KR102113101B1 - A thermoresponsive peptide structure and uses thereof - Google Patents

Abstract

The present invention relates to a peptide structure that responds to a specific temperature, and provides a peptide structure having a specific structure.

Description

A temperature sensitive peptide structure and use thereof {A THERMORESPONSIVE PEPTIDE STRUCTURE AND USES THEREOF}

The present invention relates to a peptide structure forming a self-assembled structure according to temperature and various uses thereof.

Peptide self-assembly has recently been spotlighted for its suitability and various applications in medicine and life.

It is suitable for living body to use peptide, which is a substance existing in the body, so it has no unnecessary toxicity and can be decomposed naturally in the body over time to minimize side effects.

The various structures made by self-assembly make its function and application wide. Self-assembled structures have been studied in various directions, such as drug delivery systems that deliver drugs to specific cells or drugs by modifying the structure of proteins.

Self-assembly means that a molecule forms a specific structure or a large unit by gathering molecules due to attraction forces and repulsive forces between molecules.

Various amino acid chains constituting a protein or peptide have various properties such as hydrophobicity, hydrophilicity, positive charge, negative charge and aromaticity, and thus form a specific structure according to the sequence of the chain.

Amino acid chains can form secondary structures such as α-helices, β-sheets, and micelles, vesicles, and nanofibers through secondary bonds such as hydrophobic bonds, hydrogen bonds, electrostatic attraction, and pi-pie bonds. Various structures such as hydrogels can be formed (Korean Patent KR 10-1861110).

Since the force acting on the self-assembly is mainly indirect, such as hydrophobic, electrostatic attraction, or hydrogen bonding, not covalent bonding, it is possible to change the self-assembled structure with external stimuli that interfere or interfere with it.

Recently, studies on self-assembly of peptides that respond to various environments and enzymes in cells have been conducted.

Studies to induce intramolecular bond breakage by enzymes or to induce changes due to pH have been actively conducted, but studies using temperature changes in the body are still insufficient.

The inventors have developed a self-assembled peptide construct that self-assembles in response to a specific temperature.

In particular, the method of using the conventional drug delivery method, the polymeric ELP (elastin-like polypepetide) is poor in stability and storage, the manufacturing process is complicated, and it is possible to cause an inflammatory reaction, thus making it less useful.

However, the self-assembled structure of the present invention can be easily introduced into a cell, can easily control temperature sensitivity, and has a high degree of freedom in peptide design, so its potential utilization is very high.

The present invention is to solve the problems of the above-described prior art, it is an object to provide a peptide structure capable of realizing a specific effect by changing the structure according to the in vivo conditions.

According to an embodiment of the present invention, a self-assembled peptide structure represented by the following structural formula 1 is provided.

[Structural Formula 1]

HA _n

In the above formula, H is composed of one or more hydrophobic amino acids, A is VPGXG (SEQ ID NO: 1), X is any amino acid except proline, and n is 1 to 4.

In one embodiment, the H may include one or more selected from the group consisting of tryptophan, phenylalanine, isoleucine, leucine, proline, methionine, valine, and alanine.

In one embodiment, the H may be selected from the group consisting of WFF, FFF, YFF, LFF, VFF and AFF.

In one embodiment, A is VPGVG (SEQ ID NO: 2), and n may be 4.

In one embodiment, the peptide structure may further include a target-directed peptide.

In one embodiment, the target-directed peptide is one or more from the group consisting of RGD peptide, α-helix peptide, mitochondrial target peptide, Fc receptor binding peptide and beta endorphin receptor ligand Can be selected.

In one embodiment, the structure of the peptide may be changed according to temperature.

In one embodiment, the peptide structure may be a linear (linear-type) or a rear-type (lariat-type).

In one embodiment, the retarded structure may be formed by a target-directed peptide connecting the N-terminus and C-terminus of A.

In one embodiment, the peptide structure may carry a drug through a self-assembled structure.

In one embodiment, the drug may be a low molecular weight drug, a gene drug, a protein drug or a mixture thereof.

According to another aspect of the present invention, a drug delivery composition comprising the peptide construct is provided.

According to the present invention, the peptide construct is self-assembled at a specific temperature, and thus can be utilized as a target-specific drug delivery system in consideration of target characteristics.

It should be understood that the effects of the present invention are not limited to the above-described effects, and include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the invention.

FIG. 1A shows the TD-Abs measurement according to the number of phenylalanine bound to MELPs, and FIGS. 1B and 1C show AFM images (b) and thermal-ramp CD values (c) of FFF-ELPs.
2 shows the hydrophobicity and thermal sensitivity of NAA-ELPs through RP-HPLC analysis (a) and TD-Abs measurement (b and c).
3A shows a schematic diagram of NAA-ELP-RGDs, and FIG. 3B shows NAA-ELP-RGDs thermal sensitivity and self-combination through TD-Abs measurement (b), CD spectrum (c), and AFM image (d). It was observed.
4A and 4B are observations of decomposition through cooling of NAA-ELP-RGDs through TD-Abs measurement, FIG. 4C shows optical density of FFF-ELP-RGDs thermal coacervate, and FIG. 4D is FFF- It shows the rate of temperature dependent degradation of ELP-RGDs.
5A shows a schematic diagram of NAA-ELP-EAKs, and FIGS. 5B and 5C show temperature sensitivity of NAA-ELP-EAKs through DLS (b) and CD spectrum (c).
FIG. 6A is a diagram illustrating that NAA-ELP-EAKs can be used as a drug carrier using temperature sensitivity, and FIGS. 6B to 6D are fluorescence emission spectrum measurement values of NAA-ELP-EAKs (b), intracellular pyrene The fluorescence distribution (c and d) is shown.
7 and 8 are diagrams showing various embodiments of the self-assembled peptide structure of the present invention.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and thus is not limited to the embodiments described herein.

When a part is said to “include” a certain component, this means that other components may be further provided instead of excluding other components, unless specifically stated otherwise.

Unless otherwise defined, molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and routine techniques commonly used in the field of recombinant DNA within the capabilities of those skilled in the art can be performed. These techniques are known to those skilled in the art and are described in many standardized textbooks and references.

Unless defined otherwise herein, all technical and scientific terms used have the meaning as commonly understood by one of ordinary skill in the art.

Various scientific dictionaries including terms included herein are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, several methods and materials are described. Depending on the context used by those skilled in the art, the present invention is not limited to specific methodologies, protocols, and reagents because it can be used in various ways.

As used herein, a singular form includes a plurality of objects unless the context clearly dictates otherwise. Further, unless otherwise indicated, nucleic acids are written in left-to-right, 5 'to 3' directions, and amino acid sequences are written in left-to-right, amino to carboxyl directions, respectively. Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, a self-assembled peptide structure represented by the following structural formula 1 is provided.

[Structural Formula 1]

HA _n

In the above formula, H is composed of one or more hydrophobic amino acids, A is VPGXG, X is any amino acid except proline, and n is 1-4.

Since the peptide structure has temperature sensitivity, it can be used for various purposes.

The “temperature sensitivity” refers to a property in which the peptide structure is coacervated or decomposed according to a change in temperature, and the rate of aggregation or decomposition can also be varied.

The temperature at which the structure of the peptide is transitioned is called a specific temperature (T _t ), and the specific temperature may be determined according to the structure and structure of the peptide structure of the present invention.

Since the bioactive substance can be administered at an optimal rate in a desired temperature range through the temperature-sensitive peptide structure, it can be usefully used as a drug delivery system through the above properties.

The peptide (peptide) means a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds).

The general rule for naming the peptides can be based on a 3-letter or 1-letter amino acid code, unless specifically indicated. For example, the central portion of the amino acid structure is indicated by a three-letter code (eg, Ala, Lys), and the D-stereotype (eg, D-Ala, D-Lys) is specified by writing “D-” in front of the three-letter code. If not indicated, L-stereotype may be assumed. The amino acid residues constituting the peptide may be natural or non-natural amino acid residues.

The present inventors devised a peptide platform for preparing elastin-like protein amphiphiles (ELPA) that exhibits temperature sensitivity through the substitution of a single N-terminal amino acid in the final stage of peptide synthesis, and the secondary structure of the ELPA is dependent on temperature. The mechanism was observed by observing the change.

The A refers to a peptide platform, which is based on a miniaturized elastin-like peptide (MELP), and can be conjugated with various bioactive peptide sequences.

The H may be one or more selected from the group consisting of tryptophan, phenylalanine, isoleucine, leucine, proline, methionine, valine, and alanine, and may be selected from the group consisting of WFF, FFF, YFF, LFF, VFF and AFF, It is not limited.

Although it is possible to control the temperature sensitivity by substituting X among the peptide platforms VPGXG, it is necessary to repeatedly synthesize the entire peptide until the desired specific temperature (T _t ) range, which takes a long time to produce, thereby causing the peptide structure and As the complexity of composition increases, the scope of use may be reduced.

On the other hand, the self-assembled peptide structure can easily control temperature sensitivity through substitution of the hydrophobic amino acid (H).

The substitution of the hydrophobic amino acid is to replace and change the N-terminal hydrophobic amino acid of the peptide structure with another hydrophobic amino acid, and any amino acid substitution method used in the art can be used without limitation.

The target-directed peptide may be one or more selected from the group consisting of RGD peptide, α-helix peptide, mitochondrial target peptide, Fc receptor binding peptide and beta endorphin receptor ligand.

The target-directed peptide, such as RGD peptide (FIG. 3A), is a cell adhesion region commonly present in many extracellular matrix proteins, collagen, osteopontin (OPN), vitronectin, fibronectin, von Willebrand factor, laminin, RGD peptides may be included in tenacin, fibrinogen, thrombospondin, and the like.

The self-combining peptide structure to which the RGD peptide is bound decomposes slowly in an environment higher than a specific temperature, whereas it rapidly decomposes in an environment lower than a specific temperature (FIG. 4C), which is useful in situations in which the dosage of a sample needs to be adjusted according to temperature. Can be used.

The α-helix peptide plays a key role in various biological recognition processes inside and outside the cell, such as protein-DNA, protein-RNA and protein-protein interaction, and there is no limitation on the amino acid sequence of the α-helix peptide that can be used, An α-helical peptide consisting of A (EAAAK) ₂ A (EAKs, SEQ ID NO: 3) can be used (FIG. 5A).

The self-combining peptide structure to which the EAKs are bound exhibits a property of rapidly degrading in an environment higher than a specific temperature.

In particular, WFF-ELP-EAKs can be decomposed at a high temperature range of 37 to 43 ° C during body temperature to release bioactive substances (FIG. 6A), and thus can be used as a drug delivery system applicable to a living body.

The peptide structure may be linear (linear-type) or a reliat type (lariat-type), the retarded structure may be formed by a target-directed peptide connecting the N- and C- terminus of the A have.

The structure or form of the peptide structure is not particularly limited, but may exhibit different temperature sensitivity according to structural properties.

For example, the retarded structure is a cyclic-strcuture-connected specific region of the peptide platform indicated by the 'A', and the stability and temperature response characteristics in vivo are different due to structural characteristics different from the linear structure. The structure of the peptide may be changed in consideration of actual availability.

The peptide structure may carry a drug through a self-assembled structure, and the drug may be a low molecular weight drug, a gene drug, a protein drug, or a mixture thereof.

The low-molecular-weight drug may be antipyrin, antifebrin, aspirin, or salpyrin as an antipyretic agent, and aspirin, salicylates, ibuprofen as an anti-inflammatory agent ), Flurobiprofen, Pyroccikam, Naprosen, Phenoprofen, Indomethacin, Phenylbutazone, Mesotrexate (methotrexate), mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide, cortisone or corticosteroid corticosteroid), but is not limited thereto.

The gene drug may be small interfering RNA (siRNA), small hairpin RNA (shRNA), micro ribonucleic acid (microRNA, miRNA) or plasmid deoxyribonucleic acid (plasmid DNA). However, it is not limited thereto.

The protein drugs are monoclonal antibody-based trastuzumab, rituximab, bevacizumab, cetuximab, botezomib, and elo Erlotinib, gefitinib, imatinib mesylate, sunitinib; An enzyme-based L-asparaginase; Hormone-based tritolerin acetate, megestrol acetate, flutamide, bicalutamide, goserelin; It may be a cytochrome c (cytochrome c) or p53 protein, but is not limited thereto.

According to another aspect of the present invention, a drug delivery composition comprising the peptide construct is provided.

The peptide structure can be used as a drug delivery agent because it can release a drug carried under conditions in which body temperature fluctuates due to inflammation or hypothermia, through the characteristic of forming a self-assembled structure at a specific temperature.

The drug delivery system composition may contain a pharmaceutically effective amount of a bioactive component alone or may include one or more pharmaceutically acceptable carriers, excipients, or diluents.

The 'pharmaceutically effective amount' refers to an amount sufficient for the physiologically active ingredient to be administered to an animal or a person to exhibit a desired physiological or pharmacological activity, and the age, weight, health status, sex, and administration route of the administration target And treatment periods.

In addition, the 'pharmaceutically acceptable' refers to a physiologically acceptable drug that, when administered to a human, usually does not cause an allergic reaction such as gastrointestinal disorder, dizziness, or a similar reaction. Examples of the carrier, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, particularly oral, nasal, eye, It can be formulated for injectable, transdermal or oral administration for administration via subcutaneous, intravenous, intramuscular or intraperitoneal routes. The formulation is not limited thereto, but may be in the form of powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, and sterile powder.

The drug delivery system composition can be administered through several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on several factors such as the route of administration, the patient's age, gender, weight and severity of the patient. It can be selected accordingly.

In addition, the drug delivery system composition may be administered in parallel with a known compound capable of enhancing the desired effect of the bioactive substance.

Conventionally, as a drug carrier composition, structures such as liposomes or vesicles made of lipid components and polymer particles were used. Liposomes or vesicles made of lipids have poor stability, are not easy to store, and polymer particles are The manufacturing process was complicated and could damage the material to be delivered, or cause an inflammation or an immune response.

However, the peptide structure is a self-assembled peptide structure composed of only peptides having excellent biocompatibility and no toxicity at all using a short elastin-like peptide (ELP) based on 20 or less amino acids, and thus is completely degraded in vivo. Not only is it not harmful to the human body at all, and the characteristic of recognizing a specific molecule is significantly superior to that of a conventional drug delivery carrier.

In addition, the peptide structure is economical and efficient because the manufacturing process is also performed through self-assembly. That is, the peptide construct can self-assemble in a specific temperature environment, and can also implement various pharmacological activities by acting on cells together with a target-directed peptide.

Hereinafter, the present invention will be described in more detail through examples.

Peptide design

The designed peptide structure has 6 species and the sequence is shown in Table 1 below.

Example order Sequence number WFF-ELP WFF-VPGVGVPGVGVPGVGVPGVG-COOH 4 FFF-ELP FFF-VPGVGVPGVGVPGVGVPGVG-COOH 5 YFF-ELP YFF-VPGVGVPGVGVPGVGVPGVG-COOH 6 LFF-ELP LFF-VPGVGVPGVGVPGVGVPGVG-COOH 7 VFF-ELP VFF-VPGVGVPGVGVPGVGVPGVG-COOH 8 AFF-ELP AFF-VPGVGVPGVGVPGVGVPGVG-COOH 9

To examine the structural changes according to the number of hydrophobic amino acids, sequences having different numbers of phenylalanine attached to the N-terminus of [VPGVG] ₄ were compared.

As a result of measuring temperature-dependent absorbance (TD-Abs) at 300 nm, 1 mM of miniaturized elastin-like peptide (MELP) in 150 mM NaCl aqueous solution insoluble coacervate with 3 phenylalanine residues at 25 ° C On the other hand, when a short hydrophobic amino acid (NAA, non-polar amino acid) was combined, there was no change in turbidity (FIG. 1A).

F-MELP is black, FF-MELP is gray, and FFF-MELP is blue.

In order to examine the effect on the temperature sensitivity of the peptide construct according to the type of the NAA portion, a peptide construct representing the amino acid sequences of the six examples was synthesized.

Peptide synthesis

Peptides were synthesized with Rink amide MBHA resin LL (Novabiochem, Germany) using a standard Fmoc protocol in a Tribute peptide synthesizer (Protein Technologies, USA).

Standard amino acid protecting groups except Dde-Lys (Fmoc) -OH and Fmoc-Cys (Mmt) -OH were used.

The Fmoc is Fluorenylmethyloxycarbonyl, Dde is 1- (4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl, and Mmt is 4-methoxytrityl.

For the cyclization reaction after completion of the Dde-Lys (Fmoc) -OH coupling, 20% piperidine / NMP (N-methyl-2-pyrrolidone) was used for Fmoc removal.

Bromoacetic acid (8 mg, 200 μmol) and N, N'-diisopropylcarbodiimide (DIC, 15.6 μL, 100 μmol) were mixed in 1 mL of NMP and 10 Incubated for carboxyl activation for a minute and resin was added to couple with the ε-amino group of lysine.

The mixture was shaken in a polypropylene tube for 1 hour at room temperature with frit (Restek, USA), and the resin was washed several times with NMP and dichloromethane (DCM).

For orthogonal deprotection of the cysteine Mmt protecting group, 1.5 ml of cleavage cocktail solution (DCM: triisopropylsilane: trifluoroacetic acid (TFA); 94: 5: 1) was treated several times (10 times per minute).

Intramolecular cyclization was added to 4 mL of 1% (v / v) N, N-diisopropylethylamine (DIPEA) / NMP and shaken for 3 days at room temperature, and the resin was washed several times with NMP and DMF (Dimethylformamide). Did.

1 mL of 2% (v / v) hydrazine / DMF was treated 4 times (2 minutes per treatment) to selectively remove the Dde-group of the lysine α-amino group.

2% (v / v) hydrazine / DMF and free Dde were washed with 2 mL of DMF, and the process was repeated 5 times to obtain the final product peptide.

Peptide purification

The synthesized peptide was treated with another cleavage cocktail solution (TFA: 1,2-ethanedithiol: thioanisole; 95: 2.5: 2.5) for 3 hours, and the solution was triturated with tert-butyl methyl ether to perform reversible high performance liquid chromatography (reverse It was purified by phase high performance liquid chromatography, RP-HPLC, water / acetonitrile with 0.1% TFA).

Confirm peptide molecular weight and concentration

The molecular weight of the peptide was confirmed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (Microflex LRF20, Bruker, Germany).

The peptide concentration was measured by spectrophotometry in water / acetonitrile (1: 1) (200 M ^-1 cm ^-1 ), tryptophan (5,500 M ^-1 cm ^-1 ) at 280 nm, and phenylalanine (195 M at 257.5 nm). ^A molar extinction coefficient of an amide bond (200 M ^-1 cm ^-1 ) at ^-1 cm ^-1 ) or 230 nm was used.

Circular Dichroism (CD) and UV-Vis-NIR spectroscopy

CD and UV-Vis-NIR spectra were recorded using a Chirascan Circular Dichroism spectrometer equipped with a Peltier temperature controller (Applied Photophysics., Ltd.).

The CD spectrum of the peptide was recorded from 190 to 260 nm using a 1 mm long cuvette.

The UV-Vis-NIR spectrum was recorded using a 1 cm long quartz cell.

Atomic force microscopy (AFM)

The peptide samples of the examples prepared for AFM measurement were placed on a freshly cleaved mica surface, each 1 μL, and moisture was completely evaporated.

Images were taken in a tapping mode of Nanoscope IV (Digital Instruments), and the set point was scanned at 0.8 to 1 V and the speed at 1 to 2 Hz depending on the sample.

Fluorescence spectroscopy

Steady-state fluorescence spectra were measured on a 1 cm long quartz cuvette using a PerkinElmer LS-55 fluorescence spectrophotometer.

Samples were excited at 340 nm to measure the fluorescence of Pyrene.

Excitation and emission slits with a 5 nm bandpass were used for the measurements.

Tissue culture and intracellular experiment

In order to observe the intracellular delivery of the peptide, HeLa cells (1 × 10 ⁴ ) were added to an 8-well Lab-Tek II chambered cover-glass system (Nunc) in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). And placed in 1% pen / strep and incubated overnight at 37 ° C with 5% CO ₂ .

The cells were washed with Dulbecco's phosphate-buffered saline (DPBS) and treated with a hybrid structure for 4 hours.

Thereafter, the sample solution was removed and the cells were incubated for an additional hour.

Cells were visualized with a confocal microscope (LSM 710, Carl Zeiss, Germany).

For FRAP analysis, pyrene fluorescence in small areas of cells was photobleached and observed for 45 seconds at intervals of one image per second.

The half-life recovery time (T _1/2 ) was calculated by Equation 1 below.

[Equation 1]

f (t) = α (1-e ^-kt )

α = last value of recovered intensity

T _1/2 = ln0.5 / (-k)

Experiment result

Temperature sensitivity analysis

A hydrophobic peptide-linked ELP was synthesized, and temperature sensitivity was observed.

Experiments were conducted using hydrophobic phenylalanine, and the temperature sensitivity induced when the number of phenylalanines was varied was analyzed.

Referring to FIGS. 1B and 1C, FFF-ELP bound with three phenylalanine residues was self-assembled into a micelle structure and gradually coiled into a β-helix, but turbidity did not change when a shorter peptide was bound.

Continuously coupled hydrophobic amino acids react to temperature through the phase transition mechanism of self-assembly of the micelle structure, and an increase in temperature-dependent turbidity means the formation of a higher-order superstructure from the already formed micelles.

In addition, in order to confirm the difference in self-assembly structure according to temperature sensitivity, the peptide of the above example, in which the N-terminal amino acid residue is different, was observed.

2A and 2B, the peptides of the examples were all self-assembled into a common micelle structure, and the specific temperature (T _t ) was inversely proportional to the hydrophobicity of the amino acid residue (Tt: FFF-ELP <LFF-ELP <VFF- ELP <AFF -ELP).

On the other hand, when the N-terminal amino acid is aromatic, the inverse relationship between the hydrophobicity and the transition temperature was not applied.

In particular, the hydrophobicity of 1 mM WFF-ELP is similar to that of VFF-ELP but forms insoluble coacervate at 4 ° C.

In addition, YFF-ELPs had similar hydrophobicity to AFF-ELPs, but did not coacervate at 70 ° C, and exhibited temperature-sensitive properties similar to FFF-ELPs.

Referring to FIG. 2C, in the TD-Abs measurement, the turbidity of YFF-ELP was increased in a wider temperature range than other examples, and in particular, compared to FFF-ELP, the phase transition starting temperature was almost the same, but a large difference in the rate of temperature increase Showed.

The only difference between YFF-ELP and FFF-ELP is the hydroxyl group of the tyrosine group, and since the hydroxyl group is a polar functional group, the result is a phase transition by delaying the hydrophilic-hydrophobic transition in the presence of a polar functional group. It suggests that it may cause a difference in the rate of temperature increase.

FFF-ELP ( _H FFF-ELP), in which the N-terminus was not acetylated, was synthesized to confirm the effect of the other polar functional group, the amine group.

In the TD-Abs measurement, the turbidity of _H FFF-ELPs was lower than that of FFF-ELPs and YFF-ELPs, but the phase transition starting temperature of _H FFF-ELPs was similar to that of FFF-ELPs and YFF-ELPs.

The phase transition process was delayed because the N-terminal amine group has a higher hydrophilicity than the hydroxyl group of benzene and can hold water molecules near hydrophobic residues for a long time.

The above results suggest that the inverse temperature phase transition of NAA-ELP (non-polar amino acid residues-elastin like protein) can be effectively regulated by N-terminal single amino acid substitution.

Peptide amphiphilic analog ( PAA) design

After confirming that the temperature sensitivity of the peptide could be controlled through N-terminal single amino acid substitution, it was analyzed whether the peptide could be used as a platform for the development of a temperature-sensitive peptide structure exhibiting various functionalities.

Peptide amphiphiles (PAs) are self-assembled peptide structures, consisting of a hydrophobic tail, a bioactive peptide, and a ß-sheet-forming sequence connecting other elements.

NAA-ELP contains a hydrophobic part and a twisted filament structure, so if a target-directed peptide sequence is coupled at the C-terminus, it is confirmed that a peptide amphiphile analogues (PAA) structure similar to PAs is formed. Did.

Referring to Figure 3A, the peptide Arg-Gly-Asp (RGD) was coupled to the peptide structure C-terminus of the embodiment to form a PAA structure.

The RGD peptide can bind to integrin receptors and mediate cell adhesion and internalization of target-directed substances.

In the TD-Abs measurement, the transition temperature was different according to the composition of NAA, but the temperature-sensing characteristics were similar to that of NAA-ELP (FIG. 3B).

At this time, the TD-Abs measurements of NAA-ELP-RGDs are 5 mM FFF-ELP-RGDs in black, 1.5 mM FFF-ELP-RGDs in blue, LFF-ELP-RGDs in yellow, and VFF-ELP-RGDs in orange. It is represented by.

On the other hand, FFF-ELP-RGD in the CD spectrum has a transition temperature range that is suitable for biological studies when it is 5 mM concentration (FIG. 3C).

During the phase transition process in the AFM image, FFF-ELP-RGD was folded in a ß-helix shape while maintaining self-assembly of the micelle structure (FIG. 3D).

Thermal coacervates of FFF-ELP-RGD, LFF-ELP-RGD and VFF-ELP-RGD through cooling were observed with TD-Abs.

Referring to Figure 4A, FFF-ELP-RGD, while the turbidity during coacervation rapidly increased, the turbidity during the annealing process slowly decreased.

At this time, the concentration of FFF-ELP-RGDs was black when 5 mM and blue when 1.5 mM, and the dotted line indicates the start and end of the intensity change.

Referring to FIG. 4B, in the TD-Abs measurement, there was little difference in turbidity in the coacervation and annealing process of LFF-ELP-RGD and VFF-ELP-RGD.

FFF-ELPs are blue, LFF-ELPs are yellow, and VFF-ELPs are orange, and the dotted line indicates the start and end of intensity change.

In addition, the decomposition time of FFF-ELP-RGD according to the temperature was measured.

4C and 4D, the turbidity of the FFF-ELP-RGD peptide construct decreased relatively slowly as the temperature approached the maximum aggregation point.

The above results suggest that the decomposition rate can be controlled by adjusting the environmental temperature and T _t (specific temperature), and that a coacervated PAA structure can be used as a material that releases target-directed molecules.

Design of non-linear (lariat-type) peptide structures

A nonlinear peptide construct with different structural properties and temperature sensitivity from PAA was designed.

As a target-directed peptide, an α-helical peptide sequence (A (EAAAK) ₂ A, EAKs) was used, and the N-terminal peptide was substituted with valine, leucine or tryptophan (FIG. 5A).

5B, as a result of measuring dynamic light scattering (DLS), VFF-ELP-EAKs showed little change in temperature range, but WFF-ELP-EAK biomedical purposes, particularly, high fever (37 to 43 ℃) caused a decomposition action. In addition, LFF-ELP-EAKs decomposed at higher temperatures than WFF-ELP-EAKs.

Referring to FIG. 5, in the CD spectrum, peptides of a lariat structure were generally self-assembled into a micelle structure, and there was little secondary structure change of WFF-ELP-EAKs during the decomposition process.

At this time, 37 ºC WFF-ELP-EAKs are green, and 43 ºC WFF-ELP-EAKs are red.

These results suggest that the dynamic destabilization of ELPA is due to steric hindrance induced by N-terminal residue-dependent structural changes of MELP.

Referring to FIG. 6A, a drug may be supported by utilizing a response to a temperature change of WFF-ELP-EAKs.

Pyrene phosphors were encapsulated in micelles of WFF-ELP-EAKs to visualize the temperature sensitivity of the peptide magnetic assembly containing an effective payload of small molecules.

When the distance between the pyrene groups of the amphiphilic peptide is far, only monochromatic fluorescence emission signals (375 to 405 nm) are identified, but when the self-assembled peptide nanostructure binds to the target material, it exists in the self-assembled peptide nanostructure. Each amphipathic peptide pyrene group is spatially adjacent to form an excimer through morphological changes in which the α-helical structure is significantly stabilized, and thus has fluorescence characteristics showing fluorescence intensity at different wavelengths (~ 460 nm).

Referring to Figure 6B, the fluorescence intensity ratio of ELPA at 43 ° C (I ₄₆₀ nm / I ₃₇₅ nm) was lower than at 25 and 37 ° C.

When the temperature of WFF-ELP-EAKs encapsulating pyrene is 25 ° C, blue is shown, 37 ° C is green, and 43 ° C is red.

The results suggest that the pyrene molecule is released from the micelle structure when the temperature sensitive ELPA is decomposed.

HeLa cells were treated with ELPA containing pyrene and the position of the pyrene in the cells was confirmed through confocal microscopy.

Referring to FIG. 6C, at 37 ° C., blue fluorescence of pyrene was observed throughout the cytoplasm, but did not enter the nucleus.

On the other hand, when HeLa cells were incubated at 43 ° C for 30 minutes, pyrene aggregated not only in the cytoplasm but also in the nucleus (Fig. 6D).

The above results suggest that it can be utilized as a drug delivery agent capable of selectively releasing a drug at a specific temperature by binding a target-directed peptide having a linear or lariat structure to a peptide structure.

Meanwhile, the peptide structure is not limited to the above embodiment and may be implemented in various forms. For example, the peptide structure may be implemented in a linear structure (FIG. 7) or may be implemented in a lariat structure (FIG. 8).

The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted to be included in the scope of the present invention.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A THERMORESPONSIVE PEPTIDE STRUCTURE AND USES THEREOF <130> 18PP10430 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 Val Pro Gly Xaa Gly   1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 Val Pro Gly Val Gly   1 5 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> alpha-helix peptide <400> 3 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala   1 5 10 <210> 4 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 Trp Phe Phe Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly              20 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 Phe Phe Phe Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly              20 <210> 6 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 Tyr Phe Phe Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly              20 <210> 7 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 Leu Phe Phe Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly              20 <210> 8 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 Val Phe Phe Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly              20 <210> 9 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 9 Ala Phe Phe Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly   1 5 10 15 Val Gly Val Pro Gly Val Gly              20

Claims (12)

Self-assembled peptide structure consisting of the following structural formula 1.
[Structural Formula 1]
HA _n
In the above formula, H is a peptide that is WFF, FFF, YFF, LFF, VFF or AFF,
A is VPGVG (SEQ ID NO: 2),
N is 4. delete delete delete According to claim 1,
The peptide structure is WFF- (VPGVG) ₄ , FFF- (VPGVG) ₄ , LFF- (VPGVG) ₄ and VFF- (VPGVG) ₄ selected from the group consisting of RGD or RGD at the C-terminal of any self-assembled peptide structure Peptide structure consisting of additionally binding EAKs (SEQ ID NO: 3). delete According to claim 1,
The peptide structure is a peptide structure whose structure changes with temperature. The method of claim 5,
The FFF- (VPGVG) ₄ , LFF- (VPGVG) ₄ and VFF- (VPGVG) ₄ peptide structure formed by further binding RGD to the C-terminus of any one structure selected from the group consisting of linear (linear-type) )ego,
Peptide structure formed by further combining EAKs (SEQ ID NO: 3) at the C-terminus of any one structure selected from the group consisting of WFF- (VPGVG) ₄ , LFF- (VPGVG) ₄ , and VFF- (VPGVG) ₄ Is a lariat-type peptide structure. delete According to claim 1,
The peptide structure is a peptide structure carrying a drug through a self-assembled structure. The method of claim 10,
The drug is a low molecular weight drug, gene drug, protein drug or a peptide structure thereof. A drug delivery composition comprising the peptide construct of claim 1.

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