PRODUCING METHOD OF ANTIMICROBIAL PEPTIDE WITH ENHANCED ADHESION AND USES THEREOF

Abstract

The present invention relates to a method for preparing an antimicrobial peptide with enhanced adhesion, and an antimicrobial coating method of the antimicrobial peptide with enhanced adhesion. The antimicrobial coating method of the present invention can be widely used as a method for adding an antimicrobial property to medical products and industrial products, because the method is capable of coating the antimicrobial peptide with enhanced adhesion, which has no cytotoxicity and possesses an excellent antimicrobial activity, on various surfaces with optimal efficiency.

Claims

1. An antimicrobial peptide with enhanced adhesion, wherein a linker and 3,4-dihydroxyphenylalanine (DOPA) are conjugated to an end of an antimicrobial peptide.

2. The antimicrobial peptide of claim 1, wherein the antimicrobial peptide is NKC comprising an amino acid sequence of SEQ ID NO: 1.

3. The antimicrobial peptide of claim 1, wherein the linker comprises an amino acid sequence of SEQ ID NO: 2.

4. The antimicrobial peptide of claim 1, wherein the linker minimizes mutual activity inhibition between the antimicrobial peptide and the DOPA.

5. The antimicrobial peptide of claim 1, wherein the DOPA is linked as a group of 3 to 7.

6. A method for preparing an antimicrobial peptide with enhanced adhesion, comprising conjugating a linker and 3,4-dihydroxyphenylalanine (DOPA) to an end of an antimicrobial peptide to prepare an antimicrobial peptide with enhanced adhesion.

7. An antimicrobial coating method, comprising: (a) conjugating a linker and 3,4-dihydroxyphenylalanine (DOPA) to an end of an antimicrobial peptide to prepare an antimicrobial peptide with enhanced adhesion; and (b) coating the antimicrobial peptide with enhanced adhesion on a surface.

8. The antimicrobial coating method of claim 7, wherein the coating is carried out by coating the antimicrobial peptide with enhanced adhesion at a concentration of 50 M to 200 M.

9. The antimicrobial coating method of claim 7, wherein the coating is carried out for 10 minutes to 10 hours.

10. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 is a graph showing the analysis results of adhesion according to the number of DOPA moieties conjugated to NKC.

[0046] FIG. 2 is a graph showing the analysis results of an antimicrobial activity according to the number of DOPA moieties conjugated to NKC and the concentration of the adhesive antimicrobial peptide.

[0047] FIG. 3 is diagrams showing the analysis results of coating ability according to coating time.

[0048] FIG. 4 is graphs showing the analysis results of an antimicrobial activity according to coating time.

[0049] FIG. 5A-B are diagrams showing the analysis results of coating ability according to substrates (polystyrene, titanium, PDMS). Specifically, FIG. 5a is a diagram showing the analysis results of coating ability according to XPS analysis, and FIG. 5b is a diagram showing the analysis results of AFM images.

[0050] FIG. 6 is graphs showing the analysis results of an antimicrobial activity according to substrates (polystyrene, titanium, PDMS).

[0051] FIG. 7 is a diagram showing an effect of preventing microbial adhesion by the coating of the antimicrobial peptide.

[0052] FIG. 8 is a graph identifying the persistence of an antimicrobial activity of the coated antimicrobial peptide.

[0053] FIG. 9 is graphs showing the results of the cytotoxicity of the antimicrobial peptide coating at a concentration of 100 M.

[0054] FIG. 10A-B are graphs showing the results of the cytotoxicity of the antimicrobial peptide coating at a concentration of 200 M. Specifically, FIG. 10a is a graph identifying the hemolytic activity of the antimicrobial peptide coating, and FIG. 10b is a graph identifying the cytotoxicity of the antimicrobial peptide coating against human cells.

DETAILED DESCRIPTION OF THE EMBODIMENT

[0055] Hereinbelow, the present invention will be described in detail with accompanying exemplary embodiments. However, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention.

Experimental Example 1: Manufacture of Adhesive Antimicrobial Peptides

Experimental Example 1-1: Imparting of Adhesion to Antimicrobial Peptides

[0056] In order to utilize NKC, which is a peptide exhibiting a strong antimicrobial activity against a wide range of microorganisms, as an antimicrobial coating agent, an adhesive property was imparted to antimicrobial peptides by conjugating 3,4-dihydroxyphenylalanine (DOPA), which is a non-natural amino acid known to play an important role in providing adhesion strength in mussel adhesive proteins, which are biocompatible and exhibit strong adhesion regardless of surfaces, to an end of the NKC. Herein, in order to determine the optimal number of DOPA, 1, 3, 5, or 7 DOPA moieties were conjugated to an end of the NKC, and then GGGGS, which is a flexible linking sequence, was added between NKC and the DOPA sequences to prevent the NKC and DOPA from affecting each other's activity. The amino acid sequence information of the conjugated NKC and DOPA is shown in Table 1 below.

TABLE-US-00001 TABLE1 MIC Name Aminoacidsequence* M.W. [M] NKC APKAMKLLKKLLKLQKKGI 2148.8 1 NKC-(G.sub.4S).sub.2- APKAMKLLKKLLKLQKKGIGGGGSGGGGSO 2960.5 8 DOPA.sub.1 NKC-(G.sub.4S).sub.2- APKAMKLLKKLLKLQKKGIGGGGSGGGGSOOO 3318.2 8 DOPA.sub.3 NKC-(G.sub.4S).sub.2- APKAMKLLKKLLKLQKKGIGGGGSGGGGSOOOOO 3676.3 8 DOPA.sub.5 NKC-(G.sub.4S).sub.2- APKAMKLLKKLLKLQKKGIGGGGSGGGGSOOOOOOO 4035.5 16 DOPA.sub.7 *DOPA is abbreviated as O.

Experimental Example 1-2: Identification of Antimicrobial Activity of Adhesive Antimicrobial Peptides

[0057] In order to confirm the antimicrobial activity of the adhesive antimicrobial peptides prepared in Experimental Example 1-1, the minimal inhibitory concentration (MIC) which inhibits the growth of E. coli was identified by using a method disclosed in Nature Protocols. 2008; 3(2):163-175. Briefly, a culture medium in which a single colony had been grown for 16 hours was subcultured and grown until OD.sub.600 reached 0.4 to 0.6, which corresponds to logarithmic growth. Thereafter, the number of cells was calculated according to the OD values, and the number of cells was set at 10.sup.6 cell/mL. When a Mueller-Hinton Broth (MHB) was loaded to a polypropylene 96-well plate, 90 L was added to the first row of the plate and 100 L was added to the control wells, and 50 L was added to the remaining wells. Thereafter, the peptide solution was prepared at a concentration of 640 M, and 10 L thereof was added to each well of the first row. The peptide solution was mixed with the medium using a multi-pipette, and then 50 L was transferred to the second row (B). At this time, the peptide was diluted to half concentration, and the peptide was transferred up to the seventh row by the same method so that the peptide (64 M to 1 M) was dissolved in the medium. 10.sup.6 cell/mL of Escherichia coli, which had been prepared previously, was added thereto, and then the plate was cultured for 18 hours. Thus, the final concentration range of the peptide used in the experiment became 32 M to 0.5 M. After culturing for 18 hours, the OD values were measured using a microplate reader (Tecan i-control 200 pro), and the MIC values were identified.

[0058] As a result, as shown in Table 1, it was found that the MIC value of the NKC to which DOPA was not conjugated was 1, and that the MIC value of the NKC to which a linking sequence and one or more DOPA moieties were conjugated was 8 to 16. Therefore, it was confirmed that the antimicrobial activity of the NKC with enhanced adhesion was reduced. However, the MIC values of this result were considered to be sufficient to kill bacteria, as the values were of similar levels when compared to the MIC values of another antimicrobial peptide (LL-37 (MIC: 4 M to 32 M)).

Experimental Example 2: Identification of Functionality of Coating Composition Using Adhesive Antimicrobial Peptides

Experimental Example 2-1: Identification of Coating Ability of Adhesive Antimicrobial Peptides According to Number of Conjugated DOPA Moieties

[0059] The level of adhesion according to the number of conjugated DOPA moieties is unknown. Therefore, in order to determine the optimal number of DOPA moieties, the peptides (100 M) prepared in Example 1-1 were coated on a polystyrene surface for 12 hours, and then the amount of the attached peptides was identified. Specifically, the peptide solution (100 M) was added to a polystyrene 24-well plate in an amount of 300 L, and the mixture was coated in an incubator at 37 C. for 12 hours. After the coating, the solution in the wells was pipetted to determine its volume, and the concentration of the uncoated peptides was determined by a BCA assay. Finally, the amount of the peptides was calculated considering the volume of the solution after the coating. The calculated amount was compared with the amount of the peptides in the solution before the coating, and then the amount of the peptides present in the solution and on the surface was determined.

[0060] As a result, as shown in Table 1, it was confirmed that the coating ability increased as the number of DOPA moieties increased. Particularly, the coating ability increased greatly when the number of DOPA moieties was 3 or more.

Experimental Example 2-2: Identification of Antimicrobial Activity in State where Adhesive Antimicrobial Peptides are Coated

[0061] In order to confirm the antimicrobial activity in the state where the antimicrobial peptides are coated, the experiment was carried out by an ISO 22196 method in which some steps were changed. Based on the results of Experimental Example 2-1 above, the experiment on the antimicrobial activity was carried out by only using the NKCs to which 3, 5, and 7 DOPA moieties were conjugated (i.e., NKC-L-O.sub.3, NKC-L-O.sub.5, NKC-L-O.sub.7, respectively). Each of NKC-L-O.sub.3, NKC-L-O.sub.5, and NKC-L-O.sub.7 was coated on a polystyrene surface at different concentrations of 25 M, 50 M, and 100 M, and then E. coli in logarithmic phase (10.sup.6 cell/mL) was cultured on the coated surface for 24 hours. After the culture, the culture medium was diluted to a certain concentration and smeared on a plate, and the CFU values were confirmed after 16 hours. The E. coli death level, calculated by comparing the above CFU values with CFU values of the uncoated surface, is represented using the following equation.

Antimicrobial Activity=(1CFU of Surface of Sample/CFU of Control)100

[0062] As a result, as shown in FIG. 2, the highest antimicrobial activity was exhibited when coating with NKC-L-O.sub.5 (100 M), and NKC-L-O.sub.3 or NKC-L-O.sub.5 showed the antimicrobial activity dependent on the number of DOPA moieties and the concentration of the NKC. It is considered that such results were obtained because the NKC exhibited a concentration-dependent antimicrobial activity and the coating ability was increased according to the number of DOPA moieties.

Experimental Example 3: Optimization of Coating Time for Adhesive Antimicrobial Peptide

Experimental Example 3-1: Confirmation of Coating Ability According to Coating Time

[0063] In order to optimize the coating conditions of NKC-L-O.sub.5 (100 M), which had been confirmed in Experimental Example 2-2 to have the highest antimicrobial activity, coating degree of the antimicrobial peptide according to coating time was confirmed by X-ray photoelectron spectroscopy (XPS) analysis. XPS is a spectroscopic method in which the surface of a sample is irradiated with X-rays to detect emitted photoelectrons, and the elemental composition and chemical bonding state of the sample surface can be determined based on the binding energy of the detected photoelectrons. When the peptide is coated on the surface, the content of the nitrogen which is not present on the uncoated surface is increased. Based on this, it can be determined that the peptide is present on the surface.

[0064] NKC-L-O.sub.5 (100 M) was coated on the surfaces of polystyrene and titanium for 0 (untreated group), 1, 3, 6, and 12 hours, and then a survey spectrum and a high resolution spectrum on carbon, nitrogen, oxygen, and titanium were identified using Monochromatic Al K X-ray.

[0065] As a result, as shown in FIG. 3, it was confirmed that for both polystyrene and titanium, the content of nitrogen was increased according to the coating time, and for titanium, the content of titanium was decreased. Therefore, it was confirmed that NKC-L-O.sub.5 was effectively coated according to the coating time.

Experimental Example 3-2: Confirmation of Antimicrobial Activity According to Coating Time

[0066] In order to measure the antimicrobial activity of NKC-L-O.sub.5 (100 M) according to coating time, a colony counting assay was carried out. NKC-L-O.sub.5 (100 M) was coated on a polystyrene 24-well plate for 0 (untreated group), 1, 3, 6, and 12 hours, and then E. coli in algebraic logarithmic phase (10.sup.6 cell/mL) was cultured on the coated surface for 24 hours. After the culture, an SCDLP medium (300 L) was added thereto, and then E. coli cells were recovered. Thereafter, these were diluted to a certain concentration and smeared on an LB agar plate. The plate was cultured at 37 C. for 16 hours, and then the colonies were counted.

[0067] As a result, as shown in FIG. 4, it was confirmed that NKC-L-O.sub.5 (100 M) showed a coating time-dependent antimicrobial activity up to 6 hours, and that the number of E. coli was reduced when coating for 12 hours. Therefore, it can be considered that when NKC-L-O.sub.5 at a concentration of 100 M is coated for 6 hours, the most excellent antimicrobial activity is exhibited.

[0068] Additionally, in order to confirm whether the coating time could be shortened when the concentration of the peptide was doubled, NKC-L-O.sub.5 at a concentration of 200 M was coated for 5 minutes, 10 minutes, 30 minutes, 1 hour, 3 hours, and 6 hours, and the antimicrobial activity and coating ability thereof were identified using the method specified above. As a result, when coating with NKC-L-O.sub.5 (200 M) for 10 minutes, the antibacterial activity of NKC-L-O.sub.5 (200 M) was as strong as when coating with NKC-L-O.sub.5 (100 M) for 6 hours (>99.999%) (FIG. 4).

Experimental Example 4: Characteristic Analysis of Coated Surfaces

[0069] As in Experimental Example 3-1, characteristics of the surfaces of the substrates which had been coated with NKC-L-O.sub.5 (200 M) for 10 minutes were analyzed by XPS analysis. As a result of testing three substrates (polystyrene, titanium, and PDMS), it was found that the nitrogen (N) content of the NKC-L-O.sub.5-coated polystyrene, titanium, and PDMS surfaces was increased by 14% compared to that of the uncoated surfaces, and that in the case of titanium and PDMS surfaces, the Ti content or Si content was greatly decreased after the coating (FIG. 5a). These results suggest that NKC-L-O.sub.5 is effectively coated on these three surfaces.

[0070] Additionally, the uncoated surfaces and coated surfaces were observed by atomic force microscopy (AFM). AFM is a technique wherein the morphology of surfaces are imaged while the probe touches the surfaces of samples. The surfaces of the three substrates (polystyrene, titanium, and PDMS) after and before the coating were observed by AFM. As a result, it was found that NKC-L-O.sub.5 was coated uniformly on each surface (FIG. 5b).

[0071] For the coated surfaces, the experiments were carried out as described in Experimental Example 3-2 to determine whether the antimicrobial activity was exhibited against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, which are major infectious strains. As a result, when each of these three strains was cultured on the coated surfaces for 2 hours, all of them were killed (FIG. 6).

[0072] A trace amount of bacteria remaining on the surfaces may form a biofilm. Therefore, in order to confirm whether, when coating the surfaces with NKC-L-O.sub.5, the bacteria cannot bind to and grow on the surfaces, or whether the cells remaining on the surfaces are live cells or dead cells, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus at a concentration of 10.sup.6 were cultured on the uncoated surfaces and the peptide-coated confocal chamber slide surfaces at 37 C. for 24 hours. Thereafter, the surfaces were washed with a 0.85% NaCl buffer, and the bacteria binding on the surfaces were stained with SYTO9 and propidium iodide (PI), and then these were observed by a confocal laser scanning microscope. SYTO9 is a green dye that can stain both live bacteria and dead bacteria, and PI is a dye that can only stain dead bacteria. As a result, a large amount of live bacteria was present on the uncoated surfaces, and on the NKC-DOPA5-coated surfaces, a very small amount of dead bacteria was present in a state where the morphology thereof changed (FIG. 7). These results confirmed that bacteria could not grow on the peptide-coated surfaces.

Experimental Example 5: Confirmation of Persistence of Antimicrobial Activity

[0073] In order to confirm how long the coated peptide can maintain its activity, the 24-well plate in which NKC-L-O.sub.5 had been coated was stored at 4 C. or 25 C. for 1, 3, 7, 14, 28, 56, and 84 days, and the antimicrobial activity was confirmed in the same manner as in Experimental Example 3-2. As a result, when stored at 4 C., a high antimicrobial activity was maintained up to 84 days; and when stored at 25 C., 99.2% of the antimicrobial activity was exhibited (FIG. 8).

Experimental Example 6: Confirmation of Cytotoxicity of Adhesive Antimicrobial Peptide Against Human Cell Line

[0074] In order to confirm the cytotoxicity against human cell lines, HaCaT cell lines (i.e., human keratinocytes) were cultured on the polystyrene, titanium, and silicon catheter surfaces coated with NKC-L-O.sub.5, and then cell viability was confirmed by an MTT assay. Specifically, HaCaT cells cultured in DMEM (10% FBS, 1% Pen/strep, 1% NEAA) were harvested, and the cells (510.sup.4 cells per well) were added to a 12-well cell culture plate. The plate was incubated in a CO.sub.2 incubator (37 C./5%) for 24 hours. After 24 hours, the culture medium was exchanged with serum-free DMEM, and then an uncoated surface having a size of 1 cm.sup.2 or a surface coated with NKC-L-O.sub.5 (100 M) for 6 hours was added to each well. As the negative control group, DMEM containing 2% of Triton X-100, which is a surfactant, was added to culture the cells. After culturing for 24 hours, the surface (1 cm.sup.2) and medium were removed, and a DMEM medium containing MTT was added and then cultured in a CO.sub.2 incubator (37 C./5%) for 4 hours. After the culture, the solubilization/stop mix solution was added, incubated overnight, and then the absorbance at 570 nm was measured.

[0075] As a result, as shown in FIG. 9, it was found that the cell viability of the experimental group, in which the cells were cultured on the NKC-L-O.sub.5-coated surface, was not significantly different from that of the control group, in which the cells were cultured on the uncoated surface. Therefore, it was confirmed that NKC-L-O.sub.5 is a highly safe peptide which does not affect the growth of human cell lines.

[0076] In summary, it was confirmed that the adhesive antimicrobial peptide of the present invention exhibited the most excellent antimicrobial activity when coating in the form of NKC-L-O.sub.5 at a concentration of 100 M for 6 hours, and that the adhesive antimicrobial peptide of the present invention is a highly safe antimicrobial peptide as it does not exhibit cytotoxicity under the above conditions.

[0077] Additionally, it was confirmed that when coating with NKC-L-O.sub.5 at a concentration of 200 M (i.e., the concentration increased by 2-fold), the NKC-L-O.sub.5 exhibited a strong antimicrobial activity because it was effectively coated on the surfaces within 10 minutes (FIG. 4), and that the NKC-L-O.sub.5 did not show the cytotoxicity against human cells (FIG. 10). In order to verify the hemolytic activity of the adhesive antimicrobial peptide coating, sheep blood was washed with PBS, diluted to 5% (v/v) using the same buffer. Thereafter, 100 L of the thus-prepared erythrocyte solution was placed in a 96-well plate coated with the adhesive antimicrobial peptide, and allowed to react at 37 C. for 1 hour, and then the supernatant obtained by centrifuging the reaction solution was transferred to a new 96-well plate. Thereafter, the absorbance thereof at 415 nm was measured to determine whether hemoglobin leaked due to the damage on an erythrocyte membrane. Herein, the absorbance of a sample treated in the well in which the adhesive antimicrobial peptide had not been coated was defined as 0% hemolysis, and the absorbance of a sample treated with 0.2% triton X-100 was defined as 100% hemolysis. As a result, it was confirmed that the adhesive antimicrobial peptide coating did not exhibit a hemolytic activity (FIG. 10a).

[0078] Additionally, in order to confirm the cytotoxicity against human cells when coating with NKC-L-O.sub.5 at a concentration of 200 M, HaCaT cell lines (i.e., human keratinocytes) were placed in a 24-well plate at a concentration of 80,000 cells/well using DMEM containing 10% FBS, and then cultured in the presence of 5% CO.sub.2 for 16 hours. PDMS fragments (1 cm.sup.2) coated with the adhesive antimicrobial peptide were placed in the wells so that the coated side can be in contact with the cells, and allowed to react at 37 C. for 24 hours. Thereafter, the PDMS fragments were removed, and the cell viability was measured using a CellTiter 96-cell proliferation assay kit (Promega Corp.), and then the absorbance at 595 nm was measured. As a result, it was confirmed that the biomaterial (PDMS) coated with the adhesive antimicrobial peptide did not exhibit the cytotoxicity against human cells (FIG. 10b).

[0079] In summary, it was confirmed that the adhesive antimicrobial peptide of the present invention also exhibited an excellent antimicrobial activity when coating in the form of NKC-L-O.sub.5 at concentrations of both 100 M and 200 M, and that the adhesive antimicrobial peptide of the present invention is a highly safe antimicrobial peptide as it does not exhibit cytotoxicity under the above conditions.

[0080] From the foregoing, one of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present invention as defined by the appended claims.