino acids. It is well known that melittin destroys the cell membrane of red blood cells, thereby exhibiting toxicity by hemolysis. It is known that the main mechanism of action is that after binding to a polysaccharide or a phosphorylated site on the surface of a cell membrane, an α -helical peptide permeates a double lipid membrane to form pores to allow cell lysis. The present inventors focused on the function of other mechanisms of melittin to bind to the cell membrane and permeate the double lipid membrane. Similar sequences were explored from the amino acid sequence of human proteins using 12 amino acids including the C-terminal 6 amino acids, which play the most important role in the hemolytic function of melittin peptide.
As described in example 1, 100 or more similar amino acid sequences showing 67% or more identity were obtained by NCBI (National Center for Biotechnology Information, USA, http:// WWW.ncbi.nlm.nih.gov. /) BLAST (Basic Local Alignment Search Tool), and several candidate sequences were obtained among these candidate materials by the content and arrangement form of arginine (arginine), lysine (lysine), and aspartic acid (aspartic acid), glutamic acid (glutamic acid), which are cationic amino acids, as cationic amino acids. The sequence of the peptide in which the cell membrane penetration efficiency was most excellent was finally confirmed.
As a result of the search, the finally obtained cell membrane-penetrating peptide was obtained from 10 amino acid sequences consisting of amino acid sequences 257 to 266 of the Layilin (Gene ID: 143903) protein widely known as the hyaluronic acid receptor, and the cell membrane-penetrating peptide sequence was finally determined in a form in which cysteine (cysteine) was substituted with serine (serine) in order to prevent intermolecular dimer formation of the peptide, and was named as the LayN peptide (SEQ ID NO: 1).
The following sequence formula for cell penetrating peptides was prepared based on the amino acid sequence of the LayN peptide.
The sequence formula is as follows: W-I-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 4)
The sequence formula is a sequence which is based on the basic structure of the LayN peptide and is formed by the following forms: here, X1 may be in the form of cysteine, which is a prototype sequence of the amino acid sequence of the Layilin protein, an amino acid serine substituted therefor, and alanine (alanine) in which no functional group is branched, the amino acids X2 to X6 may be in the form of cationic amino acids including arginine (arginine) and lysine (lysine), and the amino acids X7 to X8 may be in the form of anionic amino acids such as glutamic acid (glutamic acid), aspartic acid (aspartic acid), glutamine (glutamine), and asparagine (aspartic acid).
The LayN cell membrane penetrating peptide is combined with a transmembrane region (amino acids No. 236 to No. 256) of Layilin protein, so that the Layilin cell membrane penetrating peptide can be used as a peptide for simulating the function of melittin, all amino acids in an L form and an D form can be used as amino acids in the developed cell membrane penetrating peptide, and the forward direction sequence and the reverse direction sequence of the amino acid series also have cell membrane penetrating efficiency.
The cell membrane penetrating peptide used in the examples was confirmed using the LayN peptide of sequence No. 1, and the efficacy of delivering proteins and nucleic acids (siRNA) into the cell as a transporter (cargo) substance was confirmed by the examples. Furthermore, the toxicity of the peptides was confirmed in various human cell lines, and the peptides were carried out at a peptide concentration of 1.5 μm or less, which is free from a problem of cytotoxicity, and confirmed.
The cell-penetrating peptide developed by the present invention is intended to function as a mediator that binds to a mediator and transfers the substance into the interior of a cell, and therefore, a protein, a nucleic acid, a compound, a mineral, or the like can be used as the mediator to be bound without any particular limitation, and the mediator is transferred into the interior of a cell through the cell-penetrating peptide without reacting with other carriers or cell membrane receptors in the living body.
The novel cell membrane-penetrating peptides developed by the present invention can be used to develop a myriad of useful substances that can act only inside cells, and that can efficiently deliver useful bioactive substances having functions to the inside of cells, and that cannot be developed due to lack of a delivery method to the inside of cells. Furthermore, the peptides developed by the present invention are useful as cell membrane-penetrating peptides derived from amino acid sequences constituting human proteins, and are expected to be safe in the development of useful products and techniques for human application in the future.
Drawings
FIG. 1 shows the results of a flow cytometry (FACS) analysis comparing the intracellular penetration rate of the cell penetrating peptide LayN with the cell penetration rate performance of the R9 peptide, wherein A is the untreated group, B is the R9 peptide, and C is the LayN peptide treated group.
FIG. 2 shows experimental data for confirming cell permeability according to the concentration of cell-penetrating peptide LayN in adherent cell HEK293 cells by a flow cytometer, wherein A is a peptide-free treated group, B is a peptide-treated group at a concentration of 0.2. mu.M, and C is a peptide-treated group at a concentration of 1.0. mu.M.
FIG. 3 is experimental data for confirming cell permeability according to the concentration of cell penetrating peptide LayN in suspension cell Jurkat T cells using a flow cytometer, A is a peptide-free treated group, B is a peptide-treated group at a concentration of 0.2. mu.M, and C is a peptide-treated group at a concentration of 1.0. mu.M.
FIG. 4 is a photograph showing the efficiency of protein transfer by X-gal staining in an experiment for confirming that the protein is transferred into the cell in a form in which the cell-penetrating peptide, LayN, is bound to a β -galactosidase (BetaGalactosidase) protein. Panel A is a schematic diagram of the structure of a β -galactosidase fusion protein bound to a LayN peptide, with the sequence His-Tag (H6), LayN peptide, and β -galactosidase used for purification being bound from the N-terminus. Panel B shows data for confirming protein transfer efficiency by X-Gal staining after introducing the H6-LayN-. beta. -Gal fusion protein produced as described above into HEK293 cells, a human embryonic kidney cell line, at a concentration of 0.1 to 1.5. mu.M.
FIG. 5 is data showing that the cell membrane penetrating peptide LayN was allowed to bind to LacZ siRNA by electrostatic attraction, thereby confirming that expression of LacZ genetic gene was inhibited in human embryonic kidney cell line HEK293 cells transformed in morphology using pcDNA3.1/LacZ vector (vector). Panel A is a schematic diagram of the binding of LayN peptide dimer (LayN-D) to siRNA by electrostatic attraction. Panel B is data confirming the extent of binding in agarose gel (agarose gel) according to the N/P ratio of siRNA to LayN peptide dimer, the first column representing DNA size gradient marker (DNA size ladder), the second column representing the size and relative amount of free siRNA that did not bind to the band on gel (gel) as a negative control group for treating siRNA only. The third to sixth rows are data obtained when mixing is performed according to N/P ratios of 2.0, 1.0, 0.5 and 0.1, respectively. The amount of siRNA was fixed at 50nM and used. Panel D is the β -galactosidase assay data and is shown in C after being quantified.
FIG. 6 is data for confirming toxicity according to concentration in various cells of the cell membrane penetrating peptide LayN. The cytotoxicity of the LayN peptide was confirmed in human embryonic kidney cell line HEK293 cells, human cervical cancer cell line HeLa cells, human acute lymphoblastic leukemia cell line Jurkat T cells, and human keratinocyte cell line HaCaT cells, respectively. Individual cells were split 1X10 in 96-well plates (well plates)4After culturing the cells for 16 hours, the LayN peptide was treated at concentrations of 0, 0.05, 0.1, 0.2, 0.5, 1.0, and 2.0. mu.M, and then cultured for 3 hours, followed by measurement of absorbance at 420 to 480nm using WST-1 reagent, and then analyzed.
FIG. 7 is a schematic diagram of a mechanism of binding a newly developed cell membrane penetrating peptide to a mediator (cargo) to penetrate a cell membrane. Pathway a is a mechanism of transport into the cell by endocytosis (endocytosis) after being adsorbed to the cell membrane surface, and pathway B is a schematic diagram of a mechanism of transport directly into the cytoplasm after being adsorbed to the cell membrane through a pore.
Reference numerals
DNA, deoxyribonecolic acid: deoxyribonucleic acid
RNA, ribonuclear acid: ribonucleic acid
LNA, Locked Nucleic Acid: locked nucleic acid
PNA, Peptide Nucleic Acid: peptide nucleic acids
FACS, Fluorescence-activated cell sortation: flow cytometry/fluorescence activated cell sorting technology
PTD, protein transduction domain: protein transduction domains
CPPs, Cell-specific peptides: cell penetrating peptides
Detailed Description
Example 1 efficient cell penetrating peptide sequence search was performed using BLAST search.
In order to Search for similar amino acid sequences in the amino acid sequence of a protein derived from human, NCBI (National Center for Biotechnology information, USA, http:// WWW.ncbi.nlm.nih.gov /) performed BLAST (Basic Local Alignment Search Tool) Search using 8 amino acid sequences of the C-terminal part that play a decisive role in the reaction with cell membranes among 26 amino acid sequences of melittin peptide, and 100 or more sequences showing 67% or more identity were obtained. Among these candidate substances, several candidate sequences were obtained by the content and arrangement form of arginine (arginine), lysine (lysine), and aspartic acid (aspartic acid), glutamic acid (glutamic acid), which are anionic amino acids, as cationic amino acids, and finally, a lyn peptide sequence in which the cell penetrating potency was most excellent was confirmed.
As a result of the search, the finally obtained LayN peptide was obtained from 10 amino acid sequences between 257 and 266 of the Layilin (Gene ID: 143903) protein widely known as the hyaluronic acid receptor, and the LayN peptide sequence was finally determined in a form in which cysteine (cysteine) was substituted with serine (serine) in order to prevent intermolecular dimer formation.
Example 2 intracellular penetration of the LayN peptide was confirmed using a flow cytometer.
The human cervical cancer cell line HeLa cells are split into 5.5x10 cells in a 60mm culture dish5After that, culture was carried out for 24 hours in a cell culture apparatus at 37 ℃ and 5% carbon dioxide concentration using DMEM (Dulbec co's Modified Eagle's Medium) supplemented with 10% fetal bovine serum and the antibiotic Penicillin/streptomycin (Penicilin/streptomycin). After removing the medium, after two washing cycles with Phosphate Buffered Saline (PBS), the sample prepared in the medium from which fetal calf serum was removed was treated. The sample was synthesized and used with a peptide having Fluorescein Isothiocyanate (FITC) adhered to the N-terminus of each of the LayN peptide and the control R9 peptide (seq id No. 8), and was treated in cells at a concentration of 1.0 μm. In cells toThe prepared sample was treated, and after 3 hours of reaction, cells were harvested. After the cells were harvested, Trypsin-EDTA (Trypsin-EDTA) treatment was performed, the cells were removed from the culture dish, and then a third round of washing was performed using 15U/mL Heparin (Heparin) solution, and finally the cells were suspended in phosphate buffer, and the intracellular transport degree was confirmed using a flow cytometer FACSCalibur (BD).
As shown in fig. 1, it was confirmed that intracellular delivery efficiency (C) of the LayN peptide showed higher intracellular delivery potency than that of the control R9 peptide (B).
Example 3. intracellular penetration according to the concentration of the LayN peptide was confirmed in the adherent HEK293 cells and the suspension Jurkat T cells using a flow cytometer.
In order to confirm intracellular delivery efficiency according to the concentration of the LayN peptide and delivery efficiency in various cells, the delivery efficiency was confirmed in HEK293 cells, a representative human embryonic kidney cell line, which is a adherent cell, and JurkatT cells, a representative human acute lymphoblastic leukemia cell line, which is a suspension cell.
In 60mm petri dish at 5.5X105After the respective HEK293 cells and Jurkat T cells were divided, DMEM medium supplemented with 10% fetal bovine serum and the antibiotic Penicillin/streptomycin (Penicillin/streptomycin) and RPMI-1640(Roswell Park mental Institute, Rev. Losevi. Pakko Res.) medium were used for each cell line and cultured in an incubator at 37 ℃ and 5% carbon dioxide for 24 hours. After removing the medium, after two washing cycles with Phosphate Buffered Saline (PBS), the sample prepared in the medium from which fetal calf serum was removed was treated. Intracellular delivery was confirmed by treating the LayN peptide having the fluorescent substance FITC bound to the N-terminus at concentrations of 0.2 and 1.0 μm and culturing for 3 hours. The samples were processed in the same manner as used in example 2 and analyzed using a flow cytometer.
As shown in fig. 2, it was confirmed that intracellular transfer efficiency was increased according to the concentration of the LayN peptide in HEK293 cells, and as shown in fig. 3, it was also confirmed that intracellular transfer efficiency was increased according to the concentration of the LayN peptide in JurkatT cells.
Example 4. confirmation of protein delivery to the interior of the cell using β -Galactosidase (Beta Galactosidase) analysis.
Production of a LayN-. beta. -galactosidase (LayN-. beta. -Gal) fusion protein
In order to produce a fusion protein in which a cell-penetrating peptide, i.e., LayN (seq id No. 1), was bound to a β -galactosidase protein as a mediator, a genetic sequence for expressing the LayN peptide and the β -galactosidase protein was introduced into pET28c vector, thereby producing a vector for expressing the LayN- β -Gal fusion protein. In order to facilitate purification of the fusion protein, the fusion protein was produced in a form in which the His-Taq (H6) sequence was introduced into the N-terminus, and the structure of the finally produced fusion protein was in a form of H6-LayN-. beta. -Gal from the N-terminus, as shown in FIG. 4.
A vector for expressing LayN-. beta. -Gal was introduced into BL21(DE3) pLysS Escherichia coli cell line to produce a transformed cell line, and then the protein was expressed. A simplified method for protein production and purification is as follows.
The transformed BL21(DE3) pLysS E.coli cells were inoculated in 5ml LB (Luria-Bertani) medium containing the antibiotic Kanamycin (Kanamycin) and cultured once at 37 ℃ and 250 rpm. After culturing at a wavelength of 600nm and an Optical Density (Optical Density, o.d.) of about 0.6 using an ultraviolet-visible light spectroscope, culturing was performed under the same conditions in 500ml of lb medium containing Kanamycin (Kanamycin), an antibiotic. After culturing at a wavelength of 600nm and about 0.7 to 0.8O.D., 1mM IPTG (Isopropyl β -D-1-thiogalactopyranoside, Isopropyl β -D-1-thiogalactoside) was added to induce overexpression of the target protein. After four hours of culture, the cells were harvested by a centrifuge, and then a Lysis solution (Lysis buffer: 6Murea, 20mM Tris-HCl, pH8.0, 500mM NaCl; Lysis buffer: 6M urea, 20mM Tris-HCl, pH8.0, 500mM NaCl) was added thereto to disrupt the cells using an ultrasonic pulverizer. Only the protein-containing solution fraction was obtained by a centrifuge, and then a purification process was performed.
After obtaining a target protein from a protein-containing solution using a HisTrap affinity column (column), the protein-containing solution was stored at-80 ℃ by replacing the protein-containing solution with a protein storage solution containing 10% glycerol (glycerol) using a Sephadex G-25 column (column).
Introduction of the LayN-beta-Gal fusion protein into cells
Human embryonic kidney cell line HEK293 cells were split to 3X10 cells in polylysine type D (Poly-D-Lysine) treated 12-well (well) dishes4Thereafter, the samples were treated in a non-treated group, a control group having a concentration of 1.5. mu.M, and a LayN- β -Gal protein-treated group having a concentration of 0.1, 0.5, 1.0, and 1.5. mu.M, and an experiment was performed after the samples were washed with a phosphate buffer solution two times after culturing in a DMEM medium supplemented with 10% fetal bovine serum and an antibiotic Penicillin/streptomycin (Penicillin/streptomycin) at 37 ℃ for 16 hours, and the control group used a fusion protein (A10- β -Gal) prepared by replacing the LayN peptide for intracellular delivery of the target protein with the A10 peptide (SEQ ID NO: 7).
After the sample was treated, the cells were fixed for 10 minutes by three-hour incubation, three-time washing with phosphate buffer, and then adding a fixative (2% formaldehyde, 0.2% glutaraldehyde, PBS). After 10 minutes the fixative was removed and washed with phosphate buffer, X-gal staining solution was added and reacted for 30 minutes at 37 ℃. After 3 washes with phosphate buffer after staining, the degree of staining was confirmed by an optical microscope and a CCD camera (charge-coupled-device camera).
As shown in fig. 4, the intracellular introduction of the target protein by the LayN peptide showed a tendency to increase with concentration, and the intracellular delivery of the target protein into cells at a high concentration of 1.5 μm was hardly confirmed in the protein using the control peptide a10 without using the cell-penetrating peptide. From these results, it was confirmed that the LayN peptide allows intracellular delivery of a transporter (cargo) protein.
Example 5 intracellular delivery efficiency of siRNAs to LayN dimers
In order to confirm the intracellular delivery efficiency of nucleic acids via the LayN peptide, dimer LayN-D was synthesized as a continuous sequence with the LayN peptide as a monomer. The negative charge N of siRNA as nucleic acid is combined with the positive charge P of the LayN-D peptide by electrostatic attraction, and then delivered to the inside of the cell. (fig. 5A) the mixing ratio is mixed in such a manner that the ratio N/P of the negative charge and the positive charge is different, and is used as a transfer sample.
The binding of the LayN-D peptide to the siRNA was confirmed on agarose gel (agarose gel).
First, whether the LayN-D peptide was bound to siRNA was confirmed, and whether the peptide was bound or not was confirmed on agarose gel according to the mixing ratio as shown in FIG. 5-B in order to confirm the desired binding ratio. The amount of siRNA was fixed so as to impart variation in N/P ratio between siRNA and LayN-D peptide, and mixed in an amount of LayN-D according to the N/P ratio at 0.5, 1.0, 2.0, 5.0 times the concentration of siRNA, so that the reaction was carried out at room temperature for 20 minutes, followed by electrophoresis on 1.5% agarose gel, followed by staining with EtBr (ethidium bromide) and confirmation. As a result of the experiment, the N/P ratio showed the most desirable binding efficiency at 1.0 to 2.0.
Confirmation of expression-blocking potency by LacZ siRNA delivery in LacZ genetically transformed cells
The vector including the lacZ genetic gene expressing beta-galactosidase is subjected to shape-to-mass conversion in a human embryonic kidney cell line HEK293 cell, so that the LacZ siRNA is delivered to the inside of the cell by utilizing the LayN-D peptide, and the siRNA delivery efficiency is confirmed by an experiment for blocking the expression of the beta-galactosidase.
Shape and quality conversion of human embryonic kidney cell line HEK293 cell by pcDNA3.1/LacZ vector
Human embryonic kidney cell line HEK293 cells were split to 3X10 in 24-well plates treated with polylysine type D (Poly-D-Lysine)4After each, the medium was incubated at 37 ℃ in 5% DMEM supplemented with 10% fetal bovine serum and the antibiotic Penicillin/streptomycin (Penicilin/streptomycin)The culture was carried out in an incubator with a carbon oxide concentration for 18 hours. After removing the medium, the medium was washed with phosphate buffer, and then cultured for an additional 6 hours in a medium to which no serum was added. The vector pcDNA3.1/LacZ vector for LacZ expression and the vector pcDNA3.1 for control (mock) were treated and introduced in cells using liposome 2000(Lipofectamine2000) (Life Technologies Co.) reagents, respectively, and cultured for 4 hours to perform shape-mass transformation.
Intracellular introduction of the complex of the LayN-D peptide and siRNA.
The cells were treated with the LayN-D peptide mixed with LacZ siRNA in several N/P ratios in HEK293 cells that had undergone shape-shifting. The treated samples were used as shown in fig. 5-C to D, and 1. shape conversion negative control group, 2.50nMsiRNA negative control group, 3. siRNA/Lipofectamine (Lipofectamine) positive control group, 4.N/P (siRNA/LayN-D) 1.0, and 5.N/P (siRNA/LayN-D) 0.5 sample group were prepared. Each sample was treated in cells to perform 3 hours of culture, and after exchanging the medium with DMEM medium supplemented with 10% fetal bovine serum, the cells were harvested after performing additional culture for 48 hours.
Other LacZ expression blocking potency in siRNA delivery using beta-galactosidase assay
After cell harvest, RIPA solution (50mM Tris-HCl, pH7.4, 1% Np-40, 0.5% Na-deoxyholate, 0.1% SDS, 150mM NaCl, 2mM EDTA, 50mM NaF, 1mM PMSF, 0.5. mu.g/ml Leuteptin, 0.5. mu.g/ml Aprotinin, 1mg/ml Pepstatin, 0.2mM Na3VO450mM Tris hydrochloride, pH7.4, 1% ethylphenylpolyethylene glycol, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 150mM sodium chloride, 2mM ethylenediaminetetraacetic acid, 50mM sodium fluoride, 1mM phenylmethylsulfonyl fluoride, 0.5. mu.g/ml leupeptin, 0.5. mu.g/ml aprotinin, 1mg/ml pepsin inhibitor, 0.2mM sodium vanadate) were dissolved in the cells, and after centrifugation, a supernatant containing the protein was obtained, and after treatment with the same amount of 1X β -gal staining solution, a reaction was carried out at 37 ℃ for 10 minutes, and the absorbance was measured at 590nm, thereby preparing a graph (FIGS. 5-C, D).
As shown in FIGS. 5-C, D, when siRNA and LayN-D peptide were treated at an N/P ratio of 1:2(0.5), the results were similar to or slightly better than the siRNA delivery efficiency by the positive control group Lipofectamine (Lipofectamine) treatment, and from these results, the intracellular nucleic acid delivery efficiency by LayN peptide could be confirmed.
Example 6 cytotoxicity of LayN peptides in various cell lines Using WST-1 reagents
In order to confirm the cytotoxicity of the LayN peptide itself, the cytotoxicity of the LayN peptide was confirmed in various human cell lines such as human cervical cancer cell line HeLa, human embryonic kidney cell line HEK293, human keratinocyte cell line HaCaT, and acute lymphoblastic leukemia cell line Jurkat T.
For 1 × 10 in 96-well culture flasks (flash)4Each cell of the individuals was cultured in DMEM and RPMI-1640 medium supplemented with 10% fetal bovine serum and the antibiotic Penicillin/streptomycin (Penicillin/streptomycin), respectively, in an incubator at 37 degrees Celsius and 5% carbon dioxide concentration for 16 hours. As shown in FIG. 6, after the cultivation, the LayN peptide was treated in a concentration range of 0.05 to 2.0 μm, and then the cultivation was carried out for 3 hours, and the absorbance was measured at 420 to 480nm using WST-1 reagent (Roche Co.) which is a reagent for confirming cell proliferation.
As shown in FIG. 6, it was confirmed that the cytotoxicity was about 10% at about 1.0 μm and about 20% at 1.5 μm in each cell line. Based on this result, the examples used in the present invention were treated with a LayN peptide concentration of 1.5 μm or less.
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Claims (4)
1. A cell membrane penetrating peptide consisting of a peptide sequence consisting of the amino acid sequence designated as sequence No. 1.
2. The cell-membrane-penetrating peptide according to claim 1, which is a monomer of the cell-membrane-penetrating peptide and is composed of one to two tandem polymers.
3. Use of the cell membrane-penetrating peptide according to claim 1 for preparing a biologically active substance carrier by linking nucleic acids of DNA, RNA, LNA and PNA, or proteins of growth factors, cytokines, transcription factors and enzymes, or carbohydrates and minerals at the N-terminus or C-terminus of the cell membrane-penetrating peptide by chemical covalent or non-covalent bonding, or by mixing by electrostatic attraction or physical bonding, thereby delivering the substance bound to the cell membrane-penetrating peptide to the inside of the cell.
4. The use according to claim 3, wherein the bioactive substance transporter is a composition of a pharmaceutical product, a cosmetic product, or a functional food that acts inside a cell.
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| KR1020140117677A KR101695792B1 (en) | 2014-09-04 | 2014-09-04 | Novel cell membrane penetrating peptides and uses thereof |
| PCT/KR2015/009015 WO2016036060A1 (en) | 2014-09-04 | 2015-08-27 | Novel cell membrane-penetrating peptide, and use thereof as bioactive substance carrier |
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| CN109998994B (en) * | 2018-02-13 | 2021-12-07 | 四川大学 | Flexible liposome containing medicine and preparation method thereof |
| CN112533640A (en) * | 2018-05-04 | 2021-03-19 | 双亥生命科学株式会社 | Targeting of tumor-associated macrophages of type M2 using melittin-based apoptosis-inducing peptides |
| WO2019245353A1 (en) * | 2018-06-22 | 2019-12-26 | 재단법인 대구경북과학기술원 | Cytokine fusion polypeptide and cytokine library comprising same |
| CN109553659B (en) * | 2018-11-26 | 2022-06-21 | 上海华新生物高技术有限公司 | Cell penetrating peptide and transdermal interferon |
| KR20200110576A (en) * | 2019-03-15 | 2020-09-24 | 재단법인대구경북과학기술원 | Immune cells based on cytokines and immunotherapy uses thereof |
| CN110511273B (en) * | 2019-07-12 | 2021-06-01 | 福州市皮肤病防治院 | Preparation method and application of cell-penetrating polypeptide |
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| JP4573143B2 (en) * | 2008-01-25 | 2010-11-04 | 東亞合成株式会社 | Artificial peptides and their use |
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| US5672584A (en) * | 1995-04-25 | 1997-09-30 | The University Of Kansas | Cyclic prodrugs of peptides and peptide nucleic acids having improved metabolic stability and cell membrane permeability |
| KR100504305B1 (en) * | 1999-03-23 | 2005-07-28 | 제넨테크, 인크. | Secreted and Transmembrane polypeptides and Nucleic Acids Encoding the Same |
| US7579318B2 (en) * | 2005-12-06 | 2009-08-25 | Centre De La Recherche De La Scientifique | Cell penetrating peptides for intracellular delivery of molecules |
| JP5364574B2 (en) | 2006-05-05 | 2013-12-11 | モレキュラー、トランスファー、インコーポレイテッド | New reagents for transfection of eukaryotic cells |
| KR101169030B1 (en) * | 2009-01-21 | 2012-07-26 | 애니젠 주식회사 | Novel Cell Penetrating Domain and Intracellular Delivery System Comprising the Same |
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| KR20160028754A (en) | 2016-03-14 |
| CN106795205A (en) | 2017-05-31 |
| WO2016036060A1 (en) | 2016-03-10 |
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