KR20240047765A - Combination treatment method of nanoparticle inducing ferroptosis having anticancer treatment effect and radiation - Google Patents

Abstract

By using the invention according to the present application, only tumors resistant to anti-cancer treatment, more specifically, cancer cells, can be effectively killed. Furthermore, it is possible to propose a new anti-cancer treatment that targets tumors for individuals in need of cancer prevention or treatment, and can be used as a new cancer treatment method by maximizing cancer treatment efficiency.

Description

Combination treatment method of nanoparticles inducing ferroptosis with anticancer treatment effect and radiation combination treatment effect {COMBINATION TREATMENT METHOD OF NANOPARTICLE INDUCING FERROPTOSIS HAVING ANTICANCER TREATMENT EFFECT AND RADIATION}

This study relates to combination therapy of anticancer treatment using nanoparticles that induce ferroptosis and radiation therapy.

Existing anticancer drugs generally act on various metabolic pathways in actively dividing cells, inhibit nucleic acid synthesis, or exhibit cytotoxicity to show anticancer activity, so they do not act selectively only on cancer cells. It has the limitation of damaging normal cells, especially cells in tissues with active cell division, causing serious side effects such as vomiting, gastrointestinal disorders, alopecia, and leukopenia due to decreased bone marrow function.

Therefore, in order to improve these limitations, research is being actively conducted on target therapy that selects and attacks cancer cells among normal cells. Targeted therapy refers to a treatment that targets biomaterials such as specific vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), and mutated receptors whose expression is increased in cancer cells. Because it attacks selectively, it can reduce damage to normal cells. However, these targeted treatments also have limitations. First, because it targets a specific biomaterial, it does not show a therapeutic effect in patients who do not have the specific biomaterial, even if they are diagnosed with the same cancer. For example, gefitinib showed therapeutic effects only in some patients with non-small cell lung cancer, but since gefitinib targets mutations in the epidermal growth factor receptor (EGFR), It has been revealed that the treatment was effective only in patients with cellular lung cancer. In addition, even in the case of targeted therapeutics, when used continuously, cancer cells develop resistance to the therapeutic agent, which has the problem that the therapeutic effect is significantly reduced.

Therefore, interest in nano medicine using nano materials, a new method, has recently been increasing. Nanomedicine is a technology that combines nano-sized materials with medicine. It can be applied to various fields such as nanobiosensors, nanoimaging, nanodrug delivery systems, and nanotissue engineering, and can be used to treat various intractable diseases such as cancer, dementia, and cardiovascular disease. It is expected to be a breakthrough technology that can overcome the difficulties of diagnosis and treatment. In the case of the United States, a nanotechnology development strategy at the federal government level was established in 1998 and the National Nanotechnology Initiative (NNI), in which 12 federal agencies participate, was launched and more than $1 billion is being invested every year. Recently, diagnostic kits and treatments using nanoparticles have entered the clinical trial stage or have already been approved by the U.S. Food and Drug Administration, and research on nanomedicine is actively underway. In Korea, the Nanotechnology Development Promotion Act was enacted in 2002, and investments continue to be made every year in accordance with the comprehensive nanotechnology development plan. Attempts to use nanoparticles for cancer treatment are also being actively made. Since these nanoparticles mostly affect normal cells, they can cause various side effects. To improve this, methods targeting specific targets, such as passive targeting and active targeting, are being studied. However, the development of nanoparticles that can be practically used in cancer treatment and that exhibit low side effects and high therapeutic efficacy is not possible. It is still insufficient.

Meanwhile, ferroptosis is a cell death method caused by iron-mediated lipid peroxidation, in which iron meets reactive oxygen species (ROS) to induce the Fenton reaction, which This is a method in which lipid peroxides accumulate within cells and cause cell death. Recently, research on a method of killing cancer cells using ferroptosis has been reported (see "Nano Lett. (2017) 17(1): 284-291"). More specifically, using nanoparticles combining a plasmid expressing p53, which regulates oxidative stress, and a metal-organic network that induces the Fenton reaction, ferroptosis/cell programmed death ( It induced cancer cell death through a hybrid pathway (apoptosis). Polyethylenimine (PEI) used in the nanoparticles is a cationic polymer and is widely used as a gene carrier. However, because it is cytotoxic, it is actually used in the treatment of cancer. It is not suitable for this.

Accordingly, Registration Patent No. 10-2187362 has completed a nanoparticle that can be effectively used in the treatment of various cancers, has low cytotoxicity, and can specifically induce cell death through ferroptosis in cancer cells.

As a method to use the nanoparticles of the above registered patent more effectively, the applicant studied a method of combining radiation therapy with nanoparticles that can induce cell death through ferroptosis, and achieved excellent cancer treatment through this. The effect was confirmed.

Accordingly, our aim is to provide a method that can kill cancer cells more effectively by combining radiation treatment with nanoparticles that have low cytotoxicity and can specifically induce cancer cell death through ferroptosis. .

Furthermore, the combination of administration of a pharmaceutical composition containing nanoparticles as an active ingredient and radiation therapy is applied to an individual in need of cancer prevention or treatment, and the administration of a pharmaceutical composition containing nanoparticles as an active ingredient or radiation treatment alone We aim to provide a method for preventing or treating cancer that is more effective than before.

In order to solve the above problem, the first aspect of this hospital is

In the method of killing cancer cells,

Treating cancer cells with nanoparticles containing iron ions and a cancer cell-targeting hydrogel; and

Including treating the cancer cells with radiation,

A method is provided wherein the nanoparticles are in an aggregated form by combining cations of iron and anions of a cancer cell-targeting hydrogel.

In addition, the second aspect of this institution is

In the prevention and treatment of cancer,

Treating an object other than a human with a pharmaceutical composition containing nanoparticles containing iron ions and a cancer cell-targeting hydrogel as active ingredients; and

Including the step of treating radiation to an entity other than a human,

A method is provided wherein the nanoparticles are in an aggregated form by combining cations of iron and anions of a cancer cell-targeting hydrogel.

Contents common to the first and second aspects above apply equally to each aspect, and the solution herein is not limited to the above description and includes all means within the range that a person skilled in the art can understand. It should be interpreted as

By using the invention according to the present application, only tumors resistant to anti-cancer treatment, more specifically, cancer cells, can be effectively killed. Furthermore, it is possible to propose a new anti-cancer treatment that targets tumors for individuals in need of cancer prevention or treatment, and can be used as a new cancer treatment method by maximizing cancer treatment efficiency.

Figure 1 is a diagram showing the cancer cell killing mechanism (a) of iron/hyaluronic acid nanoparticles and the manufacturing process (b) of iron/hyaluronic acid nanoparticles according to the present application.
Figure 2a is a diagram showing the shape of iron/hyaluronic acid nanoparticles according to the present application observed using Energy-Filtering Transmission electron microscopy (TEM) (LIBRA 120, Carl Zeiss, Germany) operating at an acceleration voltage of 120 kV.
Figure 2b is a view analyzing the surface of iron/hyaluronic acid nanoparticles according to the present application using a field emission scanning electron microscope (FE-SEM) (JSM-7800F Prime, JEOL Ltd., Japan).
Figure 2c is a diagram showing the size of iron/hyaluronic acid nanoparticles according to the present application measured using Dynamic light scattering (DLS) zetasizer Nano ZS (Malvern Instruments, UK).
Figure 3 is a diagram comparing the colony formation results when various treatments were performed on breast cancer cells, brain cancer cells, and lung cancer cells according to Example 3.
Figure 4 is a diagram showing the results of an experiment to confirm whether programmed cell death (apoptosis) occurs due to radiation treatment when the cancer cell killing method according to the present application is applied to lung cancer cells A549 according to Example 4.

Below, with reference to the attached drawings, embodiments of the present application will be described in detail so that those skilled in the art can easily implement them. However, the present application may be implemented in various different forms and is not limited to the embodiments described herein. In order to clearly explain the present application in the drawings, parts that are not related to the description are omitted, and similar reference numerals are assigned to similar parts throughout the specification.

Throughout this specification, when a member is said to be located “on” another member, this includes not only the case where the member is in contact with the other member, but also the case where another member exists between the two members.

Throughout the specification of the present application, when a part "includes" a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary.

As used throughout the specification, the terms “about,” “substantially,” and the like are used to mean at or close to a numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to convey the understanding of the present application. Precise or absolute figures are used to assist in preventing unscrupulous infringers from taking unfair advantage of stated disclosures. The term “step of” or “step of” as used throughout the specification does not mean “step for.”

Throughout this specification, the term "combination(s) thereof" included in the Markushi format expression means a mixture or combination of one or more selected from the group consisting of the components described in the Markushi format expression, It means containing one or more selected from the group consisting of the above components.

Throughout this specification, references to “A and/or B” mean “A or B, or A and B.”

Throughout this specification, “ferroptosis” refers to a form of cell death caused by lipid peroxidation and Reactive Oxygen Species.

The present application provides a method of killing cancer cells using nanoparticle treatment and radiation treatment containing iron ions and a cancer cell-targeting hydrogel.

In one embodiment of the present application, the nanoparticles are preferably an aggregate of iron cations and anions of a cancer cell-targeting hydrogel, without the addition of additional linkers, surfactants, compounds, etc. By co-precipitation of hydrogel and iron ions, the nanoparticles may be obtained by co-precipitating the hydrogel and iron ions by adding iron ions to the hydrogel solution and then stirring, or pre-gel It may be nanoparticles obtained by forming a nano-sized composite dispersed in a continuous phase of water by stirring hydrogel and iron ions using a (pre-gel) method. The anion of the cancer cell-targeting hydrogel may be a carboxyl group, but is not limited thereto as long as it is a form of an anion functional group capable of secondary binding to iron ions.

In another embodiment of the present application, the cancer cells are derived from an entity that requires death of cancer cells, and should be interpreted to include not only cancer cells existing outside the entity but also cancer cells existing inside the entity. Specifically, the cancer cells may be cancer cells present in a specific organ of the individual, preferably in an organ where cancer has occurred.

In another embodiment of the present application, the iron ion may preferably be iron oxide, a compound of iron and oxygen, and more preferably Fe ₃ O ₄ , but is not limited thereto. The iron ion is an important substance for cell survival that is involved in oxygen transport, DNA biosynthesis, and ATP synthesis in the body, and also generates active oxygen along with ATP synthesis in mitochondria.

In another embodiment of the present application, the cancer cell-targeting hydrogel refers to any hydrogel that can selectively bind to molecules or receptors expressed on the surface of cancer cells, and the cancer cell-targeting hydrogel is described in the present technology. Any cancer cell target that is known in the field to be capable of selectively binding to CD44, ESA, HER-2/neu, KRAS, ER (estrogen receptor), etc., which are molecules or receptors overexpressed on the surface of cancer cells, or that may be discovered in the future. It may be an oriented hydrogel. The hydrogel may preferably be hyaluronic acid, carboxymethyl cellulose (CMC), alginates, carboxycellulose, chitosan, collagen, gelatin, etc., and more preferably hyaluronic acid or a derivative thereof ( derivatives). The hyaluronic acid is biodegradable, has excellent biocompatibility, and is a bio-derived high molecular weight polymer that does not induce an immune response as one of the substances in living organisms. Red blood cells have a repeating structure of glucuronic acid and N-Acetylglucosamine, which exist not only in the matrix of connective tissues such as joint synovial fluid, cartilage, and subcutaneous tissue, but also in the extracellular matrix, umbilical cord, and vitreous body of the eye. It is present on the cell surface of almost all tissues and organs except for. In addition, it has the CD44 receptor overexpressed on the surface of cancer cells, making it suitable for targeting cancer cells.

In another embodiment of the present application, the nanoparticles may preferably have a diameter of 50 to 200 nm, but are not limited thereto as long as they are of a nano size that can move into cancer cells.

In another embodiment of the present application, the nanoparticles are characterized in that they induce death of cancer cells through ferrotosis, and the nanoparticles accumulate in cancer cells to increase the concentration of iron ions in cancer cells, causing ferrotosis. may cause. The ferroptosis is iron-dependent cell death, i.e., a form of intracellular iron-dependent cell death, which may refer to cell death caused by the generation of iron-dependent reactive oxygen species and the occurrence and accumulation of lipid peroxidation. there is.

In another embodiment of the present application, the cancer is preferably breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, It may be pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, blood cancer, etc., but is not limited thereto as long as it is a type of cancer that can be recognized by the CD44 receptor.

In another embodiment of the present application, radiation may be treated along with the treatment of the nanoparticles. Radiation refers to the propagation of energetic particles or waves, and includes natural radiation such as visible light and infrared radiation, and artificial radiation generated from home appliances, diagnostic X-ray devices, and cancer treatment equipment. Among these, the radiation used in radiation therapy generally refers to high-energy radiation. As used herein, radiation refers to high energy particles or electromagnetic waves and includes alpha rays, beta rays, gamma rays, X-rays, and neutron beams. Methods for treating cancer cells using radiation include brachytherapy, stereotactic radiosurgery (SRS), intraoperative radiation therapy (IORT), proton therapy, and MRI linear accelerator. ), which involves damaging cancer cells using stereotactic body radiation therapy (SBRYT).

The most well-known principle of cancer cell death by radiation is damage to the DNA within cells by the high energy of radiation. Radiation irradiation causes double-strand breaks or single-strand breaks in DNA, leading to damage due to genetic instability. When DNA damage occurs in this way, various types of death such as apoptosis, mitotic catastrophe, radiation necrosis, senescence, and autophagy occur, ultimately leading to the death of cancer cells.

The cancer cell killing method according to the present application can be used in a cancer prevention or treatment method that involves administering a pharmaceutical composition containing gastric nanoparticles as an active ingredient to an individual with cancer and combining it with radiation therapy.

The subject may be not only a human but also a mammal such as a cow, horse, sheep, pig, goat, camel, antelope, dog, or cat, but is not limited thereto.

The route of administration of the above pharmaceutical composition is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or Includes workplace. Oral or parenteral administration is preferred. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions herein can also be administered in the form of suppositories for rectal administration.

Hereinafter, implementation examples and examples of the present application will be described in detail with reference to the attached drawings. However, the present application may not be limited to these implementations, examples, and drawings.

Example 1. Principle of cancer cell death according to the present application

The cancer cell killing method according to the present application uses a combination of iron/hyaluronic acid nanoparticles and radiation.

The cancer cell killing mechanism of iron/hyaluronic acid nanoparticles is shown in Figure 1 (a). Iron ions induce lipid peroxidation of unsaturated fatty acids present in cell membranes and also cause excessive production of reactive oxygen species generated during ATP synthesis in mitochondria, thereby increasing oxidative stress and inducing lipid peroxidation. Radiation therapy is known to induce radicals and apoptosis through DNA fragmentation. In this way, our clinic can kill cancer cells through ferroptosis and apoptosis by combined treatment of the above nanoparticles and radiation.

Example 2. Preparation of iron/hyaluronic acid nanoparticles

5 mg of sodium hyaluronate was dissolved in 50 mL of deionized (DI) water. An aqueous solution of 99.4 mg of FeCl ₂ 4H ₂ O in 10 mL of deionized water and an aqueous solution of 149.1 mg of FeCl ₃ 6H ₂ O in 10 mL of deionized water were added slowly and the entire solution was stirred for 30 min. Aqueous ammonia was added to adjust the pH to 10 and stirred for 30 minutes. After the stirring was completed, the solution was centrifuged, washed with deionized water, and then redispersed using a water sonicator.

As shown in Figure 1(b), the iron/hyaluronic acid nanoparticles (FHA NPs) of the present invention are made by reacting hyaluronic acid nanoparticles with divalent iron ions and trivalent iron ions, thereby forming cations of iron ions and hyaluronic acid. It is manufactured by combining the carboxyl group (COO ^- ) and aggregating to form a nano-sized conjugate.

Figure 2a is a diagram showing the shape of iron/hyaluronic acid nanoparticles according to the present application observed using Energy-Filtering Transmission electron microscopy (TEM) (LIBRA 120, Carl Zeiss, Germany) operating at an acceleration voltage of 120 kV.

Figure 2b is a view analyzing the surface of iron/hyaluronic acid nanoparticles according to the present application using a field emission scanning electron microscope (FE-SEM) (JSM-7800F Prime, JEOL Ltd., Japan).

Figure 2c is a diagram showing the size of iron/hyaluronic acid nanoparticles according to the present application measured using Dynamic light scattering (DLS) using a zetasizer Nano ZS (Malvern Instruments, UK).

Example 3. Clonogenic assay

Breast cancer cells (human breast adenocarcinoma cell (MCF7)), brain cancer cells (U87MG), and lung cancer cells (human lung carcinoma cell (A549)) were obtained from the Korea Cell Line Bank (Seoul, Korea). Purchased. Each cancer cell population was maintained at Rosewell Park Memorial Institute (RPMI) supplemented with 10% fetal bovine serum (FBS) (Cellsera, NSW, Australia) and 1% penicillin and streptomycin (Welgene, Korea). All cells were cultured at 37°C in a humidified atmosphere in a 5% CO ₂ incubator.

Cell lines were distributed in a volume of 1000 ㎕ in a 24-well plate (5 x 10³ cells/well) and cultured for 24 hours. After stabilizing the cells, the cells were washed once with Dulbecco's PBS (DPBS) and classified into a group not treated with nanoparticles and a group treated with 200 μg/ml nanoparticles. Radiation treatment used X-rays, and irradiation was performed at 0, 1, 3, or 6 Gray depending on the X-ray intensity. After the combination treatment, the cells were cultured at 37°C for 24 hours, washed, and fresh media was injected to observe colony formation for one month.

Number of colonies (%) according to processing method breast cancer brain cancer lung cancer Negative control group 100.0% 100.0% 100.0% Nanoparticle treatment alone 30.6% 32.1% 58.7% Radiation treatment alone (6 Gray) 20.0% 44.4% 25.2% Nanoparticle-radiation (6 Gray) combined treatment 0.6% 21.0% 13.7%

The number of colonies according to each treatment method was measured. Based on the number of colonies in the negative control group (control) of 100%, the number of colonies remaining after treatment was expressed as a percentage. As a result, the number of colonies in the group treated with any treatment decreased in all cancer cell groups, but in particular, a higher anticancer effect was confirmed in the nanoparticle-radiation combination treatment group compared to the nanoparticle treatment group alone and the radiation treatment group alone.

Therefore, the combined treatment method of nanoparticles and radiation according to the present application was confirmed to be more effective in killing cancer cells than when treated with nanoparticles or radiation separately.

Example 4. TUNEL assay

An experiment was conducted using lung cancer cells A549 to determine how effectively the cancer cell killing method according to the present invention can kill cancer cells. Cell death was confirmed by whether DNA fragments occurred (see Figure 4).

Cell lines were distributed in a volume of 1000 ㎕ in a 24-well plate (5 x 10³ cells/well) and cultured for 24 hours. After cell stabilization, cells were washed once with Dulbecco's PBS (DPBS). Depending on the experimental conditions, the untreated control group (control), the group treated with 200 μg/ml nanoparticles (NP), the group irradiated with radiation 6 Gray (X-ray), the group treated with 200 μg/ml nanoparticles and radiation 6 The experiment was conducted with a total of four test groups, including the group treated with Gray (NP + X-ray).

The experiment was performed using the invitrogen Click-iT Plus TUNEL Assay kit and nuclei (DAPI staining; 4', 6-diamidino-2-phenylindole). The results of each group were confirmed by counterstaining NA sections (TUNEL) and microfibers (actin filaments).

Among these, the TUNEL assay, which involves DNA fragmentation, is a test method that can check whether cell death occurs due to apoptosis. The TUNEL assay utilizes the fact that deoxynucleotide transferase (TdT) attaches to the DNA fragment at the 3'-hydroxyl terminus and produces fluorescence. In other words, if green fluorescence is confirmed by TUNEL assay, it means that apoptosis has occurred in the cell and a DNA fragment has been created. (see Figure 3)

The negative test group (control) and the group treated with nanoparticles alone did not show green fluorescence. Here, nanoparticles cause ferroptosis in cells, but since ferroptosis does not cause decomposition of DNA, fluorescence may not be visible.

When comparing the negative test group and the radiation-only treatment group, the radiation-only treatment group showed green fluorescence. This is because apoptosis occurs when treated with radiation.

When comparing the negative test group and the nanoparticle-radiation combination treatment group, the combination treatment group showed green fluorescence. This is because it showed both ferroptosis and apoptosis caused by nanoparticles and radiation.

In other words, the effect of programmed cell death caused by radiation could be confirmed through the TUNNEL assay. Death of cancer cells can be expected when nanoparticles or radiation are treated alone, but when nanoparticles and radiation are treated together, cancer cells are killed more effectively through two mechanisms of action: ferroptosis and apoptosis. It can be killed.

Claims (13)

In the method of killing cancer cells,
Treating cancer cells derived from an individual with nanoparticles containing iron ions and a cancer cell-targeting hydrogel; and
Including treating the cancer cells with radiation,
The method is characterized in that the nanoparticles are aggregated by combining iron positive ions and negative ions of the cancer cell-targeting hydrogel. According to paragraph 1,
The method, characterized in that the iron ion is iron oxide. According to paragraph 1,
The cancer cell targeting hydrogel is characterized in that at least one selected from the group consisting of hyaluronic acid, carboxymethyl cellulose, alginate, chitosan, collagen, gelatin, and carboxycellulose. According to paragraph 1,
A method wherein the nanoparticles induce death of cancer cells through ferroptosis. According to paragraph 1,
A method characterized in that the radiation induces death of cancer cells through apoptosis. According to paragraph 1,
The method wherein the radiation is at least one selected from the group consisting of alpha ray, beta ray, gamma ray, X-ray, and neutron beam. According to paragraph 1,
The cancer cells include breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, larynx cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, and stomach cancer. , a method characterized in that it is one or more cancer cells selected from the group consisting of bone cancer, and blood cancer. In the prevention and treatment of cancer,
Treating subjects other than humans with a pharmaceutical composition containing nanoparticles containing iron ions and a cancer cell-targeting hydrogel as active ingredients; and
Including the step of treating radiation to an entity other than a human,
The method is characterized in that the nanoparticles are aggregated by combining iron positive ions and negative ions of the cancer cell-targeting hydrogel. According to clause 8,
The method, characterized in that the iron ion is iron oxide. According to clause 8,
The cancer cell targeting hydrogel is characterized in that at least one selected from the group consisting of hyaluronic acid, carboxymethyl cellulose, alginate, chitosan, collagen, gelatin, and carboxycellulose. According to clause 8,
A method wherein the nanoparticles induce death of cancer cells through ferroptosis. According to clause 8,
A method characterized in that the radiation induces death of cancer cells through apoptosis. According to clause 8,
The above cancers include breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharynx cancer, larynx cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, and stomach cancer. , bone cancer, and blood cancer.