Patent Application
20230330429
The technology disclosed herein relates to a static magnetic field (SMF) generator for treatment of cancer, and more particularly, to a static magnetic field generator that may reduce a size of cancer cells by applying a static magnetic field of a certain size to a treatment region of the body in order to treat diseases, such as tumors and so on of the body.
Despite the development of modern medicine, cancer is still one of the most incurable diseases among human diseases. Cancer is a tissue made up of abnormal cells and occurs in one or more tissues and spreads to other tissues. More than 100 types of cancer are currently known.
90-95% of main causes of cancer are known to be genetic mutations due to environmental factors, and most of the factors are smoking, obesity, radiation, UV, stress, environmental pollution, and so on.
Cancer has a rapid growth rate and strong ability to invade surrounding tissues, and accordingly, a boundary with normal tissues is not clear, and surgical operation should be performed to remove a tumor and a surrounding tissue as much as possible while greatly reducing functional damage to the body. It is not easy to completely excise the tumor through surgical operation, and there is radiation therapy, an antimetabolic agent, anticancer drug therapy, targeted therapy, immunotherapy, or a combination therapy as an auxiliary treatment method.
However, the known methods may differ for each patient, and generally have side effects, such as hair loss, inhibition of bone marrow function, drug resistance, and organ damage. Numerous efforts have been made worldwide to overcome the side effects and to conquer cancer, but the average survival time of cancer is still short and the possibility of recurrence is high, and accordingly, the development of new treatments is urgently required.
A lot of basic medical research and clinical medical research are still being conducted, and various treatment methods are being proposed, but it is still difficult to cure cancer, and most basic medical research continues to find biological or chemical methods to inhibit a rapid growth and invasion of cancer.
The present disclosure provides a new method that may be applied to cancer treatment by a physical method without using drugs to slow down the growth of cancer cells.
An object to be achieved by the static magnetic field generator for treatment of cancer, according to an embodiment of the technical idea of the present disclosure, is to provide a static magnetic field generator that generates a static magnetic field showing a therapeutic effect on cancer cells in order to minimize side effects in treating diseases.
An object to be achieved by a static magnetic field generator for treatment of cancer, according to an embodiment of the technical idea of the present disclosure, is not limited to the object to solve the problems described above, and another object not described will be clearly understood by those skilled in the art from descriptions below.
According to an aspect of the present disclosure, a static magnetic field generator for treatment of cancer includes a static magnetic field generating unit, and a space in which a static magnetic field is formed by the static magnetic field generating unit, wherein the formed static magnetic field does not substantially act on normal cells in the space and acts on cancer cells to be killed.
The formed static magnetic field may have a magnitude range of 0.1 mT to 4 T.
According to another aspect of the present disclosure, a static magnetic field generator for treatment of cancer includes a static magnetic field generating unit, and a space in which a static magnetic field is formed by the static magnetic field generating unit, wherein a magnitude of the formed static magnetic field is configured to have a range of 0.1 mT to 4 T.
The static magnetic field may be configured to be generated by a direct current.
The static magnetic field generating unit may have at least one of a solenoid structure, a vertical or horizontal Helmholtz structure, a core coil structure, a multi-stage set coil multi-phase structure, an upper or lower coil selection Helmholtz magnetic field focusing structure, a multi-stage multiple coil multi-phase structure, and a core coil multi-frequency application rotation structure.
The static magnetic field generating unit may include a plurality of coil units, and a space in which the static magnetic field is formed may be placed in a space between the plurality of coil units.
The plurality of coil units may include a first coil unit in which a cylindrical coil is wound multiple times around a first central axis, and a second coil unit which is separated from the first coil by a preset distance and in which a cylindrical coil is wound multiple times around a second central axis coaxial with the first central axis.
Currents of a same magnitude may flow through the first coil unit and the second coil unit in the same direction, and a treatment region of a body may be placed in a space between the first coil unit and the second coil unit.
The first coil unit and the second coil unit may have coils of a same thickness which are wound around a same area in a same number of turns with same inner and outer diameters.
According to another aspect of the present disclosure, the static magnetic field generator for treatment of cancer may further include a first coil housing configured to surround and support the first coil unit, and a second coil housing configured to surround and support the second coil unit.
According to another aspect of the present disclosure, the static magnetic field generator for treatment of cancer may further include a camera configured to image the space in which the static magnetic field is formed.
According to another aspect of the present disclosure, a cancer treatment system includes a bed portion, and the static magnetic field generator defined above and configured to apply a static magnetic field to the bed portion.
According to another aspect of the present disclosure, a static magnetic field generation method for treatment of cancer is configured to generate a static magnetic field in a static magnetic field forming space by using the previously defined static magnetic field generator or the cancer treatment system.
According to another aspect of the present disclosure, a non-transitory computer readable medium includes instructions for causing a processor to perform the method defined above.
According to another aspect of the present disclosure, a static magnetic field generator includes a plurality of coil units configured to generate a static magnetic field for a treatment region of a body, wherein a treatment region of the body is placed in a space between the plurality of coil units.
Aa magnitude of the generated static magnetic field may be configured to have a range of 0.1 mT to 4 T, and a space in which the static magnetic field may be formed is placed in the space between the plurality of coil units.
The plurality of coil units may include a first coil unit in which a cylindrical coil is wound multiple times around a first central axis, and a second coil unit which is separated from the first coil by a preset distance and in which a cylindrical coil is wound multiple times around a second central axis coaxial with the first central axis.
Currents of a same magnitude may flow through the first coil unit and the second coil unit in the same direction, and a treatment region of a body may be placed in a space between the first coil unit and the second coil unit.
The first coil unit and the second coil unit may have coils of a same thickness which are wound around a same area in a same number of turns with same inner and outer diameters.
A static magnetic field generator according to an embodiment of the technical idea of the present disclosure provide a therapeutic effect that reduces sizes of cancer cells by applying only a static magnetic field to a treatment region of the body and reduces side effects.
A static magnetic field generator according to an embodiment of the present disclosure may apply a constant and uniform magnetic field to a treatment region by using a Helmholtz coil and may prevent unexpected side effects by using a low magnetic field harmless to the human body.
In particular, a size of a static magnetic field according to an embodiment of the present disclosure is harmless to the human body, and despite the static magnetic field is very small which corresponds to at least “1 of a thousand or tens of thousands” compared to a size of the discovered static magnetic field of 4 T, the static magnetic field kills cancer cells and significantly reduces invasion of cancer cells, and safety to the human body and cancer treatment efficacy may be ensured at the same time.
In addition, when a static magnetic field of 1.0 mT is applied to tumor cells illustrated in the embodiment of the present disclosure, rather than when a static magnetic field of 0.6 mT is applied, more tumors are killed, and tumor invasion is more significantly reduced, and thus, the larger the magnitude of a static magnetic field, the greater the effect on the tumors.
Furthermore, even when small static magnetic fields of the same size of, for example, 0.6 mT or 1.0 mT is applied to various types of cancer illustrated in the embodiments of the present disclosure, more tumors are killed in proportion to time/date of exposure, and tumor invasion is more significantly reduced, and thus, cancer treatment may be efficiently done by using a very low-level static magnetic field that is harmless to the human body even when applied for a long time.
Effects that may be achieved by a static magnetic field generator according to an embodiment of the technical idea of the present disclosure are not limited to the descriptions above, and other effects not described will clearly understood by those skilled in the art from descriptions below.
Embodiments will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.
The technology disclosed in the present disclosure may have various changes and various embodiments, and accordingly, specific embodiments are illustrated in the drawings and described in detail. However, this is not intended to limit the technology disclosed in the present disclosure to specific embodiments, and it should be understood that the technology disclosed herein includes all modifications, equivalents, or substitutes included in the idea and scope of the technology disclosed herein.
In describing the technology disclosed in the present disclosure, when it is determined that detailed descriptions of a related known technology may unnecessarily obscure the subject matter of the technology disclosed in the present disclosure, the detailed descriptions are omitted. In addition, numbers (for example, first, second, and so on) used in the description process of the present disclosure are only identifiers for distinguishing one component from another component.
In addition, in the present disclosure, when one component is referred to as “connected” or “coupled” with another component, the one component may be directly connected or coupled to another component, but unless specifically stated to the contrary, it should be understood that the components may be connected or coupled to each other through another component therebetween.
In addition, in the present disclosure, a component described as “˜ portion or unit” may be two or more components combined into one component, or one component may be divided into two or more for each more subdivided function. In addition, each component to be described below may additionally perform some or all of functions of another component in addition to a main function thereof, and some of the main function of each component may be exclusively performed by another component.
Expressions, such as “first” and “second” used in various embodiments, may modify various components regardless of order and/or importance and do not limit the corresponding components. For example, a first component may be referred to as a second component, and similarly, the second component may also be referred to as the first component without departing from the scope of the technology disclosed herein.
In the present disclosure, in order to check cancer cell removal and inhibition effects according to the technical idea of the present disclosure that applies a static magnetic field to cancer cells to remove and/or inhibit the cancer cells, a result of checking what kind of change occurs when weak static magnetic fields of 0.6 mT and 1.0 mT are applied to a glioblastoma cell line U87-MG is provided. This is only an example, and in particular, the technical idea of the present disclosure is applicable to all types of static magnetic field generators capable of generating density of a magnetic flux in a range of 0.1 mT to 4 T, and the magnitude of the static magnetic field is lower 30,000 times than the magnetic flux density of 3 T used in a general magnetic resonance imaging device, and accordingly, it should be noted that the magnitude of the static magnetic field is safe for the human body even when being continuously used over several days for, for example, treatment.
In addition, although glioblastoma is described as an example in the present disclosure, the technical idea of the present disclosure is not limited thereto and it will be understood that the technical idea of the present disclosure corresponds to a treatment device and a treatment method that may be applied to all types of cancer cells.
Hereinafter, a static magnetic field generator according to a preferred embodiment will be described in detail. In addition, in the present disclosure, a size of a static magnetic field may be adjusted according to a size of a current by using a coil structure, a static magnetic field generator having a structure of a Helmholtz coil may be described as an example to obtain a uniform magnetic field, and for example, a specific magnet with a fixed magnetic flux density or a special magnetic field generating device may be used as the magnetic field generator.
The static magnetic field generator according to the present embodiment may include a first coil unit 110, a second coil unit 120, a treatment space 130, and a camera. Here, the first coil unit 110 and the second coil unit 120 each have a Helmholtz coil structure.
The first coil unit 110 may have a cylindrical coil wound multiple times around a first central axis 111.
The second coil unit 120 may be separated from the first coil unit 110 by a preset distance and may have a cylindrical coil wound multiple times around a second central axis 121 coaxial with the first central axis 111. Here, a space separated by a preset distance becomes the treatment space 130 where a treatment region (a treatment portion) of the body is located. The treatment space 130 becomes a region where a uniform magnetic field is formed. The treatment space 130 is not limited in size, and in particular, may have a form of a very large space (for example, a chamber, a room, or so on) that may provide continuous treatment for several hours to several days due to very low magnetic flux density, that is, a magnetic field size in the present embodiment.
The first coil unit 110 and the second coil unit 120 may have coils of the same thickness wound around the same area in the same number of turns with the same inner and outer diameters. An average diameter of the first coil unit 110 and the second coil unit 120 may be determined as an intermediate value of the inner diameter and the outer diameter.
A current of the same magnitude and in the same direction may flow through the first coil unit 110 and the second coil unit 120, a magnetic field to treat a treatment region of the body is uniformly formed in the treatment space 130 between the first coil unit 110 and the second coil unit 120, and magnetic flux density, which is the magnitude of a magnetic field to be formed, ranges from 0.1 mT to 4 T. 0.1 mT is a minimum size of a static magnetic field required for removal and inhibition of cancer cells, and when the size of the static magnetic field exceeds 4 T, the human body may be harmed. In the present embodiment, for example, the magnitude of a magnetic flux density of 0.1 mT corresponds to a very weak static magnetic field that is “30,000 times” lower because the magnetic flux density used by a magnetic resonance imaging device is 3 T. Although a static magnetic field of 1 mT which is another exemplary size is harmless to the human body and has very small magnitude of about “ 1/4,000” compared to the magnitude of a static magnetic field of 4 T which is an upper limit of the discovered magnitude, tumors are killed and tumor invasion is significantly reduced, and even under continuous treatment, safety to the human body and cancer treatment efficacy may be ensured at the same time.
In order for the first coil unit 110 and the second coil unit 120 to configure a specific treatment device, a first coil housing 115 supporting the first coil unit 110 and a second coil housing 125a supporting the second coil unit 120 may be formed, and a member that covers the treatment space 130 and connects the first coil housing 115 to the second coil housing 125 may be placed between the first coil housing 115 and the second coil housing 125. According to the example of
In order to monitor conditions of the patient receiving treatment in the treatment space 130, a camera may be installed to capture an image of the treatment space 130, and an image captured by the camera may be transmitted to a server through a wired or wireless network.
In addition, a treatment method using a static magnetic field, according to an embodiment of the present disclosure, may be performed by following steps.
First, a step of placing a treatment region of the body in the treatment space 130 may be performed. For example, the treatment space 130 may be placed between the first coil unit 110 and the second coil unit 120. Here, the first coil unit 110 has a cylindrical coil wound multiple times around the first central axis 111, and the second coil unit 120 is separated from the first coil unit 110 by a preset distance and has a cylindrical coil wound multiple times around the second central axis 121 coaxial with the first central axis 111. In addition, the first coil unit 110 and the second coil unit 120 may be set to have coils of the same thickness wound around the same area in the same number of turns with the same inner and outer diameters, but are not limited thereto.
Next, a step of applying a current having the same magnitude to the first coil unit 110 and the second coil unit 120 in the same direction may be performed. It is preferable that the first coil unit 110 and the second coil unit 120 form a static magnetic field having magnetic flux density (a size of a static magnetic field) of 0.1 mT to 4 T.
As described above, the structure of the Helmholtz coil made according to an embodiment of the present disclosure is illustrated in
As illustrated exemplarily in
An inner diameter thereof is designed to be 130 mm by considering a margin to accommodate several cell culture dishes, each having an inner diameter of 35 mm. An outer diameter is 168.4 mm because the enameled copper wire is wound 16 times in the horizontal direction along the length of the inner diameter. Since the coil is wound several times, a diameter used when designing the coil corresponds to an average diameter. An average diameter is 149.2 mm which is obtained by summing an inner diameter and an outer diameter and dividing the summed value by 2, and an average radius is 74.6 mm which is obtained by dividing the average diameter by 2 (D=149.2 mm and R=74.6 mm).
A current I, which is supplied such that the magnitude of magnetic flux density is, for example, 1.0 mT, is 103.06 mA which is obtained by using the Bio-Savar law from the Helmholtz coil structure.
Changes caused by the static current magnetic field generator were checked from cell proliferation by directly measuring the number of cells through trypan blue staining, and the number of cells and the shape of cells were observed through microscopic observation. In addition, cell live and dead were confirmed through Calcein-AM staining, and cell apoptosis was confirmed through TUNEL staining.
Human glioblastoma cell line U87-MG, which is a brain cancer used in the example, was purchased from American type culture collection (ATCC) (Manassas, VA, USA). Dulbecco's modified eagle's medium (DMEM) (WELGENE, Korea) badge to which fetal bovine serum (FBS) (Gibco Corporation, Grand Island, NY, USA) of 10% and antibiotic-antimycotic (AA) (Gibco Corporation, Grand Island, NY, USA) of 1% were added, was used for cell culture.
U87-MG cells were cultured in an incubator of 37° C. and 5% CO2, and observed every day, and it was considered confluence when filled by 70 to 80% of an area of a cell culture dish of 100 mm, and subculture was performed by using TryPLE (Gibco Corporation, Grand Island, NY, USA). In addition, the badge was replaced with a new badge every 2 to 3 days.
In the cell live and dead observation experiment, the glioblastoma cell line U87-MG was seeded in a cell culture dish of 35 mm and cultured in an incubator of 37° C. and 5% CO2 for 24 hours. After 24 hours and 48 hours, live and dead staining were performed.
In the present embodiment, when a static magnetic field is applied, cells are planted, and then, the cells were cultured in an incubator of 37° C. and 5% CO2 without applying the static magnetic field for 24 hours. Thereafter, the static magnetic field was continuously applied for 24 hours, and on the next day, application of the static magnetic field was stopped to end the experiment.
In the present embodiment, LIVE/DEAD™ cell Imaging kit (Invitrogen, Carlsbad, CA, USA) was used for a cell live and dead staining experiment, and a protocol provided by a manufacturer was used as an experimental protocol. Before cell live and dead staining is performed, washing is done with lx PBS, and then, a LIVE/DEAD™ staining experiment was performed. After the cells are wrapped with foil to prevent reaction from occurring due to external light, the cells were placed in an incubator for 30 minutes. Thereafter, live and dead cells were observed by using a fluorescence microscope to observe a change in glioblastoma cell line U87-MG due to a static magnetic field.
When the glioblastoma cell line U87-MG was densely packed in 70 to 80% of a bottom area of the cell culture dish of 100 mm, a cell proliferation measurement experiment was started. On the day of the experiment, 1×10 4 cells were planted in a cell culture dish of 35 mm. Thereafter, the number of live cells unstained through trypan blue staining (trypan blue exclusion) was counted every 24 hours.
In the present embodiment, when a static magnetic field is applied, a static magnetic field was applied after about 12 hours after the cells were planted, and the static magnetic field was continuously applied for 72 hours after the experiment. The reason is that, when the static magnetic field is applied shortly after the cells are planted, it is not possible to check whether the cells are changed by the static magnetic field or there is a change because the cells are in poor condition, and thus, the present disclosure prevents this.
In the cell migration observation experiment, the time when the glioblastoma cell line U87-MG grew by 80 to 90% of an area of the cell culture dish of 35 mm was regarded as a dense state. Then, a scratch experiment (scratch assay) was performed in which cells on the cell culture dish were scratched in a vertical direction from top to bottom by using a pipette tip of 100 uL. After removing the scratched cells and washing is done with 1× phosphate buffered saline (PBS) to remove the existing culture fluid remaining in the cells as much as possible, newly added culture fluids had different FBS concentrations of 10%, 5%, 3%, and 1%. The reason is to minimize effects of FBS and observe only cell migration because the FBS includes an abundance of growth factors and may be affected by cell differentiation, cell proliferation, and so on. The cells were continuously imaged at 1 minute intervals for 72 hours by using a microscope equipped with a real-time cell monitoring device (live cell imaging platform).
In order to investigate an effect of a static magnetic field on cell proliferation in U87-MG cell which is a glioblastoma cell line and U87-MG+GFP cell which is transfected to produce green fluorescent protein (GFP), the number of cells was directly counted through trypan blue staining (trypan blue exclusion), and at the same time, a shape of the cell was also observed through a microscope.
The same number of 1×104 cells were planted in a culture dish of 35 mm at the beginning of an experiment for both an experimental group to which a static magnetic field was applied and a control group to which no static magnetic field was applied. The static magnetic field started to be applied after the cells were planted and then about 12 hours elapsed, and the static magnetic field was continuously applied for 72 hours until the experiment was finished.
The number of U87-MG cells in the control group and the number of U87-MG cells in the experimental group were respectively 1.31×104 and 1.24×104 after 24 hours from when the experiment starts, and the number of U87-MG cells in the control group and the number of U87-MG cells in the experimental group were respectively 1.78×104 and 1.29×104 after 48 hours from when the experiment starts, and a difference between the two groups began to appear from this time. Finally, after 72 hours from when the experiment started, the number of cells in the control group and the number of cells in the experimental group was respectively 2.69×104 and 1.23×104, and the difference therebetween was further increased.
The number of U87-MG+GFP cells in a control group and the number of U87-MG+GFP cells in an experimental group were respectively 1.31×104 and 1.33×104 after 24 hours from when the experiment started, and the number of U87-MG+GFP cells in a control group and the number of U87-MG+GFP cells in an experimental group were respectively 1.83×104 and 1.55×104 after 48 hours from when the experiment started, a difference between the two groups began to appear from this time. Finally, after 72 hours from when the experiment started, the number of cells in the control group and the number of cells in the experimental group was respectively 2.68×104 and 1.63×104, and the difference therebetween was further increased.
U87-MG cells in the experimental group increased at an average rate of 0.07×104 cells per day, U87-MG cells in the control group increased at an average rate of 0.56×104 cells per day, U87-MG+GFP cells in the experimental group increased at an average rate of 0.21×104 cells per day, and U87-MG+GFP cells in the control group increased at an average rate of 0.56×104 cells per day. Therefore, in the present embodiment, it was confirmed that a static magnetic field of 1.0 mT acts to prevent cell proliferation of U87-MG cells and U87-MG+GFP cells, which are glioblastoma cell lines. In addition, in the present embodiment, it was confirmed from the experimental results of U87-MG cells and U87-MG+GFP cells that effects of static magnetic fields of the same magnitude of 1.0 mT are different from each other depending on cells.
As may be seen from the same drawings, cell proliferation occurs very well in a control condition where no magnetic field is applied, and the cells try to fill all regions observed by the microscope, and in contrast to this, in the present embodiment, cell proliferation is reduced in a condition of SMF 1.0 mT to which a static magnetic field is applied, and cells does not fill the region compared to photographs of a microscope in the control condition, and the number of round shapes increases. That is, during cell division, the number of cells had to be increased and sizes of the cells had to be increased, but actually, the number of cells was reduced and the sizes of the cells were reduced, and there were many cells having round shapes.
In order to examine effects of a static magnetic field on cell migration of a glioblastoma cell line U87-MG, cells were continuously observed for 3 days by using a live cell imaging system.
After U87-MG cells are planted, a glioblastoma cell line, in a 35 mm cell culture dish, the culture fluid with 10% FBS added was used until the density reached 70 to 80%. At the beginning of the experiment, the concentration of FBS added to the culture fluid was changed. Migration of cells was observed in real time at different concentrations of 5%, 3% and 1%. After a U87-MG cell of a glioblastoma cell line was planted in a cell culture dish of 35 mm, the culture fluid to which FBS of 10% has been added was used until 70 to 80%, and at the beginning of the experiment, the concentrations of FBS added to the culture fluid were differently set to 5%, 3%, and 1%, and migration of cells was observed in real time. The reason why the experiment was performed with different concentrations of FBS is that FBS includes not only many growth factors necessary for cell growth but also various types of protein and so on, and thus, survival, growth, and division of cells are greatly affected.
That is, in order to perform a cell migration analysis method, quantification was performed to obtain an area surrounded by cells in a region of interest (RoI) with frames at 0 hour, 12 hours, 24 hours, 48 hours, and 72 hours by using software. After obtaining the total area of one frame by using Image J, an area of an empty part, that is, an area not filled with cells, was obtained. In addition, by subtracting these two, the area surrounded by the cells was obtained, and a ratio occupied by cells in one frame was named a cell-covered area and illustrated in a graph.
The experimental results may be checked through related drawings.
Another method used to check whether the glioblastoma cell line U87-MG is affected by a static magnetic field is cell live and dead staining, cell dead staining (TUNEL), and cell immunochemical staining (Ki-67). When a cell staining experiment was performed, the added concentration of FBS was 3%. The glioblastoma cell line U87-MG was planted in a cell culture dish of 35 mm, and the, cultured in an incubator of 37° C. and 5% CO2 for 48 hours. Staining was performed after 24 hours from when cells were planted, and another staining was performed after 48 hours from when the cells were planted.
In a method of applying a static magnetic field according to the present embodiment, the static magnetic field was not applied for 24 hours after cells were planted, and the cells were cultured in an incubator of 37° C. and 5% CO2. Thereafter, the static magnetic field was continuously applied for 24 hours, and then, the application was stopped, and the cells were stained. That is, staining was performed before the static magnetic field was applied and after the static magnetic field was applied for 24 hours.
LIVE/DEAD™ cell imaging kit (Invitrogen, Carlsbad, CA, United States) was used for a cell live and dead staining experiment, and a protocol provided by a manufacturer was used as an experimental protocol thereof. Before cell live and dead staining is performed, washing was done with 1×PBS, and then, a LIVE/DEAD™ staining experiment was performed. In addition, the cell was wrapped with aluminum foil to prevent the cell from reacting on external light, and then, the cell was reacted in an incubator of 37° C. and 5% CO2 for 30 minutes. Thereafter, live and dead cells were observed by using a fluorescence microscope. In addition, terminal deoxynucleotidyl transferase dUTP Nick-End labeling (TUNEL) staining was performed to check DNA fragmentation, which is one of characteristics of cell dead. Cells were prepared in the same way as in the cell live and dead staining experiment, and a DeadEnd™ fluorometric TUNEL system (Promega, Madison, WI, United States) product was used, and a protocol provided by a manufacturer was used as an experimental protocol. Finally, Ki-67, that was a cell proliferation marker, was examined to check there was a change when a static magnetic field was applied and when the static magnetic field was not applied through cell immunocytochemistry (ICC). A monoclonal antibody Ki-67 Antigen (MIB-1) (1:200) (Agilent, Carpinteria, CA, United States) was used as a primary antibody used in the present example, and Alexa Fluor® Plus 594 (1:200) (Invitrogen, Carlsbad, CA, United States) was used as a secondary antibody.
Calcein-AM staining was performed to examine an effect of a static magnetic field on cell live and dead of a glioblastoma cell line U87-MG. Since Calcein-AM is cell-permeable staining, the Calcein-AM selectively penetrates only live cells and stains to green fluorescence, and BOBO-3 Iodide couples to DNA of dead cells and illustrates red fluorescence, and accordingly, live cells may be easily distinguished from dead cells at the same time.
When comparing results of staining of a LIVE/DEAD™ cell imaging kit before and after a static magnetic field is applied, red fluorescence-stained portions appeared only in the cells to which the static magnetic field was applied.
Next, when comparing results of TUNEL staining to check DNA fragmentation which is one of characteristics of cell dead, green fluorescence-stained portions appeared only in the cells to which the static magnetic field was applied.
Finally, when comparing results of cell immunochemical staining to check Ki-67 which is a cell proliferation marker, a degree of expression of Ki-67 was low in the cells to which the static magnetic field was applied.
As described above, when the results obtained through the cell fluorescence staining method are combined, the glioblastoma cell line U-87MG may be affected by a static magnetic field.
After a static magnetic field is applied for 24 hours, U87-MG cells were collected in the form of pellets and protein was isolated. Total protein was extracted by using PRO-PREP™ (iNiRON Biotechnology Inc., Seongnam-si, Gyeonggi-do, Korea). The amount of protein used was 10 μg, and sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) was performed by selecting an appropriate gel percentage according to a size of the protein to be observed. Among genes related to cell dead, cleaved caspase-3, cleaved caspase-9 and cleaved PARP, which are cleaved forms, used 40 μg of protein, and phospho-Akt and phospho-PI3K, which are phosphorylated forms, and β-actin, which is a house-keeping gene, used 20 μg of protein.
Polyvinylidene fluoride (PVDF) (Whatman, Little Chalfont, Buckinghamshire, United Kingdom) was immersed in a methanol solution and activated, and then, a transfer process was performed for 1 hour and 20 minutes. Then, blocking was performed at room temperature for 1 hour with 1× tris-buffered saline with Tween 20 (TBS-T) including bovine serum albumin (BSA) (GenDEPOT, Katy, TX, United States) of 3% to minimize non-specific binding of antibody and protein, and the primary antibody was reacted at 4° C. for 16 hours. The type of used primary antibody and a dilution ratio thereof were cleaved caspase-3 (1:500), caspase-3 (1:1000), cleaved caspase-9 (1:200), PARP (1:1000) (abcam, Cambridge, United Kingdom), caspase-9 (1:1000), cleaved PARP (1:500), phospho-Akt (1:500), Akt (1:1000), phospho-PI3K (1:500), PI3K (1:1000) (Cell Signaling Technology, Inc., Danvers, MA, United States), and β-actin (1:500). Washing was done three times for 15 minutes with 1×TBS-T, and goat anti-rabbit IgG (H+L)-horseradish peroxidase (HRP) conjugate (Bio-Rad Laboratories, Inc., Hercules, CA, United States) was diluted at a ratio of 1:1000 and reacted at room temperature for 1 to 2 hours. Cleaved caspase-3, cleaved caspase-9, and cleaved PARP were diluted at a ratio of 1:500. After washing was done three times for 15 minutes with 1×TBS-T, enhanced chemiluminescence (ECL) (Bio-Rad Laboratories, Inc., Hercules, CA, United States) was sprayed on a membrane to detect a protein signal.
As a result, first, expression of protein related to cell apoptosis was confirmed.
In order to check that the glioblastoma cell line U87-MG actually undergoes cell dead and inhibits cell proliferation by a static magnetic field, a change in an expression level of cleaved caspase-3, cleaved caspase-9, and cleaved PARP were investigated through protein immunity analysis. When the cell dead is made, caspase-3 and caspase-9 genes and PARP genes are cleaved, and accordingly, caspase-3 and caspase-9 genes and PARP genes are used as important hallmarks to check cell dead. As confirmed from
In addition, protein expression related to signal transduction was confirmed.
A signal transmission path of phosphatidylinositol-3-kinase (PI3K)/AKT (Protein Kinase B) is related to proliferation, growth, differentiation, migration, and cycle of cells and play an important role, and is also used as a targeted therapy targeting a PI3K/Akt path. Therefore, it was confirmed whether the signal transmission path is affected by a static magnetic field and a change in an expression level of PI3K phosphorylation and Akt phosphorylation through protein immunity analysis. As confirmed from
As in the present embodiment, dead of tumor cells according to application of a static magnetic field was also confirmed from a Western Blot test result.
It may be seen from the same drawing that, glutathione peroxidase 4 (GPX4), FTN1 (ferritin), prostaglandin-endoperoxide synthase 2 (PTGS2), nuclear factor erythroid2-related factor 2 (NFE2L2) observed from cells with remarkably increased ferroptosis was significantly increased in a group of U87-MG tumor cells to which a static magnetic field of 1.0 mT according to the present embodiment was applied compared to a group to which the static magnetic field was not applied.
The embodiment described above relates to a change in cancer cells according to application of a static magnetic field of 1.0 mT. The present embodiment relates to a change in tumor cells according to application of a static magnetic field of 0.6 mT based on the same experimental condition, method, and so on.
As may be seen from
This effect may be clearly seen when compared with an experimental result obtained by performing the same experiment on normal cells other than tumor cells.
As may be seen from
That is, it may be seen from
1×104 cells were planted in a culture dish of 35 mm at the beginning of an experiment for each of an experimental group to which a static magnetic field was applied and a control group to which the static magnetic field was not applied. The static magnetic field was applied after about 12 hours from when cells were planted, and was continuously applied until 72 hours after the experiment was completed.
After 24 hours from when the experiment started, the number of U373-MG cells in the control group and the number of U373-MG cells in the experimental group were respectively 1.37×104 and 1.25×104, and after 48 hours from when the experiment started, the number of U373-MG cells in the control group and the number of U373-MG cells in the experimental group were respectively 1.78×104 and 1.61×104, and a difference between the two groups began to appear. Finally, after 72 hours, the number of cells in the control group and the number of cells in the experimental group were respectively 2.48×104 and 1.76×104, and the difference therebetween was further increased.
After 24 hours from when the experiment started, the number of U373-MG+GFP cells in the control group and the number of U373-MG+GFP cells in the experimental group were respectively 1.35×104 and 1.33×104, and after 48 hours from when the experiment started, the number of U373-MG+GFP cells in the control group and the number of U373-MG+GFP cells in the experimental group were respectively 1.73×104 and 1.63×104, and a difference between the two groups began to appear. Finally, after 72 hours, the number of cells in the control group and the number of cells in the experimental group were respectively 2.15×104 and 2.04×104, and the difference therebetween was further increased.
The number of U373-MG cells in the experimental group increased at an average rate of 0.25×104 cells per day and the number of U373-MG cells in the control group increased at an average rate of 0.49×104 cells per day, and the number of U373-MG+GFP cells in the experimental group increased at an average rate of 0.35×104 cells per day and the number of U373-MG+GFP cells in the control group increased at an average rate of 0.38×104 cells per day. Therefore, it may be seen that the static magnetic field of 0.6 mT acts to inhibit the cell proliferation of U373-MG cells and U373-MG+GFP cells, which are glioblastoma cell lines. In addition, it may be seen from experimental results of U373-MG cells and U373-MG+GFP cells that the effect of the static magnetic field of 0.6 mT changes depending on cells.
It may be seen from
Specifically,
In addition, a static magnetic field may be generated by using a permanent magnet instead of a DC current, and various embodiments related thereto are illustrated in
In addition, an operation and a magnetic field generation principle of each of the static magnetic field generator structures illustrated in
A summary of the embodiments described above is as follows.
According to one embodiment, it may be seen that there is a change when a static magnetic field of a certain size is applied to various types of cancer including glioblastoma cell lines. In an example for generating a static magnetic field of a uniform size, a structure of the Helmholtz coil is used, and cell proliferation may be checked by directly measuring the number of cells changed by a static magnetic field through trypan blue staining, and a change in shapes of cells may also be checked through a microscope. In addition, cell live and dead may be checked through cell fluorescence staining, the cell live and dead may be checked through calcein-AM staining, and cell dead may be checked through TUNEL staining. Finally, expression of Ki-67 may also be checked through cell immunochemical staining.
The Helmholtz coil used in the embodiment is designed with parameters, such as a diameter of a coil required for design, the number of turns of a coil, and a magnitude of a current to flow through the coil, and two circular coils are placed vertically in an upper portion and a lower portion in the z-axis direction.
As a result, for example, when a static magnetic field of 0.6 mT or 1.0 mT is applied to a glioblastoma cell line, the number of cells is hardly changed, and a shape of the cell is slightly more elongated compared to a shape of the cell when the magnetic field is not applied. In a cell migration experiment, concentrations of FBS to be added are set to 5%, 3%, and 1% in order to reduce various effects caused by the FBS as much as possible, cell migration begins to slow down after about 12 hours under conditions in a culture solution including concentration of FBS of 3%, and later, the speed of filling empty parts is also reduced gradually. Cell dead is observed while cell migration is inhibited, and accordingly, calcein-AM staining is performed to check cell live and dead, TUNEL staining is performed to check cell dead, and Ki-67 staining is performed to check a cell proliferation marker. As a result, staining results with characteristics of cell dead are obtained only when a static magnetic field is applied. In addition, a protein expression experiment was performed, and protein expression of cleaved caspase-3, cleaved caspase-9, and cleaved PARP, which are cleaved forms of genes related to cell dead, was increased in cells to which a static magnetic field was applied. In addition, protein expression of PI3K phosphorylation and Akt phosphorylation, which are phosphorylated forms, among genes related to signal transmission, was reduced in the cells to which a static magnetic field was applied.
Therefore, when summarizing all these molecular biological results, it may be seen that a static magnetic field inhibits cell proliferation and cell migration of glioblastoma cell lines and induces cell dead.
In particular, the magnitude of a static magnetic field according to an embodiment of the present disclosure ranges from 0.1 mT to 4 T, where 0.1 mT is a minimum magnitude required for removing and inhibiting cancer cells, and when exceeding 4 T, the static magnetic field may cause harm to the human body. For example, a magnetic flux density of 0.1 mT in the present embodiment corresponds to a very weak static magnetic field that is “30,000 times” lower than the magnetic flux density of 4 T used in a magnetic resonance imaging device. Although a static magnetic field of 1 mT which is another exemplary size is harmless to the human body and has very small magnitude of about “ 1/4,000” compared to the magnitude of a static magnetic field of 4 T which is an upper limit of the discovered magnitude, tumors are killed and tumor invasion is significantly reduced, and even under continuous treatment, safety to the human body and cancer treatment efficacy may be ensured at the same time.
In addition, when a static magnetic field of 1.0 mT is applied to tumor cells illustrated in the embodiment of the present disclosure, rather than when a static magnetic field of 0.6 mT is applied, more tumors are killed, and tumor invasion is more significantly reduced, and thus, the larger the magnitude of a static magnetic field, the greater the effect on the tumors.
Furthermore, even when a small static magnetic field of about 1.0 mT of the same size is applied to various types of cancer illustrated in the embodiments of the present disclosure, more tumors are killed in proportion to a duration of exposure, and tumor invasion is more significantly reduced, and thus, cancer treatment may be efficiently done by using a very low-level static magnetic field that is harmless to the human body even when applied for a long time.
Although the present disclosure is described in detail above, the scope of the present disclosure is not limited thereto, and it will be apparent to those skilled in the art that various modifications and variations may be made without departing from the technical idea of the present disclosure described in claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2022-0046520 | Apr 2022 | KR | national |
