KR101476953B1 - A novel hepsin-targeted peptide for enhancing cell permeability and its use - Google Patents
A novel hepsin-targeted peptide for enhancing cell permeability and its use Download PDFInfo
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- KR101476953B1 KR101476953B1 KR20140021749A KR20140021749A KR101476953B1 KR 101476953 B1 KR101476953 B1 KR 101476953B1 KR 20140021749 A KR20140021749 A KR 20140021749A KR 20140021749 A KR20140021749 A KR 20140021749A KR 101476953 B1 KR101476953 B1 KR 101476953B1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
Abstract
Description
The present invention relates to a hepsin target peptide having an enhanced cell permeability comprising the amino acid sequence of SEQ ID NO: 1, a nanoliposome bound to the surface of the peptide, a bioimaging system comprising the nanoliposome, a cell comprising the nanoliposome as an active ingredient Or tissues and a process for producing the nanoliposome.
The problem with the prior art in chemotherapy for cancer therapy is poor selectivity for target cells or organs of chemotherapeutic agents. In order to solve such a problem, target drug delivery technology is attracting attention in the field of diagnosis and treatment of cancer, and strategies for increasing the selective toxicity of the drug to the targeted cells or reducing the toxicity to the normal cells are being studied. Selectivity can be obtained through active targeting and passive targeting. Particularly active targets are accompanied by ligand-receptor or antigen-antibody interaction at the target cell site. Examples of ligands that can be used for active targeting include folate using folate receptor interactions with overexpressed cancer cells, ErbB2 used to target breast cancer cells, targets for prostate cancer cells prostate-specific membrane antigen (PSMA).
On the other hand, cell penetrating peptides are useful tools for intracellular delivery of drugs. Cell permeable peptides such as trans-activating transcriptional activator (Tat), antennapedia (Ant), polyarginines, And deliver the carrier and the like efficiently into the cell. The cell permeation mechanism of polyarginine in these peptides seems to be that the side chain of arginine initially binds to the phosphate group of the phospholipid bilayer and the pore is formed due to the strong collapse in the bilayer membrane. Polyarginine has also been widely used for the delivery of various drugs and carriers, but has faced limitations in terms of the lack of selectivity for cell permeable peptides.
In addition, the cell permeable peptide can transfer a macromolecule or cargo into cells, but this effect also works on normal cells. In other words, although the cell-permeable anticancer drug molecule should be delivered only to cancer cells, it may affect general cells and cause side effects. In addition, its use is limited because it may cause toxicity at certain concentrations.
Targeting molecules used for drug targeting can overcome the above problems, but it is difficult to enter into cells due to the absence of endocytosis or the large molecular weight of molecules or cargo to be delivered into cells. Therefore, a chemotherapeutic agent or a peptide drug acting on most intracellular signaling can not exhibit its efficacy despite reaching a target molecule. To solve this problem, a mechanism for intracellular entry upon drug targeting is required.
Thus, studies are being conducted to link cell-targeting peptides to cell-permeable peptides. The result of this synthesis is called a cell penetrating homing peptide and has complemented each other's deficiencies in drug delivery. Such a cell permeable target peptide can be applied to the delivery of a nucleic acid-based drug, a large protein molecule drug, or a cell-impermeable chemotherapeutic agent, which is difficult to deliver in a cell, to cancer cells. In addition, among the development of drug delivery systems, liposomal nanocarriers are the most widely used delivery vehicles for the delivery of soluble or poorly soluble drugs or therapeutic and diagnostic reagents. Liposomes can be applied through PEGylation, binding of monoclonal antibodies, or binding of lipid derivatives, proteins, peptides, and the like. In particular, liposomes can be targeted to specific cancer cells or effectively delivered into cells through techniques for modifying cell surfaces with peptides that exhibit specific functions.
However, the method of modifying a drug molecule through such a cell permeable peptide or a targeting molecule is inevitably accompanied by modification of the drug molecule itself. However, this can cause problems such as unexpected toxicity, side effects, and reduced efficacy. In addition, it is known that the affinity of two or more molecules is significantly increased in the case of a targeting molecule, as compared to the affinity for a target represented by a single molecule.
Therefore, it is necessary to use nanocarrier as a platform in which such functional peptides or target molecules can be integrated into a single entity.
Thus, the present inventors have completed the present invention by synthesizing a novel peptide exhibiting specificity for hepcidin-expressing cells and simultaneously exhibiting cell permeability.
Accordingly, an object of the present invention is to provide a hepatocyte target peptide having enhanced cell permeability, which is composed of the amino acid sequence of SEQ ID NO: 1.
It is another object of the present invention to provide a nanoliposome in which the peptide is bound to the surface.
It is another object of the present invention to provide a bioimaging system including the nanoliposome.
It is another object of the present invention to provide a composition for drug delivery in cells or tissues containing the nanoliposome as an active ingredient.
Still another object of the present invention is to provide a method for preparing a mixture of a) preparing a mixed solution of phosphatidylcholine (PC) and Tween 80; b) adding distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-mal) to the solution at a ratio of 0.2 to 4.0% of the total lipid concentration to form a liposome; And c) adding the peptide to a solution in which the liposome is formed to bind the peptide to the surface of the liposome through a thiol-maleimide reaction.
However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.
In order to accomplish the object of the present invention as described above, the present invention provides a hepatocyte target peptide having an enhanced cell permeability comprising the amino acid sequence of SEQ ID NO: 1.
The present invention also provides a nanoliposome wherein the peptide is bound to the surface.
In one embodiment of the present invention, the nanoliposome may have a size of 100 nm to 200 nm.
In another embodiment of the present invention, the liposome may be encapsulated with a fluorescent substance therein.
In another embodiment of the present invention, the fluorescent material comprises a fluorescent group selected from the group consisting of fluorescence isothiocyanate (FITC), 4 ', 6-diamidino-2-phenylindole (DIPI), Green Fluorescent Protein (GFP), Cy3, Cy5, Rhodamine and Texas red Lt; / RTI >
The present invention also provides a bioimaging system including the nanoliposome.
In another embodiment of the present invention, the system may be one that measures intracellular fluorescence.
The present invention also provides a composition for drug delivery in cells or tissues containing the nanoliposome as an active ingredient.
In one embodiment of the present invention, the composition may be a drug-encapsulated liposome.
In another embodiment of the present invention, the drug may be an anti-cancer agent.
In another embodiment of the present invention, the anticancer agent may be a cell-impermeable chemotherapeutic agent.
Also, the present invention provides a method for preparing a pharmaceutical composition comprising the steps of: a) preparing a mixed solution of phosphatidylcholine (PC) and Tween 80;
b) adding distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-mal) to the solution at a ratio of 0.2 to 4.0% of the total lipid concentration to form a liposome; And
c) adding a peptide comprising the amino acid sequence of SEQ ID NO: 1 to a solution in which the liposome is formed to bind the peptide to the surface of the liposome through a thiol-maleimide reaction.
In one embodiment of the present invention, the step a) may be carried out in chloroform, methanol or a mixed solvent thereof.
In another embodiment of the present invention, the step a) may be performed by mixing phosphatidylcholine (PC) and Tween 80 at a molar ratio of 9.5: 0.5 to 8: 2.
In another embodiment of the present invention, the step b) comprises the steps of removing the organic solvent with a vacuum rotary evaporator after addition of DSPE-PEG2000-mal and hydrating the lipid layer with distilled water or PBS (phosphate buffer saline) to form a liposome Step.
In another embodiment of the present invention, the step c) may be carried out at room temperature for 9 to 16 hours.
The present invention also provides a method for diagnosing cancer comprising administering the peptide to a subject in need thereof.
In one embodiment of the present invention, the peptide may be linked to a fluorescent substance.
In another embodiment of the present invention, the fluorescent material may be Fluorescence isothiocyanate (FITC).
In another embodiment of the present invention, the peptide may be administered in a form bound to the liposome surface.
In another embodiment of the present invention, the administration may be by oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, intraarterial injection or subcutaneous injection.
In another embodiment of the present invention, the subject may be a mammal, including a human.
In another embodiment of the present invention, the cancer may be prostate cancer, liver cancer, renal cancer, breast cancer, or ovarian cancer.
The novel peptide according to the present invention can be used for diagnosis and treatment of prostate cancer by substituting prostate specific antigen (PSA). Since the expression of heptin is not limited to prostate cancer but also occurs in breast cancer and ovarian cancer cells And can be applied as various cancer targeting molecules.
In addition, the cell permeability of the novel peptide according to the present invention has an advantage that it can be utilized as a means for delivering a cell-impermeable chemotherapeutic agent, a large protein drug, a drug carrier, etc. that require intracellular delivery.
Figure 1 shows a schematic diagram in which RIPL-Lipo is uptake into hepsin-expressing cells.
FIG. 2 is a graph showing the cell selectivity of RIPL peptides according to the present invention for RIPL-FITC and FITC on hepsin-expressing cells and heptin-non-expressing cells (**, p <0.005 versus paired group; *, p < 0.0005 versus paired).
FIG. 3 is a graph showing the degree of enhancement of cell permeability due to peptide by culturing LNCaP cells with a mixture of cell permeable peptide (R8), RIPL peptide, and target peptide (IPL) and FITC (*, p <0.05 versus paired group; **, p <0.005 versus paired; ***, p <0.0005 versus paired).
4 is a graph showing the degree of enhancement of cell permeability of RIPL-Lipo.
FIG. 5 is a graph showing cell permeability to LNCaP cells while increasing the number of peptides bound to the surface of RIPL-Lipo.
FIG. 6 is a histogram showing the cell permeability of RIPL-Lipo measured by flow cytometry on LNCaP, SK-OV-3, MCF-7, DU145, PC3 and HaCaT cells. Blue indicates RIPL-Lipo, green indicates CL, and red indicates FITC-Dextran.
FIG. 7 is a graph showing a relative comparison of MFI values on histograms of RIPL-Lipo cell permeability measured by flow cytometry on LNCaP, SK-OV-3, MCF-7, DU145, PC3 and HaCaT cells to be.
FIG. 8 is an image of fluorescence images of LNCaP, SK-OV-3, MCF-7, DU145, PC3 and HaCaT at 400 magnifications using a fluorescence microscope. Green fluorescence represents RIPL-Lipo with FITC-dextran encapsulated.
FIG. 9 is an image of a fluorescence image observed through a confocal microscope after culturing LNCaP cells and RIPL-Lipo for 10 minutes, (B) 30 minutes, (C) 1 hour, and (D) 2 hours, respectively. Green fluorescence represents RIPL-Lipo with FITC-dextran encapsulated.
Hepsin is an extracellular protease expressed at high concentrations in many tumor cells such as liver cancer, breast cancer, kidney cancer, prostate cancer and ovarian cancer. Especially, the expression level of Hepsin is high enough to clearly distinguish between prostate cancer cells and benign prostatic hyperplasia There are many differences.
Thus, the present inventors have completed the present invention by synthesizing a novel peptide which is highly effective in cell membrane permeability and capable of drug delivery in a cell, as a peptide targeting hepsin-expressing cells.
That is, the novel peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 1 according to the present invention functions as shown in the schematic diagram of FIG. 1 to enhance intracellular permeability. In this case, the peptides bound to the liposome selectively bind to the hepatocyte-expressing cells, and the polyarginine portion of the RIPL sequence binds to the protease. . Liposomes uptake through endocytosis are transiently trapped in the endosome, but because of the action of activated polyarginine, they enter the cytosol, which may increase the permeability by degrading the lysosome.
Accordingly, the present invention provides a therapeutic agent for hepatocyte, comprising a hepsin target peptide having an enhanced cell permeability, an nanoliposome bound to the surface of the peptide, a bioimaging system comprising the nanoliposome, and an nanoliposome as an active ingredient, Or a pharmaceutically acceptable salt thereof, to a cell or tissue.
The present invention also provides a process for preparing a nanoliposome comprising the steps of:
a) preparing a mixed solution of phosphatidylcholine (PC) and
b) adding distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-mal) to the solution at a ratio of 0.2 to 4.0% of the total lipid concentration to form a liposome; And
c) adding the peptide to a solution in which the liposome is formed to bind the peptide to the surface of the liposome through a thiol-maleimide reaction.
At this time, if the nanoliposome according to the present invention can be prepared, the above-mentioned preparation method can suitably change the order and / or configuration of steps and is not limited to the above step.
Preferably, the step a) may be performed by mixing phosphatidylcholine (PC) and
The step b) is a step of forming a liposome having a maleimide group bonded to its surface so that it can react with the thiol group of cysteine in the peptide. Preferably, the DSPE-PEG 2000-mal having a maleimide group is added and then the organic solvent is removed , Hydration of the lipid layer with distilled water or PBS (phosphate buffer saline) may be performed to form a lipid membrane bound to the maleimide group on the surface.
(SEQ ID NO: 1) according to the present invention may be added to the liposome surface so that the thiol-maleimide reaction can be performed. Preferably, the peptide bond is added to the surface of the liposome at 9 To 16 hours.
In addition, the peptide comprising the amino acid sequence of SEQ ID NO: 1 according to the present invention can be used for diagnosis of cancer. For this purpose, the peptide can be provided as a system in which the peptide is bound to the surface of the nanoliposome so as to be embodied in cells. In order to make it easy to observe the operation of the system under a microscope, a fluorescent substance may be enclosed in the liposome.
From the above, it can be seen that the hepatocyte peptidase having enhanced cell permeability according to the present invention or the liposome bound to the surface of the peptide can be utilized as a living body imaging system, and the system can be used as a living body cell using a flow cell analyzer or confocal microscope Lt; RTI ID = 0.0 > fluorescence. ≪ / RTI >
The size of the nanoliposome according to the present invention can be appropriately adjusted as long as it can be injected into the body, but it can be preferably formed into a spherical shape having a diameter of 100 nm to 200 nm.
In addition, in the present invention, any fluorescent or lipophilic substance which can be used in vivo and which can be bound to (binding to) a peptide or a nano-liposome can be used. Preferably, fluorescence isothiocyanate Fluorescence isothiocyanate (FITC), 4'-6-diamidino-2-phenylindole (DIPI), fluorescein isothiocyanate (FITC) A fluorescent material selected from the group consisting of Green Fluorescent Protein (GFP), Cy3, Cy5, Rhodamine, and Texas red may be used, but it is not limited thereto and can be appropriately selected and used by those skilled in the art.
In one embodiment of the present invention, the peptide and the nanoliposome bound to the surface thereof exhibit good hepcine-expressing cell (cancer cell) selectivity and cell permeability through flow cytometry and fluorescence measurement (Example 2 and Example 5).
Accordingly, the peptide of the present invention or the nanoliposome bound to the surface of the peptide exhibiting excellent cell selectivity and cell permeability can be obtained by administering the liposome-nucleic acid fluorescent nano-fusion substance to a subject in need of cancer diagnosis and measuring intracellular fluorescence Thereby providing a diagnostic method for the disease.
The diagnosis can be made by administering a peptide linked to a fluorescent substance to an individual, or by injecting a nanoliposome encapsulated in the fluorescent substance or an externally bound substance into the individual, and the administration can be carried out by oral administration, intravenous injection, intraperitoneal injection, , An arterial injection or a subcutaneous injection method. The term "subject" refers to a subject in need of diagnosis of cancer. More specifically, the term "subject" refers to a subject in need of diagnosis of cancer, and more specifically, a mammal such as a primate, a mouse, a rat, a dog, a cat, .
In addition, the diagnostic method can be used for all cancers having cancer cells expressing heptin, but can be preferably used for diagnosis of prostate cancer, liver cancer, kidney cancer, breast cancer or ovarian cancer.
Furthermore, the nanoliposome according to the present invention can be manufactured by sealing various materials therein because the inside is empty. There is no limitation on the substance that can be enclosed in the inside, but it is preferable to enclose the drug, and more preferably, it can be utilized for drug delivery in cells or tissues by enclosing an anticancer agent such as a cell-impermeable chemotherapeutic agent.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
RIPL Of peptide synthesis
The RIPL peptide (IPLVVPLRRRRRRRC) of SEQ ID NO: 1 was prepared by the following method. Peptides were synthesized by Fmoc SPPS (9-fluorenylmethyloxycarbonyl solid phase peptide synthesis) and purified by reverse phase HPLC using Vydac Everest C18 column. The amino acid units were bound to each other from the C terminus using an automatic peptide synthesizer (ASP48S, Peptron Inc.). The first amino acid with a resin attached to the C terminus of the peptide was S-trityl-L-cysteine-2-chlorotrityl resin was used. All amino acids used in peptide synthesis were protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl (t-Bu) groups and N-terminal protected with Fmoc. All these residues are removed under acidic conditions. (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) / hydroxyl-benzotriazole (HOBt) / N-methylmorpholine (NMM) as a coupling reagent. In preparative HPLC purification, elution was carried out using water-acetonitrile (acetonitrile 30-40% v / v) gradient elution with 0.1% trifluoroacetic acid. The molecular weights of the purified peptides were determined by LC / MS (Agilent HP1100 series) and lyophilized.
RIPL Of peptide Hepsin Selectivity and cell permeability assessment
RIPL peptide expression and cell permeability were evaluated by flow cytometry using fluorescence isothiocyanate (FITC) and FITC-dextran (FITC-dextran).
For evaluation of cell selectivity, hepsin-expressing cells LNCaP, SK-OV-3, MCF-7 and heptin non-expressing cells DU145, PC3 and HaCaT cells were seeded at a density of 1 × 10 6 per 6-well plate well. After the cell density reached 70-80%, the cells were incubated with FITC (RIPL-FITC) conjugated with 1 uM RIPL peptide. After 2 hours, cells were washed 3 times with phosphate buffered saline (PBS) and flow cytometry was used to measure MFI as an index of cell selectivity up to 10,000 events per histogram. As a result, as shown in FIG. 2, FITC-dextran showed a low MFI value for all cells, whereas RIPL-FITC showed a significantly higher intracellular transport than FITC-dextran, , Especially for LNCaP cells, 8.3-fold higher than FITC-dextran.
From the above, the selectivity of RIPL-FITC for hepcin was confirmed, and the enhancement of FITC-dextran cell permeability due to RIPL peptide was examined. RIPL, R8 (RRRRRRRC), and IPL (IPLVVPLC) of 28 ug / ml FITC-dextran (control), 3 uM or 6 uM with HepC2 expressing cells LNCaP, SK-OV-3 or MCF- And MFI was measured as described above. As a result, as shown in Fig. 3, intracellular migration of FITC-dextran due to R8 and RIPL peptides was increased, but IPL was the same as that of the control (CTL).
Preparation of surface-bound nanoliposomes with RIPL peptide
RIPL peptide-bound liposomes (RIPL-Lipo) were prepared by binding RIPL peptide to liposome vesicle surface via thiol maleimide reaction. First, phosphatidylcholine (PC) and
RIPL The peptide surface Combined Nanoliposome Physicochemical characterization
<4-1> Nanoliposome On the surface Combined RIPL Of peptide Quantitative analysis
The binding ratio of the RIPL peptide bound to the external maleimide group and the maleimide-substituted liposome was indirectly calculated by quantifying the amount of cysteine left without reacting in Ellman's reaction. To block unreacted maleimide after RIPL peptide binding, a 3-fold molar ratio of cysteine hydrochloride anhydrous was added. 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) was added to quantify the remaining cysteine. The binding of cystein-TNB (5-thio-2-nitrobenzoic acid) was analyzed by HPLC because the same amount of free TNB was released. In the HPLC analysis, the mobile phase was a mixture of methanol and 10 mM ammonium formate (5:95 (v / v)) at a flow rate of 1 ml / min. The column was a C18 column of shiseido and the absorbance wavelength was measured at 412 nm. In addition, since the liposome particles having a size of 160 nm are known to be composed of about 223,000 phospholipid molecules, the number of peptides bound to the surface of the nanoliposome is derived.
<4-2> Nanoliposome Particle size distribution and surface charge analysis
The particle size distribution and the surface charge of the nanoliposome prepared in the present invention were measured by a dynamic light scattering method using a Zetasizer Nano-ZS (Malvern Instrument, Worcestershire, UK). The physico-chemical properties of liposomes in which FITC-dextran is encapsulated and RIPL peptide is bound to the surface are shown in Table 1 below.
RIPL The peptide surface Combined Nanoliposome Cell permeability assessment
<5-1> Flow cytometry
In order to evaluate the enhancement of cell permeability of RIPL peptide surface-bound nanoliposomes, FITC, RIPL-FITC (cultured by mixing RIPL peptide and FITC as in Example 2), RIPL conjugated FITC (sharing RITC peptide with FITC And RIPL-Lipo were cultured together with LNCaP cells, respectively, and the MFI was measured in the same manner as in Example 2 above. As a result, as shown in Fig. 4, it was confirmed that RIPL-Lipo showed the best cell permeability.
For the selection of optimized RIPL-Lipo, cell permeability of four types of LNCaP cells regulated the number of RIPL peptides was evaluated. As a result, as shown in Fig. 5, the cell permeability to LNCaP cells was found to increase depending on the number of peptides. However, the MFI increased proportionally until the number of peptides reached 2300, and did not increase further.
In addition, MFI values were measured by flow cytometry of FITC-dextran fluorescence in which the selected permeability of RIPL-Lipo and control (CL) was sealed. LNCaP, SK-OV-3, MCF-7, DU145, PC3 and HaCaT cells were treated as in Example 2 above. However, instead of FITC-dextran, liposome corresponding to 28 ug / ml of FITC-dextran (100 ug / ml as lipid concentration) was added. As a result, as shown in FIGS. 6 and 7, RIPL-Lipo showed greater cell selectivity for the hensin-expressing cells LNCaP, SK-OV-3 and MCF-7 cells compared to the control group, , Whereas those for DU145, PC3, and HaCaT were slightly increased compared to the control group.
<5-2> Fluorescence microscopy observation
For microscopic observation, LNCaP, SK-OV-3, MCF-7, DU145, PC3 and HaCaT cells were visualized using a 400x fluorescence microscope. In addition, LNCaP cells were fixed with 3.7% formaldehyde and then focused on the Z axis through a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Jene, Germany). As shown in FIG. 8, fluorescence images of RIPL-Lipo were measured on the four cells. As a result, strong fluorescence was shown only for LNCaP, which is a hepsin-expressing cell. There was some fluorescence in the remaining cells, but it was very weak compared to LNCaP.
As shown in FIG. 9, fluorescence intensities of LNCaP cells were increased with time, as shown in FIG. 9. As a result, fluorescence intensities were increased with time, It can be observed that they are uniformly distributed.
It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> A NOVEL HEPSIN-TARGETED PEPTIDE FOR ENHANCING CELL PERMEABILITY AND ITS USE <130> PB13-11697 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> RIPL peptide <400> 1 Ile Pro Leu Val Val Pro Leu Arg Arg Arg Arg Arg Arg Arg Arg Cys 1 5 10 15
Claims (13)
Wherein the nanoliposome has a size of 100 nm to 200 nm.
Wherein the liposome is encapsulated with a fluorescent substance therein.
Wherein the fluorescent material is selected from the group consisting of fluorescence isothiocyanate (FITC), 4 ', 6-diamidino-2-phenylindole (DIPI), Green Fluorescent Protein (GFP), Cy3, Cy5, Rhodamine and Texas red. .
RTI ID = 0.0 > 1, < / RTI > wherein said system measures intracellular fluorescence.
b) adding distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-mal) to the solution at a ratio of 0.2 to 4.0% of the total lipid concentration to form a liposome; And
c) adding the peptide of claim 1 to the liposome-forming solution to bind the peptide to the surface of the liposome through a thiol-maleimide reaction.
Wherein the step a) is carried out in chloroform, methanol or a mixed solvent thereof.
Wherein the step a) comprises mixing phosphatidylcholine (PC) and Tween 80 at a molar ratio of 9.5: 0.5 to 8: 2 to prepare a solution.
Wherein the step b) comprises adding DSPE-PEG 2000-mal to remove the organic solvent, and hydrating the lipid layer with distilled water or PBS (phosphate buffer saline) to form a liposome.
Wherein the step c) is carried out at room temperature for 9 to 16 hours.
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| WO2022240055A1 (en) * | 2021-05-14 | 2022-11-17 | 주식회사 레메디 | Cargo molecule tranduction domain rmad1, variant thereof, recombinant cargo molecule, and method for tranducing cargo molecule using same |
| WO2022255708A1 (en) * | 2021-06-03 | 2022-12-08 | 주식회사 레메디 | Cargo molecule tranduction domain rmmr1, variant thereof, recombinant cargo molecule comprising same, and method for tranducing cargo molecule using same |
| WO2023128501A1 (en) * | 2021-12-27 | 2023-07-06 | 아주대학교산학협력단 | Novel cell-permeable peptide and use thereof |
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| WO2022240055A1 (en) * | 2021-05-14 | 2022-11-17 | 주식회사 레메디 | Cargo molecule tranduction domain rmad1, variant thereof, recombinant cargo molecule, and method for tranducing cargo molecule using same |
| WO2022255708A1 (en) * | 2021-06-03 | 2022-12-08 | 주식회사 레메디 | Cargo molecule tranduction domain rmmr1, variant thereof, recombinant cargo molecule comprising same, and method for tranducing cargo molecule using same |
| WO2023128501A1 (en) * | 2021-12-27 | 2023-07-06 | 아주대학교산학협력단 | Novel cell-permeable peptide and use thereof |
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