KR102309437B1 - Implementation of Surface Repeating Structure Dextran Nanogels binding to Pattern Recognition Retinoic acid-Inducible Gene-I-like Receptors - Google Patents

Abstract

The present invention relates to a dextran nanogel implementing a surface repeating structure that binds to a pattern recognition RIG-1 like receptor, and the dextran nanoparticles of the present invention have little toxicity and side effects, have long-term stability, and also have a pattern recognition receptor It mediates innate immune activation through RIG-1 like receptor (RLR), which has the effect of inhibiting the proliferation of various viruses, thereby enhancing innate immunity or antiviral pharmaceutical composition, food It can be usefully used in a composition or a feed composition.

Description

Implementation of Surface Repeating Structure Dextran Nanogels binding to Pattern Recognition Retinoic acid-Inducible Gene-I-like Receptors

The present invention relates to a dextran nanogel with a surface repeating structure that binds to a pattern recognition RIG-1 like receptor.

Immunity is divided into innate immunity, which is possessed from birth, and acquired immunity, which is acquired by living adaptively. Innate immunity, also called 'natural immunity', is characterized by non-specific responses to antigens and no special memory action.

Among the innate defense immune systems in vivo, the most important research field in recent years is the field of innate immunity induced by interferon, and the activation of interferon-mediated immunity can be a fundamental prevention method against various infectious disease pathogens. Therefore, studies on the mechanism of interferon activation and development of immunomodulatory agents capable of inducing interferon are active. In addition, immune factors (inflammatory cytokines) are secreted as one of the defense mechanisms of innate immunity following infection with pathogens, and inflammatory responses are induced by these factors, thereby providing defense against pathogens. Therefore, induction of an appropriate level of inflammatory response can also be a preventive and therapeutic method for various infectious disease pathogens, and research on the development of immune enhancing agents that can induce this is necessary.

RIG-1 like receptors (Retinoic acid-Inducible Gene-I-like Receptors, RIG-1 like receptors, RLR) are one of the important pattern recognition receptors that recognize DNA or RNA present in viruses. The RLR recognizes viral RNA in virus-infected cells and transmits signals to mitochondrial antiviral signaling protein (MAVS) and interferon regulatory transcription factor 3 (IRF3), and finally activates a pathway to generate type 1 interferon IFN-α. make it The generated type 1 interferon is secreted out of the cell and binds to the interferon receptors of all cells and transmits a new signal, leading to the generation of anti-viral genes that inhibit virus proliferation. Consequently, the generation of type 1 interferon by the RLR signaling pathway plays a key role in antiviral function.

Nanoparticles are used in various fields, and in the field of vaccine development research, customized RNA nanoparticles for Zika virus vaccine production, self-assembled nanoparticles that induce H1N1 antibody production, and immune activity-inducing DNA smart nanoparticles have been developed. . In addition, the field of cancer treatment using chitosan-containing nanoparticles as a drug delivery system is being actively studied. In addition, virus nanoparticles have been developed in the field of diagnostic analysis using nanoparticles, and are used for various purposes by fusion of each field.

Accordingly, the present inventors prepare a nanogel containing dextran, and the nanogel has almost no toxicity and side effects, has long-term stability, and is a pattern recognition receptor, RIG-1 like receptor (RIG-1 like receptor, RLR) through mediating innate immune activation, thereby completing the present invention by confirming that there is an effect that can suppress the proliferation of various viruses.

Korean Patent Registration No. 10-1770573

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating viruses.

Another object of the present invention is to provide a health functional food composition for virus prevention or improvement.

Another object of the present invention is to provide a health food composition for virus prevention or improvement.

Another object of the present invention is to provide a composition for enhancing innate immunity.

In order to achieve the above object,

The present invention relates to a nanogel composed of a macro initiator, oligo(ethylene oxide)methyl ether methacrylate as a monomer, and poly(ethylene oxide) dimethacrylate as a crosslinker; and

Virus prevention or prevention of viruses containing dextran-containing nanogels including; A therapeutic pharmaceutical composition is provided.

In addition, the present invention is a macro initiator (macro initiator), oligo (ethylene oxide) methyl ether methacrylate as a monomer (monomer), and a nanogel consisting of poly (ethylene oxide) dimethacrylate as a crosslinker; and

Virus prevention or prevention of viruses containing dextran-containing nanogels including; It provides a health functional food composition for improvement.

Further, the present invention is a macro initiator (macro initiator), oligo (ethylene oxide) methyl ether methacrylate as a monomer (monomer), and a nanogel consisting of poly (ethylene oxide) dimethacrylate as a crosslinker; and

Virus prevention or prevention of viruses containing dextran-containing nanogels including; A health food composition for improvement is provided.

Further, the present invention is a macro initiator (macro initiator), oligo (ethylene oxide) methyl ether methacrylate as a monomer (monomer), and a nanogel consisting of poly (ethylene oxide) dimethacrylate as a crosslinker; and

Enhancement of innate immunity containing dextran-containing nanogel as an active ingredient; A composition is provided.

The dextran nanogel of the present invention has little toxicity and side effects, has long-term stability, and mediates innate immune activation through RIG-1 like receptor (RIG-1 like receptor, RLR), which is a pattern recognition receptor, and thus Since it has an effect of inhibiting the proliferation of various viruses, it can be usefully used for enhancing innate immunity or for antiviral compositions, food compositions, and the like.

1 is a schematic diagram showing the preparation of the nanogel of the present invention.
2 is a result of confirming the particle size and stability of the nanogel.
3 is a result confirming the cytotoxicity of Poly IC-RITC-Dextran-Nanogel.
4 is a view confirming the cytotoxicity results of BD-NG, D-NG and RD-NG.
5 is a diagram showing the result of confirming the intracellular uptake of RD-NG.
6 is a result confirming the increase of the pattern recognition receptor according to the treatment time and concentration of RD-NG.
7 shows the results of confirming the expression of RIG-1 in cells into which Poly IC-Nanogel, B-NG, D-NG, and RD-NG were introduced at low concentrations, respectively.
8 is a result confirming whether RD-NG and RIG-1 directly bind.
9 is a result confirming the signal transduction pathway transmitted by the binding of RD-NG and RIG-1.
10 is a result of confirming the cytokine produced by the binding of RD-NG and RIG-1.
11 is a result confirming the phagocytosis of RD-NG in major organs in vivo through intravenous and intraperitoneal injection.
12 is a result of confirming the phagocytosis of RD-NG in lymph nodes through the lymphatic system by plantar injection.
13 is a result confirming the expression of RIG-1 in major organs through intravenous, intraperitoneal and plantar injection.
14 is a result of confirming the cytokine properties in serum after injection of RD-NG through various in vivo routes.
15 is a result of confirming the expression of IFN-α in serum 5 days after plantar injection of RD-NG.
16 is a result confirming the expression of IFN-α in porcine PAM cells.
17 is a result confirming the expression of IFN-α in pig BAL cells.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for virus prevention or treatment

The present invention relates to a nanogel composed of a macro initiator, oligo(ethylene oxide)methyl ether methacrylate as a monomer, and poly(ethylene oxide) dimethacrylate as a crosslinker; and

Virus prevention or prevention of viruses containing dextran-containing nanogels including; A therapeutic pharmaceutical composition is provided.

In one embodiment of the present invention, the macro initiator is characterized in that the compound represented by the formula (1):

[Formula 1]

(In Formula 1, a is an integer of 1-300).

In one embodiment of the present invention, the oligo (ethylene oxide) methyl ether methacrylate is characterized in that the compound represented by the following formula 2:

[Formula 2]

(In Formula 2, b is an integer of 1-10).

In one embodiment of the present invention, the poly (ethylene oxide) dimethacrylate is characterized in that the compound represented by the following formula (3):

[Formula 3]

(In Formula 3, c is an integer of 1-100).

In one embodiment of the present invention, the virus is influenza, avian influenza, Newcastle virus, vesicular stomatitis virus, Cossackievirus, hand, foot and mouth virus (Enterovirus-71), herpes simplex virus ( Herpes Simplex Virus), Foot and Mouth Disease (FMD), Colorado tick fever virus, Reovirus, Human Immunodeficiency Virus, Swine Fever Virus, Bovine Viral Diarrhea Virus, Swine Genital Respiratory Syndrome ( Porcine reproductive and respiratory syndrome virus), Swine Ozesky's disease, rotavirus, parvovirus, and swine epidemic diarrhea virus (Porcine epidemic diarrhea virus) is characterized in that at least one selected from the group consisting of.

The dextran-containing nanogel of the present invention can be administered in various oral and parenteral formulations during clinical administration, and when formulated, a diluent such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. It is prepared using excipients.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, and these solid preparations include at least one or more excipients, such as starch, in one or more dextran-containing nanogels of the present invention, It is prepared by mixing calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral administration include suspensions, solutions, emulsions, or syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized formulations, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

In addition, the effective dosage for the human body of the dextran-containing nanogel of the present invention may vary depending on the patient's age, weight, sex, dosage form, health status and disease degree, and is generally about 0.001-100 mg/kg/ day, preferably 0.01-35 mg/kg/day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg/day, preferably 0.7-2500 mg/day, and once a day at regular time intervals according to the judgment of a doctor or pharmacist It may be administered in several divided doses.

Health food or health functional food composition for virus prevention or improvement

The present invention relates to a nanogel composed of a macro initiator, oligo(ethylene oxide)methyl ether methacrylate as a monomer, and poly(ethylene oxide) dimethacrylate as a crosslinker; and

Virus prevention or prevention of viruses containing dextran-containing nanogels including; It provides a health food or health functional food composition for improvement.

In one embodiment of the present invention, the macro initiator is characterized in that the compound represented by the formula (1):

[Formula 1]

(In Formula 1, a is an integer of 1-300).

In one embodiment of the present invention, the oligo (ethylene oxide) methyl ether methacrylate is characterized in that the compound represented by the following formula 2:

[Formula 2]

(In Formula 2, b is an integer of 1-10).

In one embodiment of the present invention, the poly (ethylene oxide) dimethacrylate is characterized in that the compound represented by the following formula (3):

[Formula 3]

(In Formula 3, c is an integer of 1-100).

In one embodiment of the present invention, the virus is influenza, avian influenza, Newcastle virus, vesicular stomatitis virus, Cossackievirus, hand, foot and mouth virus (Enterovirus-71), herpes simplex virus ( Herpes Simplex Virus), Foot and Mouth Disease (FMD), Colorado tick fever virus, Reovirus, Human Immunodeficiency Virus, Swine Fever Virus, Bovine Viral Diarrhea Virus, Swine Genital Respiratory Syndrome ( Porcine reproductive and respiratory syndrome virus), Swine Ozesky's disease, rotavirus, parvovirus, and swine epidemic diarrhea virus (Porcine epidemic diarrhea virus) is characterized in that at least one selected from the group consisting of.

There are no special restrictions on the type of food. Examples of foods to which the dextran-containing nanogel of the present invention can be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, and ice cream. There are dairy products, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and includes both health food and health functional food in the ordinary sense.

The health food and health functional food composition containing the dextran-containing nanogel according to the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the dextran-containing nanogel may be appropriately determined according to the purpose of its use (for prevention or improvement). In general, the amount of the composition in health food and health functional food may be added in an amount of 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake for health maintenance or health control, the above amount may be less than the above range, and since there is no problem in terms of safety, the dextran-containing nanogel can be used in an amount above the above range. have.

The health food and health functional food composition of the present invention is not particularly limited in other ingredients except for containing the dextran-containing nanogel of the present invention as an essential ingredient in the indicated ratio, and contains various flavoring agents or natural carbohydrates, such as conventional beverages. It may be contained as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 of the nutraceutical composition of the present invention.

In addition to the above, the health food and health functional food composition containing the dextran-containing nanogel of the present invention are various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese) , chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health food and health functional food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage.

These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing the dextran-containing nanogel of the present invention.

Composition for enhancing innate immunity

The present invention relates to a nanogel composed of a macro initiator, oligo(ethylene oxide)methyl ether methacrylate as a monomer, and poly(ethylene oxide) dimethacrylate as a crosslinker; and

Enhancement of innate immunity containing dextran-containing nanogel as an active ingredient; A composition is provided.

In one embodiment of the present invention, the macro initiator is characterized in that the compound represented by the formula (1):

[Formula 1]

(In Formula 1, a is an integer of 1-300).

In one embodiment of the present invention, the oligo (ethylene oxide) methyl ether methacrylate is characterized in that the compound represented by the following formula 2:

[Formula 2]

(In Formula 2, b is an integer of 1-10).

In one embodiment of the present invention, the poly (ethylene oxide) dimethacrylate is characterized in that the compound represented by the following formula (3):

[Formula 3]

(In Formula 3, c is an integer of 1-100).

In one embodiment of the present invention, the composition mediates innate immune activation through increased expression of interferon alpha (IFN-α) by binding to RIG-1 like receptor (RLR). do.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

<Preparation Example 1> Preparation of RITC-DEXTRAN-Nanogel (RD-NG)

In the water layer, OEOMA 300 (oligo(ethylene oxide)methyl ether methacrylate), an ethylene oxide-based monomer having biocompatibility, PEOEMA (poly(ethylene oxide)dimethacrylate) as a crosslinking agent, CuBr2 as a catalyst, and TPMA (Tris(Tris) as a ligand) 2-pyridylmethyl)amine) and RITC-Dx (RITC-DEXTRAN) as a supporting material are added, and cyclohexane containing Span80 to be used as a surfactant in the organic layer is prepared. In addition, an aqueous solution of ascorbic acid to be used as a reducing agent was prepared according to the ratio of Table 1 below.

After mixing the water layer and the organic layer in a beaker, ultrasonication is performed in an ice bath at 0° C. for 5-10 minutes to make a milky inverse miniemulsion emulsion. Oxygen is removed by bubbling the emulsion and ascorbic acid aqueous solution as a reducing agent for 30 minutes using nitrogen, respectively. The emulsion after bubbling is injected into a vial substituted with nitrogen using a syringe substituted with nitrogen, and stirred at 700 rpm or higher in an oli bath set at 30°C. The aqueous solution of ascorbic acid is put into the vial containing the emulsion in the same way and the reaction is started. After 4 hours, the polymerization solution ^{was centrifuged at 15000 rpm and 4 o} C for 30 minutes to remove the supernatant, and the settled nanogel was taken.

After removing the supernatant, tetrahydrofuran (THF) is filled in the nanogel obtained, and after 3 hours, when the nanogel is sufficiently swollen, centrifuge again under the same conditions a total of 3 times to remove the supernatant (unreacted monomers and reactants, organic The solvent is dissolved in the supernatant and removed). In addition, after centrifugation under the same conditions to remove residual tetrahydrofuran, the operation of filling with PBS solution is performed three times. Analyze the nanogel dispersed in PBS after purification. If nanogels or micro-sized gels with a size of 400-600 nm or more exist, ^{centrifuge at 4000 rpm and 4 o} C for 15 minutes to recover the supernatant solution and remove only the settled gels.

<Comparative Example 1>

In a method similar to the method of Preparation Example 1, Basic-Nanogel (B-NG) was prepared without adding a support material, and Dextran-Nanogel (D-NG) was prepared using dextran as a support material. prepared. Polyinosinic:polycytidylic acid (Poly IC) as a support material was added at a concentration of 10 mg/ml of the entire water layer, mixed with the organic layer, and subjected to ultrasonic treatment to prepare Poly IC-Nanogel, and Poly IC) was added to 10 mg/ml of the entire water layer After the concentration, RITC-Dextran monomer was added at a ratio of 1-1.5 wt%, mixed with an organic layer, and then sonicated to prepare Poly IC-RITC-Dextran-Nanogel.

<Experimental Example 1> Confirmation of size and stability of nanogel

In order to confirm the particle stability of the nanogel prepared in Preparation Example 1, as shown in Table 2 and FIG. 2, the particle size and number after 1 day to 8 days were checked. When stored refrigerated at 4° C. for 8 days There was no change in size and number (see FIG. 2).

<Experimental Example 2> Confirmation of cytotoxicity

In order to check whether the cytotoxicity of D-NG, RD-NG, B-NG, and Poly IC-RITC-Dextran-Nanogel prepared in Preparation Example 1, MTT experiment was performed.

Specifically, 200ug of Poly IC-Nanogel and Poly IC-RITC-Dextran-Nanogel were treated in Mouse Raw264.7 cells. In addition, cytotoxicity was confirmed by treating each of 1, 10, 100 and 200 ug of B-NG and RITC-RD-NG.

As a result, as shown in FIG. 3 , Poly IC-RITC-Dextran-Nanogel containing poly IC resulted in cell death within 24 hours, confirming high toxicity to cells. On the other hand, as shown in FIG. 4 , it can be confirmed that B-NG, D-NG and RD-NG have no toxicity even when treated up to 200 μg. In addition, since RITC-Dextran is a fluorescent substance, it has advantages for observing cell phagocytosis and distribution of Nanogel in vivo.

<Experimental Example 3> Confirmation of intracellular uptake of nanogel

In order to confirm the degree of intracellular uptake of the nanogel of the present invention, Raw264.7 cells, which are mouse macropages, were treated with RD-NG of Preparation Example 1. Specifically, a cover glass was laid on a Petri dish of 60Φ and raw cells ^{were inoculated at 2.5X10 5} , and then 200ug of RITC-Dextran-Nanogel (RD-NG) of the present invention was added. Thereafter, after treatment for 0hr, 2hr, 7hr, 23hr, 48hr, and 72hr, acetone was used to fix the nucleus, and the nucleus was DAPI-stained and observed with a fluorescence microscope. As a result, it was confirmed by the red signal that Raw264.7 clearly uptaked RITC-Dextran-Nanogel from 24 hr, and as time passed, more RD-NG from Raw264.7 was uptaked into the cell. was confirmed (see FIG. 5).

<Experimental Example 4> Confirmation of increase in pattern recognition receptors in cells into which nanogels are introduced

Raw264.7 was treated with RD-NG (1ug/ul) at different concentrations of 10, 50, 100, and 200ug, and after 24 hr, western blot was performed with RIG-1, TLR-3 and As a result of confirming the increase in TLR-4 (Poly IC (polyinosinic-polycytidylic acid, PI:C) and Adenovirus used as a positive control), it was found that only RIG-1 was specifically increased in 50 ug, 100 ug, and 200 ug. (See Figure 6A). In addition, as a result of checking for 0, 2, 8, 24, 48, and 72 hrs after 200ug of RD-NG treatment on Raw264.7, RIG-1 started to increase from 8 hr, indicating that the highest expression of RIG-1 was increased at 24 hr. was confirmed (see FIG. 6B).

In addition, in order to check whether the activation of RIG-1 by RD-NG occurs even with a small amount of treatment, 0.01, 0.1, 1 and 10 ug of B-NG, D-NG and RD-NG were treated in Raw264.7 and RIG The reaction between -1 and TLR-3 was confirmed. An increase in RIG-1 was observed from 1 ug of RD-NG, and it was confirmed that it clearly induced an increase in RIG-1 than B-NG and D-NG (see FIG. 7 ).

<Experimental Example 5> Confirmation of direct binding of nanogel and RIG-1 and related signaling

In order to confirm the signal transduction related to direct binding of RD-NG and RIG-1, Raw264.7 was treated with B-NG, D-NG, and RD-NG, cells were lysed, and all phagocytosed NGs were centrifuged by centrifugation. After separation, after washing with PBS, immunoprecipitation for RIG-1 (Immunoprecipitation) was performed. As a result, it was confirmed that RD-NG binds to RIG-1 the most. In order to reconfirm the binding of RD-NG and RIG-1, the nanogel was trypsinized, and as a result of performing the same experiment, it was observed that the RIG-1 signal disappeared (see FIG. 8).

In addition, 10, 50, 100 and 200ug of RD-NG was treated in Raw264.7 for 1 hour to confirm the signaling pathway transmitted by the binding of RD-NG and RIG-1, and ERK and AKT among the upper signaling pathways , As a lower signaling pathway, phosphorylation of NFkB was confirmed by western blot. As a result, it was confirmed that phosphorylation of ERK and phosphorylation of NFkB, which is a lower signaling pathway, clearly appeared from 50 μg in the upper signaling pathway (see FIG. 9 ).

<Experimental Example 6> Confirmation of cytokine secretion by binding of nanogel and RIG-1

In order to check the cytokines generated by the combination of RD-NG and RIG-I, 0.01, 0.1, 1 ug of RD-NG was treated with Raw264.7, and then IFN-α, one of anti-dissection virus cytokines, and inflammatory ELISA for TNF-α, one of the cytokines, was performed. The secretion of IFN-α was significantly increased in RD-NG 0.1 and 1 ug, but no change was observed in the case of TNF-α (see FIG. 10).

<Experimental Example 7> Confirmation of phagocytosis of nanogels in vivo

To investigate the phagocytosis of RD-NG in immune organs in vivo, 200 μg of RD-NG was injected intravenously or intraperitoneally into C57BL/6. After 72 hours, RD-NG uptake was observed by fluorescence microscopy in the spleen, lymph nodes, thymus, liver and lungs. However, when RD-NG was injected intravenously and intraperitoneally, no signal of RD-NG was observed in all tissues observed (see Fig. 11).

To confirm the lymphatic circulation of nanoparticles, mouse footpad injections were performed. Two C57BL/B6 mice were injected; One animal was injected with 20 μl of PBS and the other with 150 μg/20 μl of RD-NG into the left paw. After 24 h, popliteal lymph nodes (pLNs) were obtained. As shown in Fig. 12A, RD-NG (red) was observed in the left pLN of mice injected with RD-NG (see Fig. 12A). Then, using the lymphatic system, several types of lymph nodes were obtained and analyzed by immunofluorescence staining to determine whether the nanoparticles migrate relative to other lymph nodes. Two C57BL/B6 mice were injected, one was injected with 20 μl of PBS into the left paw, and the other mouse was injected with 150 μg/20 μl of RD-NG into the left paw and 20 μl of PBS was injected into the right paw. . After 24 hours, pLN, mesenteric lymph node (mLN) and inguinal lymph node (iLN) were obtained. As shown in Fig. 12B, RD-NG (red) was observed in pLN, iLN and mLN obtained from mice injected with RD-NG. In particular, RD-NG was strongly detected in LNs and mLN on the left, whereas RD-NG of pLN on the right was weakly detected.

<Experimental Example 8> Confirmation of RIG-1 activation and active cytokines by nanogel in vivo

After intravenous and intraperitoneal injection of RD-NG, tissue lysates from the spleen, lymph node, liver, lung and thymus were prepared and the expression level of RIG-I was analyzed, but there was no change in RIG-I in all organs (Fig. 13A). Reference). However, 24 hours after RD-NG injection through the footpad route, pLN, mLN and spleen were obtained and whole lysates were prepared and analyzed. As a result, RIG-I expression was observed in pLN injected with RD-NG (see Fig. 13B). In addition, to confirm RIG-I expression by RD-NG circulation, three RD-NG mice were injected; One with 20 μl of PBS, one with 150 μg/20 μl of RD-NG, and the other with no injection. After 24 hours, western blotting analysis was performed on the left and right lymph nodes. As a result, RIG-I expression was more increased in the left lymph nodes injected with RD-NG than in the right lymph nodes (see FIG. 13C ).

In addition, to determine how the increase in RIG-I expression in lymph nodes by RD-NG stimulation affects the production of other cytokines, serum was collected after 24 hours of RD-NG injection by various routes, and IFN-α, IL -10 and TNF-α were measured by ELISA. When 200 μg of RD-NG was injected intravenously or intraperitoneally, no cytokine changes were observed (see Fig. 14A). However, in the serum of mice injected by the plantar route, as shown in FIG. 14B, only IFN-α was specifically increased in the serum of mice injected with RD-NG at 24 hours (see FIG. 14B).

In addition, serum was collected at intervals of 0, 1, 3, and 5 days when RD-NG was injected into the footpad of C57BL/B6, and ELISA analysis for IFN-α was performed using the serum. As a result, it was slightly increased on the 1st day, but a sharp increase was confirmed on the 5th day (see FIG. 15).

<Experimental Example 9> Measurement of IFN-α expression in pig cells

As pig cells, PAM and BAL cells were used, and spleen, tonsil, and blood PBMCs were used as primary cells. And there was no antibody recognizing porcine RIG-1, so the analysis was performed by IFN-α ELISA.

As a result, as shown in FIG. 16 , IFN-α was increased at 200 μg of RD-NG in PAM cells. In addition, as cells isolated from pig lungs with BAL cells, PBS was put into the lungs of pigs obtained aseptically after autopsy and massaged. As a result, as shown in FIG. 17 , IFN-α was increased at 200 μg of RD-NG.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is not shown in the foregoing description, but particularly in the claims, and all differences within an equivalent scope should be construed as being included in the present invention.

Claims (9)

nanogels composed of a macro initiator, oligo(ethylene oxide)methylethermethacrylate as a monomer, and poly(ethylene oxide)dimethacrylate as a crosslinker; and
RITC (rhodamine-B-isothiocyanate)-labeled dextran embedded in the nanogel; for virus prevention or treatment containing RITC-labeled dextran-containing nanogel as an active ingredient pharmaceutical composition.
nanogels composed of a macro initiator, oligo(ethylene oxide)methylethermethacrylate as a monomer, and poly(ethylene oxide)dimethacrylate as a crosslinker; and
RITC (rhodamine-B-isothiocyanate)-labeled dextran embedded in the nanogel; for preventing or improving a virus containing RITC-labeled dextran-containing nanogel as an active ingredient Health functional food composition.
nanogels composed of a macro initiator, oligo(ethylene oxide)methylethermethacrylate as a monomer, and poly(ethylene oxide)dimethacrylate as a crosslinker; and
RITC (rhodamine-B-isothiocyanate)-labeled dextran embedded in the nanogel; for preventing or improving a virus containing RITC-labeled dextran-containing nanogel as an active ingredient health food composition.
nanogels composed of a macro initiator, oligo(ethylene oxide)methylethermethacrylate as a monomer, and poly(ethylene oxide)dimethacrylate as a crosslinker; and
A composition for enhancing innate immunity containing, as an active ingredient, RITC-labeled dextran-containing nanogel containing; RITC (rhodamine-B-isothiocyanate)-labeled dextran embedded in the nanogel .
5. The method according to any one of claims 1 to 4,
The macro initiator is a composition, characterized in that the compound represented by the formula (1):
[Formula 1]

(In Formula 1, a is an integer of 1-300).
5. The method according to any one of claims 1 to 4,
The oligo (ethylene oxide) methyl ether methacrylate is a composition characterized in that the compound represented by the following formula 2:
[Formula 2]

(In Formula 2, b is an integer of 1-10). 5. The method according to any one of claims 1 to 4,
The poly (ethylene oxide) dimethacrylate is a composition characterized in that the compound represented by the following formula 3:
[Formula 3]

(In Formula 3, c is an integer of 1-100). 4. The method according to any one of claims 1 to 3,
The viruses include influenza, avian influenza, Newcastle virus, Vesicular stomatitis virus, Cossackievirus, Enterovirus-71, Herpes Simplex Virus, Foot-and-mouth disease virus (Foot). and mouth disease (FMD), Colorado tick fever virus, reovirus, human immunodeficiency virus, swine fever virus, Bovine Viral Diarrhea Virus, Porcine reproductive and respiratory syndrome virus, swine Ozesky's disease, rotavirus, parvovirus and swine epidemic diarrhea (Porcine epidemic diarrhea virus) composition, characterized in that at least one selected from the group consisting of.
5. The method of claim 4,
The composition is a RIG-1 like receptor (RIG-1 like receptor, RLR) binding to the composition characterized in that it mediates innate immune activation through increased expression of interferon alpha (IFN-α).

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