Surfactant adhesive composition

Abstract

Provided are a surfactant adhesive protein comprising an amphiphilic peptide, as a surfactant adhesive protein, at the carbon or amine terminal, a silicone oil and an anticancer composition comprising the surfactant adhesive, where the surfactant adhesive enables homogeneous dispersion of hydrophilic or hydrophobic particles in a hydrophobic or hydrophilic solvent on the basis of strong adhesive strength of the mussel adhesive protein, and the surface adhesive can be favorably used as a surface coating agent requiring antibacterial or antiviral functions as well as a cosmetic product or an ink.

Claims

1. A surfactant adhesive protein comprising an amphiphilic peptide and an adhesive protein wherein the amphiphilic peptide is incorporated into the adhesive protein, wherein said adhesive protein is derived from mussel adhesive protein selected from the group consisting of FP-1 comprising at least one of the amino acid sequences of SEQ ID NOs: 1-3, FP-2 comprising the amino acid sequence of SEQ ID No. 4, FP-3 comprising at least one of the amino acid sequences of SEQ ID NOs: 5-8, FP-4 comprising the amino acid sequence of SEQ ID NO: 9, FP-5 comprising at least one of the amino acid sequences of SEQ ID NOs: 10-13, FP-6 comprising the amino acid sequence of SEQ ID NO: 14, FP-151 comprising at least one of the amino acid sequences of SEQ ID NOs: 15-17, FP-131 comprising the amino acid sequence of SEQ ID NO: 18 and FP-251 comprising the amino acid sequence of SEQ ID NO: 19, and wherein said amphiphilic peptide is selected from the group consisting of the amino acid sequences of ARARADADARARADAD (SEQ ID NO: 20), EAEAKAKAEAEAKAKA (SEQ ID NO: 21), QQRFQWQFEQQ (SEQ ID NO: 22), AEAEAKAK (SEQ ID NO: 23), DPHHHWYHMHQH (SEQ ID NO: 24), HNWYHWWMPHNT (SEQ ID NO: 25), HWKHPWGAWDTL (SEQ ID NO: 26), HWSAWWIRSNQS (SEQ ID NO: 27), DDWSHWWRAWNG (SEQ ID NO: 28), YTSPWWLAWYDP (SEQ ID NO: 29), AWWEAFIPNSIT (SEQ ID NO: 30) and KLWKKWAKKWLKLWKA (SEQ ID NO: 31); and wherein said surfactant adhesive protein is further fused with an anticancer peptide, and the anticancer peptide is composed of an amino acid sequence selected from the group consisting of KLLLKLLKKLLKLLKKK (SEQ ID NO: 36), KLWKKWAKKWLKLWKA (SEQ ID NO: 31), LKKLAKLALAF (SEQ ID NO: 38), THRPPMWSPVWP (SEQ ID NO: 39), GWLKKIGKWKIFKK (SEQ ID NO: 40), ILPWKWPWWPWRR (SEQ ID NO: 41), KLAKLAKKLAKLAK (SEQ ID NO: 42); wherein optionally, if present, tyrosine residues among said adhesive protein are chemically modified to form DOPA (3.4- dihydroxphenylalanine) or DOPA-quinone; and wherein the anticancer peptide is for treatment of breast cancer.

2. The surfactant adhesive protein according to claim 1, wherein said amphiphilic peptide is incorporated into one or more sites selected from the group consisting of the C-terminus and N-terminus of said adhesive protein.

3. The surfactant adhesive protein of claim 1, wherein tyrosine residues among the amino acid residues of the adhesive protein are chemically modified to form DOPA (3,4-dihydroxyphenylalanine).

4. The surfactant adhesive protein of claim 3, wherein said DOPA is further chemically modified to convert to DOPA-quinone.

5. The surfactant adhesive protein of claim 4, wherein said chemical modification is conducted by tyrosinase.

6. A silicone oil comprising the surfactant adhesive protein according to claim 1 and mica particles, finely dispersed in a hydrophilic solvent.

7. The silicone oil according to claim 6, wherein said hydrophilic solvent is water or a water-containing solvent.

8. An anticancer composition comprising the surfactant adhesive protein according to claim 1, wherein the anticancer peptide is for treatment of breast cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 illustrates mica particles stably and finely dispersed by interfacially active adhesive protein in the mixture of water and silicone oil, demonstrating stable and finely dispersed phase in the mixture of water/silicone oil solution at least five hours. (A) indicates the solution of water and oil at the time of mixture, (B) indicates the solution after one hour, and (C) indicates the solution after five hours.

(2) FIG. 2 illustrates the coatability test results of mica particles dispersed in silicone oil to wet surface. When surfactant adhesive protein treated mica particles were dispersed in silicone oil, the mica particles coated with silicone oil showed excellent adhesiveness to wet surface by surface-active effect.

(3) FIG. 3 presents the anticancer activity of the surfactant adhesive protein fused with an anticancer peptide in breast cancer model. All 6 different anticancer peptide containing surfactant adhesive proteins showed anticancer activity against the most malignant breast cancer cell, the triple negative breast cancer cell line (MDA-MB-231). Among six different anticancer peptide, AMP2, AKRHHGYKRKFH (SEQ ID NO: 34), AMP4, LKKLAKLALAF ((SEQ ID NO: 38), AMP5, KLLLKLLKKLLKLLKKK (SEQ ID NO: 36), AMP6, THRPPMWSPVWP (SEQ ID NO: 39), AMP7, ILRWPWWPWRRK (SEQ ID NO: 33), AMP5, KLAKLAKKLAKLAK (SEQ ID NO: 42), AMP5 showed the strongest anticancer activity.

(4) FIG. 4 shows the cytotoxic activity of the surfactant adhesive protein fused with an anticancer peptide in kidney cells. Most anticancer peptides showed concentration-dependent cytotoxicity, but AMP6 & AMP7 are less cytotoxicity while the anticancer activity is still strong.

EXAMPLES

(5) The following examples are provided to illustrate desirable embodiment purpose in the present invention, and the exemplified embodiments are illustrative only and do not limit the scope of the invention. As exemplified in the following embodiment, it is very clear that functionally same products, composition and methods are included in the present invention.

Example 1. Construction of Vector for Interfacially Active Mussel Adhesive Protein

(6) To prepare mussel adhesive protein fused with amphiphilic peptide, we design a genetic sequence to incorporate typical amphiphilic peptide at C terminus or N-terminus of mussel adhesive protein, and the genetic sequence was synthesized by Cosmogentec Co. Ltd. (Seoul, Korea). The constructed vector was transformed into E. coli BL21 (DE3), and the fused amphiphilic peptides were listed in Table 1.

(7) TABLE-US-00001 TABLE 1 Fusion site Amphiphilic at mussel Fusion Peptide adhesive peptide Sequence protein A ARARADADARARADAD C terminus (SEQ ID No. 20) B EAEAKAKAEAEAKAKA N terminus (SEQ ID No. 21) C HWKHPWGAWDTL C terminus (SEQ ID No. 22)

Example 2. Preparation of Surfactant Mussel Adhesive Protein

(8) 2.1. Culturing of E. coli BL21(DE3)

(9) E. coli BL21 (DE) was cultured in LB media (5 g/liter yeast extract, 10 g/liter Tryptone and 10 g/liter NaCl), and IPTG was added to a final concentration of 1 mM when the optical density of the culture solution was 0.6 at 600 nm in order to induce expression of recombinantly antimicrobial peptide fused mussel adhesive protein. The E. coli BL21 (DE) culture was centrifuged at 13,000 rpm for 4 to 10 minutes to obtain the cell pellet, and this was stored at −80° C.

(10) 2.2. Confirmation of Surfactant Mussel Adhesive Protein Expression

(11) The cell pellet was resuspended in 100 μg of SDS-PAGE buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, 5% SDS, 5% β-mercaptoethanol, 0.25% bromophenol blue), denatured by boiling at 100° C. for 5 minutes. For SDS-PAGE analysis, the samples were electrophoresed on a 15% SDS-polyacylamide gel and then the protein bands detected using Coomassie blue staining.

(12) 2.3. Purification of Surfactant Mussel Adhesive Protein

(13) The cell pellets from EXAMPLE 2.1 was stirred with lysis buffer comprising 2.4 g/L Sodium phosphate monobasic, 5.6 g/L Sodium phosphate dibasic, 10 mM EDTA and 1% Triton X-100, and were broken using high pressure homogenizer. The lysates were centrifuged by centrifugal filter units as 9,000 rpm for 20 minutes and insoluble protein complex containing mussel adhesive protein was obtained. Surfactant adhesive protein eluted from the insoluble complex at an acetic acid concentration of 25 (v/v) % was centrifuged under the same conditions (9,000 rpm for 20 minutes) to gain supernatant. The obtained supernatant was centrifuged under the same conditions (9,000 rpm for 20 minutes) at pH 12.8 by adding 10N NaOH. The supernatant was neutralized at pH 6-7 using acetic acid and then centrifuged under the same conditions to take precipitated mussel adhesive protein. The precipitate was dissolved in distilled water, undergoing freezing-dried to get lyophilized mussel adhesive protein with 90% purity.

Example 3. Treatment of Tyrosinase to the Surfactant Adhesive Protein

(14) The lyophilized surfactant mussel adhesive protein was dissolved to a concentration of 1 mg/mL in 0.1M acetate buffer containing 20 mM ascorbic acid and 20 mM sodium borate and the surfactant adhesive protein solution then saturated with oxygen by adding oxygen gas to the solution for 30 minutes. Then after the addition of 40˜1 μg of tyrosinase per antimicrobial adhesive protein, preferably 40˜1 μg per adhesive protein, it was shaken for one hour under the oxygen condition. After one hour, the chemical modification reaction was terminated by adding 5% acetic acid to the solution. The terminated surfactant adhesive protein solution underwent freezing dry to obtain lyophilized powder. Through this process, the tyrosine residues of the adhesive proteins were modified to DOPA. After the dialysis of the solution, acrylation and the degree of acrylation of protein were determined with NMR or spectroscopic analysis.

Example 4. Dispersibility Test of Surfactant Protein

(15) Coating solution of surfactant mussel adhesive protein obtained from EXAMPLE 2 was prepared to measure its dispersibility.

(16) The surfactant coating solution was composed of surfactant adhesive protein dissolved to a concentration of 10 to 0.01 mg/mL in distilled water. 2 wt % of Mica particles was added to the surfactant coating solution, and 1 mL of silicone oil was added to the surfactant coating solution. As shown in FIG. 1A, when silicone oil was added to the surfactant coating solution, two phases are separated clearly. Two solutions were mixed using vortex mixer, and mixture and dispersion status of two solutions was checked after 1 and 5 hours. No phase separation of the mixture solution was confirmed after 1 and 5 hours later. Mica particles were finely dispersed and no precipitation of the mica particles was observed, confirming the dispersed phase was stabilized.

Example 5. Coatability Test with Surfactant Adhesive Protein

(17) 0.1 g of mica particles was added to 1 mg, 10 mg and 20 mg of surfactant adhesive protein dissolved in solution, respectively and incubated for 20 minutes for coating. The surfactant adhesive protein coated mica particles were isolated using manure paper and finely dispersed in 1 mL of silicone oil in petri dish. The mica particles finely dispersed silicone oil was added to wet surface coating. FIG. 2A indicated surface coating using surfactant adhesive protein, non-tyrosinase treatment, and FIG. 2B indicated the coating using said adhesive protein was tyrosinase-treated. In FIG. 2A, 1A indicated the mica coated with 1 mg of surfactant adhesive protein, 2A with 10 mg and 3A with 20 mg of surfactant adhesive protein, respectively.

(18) Coatability test was conducted by adding 100 mL of water to mica-coated surface and moving petri dish up and down to exert external stress to coated surface. As shown in FIG. 2B, 3B indicates stable coating, 2B indicates residual coated particles are rare, but 1B indicates little residual coated particles. Mica particles coated with surfactant adhesive protein without tyrosinase modification were washed out regardless of the amount of surfactant adhesive protein.

Examples 6. Anticancer Activity of Surfactant Adhesive Protein Fused with Antimicrobial Peptide

(19) MD-MBA-231 cells were purchased from ATCC (Manassas, Va.). The breast cancer cells (5,000 cells/well), MD-MBA-231, were seeded in 96 well plate and cultured in DMEM media supplemented with 20% (v/v) horse serum and 1% (v/v) penicillin/streptomycin in a humidified incubator at 37° C. and 5% CO.sub.2. After 48 hr incubation, the cancer cells were treated with the surfactant adhesive protein fused with an anticancer peptide ILRWPWWPWRRK (SEQ ID NO. 33), AKRHHGYKRKFH (SEQ ID NO. 34), KLLLKLLKKLLKLLKKK (SEQ ID NO. 36), LKKLAKLALAF (SEQ ID NO: 38), and KLAKLAKKLAKLAK (SEQ ID NO: 42), respectively. As a negative control, mussel adhesive protein without any peptide motif was used. The proteins were dissolved at a concentration of 2.6 μg/mL, 26 μg/mL, 130 μg/mL, and 260 μg/mL in a distilled water. Cell viability was determined by CCK-8 assay following manufacturer's instruction.

(20) As seen in FIG. 3, all of them had anticancer tumor activity, depending on the concentration. Most cancer cells were killed at 130 μg/mL or higher concentration while the surfactant adhesive protein having KLLLKLLKKLLKLLKKK (SEQ ID NO. 36) is a promising anticancer peptide as it has strong anticancer activity at low concentration (26 μg/mL).

Example 7. Cytotoxicity Assay of the Surfactant Adhesive Protein Fused with Antimicrobial Peptide

(21) As a model for cytotoxicity assay, Kidney Vero-E6 Cell Lines were purchased from ATCC (Manassas, Va.). The kidney cells were seeded in 96 well plate and cultured in DMEM media supplemented with 20% (v/v) horse serum and 1% (v/v) penicillin/streptomycin in a humidified incubator at 37° C. and 5% CO.sub.2. Cell viability was determined by CCK-8 assay following manufacturer's instruction. After 48 hr incubation, the kidney cells were treated with the same antimicrobial peptide containing proteins as set forth in the EXAMPLE 6.

(22) As seen in the FIG. 4, most antimicrobial peptide showed significant cytotoxic to kidney cells at 130 μg/mL or higher concentration but had little or less cytotoxicity at 26 μg/mL. Therefore, the antimicrobial peptide KLLLKLLKKLLKLLKKK (SEQ ID NO. 36) containing protein can be used for safe anti-cancer agent.