U.S. patent application number 15/495273 was filed with the patent office on 2017-09-21 for method, device and system for targetted cell lysis.
This patent application is currently assigned to TMB LABS LTD.. The applicant listed for this patent is TMB LABS LTD.. Invention is credited to John Robert DODGSON, Soiwisa SOIKUM, Lars THOMSEN.
Application Number | 20170266283 15/495273 |
Document ID | / |
Family ID | 42270755 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170266283 |
Kind Code |
A1 |
SOIKUM; Soiwisa ; et
al. |
September 21, 2017 |
METHOD, DEVICE AND SYSTEM FOR TARGETTED CELL LYSIS
Abstract
A method, device and system employs particles, such as
nanoparticles, and an electric or electro-magnetic field, to cause
cell death in target cells by non-thermal means. The method of
causing targeted cell death comprises the steps of: introducing a
particle to the interior of a target cell and exposing the target
cell to a transient electromagnetic field for a sufficient time
interval in order to cause cell death. Apparatus for performing the
method; as well as techniques of delivering particles and for
producing particles are also described.
Inventors: |
SOIKUM; Soiwisa; (Sri
Ayutthaya, TH) ; THOMSEN; Lars; (Aallborg, DK)
; DODGSON; John Robert; (London, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TMB LABS LTD. |
Hong Kong |
|
HK |
|
|
Assignee: |
TMB LABS LTD.
Hong Kong
HK
|
Family ID: |
42270755 |
Appl. No.: |
15/495273 |
Filed: |
April 24, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13643695 |
Jun 3, 2013 |
9629912 |
|
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PCT/GB2011/000645 |
Apr 26, 2011 |
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15495273 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 13/00 20130101; A61N 2/00 20130101; A61K 41/0038 20130101;
A61N 1/327 20130101; A61B 18/18 20130101 |
International
Class: |
A61K 41/00 20060101
A61K041/00; C12N 13/00 20060101 C12N013/00; A61N 1/32 20060101
A61N001/32 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 2010 |
GB |
1006841.9 |
Oct 4, 2010 |
GB |
1016582.7 |
Claims
1. A non-thermal method for inducing targeted cell death,
comprising: introducing a first dielectric particle to an interior
of a target cell; positioning a second dielectric particle on, or
adjacent to, an exterior surface of the target cell; and exposing
said target cell to a transient electromagnetic field of sufficient
strength and for a sufficient time interval to cause irreversible
electroporation (IEP) of the cell via field enhancement through the
first and second dielectric particles and across the cell membrane,
thereby inducing targeted cell death.
2. A non-thermal method for inducing targeted cell death,
comprising: introducing a first dielectric particle to an interior
of a target cell; positioning a second dielectric particle on, or
adjacent to, an exterior surface of the target cell; and exposing
said target cell to a transient electromagnetic field of sufficient
strength and for a sufficient time interval to cause irreversible
electroporation (IEP) of the cell, wherein the first and second
dielectric particles enhance the electromagnetic field across the
cell membrane, thereby inducing targeted cell death.
3. The method of claim 1, wherein the target cell is a
microorganism, a cell of a fungus, or an eukaryotic cell.
4. The method of claim 3, wherein the eukaryote cell is a mammalian
cell or a cell of another animal.
5. The method of claim 4, wherein the mammalian cell is a human
cell.
6. The method of claim 4, wherein the mammalian cell is a cell of a
neoplasia or cancer, on or in the mammal.
7. The method of claim 4, wherein the mammalian cell is a cell
infected by a virus.
8. The method of claim 3, wherein the microorganism is a bacterium
and the electromagnetic field is also enhanced across the cell wall
of the bacterium.
9. The method of claim 1, wherein at least one of the first and
second dielectric particles has a high permittivity with respect to
at least one of a cell membrane of the target cell and an
environment surrounding the membrane.
10. The method of claim 1, wherein at least one of the first and
second dielectric particles has a core comprising a conductive
metal material selected from the group consisting of iron, oxide of
iron, silver, gold, and platinum.
11. The method of claim 1, wherein a surface of the first
dielectric particle comprises a coating molecule that promotes
uptake of the first particle by the target cell.
12. The method of claim 11, wherein the coating molecule is at
least one selected from the group consisting of a ligand, antibody,
aptamer, protein, nucleic acid, and peptide species.
13. The method of claim 12, wherein uptake by the target cell is
via endocytosis.
14. The method of claim 1, wherein a surface of the second
dielectric particle comprises a coating molecule that promotes
positioning of the second particle on or adjacent to an exterior
surface of the target cell.
15. The method of claim 14, wherein the coating molecule is at
least one selected from the group consisting of a ligand, antibody,
aptamer, protein, nucleic acid, and peptide species.
16. The method of claim 1, wherein the first and second dielectric
particles independently range between 20 nm and 5 .mu.m in
size.
17. The method of claim 1, further comprising varying a strength of
said transient electromagnetic field in dependence upon time.
18. The method of claim 1, wherein the target cell is exposed to
the transient electromagnetic field by positioning first and second
electrodes that generate an electromagnetic field such that when
the electromagnetic field is generated, the target cell is within
the electromagnetic field.
19. The method of claim 1, wherein the strength of the transient
electromagnetic field is varied over time.
20. The method of claim 1, wherein the strength of the transient
electromagnetic field is varied in space, such that a varying field
gradient is applied to the targeted cell.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method, device and system
for cell lysis, more particularly the invention relates to a
method, device and system that employs particles, or nanoparticles,
together with an electric or electro-magnetic field to cause cell
death in target cells by primarily non-thermal means.
BACKGROUND OF THE INVENTION
[0002] The use of electro-magnetic fields for treatment of cancer
by causing lysis to cells that harbor, or are in close proximity to
particles responsive to such fields, is known. The techniques
involve using a micro sized particle, typically below 0.1 .mu.m,
which for example comprises an iron oxide core and is coated with a
polymer. Typically a secondary coating is applied where the
particle is activated by chemicals that show specificity for
biological targets of interest. The aim was to concentrate
particles at the site of a biological target, where a specific
chosen target can be an exclusive marker, expressed for example by
cancer cells, and presented on the extracellular side of the cell.
Target cells are then destroyed thermally by means of absorption of
energy from the electromagnetic field.
[0003] Efforts are being made to improve techniques by focusing on
factors, such as clearance of particles from blood before reaching
the target cells; binding unselectively to other than target cells;
toxicity of the particles in use and insufficient effect from
induced electric fields to cause killing of the target cells.
PRIOR ART
[0004] International Patent Application WO-A-2010/151277 (Davalos
et al) discloses a method of treating neoplasia in a subject using
irreversible electroporation in which nanoparticles are
administered to the subject in an amount sufficient to permit at
least some of the nanoparticles to come into close proximity to the
neoplastic cells. At least two electrodes are implanted into or
adjacent the neoplastic cells within the body of the subject.
Multiple electric pulses are then emitted from the electrodes into
the neoplasia. Typically a field strength of about 500 Vcm-1 to
1500 Vcm-1, is applied between the electrodes, for a duration of 90
microseconds or less. This was shown to cause predominantly
non-thermal killing of the neoplastic cells.
[0005] WO-A-2010/151277 discloses that the use of nanoparticles can
reduce the threshold field for the destruction of cells by means of
irreversible electroporation, and so allow an electric field to be
chosen, such that cells having nanoparticles in proximity, are
destroyed while those without particles remain are not destroyed.
If the nanoparticles have a coating that allows them preferentially
to associate with a neoplastic cell, rather than healthy cells,
this allows the neoplastic cell to be destroyed while the healthy
cells are undamaged.
[0006] International Patent Application WO-A-2010/151277 further
discloses that use of nanoparticles may also increase the volume of
the treated area around a pair of implanted electrodes over the
area when nanoparticles are not used. It is stated that the
mechanism of action is an enhancement of the local electric field
at the cell membrane owing to the presence of the particles. A wide
range of particle materials is mentioned, but only theoretical
results for carbon nanotubes and insulating polystyrene spheres,
and practical results for carbon nanotubes, are disclosed.
[0007] Despite the foregoing, WO-A-2010/151277 is a theoretical
discussion, showing that a relatively modest field enhancement, for
example when applied to insulating polystyrene beads located around
a cell exterior, of approximately of a factor of 2 is achieved, as
shown in Davalos FIG. 20. Carbon nanotubes were shown to aid in
killing of cells in culture, but only by a modest amount (see
Davalos FIG. 19) and only at high field strength (500 Vcm-1).
[0008] Further there is no specific guidance on the physical
properties of the particles needed to achieve effective enhanced
irreversible electroporation, save that given in Davalos FIG. 18.
FIG. 18 depicts a direct current (DC) field arrangement; and shows
a maximum area of 4 times (i.e. twice the characteristic dimension
of the particle) in which field enhancement occurs. It is seen that
the ratio of conductivity of the particle, relative to that of a
surrounding medium, has a relatively small effect. Further the
ratio of the permittivity of the particle, to that of the
surrounding medium, has substantially no effect, on the efficacy of
the method.
[0009] Therefore the field enhancement in the model described is in
fact modest and so the improvement of the method disclosed is
likewise modest. Furthermore no disclosure is made of a preferred
mode of administration of the particles.
[0010] Another disadvantage is that the electrodes in Davalos et al
need to be implanted within the body, close to or at the site of
the neoplasia, which is an invasive and in some cases impractical
procedure. No disclosure is made of a method that is capable of
treating delocalized neoplastic cells, for example within a body
fluid such as blood, for example in cases of leukemia.
[0011] The use of carbon nanotubes to reduce the field at which
reversible electroporation occurs is disclosed in International
Patent Application WO-A-2008/062378 (Raffa et al). The method has
been shown to cause cell death resulting from the formation of
irreversible pores in the membrane. The method is hereinafter
referred to as irreversible electroporation.
[0012] In a poster, shown at the Nordic Naiad Symposium,
Copenhagen, from 1-3 Nov. 2010, by Soikum and Thomsen, there is
disclosed a method for killing bacterial cells or spores, that
includes the steps of: providing at least one particle with high
electrical permittivity, in close proximity to the cell membrane or
cell wall, and subsequently applying an electric field. The
particle acts to enhance the field in the vicinity of the particle
thereby increasing the effect of the field on the cell
membrane.
[0013] Soikum and Thomsen also disclose a field enhancement effect
that arises as a result of use of a nanoparticle, for example that
obtained when using a metal nanoparticle, adjacent the cell
membrane. For example a strong field enhancement effect was
observed using a pair of gold nanospheres in proximity to each
other in a medium with the same relative permittivity as blood. A
chip technology was used to provide means for a well-defined field,
over the sample containing the target cells.
[0014] US patent U.S. Pat. No. 4,767,611 (Gordon) discloses
treatment of cancer and other diseases, including infectious
diseases, through the use of electromagnetic energy deposition
within the cell resulting in targeted cell death. The process
comprises introducing particles into the interior of cells in
living tissue. The particles are capable of affecting the
intracellular conductivity, dielectric properties, dipole content
and membrane characteristics of the cell and nucleus.
[0015] The method in US patent U.S. Pat. No. 4,767,611 differs from
other forms of thermal destruction of cells in that energy is
deposited into the cells directly, rather than into the particles
and then conducted to the cells. However, the method suffers from
disadvantages, namely: a large amount of energy needs to be
deposited in the cells to be destroyed, and heat is dissipated to
surrounding tissues, potentially causing damage to local healthy
cells.
[0016] The inventors have now improved the method for targeted cell
lysis by primarily non-thermal means. They have also improved the
associated apparatus.
SUMMARY OF THE INVENTION
[0017] According to a first aspect of the invention there is
provided a method of causing targeted cell death by non-thermal
means comprising the steps of: introducing a particle to the
interior of a target cell and exposing the target cell to a
transient electric field for a sufficient time interval in order to
cause cell death.
[0018] Targeted cell lysis is ideally achieved by the steps of:
introducing a particle to the interior of a target cell and
exposing the target cell to an electric field sufficient to cause
irreversible electroporation (IEP) to the cell. However, it is
understood that in aspects of the invention some cells may undergo
apoptosis as a result of exposure to the electric field.
[0019] Preferably the particle is a dielectric particle. Preferably
the particle has a high permittivity with respect to the cell and a
surrounding environment. In some embodiments the particle comprises
a conductive core, such as a metal, for example gold.
[0020] As a result of introducing the particle to the interior of
the cell, it has been found that when an electric field is applied
the particle enhances the effect of the electric field in the
vicinity of the particle, so reducing the field strength needed to
achieve cell death, for example by electroporation of the cell.
[0021] In preferred embodiments the particle is ideally associated
with the cell membrane, or in the case of a bacterium, the cell
wall. The particle may be adapted to bind or adhere to the cell
wall.
[0022] As a result of selecting the particle type; its location
with respect to the cell; and the specific field characteristics,
the cell experiences irreversible pore formation in the cell
membrane, at reduced field strength, so causing lysis and cell
death at lower than previously achievable electromagnetic field
magnitudes, thus reducing the risk of damage to neighbouring cells
which do not have particles within them.
[0023] Preferably particles are adapted to enter the cell through
an outer membrane. It is known in the art that small particles are
taken into cells by means of endocytosis and the invention includes
the use of particles adapted to this end. Such adaptation may also
include providing a surface coating or surface species that promote
uptake by cells. Such a coating may include surface molecules such
as protein or peptide species known in the art to promote
endocytosis. Such coatings may comprise species such as proteins,
peptides or nucleic acid species that bind to target molecules on
the exterior of the cell membrane, so acting to increase the
concentration of particles associated with the membrane, and hence
the rate of uptake of the particles by means of endocytosis not
specifically caused or enhanced by the coating of the particle.
[0024] Advantageously, by positioning a second particle in close
proximity with the first dielectric particle, further concentration
of the electric field is achieved. A second particle may also be
within the cell.
[0025] A further way of positioning a second dielectric particle in
close proximity with the first particle is to locate the second
particle on, or adjacent, an exterior surface of the targeted cell.
Upon application of an electric field, local pore forming occurs as
a result of the electric field. The effect of this on the target
cell is enhanced and this eventually leads to cell death, because
the cell quickly loses its ability to sustain its cellular membrane
potential and because nutrients leak from the pores. Eventually
pore formation becomes irreversible after a few milliseconds of
treatment and the cell dies.
[0026] When two particles are associated with a cell, in addition
to the concentration of electromagnetic field strength, there is
also an increase in a mechanical force that is applied to the cell
and in some embodiments this may rupture the cellular membrane,
(typically of the order of 10 nm thick), for example when particles
are positioned on both sides of the membrane, or render it more
susceptible to irreversible pore-forming events arising from the
field enhancement in the vicinity of the two particles.
[0027] According to another aspect of the invention particles may
have formed thereon a coating that makes them selective for the
target cell type in such a way that at least one particle binds
selectively to one or more target molecules in the target cell
membrane.
[0028] The invention provides means to achieve selective
destruction of target cells while non-target cells are left
relatively unharmed.
[0029] Preferably one or more particles are targeted to target
molecules, for example target proteins, that are specifically or
preferentially expressed by a target cell type, so promoting the
preferential association of particles with the target cell type
over non-target cells. Such target molecules may be located on the
exterior of the target cell membrane, the interior of the target
cell membrane, or at a location within the target cell. Such target
molecules may be located preferentially at a specific location or
range of locations within the target cell, for example associated
with an organelle or interior structure of the target cell, such
as, for example, the nuclear or mitochondrial membrane.
[0030] The term target molecule means any molecule, or region or
fragment of a molecule, for example a protein, present on the
exterior or interior of a target cell to which a coating, component
or region of a particle may associate or bind. The target molecule
may be a biomarker and the terms marker and target molecule are
used interchangeably.
[0031] Target molecules may be the following or regions or subunits
of them: lipid, carbohydrate, a nucleic acid (such as chromosomal
DNA and/or plasmid DNA and/or any type of RNA), a protein (for
example, from the group comprising: enzymes, structural proteins,
transport proteins, ion channels, toxins, hormones, and receptors)
or small molecules that can be bound to the cellular membrane
either in form of an agonist and/or antagonist compared to its
affinity for non-target cells.
[0032] In a preferred embodiment, such target cell molecules are
located on the exterior of the cell membrane so providing one or
more particles bound preferentially to the exterior of the target
cell, but not to the exterior of non-target cells. The particles so
associated may be adapted to enter the target cell, for example by
endocytosis, while particles are not taken up, or are taken up to a
lesser extent, by non-target cells.
[0033] Such targeting may be achieved by means of antibodies,
aptamers or other ligands provided on the surface of the particles.
Ligands for known target molecules as described in the prior art
may be used in the coating. More than one ligand species may be
provided in order to increase the capture affinity.
[0034] Such coatings allow the method of the invention to be
applied selectively to target cells in a mixed population of target
and non-target cells, allowing the field to be chosen so as to
cause death of the target cells while leaving the non-target cells
unharmed.
[0035] Such a coating may be uniform over the surface of the
particle or it may be located in a specific region of the particle,
for example in the case of an elongated structure, preferentially
at one end of the particle or preferentially remote from one end,
so allowing the particle to bind preferentially in a preferred
orientation with respect to the target cell.
[0036] The first particle may be adapted to allow it to enter the
target cell by means of endocytosis. Typically the size of the
first particle is in the range of 20 nm to 2 um, optionally to 5
um. The second particle may be adapted so as not to promote its
uptake by the cells. Typically the size of the second particle is
in the range of 20 nm to 5 um,
[0037] The first particle optionally comprises a coating adapted to
promote endocytosis, or binding to the cell membrane so as to
improve the chance of endocytosis. The second particle may have a
coating that does not promote endocytosis, or acts to delay or
hinder it.
[0038] According to another aspect the invention relates to an
apparatus for treatment of a disease condition in a subject using
particles or nanoparticles and time-varying electromagnetic or
electric fields, characterised in that a means is provided to
introduce a particle to the interior of a target cell and a means
is provided for exposing the target cell to an electric field
sufficient to cause cell death by non-thermal means,
[0039] Ideally cell death results from irreversible electroporation
of the cell. It is understood that in some embodiments cells may
undergo apoptosis as a result of exposure to the electric
field.
[0040] The invention may be used in order to treat for example
neoplasia, cancerous cells, or to treat infections caused by fungi,
vira, bacteria or other microorganisms.
[0041] Ideally the means to introduce particles to the interior of
the cell includes a particle delivery device that administers
particles to the target cells or a region of tissue comprising
them, for example topical or systemic administration. Particles so
delivered are preferably adapted to enter the target cell as
described above.
[0042] Means may be provided as part of the apparatus to provide a
first particle type, adapted as described above to enter a target
cell, and a second particle type, adapted to bind to a target
molecule on the exterior of a target cell membrane. In a preferred
embodiment, the first particle type is also targeted to a target
cell, as described above, so that it enters a target cell
preferentially. In addition entry of the first particle into
non-target cells is minimised.
[0043] Particles may be administered systemically by, for example,
administering into a body fluid such as blood, lymph, cerebrospinal
fluid, so that the fluid acts to carry the particles to the target
cells. In some embodiments the target cells may be within the body
fluid into which the particles are administered. In some
embodiments the body fluid may act as a transfer medium to carry,
or allow transmission of, the particles to the target cells. The
target cells may be bacteria, spores, vira or mammalian cells, for
example leukemic or virally-infected cells within the body fluid.
The target cells may instead be localised, for example in a local
seat of infection, a region of neoplasia or a tumour.
[0044] Particles may be administered by any means known in the art,
such as injection and/or infusion and/or electroporation through
skin and/or inhalation and/or absorption through mucal membranes
and/or via the digestive tract. Devices for administration include:
a syringe, cannula, catheter, inhaler, implanted release device,
capsule or ingestible preparation.
[0045] According to a further aspect of the invention there is
provided a means to expose the target cells to a variable electric
or electromagnetic field comprising: at least a first and a second
electrode and a control means for applying a variable potential to
the first and second electrodes, whereby target cells within the
electric field are killed and non-target cells remain unharmed. The
field strength may be chosen according to the nature of the disease
condition, the nature of the particles, and the proportions of
target and non-target cells that are to be destroyed on average in
a given treatment.
[0046] The terms electric and electromagnetic fields are used
interchangeably except where stated. The terms electric field,
electric field strength and electric field flux are also used
interchangeably to refer to the magnitude of an electric field.
[0047] The electric field is ideally a time varying field but it
will be understood that alternatively, or in addition, the electric
field may vary in space, so that a varying field gradient is
applied to the targeted cell.
[0048] In a preferred embodiment the apparatus comprises: at least
a first and second electrode adapted to be located externally to
the body of a subject or to be placed in contact with the skin of
the subject. It is an advantage of the present invention that the
field required for cell death of target cells is sufficiently low
that in contrast with the prior art electrodes may be used in some
embodiments that do not need to be implanted.
[0049] In some embodiments the apparatus may comprise one or more
electrodes adapted to be implanted within the body of the subject.
In preferred embodiments the electrodes are adapted to provide a
field localised in the vicinity of a region of target cells, such
as for example a seat of infection or a tumour. In an alternative
embodiment the electrodes are adapted to provide a field over a
larger region, so as for example to treat a delocalized condition
over a region of the subject's body.
[0050] In some embodiments the apparatus comprises means to change
the orientation of the applied field during a course of treatment.
In some embodiments the apparatus includes means to move one or
more electrodes with respect to the subject or vice versa.
[0051] According to a further aspect of the invention there is
provided a method of targeted cell lysis comprising the steps of:
causing a particle of high permittivity to associate with the
exterior of a target cell membrane and exposing the target cell to
an electric field sufficient to cause irreversible electroporation
(IEP) to the cell.
[0052] In preferred embodiments the particle is a dielectric
particle. Preferably the particle has a high permittivity with
respect to the cell and a surrounding medium.
[0053] In some embodiments the particle comprises a conductive
core, such as a metal, for example gold, or a metal oxide, for
example Fe.sub.3O.sub.4.
[0054] Particles are preferably of higher permittivity than the
mean permittivity of the composite medium environment in a region
surrounding them. Typically that region comprises one or more of
the extracellular fluid; extracellular matrix and associated
proteins; cell membranes of the target cell and surrounding cells;
cell surface molecules such as membrane proteins, glycoproteins and
sugars. The permittivity of the surrounding environment is
therefore a composite permittivity derived from the presence and
permittivities of various composite components, and therefore may
take a range of values up to that of physiological saline or
blood.
[0055] As a result of the particle becoming associated to the cell
membrane, or in the case of a bacterium, the cell wall, it has been
found that, when an electric field is applied the particle enhances
the effect of the electric field in the vicinity of the particle,
so reducing the field strength needed to achieve electroporation of
the cell.
[0056] In preferred embodiments the particle is adapted to bind to
a target molecule on the cell membrane as described above. In
preferred embodiments the particle is adapted to bind selectively
to target cell by means of binding to a target molecule that is
preferentially, or only, expressed by a target cell type.
[0057] As a result of selecting the particle type to have a high
permittivity; its location with respect to the cell; and the
specific field characteristics, the target cell experiences
irreversible pore formation in the cell membrane at reduced field
strength, so causing lysis and cell death at lower than previously
achievable electromagnetic field magnitudes, thus reducing the
degree of damage to neighbouring non-target cells.
[0058] It is additionally found that providing a second particle of
high permittivity in close proximity to the first particle, causes
a greater enhancement of an applied field than for a single
particle alone, or for a pair of particles of low permittivity.
Therefore in particularly preferred embodiments first and second
high permittivity particles are provided in proximity one to
another and to a target cell, an electric field is provided such
that death of the target cell is caused by means of damage to a
cellular structure or component, for example irreversible
electroporation of the cell membrane or the nuclear membrane.
[0059] In preferred embodiments the first particle is adapted to
enter the target cell and the second particle is adapted to bind to
the exterior of the cell membrane as described previously. In an
alternative embodiment, both the first and the second particles are
adapted to bind to the exterior of the cell membrane.
[0060] According to a further aspect, the invention relates to an
apparatus for causing targeted cell lysis, comprising: means to
provide particles to a target cell, the particles being adapted to
associate with the target cell, and means to apply an electric
field to a target cell, comprising a first and a second electrode
and a device to apply time-varying potentials to the
electrodes.
[0061] Electrodes and particles may have characteristics as
described above. Further characteristics of electrodes and
particles that are used in various embodiments of the invention are
set out below.
[0062] In a preferred embodiment the apparatus comprises a device
for providing a time varying first electric potential to the first
electrode and second electric potential to the second electrode,
and a programmable unit adapted to control the device in response
to instructions stored on a storage medium accessible by the
programmable unit.
[0063] According to a further aspect of the invention there is
provided a method for treatment of a disease in a subject by means
of destruction of target cells within the subject, comprising the
steps of: [0064] a) administering particles to the subject, either
systemically or topically in the region of the target cells, the
particles being adapted to be taken up within the target cells;
[0065] b) allowing a chosen time interval to elapse so that at
least one particle is taken up within at least one target cell; and
[0066] c) applying an electric field to a region of the subject
within which one or more target cells are located in order to cause
cell death of the targeted cells by predominantly non-thermal
means.
[0067] In preferred versions of this embodiment the one or more
particles create an enhanced electric field in their vicinity and
cause cell death of the target cell by means of irreversible pore
forming events in the cell membrane.
[0068] The particles may be adapted to be taken up into the target
cells. Particles may be adapted to be taken up selectively into
target cells, and either not taken up, or taken up to a lesser
extent by non-target cells, as described above.
[0069] Particles may be administered systemically as described
above, or topically by means known in the art such as local
injection, infusion, implantation or electroporation.
[0070] In a preferred embodiment, a second particle may be provided
that is adapted to bind to the exterior of the target cell
membrane. One or both particles may comprise a coating that makes
them selective for the target cell type in such a way that at least
one particle binds selectively to one or more target molecules in
the target cell membrane.
[0071] Optionally particles may be located and/or tracked within
the body fluid or the site of action by means known in the art, for
example MRI, ultrasound or computer tomographic (CT) scanner,
chosen according to the nature of the particles in use. In addition
the electric field may be applied at a chosen point depending on
the results from the location and tracking process, and associated
processing and display equipment may be provided in order to enable
this.
[0072] According to a further aspect of the invention there is
provided a method for treatment of disease in a subject comprising
the steps of: administrating to a subject a quantity of a first
particle type, administrating to the subject a quantity of a second
particle type; allowing at least one particle of the first particle
type and at least one particle of the second particle type to
become associated with one or more target cells, and providing an
electromagnetic field in the vicinity of the target cells so
causing death of the target cells by primarily non-thermal
means.
[0073] In preferred embodiments the particles become associated
with the target cells so as to form an arrangement of particles in
which the particles act together to enhance the electric field in
their vicinity, so causing damage to a cellular structure, such as
the cell membrane or the nuclear membrane, leading to cell
death.
[0074] In particularly preferred embodiments, the first particle
type is adapted to enter the target cell, and the second particle
type is adapted to be bound to target molecules on the exterior of
the cell. The first and second particles act together to enhance
the electric field across the cell membrane in the vicinity of the
particles, so causing cell death by means of irreversible
pore-forming events.
[0075] In preferred embodiments a delay is provided between the
administration of the first and the second particle types. The
delay is typically in excess of 10 minutes.
[0076] In preferred embodiments a delay is provided between
administration of the second particle type and application of the
electric field. The delay is typically in excess of several
minutes, ideally more than 10 minutes.
[0077] According to a further aspect of the invention there is
provided a method of treatment of disease in a subject by means of
targeted cell lysis comprising the steps of: administering a first
particle type systemically to the subject, the first particle type
being adapted to bind to a target molecule on the target cell
membrane; waiting for a time interval to allow the particles to
bind to the target cells; and exposing the target cell to an
electric field sufficient to cause irreversible electroporation of
the cell.
[0078] In preferred embodiments the particle is a dielectric
particle. Preferably the particle has a high permittivity with
respect to the cell and a surrounding medium. In some embodiments
the particle comprises a conductive core, such as a metal, for
example gold, or a metal oxide, for example Fe.sub.3O.sub.4.
[0079] In preferred embodiments the particles are adapted to bind
to target molecules on the exterior of the cell membrane, and
preferably comprise a coating as described previously.
[0080] According to another aspect the invention relates to an
apparatus and a method for treatment of a disease condition in a
subject using particles or nanoparticles and time-varying
electromagnetic or electric fields, characterised in that a means
is provided to associate a particle selectively with a target cell
in a liquid medium and a means is provided for exposing the target
cell to an electric field sufficient to cause irreversible
electroporation of the cell.
[0081] Preferably treatment of a disease in a subject by causing
death of target cells located at least partially in a body fluid
comprises the steps of: administering particles to the body fluid,
allowing the particles to become associated with target cells
within the body fluid, applying an electric or electromagnetic
field to the target cells and particles within the body fluid,
thereby causing cell death by primarily non-thermal means, for
example by irreversible electroporation of the target cell
membrane.
[0082] An example of a body fluid may be blood or a blood component
such as plasma, cerebrospinal fluid, or bone marrow. Optionally
treatment of the body fluid is performed on the fluid after it has
been removed from the human or animal body or is performed whilst
the fluid is outside the human or animal body.
[0083] In a preferred embodiment an apparatus comprises particles
adapted to bind to a target molecule on the surface of a target
cell, or to enter a target cell, as described previously; means to
administer particles into the body fluid; at least a first and a
second electrode adapted to apply an electric field to a region of
body fluid containing target cells and a device for providing time
varying potentials to the first and the second electrodes.
[0084] Preferably the electrodes apply an electric field to a
region through which a body fluid flows, so bringing target cells
into the region with the flow. In an embodiment the electrodes are
adapted to apply a field to a perfused region such as a blood
vessel in vivo, for example a blood vessel near the surface of the
body.
[0085] In an alternative embodiment the apparatus includes means
for applying an electric field to a body fluid externally to the
body. Preferably the apparatus comprises an extracorporeal flow
system comprising a flow cell through which the body fluid, such as
blood, may flow, the flow cell being adapted to apply an electric
field to the fluid. The flow system may then return the treated
fluid to the subject.
[0086] The method according to this aspect of the invention
preferably includes steps of: administering particles systemically
to the subject, allowing a time interval after administration, and
then providing an electric field in a region containing the body
fluid or through which the body fluid may flow. The method
preferably includes a repeated application of the field in order to
cause death of the target cells as they move into a region where
the electric field is applied. The method envisages that target
cells may be delocalized within the body fluid of the subject, and
present at low concentrations within the body fluid, so may be
removed gradually by repeated applications of the field over time.
It is a feature of the invention that the reduction in field
strength and electrode potentials needed to achieve irreversible
electroporation and cell death facilitates repeated applications of
the field compared with prior art apparatus and methods.
[0087] The terms lysis, cell death, cell destruction and killing
are used interchangeably.
[0088] For the avoidance of doubt the term subject is meant to
include a living organism including individual humans and animals.
The terms subject, host organism and target organism are used
interchangeably.
[0089] The mechanism of cell destruction according to the invention
is described as irreversible electroporation, or irreversible
pore-forming event, those terms are used interchangeably. It is
further understood that in embodiments of the invention cell death
may occur through exposure to transient electric fields enhanced by
particles without irreversible electroporation of the cell
membrane, rather through damage to intracellular structures,
organelles or components leading to apoptosis or necrosis.
[0090] It is further understood that features described for given
aspects of the invention or embodiments are not intended to be
employed in that aspect or embodiment only, rather they may be
combined to achieve the purposes of the invention.
[0091] In some embodiments particles may be adapted to remain
associated with target cells for an extended period after
administration. Thus in accordance with any of the aspects of the
invention herein, the method may comprise administration of
particles followed by multiple instances of application of the
electric field at intervals after administration of the
particles.
[0092] According to a further aspect of the invention there is
provided a method for causing the death of a target cell,
comprising the steps of: providing a particle arrangement of at
least a first and a second particle in proximity to the target
cell; applying an electric or electromagnetic, field to the target
cell and particle arrangement, the first and the second particles
being arranged such that they cause enhancement of the component of
the field appearing across a region of the target cell in the
vicinity of the first and second particles, so causing cell death
by primarily non-thermal means.
[0093] In a preferred embodiment the first and the second particle
are arranged so that a region or component of the target cell lies
between them.
[0094] Preferably the first and the second particle are located on
opposite sides of the target cell, of an organelle within the
target cell, for example the nucleus, or of a cell component, for
example the cell membrane, the nuclear membrane or a mitochondrial
membrane, in the direction of a component of the applied electric
field, thereby enhancing the applied field appearing across the
cell, organelle or component. A field may then be applied so as to
cause disruption of the cell, organelle or component, leading to
cell death. Cell death may be caused for example by irreversible
electroporation of a membrane.
[0095] In a preferred embodiment sufficient particles are provided
such that on average a proportion of target cells have a particle
arrangement as above. The number of particles supplied and the
applied field strength may be chosen to optimize the proportion of
target and non-target cells destroyed by the field.
[0096] The electric field may be applied in a first direction and
then subsequently in a different orientation, for example by moving
one or more electrodes used to provide the field relative to the
cell. As described further below, a plurality of electrodes may be
provided to allow the field direction to be chosen or varied during
the treatment.
[0097] A further aspect of the invention provides a method for
selectively killing a cell, the method comprising the steps of:
providing a particle arrangement of at least a first and a second
dielectric particle, where the first dielectric particle is within
the cytoplasma of the cell and the second dielectric particle is on
the extracellular side of cell.
[0098] An alternative approach is where both the first and second
dielectric particle is at the extracellular side of the cell or
both particles are in the cytoplasma of the cell. However, both
alternatives are likely to give lesser effect than the first
mentioned arrangement.
[0099] Ideally a coating is provided on at least one of the
particles so as to render it specific for the target cell type in
such a way that at least one particle binds selectively to one or
more target molecules in the cellular membrane.
[0100] Optionally by providing at least a first and a second
electrode externally to the organism hosting the target cell.
[0101] Additionally the particles are exposed to an alternating
electric field, the alternating electric field being provided by at
least the first and the second electrodes and being of sufficient
frequency and amplitude so as to cause a concentration of field
flux between the at least two particles so as to cause destruction
of molecular entities in the target cell.
[0102] This ultimately leads to break down of the cellular membrane
by causing irreversible pore forming events. The field amplitude
and frequency can be chosen so that cellular destruction is
achieved in the vicinity of the two dielectric particles and so
that adjacent cells are unaffected, because it is only the field
flux concentration between the two dielectric particles that is
strong enough to mediate molecular disarrangement for example in
the cellular membrane.
[0103] Preferred embodiments of this and other aspects of the
present invention may comprise some or all of the following
features.
[0104] In a preferred embodiment of the invention, the method may
further include the step of performing an analysis of the extent of
cellular damage, said step comprising extracting biological
material from the host organism and subjecting extracted material
to an investigation in order to determine the damage.
Alternatively, the analysis may involve a non-invasive technique
such as ultrasonic investigation, computer tomography (CT), X-ray
or magnetic resonance image analysis.
[0105] The terms "killing" relate to cause irreversible pore
forming events in the cellular membrane or in case of bacteria the
cell wall, with concomitant release of biological material from the
target cells. In the present context the term "target cell" is
related to a biological form of life comprising for example, a
microorganism, a virus, or an eukaryote cell.
[0106] In a preferred embodiment of the invention, the
microorganism is hosted inside a mammalian cell which is serving as
a reservoir for the infection.
[0107] In a preferred embodiment of the invention, the
microorganism is resistant to common chemotherapies such as
anti-biotics such as but not limited to methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus
aureus (VRSA), penicillin resistant Streptococcus, anti-biotic
resistant strains of Mycobacterium tuberculosis, penicillin
resistant Enterococcus, multi-drug resistant Pseudomonas
aeruginosa, clindamycin (or fluoroquinolone) resistant Clostridium
difficile (diarrheal disease) and multi-drug resistant Escherichia
coli.
[0108] In an embodiment of the invention, the first and a second
electrode are separated by a distance being at the most 1 m,
preferably being at the most 0.9 m, such as at most 0.8 m, 0.7 m,
0.6 m, 0.5 m, 0.4 m, 0.3 m, 0.2 m, 0.1 m, or at most 0.05 m, more
preferably being at the most 0.04 m, and even more preferably at
most 0.03 m such as at most 0.02 m, such as at most 0.01 cm.
[0109] For example the first and the second electrode may be
separated by a distance in the range of 0.01-1 m, such as in the
range of 0.01-0.05 m, 0.05-0.1 m, 0.1-0.2 m, 0.2-0.3 m, 0.3-0.4 m,
0.4-0.5 m, 0.5-0.6 m, 0.6-0.7 m, 0.7-0.8 m, 0.8-0.9 m or such as in
the range of 0.9-1.0 mm.
[0110] Typically, the first and the second electrode are separated
by a distance, which is at least 0.01 m such as at least 0.03 m or
0.05 m.
[0111] Normally, at least a part of the target cells in the
organism is positioned between the first and the second electrode.
For example, at least 1% of the target cells are positioned between
the first and the second electrode, such as at least 5, 10, 20, 30,
40, 50, 60, 70, 80, 90, 95, 97.5, 99, 99.5, 99.9 or 100% of the 1%
of target cells are destroyed by the electrical field imposed by
the first and the second electrode.
[0112] Preferably, in use, the target cells located in the organism
are positioned between at least the first and the second electrode
as part of the method. The first or the second electrode may be
attached directly to surface of the organism.
[0113] The first and/or the second electrode may have different
shapes or dimensions. For example, the first and/or the second
electrode may have a substantial form chosen from the group of a
sheet, a plate, a disc, a wire, a rod; or any combination
thereof.
[0114] In a preferred embodiment of the present invention, the
first and the second electrode may for example be a combination of
a point electrode and a sheet electrode.
[0115] In a preferred embodiment of the present invention the first
electrode and the second electrode are facing each other. For
example, they may be positioned at opposite sides of the organism
hosting the target cells creating a field that is optimal for
concentrating the electric field flux between the two
particles.
[0116] The first and second electrodes may take any appropriate
form as described in the art. The present invention offers the
advantage that the field needed for successful killing of target
cells is lower than in the prior art, so allowing a wider range of
electrode types and locations during treatment to be used.
[0117] In a preferred embodiment relating to treatment of disease
in a subject one or both of the first and second electrodes are
implanted within the body of the subject (i.e. the organism hosting
the target cells). One or both of the first and second electrodes
may be implanted in the vicinity of a group of target cells, such
as a tumour.
[0118] An implanted electrode may be positioned within, adjacent to
or around the group of target cells. In an embodiment one or both
of the first and second electrode may take the form of a probe that
may be manipulated by a clinician to apply an electric field
locally to the probe, so allowing the clinician to position the
electrode in order to apply the field to a chosen region. The probe
may be adaptable to be used during a surgical procedure that
exposes a deep-seated group of target cells, for example a tumour,
the probe then being applied to a chosen area by the clinician.
[0119] The first and second electrodes may both take the form of
probes, and both might be usable in this way. Alternatively the
second electrode may be adapted to remain in a fixed location while
the first is moved. In some embodiments the second electrode may
have an extended conducting surface, for example in contact with
soft tissue of the subject, and may in some embodiments be in
contact with the skin of the subject.
[0120] In some embodiments the electric field may be applied in a
single orientation between the first and second electrodes. In
further embodiments the electric field may be applied in further
orientations relative to the target cells or to an organism that
hosts the target cells, such as in the case of treatment of disease
in a subject. This may be achieved in some embodiments by changing
the disposition of one or more electrodes relative to each other or
to the subject, for example by moving either one or more electrodes
or moving the subject. In further embodiments more than two
electrodes may be provided in order to allow the field to be
applied in a number of different orientations.
[0121] The potential difference between the first and second
electrode may be in a range that causes an electric field flux
concentration between the said first and second particles to
generate molecular rearrangements associated with cellular
destruction as e.g. the pore forming events described
previously.
[0122] An electrode, for example a first electrode in a pair of
electrodes, may be formed from a variety of different materials.
Optionally the first electrode and a second electrode are formed
from the same material. Typically, the electrodes are formed from
metals or alloys. The first and the second electrode may for
example comprise a metal selected from the group comprising:
silver, gold, platinum, copper, carbon, iron, graphite, chromium,
nickel, cobalt, titanium, mercury or an alloy thereof.
[0123] It is also envisaged that an electrode may comprise a
conducting liquid and even essentially consist of a conducting
liquid. The conducting liquid may e.g. be mercury.
[0124] In preferred embodiments, a typical dimension of the
particles is less than 0.1 micrometre (.mu.m).
[0125] The core of embedded particles can be formed from a range of
materials capable of behaving as a dielectric such as but not
excluded to carbon, iron oxide (various forms), titanium dioxide,
cerium oxide or silicon dioxide.
[0126] In other preferred embodiments the particles may be in the
form of nanoparticles which may take the specific form of
nanotubes. Alternatively the particles may be substantially
spherical, for example cubic or octahedral, or they may be
nanorods.
[0127] The dimension or/and structure of electrodes typically
depends on the dimension of an optimal target area of a host
organism.
[0128] Advantageously the length and width of the electrodes are of
the same order of magnitude as the radius of a target area on the
target cell.
[0129] The electrodes can be formed by as little as a coating of a
few atom layers of conductive material.
[0130] In an embodiment a first and/or second electrode has a
thickness in the range of 0.001 .mu.m-2000 .mu.m, such as 0.001
.mu.m-1 .mu.m, 1 .mu.m-20 .mu.m, 20 .mu.m-200 and 200 .mu.m-2000
.mu.m.
[0131] In use, a liquid sample, in which cells are supported, is
exposed to an alternating electric field, which is provided by the
first and the second electrode. It is important that the
alternating electric field has a sufficient frequency and
sufficient amplitude and is applied for a sufficient duration of
time to cause necessary molecular destruction of the target cell so
as to cause selected killing of the target cell whereas non-target
cells remain relatively unharmed.
[0132] The term alternating electric field relates to electric
fields that change over time. The alternating electric field may
e.g. be the electric field that occurs from periodically shifting
the polarity of two electrodes between positive/negative and
negative/positive, that is connecting an AC source to the
electrodes. Also, the alternating electric field may comprise one
or more DC pulses.
[0133] Pulses may have a duration in the range 1 ns to 100 ms,
preferably in the range 10 ns to 1 ms. Pulses may generally have
durations and repetition rates, patterns and numbers of pulses as
known in the practice of electroporation, in particular for
irreversible electroporation, the use of particles in accordance
with the invention increasing the effect of each pulse or train of
pulses. Ultrashort pulses in the range 1-100 ns may be used as
known to result in apoptosis without irreversible electroporation
of the cell membrane.
[0134] In a preferred embodiment of the invention, the frequency of
the alternating electric field is at the least 10 kHz, preferably
being at least 50 kHz, and more preferably being at least 100
kHz.
[0135] In another preferred embodiment of the invention, the
frequency of the alternating electric field is at the least 100
kHz, preferably being at least 500 kHz, and more preferably being
at least 1000 kHz.
[0136] In another preferred embodiment of the invention, the
frequency of the alternating electric field is at the least 1 MHz,
preferably being at least 50 MHz, and more preferably being at
least 100 MHz.
[0137] In another preferred embodiment of the invention, the
frequency of the alternating electric field is at the least 100
MHz, preferably being at least 500 MHz, and more preferably being
at least 1 GHz.
[0138] In another preferred embodiment of the invention, the
frequency of the alternating electric field is at the least 1 GHz,
preferably being at least 500 GHz, and more preferably being at
least 1 THz.
[0139] For example, the frequency of the alternating electric field
may be at least 10 kHz, such as at least 30 KHz, 100 KHz, 300 KHz,
1 MHz, 10 MHz, 30 MHz, 100 MHz, 300 MHz, 1 GHz, 10 GHz, 30 GHz, 100
GHz, 300 GHz, such as at least 1000 GHz.
[0140] Preferably the frequency of the alternating electric field
is at most 500 GHz, such as at most 1000 GHz.
[0141] The amplitude of the alternating electric field, that is,
the maximum potential difference between the first and the second
electrode, is typically at most 100 KV, such as at most 30 KV, 10
KV, 1 KV, 300V, 100V, 30V, 10V such as at most 1 V.
[0142] The cellular destruction of the said target cells in the
host organism is strongly dependent on the design of, and the
distance between, the first and the second electrode, the electrode
structure and the materials of at least first and second particles
located within and in proximity of the extracellular side of the
cell membrane or cell wall (bacteria) and the potentials and
frequencies applied to the first and the second electrode.
[0143] In a highly preferred embodiment of the invention, the first
potential of the first electrode and the second potential of the
second electrode, and thus the alternating electric field between
the first and the second electrode, are modulated so as to yield a
cellular destruction of target cells in a target area of least 30%
of the target cells, such that at least 40% of the target cells,
preferably of at least 50% of the target cells, and more preferably
of at least 60% of the target cells, such as of at least 70%, 80%,
90%, 95%, 97.5%, 99%, 99.5% or 99.9% such as approximately of 100%
of the target cells are destroyed.
[0144] In another highly preferred embodiment of the invention, the
first potential of the first electrode and the second potential of
the second electrode, and thus the alternating electric field
between the first and the second electrode, are modulated so as to
yield a specific cellular destruction of target cells in a target
area and survival of at least 70% of the non-target cells, such
that at least 75% of the non-target cells, preferably of at least
80% of the non-target cells, and more preferably of at least 85% of
the non-target cells, such as of at least 90%, 95%, 97.5%, 99%,
99.5% or 99.9% A such as approximately of 100% of the non-target
cells survives the treatment.
[0145] In another highly preferred embodiment of the invention, the
first dielectric particle and the second dielectric particle can
mediate a field flux concentration compared to of the potential
supplied by the first and the second electrode, so that the field
flux concentration between said particles, existing in an area
between the two particles, is at the least a factor 1.1, preferably
being at least a factor 10, and more preferably being at least a
factor 100 compared to the surrounding field flux.
[0146] In another highly preferred embodiment of the invention, the
first dielectric particle and the second dielectric particle are
loaded in the extracellular fluid of the target organism e.g. for
humans that means the blood stream, in a time separated sequential
manner so that the first particle has time to accumulate in the
cytosol of the target cells before the second particle is loaded
for binding to the extracellular side of the cellular membrane or
cell wall (bacteria).
[0147] In an embodiment of the invention, the loading in the
extracellular fluid of the first and a second particles, are
separated in time by an interval being at the most 30 days,
preferably being at the most 20 days, such as at most 10 days, 9
days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1
day, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6
hours, 4 hours, or at most 2 hours, more preferably being at the
most 1 hour, and even more preferably at most 30 minute such as at
most 20 minute, such as at most 10 minutes.
[0148] Typically, the alternating electric field is provided by
modulating the polarity of the two electrodes.
[0149] The alternating electric field may have a substantial form
chosen from the group consisting of: rectangular, sinusoidal,
saw-tooth, asymmetrical triangular, symmetric triangular; or any
combination thereof.
[0150] Also, the alternating electric field, in the frequency
domain, may comprise at least a first and a second frequency
component.
[0151] In an embodiment of the invention, the duration of which the
organism is exposed to the alternating electric field is at most
3600 seconds, such as at most 3000, 2000, 1000, 500, 250, 100, 50,
40, 30, 20, 10, 5, 4, or 3 seconds, such as at most 1 second.
[0152] For example, the duration of which the organism is exposed
to the alternating electric field is in the range of 0.01-3600
seconds, such as in the range of 0.1-1, 1-5, 5-10, 10-25, 25-50,
50-100, 100-250, 250-500, 500-1000, or 1000-2000 seconds, such as
in the range of 2000-3600 seconds. In a preferred embodiment of the
invention, the duration of which the organism is exposed to the
alternating electric field is in the range of 5-100 seconds, such
as 6-90 seconds, 7-80 seconds, 8-70 seconds, 9-60 seconds and 10-50
seconds.
[0153] In a preferred embodiment of the invention, the organism is
exposed to the alternating electric field for at most 250 second,
preferably for at most 100 second such as for at most 30
seconds.
[0154] The particles are to be coated with a composition that will
enhance the binding to target cells over non-target cells in the
host organism. In preferred embodiments the coating comprises at
least one molecular component that increases the affinity of the
particle for a target cell membrane, organelle, lipid,
carbohydrate, a nucleic acid (such as chromosomal DNA and/or
plasmid DNA and/or any type of RNA), a protein (e,g, from the group
comprising enzymes, structural proteins, transport proteins, ion
channels, toxins, hormones, and receptors) or small molecule that
can be bound to the cellular membrane either in form of an agonist
and/or antagonist compared to its affinity for non-target
cells.
[0155] Another aspect of the invention relates to a device for
selectively killing of target cells in an organism, the device
comprising: a set of electrodes to be functionally associated with
the device, an electrical interface between the device and the
electrode arrangement for applying an alternating electric field
between the electrodes, an electronic circuit capable of providing
a fast switching high amplitude signal and a programmable unit.
[0156] The programmable unit ideally contains instructions,
preferably computer readable such as software, adapted to
facilitate controlling, monitoring, and/or manipulating of the
device prior to operation, under operation, and/or after
operation.
[0157] The programmable unit preferably comprises at least one
computer having one or more computer programs stored within data
storage means associated therewith, the computer system being
adapted to control a system employing first and second electrodes
arranged to apply an alternating electric field.
[0158] The programmable unit may in the context of the present
invention be chosen from the non-exhaustive group of: a general
purpose computer, a personal computer (PC), a programmable logic
control (PLC) unit, a soft programmable logic control (soft-PLC)
unit, a hard programmable logic control (hard-PLC) unit, an
industrial personal computer, or a dedicated microprocessor.
[0159] The present invention also relates to a computer program
product, such as one recorded on a data storage medium, being
adapted to enable a computer system, comprising at least one
computer having data storage means associated therewith to control,
monitor, and/or manipulate the device prior to operation, under
operation, and/or after operation.
[0160] Advantageously a computer readable medium has stored thereon
a set of routines for enabling a computer system comprising at
least one computer having data storage means associated therewith
to control, monitor, and/or manipulate the device prior to
operation, under operation, and/or after operation. The
programmable unit is ideally capable of: checking that electrodes
are functionally associated with the device, providing a voltage
protocol to the electrodes, and setting total time, amplitude and
frequency of the applied signal. Secondly being able to repeat the
voltage protocol in a number of series with a given time interval
between each exposure.
[0161] Optionally target cells are exposed whilst in a host
organism to an alternating electric field via the electrode
arrangement, said alternating electric field being provided by the
first and the second electrode and having a sufficient frequency
and a sufficient amplitude so as to cause the selective killing of
the target cells in the host organism, and optionally performing an
analysis on the exposed organism which part comprises an analysis
of the damage to target and non-target cells in the affected area
of the host organism.
[0162] The device may further comprise an electrical power supply
for supplying the high voltage needed for the first and second
electrodes to achieve the needed effect on the said first and
second particle.
[0163] In an embodiment of the invention, the programmable unit
comprising the software furthermore ensures that the device checks
that electrodes are functionally associated with the device.
[0164] According to a further aspect the invention provides a
composition comprising: a plurality of particles adapted for use in
a method and system for causing the death of a target cell by
primarily non-thermal means, for example by irreversible
electroporation, the particles being adapted to associate with the
target cells and adapted to cause an enhancement of an applied
electric or electromagnetic field in their vicinity.
[0165] In various embodiments the particles have properties as
described herein. The composition of the invention may comprise
further components to maintain the efficacy and useful life of the
composition, for example to maintain the particles in suspension,
to aid their administration to a subject, to aid their circulation
within the body fluid of a subject for example in the blood, or to
aid their absorption into the soft tissue of a subject.
[0166] According to a further aspect of the invention a system is
provided for the causing the death of a target cell comprising: at
least a first composition as described herein, comprising a
plurality of at least a first particle type, the particle being
adapted to associate with the target cell and adapted to cause an
enhancement of an applied electric or electromagnetic field in its
vicinity; and an apparatus for applying an electric field to one or
more target cells and to one or more particles associated with the
target cells, comprising at least a first and a second electrode
and a device for providing a first electric potential to the first
electrode and a second electric potential to the second electrode,
the device comprising a programmable unit adapted to control the
device in response to instructions associated with the programmable
unit.
[0167] A method as disclosed herein, carried out using the
composition and the apparatus.
[0168] In a preferred embodiment the system may additionally
comprise: a second composition comprising at least a second
particle type, the second particle type being adapted to associate
with the target cell and adapted to cause an enhancement of an
applied electric or electromagnetic field in its vicinity. The
second particle type may have any of the properties as described
herein.
[0169] In various embodiments the particles have properties as
described herein. In preferred embodiments at least one particle
type comprises a coating that makes it selective for the target
cell type in such a way that at least one particle will bind
selectively to one or more target molecules in the cellular
membrane.
[0170] According to a further aspect of the invention a system is
provided for treating a disease in a subject comprising the
composition, apparatus and method as listed above.
[0171] In a preferred embodiment the system may additionally
comprise a second composition, comprising at least a second
particle type, the second particle type being adapted to associate
with the target cell and adapted to cause an enhancement of an
applied electric or electromagnetic field in its vicinity. The
second particle type may have any of the properties as described
herein.
[0172] In a further embodiment the system additionally comprises:
means for administering the composition to the subject, the
administration being topical i.e. in the vicinity of the target
cells, systemic, or both, said means being for example (but not
being limited to): a syringe, a cannula, a catheter, an inhaler, an
implanted release device, a capsule or ingestible preparation or
means for administration using electroporation. A first means for
administration may be used with the first composition and a second
means for administration may be used with a second composition.
[0173] According to a further aspect of the invention there is
provided an apparatus and a method for a biological process, the
method comprising the steps of: providing a number of at least a
first particle type to cells in culture, the cells comprising
target cells, the particles adapted to associate selectively with
target cells; allowing the particles either to bind to target
molecules on the surface of the target cells or to be taken up
inside the target cells; and applying an electric field to the
cells in culture, so causing death of target cells by primarily
non-thermal means, for example by irreversible electroporation of
the cell membrane.
[0174] In a preferred embodiment the cell is contained within a
liquid media, the particles are mixed with the media and the field
is provided within the media by electrodes either within the media
or external to it. The electrodes may be insulated from the media.
There may be an air gap between the electrode surface and the
media.
[0175] Preferably the method comprises provision of first and
second particle types as described previously.
[0176] In a preferred embodiment the apparatus may comprise:
particles of at least a first particle type, and optionally
particles of a second particle type; a culture container adapted to
allow the provision of an electric field to the cells in culture,
and at least a first and a second electrode adapted to provide a
field to the culture container. In an alternative embodiment, the
apparatus may comprise a flow system having a flow cell through
which cells may flow in a liquid medium, the flow cell being
adapted to provide an electric field to the flowing cells, such
that target cells are destroyed selectively.
[0177] According to a further aspect of the invention there is
provided an apparatus and a method for an analytical process, the
method comprising the steps of: providing a plurality of a first
particle type to cells in a liquid sample; allowing the particles
either to bind to target molecules on the surface of the cells or
to be taken up inside the cells; and applying an electric field to
the liquid sample, so causing lysis of cells by irreversible
electroporation of the cell membrane.
[0178] This aspect relates to analytical or diagnostic processes in
which it is desired to release cell contents into a liquid sample,
for example in analysis of DNA, RNA, proteins or other constituents
of the cell. The method of the invention provides a ready means for
lysis of cells at low electric fields and hence with lower
electrode potentials than in the prior art. The particles may be
taken up into the cells or be associated with the external surface
of the cell membrane.
[0179] In a preferred embodiment the particles are selective for
target cells by binding selectively to target molecules on the
target cell membrane as described previously, so enabling selective
lysis of target cells while non-target cells remain intact. In a
preferred embodiment a first particle type is taken into the cell
and a second particle type is bound to the exterior of the cell
membrane, so reducing the threshold field for irreversible
electroporation.
[0180] In a preferred embodiment an apparatus comprises particles
adapted according to the invention; means to add particles to a
liquid sample and means to apply an electric field to the sample.
The apparatus may comprise a flow system having a flow cell within
which the liquid sample may be exposed to an electric field.
[0181] According to a further aspect the invention provides a
method for enhancing an applied electric field in a region of a
cell, comprising: providing at least one particle to the cell, the
particle having a high permittivity or conductive core and being
adapted to associate with the cell; and applying an electric field
to the cell.
[0182] Preferably the said at least one particle is adapted as
described herein. Preferably at least two particles are provided to
a cell, the particles being adapted to act together to enhance the
electric field in their vicinity. In preferred embodiments a first
particle adapted to enter the cell is provided, and a second
particle adapted to bind to a target molecule on the exterior of
the cell membrane is then provided, so as to provide an arrangement
of a first particle on the inside of the cell membrane and a second
particle external to the cell membrane, in proximity to the first,
so causing enhancement of the applied field across a portion of the
cell membrane.
[0183] The present invention will now be described, by way of
examples only, and with reference to the Figures in which:
BRIEF DESCRIPTION OF THE FIGURES
[0184] FIG. 1 shows a plot illustrating the effect of the said
method on bacterial spores. The effect is monitored as the release
of DNA due to electrolysis;
[0185] FIG. 2 shows a plot of colony forming unit (CFU) of two
populations of bacterial spores where one has been exposed to the
method and the other has not (control);
[0186] FIG. 3 shows the time course of pores formed in a cellular
membrane;
[0187] FIG. 3A shows a progression over three time points: (a), (b)
and (c); FIG. 3B shows a magnified version of time point (c);
[0188] FIG. 4 shows the field flux concentration from a microsphere
in a non-homogeneous electric field; FIG. 4A illustrates the
evolution of pores after a 1.25 V pulse; FIG. 4B illustrates the
evolution of pores after a 1.15 V pulse;
[0189] FIG. 5 shows an optimal arrangement of two particles for
creating the field flux concentration across the cellular membrane
that gives the highest effect;
[0190] FIG. 6a is a diagrammatical view of a single particle,
having a high permittivity, and how this concentrates an electric
field adjacent to cell membrane;
[0191] FIG. 6b is a diagrammatical view of a particle bound to
target molecule at the target cell surface by a ligand forming part
of a coating;
[0192] FIG. 6c is a diagrammatical view of a particle spaced from
the ligand by a linker;
[0193] FIG. 6d is a diagrammatical view of multiple particles with
high permittivity or comprising a conductive core, adjacent to a
cell membrane I;
[0194] FIG. 6e is a diagrammatical view of multiple particles with
low permittivity adjacent to a cell membrane--so concentrating
field lines in the medium between them;
[0195] FIG. 7a is a diagrammatical view of a single particle inside
a cell;
[0196] FIG. 7b is a diagrammatical view of a single particle inside
a cell away from the membrane, concentrating the field in a region
adjacent the cell membrane;
[0197] FIG. 7c is a diagrammatical view of a particle inside a cell
and adjacent (or in contact with) the cell membrane, concentrating
the field effectively at the cell membrane;
[0198] FIG. 7d is a diagrammatical view of a particle bound to a
target molecule on the inside of the cell membrane;
[0199] FIG. 7e is a diagrammatical view showing how multiple
particles, associated with the exterior of cell membrane, are taken
into the cell by endocytosis;
[0200] FIG. 7f shows an embodiment where particles are adapted to
bind to the exterior of the cell membrane to provide a population
of cells in position for endocytosis;
[0201] FIG. 7g shows multiple particles inside the cell adapted to
bind to a target molecule on the exterior of the nuclear
membrane;
[0202] FIG. 7h shows how enhancement of field lines, adjacent the
nuclear membrane leads to poration of the nuclear membrane and cell
death;
[0203] FIG. 8a is a diagrammatical view and shows how a first
particle inside and a second particle outside give additional field
enhancement;
[0204] FIG. 8b depicts the situation where a first particle, inside
the cell, is not adapted to bind to a target molecule and a second
particle, outside the cell, is bound to a target molecule;
[0205] FIG. 8c shows a first particle, inside the cell is adapted
to bind to a target molecule or target molecular region on the
inside of the cell membrane;
[0206] FIG. 8d shows a number of first particles are in various
positions within the cell. The field is enhanced primarily by the
first particle that is closest to the second particle outside the
cell;
[0207] FIG. 8e depicts how a first particle, inside the cell, is
adapted to bind to a first target molecule on the cell membrane;
and how a second particle, outside the cell, is adapted to bind to
a second target molecule;
[0208] FIG. 8f is a similar view to FIG. 8e, but depicts first and
second target molecules closer together so field enhancement is
greater than that shown in FIG. 8e;
[0209] FIG. 9a shows two particles having a high permittivity or
conductive, (such as metal particles), bind to the exterior of a
cell and produce an enhanced field in the vicinity of the cell
membrane;
[0210] FIG. 9b shows how, in some cell morphologies, two particles
on the exterior of the cell are located close together on opposite
sides of a region of the cell in the direction of a component of
the field;
[0211] FIG. 9c shows how two particles bound to the exterior of the
nuclear membrane, that are closely located, produce field
enhancement in a region of the nucleus sufficient to lead to cell
death, for example by electroporating the nuclear membrane;
[0212] FIG. 10a shows diagrammatically the significance of field
orientation relative to orientation of pairs of particles and how a
first field direction E1, for a first pair of particles, produces a
greater effect of field enhancement on the cell membrane than a
second pair of particles, for a second field orientation E2,
produces a greater effect;
[0213] FIG. 10b is a diagrammatical representation of random
provision of particles to a number of target cells in a region of
tissue and shows for a first field direction E1 certain particle
pairs or arrangements produce a greater effect than others;
[0214] FIG. 11a shows in diagrammatical form treatment of target
cells, within a region of tissue within the body, for example a
tumour, using planar electrodes external to the body;
[0215] FIG. 11b shows in diagrammatical form treatment of target
cells, with the use of electrodes, implanted in the body, and
illustrates the effect of different field orientations on different
orientation of particle arrangements;
[0216] FIG. 11c shows in diagrammatical form treatment of target
cells at a region within the body;
[0217] FIG. 11d is a diagrammatical cross section, through a body
during treatment, of target cells in a region of the body;
[0218] FIG. 11e is a diagrammatical cross section through a body
during treatment of target cells in a region of the body, showing a
movable electrode, for example a hand-held probe, which may be
separated from the skin by an insulating layer 116;
[0219] FIG. 12a is a diagrammatical overview, (showing a horizontal
cross section through the body of a patient), of an apparatus for
moving electrodes around the patient's body during treatment;
[0220] FIG. 12b is a side elevation of the apparatus in FIG. 12a
and shows a means for displacing electrodes along the length of a
patient's body as well as around the body so as to perform
treatment in accordance with the method;
[0221] FIG. 12c is a diagrammatical overview of an apparatus and
shows multiple electrodes around a region of the body, adaptable to
the contours of the body, for example by way of a flexible support
structure that is adapted to deform to conform to a patient's
body;
[0222] FIG. 12d is a diagrammatical overview of a portion of the
apparatus in FIG. 12c, that supports the multiple electrodes,
removed from the body;
[0223] FIG. 13a is a flow diagram showing steps in the method, with
a first particle type adapted to enter the target cells and a
second particle type adapted to bind to the surface of the target
cells;
[0224] FIG. 13b is a flow diagram depicting an example of the
method where a single particle type is used, the particle being
adapted to enter the target cell and reside at the surface of the
cell before endocytosis;
[0225] FIG. 14 shows a diagrammatical view of a system for treating
target cells in blood; particles are administered into the body and
allowed to associate with the target cells and blood is passed
through a flow cell where they are exposed to an electric field
before returning blood to the body;
[0226] FIG. 15a shows an alternative embodiment, with a cell
culture of attached cells and particles added to the culture, and
in which a field is applied across the cell layer, so that target
cells are affected and non-target cells are unaffected;
[0227] FIG. 15b is a diagrammatical view of a cell culture of cells
in suspension in a bioreactor, and shows particles mixed with cells
in a liquid medium, flowing through a flow cell in which cells are
exposed to a field before being returned to the bioreactor; and
[0228] FIG. 16 is a diagrammatical view of an alternative
embodiment of the invention adapted for sample processing, for
example for use in diagnostics.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0229] In the following description the terms particle,
microparticle, microsized particle and nanoparticle are used
interchangeably.
[0230] Appropriate dimensions and morphology of particles are
described by way of example only.
[0231] Example 1 The effects of varying bead concentration (2, 0.5
and 0 .mu.l) on spore electrolysis efficiency.
[0232] One hundred mg of Biobit Bacillus thuringiensis subsp.
kurstaki containing 3.2.times.10.sup.9 spores/g (Valent BioSciences
Corp, Libertyville, USA) was resuspended in 1 ml of demineralized
water and centrifuged for 90 sec. at 12000 rpm. This procedure was
repeated 4 times. The supernatant was discarded. The final solution
contains approximately 3.2.times.10.sup.8 spores. This solution was
diluted to a final concentration of 3.2.times.10.sup.5 spores/ml.
and subsequently 12 .mu.l spore sample was used for electrolysis
and PCR.
[0233] Voltage, time and frequency were kept constant (at 10 V, 30
sec and 100 KHz, respectively) variations was made in the
concentration of iron oxide beads that was added respectively 2 and
0.5 .mu.l 1 .mu.M iron oxide silica coated beads (Merck). FIG. 2
shows the results of this experiment and as apparent, the high
concentration of bead of 2 .mu.l to the 12 .mu.l spore sample
showed a decrease in C.sub.T (threshold cycle) compared to standard
lysis without beads, thus demonstrating release of amplifiable DNA
from the spores. Lowering the bead concentration to 0.5 .mu.l
decreased the effect considerably.
[0234] It other experiments it was shown that addition of more than
0.5 .mu.l silica coated iron oxide beads directly to a 20 .mu.l PCR
reaction gave more than 50% in the PCR yield. The above experiments
was carried out with a final bead addition of respectively
1/12.times.2=0.17 .mu.l bead to a 20 .mu.l PCR reaction for the
high concentration of beads and a 1/12.times.0.5=0.04 .mu.l bead to
a 20 .mu.l PCR reaction for the low concentration of beads.
Therefore, it should be expected that less PCR inhibition is
experienced in the low concentration of bead than in the high
concentration. The results are opposite that, thus the effect of
the increased beads is exceeding the PCR inhibition.
[0235] It is believed that the effect of the increased bead
concentration is to increase the likelihood of forming two particle
arrangements on opposing sides of the spore leading to an increased
field flux between the particles with concomitant molecular
disarrangements in the coating of the spores and cell wall leading
to killing of the spore.
[0236] FIG. 2 shows the spores grown on agar plates subsequent to
the lysis and spores incubated with beads and grown show no effect
whereas the number of colony forming units from the sample
undergone the electrolysis in presence of beads shows a marked
reduction (95% less CFU's compared to control).
[0237] FIG. 3 illustrates the field flux from a sphere formed
particle in a non-homogenous electrical field. The field flux
increase causes fluorescent ions to move in the most concentrated
region of the flux and electromotion overcomes diffusion and a
concentrated stream of fluorescent ions is radiating out from the
sphere. The same principles are governing smaller particles and can
be used to generate a electric flux concentration between two
particles with associated molecular rearrangement for any charged
molecule within the concentrated flux region.
[0238] A) The sequential images of microparticle concentration
evolution for a cation exchange granule with a step change in the
field to 100 V/cm, using co-ion fluorescein-dye tagged microspheres
in 10 mM Tris buffer (pH 8) at very low density (5.0.times.10E6
particles/mL). The images are taken at 0 (a), 1.35 (b), and 2.44 s
(c).
[0239] B) Ion-concentrated ejection cone. The yellow profile is the
theoretical prediction of:
R/a=( 3.left brkt-top.0.75)(1-(a-r)3)-0.5,
[0240] based on flux balance within the two bounding pole field
lines.
[0241] R: radius of jet ejecting from the sphere
[0242] r: radius of sphere
[0243] a: area of sphere
[0244] .left brkt-top.: field in volt per cm
[0245] This principle is also governing smaller particles and can
be used to create a electric flux concentration between two
particles with subsequent molecular disarrangement of charged
molecules between the two particles. In the case of a cell membrane
intercalated between the two particles it will lead to pore forming
events in the cellular membrane. (Biomicrofluidics 2008, 2,
014102)
[0246] FIG. 4 illustrates one important outcome of pore forming
events in a cellular membrane caused by a single electrical pulse.
In general high amplitude electrical pulses can cause formation of
many small pores that reverses over time and the membrane re-seals.
However, a smaller pulse creates a smaller amount of pores of and
the newly formed membrane pores can undergo a process called
"coarsening" where the resealing eventually does not occur and the
pores forms a single very large pore that eventually leads to cell
death. This information is important because it illustrates that
lower voltage can be used to cause more damage than high voltage,
so the optimal effect is not necessarily achieved by the highest
amplitude and highest frequency.
[0247] Exposing a cell to a lower voltage gives a relative higher
number of irreversible pore forming events compared to exposure
with a higher field. The higher field generates many pores but with
smaller diameter and they are reversible. The figure illustrates
pore radii during a 10 .mu.s pulse and the postshock evolution of
pores. The gray scale represents the pore radii distribution (i.e.,
the number of pores with radii between r and r+dr). Solid lines
show the 10, 20, . . . , 100th percentiles of the maximum pore
radius, illustrating the evolution of the pore radii in time. (A)
Evolution of pores after a 1.25 V pulse, which created 18,025
pores. After the pulse, all pores shrink to rm (the minimum-energy
radius of (B) Evolution of pores after a 1.15 V pulse, which
created a smaller number of pores, 2772. After the pulse, all pores
shrink to rm except the largest pore, which grows to a stable
radius of 2.23 .mu.m. (Inset) The pulse and the first 300 .mu.s
after the pulse shown on an expanded vertical scale. (Redrawn from
Biophys J. 2004 May; 86(5): 2813-2826).
[0248] FIG. 5 illustrates the optimal arrangement between two
particles where one is located in the cytoplasma of a mammalian
cell and one found on the extracellular side. A similar arrangement
is optimal for a spore but where the particles are found on
opposing sides of the spore with the same result as the effect
described for the mammalian cell.
[0249] Plot of results from theoretical model of electrical field
flux lines around two gold particles in salt water that has
chemical resemblance to human blood. The setup is made by two
electrodes with a voltage difference of 300 V separated from the
solution with a gap of air. The particles are in the solution. The
plot illustrates the field flux concentration effect that can be
achieved by positioning two gold particles in proximity of each
other. Other metals give similar results. In the model the
particles were 1 um diameter gold microspheres. Substantially
similar results were achieved for 60 nm and 40 nm diameter gold
microspheres.
[0250] FIG. 6a shows an arrangement resulting from an embodiment of
the method of the invention in which a cell 10 having a membrane
12, cytosol 14 and nucleus 16 has a particle 20 that is either
conductive or has a high permittivity is associated with the
extracellular side of the cell membrane. When an electric field, in
a direction indicated by the symbol E and shown by means of field
lines 22, is applied to the cell and the surrounding medium the
field is enhanced by the particle in a region 24 incident on the
cell membrane (note the appearance of field lines in the
extracellular medium is symbolic and not intended to be accurate
representation). With appropriate choice of field strength the
enhanced field in the region 24 is sufficient to induce
irreversible pore-forming events in the membrane in this
region.
[0251] FIG. 6b shows a preferred version of the embodiment in which
the particle 20 is bound to a target molecule 18 on the cell
surface by means of a ligand 28 provided as part of a coating on
the particle. The ligand 28 may be chosen to be specific for target
molecules 18 that are present only or preferentially on target
cells, so allowing targeted binding of particles 20 to target cells
and no, or lesser, binding to non-target cells. The ligand may in
some embodiments be adapted to bind to a specific region of a
target molecule, for example an extracellular region of a
transmembrane protein.
[0252] More than one ligand type may be present on a particle, and
the ligands may be targeted to the same or different target
molecules or molecular regions. The target molecule may be any
molecule, such as a protein, protein complex, or sugar. The target
molecular region may for example be a region of the protein or one
protein in a complex. The embodiment in FIG. 6b therefore provides
novel targeted destruction of target cells in a mixed cell
population by means of irreversible electroporation of the cell
membrane mediated by high permittivity or conductive particles.
[0253] In a further preferred version of the embodiment shown in
FIG. 6c a ligand 28 may be attached to the surface of the particle
by a linker molecule 30, which gives the advantage of controlling
the mean distance of the particle from the lipid bilayer of the
cell membrane, so controlling and optimising the effect of field
enhancement on pore formation in the membrane.
[0254] Particles in this embodiment are preferably of higher
permittivity than the mean permittivity of the environment in the
region surrounding them, that region comprising one or more of the
extracellular fluid; extracellular matrix; cell membranes of other
cells; cell surface molecule such as membrane proteins and sugars.
The permittivity of the surrounding medium is therefore a composite
permittivity derived from the presence and permittivities of the
various components, and therefore may tale a range of values up to
that of physiological saline or blood.
[0255] Particles may have characteristics as disclosed herein, for
example having a dielectric or conductive core, for example a metal
core, such as gold, and a coating comprising at least one ligand
targeted for a target molecule on the cell membrane. Such ligands
may comprise antibodies, aptamers, protein binding partners and
peptides, as known in the art.
[0256] FIG. 6d shows an arrangement of particles in an embodiment
of the invention, comprising a plurality of high permittivity
particles associated with a cell membrane 12. A number of regions
24 of enhanced field strength are formed, which together may serve
to destabilise the membrane 12 so as to reduce the threshold
applied field for irreversible pore formation. FIG. 6e shows an
arrangement of particles in a further embodiment in which a number
of particles of permittivity lower than that of the surrounding
environment are associated with, for example bound to, the cell
membrane.
[0257] In this case the field is enhanced in a region 24 located
approximately between two particles, but in most embodiments the
effect is less than that of field enhancement by one or more high
dielectric particles and ideally requires a significant number of
particles to be associated with the cell and positioned close to
one another in order that the field be enhanced significantly.
Effective reduction of the threshold for irreversible
electroporation requires more particles in general than in the case
of high permittivity particles acting together. While this
embodiment is functional, the inventive use of higher permittivity
or conducting particles in alternative embodiments is therefore an
improvement over the use of insulating or lower permittivity
particles, or carbon nanotubes, which have a relatively low
permittivity unless oriented precisely with respect to the cell
membrane, which in general will not be the case.
[0258] FIG. 7a shows an arrangement resulting from use of a further
embodiment of the method of the invention, which comprises
providing particles adapted to enter a target cell, allowing at
least one particle to enter, and then exposing the cell and at
least one particle to an electric field, so causing enhancement of
the field in the vicinity of the particle resulting in cell death
through primarily non-thermal means, in preferred embodiments
through irreversible electroporation of the cell membrane. A cell
as described before now has a particle 40 provided within it, which
may be a dielectric particle, in some embodiments of high
permittivity as described above, in alternative embodiments may be
conductive, for example comprising a metal, for example gold. As is
shown in FIG. 7b a particle within the cell close to the membrane
produces a region 24 of enhanced field in the membrane in the
vicinity of the particle, so reducing the threshold for
irreversible electroporation. The effect is greater for particles
close to the membrane than for particles further away in the
cytosol. Cells having such a particle within them may therefore be
killed while neighbouring cells without particles are undamaged. In
preferred embodiments the particles 40 are either adapted to
associate with the cell membrane 12 or are present in sufficient
quantity within the cytosol that at least one particle will be
located close to the membrane without binding to it.
[0259] FIG. 7d shows a preferred embodiment in which the internal
particle comprises a coating having at least one ligand adapted to
bind to a target molecule on the inside of the cell membrane. The
particle and ligand may be as described above, the particle being
further adapted to enter the cell through the cell membrane. Such
entry may be by means of endocytosis as known in the prior art and
described in the summary of the invention and below.
[0260] The particles may be targeted to target cells in a similar
manner as described above. For example, in FIG. 7d the target
molecule at the interior side of the membrane may be specifically
or preferentially expressed in a target cell type, so that location
of particles at the inside of the cell membrane occurs specifically
or preferentially in target cells rather than in non-target cells.
In a further embodiment as shown in FIG. 7e the particles 40 may be
targeted to associate readily with the exterior of a cell, so
providing an increased surface coverage of particles that are then
able to undergo endocytosis.
[0261] FIG. 7f shows how this might be done preferentially in
target over non-target cells. Particles 40 adapted for preferential
endocytosis by target cells have a coating comprising at least one
ligand 42 adapted to bind to a target molecule 44 which may be
preferentially expressed on target cells. This then leads to a
population of particles 40 bound to the surface. At least one
particle then preferably undergoes endocytosis as shown in FIG. 7f,
with the cell membrane forming an invagination 46 that results in
the entry of the particle and reformation of the membrane 12. FIG.
7f shows a particle adapted for endocytosis is bound to a target
molecule at the exterior of the cell membrane. After a time the
particle is taken into the cell by endocytosis and the membrane
re-forms. The target molecule is shown left in the membrane but it
may be degraded by the cellular machinery once inside the cell.
[0262] In another embodiment as shown in FIG. 7g the method of the
invention is applied to cause cell death by means of destructive
effects on the nuclear membrane resulting from enhancement of an
applied electric field by particles bound to the nuclear membrane.
FIG. 7g shows particles 40 associated with target molecules 48 on
the nuclear membrane by means of ligands 42 provides as part of a
coating on the particles. FIG. 7h shows a local field enhancement
in region 24 of the nuclear membrane caused by particle 40 bound to
it.
[0263] FIGS. 8a and 8b show a further arrangement of particles and
target cell arising from a further embodiment of the method
according to the invention. As shown in FIG. 5, it has been found
that two particles may act co-operatively to create a significantly
greater enhancement of an applied field than a single particle
alone. FIG. 8a shows a cell 10 having a first particle 40 within
the cytosol and a second particle 20 bound to the exterior of the
cell membrane 12. The two particles may be dielectric particles and
may have a high permittivity or have a conductive core, for example
comprising a metal such as gold or a metal oxide, such as
Fe.sub.3O.sub.4. The two particles cause a great enhancement of the
electric field in their vicinity, and especially between them, and
in any region of the cell membrane adjacent to them, shown here as
a region 24 between the particles.
[0264] FIG. 8b shows the case in which the first particle 40 is
adapted to enter the cell and is present within the cytosol, but is
not adapted to bind specifically within the cell, while the second
particle 20 is bound to a target molecule 18 by means of a ligand
28 as described before. Enhancement of the field is shown in FIGS.
8a, 8b by means of the increased concentration of field lines in
the vicinity of the particles compared with in the environment
surrounding them.
[0265] In preferred embodiments of the invention, first particles
40 are adapted to enter the cell, and second particles 20 are
adapted to bind to the exterior of the cell. One or both of the
first and the second particles may be targeted to target cells. The
first particles 40 may be adapted to enter target cells
preferentially as described above, and may be adapted to bind to a
specific location or range of locations with the target cell.
Alternatively, the first particles may be simply adapted to enter
both target and non-target cells. The second particles are
preferably adapted to bind to target molecules on the exterior of
the target cells as described above.
[0266] The situation resulting from the method of the invention in
this embodiment is that target cells have a particle arrangement
associated with them, comprising at least one first particle within
the cell and at least one second particle bound to the exterior of
the cell membrane, as shown in FIGS. 8a and 8b. The adaptations of
one or both of the particles to associate with or to enter target
cells selectively are such that non-target cells do not have this
particle arrangement.
[0267] FIG. 8c shows an arrangement of particles arising from a
further embodiment of the invention, in which the first particle 40
in the interior of the target cell is bound to a target molecule at
a target molecular region 58 located at the interior of the cell
membrane, by means of a ligand 68 provided as part of a coating on
the particle, while a second particle 20 is bound to a target
molecular region 18 at the extracellular side of the membrane. In
this case the target molecular region on the inside is shown as an
intracellular portion of a transmembrane protein, with the second
particle adapted to bind to extracellular portion of that
protein.
[0268] In a preferred embodiment the regions 18 and 58 are regions
of the same target molecule, such as a transmembrane protein, for
example a receptor or ion channel. This arrangement is advantageous
as the first and the second particle are both bound in proximity to
the cell membrane and to each other and highly effective field
enhancement results.
[0269] FIG. 8d shows an arrangement of particles arising from a
further embodiment of the invention, in which a number of first
particles 40 are located within the cytosol, either at random or
bound to locations with the cytosol, and a second particle 20 is
bound to a target molecule 18 on the cell membrane. Effective field
enhancement occurs between the second particle and the first
particle that is nearest to it, showing that precise location of
the first particles is not necessary provided sufficient first
particles are present within the cell that when a second particle
binds it will be in proximity to at least one first particle.
[0270] FIG. 8e shows an arrangement of particles arising from a
further embodiment of the invention, in which the first and second
particles are bound to target molecular regions 58, 18 on different
target molecules, shown as having non-binding regions 57, 17
respectively. As shown in FIG. 8f, field enhancement is most
effective when the target molecules are close together in the
membrane. It is within the scope of the invention to select the
ligands 68, 28 provided as part of a coating on the first and
second particles to achieve this situation. Certain target
molecules are known to be mobile within the membrane, and it is
within the scope of the invention that the application of a field
may induce forces on the first and/or second particle that may
cause movement of the particle relative to the target cell or cell
membrane, movement or distortion of the cell membrane, or movement
of cell components or target molecules to which the particles are
bound. Such movement may lead to increased effectiveness of causing
cell death according to the invention, for example by the first and
second particles moving to become closer together in response to
the applied field, so acting to increase the field enhancement in
their vicinity.
[0271] FIG. 8f depicts how field enhancement is optimised if target
molecules are chosen so that they are close together in the cell,
or are expressed in large numbers, thereby improving the chance
that the first and the second particles bind close together. The
first and the second target molecules may be mobile within the cell
membrane and may associate together.
[0272] FIG. 9a shows an arrangement of particles arising from a
further embodiment of the invention, in which at least two
particles 20a, 20b are bound to the exterior of a target cell.
Enhancement of the applied field occurs when the particles are
located in proximity to one another. Regions of enhanced field
intersect the cell membrane according to the position of the two
particles, the shape of the cell, and the orientation of the
particles with respect to the applied field. While only two
particles are shown it will be appreciated that in this embodiment,
the method advantageously provides sufficient particles to the
target cell that there will be on average at least one such
arrangement of particles associated with the cell.
[0273] FIG. 9b shows a particle arrangement that may arise at a
region of a target cell with higher aspect ratio, for example a
process or outgrowth, for example as in neuronal cells. FIG. 9c
shows an arrangement of particles arising from a further embodiment
of the invention, in which the particles are adapted to enter the
cell and to associate with the nuclear membrane, so causing field
enhancement across a region of the nucleus and nuclear membrane,
resulting in cell death through non-thermal means, in some
embodiments through disruption of the nuclear membrane.
[0274] In a further embodiment of the invention, the orientation of
the electric field may be controlled or varied with respect to the
target cells, a group of target cells such as a tumour, or the body
of a subject hosting the target cells. FIG. 10a shows an
arrangement of particles arising from an embodiment of the
invention, and illustrates that the orientation of the field with
respect to one or more target cells may in some embodiments affect
the degree of field enhancement. In FIG. 10a a first particle
arrangement 20a, 40a has the axis of the particle arrangement--the
line joining the two centres of the particles--aligned with the
field direction E1. This is expected to lead to a higher degree of
field enhancement in the region of the membrane near or between the
particles. A second particle arrangement 20b, 40b has its axis
perpendicular to the E1, which is expected to lead to lesser field
enhancement in the region of the membrane near or between the
particles. Field in direction E2 reverses this situation. It is
clear that for a random orientation of particles arrangements
around a target cell, the best chance of achieving high field
enhancement is through using multiple orientations of the applied
field.
[0275] FIG. 10b shows an arrangement of particles arising from a
further embodiment of the invention, the particles being provided
within a region of tissue 100 comprising a number of target cells
10. Particles targeted to these cells will in general be
distributed randomly and so within the tissue particle arrangements
80, 82, 84 resulting from the invention will be oriented randomly.
For a first field direction E1 certain particle pairs or
arrangements (80) will produce a greater effect than others (82).
For a second field direction E2 the reverse is true. Some particle
arrangements (84) will have an intermediate level of effect for
both field directions. Therefore varying the orientation of the
applied field with respect to a group of target cells, or the
tissue or body of a subject in the case of treatment of disease, is
advantageous and may be achieved by the method and apparatus of the
invention.
[0276] In accordance with the invention, the electric field may be
applied by electrodes disposed in a variety of ways around the
body. In contrast with prior art methods, the field enhancement of
the invention allows a greater variety of electrode placement to be
used. Electrodes may be located external to a region comprising the
target cells, such as a container, tissue, or body of a subject. It
is a particular advantage of the invention that the applied field
can be lower than in the prior art, and some embodiments use
electrodes placed externally to the body of a subject. For example,
FIG. 11a shows treatment of target cells within a region of tissue
100, for example a tumour within the body 102 of a subject, the
field being applied by a first electrode 110 and a second electrode
112, connected to a source of potential, such as a device according
to the invention by means of connections 114. In this embodiment
the electrodes are planar electrodes external to the body. The
electrodes are shown separated from the body by a gap 108. In an
alternative embodiment one or both electrodes may be in contact
with the skin 106. The electrodes may be flexible or adapted to
conform to the contours of the body, or may be shaped to maintain a
given separation from the body.
[0277] FIG. 11b shows treatment of target cells within a region of
tissue 100 using implanted electrodes 110 and 112, each forming
part of implanted probes 116. The probe 116 has one or more
conductive regions adapted to provide a region of electric field
within the body and one or more insulated regions 118. Probes
suitable for use in this mode are known in the prior art.
[0278] FIG. 11c shows a method and an apparatus for treatment of a
disease within a subject by means of destruction of target cells at
a region within the body. Particles are administered systemically
to a subject 150 for example by means of injection or infusion into
the blood stream. According to the method of the invention, a
composition 120 comprising a first particle type is administered,
the particles travel to the region 100 through the circulatory
system 124, and associate with the target cells. After a chosen
time interval t1, chosen to allow particles to reach their desired
locations and arrangement(s) with respect to the target cells, a
field is applied by electrodes 110 and 112, potentials on the
electrodes being provided and controlled by a device 130,
optionally under the control of a programmable unit 132. The
electrodes are shown as being external electrodes distanced from
the body, though any form or location of electrodes as disclosed
herein may be used. The field may be re-applied at intervals.
[0279] In a further embodiment, the first composition 120 is
administered as above, a chosen time interval t1 is allowed to
elapse, and then a second composition 122 comprising a second
particle type is administered. A second time interval t2 is then
allowed to elapse, and the field is applied as described above.
[0280] In preferred embodiments the first particle type is adapted
to enter cells (either all cells, or target cells selectively) as
described above and the second particle type (where used) is
adapted to bind selectively to the exterior of target cells but not
to non-target cells. In an alternative embodiment the first and
second composition both comprise the same particle type, adapted
either to enter the target cell selectively or to bind selectively
to the exterior of the target cell. In some embodiments the
particles are adapted to remain in position within or associated
with the target cells for a period of time within which multiple
applications of the electric field may be made.
[0281] FIG. 11d shows a cross section through a body 102 during
treatment of target cells in a region 100 of the body. Electrodes
110 and 112 in position A apply a field in direction E1. One or
both electrodes may be in contact with the body as shown for
electrode 110, or separated from it as shown for 112. Electrode 112
is shown also in an alternative position B that provides a field in
a second direction E2. One or both electrodes may be made movable,
for example by a clinician or automatically, moved by a motor means
(not shown) under the control of the device 130 or programmable
unit 132.
[0282] FIG. 11e shows a cross section through a body 102 during
treatment of target cells in a region 100 of the body. Electrodes
110 and 112 apply a field. Electrode 112 is movable in x and y
directions parallel to the skin surface, and may be for example a
hand-held probe, and may be separated from the skin by an air gap
or in contact with it, separated by an insulating layer 116.
[0283] FIG. 12a shows an embodiment of an apparatus usable with the
method of the invention and as part of the apparatus of the
invention, for the treatment of disease in a subject, for example
by destruction of target cells within a region 100 of a body 102.
Electrodes 110 and 112 are mounted in a structure 180 that supports
them separated from and close to the body, and is adapted to cause
them to rotate around the body as shown by the arrows, so moving
them through a range of orientations with respect to the body. This
allows the applied field between them to be moved through a range
of orientations with respect to the region 100 and the target cells
and particle arrangements, within it. For example, the structure
180 might be moved by a motor means 182 coupled to it between a
first position A and a second position B, so moving the direction
of the applied field through an angle, say 90 degrees. The angle of
rotation might be smaller or greater than 90 degrees, and may be
chosen according to the location and nature of the region 100, for
example it may be up to 180 degrees in one sense or both senses.
More than one pair of electrodes may be provided within the
structure 180, the pair that is providing the field being selected
by a switch means, in order further to control the field direction
at any time or point in the treatment process. The structure 180
might rotate about the body 102 in a horizontal plane, e.g. while
the subject is standing, or in a vertical plane, e.g. when the
subject is lying flat.
[0284] FIG. 12b shows a further embodiment of an apparatus usable
with the method of the invention and as part of the apparatus of
the invention, for the treatment of disease in a subject. Here two
or more electrodes 162 (a plurality are shown) are provided within
a structure 180 that is now adapted to move in a direction along
the body of a subject, shown here as lying flat on a treatment
surface or table 184, driven for example by motor means 182. At
least one pair of electrodes within the structure 180 are have
potentials applied to them so as to generate a field between them
as described further for FIG. 12c. The structure 180 may then move
along the body of the subject in order to subject a range of target
cells within the body to the field. It will be apparent that the
structure 180 may also rotate around the body as shown in FIG. 12a
to produce a combined motion and a combined range of electric field
directions. Motion in one or both dimensions may be controlled by a
control means provided as part of the device of the invention or
the programmable unit. In this way target cells may be exposed to a
field in both of two dimensions. A further structure 180 (not
shown) may be provided separated laterally from and parallel to the
first in order to provide control a component of field orientated
in the third dimension. Such an apparatus is applicable in cases
where target cells are not all localised within a region 100,
allowing treatment of large parts of the body without the use of
large electrodes.
[0285] FIG. 12c shows a further embodiment of an apparatus usable
with the method of the invention and as part of the apparatus of
the invention, for the treatment of disease in a subject, for
example by destruction of target cells within a region 100 of a
body 102. Here two or more electrodes are provided as part of an
electrode structure 160, which is preferably flexible and may be
shaped to, placed around or attached to the body or a body part,
for example in the manner of a belt or armband, the electrodes
themselves preferably being separated by a thin layer of insulator
from the skin 106, but in some embodiments at least one electrode
being in contact with it. The electrodes are mounted on a flexible
substrate 164 and are connected by leads 166 by means of switch
unit 170 to the device 130 and programmable unit 132. The switch
unit in use serves to connect pairs of electrode 110, 112 to the
device so as to provide the field. As different pairs around the
structure 160 are connected, so the field orientation is changed.
The switch unit may be controlled by the device or the programmable
unit to provide a desired pattern of field orientations during a
treatment, shown as for example E1 when electrodes 110a and 112a
are connected by the switch in position A, and E2 when electrodes
110b and 112b are connected by the switch in position B.
[0286] FIG. 12d shows the electrode structure 160 laying flat,
optionally provided with a fastening mechanism 168 to fasten the
structure to or around the body for example in a semi-rigid
configuration or in the manner of a flexible belt.
[0287] FIG. 13a shows a flow diagram for a method according to the
invention, for the embodiment where a first particle type is
adapted to enter the target cells and a second particle type is
adapted to bind to the surface of the target cells. It is envisaged
that multiple applications of the electric field may be made
following provision of the particles.
[0288] FIG. 13b shows a flow diagram for a method according to the
invention for the case where only a single particle type is used,
the particle being adapted to enter the target cell selectively,
for example by targeted association with the surface of the target
cell before endocytosis as described previously. In this embodiment
the location and distribution of the particles is controlled by the
timing of administration according to the method. Particles from
the first administration are allowed time interval t1 to bind the
target cell surface and then be taken into the target cell. After
interval t1 the second administration is then made and interval t2
allowed to elapse, to allow the second group of particles to bind
to the exterior of the target cells, but t2 is not long enough to
allow the particles substantially to be taken into the cells. The
field is then applied. In general in this method t2 is less than
t1.
[0289] Optionally the methods as shown in FIGS. 13a and 13b may
include steps of imaging the region 100 to determine the location
and number of particles, and hence the readiness for application of
the field. Imaging may also be used to determine the effectiveness
of the treatment.
[0290] FIG. 14 shows a method and an apparatus for treatment of a
disease within a subject by means of destruction of target cells
suspended in the subject's blood. A composition 120 comprising a
first particle type is administered systemically to a subject 150
for example by means of injection or infusion into the blood
stream, and particles then associate with target cells within the
blood. After a chosen time interval t1, chosen to allow particles
to reach the target cells and form desired particle arrangement(s)
with respect to the target cells, blood is drawn from the
circulation by means of a first cannula 146 and flowed through an
extracorporeal flow circuit 140 comprising a flow cell 142 and
preferably controlled by a pump or flow control means 144. A field
is provided in the flow cell by electrodes 110 and 112 disposed on
either side of the flow cell, such that target cells comprising
particles are destroyed by the method of the invention on passing
through the flow cell, non-target cells being substantially
unharmed. Blood is then flowed back to the subject through a second
cannula 148.
[0291] In a preferred embodiment a second composition 122
comprising a second particle type is administered after the
interval t1, and a further interval t2 allowed to elapse before the
blood is exposed to the field, the first and second particle types
being adapted as described previously.
[0292] The method may be carried out by an apparatus comprising
element as shown in FIG. 14, namely standard cannulae and
extra-corporeal blood flow components and pump or flow control
means. Flow cells for conventional electroporation are known. The
flow cell forming part of the invention may take any form adapted
to allow a sufficient flow rate of blood while applying an electric
field to the flow, and may be substantially planar or tubular, for
example formed from concentric cylinders. A suitable flow cell
comprises two parallel surfaces separated by a flow space, a
substantially planar electrode mounted on or disposed above each
surface. Electrodes are preferably insulated from the blood in the
flow space. The dimensions of the flow cell, the flow rate through
the cell, the field intensity profile are all chosen to give
effective killing of the target cells while minimising damage to
non-target cells. Flow may be continuous or may be intermittent.
Flow and field pulse profile may be controlled by the device 130 or
programmable unit 132.
[0293] In a further embodiment the method further comprises the
step of fractioning the blood and applying the method of the
invention to a blood fraction that contains the target cells. In
this embodiment blood is withdrawn from the subject and processed
by conventional apheresis techniques, the desired fraction being
withdrawn from the apheresis process to be treated using the method
and apparatus as described above.
[0294] In a further embodiment, destruction of target cells in
blood may be carried out in-situ within the body of the subject by
providing particles as above and applying an electric field to a
region of the circulatory system through which the target cells
pass, carried in the blood. Flexible electrode apparatus similar to
that shown in FIG. 12c may attached to the body and used to apply a
field to for example a region of blood vessel or other perfused
area, so as to effect gradual removal of target cells as they pass
through the field.
[0295] The invention has applications in biological processes, for
example in cell culture. By selectively destroying target cells and
leaving non-target cells unharmed, a mixed population can be
purified of the target cells, for example when these are a minority
contamination, or if the non-target cells are a desired minority
cell type, the population can be enriched in non-target cell type
by selective destruction of the target cells. Such methods have
application in for example purification of the cell therapy
product, for example in removing residual undifferentiated cells
from a population of desired differentiated cells, or for selecting
a non-target cell for further expansion by selectively removing the
majority target cells from culture.
[0296] FIG. 15a shows a further embodiment of the apparatus of the
invention, adapted for use in a biological process, here the cell
culture of attached cells in an apparatus 200. In FIG. 15a cells 10
are cultured on a surface 202 within a container 204, in a medium
206. Particles are added to the medium and allowed to associate
with the cells in a manner as described for previous embodiments. A
field is then applied across one or more cells, in preferred
embodiments across the thickness of the cell layer as shown, either
over the whole region of the cell layer, or over specific
sub-regions. The field is chosen so that target cells undergo cell
death by non-thermal means mediated by the particles, such as by
irreversible electroporation, while non-target cells are unaffected
or affected to a lesser degree. The field may be applied in some
embodiments by a first planar electrode 110 and second planar
electrode 112. A first electrode might be mounted on or formed as
part of the container 204. The second electrode is then preferably
moveable or removable so as to access the interior of the
container. The second electrode may be in contact with the medium,
and may be insulated from it. Alternatively, the second electrode
may be in electrical connection with the medium. The second
electrode may alternatively be a probe-type electrode 113, which
may be in electrical contact with the medium, the medium in some
embodiments being conductive so as to provide a common potential
over the upper surface of the cell layer. Alternatively the probe
electrode may be insulated from the medium, and may be positionable
over a region of the culture or over a single cell to localise the
effect of the field. Such a probe electrode may be formed for
example from a wire, or a tube or glass pipette filled with
conducting liquid. While a single surface and a single layer of
cells is shown in FIG. 15a, the invention is applicable to other
known forms of container with single or multiple culture
surfaces.
[0297] FIG. 15b shows a further embodiment of an apparatus
according to the invention, adapted to apply the invention to cell
culture in suspension. Cells are cultured in suspension in a
bioreactor 220. Particles are mixed with cells in medium, flowed
through a flow cell 142 in which they are exposed to a field.
Target cells are destroyed selectively as described earlier. The
suspension depleted in the target cell may then be returned to the
bioreactor. Particles may be added to the medium in the bioreactor,
or in the flow system outside the bioreactor. An incubation time
may be provided to allow particles to associate with the target
cells. A suitable flow cell may be as described with respect to
FIG. 14, for example a planar structure with electrodes on either
side of a flow space, or a tubular structure with a concentric
cylindrical electrode arrangement. More than one flow space may be
provided in parallel, each with a pair of electrode surfaces one
each side of the space, for example a stack of electrode pairs with
flow spaces between them, with common inlet and outlet manifolds.
The electrode potentials are provided by a device 130 under control
of a programmable unit 132. Flow may be continuous or intermittent,
so providing a batch process. Flow may be provided by a pump or
flow controller 144, preferably under control of a control means
associated with the device or (as shown) the programmable unit.
[0298] FIG. 16 shows an apparatus adapted to apply the method of
the invention to sample processing, for example in diagnostics, the
invention providing an improved means of cell lysis to allow
release of cell contents into a medium for analysis, for example,
DNA, RNA, proteins or other cell contents or components. A fluidic
system 230 is provided comprising an inlet port 232, a flow cell
142 having means to apply a field to the contents of the flow cell,
a supply of particles 234 connected to the flow system, and a flow
means such as a pump 144. A sample containing cells is drawn
through an inlet port 232, particles are mixed into a sample from a
reservoir 234, allowed to associate with the cells, and then a
field is applied in a flow cell 142. The field is provided by
electrodes associated with the flow cell, controlled by the device
130 and the process may be controlled by a control means associated
with the programmable unit 132, controlling the pump 144 and valves
236 and 238. The apparatus and method may be applied to lysing of
all cell types within the sample by using particles adapted to
associate with a wide range of cell types, or to lyse target cells
selectively be means of targeted particles as described above.
[0299] Particles may be targeted to target cells by means of
ligands adapted to bind to target molecules, for example cell
receptors. Receptor-mediated endocytosis is known, in which
particles bound by ligands to cell receptors are engulfed by the
cell. Particles may be adapted by means of size, shape and ligand
coating to be taken into cells by endocytosis or to be taken in to
a lesser extent while remaining bound to the cell surface. For
suitable particle sizes and morphology see for example Decuzzi and
Ferraro US2010/029785; Zhang et al. Adv. Mater. 2009 vol. 21 p.
419-424; Muro et al. Molecular Therapy 2008 vol. 16 p. 1450-58.
[0300] In preferred embodiments of the invention, particles are
used that provide suitable values of the following properties: rate
of binding to a target cell membrane; affinity of binding; rate of
endocytosis and rate of degradation of particles by lysosomes once
inside the cell. These will depend in general on the size, shape
and material of the particle and the nature and coverage of the
ligands forming part of the coating.
[0301] Embodiments may comprise particles of types adapted to the
target cell type and the mode of administration and may comprise
particles of dimensions chosen from a range. Particles below 0.1 um
diameter are taken up readily by endocytosis and may be taken up in
large numbers by target cells. Some embodiments of the invention
comprise particles that in use tend to provide up to around ten
particles within a target cell; in other embodiments up to around
several tens, or in further embodiments up to around several
hundred or over 1000 particles within a target cell. Larger
particles up to around 1 um diameter may be taken up in smaller
numbers, and some embodiments comprise particle types that in use
tend to provide up to around 10 particles in a cell, in further
embodiments 1 to 5 particles. Particles of greater than 1 um
diameter may also be taken up in small numbers by a cell and may
used in some embodiments. Preferably particles are adapted to
control the rate of endocytosis. For example, spherical particles
undergo endocytosis more readily than larger aspect ratio, for
example disc-shaped or elliptical particles, or rod-shaped
particles.
[0302] In embodiments of the invention particles have a
characteristic maximum dimension in the range 10 nm to 5 um. In a
preferred embodiment at least one particle type has a maximum
dimension of 0.1 um or less. Preferably, a first particle type
intended to be taken into the cell is of low aspect ratio, for
example spherical, and small, for example 1 um or less in largest
dimension, more preferably less than 500 nm and in some embodiments
may be less than 100 nm in largest dimension, though larger and
non-spherical particles are within the scope of the invention. In
preferred embodiments where a second particle type is provided,
intended to remain outside the cell membrane without promoting
endocytosis, the second particle is preferably large, for example
greater than 50 nm in largest dimension, more preferably greater
than 100 nm, more preferably still greater than 500 nm in largest
dimension.
[0303] In a preferred embodiment one or both of a first and a
second particle comprise a gold microsphere of order 1 um diameter.
In a further preferred embodiment one or both of the first and
second particle comprise a gold nanosphere 40 to 60 nm in diameter.
In a particularly preferred embodiment, at least a particle adapted
to associate with the exterior of the cell membrane comprises a
gold nanorod.
[0304] The first or the second particle types in some embodiments
are non-spherical. For example, particles may be polyhedral, for
example metal nanoprisms such as gold nanoprisms, elongated, for
example elliptical, rod-like, tubular or may be formed from a
cluster or agglomeration of smaller particles. A first particle
adapted to enter the target cell and a second particle adapted to
bind outside may be different sizes and different morphologies,
preferably with the second particle having a larger maximum
dimension and higher aspect ratio than the first.
[0305] In preferred embodiments, the method of the invention
comprises time interval(s) according to the nature of the particles
in use, to allow sufficient time for the particles to reach their
intended location and binding configuration but preferably not so
long that internal particles suffer lysosomal degradation or
particles intended to remain external undergo unwanted
endocytosis.
[0306] Embodiments of the invention comprise particles formed from
materials as described herein and materials similar to these as may
be understood by the skilled person. Preferably particles have a
core comprising a material that is a dielectric, ideally having a
high relative permittivity, or that is conductive or
semiconductive. Preferably particles comprise a material having a
high permittivity compared with the effective permittivity of the
environment or medium in which they are intended to be located in
use (in this context, the terms medium and environment are used
interchangeably herein). However, in embodiments, the invention is
not limited to high permittivity particles, rather may derive its
effect from the co-operative effects of lower permittivity
particles.
[0307] The material may have a permittivity selected from within a
range according to the embodiment. A typical relative permittivity
of a cell membrane is around 11.5 (see e.g. Raffa et al
WO-A-2008/062378). Therefore in some embodiments a particle
comprises a material having a relative permittivity of greater than
or approximately equal to 11. In further embodiments a particle
comprises a material having a relative permittivity of 20 or above,
and in further embodiments greater than or approximately equal to
that of blood or physiological saline (typical value 88). In
further embodiments a particle may comprise a conductive material,
for example having a semiconductive or conductive core, and in
preferred embodiments the conductivity will be greater than or
approximately equal to that of then environment in which it is
intended to be located. Magnetic particles as used in magnetic
separations comprise appropriate materials for use in embodiments
of the invention, for example commercially-available magnetic
separation particles, for example comprising a core of Fe3O4.
[0308] In alternative embodiments, particles of lower permittivity
than the surrounding medium or environment are used, for example
contributing to field enhancement by localisation external to the
cell membrane as described in relation to FIG. 6e. In these
embodiments, the relative permittivity of a core material of the
particle may be less than around 88, preferably less than 20, and
may have a value less than 5. For example, particles comprising
SiO2 or similar low permittivity oxides, or other dielectrics, may
be applied in some embodiments of the invention, with the
appropriate applied electric field strength for those particles to
achieve cell death ideally by means of irreversible
electroporation.
[0309] The invention ideally comprises particles, and apparatus and
compositions containing particles, that may be selected by a
skilled person based on the foregoing, together with choice of an
appropriate field strength and waveform.
[0310] The term cell or target cell refers to a biological form of
life comprising for example, a microorganism, a virus, or an
eukaryote cell.
[0311] The eukaryote cell may e.g. be a plant cell, a plant spore,
an animal cell such as mammal cell.
[0312] The mammal cell may e.g. be a human cell such as:
[0313] A keratinizing epithelial cell, such as a keratinocyte of
epidermis, basal cell of epidermis (stem cell), keratinocyte of
finger and toe nails, basal cell of nail bed (stem cell), hair
shaft cell, hair root sheath cell, hair matrix cell (stem cell).
Additionally a cell may be a wet stratified barrier epithelia such
as, surface epithelial cell of stratified squamous epithelium of
cornea, tongue, oral cavity, esophagus, anal canal, distal urethra,
vagina or basal cell of these epithelia (stem cell), or cell of
urinary epithelium (lining bladder and urinary ducts).
[0314] Other types of cell are an epithelial cell specialized for
exocrine secretion such as cells of the salivary glands, cell of
von Ebner's gland in tongue, cell of mammary gland, cell of
lacrimal gland, cell of ceruminous gland of ear, cell of eccrine
sweat gland, cell of apocrine sweat gland, cell of gland of Moll in
eyelid, cell of sebaceous gland, cell of Bowman's gland in nose,
cell of Brunner's gland in duodenum, cell of seminal vesicle, cell
of prostate gland, cell of bulbourethral gland, cell of Bartholin's
gland, cell of Littre's gland, cell of endometrium of uterus,
isolated goblet cell of the respiratory and digestive tracts,
mucous cell of the lining of the stomach, zymogenic cell of gastric
gland, oxyntic cell of pancreas, Paneth cell of small intestine,
type II pnemocyte of lung.
[0315] Other types of cell are cell specialized for secretion of
hormones such as cells of anterior pituitary secreting growth
hormone, follicle-stimulating hormone, luteinizing hormone,
prolactin, adrenocorticotropic hormone, thyroid-stimulating hormone
or cell of intermediate pituitary secreting melanocyte-stimulating
hormone, or cell of posterior pituitary secreting oxytocin or
vasopressin, or cell of gut and respiratory tract secreting
serotonin, endorphin, somatostatin, gastrin, secretin,
cholecystokinin, insulin, glucagon or bombesin.
[0316] Other types of cell are cell of the thyroid gland secreting
thyroid hormone or calcitonin, a cell of the parathyroid gland
secreting parathyroid hormone or an oxyphil cell, a cell of adrenal
gland secreting epinephrine, norepinephrine, steroid hormones such
as mineralocorticoids or glucocorticoids, a cell of gonads
secreting testosterone (Leydig cell of testis) estrogen (theca
interna cell of ovarian follicle), progesterone (corpus luteum cell
of ruptured ovarian follicle), a cell of juxtaglomerular apparatus
of kidney secreting rennin, an epithelial absorptive cell in the
gut, exocrine glands, and urogenital tract such as brush border
cell of intestine (with microvilli), striated duct cell of exocrine
glands, gall bladder epithelial cell, brush border cell of proximal
tubule of kidney, distal tubule cell of kidney, nonciliated cell of
ductulus efferens, epididymal principal cell or epididymal basal
cell.
[0317] Other types of cell are a cell specialized for metabolism
and storage such as hepatocyte, liver lipocyte or fat cell, an
epithelial cell serving primarily a barrier function, lining the
lung, gut, exocrine glands, and urogenital tract such as type I
pneumocyte cell (lining air space of lung), pancreatic duct cell
(centroacinar cell), nonstriated duct cell of sweat gland, salivary
gland, mammary gland etc. (various), parietal cell of kidney
glomerulus, podocyte of kidney glomerulus, cell of thin segment of
loop of Henle (in kidney), collecting duct cell (in kidney), duct
cell of seminal vesicle, prostate gland, etc. (various), an
epithelial cell lining closed internal body cavities such as
vascular endothelial cells of blood vessels and lymphatics
(fenestrated, continuous, splenic), synovial cell (lining joint
cavities, secreting largely hyaluronic acid), serosal cell (lining
peritoneal, pleural, and pericardial cavities), squamous cell
lining perilymphatic space of ear, cells lining endolymphatic space
of ear, squamous cell columnar cells of endolymphatic sac (with
microvilli or without microvilli), "dark" cell, vestibular membrane
cell, stria vascularis basal cell, stria vascularis marginal cell,
cell of Claudius, cell of Boettcher, choroid plexus cell (secreting
cerebrospinal fluid).
[0318] Other types of cell are squamous cell of pia-arachnoid,
cells of ciliary epithelium of eye (pigmented or nonpigmented),
corneal "endothelial" cell, a ciliated cell with propulsive
function of respiratory tract, of oviduct and of endometrium of
uterus (in female), of rete testis and ductulus efferens (in male)
or a cell of a central nervous system (ependymal cell lining brain
cavities), a cell specialized for secretion of extracellular matrix
such as epithelial ameloblast (secreting enamel of tooth),
epithelial planum semilunatum cell of vestibular apparatus of ear
(secreting proteoglycan), interdental cell of organ of Corti
(secreting tectorial "membrane" covering hair cells of organ of
Corti), nonepithelial (connective tissue) such as fibroblasts
(various--of loose connective tissue, of cornea, of tendon, of
reticular tissue of bone marrow, etc.), pericyte of blood
capillary, nucleus pulposus cell of intervertebral disc,
cementoblast/cementocyte (secreting bonelike cementum of root of
tooth), odontoblast/odontocyte (secreting dentin of tooth),
chondrocytes of hyaline cartilage of fibrocartilage of elastic
cartilage, osteoblast/osteocyte, osteoprogenitor cell (stem cell of
osteoblasts), hyalocyte of vitreous body of eye or stellate cell of
perilymphatic space of ear.
[0319] Other types of cell are: a contractile cell such as skeletal
muscle cells (red (slow), white (fast), inter-mediate, muscle
spindle--nuclear bag, muscle spindle--nuclear chain, satellite cell
(stem cell), or heart muscle cells (ordinary, nodal, Purkinje
fiber), or smooth muscle cells (various), or myoepithelial cells of
iris or of exocrine glands.
[0320] Other types of cell are a cell related to blood or the
immune system such as red blood cell, megakaryocyte, macrophages
and related cells (monocyte, connective-tissue macrophage
(various), Langerhans cell (in epidermis), osteoclast (in bone),
adendritic cell (in lymphoid tissues), microglial cell (in central
nervous system)), neutrophil, eosinophil, basophil, mast cell, T
lymphocyte (helper T cell, suppressor T cell, killer T cell), B
lymphocyte (IgM, IgG, IgA, IgE), killer cell or stem cells and
committed progenitors for the blood and immune system (various), a
cell with sensory and transducing functions such as photoreceptors
(rod, cones [blue sensitive, green sensitive, red sensitive],
hearing (inner hair cell of organ of Corti, outer hair cell of
organ of Corti), acceleration and gravity (type I hair cell of
vestibular apparatus of ear, type II hair cell of vestibular
apparatus of ear), taste (type II taste bud cell), smell (olfactory
neuron, basal cell of olfactory epithelium (stem cell for olfactory
neurons)), blood pH (carotid body cell [type I, type II]), touch
(Merkel cell of epidermis, primary sensory neurons specialized for
touch (various)), temperature (primary sensory neurons specialized
for temperature [cold sensitive, heat sensitive]), pain (primary
sensory neurons specialized for pain (various)), configurations and
forces in musculoskeletal system (proprioceptive primary sensory
neurons (various)).
[0321] Other types of cell are an autonomic neuronal cell
(cholinergic (various), adrenergic (various), peptidergic
(various)), a supporting cells of sense organs and of peripheral
neurons such as supporting cells of organ of Corti (inner pillar
cell, outer pillar cell, inner phalangeal cell outer phalangeal
cell, border cell, Hensen cell), or a supporting cell of vestibular
apparatus, or a supporting cell of taste bud (type I taste bud
cell), or a supporting cell of olfactory epithelium, or a Schwann
cell, or a satellite cell (encapsulating peripheral nerve cell
bodies) or a enteric glial cell; a neuronal or glial cells of the
central nervous system such as neurons (huge variety of
types--still poorly classified), glial cells (astrocyte (various),
oligodendrocyte).
[0322] It is appreciated that reference to cell includes the
following cells: a lens cell such as anterior lens epithelial cell,
lens fiber (crystallin-containing cell), or pigment cell such as
melanocyte or retinal pigmented epithelial cell, a germ cell such
as oogonium/oocyte, spermatocyte, or spermatogonium (stem cell for
spermatocyte), a nurse cell such as ovarian follicle cell, sertoli
cell (in testis), or thymus epithelial cell, Other types of cell
are n interstitial cell such as interstitial cells of Cajal in the
gastro-intestinal system, or interstitial cell of the kidney or
other organs with pacemaker functions.
[0323] The microorganism may e.g. be selected from the group
consisting of an archeal microorganism, a eubacterial microorganism
or a eukaryotic microorganism, the microorganism may be selected
from the group consisting of a bacterium, a bacterial spore, a
virus, a fungus, and a fungal spore.
[0324] In a preferred embodiment of the invention, the
microorganism is hosted inside a mammalian cell which is serving as
a reservoir for the infection.
[0325] In a preferred embodiment of the invention, the
microorganism is resistant to common chemotherapies such as
anti-biotics such as but excluded too methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus
aureus (VRSA), penicillin resistant Streptococcus, anti-biotic
resistant strains of Mycobacterium tuberculosis, penicillin
resistant Enterococcus, multi-drug resistant Pseudomonas
aeruginosa, clindamycin (or fluoroquinolone) resistant Clostridium
difficile (diarrheal disease) and multi-drug resistant Escherichia
coli.
[0326] Further applications of the invention are within biological
processes, cell biology research, detection of cells in a sample
and diagnostics. The following features and details may apply to
each of the embodiments described above, and combinations of them
may be used in any given embodiment as will be apparent to the
skilled person.
[0327] Although reference has been made to cell lysis, it is
understood that the method apparatus and systems herein described
are capable of being modified and used to arrest cell reproduction,
specifically uncontrolled cell reproduction that is encountered in
tumours.
[0328] Ideally a control means, operating under control of
instructions in the form of software and using data derived from a
look-up table, the control means applies a variable potential to
the first and second electrodes, in accordance with the data and
under instruction from the software, whereby in use, target cells
within the electric field are killed and non-target cells remain
unharmed.
[0329] A preferred embodiment of the apparatus may be portable and
worn as a belt and is usable with the method of the invention.
[0330] Furthermore it is understood that references herein to a
method are taken to encompass a corresponding apparatus and vice
versa.
[0331] Other aspects of the invention provide for the following
embodiments:
[0332] 1. Wherein a plurality of cells are killed by apoptosis.
[0333] 2. Wherein the particle is adapted to bind or adhere to the
cell wall.
[0334] 3. Wherein the cell membrane is of the order of 10 nm
thick.
[0335] 4. Wherein the target molecule is a biomarker.
[0336] 5. Wherein the target molecule is from the groups
comprising: lipids, carbohydrates, nucleic acids or proteins.
[0337] 6. Wherein the nucleic acid comprises chromosomal DNA and/or
plasmid DNA, and/or any type of RNA.
[0338] 7. Wherein the protein is from the group comprising:
enzymes, structural proteins, transport proteins, in channels,
toxins, hormones or receptors.
[0339] 8. Wherein the cell targeting is achieved by means of
antibodies, aptamers, and/or ligands on the surface of the
particle.
[0340] 9. Wherein the ligand comprises more than one species to
increase capture affinity.
[0341] 10. Wherein for an elongated particle, the coating is
located in a specific region at one end of the particle.
[0342] 11. Wherein apparatus for treatment of a disease condition
in a subject using particles or nanoparticles and time-varying
electromagnetic or electric fields, characterised in that a means
if provided to introduce a market to the interior of a target cell
and a means is provided for exposing the target cell to an electric
field sufficient to cause irreversible electroporation of the
cell.
[0343] 12. Wherein the apparatus is used to treat neoplasia,
cancerous cells or infections, such as those caused by fungi, viral
or other microorganisms.
[0344] 13. Wherein the means to introduce particles to the interior
of the cell includes a particle delivery device that administers
particles to the target cells.
[0345] 14. Wherein the means of administering is through topical or
systemic administration.
[0346] 15. Wherein the apparatus incorporates means to deliver a
first particle type adapted to enter a target cell, and a second
particle type adapted to bind to a target molecule on the exterior
of a target membrane.
[0347] 16. Wherein the particles are administered systemically
through body fluid such as blood, lymph, cerebrospinal fluid.
[0348] 17. Wherein the target cells comprise bacteria, spores, vira
or mammalian cells such as leukemic or virally infected cells
within the body fluid.
[0349] 18. Wherein the target cells are localised such as in a
local seat of infection, or a region of neoplasia or a tumour.
[0350] 19. Wherein the particles are administered by injection
and/or infusion, and/or electroporation through skin and/or
inhalation, and/or absorption through mucal membranes, and/or via
the digestive tract.
[0351] 20. Wherein devices for administration include a syringe,
cannula, catheter, inhaler, implanted release device, capsule
and/or ingestible preparation.
[0352] 21. Wherein there is provided a means to expose the target
cells to a variable electric or electromagnetic field comprising:
at least a first and a second electrode and a control means for
applying a variable potential to the first and second electrodes,
whereby target cells within the electric field are killed and
non-target cells remain unharmed. The field strength may be chosen
according to the nature of the disease condition, the nature of the
particles, and the proportions of target and non-target cells that
are to be destroyed on average in a given treatment.
[0353] 22. Wherein there is provided a method for treatment of a
disease in a subject by means of destruction of target cells within
the subject, comprising the steps of: [0354] a) administering
particles to the subject, either systemically or topically in the
region of the target cells, the particles being adapted to be taken
up within the target cells; [0355] b) allowing a chosen time
interval to elapse so that at least one particle is taken up within
at least one target cell; and [0356] c) applying an electric field
to a region of the subject within which one or more target cells
are located in order to cause cell death of the targeted cells by
predominantly non-thermal means.
[0357] 23. Wherein particles are tracked within the body fluid by
tracking means such as MRI, ultrasound, computer tomographic
scanner.
Wherein a delay of greater than 10 minutes is provided between
administration of the first and second particles
[0358] 24. Wherein targeted cell lysis--comprising a particle
inside cell and exposing to field sufficient to cause IEP
[0359] 25. Wherein a device for treatment of a disease condition
using particles and an electric field, characterised in that means
is provided to introduce a particle inside a target cell and means
is provided for exposing the target cell to a field sufficient to
cause IEP.
[0360] 26. Wherein a second particle outside the cell.
[0361] 27. Wherein particles may have a coating that makes them
selective for the target cell--one binds at membrane, one bind
inside the cell.
[0362] 28. Wherein a composition comprising plurality of particles
adapted for use in method and system for causing death of target
cell, eg. by IRE, particles adapted to enhance field.
* * * * *