U.S. patent application number 14/696270 was filed with the patent office on 2015-10-22 for electroporation device.
The applicant listed for this patent is OncoSec Medical Incorporated. Invention is credited to Punit Dhillon, Brian McCluskey.
Application Number | 20150297887 14/696270 |
Document ID | / |
Family ID | 50545482 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150297887 |
Kind Code |
A1 |
Dhillon; Punit ; et
al. |
October 22, 2015 |
ELECTROPORATION DEVICE
Abstract
An electroporation device produces electric signals that may be
adjusted in response to a cover area of electrodes, so that the
electric signals are tolerable when delivered to cells within the
cover area. The electroporation device can include an applicator, a
plurality of electrodes extending from the applicator, a power
supply in electrical communication with the electrodes, and a guide
member coupled to the electrodes. The electrodes are associated
with a cover area. The power supply is configured to generate one
or more electroporating signals to cells within the cover area. The
guide member can be configured to adjust the cover area of the
electrodes. In some embodiments, the electrical signals may include
opposing waveforms that produce a resultant interference waveform
to effectively target the cover area, and each waveform may be a
unipolar waveform or a bipolar waveform.
Inventors: |
Dhillon; Punit; (San Diego,
CA) ; McCluskey; Brian; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OncoSec Medical Incorporated |
San Diego |
CA |
US |
|
|
Family ID: |
50545482 |
Appl. No.: |
14/696270 |
Filed: |
April 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14062582 |
Oct 24, 2013 |
9020605 |
|
|
14696270 |
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|
61718561 |
Oct 25, 2012 |
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61767078 |
Feb 20, 2013 |
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61791968 |
Mar 15, 2013 |
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Current U.S.
Class: |
604/20 ;
604/501 |
Current CPC
Class: |
A61F 2007/0075 20130101;
A61N 1/0502 20130101; A61F 7/007 20130101; A61N 1/327 20130101 |
International
Class: |
A61N 1/32 20060101
A61N001/32; A61F 7/00 20060101 A61F007/00; A61N 1/05 20060101
A61N001/05 |
Claims
1. A method of electroporating cells using the device of claim 1,
the method comprising: administering selected molecules into the
cells within the cover area; contacting the cells with the
electrodes; and delivering the one or more electroporating
signals.
2. The method of claim 1 further comprising adjusting the cover
area of the electrodes.
3. The method of claim 1, wherein the one or more electroporating
signals are each associated with an electrical field, and wherein
delivering the one or more electroporating signals further
comprises maintaining the electrical field within a predetermined
range so as to substantially prevent permanent damage in the cells
within the cover area.
4. The method of claim 1, wherein the one or more electroporating
signals are each associated with an electrical field, and wherein
delivering the one or more electroporating signals further
comprises maintaining the electrical field within a predetermined
range so as to substantially minimize pain.
5. The method of claim 1 further comprising adjusting a temperature
of the cells to about 4.degree. C. to about 45.degree. C.
6. An electroporation device comprising: an applicator; a plurality
of electrodes extending from the applicator; a power supply in
electrical communication with the electrodes; and a camera coupled
to the applicator and positioned adjacent the electrodes.
7. The device of claim 6 further comprising a device memory in
electronic communication with the camera, wherein the device memory
is configured to store an electroporation treatment database.
8. An electroporation device comprising: an applicator; a plurality
of electrodes extending from the applicator; a power supply in
electrical communication with the electrodes; and a cooling/heating
element coupled to the applicator and positioned adjacent the
electrodes, wherein the power supply provides a first electrical
signal to a first electrode and a second electrical signal to a
second electrode, wherein the first and second electrical signals
combine to produce a wave having a beat frequency, wherein the
first and second electrical signals each have at least one of a
unipolar waveform and a bipolar waveform, wherein the first
electrical signal has a first frequency and a first amplitude,
wherein the second electrical signal has a second frequency and a
second amplitude, wherein the first frequency is different from or
the same as the second frequency, and wherein the first amplitude
is different from or the same as the second amplitude.
9. The device of claim 8, wherein the cooling/heating element
includes a Peltier cooler.
10. The device of claim 9, wherein the cooling/heating element is
configured to adjust a temperature of tumor cells to about
4.degree. C. to about 45.degree. C.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/062,582, filed Oct. 24, 2013, which claims priority to
U.S. Provisional Application No. 61/718,561, filed Oct. 25, 2012,
U.S. Provisional Application No. 61/767,078, filed Feb. 20, 2013
and U.S. Provisional Application No. 61/791,968, filed Mar. 15,
2013, the contents of all of which are fully incorporated
herein.
TECHNICAL FIELD
[0002] This invention relates to an electroporation device
configured to deliver one or more electroporating signals in a
tolerable manner.
BACKGROUND
[0003] In the 1970's, it was discovered that electrical fields
could be used to create pores in cells without causing permanent
damage to the cell. This discovery made it possible for large
molecules, ions, and water to be introduced into a cell's cytoplasm
through the cell wall. In some instances, electroporation can be
used in topical treatments, such as for head and neck cancer, to
introduce chemicals and other compounds into the tumor. During
these procedures, the patient may not be under general anesthesia
so pain and involuntary muscle movement should preferably be
minimized.
[0004] Some electroporation devices can produce pulse trains that
induce electroporation within a cell's wall to allow the
introduction of large molecules, ions, and water into the cell's
cytoplasm. However, their electric field or signal frequency
(generally about 3.3 Hz) necessary to create the electroporation
effect might cause the patient to experience significant pain while
receiving treatment. The pain may be at least in part a result of
an inverse effect that the frequency or electric field has on skin
impedance when an electromagnetic wave is traveling through flesh.
For example, skin impedance at 50 Hz is approximately 32000.OMEGA.
while skin impedance at 4000 Hz is reduced to approximately
40.OMEGA. It has been observed that the higher the impedance, the
greater the pain.
[0005] Therefore, there is a need in the art for an electroporation
device that delivers a strong enough pulse for delivering an agent
for treatment, but prevents pain due to cell structure
impedance.
SUMMARY
[0006] The present invention is directed to an electroporation
device comprising an applicator, a plurality of electrodes
extending from the applicator, a power supply in electrical
communication with the electrodes, and a guide member coupled to
the electrodes. The electrodes may be associated with a cover area.
The power supply may be configured to generate one or more
electroporating signals to cells within the cover area. The guide
member may be configured to adjust the cover area of the
electrodes. The guide member may be slidably coupled to the
applicator.
[0007] The guide member may be slidably coupled to the applicator
and the applicator may be associated with an applicator end.
Sliding the guide member toward the applicator end may decrease the
cover area.
[0008] The guide member may be slidably coupled to the applicator.
The applicator may be associated with an applicator end. Sliding
the guide member away from the applicator end may increase the
cover area.
[0009] At least a portion of the electrodes may be positioned
within the applicator in a conical arrangement. The one or more
electroporating signals may be each associated with an electric
field. The device may further comprise a potentiometer coupled to
the power supply and electrodes. The potentiometer may be
configured to maintain the electric field substantially within a
predetermined range.
[0010] The one or more electroporating signals may be each
associated with an electric field. The device may further comprise
a potentiometer coupled to the power supply and the electrodes. The
potentiometer may be configured to maintain the electric field to
about 1300 V/cm.
[0011] The power supply may be associated with an output power. The
device may further comprise a potentiometer coupled to the power
supply and the electrodes. The potentiometer may be configured to
adjust the output power in response to the cover area of the
electrodes.
[0012] The power supply may be associated with an output power. The
device may further comprise a potentiometer coupled to the power
supply and the electrodes. The potentiometer may be configured to
reduce the output power in response to a reduced cover area of the
electrodes.
[0013] The power supply may be associated with an output power. The
device may further comprise a potentiometer coupled to the power
supply and the electrodes. The potentiometer may be configured to
increase the output power in response to an increased cover area of
the electrodes.
[0014] The one or more electroporating signals may be each
associated with an electric field. The device may further comprise
a potentiometer coupled to the power supply and the electrodes. The
potentiometer may be configured to maintain the electric field
within a predetermined range so as to substantially prevent
permanent damage in the cells within the cover area.
[0015] The one or more electroporating signals may be each
associated with an electrical field. The device may further
comprise a potentiometer coupled to the power supply and the
electrodes. The potentiometer may be configured to maintain the
electrical field within a predetermined range so as to
substantially minimize pain.
[0016] The power supply may provide a first electrical signal to a
first electrode and a second electrical signal to a second
electrode. The first and second electrical signals may combine to
produce a wave having a beat frequency. The first and second
electrical signals may each have at least one of a unipolar
waveform and a bipolar waveform. The first electrical signal may
have a first frequency and a first amplitude. The second electrical
signal may have a second frequency and a second amplitude. The
first frequency may be different from or the same as the second
frequency. The first amplitude may be different from or the same as
the second amplitude.
[0017] The power supply may be associated with an output power. The
device may further comprise a potentiometer coupled to the power
supply and the electrodes. The guide member may be slidably coupled
to the applicator. The potentiometer may be configured to adjust
the output power in response to the cover area of the
electrodes.
[0018] The potentiometer may be configured to reduce the output
power in response to a reduced cover area of the electrodes. The
potentiometer may be configured to increase the output power in
response to an increased cover area of the electrodes. The one or
more electroporating signals may be each associated with an
electric field and the potentiometer may be configured to maintain
the electric field to about 1300 V/cm.
[0019] The power supply may provide a first electrical signal to a
first electrode and a second electrical signal to a second
electrode. The first and second electrical signals may combine to
produce a wave having a beat frequency. The first and second
electrical signals may each have at least one of a unipolar
waveform and a bipolar waveform. The first electrical signal may
have a first frequency and a first amplitude. The second electrical
signal may have a second frequency and a second amplitude. The
first frequency may be different from or the same as the second
frequency. The first amplitude may be different from or the same as
the second amplitude.
[0020] The present invention is also directed to method of
electroporating cells using an electroporation device. The
electroporation device may comprise an applicator, a plurality of
electrodes extending from the applicator, a power supply in
electrical communication with the electrodes, and a guide member
coupled to the electrodes. The electrodes may be associated with a
cover area. The power supply may be configured to generate one or
more electroporating signals to cells within the cover area. The
guide member may be configured to adjust the cover area of the
electrodes.
[0021] The method may comprise administering selected molecules
into the cells within the cover area, contacting the cells with the
electrodes, and delivering the one or more electroporating signals.
The method may further comprise adjusting the cover area of the
electrodes.
[0022] The one or more electroporating signals may be each
associated with an electric field. Delivering the one or more
electroporating signals may further comprise maintaining the
electrical field within a predetermined range so as to
substantially prevent permanent damage in the cells within the
cover area.
[0023] The one or more electroporating signals may be each
associated with an electrical field. Delivering the one or more
electroporating signals may further comprise maintaining the
electrical field within a predetermined range so as to
substantially minimize pain. The method may further comprise
adjusting a temperature of the cells to about 4 .degree. C. to
about 45 .degree. C.
[0024] The present invention is also directed to an electroporation
device comprising an applicator, a plurality of electrodes
extending from the applicator, a power supply in electrical
communication with the electrodes, and a camera coupled to the
applicator and positioned adjacent the electrodes. The device may
further comprise a device memory in electronic communication with
the camera. The device memory may be configured to store an
electroporation treatment database.
[0025] The present invention is also directed to an electroporation
device comprising an applicator, a plurality of electrodes
extending from the applicator, a power supply in electrical
communication with the electrodes, and a cooling/heating element
coupled to the applicator and positioned adjacent the electrodes.
The power supply may provide a first electrical signal to a first
electrode and a second electrical signal to a second electrode. The
first and second electrical signals may combine to produce a wave
having a beat frequency. The first and second electrical signals
may each have at least one of a unipolar waveform and a bipolar
waveform. The first electrical signal may have a first frequency
and a first amplitude. The second electrical signal may have a
second frequency and a second amplitude. The first frequency may be
different from or the same as the second frequency. The first
amplitude may be different from or the same as the second
amplitude.
[0026] The cooling/heating element may include a Peltier cooler.
The cooling/heating element may be configured to adjust a
temperature of tumor cells to about 4 .degree. C. to about 45
.degree. C.
[0027] Other aspects of the invention will become apparent by
consideration of the detailed description and accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a cross-sectional view of an applicator of the
electroporation device according to one embodiment.
[0029] FIG. 2 is a perspective view of an electroporation device
illustrating an applicator according to another embodiment.
[0030] FIG. 3 is a schematic of circuitry of the electroporation
device of FIG. 2.
[0031] FIG. 4a is a perspective view of one embodiment of an
applicator of the electroporation device of FIG. 2, illustrating a
disposable needle array tip.
[0032] FIG. 4b is a cross-sectional view of the applicator of FIG.
4a.
[0033] FIG. 5 is a cross-sectional view of an applicator of the
electroporation device of FIG. 2, illustrating a retractable
shield.
[0034] FIG. 6 is a cut-away view of an applicator of the
electroporation device according to yet another embodiment.
[0035] FIG. 7 is a plan view of an applicator of the
electroporation device according to still another embodiment.
[0036] FIG. 8 is a schematic of one embodiment of an electrode
needle array of the electroporation device of FIG. 2, illustrating
partially insulated electrode needles.
[0037] FIG. 9 is a schematic of a 4.times.4 mapping array for
needles of the electroporation device of FIG. 2, illustrating 9
treatment zones.
[0038] FIG. 10a is a schematic of a pulse sequence for a 2.times.2
treatment zone of the electroporation device of FIG. 2.
[0039] FIGS. 10b-10d illustrate a pulse sequence for a 6-needle
array of the electroporation device of FIG. 2.
[0040] FIG. 11 is a graph plotting waveforms produced by the
electroporation device of FIG. 2.
[0041] FIGS. 12a-12c are schematic illustrations of opposing
unipolar waveforms and the resultant unipolar interference
waveform, produced by the electroporation device of FIG. 2.
[0042] FIGS. 13a-13c are schematic illustrations of two waveforms
and the resultant interference waveform, produced by the
electroporation device of FIG. 2.
[0043] FIG. 14 shows photonic emission at 24 hours for cells mixed
with DNA encoding luciferase.
[0044] FIG. 15 shows photonic emission at 24 hours for cells mixed
with DNA encoding luciferase and then treated with interference
electroporation.
[0045] FIG. 16 shows photonic emission of mice at day 1 following
injection with plasmid DNA.
[0046] FIG. 17 shows photonic emission of mice at day 1 following
injection with plasmid DNA and treatment with interference
electroporation.
[0047] FIG. 18 shows photonic emission of mice at day 2 following
injection with plasmid DNA.
[0048] FIG. 19 shows photonic emission of mice at day 2 following
injection with plasmid DNA and treatment with interference
electroporation.
[0049] FIG. 20 shows photonic emission of mice at day 5 following
injection with plasmid DNA.
[0050] FIG. 21 shows photonic emission of mice at day 5 following
injection with plasmid DNA and treatment with interference
electroporation.
[0051] FIG. 22 shows photonic emission of mice at day 7 following
injection with plasmid DNA.
[0052] FIG. 23 shows photonic emission of mice at day 7 following
injection with plasmid DNA and treatment with interference
electroporation.
[0053] FIG. 24 is a graph plotting time after plasmid DNA delivery
and average photon emission.
[0054] FIG. 25a shows representative hematoxylin and eosin
(H&E) staining of tissue sections from mice injected with
plasmid DNA; and FIG. 25b shows representative hematoxylin and
eosin (H&E) staining of tissue sections from mice injected with
plasmid DNA and receiving interference electroporation
treatment.
DETAILED DESCRIPTION
[0055] The inventors have discovered a new type of electroporation
device that can cover or accommodate tumors of various sizes. The
electroporation device may adjust electric signals in response to a
cover area of electrodes, so that the pain from the electric
signals may be tolerable when the electric signals are delivered to
cells within the cover area. The electric signals may include
opposing waveforms that produce a resultant interference waveform
to effectively target the cover area. Each opposing waveform may be
a unipolar waveform or a bipolar waveform. The resultant
interference waveform may be shaped to the tumor and the voltage
may vary across the tumor, for example, less voltage at a periphery
of the tumor and more voltage at a central portion of the
tumor.
[0056] Moreover, the electroporation device may include a camera to
measure a size of the tumor and to better place the electrode
needles. Furthermore, the electroporation device may include a
cooling/heating element to adjust a temperature of a surface of the
tumor. The invention thus provides an apparatus and a method for
the therapeutic application of electroporation while minimizing
tissue damage and the pain experienced by the patient.
I) ELECTROPORATION DEVICE
[0057] The electroporation device 10 of the present invention
includes a housing 14 (FIG. 2) containing circuitry (FIG. 3), an
electrode applicator 22, 200 removably coupled to the housing 14
(FIG. 2), and a foot pedal 26 coupled to the housing 14 and in
electrical communication with the circuitry 18 (FIG. 3). A remote
therapy activation connection may be provided to accommodate the
foot pedal 26 for activating pulses to the electrode applicator 22,
200. The foot pedal 26 may permit a physician to activate the
electroporation device 10 while freeing both hands for positioning
of the electrode applicator 22, 200 in a patient's tissue.
Indicator lights for fault detection, power on, and completion of a
therapy session may be provided for convenience. Other indicator
lights may be provided to positively indicate that the electrode
applicator 22, 200 is connected to the electroporation device 10
and to indicate the type of needle array. A standby/reset button
may be provided to pause the electroporation device 10 and reset
all functions of the electroporation device 10 to a default state.
A ready button may be provided to prepare the electroporation
device 10 for a therapy session. A "therapy in process" indicator
light may indicate that voltage pulses are being applied to the
electrode applicator 22, 200. In addition, the electroporation
device 10 may have audio indicators for such functions as a button
press, a fault state, commencement or termination of a therapy
session, indication of therapy in process, etc. In some
embodiments, the electroporation device 10 can be coupled to a
feedback sensor that detects heart beats. Applying pulses near the
heart may interfere with normal heart rhythms. By synchronizing
application of pulses to safe periods between beats, the
possibility of such interference may be reduced.
[0058] Referring to FIG. 1, the illustrated electrode applicator
200 includes a body 30, a first electrode 34 having a first set of
electrode needles 38, and a second electrode 42 having a second set
of electrode needles 46. During operation, the user can manually
manipulate the electrode applicator 22 to place the electrode
needles 38, 46 in physical contact with the target area of the
tissue. The illustrated electroporation device 10 includes a
potentiometer 228 that is configured to adjust an electric output
power in response to a cover area 220 of the electrodes 34, 42,
thereby maintaining an electric field of the electric signals
substantially within a predetermined range.
[0059] A) Electrode Applicator
[0060] Referring to FIG. 1, in the illustrated embodiment, a guide
member 204 is coupled to the electrodes 34, 42. The electrodes 34,
42 are associated with the cover area 220. A power supply is
configured to generate one or more electroporating signals 94, 110
to cells within the cover area 220. The guide member 204 is
configured to adjust the cover area 220 of the electrodes 34, 42.
In the illustrated embodiment, the guide member 204 is in the form
of a ring. In other embodiments, the guide member 204 can instead
include portions of a ring, arcuate members, and the like that can
suitably adjust the cover area 220 of the electrodes 34, 42.
[0061] In the illustrated embodiment, the guide member 204 is
coupled to the electrode applicator 200. The electrode applicator
200 is associated with an applicator end 212, and sliding the guide
member 204 toward the applicator end 212 decreases the cover area
220 of the electrodes 34, 42. For example, each electrode 34, 42
may be needle-shaped, and include spring tension at an end distal
to the applicator end 212. In the illustrated embodiment, the
electrodes 34, 42 are positioned within the electrode applicator
200 in a conical arrangement. The conical arrangement of the
electrodes 200 is associated with an apex or tip 216, where
individual electrodes 34, 42 are connected to one another in a
tight bundle, and the cover area 220 positioned away from the apex
or tip 216. In some embodiments, the cover area 220 may assume any
geometric form, including, but not limited to, a circle associated
with a diameter 224, an oval, an ellipse, a lens, a squircle, a
polygon, a symbol, or a combination thereof.
[0062] In the illustrated embodiment, for example, as the guide
member 204 is moved toward the applicator end 212, the
spring-tensioned electrodes 34, 42 are drawn radially inward within
the conical arrangement, thereby reducing the base diameter 224. On
the other hand, sliding the guide member 204 away from the
applicator end 212 increases the base diameter 224, thereby
increasing the cover area 220. Although FIG. 1 illustrates the
guide member 204 as being slidably coupled to the electrode
applicator 200, in other embodiments, the guide member 204 may be
coupled to the electrode applicator 200 using other mechanisms.
[0063] In the illustrated embodiment, the potentiometer 228 is
coupled to the power supply and the electrodes 34, 42. The power
supply is associated with an output power, and the potentiometer
228 is configured to adjust the output power in response to the
cover area 220 of the electrodes 34, 42. For example, in the
illustrated embodiment, sliding the guide member 204 toward and
away from the applicator end 212 can provide feedback to the power
supply regarding the associated base diameter 224 or cover area
220, so that the output power can be adjusted accordingly. In the
illustrated embodiment, the potentiometer 228 is electrically
insulated from the guide member 204 and supports the guide member
204 during travel. The potentiometer 228 is configured to reduce
the output power in response to a reduced base diameter 224 or
cover area 220 of the electrodes 34, 42, and increase the output
power in response to an increased base diameter 224 or cover area
220 of the electrodes 34, 42. In other embodiments, the
potentiometer 228 may be configured to adjust the output power
using other mechanisms.
[0064] The electroporating signals 94, 110 are each associated with
an electric field, and in some embodiments the potentiometer 228 is
configured to maintain the electric field substantially within a
predetermined range so as to substantially prevent permanent damage
in the cells within the cover area 220 and to minimize the amount
of pain experienced by the user. For example, the potentiometer 228
can be configured to maintain the electric field to about 1.300
kV/cm. In some embodiments, the potentiometer is configured to
maintain the electric field to at least 375 V/cm, at least 450
V/cm, at least 525 V/cm, at least 600 V/cm, at least 675 V/cm, at
least 750 V/cm, at least 825 V/cm, at least 900 V/cm, at least 975
V/cm, at least 1.000 kV/cm, at least 1.075 kV/cm, at least 1.150
kV/cm, or at least 1.225 kV/cm. In further embodiments, the
potentiometer 228 is configured to maintain the electric field to
no more than 1.300 kV/cm, no more than 1.225 kV/cm, no more than
1.150 kV/cm, no more than 1.075 kV/cm, no more than 1.000 kV/cm, no
more than 925 V/cm, no more than 850 V/cm, no more than 775 V/cm,
no more than 750 V/cm, no more than 675 V/cm, no more than 600
V/cm, no more than 525 V/cm, or no more than 450 V/cm. In other
embodiments, the potentiometer 228 may be configured to maintain
the electric field as other values.
[0065] FIGS. 2 and 5 illustrate an electrode applicator of an
electroporating device according to another embodiment. This
embodiment employs much of the same structure and has many of the
same properties as the embodiment of the electroporation device
described above in connection with FIG. 1. Accordingly, the
following description focuses primarily upon the structure and
features that are different than the embodiment described above in
connection with FIG. 1. Structure and features of the embodiment
shown in FIG. 1 that correspond to structure and features of the
embodiment of FIGS. 2 and 5 are designated hereinafter with like
reference numbers.
[0066] The guide member 206 in this embodiment is a retractible
shield. The retractible shield 206 may be restricted by a friction
O-ring (not shown) near a distal end of the body 30 of the
electrode applicator 22, and can be slid fore and aft along the
body 30 to protect or expose the first and second electrodes 34,
42. Thus, the retractible shield 206 is configured to adjust the
cover area 208 of the first and second electrodes 34, 42 to
accommodate tumors of various sizes.
[0067] Because it is possible for a number of different electrode
applicator 22 designs to be attached to the electroporation device
10, the electrode applicator 22 includes an electrically erasable
programmable read-only memory (EEPROM) chip 50 (FIG. 3) with
profile data stored thereon. The profile data is unique to each
specific applicator and may include information regarding the
model, the make, the number of electrodes present, and instructions
for a desired treatment. In use, the electroporation device 10 can
read the EEPROM chip 50 to assure the proper settings are being
used for each particular type of electrode applicator 22.
[0068] B) Integrated Wide-Angle Camera Applicator
[0069] Referring to FIG. 6, in the illustrated embodiment, a small
or miniature wide-angle camera 300 is embedded or integrated in the
electrode applicator 22 between the electrode needles 38, 46. In
some embodiments, this camera 300 may interface with an onboard
electroporator software to acquire details regarding a tumor, such
as type, size, shape, color, condition, and progression during
electroporation therapy (EPT). The electroporator software may
display an image transmitted from the camera 300, so that a
clinician or medical professional performing EPT can determine
tumor treatment coverage. In further embodiments, the tumor may be
displayed with a visible grid. The clinician may then utilize
visual analysis algorithms to measure a size of the tumor and to
better place the electrode needles 38, 46, thereby ensuring a
complete electroporation coverage. As explained below, in some
embodiments, the electroporator software may also record a patient
number (not necessarily including patient's personal information),
dates of treatments, waveform parameters, needle placement, and
therapeutic agent dosage.
[0070] C) Integrated Thermoelectric Cooler/Heater Applicator
[0071] Referring to FIG. 7, in the illustrated embodiment, a small
or miniature thermoelectric cooling/heating element 400 is included
or integrated in the electrode applicator 22. The illustrated
thermoelectric cooling/heating element 400 is located at a distal
end of the electrode applicator 22. In some embodiments, the
thermoelectric cooling/heating element 400 may provide non-contact
or radiant pre-cooling/heating to a surface of the tumor. In other
embodiments, however, the thermoelectric cooling/heating element
400 may provide contact cooling/heating to a surface of the
tumor.
[0072] Lowering a temperature of the tumor cells before
electroporation to about 4 .degree. C. may improve the transfection
of the therapeutic agent about ninefold. Thus, the thermoelectric
cooling/heating element 400 can reduce the temperature of the solid
tumor body, and thereby improve transfection. In some embodiments,
the thermoelectric cooling/heating element 400 can reduce the
temperature of the tumor cells before EPT to about 45 .degree. C.,
to about 44 .degree. C., to about 43 .degree. C., to about 42
.degree. C., to about 41 .degree. C., to about 40 .degree. C., to
about 39 .degree. C., to about 38 .degree. C., to about 37 .degree.
C., to about 36 .degree. C., about 35 .degree. C., about 34
.degree. C., about 33 .degree. C., about 32 .degree. C., about 31
.degree. C., about 30 .degree. C., about 29 .degree. C., about 28
.degree. C., about 27 .degree. C., about 26 .degree. C., about 25
.degree. C., about 24 .degree. C., about 23 .degree. C., about 22
.degree. C., about 21 .degree. C., about 20 .degree. C., about 19
.degree. C., about 18 .degree. C., about 17 .degree. C., about 16
.degree. C., about 15 .degree. C., about 14 .degree. C., about 13
.degree. C., about 12 .degree. C., about 11 .degree. C., about 10
.degree. C., about 9 .degree. C., about 8 .degree. C., about 7
.degree. C., about 6 .degree. C., about 5 .degree. C., or about 4
.degree. C. In some embodiments, the thermoelectric cooling/heating
element 400 can increase the temperature of the tumor cells before
electroporation to about 38 .degree. C., about 39 .degree. C.,
about 40 .degree. C., about 41 .degree. C., about 42 .degree. C.,
about 43 .degree. C., about 44 .degree. C., or about 45 .degree. C.
Thus, the thermoelectric cooling/heating element 400 can adjust the
temperature of the tumor cells before EPT to about 4 .degree. C. to
about 45 .degree. C., to about 33 .degree. C. to about 45 .degree.
C., to about 4 .degree. C. to about 40 .degree. C., or to about 33
.degree. C. to about 40 .degree. C.
[0073] In some embodiments, the thermoelectric cooling/heating
element 400 may include a Peltier cooler. A Peltier cooler is a
solid-state active heat pump creating a heat flux between a
junction of two different types of materials. The heat is thereby
transferred from one material to the other, with consumption of
electrical energy, depending on the direction of the current. In
other embodiments, the thermoelectric cooling/heating element 400
may utilize other cooling mechanisms. In operation, the clinician
may move the distal end of the electrode applicator 22 close to the
tumor before beginning EPT, and lower the temperature of the tumor
tissue through radiant cooling. The reduced tumor temperature may
increase the percentage of therapeutic agent transfer into the
cells. In some embodiments, the thermoelectric cooling/heating
element 400 may be powered by the same power supply as the
electrode applicator 22.
[0074] D) Electroporation Treatment Database
[0075] In some Embodiments, an Electroporation Device Memory (not
Shown) May Store Data such as tumor type, photographic record of
tumor size, color, shape, tumor progression, therapeutic agent
dosage, electroporation parameters, and needle insertion placement.
In further embodiments, this data may be collected into the
electroporation device memory and downloaded, e.g., periodically,
via a wireless connection to a cloud storage database (not shown).
This may facilitate the creation of a novel database which can be
developed to answer questions about future EPT protocols or aspects
of the treatment unknown at this time. In some embodiments, this
information may not include patient personal information, but
instead include a patient number. In further embodiments, patient
sex, age, location, treating physician, tumor type, photographic
record of tumor size, color, shape, tumor progression, therapeutic
agent dosage, EPT parameters, needle insertion placement, etc., may
be collected and included in the database.
[0076] E) Electrical Signals
[0077] The electroporation device 10 as disclosed herein is
operable to provide an unlimited variety of electric signals so
long as the pain from the electric signals is tolerable. In some
embodiments, the electroporation device 10 is operable to
separately apply pulses of high amplitude electric signals to at
least two pairs of the first and second electrodes 34, 42. The
electric signals may be applied proportionately to the distance
between the electrodes of a pair to generate a nominal field
strength of about 10 V/cm to about 1500 V/cm in the cells and
effect introduction of selected molecules into the cells without
permanently damaging the cells. In some embodiments, the electric
signals may be applied simultaneously. In further embodiments, the
electric signals may be applied to some, but not all
electrodes.
[0078] Referring to FIG. 11, in some embodiments, the
electroporation device 10 sends multiple, independent electric
signals during operation to selected electrode needles 34, 42 that,
when in contact with tissue, can cause electroporation in the cell
wall. That is, a power supply (not shown) may provide a first
electrical signal 94 to the first electrode 34 and a second
electrical signal 110 to the second electrode 42. When the first
and second electrodes 34, 42 are in electrical contact with a
biological sample, the first electrical signal 94, which has a
first frequency (corresponding to the wavelength 18 in FIG. 11),
and the second electrical signal 110, which has a second frequency
(corresponding to the wavelength 114 in FIG. 11), different or the
same from the first frequency (the first and second signals may
have amplitudes that are different or the same), combine to produce
a resultant wave 130 that may include a beat frequency and an
embedded frequency to effect introduction of selected molecules
into cells of the sample without permanently damaging the cells and
minimizing pain. In other embodiments, however, the power supply of
the electroporation device 10 may adjust the electrical signals for
all electrodes 34, 42 in unison. That is, the electrical signals
for one electrode may not be independent of other electrodes, and
the electrical signals 94, 110 may not produce a beat
frequency.
[0079] The nature of the tissue, the size of the selected tissue,
and its location determine the nature of the electric signals 94,
110 to be generated. It is desirable that the field be as
homogenous as possible and of the correct amplitude. An excessive
field strength may result in lysis of cells, whereas a low field
strength may result in a reduced efficiency of delivering agents
into the cell. This is especially true in the present invention
where the resultant or resulting wave 130 (e.g., the waveform
experienced by the patient during therapy) is the result of the
interference of the first and second electrical signals 94, 110. As
such, any minor variances in the first and second signals 94, 110
could result in major variances in the resulting wave 130.
[0080] As illustrated in FIG. 11, the first electrical signal 94
may include a sinusoidal, cosinusoidal or pulsed electrical wave
having a first frequency (corresponding to the wavelength 98 in
FIG. 11) and the first amplitude 102. The electrical signal may be
monopolar or bi-polar dependent upon the specific treatment being
administered. In some embodiments, the first frequency is generally
between about 500 Hz and about 10,000 Hz. In other embodiments, the
first frequency is between about 600 Hz and about 9,000 Hz. In
still other embodiments, the first frequency is between 700 Hz and
about 8,000 Hz. In still other embodiments, the first frequency is
between about 800 Hz and about 7,000 Hz. In still other
embodiments, the first frequency is between about 900 Hz and about
6,000 Hz. In still other embodiments, the first frequency is
between about 1,000 Hz and about 5,000 Hz. In still other
embodiments, the first frequency is between about 2,000 Hz and
about 4,000 Hz. Furthermore, the first amplitude 102 is generally
between about 150 V and about 3,000 V. In other embodiments, the
first amplitude 102 is between about 250 V and about 2,000 V. In
still other embodiments, the first amplitude 102 is between about
350 V and about 1,000 V. In still other embodiments, the first
amplitude 102 is between about 450 V and about 900 V. In still
other embodiments, the first amplitude 102 is between about 550 V
and about 800 V. In the case of a pulsed electrical wave, the first
electrical signal 94 may produce from about 500 pulses per second
to about 10,000 pulses per second.
[0081] As illustrated in FIG. 11, the second electrical signal 110
may include a sinusoidal or cosinusoidal electrical wave having a
second frequency (corresponding to the wavelength 114 in FIG. 11)
different from the first frequency and a second amplitude 118
substantially similar to the first amplitude 102. The second
electrical signal 110 may be monopolar or bi-polar dependent upon
the type of treatment being administered. In some embodiments, the
second frequency is generally between about 500 Hz and about 10,000
Hz. In other embodiments, the second frequency is between about 600
Hz and about 9,000 Hz. In still other embodiments, the second
frequency is between 700 Hz and about 8,000 Hz. In still other
embodiments, the second frequency is between about 800 Hz and about
7,000 Hz. In still other embodiments, the second frequency is
between about 900 Hz and about 6,000 Hz. In still other
embodiments, the second frequency is between about 1,000 Hz and
about 5,000 Hz. In still other embodiments, the second frequency is
between about 2,000 Hz and about 4,000 Hz. Furthermore, the second
amplitude 118 is generally between about 150 V and about 3,000 V.
In other embodiments, the second amplitude 118 is between about 250
V and about 2,000 V. In still other embodiments, the second
amplitude 118 is between about 350 V and about 1,000 V. In still
other embodiments, the second amplitude 118 is between about 450 V
and about 900 V. In still other embodiments, the second amplitude
118 is between about 550 V and about 800 V. In the case of a pulsed
electrical wave, the second electrical signal 110 may produce from
about 500 pulses per second to about 10,000 pulses per second.
[0082] Illustrated in FIG. 11, the resultant wave 130 is the result
of the combined interference of the first electrical signal or wave
94 and the second electrical signal or wave 110. Governed by the
laws of wave interference, the resultant wave 130 may include a
beat frequency, defined as the frequency of the oscillation of the
envelope of the resultant wave 130 (corresponding to a wavelength
138 in FIG. 11). The resultant wave 130 may also include an
embedded frequency defined as the frequency of the carrier wave
(corresponding to a wavelength 142 in FIG. 11). In the illustrated
embodiment, the beat frequency is between about 2 Hz and about
3,000 Hz. In other embodiments, the beat frequency is between about
50 Hz and about 2,000 Hz. In still other embodiments the beat
frequency is between about 100 Hz and about 1,000 Hz. In still
other embodiments, the beat frequency is between about 200 Hz and
about 900 Hz. In still other embodiments, the beat frequency is
between about 300 Hz and about 800 Hz. In still other embodiments,
the beat frequency is between about 400 Hz and about 700 Hz.
Furthermore, the embedded frequency is from about 500 Hz to about
10,000 Hz. In other embodiments, the embedded frequency is between
about 600 Hz and about 9,000 Hz. In still other embodiments, the
embedded frequency is between 700 Hz and about 8,000 Hz. In still
other embodiments, the embedded frequency is between about 800 Hz
and about 7,000 Hz. In still other embodiments, the embedded
frequency is between about 900 Hz and about 6,000 Hz. In still
other embodiments, the embedded frequency is between about 1,000 Hz
and about 5,000 Hz. In still other embodiments, the embedded
frequency is between about 2,000 Hz and about 4,000 Hz. An
amplitude 140 of the resultant wave 130 is generally equal to the
sum of the amplitudes of the interfering waves (e.g., the first
wave and the second wave corresponding to the first and second
electrical signals 94, 110). In the case of a pulsed wave, the beat
frequency of the resultant wave 130 is from about 2 pulses per
second (Hz) to about 3,000 pulses per second (Hz).
[0083] It is to be understood that although the above described
wave forms are sinusoidal or cosinusoidal in nature, square waves,
saw tooth waves, step waves, and the like may also be produced by
the electroporation device 10. Referring also to FIG. 12, each
opposing waveform may be a unipolar waveform or a bipolar waveform.
When each opposing waveform is unipolar (e.g., square wave) as
illustrated in FIGS. 12a and 12b, the result interference waveform
is also unipolar as illustrated in FIG. 12c. The amplitude of the
illustrated resultant wave is generally equal to the sum of the
amplitudes of the opposing waves, and the beat frequency may be
defined as the pulse frequency of the resultant square wave.
[0084] In some embodiments, the resultant wave 130 may be the
result of the interference of more than two wave forms. The
specific waveforms produced by the electroporation device 10 are
designed to produce the desired electroporation effect in the cell
wall while minimizing the amount of pain experienced by the user,
minimizing tissue damage, and providing maximum pore formation for
introducing an agent into a cell.
[0085] As illustrated in FIGS. 13a-13c, two separate waveforms
(i.e., signals 1 and 2) may combine or interfere such that the
resultant waveform has a specific shape. The shape of the resultant
waveform may be similar to (or the same as) the shape of the tissue
or cells targeted for electroporation. Accordingly, the
electroporation effect is directed to the targeted tissue or cells
and not surrounding or other tissues, thereby minimizing tissue
damage in the user. The waveforms may be varied such that the
resultant waveform has any desired shape, and therefore, the
electroporation effect may be produced in tissues or areas of
treatment having different shapes and sizes. The electroporation
effect may be shaped to the tissue or area of treatment.
[0086] Additionally, the voltage at the intersection of the two
waveforms may be the sum of the voltages of the two waveforms. For
example, if both the first and second waveforms have a voltage of
650 V, then at the intersection of the first and second waveforms,
the voltage would be 1300 V. The waveforms may each have a lower
voltage, but combine or interfere to provide the higher voltage
needed for maximum pore formation for introducing the agent into a
cell. These lower voltage waveforms have a higher frequency, which
in turn, reduces impedance through the tissue, decreases tissue
damage, and minimizes pain experienced by the user.
[0087] The waveforms may also combine such that the resultant
waveform has a shape matching the targeted tissue and the voltage
varies across the targeted tissue. For example, if the targeted
tissue is a tumor, the resultant waveform may have a shape similar
to (or the same as) a shape of the tumor, but because the voltage
varies across the tumor shape, different portions of the tumor are
exposed to more or less voltage (e.g., less voltage at a periphery
portion of the tumor and higher voltage at a central portion of the
tumor). The electroporation effect may be shaped to the tissue or
area of treatment while a spectrum of voltages is present in the
tissue or area of treatment. This in turn allows the
electroporation effect to be directional to minimize tissue damage
and pain experienced by the user while maximizing pore formation
for the introduction of the agent into the cell.
[0088] F) Signal Generation
[0089] Illustrated in FIG. 3, the circuitry 18 of the
electroporation device 10 includes an AC power module 54, a first
waveform generator 58, a second waveform generator 62, and a
control module 66. In some embodiments, the circuitry 18 of the
electroporation device 10 produces a beat wave 130 (FIG. 11). The
beat wave 130 is designed to produce electroporation in the cell
while minimizing the amount of pain experienced by the patient.
[0090] The AC power module 54 of the circuitry 18 receives
electricity from a power source (e.g., from a wall socket, a
generator, and the like), conditions the signal, and isolates the
signal into multiple power sources to be used as electrical power
throughout device. More specifically, the AC power module 54
produces a low-voltage DC power supply 70 suitable for use by the
control module 66, and a high voltage power supply 74 (e.g., up to
several thousand volts) suitable for waveform generation. In the
illustrated construction, the AC power module 54 utilizes a large
toroidal transformer to isolate and condition the power source
signal.
[0091] The AC power module 54 also includes a plurality of
capacitors 78 to store the high voltage power supply 74 for use by
the first and second waveform generators 58, 62. In the illustrated
embodiment, pulse width modulation is used to control the high
voltage power supply 74 so it better accommodates therapy and
delivery requirements.
[0092] The control module 66 of the interior circuitry 18 includes
a microprocessor 82 and a custom programmable logic array (PLA) 86.
The PLA 86 of the current invention independently controls the
first and second waveform generators 58, 62. Stated differently,
the PLA 86 is pre-programmed with multiple sets of waveform
profiles, each corresponding to a unique therapy or treatment.
During operation, the microprocessor 82 sends a signal to the PLA
86 instructing it to produce a particular waveform profile. The PLA
86 then independently controls the first and second waveform
generators 58, 62 until the signal from the microprocessor 82 is
stopped. The PLA 86 also receives feedback from the produced
waveforms (e.g., through a voltage and current monitor 88), to
assure the waveforms are within acceptable parameters. In the event
the waveforms are unacceptable or a fault is detected, the PLA 86
may shut down automatically.
[0093] Utilizing the PLA 86 independent of the microprocessor 82
ensures that the generated waveforms are not affected by the other
services required by the microprocessor 82 during operation of the
device. The separation also ensures the system reacts immediately
to any faults or error inputs in a safe fashion.
[0094] The microprocessor 82 acts as a system controller, sending
and receiving signals from various devices in the electroporation
device 10. One such function of the microprocessor 82 includes
determining the type of electrode applicator 22 in use. When the
electrode applicator 22 is connected to the housing 14, the
microprocessor 82 reads the profile data stored in the EEPROM chip
50 and uses that data to determine the proper waveform profiles the
PLA 86 should produce. The microprocessor 82 also receives a signal
from the foot pedal 26 and is capable of outputting data regarding
the treatment to a printer or other output device 90.
[0095] The first waveform generator 58 receives high voltage
electrical power from the capacitors 78 of the AC power module 54,
along with input from the PLA 86, to produce a first electrical
signal 94 having a first frequency (corresponding to a wavelength
98 in FIG. 11) and a first amplitude 102 (see FIG. 11). In the
illustrated construction, the first waveform generator 58 utilizes
an insulated gate bipolar transistor or IGBT to produce the first
electrical signal 94.
[0096] The first waveform generator 58 is electrically connected to
a solid-state high-voltage relay 106 to control and output the
first electrical signal 94 to the desired electrode needles 38 of
the first electrode 34. In the illustrated embodiment, after the
waveform is produced (e.g., by the IGBT), the waveform may be
checked by the PLA 86 before being passed on by the high-voltage
relay 106.
[0097] The second waveform generator 62 is substantially similar to
the first waveform generator 58. The second waveform generator 62
receives high voltage electrical power from the capacitors 78 of
the AC power module 54, along with input from the PLA 86, to
produce a second electrical signal 110 having a second frequency
(corresponding to a wavelength 114 in FIG. 11), different from the
first frequency, and a second amplitude 118. In the illustrated
construction, the second waveform generator 62 utilizes an
insulated gate bipolar transistor or IGBT to produce the second
electrical signal 110.
[0098] The second waveform generator 62 is electrically connected
to a solid-state high-voltage relay 122 to control and output the
second electrical signal 110 to the desired electrode needles 46 of
the second electrode 42. In the illustrated embodiment, after the
waveform is produced (e.g., by the IGBT), the waveform may be
checked by the PLA 86 before being passed on by the high-voltage
relay 122.
[0099] G) Needle Array
[0100] 1) Disposable Needle Array Tips
[0101] The electroporation device 10 may include disposable needle
array tips. The whole needle array shown in FIG. 5 may be
disposable, including the cable and the connector. However, it may
be more desirable to make the needle array tip an independent
component that is detachable from the body 30 and the cable. Hence,
a needle array tip may be disposed after use similar to the
disposable needles used in injection of a fluid drug. Such
disposable needle array tips can be used to eliminate possible
contamination due to improper sterilization when reusing a needle
array tip.
[0102] FIG. 4a shows one embodiment 1800 of the electroporation
applicator according to this aspect of the invention. The
electroporation applicator 1800 includes an applicator handle 1810,
a detachable needle array tip 1820, and an applicator cable 1812
connected to the applicator handle 1810. The detachable needle
array tip 1820 can be engaged to and detached from one end of the
applicator handle 1810. When engaged to the applicator handle 1810,
the detachable needle array tip 1820 can receive electrical signals
from the electroporation device 10 through the applicator cable
1812.
[0103] FIG. 4b shows structure details of the applicator handle
1810 and the detachable needle array tip 1820. The applicator
handle 1810 includes a main body 1811A and a distal end 1811B
formed on one end of the main body 1811A. The other end of the main
body 1811A is connected to the applicator cable (not shown, see
FIG. 4a). The main body 1811A includes two or more conducting wires
1815 for transmitting electrical signals to the detachable needle
array tip 1820. These signals may include needle voltage setpoint,
pulse length, pulse shape, the number of pulses, and switching
sequence. In one embodiment, when the detachable needle array tip
1820 is used to deliver a liquid substance, one or more electrode
needles may be made hollow for transmitting the liquid substance
and one or more liquid channels may be accordingly implemented in
the applicator handle 1810. The liquid channel may be integrated
with one of the conducting wires 1815 by, for example, using a
metal-coated plastic tube or a metal tube. Alternatively, the
liquid substance may be delivered to a target by using a separate
device, for example, prior to application of the electrical pulses.
The distal end 1811B has an opening 1813 for engaging the needle
array tip 1820. A plurality of connector holes 1814 are formed for
receiving connector pins in the detachable needle array tip
1820.
[0104] The detachable needle array tip 1820 has a plurality of
electrode needles 1822 forming a desired needle array, a support
part 1823A that holds the electrode needles 1822, and a connector
part 1823B for engaging to the applicator handle 1810. In one
embodiment, when the detachable needle array tip 1820 is also used
to deliver the liquid substance, at least one electrode needle is
hollow and is connected to a liquid channel in the applicator
handle 1810 for receiving the liquid substance. The connector part
1823B is shaped to be inserted into the opening 1813 in the distal
end 1811B of the applicator handle 1810. A locking or snapping
mechanism may be optionally implemented to secure the detachable
needle array tip 1820 to the applicator handle 1810. A plurality of
connector pins 1825 corresponding to the electrode needles 1822 are
formed in the connector part 1823B for engaging to the respective
connector holes 1814 in the distal end 1811B.
[0105] The detachable needle array tip 1820 may include a
contamination shield 1824 formed on the support part 1823A for
preventing the applicator handle 1810 from directly contacting any
substance during an electroporation process. A removable plastic
cover 1826 may also be formed on the support part 1823A to seal the
electrode needles 1822 and maintain the sterility of the needles
1822 prior to use.
[0106] In some embodiments, the applicator handle 1810 may be
configured to receive a detachable needle array tip 1820 with other
numbers of electrode needles 1822. In further embodiments, an
electrical identification element or EEPROM 50 may be implemented
to allow the electroporation device 10 of FIG. 2 to determine the
number of the electrode needles in an attached needle array tip.
This identification element 50 may also be configured to generate
proper electrical signal parameters corresponding to an identified
needle array tip. A desired needle array addressing scheme may be
selected accordingly to address the electrode needles.
[0107] 2) Needle Arrays with Partially Insulated Electrode
Needles
[0108] The electroporation device 10 may include needle arrays with
partially insulated electrode needles. Each electrode needle in the
fixed and disposable needle arrays shown in FIGS. 1, 4a, 4b, 5, 6,
7, and 8 may be partially covered with an insulator layer in such a
way that only a desired amount of the tip portion is exposed. The
pulsed electric fields generated by such a partially insulated
needle array are primarily concentrated in regions between and near
the exposed tip portions of the electrode needles during a
treatment, and are small in regions between and near the insulated
portions. A partially insulated needle array can be used to confine
the electroporation in a targeted area with a tumor and
significantly shield the skin and tissues beyond the target area
from the electroporation process. This provides protection to the
uninvolved skin and tissues, which are at risk because certain
drugs may cause undesired or even adverse effects when injected
into uninvolved surface tissue above the target area.
[0109] FIG. 8 shows one embodiment of a partially insulated needle
array 1900. A support portion 1910 is provided to hold multiple
electrode needles 1920 in a predetermined array pattern. Each
electrode needle 1920 has a base portion 1922 that is covered with
a layer of electrically insulating material such as Teflon and a
tip portion 1924 that is exposed. When electrical voltages are
applied to the electrode needles 1920, the generated electrical
fields in regions among and near the exposed tip portions 1924 are
sufficiently strong to cause electroporation but the electrical
fields in regions among and near the insulated base portions 1922
are either negligibly small or completely diminished so that
electroporation cannot be effected due to the shielding of the
insulation. Therefore, electroporation is localized or confined in
regions where the exposed tip portions 1924 are positioned.
[0110] The lengths of the insulated base portion 1922 and the
exposed tip portion 1924 may be predetermined or may be adjustable
based on the location of a specific target area in a body part. In
one implementation, each needle electrode may be pre-wrapped with a
suitable insulating layer to cover most of the electrode needle
with a minimal usable exposed tip portion. A user may adjust a
desired amount of the exposed tip portion as needed in a
treatment.
[0111] The partially insulated electrode needles shown in FIG. 8
can be used for both the fixed needle array as shown in FIG. 5 and
the disposable needle array shown in FIGS. 4a and 4b.
[0112] 3) Needle Array Addressing
[0113] The electroporation device 10 of FIG. 2 is designed to
accommodate electrode applicators 22 having varying numbers of
electrode needles 38, 46. Accordingly, an addressing scheme has
been developed that, in the preferred embodiment, permits
addressing up to 16 different needles, designated A through P,
forming up to 9 square treatment zones and several types of
enlarged treatment zones. A treatment zone comprises at least 4
needles in a configuration of opposing pairs that are addressed
during a particular pulse. During a particular pulse, two of the
needles of a treatment zone are of positive polarity and two are of
negative polarity.
[0114] FIG. 9 shows a preferred 4.times.4 mapping array for needles
forming 9 square treatment zones numbered from the center and
proceeding outward radially and then clockwise. In the preferred
embodiment, this mapping array defines 4-needle, 6-needle,
8-needle, 9-needle, and 16-needle electrode configurations. A
4-needle electrode comprises needles placed in positions F, G, K,
and J (treatment zone 1). A 9-needle electrode comprises needles
placed in positions defining treatment zones 1-4. A 16-needle
electrode comprises needles placed in positions defining treatment
zones 1-9.
[0115] FIG. 10a shows a pulse switching sequence for a 2.times.2
treatment zone or mapping array in accordance with one embodiment
of the invention. During any of four pulses comprising a cycle,
opposing pairs of needles are respectively positively and
negatively charged, as shown. Other patterns of such pairs are
possible, such as clockwise or counterclockwise progression. For
example, for a 9-needle electrode configuration, a preferred cycle
comprises 16 pulses (4 treatment zones at 4 pulses each). For
example, for a 16-needle electrode configuration, a preferred cycle
comprises 36 pulses (9 treatment zones at 4 pulses each).
[0116] A 6-needle electrode can comprise a circular or hexagonal
array as shown in FIGS. 10b-10d. Alternatively, a 6-needle
electrode can be defined as a subset of a larger array, such as is
shown in FIG. 9. For example, with reference to FIG. 9, a 6-needle
electrode can be defined as a 2.times.3 rectangular array of
needles placed in positions defining treatment zones 1-2 (or any
other linear pair of treatment zones), or a hexagonal arrangement
of needles B, G, K, N, I, E (or any other set of positions defining
a hexagon) defining an enlarged treatment zone (shown in dotted
outline in FIG. 9). Similarly, an 8-needle electrode can comprise
an octagon, or a subset of the larger array shown in FIG. 9. For
example, with reference to FIG. 9, an 8-needle electrode can be
defined as a 2.times.4 array of needles placed in positions
defining treatment zones 1, 2 and 6 (or any other linear triplet of
treatment zones), or an octagonal arrangement of needles B, C, H,
L, O, N, I, E (or any other set of positions defining an octagon)
defining an enlarged treatment zone.
[0117] FIGS. 10b-10d show a hexagonal arrangement and one possible
activation sequence. FIG. 10b shows a first sequence, in which
needles G and K are positive and needles I and E are negative
during a first pulse, and have reversed polarities during a next
pulse; needles B and N, shown in dotted outline, are inactive. FIG.
10c shows a second sequence, in which needles K and N are positive
and needles E and B are negative during a first pulse, and have
reversed polarities during a next pulse; needles G and I are
inactive. FIG. 10d shows a third sequence, in which needles N and I
are positive and needles B and G are negative during a first pulse,
and have reversed polarities during a next pulse; needles K and E
are inactive. In some embodiments, a total of 6 pulses may be
applied in a cycle of sequences. A similar activation sequence can
be used for an octagonal arrangement, which may apply other numbers
of pulses.
[0118] Regardless of physical configuration, the preferred
embodiments of the invention may use at least two switched pairs of
electrodes (for example, as shown in FIG. 10a) in order to achieve
a relatively uniform electric field in tissue undergoing EPT. The
electric field intensity should be of sufficient intensity to
effect the process of electroporation, to allow incorporation of a
treatment agent.
[0119] H) "Sweet Spot" Manipulation
[0120] The electroporation device 10 may produce a "sweet spot"
during use. The "sweet spot" can be defined as the area in the
tissue where the first electric signal 94 interferes with the
second electric signal 110 to produce the resultant wave 130. In
some embodiments, the electroporation device 10 is designed to move
the "sweet spot" with respect to the needles 38, 46 of the
electrode applicator 22. More specifically, the electroporation
device 10 may adjust the amplitude, frequency, and pulse time of
the signals being produced at each individual electrode needle 38,
46 to move, with respect to the electrode needles 38, 46, the exact
location where the resultant wave 130 is produced in the tissue. As
such, the device 10 is able to treat multiple areas of the tissue
without the need to continuously move the applicator 22. Although a
unipolar resultant result interference waveform can effectively
target the sweet spot, a bipolar resultant interference waveform
may more effectively target or move the sweet spot compared to a
unipolar resultant interference waveform, depending on the usage
requirements or preferences for the particular electroporation
device 10.
II) THERAPEUTIC METHOD
[0121] The electroporation device 10 may be used in a therapeutic
method. The therapeutic method of the invention includes
electrotherapy, also referred to herein as electroporation therapy
(EPT), for the delivery of an agent or molecule to a cell or
tissue. The term "agent" or "molecule" as used herein refers to,
for example, drugs (e.g., chemotherapeutic agents), nucleic acids
(e.g., polynucleotides), peptides, and polypeptides, including
antibodies. The term polynucleotides include DNA, cDNA, and RNA
sequences. It should be understood that the electroporation of
tissue can be performed in vitro, in vivo, or ex vivo.
Electroporation can also be performed utilizing single cells, e.g.,
single cell suspensions, in vitro, or ex vivo in cell culture.
[0122] Drugs contemplated for use in the method of the invention
are typically chemotherapeutic agents having an antitumor or
cytotoxic effect. Such drugs or agents include bleomycin,
neocarcinostatin, suramin, doxorubicin, carboplatin, taxol,
mitomycin C, and cisplatin. Other chemotherapeutic agents will be
known to those of ordinary skill in the art (see, for example, The
Merck Index). In addition, "membrane-acting" agents are also
included in the method of the invention. These agents may also be
agents as listed above, or alternatively, agents which act
primarily by damaging the cell membrane. Examples of
membrane-acting agents include N-alkylmelamide and para-chloro
mercury benzoate. The chemical composition of the agent will
dictate the most appropriate time to administer the agent in
relation to the administration of the electric pulse. For example,
while not wanting to be bound by a particular theory, it is
believed that a drug having a low isoelectric point (e.g.,
neocarcinostatin, IEP=3.78), would likely be more effective if
administered post-electroporation in order to avoid electrostatic
interaction of the highly charged drug within the field. Further,
such drugs as bleomycin, which have a very negative log P, (P being
the partition coefficient between octanol and water), are very
large in size (MW=1400), and are hydrophilic, thereby associating
closely with the lipid membrane, diffuse very slowly into a tumor
cell and are typically administered prior to or substantially
simultaneous with the electric pulse. In addition, certain agents
may require modification in order to enter the cell allow more
efficiently. For example, an agent such as taxol can be modified to
increase solubility in water which would allow it to enter the cell
more efficiently. Electroporation facilitates entry of bleomycin or
other similar drugs into the tumor cell by creating pores in the
cell membrane.
[0123] In one embodiment, the invention provides a method of
electroporating cells using the electroporation device 10,
comprising administering selected molecules into the cells within
the cover area 220, contacting the cells with the electrodes 34,
42, and the electroporating signals 94, 110. In some embodiments,
the cover area 220 of the electrodes 34, 42 may be adjusted before
contacting the cells with the electrodes 34, 42. In further
embodiments, the electric field associated with the electroporating
signals 94, 110 may be maintained within a predetermined range so
as to substantially prevent permanent damage in the cells within
the cover area 220, and to minimize pain.
[0124] In another embodiment, the invention provides a method for
the therapeutic application of electroporation to a tissue of a
subject for introducing molecules into cells therein, comprising
providing an array of electrodes, at least one of the electrodes
having a needle configuration for penetrating tissue; inserting the
needle electrode into selected tissue for introducing molecules
into the tissue; positioning a second electrode of the array of
electrodes in conductive relation to the selected tissue; applying
a first electric signal to the first electrode and applying a
second electric signal to the second electrode such that a
resultant electrical signal or wave is formed in the tissue from
the wave interference between the first wave and the second wave.
The resultant wave has a beat frequency sufficient to cause
electroporation in the cell wall, and the embedded frequency is
sufficient to minimize pain.
[0125] In addition to minimizing pain and tissue damage, the method
of the invention may increase the uptake of the agent by cells
relative to a method that does not employ EPT. Uptake or
introduction of the agent to the cells may be increased by about
0.5-fold to about 50-fold, about 0.5-fold to about 45-fold, about
0.5-fold to about 40-fold, about 0.5-fold to about 35-fold, about
0.5-fold to about 30-fold, about 0.5-fold to about 25-fold, about
1-fold to about 50-fold, about 1.5-fold to about 50-fold, about
2-fold to about 50-fold, about 2.5-fold to about 50-fold, or about
3-fold to about 50-fold. Uptake of the agent by the cells may be
increased by 0.5-fold to about 6-fold, about 0.75-fold to about
6-fold, about 1-fold to about 6-fold, about 1.25-fold to about
6-fold, about 1.5-fold to about 6-fold, about 1.75-fold to about
6-fold, about 2-fold to about 6-fold, about 2.25-fold to about
6-fold, about 2.5-fold to about 6-fold, about 2.75-fold to about
6-fold, about 3-fold to about 6-fold, about 3.25-fold to about
6-fold, about 0.5-fold to about 5.75-fold, about 0.5-fold to about
5.5-fold, about 0.5-fold to about 5.25-fold, about 0.5-fold to
about 5-fold, about 0.5-fold to about 4.75-fold, about 0.5-fold to
about 4.5-fold, about 0.5-fold to about 4.25-fold, about 0.5-fold
to about 4-fold, or about 0.5-fold to about 3.75-fold. Uptake of
the agent by the cells may also be increased by about 0.75-fold to
about 5.75-fold, about 1-fold to about 5.5-fold, about 1.25-fold to
about 5.25-fold, about 1.5-fold to about 5-fold, about 1.75-fold to
about 4.75-fold, about 2-fold to about 4.5-fold, about 2.25-fold to
about 4.25-fold, about 2.5-fold to about 4-fold or about 2.75-fold
to about 3.75-fold.
[0126] Uptake of the agent by the cells may be increased by about
18-fold to about 30-fold, about 18-fold to about 29-fold, about
18-fold to about 28-fold, about 18-fold to about 27-fold, about
18-fold to about 26-fold, about 18-fold to about 24-fold, about
19-fold to about 30-fold, about 20-fold to about 30-fold, about
21-fold to about 30-fold, about 22-fold to about 30-fold or about
23-fold to about 30-fold. Uptake of the agent by the cells may also
be increased by about 19-fold to about 29-fold, about 20-fold to
about 28-fold, about 21-fold to about 27-fold, about 22-fold to
about 26-fold, about 23-fold to about 25-fold, or about 23-fold to
about 24-fold. Uptake of the agent by the cells may be increased by
about 18-fold, about 19-fold, about 20-fold, about 21-fold, about
22-fold, about 23-fold, about 24-fold, about 25-fold, about
26-fold, about 27-fold, about 28-fold, about 29-fold, or about
30-fold.
[0127] In another embodiment, uptake or introduction of the agent
to the cells may be increased by about 50% to about 5000%, about
50% to about 4500%, about 50% to about 4000%, about 50% to about
3500%, about 50% to about 3000%, about 50% to about 2500%, about
100% to about 5000%, about 150% to about 5000%, about 200% to about
5000%, about 250% to about 5000%, or about 300% to about 5000%.
Uptake of the agent by the cells may be increased by about 50% to
about 600%, about 75% to about 600%, about 100% to about 600%,
about 125% to about 600%, about 150% to about 600%, about 175% to
about 600%, about 200% to about 600%, about 225% to about 600%,
about 250% to about 600%, about 275% to about 600%, about 300% to
about 600%, about 325% to about 600%, about 50% to about 575%,
about 50% to about 550%, about 50% to about 525%, about 50% to
about 500%, about 50% to about 475%, about 50% to about 450%, about
50% to about 425%, about 50% to about 400%, or about 50% to about
375%. Uptake of the agent by the cells may also be increased by
about 75% to about 575%, about 100% to about 550%, about 125% to
about 525%, about 150% to about 500%, about 175% to about 475%,
about 200% to about 450%, about 225% to about 425%, about 250% to
about 400%, or about 275% to about 375%. Uptake of the agent by the
cells may be increased by about 345%, about 346%, about 347%, about
348%, about 349%, about 350%, about 351%, about 352%, about 353%,
about 354%, about 355%, about 356%, about 357%, about 358%, about
359%, about 360%, about 361%, about 363%, about 364%, about 365%,
about 366%, about 367%, about 368%, about 369%, about 370%, about
371%, about 372%, about 373%, about 374%, about 375%, about 376%,
about 377%, about 378%, about 379%, or about 380%.
[0128] Uptake of the agent by the cells may also be increased by
about 1800% to about 3000%, about 1800% to about 2900%, about 1800%
to about 2800%, about 1800% to about 2700%, about 1800% to about
2600%, about 1800% to about 2500%, about 1800% to about 2400%,
about 1900% to about 3000%, about 2000% to about 3000%, about 2100%
to about 3000%, about 2200% to about 3000%, or about 2300% to about
3000%. Uptake of the agent by the cells may be increased by about
1850% to about 2950%, about 1900% to about 2900%, about 1950% to
about 2850%, about 2000% to about 2800%, about 2050% to about
2750%, about 2100% to about 2700%, about 2150% to about 2650%,
about 2200% to about 2600%, about 2250% to about 2550%, about 2300%
to about 2500%, or about 2350% to about 2450%. Uptake of the agent
by the cells may be increased by about 2300%, about 2310%, about
2320%, about 2330%, about 2340%, about 2350%, about 2360%, about
2361%, about 2362%, about 2363%, about 2364%, about 2365%, about
2366%, about 2367%, about 2368%, about 2369%, about 2370%, about
2371%, about 2372%, about 2373%, about 2374%, about 2375%, about
2376%, about 2377%, about 2378%, about 2379%, about 2380%, about
2381%, about 2382%, about 2383%, about 2384%, about 2385%, about
2386%, about 2387%, about 2388%, about 2389% or about 2390%.
[0129] Accordingly, the method of the invention minimizes the pain
experienced by the subject by decreasing impedance due to cell
structure (e.g., skin) while increasing the efficiency of
introducing the agent into a cell(s). The method of the invention
advantageously provides effective delivery of the agent to within
the cells, but unlike other electrotherapy methods, minimizes (or
reduces) the pain experienced by the subject due to impedance.
[0130] It may be desirable to modulate the expression of a gene in
a cell by the introduction of a molecule by the method of the
invention. The term "modulate" envisions for example the
suppression of expression of a gene when it is over-expressed, or
augmentation of expression when it is under-expressed. Where a cell
proliferative disorder is associated with the expression of a gene,
nucleic acid sequences that interfere with the gene's expression at
the translational level can be used. This approach utilizes, for
example, antisense nucleic acid, ribozymes, or triplex agents to
block transcription or translation of a specific mRNA, either by
masking that mRNA with an antisense nucleic acid or triplex agent,
or by cleaving it with a ribozyme.
[0131] It is to be understood that the above described treatment
may be utilized on various forms of solid cancer types, such as,
but not limited to, sarcomas, carcinomas, and lymphomas. Solid
tumors may be located throughout the body such as in the neck,
lungs, skin, brain, prostate, liver, pancreatic, gall bladder,
stomach, and lymph nodes. These tumors may further metastasize to
other locations throughout the body. The electroporation device 10
can be used to introduce agents such as bleomycin to kill the tumor
by necrosis and further stimulate the immune system to prevent
metastasis.
[0132] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262:40, 1990). In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will not
translate a mRNA that is double-stranded. Antisense oligomers of
about 15 nucleotides are preferred, since they are easily
synthesized and are less likely to cause problems than larger
molecules when introduced into the target cell. The use of
antisense methods to inhibit the in vitro translation of genes is
well known in the art (Marcus-Sakura, Anal. Biochem., 172:289,
1988).
[0133] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1(3):227, 1991;
Helene, C., Anticancer Drug Design, 6(6):569, 1991).
[0134] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0135] There are two basic types of ribozymes, namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
18-based recognition sequences are preferable to shorter
recognition sequences. Therefore, "hammerhead"-type ribozymes are
preferable to tetrahymena-type ribozymes for inactivating a
specific mRNA species.
[0136] The invention also provides gene therapy for the treatment
of cell proliferative or immunologic disorders mediated by a
particular gene or absence thereof. Such therapy would achieve its
therapeutic effect by introduction of a specific sense or antisense
polynucleotide into cells having the disorder. Delivery of
polynucleotides can be achieved using a recombinant expression
vector such as a chimeric virus, or the polynucleotide can be
delivered as "naked" DNA for example.
[0137] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
When the subject is a human, a vector such as the gibbon ape
leukemia virus (GaLV) can be utilized. A number of additional
retroviral vectors can incorporate multiple genes. All of these
vectors can transfer or incorporate a gene for a selectable marker
so that transduced cells can be identified and generated.
[0138] Therapeutic peptides or polypeptides may also be included in
the therapeutic method of the invention. For example,
immunomodulatory agents and other biological response modifiers can
be administered for incorporation by a cell. The term "biological
response modifiers" is meant to encompass substances which are
involved in modifying the immune response. Examples of immune
response modifiers include such compounds as lymphokines
Lymphokines include tumor necrosis factor, interleukins 1, 2, and
3, lymphotoxin, macrophage activating factor, migration inhibition
factor, colony stimulating factor, and alpha-interferon,
beta-interferon, and gamma-interferon and their subtypes.
[0139] Also included are polynucleotides which encode metabolic
enzymes and proteins, including antiangiogenesis compounds, e.g.,
Factor VIII or Factor IX. The macromolecule of the invention also
includes antibody molecules. The term "antibody" as used herein is
meant to include intact molecules as well as fragments thereof,
such as Fab and F(ab').sub.2.
[0140] Administration of a drug, polynucleotide or polypeptide, in
the method of the invention can be, for example, parenterally by
injection, rapid infusion, nasopharyngeal absorption, dermal
absorption, and orally. In the case of a tumor, for example, a
chemotherapeutic or other agent can be administered locally,
systemically, or directly injected into the tumor. When a drug, for
example, is administered directly into the tumor, it is
advantageous to inject the drug in a "fanning" manner. The term
"fanning" refers to administering the drug by changing the
direction of the needle as the drug is being injected or by
multiple injections in multiple directions like opening up of a
hand fan, rather than as a bolus, in order to provide a greater
distribution of drug throughout the tumor. As compared with a
volume that is typically used in the art, it is desirable to
increase the volume of the drug-containing solution, when the drug
is administered (e.g., injected) intratumorally, in order to ensure
adequate distribution of the drug throughout the tumor. For
example, in the EXAMPLES using mice herein, one of skill in the art
typically injects 50 .mu.l of drug-containing solution, however,
the results are greatly improved by increasing the volume to 150
.mu.l. In human clinical studies, approximately 20 ml would be
injected to ensure adequate perfusion of the tumor. Preferably, the
injection should be done very slowly all around the base and by
fanning Although the interstitial pressure is very high at the
center of the tumor, it is also a region where very often the tumor
is necrotic.
[0141] Preferably, the molecule is administered substantially
contemporaneously with the electroporation treatment. The term
"substantially contemporaneously" means that the molecule and the
electroporation treatment are administered reasonably close
together with respect to time, i.e., before the effect of the
electrical pulses on the cells diminishes. The administration of
the molecule or therapeutic agent depends upon such factors as, for
example, the nature of the tumor, the condition of the patient, the
size and chemical characteristics of the molecule, and the
half-life of the molecule.
[0142] Preparations for parenteral administration include sterile,
aqueous, or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Besides the inert diluents, such
compositions can also include adjuvants, wetting agents,
emulsifying, and suspending agents. Further, vasoconstrictor agents
can be used to keep the therapeutic agent localized prior to
pulsing.
[0143] Any cell can be treated by the method of the invention. The
illustrative examples provided herein demonstrate the use of the
method of the invention for the treatment of tumor cells, e.g.,
pancreas, lung, head and neck, cutaneous and subcutaneous cancers.
Other cell proliferative disorders are amenable to treatment by the
electroporation method of the invention. The term "cell
proliferative disorder" denotes malignant as well as non-malignant
cell populations which often appear to differ from the surrounding
tissue both morphologically and genotypically. Malignant cells
(i.e., tumors or cancer) develop as a result of a multi-step
process. The method of the invention is useful in treating
malignancies or other disorders of the various organ systems,
particularly, for example, cells in the pancreas, head and neck
(e.g., larynx, nasopharynx, oropharynx, hypopharynx, lip, throat,)
and lung, and also including cells of heart, kidney, muscle,
breast, colon, prostate, thymus, testis, and ovary. Further,
malignancies of the skin, such as basal cell carcinoma or melanoma
may also be treated by the therapeutic method of the invention (see
Example 2). Preferably the subject is human; however, it should be
understood that the invention is also useful for veterinary uses in
non-human animals or mammals.
[0144] In yet another embodiment, the invention provides a method
for the therapeutic application of electroporation to a tissue of a
subject for damaging or killing cells therein while minimizing the
amount of pain experienced by the patient. The method includes
providing an array of electrodes; positioning a second electrode of
the array of electrodes in conductive relation to the selected
tissue; and applying a first electric signal to the first electrode
and applying a second electric signal to the second electrode such
that a resultant electrical signal or wave is formed from the wave
interference between the first signal and the second. The method
may utilize a low voltage and a long pulse length, e.g., a nominal
electric field from about 25 V/cm to 75 V/cm and pulse length from
about 5 .mu.sec to 99 msec.
[0145] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
III) EXAMPLES
Example 1
Interference Electroporation Treatment In Vitro
Materials and Methods
[0146] Plasmid.
[0147] The plasmid gWiz-Luc, which encoded luciferase, was acquired
from Aldevron (Fargo, N. Dak.).
[0148] Electroporation.
[0149] B16 F10 cells (2.5.times.10.sup.6 cells/mL) were prepared in
complete medium (i.e., Mcoy's 5A medium with 10% Fetal Bovine Serum
(FBS)). Cells were split into two groups, Group 1 and Group 2.
Group 1 cells were mixed with DNA, but received no interference
electroporation (IEP) treatment. Specifically, 1 mL of cell
suspension was transferred to a cuvette and 50 .mu.L gWiz-Luc (2
mg/mL) was added to this cuvette to arrive at a final concentration
of 100 .mu.g/mL of DNA. The cells and DNA were mixed by pipette
before placement into a cuvette holder. The cells received no IEP
treatment and the cell suspension was subsequently transferred to a
6-well plate. The cells were incubated at 37 degrees Celsius, 5%
CO.sub.2 before imaging as described below.
[0150] Group 2 cells were mixed with DNA and then received IEP
treatment. Specifically, two cuvettes were prepared, in which each
cuvette received 1 mL of cell suspension and 50 .mu.L gWiz-Luc (2
mg/mL) to arrive at a final concentration of 100 .mu.g/mL of DNA. A
pipette was used to mix the cell suspension and DNA before
placement of each cuvette into a cuvette holder. The parameters for
IEP were 650V/cm each channel.times.2 channels, 100 .mu.s, 1 KHz, 6
pulses. After IEP, 140 .mu.L of cell suspension was transferred
from each cuvette into a respective well of a E-well plate. Each
well contained 2 mL complete medium. This was done in triplicate
for each cuvette (i.e., 3 wells for each cuvette). The cells were
incubated at 37 degrees Celsius, 5% CO.sub.2 before imaging as
described below.
[0151] Cell Imaging.
[0152] 24 hours (hr) after electroporation, medium was removed from
each well of the 6-well plates and 300 .mu.L pre-warmed medium
containing D-luciferin (250 .mu.g/ml, Goldbio, St. Louis, Mo., USA)
was added to each well. Cells were then incubated at 37 degrees
Celsius for 5 minutes (min) before imaging. Imaging was done with
an IVIS Spectrum system (Caliper Life Sciences, Hopkinton, Mass.,
USA) and assessment of photonic emissions from the cells was
performed about 8 min to about 10 min after incubation of the cells
with D-luciferin.
Results
[0153] To determine if interference electroporation (IEP) could
increase the efficiency of molecule uptake by cells, an in vitro
system was utilized, in which cells were split into two different
treatment groups. The two treatment groups were as follows: Group 1
was cells mixed with DNA, but receiving no IEP treatment; and Group
2 was cells mixed with DNA and receiving IEP treatment. The DNA
used was a plasmid that encoded luciferase and DNA uptake was
indirectly measured by luciferase activity. Luciferase acts upon
its substrate D-luciferin to cause the emission of light or
photons.
[0154] FIGS. 14 and 15 show the captured images of photonic
emission (p/sec/cm.sup.2/sr) from Groups 1 and 2, respectively, 24
hours after electroporation. The measurement for each well and the
average emission for Groups 1 and 2 are shown in Table 1. For each
of Groups 1 and 2, the average emission was calculated by averaging
the measurements from the six wells depicted in FIGS. 14 and 15,
respectively. For FIG. 15, each row of three wells corresponded to
one of the two cuvettes described above that received IEP
treatment.
TABLE-US-00001 TABLE 1 Measured Photon Emission (p/sec/cm.sup.2/sr)
Well 1A Well 1B Well 1C Well 2A Well 2B Well 2C Average Group 1
2.93E+05 2.53E+05 2.43E+05 3.61E+05 3.61E+05 2.32E+05 2.90E+05
Group 2 8.55E+06 6.69E+06 6.74E+06 1.10E+07 8.58E+06 9.13E+06
8.44E+06
[0155] As shown in FIGS. 14 and 15 and Table 1, IEP treatment
increased photonic emission by 2374% as compared to cells that did
not receive IEP treatment. These data indicated that IEP treatment
significantly increased DNA uptake by cells. Regular
electroporation worked at least as well as IEP (data not
shown).
Example 2
Interference Electroporation Treatment In Vivo
Materials and Methods
[0156] Plasmid.
[0157] The plasmid gWiz-Luc, which encoded luciferase, was obtained
from Aldevron (Fargo, N. Dak.). The gWiz-Luc preparation had
endotoxin levels less than 100 EU/mg.
[0158] Cells.
[0159] B16.F10 melanoma cells were maintained in McCoy's 5A media
(Mediatech, Manassas, Va.) supplemented with 10% fetal bovine serum
(Life Technologies, Grand Island, N.Y.) and 1% Gentamycin at 37
degrees Celsius and 5% CO.sub.2 humidified air.
[0160] Mice.
[0161] Female C57BL/6J mice (6-8 weeks old) were purchased from
Jackson Laboratories (Bar Harbour, Me.).
[0162] Tumor Establishment.
[0163] 50 .mu.L of B16.F10 melanoma cells (1.times.10.sup.6 cells)
were introduced by subcutaneous (s.c.) injection into the shaved
left flank of the C57BL/6J mouse in order to establish the tumor.
Each mouse was monitored closely for tumor development and tumor
volume was measured using digital calipers. Tumor volume was
calculated by using the formula for the volume of an ellipsoid:
v=.pi.ab.sup.2/6, where a is the long diameter and b is the short
diameter. Tumors were allowed to grow to a diameter of 3
millimeters (mm) to 5 mm (about 5 days after s.c. injection) before
delivery of plasmid DNA as described below.
[0164] Electroporation.
[0165] A tumor was established in each mouse as described above and
after the tumor reached a diameter of 3 mm to 5 mm, 50 .mu.g of
plasmid DNA (2 mg/mL) was injected directly into the tumor using a
syringe with a 25 gauge needle. The electrode was placed around the
tumor and pulses were applied using an interference electroporation
(IEP) protocol. The IEP protocol employed a 4-needle electrode with
the following parameters: (1) 650 V/channel, 100 .mu.s, 6 pulses,
900 .mu.s gap.
[0166] Imaging.
[0167] Each mouse was anesthetized and D-Luciferin was injected
into the tumor. Imaging was done with an IVIS Spectrum system
(Caliper Life Sciences, Hopkinton, Mass., USA). Photonic emissions
from the tumor of each mouse was imaged at day 1, day 2, day 5, and
day 7 after electroporation treatment. A portion of the mouse that
did not contain the tumor was also imaged and served as a control
for background photonic emissions.
Results
[0168] The data above demonstrated that interference
electroporation (IEP) increased the efficiency of DNA uptake by
cells in vitro. To further examine the capabilities of IEP, DNA
uptake in vivo was examined. In particular, tumors were established
in two groups of mice and plasmid DNA was administered to each
tumor by injection. Group 1 received no IEP treatment and contained
four mice. Group 2 also contained four mice and received IEP
treatment with the parameters: 650 V/channel, 100 .mu.s, 6 pulses,
900 .mu.s gap. Each group of mice was imaged at day 1, day 2, day
5, and day 7 after DNA administration to measure photonic emissions
from the tumors.
[0169] FIGS. 16, 18, 20, and 22 show the captured images of the
photonic emissions (p/sec/cm.sup.2/sr) from the tumors of Group 1
mice at day 1, day 2, day 5, and day 7, respectively. FIGS. 17, 19,
21, and 23 show the captured images of the photonic emissions from
the tumors of Group 2 mice at day 1, day 2, day 5, and day 7,
respectively. The solid circle on the hind region of each mouse
indicated the area imaged for the tumor. An additional area was
imaged on one mouse in each of Group 1 and 2 to provide a control
for background photonic emission (see solid circle on the head
region of the mouse). Tables 2-5 list the measurements obtained for
each mouse on day 1, day 2, day 5, and day 7, respectively, for
Groups 1 and 2. The average photonic emission in Tables 2-5 is an
average of the photonic emission of the four mice in each of Groups
1 and 2.
TABLE-US-00002 TABLE 2 Measured Photon Emission (p/sec/cm.sup.2/sr)
on Day 1 Mouse 1 Mouse 2 Mouse 3 Mouse 4 Average Group 1 4.88E+05
2.10E+05 2.11E+05 5.41E+05 3.62E+05 Group 2 8.79E+05 1.49E+06
4.41E+05 2.37E+06 1.29E+06
TABLE-US-00003 TABLE 3 Measured Photon Emission (p/sec/cm.sup.2/sr)
on Day 2 Mouse 1 Mouse 2 Mouse 3 Mouse 4 Average Group 1 2.64E+05
1.06E.+-.05 1.18E+05 3.52E+05 2.10E+05 Group 2 4.51E+05 1.18E+06
9.55E+04 1.31E+06 7.59E+05
TABLE-US-00004 TABLE 4 Measured Photon Emission (p/sec/cm.sup.2/sr)
on Day 5 Mouse 1 Mouse 2 Mouse 3 Mouse 4 Average Group 1 1.26E+05
2.52E+04 2.50E+04 1.89E+05 9.12E+04 Group 2 1.01E+05 2.54E+05
3.76E+04 8.89E+05 3.21E+05
TABLE-US-00005 TABLE 5 Measured Photon Emission (p/sec/cm.sup.2/sr)
on Day 7 Mouse 1 Mouse 2 Mouse 3 Mouse 4 Average Group 1 1.33E+05
2.79E+-04 2.12E+04 6.71E+04 6.23E+04 Group 2 5.11E+04 9.44E+04
4.78E+04 7.39E+05 2.33E+05
[0170] FIG. 24 shows a comparison of the average photonic emission
for Groups 1 and 2 at day 1, day 2, day 3, and day 4. These data
indicated that at each day, IEP treatment increased photonic
emission from the tumors as compared to mice that did not receive
IEP treatment. Particularly, IEP treatment increased photonic
emission by 357%, 361%, 351%, and 374% on day 1, day 2, day 5, and
day 7, respectively. Accordingly, these dated indicated that IEP
treatment significantly increased DNA uptake by targeted cells
(i.e., the tumor cells) in an animal. Regular electroporation
worked at least as well as IEP (data not shown).
Example 3
Interference Electroporation Treatment does not Damage Tissue
Materials and Methods
[0171] The plasmid gWiz-GFP, which encoded green fluorescent
protein (GFP), was obtained from Aldevron (Fargo, N. Dak.). The
gWiz-GFP preparation had endotoxin levels less than 100 EU/mg.
[0172] Cells.
[0173] B16.F10 melanoma cells were maintained in McCoy's 5A media
(Mediatech, Manassas, Va.) supplemented with 10% fetal bovine serum
(Life Technologies, Grand Island, N.Y.) and 1% Gentamycin at 37
degrees Celsius and 5% CO.sub.2 humidified air.
[0174] Mice.
[0175] Female C57BL/6J mice (6-8 weeks old) were purchased from
Jackson Laboratories (Bar Harbour, Me.).
[0176] Tumor Establishment.
[0177] 50 .mu.L of B16.F10 melanoma cells (1.times.10.sup.6 cells)
were introduced by subcutaneous (s.c.) injection into the shaved
left flank of the C57BL/6J mouse in order to establish the tumor.
Each mouse was monitored closely for tumor development and tumor
volume was measured using digital calipers. Tumor volume was
calculated by using the formula for the volume of an ellipsoid:
v=.pi.ab.sup.2/6, where a is the long diameter and b is the short
diameter. Tumors were allowed to grow to a diameter of 3
millimeters (mm) to 5 mm (about 5 days after s.c. injection) before
delivery of plasmid DNA as described below.
[0178] Electroporation.
[0179] A tumor was established in each mouse as described above and
after the tumor reached a diameter of 3 mm to 5 mm, 50 .mu.g of
plasmid DNA (2 mg/mL) was injected directly into the tumor using a
syringe with a 25 gauge needle. The electrode was placed around the
tumor and pulses were applied using an interference electroporation
(IEP) protocol. The IEP protocol employed a 4-needle electrode with
the following parameters: 650 V/channel, 100 .mu.s, 6 pulses, 900
.mu.s gap.
[0180] Staining.
[0181] Tumor sections were stained with hematoxylin and eosin
(H&E) stain.
Results
[0182] The above data demonstrated that IEP increased the uptake of
DNA by cells both in vitro (i.e., tissue culture) and in vivo
(i.e., in established tumors in mice). The effect of IEP on cell
biology was further examined by H&E staining of tumor sections
after electroporation. In particular, the staining was utilized to
determine if IEP caused tissue damage.
[0183] Tumors were established in two groups of mice and plasmid
DNA was administered to each tumor by injection. Group 1 received
no IEP treatment. Group 2 received IEP after injection. After 7
days, mice were sacrificed and the respective tumors were sectioned
for H&E staining Representative staining for Groups 1 and 2 is
shown in FIGS. 25a and 25b, respectively.
[0184] As seen in FIGS. 25a and 25b, similar cell morphology was
observed in the stained tumor tissue sections from Groups 1 and 2.
These data indicated that tissue receiving IEP treatment was not
damaged by the electrical pulses from the IEP treatment.
Example 4
EPT for Treatment of Tumors In Vivo
[0185] A single treatment procedure will involve an injection of
bleomycin (0.5 units in 0.15 ml saline) intratumorally, using
fanning, followed by application of six electrical pulses of the
first electrical signal and the second electrical signal,
simultaneously, using needle array electrodes as described in the
present application, arranged along the circumference of a circle 1
cm in diameter.
[0186] The needle arrays of variable diameters (e.g., 0.5 cm, 0.75
cm and 1.5 cm) can also be used to accommodate tumors of various
sizes. Stoppers of various heights can be inserted at the center of
the array to make the penetration depth of the needles into the
tumor variable. A built-in mechanism will allow switching of
electrodes for maximum coverage of the tumor by the pulsed field.
The electrical parameters will be: 780 V/cm center field strength
and 6.times.99 .mu.s pulses spaced at 1-second intervals.
Example 5
Clinical Trials for Basal Cell Carcinomas and Melanomas
[0187] The effectiveness of bleomycin-EPT on tumors will be
assessed similar to Example 1.
Example 6
EPT for Head and Neck Cancers
[0188] A single-center feasibility clinical study will be conducted
in which the efficacy of the EPT procedure in combination with
intralesional bleomycin will be compared to that for traditional
surgery, radiation, and/or systemic chemotherapy. Approximately 50
study subjects will be enrolled in the study. All study subjects
will be assessed prior to treatment by examination and biopsy.
Patients will be treated with bleomycin intratumoral injection and
needle arrays of different diameters with six needles. The voltage
will be set to achieve a nominal electric field strength of 1300
V/cm (the needle array diameter is multiplied by 1300 to provide
the required voltage). The pulse length will be 100 .mu.s.
Postoperative assessment of study subjects will be weekly for 4-6
weeks, and monthly thereafter for a total of 12 months.
Approximately 8 to 12 weeks following therapy, a biopsy of the
tumor site will be performed. Use of CT or MRI scans will be
utilized in accordance to standard medical follow-up evaluation of
HNC subjects.
[0189] Tumor evaluation will include measuring the tumor diameter
(in centimeters) and estimating its volume (in cubic centimeters).
Prior to intratumoral administration of bleomycin sulfate, the
tumor site will be anesthetized with 1% lidocaine (xylocalne) and
1:100,000 epinephrine. The concentration of bleomycin sulfate
injected will be 4 units per milliliter, up to a maximum dose of 5
units per tumor. If more than one tumor per subject is treated, a
total of 20 units per subject should not be exceeded. The dose of
bleomycin administered will be 1 unit/cm.sup.3 of calculated tumor
volume. Approximately ten minutes subsequent to the injection of
bleomycin sulfate, the applicator will be placed on the tumor and
electrical pulses initiated. In this study, success will be defined
as significant tumor regression in a period of 16 weeks or less
without major side effects seen with traditional therapy. There are
four possible response outcomes:
[0190] Complete Response (CR): Disappearance of all evidence of
tumor as determined by physical examination, and/or biopsy.
[0191] Partial Response (PR): 50% or greater reduction in tumor
volume.
[0192] No Response (NR): less than 50% reduction in tumor
volume.
[0193] If the tumor increases (25% tumor volume) in size, other
therapy, if indicated, will be instituted per subject's desire.
Example 7
Low Voltage Long Pulse Length (LVLP) EPT
[0194] Electroporation response of MCF-7 will be carried out at
both high voltage/short pulse length (HVSP) and low voltage/long
pulse length (LVLP) using an XTT assay. XTT is a tetrazolium
reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)
carbonyl]-2H-tetrazolium hydroxide, which is metabolically reduced
in viable cells to a water-soluble formazan product. Therefore,
only the cells which are live convert XTT to formazan. The
metabolic conversion of XTT to formazan after 70 hours will be
measured spectrophotometrically at 450 nm. (M. W. Roehm, et al., An
Improved Colorimetric Assay for Cell Proliferation and Viability
Utilizing the Tetrazolium Salt XTT, J. Immunol. Methods 142:2,
257-265, 1991.) The percent cell survival values will be calculated
using a formula from the O.D. values of the sample. (Control, with
100% cell survival (D-E) and control with 0% cell survival (D-E
with SDS).) The experiments with HVSP will be done to permit direct
comparison with the LVLP mode of EPT.
Example 8
Cytotoxicity of Drugs with EPT In Vitro
[0195] Cells will be obtained from ATTC (American Type Tissue
Collection, Rockville, Md., USA) and maintained by their
recommended procedures. The cells will be suspended in appropriate
medium and uniformly seeded in 24/96 well plates. One of bleomycin,
cisplatin, mitomycin C, doxorubicin and taxol will be added
directly to the cell suspensions at final concentrations of about
1.times.10.sup.-4 (1E-4) to 1.3.times.10.sup.-9 (1.3E-9). The
electrical pulses generated by a BTX T820 ElectroSquarePorator will
be delivered to the cell suspensions in microplates using a BTX
needle array electrode as described herein. Depending on the
experiment, six pulses of either 100 .mu.s or 10 ms and at various
nominal electric fields of either high voltage or low voltages will
be applied between two opposite pairs of a six-needle array using
EPT-196 needle array switch. The microplates will be incubated for
either 20 hrs or 70 hrs and the cell survival will be measured by
the XTT assay.
Example 9
Unipolar Waveform Prototype
[0196] Prototypes will be assembled, e.g., using off-the-shelf
instruments. The prototypes will be used to produce two opposing
waveforms or signals that create an interference waveform. As
illustrated in FIGS. 12a and 12b, in one prototype, each opposing
waveform may be unipolar, and, in combination, the resultant
interference waveform may also be unipolar as illustrated in FIG.
12c. In other prototypes, each opposing waveform may be bipolar as
illustrated in FIG. 11, and the resultant interference waveform may
therefore be bipolar.
[0197] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
[0198] For example, a variable valve lift (VVL) apparatus or a
continuously variable valve lift (CVVL) apparatus implemented so
that the valve is operated with another lift in accordance with
revolutions per minute of an engine, and a variable valve timing
(VVT) apparatus which opens and closes the valve at proper timing
in accordance with revolutions per minute of the engine have been
researched.
[0199] For example, a variable valve lift (VVL) apparatus or a
continuously variable valve lift (CVVL) apparatus implemented so
that the valve is operated with another lift in accordance with
revolutions per minute of an engine, and a variable valve timing
(VVT) apparatus which opens and closes the valve at proper timing
in accordance with revolutions per minute of the engine have been
researched.
* * * * *