KR101657040B1

KR101657040B1 - Nanofibril conjugate for detection of metal ion and preparation method thereof

Info

Publication number: KR101657040B1
Application number: KR1020150074068A
Authority: KR
Inventors: 이은지; 김인혜; 김용재; 김건희
Original assignee: 충남대학교산학협력단; 한국기초과학지원연구원
Priority date: 2015-05-27
Filing date: 2015-05-27
Publication date: 2016-09-19

Abstract

FIELD OF THE INVENTION The present invention relates to nanofibril complexes and methods for their preparation, and more particularly to nanofiber complexes comprising pyrene-linked peptides composed of alkyl chains; And a histidine-rich peptide coupled to the peptide. The nanofiber complex through the present invention is expected to be useful for the detection of various kinds of metal ions including silver ions.

Description

TECHNICAL FIELD The present invention relates to a nanofibrillar complex for metal ion detection and a method for producing the nanofibril complex,

FIELD OF THE INVENTION The present invention relates to nanofibril complexes and methods for their preparation, and more particularly to nanofiber complexes comprising pyrene-linked peptides composed of alkyl chains; And a histidine-rich peptide coupled to the peptide.

Self-assembly of peptide amphiphiles (PAs) can be carried out in aqueous solution using micelles, barrels, vesicles, nanofibrils (NFs), ribbons, Thereby forming various nanostructures such as a tube. The peptides have been recognized as attractive biomaterials due to their properties including excellent biodegradability, biocompatibility and bioactivity. Through the understanding of the self-assembling properties of amphipathic peptides, molecular-based tissue nanostructures have provided new opportunities for the development of new functional materials for applications such as tissue engineering, regenerative medicine, target therapy delivery and diagnosis. For example, of the amphipathic peptide-based nanostructures, a one-dimensional sheet rich amphipathic peptide fibril structure is known to play an important role in neurodegenerative disease. In addition, the intrinsic propensity of the amphipathic peptide to form core-shell aggregation in an aqueous solution has the advantage of easily imparting desired chemical and biological activity to a specific region of the nanostructure. However, since the nanofibrils change the lamination of molecules in the fiber core by showing a photoreaction in the physiological environment, many studies have not been made.

Accordingly, the present invention provides a novel molecular system for detecting biologically valuable metal ions using a peptide building block and an amphipathic peptide.

The present invention relates to pyrene-labeled peptide amphiphiles composed of hydrophobic, linear or branched alkyl chains; And a hydrophilic histidine-rich peptide coupled to the peptide. &Lt; RTI ID = 0.0 > [0002] < / RTI >

The present invention also provides a method for detecting a metal ion in a nanofibrillin complex comprising pyrene butyric acid and N-terminal histidine-rich peptide covalently bonded to a linear or branched dodecanoic acid. And a manufacturing method thereof.

The present invention also relates to a composition for cell imaging comprising the complex.

The present invention also relates to an antimicrobial composition comprising metal ions bound to the complex.

The nanofiber complex through the present invention is expected to be useful for the detection of various kinds of metal ions including silver ions.

Figure 1 is a visualization of a water soluble self-assembled nanostructure of an amphipathic peptide.
(c) TEM image of NFs 2, (d) cryo-TEM image of NFs 2, (e) hydrodynamic diameter of TEM image of NFs 1, (b) cryo-TEM image of NFs 1, And (f) the emission spectrum before and after addition of Cu ²⁺ at 0.1 mM of NFs in water.
Figure 2 shows the result of sensing of Cu ²⁺ using nanofibrils.
(a) Clean (unstained) TEM image, (b) clean (unstained) of NFs 2 TEM image, (c) the NFs 1 Cu ²⁺ - MALDI-TOF mass spectrum of the nano-fibril composite, (d) Cu ²⁺ (E) TEM image of NFs 1 combined with Cu ²⁺ particles, and (f) TEM image of NFs 2 combined with Cu ²⁺ particles.
Figure 3 shows the results of supramolecular fluorescence nanoprobe behavior measurement of an amphipathic peptide for metal ions.
(a) the fluorescence intensity of NFs 2 due to addition of metal ions, (b) the fluorescence intensity ratio measured at 396 nm ( F _metal / F _{metal free} ), (c) coexistence of various metal ions measured at 340 nm under a fluorescence spectrum, and (d) of Cu ²⁺ ions is TEM image of the NFs associated with the nanoparticles.
Figure 4 is a cell-imaging result.
(a) a bright field, (b) 10 μM NFs 2 and HeLa cell lines, and (c) 150 μM AgNO ₃ and NFs 2.
Figure 5 shows the result of antimicrobial activity measurement.
(a) the growth curve of E. coli in 10 ug / mL Ag nanoparticles (NPs) -NFs 2, (b) turbidity measurement of the culture of E. coli O157: H7, (c) GFP expression results using microchips, Growth curves of Bacillus subtilis in 10 ug / mL Ag silver nanoparticles - NFs 2.

In the present invention, the histidine-rich peptide may have an amino acid sequence composed of histidine and glycine, though not limited thereto. Histidine-rich peptides are molecular recognition scaffolds for binding metal ions to the active site of a protein because of its highly efficient imidazole-nitrogen donor atom at the histidyl residue, It has attracted attention. For example, in the present invention, the histidine-rich peptide may be an amino acid sequence of HGGGHGHGGGHG (HG12). The HG12 peptide is immobilized on a previously prepared nanotube to form a complex with Cu2 ⁺ or Ni2 ⁺ ions, thereby leading to the formation of nanotubes coated with metal nanoparticles under a reducing agent.

In the present invention, the pyrene is not limited but may be linked to the N-terminal of the histidine-rich peptide.

In the present invention, the complex is not limited, but may exhibit fluorescence by binding with a metal.

In the present invention, the structure of the pyrene-linked peptide is not limited, but it may have a structure of the formula (II) wherein pyrene is combined with a hydrophobic linear peptide and a pyrene-linked peptide of the formula I or a hydrophobic branching peptide as follows.

(I)

&Lt; RTI ID = 0.0 &

But not the structure of the complex is limited in the present invention, for example to and can have the form of formula (III) or (IV), those having a branched structure of Formula IV as in the embodiment of the invention with Ag ⁺ And is more efficient in exhibiting antibacterial activity through binding with the same metal ion.

(III)

(IV)

In the present invention, the metal ion is not particularly limited, but Ag ⁺ , Ca ²⁺ , Co ²⁺ , Cu ²⁺ , Fe ²⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Pb ²⁺ or Zn ²⁺ Can be used, and Ag ⁺ can be preferably used.

In the present invention, the preparation method is not limited, but may further include a step of purifying a pyrene-labeled peptide using a standard Fmoc (9-fluorenylmethoxycarbonyl) method.

In the present invention, the antimicrobial composition may have antimicrobial properties against Gram-negative or Gram-positive bacteria, though not limited thereto. The Gram-negative or Gram-positive group is not particularly limited as long as it is a bacterium having the corresponding property. For example, as used in one embodiment of the present invention, Gram-negative bacteria include Escherichia coli O157: Gram-positive bacteria can be Bacillus subtilis ( Bacillus subtilis ).

Hereinafter, the present invention will be described in detail with reference to examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited by the following examples.

[Example 1] Synthesis and purification of an amphipathic peptide

Amphiphilic peptides were synthesized on Rink amide MBHA resin (100-200 mesh, Merck) using standard Fmoc (9-fluorenylmethoxycarbonyl) method.

Pyrene butyric acid (Alfa Aesar) is shared at the N-terminus of the HGGGHGGGGGHG (HG12) peptide using linear or branched dodecanoic acid of the following general formula (I) and general formula Lt; RTI ID = 0.0 > (IV) < / RTI >

(I)

&Lt; RTI ID = 0.0 &

(III)

(IV)

Amphiphilic Peptide 1 (PA 1) Synthesis

The pyrene labeled amphipathic peptide 1 was prepared using conventional solid phase peptide synthesis procedure using a ^{CEM Focused Microwave TM Synthesis System (Discover} ) on Rink amide MBHA resin. The resin was washed with dichloromethane (DCM; Daejung Chemicals), placed in a shaking incubator with a 1: 1 mixture of DMF (N, N-dimethylformamide; Fisher) and DCM .

Then, a mixture of Fmoc-His (Trt) -OH (5 equiv .; Beadtech) and Fmoc-Gly-OH (5 equiv .; Merck) was ligated to the N-terminus of the peptide in the resin having the sequence of GHGGGHGHGGGH-Resin . Amphipathic peptide 1 was synthesized by attaching Fmoc-12-aminododecanoic acid directly to HG 12 peptide and 1-pyrene butyric acid.

After the above procedure, the resin was dissolved in TFA (trifluoroacetic acid, 99%; Sigma-Aldrich): 1,2-ethanedithiol (98%; Merck): thioanisole 95: , And 5: 2.5. The mixed solution was triturated with tert-butyl methyl ether.

Amphiphilic peptide 1 was purified by reverse phase HPLC on a C18 column using a linear gradient with 0.1% TFA in water and 0.1% TFA in acetonitrile.

Amphiphilic Peptide 2 (PA 2) Synthesis

The pyrene labeled amphipathic peptide 2 was prepared using conventional solid phase peptide synthesis procedure using a ^{CEM Focused Microwave TM Synthesis System (Discover} ) on Rink amide MBHA resin. The resin was washed with dichloromethane (DCM; Daejung Chemicals), placed in a shaking incubator with a 1: 1 mixture of DMF (N, N-dimethylformamide; Fisher) and DCM .

Then, a mixture of Fmoc-His (Trt) -OH (5 equiv .; Beadtech) and Fmoc-Gly-OH (5 equiv .; Merck) was ligated to the N-terminus of the peptide in the resin having the sequence of GHGGGHGHGGGH-Resin . Fmoc-Lys (Fmoc) -OH (Sigma-Aldrich) was coupled to GHGGGHGHGGGH-Resin to synthesize amphipathic peptide 2 as a branched alkyl chain, and Fmoc-12-aminododecanoic acid was further reacted with HG 12 peptide And 1-pyrenebutyric acid.

Amphiphilic peptide 2 was purified by reverse phase HPLC on a C18 column using a linear gradient with 0.1% TFA in water and 0.1% TFA in acetonitrile.

The obtained product was purified to obtain amphipathic peptides each having a purity of 95% or more. The molecular weights of the purified peptides were determined by MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight; Bruker Ultraflextreme) mass spectrometry.

[Example 2] Self-assembly behavior of an amphipathic peptide on an aqueous solution

The purified amphiphilic peptide was measured using a TEM (JEM-0311 HR, 300 kV and JEM-1400, 120 kV) to observe self-assembling behavior in an aqueous solution (Fig. 1). Observation of aggregates of the amphipathic peptide 1 of the formula 1 containing a linear alkyl chain revealed micrometer-unit length nanofibrils (NFs) with a constant diameter around 9.5 nm on average. The presence of water soluble NFs with an average diameter of 4.2 nm was confirmed by cryo-TEM (cryo-TEM; JEM-0311 HR; 300 kV, temperature range) measurement. On the other hand, when the agglomerates of the amphipathic peptide 2 of Formula 2 containing the alkyl chain composed of two branches were observed, both the average diameter and the length of the NFs were decreased as compared with the case of containing the linear alkyl chain. The hydrodynamic diameters (RH) were 6.6 μm and 4.9 μm, respectively. The diameters of TEM of NFs of amphipathic peptide 2 decreased to 8.2 and to amphipathic peptide 1, whereas diameters measured with cryo-TEM increased to 5.7 nm, which was contradictory.

The unpredictable increase in the diameter of the hydrophobic core can be interpreted as a new phenomenon due to the change in the internal molecular stacking due to the introduction of the branched dodecanooxy terminus. Changes from linear alkyl chains to branched alkyl chains result in stereoselective constraints between the HG12 peptides, resulting in enhanced aggregation between the pyrenyl group and the [beta] -sheet structure of the peptide, resulting in the formation of nanofibrils (NFs ) Was decreased. From the spectrum of FIG. 1 (f), it can be seen that the amphipathic peptide 1 into which the linear alkyl-chain was introduced exhibited the general interdigitated bilayer assembly structure while the amphipathic peptide 2 had the inherent bilayer structure there was.

[Example 2] Sensing of Cu ²⁺ using nanofibrils

The phenomenon in which the histidine-rich peptide HG12-coated nanofibrils (NFs) of the above example recognized metal ions was tested using Cu ²⁺ .

It was confirmed that the fiber structure having a high aspect ratio was maintained while a significant quenching phenomenon occurred after the addition of the paramagnetic Cu ²⁺ ion (Fig. 1 (f)). In particular, the lambda _max of amphipathic peptide 2 shows a shift from a dynamic excimer to a partially overlapping stable dimer, such as sandwich, by blue-shifting from 478 nm to 455 nm there was. This means that HG12 peptides capture Cu2 ⁺ ions and induce closer molecular interactions between the pyrenyl groups. TEM images of the nanofibrils of amphipathic peptides 1 and 2 were measured and it was confirmed that there was a significant improvement in mass contrast without staining (FIG. 2).

The association with NFs in the presence of Cu ²⁺ ions was performed using MALDI-TOF / TOF mass spectrometry. As can be seen from FIG. 2 (c), the ratio of m / z before and after Cu ²⁺ ion binding increased from 1491.9 to 1553.8 for amphipathic peptide 1 and increased from 2089.0 to 2136.0 for amphipathic peptide 2 .

In addition, in order to verify that the Cu ²⁺ ion is bound to NFs, FTIR (Fourier transform infrared) measurement can be used to confirm the oscillation of the NH and C═C bonds of the histidine side chain as shown in FIG. 2 (d). The Cu ²⁺ ions according to the addition of the frequency v of the _NH NH is decreased from 3500 to 3490 cm ^-1, C = C of the frequency v _{C = C} is increased by from 1494 to 1508 cm ^-1, wherein the Cu ²⁺ ions Is chelated with the histidine residue of HG12. Also, it was predicted that the amide II band peak at 1543 cm ^-1 of amphipathic peptide 1 was greatly reduced, making the β-structure of HG12 very unstable.

The direct confirmation of the formation of NFs bound to Cu ²⁺ ions was made by adding NaBH 4 and visual confirmation by reduction with metal ions. As shown in FIGS. 2 (e) and 2 (f), the formation of nanocrystals can be confirmed through addition of the substance. In the case of using amphipathic peptide 1 and amphiphilic peptide 2, the diameters are about 11.6 nm and 21.6 nm, .

Example 3 Supramolecular fluorescent nanoprobe behavior of an amphipathic peptide to a metal ion

In order to confirm the metal binding affinity of the water soluble NFs, Ag ⁺ , Ca ²⁺ , Co ²⁺ , Cu ²⁺ , Fe ²⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Pb ²⁺ or Zn ²⁺ And the affinity was measured by using various kinds of metal ions.

The critical micellar concentration (CMC) of the amphipathic peptide in water was measured using a fluorescence spectrometer (Perkin Elmer LS-55), and the ability of NFs as a metal complex was measured in a dilute solution . As a result of measuring the intensity ratio ( I ₃₉₆ / I ₃₇₈ ) of emission peaks at two wavelengths, it was found to be very sensitive to the polarity of the medium (FIG. 3). From the low measured values, the amphipathic peptides of the present invention were found to be superior to supramolecular 1D nanoprobes in a dilute solution. The amphipathic peptides 1 and 2 were found to exhibit 7.5 袖 M and 6.1 袖 M, respectively, It can be used as

For amphiphilic peptide 1 among the metal ions used in this example, chelated quenching phenomenon occurred in the case of Cu ²⁺ , Fe ²⁺ , and Ni ²⁺ as the metal ion concentration increased.

In particular, as shown in FIG. 3 (a), when 7.6 μM of amphiphilic peptide 2 was used in water, it was confirmed that fluorescence increased dramatically with addition of ion (Ag ⁺ ). As can be seen from FIG. 3 (b), silver ions (Ag ⁺ ) exhibited the greatest improvement in fluorescence emission, while Cu ²⁺ ions produced the greatest quenching effect.

Cu ²⁺ ion was added to the experimental group to which each ion was added. The result was that the ion (Ag ⁺ ) interferes with the quenching effect due to the binding of the Cu ²⁺ ion to the amphipathic peptide 2 (FIG. 3 (c) AgNO ₃ was added to the amphiphilic peptide 2, and the reduction reaction was continuously induced with NaBH _{4. As} a result, a spherical agglomerate was formed in black contrast as shown in the TEM image of FIG. 3 (d) Could know.

From the above results, it was confirmed that the amphiphilic peptide 2 can selectively enhance the fluorescence of the silver ion (Ag ⁺ ), and that the silver nanoparticle has the ability to convert the silver nanoparticles into hybridized NFs. Respectively.

[Example 4] Cell-imaging < RTI ID = 0.0 >

HeLa cells were cultured in DMEM (Dulbecco's modified Eagle's medium) and 10% FBS (fetal bovine seume). All cells were supplied with an antibiotic-antifungal solution mixed with 100 units / mL penicillin, 0.1 mg / mL streptomycin and 0.25 mg / mL amphotericin B and incubated at 37 ° C in standard culture conditions were incubated for 12 hours in a 5% CO ₂ and 95% humidity. To prevent the formation of insoluble AgCl precipitate, a buffer containing no chloride ion of pH 6.8 (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) containing 1.0 mM sodium nitrate was used. Cells were first loaded with 1OuM of amphipathic peptide 2 (PA 2) in HEPES at 37 ° C for 1 hour. Then, free PA 2 was removed by washing three times with 20 mM HEPES buffer solution of pH 6.8 containing 1.0 mM sodium nitrate. The cells were then incubated for 30 min in 20 mM HEPES containing 150 μM silver nitrate.

The formation of biocompatible amphipathic peptide-based NFs has enabled the pursuit of potential biomedical applications. In the present invention, although the specific data are not disclosed, a standard survival experiment was performed on HeLa cells using 10 [mu] M of NF, indicating a high cell survival rate of about 93%. In particular, the amphiphilic peptide can be utilized in the field of cell imaging through selective fluorescence emission enhancing ability of NFs using Ag ⁺ ions confirmed from the above embodiments. Nanofibrils on which Ag ⁺ ions are immobilized allow us to investigate whether hybrid NFs have the potential to induce an antibacterial effect. However, since the reduced form of Ag compared to Ag ⁺ ion shows much less toxicity in animal cells, the process of reducing the form of Ag ⁺ ion adhering to the surface of NFs was further performed.

Whole HeLa cells were cultured in 10 μM amphipathic peptide 2 at 37 ° C for 1 hour without Ag ⁺ ion. Then, 20 mM HEPES buffer solution (pH 6.8) containing sodium nitrate (NaNO ₃ ) was added instead of sodium chloride (NaCl) , And it was observed whether it exhibited fluorescence (Fig. 4 (b)).

As shown in FIG. 4 (c), when the cells were incubated in 150 μM AgNO ₃ for 1 hour at a temperature of 37 ° C., a prominent increase in fluorescence was obtained in the cells 4.1-fold. These results indicate that amphipathic peptide 2 can penetrate cells and exhibit fluorescence signals through interaction with intracellular silver ions (Ag ⁺ ). Although amphipathic peptide 2 does not contain a cell permeable peptide such as 'TAT', arginine- and lysine-rich peptides, the presence of HG12, which is bound externally to NFs, . It is known that histidine-containing peptides can interact with cell membranes through electrostatic action and can penetrate cell membranes under mild acid conditions.

[Example 5] Measurement of antimicrobial activity

The antimicrobial activity of the Ag nanoparticles -NFs was investigated by the inhibition of growth of Gram-negative bacteria Escherichia coli 157: H7 and Gram-positive bacteria Bacillus subtilis .

(AgNPs-NFs) (amphipathic peptide 2) into which Ag nanoparticles (AgNPs), NFs (amphipathic peptide 2) and Ag nanoparticles were introduced were each kept in air and added to LB (Luria-Broth) / mL, Escherichia coli 157: H7 and Bacillus subtilis were inoculated and cultured, and then the microorganisms were injected into the culture medium. Hereinafter, the growth curve of the microorganism is shown by measuring the absorbance at 600 nm with time (Figs. 5 (a) and (d)). As can be seen from the figure, it can be confirmed that the presence of AgNPs-NFs significantly inhibits the growth of the microorganism as compared with the control group without any treatment. As a result of examination of the turbidity of the culture solution against E. coli after 7 days, it can be seen that when AgNPs-NFs is present as shown in FIG. 5 (b), it remains as a transparent medium. From this, it can be inferred that microorganisms hardly survived in the presence of the substance. As a result, it was confirmed that AgNPs gave long-lasting antibacterial activity through connection with NFs. On the other hand, the experimental group treated with AgNPs or NFs alone did not inhibit microbial growth as a result.

FIG. 5 (c) shows the GFP (green fluorescence protein) expression after 12 hours of the experiment, and E. coli was grown in the remaining three experimental groups except for AgNP-NF after 24 hours . From these results, it can be seen that AgNPs-NFs has a very efficient antimicrobial activity in the Gram-negative bacterium Escherichia coli compared to AgNPs, and this result is consistent with the results shown in FIGS. 5 (a) and 5 (b).

Claims

A pyrene-labeled peptide consisting of a hydrophobic linear or branched alkyl chain; And a hydrophilic histidine-rich peptide coupled to the peptide, the nanofibril complex comprising:
Wherein the pyrene-linked peptide has the structure of Formula (I) or Formula (II).
(I)

&Lt; RTI ID = 0.0 &

.

The method according to claim 1,
Wherein the histidine-rich peptide has an amino acid sequence of HGGGHGHGGGHG.

The method according to claim 1,
Wherein said pyrene is linked to the N-terminal of a histidine-rich peptide.

The method according to claim 1,
Wherein said complex is combined with a metal to exhibit fluorescence.

delete

Covalently bonding pyrene butyric acid and a N-terminal histidine-rich peptide to a linear or branched dodecanoic acid, to prepare a nanofiber complex of claim 1 for metal ion detection. Way.

The method according to claim 6,
Wherein the metal ion is Ag ⁺ , Ca ²⁺ , Co ²⁺ , Cu ²⁺ , Fe ²⁺ , Mg ²⁺ , Mn ²⁺ , Ni ²⁺ , Pb ²⁺ or Zn ²⁺ .

8. The method according to claim 6 or 7,
Wherein the preparation method further comprises a step of purifying the pyrene-labeled peptide using a standard Fmoc (9-fluorenylmethoxycarbonyl) method.