KR102473102B1 - Core-shell Structured Nanoparticles Conjugated with Immune Adjuvants and use thereof - Google Patents

Abstract

It relates to nanoparticles of a core-shell structure conjugated with an immunoadjuvant and uses thereof. In one aspect, the nanoparticles conjugated with an immunoadjuvant enhance the activity of immune cells, so when used in combination with irreversible electroporation, anti-tumor As a synergistic effect of promoting the immune response appears, it is expected to have an excellent effect as an anti-cancer immunotherapy. In addition, since the nanoparticles having an iron-zinc oxide/gold structure can be imaged through a magnetic resonance imaging device and a computed tomography device, they can also be used as a contrast agent for living body imaging.

Description

Core-shell Structured Nanoparticles Conjugated with Immune Adjuvants and use thereof {Core-shell Structured Nanoparticles Conjugated with Immune Adjuvants and use thereof}

It relates to immunoadjuvant-conjugated core-shell nanoparticles and uses thereof.

Immunotherapy has recently been in the limelight because it can activate the body's immune system to target and kill cancer cells. Anticancer treatment has been the mainstream of surgery, anticancer drugs, radiation, etc., but recently, immunotherapy, a fourth-generation treatment, is attracting attention. To date, various forms of cancer immunotherapy, including cytokine therapy, immune checkpoint inhibitor therapy, chimeric antigen receptor T-cell (CAR-T) therapy, and cancer vaccines, have shown positive effects in clinical practice.

However, most of the current immunotherapeutic agents have limitations in clinical application due to immunotoxicity problems such as expensive treatment costs, differences in efficacy between individuals, and cytokine release syndrome. Among the immunotherapy strategies mentioned above, cancer vaccine strategies have many advantages. A cancer vaccine encapsulated with a cancer antigen has an advantage because it induces an antigen-specific immune response against cancer rather than inducing a non-specific immune response like conventional anticancer immunotherapies. In addition, since cancer vaccines have a long-term immune memory effect, they are effective in preventing cancer recurrence. However, the heterogeneity of patient tumors makes it difficult to develop cancer-specific antigens, and cancer cell vaccines are difficult to manufacture and difficult to determine an appropriate dose. Therefore, there is a need for a treatment strategy capable of effectively generating tumor-associated antigens in vivo.

Recently, research results have been reported that tumor resection such as photothermal therapy, photodynamic therapy, and radiation therapy can induce anticancer immune responses by effectively generating tumor-associated antigens. However, in the case of a method of inducing an immune response using light, it is difficult to deliver a light source to a tumor site located deep in the body, so there is a limit to clinical application. In addition, treatment using radiation and heat damages blood vessels and lymph nodes around the treatment site, which may inhibit the effect of anticancer immune response through tumor-associated antigens.

Irreversible electroporation is a new interventional treatment that necrosis cancer cells by making holes in the cell membrane using an electrode that delivers high voltage for a short period of time into the tumor. In particular, it is a new medical technology that is expected to be an effective local treatment because it can be treated without damage to blood vessels / lymph nodes and the treatment area is not limited to patients who are unable or unsuitable for treatment due to proximity to major blood vessel / lymph node structures or adjacent organs. (Korean Registered Patent No. 10-1780269), anti-cancer immunotherapy that induces an anti-cancer immune response using tumor-related antigens generated from cancer cells necrotic by irreversible electroporation using immune-enhancing nanoparticles there is no research

One aspect relates to magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; and a second layer containing gold formed on the surface of the first layer to provide a multi-layered nanoparticle.

Another aspect is magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; and a multi-layered nanoparticle comprising a gold-containing second layer formed on the surface of the first layer or a multi-layered nanoparticle further comprising a polymer layer formed on the surface of the second layer; and a drug, wherein the drug is encapsulated inside the multi-layered nanoparticles or bound to a polymer layer of the multi-layered nanoparticles.

Another aspect relates to magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; and a gold-containing second layer formed on the surface of the first layer as a contrast agent comprising nanoparticles having a multi-layer structure, wherein the contrast medium is magnetic resonance imaging (MRI), X-ray, or computer. It is to provide a contrast agent that is used in computed tomography (CT).

Another aspect relates to magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; a second layer containing gold formed on a surface of the first layer; and a multi-layered nanoparticle comprising a polymer layer formed on the surface of the second layer; And to provide an anticancer adjuvant comprising an immunoadjuvant bound to the polymer.

One aspect relates to magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; and a second layer containing gold formed on the surface of the first layer.

The term "multilayer structure" may mean a structure having a plurality of layers and including a "core-shell structure". The term “shell” may be used interchangeably with the terms “shell” and “shell layer”. In one embodiment, the multi-layered nanoparticles are spheric, triangular, cubic, hexagonal, oval, helical, and porous. It may be any one selected from the group consisting of, but is not limited thereto, and may include all multilayer structures within the range in which the effect of the present invention is maintained.

The "magnetic nanoparticle" is a material constituting a core in a multilayer structure and may be any nanoparticle known to be used as an MRI contrast agent, and in one embodiment, iron-zinc oxide nanoparticles, for example , (Zn _x Fe _1-x ) Fe ₂ O ₄ . The iron-zinc oxide nanoparticles may be prepared according to any method for preparing magnetic nanoparticles known in the art. For example, by thermally decomposing the precursor FeL ₃ (L = CO ₅ , NO ₃ , acetylacetonate, etc.), (Zn _x Fe _1-x ) Fe ₂ O ₄ of uniform size is directly synthesized. method can be used. In addition, as a method for producing hollow iron-zinc oxide (hollow (Zn _x Fe _1-x )Fe ₂ O ₄ ) nanoparticles, through a substitution reaction between phosphoric acid or nitric acid and iron-zinc oxide, iron-zinc oxide A method of introducing phosphorus or nitrogen into the nanoparticles and then dissolving them again may be used.

The “first layer” is a layer formed on the surface of the magnetic nanoparticles, and in one embodiment, the first layer containing silicon dioxide is surface-modified cationicly to form a second layer containing gold. may have been For example, to form a gold layer on the surface of the silicon dioxide layer, [3-(2-aminoethylamino)propyl]trimethylsilane ([3-(2-Aminoethylamino)propyl]trimethoxysilane, AEAPTMS), 3-aminopropyl It may be surface-modified with triethoxysilane ((3-Aminopropyl)triethoxysilane, APTES) or 3-aminopropyltrimethoxysilane (APTMS).

The "second layer" is a layer formed on the surface of the first layer, and in one embodiment, the second layer containing gold has a size of 0.5 nm to 10 on the cationic surface-modified first layer. nm, eg, 0.5 nm to 7 nm, 0.5 nm to 5 nm, 0.5 nm to 4 nm, 0.5 nm to 3 nm, 0.5 nm to 2.5 nm, 0.5 nm to 2 nm, 1 nm to 10 nm, 1 nm to 7 nm, 1 nm to 5 nm, 1 nm to 4 nm, 1 nm to 3 nm, 1 nm to 2.5 nm, 1 nm to 2 nm, 1.5 nm to 10 nm, 1.5 nm to 7 nm, 1.5 nm to 5 nm, 1.5 nm to 4 nm, 1.5 nm to 3 nm, and 1.5 nm to 2.5 nm gold nanoparticles may be first combined, and then a gold shell layer may be formed using the gold nanoparticles. When the size of the gold nanoparticles is less than 0.5 nm, the surface of the second layer may not be formed, and when the size is greater than 10 nm, the surface of the second layer may not be uniformly formed. Also, the gold nanoparticles bonded to the surface-modified first layer may be bonded at a density of 8 or more per 100 nm ² of the surface of the first layer. When the number of gold nanoparticles per 100 nm ² of the surface of the first layer is 8 or more, the surface of the second layer can be uniformly synthesized. When the number is less than 8, the surface is not formed or the surface is not formed smoothly, and each nanoparticle The size deviation of can be large.

In one embodiment, the multi-layered nanoparticles may further include a polymer layer formed on the surface of the second layer. The polymer layer may be made of a biocompatible polymer, for example, polyvinyl alcohol, polylysine, polyacrylic acid, polyacrylamide, polyurethane, poly(acrylonitrile-co-acrylic acid), polyethylene glycol, It may be at least one selected from the group consisting of polyethyleneimine, chitosan, dextran, and cellulose, but is not limited thereto.

In one embodiment, the multi-layered nanoparticles have a size of 1 nm to 300 nm, for example, 1 nm to 250 nm, 1 nm to 200 nm, 1 nm to 150 nm, 1 nm to 100 nm, 1 nm to 50 nm, 5 nm to 300 nm, 5 nm to 250 nm, 5 nm to 200 nm, 5 nm to 150 nm, 5 nm to 100 nm, 5 nm to 50 nm, 10 nm to 300 nm, 10 nm to 250 nm, 10 nm to 200 nm, 10 nm to 150 nm, 10 nm to 100 nm, 10 nm to 50 nm, 15 nm to 300 nm, 15 nm to 250 nm, 15 nm to 200 nm, 15 nm to 150 nm , 15 nm to 100 nm, 15 nm to 50 nm, 20 nm to 300 nm, 20 nm to 250 nm, 20 nm to 200 nm, 20 nm to 150 nm, 20 nm to 100 nm, 20 nm to 50 nm, 25 nm to 300 nm, 25 nm to 250 nm, 25 nm to 200 nm, 25 nm to 150 nm, 25 nm to 100 nm, 25 nm to 50 nm, 30 nm to 300 nm, 30 nm to 250 nm, 30 nm to It may be 200 nm, 30 nm to 150 nm, 30 nm to 100 nm, or 30 nm to 50 nm. When the size of the multi-layered nanoparticles is less than 1 nm, it is difficult to form magnetic nanoparticles and core/shell structures and may not have applicable magnetic properties, and when the size exceeds 300 nm, mass production is difficult and the nanoparticles as MRI contrast agents Efficiency is reduced and stability of the nanoparticles may be deteriorated.

Another aspect is magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; and a multi-layered nanoparticle comprising a gold-containing second layer formed on the surface of the first layer or a multi-layered nanoparticle further comprising a polymer layer formed on the surface of the second layer; and a drug, wherein the drug is encapsulated inside the multi-layered nanoparticles or bound to a polymer layer of the multi-layered nanoparticles. In the drug delivery system, the multi-layered nanoparticles, magnetic nanoparticles, the first layer, the second layer, and the polymer layer are as described above.

The drug may be a chemical drug, protein, peptide, or nucleotide, and may be, for example, an immunoadjuvant or an anticancer agent.

The drug may be encapsulated in the inner cavity of the multi-layered nanoparticle, bound to the surface of a material forming each inner layer, or encapsulated between layers, or bound to the polymer layer. can In one embodiment, the drug may be bound to the polymer through a covalent bond or a non-covalent bond, and the non-covalent bond may be an ionic bond, a coordinate bond, a hydrophobic interaction, a van der Waals bond, or a combination thereof. It may be, specifically, it may be an ionic bond. The polymer may be a cationic or anionic polymer, and when the polymer is cationic, a drug bound thereto may be anionic, and when the polymer is anionic, a drug bound thereto may be cationic. In one embodiment, the polymer may be a cationic polymer, such as polyethyleneimine, and the drug may be an anionic immunoadjuvant, such as CpG oligonucleotide.

Another aspect relates to magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; and a second layer containing gold formed on the surface of the first layer. In the contrast medium, the multi-layered nanoparticles, magnetic nanoparticles, first layer, and second layer are as described above.

According to one aspect, the multi-layered nanoparticles, including iron-zinc oxide and gold, are imaged through Computed Tomography (CT), X-ray, or Magnetic Resonance Imaging (MRI). Where possible, it can be used as a contrast agent for computed tomography, X-ray, or magnetic resonance imaging.

Another aspect relates to magnetic nanoparticles; A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles; a second layer containing gold formed on a surface of the first layer; and a multi-layered nanoparticle comprising a polymer layer formed on the surface of the second layer; And it provides an anticancer adjuvant comprising an immunoadjuvant bound to the polymer. In the anticancer adjuvant, the multi-layered nanoparticles, magnetic nanoparticles, the first layer, the second layer, and the polymer layer are as described above.

The term "immune adjuvant" may be used interchangeably with the term "immune adjuvant" or "immune adjuvant" and refers to an adjuvant used when a sufficient immune response cannot be obtained with vaccine antigen alone. can do. The term “immune enhancement” can mean inducing an initial immune response or measurably increasing an existing immune response to an antigen. In one embodiment, the immunoadjuvant is aluminum salts (aluminum phosphate or hydorxide), emulsions (MF59, AS03, AF03, SE), DsRNA analogues (poly(I:C)), Lipid A analogues (MPL, GLA) , Flagellin, Imidazoquinolines (Imiquimod, R848), CpG ODN, Saponins (QS21), C-type lectin ligands (TDB), CD1d lignads (α-galactosylceramide), AS01 (liopsone, MPL, QS21), AS02 (emulsion, MPL, QS21), AS04 (Alum, MPL), AS15 (liposone, MPL, QS21, CpG), GLA-Se (emulsion, GLA), IC31 (CpG, cationic peptide), CAF01 (TDB, cationic liposome), and ISCOMs (saponin , phospholipid) may be one or more selected from the group consisting of. In certain embodiments, the immunoadjuvant can be a CpG oligonucleotide. In one aspect, the immunoadjuvant-coupled nanoparticles may enhance immunotherapeutic effects and enhance secondary preventive immune functions by the immunoadjuvant.

The term "anti-cancer adjuvant" may refer to an agent capable of improving, improving or increasing the anti-cancer effect of anti-cancer treatment. For example, it does not exhibit significant anticancer activity alone, but may be used in combination with other anticancer treatments such as anticancer drugs, radiation therapy, and immunotherapy to improve, enhance, or increase the anticancer effect. In one embodiment, the anti-cancer adjuvant may be used in combination with immuno-anticancer therapy to enhance immune cell activity, thereby enhancing the therapeutic effect of immuno-anticancer therapy. Specifically, it may exhibit improved immune enhancing efficacy compared to the case of performing immunotherapy alone.

In one embodiment, the immuno-anticancer therapy may be at least one selected from the group consisting of cytokine therapy, immune checkpoint inhibitor therapy, chimeric antigen receptor T-cell (CAR-T) therapy, and cancer vaccine.

In another embodiment, the immuno-anticancer therapy may induce an anticancer immune response by generating tumor-associated antigens (TAAs) in a subject, and the tumor-associated antigens are generated by cancer cell death. it may be In a specific embodiment, the tumor-associated antigen may be generated by photothermal therapy, photodynamic therapy, radiation therapy, irreversible electroporation, or a combination thereof, specifically, by irreversible electroporation. may be occurring. The irreversible electroporation is an interventional procedure performed through a minimally invasive method using ultrasound or CT images, and can be applied regardless of tissue depth or treatment site, so that it can be applied to intractable or metastatic cancer treatment that cannot be operated. can In the irreversible electroporation method, a tumor-associated antigen is generated in vivo by killing cancer cells by piercing a cell membrane using an electrode that transmits a high voltage, thereby inducing an antigen-specific immune response. The anticancer adjuvant may be administered in combination with irreversible electroporation simultaneously, separately or sequentially to enhance the immune cell activation effect by irreversible electroporation. The irreversible electroporation may be performed under conditions of 1 to 2500 voltage, 1 to 99 pulses, and 1 to 1000 microseconds (microsecond wide). In the irreversible electroporation method, the range of cancer cell killing effect may vary depending on the voltage and pulse number, and specifically, the range of cancer cell killing effect may increase as the voltage and pulse number increase.

In one embodiment, the anticancer adjuvant may be absorbed by immune cells to enhance immune cell activity. The immune cells may be any one selected from the group consisting of macrophages, dendritic cells, B lymphocytes, T lymphocytes, mast cells, monocytes, eosinophils, natural killer cells, basophils, and neutrophils, and in certain embodiments, immune cells may be macrophages or dendritic cells.

The therapeutically effective amount and effective dosage of the anticancer adjuvant may vary depending on the type of specific immunoadjuvant, and can be appropriately selected by experts in the art through conventional clinical trials depending on the type of immunoadjuvant, and the patient's Depending on race, gender, age, cancer progression, etc., it may be appropriately added or subtracted according to the expert's judgment. The term "treatment-effective amount" is sufficient to produce a desired effect in a patient suffering from cancer, including improvement of the condition (eg, one or more symptoms) of the cancer patient, delay of disease progression, and the like. means quantity.

The anti-cancer adjuvant may be formulated as an injection, and when formulated as an injection, it may include a non-toxic buffer solution isotonic with blood as a diluent, for example, a phosphate buffer solution of pH 7.4. The anticancer adjuvant may include other diluents or additives in addition to the buffer solution. Excipients and excipients that can be added to these injections may use those well known to those skilled in the art.

Another aspect includes forming a first layer including silicon dioxide on the surface of the magnetic nanoparticles; Forming a second layer containing gold on the surface of the first layer; provides a method for producing nanoparticles having a multi-layer structure including. In one embodiment, the method may further include forming a polymer layer on the surface of the second layer. In the above method, the multi-layered nanoparticles, magnetic nanoparticles, the first layer, the second layer, and the polymer layer are as described above.

In one embodiment, the method may further include the step of cationic surface modification of the first layer after the step of forming the first layer.

In one embodiment, the forming of the second layer may include binding gold nanoparticles having a size of 0.5 nm to 10 nm to the cationic surface-modified first layer. Also, in the combining of the gold nanoparticles, the number of gold nanoparticles per 100 nm ² of the surface of the first layer may be 8 or more.

In one embodiment, the method may further include binding a drug to the polymer after the step of forming the polymer layer. In certain embodiments, the drugs may be immunoadjuvants or anti-cancer agents.

Nanoparticles conjugated with immune adjuvant according to one aspect enhance the activity of immune cells, so when used in combination with irreversible electroporation, a synergistic effect of promoting anti-tumor immune response appears, which is expected to be excellent as an anti-cancer immunotherapy. It is expected. In addition, since the nanoparticles having an iron-zinc oxide/gold structure can be imaged through a magnetic resonance imaging device and a computed tomography device, they can also be used as a contrast agent for living body imaging.

1 is a schematic diagram showing the principle of an immunotherapy method using immunoadjuvant-conjugated core/shell nanoparticles and irreversible electroporation according to one embodiment.
Figure 2 is a photograph taken with a transmission electron microscope of the nanoparticles prepared in Examples 1.1 to 1.5. (Nanoparticle 1: iron-zinc oxide nanoparticles (Example 1.1), nanoparticle 2: iron-zinc oxide/silicon dioxide nanoparticles (Example 1.2), nanoparticle 3: iron-zinc oxide/silicon dioxide/2 nm gold Nanoparticle (Example 1.3), Nanoparticle 4: Iron-zinc oxide/silicon dioxide/gold nanoparticle (Example 1.3), Nanoparticle 5: Iron-zinc oxide/silicon dioxide/gold/cationic polymer nanoparticle (Example 1.3) Example 1.4), nanoparticle 6: iron-zinc oxide/silicon dioxide/gold/cationic polymer/immunoadjuvant nanoparticles (Example 1.5))
FIG. 3 is a graph measuring the size and zeta potential of nanoparticles prepared in Examples 1.1 to 1.5 with respect to the photograph of FIG. 2 . (Nanoparticle 1: iron-zinc oxide nanoparticles (Example 1.1), nanoparticle 2: iron-zinc oxide/silicon dioxide nanoparticles (Example 1.2), nanoparticle 3: iron-zinc oxide/silicon dioxide/2 nm gold Nanoparticle (Example 1.3), Nanoparticle 4: Iron-zinc oxide/silicon dioxide/gold nanoparticle (Example 1.3), Nanoparticle 5: Iron-zinc oxide/silicon dioxide/gold/cationic polymer nanoparticle (Example 1.3) Example 1.4), nanoparticle 6: iron-zinc oxide/silicon dioxide/gold/cationic polymer/immunoadjuvant nanoparticles (Example 1.5))
4 is a result of performing gel electrophoresis to evaluate the conjugation ratio of the nanoparticles prepared in Example 1.4 and the immunoadjuvant.
Figure 5 shows the degree of expression of CD80 and CD86 on the cell surface after treating bone marrow-derived dendritic cells (BMDC) with PBS (control), immunoadjuvant, and nanoparticles conjugated with immunoadjuvant. This is a comparison graph.
6 is a graph showing cell viability after irreversible electroporation was performed under various voltage and pulse conditions in order to confirm the cancer cell killing effect of irreversible electroporation.
FIG. 7 is a photograph and a graph of measuring the range of cancer cell death after irreversible electroporation was performed by varying the number of pulses under the same voltage condition in order to confirm the range of cancer cell killing effect of irreversible electroporation.
8 is a graph showing the results of measuring the amount of ATP released extracellularly and the expression level of calreticulin expressed in the cell membrane after performing irreversible electroporation on cancer cells.
9 is a result of confirming the tumor size suppression effect in a tumor model in order to confirm the combined effect of immunoadjuvant-conjugated nanoparticles and irreversible electroporation.
10a to 10c show changes in the brightness of the nanoparticles according to varying the surface gold concentration and the iron oxide concentration in the center of the nanoparticles prepared in Example 1.3 using computed tomography, X-ray, and magnetic resonance imaging devices, respectively. This is the result of checking

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of nanoparticles with immunoadjuvant conjugated core/shell structure

1.1 Preparation of iron-zinc oxide nanoparticles

After putting 423.6 mg iron (III) acetylacetonate and 120 mg zinc chloride in a glass reactor, 1.2 ml oleic acid, 4.8 ml oleylamine, and 4.8 ml trioctylamine was added. A glass reactor containing the reactants was connected to the Schlenk line and stirred for 3 minutes at 300 rpm in a vacuum state. The temperature of the reactant was raised to 200 °C for 25 minutes using a temperature controller. After maintaining the reaction at 200 °C for one hour, the temperature of the reaction was raised to 330 °C for one hour. After maintaining at 330 °C for one hour, the temperature of the reactant was lowered to 25 °C for one hour. Toluene and ethanol were added to the reaction mixture at a ratio of 1:15.625, and centrifugation was performed at 1,600g for 5 minutes to obtain a reaction mixture. After adding 8 ml toluene and 30 μl oleylamine to the reaction mixture, the mixture was redispersed, centrifuged at 650 g for 3 minutes, redispersed in 4 ml ethanol, and centrifuged at 1,600 g for 5 minutes. The supernatant was removed and iron-zinc oxide nanoparticles were dispersed in 4 ml toluene.

1.2 Fabrication of nanoparticles with iron-zinc oxide/silicon dioxide structure

770 mg Igepal CO-520 and 12.6 ml cyclohexane were placed in a glass bottle and mixed until clear. After putting 100 μl iron-zinc oxide nanoparticles prepared in 1.1 and 105 μl ammonia water (NH ₄ OH), 30 μl tetraethyl orthosilicate (TEOS) was added. The reactants were mixed for 1 minute and reacted for 48 hours to form a silicon dioxide layer on the iron-zinc oxide nanoparticles.

In order to synthesize a gold structure on the iron-zinc oxide/silicon dioxide nanoparticles, the surface of the iron-zinc oxide/silicon dioxide must be cationically modified, so the following process was performed. After adding 10 μl of [3-(2-aminoethylamino)propyl]trimethoxysilane (AEAPTMS) to the iron-zinc oxide/silicon dioxide nanoparticles, they were incubated at 100 °C for 90 minutes. Stir at rpm. A 50 mM tetramethylammonium hydroxide solution was added to remove unreacted compounds. After shaking the solution to separate into a brown layer with nanoparticles and a white layer without nanoparticles. Nanoparticles containing iron-zinc oxide were purified from the brown layer using a MACS LS column. The obtained AEAPTMS conjugated iron-zinc oxide/silicon dioxide nanoparticles were dispersed in 1 ml of final dimethyl sulfoxide.

1.3 Fabrication of iron-zinc oxide/silicon dioxide/gold structure nanoparticles

In order to uniformly synthesize the iron-zinc oxide/silicon dioxide/gold structure, 2 nm gold particles were first bonded to the iron-zinc oxide/silicon dioxide nanoparticles. Specifically, 664 μl of 1 M sodium hydroxide (NaOH) solution and 16 μl of tetrakis (hydroxymethyl) phosphonium chloride solution were added to 60 ml of ultrapure water and stirred at 500 rpm for 10 seconds. did 2.4 ml of a 1% (weight percentage/weight percentage) solution of Au(III) chloride trihydrate was added to the stirring solution. After concentrating the solution at 3,000 g for 30 minutes using a centrifugal filter unit, the solution was stored for 1 hour. 0.1 ml of AEAPTMS-conjugated iron-zinc oxide/silicon dioxide nanoparticles obtained in Example 1.2 was reacted with 1.9 ml of concentrated 2 nm gold nanoparticles at 200 rpm for 5 hours to form 2 nm gold nanoparticles. conjugated nanoparticles were prepared. In order to remove unreacted gold nanoparticles with a size of 2 nm, only iron-zinc oxide nanoparticles to which gold nanoparticles are attached were separated using a MACS LS column.

To synthesize the gold shell layer of nanoparticles, 100 mg potassium carbonate and 60 mg Au(III) chloride trihydrate were dissolved in 800 ml ultrapure water. After the solution was stirred at 400 rpm for 24 hours, nanoparticles with gold nanoparticles and ultrapure water were dissolved in 20 ml of 1 mg/ml bis(p-sulfonaphophenyl)phenylphosphenine (Bis(p-Sulfonatophenyl) Phenylphosphine) solution was added. As the ratio of nanoparticles increased, the thickness of the gold shell layer decreased. 22 ml of a 130 μg/ml hydroxylamine hydrochloride (NH2OH·HCl) solution was added to the reaction mixture. The color of the reactant was changed to red when the gold shell layer was formed, and then the color of the solution gradually changed to blue as the thickness of the gold shell layer increased. After the reaction was completed, each was transferred to a 50 ml conical tube and centrifuged at 1,500 g for 60 minutes to remove the supernatant. It was dispersed with 1 ml of p-Sulfonatophenyl)Phenylphosphine) solution. Only core/shell structured nanoparticles containing iron-zinc oxide were obtained using a MACS LS column.

1.4 Preparation of nanoparticles conjugated with cationic polymers

Polyethleneimine (PEI), a cationic polymer, was conjugated to nanoparticles having an iron-zinc oxide/silicon dioxide/gold structure in order to combine the anionic immunoadjuvant with the nanoparticles. Specifically, 0.1 ml of the iron-zinc oxide/silicon dioxide/gold structure nanoparticles prepared in 1.3 was added to a 10 mg/ml polyethyleneimine solution and reacted for 12 hours. Purification was performed using a MACS LS column to remove unreacted polyethyleneimine. The obtained nanoparticles were finally dispersed in 1 ml of ultrapure water.

1.5 Preparation of nanoparticles conjugated with immunoadjuvant

A cytosine-phosphate-guanine (CpG) oligonucleotide having the nucleotide sequence of 'TCCATGACGTTCCTGACGTTTTTTTAAAAAAAAAAAAAA' was added to the nanoparticles prepared in 1.4 above to a final volume of 3 mM, and then the reaction was It was stirred slowly for 2 hours. It was purified using centrifugation to remove unreacted nucleotide sequences. The obtained nanoparticles were finally dispersed in 0.1 M phosphate buffered saline and stored at 4°C until use. Hereinafter, the nanoparticles prepared in Example 1.5 are referred to as immunoadjuvant-conjugated nanoparticles.

Experimental Example 1. Analysis of physicochemical properties of core/shell structured nanoparticles

1.1 Confirmation of nanoparticle shape at each stage

In order to evaluate whether the nanoparticles were successfully prepared, the shapes of the nanoparticles prepared in Examples 1.1 to 1.5 were observed with a transmission electron microscope.

Specifically, the shape of the nanoparticles was observed using a transmission electron microscope (HITACHI, Japan). The size of the nanoparticles was measured based on a picture obtained by a transmission electron microscope. Figure 2 is a photograph taken with a transmission electron microscope of the nanoparticles prepared in Examples 1.1 to 1.5. FIG. 3 is a graph measuring the size and zeta potential of nanoparticles prepared in Examples 1.1 to 1.5 with respect to the photograph of FIG. 2 . (Nanoparticle 1: iron-zinc oxide nanoparticles (Example 1.1), nanoparticle 2: iron-zinc oxide/silicon dioxide nanoparticles (Example 1.2), nanoparticle 3: iron-zinc oxide/silicon dioxide/2 nm gold Nanoparticle (Example 1.3), Nanoparticle 4: Iron-zinc oxide/silicon dioxide/gold nanoparticle (Example 1.3), Nanoparticle 5: Iron-zinc oxide/silicon dioxide/gold/cationic polymer nanoparticle (Example 1.3) Example 1.4)), nanoparticle 6: iron-zinc oxide/silicon dioxide/gold/immunoadjuvant nanoparticles (Example 1.5))

As a result, as shown in FIGS. 2 and 3, the iron-zinc oxide nanoparticles had an average size of about 13 nm, and it was confirmed by transmission electron microscopy that the size increased as each layer was synthesized. Conjugation of the immunoadjuvant, which could not be confirmed in , was confirmed through size and zeta potential using dynamic light scattering (Malvern, USA).

In addition, in order to quantify the immunoadjuvant conjugated to the nanoparticles, gel electrophoresis was performed to confirm the degree of conjugation between the nanoparticles and the immunoadjuvant at various ratios.

Specifically, for the same amount of immunoadjuvant having a constant concentration (w / w), the concentration (w / w) of the nanoparticles prepared in Example 1.4 was varied and reacted at room temperature for 2 hours, and then applied to an agarose gel. By loading, the degree of bonding according to the presence or absence of a band was confirmed. 4 is a result of performing gel electrophoresis to evaluate the conjugation ratio of the nanoparticles prepared in Example 1.4 and the immunoadjuvant.

As a result, as shown in FIG. 4, it was confirmed that as the concentration of the nanoparticles increased, the bands became invisible due to conjugation with the immunoadjuvant.

1.2 Confirmation of immune cell activity enhancement by nanoparticles conjugated with immunoadjuvant

In order to confirm whether the immunoadjuvant-conjugated nanoparticles prepared in Example 1.5 activate immune cells, cell activity expressed on the surface of immune cells after treatment of immune cells with immunoadjuvant-conjugated nanoparticles The expression levels of the markers CD80 and CD86 were confirmed.

Specifically, after treating bone marrow-derived dendritic cells (BMDC) with PBS (control), immunoadjuvant, and nanoparticles conjugated with immunoadjuvant, expression on the surface of bone marrow-derived dendritic cells 24 hours later Various markers were stained and analyzed by FACS after 1 hour. Figure 5 shows the degree of expression of CD80 and CD86 on the cell surface after treating bone marrow-derived dendritic cells (BMDC) with PBS (control), immunoadjuvant, and nanoparticles conjugated with immunoadjuvant. This is a comparison graph.

As a result, as shown in Figure 5, When comparing the immunoadjuvant and the nanoparticles conjugated with the immunoadjuvant, it was confirmed that the activity of bone marrow-derived dendritic cells was expressed higher by the nanoparticles. This means that nanoparticles conjugated with immunoadjuvant can increase the reaction with TLR-9 in the endosome of immune cells, and can be used as an anticancer adjuvant to enhance immune cell activity in combination with immunocancer therapy. do.

Experimental Example 2. Confirmation of immunotherapeutic effect by irreversible electroporation

2.1 Confirmation of cancer cell killing effect through irreversible electroporation

In order to confirm whether irreversible electroporation has an effect of killing cancer cells, the number of cells was measured after irreversible electroporation was performed on cancer cells.

Specifically, CT-26 cancer cells were treated with irreversible electroporation, cultured for 4 hours, and evaluated using Cell counting-8 (CCK-8) assay kit. After treatment with a 10% CCK-8 solution in the cell medium treated with the irreversible electroporation method, the cells were incubated at 37° C. for 2 hours, and the optical density was measured at 450 nm using a microplate reader. 6 is a graph showing cell viability after irreversible electroporation was performed under various voltage and pulse conditions in order to confirm the cancer cell killing effect by irreversible electroporation. FIG. 7 is a photograph and a graph showing the extent of cancer cell death after irreversible electroporation was performed by varying the number of pulses under the same voltage condition in order to confirm the range of cancer cell killing effect of irreversible electroporation.

As a result, as shown in FIG. 6, it was found that the cell viability was reduced in cancer cells subjected to irreversible electroporation compared to the control group, and the cell viability was reduced to less than 20% under high voltage and high pulse conditions. . This means that the irreversible electroporation method that damages the cell membrane using a high voltage has an excellent cancer cell killing effect.

Also, as shown in Figure 7, It was confirmed that the range of cancer cell killing effect varies according to the number of irreversible electroporation pulses, which means that the range of cancer cell killing effect can be controlled by irreversible electroporation pulses.

2.2 Confirmation of immunogenic cell killing effect induced by irreversible electroporation

In order to confirm whether irreversible electroporation can induce immunogenic apoptosis of cancer cells, irreversible electroporation is performed on cancer cells, and then the amount of ATP released outside the cell and calreticulin expressed on the cell membrane The expression level of was measured.

Specifically, for ATP analysis, CT-26 cancer cells were treated with irreversible electroporation, and then optical density was measured at 570 nm luminometer using an ATP assay kit to check the amount of ATP released within the cells at 1 hour and 6 hours. . In addition, to confirm the expression level of calreticulin, CT-26 cancer cells were treated with irreversible electroporation and then calreticulin antibody was attached 1 hour, 6 hours, 24 hours, and 48 hours later. After staining for 1 hour, it was analyzed through FACS. 8 is a graph showing the results of measuring the amount of ATP released extracellularly and the expression level of calreticulin expressed in the cell membrane after performing irreversible electroporation on cancer cells.

As a result, as shown in FIG. 8, ATP released extracellularly showed a greater amount of release at 1 hour than at 6 hours, and a larger amount of ATP could be confirmed as the pulse went higher at the same voltage of 1000. In the case of calreticulin, it was confirmed that the expression on the surface of cancer cells increased over time. This is because when cancer cells are treated with irreversible electroporation, ATP initially and calreticulin over time can affect the activation of immune cells, resulting in cancer cells necrotic by irreversible electroporation. It means that an anticancer immune response can be induced using a tumor-associated antigen.

Experimental Example 3. Confirmation of tumor growth inhibitory effect by injection of nanoparticles conjugated with immunoadjuvant and irreversible electroporation in tumor animal models

3.1 Formation of tumor animal models

In order to confirm the effect of immunoadjuvant-conjugated nanoparticles and irreversible electroporation, a colorectal cancer model was formed using BABL/c and CT-26 cancer cells.

Specifically, 7-week-old BABL/c was subjected to respiratory anesthesia, and the hair on the upper part of the right hip was removed. Then, 2x10 ⁶ CT-26 cancer cells were injected per animal. As shown in FIG. 9, on the 8th day after cancer cell injection, the average tumor size grew to 100 mm ³ , and treatment using immunoadjuvant-conjugated nanoparticles and irreversible electroporation was started.

3.2 Confirmation of tumor growth inhibitory effect

In order to confirm whether the anti-cancer treatment effect is enhanced when the immunoadjuvant-conjugated nanoparticle injection and irreversible electroporation are combined, tumor growth was evaluated in a tumor animal model.

Specifically, nanoparticles conjugated with immunoadjuvant were injected on days 9, 11, and 14 after cancer cell injection, and irreversible electroporation was performed on day 10. 9 is a result of confirming the tumor size suppression effect in a tumor model in order to confirm the combined effect of immunoadjuvant-conjugated nanoparticles and irreversible electroporation.

As a result, as shown in FIG. 9 , tumors continued to grow in the control group that was not treated with anything. On the other hand, in the group treated with irreversible electroporation, it was confirmed that tumor growth was slightly more inhibited than the control group. In the group in which the injection of the nanoparticles conjugated with the immunoadjuvant and the irreversible electroporation method were combined, it was confirmed that the tumor growth inhibition ability was remarkably improved compared to the other groups.

Experimental Example 4. Confirmation of imaging effect using nanoparticles

To confirm the imaging effect of the synthesized nanoparticles, a computed tomography device (Skyscan 1172, Aartselaar, Belgium), an X-ray system (Portable digital fluoroscopy system (mini C-arm), Fluoroscan InSight; Hologic Inc, Bedford, MA, USA) and a magnetic resonance imaging device (9.4T MRI System, Agilent Technologies, Santa Clara, CA) were used to confirm the degree of imaging.

Specifically, nanoparticle 4: iron-zinc oxide/silicon dioxide/gold nanoparticles (Example 1.3) was transferred together with 2% agarose gel into an Eppendorf tubes® tube or PCR® tube, and finally a 1% agarose gel was obtained. nanoparticles of various concentrations were prepared. The prepared nanoparticles were compared with distilled water as a control for the brightness of the nanoparticles using computed tomography, X-ray, and magnetic resonance imaging, respectively. 10a to 10c show changes in the brightness of nanoparticles according to varying the concentration of gold on the surface of the nanoparticles prepared in Example 1.3 and the concentration of iron oxide in the center of the nanoparticles using computed tomography, X-ray and magnetic resonance imaging devices, respectively. This is the result of checking

As a result, as shown in FIGS. 10A to 10C , it was confirmed that as the gold concentration on the surface of the nanoparticles increased, a higher Hounsfield unit (HU) was displayed in the computed tomography apparatus. In addition, as the gold concentration on the surface of the nanoparticles increased, the X-ray opacity increased and it was confirmed that the color was darker in the X-ray system. As the concentration of iron oxide in the center of the nanoparticles increased, magnetic resonance imaging It was confirmed that it was made darker on the device. The above result means that the iron-zinc oxide/silicon dioxide/gold nanoparticles prepared in Example 1.3 can be used as a contrast agent, and in particular, can be used as a negative (T2) contrast agent in a magnetic resonance imaging device.

Claims (21)

As an anticancer adjuvant used in combination with irreversible electroporation (IRE),
The anticancer adjuvant includes a multi-layered nanoparticle to which an immunoadjuvant is conjugated,
The multi-layered nanoparticles,
magnetic nanoparticles;
A first layer containing silicon dioxide formed on the surface of the magnetic nanoparticles;
a second layer containing gold formed on a surface of the first layer; and
Including; a polymer layer formed on the surface of the second layer;
The immunoadjuvant is an anticancer adjuvant used in combination with irreversible electroporation, which is conjugated to the polymer. delete The anticancer adjuvant according to claim 1, wherein the first layer is cationicly surface-modified, used in combination with irreversible electroporation. The method according to claim 3, wherein the surface modification is [3- (2-aminoethylamino) propyl] trimethylsilane ([3- (2-Aminoethylamino) propyl] trimethoxysilane, AEAPTMS), 3-aminopropyltriethoxysilane ((3 -Aminopropyl)triethoxysilane, APTES), and 3-aminopropyltrimethoxysilane ((3-Aminopropyl)trimethoxysilane, APTMS) by at least one selected from the group consisting of, an anti-cancer adjuvant used in combination with irreversible electroporation. The method according to claim 3, wherein the second layer is formed by binding gold nanoparticles having a size of 0.5 nm to 10 nm to the cationic surface-modified first layer, anticancer adjuvant used in combination with irreversible electroporation . The method according to claim 1, wherein the size of the nanoparticles of the multilayer structure is 1 nm to 300 nm, anti-cancer adjuvants used in combination with irreversible electroporation. The method according to claim 1, wherein the multi-layered nanoparticles are composed of spheric, triangular, cubic, hexagonal, oval, helical, and porous shapes. Any one selected from the group, an anti-cancer adjuvant used in combination with irreversible electroporation. delete delete delete delete The anticancer adjuvant according to claim 1, wherein the polymer is a cationic polymer, used in combination with irreversible electroporation. The method according to claim 1, wherein the immunoadjuvant is aluminum salts (aluminum phosphate or hydorxide), emulsions (MF59, AS03, AF03, SE), DsRNA analogues (poly(I:C)), Lipid A analogues (MPL, GLA), Flagellin, Imidazoquinolines (Imiquimod, R848), CpG ODN, Saponins (QS21), C-type lectin ligands (TDB), CD1d lignads (α-galactosylceramide), AS01 (liopsone, MPL, QS21), AS02 (emulsion, MPL, QS21) ), AS04 (Alum, MPL), AS15 (liposone, MPL, QS21, CpG), GLA-Se (emulsion, GLA), IC31 (CpG, cationic peptide), CAF01 (TDB, cationic liposome), and ISCOMs (saponin, An anti-cancer adjuvant used in combination with irreversible electroporation, which is at least one selected from the group consisting of phospholipid). delete delete The anticancer adjuvant according to claim 1, wherein the irreversible electroporation method induces an anticancer immune response by generating tumor-associated antigens (TAAs) in a subject. The anticancer adjuvant according to claim 16, wherein the tumor-associated antigen is generated by cancer cell death, used in combination with irreversible electroporation. delete The method according to claim 1, wherein the irreversible electroporation is performed under the conditions of 1 to 2500 voltage, 1 to 99 pulses, and 1 to 1000 microseconds (microsecond wide), irreversible electroporation Anti-cancer adjuvant used in combination with the law. The anticancer adjuvant used in combination with irreversible electroporation according to claim 1, wherein the anticancer adjuvant is uptaken by immune cells to enhance immune cell activity. The method according to claim 20, wherein the immune cell is any one selected from the group consisting of macrophages, dendritic cells, B lymphocytes, T lymphocytes, mast cells, monocytes, eosinophils, natural killer cells, basophils, and neutrophils, irreversible Anticancer adjuvant used in combination with electroporation.

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