JP5007446B2 - Carbon nanohorn complex, antibacterial agent containing the complex, and microorganism killing method - Google Patents

Description

  The present invention relates to a carbon nanohorn complex, an antibacterial agent containing the complex, and a microorganism killing method.

  In recent years, interest in carbon nanoparticles such as fullerenes, carbon nanotubes (CNT), and carbon nanohorns (CNH) has increased as functional nanoparticles. The carbon nanohorn is not a constant tube diameter like the carbon nanotube, for example, it is a very characteristic substance in which the tube-shaped nanocarbon material with a conical cap (horn) having a diameter of about 80 nm is agglomerated in a spherical shape. It is. The characteristic structure possessed by carbon nanohorn attracts attention and is expected to be applied to electronic materials, catalysts, drug carriers and the like.

  Since carbon nanohorn is inherently hydrophobic, it is difficult to disperse in an aqueous solvent and difficult to use in an aqueous solution system. However, as a solution to such a problem, for example, carbon A hydrophilic carbon nanohorn having a functional group added to the carbon structure constituting the nanohorn has been reported (Patent Document 1).

  There has also been a report that the dispersibility of the carbon nanohorn structure in an aqueous solvent is enhanced by a polyethylene glycol-doxorubicin complex (Non-Patent Document 1).

  Furthermore, as a drug carrier in a drug delivery system, a drug carbon nanohorn complex in which a steroidal drug or a metal-containing drug is adsorbed or encapsulated in an oxidized pore-opened carbon nanohorn has also been reported (Patent Document 2).

  On the other hand, MRSA (Methicillin Resistant Staphylococcus Aureus; methicillin-resistant Staphylococcus aureus), VRSA (Vanmycin Resistant Staphylococcus Aureus; The emergence of multidrug-resistant bacteria that do not work at all has become a major social problem, and the development of new antibacterial agents is desired.

  In addition, the main antibiotics currently used have a defect that kills not only harmful bacteria but also useful bacteria because the antibacterial spectrum is too wide.

Furthermore, with the emergence of various infectious diseases caused by viruses, there is a strong demand for the establishment of effective treatments. Antiviral agents that have been developed so far basically show medicinal effects by inhibiting the virus growth process (enzyme reaction group), but it is a matter of time that the virus disappears due to its rapid evolution. . In addition, since the antiviral agent itself is not selective, normal cells and tissues are damaged and strong side effects are caused.
JP 2003-95624 A JP-A-2005-343885 Murakami, T. et al. Mol. Pharmaceutics 3, 407-414 (2006) Murakami, T. et al. Mol. Pharmaceutics 1, 399-405 (2004)

  The present invention is a novel carbon that selectively kills harmful microorganisms such as bacteria, fungi, and viruses while suppressing the appearance of multi-drug resistant bacteria by a mechanism completely different from conventional antibiotics and antiviral agents. It is an object to provide a nanohorn complex, an antibacterial agent containing the complex, and a method for sterilizing microorganisms.

  As a result of intensive studies to solve the above problems, the inventors of the present invention are excellent in that the novel carbon nanohorn complex synthesized for the first time by the inventors binds to and kills the cell walls of microorganisms such as harmful bacteria. And found to have antibacterial action. The present invention has been completed based on such findings.

The present invention provides a carbon nanohorn complex shown in the following items 1 to 6, an antibacterial agent containing the complex, and a method for sterilizing microorganisms.
Item 1. A carbon nanohorn complex formed by adsorbing a phospholipid-polyethylene glycol-molecule recognition element conjugate to a carbon nanohorn.
Item 2. The carbon nanohorn complex according to claim 1, wherein the molecular recognition element is a lectin or an antibody.
Item 3. Item 3. The carbon nanohorn complex according to Item 1 or 2, wherein the phospholipid is selected from the group consisting of monoacyl or diacyl phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, diacylphosphatidylethanolamine, and sphingomyelin.
Item 4. An antibacterial agent comprising the carbon nanohorn composite according to Item 1 as an active ingredient.
Item 5. An antiviral agent comprising the carbon nanohorn complex according to Item 1 as an active ingredient.
Item 6. The carbon nanohorn complex according to Item 1 is brought into contact with a microorganism to form a microorganism capture complex, and the capture complex is irradiated with visible (600 nm to 700 nm) to near infrared light (700 nm to 2500 nm), thereby A method for killing microorganisms characterized by killing.

  According to the present invention, only specific microorganisms (bacteria, fungi, viruses, etc.) to which the molecular recognition element binds can be killed, and useful microorganisms are not affected, so that the balance of microorganisms is not destroyed.

  The novel carbon nanohorn complex of the present invention is capable of selectively killing harmful microorganisms such as bacteria, fungi and viruses by a mechanism completely different from conventional antibiotics and antiviral agents. It is useful as an active ingredient for antibacterial agents and antiviral agents.

  The inventor has discovered that when the composite is irradiated with a near-infrared laser, an exothermic reaction is caused.

  Laser irradiation increases the particle size of the complex over time, so when administered to animals such as humans (especially mammals), laser irradiation increases the particle size of the complex and discharges it outside the body. It is possible to promote.

  In addition, by applying the complex of the present invention and a near-infrared laser, we succeeded in observing the selective killing of microorganisms such as bacteria, fungi and viruses in real time.

  The carbon nanohorn (hereinafter sometimes abbreviated as CNH), which is a raw material for the carbon nanohorn composite of the present invention, has a diameter of about 80 nm, and a structure in which a tube-shaped nanocarbon material with a conical cap is mostly aggregated in a spherical shape. Have

  In the present invention, microorganisms recognized by the molecular recognition element include Escherichia coli (including pathogenic Escherichia coli such as O-157), Salmonella, Campylobacter, Staphylococcus aureus, Clostridium perfringens, Bacillus cereus, Vibrio parahaemolyticus, and Yersinia. Multidrug-resistant bacteria such as food poisoning bacteria, MRSA, VRSA, VRE, spirochetes, gonorrhea, tuberculosis, rickettsia, chlamydia, mycoplasma, anthrax, cholera, diphtheria, tetanus, plague, shigella, streptococci (A Group β-streptococci, pneumococci, etc.), Staphylococcus aureus (MSSA), Staphylococcus epidermidis, enterococci, Listeria, meningococcus, Klebsiella (Klebsiella pneumoniae), Proteus, Bordetella pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter , Acinetobacter, Enterobacter, Clostridium, Scarlet fever, Trachoma, Legione , Candidiasis, cryptococcosis, ringworm, histoplasmosis, include the bacteria that cause infections, such as Pneumocystis pneumonia bacteria. Examples of the microorganism recognized by the molecular recognition element include adenovirus, coronavirus, herpes virus, influenza virus (type A, type B), measles virus, vaccinia virus, HIV, dengue virus, and the like. And viruses shown in Tables 1 to 3 below.

  CNH to which a carboxyl group is added (hereinafter sometimes abbreviated as CNH-COOH) may be produced by oxidizing a carbon atom to COOH using a nitric acid aqueous solution, for example, according to a conventionally known method (Patent Document 1). . A specific production example of CNH—COOH is shown in Reference Example 1 described later. CNH can be produced by a conventional method such as irradiating laser light to carbon at room temperature.

  Examples of the phospholipid constituting the CNH complex of the present invention (hereinafter sometimes abbreviated as “PL”) include glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and diphosphatidylglycerol (cardiolipin). And sphingophospholipids such as sphingomyelin. Among these, phosphatidylethanolamine is preferable.

  The molecular recognition element constituting the CNH complex of the present invention is not particularly limited as long as it can recognize cell walls of bacteria, fungi and the like, and examples thereof include lectins, antibodies, peptides, virus receptors and the like.

  Examples of the lectin include L-fucose binding lectin, D-galactose or N-acetyl-D-galactosamine binding lectin, D-mannose binding lectin (such as lectin derived from concanavalin A, kidney bean or broad bean), di-N-acetyl key. Tobiose binding lectin (lectin derived from WGA, PWM, DSA, etc.), sialic acid binding lectin (lectin derived from horseshoe crab, etc.) and the like can be mentioned.

  Examples of the antibody include an antibody that selectively recognizes MRSA, an antibody that recognizes LPS (lipopolysaccharide) such as Escherichia coli and Salmonella.

  Examples of the peptide include LPS such as Escherichia coli, a peptide that recognizes lipid A, and the like (Nagaoka, I et al. J. Immunol. 167, 3329-3338 (2001) and JP-A-2002-311029).

  As the polyethylene glycol (PEG) constituting the CNH complex of the present invention, for example, one having a molecular weight of about 300 to 10,000 can be used, preferably having a molecular weight of about 500 to 8,000, more preferably about 1,000 to 4,000.

  The PL-PEG- (molecular recognition element) conjugate of the present invention includes a PL-PEG derivative having a linking group at one end, such as PL-PEG- (N-hydroxysuccinimide) and PL-PEG- (maleimide). It can be produced by reacting with a molecular recognition element. Alternatively, PL-PEG-biotin or a PL-PEG-avidin-based material (eg, avidin, streptavidin, neutravidin, etc.) can be used to link the molecular recognition element via the biotin-avidin-based material bond. When the molecular recognition element has a sugar chain such as sialic acid, glycosaminoglycan, and saccharide (carbohydrate), a glycosyltransferase may be used, and these sugar chains are biotinylated or (strept) avidinized to form biotin. / You may make it couple | bond with CNH using an avidin bond.

  The CNH complex of the present invention comprises CNH or a CNH derivative such as carboxylated CNH (CNH-COOH) and a PL-PEG- (molecular recognition element) conjugate in an appropriate solvent such as water or alcohol. It can be obtained by mixing by stirring, shaking and the like.

  As the CNH derivative, in addition to the carboxyl group, a CNH derivative in which a carbonyl group, a hydroxyl group, an ether group, an imino group, a nitro group, a sulfone group, or the like is bonded to CNH may be used.

  As shown in FIG. 1, the CNH complex of the present invention has a structure in which a large number of phospholipid (PL) -polyethylene glycol (PEG) -molecular recognition element conjugates are adsorbed on the carbon structure constituting CNH. Yes. It is considered that the alkyl chain of PL is adsorbed on the surface of CNH-COOH by hydrophobic interaction. PEG enhances the dispersibility of CNH-COOH in an aqueous solvent, and the molecular recognition element selectively binds to cell walls of cells, fungi and the like.

FIG. 1 exemplifies adsorption of bacteria, fungi and the like, while FIG. 7 schematically shows how a virus adsorbs to the CNH complex of the present invention and is killed by light. FIG. 7 shows the case of recognizing the surface protein of a virus using an antibody. If a molecular recognition element that binds to a site where the virus recognizes a cell / target of infection is conjugated, the mutation of the virus Even if it occurs, it is preferable because an infectious virus can be surely killed. The molecular recognition elements that bind to the site where the virus recognizes the cell / infection target are shown in Tables 1 to 3 below. Tables 1 to 3 are described in the following documents: Principles of Virology 2nd Ed: Molecular Biology, Pathogenesis, and Control, LW Enquist, RM Krug, VR Racaniello, AM Skalka, SJ Flint, S Jane Flint, SJ Flint, ASM press.
In Tables 1 to 3, molecular recognition elements for each target virus (Virus) are exemplified as receptors (Receptors) and coreceptors (Coreceptors). “Type of molecule” indicates the type of molecule of the molecular recognition element for the virus.

  The PL-PEG-molecular recognition element conjugate is adsorbed to such an extent that CNH or a derivative thereof is appropriately dispersed in an aqueous solution.

  The CNH complex of the present invention is produced, for example, by the following steps. A specific production example is shown in Production Example 1 described later.

  CNH-COOH and 3- (N-succinimidyloxyglutaryl) aminopropyl (NHS) -PEG-PL conjugate are added to PBS buffer and sonicated for 1 hour in an ice bath. NHS-PEG-PL-CNH-COOH is obtained by removing unreacted materials by ultrafiltration and washing with PBS buffer. A molecular recognition element is added to the obtained aqueous solution of NHS-PEG-PL-CNH-COOH and stirred for several hours. Unreacted molecular recognition elements are removed by suction filtration and washed with distilled water. Finally, the obtained black solution is pre-frozen with liquid nitrogen and freeze-dried for 48 hours, thereby obtaining the CNH complex ([molecular recognition element-PEG-PL] .CNH or a derivative thereof) of the present invention. .

  The CNH composite of the present invention is excellent in dispersion stability in an aqueous solution, as is apparent from the results of the dispersion stability test described later (FIGS. 3 and 8).

  From the results of the structural analysis test, it can be seen that the size of one CNH complex of the present invention is a spherical nanoparticle of about 80 nm (FIG. 3).

  The CNH complex of the present invention can be used by aggregating about 2 to several.

  When aggregated and used, the molecular recognition element may be crosslinked using an appropriate molecule such as glutaraldehyde. Alternatively, an aggregate can be formed by irradiating the composite of the present invention with laser light. It should be noted that even such an aggregate is light and therefore has good dispersibility in water.

  The CNH complex of the present invention can be bound to a specific cell wall by appropriately selecting a molecular recognition element that binds to the cell wall of bacteria or the like according to the type of the cell wall.

  The CNH complex of the present invention has a property of generating heat upon light irradiation. By selecting a molecular recognition element, CNH bound to the cell wall of a specific bacterium can generate heat and kill bacteria by irradiating laser light from the outside.

The CNH complex generates heat by absorbing light in a wide wavelength range of about 400 to 1100 nm (visible to near infrared region) (FIG. 4). Further, the CNH complex generates heat by absorbing light having a wavelength of 1100 nm or more.
Therefore, the wavelength of the light to be used may be appropriately selected depending on the application. For example, in order to generate heat in the CNH complex in the human body, light having a wavelength of about 650 to 2500 nm, preferably about 700 nm to 1200 nm (near infrared region) passing through the human body may be irradiated. The irradiated light is preferably near infrared (about 700 to 2500 nm), but red visible light having a wavelength of 600 nm or more, preferably 650 nm or more is also excellent in the permeability of the living body.

  The output of the laser may be appropriately selected depending on the wavelength of light to be used, its application, and the like. However, when using light in the near infrared region, for example, it should be 3 W or more, preferably 5 W or more (see FIG. 4).

  Since the CNH complex of the present invention has an excellent antibacterial action as shown in the following test examples, it is useful as an antibacterial agent, and bacteria such as staphylococci, enterococci, and E. coli, fungi such as ringworm, It can be suitably used as a prophylactic and therapeutic agent for diseases caused by viruses, and can also be suitably used as a detection agent for these bacteria and viruses, an antibacterial agent for food, and an antiviral agent.

  The present invention provides medical and food antibacterial agents and antiviral agents comprising a CNH complex as an active ingredient.

  The antibacterial agent of the present invention may be composed of only a carbon nanohorn complex, or a composition prepared in a form suitable for a desired use by a conventionally known method in combination with any carrier or additive. There may be.

  The form of the medical antibacterial agent of the present invention is not particularly limited, but solid preparations such as tablets, powders, granules, pills, powder syrups and capsules (hard capsules and soft capsules); creams, Paste or gel preparations such as ointments and gels; liquid preparations such as liquids, suspensions, emulsions, syrups, and elixirs.

  The blending amount of the antibacterial agent of the present invention is not particularly limited as long as it is contained in a proportion exhibiting an antibacterial effect, and is 0.001 to 50% by weight, preferably 0.01 to 10% by weight in 100% by weight of the preparation. More preferably, it can be appropriately set and prepared in the range of 0.1 to 5% by weight.

  The antibacterial agent and antiviral agent may be contained as long as the CNH complex exhibits an effect, and other components can be blended within a range not impeding this effect. Such other components are not limited as long as they are pharmacologically and pharmaceutically acceptable. For example, excipients, binders, dispersants, thickeners, lubricants, pH adjusters, solubilization In addition to carriers generally used in the preparation of preparations such as agents, antibiotics, other antibacterial agents, bactericides, preservatives, enzymes, chelating agents, colorants (dyes, pigments, etc.), moisturizers, surfactants, Antioxidants, fragrances, flavoring agents, flavoring agents, solvents and the like are included.

  The method of using the antibacterial agent can be a method in which the preparation is taken into the body by oral administration, application, drip, injection or the like, or a local application to the affected area.

  Since the amount used varies depending on the dosage form and administration (use) method, etc., it cannot be specified unconditionally. For example, an appropriate daily dose is converted into the above-mentioned dose of the CNH complex of the present invention. In general, it can be appropriately set according to the age and symptoms of the patient in the range of 1 ng to 100 mg, preferably about 10 ng to 50 mg per 1 kg of adult. These preparations are divided into 1 to several times a day. It is good to do.

  The antibacterial agent of the present invention can also be suitably used as a food antibacterial agent.

  Hereinafter, the present invention will be further clarified by giving production examples, formulation examples, and test examples of the carbon nanohorn composite of the present invention, but the present invention is not limited thereto.

Production Example 1
Synthesis of carboxylated carbon nanohorn Carbon nanohorn (50 mg) was added to a 70% nitric acid aqueous solution (200 mL) and heated to reflux (130 ° C., 15 minutes). Distilled water (600 mL) was gradually added to the resulting solution in an ice bath. Next, suction filtration was performed using distilled water until the solution became neutral (PTFE membrane, pore size = 100 nm). The obtained PTFE film on which the carboxylated carbon nanohorn (CNH-COOH) was deposited was placed in distilled water (50 mL) and subjected to ultrasonic treatment for several minutes. The resulting black CNH—COOH aqueous solution was pre-frozen with liquid nitrogen and freeze-dried for 48 hours. The FT-IR, the resulting CNH-COOH in a -COOH group (1724 cm ^-1) and -COO ^- group (1571 cm ^-1) was confirmed to be displayed (FIG. 2).

Formulation Example 1
Synthesis of molecular recognition element-carbon nanohorn complex CNH-COOH (1 mg), [3- (N-succinimidyloxyglutaryl) aminopropyl (NHS)]-[polyethylene glycol-carbamyl (molecular weight of PEG chain = 2,000)]-[distearoylphosphatidylethanolamine] conjugate (NHS-PEG-PL) is added to PBS buffer (pH 7.3, 10 mL) and sonicated in an ice bath (<8 ° C) for 1 hour. gave. Unreacted substances were removed by ultrafiltration (fractionated molecular weight = 100,000, centrifugation conditions: 6,000 rpm, 15 min, 4 ° C.), and washed 3 times with PBS buffer (5 mL). Next, wheat germ-derived lectin (WGA) (1 mg) was added to the obtained NHS-PEG-PL-CNH-COOH aqueous solution (10 mL), and the mixture was stirred for 3 hours.
Unreacted WGA was removed by suction filtration (polycarbonate membrane, pore size = 50 nm) and washed with distilled water (100 mL). Finally, the resulting black solution was pre-frozen with liquid nitrogen and lyophilized for 48 hours.

  The chemical formula of the WGA-PEG-PL conjugate is shown below.

  Fluorescent molecule labeled WGA-CNH complex (Alexa633-WGA-PEG-PL-CNH-COOH) and LPS core antibody-CNH complex (anti LPS core-PEG-PL-CNH-COOH) It was synthesized by the same method as PL-CNH-COOH.

On the other hand, (aminopropyl polyethylene glycol-carbamyl distearoylphosphatidylethanolamine (NH ₂ -PEG-PL) -CNH-COOH (hereinafter referred to as NH ₂ -PEG-PL-CNH-COOH) is NHS-PEG-PL-CNH Obtained by the same method as -COOH.

  All CNH complexes were confirmed to be stable in dispersion in PBS buffer for 3 months or more at 4 ° C. (FIG. 3a).

Analysis Example 1 (Nanostructure analysis of CNH composite)
Structural analysis of CNH composites includes scanning electron microscope (SEM) (acceleration voltage: 10 keV), transmission electron microscope (TEM) (acceleration voltage: 200 or 300 keV), atomic force microscope (AFM) (cantilever: tapping Mode) (FIG. 3).

Test example 1 (light absorption characteristics of CNH)
The light absorption characteristics of CNH, CNH-COOH and WGA-PEG-PL-CNH-COOH were evaluated by UV-Vis-NIR spectral analysis (JASCO, V-630) at room temperature. did.
Next, various CNH samples (CNH and CNH-COOH = 50 μg / mL, WGA-PEG-PL-CNH-COOH = 150 μg / mL) are dispersed in PBS buffer, and quartz cell (optical path length = 1 cm) Absorbance was monitored in (Fig. 4b). Absorbance monitoring was performed using laser light in the infrared region (NIR (1064 nm)) that is harmless to the living body (FIGS. 4a and b).

As a result, it was found that the CNH complex (WGA-PEG-PL-CNH-COOH) of the present invention has a wide light absorption property of Vis-NIR (400 to 1100 nm). Although not shown in FIG. 4, the CNH composite of the present invention generates heat by absorbing light having a wavelength of 1100 nm or more.
FIG. 4a is a UV-Vis-near infrared spectrum analysis of the CNH complex of the present invention.
FIG. 4 b shows light absorption characteristics (near infrared region (1,064 nm)) of WGA-PEG-PL-CNH-COOH at various concentrations.

Test Example 2 (CNH temperature change measurement)
The CNH complex WGA-PEG-PL-CNH-COOH (25 or 200 μg / mL) of the present invention was dispersed in a PBS buffer and irradiated with a 1064 nm laser at various outputs (1 to 5 W). As a control experiment, an experiment without CNH complex was also conducted. The temperature of the solution was measured with a mercury thermometer every other minute during laser irradiation. All CNH samples were filtered (pore size = 5 μm) before laser irradiation (FIGS. 4c and d).

  FIG. 4c is a temperature change when the WGA-PEG-PL-CNH-COOH is irradiated with laser light (1,064 nm) of each output (1 to 5 W).

As a result, due to the presence of CNH in the system, exothermic phenomenon and bumping phenomenon were observed when laser light (1,064 nm) was irradiated.
FIG. 4d is a photograph of WGA-PEG-PL-CNH-COOH (200 μg / mL) before laser beam irradiation (left panel) and after laser beam irradiation (right panel). The top panel is a reduced photo. The lower panel is an enlarged photo.

Test Example 3 (Adsorption experiment of fluorescently labeled WGA-CNH complex to bacteria)
The selective adsorption experiment of Alexa 633-WGA-PEG-PL-CNH-COOH to bacteria was evaluated by an inverted fluorescence microscope [object lens: 100 ×, EB-CCD high sensitivity camera, optical filter set (excitation wavelength: 510 -560 nm, absorption wavelength:> 690 nm)] (Figure 5).
5a-c used S. cerevisiae (yeast), df; E. colij (E. coli). All scale bars are 20 μm and all magnifications are x100.

  5a and 5d are phase contrast micrographs. FIGS. 5b and 5e are fluorescence micrographs after adsorption of Alexa 633-WGA-PEG-PL-CNH-COOH to bacteria. Figures 5c and f are fluorescence micrographs of control experiments (without Alexa 633-WGA-PEG-PL-CNH-COOH).

  As a result of the adsorption test, it was found that Alexa 633-WGA-PEG-PL-CNH-COOH was selectively adsorbed and shined on S. cerevisiae (FIG. 5b). On the other hand, for E. coli, almost no fluorescence of Alexa 633-WGA-PEG-PL-CNH-COOH was observed (FIG. 5e).

Test Example 4 (Selective sterilization test)
Pretreatment Yeast (S. cerevisiae) was cultured in YPD medium, and E. coli was cultured in LB medium for 24 hours. These bacterial solutions (1 mL) were collected by centrifugation (4,500 rpm, 5 min, 4 ° C.) and washed 3 times with PBS buffer (1 mL). Next, each CAN complex (100 μg / mL) dispersed in PBS buffer is centrifuged (4,500 rpm, 5 min, 4 ° C), and the supernatant solution (1 mL) is recovered, and the filter (pore size = 5 μm). The obtained uniformly dispersed CAN complexes (0.5 mL) were added to the washed bacterial solution (0.5 mL), incubated at 4 ° C for 1 hour, and then washed 3 times with PBS buffer (1 mL). (Centrifuge conditions: 4,500 rpm, 5 min, 4 ° C). Finally, an aqueous propidium iodide solution (43 μM, 5 μL) for staining dead bacteria was added to the obtained solution (500 μL) and incubated at 4 ° C. for 15 minutes.

-Selective sterilization test The selective sterilization test was performed by a device in which a single laser beam (1,064 nm) was attached to a fluorescence microscope. The laser beam was focused by an objective lens (× 40) on the cover glass. Images were taken with a color CCD camera equipped with a 3-band filter (Fig. 6). All scale bars are 25 μm. All magnifications are 20 times. Laser power is 1 W. The wavelength is 1,064 nm. The upper panel used S. cerevisiae and the lower panel used E. coli.
Figure 6a; WGA-PEG-PL-CNH-COOH.
Figure 6b; Control (without WGA-PEG-PL-CNH-COOH).
Figure _{6c; NH 2 -WGA-PEG-} PL-CNH-COOH ( without molecular recognition elements).
FIG. 6d; Anti LPS core-PEG-PL-CNH-COOH.

As a result of the selective sterilization test, when WGA-PEG-PL-CNH-COOH was used, only S. cerevisiae could be selectively sterilized (FIG. 6a). In a control experiment (without WGA-PEG-PL-CNH-COOH), none of the bacteria were killed (FIG. 6b). Further, even when using _{NH 2 -WGA-PEG-PL-} CNH-COOH having no molecular recognition element, one of the bacteria also been killed (Figure 6c).
On the other hand, when Anti LPS core-PEG-PL-CNH-COOH was used, only E. coli could be selectively sterilized (FIG. 6d).

Formulation Example 2
Synthesis of virus recognition element-carbon nanohorn complex
CNH-COOH (1 mg) and 3- (N-succinimidyl osigglutaryl) aminopropyl polyethylene glycol carbamyl distearoylphosphatidylethanolamine (NHS-PEG-PL) (PEG chain molecular weight = 2,000) in PBS buffer (PH 7.3, 10 mL) and sonicated in an ice bath (<8 ° C.) for 1 hour. Unreacted NHS-PEG-PL was removed by ultrafiltration (fractionated molecular weight = 100,000, centrifugation conditions: 6,000 rpm, 15 min, 4 ° C.), and washed 3 times with PBS buffer (5 mL). Next, T7 tag antibody (T7 tag Ab) (50 μg) was added to the washed NHS-PEG-PL-CNA-COOH solution (10 mL) and stirred for 3 hours. Unreacted T7 tag antibody was removed by suction filtration (polycarbonate membrane, pore size = 50 nm) and washed with distilled water (100 mL). Finally, the resulting black solution was pre-frozen with liquid nitrogen and lyophilized for 48 hours.

Test Example 5 (particle size measurement)
T7 tag Ab-PEG-PL-CNH-COOH aqueous solution (300 μg / mL) after filtration (cellulose acetate membrane, pore size = 200 nm), dynamic light scattering particle size distribution analyzer (HORIBA, LB-550 ) To analyze the particle size. The measurement was performed on the date of manufacture (first date), 5 days, 20 days, and 30 days. The results are shown in FIG. FIG. 9 shows the relationship between the laser irradiation time and the particle size. As a result of FIG. 9, it was found that the particle diameter increases as the laser irradiation time increases. The photo on the right shows the solution after irradiation with NIR laser for 60 minutes. It can be seen that no precipitate is formed. Presumably, CNH is very light and there is a water-soluble carboxyl (-COOH) group on the CNH surface, so no precipitation occurs. As the particle size increases, the nano-risk also decreases. Therefore, the complex of the present invention that has high dispersibility (solubility) and does not produce a precipitate is easy to use as a raw material for pharmaceuticals, foods, cosmetics and the like.

Test Example 6 (antiviral activity test)
Various CAN complexes (0 to 300 μg / mL) (0.25 mL) dispersed in PBS buffer were added to T7 phage solution (4.8 × 10 ¹⁰ pfu / mL) (0.25 mL). Next, in order to stain the dead T7 phage, an aqueous propidium iodide solution (43 μM, 5 μL) was added and incubated at 4 ° C. for 5 minutes. The antiviral activity test was performed with a device equipped with a single laser beam (1,064 nm) attached to a fluorescence microscope. The laser beam was focused by an objective lens (× 40) on the cover glass. Images were taken with a color CCD camera equipped with a 3-band filter. The results are shown in FIG. The results of FIG. 10 reveal the following:
(i) When the CNH concentration is high, the T7 phage can be killed regardless of the presence or absence of the virus recognition element.
(ii) The T7 phage can be killed more effectively by the effect of the virus recognition element.
(iii) The T7 phage can be killed by the effect of the CNH complex.

The conceptual diagram of the cell sterilization by the carbon nanohorn composite_body | complex of this invention. FT-IR analysis result of CNH-COOH The dispersion stability and nanostructure analysis result of the carbon nanohorn composite of the present invention. a; Photograph after incubation of CNH (100 μg / mL), CNH-COOH (100 μg / mL) and WGA-PEG-PL-CNH-COOH (300 μg / mL) in PBS buffer at 4oC for 24 hours . b: SEM photograph of WGA-PEG-PL-CNH-COOH (scale bar: 100 nm). c: TEM photograph of CNH-COOH (acceleration voltage = 300 keV) (scale bar: 20 nm). d: TEM photograph of WGA-PEG-PL-CNH-COOH (acceleration voltage = 200 keV) (scale bar: 20 nm). 0.04% (w / v) ammonium molybdate was added to observe surface proteins and PEG. e; AFM photograph of WGA-PEG-PL-CNH-COOH (scale bar: 500 nm) f; AFM photograph of WGA-PEG-PL-CNH-COOH (height profiling) The light absorption characteristics of the CNH complex of the present invention and the exothermic reaction due to laser irradiation (1,064 nm) are shown. a: UV-Vis-near infrared spectrum analysis of CNH. b: Light absorption characteristics of each concentration of WGA-PEG-PL-CNH-COOH (near infrared region (1,064 nm)). c: Temperature change when WGA-PEG-PL-CNH-COOH was irradiated with a laser (1,064 nm) of each output (1 to 5 W). d: Photographs of WGA-PEG-PL-CNH-COOH (200 μg / mL) before laser irradiation (left panel) and after laser light irradiation (right panel). The top panel is a reduced photo. The lower panel is an enlarged photo. Evaluation of adsorption property to bacteria of fluorescent Alexa 633-WGA-PEG-PL-CNH-COOH by a phase contrast microscope and a fluorescence microscope (scale bars are all 20 μm, magnification is all x100). ac. S. cerevisiae (yeast) df; E. coli j (E. coli) a and d; phase contrast microscope. b and e; Fluorescence micrograph after adsorption of Alexa 633-WGA-PEG-PL-CNH-COOH to bacteria. c and f; fluorescence micrograph of control experiment (without Alexa 633-WGA-PEG-PL-CNH-COOH) Real-time bactericidal test results (fluorescence micrographs) (scale bars are all 25 μm, magnification is all 20 times, laser power is 1 W. Wavelength: 1,064 nm. (Upper panel; S. cerevisiae (yeast), lower panel; E) . coli (E. coli)) a;. Alexa 633- WGA-PEG-PL-CNH-COOH.b; control (no Alexa 633-WGA-PEG-PL -CNH-COOH) .c; NH 2 -WGA-PEG- PL-CNH-COOH (without molecular recognition element) d; Anti LPS core-PEG-PL-CNH-COOH. Conceptual diagram [sterilization with T7 tag Ab-CNH complex and NIR laser (1064 nm)]. T7 tag Ab selectively binds to the virus head (g10 region). PEG chain enhances water dispersibility in CAN-COOH. PL is adsorbed on the wall of CNH-COOH by hydrophobic interaction. Evaluation of dispersion stability of T7 tag Ab-PEG-PL-CNH-COOH. From the results of FIG. 8, it was found by dynamic light scattering particle size distribution measurement that T7 tag Ab-PEG-PL-CNH-COOH has high dispersion stability for at least 30 days. Particle size change of T7 tag Ab-PEG-PL-CNH-COOH by NIR laser Real-time antiviral activity test. a, T7 tag Ab-PEG-PL-CNH-COOH (300 μg / mL). b, NH ₂ -PEG-PL-CNH-COOH (no virus recognition element) (300 μg / mL). c, T7 tag Ab-PEG-PL-CNH-COOH (30 μg / mL). d, NH ₂ -PEG-PL-CNA-COOH (no virus recognition element) (30 μg / mL). e, Control experiment (without virus recognition element-CNH complex). All scale bars are 25 μm. All magnifications are x20. Laser power: 1 W. Wavelength: 1,064 nm.

Claims (5)

A carbon nanohorn complex formed by adsorbing a phospholipid-polyethylene glycol-molecule recognition element conjugate to a carbon nanohorn. The carbon nanohorn complex according to claim 1, wherein the molecular recognition element is a lectin or an antibody. The carbon nanohorn complex according to claim 1 or 2, wherein the phospholipid is selected from the group consisting of monoacyl or diacyl phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, diacylphosphatidylethanolamine, and sphingomyelin. An antibacterial agent comprising the carbon nanohorn complex according to claim 2 as an active ingredient. An antiviral agent comprising the carbon nanohorn complex according to claim 2 as an active ingredient.

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