TECHNICAL FIELD

This invention relates to electroporation of cells in vivo, particularly cells of a patient's tissues. More specifically, this invention relates to novel devices and methods for delivering molecules to cells situated at, near and/or adjacent to a predetermined insertion track site of an elongate single-needle electrode. Still more specifically, the invention concerns the electroporated delivery of substances into cells along and in the vicinity of the needle track made by insertion of the electrode from the surface of a tissue and into the tissue to a depth of from 3 millimeters to 3 cm, which tissues can comprise any tissues, including without limitation skin, striated and smooth muscle, mucosa, and organs.

BACKGROUND

The following description includes information that may be useful in understanding the present invention. It is not an admission that any such information is prior art, or relevant, to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.

Electroporation has been applied to delivering molecules to subsurface tissues using various multiple-electrode designs such as arrays of two or more electrodes that typically are designed as needle electrodes for insertion into said tissue. Generally, such arrays define a treatment zone lying between the needle electrodes of the array. Such treatment zones therefore comprise a three dimensional volume of tissue wherein cells within the treatment zone are exposed to an electric field of an intensity sufficient to cause temporary or reversible poration, or even sometimes irreversible poration, of the cell membranes to those cells lying within and or near the three dimensional volume.

Current practices for electroporating cells in tissue include use of significant voltages in order to impart through the three dimensional treatment zone a relatively uniform electric field. By “relatively uniform” is meant that electric lines of force coincident with application of an electric pulse sufficient to cause poration is imparted across the cells somewhat evenly throughout the three dimensional treatment zone volume. Ultimately, a large number of electrode needles combined with large injection volumes and high electrical fields have been necessary to ensure a sufficient overlap between an injected drug and the tissue volume experiencing the electrical field since typically, the injection bolus that is delivered to the tissues quickly spreads from the injection site. Use of high electric fields and large electrode arrays has several drawbacks. For example, use of many needles and high electric field (voltages) causes more pain while high injection volume makes dosing difficult to control as it causes waste of the drug (most of the drug is not getting into the cells as it will be outside the treatment zone). Also, use of such multiple needle devices is cumbersome and a cause for apprehension from the standpoint of the patient.

Besides the invasive aspect of a device with multiple needles, typical electroporation techniques, as stated above, result in variability in electroporation of cells within a treatment zone. This is a drawback to medical use of electroporation in that dispersion of treatment molecules of the injected bolus into surrounding tissue results in loss of control as to the amount of such treatment molecule that is ultimately transfected into cells within the treatment zone by the electroporation event. Thus, a need exists in the electroporation arts for a device and method to narrow or refine control over “dosing” of treatment molecules into specific and well defined delivery sites within a patient's tissue. Likewise, there is still a need in the art for methodologies and devices that can electroporate with less invasiveness and impart less pain from the electric field pulse employed in the delivery of therapeutic substances to various tissues including skin, muscle, mucosa and organs.

SUMMARY

In a first embodiment, this invention provides for electroporation of cells in situ, particularly cells that are located subcutaneously, intradermally, subdermally, and/or intramuscularly (particularly skeletal muscle, striated, and smooth muscle, e.g., heart, muscle). In a related embodiment, the invention provides for the electroporation of cells near and/or adjacent to the track made by insertion of the single elongate needle electrode into tissue. For example, cells that become electroporated using the invention device are those situated within a radius from the needle track anywhere from between 0.0 and 5 mm so as to comprise a generally cylindrical treatment zone imparted by the novel design and pulsing of and of the electric field imparted into the tissue by the single-needle electrode.

In a second embodiment, the invention provides for any number of structural arrangements providing for at least two opposite electrode leads (i.e., at least one anode and at least one cathode) situated in association with a single elongate electrically inert shaft, which shaft itself can comprise electrodes and an electrically inert material, such as a medically acceptable plastic or polycarbonate, filling the space between the electrodes a 0.05 mm to a 1.5 mm between, or can comprise just elongate opposing spaced electrodes. In either embodiment, the electrodes of the tissue penetrating single needle electrode or electrode containing shaft have spaced dimensions of between 0.05 mm and 1.5 mm. In a related embodiment, the electrodes themselves can have a length exposed along the elongate shaft anywhere from the whole needle length to just a section of the needle, such as near the shaft penetration tip. Further, the electrodes can have cross sectional dimensions of between 0.005 and 0.80 mm. In yet another structural arrangement embodiment, the single needle electrode can comprise a hypodermic needle comprising at least two elongate electrodes spaced along at least a portion of the length of the hypodermic needle exterior. For example, the hypodermic needle can include at least two electrodes (i.e., an anode and a cathode) running along a portion of the length of the needle. (SeeFIG. 1.) In working embodiments, each electrode is connected to a source of electric energy for generating an electric field between opposite poles, i.e., one electrode is an anode and the other a cathode electrode. In other examples, multiple electrodes can be formed on the exterior of a hypodermic injection needle such as disclosed inFIG. 3Acomprising multiple straight and parallel electrodes, or as depicted inFIGS. 2 and 4comprising multiple electrodes spiraled around the injection needle. In still further embodiments, the single-needle electrodes can be manufactured using any number of well understood methods including etching and layering per Micro electro-mechanical systems (MEMS) technologies. In such manufacturing methods, micromachining processes are used to add or strip away layers of substances important to the proper annealing, insulation, and conduct of electric pulses and circuitry.FIGS. 13A, B, C, D and E are photographs of the embodiment wherein the electrodes are etched on to the delivery needle shaft. Specifically, gold electrode layering has been coated above a layer of and inert substance (parylene) which itself had been layered over the hypodermic needle shaft. Additional methods for manufacturing the elongate electrodes include extrusion technologies wherein the electrode leads are formed into and or along the shaft of an electrically inert composition having insulating qualities, such a plastic, a polyester derivative, or polyvinylchloride (PVC), or insulative carbon fiber. As shown inFIGS. 14A and B, an elongate hollow needle can be extruded with electrode component, such as for example, wire either along opposite sides of the hollow shaft or in a spiral fashion as shown inFIG. 14B. Further still, the needle shaft can also comprise sections with no exposed electrodes. For example, one end of the needle shaft connects to a hub forming a connector for connecting to a source of fluid, such as for example, a syringe. Insulation near or along such section of the shaft may provide for additional lessening of electric stimulus sensation noticeable by the patient. In yet a further embodiment with respect to any such electrode configuration described herein, each of the electrodes are individually energizable so that any combination of the electrodes may be energized in pairs (i.e., a cathode and anode) simultaneously together, or in any given sequence, and further using any type of pulse including without limitation monopolar, bipolar, exponential decaying, or pulse train combinations of any of the former.

In a third embodiment, the invention provides for use of relatively low voltage and/or low current, which in turn not only provides sufficient electrical energy for causing reversible poration of cells in the treatment zone, but also allows for a low pain level experienced by subjects during application of electric pulses into the surrounding tissue, said application using nominal electric field strengths of generally between 1 and 100 V, typically between 2 and 50V, an more preferably between 3 and 25V. In a related aspect, electric current employed in the invention device and methods uses generally between 1-400 mAmps, typically between 5-200 mAmps, and more preferably between 20 and 100 mAmps. In a related embodiment, the amperage chosen depends on the total surface area of the electrodes. For example, the device may employ a range between 10 to 40, or 25 to 100, or 50 to 150, or 125 to 200, or 175 to 250, or 225 to 300, or 250 to 300 or 300 to 400 mAmps depending upon the total electrode surface area of each electrode. The smaller the surface area, the lower the amperage necessary to achieve an electroporating electric filed in the in situ tissue. Pulses can be applied for between 1 and 1000 millisec.

In another embodiment, the invention provides for delivery of treatment molecules at various concentrations (e.g., for example, between 0.05 μg-3 mg/ml) and preferably at low bolus volumes (e.g., for example, generally 1 μl to 1 ml). In a related embodiment, using a structural embodiment inclusive of a delivery tube associated with the single needle electrode shaft, the volume of treatment molecules immediately following injection into the tissue (such as a controlled injection wherein the injectate is delivered during insertion of the needle) surprisingly remains to a substantial level in the vicinity of the injection needle track. Treatment molecules are contemplated to include therapeutic drugs, e.g., small molecules, organic compounds, as well as proteins, and nucleic acids encoding polypeptides having either a biologic activity or that will induce an immune response in the host once such polypeptide is expressed in the electroporated cell. The polypeptides once expressed in the cell are available for interacting with cellular metabolic machinery and immune system pathways.

In yet another embodiment, electrical energy used to pulse the tissue provides for a unique electric field that is unlike prior applied fields used for electroporation of similar tissues. Specifically, prior art electric fields intentionally and inherently impart what has been recognized in the electroporative arts as a “uniform” electric field meaning that the applied electrical energy is of sufficient strength to impart a nominal field strength and a relatively even voltage drop across the treatment zone created by widely separating the electrodes a given distance apart from one another and placing the target treatment zone optimally central between said spaced electrodes. Such electrode array designs when pulsed in tissue tend to electroporate cells primarily within the zone bordered by the electrodes generally in the vicinity of the electric lines of force and to a smaller degree a zone of cells situated just adjacent and surrounding the three dimensional treatment zone.

In contrast, the current invention uses electric fields that comprise a generally cylindrical or columnar “non-uniform” field that is created about the length of the needle shaft thereby creating a treatment zone of cells lying within an area close enough to the centrally placed electrodes to be subjected to an electroporation field “outside” the immediate location of the electrodes, of sufficient strength to porate said cells. Such a treatment zone is completely external to and surrounding the central needle and electrodes and the non-uniform field dissipates relative to the distance outward from the electrode/needle. Generally, it is thought that the dissipation in electrical energy as the distance from the single needle electrode increases is parallel to the dissipation found in other physical phenomenon wherein energy, here energy sufficient to reversibly porate cells, dissipates at an exponential rate. However, such dissipation rate if applicable does not negatively affect the functioning of the invention device or the intended outcome of delivering substances into cells in a defined zone. Thus, since electrical energy necessary to cause cell poration dissipates with the distance from the electrical field source, the area around the needle tract that is susceptible to electroporation is inherently confined to a central core correlating to the length of the needle track and laterally to a given radius forming therefore a generally cylindrical treatment zone of variable radii depending upon the pulse energy imparted to the electrodes. In a further related embodiment, the more energy used to pulse, the greater the potential to damage cells directly in contact with the electrodes. It is yet a further intention of the invention methods to employ the ability to cause such damage for the purpose of further stimulating the immune system. Thus, treatment regimens can be used that intentionally impart a greater rather than a lesser energy so as to provide a stimulus for immune response activity around the treatment site.

In other embodiments, the device can be used to deliver drugs, natural polypeptides having a biologic activity, and genes encoding such polypeptides that can be expressed in situ in cells within the treatment zone for treating disorders or for modulating an immune response in the host and/or for treating a variety of diseases including but not limited to diseases caused by pathogenic organisms and viruses and cancers.

Other features and advantages of the invention will be apparent from the following drawings, detailed description, and appended claims.

