KR101923520B1 - Self-assembled peptide nanostructures with structural diversity by thermodynamic control - Google Patents

Abstract

The present invention relates to a self-assembled peptide nanostructure, wherein a lysine containing a hydrocarbon chain is inserted between tryptophan, thereby improving the flexibility and hydrophobic packing effect of the self-assembled peptide nanostructure, There is an effect of providing a self-assembled nanostructure having a wider range of structural diversity

Description

[0001] SELF-ASSEMBLED PEPTIDE NANOSTRUCTURES WITH STRUCTURAL DIVERSITY BY THERMODYNAMIC CONTROL [0002]

The present invention relates to a self-assembled peptide nanostructure, and more particularly, to a self-assembled peptide nanostructure having various structures by thermodynamic or dynamic control.

Self-assembly peptide nanostructures (SPNs) are a major concern in nanostructure morphology, surface functionalization of nanostructures, structural regulation of peptide chains, and utilization of various SPNs. In the field of nanotechnology, the regulation of nanostructured morphology and substructure is the starting point required for SPNs investigation. In particular, cyclic peptides provide the possibility of assembling unique, stable molecular assemblies that can not be expected in conventional linear peptides. One of the most important topological benefits of cyclic peptides is that they have a morphologically flexible structure that limits the degree of freedom of the molecule and can minimize the disadvantage of entropy involving molecular self-assembly or target substance binding.

The vesicles can be defined as fluid-filled sacs formed by the self-assembly of molecular building blocks in bilayer structures. These building blocks are typically amphiphilic molecules. The geometric laminate structure indicates that the structure of the vesicles can be formed when the volume fraction of the hydrophilic segment is somewhat greater than the volume fraction of the hydrophobic segment. Amphiphilic molecules can form vesicles containing lipids, low molecular weight amphipathic materials, block copolymers, amphipathic dendrimers or hyperbranched polymers, rod coils, self-assembling peptides.

Self-assembly generally involves the process at the point of reaching a thermodynamic equilibrium state, that is, at a global minimun of the potential energy surface. However, when the dynamically captured metastable state, that is, the local minimun at the potential energy surface becomes irreversible at the experimental time scale, it can be used to expand the morphology and structural space of the nanodisperse . Although many studies have investigated the subject of thermodynamic and dynamic regulation in block copolymers and other building blocks, the regulation of peptide assemblies has not yet been found.

We report the results of thermodynamic, dynamic control and phase effects of peptide vesicles based on self-assembling apheresis peptides (cyclopeptisomes). In contrast to the results in linear peptides, strong aromatic interactions were observed only in the ring formation of the peptides. As a result, the formation of stable vesicles is only possible with cyclic peptides, and by proposing a vast possible structural space of the cyclopeptidomes according to a control method based on dynamic and thermodynamic principles, .

Zhan, S. G. Nat.Biotechnol.2003,21,1171. Klok, H. A. Angrew. Chem. Int. Ed. 2002, 42, 1509. Gazit, E. Chem.Soc.Rev.2007,36,1263.

An object of the present invention is to provide a self-assembled nanostructure having reversible behavior according to thermodynamic conditions and stability such as colloid by placing lysine between tryptophan in a hydrophobic segment composed of tryptophan.

In order to achieve the above object, the present invention provides a self-assembled peptide nanostructure comprising a hydrophobic segment composed of a tryptophan and lysine (K) located between the tryptophan.

The hydrophobic segment may be composed of the following sequence 1.

[SEQ ID NO: 1]

WWKWW

The self-assembled peptide nanostructure may be a cyclic peptide,

The self-assembled peptide nanostructure may be represented by the following structural formula (1).

[Structural formula 1]

The self-assembled peptide nanostructure may be controlled by thermodynamic or kinetic control, and the self-assembled peptide nanostructure may be formed by dynamic control under a quiet environment, and the co-solvent may be formed by HFIP hexafluoroisopropanol), and the concentration of HFIP may be 20 to 40%.

According to the present invention, by inserting and positioning lysine containing a hydrocarbon chain between tryptophan, flexibility and hydrophobic packing effect of the self-assembled peptide nanostructure is improved.

Also, there is an effect of providing a self-assembled parcel having a wider range of structural diversity than conventional ones through dynamic and thermodynamic control.

In addition, dynamic and thermodynamic control can be finely adjusted for self-assembly in a quiet environment, and uniform and round shaped vesicles can be provided. Due to the solid structure of the vesicles, System can be provided.

1 shows a schematic diagram of thermodynamic and dynamic control of vesicles according to an aspect of the present invention.
Figure 2 shows the structure of linear and cyclic peptides according to an embodiment of the present invention.
FIG. 3 shows behavioral characteristics of a self-assembled peptide nanostructure according to an embodiment of the present invention in accordance with a critical concentration (CAC), wherein a) is a linear peptide (LP) in pure water and b) (MCP), c) is a linear peptide (LP) in PBS, and d) is a cyclic peptide (MCP) in PBS. (PBS: 20 mM potassium phosphate, 150 mM KF, pH 7.4)
Figure 4 shows the analysis results of the circularly polarized dichroism spectroscopy (CD) of a digitized peptide nanostructure according to an embodiment of the present invention. (A) Thermal ramp CD data for LP, b) Thermal ramp CD data For MCP, forward scan, c): Thermal ramp CD data for MCP, backward scan, d): Molecular ellipticity graph of MCP at 229 nm with temperature. Peptide concentration is 20 μM.
FIG. 5 is a TEM image showing the self-assembly behavior of LP in pure water according to an embodiment of the present invention. (a) [LP] = 2 [mu] M, b) [LP] = 5 [mu] M)
Figure 6 shows a TEM image of a self-assembly of an MCP building block in a parcel according to an aspect of the present invention. b) was sonicated in PBS buffer (PBS; 20 mM potassium, 150 mM KF, pH 7.4), and c) and d) were prepared from pure water by a 100 nm pore membrane As shown in Fig. a) ~ d): [MCP] = 20 [mu] M)
FIG. 7 is a result of measuring the MCP type magnetic susceptibility in HFIP through the circularly polarized dichroism spectroscopy (CD) for the MCP according to an embodiment of the present invention.
FIG. 8 shows TEM results of thermodynamic control results of vesicles according to an embodiment of the present invention (a): results obtained by flash injection of an MCP of HFIP using an excess amount of water. b): This is the result of ultrasonic treatment of parasitic by flash injection. c): The result of dialysis of MCP in 30% HFIP. d): the result of dialysis of MCP in 50% HFIP.
Figure 9 shows TEM measurements of thermodynamic control results of vesicles according to an embodiment of the present invention (ultrasound treatment of lyophilized MCP from 30% HFIP under reduced pressure).
FIG. 10 shows fluorescence analysis results of a cyclopeptisome according to an embodiment of the present invention and a liposome as a comparative example. (a): Liposome, b): Cyclopeptisome, fluorescence emission was observed at 535 nm. Inset: The release profile of the initial point. Liposomes were prepared using 1-α-phosphatidylcholine extracted from egg yolk. c): Observation of intracellular delivery of Cyclopeptisome and captured FAM molecules (Left, bright-field, Right fluorescence image)
11 is a graph illustrating the Zeta Potential Distribution (Zeta potential value = +33.1 mV) of water in an MCP according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

A self-assembled peptide nanostructure according to one aspect of the present invention comprises a hydrophobic segment composed of a tryptophan, a charged hydrophilic segment and a linker segment, and a lysine (K) positioned between the tryptophan.

The hydrophobic segment may be composed of SEQ ID NO: 1 below.

[SEQ ID NO: 1]

WWKWW

In the above sequence, W is tryptophan and K is lysine.

The charged hydrophilic segment may be composed of SEQ ID NO: 2 below.

[SEQ ID NO. 2]

TRQARRNRRRRWRR

The self-assembled peptide nanostructure may be a linear peptide or a cyclic peptide, and is preferably a cyclic peptide. Strong interactions of aromatic hydrophobic segments can only be found in cyclic peptides, and linear peptides tend to aggregate nonspecifically as the concentration increases, even though they tend to form vesicles, while cyclic peptides form stable vesicles at high concentrations can do.

The self-assembled peptide nanostructure can be represented by the following structural formula 1.

[Structural formula 1]

The self-assembled peptide nanostructure represented by Formula 1 can be formed by a head-to-tail cyclization reaction of a linear peptide represented by Formula 2 below.

[Structural formula 2]

In addition, the self-assembled peptide nanostructure may be thermodynamically or kineticly controlled to control the structure, and the self-assembled peptide nanostructure may be formed by dynamic control under a quiet environment, HFIP (hexafluoroisopropanol), and the concentration of HFIP may be 20 to 40%.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1. Synthesis of peptides

Prepare 2-Chlorotrityl resin (Novabiochem, Germany) and Fmoc-Ebes-OH (Anaspec, USA). Peptides are synthesized on a 0.01 mmol scale using standard peptide protocols using a Tribute peptide synthesizer (Perotein Technologies, USA).

For peptide synthesis, a standard amino acid protecting group group with the exception of Dde-Lys (Fmoc) -OH is used. During head-to-tail cyclization, the N-terminal Fmoc-group is deprotected after the final amino acid synthesis is complete. Acetic acid / trifluoroethanol (TFE) / methylene chloride (MC) were mixed at a ratio of 2: 2: 6 to separate the protected peptide fragments (20 mMol) from the resin And treated with a mixed solvent (cleavage cocktail). After 1 to 2 hours, the resin is removed by filtration and the filtrate is recovered (4 mL x 2). The above procedure using the above-mentioned mixed solvent (cleavage cocktail) is repeated three times for the complete removal of the resin. Hexane was added to the filtrate, and acetic acid remaining in the filtrate was removed by azeotrope. The peptide coupled with the protecting group is recovered in the form of a white powder by repeating the process of adding MC and hexane and evaporating it.

Peptides are synthesized as cyclic peptides by head-to-tail cyclization of N-terminal amino and C-terminal carboxylic acid groups. In a typical cyclization reaction, 20 μmol (1 equiv.) Of the peptide fragment bound with the protecting group and 4 equiv. Of DIPEA are added to DMF (20 mL) and transferred to a syringe. Then add HATU 1 equiv. To DMF (20 mL) and transfer it to another syringe. To obtain diluted conditions at high magnification, the DMF solution containing the peptide and the DMF solution containing HATU were added to a mixed solution of 0.1 equiv. HATU and 1 equiv. Of HOBt in DMF (20 mL) using a syringe pump 0.0 > mL / min < / RTI > while stirring the solution. When the addition is completed, the reaction mixture solution is stirred for 12 hours. DMF is evaporated and the remainder is dissolved in MC and tert-butyl methyl ether / nucleic acid is added to the mixture to pulverize the peptide fragment bound with the cyclized protecting group. This process is repeated four times. The Dde group of the lysine group of peptides is selectively removed using a mixture of 2% (v / v) hydrazine / DMF and repeated four times every 2 minutes. The mixture of 2% (v / v) hydrazine / DMF and free Dde are removed by evaporation. The peptide fragments were powdered with MC and tert-butyl methyl ether / nucleic acid and removed with a protector mixture of trifluoroacetic acid (TFA) / diisopropylsilane (TIS) / water in a ratio of 95: 2.5: 2.5 Treat the cleavage cocktail for 3 hours. It is then pulverized with tert-butyl methyl ether. The peptide is purified by reverse phase HPLC (water / acetonitrile with 0.1% TFA). The molecular weight of the synthesized peptide was confirmed using a MALDI-TOP mass spectrometer. The concentration of synthesized peptide was determined by using a tryptophan molar extinction coefficient value (2205 M ^-1 cm ^-1 ) at 280 nm and using water / acetonitrile (1: 1) using the spectrophotometric method.

Example 2. Formation of a vesicle

The concentration of the peptide prepared in Example 1 during the formation of the vesicle is 20 [mu] M. The structure of self-assembled vesicles is controlled by the following various protocols.

Sonication

Peptide vesicles were sonicated for 20 min (~ 180 W; 221,510 J) using a cup-horn sonicator (Qsonica, USA).

Extrusion

Peptide vesicles are extruded through a mini-extruder (Avanti ^® Polar Lipids, Inc.) 10 times through a polycarbonate membrane (Whatman) with a pore size of 0.1 μm.

Flash Injection

Peptide vesicles are lyophilized and then dissolved in HFIP. The peptide solution (400 μM, 20 μL) is injected into an excess of distilled water (400 μL) momentarily and then the HFIP is evaporated. The final peptide concentration is 20 μM.

Dialysis

Peptide vesicles were lyophilized and hydrated with 30% HFIP (HFIP / Water, vol%). The peptide solution is transferred to a dialysis membrane (Spectra / Por ^® 6 standard RC Pre-wetted Dialysis Tubing, 1,000 Da MWCO, Spectrum Laboratories, USA) and dialyzed against distilled water.

Sonication of the Lyophilized Peptide from 30% HFIP

Peptide vesicles are lyophilized under reduced pressure and then dissolved in 30% HFIP (HFIP / Water, vol%). After the peptide solution is lyophilized, the peptide is rehydrated by distilled water. The solution containing the peptide is then sonicated for 20 minutes using a Cup-horn sonicator.

Experimental Example 1. Characteristic Experiment

(1) Measurement of circular dichroism spectroscopy (CD)

Circular dichroism was measured using a Chriscan ^TM Circular Dichroism spectrometer (Applied Photophysics, USA) equipped with a Peltier temperature controller.

The CD spectra of the samples were recorded at 260-190 nm using a 2 mm path length cuvette. The molar ellipticity was calculated for each amino acid residue.

(2) Transmission electron microscopy (TEM) analysis

2 [mu] l of the sample was completely dried on a carbon coated copper lattice. After the addition of 3 μl of 1% (w / v) uranyl acetate, the excess solution was removed using filter paper after 1 minute. The excess solution was wicked off using filter paper. The JEOL-JEM 2010 instrument The sample was observed at 120 kV using. The obtained data was analyzed using Digital Micrograph ^TM software.

(3) Leakage Experiment

A vesicle containing 5 (6) -carboxyfluorescein (FAM) is prepared under an excess of FAM fluorescent material, and the untrapped FAM is removed. The vesicles are treated with 1% (vol%) Triton X-100 at room temperature with gentle shaking. Fluorescence emission from the sample is recorded on a 384-well plate using a Victor X5 plate reader (Perkin Elmer, USA). Samples are excited at 495 nm and high emission filters are installed at 535 nm. To analyze the time-course emission data, the percentage of FAM released at a particular time is calculated using the following equation.

Equation 1.

% release = 100 (I _t - I ₀ ) / (I _∞ - I ₀ )

In the above Equation 1, I _t is the fluorescence intensity at a specific time, I ₀ is the fluorescence intensity before the surface treatment, and I _∞ is the fluorescence intensity at the time when the defoliation is completed.

(4) Tissue culture and intercellular delivery experiments

HeLa cells (1 × 10 ⁴ ) were dispensed into an 8-well Lab-Tek Ⅱ chamber cover glass system (Nunc, USA) and incubated with 10% fetal bovine serum (FBS) ) And DMEM (Dulbecco's modified Eagle's medium) containing 1% pen / strep (penicillin / streptomycin) at 5% CO ₂ and 37 ° C overnight (12 hours). The cultured cells were treated with Opti-MEM (ThermoFisher, USA) and treated with vesicles (20 μM) containing 5 (6) -carboxyfluorescein (FAM) for 16 hours . After the period, the sample solution was removed and replaced with Opti-MEM. Cells can be visually observed through a confocal microscope (LSM 8800, Carl Zeiss, Germany).

(5) Measurement of Zeta Potential Distibution

The zeta potential distribution was measured by dissolving the MCP prepared in Example 1 in water and the Zeta potentiol value was measured as +33.1 mV. The results are shown in Fig.

Results and review

1 and 2, a linear peptide (LP) or a marcrocyclic peptide (MCP) building block composed of a charged hydrophilic segment, a linker segment and a tryptophan-rich hydrophobic segment was prepared. In the new MCP, the lysine linked to the ε-amino group is located in the middle of the tryptophan-rich hydrophobic segment to improve the flexibility of the hydrophobic segment. The bendability of hydrophobic segments is important in the formation of cyclopeptidoses because it reduces the rigidity of the polypeptide chains and enhances the internal hydrophobic packing of the molecular assembly and chain entanglement. The hydrophilic peptide segment is derived from the Rev protein of human immunodeficiency virus type 1 (HIV-1), binds the RRE RNA of HIV-1 and controls the release of nuclear cytoplasmic HIV-1 RNA. Rev peptides contain enormous arginine and hydrophilic segments are positively charged.

The tryptophan-rich hydrophobic segment, which is composed of tryptophan, induces self-assembly of LP and MCP due to the hydrophilic nature of amino acids and the ability to laminate π-π. Because the indole chromophore in tryptophan is highly sensitive to the local environment, the inherent fluorescence of amino acids is used to monitor the process of self-assembly of molecules. The peptides were dissolved in pure water and the fluorescence emission of tryptophan excited at 280 nm was monitored. Referring to Figs. 3 (a) and 3 (b), the fluorescence intensities of the building blocks increase with increasing concentration (concentration dependent). The maximum emission is observed at about 360 nm. Plotting the emission intensity value at 360 nm as a function of peptide concentration confirms that the fluorescence value is suddenly increased. This discontinuity is represented by the critical aggregation concentration (CAC). The CAC is calculated using the intersection of the regression straight line of the linear dependent area. The calculated LP and MCP CACs are 1.7 and 1.4 μM, respectively. Therefore, the linear and annular building blocks have no significant difference in aggregation tendency. The CAC measurement was performed in a physiological buffer, PBC (phosphate buffered saline, CAC of LP and MCP was 0.5 and 0.7 μM, respectively.) As the ionic strength increases, the hydrophobic interaction increases, The decrease in the value of CAC between peptides is not significantly different. The decrease in CAC values in high ionic strength solutions indirectly demonstrates that they play an important role in the self-assembly of these peptide building blocks.

To further illustrate the structural characteristics of such an assembly, a circularly dichroism spectroscopy (CD) measurement was performed. Circular dichroism spectroscopy (CD) measurements were performed at the CAC concentrations of LP and MCP. Referring to Fig. 4, the LP spectral dichroism spectra show a strong negative minimum at 200 nm (Fig. 4a), indicating that the peptides do not have a clearly defined secondary structure. A negligible change in the LP spectral dichroism spectra of LP in the subsequent temperature ramp indicates that the peptide is not thermodynamically resistant or conditioned at low temperatures. In contrast to LP, the circularly dichroic spectroscopic spectrum of MCP shows a specific maximum at 239 nm (Figure 4b). A pair of excitons indicate the interaction between aromatic chromophores, in particular tryptophan. Therefore, it can be seen that the interaction between the tryptophan residues of MCP is significantly stronger than the interaction between the tryptophan residues of LP. The bands at 229 nm gradually disappear as the temperature increases due to the thermodynamic instability of the MCP assembly (Figure 4b). In particular, the MCP assembly exhibits reversible thermodynamic behavior (Figures 4c, d)

Next, the shapes of the LP assemblies were examined. LP was dissolved in pure water and then strongly sonicated using a high intensity Cup horn sonicator. Since water is an optional solvent for hydrophilic segments, tryptophan-rich hydrophobic segments can form the core of molecular assemblies. 5, transmission electron microscopy (TEM) analysis at a CAC concentration of LP is observed as a spherical object having a diameter of about 40 to 45 nm (Fig. 5a). Considering the molecular length of the fully extended LP, The observed nanoparticles may be vesicles. In further observations at somewhat higher concentrations, LP aggregated into irregular bodies (Figure 5b). As shown in the circularly dichroic spectra, the weak correlation between tryptophan at high concentrations was consistent with the heterogeneous LP response It can be a forming cause

Next, the MCP is dissolved in distilled water and ultrasonicated in the same manner as the LP. Referring to FIG. 6A, it can be confirmed that a vesicle is formed through TEM analysis. The size of the parcel is larger than the parcel of LP. MCP did not form irregular aggregates at high concentrations. The cyclic structure of unnatural MCP is the reason for strong aromatic interactions and continuous stable molecular assembly. In particular, the thickness of the vesicle wall was not constant, and the shape of the vesicle was polygonal rather than spherical (polygonal vesicles). These results indicate that the MCP has a molecular structure and composition suitable for forming vesicles in water. However, the inhomogeneity of the observed shape indicates that the vesicles are dynamically grabbed and stable. The hydrophobic action becomes stronger and the self-assembly is performed under physiologically appropriate buffered saline conditions in which the charge repulsion in the hydrophilic segment becomes weaker due to charge screening (Fig. 6b). The vesicles formed under these conditions are very irregular It is a form of corrugated vesicles that are far from perfect spheres. The formation of these corrugated vesicles implies an unreliable rearrangement between tryptophan due to enhanced crystallization of the hydrophobic segment at high ionic strength. Therefore, the corrugated vesicles are dynamically captured compared to the polygonal vesicles. Next, prepare a parcel by extrusion. (Fig. 6c, d) The unstable energy supplied to the molecular rearrangement (the detoxifying energy) was not uniformly distributed in the extrusion process, resulting in a mixed vesicle distribution . The round vesicles must be present in thermodynamic equilibrium. Therefore, vesicles having various shapes and roughness can be produced by the nature of the solvent and the manufacturing method.

By the possibility of structural regulation, we have studied the structural space of additional cyclopeptides and have attempted to invent a method for producing more uniform vesicles. The modified co-solvent method using hexafluoroisopropanol (HFIP), a general solvent, was used. Quiet methods (or solvent exchange methods) are commonly used to fine tune the thermodynamic and dynamic parameters of block copolymer assembly. HFIP is a strong inducer of alpha-helix. Therefore, MCP is expected to have an alpha helical form despite the thermodynamically stable form of the peptide. These results indicate that the MCP form promoting recombination into a thermodynamically stable form that forms a self-assembled structure is completely disrupted.

In order to quantify the conformational susceptibility of MCPs in HFIP, the secondary structure of molecules and peptides dissolved in a mixture of water and HFIP was investigated by means of circular dichroism spectroscopy. The conformation of MCP in pure water is almost random coil form. There is considerable migration of MCP in the secondary structure when the concentration of HFIP reaches 20%. Referring to FIG. 7, the minimum value of the double sound at 204 nm and 221 nm indicates that the MCP is mainly in alpha-helix form. The ellipticity increases slightly at a concentration of 40% HFIP 221 nm. When the concentration of HFIP is increased to 80%, a larger increase results in the similarity of helix stabilization. These results indicate that HFIP at approximately 20-40% concentration is a significant concentration that interferes with the form of significant peptides and the tendency to aggregate.

First, MCP was dissolved in HFIP, and then flash injection was performed using an excess amount of water. Very round vesicles distributed in uneven size were obtained through a rapid solvent exchange method. 8, it is interesting to note that most of the vesicles are surrounded by small strata, which represent a morphological transition from the corrugations while the vesicles are formed (Fig. 8a). Ultrasonic processing of such vesicles results in a small vesicle (Fig. 8b). Thus, the round vesicles formed by the flash incision are more likely to have a deep local minima than the global minimun on the potential energy surface. Value.

For the gradual conversion of the solvent from HFIP to water, the MCP was dissolved in 30% HFIP and dialyzed with pure water. This slow, persistent solvent conversion results in the formation of fairly uniform, rounded vesicles (Figure 8c). For further degradation of the MCP building block, the MCP is dissolved in 50% HFIP and dialyzed with pure water . As a result, a very round vesicle was formed (Figure 8 d). An additional elongated vesicle fusion was found. This is probably due to enhanced mixing of hydrophobic domains affected by common solvents.

Next, a simpler quiet method was devised. After dissolving the MCP in 30% HFIP, the crushed peptide form was frozen with the expectation that it would remain frozen. The dried samples were rehydrated with water and then strongly sonicated (ca. 2 × 10 ⁵ Jules). Referring to Figure 9, very round and uniform peptide vesicles were obtained by this method. These vesicles exhibit a global minimun because the MCP has been broken down by HFIP and ultrasonication.

A time-dependent emission profile of FAM treated with Triton X-100 (Triton X-100) on the membrane of pulverized vesicles after recognition of fluorescent markers (FAM) in vesicles under stable experimental conditions to form uniform vesicles in thermodynamic equilibrium state, Were observed. Typical liposomal release trends are very rapid. 10, the release of the peptide enclosed in the capsule is completed within approximately 20 seconds (a, inset in FIG. 10). In contrast, the release of cyclopeptidomes is relatively slow. ) The time window of cyclopetiomesolysis is approximately one hour. This slow release kinetic indicates that the cyclopeptidomes are structurally more robust than liposomes.

All rich-arginine peptides have cell penetration ability. Because the MPC cyclopeptide is surrounded by a rich-arginine hydrophilic segment, the cyclopeptidom can mediate the intracellular transport of entrapped molecules. To address these questions, HeLa cells were treated with MCP cyclopeptidomes harboring FAM molecules, and the intracellular distribution of fluorescence was monitored through a confocal microscope (CLSM). Referring to FIG. 10C, all cells showed bright green fluorescence of FAM. This demonstrates an effective intracellular delivery of cyclopeptidomes.

conclusion

Linear and cyclic peptides were synthesized by vesicles and self - assembly behavior was investigated by dynamic and thermodynamic control. The peptide vesicles showed high colloidal stability as shown in the zeta potential measurement (Fig. 11), and no recognizable precipitates were found after storage for several months at room temperature. Comparison of the phase effects of self-assembly shows that strong interaction of the aromatic hydrophobic segments is found only in the cyclic peptide. This is due to the flexible structure and limited molecular degrees of freedom. As in the results, linear peptides aggregated nonspecifically as the concentration increased, even though they tend to form vesicles. Conversely, the cyclic peptides formed stable vesicles even at high concentrations. Depending on the method of adjustment, various forms of polygonal, corrugated, round, round-patched and round-fused vesicles were found in cycle peptides. These results indicate that the structural space of the peptide vesicles or cyclopeptidomes is very large. At present no relevant structural studies of cyclopeptidases have been found in related field studies. The wider soluble structural space of the cyclopeptidomes will be investigated using modulation and preparation methods of peptide structures, and their various physico-chemical and biological systems will be investigated. Because the cyclopeptidomes are surrounded by bioactive peptides, the cyclopeptidomes can be used in a variety of applications including intracellular delivery and molecular recognition. In addition, the structural robustness of the cyclopeptidomes will enable the development of nanotransport of slow release kinetic.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A hydrophobic segment of Sequence 1 below; And
A charged hydrophilic segment of SEQ ID NO: 2,
The lysine (K) of SEQ ID NO: 1 below is linked to tryptophan through an epsilon amino group, a cyclic self-assembled peptide nanostructure.
[SEQ ID NO: 1]
WWKWW
[SEQ ID NO. 2]
TRQARRNRRRRWRR
delete delete delete The method according to claim 1,
Self-assembled peptide nanostructures capable of regulating structure by thermodynamic or kinetic control.
6. The method of claim 5,
Wherein the self-assembled peptide nanostructure is formed by dynamically controlling the self-assembled peptide nanostructure under a quiet environment.
The method according to claim 6,
Wherein the co-solvent is hexafluoroisopropanol (HFIP).
8. The method of claim 7,
Wherein the concentration of the HFIP is 20 to 40% (HFIP / water, vol%).

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