U.S. patent application number 10/810829 was filed with the patent office on 2004-10-14 for electrodes coated with treating agent and uses thereof.
Invention is credited to King, Alan D., Walters, Richard E..
Application Number | 20040203124 10/810829 |
Document ID | / |
Family ID | 25444527 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040203124 |
Kind Code |
A1 |
King, Alan D. ; et
al. |
October 14, 2004 |
Electrodes coated with treating agent and uses thereof
Abstract
An object of the invention is to provide a method for delivery
of macromolecules into biological cells in the tissues of a patient
and includes the steps of: (a) providing electrodes (16) in an
electrode assembly (12), wherein the electrodes have fixed
electrode surfaces (42) which are coated with at least one static
layer of electrode releasable molecules (44) to be delivered; (b)
providing a waveform generator (15) for generating electric fields;
(c) establishing electrically conductive pathways between the
electrodes (16) and the waveform generator (15); (d) locating the
electrodes (16) such that the biological cells are situated
therebetween, and (g) providing electric fields in the form of
pulse waveforms from the waveform generator (15) to the electrodes
(16), such that molecules in the at least one static layer of the
electrode releasable molecules (44) on the electrodes (16) are
delivered into the biological cells. The electrode releasable
molecules (44) can be either electric field separable molecules
and/or solvent separable material. Another object of the invention
is to provide an apparatus for carrying out the method of the
invention. The static-coated electrode assembly (12) can be
provided in a sterile package (24), from which the electrode
assembly (12) is removed prior to use. The statically-coated
electrode assembly (12) can be in a form of a disposable assembly
(12) which is removable and replaceable from an electrode assembly
holder (13).
Inventors: |
King, Alan D.; (Takoma Park,
MD) ; Walters, Richard E.; (Columbia, MD) |
Correspondence
Address: |
Marvin S. Towsend
Patent Attorney
8 Grovepoint Court
Rockville
MD
20854
US
|
Family ID: |
25444527 |
Appl. No.: |
10/810829 |
Filed: |
March 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10810829 |
Mar 29, 2004 |
|
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|
09920861 |
Aug 3, 2001 |
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6713291 |
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Current U.S.
Class: |
435/173.6 ;
435/446 |
Current CPC
Class: |
A61P 43/00 20180101;
A61N 1/306 20130101; A61N 1/325 20130101; A61H 39/002 20130101 |
Class at
Publication: |
435/173.6 ;
435/446 |
International
Class: |
C12N 013/00; C12N
015/01 |
Claims
1. (cancelled)
2. (cancelled)
3. (cancelled)
4. (cancelled)
5. (cancelled)
6. (cancelled)
7. (cancelled)
8. (cancelled)
9. (cancelled)
10. (cancelled)
11. (cancelled)
12. (cancelled)
13. (cancelled)
14. (cancelled)
15. (cancelled)
16. (cancelled)
17. (cancelled)
18. (cancelled)
19. (cancelled)
20. (cancelled)
21. (cancelled)
22. (cancelled)
23. (cancelled)
24. (cancelled)
25. An electrode which includes a coating having at least one
static layer of electrode releasable molecules to be delivered into
biological cells:
26. A plurality of electrodes of claim 25 which form an electrode
assembly.
27. The electrode assembly of claim 26 wherein said plurality of
electrodes are arranged in at least two parallel rows of
electrodes.
28. The electrode assembly of claim 27 wherein said parallel rows
of electrodes include needle electrodes.
29. The electrode of claim 25 wherein said molecules in said static
coating are in a solid phase.
30. The electrode of claim 25 wherein said molecules in said static
coating are in a gel.
31. The electrode of claim 25 wherein said electrode includes a
fixed electrode surface which is coated with said static layer of
electrode releasable molecules.
32. The electrode of claim 31 wherein: said fixed electrode surface
42 includes a fixed surface matrix, and said molecules in said
static coating are in a liquid fixed on said fixed surface
matrix.
33. The electrode of claim 31 wherein said fixed electrode surface
includes solid surface particles.
34. The electrode of claim 33 wherein said solid surface particles
are metal particles.
35. The electrode of claim 31 wherein said fixed electrode surface
includes a liposome matrix.
36. The electrode of claim 31 wherein said fixed electrode surface
includes a solid polymer matrix.
37. The electrode of claim 25 wherein said molecules in said static
coating are macromolecules.
38. The electrode of claim 25 wherein said macromolecules in said
static coating include a polynucleotide vaccine.
39. The electrode of claim 25 wherein said macromolecules in said
static coating include a solid phase polynucleotide vaccine.
40. The electrode of claim 25 wherein said macromolecules in said
static coating include a DNA vaccine.
41. The electrode of claim 25 wherein said macromolecules in said
static coating include a solid phase DNA vaccine.
42. The electrode of claim 25 wherein said macromolecules in said
static coating include an RNA vaccine.
43. The electrode of claim 25 wherein said macromolecules in said
static coating include a solid phase RNA vaccine.
44. The electrode of claim 25 wherein said macromolecules in said
static coating include a protein-based vaccine.
45. The electrode of claim 25 wherein said macromolecules in said
static coating include a solid phase protein-based vaccine.
46. The electrode of claim 25 wherein said macromolecules in said
static coating include an organ treating agent.
47. The electrode of claim 46 wherein said organ treating agent
includes a deep tissue tumor treating agent.
48. The electrode of claim 25 which is in a form of a needle
electrode.
49. The electrode of claim 25, wherein coating of said electrode
with said static layer of molecules to be delivered to the
biological cells is carried out by the following steps: preparing a
liquid medium in which a quantity of said molecules are carried,
contacting said electrode with the prepared medium, and removing
said electrode from the medium and drying off the medium, such that
a static coating of said molecules remains on said electrode.
50. The electrode of claim 25 wherein coating of said electrode
with said static layer of molecules to be delivered to the
biological cells is carried out by the following steps: preparing a
liquid medium in which a quantity of said molecules are carried,
contacting said electrode with the prepared medium, applying pulse
waveforms to said electrode, such that a portion of said molecules
are bound to said electrode, and removing said electrode from the
medium and drying off the medium, such that a coating of said
molecules remains on said electrode.
51. (Cancelled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority based upon copending PCT
International Application Number PCT/US00/00014, filed 12 Jan.
2000, which is based upon copending U.S. Provisional Application
Serial No. 60/117,755, filed 28 Jan. 1999, and which was published
on 3 Aug. 2000 with PCT International Publication Number WO
00/44438.
TECHNICAL FIELD
[0002] The present invention relates generally to methods and
apparatus for delivery of macromolecules into cells. More
specifically, the present invention provides methods and apparatus
for delivering substances, such as macromolecules, e. g. deep tumor
tissue treating agents, polynucleotide vaccines (DNA vaccine and/or
RNA vaccine) and protein-based vaccines, into cells in tissues.
BACKGROUND ART
[0003] The first DNA vaccination procedure in the prior art was
called naked DNA vaccination because a liquid solution of DNA was
injected into the muscle of mice with no additives to enhance
transfection. This method does transfect a few cells and does
induce an immune response to the expressed antigen in mice.
However, in humans and primates, the method does not work well.
[0004] In the prior art, an improvement in DNA vaccine efficiency
was obtained by the use of a biolistic method for DNA delivery. The
biolistic method is done by coating metal microbeads with DNA and
shooting the particles into skin after accelerating the particles
to a chosen velocity. This method works much better than naked DNA.
Part of the reason is that the DNA coated particles are injected
into the skin to a depth that increases the chance of transfecting
Langerhans cells. However, the biolistic method has some
disadvantages. First, it causes some skin damage that may scar in
some individuals. Second, in spite of the increased efficiency,
more efficiency is needed. Third, the ballistic particle remains
inside the patient after treatment. In this respect, it would be
desirable if a method for delivering DNA to biological cells were
provided which does not cause skin damage that results in scarring.
Also, it would be desirable if a method for delivering DNA to
biological cells were provided which does not leave a residue of
ballistic particles in cells that are treated. As a matter of
interest, the following U.S. patents disclose biolistic methods:
U.S. Pat. Nos. 5,036,006 and 5,478,744.
[0005] A number of additional approaches to delivering
macromolecules to biological cells are disclosed in the prior art
and are represented by the following U.S. patents or other
publications as follows.
[0006] U.S. Pat. No. 5,019,034 of Weaver et al discloses a process
for electroporation of tissues in which electrodes are placed on
top of the tissue surface, such as skin, of a patient. Molecules
that are used for treating the skin are placed in reservoirs on top
of the skin surface, and the treatment molecules must penetrate
into the skin tissues transdermally. That is, the treatment
molecules must pass from outside the skin to inside the skin. Not
only is the surface layer of the skin relatively impermeable, if
the layers of the skin to be treated are near the basal lamina of
the epidermis, then the treatment molecules must traverse
considerable skin tissue before the cells to be treated are reached
by the treatment molecules Such a treatment method is inefficient
for treating cells near the basal lamina. Rather than using
electrodes that are placed on the skin surface and have treatment
molecules pass through the skin transdermally to treat biological
cells near the basal lamina of the epidermis, it would be desirable
if an electroporation method were provided for delivering molecules
to biological cells in the epidermis, near the basal lamina,
without having the treatment molecules pass through the skin
transdermally.
[0007] U.S. Pat. No. 5,273,525 of Hofmann discloses an apparatus
for electroporation of drugs and genetic material into tissues
which employs relatively long hollow hypodermic needle for placing
the drugs and genetic material in the vicinity of the tissues to be
electroporated. Whenever a hollow hypodermic is employed in a
tissue, the tissue is cut with a circular cut by the hollow
hypodermic needle. As a result, when a patient receives hypodermic
injection, the patient has considerable pain. To avoid such a
circular cut, and to avoid the considerable pain involved, it would
be desirable if a method for delivering molecules to biological
cells were provided which does not employ a hypodermic needle.
[0008] U.S. Pat. No. 5,318,514 of Hofmann discloses an applicator
for the electroporation of drugs and genes into cells. The
applicator includes a plurality of needle electrodes which can be
penetrated into the skin of a patient. Material to be
electroporated into the skin is retained in a fluid reservoir which
wets an open cell foam elastomer carrier for the fluid. Because the
material to be electroporated is retained in a fluid, in both the
reservoir and the open cell foam elastomer, careful control of the
amount of the material at the electrode surfaces is difficult. It
is difficult to control how much fluid flows down from the
reservoir and the open cell foam elastomer to the surfaces of the
needle electrodes, and, thereby, it is difficult to control how
much of the treatment molecules is actually present on the surfaces
of the electrodes 16 as the electroporation process is being
carried out on the patient. Moreover, the presence of the fluid
medium can have a flushing or washing effect on the tissues that
are electroporated in such a way that the electroporation process
is interfered with. In these respects, it would be desirable if an
electroporation method for delivering molecules to biological cells
were provided which does not employ a fluid medium that flows down
onto the electrodes as the electroporation process is being carried
out on the patient.
[0009] Other disclosures relating to the use of electroporation to
mediate gene transfer into epidermal cells are found in an article
by Reiss et al entitled "DNA-mediated gene transfer into epidermal
cells using electroporation" in Biochem. Biophys. Res. Commun.,
Vol. 137, No. 1, (1986), pages 244-249 and in an article by
Titomirov entitled "In vivo electroporation and stable
transformation of skin cells of newborn mice by plasmid DNA" in
Biochim. Biophys. Acta., Vol. 1088, No. 1, (1991), pages
131-134.
[0010] U.S. Pat. No. 5,389,069 of Weaver discloses a method and
apparatus for in vivo electroporation of tissues which employs a
relatively long hollow cylindrical needle for providing treating
substances deep into tissues. As mentioned above, avoiding the use
hollow cylindrical needles would be desirable to avoid the pain
involved therewith.
[0011] U.S. Pat. Nos. 5,580,859 and 5,589,466, both of Felgner et
al, disclose a method of delivering macromolecules into muscles and
skin of a patient by an injection method. Their method does not
employ electroporation.
[0012] U.S. Pat. No. 5,697,901 of Eriksson discloses gene delivery
into tissues by the use of a gene-carrying fluid medium that is
pressurized in conjunction with hollow microneedles. Problems of
control and flushing using fluid media have been discussed
hereinabove. An electroporation step is not employed in the
Eriksson patent. As a matter of interest, Eriksson addresses the
subject of pain in two respects. There is a statement that the
hollow microneedle system can be used for treating pain. There is a
statement that pain in wounds can be relieved by cooling. It is
noted by the present inventors herein that Eriksson does not
discuss his treatment method per se as being of a pain free or
reduced pain treatment method. The present inventors theorize that
the pressurized fluid injection method that is employed by Eriksson
is not conducive to a pain free or reduced pain treatment method.
In this respect, it would be desirable to provide a gene therapy
treatment method that employs micro-sized needles, but that does
not employ a pressurized fluid injection step for injecting fluid
into a patient.
[0013] In an article by Henry et al entitled "Microfabricated
Microneedles: A Novel Approach to Transdermal Drug Delivery" in
Journal of Pharmaceutical Sciences, Vol. 87, No. 8, August 1998,
pages 922-925, there is a disclosure that an array of microneedles
are employed to penetrate the epidermis to leave micro-sized
perforations to facilitate transdermal permeability of
fluid-carried treatment agents into the microperforated epidermis.
Because the microneedles are inserted only a microscopic distance
into the epidermis, use of the microneedles is potentially
nonpainful. There is no disclosure that the microneedles are to be
used as electrodes. Also, an electroporation step is not disclosed
in the Henry et al article.
[0014] The following U.S. patents may be of interest for their
disclosure of the use of relatively long electrodes in treating
biological cells: U.S. Pat. No. 5,439,440 of Hofmann; U.S. Pat. No.
5,468,223 of Mir; U.S. Pat. No. 5,674,267 of Mir et al; U.S. Pat.
No. 5,702,359 of Hofmann et al; U.S. Pat. No. 5,810,762 of Hofmann;
and U.S. Pat. No. 5,873,849 of Bernard. It is noted that none of
the patents listed in this paragraph disclose the use of relatively
long electrodes which have fixed electrode surfaces which are
coated with a static layer of electrode releasable molecules for
treating the biological cells either when an electric field is
applied to the electrodes or when the static layer dissolves off of
the electrodes in a solvent near the biological cells.
[0015] Further with respect to the issue of reduced pain treatment,
it is noted that two important electrical parameters in
electroporation are closely related to a perceived pain in vivo.
One parameter is absolute voltage experienced by the in vivo
tissue. Another parameter is the pulse width experienced by the in
vivo tissue. In these respects, it would be desirable to provide an
electroporation method for delivering molecules to biological cells
which applies relatively low absolute voltage to cells undergoing
electroporation and which can be used, if desired, to apply pulses
having relatively short pulse width to the cells undergoing
electroporation.
[0016] Still other features would be desirable in a method and
apparatus for delivery of macromolecules into epidermal cells. For
example, when electrodes are penetrated into the epidermis, the
conductive base electrode portions and the conductive tips of the
electrodes may exhibit electrical characteristics which are
undesirable with respect to the electroporation process in general
and the biological cells that are treated in particular. In this
respect, it would be desirable if a method and apparatus for
delivery of macromolecules into epidermal cells were provided which
render nonconductive the base portions and tip portions of the
electrodes.
[0017] Once electrode assemblies having a plurality of needle
electrodes have been employed on a patient, it may be a difficult
task to clean and sterilize them for a subsequent use. In this
respect, it would be desirable if a method and apparatus for
delivery of macromolecules into cells were provided in which the
electrode assemblies are disposable.
[0018] When disposable electrode assemblies are employed, it would
be desirable if the disposable electrode assemblies are packaged in
sterile packaging.
[0019] Thus, while the foregoing body of prior art indicates it to
be well known to use electroporation to deliver molecules to
biological cells, the prior art described above does not teach or
suggest a method and apparatus for delivery of macromolecules into
cells which has most of the following combination of desirable
features: (1) does not cause skin damage that results in scarring;
(2) does not leave a residue of ballistic particles in cells that
are treated; (3) provides an electroporation method for delivering
molecules to biological cells in the epidermis, near the basal
lamina, without having the treatment molecules pass through the
skin transdermally; (4) does not employ a hypodermic needle; (5)
does not employ a fluid medium that flows down onto the electrodes
as the electroporation process is being carried out on the patient;
(6) does not employ a pressurized fluid injection step for
injecting fluid into a patient; (7) applies relatively low absolute
voltage to cells undergoing electroporation; (8) if desired, can be
used to apply pulses having relatively short pulse width to the
cells undergoing electroporation; (9) renders the base portions and
tip portions of the electrodes nonconductive; (10) provides
disposable electrode assemblies; (11) provides electrode assemblies
which are packaged in sterile packaging: and (12) permits treatment
of tissues using coated long electrodes which have electrode
releasable material which includes a tissue treating agent. The
foregoing desired characteristics are provided by the unique
electrodes coated with treating agent and uses thereof, of the
present invention as will be made apparent from the following
description thereof. Other advantages of the present invention over
the prior art also will be rendered evident.
DISCLOSURE OF INVENTION
[0020] It is noted that aspects of the invention have been
disclosed in copending PCT International Application Number
PCT/US00/00014, filed 12 Jan. 2000, which is based upon copending
U.S. Provisional Application Serial No. 60/117,755, filed 28 Jan.
1999. The PCT International Application Number PCT/US00/00014 was
published on 3 Aug. 2000 with PCT International Publication Number
WO 00/44438. In addition to currently disclosing some of those
aspects of the invention previously disclosed in the
above-mentioned PCT and U.S. Provisional applications, the present
application discloses additional invention aspects.
[0021] In accordance with one aspect of the invention, a method is
provided for delivery of molecules into biological cells which
includes the steps of:
[0022] (a) providing electrodes in an electrode assembly, wherein
the electrodes have a fixed electrode surface,
[0023] (b) coating the fixed electrode surfaces of the electrodes
with at least one static layer of electrode releasable molecules to
be delivered,
[0024] (c) attaching the electrode assembly having the statically
coated electrodes to an electrode assembly holder,
[0025] (d) providing a waveform generator for generating electric
fields,
[0026] (e) establishing electrically conductive pathways between
the electrodes and the waveform generator,
[0027] (f) locating the electrodes such that the biological cells
are situated therebetween, and
[0028] (g) providing electric fields in the form of pulse waveforms
from the waveform generator to the electrodes, such that molecules
in the at least one static layer of the electrode releasable
molecules on the electrodes are delivered into the biological
cells.
[0029] On the one hand, when the static layer of electrode
releasable molecules does not include solvent separable material,
then substantially all of the static layer of electrode releasable
molecules are electric field separable molecules. In such a case,
the electric field separable molecules are both driven off of the
electrodes and delivered into the biological cells by the applied
electric fields.
[0030] On the other hand, when the static layer of electrode
releasable molecules does include solvent separable material, such
as solvent separable solid material, then the static layer of
electrode releasable molecules includes both solvent separable
solid material and electric field separable molecules. In such a
case, a solvent dissolves the solvent separable material thereby
releasing the electric field separable molecules from the
electrode, and the electric field separable molecules are delivered
into the biological cells by the applied electric fields. The
solvent includes body fluids which are present in body tissues.
[0031] Often, the electrode releasable molecules are in a form of a
static coating on the fixed electrode surface. In this respect, the
term "static" means that the coating remains stationary on the
fixed electrode surface when either not in tissues or not under the
influence of an electric field. However, such a static coating
moves off of the fixed electrode surface either when it is
dissolved off of the fixed electrode surface or when it is driven
off of the fixed electrode surface under the influence of either a
solvent or a suitable electric field, respectively.
[0032] A number of benefits can be realized by employing the static
coated electrodes of the present invention. For example, a
pre-measured quantity of a static layer of electrode releasable
molecules can be retained on the fixed electrode surfaces. Such a
pre-measured quantity of the static layer of electrode releasable
molecules can serve as a pre-measured dose of material to be
delivered to the biological cells. Moreover, the static coated
electrodes can be coated with a concentrated quantity of the
electrode releasable molecules. In addition, the static coated
electrodes can be pre-packaged so that when they are removed from
their package, they are rapidly ready for use, without the need for
conventional preparatory steps such as dilution and hypodermic
injection.
[0033] Also, in accordance with aspects of the present invention,
an electrode includes an electrode underbody and a fixed electrode
surface which lies on top of the electrode underbody. The fixed
electrode surface can be implemented in a wide variety of
embodiments. For example, most simply, the simple surface of the
electrode itself can serve as the fixed electrode surface which
lies on top of the electrode underbody. The fixed electrode surface
can be a smooth electrode surface, can be an oxidized metal surface
(e. g. oxides of silver, nickel, and copper), can include fixed
metal particles, and can be a roughened surface.
[0034] Also, in accordance with aspects of the present invention,
the electrode releasable material on the fixed electrode surface
can be in a form of a gel coating, a solid layer of nonpolymeric
material, and a polymer layer.
[0035] Varieties of the fixed electrode surface and the static,
electrode releasable material can be mixed and matched.
[0036] Some specific examples of combinations of the fixed
electrode surface and the electrode releasable material include: a
fixed electrode surface having metal oxides and electrode
releasable material including DNA; a fixed electrode surface being
a smooth surface and the electrode releasable material in the form
of a solid coating; a fixed electrode surface having an etched
rough surface and the electrode releasable material as either a
solid nonpolymeric layer, a gel layer, or a polymeric layer.
[0037] The pulse waveforms may be provided by applying a sequence
of at least three single, operator-controlled, independently
programmed, DC electrical pulses, to the biological cells. The
sequence of at least three DC electrical pulses has one, two, or
three of the following characteristics (a) at least two of the at
least three pulses differ from each other in pulse amplitude, (b)
at least two of the at least three pulses differ from each other in
pulse width, and (c) a first pulse interval for a first set of two
of the at least three pulses is different from a second pulse
interval for a second set of two of the at least three pulses.
[0038] Additionally, the method can include a step of providing the
electrode assembly holder with electrically conductive pathways
between the electrode assembly and the waveform generator.
[0039] In addition, the method can include a step of providing the
electrode assembly in a sterile package. In such a case, the
electrode assembly is removed from the sterile package prior to
use.
[0040] Further, the method can include the steps of providing the
electrodes with electrically insulated outer surface electrode tip
portions and electrically insulated outer surface electrode base
portions.
[0041] The molecules in the at least one static layer of molecules
in the electrode coating preferably include macromolecules. The
macromolecules in the electrode coating can include a tissue
treating agent, a polynucleotide vaccine (DNA vaccine and/or RNA
vaccine), or a protein-based vaccine, among others.
[0042] With a variation of the method of the invention, the
molecules can be delivered to Langerhans cells in epidermal tissue
of a patient with reduced sensation (reduced pain or nearly
painless or pain free) to the patient. To provide reduced sensation
delivery of molecules to the patient, the following conditions are
maintained (a) the pulse waveforms have an absolute applied voltage
in a range of 0.1 to 300 volts; (b) the electrodes of opposite
polarity are separated by a separation distance in a range of from
50 to 500 microns; and (c) the electrodes are penetrated into the
epidermal tissue a distance up to and slightly beyond the basal
lamina layer of the epidermal tissue.
[0043] With another variation of the method of the invention, the
molecules can be delivered to a tissue which is deeply located
under healthy tissue. With such a variation of the method of the
invention, the electrodes are long enough to penetrate through the
healthy tissue and into the tumor. The fixed electrode surface
portions of the electrodes that penetrate the tumor are coated with
electrode releasable material that includes a deep tumor tissue
treating agent.
[0044] The pulse waveforms which drive the molecules of the
electrode releasable coating molecules off of the electrodes are
electrophoresis waveforms. The pulse waveforms which deliver the
driven-off molecules into the biological cells are electroporation
waveforms. For a static layer of electric field separable
molecules, common pulse waveforms both drive the coating molecules
off of the electrodes and deliver the driven-off molecules into the
biological cells.
[0045] The biological cells can be in vivo, ex vivo, or in vitro.
More specifically, the biological cells can be in epidermal tissue
and can be Langerhans cells in the epidermal tissue. Also, the
biological cells can be in deep tissues, and can be in tumors in
deep tissues.
[0046] In accordance with another aspect of the invention, an
apparatus is provided for delivery of molecules into biological
cells and includes a waveform generator which provides pulse
waveforms. An electrode assembly holder is provided, and an
electrode assembly is mechanically supported by the electrode
assembly holder. The electrode assembly holder is also electrically
connected to the waveform generator through electrically conductive
pathways. The electrode assembly includes electrodes which are
coated with at least one static layer of molecules to be delivered
into the biological cells.
[0047] The electrode assembly can be removable and replaceable from
the electrode assembly holder. In this respect, the electrode
assembly includes electrode-assembly-conductive strips. The
electrode assembly holder includes holder conductors which are
registrable with the electrode-assembly-conductive strips when the
electrode assembly is mechanically connected to the electrode
assembly holder. Also, the electrode assembly holder includes
electrically conductive pathways between the holder conductors and
the waveform generator.
[0048] With the apparatus, a sterile package can be provided for
the electrode assembly. The sterile package is removed from the
electrode assembly after the electrode assembly is mechanically
supported by the electrode assembly holder and is electrically
connected to the waveform generator.
[0049] With the apparatus, if desired, the waveform generator
provides pulse waveforms which include a sequence of at least three
single, operator-controlled, independently programmed, DC
electrical pulses, to the biological cells. The sequence of at
least three DC electrical pulses has one, two, or three of the
following characteristics (a) at least two of the at least three
pulses differ from each other in pulse amplitude, (b) at least two
of the at least three pulses differ from each other in pulse width,
and (c) a first pulse interval for a first set of two of the at
least three pulses is different from a second pulse interval for a
second set of two of the at least three pulses.
[0050] The electrodes can include electrically insulated outer
surface electrode tip portions and electrically insulated outer
surface electrode base portions. The electrodes are coated with at
least one static layer of molecules, which may include
macromolecules, which may include a tissue treating agent, a
polynucleotide vaccine (a DNA vaccine and/or a RNA vaccine) and/or
a protein-based vaccine.
[0051] The static layer of electrode releasable macromolecules on
the electrodes (e. g. a tissue treating agent, a polynucleotide
vaccine, or a protein-based vaccine, among others) can be in a
variety of forms prior to using the electrodes on a patient. More
specifically, the static layer of macromolecules can be in a solid
form, coating the solid electrodes. Also, the static layer of
macromolecules can be in a gel form or can be in a form of a liquid
fixed on a fixed surface matrix of the electrodes. The fixed
surface matrix can be solid surface particles (e. g. metal
particles), a liposome matrix, or a solid polymer matrix.
[0052] In accordance with yet another aspect of the invention, a
packaged sterile electrode assembly is provided which includes a
sterile electrode assembly which includes electrodes which are
coated with at least one static layer of molecules to be delivered
into biological cells. The electrode assembly includes
electrode-assembly-conductive strips for connection to
complementary electrically conductive pathways leading to the
waveform generator. In addition, an internally sterile package
encloses the sterile electrode assembly contained therein.
[0053] With the packaged sterile electrode assembly, the electrodes
can include electrically insulated outer surface electrode tip
portions and electrically insulated outer surface electrode base
portions.
[0054] With the packaged sterile electrode assembly, the electrodes
are coated with macromolecules which can include a solid phase
polynucleotide (DNA vaccine and/or RNA vaccine) and/or a solid
phase protein-based vaccine. Also, the polynucleotide vaccine or
protein-based vaccine can be in a gel form or can be in a form of a
liquid fixed on a fixed surface matrix of the electrodes, prior to
using the electrodes on a patient. The fixed surface matrix can be
solid surface particles (e. g. metal particles), a liposome matrix,
or a solid polymer matrix.
[0055] In accordance with the invention, transfection of cells with
DNA in vivo, using electric field mediated transfection, is an
efficient process. Additionally, electric fields can be used for
the delivery of other macromolecules such as RNA and proteins into
cells. In the prior art, the electric field delivery has one
disadvantage, that being the pain induced by the high voltage
electrical pulses required for the transfection. In contrast, as
described herein, a method is provided for delivering
macromolecules (DNA, RNA, and protein) to cells, in tissues in
vivo, using painless (or nearly painless) and efficient electric
field mediated delivery.
[0056] A number of applications of the method and apparatus for
delivery of macromolecules into cells, of the invention, are
contemplated. Briefly, such applications include treating organ
tissues, polynucleotide vaccination, protein vaccination, and gene
therapy.
[0057] For treating deep tumor tissues, it is important to maximize
delivery of the deep tumor tissue treating agent to the tumor
tissue and to minimize deliver of the agent to healthy tissue.
[0058] For DNA vaccination, there are two overriding requirements.
One is gene expression in vivo and the other is that
antigen-presenting cells must either obtain antigen from a nearby,
transfected cell or express the antigen themselves. The highest
concentration of accessible antigen presenting cells resides in the
skin as cells called Langerhans cells. These cells are part of a
very effective group of antigen presenting cells called dendritic
cells. Electroporation is a viable alternative method for
transfecting selected cells in vivo.
[0059] Proteins also can be introduced into cells using electric
field mediated delivery. In conventional vaccination, proteins are
delivered outside cells using a hypodermic needle. This type of
delivery is inefficient in inducing a cell mediated cytotoxic
lymphocyte immune response. Some infectious diseases require a
cytotoxic lymphocyte response as a component of the immune response
for efficient clearance of the infection. Delivery of proteins into
cells promotes the induction of that response.
[0060] Delivery of therapeutic genetic medicine into cells for the
purpose of making those cells express a missing protein is the
basis of gene therapy. Using relatively short electrodes of the
invention, the method and apparatus of the invention can be used to
deliver therapeutic DNA into cells on the surface of any accessible
organ in addition to the skin. Using relatively long electrodes of
the invention, the method and apparatus of the invention can be
used to deliver therapeutic DNA into cells deep into tissues and
organs.
[0061] The method of the invention can be used in a method for
painless, effective delivery of macromolecules to epidermal
tissues, in vivo, for the purpose of vaccination (or treatment),
DNA vaccination, gene therapy, or other reasons.
[0062] An electrode with at least one of two characteristics is
used for delivery of macromolecules into cells in epidermal tissue.
One of the two characteristics is an electrode length short enough
that it does not penetrate to a depth in tissue with nerve endings.
Another characteristic is that inter-electrode distances are small
enough to allow pulse parameters (voltage and pulse width) to be
used that are painless. Only one or the other of these
characteristics is needed in any given epidermal application,
however, they may be used together.
[0063] The above brief description sets forth rather broadly the
more important features of the present invention in order that the
detailed description thereof that follows may be better understood,
and in order that the present contributions to the art may be
better appreciated. There are, of course, additional features of
the invention that will be described hereinafter and which will be
for the subject matter of the claims appended hereto.
[0064] In this respect, before explaining preferred embodiments of
the invention in detail, it is understood that the invention is not
limited in its application to the details of the construction and
to the arrangements of the components set forth in the following
description or illustrated in the drawings. The invention is
capable of other embodiments and of being practiced and carried out
in various ways. Also, it is to be understood, that the phraseology
and terminology employed herein are for the purpose of description
and should not be regarded as limiting.
[0065] As such, those skilled in the art will appreciate that the
conception, upon which disclosure is based, may readily be utilized
as a basis for designing other structures, methods, and systems for
carrying out the several purposes of the present invention. It is
important, therefore, that the claims be regarded as including such
equivalent constructions insofar as they do not depart from the
spirit and scope of the present invention.
[0066] In view of the above, it is an object of the present
invention to provide a new and improved method and apparatus for
delivery of macromolecules into cells which does not cause skin
damage that results in scarring.
[0067] Another object of the present invention is to provide a new
and improved method and apparatus for delivery of macromolecules
into cells which does not leave a residue of ballistic particles in
cells that are treated.
[0068] Even another object of the present invention is to provide a
new and improved method and apparatus for delivery of
macromolecules into cells that provides an electroporation method
for delivering molecules to biological cells in the epidermis, near
the basal lamina, without having the treatment molecules pass
through the skin transdermally.
[0069] Still a further object of the present invention is to
provide a new and improved method and apparatus for delivery of
macromolecules into cells which does not employ a hypodermic
needle.
[0070] Yet another object of the present invention is to provide a
new and improved method and apparatus for delivery of
macromolecules into cells that does not employ a fluid medium that
flows down onto the electrodes as the electroporation process is
being carried out on the patient.
[0071] Still another object of the present invention is to provide
a new and improved method and apparatus for delivery of
macromolecules into cells which does not employ a pressurized fluid
injection step for injecting fluid into a patient.
[0072] Yet another object of the present invention is to provide a
new and improved method and apparatus for delivery of
macromolecules into cells that applies relatively low absolute
voltage to cells undergoing electroporation.
[0073] Still a further object of the present invention is to
provide a new and improved method and apparatus for delivery of
macromolecules into cells that can be used, if desired, to apply
pulses having relatively short pulse width to the cells undergoing
electroporation.
[0074] Yet another object of the present invention is to provide a
new and improved method and apparatus for delivery of
macromolecules into cells which renders the base portions and tip
portions of the electrodes nonconductive.
[0075] Still a further object of the present invention is to
provide a new and improved method and apparatus for delivery of
macromolecules into cells that provides disposable electrode
assemblies.
[0076] Yet another object of the present invention is to provide a
new and improved method and apparatus for delivery of
macromolecules into cells which electrode assemblies are packaged
in sterile packaging.
[0077] Still another object of the present invention is to provide
a new and improved method and apparatus for delivery of
macromolecules into cells which permit treatment of tissues using
coated long electrodes which have electric field separable material
which includes a tissue treating agent.
[0078] These together with still other objects of the invention,
along with the various features of novelty which characterize the
invention, are pointed out with particularity in the claims annexed
to and forming a part of this disclosure. For a better
understanding of the invention, its operating advantages and the
specific objects attained by its uses, reference should be had to
the accompanying drawings and descriptive matter in which there are
illustrated preferred embodiments of the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0079] The invention will be better understood and the above
objects as well as objects other than those set forth above will
become more apparent after a study of the following detailed
description thereof. Such description makes reference to the
annexed drawing wherein:
[0080] FIG. 1 is a schematic illustration of the overall apparatus
of the invention.
[0081] FIG. 2 is a schematic illustration of relatively short
electrodes coated with a static layer of macromolecules, with the
electrodes penetrating an epidermal skin layer, and with the
macromolecules being driven by pulse waveforms off of the
electrodes to deliver macromolecules into epidermal cells.
[0082] FIG. 3 is a schematic illustration of tip portions of the
electrodes.
[0083] FIG. 4 is a schematic illustration of relatively long
electrodes coated with a static layer of macromolecules, with the
electrodes penetrating through healthy tissue into the tissue of a
tumor.
[0084] FIG. 5 schematically shows an electrode which has a fixed
electrode surface that is coated with a static layer of electrode
releasable molecules.
[0085] FIG. 6 schematically shows apparatus used for coating the
electrodes with macromolecules.
MODES FOR CARRYING OUT THE INVENTION
[0086] A method and apparatus are provided for delivery of
macromolecules into cells, and with reference to the drawings, said
method and apparatus are described below. The method for delivery
of molecules into biological cells employs the apparatus set forth
and includes the steps of
[0087] (a) providing electrodes 16 in an electrode assembly 12,
wherein the electrodes have a fixed electrode surface 42,
[0088] (b) coating the fixed electrode surfaces 42 of the
electrodes 16 with at least one static layer of electrode
releasable molecules 44 to be delivered,
[0089] (c) attaching the electrode assembly 12 having the
statically coated electrodes 16 to an electrode assembly holder
13,
[0090] (d) providing a waveform generator 15 for generating
electric fields,
[0091] (e) establishing electrically conductive pathways between
the electrodes 16 and the waveform generator 15,
[0092] (f) locating the electrodes 16 such that the biological
cells are situated therebetween, and
[0093] (g) providing electric fields in the form of pulse waveforms
from the waveform generator 15 to the electrodes 16, such that
molecules in the at least one static layer of the electrode
releasable molecules 44 on the electrodes 16 are delivered into the
biological cells.
[0094] Referring to FIG. 5, an electrode 16 includes an electrode
underbody 40 and a fixed electrode surface 42 which lies on top of
the electrode underbody 40. A static layer of electrode releasable
molecules 44 is located on top of the fixed electrode surface 42.
It is noted, however, that in different embodiments, the fixed
electrode surface 42 and the static layer of electrode releasable
molecules 44 can be intermingled.
[0095] In some embodiments of the invention, the static layer of
electrode releasable molecules 44 is homogeneous and includes
substantially only electric field separable molecules. In other
embodiments, the electrode releasable molecules 44 include only a
solvent separable material. In still other embodiments, the
electrode releasable molecules 44 include both electric field
separable molecules and solvent separable material. Generally, the
solvent separable material is in a solid state. Alternatively, the
solvent separable material can be in a gel state.
[0096] With reference to FIG. 4, in one variation of the method,
using long needle electrodes 34 which are coated with a static
layer of electrode releasable molecules 44, e. g. macromolecules 18
which include a deep tumor tissue treating agent, a deep tumor
tissue 38 is treated with the macromolecules 18 which are driven
off of the long needle electrodes 34 by an imposed electric
field.
[0097] With reference to FIG. 4, a specific example of the above
procedure is the use of plasmids expressing lymphokines for cancer
immunotherapy. In one example, plasmids expressing interleukin-2
are coated onto the long needles 34 using the described method.
More specifically, the macromolecules 18 in FIG. 4 include the
plasmids expressing interleukin-2. The long needles 34 are then
inserted through healthy tissue 33 into selected tumor tissue 38.
Then, the electrophoretic and electroporation pulses administered.
This method is used to induce a local immune response to the tumor
tissue 38 in an effort to generate a generalized immune response.
Plasmids expressing other lymphokines can be used in this
procedure. Examples are interleukin-12, interleukin-4,
interferon-gamma and a variety of others. Plasmids expressing other
immune system molecules such as co-stimulatory molecules or
adhesion molecules can also be used.
[0098] In another variation of the method, referring to FIG. 2, the
statically-coated molecules are delivered with reduced sensation in
a patient to Langerhans cells 22 in the epidermis 20 of a patient.
The pulse waveforms have an absolute applied voltage in a range of
from 0.1 to 300 volts. Electrodes 16 of opposite polarity are
separated by a separation distance in a range of from 50 to 500
microns. The statically-coated electrodes 16 are penetrated into
the epidermal tissue up to and slightly beyond the basal lamina
layer of the epidermal tissue.
[0099] The pulse waveforms which drive the coating molecules off of
the electrodes 16 are electrophoresis waveforms. The
electrophoresis waveforms can be in a range of from 0.1 to 100
volts/cm. The pulse waveforms which deliver the driven-off
molecules into the biological cells are electroporation waveforms.
The electroporation waveforms can be in a range of from 100 to
10,000 volts/cm. Common pulse waveforms both drive the coating
molecules off of the electrodes 16 and deliver the driven-off
molecules into the biological cells.
[0100] The biological cells to which the molecules are delivered
can be in vivo, ex vivo, or in vitro. More specifically, as shown
in FIG. 2, the biological cells can be in the epidermis 20
(epidermal tissue) and can be Langerhans cells 22 in the epidermal
tissue.
[0101] The statically-coated molecules driven off of the electrodes
16 by electrophoresis electrical pulses are delivered to the cells
by electroporation pulses. In accordance with an exemplary
protocol, the pulse waveforms are provided by the waveform
generator 15 by applying a sequence of at least three single,
operator-controlled, independently programmed, DC electrical
pulses, to the biological cells. The sequence of at least three DC
electrical pulses has one, two, or three of the following
characteristics (1) at least two of the at least three pulses
differ from each other in pulse amplitude, (2) at least two of the
at least three pulses differ from each other in pulse width, and
(3) a first pulse interval for a first set of two of the at least
three pulses is different from a second pulse interval for a second
set of two of the at least three pulses.
[0102] The electrode assembly holder 13 is provided with
electrically conductive pathways, which includes conductors 21,
between the electrode assembly 12 and the waveform generator
15.
[0103] The electrode assembly 12 can be provided in a sterile
package 24 which is removed from the electrode assembly 12 prior to
use.
[0104] Preferably, for electrodes 16 used with epidermal tissue,
the electrodes 16 have conical tips, whereby they are referred to
as needle electrodes. The electrodes 16 can be provided with
electrically insulated outer surface electrode tip portions 17 and
electrically insulated outer surface electrode base portions 19.
The electrically insulated outer surface electrode base portions 19
minimize current flowing across the upper skin surface when the
pulse voltage is applied. Moreover, DNA binds poorly to the
electrically insulated outer surface electrode base portions 19.
The outer surface area of the electrodes 16 between the
electrically insulated outer surface electrode tip portions 17 and
the electrically insulated outer surface electrode base portions 19
does not have an outer insulation layer and is a good surface for
DNA binding. The outer surface areas between the electrically
insulated outer surface electrode tip portions 17 and the
electrically insulated outer surface electrode base portions 19 is
conductive and can be referred to as an active electrode area. The
electrically insulated outer surface electrode tip portions 17
prevent large local electric field intensity which may cause
burning in the tissue.
[0105] An electrode assembly 12 that is suitable for delivering DNA
vaccines to Langerhans cells 22 in the epidermis 20 of the forearm
has the following characteristics:
[0106] (a) electrode length--130 microns
[0107] (b) electrode material resistivity--less than 0.1 ohm-cm
[0108] (c) insulation at tip--extending upward 10 microns from tip
end
[0109] (d) insulation at base--extending downward 55 microns from
electrode carrier
[0110] (e) electrode tip flatness--less than 1 square micron
[0111] (f) electrode diameter at base--43 microns
[0112] (g) electrode spacing in a conductive row--130 microns
[0113] (h) number of electrodes in a conductive row--35
[0114] (i) space between conductive rows--260 microns
(2.times.130)
[0115] (j) number of conductive rows--25.
[0116] For epidermal applications, the lengths of the electrodes 16
are determined by the thickness of the epidermis 20. The thickness
of the epidermis 20 varies in different parts of the human body.
For example, the thickness of the epidermis 20 on the medial
forearm or the lateral upper arm above the deltoid muscle is
considerably thinner than the thickness of the epidermis 20 on the
heel or sole of the foot.
[0117] Referrring to FIG. 5, molecules in the static layer of
electric field separable molecules 44 preferably include
macromolecules 18. The macromolecules 18 in the electrode coating
can include a deep tumor tissue treating agent, a polynucleotide
vaccine (e.g a DNA vaccine or an RNA vaccine), and/or a
protein-based vaccine. The polynucleotide vaccine and the
protein-based vaccine can be in the form of a solid phase DNA
vaccine or protein-based vaccine applied to the electrodes 16.
Also, the polynucleotide vaccine or protein-based vaccine can be in
a gel form or can be in a form of a liquid fixed on a fixed surface
matrix of the electrodes. The fixed surface matrix can be solid
surface particles (e. g. metal particles), a liposome matrix, or a
solid polymer matrix.
[0118] Preferably, the electrode assembly 12 is removable and
replaceable from the electrode assembly holder 13. The electrode
assembly 12 includes electrode-assembly-conductive strips. The
electrode assembly holder 13 includes holder conductors which are
registrable with the electrode-assembly-conductive strips when the
electrode assembly 12 is mechanically connected to the electrode
assembly holder 13. The electrode assembly holder 13 includes
electrically conductive pathways between the holder conductors and
the waveform generator 15.
[0119] As stated above, there are three main components required
for the delivery of macromolecules into cells in tissue. They are a
waveform generator 15, an electrode assembly holder 13, and a
statically-coated electrode assembly 12. The waveform generator
supplies the electrical pulses necessary for generating the
electric field in the tissue. The electrode assembly 12 contains
the electrodes 16, and the polynucleotide or protein macromolecules
are applied to the electrodes 16 as a static coating. The electrode
assembly holder 13 connects the electrode assembly 12 to the
waveform generator 15.
[0120] The statically-coated electrode assembly 12 can be in the
form of an electrode array can be in the form of a disposable,
one-time-use electrode array which has the macromolecules
pre-loaded onto the electrodes. In this respect, the pre-loaded
electrode array can be provided as a sterile package. To use such
an electrode array, the sterile package is opened, and the
electrode array is connected to the electrode array holder. The
electrode assembly holder is grasped by a person, and portions of
the electrode assembly are forced into the selected tissue of a
patient. For epidermal tissue, preferably, the tips of the
electrodes in the statically-coated electrode assembly are located
in the region of the Langerhans cells, which are dendritic cells of
the epidermis.
[0121] Then, a pulse waveform is sent from the waveform generator,
through the electrode assembly holder, and to the statically-coated
electrode assembly. The pulse waveform drives pre-loaded
macromolecules off of the statically-coated electrode assembly and
into the epidermis. In the epidermis, the pulse waveform
electropermeabilizes the target epidermal cells so that the
macromolecules enter the target cells.
[0122] As illustrated in FIG. 2, an electrode assembly 12 includes
a non-conductive electrode carrier 14 and a plurality of individual
needle electrodes 16 supported by the electrode carrier 14. The
active areas 29 of the electrodes 16 are statically coated with
macromolecules which are illustrated as small "x's" 18 on the
surfaces of the electrodes 16. Under the influence of the pulse
waveforms, some of the macromolecules 18 are driven off of the
electrodes 16 by electrophoresis voltage and enter the epidermis 20
and are delivered to the dendritic Langerhans cells 22 and the
living epithelial cells 23 in the living epidermis above the basal
lamina 25 in the epidermis 20 by electroporation voltage.
[0123] The waveform generator 15 produces the pulses for the
protocol. The output of the waveform generator can be conventional
with a single selection of pulse parameters such as voltage, pulse
width, interval between pulses, number of pulses and the vector of
the pulse. Alternatively, the output of the waveform generator can
be programmable with the ability to change any of the parameters
(voltage, pulse width, interval between pulses, number of pulses,)
from pulse to pulse. The variable output is needed for optimal
performance because a different electric field is required for
macromolecule movement off of the electrodes 16 than the electric
field required for electric field mediated delivery of
macromolecules into cells. A suitable programmable pulse generator
is the PulseAgile (Registered in U.S. Patent and Trademark Office),
Model PA-4000 Electroporation System made by Cyto Pulse Sciences,
Inc., P. 0. Box 609, Columbia, Md. 21045. It is noted that the
Model PA-4000 delivers rectangular waves of various amplitudes
(voltages), width, and intervals.
[0124] In addition to programmable control of voltage, pulse width,
interval between pulses and number of pulses from pulse to pulse,
programmable control of two other parameters is desired. One is
control of the direction or vector of the applied electric field.
The other is control of electrode selection. In one application,
electric field direction could be reversed to insure better
distribution of the macromolecule. In another application,
individual pairs of electrode arrays could be sequentially
selected.
[0125] A suitable device for electrode selection and the selection
of electrode field direction is the programmable pulse switch,
which is an optional component of the above-mentioned PA-4000
Electroporation System.
[0126] The statically-coated electrode assembly 12 serves two
functions. It delivers the macromolecule to the desired site and it
delivers the electric field to the tissue.
[0127] The statically-coated electrode assembly 12 includes:
[0128] 1. a non-conductive electrode carrier 14.
[0129] 2. an array of needle electrodes 16 fabricated on the
electrode carrier 14, wherein the needle electrodes 16 are
statically coated.
[0130] 3. Electrode-assembly-conductive strips for electrical
connection to the holder conductors on the electrode assembly
holder 13 to connect electrically to the waveform generator 15.
[0131] In carrying out the method of the invention for epidermal
use, the tips of the statically-coated needle electrodes 16 are
pressed against the epidermis 20 of a patient with the needles
puncturing the stratum corneum 27 and extending into the epidermis
20 and the upper dermis 31 as shown in FIG. 2.
[0132] The electrode needles can have many shapes. Examples of
needle electrode shapes are: cylindrical needles, flat needles,
cone shaped needles, and blade needles. The needles can be pointed
rounded or blunt. Each of these shapes can be single or multiple
per electrode row.
[0133] The purposes of the electrode assembly holder are to
establish an electrical connection between the waveform generator
and the electrode assembly and to provide a support for the
electrode assembly when the electrode assembly is applied to the
patient. It provides a mechanical connection for application to the
patient. It also provides a means of delivering the electrode
assembly to the patient's tissue while maintaining sterility of the
electrode assembly.
[0134] The statically-coated electrode assembly can have the
following optional features. It can have means to provide proper
pressure on the electrode assembly to the tissue. It can have
indicators that indicate correct application pressure, on-going
electrical delivery, and completion of electrical delivery. It can
have a switch for initiation of the pulse protocol. It can have a
means for automatically initiating a pulse protocol when proper
pressure is applied to the electrode assembly holder.
[0135] As stated above, macromolecules, including DNA and protein
macromolecules, need to be driven off of the statically-coated
coated electrodes 16 by electrophoresis voltages so that they can
move through the extra-cellular spaces of tissue prior to the
application of electroporation pulses for delivering the
macromolecules into the targeted biological cells in the
tissue.
[0136] As stated above, the macromolecules 18 in the static layer
of electric field separable molecules 44 are initially bound to the
fixed electrode surfaces 42 of the electrodes 16. In a mechanical
approach to coating the electrodes 16 with a static coating of
macromolecules 18, a relatively high concentration of
macromolecules 18 is dissolved or suspended in a solvent or liquid
carrier. The electrodes 16 are then dipped into the solution or
suspension. Then, the solvent or liquid carrier is evaporated,
leaving a static coating of macromolecules 18, in this case a solid
coating of macromolecules 18, on the electrodes 16. Alternatively,
the electrodes 16 are coated by spraying. Other mechanical means of
coating the electrodes 16 are possible.
[0137] Macromolecules such as DNA bind with good efficiency to many
surfaces. The physical and chemical properties of the material can
be used to enhance binding to electrode surfaces to provide the
statically-coated electrodes 16.
[0138] Molecules tend to bind to each other through various
molecular interactions, each having a different binding strength.
These same forces are active between solid substrates and soluble
molecules as well as among molecules in solution. The molecular
interactions are:
[0139] 1. Solvation: Solvent binding. An interaction between the
components of a molecule and the solvent molecules.
[0140] 2. Hydrophobic interaction: A solute-solute interaction as a
consequence of the inability to interact with the solvent; an
avoidance interaction
[0141] 3. Van der Walls forces are weak attractions that exist
between all molecules. It is effective only at short distances and
can be stronger if interactions based upon complementary shape
[0142] 4. Hydrogen bonds are bonds formed between hydrogen and
other molecules such as nitrogen and oxygen.
[0143] 5. Ionic bonds are attractions based upon attraction of
oppositely charged portions of molecules.
[0144] 6. Covalent bonds are the strongest of molecular bonds.
[0145] More specifically with respect to DNA, DNA is both sparingly
soluble in water and charged. The organic rings within the
nucleotides impart the hydrophobic properties to DNA. The phosphate
molecules in the DNA polymer., impart a net negative charge.
[0146] The strongest bond between an electrode surface and DNA is
the hydrophobic bond. When an electrode has a positive charge, DNA
moves towards the electrode thereby enhancing the interaction of
DNA with the conductive hydrophobic surface. For delivery of the
DNA to biological tissues, the electrical charge is reversed.
Migration of macromolecules 18 from the electrode surface occurs as
soon as the repelling force of like charges exceeds the force of
the hydrophobic and other molecular interaction.
[0147] DNA can be coated onto specific sites by binding the DNA to
metal (such as an electrode surface.) or another conductive
material through the use of a positive charge. Subsequently, for
driving the DNA off of the electrode surface and for subsequently
delivering the DNA to biological cells, a negative charge is
applied to the same surface. DNA, being negatively charged, will
migrate in an electric field toward the positive electrode. This
phenomenon is called electrophoresis. If the positive electrode is
a hydrophobic surface as are most metals, the positive charge and
the hydrophobic interaction will work together to hold the DNA to
the surface.
[0148] Most macromolecules have a net charge in solution at a pH
other than its iso-electric point. DNA, for instance, is negatively
charged at physiological pH. This means that a DNA molecule will
migrate towards a positive electrode. This property is used to
bring the macromolecule in contact with the electrode where binding
of the DNA to the electrode surfaces occurs via the other molecular
interactions listed. DNA, for instance, can bind because it is
hydrophobic.
[0149] Electrical coating takes advantage of the charge of the
macromolecules. A stated above, DNA is negatively charged and
therefore migrates to a positively charged electrode. Reference is
made to FIG. 6 which illustrates an apparatus used for coating the
electrodes 16 with a static coating. In one coating process, DNA is
added to a buffer solution and then placed into a chamber with an
electrode that serves as the cathode. Preferably this electrode is
separated from the buffer by a gel interface to prevent metal of
the cathode coming into contact with the DNA. The electrode device
is inserted into the liquid, and a positive charge is applied to
the electrode device drawing the DNA to the surface of the
electrode device. The DNA attaches to the surface of the electrode
device by hydrophobic or other interaction to provide a
statically-coated electrode until the DNA is expelled by a reverse
charge. The DNA is dried on to the device with or without a
protectant, such as sugars, and with or without other carrier
molecules. Substances can also be added to the static coating on
the electrodes which promote uptake of the treating material into
the target cells.
[0150] The amount of statically-coated macromolecule on the
electrode assembly varies depending upon the application. For DNA
immunization, for example, the electrode assembly is loaded with
0.01 to 100 micrograms of plasmid DNA.
[0151] Sterile materials and a sterile local environment can be
used in the manufacture of the electrode assembly with the
macromolecule. Alternatively, the assembly can be sterilized after
manufacture.
[0152] A typical sequence of steps in administering macromolecules
18 to a patient using the method and apparatus of the invention are
described as follows. In a clinic, the waveform generator 15 would
be connected to the electrode assembly holder 13. For an individual
application, an electrode assembly 12 whose electrodes 16 have been
loaded with a static coating of the desired macromolecule is
selected. The electrode assembly 12 is then mechanically connected
to the electrode assembly holder 13. As the electrode assembly
holder 13 is grasped by an operator, the statically-coated
electrodes 16 are pressed onto the patient's tissue (e. g. skin).
For skin, the statically-coated electrodes 16 penetrate into the
epidermis 20 and extend substantially only to the basal lamina
layer. After the statically-coated electrodes 16 have been located
thusly in the epidermis 20, the macromolecular delivery process is
started, and the selected pattern of electric fields is initiated.
After completion of the delivery protocol, the electrodes 16 are
removed from the epidermis 20, and the electrode assembly 12 is
discarded.
[0153] As stated above, when a solvent separable solid material is
not employed in the electrode releasable molecules 44, the
electrical protocol is designed to drive the statically-coated
macromolecules off of the electrodes 16 into the tissue, followed
by delivery of the macromolecules into cells in the tissue. For
DNA, a typical sequence of electrical pulses is as follows. First,
a series of low voltage (electrophoresis) pulses are applied to the
electrodes 16 to remove the DNA from all negatively charged
electrodes. Typically, alternating rows of electrodes are
negatively charged. Next, higher voltage electroporation pulses are
applied to the electrodes 16 to drive the DNA into cells. Next,
electrode polarity is reversed and low voltage pulses delivered
with opposite polarity to remove the DNA from the remaining
electrodes. Higher voltage electroporation pulses are then applied
to force the DNA into cells.
[0154] A significant use of this macromolecular delivery system is
to deliver macromolecules to skin. For this use, electrode needle
length is chosen to allow penetration of the electrode to the
stratum basalis and basement membrane (basal lamina). Some slight
penetration into the dermis may occur. For this use on a patient's
arm, an electrode length of 130 microns is selected. This depth
allows treatment of cells of the epidermis. For a DNA vaccine or
gene therapy, the cells transfected by this delivery method are
dendritic cells (skin Langerhans cells 22) and epithelial
cells.
[0155] Aside from administering macromolecules to biological cells
in the epidermis, the method and apparatus of the invention can be
used in other biological environments, such as tissues during
surgery and with plants.
[0156] A wide variety of methods can be employed for manufacturing
the electrode assembly 12 of the invention, prior to application of
the static coating. A number of examples are presented below.
[0157] Standard microchip manufacturing processes can be adapted to
make the conductive microneedles on a non-conductive support, as in
accordance with the invention. In one example, a blank consisting
of a silicon or other non conductive layer and a metal layer would
be used. The mask would be designed to encourage more etching
between rows than within rows, resulting in conductive rows of
electrodes with nonconductive spaces between rows.
[0158] Another method of construction of an electrode assembly is
by adapting the known technique of extrusion micro-fabrication, and
an example follows. Electrode material and adjacent insulating
material are prepared by mixing a ceramic, metal or other powder
with a thermoplastic binder. The individual components are
assembled and warm pressed to stick together. The resulting rod is
extruded to reduce its size. Following the extrusion, the new rods
are assembled in a rod composed of a multiple of the extruded rod.
This newest rod is re-extruded to reduce the size of the multiple
rods to the size of the first extruded rod. After the size is
reduced to the desired size the parts can be heated to remove the
binder. A second, higher heat is used to sinter the metal or
ceramic powders together. The rods are cut into disks before or
after the sintering. Differential sand blasting or other mechanical
or chemical techniques can be used to raise the needles above the
surface of the insulator.
[0159] Another method for manufacture would be to use laser milling
techniques to remove material from a sandwich composed of
conductive and nonconductive layers.
[0160] For some of the arrays of electrodes, the distance between
the electrodes is large enough for mechanical assembly. An example
of such assembly follows. Wire of the desired metal composition and
diameter is arranged on spools for assembly. The wires are fed into
an apparatus that aligns the wire to the correct distance apart.
Ceramic or plastic material is injected into a flow through system
that results in complete filling of the gap between the electrodes
and forms the shape of the outside rim of the electrode. The
plastic or ceramic is hardened and cut into discs. The resulting
disks are differentially eroded, taking advantage of the softer
matrix. The erosion can be done using mechanical methods, chemical
methods or a combination of methods. The surface erosion leaves
needles of the desired length protruding above the supporting
matrix.
[0161] Another manufacturing technique for an electrode assembly is
described as follows. Stainless steel needles 30 mm in length and
120 microns in diameter are obtained. One source is from an
acupuncture supply company. Seirin No. 02 needles are an example.
The needles are cut from the handle if one is present. A number of
needles are selected for each row of the device. Thirty-five
needles per row are used for this example. The needles are
carefully placed side by side with the tips of the needles in line.
This step requires care and a jig made of a microscope slide glued
at 90 degrees on top of another microscope slide is a tool to help
in the alignment. The slide also is be used to check the alignment
on a microscope. The needle row (needle bundle) is taped together
with 50 micron thick tape. Two or more of the needle bundles are
stacked to form an electrode array with the tips of each bundle
aligned with the next bundle. The needles are silver soldered to a
wire, and alternating needle bundles are connected together
electrically. An overall support structure is provided to support
the electrode array of needle bundles.
[0162] As example of making an electrode assembly which includes
long needles is as follows. One embodiment of long needle
electrodes can be made in the following manner. Several 1/8 inch
thick Lexan sheets are cut to the dimension of 1 cm by 1.5 cm. Two
parallel rows are markded 5 mm apart. Drill points 2 mm apart are
marked along each of the parallel lines that are 5 mm apart. Holes
are drilled at each marked point, 0.35 mm in diameter.
[0163] Stainless-steel acupuncture needles, such as Seirin No. 8
needles, are selected. Needles are inserted through one of the
three Lexan pieces. Lengths of nickel capillary tube, 2 mm in
length, are cut wherein the capillary tube has an inner diameter of
0.020 inches. A cut capillary tube is slid onto each of the
needles. Needles are inserted through the remaining two of the
Lexan pieces. The Lexan piece nearest the points is placed on a
spacer that leaves 0.5 cm of needle extending beyond the Lexan
piece. Using an epoxy adhesive, the needles are fixed in place to
the Lexan piece nearest the point. The second piece is moved 0.5 cm
from the first piece. The space formed by three sides between the
two Lexan pieces is sealed, and a high heat plastic or epoxy is
poured in to fill the space. The nickel pieces are soldered to the
stainless steel by filling the space between the nickel tubing and
the needle. A high-voltage wire is soldered to connect one of the
rows of needles at the site of the nickel capillary tubing. A
high-voltage wire is soldered to the other row of needles at the
site of the nickel capillary tubing. The space between the last two
Lexan pieces is sealed on three sides, and the space is filled with
a high heat epoxy. The acupuncture needle handles are removed, and
the needles are rinsed to a point flush with the top of the last
Lexan piece. The top is sealed with an electrically resistant
epoxy. Banana plugs are placed on the other end of the high-voltage
wires. The needles can be insulated by adding a small amount of
epoxy or Teflon paint to the ends of the needles. In addition,
other selected areas of the needle may be insulated.
[0164] The long-needle electrodes can be coated in many ways. The
following is one method. The needles are immersed in a solution of
DNA that has 0.3 micrograms per microliter of DNA in a TE buffer.
The needles are suspended in the solution without touching the
bottom of the container. The container holding the liquid has a
stainless-steel bottom that serves as a negative electrode. All of
the needles are then connected to a common positive pole of a power
supply. A voltage of 1.5 V is applied to the needles for several
minutes. The needles are then removed from the solution and can be
used immediately because the DNA forms a static coating on the
electrodes. Alternatively the DNA can be dried on the needles. A
statically-coated protectant such as sucrose may be added to the
needles by dipping in a sucrose solution prior to drying the
DNA.
[0165] The capacity of the needles for retaining statically-coated
surface DNA can be extended in several methods. One method is
simply to apply multiple layers of statically-coated DNA to the
surface of the needles. Another method is to make the surface area
of the needle greater by adding a porous sintered metal surface.
Another method is to add a polymer or a gel to the electrode
surface to provide a three-dimensional surface to the metal
electrode surface for the statically-coated DNA to adhere to.
[0166] The statically-coated DNA can be protected from the
influence of environmental DNAses by embedding the DNA in
liposomes, adding DNAse inhibitors or coating the needle in a
protectant. Sterile, desiccated packaging will also protect the
statically-coated needles.
[0167] An example of using statically-coated, long needle
electrodes is described as follows. The needle electrodes
containing the statically-coated DNA are inserted into tissue for
use. The small voltage electrophoretic potential may be applied for
a period of seconds to minutes to move the DNA off of the needles
and into the tissue. Electroporation pulses can then be added to
drive the DNA into cells of the tissue. More electrophoretic pulses
may be needed to help move the DNA into cells. After a resting
period of several seconds, polarities can be reversed on the
needles and the same protocol can be repeated.
[0168] In accordance with another aspect of the invention, polymers
can be used to modify adhesion of DNA to metal electrodes. As
described above, DNA can be coated onto a metal electrode surface
using electric charge to facilitate the binding of the DNA to the
electrode surface. However, such a DNA binding can be a high
affinity binding which makes driving the DNA off from the electrode
using electric fields difficult. Consequently, in accordance with
the invention, methods are provided for controlling the adhesion of
DNA to fixed electrode surface 42 and easing the release of DNA
from the fixed electrode surface 42 using electric fields.
[0169] In accordance with an aspect of the invention, a
water-insoluble adhesion controlling polymer 52 can be coated onto
the electrode underbody 40 to form the fixed electrode surface 42.
Then, the electrode releasable molecules 44 are coated onto the
fixed-surface adhesion controlling polymer 52. In such a case, when
the electrodes are placed next to the biological cells, the
water-insoluble adhesion controlling polymer 52 remains fixed on
the electrode underbody 40, and the electrode releasable molecules
44, which are electric field separable molecules, are driven off of
the water-insoluble adhesion controlling polymer 52.
[0170] Also, in accordance with an aspect of the invention, a
water-insoluble adhesion controlling polymer 52 can be mixed
together with electrode releasable molecules 44, and the mixture
can be coated onto the electrode underbody 40 to form a fixed
electrode surface 42 which is intermingled with the electrode
releasable molecules 44. In such a case, when the electrodes are
placed next to the biological cells, the water-insoluble adhesion
controlling polymer 52 remains fixed on the electrode underbody 40,
and the intermingled electrode releasable molecules 44, which are
electric field separable molecules, are driven off of the
water-insoluble adhesion controlling polymer 52 and driven into the
biological cells.
[0171] Alternatively, in accordance with the invention, the
adhesion controlling polymer 52 can be water soluble and can be
applied to the surface of the electrode underbody 40 which serves
as the fixed electrode surface 42. Then, the electrode releasable
molecules 44 are coated onto the adhesion controlling polymer 52.
In such a case, when the electrodes are placed next to the
biological cells, the water-soluble adhesion controlling polymer 52
is dissolved by the body fluids, and both the water-soluble
adhesion controlling polymer 52 and the electrode releasable
molecules 44 are released from the fixed electrode surface 42.
Under the influence of applied electric fields, the electrode
releasable molecules 44 are driven into the biological cells.
[0172] Alternatively, in accordance with the invention, the
adhesion controlling polymer 52 can be water soluble and can be
mixed with the electrode releasable molecules 44. The mixture is
applied to the surface of the electrode underbody 40 which serves
as the fixed electrode surface 42. In such a case, when the
electrodes are placed next to the biological cells, the
water-soluble adhesion controlling polymer 52 is dissolved by the
body fluids, and both the water-soluble adhesion controlling
polymer 52 and the electrode releasable molecules 44 are released
from the fixed electrode surface 42. Under the influence of applied
electric fields, the electrode releasable molecules 44 are driven
into the biological cells.
[0173] Alternatively, all of the following can be used to coat the
electrode underbody 40: a water-insoluble adhesion controlling
polymer 52, a water soluble adhesion controlling polymer 52, and
electrode releasable molecules 44. In such a case, the
water-insoluble molecules remained fixed on the fixed electrode
surface 42, and the water-soluble adhesion controlling polymer 52
and the electrode releasable molecules 44 are released from the
fixed electrode surface 42 when the electrodes are placed next to
the biological cells. The electric fields then drive the electrode
releasable molecules 44 into the biological cells.
[0174] In general, in one class of methods, polymers are added to
the electrode underbody 40 prior to applying the electrode
releasable molecules 44, e. g. DNA. In another class of methods,
polymers are mixed with electrode releasable molecules 44, e. g.
DNA, prior to coating the electrode underbody 40 with the
mixture.
[0175] For in vivo delivery of DNA, the polymer needs to be
biocompatible. A partial list of biocompatible polymers follows:
poly(vinyl alcohol), poly(methacrylic acid), poly(ethoxazoline),
poly(hydroxybutyrate), poly(caprolactone),
poly(2-hydroxymethacrylate) and poly(acrylic acid). Co-polymers
with components of these polymers may also be biocompatible. Many
other polymers are biocompatible and may be appropriate for
use.
[0176] Co-polymers may be especially useful if modified properties
are desired. For example, the addition of segments of cationic
polymers in non-ionic polymers may promote binding of DNA to the
polymer. Other ionic co-polymers may be used to impart an
environmental sensitivity to the polymer. Examples are pH
sensitivity, temperature sensitivity, chemical sensitivity, solvent
sensitivity or electric field sensitivity. All of these
modifications can be used to control the release of biopolymers
(DNA, RNA, proteins) mixed with or associated with the additional
polymer on the surface of an electrode.
[0177] One example of the use of a polymer to assist loading of DNA
onto a needle surface is as follows: Poly vinyl alcohol is
dissolved in water by heating. The amount of polymer used in this
example is a 2% polymer in water (w/v). The electrode is dipped in
the polymer and the electrode is dried at 70 degrees centigrade.
Next, 10 microliters of DNA at 2.5 micrograms per microliter is
added to the needle surface by pipetting. The DNA is dried onto the
surface at 70 degrees centigrade. This process can be done in one
step by mixing the DNA and polymer prior to application. The needle
electrode is then stored dry until used. Another advantage of
adding polymers to electrode metal surfaces with the DNA is to
reduce electrolysis at electrode surfaces touching cells and DNA. A
polymer that remains on the metal surface as a hydrated polymer can
keep the metal separated from living cells and from the DNA. Such a
polymer separates electrode surfaces, which may have sites with
active surface electrolysis, from the living cells and from the
DNA, thereby reducing the chance of damage to either the cells or
the DNA.
[0178] Even if polymers are used to assist in initial release of
biopolymers, e. g. DNA, from the surface of the electrode, pulsed
electric fields are still needed for subsequent phases of the
delivery of the biopolymers into the biological cells. Pulses whose
main purpose is electrophoresis may be used to move the biopolymer
from next to the needle electrode to surrounding tissue. An
electroporation pulse is needed to make the surrounding cells
permeable to the biopolymers. And an additional set of pulses, used
for electrophoresis, may be needed to move the biopolymer into the
cells.
[0179] Although aspects of the invention have been described
hereinabove in greater detail, here is a relatively brief review of
aspects of the invention.
[0180] A method is provided for delivery of molecules into
biological cells. The method includes providing electrodes in an
electrode assembly wherein the electrodes have fixed electrode
surfaces which are coated with at least one static layer of
electric field separable molecules to be delivered. A waveform
generator is provided for generating electric fields, and
electrically conductive pathways are established between the
electrodes and the waveform generator. The electrode assembly
having the statically coated electrodes can be attached to an
electrode assembly holder for establishing electrically conductive
pathways between the electrodes and the waveform generator. The
electrodes are located such that the biological cells are situated
therebetween, and electric fields are provided in the form of pulse
waveforms from the waveform generator to the electrodes, such that
molecules in the at least one static layer of the electric field
separable molecules on the electrodes are driven off of the
electrodes and delivered into the biological cells.
[0181] An electrode surface itself can serve as the fixed electrode
surface. The fixed electrode surface can include an oxidized metal
surface. The oxidized metal surface can include an oxide of silver,
an oxide of nickel, or an oxide of copper. The fixed electrode
surface can include fixed metal particles, or a roughened
surface.
[0182] The electric field separable material on the fixed electrode
surface can include a gel coating, a solid layer of nonpolymeric
material, or a polymer layer.
[0183] In accordance with another aspect of the invention, a method
is provided for immunotherapy which includes the steps of (a)
obtaining statically-coated electrodes which are statically-coated
with an immuno-stimulating material, (b) inserting the
statically-coated electrodes into a tissue to be treated, (c)
applying electrophertic and electroporation pulses to the
statically-coated electrodes such that the immuno-stimulating
material is driven off of the statically-coated electrodes and into
cells in the tissue.
[0184] In accordance with another aspect of the invention, an
electrode is provided which includes a coating having at least one
static layer of electric field separable molecules to be delivered
into biological cells. The electrode can include a fixed electrode
surface which is coated with the static layer of electric field
separable molecules. The molecules in the static coating can be in
a solid phase, a gel.
[0185] The fixed electrode surface 42 can include a fixed surface
matrix, and the molecules in the static coating are in a liquid
fixed on the fixed surface matrix. The fixed surface matrix can
include solid surface particles. The solid surface particles can be
metal particles. The fixed surface matrix can include a liposome
matrix or a solid polymer matrix.
[0186] The molecules in the static coating can be macromolecules,
and the macromolecules can include a polynucleotide vaccine, such
as a solid phase polynucleotide vaccine, a DNA vaccine, a solid
phase DNA vaccine, a RNA vaccine, or a solid phase RNA vaccine. The
macromolecules in the static coating can include a protein-based
vaccine, such as a solid phase protein-based vaccine. The
macromolecules in the static coating can include an organ treating
agent. The organ treating agent can include a deep tumor tissue
treating agent.
[0187] The electrode can be in a form of a needle electrode.
[0188] In accordance with another aspect of the invention, the
coating of the electrode by the static coating molecules is carried
out by the following steps: preparing a liquid medium in which a
quantity of the molecules are carried; contacting the electrodes
with the prepared medium; removing the electrodes from the medium;
and drying off the medium, such that a static coating of the
molecules remains on the electrodes.
[0189] In accordance with another aspect of the invention, the
coating of the electrode by the static coating molecules is carried
out by the following steps: preparing a liquid medium in which a
quantity of the molecules are carried; contacting the electrodes
with the prepared medium; applying pulse waveforms to the
electrode, such that a portion of the molecules are bound to the
electrode; removing the electrode from the medium; and drying off
the medium, such that a coating of the molecules remains on the
electrode.
[0190] In accordance with another aspect of the invention, an
apparatus is provided for delivery of molecules into biological
cells. The apparatus includes a waveform generator which provides
pulse waveforms. An electrode assembly holder is provided, and an
electrode assembly having a plurality of electrodes is mechanically
supported by an electrode assembly holder. The electrode assembly
is electrically connected to the waveform generator through
electrically conductive pathways. The electrode assembly includes
electrodes which are coated with at least one static layer of
molecules to be delivered into the biological cells.
[0191] It is apparent from the above that the present invention
accomplishes all of the objects set forth by providing a method and
an apparatus for delivery of macromolecules into cells that do not
cause skin damage that results in scarring. With the invention, a
method and an apparatus for delivery of macromolecules into cells
are provided which do not leave a residue of ballistic particles in
cells that are treated. With the invention, an electroporation
method for delivering molecules to biological cells in the
epidermis, near the basal lamina, does not have the treatment
molecules pass through the skin transdermally. With the invention,
a method and an apparatus for delivery of macromolecules into cells
are provided which do not employ a hypodermic needle. With the
invention, a method and an apparatus for delivery of macromolecules
into cells are provided which do not employ a fluid medium that
flows down onto the electrodes as the electroporation process is
being carried out on the patient.
[0192] With the invention, a method and an apparatus for delivery
of macromolecules into cells are provided which do not employ a
pressurized fluid injection step for injecting fluid into a
patient. With the invention, relatively low absolute voltages are
applied to cells undergoing electroporation. With the invention,
pulses that are applied to the cells can have, if desired,
relatively short pulse width to the cells undergoing
electroporation. With the invention, a method and an apparatus for
delivery of macromolecules into cells are provided which can
employ, if desired, electrodes in which the base portions and tip
portions of the electrodes are nonconductive. With the invention, a
method and an apparatus for delivery of macromolecules into cells
provide disposable electrode assemblies. With the invention, a
method and an apparatus for delivery of macromolecules into cells
are provided in which electrode assemblies are packaged in sterile
packaging. With the invention, a method and an apparatus for
delivery of macromolecules into cells are provided which permit
treatment of tissues using coated long electrodes which have
electric field separable material which includes a tissue treating
agent.
* * * * *