U.S. patent application number 16/272685 was filed with the patent office on 2019-06-06 for contactless electropermeabilization electrode and method.
This patent application is currently assigned to VGX Pharmaceuticals, LLC. The applicant listed for this patent is VGX Pharmaceuticals, LLC. Invention is credited to Kate BRODERICK, Michael P. FONS, Thomas Joseph KARDOS, Stephen Vincent KEMMERRER, Rune KJEKEN, Jay MCCOY.
Application Number | 20190167983 16/272685 |
Document ID | / |
Family ID | 42982880 |
Filed Date | 2019-06-06 |
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United States Patent
Application |
20190167983 |
Kind Code |
A1 |
KARDOS; Thomas Joseph ; et
al. |
June 6, 2019 |
CONTACTLESS ELECTROPERMEABILIZATION ELECTRODE AND METHOD
Abstract
Devices and methods for delivering an electropermeabilizing
pulse of electric energy to a tissue surface to enable delivery
into cells in the tissue therapeutic substances. The device
incorporates a source capable of generating a sufficient voltage
potential to deliver a spark across a gap and delivers same to the
tissue surface.
Inventors: |
KARDOS; Thomas Joseph;
(Aliso Viejo, CA) ; KEMMERRER; Stephen Vincent;
(San Diego, CA) ; KJEKEN; Rune; (Oslo, NO)
; BRODERICK; Kate; (San Diego, CA) ; MCCOY;
Jay; (San Diego, CA) ; FONS; Michael P.; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VGX Pharmaceuticals, LLC |
Plymouth Meeting |
PA |
US |
|
|
Assignee: |
VGX Pharmaceuticals, LLC
Plymouth Meeting
PA
|
Family ID: |
42982880 |
Appl. No.: |
16/272685 |
Filed: |
February 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13263802 |
Oct 10, 2011 |
10232173 |
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PCT/US2010/031431 |
Apr 16, 2010 |
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16272685 |
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61212803 |
Apr 16, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61N 1/325 20130101;
A61N 1/0476 20130101; A61N 1/0416 20130101; A61N 1/327 20130101;
A61M 2037/0007 20130101 |
International
Class: |
A61N 1/32 20060101
A61N001/32; A61N 1/04 20060101 A61N001/04 |
Claims
1. A method of enabling the delivery of therapeutic substances into
cells of tissue of a mammal by using a spark electropermeabilizing
device to deliver an electropermeabilizing electric voltage
potential to the surface of a mammalian tissue, comprising:
generating a voltage potential sufficient to jump a gap of
predetermined distance; delivering said generated voltage potential
in the form of a spark that can jump said gap to the surface of the
mammalian tissue without causing macroscopic damage to an integrity
of the surface of the mammalian tissue; and delivering the
therapeutic substances to cells within the mammalian tissue.
2. The method of claim 1, wherein the delivering step comprises:
delivering the voltage potential across a spark-gap regulator
having a space gap; wherein the spaced gap is the gap of
predetermined distance.
3. The method of claim 1, wherein the generated voltage potential
is from 0.9 kV to 109 kV.
4. The method of claim 1, wherein the delivered voltage potential
further comprises a total electric charge of about between
2.8.times.10E-8 and 2.5.times.10E-6 Coulombs.
5. The method of claim 1, wherein the delivered voltage potential
imparts a total energy to the surface of a mammalian tissue about
between 0.000025 and 0.27 Joules.
6. The method of claim 1, wherein the delivered voltage potential
has a pulse length of about between 5 nanoseconds and 5
microseconds.
7. The method of claim 1, wherein the delivering step is repeated a
multiplicity of times between 2 and 20 pulses.
8. The method of claim 1, wherein the therapeutic substance
comprises a polynucleotide encoding an expressible polypeptide.
9. The method of claim 1, wherein the therapeutic substance
comprises a polypeptide.
10. The method of claim 1, wherein the energy source of the
electric voltage potential is selected from one of a 1, 3, 9 or 12V
battery, a piezoelectric crystal, a charged induction coil, a van
de Graaff generator, and a charged capacitor.
11. The method of claim 2, further comprising: discharging from the
electrode directly to the surface of the mammalian tissue.
12. The method of claim 2, further comprising: providing the
spark-gap regulator having a housing comprising an electrically
inert material; providing a supply electric lead and a receiving
electric lead that are each placed within the housing and that are
electrically isolated from one another; providing a space gap
between the supply electric lead and the receiving electric lead
inside the housing; and adjusting an atmospheric pressure in the
housing to a predetermined value.
13. The method of claim 12, wherein the space gap has a measurement
of between 0.01 cm and 4 cm.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS SECTION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 13/263,802, filed Oct. 10, 2011, which is a
371 National stage entry of International Application No.
PCT/US2010/031431, filed Apr. 16, 2010, and claims the benefit of
U.S. Provisional Application No. 61/212,803, filed Apr. 16, 2009,
the entire contents of each of which are incorporated herein by
reference.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to delivery of therapeutic
substances including macromolecules, such as polynucleotides and
polypeptides, into mammalian cells, particularly cells lying
adjacent or otherwise near tissue surfaces, using a novel
electropermeabilization system.
BACKGROUND
[0003] The following description includes information that may be
useful in understanding the present disclosure. 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.
[0004] Electropermeabilization of mammalian cells is a technique
that has been used for delivery of therapeutic substances including
small molecules, such as anticancer agents bleomycin and cisplatin,
and macromolecules such as nucleic acids and proteins. Typically,
delivery of such substances into cells is brought about by
injecting the substance into the tissues containing the cells,
which injection merely places such substance into the interstitial
spaces between the cells, followed by physically contacting the
tissues with a metallic electrode of one configuration or another
and applying an electric potential across the electrodes. Usually,
the electrode is manifest in the form of at least two opposing
needle-like tissue piercing rods or tubes comprising an anode and a
cathode. Other forms of tissue contacting electrodes include
non-penetrating electrodes such as planar pads as found in
"caliper" electrode devices such as disclosed in U.S. Pat. No.
5,439,440 and meander electrodes such as disclosed in U.S. Pat. No.
6,009,345. Still other electrode types have included minimally
invasive electrodes such as disclosed in U.S. Pat. No.
6,603,998.
[0005] With regard to the electrodes as mentioned above, all
operate within a paradigm well understood in the electrical arts to
require express and direct contact between the electrode and the
tissue undergoing electropermeabilization. Further, the electrical
potential placed across the positive and negative poles, often
expressed as "field strength" in Volts/centimeter, has been in the
vicinity of tens to hundreds of volts per centimeter, i.e., voltage
potential between the positive and negative poles spaced apart in,
or on, the tissue a given distance. Typically, distances between
electrodes are from tenths of centimeters to full centimeters of
length. In most disclosures concerning electropermeabilization, the
voltages required to provide a field strength sufficient for cell
poration in the tissues are anywhere from one Volt for skin tissues
to upwards of five or six hundred volts for cells lying in deeper
body tissues. The various levels of voltage applied are typically
dependent upon the spacing of the positive and negative electrodes
and the electric resistance of the tissue undergoing treatment.
[0006] There have been many recent advances in the art of
electropermeabilization wherein low voltage potentials have been
applied to skin tissues. In many of these cases, the low voltage
applied has been tied to very lengthy time periods for applying the
electrical energy. In some cases, the electrical energy has been
applied in a direct current form understood in the arts as
providing an electrophoresis or iontophoresis effect wherein
substances are moved through the tissue slowly. In such conditions
and particularly with small molecules, the electric pulses only
provide for the molecules to be moved through tissue interstitial
space, not inside the cells within the tissue. Even where the low
voltage has been applied for short periods of time, the electrodes
comprise the typical complex two pole array arrangement, i.e., for
example, at least one each of independently chargeable cathode(s)
and anode(s) placed in contact with the tissues. Other recent
disclosures discuss the use of very high voltages, in the 10,000
plus Volt range, for very short time periods to achieve delivery of
substances into cells but none the less require contact of the
electrodes with the tissue.
[0007] Whether using low or high voltages, tissue contacting
electrodes systems are subject to practical limitations primarily
concerning safety and comfort, or lack thereof, to the mammal
undergoing treatment. There is also the practicality or
impracticality of manufacturing complex miniaturized arrays
containing both anodes and cathodes often organized to be pulsed
independently of one another in various sequences and direction of
pulsing. Use of high voltages with tissue piercing and surface
touching electrodes can be dangerous for the potential of severe
electric shock if conditions include high amperage over a time
greater than 10 millisec. Use of low voltages over an extended
period of time, though typically not dangerous, has the potential
of being uncomfortable to the patient mammal or otherwise requires
complex manufacturing processes. In addition, there are concerns
that voltage facilitated systems or delivery methods result in low
levels of efficacy.
[0008] Still other issues are of concern in delivery of substances
to skin or tissue surface cells. For example, some systems disclose
methods of delivering the substance through the skin surface, i.e.,
the stratum corneum, followed by delivery of the electric potential
with the typical tissue penetrating or surface contacting
electrodes. With regard to such substance delivery, instead of
direct injection some systems attempt to draw the substance
directly through the stratum corneum by iontophoresis and/or
electrophoresis by applying various means to first ablate the
stratum corneum before providing the substance and electric
potential. For example, one system uses a laser beam to poke holes
in the stratum corneum (U.S. Pat. No. 6,527,716). Another uses a
high intensity tissue ablating spark not unlike a cauterizing
surgical instrument (U.S. Pat. No. 6,611,706). In each of such
systems, the methodology relies on physically disrupting the
stratum corneum in order to deliver the substance and further aid
in the transmission of the electrical energy from the tissue
contacting electrodes into the tissue.
[0009] Thus, there is in the art a need to advance delivery of
substances into cells using electropermeabilization in a manner
that avoids electrical hazards, discomfort to the treated patient,
damage to the tissues, and complex manufacturing.
SUMMARY
[0010] Disclosed herein is a novel methodology and
electropermeabilization (or electroporation) device that delivers
an electric potential having a sufficient field strength for
causing cell permeability (or reversible pore formation) in
skin-based or tissue surface-based cells without either causing
potential hazardous electrical conditions, physical damage to the
tissue surface, or noticeable discomfort. Further, the novel
electrode is capable of delivering the necessary electric potential
without being invasive, i.e., not penetrating the skin.
[0011] In one embodiment, there are disclosed methods of delivering
therapeutic substances, which can include drugs, small molecules,
and macromolecules to cells associated with tissue surfaces of a
mammal, such as that of skin. With respect to skin, the current
method can be used to deliver such substances into cells of
epidermal and dermal tissue. As defined herein, macromolecules
include large steroidal chemical compounds, as well as
polynucleotides such as DNA, RNA, siRNA and the like, and
polypeptides such as proteins comprising chains of amino acids
between 8 and 3000 amino acid units. In an embodiment,
polynucleotides include single and double stranded moieties, as
well as both linear and circular polynucleotide sequences encoding
polypeptides comprising whole functional proteins and fragments
thereof, including short epitopes.
[0012] In one embodiment, there are disclosed methods of imparting
a sufficient electric energy pulse (or electropermeabilizing
electric voltage potential) to a tissue surface to cause reversible
poration in cells of the tissue for the cellular delivery of a
therapeutic substance into the porated cells. In another
embodiment, there are disclosed methods of delivering the pulse of
electric energy (or delivering or discharging the electric voltage
potential) without the need for a tissue penetration, and
preferably without contacting the tissue with the electrode or
"non-contact." Thus, the electrode advances the art of imparting
electroporative electric energy pulses to tissues for the purpose
of causing electropermeabilization in that there need not be any
trauma to the tissue, such as penetrating the tissue by a
needle-like member, or alternatively, compressing, scratching,
burning, or ablation of the skin surface other than the
subcutaneous placement preelectropermabilization of the substance
to be delivered to the cells.
[0013] In still another embodiment, the electropermeabilization
system advances the delivery of electric energy by providing a
novel route to electropermeabilization of cells for the direct
delivery into the cells of the therapeutic substances in that
present implementations provide a simplification of the electrode
circuitry from the prior art requirement of two electrodes of
opposing polarities at the delivery site in a tissue to instead,
use of only a single electrode of a singular polarity at the
delivery site that works in concert with (or otherwise discharging
against) the polarity of the quiescent or grounded tissue of the
subject being treated whether that is more positive or more
negative relative to the delivering electrode polarity. In a
further embodiment, the energy imparted to the tissue is dependent
upon a combination of the potential of the voltage discharged and
the distance or "gap" between the electrode tip and the tissue
surface. In a related embodiment, the gap, rather than exist
exclusively between the non-contact electrode and the tissue
surface, can include a "spark-gap regulator" element located
between the non-contact electrode and the device circuitry. The
spark-gap regulator allows for discharge of the voltage potential
in a predeterminable fashion limiting the amount of current
relative to time. Furthermore, in this embodiment, the distal end
of the electrode can be in contact with the surface of the tissue,
i.e., can contact the surface of skin. In a related embodiment, the
singular polarity of the non-contact electrode allows for reference
not to "field strength" as is common in prior electroporation
systems requiring electrodes of opposing polarity, but to "total
energy" imparted to the tissue. In a further embodiment, the pulse
of electric energy provided by the non-contact electrode can have a
total electrical charge imparted into the tissue of between
2.8.times.10E-8 and 2.5.times.10E-6 Coulombs to achieve
electropermeabilization that is equivalent to between about 0.025
mJ and 270 mJ (millijoules) of energy, as further disclosed in
Table I.
[0014] In another embodiment, an alternative single polarity
electrode may comprise a tissue contacting array of noninvasive or
alternatively minimally invasive electrodes comprising needle-like
projections, in each case, all of a singular polarity. In an
embodiment, the array can be constructed from a single electrically
conductive material. As used herein a "single polarity electrode"
or a "singular polarity electrode" is one that is constructed so as
to possess only one pole, namely either anode or cathode, as the
case may be. Where such a singular polarity electrode comprises an
array of needle like projections, all such projection electrodes
are pulsed in one pole from the electric energy generation source
to the tissue. As used herein, a noninvasive electrode is an
electrode comprising an array of needle-like projections that do
not penetrate through a stratum corneum layer of skin tissue. As
used herein, a minimally invasive electrode is an electrode array
comprising needle-like projections that penetrate through a stratum
corneum layer to a depth of subdermal tissue, i.e., about 1-2 mm.
In this alternate embodiment, the electrode does contact tissue
surface but energy imparted from the singular electrode derives
from an electrical pulse driven across a spark-gap regulator as
disclosed herein.
[0015] In some embodiments where the electrodes are minimally
invasive or contact the surface of the tissue, the energy source
can be one that is capable of generating a sufficient electric
voltage potential over a period of time less than 1 millisecond,
and preferably less than 100 microseconds. In preferred
embodiments, the energy source is a piezoelectric crystal. In such
embodiments, only one gap is necessary between the energy source
and the tissue--such gap is between the electrode's distal end and
the tissue. In some embodiments, the electrode can have a spark-gap
regulator.
[0016] In another embodiment, there provided methods of delivering
the electroporative pulse of energy in a regulated manner wherein
the total energy discharge is controlled by a "spark-gap
regulator". In an embodiment, the spark-gap regulator comprises an
electrically neutral or nonconductive housing, such as clear
plastic, encasing two electric leads separated by a "gap" of a
predetermined measurement and of a predetermined electrical
resistance rating and optionally under either positive or negative
atmosphere pressure conditions, such as for example a vacuum or
atmospheric pressure. In related embodiments, the spark-gap
regulator provides the ability for the delivery system to be
equipped with any of a variety of spark-gap regulators each
manufactured to preset voltage thresholds for use in setting
predetermined delivery parameters including such as minimum and
peak voltages, current range, and total and/or net charges/energies
(Coulombs or Joules) imparted into the tissue. It is contemplated
that each of these parameters can affect the immune outcome for a
particular disease or disorder being treated in the mammal.
[0017] In still another embodiment, the spark-gap regulator can be
constructed to provide for any number of electrical energy levels
(or electric voltage potentials) to be dischargable across the gap
and thereby regulate the discharge of the voltage potential applied
to the tissue via the electrode. In some instances the electrodes
include: contacting single polarity electrode array, or
alternatively, the non-contact electrode.
[0018] In yet other embodiments, the energy source for providing a
single polarity potential can comprise any of a 1 to 12 V battery,
a charged capacitor, a charge coil, a piezoelectric crystal, or a
Van de Graaff generator.
[0019] In further embodiments, there is provided methods of
eliciting an immune response in a mammalian tissue by single
polarity electropermeabilization.
[0020] Other features and advantages will be apparent from the
following drawings, detailed description, and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The foregoing summary, as well as the following detailed
description of illustrative embodiments, is better understood when
read in conjunction with the appended drawings. For the purpose of
illustrating the embodiments, there are shown in the drawings
example constructions of the embodiments; however, the embodiments
are not limited to the specific methods and instrumentalities
disclosed. In the drawings:
[0022] FIG. 1 is a schematic drawing depicting examples of elements
of a device in accordance with the present disclosure;
[0023] FIG. 2 is a close up representation of the non-contact
singular polarity electrode separated by a "gap" from the tissue
surface;
[0024] FIGS. 3A, B, and C are schematic depictions of three shapes
that may be used for the non-contact singular polarity
electrode;
[0025] FIGS. 4A and B are schematic drawings depicting examples of
elements of alternate designs of the device of FIG. 1;
[0026] FIG. 5 is a three dimensional view of a spark-gap
regulator;
[0027] FIGS. 6A, B, and C are graphs depicting the pulse
characteristics of electrostatic discharges from various energy
sources;
[0028] FIGS. 7A to F are drawings of various example electric
circuits generally laying out the charge generation elements of
alternate embodiments;
[0029] FIGS. 8A and B are graphs showing pulse discharges using a
Van de Graaff generator; and
[0030] FIG. 9 is a graph showing titers of antibodies to influenza
protein (NP) following dermal injection and pulsed using the
spark-gap method of the disclosure.
DETAILED IMPLEMENTATIONS OF THE DISCLOSURE
[0031] Provided herein are novel electropermeabilization (or
electroporation) systems and methods for imparting
electropermeabilizing electric pulses of energy to surfaces of
tissues using a non-contact electrode, wherein the electrode does
not contact the tissue, or a contact electrode wherein the
electrode does contact the tissue with minimal penetration of skin,
and preferable only surface contact.
[0032] FIG. 1 is a schematic drawing depicting examples of elements
of a device 10 resting on a tissue surface 15 wherein there has
been placed a bolus of therapeutic substance 16. The device has
housing 11 containing an electric energy source 14, circuitry 13
and non-contact singular polarity electrode 12. The housing 11
forms a cowling around and extending a predetermined measurement
greater than a length of the electrode 12.
[0033] FIG. 2 is a close up representation of the non-contact
singular polarity electrode 12 separated by "gap" 17 from the
tissue surface 15.
[0034] FIGS. 3A, B, and C are schematic depictions of three shapes
that are useful for the non-contact singular polarity electrode. In
FIG. 3A, the tip of the electrode is spherical. In FIG. 3B, the tip
is pointed. In FIG. 3C, the tip is flat.
[0035] The term "sufficient voltage potential" or "voltage
potential sufficient" when referring to the energy source and the
voltage potential such source generates, the term refers to a
minimum amount of voltage potential that is required to generate a
spark that is able to jump across the gap in the electrode (i.e.,
the space gap of the spark-gap regulator), or the gap between the
electrode and the surface of the tissue when spark-gap regulator,
to deliver a desired charge to the tissue.
[0036] The term "predetermined distance" or "desired distance" when
referring to the gap in the electropermeabilization system, refers
to either the space gap of the spark-gap regulator or the gap
between the distal end of the electrode and the tissue surface
(when no spark-gap regulator is present). However, if both the
space gap and the gap between electrode and tissue are provided in
said system, neither gap will be larger than that of the
predetermined distance. In embodiments where both gaps are present,
the gap between the distal end of the electrode and the surface of
the tissue will be the same or shorter than the space gap of the
spark-gap regulator.
[0037] In a first embodiment, the device 10 can comprise an
electrode 12 that need not contact the tissue surface 15 in order
to impart into the tissue a pulse of electric energy sufficient to
cause electropermeabilization of cells in the tissue. This ability
to provide sufficient energy into the tissue without the electrode
12 touching the tissue 15 is a novel concept throughout the field
of the electroporative arts. Moreover, the energy can be supplied
to the tissue via the electrode 12 without causing any discernable
damage, such as burning or ablation of the tissue surface 15.
Rather, as concerns imparting energy to the stratum corneum, for
example, the energy is imparted to the tissue with the stratum
corneum remaining macroscopically intact. Specifically, the
electric energy is applied directly to the tissue surface 15 at a
location directly above or otherwise in the vicinity of a
previously delivered bolus of therapeutic substance that has been
injected just under the tissue surface 15. In an embodiment, the
bolus is injected into a mammalian patient, such as a human, at any
of subepidermal, subcutaneous, or intradermal locations. The
electric discharge can be directed as single, or alternatively, a
plurality of individual pulses to various portions of the tissue
area containing the bolus. By macroscopically intact, it is meant
that there is no discernable macroscopic histological damage or
alteration to the stratum corneum.
[0038] In this first embodiment, the non-contact electrode 12
operates as a point discharge source wherein electric energy pulsed
to the electrode from the system circuitry is discharged to the
tissue surface 15 across a space (air) gap 17 of a predetermined
measure. Specifically, the electric pulse is discharged via a spark
jumping across the gap 17 into the tissue after which the electric
energy dissipates through the tissue. Thus, the electrode 12
imparts a singular polarity static discharge into the tissue. The
energy pulses can comprise from 1 to over 100 individual sparks
imparted to the tissue surface 15 with the electrode tip resting
from anywhere of 0.01 cm to 10 mm above the tissue surface. In an
embodiment, the total energy passed to the tissues via the spark is
sufficient to cause electropermeabilization of cells within the
tissue but is not so great as to cause any discernable damage to
the tissue or the tissue surface 15. Generally, the electric energy
can be described as static electric pulses. Typically, in order to
achieve transfer of the energy pulse from the electrode across an
air gap 17 to the tissue surface 15 requires voltage potentials
that are in the range of kilovolts. Although high voltage
potentials alone suggest the possibility of danger or discomfort
potential for the treated mammal, since the generation of high
voltages is of a static electric nature, and because the discharge
across the air gap 17 occurs in an extremely short time frame
(nanoseconds), there is little current generated in the tissue to
cause tissue damage despite amperages reaching meaningful values.
Thus, the system provides for discharge of high voltage potential
with little danger of injury to the patient. In an embodiment, the
voltage potential can range from about between 5 kVolts to over 100
kVolts. The time frame of discharge can be about between 5
nanoseconds to 5 microseconds where the gap 17 between electrode
and tissue surface is between 0.01 cm and 1 cm. Where, as in this
embodiment, the gap 17 between electrode 12 and tissue surface 15
acts as a defacto spark-gap regulator, i.e., there is no separate
spark-gap regulator in the upstream circuitry, the gap 17 can be up
to 1 cm without the resultant current flow becoming too great for
both safety and sensation concerns. Within this gap range, in each
instance the imparting of electric energy is barely
perceptible.
[0039] Various sources of electric energy can be used to generate
the voltage potentials for the non-contact electrode 12. For
example, a Van de Graaff apparatus can be used to generate static
electric potentials wherein a metal dome acts as a capacitor for
building charge of between 5 kVolts and 100 kVolts that can be
channeled through the circuitry 13 to the electrode for a discharge
period of between 5 nanoseconds and 5 microseconds. Alternatively,
a piezoelectric crystal can be used wherein an impact mechanism can
create high voltage short duration pulses of 20 to 100 nanoseconds.
In yet another alternative, a high voltage Tesla coil with a
transformer and switch can be arranged wherein a primary coil can
be used to induce a secondary coil to a 5 kVolts to 100 kVolts
potential over a period of 40 to 100 nanoseconds. In still another
alternative, a 1, 3, 9, or a 12 Volt battery, for example, can be
used to charge a capacitor to build voltage potentials of 100 volts
to 1 kilovolt which can be discharged across the primary coil of a
non-contact electrode 12 to the tissue surface 15 or alternatively
via a spark-gap regulator (see, FIGS. 4A and 5) to a single
polarity tissue contacting electrode 12. Finally, an alternating
current source can be used in connection with a transformer to
build the static voltage potential and operate as disclosed herein
in singular polarity form. One of ordinary skill in the electric
arts will understand details of how appropriate circuitry can be
arranged to use these and other electric energy generators to build
an appropriate voltage potential and to send the potential to the
non-contact electrode 12 directly or first through a spark-gap
regulator for added electrical discharge control.
[0040] In some embodiments where the electrodes are minimally
invasive or contact the surface of the tissue, the energy source
can be one that is capable of generating a sufficient electric
voltage potential over a period of time less than 1 millisecond,
and preferably less than 100 microseconds. In preferred
embodiments, the energy source is a piezoelectric crystal.
[0041] Using any of the different electric energy sources noted
herein, the discharge of the electric energy will occur naturally
across the spark-gap regulator, or otherwise across the gap 17 from
the non-contact electrode 12 to the tissue surface 15, in an
oscillatory fashion such that the polarity of the pulse actually
reverses in nanosecond time frames. Such discharge likely moderates
the power intensity of the pulse thereby keeping the sparked
voltage potential from burning, ablating or otherwise damaging the
tissue surface 15.
[0042] FIGS. 4A and B are schematic drawings depicting examples of
elements of alternate designs for the device wherein the singular
polarity electrode comprises either a tissue contacting single
polarity electrode array 19 that is connected to a spark-gap
regulator 18 as shown in FIG. 4A, or alternatively comprises a
non-contact electrode 12 connected to a spark-gap regulator 18 as
depicted in FIG. 4B.
[0043] With respect to the regulation of the total energies
imparted to the tissue using the non-contact electrode, the
energies sufficient for electropermeabilization can be tailored by
adjusting the measure of the gap between the tip of the non-contact
electrode and the tissue surface, or alternatively the measure of
the gap of a spark-gap regulator, or alternatively adjusting the
combination of a gap in the spark-gap regulator within the
circuitry and the gap between the tissue surface and non-contact
electrode. As an example, the system electronics and electrode can
be arranged such that the gap between the non-contact electrode and
the tissue surface is not the only location for regulation of
voltage discharge. Rather, the non-contact electrode can have a
spark-gap regulator upstream from the non-contact electrode, as
depicted in FIG. 4B, so as to set the discharge regulation away
from the non-contact electrode/treatment zone. This is similar to
the alternate arrangement wherein the single polarity tissue
contacting electrode array is connected to a spark-gap regulator
upstream as shown in FIG. 4A.
[0044] Spark-gap regulation for electroporation allows for delivery
of extremely short and high intensity voltage pulses without danger
to the treated mammal and without macroscopically affecting the
stratum corneum. The minimal gap distance can be calculated for
setting voltages for use in electroporation. For example, at 1
atmosphere (760 torr) and room temperature (20 degrees Celsius),
based on Paschen's Law and the Townsend breakdown mechanism in
gases, as understood by one of ordinary skill in the art, where N
is the density of air, "d" is the gap measurement, under the
formula Voltage=K(Nd), the breakdown voltage necessary to cross a
gap of the measurements is disclosed in Table I.
[0045] FIG. 5 is a three dimensional view of a spark-gap regulator
wherein electric leads 22 and 23 are separated by a gap 24 of a
predetermined measurement inside housing 25. As shown in FIG. 5,
the spark-gap regulator is a device comprising a housing 25, which
is constructed of an electrically inert material such as glass,
Plexiglas or clear plastic, enclosing each of two wires, a lead
wire 20 and a receiving wire 21 placed so that there is a gap 24
separating terminal ends 22 and 23, respectively, thereof. The
housing is constructed so that there can be a vacuum space, if
desired, comprising the gap 24. This vacuum aspect provides for the
allowance of any discharge of electric energy between the terminal
ends 22 and 23 of the wires across the gap to occur without
atmospheric molecules influencing the resistance of electric charge
transfer across the gap. In this manner, the gap can be constructed
to have any length measurement practical for use in sparking charge
from the lead to the receiving wire and thereby specifically
controlling the amount of energy that can discharge across the gap.
Typically, the gap can measure between 0.01 and 4 cm.
[0046] FIGS. 6A, B, and C are graphs depicting the pulse
characteristics of electrostatic discharges from various energy
sources. FIG. 6A is a graph of the discharge from a Van de Graaff
generator, FIG. 6B is a graph of the discharge from a piezoelectric
crystal, and FIG. 6C is a graph of the discharge from a spark coil.
Each of the Figures illustrates time vs. voltage.
[0047] As depicted in FIG. 6A, the discharge of a pulse from a Van
de Graaff generator occurred across about 40 nanoseconds in a
sinusoidal fashion, each polarity having a pulse of between about 2
and 5 nanoseconds. The discharge of a piezoelectric crystal
provided a similar sinusoidal discharge as shown in FIG. 6B. In
this case, the bipolarity of the sinusoidal discharge occurred in a
little over 10 nanoseconds. In still another example of electric
pulse source discharge, a discharge across an air gap using a spark
coil (step up transformer) is shown in FIG. 6C. In this instance,
the discharge is even shorter on the order of 10 nanoseconds but
here, the discharge is in a single broad sinusoidal spike. Thus,
the spark coil generated discharge has inherently longer singular
pulses of either polarity. The longer the single pulse, the more
association with damage to tissue is observed. In an embodiment,
the Van de Graaff and piezoelectric generated potential and
discharge provides superior results in causing no discernable
effect on the tissue surface while generation from a spark coil has
the potential of being associated with effects on tissue if the
total energy of the potential is higher than a predeteminable
level. Therefore, the sparks, however generated, are those that can
be generated and discharged into tissue surface without either
purposely or inadvertently causing damage to the integrity of the
stratum corneum.
[0048] FIGS. 7A to F are drawings of various electric circuits
generally laying out the charge generation elements of alternate
embodiments. In FIG. 7A, a Van de Graaff generator system is
depicted wherein charge buildup is delivered to the patient tissue
directly with a non-contact single polarity electrode and a ground
plate. This system can also include a spark-gap regulator if
desired. In FIG. 7B, a battery power source can be arranged to step
up voltage and send charge through a spark-gap regulator to the
patient tissue. In FIG. 7C, a simpler circuit is disclosed wherein
the charge generated is sent through a spark-gap regulator to the
patient tissue. In FIG. 7D, a circuit wherein the charge is
generated using a piezoelectric crystal is disclosed. Here the
charge can be sent directly to the non-contact electrode or can be
sent through a spark-gap regulator before being sent to the patient
tissue. In FIG. 7E, a circuit is disclosed depicting a high voltage
Field Effect Transistor (FET) switched coil. In FIG. 7F, a circuit
is disclosed depicting a manually switched coil.
[0049] A Van de Graaff generator circuit can be relatively simple,
using as basic components a circuit comprising a Van de Graaff
generator attached to the non-contact electrode element such that
the charge potential generated is regulated in its delivery to the
tissue by gapping across the space between the non-contact
electrode and the tissue surface. Alternatively, the charge
potential can be sent to a spark-gap regulator and then on to the
tissue via either the non-contact electrode or the tissue
contacting single polar array electrode. There need not be any
counter electrode. Rather, the system inherently provides for
delivery of energies that will discharge through the body tissue
into the environment. An alternative to using no counter electrode
can be the use of a ground plate or an electrically conductive foot
pad to assist complete discharge of energy through the body.
Further, given that there is a lower net total charge imparted to
the tissue via the Van de Graaff generated pulse (likely associated
with the oscillatory nature of the static discharge, as shown in
FIG. 6A), there is even less concern for needing a counter
electrode to dissipate the imparted electric potential.
[0050] FIGS. 7B and C depict circuits that incorporate spark-gap
regulators. These circuits are compatible with either the
non-contact electrode or the single polarity tissue contacting
electrode. In FIG. 7B, batteries "Bat 1" and "Bat 2" are connected
to the primary coils of "xfr 1" and "xfr 2" by switch SW1 wherein
the secondary windings of xfr 1 and xfr 2 are greater than their
respective primary windings by the same ratio with respect to the
voltage in the secondary windings as compared to the voltage of Bat
1 and Bat 2. These secondary windings are rectified by diode D1,
and connect to additional coils L1 and L2 which further increase
the voltage sufficient to jump the gap in the "spark-gap" unit
which drives the primary coil of "xfr 3" to induce the final
voltage seen by the spark electrode on the patient who is grounded
to the environment. In this configuration, the spark-gap can be as
little as 0.01 cm. In FIG. 7C switch SW 1 transfers battery voltage
to the primary coil of xfrl and the secondary coil of xfr 1, which
is larger than the primary coil, and connects to a spark-gap
regulator embedded within the primary coil of the output
transformer, thereafter leading to the patient.
[0051] FIG. 7D is a drawing representing a circuit employing a
piezoelectric crystal. Like the Van de Graaff generator, the charge
pulse can be sent directly to the non-contact electrode or can be
sent through a spark-gap regulator to a single polarity tissue
contacting electrode. Also, as with the Van de Graaff generator,
there need not be a return electrode but alternatively a grounding
means, such as a conductive pad for example, on which the treated
mammal stands can be used if desired.
[0052] FIGS. 7E and F depict coil voltage potential generator
circuits. In FIG. 7E a high voltage field effect transistor (FET)
switched coil is disclosed. Here, when a pulse is applied to the
base of the FET 1 as diagrammed by closing a switch, the coil L1 is
switched to ground and current begins to flow from the battery
(Bat) through L1. When FET 1 turns off, current tries to continue
through the inductive coil of L1 but cannot, and will trigger a
spark through the patient until the energy stored up in L1
dissipates. Similarly, FIG. 7F shows a manually switched coil
wherein the same mechanism occurs when the switch is opened after
the coil has built up charge.
[0053] FIGS. 8A and B are graphs showing pulse discharges using a
Van de Graaff generator. In FIG. 8A, the pulse is depicted wherein
there are 100 nanoseconds per division in the graph. There are
about 500 nanoseconds of a diminishing pulse at about 1 Volt per
division. This translates to 10 Amps per division or a 25 Amp
maximum current. This example has an initial pulse spike lasting
about 30 nanoseconds. In FIG. 8B, the same section of the pulse is
shown in 10 nanosecond increments showing the about 30 nanosecond
nature of the initial pulse spike.
[0054] In further related embodiments, as shown in the example of
FIGS. 8A and B, the discharge from a Van de Graaff generator is
actually an oscillating discharge wherein there is a reversal of
the spark polarity with reversals of charge flow in diminishing
switchbacks until the energy, as measured in volts, falls below
that value allowing jumping of the charge across the gap. The
oscillating charge flow across the gap can occur lower than the
initial gap cutoff value due to the charge flow across the air gap
becoming a plasma. The observed discharge then continues in
oscillations running out to about 1 millisecond even though the
initial first pulse occurred in less than 40 nanoseconds.
[0055] In still other embodiments, the discharge of the electric
potential can be sent into the tissue using an alternate to the
non-contacting electrode, namely through a singular polarity tissue
contacting electrode. In this alternate embodiment, rather than a
spark directly to the tissue surface, the energy is directed to a
single polarity array of needle-like projections after passing
across an upstream spark-gap regulator. In an embodiment, the
needle-like projections are either of a non-invasive type or
alternately, of a minimally invasive type. Further, the singular
polarity electrode can be fashioned from a simple single block of
electrically conductive material. In a related embodiment, the
array of "pins" or needle-like projections can comprise an array of
various dimensions such as, for example, 1 to 100 pins arranged in
a grid such as a 4.times.4 array or alternatively a 10.times.10, or
any other configuration or shape, such as a square grid or a
circular grid.
[0056] In either alternate embodiment of the electrode, i.e.,
either the singular polarity non-contact spark electrode, or the
tissue contacting singular polarity array, as one of skill in the
electric arts will recognize, there is a need for the voltage
potential to discharge through the tissue and, ostensibly, find its
way to a zero potential. This can be accomplished by either placing
a discharge plate, essentially an opposite polarity electrode, in
contact with the treated mammal, or preferably, providing for the
imparted charge to become grounded in the tissue of the treated
animal itself. Not in contrast with this physical need for the
voltage potential to fully discharge from the site of entering the
tissue, there is not a requirement for there to be located an
electrode of opposite potential near the site of delivery of the
singular polarity discharge that is delivered from the electrodes.
The treated mammal should be grounded sufficiently to allow the
static charge imparted to the mammal to reach ground potential.
This can be accomplished by the imparted potential dissipating
throughout the mammal's body tissues, or alternatively, a remote
electrically conductive material can be placed in contact with the
mammal's body, such as an electrically conductive foot pad.
[0057] For calculating the values in Table I, the majority of the
discharge, whether using a Van de Graaff generator, a piezoelectric
crystal, or a Tesla coil, occurs at the front end of the pulse (as
shown in FIGS. 8A and B). Thus, taking the values obtained from the
front end pulse spike, Amps, Coulombs, and total energy may be
determined that is imparted into the tissue occurring from a
variety of starting voltage potentials. For example, a positive
charge having a 20 Amp peak current and lasting approximately 40
nanoseconds (Amps.times.pulse length=Coulombs) equals approximately
8.0.times.10E.sup.-7 Coulombs for a 10 mm gap. Each of the values
in the Table I were calculated similarly as one of ordinary skill
in the art will understand.
TABLE-US-00001 TABLE I Total Net Minimum Representative Charge
Energy KVolts to Current from to Gap in breach gap Pulse Width
Across gap electrode tissue millimeters (breakdown) (nanoseconds)
(Amps) (Coulombs) (Joules) 0.1 0.9 7 4 2.8 .times. 10E-8 0.000025 1
4.3 10 11 1.1 .times. 10E-7 0.00047 2 7.6 12 15 1.8 .times. 10E-7
0.0014 5 16.4 29 16 4.6 .times. 10E-7 0.0076 10 (1 cm) 30.3 40 20
8.0 .times. 10E-7 0.024 15 43.8 40 22 8.8 .times. 10E-7 0.039 20 (2
cm) 57.0 40 29 1.2 .times. 10E-6 0.066 25 70.2 40 35 1.4 .times.
10E-6 0.098 30 (3 cm) 83.2 45 38 1.7 .times. 10E-6 0.14 35 96.1 40
60 2.4 .times. 10E-6 0.23 40 (4 cm) 109.0 35 70 2.5 .times. 10E-6
0.27 Values based on 1 Atm (760 torr) at 20 degrees C, based on
Paschen's Law, Townsend breakdown mechanism in gases, V = K (Nd), N
= air density, d = gap; Current measured with Pearson Model 411
Current Meter, calibrated at 0.1 V/Amp. Spark generated via Van de
Graaff generator. Formula Q (Coulombs) = amps .times. t (seconds);
E (Joules) = Q .times. V.
[0058] Specifically, the minimum electric energy necessary to cross
a gap of a given distance using a Van de Graaff generator is
disclosed in Table I. With that minimum voltage potential, the
minimum pulse length and the minimum current can be calculated as
disclosed. Further, the total charge, in Coulombs, discharged
across the gap can be calculated. Thus, there is an ability to
graph the actual discharge, and calculate the net energy, in
Joules, imparted into the tissue. From this Table I, the minimal
energies capable of being delivered to the tissue surface for any
given gap employed in the device can be determined whether the
electrode to tissue surface gap and/or the upstream spark-gap
regulator is used. This spark-gap regulation is useful for the
alternate embodiments, e.g., the non-contact electrode and the
tissue contacting electrode array. In both instances, singular
polarity voltage potential discharge can be administered with a
known total energy delivery on demand. Since that discharge will
take place over a pulse period of approximately between 5
nanoseconds and 5 microseconds, there is no potential harm to the
treated mammal. This short pulse time period allows for current of
4 to 70 Amps at very low total energies between about 0.00001 and
0.5 Joules without any significant danger to the treated mammal
despite the high voltage levels associated with discharge across
the gap.
[0059] For example, a Van de Graaff generator, such as for example,
one chargeable to between 75 kVolts and 100 kVolts, was charged to
100 kVolts and the charge discharged through the spark-gap was
calculated to be about 10.sup.-5 Coulombs. Specifically, as charge
builds up on the large metal head of the Van de Graaff generator,
which acts as a capacitor across the dielectric of air over a
spark-gap of approximately 4 cm, a breakdown will occur over this
gap once the voltage becomes over about 100 kVolts. Upon the
breakdown and charge beings delivery across the gap, air ionizes
between the electrode head and the skin/tissue of the mammal
subject, and current begins to flow until the charge from the Van
de Graaff equalizes with the static potential of the tissue.
However, since the flowing current has inertia, the equal potential
point between the Van de Graaff head and tissue will reverse, and
build up an opposite relative charge as compared to the time when
the breakdown initiated. There will be several oscillations of
potential reversal as current flows back and forth, until the
potential difference drops below a minimum to keep the spark-gap
air ionized, at which time the spark event terminates.
[0060] In a similar fashion, calculations of energies imparted into
the tissue can be made for voltage potentials generated from both
piezoelectric crystals and Tesla coils as shown in Tables II and
III, respectively, below. In these tabular calculations conditions
for calculating were 1 atmosphere (760 torr), 20 degrees Celsius,
based on Paschen's Law, Townsend breakdown mechanism in gases; V=K
(Nd), N=air density, d=gap; current measured with Pearson Model 411
Current Meter, calibrated at 0.1 V/Amp. Q (Coulombs)=I
(amps).times.t (seconds), E (Joules)=Q.times.Volts.
TABLE-US-00002 TABLE II* Initial pulse Kvolts width Current Charge
Energy Gap (mm) (breakdown) (nanosec) (Amps) (Coulombs) (Joules) 1
4.3 10 6 6.0 .times. 10E-8 0.00026 2 7.6 14 10 1.4 .times. 10E-7
0.0011 5 16.4 17 15 2.5 .times. 10E-7 0.0041 10 (1 cm) 30.3 30 18
5.4 .times. 10E-7 0.016 *Piezoelectric crystal generator
TABLE-US-00003 TABLE III** Initial pulse Kvolts width Current
Charge Energy Gap (mm) (breakdown) (nanosec) (Amps) (Coulombs)
(Joules) 1 4.3 13 2 2.8 .times. 10E-8 0.00012 2 7.6 14 2.5 3.5
.times. 10E-8 0.0026 5 16.4 15 6 9.0 .times. 10E-8 0.0015 10 (1 cm)
30.3 20 14 2.8 .times. 10E-7 0.0085 **Tesla coil generator
[0061] For example, calculations on total energy imparted were
carried out using a Pearson inductive current monitor (Model 411,
Pearson Electronics, Inc., Palo Alto, Calif. USA) that develops a
0.1 Volt/Amp voltage current ratio, and a Tektronix TDS210
oscilloscope (Tektronix, Inc., Beaverton, Oreg., USA) with a
piezoelectric crystal that was capable of generating a 1 cm spark
length, resulted in the generation of 25 Amps over a 20 nanosecond
pulse to deliver about 10.sup.-6 Coulombs of electric charge.
Specifically, I=dQ/dt with 1 Coulomb=1 Amp.times.1 Sec results in
25 Amps.times.20.times.10.sup.-9 sec=0.5.times.10.sup.-6
Coulombs.
[0062] In another example, using the formula I=delta Q/delta t,
which can also be written as dQ=I.times.dt=V/R.times.dt, where
I=current, Q=energy in Coulombs, V=volts, R=resistance in ohms, and
t=time, using a piezoelectric crystal, a current of 10 Amps is
generated over a 100 nanosecond pulse and a total charge transfer
of 10.sup.-6 Coulombs. Specifically, the tip of the piezoelectric
generator, which is conductive, such as an electrically conductive
metal, and in contact with one side of the piezoelectric crystal,
is brought within about 1 cm of the skin/tissue of the mammal
subject. The other side of the piezoelectric crystal is connected
to another electrical conduit that is subsequently grounded to the
environment of the subject mammal wherein the grounded subject is
in electrical communication with this second side of the crystal
circuit. Upon mechanical impulse being applied to the crystal, a
high voltage pulse between 15 and 35 kVolts is generated. This
pulse, directed to the electrode head, and positioned about 1 cm
from the tissue will be at an electrical potential with respect to
the grounded subject that is sufficiently high to ionize the air
gap between the nearest point of the test subject tissue surface to
the electrode. Further, current will flow until the current built
upon the crystal by the mechanical impulse dissipates.
[0063] As noted above, FIGS. 6A, B and C show the discharge of
voltages from three different sources of power generation. In FIG.
6A, discharge from a Van de Graaff is shown wherein the discharge
actually comprises an oscillating wave form. Thus, the total energy
imparted to the tissue not only occurs in less than about 40
nanoseconds, it is actually a net of opposite polarities
oscillating into the tissue. Further, the pulse period for each
pulse polarity is extremely short, namely about 2 to 5 nanoseconds.
Similarly, as shown in FIG. 6B, discharge of a piezoelectric
crystal naturally occurs in a somewhat oscillatory fashion, again
each polarity of the pulse being very short, about 2-5 nanoseconds.
Still further, as shown in FIG. 6C, the discharge from a spark coil
also exhibits an oscillation in the actual discharge but in this
instance, the waveform comprises single long pulse periods in
either pole of the oscillation, a phenomenon different from the
discharge occurring from the Van de Graaff or piezoelectric crystal
generators. The discharge from a spark coil generator, if too high
a voltage potential, can cause tissue damage. Thus, when using a
spark coil as a generation source, the system is tailored to
discharge only energies sufficient for electropermeabilization and
not damage tissue.
Examples
A. Non-Contact Electrode Experiment
[0064] A device comprising a Van de Graaff generator as a power
source and a non-contact electrode was placed 1 cm above a
mammalian tissue surface (guinea pig) was pulsed with either 4 or 8
spark discharges across the 1 cm gap and above a 50 ul (microliter)
bolus of previously delivered intradermal injection of green
florescent protein (2 mg/ml dna plasmid in PBS) expressing plasmid
(plasmid pgw12-GFP from Aldevron, N. Dakota). The spark-gap used
with the Van de Graaff generator is approximately 1 cm, resulting
in repeated sequence of 4 or 8 sparks of approximately 30 kVolts,
resulting in delivery of approximately 25 milliJoules of energy
during each spark event. If electropermeabilization has taken place
and the plasmid entered the cells in the epidermal tissue, the
protein encoded by the plasmid will be expressed and the green
florescent protein will fluoresce under UV light.
[0065] In this experiment, the non-contact electrode was placed
directly above the site where a 50 ul bolus of GFP (2 mg/ml dna
plasmid in PBS) was injected intradermally. The intensity of
expression of the GFP is substantial where only four spark
discharges were administered. Similar results were obtained where
eight spark discharges were administered.
B. Single Polarity Non-Invasive Tissue Contacting Array
[0066] A device including a Van de Graaff generator and a
non-invasive single polarity tissue contacting 4 by 4 array to
deliver either 4 or 8 pulses to tissue was obtained. Guinea pigs
were obtained and treated in four repeat experiments by a 50 ul
bolus intradermal injection of GFP (2 mg/ml dna plasmid in PBS)
followed by pulsing the tissue surface using the single polarity
contact electrode (30 kVolts, gap of 1 cm, 4 to 8 pulses of about
25 milliJoules energy). The pulses were effectively pulsed to the
array wherein each needle or pin received an equivalent charge to
dissipate into the tissue. The GFP was successfully electroporated
into the epidermal tissues whether 4 pulses or 8 pulses were
administered.
C. Single Polarity Invasive Tissue Contacting Electrode
[0067] A device including a Van de Graaff generator and a tissue
penetrating electrode was tested in guinea pigs using 280 ul of 0.1
mg/ml GFP delivered in a single bolus via delivery into the tissue
from the tissue penetrating electrode itself. The single spark onto
an invasive electrode provided sites of electroporation to the
tissue cells.
D. Electropermeabilization System Using Piezoelectric Crystal
[0068] A device comprising a piezoelectric crystal as a power
source and a contact electrode array is placed 1 cm above a
mammalian tissue surface and is pulsed with either 4 or 8 spark
discharges across the 1 cm gap and above a 50 ul (microliter) bolus
that is previously delivered by intradermal injection of green
florescent protein (2 mg/ml dna plasmid in PBS) expressing plasmid
(plasmid pgw12-GFP from Aldevron, N. Dakota). This gap should
result in repeated sequence of 4 or 8 sparks, resulting in delivery
of approximately 25 milliJoules of energy during each spark event.
If electropermeabilization has taken place and the plasmid entered
the cells in the epidermal tissue, the protein encoded by the
plasmid will be expressed and the green florescent protein will
fluoresce under UV light.
[0069] Application of the methods and devices described herein are
well suited for cellular delivery of therapeutic molecules to cells
for eliciting immune responses or for other treatments. This
technology is well suited for DNA based vaccine delivery and for
gene-based therapies. For example, a therapeutic amount of
substance comprising a polynucleotide encoding an antigen
polypeptide or a formulation of the polynucleotide and biologic
salts as one of skill in the pharmaceutical arts is well versed,
can be injected into epidermal, dermal, or subdermal tissues
followed by delivery to the tissue of a discharge of electric
energy from the device via either the non-contact electrode or
alternatively the singular polarity tissue contacting array, and
the injected polynucleotide will be electroporated into the cells
of that tissue. Moreover, the use of the spark-gap method allows
for electropermeabilization of targeted tissue, specifically the
upper most layers of the skin tissues. GFP expression only occurs
in the top most layers of the skin.
Immune Experiment
[0070] In further experiments, animals injected with influenza
antigen (NP) and pulsed using the apparatus, were studied for the
expression of antibodies to the antigen. Specifically, the test
animals (guinea pigs) were given intradermal injections of the
antigen (50 ul bolus comprising 1 mg/ml NP plasmid) followed by
electropermeabilization using the spark-gap apparatus (each animal
receiving 10 sparks per injection site). Titers were followed out
to ten weeks as shown in FIG. 9, which is a graph showing titers of
antibodies to influenza protein (NP) following dermal injection and
pulsed using the spark-gap method. The data of the spark-gap pulsed
animals is compared to immune response elicited from delivery of
antigen to muscle pulsed using a non spark-gap system, and to
dermal injection of antigen without pulsing as control. The animals
were boosted with an injection of antigen at week 4. Titers reached
significant levels by week 5. These titers were superior to those
logged for muscle delivered and electroporated antigen.
[0071] While embodiments may have many different forms, there is
shown in the drawings and as herein described in detail various
implementations with the understanding that the present disclosure
is to be considered exemplary and is not intended to limit the
invention to the embodiments illustrated. The scope of the
invention will be measured by the appended claims and their
equivalents.
[0072] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
have been described in terms of various implementations, it will be
apparent to those of skill in the art that variations may be
applied to the compositions and methods and in the steps or in the
sequence of steps of the method described herein without departing
from the spirit and scope of the invention. More specifically, the
described embodiments are to be considered in all respects only as
illustrative and not restrictive. All similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit and scope of the invention as defined by the
appended claims.
[0073] All patents, patent applications, and publications mentioned
in the specification are indicative of the levels of those of
ordinary skill in the art to which the invention pertains. All
patents, patent applications, and publications, including those to
which priority or another benefit is claimed, are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0074] The implementations illustratively described herein suitably
may be practiced in the absence of any element(s) not specifically
disclosed herein. Thus, for example, in each instance herein any of
the terms "comprising", "consisting essentially of", and
"consisting of" may be replaced with either of the other two terms.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and there is no
intention that use of such terms and expressions imply excluding
any equivalents of the features shown and described in whole or in
part thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by various implementations and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims.
* * * * *