Patent Application
20220202489
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The present invention relates generally to treatment of cancer cells and specifically use of cold atmospheric plasma in combination with magnetic field generation to target cancer cells.
Cancer is a well-known health issue. There is a large amount of research geared toward effective treatment of cancer. One area of the research has been directed toward methods of eradicating cancerous cells. Many known methods are problematic because although they result in effective eradiation of cancer cells they also kill healthy cells.
It has been reported since the late 1970s that magnetic fields appear to have a strong effect on biological systems. Research of an electromagnetic field effect on biological systems advanced after Wertheimer and Leeper [1979] found that the likelihood of developing leukemia in children increased as they were present in 60 Hz electromagnetic fields. As the research progressed, it appeared as though the vibrational energy levels in the ion-protein complex were pumping into the system, which was creating parametric resonance. This occurs when the atoms shake slightly. This “shaking” is an anomaly that can change ion flux through the cell membrane or cell mobility. It has been shown that extremely low frequency (ELF) magnetic fields influence physiological processes such as plasma membrane structure modification and the initiation of the signal cascade pathways interference in different organisms. Cell membrane morphology modification by ELF was again reaffirmed by Ikehara et al. [2003], who found that exposure to the ELF magnetic field has reversible effects on the N—H inplane bending and C—N stretching vibrations of peptide linkages, and changes the secondary structures of α-helix and β-sheet in cell membrane proteins.
In the past few decades, cold atmospheric plasma (CAP) has been widely used in various fields such as material processing, bacterial inactivation, wound healing, cut coagulation, cancer therapy, and viral destruction. The temperature of heavy species in CAP is usually close to room temperature, allowing its application to living tissue treatment. Although plasma can selectively kill cancer cells, long time exposure can still damage the normal cells around the tumor.
As proved by numerous studies, cold atmospheric plasma can kill various kinds of cancer but enhancing the efficiency of cold atmospheric plasma on cancer therapy has not been widely studied.
Cells are also being tested in order to examine how static magnetic fields (SMFs) affect apoptosis. Based on the findings of Fanelli et al. [1999], SMFs (0.6-6 mT) exert a strong and reproducible effect of reducing U937 and CEM (normal cell lines) apoptosis. This effect is mediated by the ability of magnetic fields to increase Ca2+ influx since its inhibition abrogated the antiapoptotic effect of the magnetic field. On the other hand, Raylman et al. [1996] showed the growth of three cancerous cell lines (HTB 63, HTB 77 IP3, and CCL 86) exhibited a significant reduction in viability after lengthy exposures (64 hours) to very high uniform static magnetic fields at 7 T. Potenza et al. [2004] reported that alterations in terms of increased Escherichia coli cell proliferation and changes in gene expression with a long incubation time (up to 50 hours) were induced by static magnetic field.
Thus, there is a need for a combined cold atmospheric plasma and magnetic field based system for eliminating cancer cells. There is a further need for incorporation of a magnetic field to selectively apply the plasma to an infected area. There is a further need for a cold atmospheric plasma based system that preserves healthy cells while eliminating cancer cells.
According to one example, a system for treatment of an area having cancerous cells is disclosed. The system includes a plasma device to generate a cold atmospheric plasma jet directed at the area having cancerous cells. A magnet generates a magnetic field directed at the area having cancerous cells. A controller is coupled to the plasma device to control the plasma jet generated by the plasma device.
Another example is a method of eradicating cancerous cells in an area. A gas is ionized to create a cold atmospheric plasma jet. The plasma jet is directed toward the area of cancerous cells. A magnetic field is generated in the area of cancerous cells.
Another example is a system for treatment of an area having cancerous cells. A plasma device generates a cold atmospheric plasma jet directed at the area having cancerous cells. The system includes a particle container containing nanoparticles. A magnet generates a magnetic field to magnetize the nanoparticles. An injector injects the nanoparticles into the area having cancerous cells. A controller is coupled to the plasma device and the magnet to control the plasma jet generated by the plasma device and control the magnetic field generated by the magnet.
Additional aspects of the invention will be apparent to those of ordinary skill in the art in view of the detailed description of various embodiments, which is made with reference to the drawings, a brief description of which is provided below.
While the invention is susceptible to various modifications and alternative forms, specific 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 invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
The cold plasma emitter device 104 includes a power supply 112, a gas source 114, and a delivery mechanism 120. In this example, the delivery mechanism 120 is an elongated syringe having a main body 121. The body 121 may be made by glass or a rigid plastic, but also can be made of a flexible material. A proximal end of the body 121 is sealed 124 and an opposite distal end has a discharge area 122. The distal discharge end 122 of the syringe body 121 has a narrowed neck and a distal opening or nozzle 129. A central electrode 126 is located at the center of the body 121 at the interior of the body 121 at the central longitudinal axis of the syringe 120. The central electrode 126 enters the syringe 120 at the sealed proximal end of the body 121 and extends the length of the body 121 to approximately the discharge end 122. A sealing plug 124 (such as rubber) is located over the open end of the syringe 120 to prevent the gas from escaping from the inside of the syringe 120. The electrode 126 is entirely surrounded by insulation except at its distal end, which is exposed and in contact with gas and plasma. The insulation allows the power to be focused at the exposed distal end to lead to the discharge at the end. The central electrode 126 and surrounding insulation, has a proximal end that extends to the outside of the syringe 120 through an opening in the plug 124. The plug opening forms a friction fit with the insulation, so that gas does not escape from the syringe 120. The central electrode 126 is positioned inside the body 121 of the syringe 120, except for the portion of the proximal end of the electrode 126 that extends into and through the plug.
In this manner, the plug opening holds the electrode 126 and insulation in position within the syringe 120, with the distal end of the electrode 126 facing the distal nozzle 129 of the syringe body 121. In addition, an annular outer ring electrode 128 is located about a portion of the narrow neck at the outside of the syringe 120. The electrodes 126 and 128 are high voltage electrodes. The central electrode 126 may be, for instance, a wire, and the insulation can be a ceramic insulation. The high voltage power supply 112 is electrically connected to the electrodes 126 and 128 and provides a high voltage supply to the electrodes 126 and 128 through cables. The controller 108 regulates the voltage and frequency that is applied to the central electrode 126 and the ring electrode 128 and therefore controls the intensity of a plasma jet 130 emitted by the nozzle 129.
The gas source 114 is in gas communication with the delivery device 120 through a supply tube. The supply tube is connected to a port located on the plug 124 of the syringe 120. The supply tube 118 may also be connected to the syringe 120 through an adapter. The gas source 114 can be pressurized, so that gas travels through the supply tube 118 into the inside space of the syringe body 121. A separate gas controller (not shown) may be provided to control the flow rate of the gas in the supply tube 118, or the gas controller may be integrated with the controller 108. The gas then continues through the syringe 120 and exits the syringe 120 through the neck and nozzle 129 at the discharge end 122 as the jet or stream flow 130.
As the gas enters the discharge area 122 and the neck of the syringe 120, the electrodes 126 and 128 excite the gas, thereby ionizing the gas to form a cold plasma jet. In this example, the gas is helium, though other gases such as nitrogen may be used. Thus, as the gas is discharged out of the distal nozzle 129 of the syringe 120, it is a cold plasma jet. The cold plasma jet or stream flow 130 diffuses over time. In accordance with this example, the plasma is provided at a flow rate of 17 liters per minute, with the voltage supply being 5 kV and 30 kHz. At that configuration, the plasma will have a high ionization as it exits the syringe 120. Accordingly, the syringe 120 is preferably placed at a predetermined distance from the target cells of the patient 102 being treated. The syringe 120 allows the plasma to be targeted at desired cancer cells in the skin to selectively eradicate the cancerous cells and reduce tumor size. The syringe 120 may be utilized, for instance, to treat any cancer type that is close to the skin and can be applied without surgery, such as breast, colon, lung, bladder, or oral. With surgery, the system 100 may be applied to any tumor. In this example, the flow rate may be 10-17 liters/min., with a voltage of 2-5 kV and frequency of 30-35 KHz, and a nozzle 129 of 3-5 mm diameter and a distance between the central electrode 126 and the ring electrode 128 of 5-10 mm. The plasma preferably has a density of about 3×10 to 9×10-cm3, such as discussed in “Temporary-resolved measurement of electron density in small atmospheric plasmas,” to Shashurin et al, Applied Physics Letters 96, 171502 (2010), which is hereby incorporated by reference. At the predetermined distance, the plasma will have diffused to a desirable level. However, the intensity of the plasma will continue to decrease as the target area is moved further from the syringe 120, and the plasma will be essentially entirely dissipated at a distance of 5 cm from the syringe 120 in this example. The plasma is well collimated the entire length up to about 5 cm from the syringe 120. The plasma jet stream is discontinuous and represents a series of propagating plasma bundles.
It should be apparent, however, that other suitable settings may be utilized. Preferably, however, the power supply 112 has a voltage from about 2-5 kV with a frequency of about 30 kHz, and the gas has a flow rate of about 2-17 l/min.
The magnetic field generator 106 includes an electromagnet 142 that is coupled to a power regulator 144 to generate a magnetic field 146 around the area of the patient 110. The electromagnet 142 may be moved to focus the magnetic field in the area where the plasma jet stream 130 from the syringe 120 is focused. The strength of the magnetic field 146 may be controlled by the controller 108.
As will be explained below, the plasma jet 130 in combination with the magnetic field 146 in
The controller 108 is coupled to a magnetic field generator 172 that controls an electromagnet 174. The electromagnet 174 generates a magnetic field 176 in a particle chamber 178. The particle chamber 178 holds nanoparticles 180 that are magnetized by the magnetic field 174. The magnetized magnetic nanoparticles 180 are delivered to the area of the patient 102 via an injector 182. The magnetic nanoparticles 180 are thus delivered to emit a magnetic field on cancerous cells in conjunction with the plasma jet 130.
The treatment system 170 allows generating the magnetic field in areas with high concentrations of cancerous cells. Thus, the effect of the magnetic field on surrounding areas with normal cells is bypassed. The magnetic nanoparticles 180 target only the cancer cells in the injection region. The magnetic nanoparticles 180 may be further guided by a magnet 190 to a specific location for a more focused treatment. Alternatively, the magnetic nanoparticles 180 may be conjugated with a targeting antibody that may be injected in the area of cancerous cells. Such an arrangement may allow a synergetic effect of plasma, magnetic field, and a drug carried by the magnetic nanoparticles.
The cold plasma device in this example produces the plasma jet 202. In this example, the cold plasma device has a configuration of central powered electrode of 1 mm diameter coating with 2 mm ceramic layer and a grounded outer electrode wrapped around the outside of a 4.5 mm diameter quartz tube. The electrodes are connected to a secondary of high voltage resonant transformer with voltage up to 10 kV and a frequency of 30 kHz. The plasma discharge is driven by alternating current (AC) high voltage. The output voltage is set to 3.16 kV. The feeding gas helium (Airgas, Alexandria, Va.) is set at a flow rate of 4.7 l/min. The distance between the cold atmospheric plasma nozzle emitting the plasma jet 202 and the plate 204 was set to 3.5 cm in this example.
The permanent magnet 210 is used to provide a static magnetic field. The magnetic field strength is measured by a gauss meter (GM08 by Hirst Magnetic Instruments, Falmouth, UK). The magnetic fields at the vertex, quarter, center, and end points (spots A, B, C, and D) on the magnet 210 were tested. In this example, various cancerous and healthy cells were tested as well as different conditions such as without a magnetic field to test the effectiveness of the plasma and magnetic field in eradicating cancer cells. Different areas of the magnet 210 for location of the cells shown as spots A, B, C, and D in
In this example, human MDA-MB-231 breast cancer cells were used. In order to show the selective effect of plasma, wild type mouse dermal fibroblasts (WTDF) were also tested under the same conditions. The cells were cultured in Dulbecco's Modified Eagle Medium (Life Technologies) supplemented with 10% (v/v) fetal bovine serum (Atlantic Biologicals, Frederick, Md.) and 1% (v/v) Penicillin and Streptomycin (Life Technologies, Grand Island, N.Y.). Cultures were maintained at 37° C. in a humidified incubator containing 5% (v/v) CO2 (Airgas, Alexandria, Va.). Cells were observed under a Nikon Eclipse TS100 inverted microscope (Nikon Instrument, Md.).
The testing involved both direct and indirect treatment using cold atmospheric plasma. The direct treatment involved pre-seeding cells in 96-well plates such as the plate 204. The 96-well plate 204 may be a Costar 96-well plate available from Sigma-Aldrich, St. Louis, Mo. After 24 hours of incubation the culture media in the wells was replaced by 100 μl fresh culture media. The cells were treated directly under the plasma jet from the plasma device alone and in combination with applying the magnetic field. The magnet 210 was stationary at all times during the treatment. The tip of the plasma jet 202 was aligned with the magnet 210 at a desired spot. The 96-well plate 204 was placed on the magnet 210 and the plate 204 was moved from well to well 206 for exposure to the plasma jet. Since the size of the plasma jet is slightly bigger than the area of each well 206, the cells were plated in every other row and every other column to avoid triple or quadric plasma treatment.
The indirect treatment using cold atmospheric plasma involved warming the cell culture media up to 37° C. and adding the media in blank 96-well plates (100 μl per well) and then treating the cell media by plasma with and without the magnetic field. After treatment, the CAP-stimulated media was immediately transferred to affect the cells, which had been pre-cultured in a 96-well plate for 24 hours (the old media was discarded).
In order to compare the cell activity of plasma treatment with and without the application of a magnetic field, cell viability was monitored using the MTT assay (Sigma-Aldrich, St. Louis, Mo.), which is a colorimetric assay for measuring the activity of mitochondria and cellular dehydrogenase enzymes that reduce 3-[4,5-dimethylthiazol-2-yl]-2,5-dyphenyltetrazolium bromide, MTT, to its insoluble formazan, giving a purple color.
The cells were plated at a confluence of 30000 ml−1, and then incubated for one day to ensure a proper cell adherence and stability. Before treatment, cells were replaced with fresh media, and treated with direct or indirect cold atmospheric plasma followed by an additional incubation at 37° C. for 72 hours. After the incubation, 100 μl of MTT solution per well (7 mg Thiazolyl Blue Tetrazolium Blue in 10 ml medium for one 96-well plate) was added into each well. Reactions were maintained for three hours at 37° C. The MTT solution was aspirated and 100 μl of MTT solvent (0.4% (v/ v) HCl in anhydrous isopropanol) was added to each well to dissolve formazan crystals. Reactions were monitored by a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, Vt.) at a wavelength of 570 nm. The entire set of experiments was repeated four times in duplicates.
In this study, the spectra of the plasma jet alone and with the presence of a static magnetic field were measured to detect the difference of reactive species variation in these two experimental conditions. The spectrometer and the detection probe were purchased from Stellar Net of Tampa, Fla. Integration time of the collecting data was set to 100 ms.
5,6-Chloromethyl-29, 79-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) was purchased from Invitrogen for the general intracellular reactive oxygen species (ROS) measurement. The MDA-MB-231 cells were plated in 96 well plates with 100 μl media and treated as required. Two hours after the treatment, 10 μl of 10X CM-H2DCFDA solution in PBS was added in each well to reach the final concentration of 10 nM. 30 minutes later, the intensity of the fluorescence was read by a Synergy H1 Hybrid Multi-Mode Microplate Reader at an excitation wavelength of 492 nm and emission wavelength of 527 nm. The sensitivity of the reader was set to 100.
A hydrogen peroxide (H2O2) detection kit from Sigma-Aldrich of St. Louis, Mo. was used. The experiment was performed according to the detailed protocols given on the official website. Cells were plated in black clear-bottom 96 well plates. Immediately after the required treatment, the fluorescence intensity of the H2O2 was obtained with a microplate reader (Synergy H1 Hybrid Multi-Mode) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Sensitivity of the reader was set to 60.
Results of four repetitions of each experiment were plotted using Microsoft Excel software (2011) as mean ±standard deviation. Student t-test (for comparison between two groups) and one way ANOVA (for comparison between three groups) were used to check the statistical significance (p<0.05).
The application of cold atmospheric plasma and a static magnetic field were integrated using the testing system 200 shown in
The values of the magnetic field strengths at different spots A, B, C, and D in
U87 cells, the most robust among the four cancer cell lines explained below, were used to test the optimal spot on the magnet 210 having the most significant effect on the cancer cell destruction.
The spectra of a normal helium plasma jet alone and with a static magnetic field at Spot C in
To understand the interaction between the static magnetic field and plasma, cells were isolated from the treatment. Previous studies have demonstrated that the chemical components of plasma-stimulated culture media are modified by the plasma treatment, and this activated media is also capable of inducing the death of cancer cells. The way that cells treated by plasma-activated media, rather than plasma directly, is termed indirect treatment, while the cells treated by plasma jet is termed direct treatment. The indirect treatment offers the possibility of cell isolation from the system of a static magnetic field and plasma.
Cell viability assay was performed to understand the interaction between the static magnetic field, the plasma jet, and the cells. In this study MDA-MB-231 human breast cancer cells were used. In order to show the selective effect of the plasma treatment, wild type mouse dermal fibroblasts (WTDF) cells were also tested under the same conditions. All the cells were also plated at a density of 3000/well and incubated for 24 hours before treatment. The MTT assay was performed at 72 hours after treatment. The viability of cancerous and normal cell lines directly and indirectly treated by helium plasma jet only, a plasma jet with a static magnetic field (SMF), and a plasma jet with a copper board at Spot C on the magnet 210 was assessed.
The cold atmospheric plasma jet, a weakly ionized gas, may be intensified by coupling it to the conductive plate of the magnet 210. Therefore, with magnet 210 being a conductor, the distance between the nozzle of the plasma device and the magnet surface is delicate. If the distance is too far, the intensity of the plasma jet will be significantly reduced so that very little amount of reactive species can reach the media or cells, while if it is too close, the plasma jet will be enhanced at the tip of the jet where it is in contact with the media. The plasma jet coupled to a conducting plate may lead to a much higher amount of the ionized species than the plasma treatment without a magnet, making the data incomparable. Therefore, a non-magnetic ferrite bar of the same material was used as a conductor to replace the magnet 210. The non-magnetic ferrite bar eliminates the factor of the plasma jet enhancement from a conducting plate.
The results of the testing are shown in
A one-way ANOVA test was performed between the cell viability by direct treatment of cold atmospheric plasma and treatment of cold atmospheric plasma with a static magnetic field and treatment of cold atmospheric plasma alone. The p value of this ANOVA test is 1.798E-11, indicating that there are statistical differences between the three treatments. Thus, a student t-test was performed between the three groups of treatments to determine where the significance lay. As presented in
The same calculation was also performed on the indirect treatments as shown in
In order to prove that the effect of the magnetic field alone does not have the ability to activate the media or induce cell death, the cell culture media and the cells were placed in the static magnetic field for 30 seconds respectively. The magnetic field treated media was then transferred to infect the pre-plated cells immediately (indirect treatment). Untreated MDA-MB-231 cells were used as a negative control. Cell viability obtained after 72 hours of incubation is shown in
To further illustrate the static magnetic field does not change the plasma configuration, the generation of H2O2 in the culture media treated by plasma alone and plasma with application of the magnetic field was measured (data was normalized to cold atmospheric plasma treatment).
The cold atmospheric plasma treatment may lead to an increased level of free radicals, which has an impact on cellular activity and may explain the decrease of cell viability. Therefore, to determine if the reactive oxygen species pathways are involved in the mechanism of the plasma and magnetic field treatment further decreasing the cell viability, the production of intracellular reactive oxygen species (ROS) was assessed in cells treated by cold atmospheric plasma alone and cold atmospheric plasma with the application of a magnetic field.
The results shown in
An MTT assay was used again to compare the cell viability of plasma treatment with or without a magnetic field in the way of indirect treatment as shown in
The data presented in the above testing shows that plasma alone, and in combination with a static magnetic field, can selectively induce cancerous cell death. The interaction between cells and plasma has been intensively investigated. In terms of mechanism of cancer therapy, the majority favors the theory of reactive oxygen and nitrogen species (ROS and RNS) generated by plasma (extracellular ROS/RNS) and the intracellular ROS signaling and apoptotic pathways they induce. The intracellular ROS generation is promoted by plasma, which could cause cell death by impairing the function of intracellular regulatory factors. Recent studies have emphasized the importance of H2O2 formation in the culture media treated by plasma. The H2O2 is majorly formed by two ●OH radicals. The toxicity of plasma is highly dependent on H2O2, which has a dominant role in the mechanism of cell death. Reactive nitrogen species (RNS) especially NO and peroxynitrite (ONOOH) are also considered important species that lead to cell death. Peroxynitrite formation in the plasma activated media is through the reaction of NO2− with H2O2 and H+. ONOOH is a powerful oxidant and nitrating agent that is known to be much more damaging to the cells than NO or superoxide because cells readily remove superoxide and NO to reduce their harmful effects, while fail to neutralize ONOOH.
In the treatment system 100 in
The consumption of H2O2 by cells over time has been studied. Each cell line consumed H2O2 at different rates. The concentration of H2O2 halved when stored in room temperature in comparison to −80° C. after three days. However there has been a lack of investigation in the H2O2 decay at different time intervals within one day (0, 30 minutes, 1 hour, 6 hours, and 24 hours). Possible future experiments could help discover more about the role of ROS by treating media with plasma alone and plasma with a magnetic field, then adding H2O2 to the cells after different time intervals from 0 to 3 days. Cell viability of each post-delayed addition of media can be measured to support the decay of H2O2.
As described above, the culture media activated by a plasma jet alone as well as the plasma jet with a static magnetic field was used to affect the cells prepared in the 96-well plates. The indirect treatment isolates cells from the system of the magnetic field and the plasma, showing the interaction is solely between the magnetic field and cells. The results of indirect treatment experiments above have shown that the cold atmospheric plasma and magnetic field activated media can also increase the cell death rate comparing to the plasma activated media alone. However, the cell death rate of indirect treatment is statistically significantly lower than that of direct treatment, suggesting that the mechanism of cells killed by plasma in a static magnetic field could be an outcome of two separate reactions: the magnetic field with cells and the magnetic field with the plasma-activated media.
Previous studies have shown both static and extremely low frequency magnetic fields can interact with biological systems. The possible mechanism of this is the calcium and potassium ions specifically activated by a magnetic field to enhance their transport through membrane ion channels, thereby altering signaling mechanisms and cellular function. While others have demonstrated that prolonged exposure in the static magnetic field may inhibit human cancer cell growth and increase normal cell survival, in the above described tests, a 30-second static magnetic field treatment alone does not induce cell death. Thus the role of static magnetic field effect on the cells may be ruled out of the exploration of mechanism because the cells were not incubated with a static magnetic field for a long period of time as the above studies did.
Cancer cells and normal cells differ in their cell-cell communication, characteristic cell death, repair mechanisms, or other cellular activities. As normal cells, WTDF cells were studied to demonstrate the selectivity of plasma treatment. The breast cancer cells have an 8.5 times higher odds to be killed by plasma treatment than the WTDF cells. To insure plasma and the magnetic field treatment only affect the viability of cancer cells, plasma can be assembled with an endoscope, targeting only an area with a tumor.
Finally, in the system of cells, plasma, and magnetic fields, as discussed above, plasma will generate extracellular ROS and RNS species in media, such as ●OH, H2O2, O2−, NO2−, NO3−, and ONOOH. Plasma-produced ROS (or their reaction products) in media then can either diffuse through the plasma membrane or react with the plasma membrane to produce intracellular ROS. Once ROS enter cells, they can damage intracellular components, or promote or inhibit intracellular signaling pathways. Therefore, one possible way to explore the mechanism of plasma and magnetic field synergy is to monitor the intracellular ROS production. However, the matching ROS level in the cells treated by plasma alone and plasma with a magnetic field leads to a second possibility. These radicals or ions could also be activated by the static magnetic field so that their reaction rate with cells is enhanced. This phenomenon is termed parametric resonance, a phenomenon observed in atomic spectroscopy. This model focuses on the magnetic effect in molecules instead of the ion channel, as in original ion cyclotron resonance hypothesis of Liboff.
Combining cold atmospheric plasma and a static magnetic field achieves an enhanced killing effect on cancer cells. In the system of plasma, cells, and magnetic fields, the magnetic field enhances the efficiency of plasma on cancer therapy through interfering with the cell biological system and reactive species instead of interacting with plasma jet. In addition, the magnetic field may be used to guide the plasma-cell interaction region. As such it has promise to enhance selectivity of the region exposed to the treatment.
Each of these embodiments and obvious variations thereof is contemplated as falling within the spirit and scope of the claimed invention, which is set forth in the following claims.
The present application claims priority to U.S. Provisional Application No. 62/187,500, filed on Jul. 1, 2015, which is hereby incorporated by reference in its entirety.
This invention was made with U.S. government support under Grant No. 1465061 awarded by the National Science Foundation. The Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62187500 | Jul 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15741183 | Dec 2017 | US |
| Child | 17647118 | US |
