PRIORITY CLAIM

[0001] The present application claims priority to U. S. Provisional Patent Application Serial No. 62/889,138, filed August 20, 2019. The entirety of this application is hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

[0002] The subject matter of this application was made with support from the United States government under a contract awarded by the National Science Foundation, Grant Numbers 1465061 and 1747760. The United States government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates generally to cancer cell treatments, and more particularly, using a nano-second pulsed magnetic field for sensitizing cancer cells for treatment.

BACKGROUND

[0004] Cancer is one of the most fatal diseases in modem medicine. For example, Melanoma is a common skin and mucosa tumor, with a high mortality rate so far. Melanoma is also one of the fastest growing malignant tumors. The current therapy is mainly based on surgical treatment, radiotherapy, and chemotherapy. Such previous modalities tend to affect the growth of cancer cells via directly causing apoptosis or other cell deaths. For example, cold atmospheric plasma (CAP) is a potential method to treat melanoma based on its abundant reactive species components. The CAP treatment kills cancer cells through the direct transportation of these reactive species into the extracellular environment. In one study, CAP- treated pancreatic adenocarcinoma cells (PA-TU-8988T) could quickly enter into a specific state, in which cells were very sensitive to the cytotoxicity of reactive species, such as H202 and N02. Such activation is partially due to a physical effect such as the electromagnetic field. [0005] Several novel modalities have been introduced in recent years to selectively treat cancer cells. The reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important role in the anti-cancer capacity of several modalities, such as cold atmospheric plasma (CAP). These treatments cause apoptosis or other cell deaths in cancerous cells. However, all of these treatments present a challenge because healthy cells may also be affected resulting in undesirable apoptosis or other cell deaths.

[0006] Existing experimental evidence shows electromagnetic fields can regulate the expression of genes involved in migration and invasion and disrupt the mitotic spindle similar effect is also observed during treatment with cold atmospheric plasma (CAP). The biological effects of electro-magnetic fields have been studied for decades. As an example, the tumor treating fields approved in 2011 by the U.S. Food and Drug Administration for the treatment of recurrent glioblastoma is an example of the medical application of electro-magnetic fields. The tumor-treating fields are generated via transducer arrays and produce an alternating electromagnetic field with low-intensity, intermediate-frequency (200 kHz). These electromagnetic fields can penetrate the scalp and effectively inhibit the migration of glioblastoma cells, disrupt the formation of their mitotic spindles, and block their proliferation. Furthermore, various studies found that the biomedical use for low intensity and frequency pulsed-electromagnetic fields (LIFP-EMF) can generate 0.3-5mT peak-to-peak magnitude at a pulse frequency of 20-50Hz, which could induce apoptosis in cancer cells. Low intensity and frequency pulsed-electromagnetic fields can block the development of neovascularization that is required for blood supply to support tumor growth and induce genetic instability to decrease the stringency of late-cycle (G2) checkpoint. Moreover, studies have found that EMF is innocuous and even beneficial to normal cells. EMF has been demonstrated to lower the number of metastatic tumor sites and slow tumor growth rate when compared to the control group, without showing harmful side effects.

[0007] Pulsed magnetic fields have been used as a medical modality for years. The low- intensity pulsed magnetic field was an effective treatment for osteoporosis. Compared with the continuous magnetic field, the pulsed magnetic field owned a faster rising-edge, which causes a stronger induced electric field and a wider spectrum without significant thermal effect. In contrast to a CAP treatment, the pulsed magnetic field only affected cancer cells via its physical effect. The previous studies tended to directly affect the growth of cancer cells by causing apoptosis or other cell death pathways.

[0008] There is a need for a method to sensitize cancer cells with a pulsed magnetic field before a conventional cancer treatment. There is another need for a system that allows the adjustment of magnetic field strength and pulse frequency to control the activation level of cancer cells exposed to a pulsed magnetic field. SUMMARY

[0009] One disclosed example is a system for treatment of an area having cancerous cells. The system includes a magnetic field generator to generate a nano-pulsed magnetic field directed at the area having cancerous cells. A controller is coupled to the plasma device to control the nano-pulsed magnetic field generated by the magnetic field generator.

[0010] A further implementation of the example system is an embodiment where the nano second pulsed magnetic field has a maximum DC output voltage of 12kV, a rise time less than 10 nanoseconds, and a pulse width of at least 50 nanoseconds. Another implementation is where the controller is coupled to the nano-pulsed magnetic field generator and directs the frequency, pulsing, strength and area of the magnetic field. Another implementation is where the area is within a patient and the area includes healthy cells and wherein the system is an in vivo treatment system. Another implementation is where the area is a cell holder of a plate well and the treatment is an in vitro treatment system. Another implementation is where the cancerous cells are melanoma cancer cells. Another implementation is where the system includes a treatment device operable to apply a treatment to the sensitized cancer cells after the cancer cells are sensitized by the nano-pulsed magnetic field. Another implementation is where the treatment is one of applying an oxygen or a nitrogen reactive species, applying radiation, or applying H202. Another implementation is where the treatment includes administration of a chemotherapeutic. Another implementation is where the treatment is chemotherapy, and the treatment device adjusts the treatment below a baseline level in response to the sensitized cells. [0011] Another disclosed example is a method of sensitizing cancerous cells in an area. A nano-pulsed magnetic field directed at the area having cancerous cells is generated via a magnetic field generator. The generation of the nano-pulsed magnetic field generated by the magnetic field generator is controlled via a controller.

[0012] A further implementation of the example method is an embodiment where the nano second pulsed magnetic field has a maximum DC output voltage of 12kV, a rise time less than 10 nanoseconds, and a pulse width of at least 50 nanoseconds. Another implementation is where the controller is coupled to the nano-pulsed magnetic field generator and directs the frequency, pulsing, strength and area of the magnetic field. Another implementation is where the area is within a patient and the area includes healthy cells and wherein the method is an in vivo treatment. Another implementation is where the area is a cell holder of a plate well and the treatment is an in vitro treatment system. Another implementation is where the cancerous cells are melanoma cancer cells. Another implementation is where a treatment is applied to the sensitized cancer cells after the cancer cells are sensitized by the nano-pulsed magnetic field. Another implementation is where the treatment is one of applying an oxygen or a nitrogen reactive species, applying radiation, or applying H202. Another implementation is where the treatment includes administration of a chemotherapeutic. Another implementation is where the treatment is chemotherapy, and wherein the treatment is adjusted below a baseline level in response to the sensitized cells.

[0013] The above summary is not intended to represent each embodiment or every aspect of the present disclosure. Rather, the foregoing summary merely provides an example of some of the novel aspects and features set forth herein. The above features and advantages, and other features and advantages of the present disclosure, will be readily apparent from the following detailed description of representative embodiments and modes for carrying out the present invention, when taken in connection with the accompanying drawings and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The disclosure will be better understood from the following description of embodiments together with reference to the accompanying drawings.

[0015] FIG. 1A is an example in vitro test system to demonstrate use of a magnetic field generator to generate a pulsed magnetic field to sensitize in vitro cancer cells;

[0016] FIG. IB is a block diagram of an example in vivo magnetic field system for sensitizing an area of a subject with cancer cells with a pulsed magnetic field;

[0017] FIG. 2 is a circuit diagram of the magnetic field system in FIG. 1 A;

[0018] FIG. 3 is a trace diagram of the generated pulsed magnetic field from the magnetic field generator in FIG. 1 A;

[0019] FIG. 4 is a diagram showing the different states of cancer cells after being sensitized from the electro-magnetic field;

[0020] FIGs. 5A-5B are graphs of test results that compare the effects of different durations of the application of magnetic fields with treatment with a control group;

[0021] FIGs. 6A-6B are graphs of test results that compare the effects of different durations of the application of magnetic fields; and

[0022] FIGs. 7A-7D are graphs of test results that compare the effects of different durations of the application of magnetic fields.

[0023] The present disclosure is susceptible to various modifications and alternative forms. Some representative embodiments have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

DETAILED DESCRIPTION

[0024] The present inventions can be embodied in many different forms. Representative embodiments are shown in the drawings, and will herein be described in detail. The present disclosure is an example or illustration of the principles of the present disclosure, and is not intended to limit the broad aspects of the disclosure to the embodiments illustrated. To that extent, elements and limitations that are disclosed, for example, in the Abstract, Summary, and Detailed Description sections, but not explicitly set forth in the claims, should not be incorporated into the claims, singly or collectively, by implication, inference, or otherwise. For purposes of the present detailed description, unless specifically disclaimed, the singular includes the plural and vice versa; and the word “including” means “including without limitation.” Moreover, words of approximation, such as “about,” “almost,” “substantially,” “approximately,” and the like, can be used herein to mean “at,” “near,” or “nearly at,” or “within 3-5% of,” or “within acceptable manufacturing tolerances,” or any logical combination thereof, for example.

[0025] This disclosure is directed toward a treatment method and system using the generation of a nanosecond-pulsed magnetic field (NMF) to sensitive a cancerous cell line prior to a standard cancer treatment. For example, the cancerous cell line may be a melanoma cell line B16-F10 to the cytotoxicity of an important ROS such as H202. The magnetic field strength and the frequency are key factors to control the activation level of the disclosed NMF treatment. The disclosed principles use electromagnetic modality in cancer treatment particularly for the treatment on deep tumors such as brain tumors or liver tumors. In this example, a nano-pulsed magnetic field activates pancreatic cancer cells and melanoma cells to increase the effectiveness of conventional treatments such as reactive species.

[0026] FIG. 1 A shows an example experimental in vitro test system 100 for sensitization of cancer cells via electro-magnetic field. The test system 100 includes a magnetic field generation system 110 that allows the application of a pulsed magnetic field to sensitize cancer cells for effective reactive oxygen species treatment. [0027] The test system 100 includes a conductive plate 112 that supports a test sample well plate assembly 114. The magnetic field generation system 110 includes a high voltage input 120 and an impulse output 124. A coaxial cable 130 is connected to the system 110. The coaxial cable 130 is suspended over the test sample well plate 114.

[0028] An inset 132 is shown in FIG. 1A that shows a top down view of the test sample well plate 114 in relation to the coaxial cable 130. The test sample well plate 114 includes a holder plate 140 that includes a grid arrangement of sample wells 142. In this example, the plate 114 is a 96 well plate such as a 62406-081 type plate manufactured by Falcon-Coming. An inset 152 shows a side view of the well plate 114 in relation to the coaxial cable 130 along the line A-A in the inset 132. A culture medium is placed in each of the wells 142 of the plate 114 along with the cells to be tested.

[0029] In this example, the magnetic field generation system 110 generates a transmission line, type pulsed magnetic field. In this example, the magnetic field generation system 110 generates a nano-pulsed magnetic field with a rise time of less than 10 ns, a pulse width of 110 ns, and a magnetic intensity up to 6 mT. The operation frequency could be modulated by controlling a power supply as will be explained. In this example, the nanosecond-pulsed square wave current generating device worked at a coaxial cable energy storage mode. The maximum output DC voltage is 12 kV in this example, but the output DC voltage may range between 5- 12 kV. In this example, the rise time may range between 2 and 10 ns, the pulse width may range between 50 and 110 ns, the magnetic intensity may range between 1 and 6 mT.

[0030] FIG. IB is a block diagram of an in vivo cancer treatment system 150 that subjects an area of normal cells and cancerous cells on a subject 152 to an electro-magnetic field generated by a magnetic field generator 154. In this example, the subject 152 is a test subject such as a laboratory animal, but it is to be understood the system 150 may be used as part of the treatment for patients with cancerous cells. The magnetic field generator 154 emits a nano- pulsed electro-magnetic field 170 through a coaxial cable 158.

[0031] A controller 156 allows a user to control the intensity, frequency, and duration of the nano-pulsed magnetic field generated by the magnetic field generator 154 to selectively sensitize the cancerous cells in the subject 152. As will be explained, the controller 156 is operative to control the field generator 154. Thus, the controller 156 allows control of the plasma generated pulsed electro-magnetic field to increase sensitivity of cancerous cells. [0032] A treatment system 160 such as a medicine dispensary for chemotherapy or radiation device is provided for treatment of cancerous cells in the subject 152 after the application of the pulsed electro-magnetic field generated from the magnetic field generator 154. In this example, the treatment system 160 provides reactive H202 to change the melanoma cancer cells to an apoptotic state. However, the treatment system 160 may provide reactive oxygen species or reactive nitrogen species to change the cancer cells to an apoptotic state in the area on the patient 152. Alternatively, the treatment system 160 may generate radiation to change the cancer cells to an apoptotic state.

[0033] A treatment system 160 such as a medicine dispensary for chemotherapy or radiation device is provided for any standard treatment of cancerous cells in the subject 152 after the application of the pulsed electro-magnetic field generated from the cold atmospheric plasma device 154. In this example, the treatment system 160 provides a chemotherapeutic such as H202 reactive species to change the cancer cells to an apoptotic state. However, the treatment system 160 may provide reactive oxygen species or reactive nitrogen species to change the cancer cells to an apoptotic state in the area on the patient 152. Alternatively, the treatment system 160 may generate radiation to change the cancer cells to an apoptotic state. In some examples, the treatment system 160 may allow administration of chemotherapeutics such as by injection or orally. Of course, other well-known routes may be taken in the administration of chemotherapeutics. For example, other chemotherapeutics such as Afmitor (Everolimus), Afmitor Disperz (Everolimus). Avastin (Bevacizumab). Bevacizumab, BiCNU (Carmustine), Carmustine, Carmustine Implant, Everolimus. Gliadel Wafer (Carmustine Implant), Lomustine, Mvasi (Bevacizumab), or drug combinations such as PCV may be used for brain cancer cells. Other chemotherapeutics may be used for other types of cancerous cells in conjunction with the sensitization of the cancerous-cells by the described pulsed-magnetic field.

[0034] The field generator 154 includes a power supply 162, a frequency generator 164, a power switching device 166, and the coaxial cable 158. A pulsed power signal is supplied to the coaxial cable 158 to produce a pulsed magnetic field. The high voltage power supply 162 is electrically connected to the co-axial cable through the power switching device 166. The frequency generator 164 is coupled to the controller 156 and the power switching device 166. The frequency generator 164 thus switches the switching device 166 on and off to control the frequency of the power supplied to the co-axial cable 158 to regulate the emitted pulsed magnetic field. The controller 156 is coupled to the high voltage power supply 162 and regulates the discharge voltage and frequency that is applied to the coaxial cable.

[0035] The generated pulsed magnetic field may be utilized, for instance, to sensitize cells of any cancer type that is close to the skin and can be applied without surgery, such as for breast, colon, lung, bladder, or oral cancers. With surgery, the system 150 may be applied to any tumor.

[0036] FIG. 2 A is a circuit diagram 200 of the magnetic field generation system 110 in FIG. 1A. The main components in the circuit diagram 200 include a DC voltage source 210, a current limiting resistor 212, the coaxial cable 130, a switch 214, and a matching impedance in the form of a matching resistor 216. The matching impedance of the resistor 216 is selected to match the impedance of the coaxial cable 130. In this example, the output voltage of the DC voltage source 210 is coupled to one lead of the resistor 212. The other lead of the resistor 212 is coupled to an interior conductor 220 of the coaxial cable 130. An exterior conductor 222 of the coaxial cable 130 is radially located outside the interior conductor 220. The exterior conductor 222 is coupled to one end of the switch 214. The switch 214 is activated by a control circuit 230 that switches the switch 214 on and off at a set frequency to generate the magnetic field from the coaxial cable 130. The other end of the switch 214 is grounded with the DC voltage source 210 to complete the circuit.

[0037] FIG. 3 is a voltage trace graph of the magnetic field waveform 300 generated by the example magnetic field generation system 110 in FIG. 1A plotting field strength against the time of the pulse. When the distance between the coaxial cable 130 and the test cells varied, different magnetic field intensities were obtained. The time dependence of the magnitude of the magnetic field during the pulse is thus shown by the waveform 300 [0038] FIG. 4 shows different stages of the cancerous cells during the process involving the sensitivity treatment involving the magnetic field generated by the magnetic field generator system 110 in FIG. 1A and subsequent treatment such as through application of reactive species. In this example, the magnetic field generator system 110 generates a nano-pulsed magnetic field to treat melanoma cells through a barrier of a plastic casing on the 96-well plate 114.

[0039] A first stage 400 occurs with the application of a magnetic field treatment via a system such as the magnetic field generator system 110 in FIG. 1 A. The nano-pulsed magnetic field is generated by the magnetic field generator system 110 and applied to unsensitized cancer cells 402. A second stage 410 occurs immediately post application of the magnetic field sensitivity treatment. In the second stage 410, the cancer cells 402 are transformed into sensitized cells 414 based on the treatment of the nano-pulsed electro-magnetic field from the first stage 410. A third stage 420 occurs with the application of a cancer treatment such as a reactive species 422 via a pipette 424. The reactive species 422 begins to diminish after some time in the third stage 420. The sensitized cells 414 begin to transition to apoptotic cells 432 due to the application of the reactive species 422. A fourth stage 430 occurs after a pre determined time from the application of the reactive species treatment. In the fourth stage 430, the reactive species 422 has largely disappeared, but the sensitized cells 414 have become apoptotic cells 432 due to the increased sensitivity that is created by the nano-pulsed magnetic field. This process thus increases the effectiveness of the reactive species treatment.

[0040] A control group without the application of the nano-pulsed magnetic field is shown in a first stage 450. The first stage 450 includes cancer cells 452 in a medium 454. A second stage 460 occurs with the application of a cancer treatment such as a reactive species 462 via a pipette 464. The reactive species 462 begins to diminish after some time in a third stage 470. The third stage 470 occurs after a pre-determined time from the application of the reactive species treatment. In the third stage 470, the reactive species 462 has largely disappeared. Only a limited number of the cancer cells 452 transition to apoptotic cells 472 due to the application of the reactive species 462.

[0041] In one experiment conducted using the test system 100 in FIG. 1A, a human pancreatic adenocarcinoma cell line (PA-TU-8988T) was examined. The cells were cultured using DMEM (11965-118, Life Technologies) supplemented with 1% (v/v) penicillin and streptomycin (15140122, Life Technologies) and 10% (v/v) fetal bovine serum (E17063, Atlanta Biologicals). A Murine melanoma cell line (B16-F10) was cultured using RPMI-1640 medium (80614171, ATCC) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin. In each experiment, 3 x 10 ³ cells were seeded in each well of the 96-well plate 114 and were cultured for 24 hours under the standard culture conditions (a humidified, 37°C, 5% C02 environment) prior to treatment by the nano-pulsed magnetic field. The initial media was removed before the nano-pulsed magnetic field treatment and new media was renewed after the treatment.

[0042] The activation phenomena in cancer cells was monitored by comparing the cytotoxicity of H202 on activated cells to the cytotoxicity of H202 on the cells without such activation. The H202-containing DMEM was made by adding 9.8 M H202 standard solution (216763, Sigma-Aldrich) in the DMEM or RPMI with a designed concentration. The H202- containing medium was transferred to affect the growth of cancer cells in the wells. The corresponding control was just cultured in the new DMEM or RPMI accordingly. The cancer cells were cultured 2 days after treatment. The cell viability was measured by using MTT (3- (4,5-Dimethyl-2-thiazol)-2,5-Diphenyl-2H-tetrazolium Bromide) assay according to the standard protocols (Sigma-Aldrich, M2128). The 96-well plates were read (570 nm) by a microplate reader (HI, Hybrid Technology). The measured absorbance at 570 nm was further processed to be relative cell viability via the division between the experimental group and the control group. All experiments were independently repeated for at least three times in sextuplicate.

[0043] To investigate the activation effect, the following protocol was employed with three experimental cases. The comparison between these cases reveals the activation of these cancer cells due to the nano-pulsed magnetic field. The first experimental case employed treatment with the nano-pulsed magnetic field generated by the system 100 in FIG. 1A. In this experimental case, the cancer cells were immersed in the 50 pL/well DMEM or RPMI during the treatment. After that, DMEM or RPMI was quickly (<20 s) removed and was renewed by the 50 pL/well of new DMEM or RPMI. The cancer cells were activated by the nano-pulse magnetic field in this case.

[0044] The second experimental case is where the nano-pulsed magnetic field treatment is supplemented with H202 treatment. The cancer cells were first activated by a nano-pulsed magnetic field in the 50 pL/well medium, followed by a quick remove (<20 s) of DMEM or RPMI after the treatment. Then, a 50 pL/well H202-containing DMEM or RPMI has used to culture the NMF-treated cancer cells until the final cell viability assay.

[0045] The third experimental case is the application of a H202 treatment alone to the cancer cells. The cancer cells were just cultured in 50 pL of H202-containing DMEM or RPMI before the final cell viability assay. In all cases, cancer cells were cultured for 3 days before the final cell viability assay. The cell viability was observed and recorded. The occurrence of a noticeable activation is defined as when the cell viability ratio between the second experimental case and the third experimental case was less than 0.95.

[0046] The cytotoxicity of H202 on two cancer cell lines was investigated. To reveal the activation state, weak cytotoxicity of just H202 treatment on cancer cells is desired. In this way, the activated cancer cells show observable stronger vulnerability to H202 treatment. The H202-containing DMEM and RPMI with different concentrations were prepared first and were immediately used to affect the PA-TU-8988T cells and the B16F10 cells, respectively. Based on the cytotoxicity, the concentration of the H202-containing medium was set to be 5 pM/10 mM and 20 pM/40 pM for PA-TU-8988T cells and B16F-F10 cells, respectively. Under these conditions, H202 did not cause noticeable cytotoxicity on these two cell lines.

[0047] The potential activation effect was investigated on the PA-TU-8988T cells due to the nano-pulsed magnetic field treatment. The strength of a 1 Hz magnetic field was increased from 0.119 mT to 1.176 mT. The nano-pulsed magnetic field treatment lasted two hours. As shown in FIG. 5A, the nano-pulsed magnetic field treatment alone did not cause a noticeable inhibition on PA-TU-8988T cells, except in one single experimental condition (0.119 mT, 10 mM H202). The nano-pulsed magnetic field treatment also did not noticeably enhance the cytotoxicity of H202. Thus, the obvious activation effect has not been observed on the nano- pulsed magnetic field treated PA-TU-8988T cells.

[0048] FIG. 5A is a graph of the results of the comparison of a reactive treatment of 5 pM H202 and a lHz nano-pulsed magnetic field treatment with only the nano-pulsed magnetic field treatment, or only the reactive treatment on the PA-TU-8988T cells. FIG. 5B is a graph of the results of the comparison of reactive treatment of 10 pM H202 and a lHz nano-pulsed magnetic field treatment with only the magnetic field treatment, or only the reactive treatment on the PA-TU-8988T cells.

[0049] In these tests, the effect of a nano-pulsed magnetic field on the cytotoxicity of H202 on PA-TU-8988T cells is demonstrated. The cancer cells were treated by 1 Hz of the generated nano-pulse magnetic field for two hours. The H202 treatment with different concentrations was performed including application of 5 pM H202 and application of 10 pM H202. The results were shown as the mean ± standard deviation (s.d.).

[0050] FIG. 5A shows bars 502a, 504a, 506a, 508a, 510a, 512a, 514a, and 516a that represent the measured cell viability based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively. Bars 502b, 504b, 506b, 508b, 510b, 512b, 514b, and 516b represent the cell viability measurements based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively followed by the application of 5 pMH202. Bars 502c, 504c, 506c, 508c, 510c, 512c, 514c, and 516c represent the measured cell viability based on the application of 5 pM H202 alone.

[0051] The cell viability ratios of the bars 502b, 504b, 506b, 508b, 510b, 512b, 514b, and 516b relative to the respective bars 502a, 504a, 506a, 508a, 510a, 512a, 514a, and 516a were determined and used to assess the activation level. Thus, the ratio between bars 502b and 502a was .98; the ratio between bars 504b and 504a was 1.03; the ratio between bars 506b and 506a was 1.11; the ratio between bars 508b and 508a was .95; the ratio between bars 510b and 510a was 1.02; the ratio between bars 512b and 512a was .97; the ratio between bars 514b and 514a was .97; and the ratio between bars 516b and 516a was .97.

[0052] FIG. 5B shows bars 552a, 554a, 556a, 558a, 560a, 562a, 564a, and 566a that represent the measured cell viability based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively. Bars 552b, 554b, 556b, 558b, 560b, 562b, 564b, and 566b represent the cell viability measurements based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively, followed by the application of 10 mM H202. Bars 552c, 554c, 556c, 558c, 560c, 562c, 564c, and 566c represent the measured cell viability based on solely the application of 10 pM H202 alone. The cell viability ratios of the bars 552b, 554b, 556b, 558b, 560b, 562b, 564b, and 566b relative to the respective bars 552a, 554a, 556a, 558a, 560a, 562a, 564a, and 566a were determined and used to assess the activation level. Thus, the ratio between bars 552b and 552a was .94; the ratio between bars 554b and 554a was 1.09; the ratio between bars 556b and 556a was .98; the ratio between bars 558b and 558a was .98; the ratio between bars 560b and 560a was 1.08; the ratio between bars 562b and 562a was 1.02; the ratio between bars 564b and 564a was .97; and the ratio between bars 566b and 566a was 1.09.

[0053] As shown specifically in bars 552b and 552c representing the second case of the H202 treatment combined with the nano-pulsed magnetic field compared with the third case of the H202 treatment alone in FIG. 5B, this case shows a noticeable activation effect where the ratio between the bars 552b and 552c was < 0.95. Thus, the magnetic field intensity of .119 shows the conditions for the greatest effect of the nano-pulsed magnetic field on the cancerous cells.

[0054] The potential activation effect from application of a nano-pulsed magnetic field was also investigated on B16-F10 cells. FIG. 6A-6B show the results of this investigation. FIG. 6A is a graph of the results of application of a lHz nano-pulsed magnetic field treatment for one hour at different intensities to the B16-F10 cells. 20 pM of the H202 concentration was applied to the cancerous cells after the application of the nano-pulsed magnetic field. FIG. 6B is a graph of the results of a lHz nano-pulsed magnetic field treatment for two hours at different intensities to the B 16-F 10 cells. 40 pM of the H202 concentration was applied to the cancerous cells after the application of the nano-pulsed magnetic field.

[0055] The H202-containing RPMI (20pM or 40pM) was used to affect the growth of B 16- F10 cells with or without the activation by NMF (1 Hz) last 1 hr or 2 hr. The magnetic field strength was increased from 0.119 mT to 1.176 mT.

[0056] FIG. 6 A shows bars 602a, 604a, 606a, 608a, 610a, 612a, 614a, and 616a that represent the measured cell viability based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively. Bars 602b, 604b, 606b, 608b, 610b, 612b, 614b, and 616b represent the cell viability measurements based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively followed by the application of 20 mM H202. Bars 602c, 604c, 606c, 608c, 610c, 612c, 614c, and 616c represent the measured cell viability based on the application of 20 pM H202.

[0057] The cell viability ratios of the bars 602b, 604b, 606b, 608b, 610b, 612b, 614b, and 616b relative to the respective bars 602a, 604a, 606a, 608a, 610a, 612a, 614a, and 616a were determined and used to assess the activation level. Thus, the ratio between bars 602b and 602a was .71; the ratio between bars 604b and 604a was .72; the ratio between bars 606b and 606a was .72; the ratio between bars 608b and 608a was .95; the ratio between bars 610b and 610a was .68; the ratio between bars 612b and 612a was .98; the ratio between bars 614b and 614a was .97; and the ratio between bars 616b and 616a was .95.

[0058] FIG. 6B shows bars 652a, 654a, 656a, 658a, 660a, 662a, 664a, and 666a that represent the measured cell viability based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively. Bars 652b, 654b, 656b, 658b, 660b, 662b, 664b, and 666b represent the cell viability measurements based on the application of different respective magnetic field intensities of .119, .176, .298, .358, .392, .527, .894, and 1.176 mT respectively followed by the application of 40 pM H202. Bars 652c, 654c, 656c, 658c, 660c, 662c, 664c, and 666c represent the measured cell viability based on solely the application of 40 pM H202.

[0059] The cell viability ratios of the bars 652b, 654b, 656b, 658b, 60b, 662b, 664b, and 666b relative to the respective bars 652a, 654a, 656a, 658a, 660a, 662a, 664a, and 666a were determined and used to assess the activation level. Thus, the ratio between bars 652b and 652a was .68; the ratio between bars 654b and 654a was .73; the ratio between bars 656b and 656a was .85; the ratio between bars 658b and 658a was .78; the ratio between bars 660b and 660a was .92; the ratio between bars 662b and 662a was .85; the ratio between bars 664b and 664a was 1.01; and the ratio between bars 666b and 666a was 1.10.

[0060] As shown in the results in FIGs. 6A-6B, the nano-pulsed magnetic field treatment alone just slightly inhibited the growth of B16-F10 cells when the magnetic field strength increased from 0.119 mT to 0.392 mT. However, the H202 treatment on the nano-pulsed magnetic field treated B16-F10 cells caused noticeable stronger cytotoxicity compared with the B16-F10 cells without magnetic field treatment. For the H202 (20pM) treatment in FIG. 6A, the ratio of the cell viability from the H202 treatment and magnetic field application (Case 2) to cell viability of just the magnetic field treatment (Case 3) tended to decrease as the magnetic field strength increased. The ratio of cell viability from Case 2 to Case 3 reflected the activation level. Thus, a low magnetic field facilitated the activation of B16-F10 cells. Just extending the magnetic field treatment time did not noticeably change activation level on B16- F10 cells as shown in comparing the results in FIG. 6 A to FIG. 6B.

[0061] The nano-pulsed magnetic field treatment effectively enhanced the cytotoxicity of H202 on B16-F10 cells. A ratio of Case2/Case 3 of 0.68 could be achieved when the 20mM H202-treated B16-F10 cells were pre-treated by the nano-pulsed magnetic field for one hour with a magnetic field strength of 0.392 mT (as shown by the bars 610b and 610c) or by nano- pulsed magnetic field for two hours with a magnetic field strength of 0.119 mT (as shown by the bars 652b and 652c). In these two cases, the H202 inhibition rate of cells growth increased from 0% to 32% and from 3% to 34%, respectively as shown in FIGs. 6A-6B.

[0062] FIG. 7A-7D are test results for the effect of frequency on the activation effect of nano-pulsed magnetic fields on cancerous cells. The magnetic field strengths of the magnetic field treatment were 0.119mT, 0.176mT, 0.298mT, and 0.358mT at different frequencies (1 Hz, 5 Hz, 10Hz, and 20 Hz) for each set of results. The magnetic field treatment lasted one hour in each experiment.

[0063] FIG. 7A is a graph of the results of tests performed at a field intensity of .119 mT. FIG. 7A shows bars 702a, 704a, 706a, and 708a, that represent the measured cell viability based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively. Bars 702b, 704b, 706b, and 708b represent the cell viability measurements based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively, followed by the application of 20 mM H202. Bars 702c, 704c, 706c, and 708c represent the measured cell viability based on the application of 20 mM H202. The cell viability ratios of the bars 702b, 704b, 706b, and 708b relative to the respective bars 702a, 704a, 706a, and 708a were determined and used to assess the activation level. Thus, the ratio between bars 702b and 702a was .68; the ratio between bars 704b and 704a was .72; the ratio between bars 706b and 706a was .85; and the ratio between bars 708b and 708a was .79. [0064] FIG. 7B is a graph of the results of tests performed at a field intensity of .176 mT. FIG. 7B shows bars 712a, 714a, 716a, and 718a, that represent the measured cell viability based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively. Bars 712b, 714b, 716b, and 718b represent the cell viability measurements based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively, followed by the application of 20 mM H202. Bars 712c, 714c, 716c, and 718c represent the measured cell viability based on the application of 20 mM H202. The cell viability ratios of the bars 712b, 714b, 716b, and 718b relative to the respective bars 712a, 714a, 716a, and 718a were determined and used to assess the activation level. Thus, the ratio between bars 712b and 712a was .64; the ratio between bars 714b and 714a was .69; the ratio between bars 716b and 716a was .86; and the ratio between bars 718b and 718a was 1.02. [0065] FIG. 7C is a graph of the results of tests performed at a field intensity of .298 mT. FIG. 7C shows bars 722a, 724a, 726a, and 728a, that represent the measured cell viability based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively. Bars 722b, 724b, 726b, and 728b represent the cell viability measurements based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively, followed by the application of 20 mM H202. Bars 722c, 724c, 726c, and 728c represent the measured cell viability based on the application of 20 pM H202. The cell viability ratios of the bars 722b, 724b, 726b, and 728b relative to the respective bars 722a, 724a, 726a, and 728a were determined and used to assess the activation level. Thus, the ratio between bars 722b and 722a was .66; the ratio between bars 724b and 724a was .84; the ratio between bars 726b and 726a was .99; and the ratio between bars 728b and 728a was .90. [0066] FIG. 7D is a graph of the results of tests performed at a field intensity of .358 mT. FIG. 7D shows bars 732a, 734a, 736a, and 738a, that represent the measured cell viability based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively. Bars 732b, 734b, 736b, and 738b represent the cell viability measurements based on the application of different respective magnetic field frequencies of 1 Hz, 5 Hz, 10Hz, and 20 Hz respectively, followed by the application of 20 pM H202. Bars 732c, 734c, 736c, and 738c represent the measured cell viability based on the application of 20 pM H202. The cell viability ratios of the bars 732b, 734b, 736b, and 738b relative to the respective bars 732a, 734a, 736a, and 738a were determined and used to assess the activation level. Thus, the ratio between bars 732b and 732a was .94; the ratio between bars 734b and 734a was 1.15; the ratio between bars 736b and 736a was 1.10; and the ratio between bars 738b and 738a was 1.13. [0067] The cell viability ratio of grey bar/orange bar was marked in FIGs. 7A-7D, which was used to assess the activation level. A series of marked areas show the cases with noticeable activation phenomenon (Case 2 (grey bar)/ Case 3 (orange bar) < 0.95). The results were shown as the mean ± s.d.. Thus, in FIG. 7A, all the tested frequencies showed noticeable activation phenomenon as highlighted by a marked area 740. In Fig. 7B, the 1, 5, and 10 Hz frequencies showed noticeable activation phenomenon as highlighted by a marked area 742. In Fig. 7C, the 1, 5, and 20 Hz frequencies showed noticeable activation phenomenon as highlighted by marked areas 744 and 746. In Fig. 7D, the 1 Hz frequency showed noticeable activation phenomenon as highlighted by a marked area 748. [0068] A nano-pulsed magnetic field activates a melanoma cell line B16-F10 after one or two hours of pretreatment with a frequency of 1 ~ 20 Hz and a magnetic field strength of 0.119 ~ 0.527 mT. The magnetic field activated melanoma cells were much more vulnerable to the cytotoxicity of H202 than the melanoma cells without the magnetic field treatment. The activation level could be modulated by controlling the magnetic field strength and the frequency of the applied nano-pulsed magnetic field. The nano-pulsed magnetic field treatment with weaker magnetic field strength and a smaller magnetic frequency generates a stronger activation effect on melanoma cells. The nano-pulsed magnetic field treatment did not activate the pancreatic cancer cell line PA-TU-8988T cells, which demonstrated that the magnetic field- based activation was cellular specific to cells such as B16-F10.

[0069] The above describe methods using nano-pulsed magnetic field enhance the anti cancer efficacy of using ROS-based modalities through a physical activation on the cancer cells. The activation by nano-pulsed magnetic field relates to the effect of a nano-pulsed magnetic field on the cellular functions. A magnetic field can affect the function of different channels on the cytoplasmic membrane, such as Calcium (Ca2+) channels. The functional change of ion channels may facilitate the damage from ROS. More importantly, the magnetic field can penetrate the skin and may deliver its activation effect on the deep tumors, which may enhance the efficacy of the chemotherapy.

[0070] Therefore, a combination treatment with nano-pulsed magnetic field and a cancer treatment such as ROS enhances efficacy, minimizes the necessary dose and thus reduces side effects. The sensitization of cancerous cells using a nano-pulsed magnetic field with other forms of treatments to eradicate cancerous cells may incorporate the disclosed principles. Such treatments may include various standard care chemotherapy drugs, RNS or ROS treatment, or radiation treatment. The chemotherapy or radiation or other treatment is not administered by the same device as the nano-pulsed magnetic field generation system. The treatment may not be a separate system but may be chemotherapeutics taken, for example, by oral administration. Other treatments may include via injection and various routes for injection like intravenous, intratumoral, intramuscular, subcutaneous. Although the examples above relate to melanoma cells, other similar types of cancerous cells may be treated. Thus, the standard treatment for such cells may be reduced below a baseline level to account for the sensitized cells. For example, a relatively smaller volume of reactive species or chemotherapeutics may be used in conjunction with the electro-magnetic field treatment in comparison with the baseline volume required if the electro-magnetic field treatment is not applied. [0071] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. [0072] While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein, without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments. Rather, the scope of the invention should be defined in accordance with the following claims and their equivalents.

[0073] Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur or be known to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.