Abstract:
The present invention provides a system and method of directing, focusing, or concentrating electrical charges within a defined electric field so that these charges can be used to exert forces on cells and tissues in vivo and/or cell cultures in vitro. The present invention reduces and/or eliminates the damage at a target site that would normally be caused by an electrode that acts as a current source or sink to accomplish the same task.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of prior filed International Application, Ser. No. PCT/US2007/008662 filed Apr. 9, 2007, which claims priority to U.S. provisional patent application No. 60/744,424 filed Apr. 7, 2006 and U.S. provisional patent application No. 60/745,530 filed Apr. 25, 2006 which is hereby incorporated by reference into this disclosure. 
     
    
     GOVERNMENT SUPPORT 
       [0002]    This invention was made with government support under Grant No. DGE-0221681 awarded by the National Science Foundation. The government has certain rights in the invention. 
     
    
     BACKGROUND OF THE INVENTION 
       [0003]    Electroporation is known in the art as a method of treating a tissue in order to transiently increase the tissue&#39;s permeability to enhance molecular transport either for drug delivery or for sampling of interstitial fluids. Electroporation, or electropermeabilization, ellicits a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. Electroporation and electropermeabilization are known in the art. Some chemotherapeutic drugs used in cancer therapy have poor access into the tumor cells. Therefore, electropermeabilization offers an approach for increased drug delivery into the cells and thus better antitumor effectiveness. Tissues can also be electropermeabilized and thus the antitumor effectiveness of chemotherapeutic drugs potentiated by increasing drug delivery into the cells. It is known that electroporation can be performed both in vivo and in vitro. 
         [0004]    Forces resulting from applied electric fields can be used on biological entities such as cells and tissues for many purposes that include cell breakdown, electrophoretic extraction or insertion of molecules, including electroporation or electropermeability, in addition to other forms of molecular manipulation know in the art. 
         [0005]    In a system to apply electric fields to biological entities, a set of electrodes are used to establish an electric field within a biological entity. Currently, there is no system to direct, or focus, the established electric field intensity in in vivo and in vitro situations to a specific location within an electric field defined by the set of electrodes. 
       SUMMARY OF INVENTION 
       [0006]    The present invention consists of a system and method of focusing electric fields for the purpose of exerting electrical forces on biological entities. 
         [0007]    In accordance with the present invention, a system and method are provided for focusing an electric field intensity within a defined electric field to exert electrical forces on a biological entity. In a particular embodiment, the system includes, at least two active electrodes to establish an electric field within the biological entity and at least one passive electrode positioned within the path of the electric field to focus the electric field intensity of the established electric field at a desired location within the biological entity. 
         [0008]    The system further includes a power source coupled to the at least two active electrodes to establish the electric field and software to control the field attributes, such as duration and intensity level. In this configuration, one of the electrodes is considered the source electrode and another of the electrodes is the sink electrode. 
         [0009]    The system of the present invention is operation in both an in vivo and in vitro environment. In the case of an in vivo application, the biological entity may be a cell or group of cells, or a cellular tissue. In the case of an in vitro application, the biological entity may be a cell culture. 
         [0010]    Various embodiments of the passive electrode are within the scope of the present invention. These embodiments include, but are not limited to, a passive electrode that is fabricated of a material from the group includes metals, semiconductors, nanoparticles, plastics, polymers, bio-polymers, fluidistic substances, bio-molecules, antibodies, proteins, DNA, RNA, non-conductive materials and high relative permittivity materials. 
         [0011]    Additionally, the geometric configuration of the passive electrode may take on various forms. These forms include, but are not limited to, a star geometry, a four-comers geometry, a group of conductive needles, or a sphere for positioning below a surface level of the biological entity. The system of claim  1 , wherein the passive electrode is shaped in a star geometry. 
         [0012]    As such, the method of the present invention accomplishes the task of focusing the electric field within the biological entity by using a passive electrode(s) that does not act as a current source or sink for the field as defined by the set of electrodes used to generate the electric field. Accordingly, the present invention provides several advantages over existing technologies. The present invention provides a means of directing electric fields for application to living cells and tissues, facilitates in vivo electroporation in tissues that are in locations that are difficult to access, provides new applications and protocols for existing proprietary electroporation and/or electrofusion technologies, reduces and/or eliminates direct harm to cells and tissues at desired treatment sites caused by direct contact with active electrodes, and greatly improves the directional focus of the electric field. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0013]    The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
           [0014]    For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which: 
           [0015]      FIG. 1  is an illustrative example of an embodiment of the present invention wherein the electric field running between two electrodes is concentrated in the perpendicular direction of the applied electric field when using highly conductive materials. 
           [0016]      FIG. 2  is an illustrative example of an embodiment of the present invention wherein electric fields are concentrated in the direction of the applied electric field when using materials with relatively high permittivity. 
           [0017]      FIG. 3  illustrates the results corresponding to the magnitude of the electric field intensities resulting from the simulation wherein there is no passive electrode. 
           [0018]      FIG. 4  illustrates an embodiment of the present invention showing the results corresponding to the magnitude of the electric field intensities resulting from a simulation wherein there is a passive electrode with a “star” geometry is positioned within the field. 
           [0019]      FIG. 5  illustrates an embodiment of the present invention showing the results corresponding to the magnitude of the electric field intensities resulting from a simulation wherein there is a passive electrode with a “four corners” applicator geometry. 
           [0020]      FIG. 6  illustrates actual results for a prototype “star” geometry applicator in accordance with the present invention. 
           [0021]      FIG. 7  illustrates the results of improved electroporation at the site of an implantable device in accordance with the present invention. 
           [0022]      FIG. 8  illustrates the results of improved electroporation for a needle electroporation applicator in accordance with the present invention. 
           [0023]      FIG. 9  illustrates a simulation of an implantable electroporation device that concentrates electric fields under living tissue employing a sphere. 
           [0024]      FIG. 10  illustrates a simulation of an implantable electroporation device that concentrates electric fields under living tissue employing a hollow sphere. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0025]    The method in accordance with the present invention takes advantage of a set of electric phenomena that occurs with changes in materials through an electric field&#39;s path. The electric field is redirected or concentrated in different places depending on the materials and different design geometries. How the method uses materials that vary in geometries and electrical characteristics such as conductivity and permittivity and different arrangements and geometries can be better explained by simple abstractions such as those in  FIG. 1 , where the electric field running between two electrodes is concentrated in the perpendicular direction of the applied electric field when using highly conducting materials, and in  FIG. 2 , where the electric field is concentrated in the direction of the applied electric field when using materials with relatively high permittivity. 
         [0026]    With specific reference to  FIG. 1 , a 2D simulation illustrates an electric field running from the left electrode  10  to the right electrode  15 . In this exemplary embodiment, the circles  20  in between the electrodes are made of platinum with the background being a vacuum. Platinum is an exemplary material and other conductive materials are within the scope of the present invention. As illustrated with reference to the accompanying legend  25 , the highest electric field is shown as red shading and the lowest electric field is shown as blue shading. As such, the highly conductive circles  20  are effective in concentrating the electric field in the vertical direction (y)  30  when the applied electric field is running in the horizontal direction (x)  35 . 
         [0027]    With specific reference to  FIG. 2 , a 2D simulation illustrates an electric field running from the left electrode  10  to the right electrode  15 . In this exemplary embodiment, the circles  20  in between the electrodes are made of mica with the background being a vacuum. Mica is an exemplary material and other materials having a substantially high permittivity are within the scope of the present invention. As illustrated with reference to the accompanying legend  25 , the highest electric field is shown as red shading and the lowest electric field is shown as blue shading. As such, the high relative permittivity of the circles of mica  20  are effective in concentrating the electric field in the horizontal direction (x)  35  when the applied electric field is running in the same horizontal direction (x)  35 . 
         [0028]    The system in accordance with the present invention consists of a set of electrodes, having at least one current source electrode and at least one current sink electrode and a power source, a passive electrode and supporting software. The set of electrodes and associated power source are used to establish an electric field either in vivo or in vitro. The passive electrode is then placed in close proximity to the established electric field. The passive electrode is used to direct, concentrate, and/or focus charge at the location of the passive electrode and away from the current source and sink electrodes that define the shape of the overall electric field. This focused charge in an in vivo and/or in vitro application dramatically reduces, if not eliminates, the adverse effects of Joule heating and redox reactions that would occur at the location of the passive electrode if a current source or sink electrode were used instead of the passive electrode. 
         [0029]    In accordance with the present invention, a passive electrode refers to a device that does not contain a source of electrical energy. 
         [0030]    With reference to  FIG. 3 ,  FIG. 4  and  FIG. 5 , an electrostatic simulation is illustrated as proof of concept of the invention. In this embodiment, a 5 cm plastic well with PBS solution is provided and four active electrodes  50  are used to apply current to the solution. 
         [0031]    In  FIG. 3 , a passive electrode is not provided and the corresponding magnitudes of the electric field intensities that result from the simulation are illustrated, along with the legend  25  identify the associated colors. As shown, the lowest field intensity value is drawn in dark blue  55  (starting at 0V/m), and the most intense is drawn in red  60  (ending at 1.5V/m). It is expected that the areas where the electric field intensity is highest are the areas where charge will be focused and electroporation/electrofusion will be facilitated. 
         [0032]    In  FIG. 4 , a passive electrode  65  with a “star” geometry is used and the corresponding magnitudes of the electric field intensities that result from the simulation are illustrated, along with the legend  25  identify the associated colors. As shown, the lowest field intensity value is drawn in dark blue  55  (starting at 0V/m), and the most intense is drawn in red  60  (ending at 1.5V/m). It is expected that the areas where the electric field intensity is highest are the areas where charge will be focused and electroporation/electrofusion will be facilitated. 
         [0033]    In  FIG. 5 , a passive electrode  70  with a “four corners” geometry is used and the corresponding magnitudes of the electric field intensities that result from the simulation are illustrated, along with the legend  25  identify the associated colors. As shown, the lowest field intensity value is drawn in dark blue  55  (starting at 0V/m), and the most intense is drawn in red  60  (ending at 1.5V/m). It is expected that the areas where the electric field intensity is highest are the areas where charge will be focused and electroporation/electrofusion will be facilitated. 
         [0034]    The simulation results illustrated in  FIG. 3-FIG .  6 , are shown in a 2 dimensional space, but applications and design of applications are accomplished in a 3 dimensional space. The geometries presented here serve to indicate concept feasibility. Actual applicator design will be adjusted to the reality of cell and/or tissue 3D space. 
         [0035]      FIG. 6  illustrates how the concept of the present invention is proven by generating electroporation in the zone where the electric fields should be higher according to the simulation in  FIG. 4 , as previously described.  FIG. 6  illustrates actual results for a prototype “star” geometry passive electrode as described in the simulations presented above. The “greenish” color is the result of electroporation and subsequent florescence of an electroporation sensitive biomarker. The four peripheral green circular areas in the figure indicate the electroporation that has occurred at each of the four current source/current sink active electrodes. The color region in the middle of the figure reflects electroporation at the passive “star” configured electrode. The absence of color in the remaining area of the figure illustrates that electroporation is limited to the targeted electroporation site at the location of the passive electrode. 
         [0036]    The materials that compose the active electrodes and/or the passive electrodes include but are not limited to the following: 
         [0037]    (1) Metals, (2) Semiconductors, (3) Nanoparticles, (4) Non-metals such as plastic, Polymers, Bio-polymers, (5) Fluidistic substances, (6) Bio-molecules such as antibodies, proteins, DNA, and RNA, (7) Non-conductive materials and (8) High relative permittivity materials. 
         [0038]    Additionally, the passive electrode can be anything that is in the path of the electric field, and may be native to the biological system that is being treated and thus does not need to be introduced prior to the electric field being established. The passive electrode is positioned in the path of the electric fields between the sink and the source electrode(s). Passive electrodes can be inside or outside the biological entity, and are not limited to foreign entities, but in additional may be intrinsic to the entity. For example, the active electrodes may be positioned such that a bone within the biological entity becomes the passive electrode. 
         [0039]    The passive electrode may take the form of a fluid which can change its shape, volume and conformation to change its field distribution characteristics. When the passive electrode is a fluid, the fluid will take on different concentration profiles and 3D shapes over time. It is also know that a fluid passive electrode can change its position or be induced to change its position relative to the active electrodes as necessary. 
         [0040]    The passive electrode in accordance with the present invention may be inside or outside of the biological entity. In addition, it can remain permanently with the biological entity or be removed after it has served its purpose. Alternatively, the passive electrode may become an active electrode. For example, a passive electrode that experiences a chemical reaction that results in a potential difference, or a passive electrode that is grounded after it has served its purpose would result in a change from a passive electrode to an active electrode. Additionally, the electrical characteristics of the passive electrode may change so that the electric field is affected differently. For example, the passive electrode may be fabricated of a material that changes conductivity under temperature conditions. 
         [0041]    When utilizing an in vivo application, the device and components could be located in any tissue of a living being not restricted to a single geographic localization. 
         [0042]    Possible geometries are not limited to those in the proof of concept device; such geometry was only used to demonstrate the principle behind the application of electric fields by passive electroporation on living cells and biomolecules. 
         [0043]    With reference to  FIG. 7 , in an exemplary embodiment it is shown that improved electroporation can be accomplished at the site of an implantable device. The concentration of electric fields can be induced at a specific site by using micro-metallic beads or composed material beads. In this embodiment an applied electric field is concentrated in living tissue near the site of an implantable device in vivo. In this embodiment, the electric fields are concentrated at the place of interest. These electric fields are concentrated in the larger green volume  85  in between the two electrodes  90  in the image because of micro-beads of conductive material that are suspended in a hydrogel or other type of biocompatible matrix. 
         [0044]    With reference to  FIG. 8 , in an exemplary embodiment it is shown that improved electroporation can be accomplished utilizing a plurality of needles as the passive applicator. By using a combination of materials as composing the needles, it is possible to direct currents in specific directions or depths.  FIG. 8  shows an electric field as it is applied from electrodes  95  located at the left and right in the volume. In this embodiment, passive conductive needles  100  are added in between the active electrodes, with no other changes to geometries or variables, the charges are concentrated at the location of the passive needles. This configuration improves current state of the art in vivo electroporation devices. 
         [0045]    With reference to  FIG. 9  and  FIG. 10 , in an exemplary embodiment it is shown that improved electroporation can be accomplished utilizing an implantable electroporation device.  FIG. 9  and  FIG. 10  show a simulation of an implantable electroporation device  105  that concentrates electric fields under living tissue. The first configuration, illustrated in  FIG. 9 , is an implanted sphere  105  that serves to concentrate charges established by the electrodes  110  under living tissue conditions. Additionally, as illustrated in  FIG. 10 , the sphere can be either hollow or filled with a different material, and the surface of the sphere  105  may have a set of shell particles  115  that help concentrate charges as shown by the higher electric fields (red dots around surface of sphere). Different configurations of this device can achieve many applications. Additionally the passive electrode may take the form of a container for any agent. For example, the passive electrode may be a hollow sphere containing a drug for delivery to the biological entity. 
         [0046]    In an additional embodiment, the passive electrode may function as a scaffold for biological structures. For example, the passive electrode may function as a scaffold for bone growth when used in vivo. 
         [0047]    In an additional embodiment, the passive electrode is labeled to allow the identification of the position of the passive electrode within the entity at any point in time. The passive electrode may be fluorescent labeled, radio-labeled, magnetically labeled or contrast agent labeled, in addition to other such labeling techniques known in the art. 
         [0048]    It will be seen that the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween. Now that the invention has been described,