KR102219828B1 - A pH SENSITIVE PEPTIDE STRUCTURE AND USES THEREOF - Google Patents

Abstract

The present invention relates to a pH-sensitive peptide structure, wherein the peptide structure is self-assembled and inversely self-assembled according to pH, so that drug delivery is possible and can be used as a contrast agent.

Description

pH-sensitive peptide structure, and use thereof {A pH SENSITIVE PEPTIDE STRUCTURE AND USES THEREOF}

The present invention relates to a peptide structure that forms a self-assembled structure depending on pH and various uses thereof.

In the past years, a wide range of tumor-specific therapeutic proteins, including antibody fragments and ligands for cell surface receptors, have been developed and tested in clinical practice. Peptide self-assembled structures are attracting much attention because of their potential for various biological applications in the medical field or nanotechnology field. Peptide self-assembly structure is composed of short units of protein, so biological stability is high in nature. In some cases, the peptide monomer can form a β-sheet secondary structure, and through secondary bonds such as hydrophobic bonds, hydrogen bonds, electrostatic attraction, and pi pi bonds, various types of micelles, vesicles, nanofibers, hydrogels, etc. Structure can be formed. In addition, peptides are capable of specific binding to specific proteins depending on their sequence.

In the last 20 years, research has been conducted to realize a more precise and stable functional structure by giving various functions as well as various structures according to the properties of such peptides. In the field of drug delivery systems in the medical field, peptide constructs are being studied as safe and highly efficient therapeutic agents for cancer cells. _{Cancer cells express more α v} β ₃ integrin receptor protein on the cell surface than normal cells, have a slightly lower pH, and have peptides or protein enzymes such as glutathione in the cell.

In many studies, a selective peptide construct is designed in consideration of the characteristics of cancer cells. In order for the peptide structure to accurately realize the therapeutic effect inside the cell, not only the ability to specifically bind to cancer cells, but also the process of releasing the drug or the process of destroying the cell due to the structural change of the structure are required. Conditions such as the difference in pH outside and inside the cell, the difference in the concentration of glutathione, and the temperature difference are considered.

Recently, in the case of a peptide structure, a method of destroying cells by inducing a structural change due to a cancer cell-specific change factor as well as the destruction of cancer cells due to drug delivery has been proposed, and this is collectively referred to as an inverse-self-assembled structure. The present inventors have developed a peptide nanostructure that can specifically bind to a specific receptor protein, and reverse-self-assembly by reacting to acidic pH when the structure bound to the receptor enters the cell through intracellular transfection.

Korean Patent Publication No. 10-2016-0127827

An object of the present invention is to provide a pH-sensitive peptide structure.

Another object of the present invention is to provide a composition for drug delivery comprising the pH-sensitive peptide structure.

Another object of the present invention is to provide a contrast agent composition comprising a pH-sensitive peptide structure.

One aspect of the present invention for achieving the above object is a peptide structure comprising a hydrophobic amino acid, histidine, and a target-oriented peptide, wherein the hydrophobic amino acid, histidine and target-oriented peptide are arranged in the order of the following Structural Formula 1 or Structural Formula 2. , to a pH-sensitive peptide structure.

[Structural Formula 1]

X _n -H _m -R _l

[Structural Formula 2]

X _n -R _l -H _m

In the above formula, X is a hydrophobic amino acid or a conjugate of two or more hydrophobic amino acids;

H is histidine;

R is a target-directing peptide;

N is 3 to 8;

M is 6 to 10;

The l is a peptide structure of 1 to 5.

Specifically, X may be one or more selected from the group consisting of isoleucine, leucine, valine, isoleucine, and a conjugate of leucine.

In addition, specifically, the peptide structure may be composed of one or more amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14.

In an embodiment of the present invention, a peptide structure consisting of one or more amino acid sequences of SEQ ID NOs: 1 to 14 was prepared, and it was confirmed that the structure of the peptide structure was modified according to pH.

Specifically, the peptide structure may be characterized in that at least one of the structures represented by the following Chemical Formulas 1 to 14.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

In addition, specifically, the target-directed peptide of the present invention may be one or more selected from the group consisting of RGD peptide, mitochondrial target peptide, Fc receptor binding peptide, and beta endorphine receptor ligand. .

In the present invention, "target-oriented peptide" refers to a peptide or antibody capable of binding to the cell surface of a target site so that the peptide construct of the present invention can move to a target site in vivo.

The target site in the living body may vary as necessary, and may be a target part of a therapeutic agent or drug, such as cancer cells or inflammatory cells. Through the target-oriented peptide, the peptide construct of the present invention may be introduced into a target site such as cancer cells and inflammatory cells, and the target-oriented peptide may be appropriately changed according to the target site.

In one embodiment, one or more amino acids may be additionally included in the pH-sensitive peptide structure.

The peptide structure may further include an amino acid having an _{amine group (-NH 2 ).} For example, lysine is involved in forming a cyclic peptide structure using an amine group.

The peptide structure may further include glycine (G). For example, glycine (G) acts as a linker between blocks. Or it is possible to prevent the formation of enantiomers in the cyclic structure.

The peptide structure may further include tryptophan (W). For example, tryptophan (W) can absorb light of a specific wavelength in the aromatic ring portion, and thus can be used for peptide purification.

More particularly, the tryptophan, in a more contained peptide structure an additional glycine or lysine, the Structure 1 or combined or in two of X _n added to the tryptophan at the top, of the formula 1 R _l at the bottom or of the formula 2 H _It may be a form in which glycine or lysine is additionally bound to the bottom of m, but is not limited thereto, and an appropriate amino acid may be added or changed as necessary.

The structure of the peptide structure may change according to pH, and may be reverse self-assembly under conditions of pH 6.0 or less.

The present inventors have determined the number and type of amino acids in the hydrophobic block, the number of histidine and

The structural change of the peptide was observed according to the binding position, linear or cyclic form, and the mechanism was investigated by observing the secondary structure change according to pH with CD.

The peptide structure is an RGD block, an H block, and a hydrophobic amino acid (I, L, V) block

It can be composed of.

The peptide structure is a species consisting of only two types of moieties (hydrophilic and hydrophobic)

Since the structure is complex compared to the original peptide, it can be self-assembled into various structures, and it can exhibit target orientation and pH sensitivity.

In the case of the target-directed peptide, such as an RGD block, it can specifically bind to integrins expressed in large amounts on the surface of cancer cells, and can be introduced into cells by inducing endocytosis through binding.

In addition, the peptide structure has hydrophilic properties, so it is easy to use in vivo.

Can be dissolved. The target-directed peptide may be linked by repeating one or more times in consideration of the type and number of other hydrophobic amino acids, thereby controlling the total hydrophilicity.

In the case of the histidine block, when the pKa of the imidazole partial nitrogen of the residue is 6.04 and the pH is higher than pKa, it is electrically neutral, whereas when the pH is lower than pKa, a +1 charge may be displayed.

Therefore, the peptide structure has strong hydrophobic properties at pH 7.4 in vivo.

On the other hand, at pH 4.5 to 6, which is a condition in the endosomes or lysosomes of cancer cells, it can be charged and have hydrophilicity.

That is, in the peptide structure, the length of the hydrophilic amino acid chain is

It gets longer, and the Flurry Huggins constant changes, which can lead to structural changes.

In addition, specifically, the peptide structure may be capable of drug delivery through self-assembly and inverse self-assembly structure change.

While the peptide structure stably forms a self-assembled structure in the bloodstream, it has strong hydrophilicity and reverse-self-assembly in acidic pH conditions, and thus can release drugs carried in low pH conditions such as cancer tissues. It can be used as a drug delivery vehicle.

The peptide structure forms a self-assembled structure at the pH of the blood so that it can be safely transported to the target tissue without loss of the supported drug, and can release the supported drug after reaching the target tissue, which is an acidic pH condition. When administered, the drug delivery effect is excellent and can be used for drug delivery.

Another aspect of the present invention relates to a composition for drug delivery comprising the peptide structure.

Specifically, 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 salipyrin as antipyretics, and aspirin, salicylates, ibuprofen ), Flurobiprofen, Pyroccikam, Naprocene, Fenoprofen, Indomethacin, Phenylbutazone, Mesotrexate (methotrexate), mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide, cortisone, or corticosteroids. corticosteroid), but is not limited thereto.

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

The protein drugs are monoclonal antibodies, trastuzumab, rituximab, bevacizumab, cetuximab, bortezomib, and Elo. Erlotinib, gefitinib, imatinib mesylate, sunitinib; L-asparaginase of the enzyme family; Hormone-based tritolerin acetate, megestrol acetate, flutamide, bicalutamide, goserelin; It may be a cytochrome c or p53 protein, but is not limited thereto.

Another aspect of the present invention relates to a contrast medium composition comprising the peptide structure.

Since the peptide structure is bound to a target-directed peptide, a contrast material may be released using a pH change at a target site. For example, since a contrast material can be released from cancer tissues having a relatively low pH, it can be used as a contrast agent for diagnosing various types of cancer.

The contrast agent is a paramagnetic material as an MRI contrast agent, in the form of a complex compound of gadolinium and manganese, and may include at least one selected from the group consisting of Gd-DTPA, Gd-DTPA-BMA, GdDOTA, Gd-DO3A, and iron oxide as a superparamagnetic material. And, as a PET contrast agent, ^{at least one selected from the group consisting of radioactive isotopes 18} F, ¹²⁴ I, ⁶⁴ Cu, ⁹⁹ mTc and ¹¹ In may be included, and may be introduced into the peptide structure as a chelate DOTA and DTPA complex. .

The contrast agent composition may further include a pharmaceutically acceptable carrier.

Carriers used in the contrast agent composition according to the present invention include carriers and vehicles commonly used in the field of medicine, specifically ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (E.g., various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, camium hydrogen phosphate, sodium chloride and zinc salts) , Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool paper, but are not limited thereto. The contrast agent composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

By administering the contrast agent composition according to the present invention to a living body or a sample, detecting a signal emitted from the living body or sample by a nanostructure exhibiting fluorescence, and obtaining an image, and analyzing the form of light emission in the living body or sample It can be used for diagnosis of diseases.

The “sample” refers to a tissue or cell isolated from a subject to be diagnosed. In addition, the step of injecting the contrast agent composition into a living body or a sample may be administered through a route commonly used in the pharmaceutical field, for example, parenteral administration such as intravenous, intraperitoneal, intramuscular, subcutaneous or local routes. Can be used.

According to the technology disclosed by the present specification, the following effects occur.

Since the peptide construct disclosed in the present specification is inversely self-assembled under a predetermined pH condition, it can be utilized as a target-specific drug delivery system and contrast agent in consideration of the characteristics of the target.

In addition, since the peptide structure of the present invention can form a multidimensional structure through self-assembly, it can be used as a drug delivery system, a biomimetic device, a nanosensor material, a bioimaging material, and the like.

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

1 is a graph showing the results of HPLC purification of a peptide structure according to an embodiment of the present invention (horizontal axis: time (time, minutes), vertical axis: intensity (intensity, au)).
Figure 2 shows the MALDI-TOF measurement results of the peptide structure according to an embodiment of the present invention (horizontal axis: time (time, minutes), vertical axis: intensity (intensity, au)).
3 is a view of the peptide structure (Linear I ₆ H ₆ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
4 is a view of the peptide structure (Cyclic I ₆ H ₆ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
5 is a view of the peptide structure (Cyclic I ₈ H ₆ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
6 is a view of the peptide structure (Cyclic I ₆ H ₁₀ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
7 is a view of the peptide structure (Cyclic I ₆ H ₁₀ RGD buffer) according to an embodiment of the present invention through AFM (SABS buffer pH 4.5, KPBS buffer pH 7.5 from the left).
8 is a view of the peptide structure (Cyclic I ₆ RGDH ₆ ) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
9 is a view of the peptide structure (Cyclic L ₆ H ₁₀ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
10 is a view of the peptide structure (Cyclic V ₆ H ₁₀ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
11 is a view of the peptide structure (Cyclic I ₃ L ₃ H ₁₀ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
12 is a view of the peptide structure (Cyclic L ₃ I ₃ H ₁₀ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
13 is a view of the peptide structure (Cyclic (IL) ₃ H ₁₀ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
14 is a view of the peptide structure (Cyclic (LI) ₃ H ₁₀ RGD) according to an embodiment of the present invention through AFM (from the left, pH 4.5, pH 6, pH 7.5).
15 is a view of the peptide structure according to an embodiment of the present application through TEM.
16 is a view of the peptide structure according to an embodiment of the present application through a Circular Dichroism spectrometer.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

One. Peptide design

The designed peptide structure is 14 types, and the amino acid sequence is shown in Table 1 below.

Peptide structure Amino acid sequence Sequence number Linear I ₆ H ₆ RGD W-IIIIII-G-HHHHHH-G-RGDRGDRGD-K One Linear I ₈ H ₆ RGD W-IIIIIIII-G-HHHHHH-G-RGDRGDRGD-K 2 Linear I ₆ H ₁₀ RGD W-IIIIII-G-HHHHHHHHHH-G-RGDRGDRGD-K 3 Linear I ₆ RGDH ₆ W-IIIIII-G-RGDRGDRGD-G-HHHHHH-K 4 Cyclic I ₆ H ₆ RGD W-IIIIII-G-HHHHHH-G-RGDRGDRGD-G 5 Cyclic I ₈ H ₆ RGD W-IIIIIIII-G-HHHHHH-G-RGDRGDRGD-G 6 Cyclic I ₆ H ₁₀ RGD W-IIIIII-G-HHHHHHHHHH-G-RGDRGDRGD-G 7 Cyclic I ₆ RGDH ₆ W-IIIIII-G-RGDRGDRGD-G-HHHHHH-G 8 Cyclic L ₆ H ₁₀ RGD W-LLLLLL-G-HHHHHHHHHH-G-RGDRGDRGD-G 9 Cyclic V ₆ H ₁₀ RGD W-VVVVVV-G-HHHHHHHHHH-G-RGDRGDRGD-G 10 Cyclic I ₃ L ₃ H ₁₀ RGD W-IIILLL-G-HHHHHHHHHH-G-RGDRGDRGD-G 11 Cyclic L ₃ I ₃ H ₁₀ RGD W-LLLIII-G-HHHHHHHHHH-G-RGDRGDRGD-G 12 Cyclic (IL) ₃ H ₁₀ RGD W-ILILIL-G-HHHHHHHHHH-G-RGDRGDRGD-G 13 Cyclic (LI) ₃ H ₁₀ RGD W-LILILI-G-HHHHHHHHHH-G-RGDRGDRGD-G 14

In the case of the histidine (H) block, the pKa of the imidazole partial nitrogen at the histidine residue is 6.04, which is electrically neutral when the pH is above pKa, whereas when the pH is lower than pKa, it has a +1 charge. As a result, the hydrophobic property becomes stronger at pH 7.4, which is in vivo, and the peptide structure enters the cancer cell and becomes charged when the internal condition of the endosome or lysosome, pH 4.5 ~ 6, has a hydrophilic property, resulting in the hydrophilic amino acid chain of the peptide. As the length becomes longer, the Flurry Huggins constant changes, causing a structural change in the structure.

In order to examine the structural change according to the number of histidine (H), the sequence of 6 and 10 of histidine was compared. In addition, in order to examine the structural changes according to the positions of the RGD and H blocks, sequences with different positions such as _{I 6} H ₆ RGD and I ₆ RGDH _{6 were also designed.}

_{In the case of the RGD block, it can specifically bind to α v} β ₃ integrin, which is frequently expressed on the surface of cancer cells, and has the function of inducing endocytosis due to the binding so that the peptide nanostructure can enter the cell. In addition, it has hydrophilic properties, so it has the function of allowing peptides to be well dissolved in living conditions.

In addition, structural changes according to the hydrophobic isoleucine (I), leucine (L) or valine (V) block were confirmed. The hydrophobic blocks have a function of forming a core due to a hydrophobic effect when dissolved in a solution in vivo to form a specific nanostructure such as a micelle vesicle tube. These have the property of forming a β-sheet and are predicted to cause a more rapid structural change depending on the pH.

In addition, in order to examine the structural change according to the number of isoleucine (I), _{sequences such as I 6} H ₆ RGD and I ₈ H ₆ RGD were compared.

In addition, in order _{to confirm the possibility of a wider application of the H 10} RGD platform, amino acids that are hydrophobic like L and V and form β-sheets (cyclic L ₆ H ₁₀ RGD, cyclic V ₆ H ₁₀ RGD, ) And compared with cyclic I ₆ H _{10 RGD.}

In addition, in order to prove whether it is possible to utilize a combination of hydrophobic amino acids, amino acids combining I or L (cyclic I ₃ L ₃ H ₁₀ RGD, cyclic L ₃ I ₃ H ₁₀ RGD, cyclic (IL) ₃ H ₁₀ RGD, The pH sensitivity of cyclic (LI) ₃ H ₁₀ RGD) was confirmed.

In addition, while lysine (K) was devising methods for synthesizing a linear peptide in a cyclic form, it was arranged to try a method of utilizing the amine group of lysine.

Glycine (G) acts as a linker between blocks. Alternatively, trityl resin was used when synthesizing a cyclic peptide, and glycine was used as the first sequence to prevent the formation of enantiomers.

Tryptophan (W) was arranged for peptide purification because the aromatic ring portion absorbs light of 280 nm wavelength.

After the peptide synthesis is complete, a purification process is performed using HPLC to purify only the desired final product.If 280nm wavelength light is finally projected onto the solution flowing out of the column, tryptophan is light when the peptide dissolved in the solution passes through the light. By absorbing, the signal of HPLC can be generated.

2. Synthesis and Experiment Method

2-1. Peptide synthesis

Linear peptides were synthesized through solid phase peptide synthesis on rink amide resin. Using Fmoc chemistry, one amino acid was sequentially synthesized from the N-terminal in sequence.

Resin was synthesized with 100umol as 1 equivalent. When Fmoc was first removed from the resin, an NMP solution in which 20% by volume of piperidine was dissolved was added to the resin, reacted for about 10 minutes, and washed with DMF and NMP. Subsequently, the amino acids to be synthesized were dissolved in NMP with HCTU, HOPt, and DIPEA, and incubated for about 5 minutes. Reagents and dissolved amino acids were added to NMP to the resin from which Fmoc was removed, and reacted on a shaker for about 60 minutes. When synthesizing the next amino acid, Fmoc was removed in the same manner, and the next amino acid was synthesized, and sequentially repeated in sequence until all synthesis was completed.

After the synthesis was completed, reagent K solution, a TFA solution in which 2.5% H20 and 2.5% TIS were dissolved in the resin, was treated for 2 hours or more to remove the resin and the protecting group attached to the peptide. To remove reagent K, it was volatilized with argon gas, and TBME was added to the remaining reagent K solution. Thereafter, the solution was centrifuged for 3 minutes to precipitate the peptide, and the upper layer solution was removed, and the process was repeated three times to obtain the final product, the peptide.

Cyclic peptides were synthesized through solid phase peptide synthesis on trityl resin. Using Fmoc chemistry, one amino acid was sequentially synthesized from the N-terminal in sequence.

Resin was synthesized with 100umol as 1 equivalent. When Fmoc was first removed from the resin, an NMP solution containing 20% piperidine by volume was added to the resin, reacted for 10 minutes, and washed with DMF and NMP.

The amino acids to be synthesized sequentially were dissolved in NMP with HCTU, HOBt, and DIPEA, and then incubated for about 5 minutes. In the case of cyclic peptides, the first amino acid to be synthesized in resin was synthesized as glycine due to concern about the occurrence of enantiomers.

Reagents and dissolved amino acids were added to NMP to the resin from which Fmoc was removed, and reacted on a shaker for about 60 minutes.

Subsequently, when synthesizing amino acids, Fmoc was removed in the same manner, and the next amino acid was synthesized, and it was sequentially repeated until all of the synthesis was completed.

Synthesis was completed to the last sequence, Fmoc was removed, and a cyclization process was performed to make a cyclic form.

TFA: TFE: MC = 2: 2: 6 The solution was treated with the peptide and separated from the resin, and the peptide dissolved in the solution was obtained in a solid state through distillation under reduced pressure.

A solution in which 20 μmol of the peptide was dissolved in 20 mL of DMF together with 4 equivalents of DIPEA and 1 equivalent of HATU in 20 mL of DMF were prepared in separate syringes.

A solution in which 1 equivalent of HOBt and 0.1 equivalent of HATU were dissolved in 20 mL of DMF was prepared in a round bottom flask. Using a syringe pump, the solution in the two syringes prepared above was flowed into the solution in a round bottom flask at a rate of 0.06 mL/min.

When all the solution flowed into the round flask solution in the syringe, the reaction was terminated. After the reaction was completed, DMF was removed as much as possible by distillation under reduced pressure to obtain a solid peptide.

The remaining DMF was dissolved in 1 mL of MC, transferred to a 50 mL tube, 1 mL of TBME and about 50 mL of Hexane were sequentially added, and sonication was performed each time each solution was added.

Thereafter, the solution was centrifuged for 3 minutes to precipitate the peptide, and the upper layer solution was removed, and the final product, the peptide, was obtained by repeating this process three times.

Reagent K solution was treated for 2 hours or more to remove the protective groups remaining on the peptide.

To remove reagent K, it was volatilized with argon gas, and TBME was added to the remaining reagent K solution.

The solution was centrifuged for 3 minutes to precipitate the peptide, and the upper layer solution was removed, and the process was repeated three times to obtain the final product, the peptide.

2-2. Peptide purification

The synthesized peptide was purified by HPLC. The peptide sample obtained in a solid state by dividing by 5 to 10 umol was _{dissolved by sonication treatment in 1 mL of H 2} O: ACN = 1: 1 solution, and filtered through a syringe filter.

_{Sonication treatment was performed by adding 4 mL of H 2} O to the filtered sample, followed by injection into HPLC equipment for purification. TFA 0.1% H2O solution and TFA 0.1% ACN solution were used as mobile phases, and C 4 and C 18 columns were used as stationary phases.

It was purified by flowing 0.1% TFA/ACN solvent from 5% to 50% gradient at 25°C through a C4 column for 60 minutes. Cyclic I ₃ L ₃ H ₁₀ RGD, Cyclic L ₃ I ₃ H ₁₀ RGD, Cyclic (IL) ₃ H ₁₀ RGD, Cyclic (LI) ₃ H ₁₀ RGD uses 0.1% TFA/ACN solvent at a 10-37% gradient. It was purified by flowing it at 25° C. through a C4 column for minutes (FIG. 1).

2-3. Confirmation of peptide molecular weight and purification

During or after synthesis with MALDI-TOF, samples were measured to confirm synthesis and purification.

The sample was solidified by cocrystallization with CHCA metrix in a 1:1 volume ratio, and measured in a linear mode (FIG. 2).

2-4. Peptide sample preparation according to pH

To prepare a peptide self-assembly sample according to pH7, each peptide was dissolved in HFIP and the solvent was removed using a speed bag.

The pH was measured using a pH paper. In the case of a peptide sample dissolved in a pH 4.5 aqueous solution, an aqueous solution having a pH of 4.5 was added by mixing water and HCl in an EP tube in which only the peptide remained after the solvent was removed.

Peptide samples dissolved in pH 7.5 aqueous solution were mixed with water and NH3 in an EP tube in which only peptide remained, and an aqueous solution adjusted to pH 7.5 was added.

The final concentration of the peptide prepared for each pH was 20 μM, and sonication was applied for 10 minutes or more using a sonicator and incubated at room temperature for 1 day.

In the case of Cyclic I6H10RGD, samples were prepared on pH 4.5 SABS (Sodium acetate buffer 15mM) and pH 7.5 KPBS (Potassium Phosphate buffer 15mM) to determine whether the structure is maintained in vivo.

After dissolving the peptide in HFIP in the same way, the solvent was removed using a speed bag, and each buffer was added.

The final concentration of the peptide was 20 μM, and sonication was applied for 10 minutes or more using a sonicator and incubated at room temperature for 1 day.

2-5. Atomic force microscopy (AFM) measurement

20 μL of each peptide sample prepared for AFM measurement was placed on fresh mica cotton, and the moisture was completely evaporated.

Images were obtained in non-contact mode using Park NX10 equipment, and scans were performed for set points of 8 to 14 nm, Z servo gain of 1.5 to 4, and speed of 0.1 to 0.5 Hz, depending on the sample.

2-6. TEM (Transmission Electron Microscopy) measurement

Peptide samples for each pH prepared for TEM measurement were placed on a carboncoated copper grid of 2 μL each, and moisture was completely evaporated. Since the organic sample is difficult to observe in TEM due to its low electron density, 2 μL of 2% (w/v) uranyl acetate, which has a high electron density, is treated on the carbon-coated copper grid on which the peptide sample is placed, and after 1 minute, completely Absorbed. Samples were observed at 120 kV voltage with JEOL-JEM 2010.

2-7. CD (Circular dichroism) measurement

The prepared peptide samples for each pH were measured in a cuvette having a path length of 100 Μ each using a Chirascan Circular Dichroism spectrometer instrument (260 to 190 nm). Molar ellipticity was calculated by dividing by the peptide concentration and the number of amino acids.

3. AFM experiment results

Before synthesizing cyclic peptides, linear peptides were first synthesized and observed through AFM whether the nanostructure was changed sensitive to pH.

3-1. Linear I ₆ H ₆ RGD

In the case of Linear I ₆ H ₆ RGD, an amorphous structure, which is judged to be difficult to self-assembly at pH 4.5, was observed (FIG. 3).

On the other hand, _{uniform micelles having a diameter of about 20 nm were formed in H 2} O phase with a pH of 6, and vesicles having various sizes of 40 to 80 nm in diameter or vesicles having various sizes of 40 to 80 nm were fused to each other when the pH was 7.5. One structure was formed.

3-2. Cyclic I ₆ H ₆ RGD

Since it was confirmed that the peptide designed based on the results of linear I ₆ H ₆ RGD has sufficient pH sensitivity, the structural change according to the pH of the cyclic peptide, which is expected to show a more rapid structural change, was observed.

In the case of Cyclic I ₆ H ₆ RGD, a self-assembled structure was hardly observed in a pH 4.5 environment, but very rarely micelle structures with a diameter of around 20 nm or a vesicle structure of 100 nm or more were observed.

In the pH 6 environment, the number and density of self-assembled structures were much higher than that of pH 4.5, and micelle structures of around 20 nm in diameter were mainly observed, and vesicle structures of around tens of 100 nm in diameter were observed.

Similar sized micelles and vesicles were observed in the pH 7.5 environment as in the pH 6 environment (FIG. 4).

3-3. Cyclic I ₈ H ₆ RGD

In the case of Cyclic I ₈ H ₆ RGD, the structural difference according to pH was more clear than that of Cyclic I ₆ H _{6 RGD.} In the pH 4.5 environment, micelles or cylindrical micelles having a diameter of about 20 nm were observed, and at pH 6, plate-like structures having various sizes of several µm were formed.

In the pH 7.5 environment, a larger and atypical cluster structure was formed. In the case of Cyclic I ₈ H ₆ RGD, the sensitivity to pH is very high, but in a high pH environment, the number of I is large, so the hydrophobic bond is very large, so it is judged that the shape is excessively atypical and high-dimensional self-assembly is not possible (FIG. 5).

3-4. Cyclic I ₆ H ₁₀ RGD

Similarly, in the case of Cyclic I ₆ H ₁₀ RGD, the difference in structure according to pH was more clear than that of _{Cyclic I 6} H _{6 RGD.}

In the pH 4.5 environment, micelles having a diameter of about 20 nm were observed, and micelles having a diameter of about 20 nm were observed even at pH 6 (FIG. 6).

In the pH 7.5 environment, an elliptical structure with a length of several µm was formed, which is estimated to be fusion between vesicles and vesicles having various diameters ranging from several tens of nm to about 500 nm.

It was judged that the reason why the structural difference according to the pH of the Cyclic I ₆ H ₁₀ RGD is more clear than that of the Cyclic I ₆ H _{6 RGD is that the number of H is 4 more.}

Cyclic I ₈ H ₆ RGD has a stable vesicle structure at a higher pH than that of RGD because the number of I is small, so it does not form an atypical structure even at high pH, and it is considered to be due to proper hydrophobic bonding.

3-5. Cyclic I ₆ H ₁₀ RGD buffer

It was judged that the formation of a homogeneous structure compared to other peptides and the formation of micelles and vesicles showed various application possibilities. Therefore, it was observed whether the structure was formed in the SABS buffer of pH 4.5 and the KPBS buffer of pH 7.5, similar to those in vivo.

As expected, micelles with a diameter of around 20 nm were observed in the SABS buffer as in the aqueous environment of pH 4.5, and vesicles of various sizes of tens to hundreds of nm in diameter were observed as in the aqueous environment of KPBS buffer pH 7.5.

That is, _{it is determined that Cyclic I 6} H ₁₀ RGD peptide can be applied in vivo by maintaining its structure and function even in the in vivo environment (FIG. 7).

3-6. Cyclic I ₆ RGDH ₆

In the case of Cyclic I ₆ RGDH ₆ , an amorphous structure with a size of several nm and a micelle structure of about 20 nm, which does not seem to form well in a pH 4.5 environment, were observed.

In the pH 6 environment, the number and density of self-assembled structures were much higher than that of pH 4.5, and micelle structures around 20 nm were mainly observed.

In the pH 7.5 environment, _{amorphous structure of several μm size similar to that of Cyclic I 8} H ₆ RGD was observed (FIG. 8).

Although only the direction of the H block position with respect to the hydrophobic block is different _{, the structure change according to pH of Cyclic I 6} RGDH ₆ is clear compared to Cyclic I ₆ H ₆ RGD, so the environment or orientation around the hydrophobic block controls self-assembly. It seems to be an important factor that can be done.

Since the structure of all cyclic peptides changed according to pH, additional peptides were synthesized to find out the possibility of generalization of the designed sequence.

Among the four peptides, Cyclic I ₆ H ₁₀ RGD peptide had a clear structural change according to pH, and the solubility was homogeneous between micelles and vesicles according to pH, so the hydrophobic peptide moiety part was intended to investigate the expandability of the _{H 10 RGD platform. Cyclic L 6} H ₁₀ RGD and Cyclic V ₆ H ₁₀ RGD were synthesized in which (I) was replaced with L and V.

3-7. Cyclic L ₆ H ₁₀ RGD

In the case of Cyclic L ₆ H ₁₀ RGD, a micelle structure with a diameter of about 20 nm or a vesicle structure of about several tens of nm was observed in a pH 4.5 environment.

In the pH 6 environment, the number and density of self-assembled structures were much higher than that of pH 4.5, and micelles of about 20 nm, vesicles of about several tens of nm, and cluster structures were variously observed (FIG. 9).

In the pH 7.5 environment, an atypical structure of several µm size similar to _{that of Cyclic I 8} H ₆ RGD and Cyclic I ₆ RGDH _{6 was observed.}

The reason that the larger or atypical structure was observed compared to the Cyclic I ₆ H _{10 RGD was judged because L has more hydrophobic properties than I.}

3-8. Cyclic V ₆ H ₁₀ RGD

In the case of Cyclic V ₆ H ₁₀ RGD, a vesicle or long vesicle structure with a diameter of 100 ~ 200 nm was observed in a pH 4.5 environment.

In the pH 6 environment, micelles with a diameter of about 20 nm and vesicle structures of about several tens of nm were observed in various ways. In the pH 7.5 environment, a cylindrical micelle structure having a diameter of about 20 nm was observed (Fig. 10).

Cyclic V ₆ H ₁₀ RGD, unlike other peptides, formed a larger structure when the pH was low.

The above results are contrary to the formation of a smaller self-assembled structure such as micelles as the volume or length ratio of the hydrophobic chain decreases from the viewpoint of packing parameters, but V is a unique secondary β-sheet stacking different from other amino acids. It was estimated because

Cyclic L ₆ H ₁₀ RGD and Cyclic V ₆ H ₁₀ RGD were also determined to be able to generalize the pH sensitivity of the _{Cyclic X n} -H _m -R ₁ peptide, since the structure was remarkably changed according to the pH as predicted.

3-9. Cyclic I ₃ L ₃ H ₁₀ RGD, Cyclic L ₃ I ₃ H ₁₀ RGD, Cyclic (IL) ₃ H ₁₀ RGD and Cyclic (LI) ₃ H ₁₀ RGD

In the case of Cyclic I ₃ L ₃ H ₁₀ RGD, Cyclic L ₃ I ₃ H ₁₀ RGD, Cyclic (IL) ₃ H ₁₀ RGD and Cyclic (LI) ₃ H ₁₀ RGD, it was also confirmed that the structure changes according to pH. Specifically, a vesicle-like structure was formed in the environment of pH 4.5 and pH 6, and a much larger and irregular structure was formed in the environment of pH 7.5 (FIGS. 11, 12, 13 and 14).

4. TEM experiment results

Considering the AFM data, Cyclic I ₆ H ₁₀ RGD was judged to form homogeneous micelle and vesicle structures better than other peptides, and the structure was additionally observed with TEM. In the pH 7.5 environment, like AFM, vesicles having a diameter of several tens of nm were well formed (FIG. 18).

However, in a pH 4.5 environment, the micelle structure was not well observed similar to AFM, but small structures with a diameter of 20 nm or less were observed, confirming the pH sensitivity of _{Cyclic I 6} H _{10 RGD.}

5. CD test results

CD was measured to explain with a more clear mechanism how the structure changes according to the pH of the peptide.

As a result of measuring the CD for the Cyclic X ₆ H ₁₀ RGD peptide, a secondary structure of a random coil was formed in an environment of pH 4.5 and 6, whereas a secondary structure of a β-sheet was formed in an environment of pH 7.5 (FIG. 19).

Referring to the above results, as the pH increases, the histidine loses charge and the hydrophobic property becomes strong, so it affects the formation of the secondary structure of the hydrophobic amino acid, thereby facilitating the formation of β-sheets, thereby forming a standardized high-dimensional structure. It can be judged by letting go.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it 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 embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A pH SENSITIVE PEPTIDE STRUCTURE AND USES THEREOF <130> 19PP30240 <150> KR 10-2018-0042402 <151> 2018-04-11 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Linear I6H6RGD <400> 1 Trp Ile Ile Ile Ile Ile Ile Gly His His His His His His Gly Arg 1 5 10 15 Gly Asp Arg Gly Asp Arg Gly Asp Lys 20 25 <210> 2 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Linear I8H6RGD <400> 2 Trp Ile Ile Ile Ile Ile Ile Ile Ile Gly His His His His His His 1 5 10 15 Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Lys 20 25 <210> 3 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Linear I6H10RGD <400> 3 Trp Ile Ile Ile Ile Ile Ile Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Lys 20 25 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Linear I6RGDH6 <400> 4 Trp Ile Ile Ile Ile Ile Ile Gly Arg Gly Asp Arg Gly Asp Arg Gly 1 5 10 15 Asp Gly His His His His His His Lys 20 25 <210> 5 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Cyclic I6H6RGD <400> 5 Trp Ile Ile Ile Ile Ile Ile Gly His His His His His His Gly Arg 1 5 10 15 Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 6 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Cyclic I8H6RGD <400> 6 Trp Ile Ile Ile Ile Ile Ile Ile Ile Gly His His His His His His 1 5 10 15 Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 7 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Cyclic I6H10RGD <400> 7 Trp Ile Ile Ile Ile Ile Ile Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 8 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Cyclic I6RGDH6 <400> 8 Trp Ile Ile Ile Ile Ile Ile Gly Arg Gly Asp Arg Gly Asp Arg Gly 1 5 10 15 Asp Gly His His His His His His Gly 20 25 <210> 9 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Cyclic L6H10RGD <400> 9 Trp Leu Leu Leu Leu Leu Leu Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 10 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Cyclic V6H10RGD <400> 10 Trp Val Val Val Val Val Val Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 11 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Cyclic I3L3H10RGD <400> 11 Trp Ile Ile Ile Leu Leu Leu Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 12 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Cyclic L3I3H10RGD <400> 12 Trp Leu Leu Leu Ile Ile Ile Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 13 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Cyclic (IL)3H10RGD <400> 13 Trp Ile Leu Ile Leu Ile Leu Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25 <210> 14 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Cyclic (LI)3H10RGD <400> 14 Trp Leu Ile Leu Ile Leu Ile Gly His His His His His His His His 1 5 10 15 His His Gly Arg Gly Asp Arg Gly Asp Arg Gly Asp Gly 20 25

Claims (11)

As a peptide construct comprising a hydrophobic amino acid, histidine, and a target-directed peptide,
The peptide structure is a peptide structure consisting of one or more amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14.
delete delete The method of claim 1,
The peptide structure is a peptide structure, characterized in that at least one of the structures represented by the following formulas 1 to 14.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

The method of claim 1,
The target-directed peptide is one or more selected from the group consisting of RGD peptide, mitochondrial target peptide, Fc receptor binding peptide, and beta endorphin receptor ligand.
delete The method of claim 1,
The peptide structure will change the structure according to the pH, the peptide structure.
The method of claim 1,
The peptide structure is to be reverse self-assembly under conditions of pH 6.0 or less, the peptide structure.
The method of claim 1,
The peptide structure is capable of drug delivery through self-assembly and inverse self-assembly structure change.
A composition for drug delivery comprising the peptide structure of claim 1.
Comprising the peptide structure of claim 1, contrast agent composition.

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