Patent Description:
Biofilms are formed by attaching bacteria to surfaces of surgical instruments and implantable medical devices, and during this process, an immune response to a host or resistance to antimicrobial substances is acquired. In this case, problems such as failure to treat patients, prolonged treatment, and complications may arise. Various antimicrobial agents and antimicrobial strategies have been developed to prevent biofilm formation through surgical infection; however, various problems such as increased resistance to antimicrobial agents, toxicity to surrounding tissues, and a limited lifespan of antimicrobial agents are emerging.

Certain strains of pathogens infecting the human body are parasitic inside host cells and continue to proliferate while controlling metabolic activity of the host. It has been revealed that Mycobacterium tuberculosis, typhoid bacteria, chlamydia and listeria are representative intracellular bacteria, and E. coli and Staphylococcus, commonly known as extracellular bacteria, also invade host cells and are parasitic in the host cells. In order to inhibit the infection of these intracellular bacteria, antimicrobial agents need to reach the inside of the cell by diffusion or inclusion; however, more than two-thirds of existing antimicrobial agents have limitations, in that they show no effect on the intracellular bacteria.

Antibiotic peptides (antimicrobial peptides; AMPs) are amino acid chains produced as part of an innate immune system of living organisms in nature and generally exhibit antibiotic activity against bacteria, viruses, and fungi. In particular, the antibiotic peptides break down or puncture bacterial cell membranes, resulting in loss of intracellular materials, and react with substances in bacterial cells to inhibit synthesis of bacterial nucleic acids or proteins. Accordingly, the antibiotic peptides have drawn attention as a next generation antibacterial agent.

Meanwhile, mussels, which are marine life organisms, produce and secrete adhesive proteins to securely attach themselves to wet solid surfaces such as rocks in the ocean, and thus do not get affected by waves and a buoyancy effect of seawater. The mussel adhesive proteins are known as a powerful natural adhesive and have flexibility to bend while exhibiting a tensile strength of approximately twice that of epoxy resins compared to chemical synthetic adhesives. In addition, the mussel adhesive proteins can adhere to a variety of surfaces, including plastics, glass, metals, Teflon, and biomaterials, and even to wet surfaces in a few minutes, which still remains a problem which has not yet been solved in the field of chemical adhesive development.

To obtain <NUM> of a naturally extracted adhesive material from mussels, however, about <NUM>,<NUM> mussels are required. Despite the excellent physical properties of mussel adhesive proteins, many restrictions apply to the industrial use of naturally extracted mussel adhesive proteins. As an alternative, studies on the production of mussel adhesive proteins using genetic recombination techniques have been conducted using Mefp (Mytilus edulis foot protein)-<NUM>, Mgfp (Mytilus galloprovincialis foot protein)-<NUM>, Mcfp (Mytilus coruscus foot protein)-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM> and the like.

Meanwhile, nanoparticles, capable of invading animal cells through a phagocytosis or pinocytosis pathway, have drawn attention as a strategy for inhibiting intracellular infection. In this regard, studies on improvement of the stability of antimicrobial agents and release of selective antimicrobial agents by carrying the antimicrobial agents on nanoparticles based on materials sensitive to external stimuli, such as temperature or pH, have been reported. However, most of the studies showed that the antimicrobial effects were due to the antimicrobial agents carried by the nanoparticles, and there have been no reports on a material in which the nanoparticles themselves have antimicrobial properties.

<CIT> describes a nanofiber scaffold for tissue engineering.

<CIT> describes a mussel adhesive protein fused to an antimicrobial peptide.

A first aspect of the invention provides an antimicrobial nanoparticle having inherent antimicrobial activity comprising:.

It is preferable that the antibiotic peptide be at least one peptide selected from the group consisting of: an A7 peptide, such as having the amino acid sequence ILKWKWKWWKWRR; a Tet-<NUM> peptide, such as having the amino acid sequence KRWRIRVRVIRKC; a minTBP peptide, such as having the amino acid sequence RKLPDAP; and an MP196 peptide such as having the amino acid sequence RWRWRW.

It is preferable that the coordinatable metal be at least one selected from the group consisting of titanium, vanadium, chrome, manganese, iron, cobalt, nickel, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum and gold.

It is preferable that an antimicrobial agent is additionally loaded in the antimicrobial nanoparticle.

It is preferable that the microbial agent be at least one selected from the group consisting of penicillins, cephalosporins, β-lactams, macrolides, glycopeptides, lincosamides, quinolones, rifamycin, chloramphenicol, polymycin, trimethoprim, streptogamine, oxazolidinone, gentamicin and baccitracin.

An antimicrobial effect is achieved under a condition of pH <NUM> to pH <NUM>, which is a bacterial infection environment.

A second aspect of the invention provides a method for preparing an antimicrobial nanoparticle according to the first aspect of the invention, comprising: dissolving an antimicrobial adhesive protein comprising an antibiotic peptide linked to a mussel adhesive protein derivative of which a tyrosine residue in the mussel adhesive protein is modified to a catechol derivative, in a mixed solvent of water and ethanol; preparing a spinning solution by adding a salt of a metal capable of coordinating with the mussel adhesive protein derivative; and electrospinning the spinning solution, wherein the mussel adhesive protein is a protein selected from the group consisting of Mytilus edulis foot protein (Mefp)-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mytilus galloprovincialis foot protein (Mgfp)-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mytilus coruscus foot protein (Mcfp)-<NUM>,Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, foot protein (fp)-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM> and fp-<NUM>, or a fusion protein to which at least one protein selected from the group is linked, and
wherein the catechol derivative is at least one selected from the group consisting of <NUM>,<NUM>-dihydroxyphenylalanine (DOPA), DOPA o-quinone, dopamine, norepinephrine, epinephrine, and epigallocatechin gallate.

It is preferable that the mixed solvent comprises water and ethanol in a weight ratio of <NUM>:<NUM> to <NUM>:<NUM>.

It is preferable that the metal salt be added so as that a molar ratio of a metal ion and a catechol derivative is <NUM>:<NUM> to <NUM>:<NUM>.

It is preferable that the metal be at least one selected from the group consisting of titanium, vanadium, chrome, manganese, iron, cobalt, nickel, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum and gold.

According to still another aspect, an antimicrobial composition, comprising the antimicrobial nanoparticle of the first aspect of the invention is provided.

It is preferable that the antimicrobial nanoparticle be comprised in an amount of <NUM>µg/mL to <NUM>µg/mL.

The present invention allows an antibiotic peptide to inhere in a mussel adhesive protein so that the protein can be used as a bioadhesive material exhibiting an antimicrobial effect. The mussel adhesive protein may be coated on various surfaces using excellent adhesion properties thereof regardless of types of the surfaces. By preparing the same in the form of a nanoparticle, intracellular infections can be effectively prevented. In particular, the antimicrobial nanoparticle according to the present invention, when utilized for intracellular infections, is present in an inactive form in an infected animal and thus does not have any effect thereon, while having an intrinsic antimicrobial activity of killing bacteria, that is, infected endogenous bacteria in animal cells.

The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein.

According to the present invention, an antimicrobial nanoparticle, an antimicrobial composition, and a preparation method therefor are provided.

The antimicrobial adhesive protein provided herein refers to a protein having antimicrobial activity itself and having an antibiotic peptide connected to a mussel adhesive protein.

The mussel adhesive proteins disclosed in <CIT>, <CIT>, <CIT>, or the like may be used. A detailed preparation process may be the same as that disclosed in <CIT> and <CIT>.

The mussel adhesive protein is selected from the group consisting of Mytilus edulis foot protein (Mefp)-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mytilus galloprovincialis foot protein (Mgfp)-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mytilus coruscus foot protein (Mcfp)-<NUM>,Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, foot protein (fp)-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM> and fp-<NUM>, or a fusion protein to which at least one protein selected from the group is linked.

Suitable mussel adhesive proteins disclosed in <CIT> and <CIT>. Preferably, the mussel adhesive protein may include fusion proteins such as fp-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM>, and the like. In addition, the mussel adhesive protein may include a polypeptide, in which <NUM> to <NUM> or more decapeptides are linked in tandem, where the decapeptides recur about <NUM> times in fp-<NUM>, and may preferably be an fp-<NUM> variant polypeptide in which <NUM> decapeptides of SEQ ID NO:<NUM> are connected in tandem.

The present inventors developed an fp-<NUM>, a novel mussel adhesive protein in which a structure of their previous study, in which <NUM> repeating amino acids of the fp-<NUM> recur <NUM> times, is connected to N- and C-termini of fp-<NUM> at a genetic level, and successfully expressed the recombinant mussel adhesive protein in E. coli, thereby finding feasibility of mass production thereof and significantly high industrial applicability through simple purification processes (<CIT> and <CIT>).

Meanwhile, the antibiotic peptide may be at least one peptide selected from the group consisting of an A7 peptide, a Tet-<NUM> peptide, a minTBP peptide, an MP196 peptide, a PTP7 peptide and temporin 1CEa as disclosed in Table <NUM>, but is not limited thereto.

For example, the antibiotic peptide may consist of an amino acid sequence of SEQ ID NO:<NUM>.

According to the present invention, an antimicrobial nanoparticle having inherent antimicrobial activity is provided, and the antimicrobial nanoparticle include a metal capable of coordinating with a derivative of a mussel adhesive protein derivative, of which a tyrosine residue in the mussel adhesive protein is modified to a catechol derivative.

As used herein, the expression "antimicrobial activity" may include an antibiotic effect of inhibiting growth of microorganisms or strains and a bactericidal effect of removing existing microorganisms or strains.

The mussel adhesive protein derivative, of which the tyrosine residue is modified to a catechol derivative,
may refer to a "modified mussel adhesive protein" and a modification method is not particularly limited.

The catechol derivative is a compound containing a dihydroxyl group and is coordinatable with a metal. The catechol derivative is preferably at least one selected from the group consisting of <NUM>,<NUM>-dihydroxyphenylalanine (DOPA), DOPA o-quinone, dopamine, norepinephrine, epinephrine, epigallocatechin gallate and derivatives thereof.

For example, the catechol derivative may have <NUM>% to <NUM>% of the entire tyrosine residue modified to a catechol derivative, particularly to DOPA.

The coordinatable metal is any metal coordinatable with the catechol derivative and may be a typical metal or a transition metal. For example, the metal may be at least one selected from the group consisting of titanium, vanadium, chrome, manganese, iron, cobalt, nickel, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum and gold. The metal is preferably an iron (III).

A salt of the metal, which can be used here, may be, for example, iron (II) chloride (FeCl<NUM>), iron (III) chloride (FeCl<NUM>), titanium tetrachloride (TiCl<NUM>), titanium (III) chloride (TiCl<NUM>), cobalt (III) chloride (CoCl<NUM>), Cobalt (II) chloride (CoCl<NUM>), nickel chloride (NiCl<NUM>), silver chloride (AgCl), and the like, but is not limited thereto.

The catechol derivative and the metal coordinate with each other to form a metal-catechol derivative complex. Preferably, the metal-catechol derivative complex may be an Fe(III)-DOPA complex. According to an exemplary embodiment, the DOPA and the Fe(III) form a crosslink through a coordination bond. As the DOPA and the Fe(III) are already present in the human bodies and are advantageous due to excellent biocompatibility.

The DOPA is known to is known to form mono-, bis-, or tris-cross-links, while reacting with the Fe(III), depending on pH. Further, the DOPA is known to exhibit unique colors of Fe(III)-DOPA complexes depending on bonding aspects. According to an exemplary embodiment, a reagent containing Fe(III), that is, FeCl<NUM>, may be used to provide Fe(III) metals. Preferably, an FeCl<NUM> solution may be added so as that a molar ratio of Fe(III) to DOPA is <NUM>:<NUM> or a ratio of Fe(III) is less than that.

Further, the antimicrobial nanoparticle may have an antimicrobial agent additionally loaded and may be applied as a multi-drug form by for example, carrying an antimicrobial drug. The antimicrobial drug may be penicillins such as penicillin, methicillin, oxacillin, naphcillin, ampicillin, carboxyphenicillin, amoxicillin, piperecillin, or the like; cephalosporins such as cephalosporin, cephazoline, ceftazidime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, or the like; other beta-lactams such as carbapenem, meropenem, sulbactam, clavulanate, tazobactam, or the like; aminoglycosides such as streptomycin, neomycin, gentamicin, tobramycin, amikacin, sisomicin, astromicin, isepamicin, arbekacin, or the like; macrolides such as erythromycin, clarithromycin, or the like; tetracyclines such as tetracycline, metacycline, minocycline, tigecycline, doxycycline, or the like; glycopeptides such as vancomycin, teicoplanin, or the like; lincosamides such as lincomycin, clindamycin, or the like; quinolones such as nalidixic acid, oxolinic acid, fluoroquinolone, ciprofloxacin, norfloxacin, levofloxacin, or the like; rifamycin, chloramphenicol, polymyxin, trimethoprim, streptogramin, oxazolidinone, bacitracin and mixtures thereof, but is not limited thereto.

For example, it is preferable that the antibiotic agent be at least one selected from the group consisting of penicillins, cephalosporins, β-lactams, macrolides, glycopeptides, lincosamides, quinolones, rifamycin, chloramphenicol, polymycin, trimethoprim, streptogamine, oxazolidinone, gentamicin and bacitracin.

The antimicrobial nanoparticle of the present invention has inherent antimicrobial activity at pH <NUM> to pH <NUM>, preferably at pH <NUM> to pH <NUM>, for example, at pH <NUM> to pH <NUM>, the nanoparticle is dissolved, thereby exhibiting antimicrobial activity.

That is, the nanoparticle maintain the nanoparticle form in body fluids having a pH of <NUM>, thereby exhibiting stable properties, whereas in an acidic environment (pH of about <NUM>) formed by producing acetic acids and lactic acids, metabolites produced due to bacterial infections, the nanoparticle loses its form and may induce positive charges of an original antimicrobial adhesive protein.

Accordingly, the nanoparticle based on the antimicrobial adhesive protein MAP-cAMP is present in an inactive form and dissolved at a time point where antimicrobial activity is required, thereby establishing technology enabling expression of the antimicrobial activity. This in particular enables an application of the nanoparticle in killing infected bacteria inside cells without affecting infected host animal cells.

The nanoparticle may have an average diameter of <NUM> to <NUM>, preferably <NUM>. The nanoparticle with this size is appropriate to migrate to a target cell and can be delivered via various routes including injection, oral administration, and dermal administration, when introduced to the human body. The loaded drug may be appropriately delivered to human or other mammals having a treatment-effective disease or symptoms thereof via injection or other routes, but preferably via parenteral administration.

The term "parenteral" refers to intramuscular, intraperitoneal, intraabdominal, subcutaneous, intravenous and intraarterial. Accordingly, the nanoparticle disclosed herein may representatively be formulated into an injection formulation.

The injectable nanoparticle may be injected or inserted into a human body or other mammals' bodies via any appropriate administration route, preferably via an injection with a subcutaneous needle. For example, the nanoparticle may be administered via injection to artery, vein, urogenital organs, under the skin, muscle, under the skin, skull, pericardium, pleura, or other body cavities or available spaces. Alternately, the nanoparticle may be introduced into, for example, joint during arthroscopic surgery, urogenital organs, vas, palate, pleura, or any body cavities or available spaces via a catheter or a syringe during an operation, surgical treatment, diagnosis or interventional procedure.

The nanoparticle can be used for tuberculosis, hepatitis, typhoid fever, food poisoning, cholera, dysentery and other infectious diseases, but is not limited thereto.

Further, according to the present invention, a method for preparing an antimicrobial nanoparticle having inherent antimicrobial activity as described above.

The antimicrobial nanoparticle preparation method of the present invention includes dissolving an antimicrobial adhesive protein comprising an antibiotic peptide linked to a mussel adhesive protein derivative, of which a tyrosine residue in the mussel adhesive protein is modified to a catechol derivative, in a mixed solvent of water and ethanol; preparing a spinning solution by adding a salt of a metal capable of coordinating with the mussel adhesive protein derivative; and electrospinning the spinning solution, wherein the mussel adhesive protein is a protein selected from the group consisting of Mytilus edulis foot protein (Mefp)-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mytilus galloprovincialis foot protein (Mgfp)-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mytilus coruscus foot protein (Mcfp)-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, foot protein (fp)-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM> and fp-<NUM>, or a fusion protein to which at least one protein selected from the group is linked, and
wherein the catechol derivative is at least one selected from the group consisting of <NUM>,<NUM>-dihydroxyphenylalanine (DOPA), DOPA o-quinone, dopamine, norepinephrine, epinephrine, and epigallocatechin gallate.

The electrospinning is a technique forming a nanoparticle using electrical attraction and repulsion occurring when charging a polymer solution or a molten polymer with a predetermined voltage. According to electrospinning, nanoparticles having various diameters of several nanometers to several thousands of nanometers can be prepared with simple equipment and may be applied to a variety of substances.

Use of the water-based solvent, instead of an organic solvent, may eliminate a toxic effect of the solvent remaining during the electrospinning. In order to improve evaporation of the water-based solvent, an organic solvent may be further mixed, and preferably, <NUM>% to <NUM>% (v/v) of ethanol may be additionally mixed with respect to distilled water. That is, the mixed solvent includes water and ethanol mixed preferably in a weight ratio of <NUM>:<NUM> to <NUM>:<NUM>, more preferably in a weight ratio of <NUM>:<NUM>.

Meanwhile, it is preferable that a salt of the metal is added so as that a molar ratio of a metal ion and a catechol derivative is <NUM>:<NUM> to <NUM>:<NUM>. The metal maybe at least one selected from the group consisting of titanium, vanadium, chrome, manganese, iron, cobalt, nickel, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum and gold.

Electrospinning of thus-prepared spun solution is not particularly limited, but for example, may involve discharging the spun solution using a syringe pump at a speed of <NUM>/h to <NUM>/h and applying a high voltage of <NUM> kV to <NUM> kV when passing through a needle having a diameter of <NUM> to <NUM> to generate a nanoparticle. Thus-prepared nanoparticle may be collected on a stirred tank containing aluminum phosphate buffered saline (PBS; pH <NUM>), and the like, or an aluminum foil.

Further, according to the present invention, an antimicrobial composition containing the antimicrobial nanoparticle having the inherent antimicrobial activity as described above is provided.

The antimicrobial nanoparticle is contained preferably at a concentration of <NUM> ug/mL to <NUM> ug/mL, more preferably at a concentration of <NUM> ug/mL. When the concentration of the antimicrobial nanoparticle is less than <NUM> ug/mL, the antimicrobial activity may be insufficient, whereas difference in effects may not be significant compared to the concentration when the concentration exceeds <NUM> ug/mL, thereby making it not preferable in terms of process economy.

A strain, in which the antimicrobial nanoparticle and antimicrobial composition of the present disclosure can exhibit an antibiotic effect of inhibiting growth of microorganisms or strains and a bactericidal effect of removing existing microorganisms or strains, is not particularly limited, but may be, for example, E. coli, and the like.

Hereinafter, the present invention will be described in more detail with reference to specific exemplary embodiments. The following exemplary embodiments are merely examples to help understanding of the present invention.

To the extent that any of the following examples fall outside the scope of the accompanying claims, they are included as comparative examples only.

A mussel adhesive protein fp-<NUM> (Mytilus mussel foot protein type <NUM>) variant composed of <NUM> times-repeated decapeptides (AKPSYPPTYK) was prepared according to a known process (see<NPL>).

A primer of a sequence for an A7 peptide (<NPL>), a pseudopeptide derived from indolicidin, an antimicrobial peptide, was prepared to connect to thus-prepared mussel adhesive protein fp-<NUM> using a polymerase chain reaction (Table <NUM>).

A product, MAP-cAMP gene was transformed into an E. coli TOP10 strain using a pET-22b(+) vector containing a T7 promoter as a plasmid carrier. Further, a cloned recombinant vector was transformed back into an E. coli BL21 (DE3) strain to express a fusion protein.

coli BL21 transformed into the MAP-cAMP gene was cultured in an LB liquid medium containing <NUM> ug/mL of ampicillin at <NUM> and <NUM> rpm. When optical density for <NUM> (OD<NUM>) reached <NUM> to <NUM>, <NUM> isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured at the same conditions. Thus-cultured cell was centrifuged for <NUM> under conditions of <NUM> and <NUM>,<NUM>>g, and resuspended in an elution buffer (<NUM> Tris-HCl, and <NUM> sodium phosphate, pH <NUM>) to lyse the cell at <NUM> Kpsi.

To obtain cell debris from thus-obtained cell lysate, a resultant was centrifuged for <NUM> under conditions of <NUM> and <NUM>,<NUM>> g. <NUM>%(v/v) acetic acid was used to extract a target fusion protein, and the fusion protein lastly purified was lyophilized and stored at -<NUM>.

The preparation and purification of the protein was analyzed using <NUM>%(w/v) SDS-PAGE, and consequently confirmed successful expression of the fusion protein (<FIG>). A concentration of the protein was measured using Bradford assay (Bio-Rad). Compared to MAP, the control protein, the prepared fusion protein MAP-cAMP exhibited a higher positive charge in a solution of pH <NUM> (<FIG>).

To measure the antimicrobial activity of the MAP-cAMP, the antimicrobial adhesive protein prepared in <NUM>. , the protein was dissolved in distilled water at concentrations of <NUM>/mL, <NUM>/mL, <NUM>/mL and <NUM>/mL and applied to a surface in a predetermined amount so as to cover a cover slip formed of polystyrene, followed by drying for an hour at <NUM> to coat the surface. Thus-prepared MAP-cAMP-coated surface was applied to a solid medium and a liquid medium, which were inoculated with bacteria, to confirm the antimicrobial effect. An uncoated material was used as a negative control.

The surfaces coated with <NUM>/mL, <NUM>/mL, <NUM>/mL and <NUM>/mL MAP-cAMP as described above was cultured in an agar plate medium spread with Escherichia coli, a gram-negative bacterium in a log phase, at <NUM> for <NUM> day.

The antimicrobial effect was analyzed by measuring an inhibition area using a graphic program (ImageJ). As a result, all MAP-cAMP-coated surfaces showed bacterial growth inhibition, which indicates that the higher the coating concentration, the larger the inhibition area (<FIG> and <FIG>).

The cover slip formed of polystyrene used in (<NUM>) was applied to <NUM>µL of an LB liquid medium inoculated with Escherichia coli, a gram-negative bacterium in a log phase, and cultured for <NUM> hours to measure optical density for <NUM> (OD<NUM>). A <NUM>-well culture plate was used for the culturing of the bacterial strain at <NUM> and <NUM> rpm.

As a result, a minimum coating concentration of the MAP-cAMP was shown to be <NUM>/mL for <NUM>% inhibition of the growth of the E. coli and that was shown to be <NUM>/mL for <NUM>% inhibition of the growth of the E. coli (<FIG>).

The MAP-cAMP, the fusion protein prepared in <NUM>, was subject to an in vitro enzymatic reaction using a mushroom tyrosinase to convert a tyrosine residue of the MAP-cAMP to dihydroxyphenylalanine (DOPA). Specifically, <NUM>/mL of an MAP-cAMP solution and <NUM> ug/mL of the tyrosinase were reacted in a buffer solution (<NUM> of sodium phosphate, <NUM> of boric acid, and <NUM> of ascorbic acid; pH <NUM>) for <NUM> hour, and dialyzed with <NUM>% acetic acid.

Electrospinning was used to prepare a nanoparticle using the mMAP-cAMP prepared in (<NUM>). Specifically, <NUM> wt% to <NUM> wt% mMAP-cAMP was dissolved in a solvent of distilled water:ethanol (<NUM>:<NUM>), and an FeCl<NUM> solution was added such that a molar ratio of Fe<NUM>+:DOPA is <NUM>:<NUM>. The mixed solution was then electrospun. The electrospinning was performed by applying a high voltage of <NUM> kV to <NUM> kV when passing through a needle having a diameter of <NUM> while discharging the solution at a flow rate of <NUM>/h using a syringe pump, thereby forming a nanoparticle. Thus-prepared nanoparticle was collected into a stirring water bath containing phosphate buffered saline (PBS; pH <NUM>) or an aluminum foil. Compared to a conventional solution phase, the MAP-cAMP showed a reduced positive charge as it becomes a nanoparticle and showed a surface potential similar to a charge pattern of the nanoparticle of the control MAP (<FIG>).

Accordingly, the nanoparticle based on the antimicrobial adhesive protein disclosed herein is present in an inactive form and dissolved at a time point where antimicrobial activity is required, thereby establishing technology enabling expression of the antimicrobial activity. In this regard, the nanoparticle can be used in killing of infected bacteria inside cells without affecting infected host animal cells.

To prepare an Fe(III)-DOPA nanoparticle loaded with gentamicin (GENT), a GENT solution was added to the mixed solution, and the mixed solution was directly electrospun into PBS. Dialysis was performed three times against the PBS (pH <NUM>) using a molecular weight cut off (MWCO) <NUM> membrane to remove unloaded GENTs, thereby obtaining a purple pure GENT-loaded Fe(III)-DOPA complex nanoparticle.

A DLS analysis showed that the MAP-cAMP-NP and (MAP-cAMP@GENT)-NP had average diameters of about <NUM> and about <NUM>, respectively (<FIG>).

To examine whether the MAP-cAMP-NP, the antimicrobial nanoparticle obtained in <NUM>. (<NUM>), exhibits antimicrobial activity, the nanoparticle was applied to <NUM>µL of an LB liquid medium inoculated with <NUM>µL of E. coli, a gram-negative bacterium and cultured for <NUM> hours to measure optical density for <NUM> (OD<NUM>). A <NUM>-well culture plate was used for the culturing of the bacterial strain at <NUM> and <NUM> rpm.

As a result, the antimicrobial nanoparticle MAP-cAMP-NP was shown to exhibit a higher positive charge in a solution of pH <NUM>, an acidic environment, than in a solution of pH <NUM>, a neutral environment (<FIG>). It can be seen that the positive charge of the nanoparticle, which was reduced while becoming a nanoparticle, is dissolved in an acidic environment, such as a bacterial infection environment, to restore an original positive charge. Further, SEM observation shows that the antimicrobial nanoparticle MAP-cAMP-NP exhibits a disintegration pattern of the existing nanoparticle form in an acidic environment (<FIG>).

To measure the antimicrobial activity of the antimicrobial nanoparticle MAP-cAMP-NP obtained in <NUM>. (<NUM>), the nanoparticle was applied to the liquid medium inoculated with bacteria at concentrations of <NUM> pg/mL, <NUM> ug/mL, <NUM> ug/mL, <NUM> ug/mL, <NUM>µg/mL and <NUM> ug/mL.

Specifically, the nanoparticle of each concentration was applied to <NUM>µL of an LB liquid medium inoculated with <NUM>µL (about <NUM>×<NUM><NUM> CFU) of gram negative E. coli and cultured for <NUM> hours and <NUM> hours to measure optical density for <NUM> (OD<NUM>). A <NUM>-well culture plate was used for the culturing of the bacterial strain at <NUM> and <NUM> rpm.

Claim 1:
An antimicrobial nanoparticle having inherent antimicrobial activity comprising:
an antimicrobial adhesive protein comprising an antibiotic peptide linked to a mussel adhesive protein derivative of which a tyrosine residue in the mussel adhesive protein is modified to a catechol derivative; and
a metal capable of coordinating with the mussel adhesive protein derivative,
wherein the antimicrobial nanoparticle has an antimicrobial effect at a pH of from <NUM> to <NUM>,
wherein the mussel adhesive protein is a protein selected from the group consisting of Mytilus edulis foot protein (Mefp)-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mefp-<NUM>, Mytilus galloprovincialis foot protein (Mgfp)-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mgfp-<NUM>, Mytilus coruscus foot protein (Mcfp)-<NUM>,Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, Mcfp-<NUM>, foot protein (fp)-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM>, fp-<NUM> and fp-<NUM>, or a fusion protein to which the at least one protein selected from the group is linked, and
wherein the catechol derivative is one selected from the group consisting of <NUM>,<NUM>-dihydroxyphenylalanine (DOPA), DOPA o-quinone, dopamine, norepinephrine, epinephrine, and epigallocatechin gallate.