KR20180064585A - Antimicrobial nano-complex, and uses thereof - Google Patents

Abstract

The present invention relates to: an antimicrobial nano structure, which comprises: gold nanoparticles, an aptamer bonded to the surface of the gold nanoparticles and antimicrobial nano structure comprising antimicrobial peptides (AMPs) bound to the aptamer; a manufacturing method thereof; and an antimicrobial composition comprising the same, wherein the nano structure specifically binds to an antimicrobial peptide to be delivered by various tagged platamers or antimicrobial peptide-specific aptamers bound to gold nanoparticles to effectively deliver the antimicrobial peptide into cells, and uses the gold nanoparticles having very low cytotoxicity to be harmless to the human body, thereby being useful for antibacterial uses.

Description

Antimicrobial nanostructures and uses thereof < RTI ID = 0.0 >

The present invention relates to an antibacterial nanostructure, a method for producing the antibacterial nanostructure, and a composition for antibacterial use comprising the antibacterial nanostructure. Specifically, the antibacterial nanostructure comprises an antimicrobial peptide- To an improved antibacterial nanostructure and a method for producing the same.

Increased frequency of infection in hospitals and the spread of multidrug-resistant bacteria are threatening global health and burdening the healthcare system. In particular, the bacteria remaining in the host cell prevent the antimicrobial compound from being absorbed by the infected host cell, thereby significantly reducing the therapeutic effect of the compound. Therefore, in view of the overall sharp increase in the occurrence of such bacterial resistance, the need for a novel therapeutic strategy is evident, which can be performed by a new class of antimicrobial compounds and an efficient intracellular drug delivery system.

On the other hand, among many intracellular pathogenic bacteria, Salmonella infection is a major public health concern because Salmonella is a major enteric pathogenic disease affecting both humans and animals. In particular, S. typhimurium, the serotype of S. typhimurium, is the most characteristic strain in the genus, and the pathogen is a polymorphonuclear leukocyte that passes through the intestinal monolayer and ultimately enters the systemic site , It colonizes the intestinal tract and adequately controls the integrity of the epithelial tight junctions for physical movement that can penetrate into the intestinal epithelium that can access the epithelium. Currently, although antibiotics are being used to treat invasive Salmonella infections, multi-durg resistance (MDR) remains a major concern.

Many new classes of antimicrobial compounds are being developed to treat multidrug-resistant (MDR) bacteria, among which antimicrobial peptides (AMPs) are regarded as promising drug candidates due to their wide activity range and reduced resistance by target cells . Many types of organisms present in the natural world produce antimicrobial peptides outside the body as part of a biological defense system. These antimicrobial peptides are composed of relatively short amino acid sequences (from about 10 to about 100 amino acid residues) and have a net charge of +2 to +9. However, more than 300 species, including Magainin, Cecropin, Defensin, Buforin, Protegrin, and Tachyplesin, have been discovered by researchers around the world, and a series of antimicrobial peptides produced by almost all living organisms, +), Gram-negative bacteria (Gram-), fungi, and tumor cells. Antimicrobial peptides found in eukaryotic multicellular organisms have alpha-helix, beta-sheet or random coil structures.

When these peptides bind to the cell membrane, they form an ion channel in the cell membrane to inhibit the energy production of the microorganism, or to make a large hole in the cell membrane, resulting in death of the cell. Unlike conventional antibiotics, which inhibit the cell wall and the synthesis of intracellular polymers, the microorganisms are very resistant to antimicrobial peptides because they have a mechanism of destroying the cell membrane by a nonspecific and effective physical method in a short time, To date, antimicrobial peptides have been reported not to produce resistance. The expression of these antimicrobial peptides in vivo or in vitro may be continuous, induced by bacterium-derived substances such as inflammatory cytokines, bacteria, and lipopolysaccharides (LPS) that stimulate innate immune responses do.

In addition, the mechanism of action of the antimicrobial peptides is believed to include binding of the lipopolysaccaride and lipoteichoic acid to the antimicrobial peptide, pore formation, or subsequent membrane break through other processes. In this case, the bacterial cell membrane is composed of phosphatidylglycerol and phosphatidylethanolamine, and is negatively charged. The amino acid residue charged by the amount of the antibacterial peptide mediates the electrostatic interaction with the microorganism through the negative charge exposed on the surface thereof do. This can alter the secondary structure of the antimicrobial peptide and promote their antimicrobial effect.

However, the application of antimicrobial peptides in clinical practice has been hampered by the lack of an effective delivery system. Systemic delivery of antimicrobial peptides is also challenging because antimicrobial peptides are degraded prior to reaching the site of infection and can not be administered orally, and the host immune system is rapidly identified and subject to immunity. For this reason, high antimicrobial peptide dosages are required, which can result in significant cost and more serious and serious side effects to achieve a therapeutic effect.

Over the past decade, researchers have studied liposomes, chitosan, lactide-co-glycolic acid (PLGA) -nano-particles, and lactic acid bacteria for the delivery of antimicrobial peptides. However, these molecules have limitations due to complexity and intrinsic cytotoxicity for loading AMPs. Therefore, there is an urgent need to study a structure for easily and efficiently delivering recombinant proteins to mammalian life forms without cytotoxicity (see Korean Patent Publication No. 10-2013-0118518).

DISCLOSURE OF THE INVENTION The present invention has been made to overcome the above-mentioned problems in the prior art. The present inventors have studied a structure for easily and efficiently delivering a recombinant protein to a mammalian organism without cytotoxicity, and a gold nanoparticle- (AuNP-Apt) complex-based delivery system and completed the present invention.

Accordingly, an object of the present invention is to provide an antimicrobial composition comprising a nanostructure comprising gold nanoparticles, an Aptamer bonded to the surface of the gold nanoparticles, and an antimicrobial peptide (AMPs) And a method for producing the nanostructure.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a nanostructure comprising gold nanoparticles, an Aptamer bonded to the surface of the gold nanoparticles, and an antimicrobial peptide (AMPs) And an antimicrobial composition.

In one embodiment of the present invention, the nanostructure may have a size of 10-20 nm.

In another embodiment of the present invention, the aptamer can be labeled with a label.

In another embodiment of the present invention, the antimicrobial peptide may be labeled with a label.

In another embodiment of the present invention, the aptamer can specifically bind to the labeling substance.

In another embodiment of the present invention, the labeling substance may be histidine (His).

The present invention also provides a method for producing a nanostructure comprising the steps of binding gold nanoparticles and an aptamer, and binding the antimicrobial peptide (AMPs) to the abtamer.

The present invention relates to an antimicrobial nanostructure comprising gold nanoparticles, an Aptamer bonded to the surface of the gold nanoparticles, and an antimicrobial peptide (AMPs) bound to the antimicrobial tamper, a method for producing the same, Wherein the nanostructure is specifically bound to an antimicrobial peptide to be delivered by various tagged platamers or antimicrobial peptide-specific aptamers bound to gold nanoparticles so that the antimicrobial peptide can be effectively delivered into cells, The gold nanoparticles having cytotoxicity are harmless to the human body and are expected to be useful for antibacterial use.

1 is a gold nanoparticle-a simplified schematic diagram of the ^His AMP pathway of infection in a mammalian cell-aptamer loaded on ^{His His} AMP and thus according Salmonella.
FIG. 2A is an analysis of the masses of synthetic antimicrobial peptides A3-APO and A3-APO ^His using matrix-assisted laser desorption ionization (MALDI) mass spectrometry.
2B is a graph showing cytotoxicity of AMP and AMP ^His in HeLa cells.
FIG. 2C is a graph showing the loading capacity of the gold nanoparticle-platemarin ^His complex. FIG.
FIG. 3 is a graph showing the antimicrobial peptide transporting ability of the gold nanoparticle-plastomer ^His through immunoassay and confocal laser scanning microscopic analysis.
FIG. 4 shows a circular dichroism (CD) spectrum of A3-APO and A3-APO ^His peptides.
FIG. 5 is a view showing the membrane depolarization of A3-APO and A3-APO ^His peptides.
FIG. 6 shows the breakdown of Salmonella cell membrane of A3-APO and A3-APO ^His peptides through SYTOX Green staining.
FIG. 7 shows the effect of A3-APO and A3-APO ^His peptides on GUVs (PE / PG, m / m).
Figure 8a is a diagram showing the mode of action of an artificial giant unilinear endoplasmic reticulum.
8B is a SEM micrograph of the S. typhimurium cells.
FIG. 9 shows antimicrobial effect of gold nanoparticle-platamater-AMP complex on S. typhimurium-infected HeLa cells through measurement of viable cell count of Salmonella.
Figure 10a is in the growth of the Salmonella typhimurium (S. Typhimurium) cells from the HeLa cells with gold nanoparticles - a diagram showing the effect of the APO-A3 loaded in the aptamer ^His.
Figure 10b A3-APO, and APO ^His-A3 and gold particles in serum - a diagram showing the stability of the bond between the aptamer ^His.
FIG. 11 is a graph showing the antimicrobial effect of the gold nanoparticle-plastomer-AMP complex against S. typhimurium-infected HeLa cells through measurement of the number of viable HeLa cells.
Figure 12 is a visualization of antimicrobial peptides (AMPs) delivered into cells through a gold nanoparticle-platamater-AMP complex in mice.
13 is a Salmonella typhimurium (S. Typhimurium) - the antimicrobial effect of the aptamer complexes -AMP - via the survival time and survival rate of infected mice, Salmonella typhimurium (S. Typhimurium) - gold particles on the infected mice It is also confirmed.
Figure 14 shows the results of a gold nanoparticle-platamer-AMP for S. typhimurium-infected mice through the measurement of viable bacterial counts in mesenteric lymph nodes (MLNc), Spleen, and Liver. The antimicrobial effect of the complex was confirmed.
15 is Vibrio vulnificus (V. vulnificus) - the antimicrobial effect of the aptamer complexes -AMP - via the survival time and survival rate of infected mice, Vibrio vulnificus (V. vulnificus) - gold particles on the infected mice It is also confirmed.

Based on the fact that the gold nanoparticle-platemater complex exhibits low toxicity and high efficiency of delivery of antimicrobial peptides (AMPs), the present inventors have specifically confirmed the antimicrobial / sterilizing effect of the gold nanoparticle-abatumer-AMPs , Thereby completing the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides an antimicrobial composition comprising a nanostructure comprising gold nanoparticles, an Aptamer bonded to the surface of the gold nanoparticles, and an antimicrobial peptide (AMPs) bonded to the electrode.

The term "nanoparticle" in the present invention means particles of various materials having a diameter of nano unit, and the nanoparticles are not particularly limited as long as they are nanoparticles.

The term "gold nanoparticles" in the present invention means metal particles of gold having a diameter of nano unit, and such small particle size facilitates penetration of the nanoparticles of the present invention into cells of interest (for example, human cells) To allow penetration of the protein carrier into the cell.

Gold nanoparticles are not only easy to manufacture in the form of stable particles, but they can also be varied in size from 0.8 nm to 200 nm to suit their application. In addition, gold can be combined with various kinds of molecules such as peptides, proteins, nucleic acids, etc. to change its structure and reflects light at various wavelengths, so that its position in a cell can be easily confirmed. Furthermore, gold nanoparticles are harmless to human body, unlike heavy metals such as manganese, aluminum, cadmium, lead, mercury, cobalt, nickel, and beryllium, and have high biocompatibility and have very little cytotoxicity.

When the diameter of the gold nanoparticles is more than 100 nm, the nanoparticles lose their properties and the bond between the gold surface and the functional groups such as the thiol group becomes weaker than the nanomaterial, Since the gold nanoparticles induce cytotoxicity, the gold nanoparticles of the present invention preferably have a diameter of 10 to 20 nm.

In the present invention, "Aptamer " means a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and capable of binding with high affinity and specificity to a target molecule , And SELEX (Systematic Evolution of Ligands of Exponential Enrichment) can be developed for various desired target substances (proteins, saccharides, dyes, DNAs, metal ions, cells, etc.).

The term "antimicrobial peptide " in the present invention means a peptide produced by many kinds of living organisms in nature as part of a biological defense system, but may be produced by an artificial method. These antimicrobial peptides are relatively short amino acids (From about 10 to about 100), and any kind can be used, and it is not particularly limited.

In addition, in the present invention, an aptamer that binds to gold nanoparticles may be any of various tagged platamers or antimicrobial peptide-specific platamers, and is not particularly limited.

The nanostructure according to the present invention can specifically bind an antimicrobial peptide to be delivered with various tagged platamers or antimicrobial peptides (AMPs) -specific aptamers bound to gold nanoparticles, thereby effectively delivering the antimicrobial peptide into cells, And can be usefully used for applications.

The present invention also provides a method for producing a nanostructure comprising the steps of binding gold nanoparticles and an aptamer, and binding the antimicrobial peptide (AMPs) to the abtamer.

The present invention also provides a method of delivering an antimicrobial peptide comprising the step of administering the construct to a subject. In the present invention, the term "individual" refers to a subject in need of diagnosis or treatment of a disease, and more specifically refers to a human or non-human primate, mouse, rat, dog, And the like.

In one embodiment of the invention, a gold nanoparticle-plumerator-peptide complex is prepared (see Example 1-3), and the viable cell count and viability of the infected cell in vitro or in vivo by the nanostructure 4 to 6). As a result, it was confirmed that the antimicrobial peptide was effectively delivered into the cells by the nanostructure of the present invention.

Therefore, the gold nanoparticle-plumper-peptide complex according to the present invention does not exhibit toxicity to cells and has high permeability into cells, and thus can be used for various purposes and applications requiring high delivery efficiency of an antibacterial peptide.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Peptide synthesis and purification

Peptides were synthesized by previously known procedures (J. K Lee, et al., A helix-PXXP-helix peptide with anti-bacterial activity without cytotoxicity against MDRPA-infected mice, Biomaterials 35 (2014) 1025-10390).

Next, to generate an N-terminal fluorescent-labeled peptide, the resin-bound peptide was treated with 20% (v / v) piperidine under dimethylformamide (DMF) to remove the Fmoc protecting group from the N- , The resin-bound peptide was reacted with rhodamine-SE (3-4 equivalents) under DMF containing 5% (v / v) diisopropylethylamine. After that, the mixture was gently mixed in the dark for 24 hours, and the resin was washed thoroughly in the order of DMF and dichloromethane (DCM), and the peptide was cleaved from each resin and precipitated and extracted with ether. The extracted crude peptide was purified by reversed-phase prep HPLC using a Jupiter C18 column (250 x 21.2 mm, 15 mm, 300 A) with a 0-60% acetonitrile gradient appropriate to water containing 0.05% trichloroacetic acid. Lt; / RTI > Peptide purity was measured by analytical reversed-phase HPLC on a Jupiter proteo C18 column (250 x 4.6 mm, 90 A, 4 μm) and molecular mass was determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI II, Kratos Analytical Ins.) Respectively.

1-2. Synthesis of gold nanoparticle-abatumomer conjugate (AuNP-Apt conjugates)

Citrate-reduced gold nanoparticles (AuNPs) (15 nm in diameter) were purchased from BBI Life Sciences (UK) and the histidine-tagged DNA extender (5'-GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC- A10-Thiol-3 ') was purchased from Bioneer (Korea). The DNA aptamer was treated with 1 N dithiothreitol (DTT) for 30 minutes at room temperature (RT) to cleave the disulfide bond, a His-tagged DNA plasmid oligonucleotide. Extracted three times with ethyl acetate to remove excess DTT and unwanted thiol fragments from the free thiol-modified oligonucleotide mixture. The cleaved oligonucleotides were purified using ethanol precipitation. Gold nanoparticles (AuNP) were mixed with the oligonucleotide at a ratio of 1: 100, and citrate-HCl buffer (0.5 M, pH 3) was added to a final concentration of ~10 mM. After brief vortexing, the samples were incubated at room temperature for 3 minutes. Next, the pH of the gold nanoparticle (AuNP) solution was adjusted to neutrality using HEPES buffer (0.5 M, pH 7.6). After further incubation at room temperature for 5-10 minutes, the gold nanoparticle (AuNP) -oligo mixture was centrifuged at 13000 x g and the supernatant was removed. The final mixture was redispersed in 5 mM HEPES buffer. The concentration of functionalized gold nanoparticles (AuNP) was measured by UV-Vis spectroscopy. The concentration of nanoparticles was measured through Beer's law (A = εbc) using absorbance values. The wavelengths of the maximum absorption (λ) and extinction coefficient (ε) used for the 15 nm particle size are: λ = 524 nm, ε = 2.4 × 10 ⁸ L /

1-3. Manufacture of gold nanoparticle-abtamer-peptide complex (AuNP-Apt-peptide complex)

Abtamer-bound gold nanoparticles (AuNPs) were pre-incubated at 80 ° C for 5 minutes to prevent the formation of secondary structures. AuNP-Apt ^His (1 nM) and the purified His-tagged peptide were equilibrated with 1 x AMP (1 mM) buffered with 1 x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , in binding buffer (200 mM Tris-Cl , pH 8.8, 200 mM NaOH, and 1 mM MgCl ₂₎ and incubated at room temperature for 10 minutes. The pH of each solution was adjusted with hydrogen chloride.

1-4. Evaluation of Binding Capacity

Evaluation of binding potency between antimicrobial peptides (AMPs) and AuNP-Apt ^His conjugates was performed according to a previously known procedure (SM Ryou, et al., Gold nanoparticle-DNA aptamer composites as a universal carrier for in vivo delivery of biologically functional proteins, J. Control Release 196 (2014) 287-294).

1-5. Antibacterial evaluation

Salmonella enterica Typhimurium ATCC14028 cells were cultured at 37 ° C in Luria-Bertani (LB) medium and the minimum inhibitory concentration (MIC) of each peptide was measured by microdilution analysis. In other words, to produce a culture medium containing 5 x 10 ⁵ colony forming unit (CFU) / ml of bacteria in each mid-logarithmic phase, A twofold serial dilution was added and the samples were incubated at 37 ° C for 18-24 hours, at which time the MIC was confirmed at the lowest peptide concentration based on the OD ₆₀₀ measurements of the culture.

1-6. Animal cell culture

HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v / v) fetal bovine serum (Welgene) and 1% (v / v) penicillinestreptomycin (Welgene) ) (Welgene, Korea) at 37 ° C and 5% (v / v) CO ₂ .

1-7. Cytotoxicity Assessment

HeLa (2 x 10 ⁴ / well) cells were cultured in 96-well culture dishes for 18-24 hours. The cultured cells were further incubated with the peptide for 24 hours, and viability was evaluated using the CellTiter-Glo® Luminescent Cell Viability Assay kit.

1-8. Live and dead cell assay

To analyze live and dead cells, S. typhimurium ATCC 14028 cells were cultured with the peptide and monitored using a fluorescence microscope (LX71, Olympus, Tokyo, Japan). A3-APO and A3-APO ^His peptides were added to the cells using the same procedure as used for the antimicrobial assay. The peptides were added to each 500 μl cell suspension (1 × 10 ⁷ CFU / mL) with each MIC. After 20 minutes of incubation, the cells were centrifuged at 8000 xg for 5 minutes and the cells on the slides were observed with a microscope (LX71, Olympus).

1-9. Flow cytometry

Salmonella typhimurium (S. Typhimurium) is incubated at 37 ℃, LB medium, and cells were suspended in LB medium ^{(5 x 10 6 CFU / ml} ). The cells were harvested by centrifugation (10,000 xg, 5 min) and cultured at 37 ° C for 20 min. The peptides were suspended in FxCycle ™ PI / RNase staining solution (Life Technologies, Carlsbad, Calif., USA) . Cells were then incubated at room temperature for 20 minutes and flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, Calif., USA).

1-10. Circular dichroism analysis

Analysis of circular dichroism was performed by previously known procedures (JK Lee, et al., A helix-PXXP-helix peptide with anti-bacterial activity without cytotoxicity against MDRPA-infected mice, Biomaterials 35 (2014) 1025-1039 ',' M. Cassone, et al., Scope and limitations of the designer proline-rich antibacterial peptide dimer, A3-APO, alone or in synergy with conventional antibiotics, Peptides 29 (2008) 1878-1886 ').

1-11. Membrane depolarization analysis

The membrane depolarization assay was performed according to a previously known procedure (J. K. Lee, et al., A helix-PXXP-helix peptide with antibacterial activity without cytotoxicity against MDRPA-infected mice, Biomaterials 35 (2014) 1025-10390).

1-12. SYTOX Green dyeing

SYTOX Green staining was performed according to previously known procedures (J. K. Lee, et al., A helix-PXXP-helix peptide with anti-bacterial activity without cytotoxicity against MDRPA-infected mice, Biomaterials 35 (2014) 1025-10390).

1-13. Giant liposome analysis

Giant liposomal analysis was performed according to previously known procedures (T. M. Domingues, K. A. Riske, A. Miranda, Revealing the lytic mechanism of the antimicrobial peptide gomesin by observing giant unilamellar vesicles, Langmuir 26 (2010) 11077-11084.).

1-14. Confocal laser scanning microscopy (CLSM) analysis

Confocal laser scanning microscopy analysis was performed according to previously known procedures (JK Lee, et al., A helix-PXXP-helix peptide with anti-bacterial activity without cytotoxicity against MDRPA-infected mice, Biomaterials 35 (2014) 1025-10390 ).

1-15. Scanning electron microscopy (SEM) analysis

Scanning electron microscopy analysis was performed according to previously known procedures (J. K Lee, et al., A helix-PXXP-helix peptide with anti-bacterial activity without cytotoxicity against MDRPA-infected mice, Biomaterials 35 (2014) 1025-10390).

1-16. Gold nanoparticles - Abtamer  Complex Combined Of peptide  Visualization of peptide uptake by AuNP-Apt conjugates

HeLa cells cultured on lysine-coated 10 mm cover slip were treated with gold nanoparticle-abtamer-AMP complex (AuNP-Apt-AMP complex) and incubated for 1 hour. After incubation, cells were fixed with 4% paraformaldehyde (Sigma, USA) and cells were immunostained with Alexa 488 labeled anti-His and rabbit IgG (excitation 495 nm and emission 519 nm). Alexa488 (495 nm excitation, 519 nm emission) fluorescence was detected through a confocal microscope (Carl Zeiss ZEN 2011, Germany).

1-17. Evaluation of peptide stability of peptide

Hypophilic peptide (16 μM) and gold nanoparticle-abtamer-peptide (16 μM) complexes were cultured in DMEM containing 10 or 100% (v / v) fetal bovine serum (FBS) , 12, 24, or 48 hours later, each peptide and serum were separated by tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) Respectively.

1-18. Western blot analysis

Tricine-SDS-PAGE of the prepared sample was performed and transferred to the membrane. Membranes were immunoblotted with anti-His (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Qiagen) and anti-GAPDH (Ab Frontier, Seoul, Korea) antibodies. The signal was measured with an electrochemiluminescent reagent. Band strength was measured with Quantity One (Bio-Rad, Richmond, CA, USA).

1-19. ( In vitro ) Measuring live cell count in intracellular bacteria cell

HeLa (5 x 10 ⁴ / well) cells were cultured in a 24-well culture dish for 18-24 hours. The cultured HeLa cells were infected with S. typhimurium ATCC14028 for 1 hour at a multiplicity of infection (MOI) of 1: 100. Cells were washed with DMEM containing 50 μg / ml gentamycin (GIBCO ™, Invitrogen, USA) and resuspended in 10% (v / v) FBS and gold nanoparticle-aptamer-AMP complex (AuNP- Or in DMEM containing control reagent for 1 or 6 hours. The infected cells were washed three times with 1 x PBS to remove remaining extracellular bacteria and antimicrobial peptides, dissolved in PBS containing 1% (v / v) Triton X-100, and the intracellular bacteria released from the cells . Bacterial cells were diluted with PBS (1:25) and plated on LB plates to determine the number of CFUs.

1-20. ( In vitro ) Evaluation of infected cell viability

HeLa (2 x 10 ⁴ / well) cells were cultured in 96-well culture dishes for 18-24 hours. The cultured HeLa cells were either buffered or infected with S. typhimurium at a multiplicity of infection (MOI) of 1: 100 for 1 hour and then incubated with 50 μg / ml gentamicin (GIBCO , Invitrogen, USA). The cells were then cultured for 0, 6, 12, or 24 hours in DMEM containing 10% (v / v) FBS and gold nanoparticle-abtamer-AMP complex (AuNP-Apt-AMP complex) The infected cells were then recovered with trypsin-ethylenediaminetetraacetic acid and viability was measured by trypan blue dye exclusion staining.

1-21. Animal preparation

18-20 g 6-week-old female FvB mice (DBL, Korea) were prepared, and the animal breeding room maintained a 30-40% humidity and a temperature of 22 ± 1 ° C. Indoor lighting was controlled by 12-hour light-dark cycle. The animal protocol was approved by the Chung Ang University Animal Experiment Center for animal experiments, and the animals were processed as described in the protocol.

1-22. ( In vivo ) Evaluation of peptide stability

Hydrophilic AMP (8 mg / kg) and gold nanoparticle-aptamer-AMP (8 mg / kg) complex were intravenously injected (i.v.). After 0, 6, 12, 24, or 48 hours of injection, the mice were sacrificed and the spleen and liver were removed and fixed in 4% paraformaldehyde at 4% (v / v) overnight. The fixed organs were dehydrated for 2 days at 4 ° C, 30% (v / v) sucrose, and then embedded in the optimal cleavage temperature compound. Frozen blocks were cut to 10 mm, and after hot air drying, the fragments were immunostained with Alexa 488 labeled anti-His and rabbit IgG antibodies. Alexa488 (495 nm excitation, 519 nm emission) fluorescence was detected through a confocal microscope (Carl Zeiss ZEN 2011, Germany).

1-23. ( In vivo ) Measuring live cell count in intracellular bacteria cell

The inoculum of S. Typhimurium ATCC14028 was cultured on LB on mid-log and washed with PBS. Mice were prevented from consuming food and water for 4-18 hours before inoculation. The mice were then infected with 100 μl PBS containing 1-5 × 10 ² CFU of salmonellal intraparenchymal. Four hours after bacterial challenge, gold nanoparticles-aptamer-AMP (8 mg / kg) complex and control reagent were intravenously injected iv once a day.

Bacterial administration (bacterial challenge) 3 days, the control group, A3-APO ^His -, AuNP-Apt -HPN3 ^{His His} - and -A3-AuNP-Apt ^{His His} APO were sacrificed treated mice mesenteric lymph nodes, spleen, and liver . After organ weighing and individual homogenization, they were dissolved in PBS. The homogenate was plated on Salmonella-Shigella agar (Neogen, Lansing, MI, USA) and incubated overnight at 37 ° C so that the colonies could be counted. The results are expressed as the number of CFU per gram, and the analysis was repeated twice.

1-24. ( In vivo ) Bacterial administration and treatment with gold nanoparticle-abtamer-AMP complex

Four hours after bacterial challenge, gold nanoparticle-platemaker-AMP (8 mg / kg) complex and control reagent were intravenously injected (i.v.) once a day for five consecutive days. The injected mice were observed for 14 days after infection, and the moribund cases were sacrificed immediately.

Example  2: Gold nanoparticles - Abtamer  Antibacterial through complex Peptides  (AMP) < / RTI > Transmission ability  Confirm

To confirm whether gold nanoparticles (AuNP-Apt ^His ) bound to histidine-tagged DNA plasmids could transfer antibiotic activity by transferring the hexa-histidine (His) tagged antimicrobial peptide (AMP ^His ) into mammalian cells , And the experiment was conducted as follows. A schematic diagram of the experiment was shown in Fig.

2-1. Identification of antimicrobial activity of peptides

First, the bactericidal activity and cytotoxicity of the C-terminal histidine-tag A3-APO (A3-APO ^His ) were evaluated. A3-APO is a new class of synthetic peptides derived from natural insect products. It is a major peptide and has a bactericidal activity against Salmonella enterica serotype Typhimurium in vitro and in vivo (Table 1 and Fig. 2A below). Even if labeled with hexa histidine, the random coil structure of A3-APO is not altered, and still has a bactericidal activity against Salmonella compared with the case of His-unlabeled. In addition, A3-APO ^His did not show cytotoxicity to HeLa cells at a concentration of 200 μM (Fig. 2B). At this time, the histidine-tag HPN3 (HPN3 ^His ) used as a negative control did not show any bactericidal action or cytotoxicity. Further, when the treatment for 20 minutes, the Salmonella typhimurium (S. Typhimurium) cells with APO-A3 and A3-APO ^His peptide of the minimum inhibitory concentration (MIC), propidium iodide-sensitive cells, the number of significantly increased As a result, the death of bacterial cells was rapidly induced by A3-APO and A3-APO ^His peptides.

2-2. Confirmation of intracellular delivery of antimicrobial peptide (AMP) through gold nanoparticle-platameric complex

First, the histidine-tagged AMPs the Simple Mixed Culture with gold nano-particles and then loading the aptamer ^His, was applied to HeLa cells, and histidine from the reaction mixture - the tag about 40 to 50% of the gold particles of the AMPs-aptamer ^His (Fig. 2C).

Thus, according to the method of Example 1-16, AMPs and a final concentration of ^AuNP-His Apt by a 16 mM and 1 nM, respectively, a buffer, a glass A3-APO ^His, gold nanoparticle-aptamer ^His, gold nano particle-aptamer ^His -A3-APO ^His, or gold nanoparticle-aptamer ^{His His} -HPN3 confirming the fluorescent image (scale bar = 20 μm) of the HeLa cells were incubated for 1 hour by the addition of the results, shown in Figure 3 HeLa cells cultured with gold nanoparticle-aptamer ^His loaded with histidine-tagged AMPs (A3-APO ^His or HPN3 ^His ) showed strong fluorescence signals on immunoassay and confocal laser scanning microscopy, But not fluorescence signals in control cells treated with only gold nanoparticle-platamater ^His or A3-APO ^His peptide.

Example 3: A3-APO and A3-APO ^His Confirm the mechanism of action of

In order to confirm the mechanism of action of A3-APO and A3-APO ^His peptides based on the antimicrobial activity and the intracellular delivery ability of the antimicrobial peptide (AMP) through the gold nanoparticle-platamater complex, To (1-15).

First, to observe the membrane-binding interactions of A3-APO and A3-APO ^His peptides, anionic sodium dodecyl sulfate (SDS), which forms a membrane-mimetic environment through aggregation and pore formation during binding to phospholipids, Micelles were used. As a result, as shown in Fig. 4, the circular dichroism (CD) spectrum of the two peptides showed no self-association such as 30 mM SDS micelles in the membrane-mimetic environment, It was confirmed that the chemical shift value of the residue slightly changed.

In addition, as shown in Fig. 5, when A3-APO and A3-APO ^His peptides were added to S. typhimurium cells, outer membrane splitting was observed at DisC3-5 fluorescence intensity, No change in fluorescence intensity was observed. More specifically, APO-A3 and A3-APO ^His showed that it does not share the same mechanism acting on the outer membrane of Salmonella typhimurium (S. Typhimurium). A3-APO peptide induced rapid pore formation with rapid reaction, whereas A3-APO ^His did not show such a phenomenon, but instead showed increased membrane-depolarization activity.

As a result of confirming whether or not the peptide can cause cleavage of cytoplasmic membrane of SYTOX green-loaded S. typhimurium cells, it was confirmed that A3-APO and A3-APO ^His were able to induce cleavage of cytoplasmic membrane of Salmonella typhi It was confirmed that inducing internal membrane division of S. typhimurium and membrane breakage was found based on increase of SYTOX green fluorescence intensity. At this time, the intracellular SYTOX fluorescent dye signal does not change when the cell membrane is damaged, but increases when the dye binds to the exposed nucleic acid due to membrane breakage.

Further, confirmation of the morphological change and localization of the peptide using one large single-layered vesicle (GUVs) containing phosphatidylethanolamine / phosphatidylglycerol (PE / PG, 7: 3, v / v) As shown in FIG. 7, A3-APO ^His was found to destroy the GUV membrane to a greater extent than A3-APO. In particular, the A3-APO ^His peptide contained hexahistidine, which positively charged at the C- And the surrounding of the lipid in the GUV membrane. That is, although the A3-APO ^His antimicrobial peptide could function through a kind of "carpet-like" behavioral mechanism, the A3-APO peptide could not penetrate the cell membrane due to temporary pore formation (FIG. 8A).

Furthermore, using the confocal laser scanning microscope (CLSM) Salmonella typhimurium (S. Typhimurium) when cultured Rhodamine labeled-by monitoring the position of the peptide A3-APO in live Salmonella typhimurium (S. Typhimurium) cells and A3-APO results confirm the mechanism of ^His, rhodamine-labeled A3-APO ^His peptide as opposed to, rhodamine-labeled A3-APO peptides were found to accumulate on the cell surface, rather than the cytoplasm, which in the A3-APO antimicrobial peptide In the case of A3-APO ^His peptide, it means to strongly interact with the cell surface that destroys the cell membrane, while it means to migrate from the S. typhimurium membrane to the cytoplasm interacting with nucleic acid and related protein. Thus, these peptides appear to have played a role through other mechanisms leading to apoptosis of S. typhimurium.

In addition, A3-APO ^His peptides were found to cause membrane breakage in the cytoplasmic membrane by scanning electron microscopy (SEM) and confirming the cultured Salmonella ATCC14208 cells by adding A3-APO or A3-APO ^His of each MIC. , But the A3-APO peptide did not affect the cell membrane. In other words, it was confirmed that A3-APO ^His peptide can destroy the cytosolic membrane to kill bacteria, whereas A3-APO peptide can penetrate cells only by forming temporary pores in the membrane (FIG. 8B).

Example  4: ( in vitro ) Salmonella Tiffy myrium  ( S. Typhimurium ) - Confirmation of antimicrobial effect of gold nanoparticle-platamer-AMP complex on infected mammalian cells

In order to confirm the influence of gold nanoparticle-platamer ^His- A3-APO ^His on S. typhimurium-infected cells, the experiment was carried out by the method of Example 1-19.

The aptamer cell added -HPN3 ^{His His-As} a result, the gold nano-particles as shown in Fig. 9 - aptamer ^His -A3-APO when the addition of ^His, gold nanoparticle-aptamer ^His or gold nanoparticles , The number of cells of S. typhimurium cells was about 30-50% lower than that of gold nanoparticles, and the bactericidal effect of gold nanoparticle-aptamer ^His -A3-APO ^His was dose-dependent 10a). In addition, it was confirmed that the treatment of A3-APO ^His for 1 hour did not affect the number of surviving salmonella cells in the cell, but when the same treatment was performed for 6 hours, the viable cell count was reduced by about 20% , The bactericidal effect of this A3-APO3 ^His appears to be due to its low permeability to mammalian cells, as reported for A3-APO.

Therefore, it is expected that the gold nanoparticle-platamater ^His conjugate effectively transfers the antimicrobial peptide through the plasma membrane of mammalian cells, thereby improving the bactericidal action of A3-APO ^His on intracellular bacteria.

Thus, the gold nano-particles, and incubated for an additional 48 hours in the serum was measured for the amount of intact A3-APO ^His to confirm whether or not to improve the stability of the ^A3-His APO when loaded into the aptamer ^His. As a result, the gold nano-particles, if not loaded in the aptamer ^His was a full A3-APO ^His quickly and progressively confirm the reduced, and consequently determine the serum culture After 48 hours, about 60% of the peptide disappeared (Fig. 10b ). Decomposition (degradation) of the other hand, the loaded A3-APO ^His in the serum of gold nanoparticles -Apt ^His complex is A3-APO ^His This result indicates that the gold nanoparticle-aptamer ^His can improve not only the permeability of the host cell but also the stability of A3-APO ^His (see, for example, do.

Gold nanoparticle-pressure based on the fact that the transfer of A3-APO ^His induces death of the bacterial cells through Tamer ^His, gold nanoparticle-aptamer ^His reduction in the number of cells within the bacteria through -A3-APO is ^His It was confirmed by experimenting with the method of Example 1-20 that the viability of S. Typhimurium-infected HeLa cells could be increased by the method of Example 1-20.

As a result, as shown in Fig. 11, the cells treated with the gold nanoparticle-platamater ^His -A3-APO ^His proliferated continuously, resulting in an increase in the number of viable HeLa cells by about 80% within 24 hours , These results were similar to those of HeLa cells infected with Salmonella. In contrast, cells treated with gold nanoparticles-platemer ^His or gold nanoparticles-platamag ^His -HPN3 ^His showed about 40% reduction in the number of viable HeLa cells, similar to untreated cells. In addition, the number of viable cells was reduced by about 15% when treated with A3-APO ^His , as compared to that treated with gold nanoparticle-platamater ^His or gold nanoparticles-platamag ^His -HPN3 ^His , Consistent with the low level of intracellular bactericidal activity of A3-APO ^His on S. Typhimurium-infected HeLa cells.

Taken together, the increase in intracellular bactericidal activity of A3-APO ^His by the gold nanoparticle-platemer ^His complex increases the viability of S. typhimurium-infected mammalian cells, The separation of A3-APO ^His from the gold nanoparticle-platemer ^His complex in HeLa cells was performed using HeLa cells treated with A3-APO ^His loaded on Cy5-labeled gold nanoparticles-aptamer ^His Confocal fluorescence images were confirmed.

Example  5: ( in vivo ) Salmonella Tiffy myrium  ( S. Typhimurium ) - Gold nanoparticles for infected mice - Abtamer Confirmation of sterilization effect of -AMP complex

Based on the results of the above examples, it was also examined whether the system could be applied to a living organism. To this end, similar to Salmonella typhimurium (S. Typhimurium) and the typhoid fever caused by human Salmonella typhimurium (S. Typhimurium) - was used to infect a mouse model.

First, acute infection FvB / N induced by Salmonella typhimurium (S. Typhimurium) injected intraperitoneally (ip) to 1-5 x 10 ² CFU per mouse, eight hours after the inoculation, mesenteric lymph nodes, spleen, and liver Lt; RTI ID = 0.0 > Typhimurium < / RTI > Four hours after the inoculation, the growth of S. typhimurium and its establishment in these organs were confirmed.

In addition, the gold nanoparticle-aptamer ^His -A3-APO to optimize the effect of ^His, the gold nanoparticles in the mouse-aptamer ^His -A3-APO ^His 48 hours of the (8 ㎎ / ㎏) intravenous (iv) , The half-life of A3-APO ^His was measured by analyzing the relative amount of intact A3-APO3 ^His in each organ. At this time, the presence of A3-APO3 ^His was measured by immunostaining and confocal laser scanning microscopy, and the half-life of the measured A3-APO3 ^His was 28 hours, which was much longer than that of other related proline-rich peptides ). Considering that A3-APO ^His was not detected in the mouse organs when injected alone, it was found that the gold nanoparticle-aptamer ^His enhanced the cell permeability as well as the stability of A3-APO ^His in mice.

Next, in order to confirm whether or not the gold nanoparticle-platemer ^His complex can increase the intracellular bactericidal ability of A3-APO ^His in S. Typhimurium-infected mice, 23 and 1-24.

First, Salmonella typhimurium (S. Typhimurium) cells 4 hours after inoculation, a buffer, APO-A3, A3-APO ^His, gold nanoparticle-aptamer ^His, gold nanoparticle-aptamer -HPN3 ^{His His,} or gold nano The survival rate of each group of mice was measured for 10 days, intravenously (iv) once a day for consecutive 5 days, with the particle-platelet ^His -A3-APO ^His .

As a result, the mean survival (median survival duration) of the mouse as shown in FIG. 13, buffer, A3-APO, A3-APO ^His, gold nanoparticle-aptamer ^His, or gold nanoparticle-aptamer ^His If one to three times each injection -HPN3 ^His 5.1-5.7 il was the other hand, gold nanoparticle-aptamer for ^His treated mice in -A3-APO ^His, all mice survived in the measurement period.

Thus, in order to confirm whether 100% survival of mice treated with gold nanoparticle-platamater ^His -A3-APO ^His was due to complete removal of intracellular S. typhimurium cells, mesenteric lymph nodes, Salmonella typhimurium (S. Typhimurium) measured the number of cells that survive in mouse organs, such as the spleen, and liver.

As a result, as shown in Figure 14, immunization Three days later, when the visible signs of morbidity (morbidity) of infected mice did not show a buffer, A3-APO, A3-APO ^His, gold nanoparticle-aptamer ^His, or gold nanoparticle-aptamer ^His -HPN3 gold nanoparticles as compared to the mice treated with a ^His-for aptamer ^{His His} -A3-APO-treated mouse, a Salmonella typhimurium survival in all three organs Solarium (S. typhimurium) The number of cells was reduced by ~ 93-98%, and in the organs of infected mice injected with gold nanoparticles-aptamer ^His -A3-APO ^His once a day for five consecutive days, colonization of S. Typhimurium Was not observed, and mice were sacrificed after 7 days of inoculation.

Taken together, intravenous (iv) injection of A3-APO ^His -loaded gold nanoparticle-platemer ^His complex completely removes the intracellular S. typhimurium cells and allows infected mice to survive I can see that it can.

Example  6: ( in vivo ) Vibrio Bulfinicus  ( Vibrio vulnificus ) - Determination of the bactericidal effect of gold nanoparticle-platamer-AMP complex on infected mice

Based on the results of this example, it was also examined whether this system could be applied to infections of other infectious bacterium, V. vulnificus . For this purpose, a mouse model of infection with V. vulnificus , a germ causing sepsis and food poisoning in humans, was used, and platamol HPA3P and HPA3P ^His having a specific inhibitory effect on V. vulnificus Respectively.

Vibrio vulnificus (V. vulnificus) cell inoculation After 2 hours, buffer, HPA3P ^His, gold nanoparticle-aptamer ^His, or gold nanoparticle-aptamer ^{His His} -HPA3P a one-time intravenous injection (iv) infection in mice And the survival rate of each group of mice was measured for 5 days.

Whereas when injected once with aptamer ^His, 24 ~ 40 hours, - As a result, the average survival time of the mice (median survival duration) is, the buffer, HPA3P ^His, or gold particles as shown in Fig. 15 gold nanoparticle-aptamer for the mice treated with -HPA3P ^{His His,} all mice survived in the measurement period.

Taken together, the above information, ^His HPA3P the loading gold nanoparticle-IV of aptamer ^His composite (iv) injection of cells Vibrio vulnificus (V. vulnificus) cells found to be able to survive the infection, the mouse there was.

Therefore, when the gold nanoparticle-platemer ^His complex is used as a carrier for an antibacterial peptide, the toxicity can be lowered and the delivery efficiency can be increased, suggesting that the gold nanoparticle-platemer ^His complex can be applied to the treatment using an antibacterial substance.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (7)

Gold nanoparticles;
An aptamer bonded to the surface of the gold nanoparticles; And
And an antimicrobial peptide that is bound to the platemaker.
The method according to claim 1,
Wherein the gold nanoparticles have a size of 10-20 nm.
The method according to claim 1,
Characterized in that the aptamer is labeled with a label.
The method according to claim 1,
Wherein said antimicrobial peptide is labeled with a label.
5. The method of claim 4,
Wherein the aptamer specifically binds to the labeling substance.
4. The method according to claim 3 or 4,
Wherein the labeling substance is histidine (His).
Combining gold nanoparticles with an aptamer; And
And binding the antimicrobial peptide to the squamometer.

Images