KR20220140227A - Antiviral-antibiotic organic-inorganic hybrid nanoparticle and its method of preparing the same, and article including the same - Google Patents

Abstract

Disclosed are organic-inorganic hybrid nanoparticles, a method for manufacturing the same, and an article thereof. The disclosed organic-inorganic hybrid nanoparticles include hematite, and lysine conjugated to the hematite. In the present invention, lysine molecules form strong ionic bonds on the surface of hematite to form chemically stable organic molecule-inorganic metal oxide hybrid nanoparticles.

Description

Antiviral-antibiotic organic-inorganic hybrid nanoparticle and its method of preparing the same, and article including the same

Disclosed are an organic-inorganic hybrid nanoparticle having antiviral-antibacterial properties, a method for preparing the same, and an article. More specifically, organic-inorganic hybrid nanoparticles having a property of adsorbing and killing viruses and bacteria while having biocompatibility, and at the same time maintaining a colloidal state by being well dispersed in water, and a method for manufacturing the same; and articles are disclosed.

Iron oxide nanoparticles have been widely used in the field of bio-medical science because of their excellent biocompatibility, relative safety to the human body, and various advantages such as magnetic properties described below. Representative examples include contrast agent, drug delivery, cell labeling, and hyperthermia therapy of Magnetic Resonance Imaging (see Non-Patent Documents 1 to 3). .

However, all the actions and functions listed above are fundamentally possible only when the iron oxide nanoparticles are magnetic nanoparticles that are attracted to magnets. As magnetic iron oxides used for this purpose, magnetite (Fe ₃ O ₄ ) and maghemite (gamma-Fe ₂ O ₃ ) are known (see Non-Patent Document 1). These nanoparticles exhibit superparamagnetism at room temperature and thus have a characteristic of strongly responding to magnetic fields. Hematite (=α-Fe ₂ O ₃ ), an iron oxide that hardly responds to magnetic fields, cannot be used for the above purposes, so the accumulated research results are much less. In addition, since iron oxide is generally not soluble in water, research and development for use in drug manufacturing has been conducted by coating various organic molecules on the surface of magnetite/maghemite nanoparticles to increase solubility. Among them, lysine is not only excellent in its own water-solubility (1.5 kg/L at room temperature) (see Non-Patent Document 4), but also attaches to the surface of the metal oxide to increase the solubility of the metal oxide. plays a role in enhancing As lysine is a kind of amino acid that is a component of the human body, it has excellent biocompatibility and is not toxic to the human body, so there are prior art documents about magnetite/maghemite nanoparticles conjugated with lysine (lysine-grafted) (Non-Patent Document 5~ 7).

On the other hand, another application field of iron oxide is an antiviral-antibacterial drug material, and basic research results in this field have been reported intermittently (see Non-Patent Documents 8 to 10). However, almost all life science-related iron oxide research has already focused on magnetite/maghemite, and the properties of their materials are relatively well known. See Non-Patent Documents 8 to 10). Until now, it has been reported that iron oxide has antiviral-antibacterial properties, but there is no evidence of superior advantages over existing drugs. did not reach

Separately, there is a literature researching iron oxide as a candidate material as a therapeutic agent for anemia (see Non-Patent Documents 2-3). In this case, other iron compounds that are well absorbed by the human body than iron oxide have better efficacy and are commercialized through long research and development. could not be continued.

In addition, there is a study of coating various amino acids on iron oxide for the purpose of controlling its mobility in electrophoresis (see Non-Patent Document 11). However, in the study for this purpose, the lysine concentration range in the lysine solution for coating was limited to a very low value (0.01mM ~ 10mM), so it did not lead to the development of an antiviral-antibacterial substance.

[Prior art literature]

[Non-patent literature]

1. D. Chang et al. Frontiers in Pharmacology 9, 831 (2018)

2. E. Alphandery et al. Nanotoxicology 13, 573 (2019)

3. D. Bobo et al. Pharm. Res. 33, 2373 (2016)

4. https://en.wikipedia.org/wiki/lysine

5. R. Contreras-Montoya et al. Crystal Growth&Design 20, 533 (2020)

6. C. Mandawala et al. J. Materials chemistry B, Royal Society of Chemistry, 2017, 10.1039/C6TB0348F. hal-01586778

7. H. Zhang, https://pubmed.ncbi.nlm.nih.gov/20641901

8. S. Tong et al. Mater. Today 31, 86 (2019)

9. Y. Abo-zeid et al. WIREs Nanomed Nanobiotechnol 12, e1592 (2020)

10. R. Kumar et al. J. Infect. Chemother. 25, 325 (2019)

11. A. Ben-Taleb et al. Materials Chemistry and Physics 37, 68 (1994)

One embodiment of the present invention provides an organic-inorganic hybrid nanoparticle having a property of adsorbing and killing viruses and bacteria while having biocompatibility, and at the same time being well dispersed in water to maintain a colloidal state. .

Another embodiment of the present invention provides a method for preparing the organic-inorganic hybrid nanoparticles.

Another embodiment of the present invention provides an article including the organic-inorganic hybrid nanoparticles.

One aspect of the present invention is

hematite; and

It provides an organic-inorganic hybrid nanoparticle comprising lysine conjugated to the hematite.

The iron atom of the hematite may be bonded to an oxygen atom in the carboxyl group of the lysine by an ionic bond.

The content of the lysine in the organic-inorganic hybrid nanoparticles may be 1 to 4% by weight.

The content of the lysine in the organic-inorganic hybrid nanoparticles may be 2-3% by weight.

The hematite may have an average particle diameter of 10 to 200 nm.

Another aspect of the present invention is

It provides an article comprising the organic-inorganic hybrid nanoparticles.

The article may be a non-woven fabric, a composite non-woven fabric or a non-woven fabric laminate.

Another aspect of the present invention is

Dissolving lysine in a solvent to prepare a lysine solution (S10);

Stirring after adding hematite to the lysine solution (S20); and

It provides a method for producing organic-inorganic hybrid nanoparticles comprising the step (S30) of removing the solvent from the resultant obtained in the step (S20).

The solvent may include water.

The lysine concentration in the lysine solution in the step (S10) may be 0.03 ~ 4M.

The lysine concentration in the lysine solution in the step (S10) may be 0.05 ~ 2M.

The steps (S10) and (S20) may be performed at room temperature and under normal pressure.

In the step (S10) and the step (S20), no substance may be added except for the lysine, the solvent, and the hematite.

Organic-inorganic hybrid nanoparticles according to an embodiment of the present invention have the following advantages.

(1) Lysine molecules form strong ionic bonds on the hematite surface to form chemically stable organic-inorganic metal oxide hybrid nanoparticles.

(2) In general, metal oxide is hardly soluble in water, so hematite also has this weakness. By conjugating lysine, the solubility is dramatically increased, so that it can be maintained in a colloidal state for a long time.

(3) Both lysine and hematite have excellent biocompatibility and almost no cytotoxicity.

(4) It exists in the form of a powder that can be easily stored for a long time at room temperature and pressure.

(5) Hematite has the highest isoelectric point (IEP) value among various iron oxides. The IEP value of lysine is also the second highest after arginine among 20 amino acids. As will be described later, a high IEP value is overwhelmingly advantageous in adsorbing and killing negatively charged viruses or bacteria.

(6) Lysine has the characteristic of zwitterion (dipolar ion), and thus has various physiological actions and can inhibit movement by adsorbing organic substances (see References 1-2).

(7) There is no need for a separate catalyst, surfactant, adhesive, etc., and harmful chemicals are not used in the manufacturing process.

(8) The manufacturing process is performed only at room temperature to 90°C or lower, and energy consumption is extremely low through all processes.

(9) In the manufacturing process, special conditions such as high temperature, high pressure, high vacuum, or low temperature below 0℃ are not required, so all processes are very safe in terms of facilities and working conditions.

(10) Since the invented organic-inorganic hybrid nanoparticles themselves have antiviral-antibacterial ability, it is sufficient to maintain normal cleanliness without extreme conditions of sterility-freedom in the manufacturing process.

(11) No harmful chemicals are emitted in the manufacturing process.

1 is a schematic diagram showing the structure of lysine.
2 is a plan view of the (0001) surface of hematite.
3 is a side view of organic-inorganic hybrid nanoparticles showing a state in which lysine is adsorbed on the (0001) surface of hematite.
4 is an XRD graph of hematite prepared in Preparation Example 1.
5 is an XRD graph of lysine-grafted hematite prepared in Examples 1-5 and 1-8.
6 is an XRD graph of a typical hematite disclosed in Reference 19.
7 is a real photograph of hematite and lysine-conjugated hematite powders prepared in Preparation Examples 1 and 1-5.
8 is an SEM image of hematite and lysine-conjugated hematite prepared in Preparation Example 1 and Examples 1-5.
9 is an N ₂ isothermal adsorption curve and a pore size distribution curve of hematite and lysine-conjugated hematite prepared in Preparation Example 1 and Examples 1-5.
10 is a photograph comparing the sedimentation rate of lysine-conjugated hematite nanoparticles prepared by dispersing lysine-conjugated hematite prepared using lysine solutions of various lysine concentrations in distilled water, and then standing for a certain time to make a colloidal solution. to be.
11A to 11L are SEM images of lysine-conjugated hematite-coated SMS composite nonwoven fabrics prepared using lysine solutions of various lysine concentrations.
12 is a photograph of cytotoxicity of lysine-conjugated hematite prepared in Examples 1-5 to MDCK host cells.
13 is a graph showing cytotoxicity and cell viability of lysine-conjugated hematite prepared in Example 1-5 to MRC-5 host cells.
14 is a lysine-conjugated hematite prepared in Examples 1-5, showing the results of the antibacterial efficacy test against Escherichia coli (E. coli).
15 is a lysine-conjugated hematite-coated SMS composite nonwoven fabric prepared in Examples 1-5, showing the experimental results of measuring virus inactivation over time.

Hereinafter, organic-inorganic hybrid nanoparticles according to an embodiment of the present invention will be described in detail.

As used herein, "room temperature" means 10 ~ 40 ℃ (eg, 25 ℃).

Also, in this specification, "normal pressure" means 724 to 770 mmHg (eg, 760 mmHg).

Also, in this specification, "non-woven fabric" means a single non-woven fabric.

In addition, in the present specification, "non-woven fabric composite" is not a non-woven fabric laminate manufactured through a separate lamination (lamination) post process after two or more kinds of non-woven fabrics are individually prepared, but two or more kinds of non-woven fabrics are one It means a nonwoven fabric that is manufactured in a continuous process in each of the devices and integrated. Therefore, in this specification, "composite non-woven fabric" may also be referred to as "monolithic non-woven fabric".

In addition, as used herein, the term "non-woven fabric laminate" refers to a multilayer nonwoven fabric manufactured through a separate lamination (lamination) post process after two or more types of nonwoven fabrics are separately manufactured.

The organic-inorganic hybrid nanoparticles according to an embodiment of the present invention include hematite and lysine grafted to the hematite.

The lysine has the chemical structure of FIG. 1 as C ₆ H ₁₄ N ₂ O ₂ .

The hematite has a chemical formula of α-Fe ₂ O ₃ , and has the (0001) surface structure of FIG. 2 . In FIG. 2 , a dotted line indicates a unit cell on the (0001) surface.

The iron atom on the surface of the hematite, as shown in FIG. 3, may be bonded to an oxygen atom in the carboxyl group of the lysine by an ionic bond.

3 is a side view of the organic-inorganic hybrid nanoparticles 100 (ie, lysine-conjugated hematite) showing a state in which lysine 110 is adsorbed to the (0001) surface of the hematite 120 . Figure 3 (a) is a view showing the lysine 110 is standing (upright configuration), Figure 3 (b) is a view showing the lysine 110 is lying (flat configuration).

Hereinafter, the hematite and lysine will be described in more detail.

Hematite, which exhibits little attraction to magnets, is iron oxide commonly found in nature. Unlike the crystal structure of magnetite/maghemite, which is strongly attracted to magnets, belongs to the cubic crystal system. The crystal structure belongs to the trigonal crystal system (Reference 3).

From a scientific point of view, the fundamental unit of magnetism, spin, has a so-called “canted antiferromagnetic order”, so that the effective magnetic moment per iron atom is 0.002 μ _B (Bohr magneton). (Reference 3), and the saturation magnetization value of magnetite/maghemite (85~95 Am ² /kg (Reference 4 and Non-Patent Document 5), about 1.2 μ _B per iron atom). It is so small that it cannot be used for the medical purposes listed above.

However, as a pharmaceutical material for antiviral-antibacterial purposes, hematite has excellent advantages as described below compared to magnetite/maghemite. A critically important item required as an antiviral-antibacterial material is its electrostatic properties. The isoelectric point (IEP) of hematite is known to be 9.1 (Reference 5) or 8.4-8.5 (Reference 6). Under normal conditions, the pH of the human body is about 7.4, and hematite with an IEP value higher than this has a positive (+) charge. It is well known that viruses and bacteria are generally negatively charged (Ref. 7, eg IEP = 3.9 for MS2 bacteriophage). This allows hematite to strongly attract and adsorb negatively charged viruses or bacteria, as well as kill them by the mechanism described later. On the other hand, the IEP of magnetite is 6.0-6.8 (Reference 5) or 6.5-6.8 (Reference 6), and the IEP of maghemite is 3.3-6.7 (Reference 6). Accordingly, both magnetite/maghemite are negatively charged under normal physiological conditions. Since IEP is defined on a logarithmic scale, hematite, which has an IEP value of 2 or more than that of magnetite/maghemite, tends to be positively charged by 100 times or more than that of magnetite/maghemite.

If lysine with IEP=9.7 (Reference 8) is added here, the tendency to take on a positive charge increases by that much, so lysine-grafting can greatly increase the drug efficacy. Although lysine can reinforce the positively charged tendency of all iron oxides, it does not change the fact that lysine-conjugated hematite has an overwhelming advantage over lysine-conjugated magnetite/maghemite anyway.

Another essential item for antiviral-antibacterial efficacy is the water-solubility of the drug ingredient, since it must exist in a dispersed form in water during the manufacturing process and in the process of acting as a drug. Iron oxide nanoparticles are hardly dispersed in water, and even with active stirring, they cannot maintain a colloidal state and precipitate rapidly. Lysine itself has excellent water solubility (1.5 kg/L, non-patent document 4), and when grafted onto the surface of hematite nanoparticles, disperses the nanoparticles in water to maintain the colloidal solution. . When lysine is conjugated by soaking hematite nanoparticles in a lysine solution, the decisive important point found by the present inventors is that the lysine concentration must be very high (0.05M-2M). At such a high concentration, lysine strongly ionically bonds to hematite nanoparticles, so that the IEP value of the nanoparticles and the properties of maintaining a colloidal state by being dispersed in water are remarkably increased.

When testing the antiviral-antibacterial efficacy, it takes more than 5 days, including the incubation period (usually 2 to 3 days) and the pretreatment time. % or more) should remain without precipitation. If the concentration of lysine in the lysine solution is less than 0.05M, the amount of lysine conjugated to the nanoparticles is small, and the hydrophilicity of the nanoparticles is weak, so that they cannot be sufficiently dispersed and the particles settle over time. From 0.05M to 0.2M, the amount of lysine conjugated to the nanoparticles increases, so that the nanoparticles are well dispersed in water and maintain a colloidal state for a long time (more than 50% even after 7 days). When the lysine concentration exceeds 0.2M and increases to 2M, the surface of the nanoparticles is already almost covered with lysine and the hydrophilicity is sufficiently increased, so the dispersion property in water is not particularly improved and the weight ratio of lysine in the nanoparticles is almost saturated. . As will be described later, when the lysine concentration is 0.05M to 2M, the weight ratio of lysine in the nanoparticles is 2-3%. When the concentration of lysine is excessively increased (2~4M), the water solubility of the nanoparticles does not increase any more, and the interaction between lysine molecules becomes stronger and agglomeration occurs. 4%) can be increased. Concomitantly, the color of the solution becomes cloudy, and the adhesion to hematite may be rather weakened due to the cohesive force between lysine. In this range, the weight ratio of lysine among nanoparticles can be slightly increased (from 3% to 4%), but even if it is increased, it does not help to improve the properties of nanoparticles because lysine already covers the surface of the nanoparticles. In addition, dissolving lysine in water at an ultra-high concentration of 4M or more is not practically easy and only wastes lysine, so there is no practical benefit. In the existing literature (Non-Patent Documents 5, 11 and Reference 9), since the experiment was usually conducted at a much lower lysine concentration, it did not show practicality as an antiviral-antibacterial drug material. For example, in Non-Patent Document 5, the lysine concentration is 0.1mM to 10mM, in Non-Patent Document 11, the lysine concentration is 0.01mM to 10mM, and in Reference 9, the lysine concentration is 0.1mM to 3mM, so the concentration range of the present invention is It is much lower than the minimum value of 0.05M.

On the other hand, considering only the isoelectric point, the IEP value of arginine among amino acids is the highest as 10.8 (Reference 8), which is advantageous. However, arginine has a very low solubility in water (149 g/L at 20 ° C, Reference 10) compared to lysine, and when an experiment was performed to dissolve iron oxide nanoparticles by coating arginine in water, iron oxide nanoparticles were well It did not disperse and precipitated quickly, making it unsuitable as a pharmaceutical material.

As described above, the chemical mechanism of lysine bonding with hematite is that an oxygen (O) anion in a carboxylic group of lysine forms an ionic bond with an iron (Fe) cation on the surface of hematite. Atomic and electronic structure calculations based on quantum mechanics were performed to investigate the structure of lysine attached to the hematite surface, the form of the chemical bond, and the adsorption energy.

As described above, FIG. 1 shows the structure of a lysine molecule, and FIG. 2 shows a (0001) surface of hematite with iron atoms exposed on the surface (Fe-terminated). The number of Fe atoms present on the (0001) surface of hematite is a value that maintains 2:3, the stoichiometry of Fe and O of the entire nanoparticles, and is consistent with the known structure (Reference 11).

As a method for calculating the adsorption structure and adsorption energy, the so-called Generalized Gradient Approximation (GGA) calculation method based on Density Functional Theory (Reference 12) is widely known. In addition, when an iron atom, which is a transition metal, exists, it is necessary to add the so-called Hubbard U term (Reference 13), so the universal calculation method (GGA+U) method combining the two equations is adopted. did. As the U value of iron (Fe), U=4 eV, which is commonly used, was selected.

이었다. 계산된 흡착에너지는 도 3의 (a) 및 (b) 각각의 경우에 라이신 분자당 -1.35eV(=-130kJ/mol) 및 -1.45eV(=-140kJ/mol)이었다. 도 3의 (a) 및 (b) 구조 간의 흡착에너지 차이가 매우 작아 주위 환경에 따라 두가지 형태가 모두 가능함을 알 수 있다. 이러한 크기의 흡착에너지 값(약 1.4eV)은 보통 이온결합에너지의 전형적인 값이며, 라이신이 헤마타이트 나노입자의 표면에 안정되게 결합되어 있음을 알 수 있다. 계산된 원자 및 전자구조를 분석해보면, 도 3의 (a) 및 (b)의 경우 모두 수소결합 (hydrogen bonding)(결합에너지는 전형적으로 약 0.2eV)이 아울러 존재하여 구조 안정성을 증가시킨다. As described above, FIG. 3 shows a structure in which lysine is attached to the surface of hematite nanoparticles. The oxygen atom (negatively charged) in the carboxylic group in lysine forms an ionic bond with the iron atom (positive) of hematite, and the O-Fe bond length obtained in the calculation is 1.95

It was. The calculated adsorption energies were -1.35 eV (=-130 kJ/mol) and -1.45 eV (=-140 kJ/mol) per lysine molecule in each case of FIGS. 3 (a) and (b). It can be seen that the difference in adsorption energy between the structures (a) and (b) of FIG. 3 is very small, so that both types are possible depending on the surrounding environment. The adsorption energy value (about 1.4 eV) of this size is a typical value of the ionic binding energy, and it can be seen that lysine is stably bound to the surface of the hematite nanoparticles. Analyzing the calculated atomic and electronic structures, in both (a) and (b) of FIG. 3, hydrogen bonding (bonding energy is typically about 0.2 eV) also exists, increasing structural stability.

The mechanism (mechanism) showing the antiviral-antibacterial effect of metal-oxide nanoparticles is known as follows (References 14, 15, Non-Patent Document 9), and lysine-conjugated hematite nanoparticles It will be described with a specific example.

(1) electrostatic interaction

Hematite nanoparticles are positively charged under normal conditions by Fe ³⁺ ions on the surface. And, lysine bound to the surface (IEP = 9.7) also has a positive (+) charge, which is due to the tendency of the amine group (NH ₂ group) to be ionized into NH ₃ ⁺ .

Since viruses and bacteria are negatively charged (Reference 7), they are preferentially attracted to and adsorbed to the inventive material (ie, lysine-conjugated hematite nanoparticles) and cannot move, and are discharged by contact between positive and negative charges ( discharge) occurs, and the membrane part of the virus/bacteria is depolarized and damaged and destroyed. Since this is a phenomenon similar to an electric short-circuit, it causes two effects, namely, breakup of chemical bonds in components constituting the film and destruction of the film by generation of heat. Quantitatively, when a negatively charged virus contacts the positively charged inventive material, the potential difference is typically several tens of mV. If the potential difference is 50 mV, the energy generated every time one electron (e) is discharged is 50 meV (=8 × 10 ^-21 J), and when converted to water (H ₂ O), the temperature of one water molecule is raised by about 64°C. corresponds to the column. That is, as the electrons are discharged and depolarized, a kind of Joule heat is generated in the contact part, and the temperature of the surrounding molecules rises, contributing to the structural destruction of the contact part.

(2) Generation of Reactive Oxygen Species

When the virus/bacterial membrane is destroyed and the inside is exposed, and when Fe ³⁺ and lysine on the surface of the material of the present invention come into contact with the inside of the virus/bacterium, Fe ³⁺ or NH ₃₊ causes oxidation, such as reactive oxygen (Reactive Oxygen ⁾ . Species, ROS) (Ref. 16). When active oxygen is generated inside the virus/bacterium, it actively reacts with the internal components, causing fatal harm.

(3) Disturbance of physiological action due to excess of metal ions

Since Fe ³⁺ ions are excessively present in the virus/bacterium, the physiological action of the virus/bacterium is disturbed, and its homeostasis is broken, leading to inactivation and death.

It should be noted here that metal oxide nanoparticles should not have cytotoxicity as an essential condition for use as a pharmaceutical ingredient. The advantage of iron oxide nanoparticles is the fact that the toxicity is remarkably low compared to oxides of other metals (Cu, Ag, Al, Ti, Zn, etc.) with antiviral-antibacterial properties. Iron ions (Fe ²⁺ or Fe ³⁺ ) themselves exist in relatively large amounts in living things in the form of red blood cells, and have been deeply involved in life phenomena, and it is well known that they are not toxic under normal circumstances. Therefore, as a drug to be administered to the human body, it has already been approved for use by the FDA, etc. as an MRI contrast agent or an anemia treatment agent. When used for antiviral-antibacterial purposes, as shown in the evaluation examples to be described later, a specific part of a virus or bacteria (e.g., a spike protein of a virus) that is not toxic to normal cells and has a different physiological structure and function. )) to adsorb, inactivate, and kill them by the mechanisms previously described.

The content of the lysine in the organic-inorganic hybrid nanoparticles may be 1 to 4% by weight. If the content of the lysine in the organic-inorganic hybrid nanoparticles is less than 1% by weight, the antiviral-antibacterial effect is insignificant, and if it exceeds 4% by weight, it is not practically easy to manufacture and does not increase the antiviral-antibacterial efficacy. There is no practicality.

Specifically, the content of the lysine in the organic-inorganic hybrid nanoparticles may be 2-3% by weight. When the content of the lysine in the organic-inorganic hybrid nanoparticles is within the above range (2 to 3% by weight), excellent antibacterial and antiviral effects can be obtained.

The hematite may have an average particle diameter of 10 to 200 nm.

Another aspect of the present invention provides an article including the organic-inorganic hybrid nanoparticles.

The article may be a non-woven fabric, a composite non-woven fabric or a non-woven fabric laminate. For example, it is possible to use the organic-inorganic hybrid nanoparticles as an antiviral-antibacterial filter by coating the fibers. Accordingly, the article may be an SMS composite nonwoven or a Spunbond-Meltblown-Spunbond nonwoven fabric composite or laminate.

Hereinafter, there is provided a method for producing organic-inorganic hybrid nanoparticles according to an embodiment of the present invention.

The method for producing organic-inorganic hybrid nanoparticles according to an embodiment of the present invention comprises the steps of dissolving lysine in a solvent to prepare a lysine solution (S10), adding hematite to the lysine solution and then stirring (S20) and and removing the solvent from the resultant obtained in step (S20) (S30).

The solvent may include water.

The lysine concentration in the lysine solution in the step (S10) may be 0.03 ~ 4M. If the lysine concentration in the lysine solution in the step (S10) is less than 0.03M, the amount of lysine conjugated to the nanoparticles in the next step (S20) is too small so that the nanoparticles precipitate quickly in water, and the antiviral-antibacterial effect is also insignificant. , dissolving at a high concentration of 4M or more is not practically easy, and only lysine is wasted, so it is not practical.

Specifically, the lysine concentration in the lysine solution in the step (S10) may be 0.05 ~ 2M. If the concentration of lysine in the lysine solution in the step (S10) is in the above range (0.05 to 2M), it is possible to obtain organic-inorganic hybrid nanoparticles exhibiting excellent antiviral-antibacterial efficacy.

More specifically, the lysine concentration in the lysine solution in the step (S10) may be 0.15 ~ 0.2M. If the concentration of lysine in the lysine solution in the step (S10) is in the above range (0.15-0.2M), organic-inorganic hybrid nanoparticles having excellent fiber coating properties can be obtained as described below.

The steps (S10) and (S20) may be performed at room temperature and under normal pressure.

In addition, in the step (S10) and the step (S20), any substance other than the lysine, the solvent, and the hematite may not be added.

The step (S30) may be performed by centrifugation and heat drying.

Hereinafter, the present invention will be described with reference to the following examples, but the present invention is not limited only to the following examples.

Example

Preparation Example 1: Preparation of hematite

In order to synthesize Fe(OH) ₃ used as a raw material powder, 32.4 g of Fe(NO ₃ ) ₃ and 30 g of NaHCO ₃ were dissolved in 250 ml water by stirring for 10 minutes, respectively, and then Fe(NO ₃ ) ₃ solution and NaHCO ₃ solution was mixed. At this time, a brown precipitate is generated in the mixed solution, and the precipitate is first removed from water through a centrifugal separator, and then washed with distilled water at least once to remove unreacted residue to obtain Fe(OH) ₃ in the form of brown powder. The above process was performed at room temperature and under normal pressure (25° C., 1 atm). The thus obtained Fe(OH) ₃ was put into 250 ml of distilled water, heated to 90° C., maintained for 7 hours, and then water was removed through a centrifugal separator, washed once with distilled water, and dried to obtain hematite (α-Fe ₂ O ₃ ) was obtained.

Examples 1-1 to 1-8 and Comparative Examples 1-1 to 1-6: Preparation of organic-inorganic hybrid nanoparticles

Lysine 43.85g was completely dissolved in 1.5L distilled water to prepare an aqueous solution of lysine. Then, 30 g of hematite obtained in Preparation Example 1 was added to the lysine aqueous solution and stirred for 24 hours. The above process was performed at room temperature and under normal pressure (25° C., 1 atm). From the resultant obtained in this way, water was removed through a centrifuge and dried at 50° C. for 24 hours to obtain lysine-conjugated hematite nanoparticles.

In Examples 1-1 to 1-8 and Comparative Examples 1-1 to 1-6, the lysine concentration (C _L ) in the lysine aqueous solution and the lysine content (W _L ) in the lysine-conjugated hematite nanoparticles are summarized below. Table 1 shows. The lysine content in the lysine-conjugated hematite nanoparticles was calculated from TGA (ThermoGravimetric Analysis) and the amount of supply and change of lysine in the solution.

Example 1-1 1-2 1-3 1-4 1-5 1-6 1-7 1-8 C _L 0.05M 0.07M 0.1M 0.15M 0.2M 0.4M 1M 2M W _L (wt%) 2.0 2.2 2.4 2.6 2.8 2.9 2.95 3.0 comparative example 1-1 1-2 1-3 1-4 1-5 1-6 C _L 0.5 mM 2 mM 0.02M 0.03M 3M 4M W _L (wt%) 0.3 0.5 0.7 1.1 3.5 4

Examples 2-1 to 2-8 and Comparative Examples 2-4 to 2-6: Preparation of SMS composite nonwoven fabric

10 g of each of the lysine-conjugated hematite nanoparticles prepared in Examples 1-1 to 1-8 and Comparative Examples 1-4 to 1-6 were put in 2.5L distilled water and stirred for 24 hours or more, so that the nanoparticles were dissolved in distilled water. An evenly dispersed colloidal solution was prepared. Thereafter, the SMS composite nonwoven fabric was immersed in the colloidal solution for about 10 minutes to adsorb the lysine-conjugated hematite nanoparticles to the SMS composite nonwoven fabric, and then vacuum-dried the SMS composite nonwoven fabric for 24 hours to obtain lysine-conjugated hematite nanoparticles. were uniformly dispersed at a high density to obtain a coated SMS composite nonwoven fabric. In the case of Comparative Examples 1-1 to 1-3, the amount of lysine was too small to be well coated on the nonwoven fabric, so Comparative Examples 2-1 to 2-3, which is a corresponding SMS composite nonwoven fabric coating test, were excluded here.

Evaluation Example 1: XRD graph analysis of hematite nanoparticles and lysine-conjugated hematite nanoparticles

The XRD graph of the hematite nanoparticles prepared in Preparation Example 1 is shown in FIG. 4, and the XRD graphs of the lysine-conjugated hematite nanoparticles prepared in Examples 1-5 and 1-8 are shown in FIG. 5, respectively. Rigaku's D/Max-2500VK/PC was used as the XRD device, and the analysis conditions were CuKα radiation speed 2° min ^-1 .

6 is an XRD graph of a typical hematite disclosed in Reference 19.

4 to 6 , the XRD graph of FIG. 4 (Preparation Example 1) and the XRD graph of FIG. 5 (Examples 1-5 and 1-8) are 24.14° (012), 33.22° (104), 35.68 ° (110), 40.88° (113), 43.46° (202), 49.38° (024), 54.02° (116), 57.46° (018), 62.4° (214), 64° (300), 69.7° ( 208), 72° (1010), and 75.42° (220) peaks coincide with the standard values of typical hematite (α-Fe ₂ O ₃ ) shown in FIG. 6 , so hematite was successfully synthesized in Preparation Example 1 as well as , It can be seen that iron oxide is maintained in a hematite state even in the lysine-conjugated hematite nanoparticles prepared in Examples 1-5 and 1-8. Since the hematite structure did not change when lysine was conjugated to hematite in high-concentration lysine solutions of 0.2M and 2M concentrations, it is obvious that the hematite structure would not change when lysine solutions of smaller concentrations were used. Therefore, although XRD graphs for all examples are not included in the present specification, it can be seen that the hematite nanoparticles always maintain the hematite structure.

Evaluation Example 2: Real pictures of hematite nanoparticles and lysine-conjugated hematite nanoparticles powder

7A is an actual photograph of the hematite powder prepared in Preparation Example 1, and FIG. 7B is an actual photograph of the lysine-conjugated hematite nanoparticle powder prepared in Examples 1-5.

Referring to FIG. 7 , the hematite (a) powder has a dark brown color, and the lysine-conjugated hematite nanoparticle (b) powder has a deep red color.

Evaluation Example 3: SEM analysis of hematite nanoparticles and lysine-conjugated hematite nanoparticles

Figure 8 (a) is an SEM image of the hematite nanoparticles prepared in Preparation Example 1 (magnification 80,000), Figure 8 (b) is the lysine-conjugated hematite nanoparticles prepared in Examples 1-5 SEM image (magnification 80,000). The SEM device used here was JEOL's JSM-6390LV.

Referring to FIG. 8 , the lysine-conjugated hematite nanoparticles prepared in Examples 1-5 have a relatively slightly smaller particle size than the hematite nanoparticles prepared in Preparation Example 1, and the particles are uniformly distributed. can be checked

Evaluation Example 4: BET analysis of hematite nanoparticles and lysine-conjugated hematite nanoparticles

9 shows the BET (specific surface area) analysis results of the hematite nanoparticles prepared in Preparation Example 1 and the lysine-conjugated hematite nanoparticles prepared in Examples 1-5. The inset of FIG. 9 shows the BJH analysis result for the pore size distribution.

In addition, the BET characteristics of the hematite nanoparticles prepared in Preparation Example 1 and the lysine-conjugated hematite nanoparticles prepared in Examples 1-5 are shown in Table 2 below.

sample Total surface area (m ² /g) Average pore diameter (nm) Total pore volume (cm ³ /g) Surface area (m ² /g) hematite nanoparticles 96.786 11.606 0.2808 91.059 Lysine-Conjugated Hematite Nanoparticles 86.911 11.372 0.2471 78.378

Referring to Table 2, it can be seen that the specific surface area of the lysine-conjugated hematite nanoparticles prepared in Examples 1-5 is slightly reduced compared to the hematite nanoparticles prepared in Preparation Example 1.

Evaluation Example 5: Comparative analysis of sedimentation rates of hematite nanoparticles and lysine-conjugated hematite nanoparticles

Photographs comparing the sedimentation rates of the hematite nanoparticles prepared in Preparation Example 1, and the lysine-conjugated hematite nanoparticles prepared in Examples 1-1 to 1-8 and Comparative Examples 1-1 to 1-6 It is shown in Figure 10. Specifically, FIG. 10 shows a colloidal solution by dispersing lysine-conjugated hematite nanoparticles prepared using a lysine aqueous solution of various lysine concentrations in distilled water for a predetermined time ((a) of FIG. 10 immediately after preparing the colloidal solution, ( b) 60 minutes after preparation of colloidal solution, (c) 24 hours after preparation of colloidal solution, (d) 7 days after preparation of colloidal solution) is a photograph comparing the sedimentation rate of lysine-conjugated hematite nanoparticles . As described above, it can be seen that hematite (Preparation Example 1) to which lysine is not conjugated does not maintain a colloidal state even with vigorous stirring and is soon precipitated.

The number described in Figure 10, together with the lysine concentration in the lysine aqueous solution, hematite prepared in Preparation Example 1, and Examples 1-1 to 1-8 and Comparative Examples 1-1 to 1-6 Lysine-conjugated It is shown in Table 3 by matching one-to-one with the hematite nanoparticles.

number in figure 10

① ② ③ ④ ⑤ ⑥ ⑦ Lysine concentration in lysine aqueous solution 0.000M 0.5 mM 2 mM 0.02M 0.03M 0.05M 0.07M 0.1M Experimental example Preparation Example 1 Comparative Example 1-1 comparative example
1-2 comparative example
1-3 comparative example
1-4 Example
1-1 Example
1-2 Example
1-3 number in figure 10 ⑧ ⑨ ⑩ ⑪ ⑫ ⑬ ⑭ Lysine concentration in lysine aqueous solution 0.15M 0.2M 0.4M 1M 2M 3M 4M Experimental example Example 1-4 Example
1-5 Example
1-6 Examples 1-7 Examples 1-8 Comparative Example 1-5 Comparative Example 1-6

Referring to Figure 10 (c) and Table 3, it can be seen that the color of the colloidal solutions prepared by using the aqueous solution of lysine having a concentration of lysine of less than 0.05M when left still for 24 hours after the preparation of the colloidal solution is slightly pale, whereas It can be seen that the color of the colloidal solutions prepared by using an aqueous lysine solution in which the concentration of lysine exceeds 0.05M is almost unchanged.

In addition, referring to FIG. 10 (d) and Table 3, when the colloidal solution was prepared and then left for 7 days, the concentration of lysine and colloidal solutions prepared using an aqueous lysine solution having a lysine concentration of less than 0.05M exceeds 0.05M The difference in the degree of precipitation between the colloidal solutions prepared using the lysine aqueous solution was clearly shown.

As described above, when testing the antiviral-antibacterial efficacy, it takes more than 5 days in consideration of the pretreatment time including the incubation period (usually 2-3 days). A significant portion (more than 50%) should remain unsettled. If the concentration of lysine in the lysine solution is less than 0.05M, the amount of lysine conjugated to the nanoparticles is small and the hydrophilicity of the nanoparticles is weak, so that they cannot be sufficiently dispersed and the particles settle over time. From 0.05M to 0.2M, the amount of lysine conjugated to the nanoparticles increases, so that the nanoparticles are well dispersed in water and maintain a colloidal state for a long time (more than 50% even after 7 days). When the lysine concentration exceeds 0.2M and increases to 2M, the surface of the nanoparticles is already almost covered with lysine and the hydrophilicity is sufficiently increased, so the dispersion property in water is not particularly improved and the weight ratio of lysine in the nanoparticles is almost saturated. . As shown in Table 1, when the lysine concentration is 0.05M to 2M, the weight ratio of lysine in the nanoparticles is 2-3%. When the concentration of lysine is excessively increased (2~4M), the water solubility of the nanoparticles does not increase any more, and the interaction between lysine molecules becomes stronger and agglomeration occurs. 4%) can be increased. Concomitantly, the color of the solution becomes cloudy and the adhesion to hematite may be rather weakened due to the cohesive force between lysine. In this range, the weight ratio of lysine among nanoparticles can be slightly increased (from 3% to 4%), but even if it is increased, it does not help to improve the properties of nanoparticles because lysine already covers the surface of the nanoparticles. And, it is not easy to dissolve lysine at an ultra-high concentration of 4M or more, and it is not practical because only lysine is wasted.

In summary, it can be seen that the concentration of lysine in the aqueous solution of lysine used to prepare lysine-conjugated hematite nanoparticles determines the characteristics of maintaining the colloidal state of the nanoparticles. On the other hand, as an additional experiment, conjugation was attempted with other types of amino acids, ie, glycine, proline, arginine, etc., to the metal oxide nanoparticles using the same conditions as for lysine, but conjugation was not performed properly unlike lysine. Therefore, in the case of these additional experiments, the obtained nanoparticles were not well dispersed in water, precipitated quickly, and it was found that they were not suitable as drug materials.

Evaluation Example 6: SEM image analysis of SMS composite nonwoven fabric coated with lysine-conjugated hematite nanoparticles

11a to 11l are SEM images of lysine-conjugated hematite nanoparticles coated with SMS composite nonwoven fabric (Spunbond-Meltblown-Spunbond nonwoven fabric composite) prepared using lysine aqueous solution of various lysine concentrations. The SEM device used here was JEOL's JSM-6390LV. 11a is an SEM image of a bare SMS composite nonwoven fabric, and FIG. 11b is a lysine-conjugated hematite nanoparticle-coated SMS composite nonwoven fabric (Comparative Example 2-4) prepared using a lysine aqueous solution having a lysine concentration of 0.03M. , Figure 11c is when the lysine concentration is 0.05M (Example 2-1), Figure 11d is when the lysine concentration is 0.07M (Example 2-2), Figure 11e is when the lysine concentration is 0.1M ( Example 2-3), Figure 11f is when the lysine concentration is 0.15M (Example 2-4), Figure 11g is when the lysine concentration is 0.2M (Example 2-5), Figure 11h is lysine When the concentration is 0.4M (Example 2-6), Figure 11i is when the lysine concentration is 1M (Example 2-7), Figure 11j is when the lysine concentration is 2M (Example 2-8) and , Figure 11k is when the lysine concentration is 3M (Comparative Example 2-5), Figure 11L shows the case where the lysine concentration is 4M (Comparative Example 2-6).

Lysine-conjugated hematite nanoparticles prepared using an aqueous solution of lysine having a lysine concentration of less than 0.03M (Comparative Examples 1-1 to 1-3) were used to coat the SMS composite nonwoven fabric. SEM images were not presented in

11, when the SMS composite nonwoven fabric is coated with lysine-conjugated hematite nanoparticles prepared using an aqueous lysine solution having a lysine concentration of 0.2M (Example 2-5), in all cases, the nanoparticles are agglomerated As the tendency is minimized, it can be seen that it is evenly and well-distributed and the most excellent coating can be confirmed. As a suboptimal method, when the SMS composite nonwoven fabric is coated with lysine-conjugated hematite nanoparticles prepared using an aqueous lysine solution having a lysine concentration of 0.15M (Example 2-4), the nanoparticles are relatively evenly dispersed and the coating is made. It can be seen that lost In terms of maintaining the colloidal state of nanoparticles in water, as shown in Evaluation Example 5, an aqueous solution of lysine having a lysine concentration in the range of 0.05M to 2M is possible to some extent. It can be seen that an aqueous solution of lysine having a concentration of 0.15M to 0.2M is optimal.

Evaluation Example 7: Lysine-conjugated hematite nanoparticles to MDCK cytotoxicity (cytotoxicity) test

The cytotoxicity of lysine-conjugated hematite nanoparticles was measured. As a sample, lysine-conjugated hematite nanoparticles (Example 1-5) prepared using an aqueous lysine solution having a lysine concentration of 0.2M were selected. The reason is that when using a 0.2M solution, the content of conjugated lysine in nanoparticles is sufficiently high (2.8 wt%), and the colloidal state is well maintained even after 7 days. The selected lysine-conjugated hematite nanoparticles were dissolved in water to obtain an aqueous solution of nanoparticles having a nanoparticle concentration of 20 mg/mL. Phosphate buffer saline (PBS) was added to the aqueous nanoparticle solution, diluted to 2%, centrifuged at 1000 rpm for 10 minutes, and the supernatant was used as a test stock solution. The concentration of lysine in the test stock solution was 80 μM.

MDCK (Madin Darby Canine Kidney) selected as a host cell is an epithelial cell extracted from a cocker spaniel kidney, a type of dog, and is widely used in bio and pharmaceutical research.

In order to evaluate the cytotoxicity of powder samples, in accordance with Chapter 11 or ISO 10993-12 Sample preparation and reference materials of 『Common Standards for Biological Safety of Medical Devices-24-KFDA Notification No. 2006-32』 After dissolution with PBS, the test was carried out according to Chapter 5 or ISO 10993-5 of 『KFDA Notification No. 2006-32 of Common Standards for Biological Safety of Medical Devices』. The experiment was carried out according to the test method by direct contact method, which is generally used, and the cytotoxicity was evaluated according to the result. The cytotoxicity test is a test method to find the maximum concentration that does not affect cell viability when serially diluted samples are processed and cultured for a certain period of time.

This test was performed according to the cytotoxicity test guideline document number KRBOP 0803 01, Crystal violet method of a testing institution (KR Biotech, Konkuk University, Seoul) prepared according to the above standards.

① In a 96 well plate, MDCK cells were cultured at 5% CO ₂ , 37°C.

② Serial dilution with the sample eluate stock solution and 2 times was added to the cultured cells and cultured for 3 days. At this time, one column was left for use as a negative control group.

③ Using a multi pipette, 50 μl of Crystal violet staining reagent was added to all wells.

④ After standing at 22±2℃ for 30 minutes, wash the plate with running water.

⑤ After drying the plate, absorbance was measured at a wavelength of 575 nm using a multi-well plate reader.

Based on the absorbance (100%) in the cell negative control group (PBS solution not containing the sample of the present invention), it was determined that there was no cytotoxicity when an absorbance of 50% or more was obtained.

As a result of the test, when MDCK cells were treated with the highest concentrations of the test stock solution, 1/2-fold dilution, and 1/4-fold dilution, respectively, cytotoxicity was not observed in all areas. A photo of the cytotoxicity test result was shown in FIG. 12 . 12 is a cytotoxicity photograph of lysine-conjugated hematite prepared in Example 1-5 against MDCK host cells. It is a photograph showing the absorbance of the negative control and this sample solution for the stock solution and the diluted solutions (1/2 and 1/4), respectively, and there is little difference in absorbance between the control and this sample solution. In addition, the cytotoxicity test results are shown in Table 4 below.

sample host cell Highest dilution without toxicity sample of the present invention MDCK 1 (No cytotoxicity even in the highest concentration of stock solution)

Evaluation Example 8: Cytotoxicity test for MRC-5 of lysine-conjugated hematite nanoparticles

The cytotoxicity of lysine-conjugated hematite nanoparticles was re-verified against another host cell, MRC-5 (human lung fibroblasts), as follows.

(1) test material

1) Cell: MRC-5 (human lung fibroblast)

2) Growth medium: DMEM (10% FBS, 1% penicillin/streptomycin)

3) Cytotoxicity test reagent: WST-1 assay kit, Takara

4) Physiological saline: PBS (phosphate buffered saline)

5) Sample: Lysine-conjugated hematite nanoparticles (Example 1-5) prepared using a lysine aqueous solution having a lysine concentration of 0.2M, and the nanoparticle concentration in a solution of these nanoparticles in water is 20 mg/mL It was.

6) Plate: 96 well cell culture plate

(2) Test method

1) After dispensing human lung fibroblast MRC-5 at 8 x 103 cells/200μL/well in a 96 well plate, 37℃, 5% CO₂ CO ₂ incubator (37℃, 5% CO ₂ ) incubated for 24 hours.

2) Samples of the present invention were stored at room temperature before testing.

3) The test sample was dissolved in sterile tertiary distilled water and stirred using a stirrer for 12 hours or more.

4) Taking a sample immediately after stirring, a 2% concentration solution of the original nanoparticle concentration (20 mg/mL) was prepared using a growth medium, and this was used as a sample stock solution. The concentration of lysine in the sample stock solution was 80 μM.

5) The sample stock solution of No. 4 was serially diluted 3 times in the growth medium as follows to prepare a test sample solution of various dilutions.

13 (a) is a photograph of a sample obtained by measuring the absorbance, and FIG. 13 (b) is a graph of the absorbance result. Specifically, Figure 13 (b) shows the cell viability at each concentration of the sample.

A sample having a nanoparticle concentration of 20 mg/mL was stirred at 800 rpm or higher for 12 hours or more, and a test solution in which the cells were treated by taking the supernatant immediately after stirring and after stopping the stirring for 30 minutes was prepared as shown in Table 5 below. The dilution concentration means the diluted ratio to the original 20 mg/mL.

dilution concentration in test solution 2% 0.66% 0.22% 0.07% 0.02% Media (μL) 980 600 600 600 600 20mg/mL sample (μL) 20 300 300 300 300

6) In a 96-well plate cultured at 8 x 103 cells/200μL/well, 200 μL of serially diluted test solution at a concentration of 2% to 0.02% was treated for 24 hours.

7) After 24 hours, the test solution dispensed in the 96 well plate was removed.

8) The sample remaining on the bottom of the plate was washed with PBS.

9) 100 μL of WST-1 medium (WST-1 10 μL in 100 μL media/well) was treated in each well, and then reacted in a CO ₂ incubator for 2 hours.

10) The absorbance of the reaction medium was measured at a wavelength of 420 nm using a microplate reader.

(3) Results

Cell viability was calculated using the WST-1 assay absorbance value.

Since the cell viability of the host cell MRC-5 is 80% or more as shown in FIG. 13(b) in all the tested concentration ranges, the CC ₅₀ value representing the concentration at which the cell viability is usually 50% is the test. It is significantly higher than the highest concentration performed (80 μM based on lysine concentration), so it is judged that there is almost no cytotoxicity of the sample of the present invention. And, this result is consistent with the conclusion that there is no cytotoxicity in the previous independent cytotoxicity test for MDCK host cells (Evaluation Example 7).

Evaluation Example 9: Antimicrobial Efficacy Test of Lysine-Conjugated Hematite Nanoparticles

The antibacterial effect of the sample against Escherichia coli was measured. The sample used was lysine-conjugated hematite nanoparticles (Example 1-5) prepared using an aqueous lysine solution having a lysine concentration of 0.2M, and nanoparticles in a solution obtained by dissolving lysine-conjugated hematite nanoparticles in water The concentration was 20 mg/mL (4 mM based on the lysine concentration). When the sample is added to the bacteria, the final sample concentration (based on the stock solution) is 80 μM, which is 2% of the above value.

Antibacterial activity was confirmed through a solid LB (Luria-Bertani) medium (or incubator: Culture Medium) after the bacteria were reacted with the sample of the present invention for 5 minutes. As a result of the experiment, it was confirmed that it showed an antibacterial effect of more than 99% against Echerichia coli. The test procedure was as follows.

1) The day before the test, the bacteria were pre-cultured in a shaking incubator at 37°C and 220 rpm.

2) On the day of the test, 1/100 of the pre-cultured bacteria were inoculated into new LB media and incubated for 2 hours to 2 hours and 30 minutes in a shaking incubator at 37°C and 220 rpm.

3) During the main culture, the OD (Optical Density: optical density or absorbance) value was measured and cultured until the OD600 (absorbance at a wavelength of 600 nm) was 0.5 to 1.0.

4) The cultured bacteria were precipitated by centrifugation.

5) The centrifuged bacteria were washed 2-3 times with PBS so that no LB media component remained.

6) After dispensing the washed bacteria by 0.5 ~ 1 x 107 CFU/mL, the bacteria were precipitated by centrifugation.

7) 1 mL of the sample was added to the precipitated bacteria, and then reacted for 1 to 5 minutes.

8) As a negative control, tap water was used instead of the sample.

9) After the reaction, the sample and the bacteria were separated by centrifugation.

10) Washing was carried out 2-3 times with PBS to remove the sample components remaining in the bacteria.

11) After resuspension by adding 1 mL of PBS to the washed bacterial precipitate, 100 uL of the bacterial solution was spread on the solid LB medium.

As a result of the test, when the Escherichia coli antibacterial efficacy of the sample of the present invention was compared with the negative control through the count of viable cells, it was confirmed that the antibacterial efficacy was greater than 99% as shown in Table 6 below. The results of the culture photograph were as shown in FIG. 14 .

sample Number of viable cells on the medium (Count) Negative control group (tap water) uncountably many sample of the present invention 99% or more reduction

Evaluation Example 10: Antiviral test on nonwoven fabric coated with lysine-conjugated hematite nanoparticles

The antiviral efficacy of the samples against HCoV-OC43 was measured. The selected sample is lysine-conjugated hematite nanoparticles (Example 1-5) prepared using an aqueous lysine solution having a lysine concentration of 0.2M, because in this case, as shown in Examples 2-5 and 11g This is because the nanoparticles are most evenly and well dispersed on the SMS non-woven fabric to maximize the efficacy. The concentration of nanoparticles in a solution obtained by dissolving the selected lysine-conjugated hematite nanoparticles in water was 20 mg/mL (4 mM based on the lysine concentration).

The purpose of this test is to verify the virus inactivation effect of the nonwoven fabric coated with the sample of the present invention. To this end, after spiking HCoV-OC43 on the nonwoven fabric selected above (Example 2-5), the degree of inactivation according to the contact time (2 hours, 4 hours, 8 hours) was confirmed. When the sample is added to the bacteria, the final sample concentration (based on the stock solution) is estimated to be 80 μM, which is 2% of the above value.

The nonwoven fabric used in this test was an SMS composite nonwoven fabric (Spunbond-Meltblown-Spunbond nonwoven fabric).

Human coronavirus OC43 (HCoV-OC43) described in Table 7 below was used as the target virus. Human coronavirus OC43 was purchased from the Korea Bank for Pathogenic Viruses.

virus cell line note Human coronavirus OC43 (KBPV-VR-8) HCT-8
(KCLB-10244) - Model virus of SARS-CoV-2 that causes COVID-19
- Viruses of the genus Betacoronaviruses
- (+)ssRNA virus
- Enveloped virus
- zoonotic virus

The procedure of the test was as follows.

(1) Cell culture medium: RPMI + 10% fetal bovine serum

(2) Virus culture medium: DMEM + 2% fetal bovine serum

HCT-8 cells (KCLB-10244) were cultured at 37° C., 5% CO ₂ in an incubator. After infecting the monolayer cells cultured in the T-150 flask with HCoV-OC43, a virus culture medium was added, and CPE (cytopathic effect) was periodically observed while culturing in an incubator at 37° C. and 5% CO ₂ . When CPE was clearly observed, the culture medium and cell debris were collected, and then the cells were disrupted by freezing and thawing three times, and the cell debris was removed by centrifugation. The centrifugal supernatant was filtered through a 0.45 μm filter and then divided and stored at -70°C.

A total of 3 sets were prepared by using 10 pieces of nonwoven fabric coated with a sample having a size of 1 x 1 cm ² as one set. In each set, 0.2 mL of HCoV-OC43 was spiked to trap the virus for 1 min. While reacting the sample in which the virus adsorption was completed at room temperature, one set was recovered every 2 hours, 4 hours, and 8 hours, and the virus present in each sample was immediately quantified. Viruses were recovered from each sample as follows. After transferring the two pieces of the reaction sample to a conical tube, 2.8 mL of virus culture medium was injected, and vigorously mixed with a vortex mixer 3 times for 1 minute each. Thereafter, the recovered solution was filtered through a 0.45 μm filter and immediately quantified. Each sample was prepared and performed in duplicate.

The virus culture solution and the recovered solution collected from each sample were filtered through a 0.45 μm filter and serially diluted 8 times with a 7-fold number. The diluted samples were inoculated into 8 wells of 0.25 mL each on a previously prepared cell plate. After culturing in an incubator at 37°C, 5% CO ₂ for 1 hour, 1 mL of virus culture medium was injected, and wells in which CPE reactions appear periodically were checked while culturing in an incubator at 37°C, 5% CO ₂ . Virus titer was analyzed by TCID50.

In this test, it was attempted to confirm the corona virus inactivation effect by the nonwoven fabric coated with the sample. To this end, the inactivation effect of Human coronavirus OC43 according to the time (2 hours, 4 hours, 8 hours) in contact with the nonwoven fabric coated with the sample was measured.

In the test to confirm the inactivation effect of the nonwoven fabric coated with the sample (2nd repetition), 51.26% of HCoV-OC43 spiked after 2 hours of reaction was inactivated, and after 4 hours of reaction, 66.18% was inactivated, and after 8 hours of reaction, HCoV-OC43 was inactivated. was inactivated by 96.01%.

Table 8 below is a summary of the results, and FIG. 15 is a graphical representation of Table 8.

Inactivation (%) EXP. One EXP. 2 Average Sample treated for 2 hours 51.26 51.26 51.26 Sample treated for 4 hours 68.84 63.52 66.18 Sample treated for 8 hours 96.10 95.91 96.01

Evaluation Example 11: Cytotoxicity, antibacterial and antiviral efficacy evaluation of lysine-conjugated hematite nanoparticles

Cytotoxicity of lysine-conjugated hematite nanoparticles prepared using lysine aqueous solutions prepared in Examples 1-1 to 1-4, Examples 1-6 to 1-8 and Comparative Examples 1-1 to 1-6 , Antibacterial and antiviral efficacy was evaluated according to Evaluation Examples 7 to 10, and the results are shown in Table 9 below with the results of lysine-conjugated hematite nanoparticles prepared using the lysine aqueous solution prepared in Examples 1-5 shown in In Table 9 below, cytotoxicity to MDCK was evaluated by cell viability at a dilution of 1/4 (ie, 20 μM based on lysine), and cytotoxicity to MRC-5 was evaluated by cell viability at a dilution of 1/80 (lysine 1 μM). The survival rate was evaluated, and the Escherichia coli antibacterial efficacy was evaluated by the reduction rate of live cells under the conditions of Evaluation Example 9, and the antiviral efficacy against HCoV-OC43 was evaluated by the virus inactivation rate after 8 hours of reaction under the conditions of Evaluation Example 10.

Example 1-1 1-2 1-3 1-4 1-5 1-6 1-7 1-8 Cell viability (%) against MDCK
(dilution factor 1/4) 99 99 99 99 99 99 98 96 Cell viability (%) for MRC-5 (dilution 1/80) 99.9 99.9 99.9 99.9 99.9 99.9 99.5 99 Escherichia coli reduction rate (%) 40 54 70 90 99 95 80 56 HCoV-OC43 virus inactivation rate (%) 25 40 60 88 96 90 74 50 comparative example 1-1 1-2 1-3 1-4 1-5 1-6 Cell viability (%) against MDCK
(dilution factor 1/4) 99 99 99 99 93 88 Cell viability (%) for MRC-5 (dilution 1/80) 99.9 99.9 99.9 99.9 97 93 Escherichia coli reduction rate (%) 2 3 8 25 30 20 HCoV-OC43 virus inactivation rate (%) One 2 5 10 20 10

Referring to Table 9, the lysine-conjugated hematite nanoparticles prepared in Examples 1-1 to 1-8 have no cytotoxicity to MDCK and cytotoxicity to MRC-5, Escherichia coli antibacterial efficacy and HCoV It was shown that the antiviral efficacy against -OC43 was excellent.

On the other hand, the lysine-conjugated hematite nanoparticles prepared in Comparative Examples 1-1 to 1-6 generally do not have cytotoxicity to MDCK and cytotoxicity to MRC-5, but Escherichia coli antibacterial efficacy and HCoV-OC43 Its antiviral efficacy was found to be very poor. The reason that cytotoxicity is almost nonexistent in all cases (that is, high cell viability) is because basically both hematite and lysine are substances with little cytotoxicity. Only when the concentration of lysine was extremely high, the cell viability was slightly reduced, but the amount of decrease was insignificant. The reason that the antibacterial and antiviral efficacy was sharply decreased in the comparative examples is because the change in the isoelectric point (IEP), the change in water solubility, and the effect of lysine aggregating with each other at ultra-high concentrations acted in a complex way.

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Although the present invention has been described with reference to the drawings and embodiments, it will be understood that these are merely exemplary, and that those skilled in the art can make various modifications and equivalent other embodiments therefrom. Accordingly, the true technical protection scope of the present invention should be defined by the technical spirit of the appended claims.

100: organic-inorganic hybrid nanoparticles 110: lysine
120: hematite

Claims (13)

hematite; and
Organic-inorganic hybrid nanoparticles comprising lysine conjugated to the hematite. According to claim 1,
The iron atom of the hematite is organic-inorganic hybrid nanoparticles combined with an oxygen atom in the carboxyl group of the lysine by an ionic bond. According to claim 1,
The organic-inorganic hybrid nanoparticles in which the content of the lysine in the organic-inorganic hybrid nanoparticles is 1 to 4% by weight. 4. The method of claim 3,
The organic-inorganic hybrid nanoparticles in which the content of the lysine in the organic-inorganic hybrid nanoparticles is 2-3% by weight. According to claim 1,
The hematite is an organic-inorganic hybrid nanoparticle having an average particle diameter of 10 to 200 nm. An article comprising the organic-inorganic hybrid nanoparticles according to any one of claims 1 to 5. 7. The method of claim 6,
The article is a nonwoven fabric, a composite nonwoven fabric or a nonwoven laminate article. Dissolving lysine in a solvent to prepare a lysine solution (S10);
Stirring after adding hematite to the lysine solution (S20); and
Method for producing organic-inorganic hybrid nanoparticles comprising the step (S30) of removing the solvent from the resultant obtained in the step (S20). 9. The method of claim 8,
The solvent is a method of producing an organic-inorganic hybrid nanoparticle containing water. 9. The method of claim 8,
The lysine concentration in the lysine solution in the step (S10) is a method of producing an organic-inorganic hybrid nanoparticle of 0.03 ~ 4M. 11. The method of claim 10,
The lysine concentration in the lysine solution in the step (S10) is a method of producing an organic-inorganic hybrid nanoparticle of 0.05 ~ 2M. 9. The method of claim 8,
The step (S10) and the step (S20) is a method for producing an organic-inorganic hybrid nanoparticle is performed at room temperature and under normal pressure. 9. The method of claim 8,
In the step (S10) and the step (S20), no material is added except for the lysine, the solvent, and the hematite.

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