Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SYSTEMS FOR TREATMENT OF DISEASE VIA APPLICATION OF MECHANICAL FORCE BY
CONTROLLED ROTATION
OF NANOPARTICLES INSIDE CELLS
Cross Reference To Related Application
[0001] This application claims the benefit of U.S. Provisional
Application No.
62/215,001, filed September 6, 2015, the content of which is hereby
incorporated by reference
herein in its entirety.
Government Support
[0002] This invention was made with government support under CA016396
awarded by
the National Institutes of Health. The government has certain rights in the
invention.
Field of the Invention
[0003] This invention relates generally to systems and methods that exert
targeted
mechanical force. In particular embodiments, the invention relates to systems
and methods for
external control of the movement of magnetic particles with high precision and
at significant
depths (e.g., 5 to 10 cm, 10 to 20 cm, or 20 to 30 cm) within a subject's
body.
Background
[0004] Investigations using magnetic nanoparticles have explored the
capability of
controlling the motion or energy of magnetic nanoparticles within cells and
tissues by remote
application of magnetic fields. So far, this has been investigated using
permanent magnets that
set nanoparticles in a longitudinal motion, using alternating magnetic fields,
or through rotating
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permanent magnets outside of the tissues of interest. In the latter scenario,
the nanoparticles
describe circular motions but do not individually rotate around their own
axis.
The combination of alternating magnetic fields and magnetic nanoparticles has
been used to
transform energy into heat. For example, hyperthermia is used as an adjunctive
treatment in
cancer therapy; here, high-frequency alternating (but not moving)
electromagnetic fields in the
kilo- to megahertz (kHz¨MHz) range have been used to kill cancer cells loaded
with engineered
magnetic nanoparticles through thermal induction. However, such treatment is
not without risks,
particularly near thermally sensitive structures such as the gut or
gallbladder if nanoparticles are
injected systemically, as the heat induction cannot be controlled spatially
with high precision and
could cause tissue necrosis. Therefore, in contrast to thermal ablation
systems, ambient
temperature increases greater than 46 C are not desirable for purposes of
remote controlling
apoptosis with magnetic fields.
Summary
[0005] It is observed that targeted cell death can be achieved by
application of
mechanical force through rotational movement of magnetic particles in the body
of a subject.
The present disclosure describes systems and methods for inducing such
mechanical force to
achieve directed disruption of lysosomes in certain cells, leading to cell
death from apoptosis.
[0006] The present disclosure describes systems and methods for external
control of the
movement of magnetic particles with high precision and at significant depths
(e.g., 5 to 10 cm,
to 20 cm, or 20 to 30 cm) within a subject's body. In particular, in certain
embodiments, the
system features an alternating current superconductor (ACSC) to greatly
enhance the magnetic
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field amplitude so that the field can penetrate deeper into a body with
sufficient amplitude to
control movement of the nanoparticles within a working volume.
[0007] Among other things, the present disclosure demonstrates use of a
dynamic
magnetic field (DMF) generator that can induce rotational movement of certain
magnetic
particles. Particular surprising and advantageous features of the described
technology include
that it can be used to rotate individual particles, and moreover can rotate
such particles about
their own axis. Additional or alternative surprising and advantageous features
include that the
present disclosure provides for manipulation of magnetic particles in a
magnetic field without
significant heat generation (e.g., without heating the particles).
[0008] The disclosure particularly demonstrates that use of magnetic
particles designed
and/or constructed to specifically bind to a target structure is surprisingly
effective, even as
compared with use of otherwise comparable (e.g., otherwise identical) magnetic
particles that
lack such specific binding, for exertion of mechanical force on the target
structure.
[0009] Specifically exemplified herein is remote induction of cell death
by application of
mechanical force through rotational movement of magnetic particles
(specifically
superparamagnetic iron oxide nanoparticles, "SPIONs") exposed to DMF
treatment. As
exemplified, particles were specifically targeted to lysosomal membrane
structures, and were
individually induced through application of DMF treatment to rotate about
their axes. The
resulting mechanical force disrupted (e.g., permeabilized) the lysosomal
membranes, releasing
lysosomal enzymes and triggering apoptosis, all without heating the particles
(or their
surroundings).
[0010] The Examples included herein demonstrate, among other things, that
shear forces
created by the generation of torques (incomplete rotation) of magnetic
particles (e.g.,
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superparamagnetic nanoparticles such as SPIONs) specifically bound to
lysosomal membranes
causes membrane permeabilization, and furthermore leads to extravasation of
lysosomal contents
into the cytoplasm. Still further, the present exemplification demonstrates
that such application
of shear forces to lysosomal membranes can induce apoptosis.
[0011] Still further, the exemplification provided herein explicitly
demonstrates that use
of specifically targeted magnetic particles (e.g., superparamagnetic
nanoparticles such as
SPIONs covalently conjugated to specific binding agents that target lysosomal
ligands, in
particular to antibodies targeting the lysosomal protein marker LAMP1 (LAMP1-
SPION)) shows
surprisingly superior effect in application of mechanical force as described
herein. The present
exemplification specifically demonstrates, for example, that remote activation
of slow rotation of
LAMP1-SPIONs significantly improved the efficacy of cellular internalization
of the
nanoparticles. LAMP1-SPIONs then preferentially accumulated along the membrane
in
lysosomes in both rat insulinoma tumor cells and human pancreatic beta-cells,
presumably due to
binding of LAMP1-SPIONs to endogenous LAMP 1. Further activation of torques by
the
LAMP1-SPIONs bound to lysosomes resulted in rapid decrease in size and number
of
lysosomes. Without wishing to be bound by any particular theory, it is
contemplated that such
rapid decrease are attributable to tearing of the lysosomal membrane by the
shear force of the
rotationally activated LAMP1-SPIONs. Regardless of mechanism, the present
exemplification
demonstrates that such remote activation resulted in an increased expression
of early and late
apoptotic markers and impaired cell growth. Findings described herein suggest,
among other
things, that DMF treatment of lysosome-targeted nanoparticles offers a non-
invasive tool to
induce apoptosis remotely.
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[0012] The present disclosure demonstrates, among other things, that use
of DMF
treatment with appropriate magnetic particles (e.g., with superparamagnetic
nanoparticles), and
particularly with specifically-targeted such particles, can apply mechanical
force in a controlled
and effective manner, for use in a wide range of applications, specifically
including biomedical
applications. In certain embodiments, provided technologies are utilized to
induce apoptosis or
otherwise to achieve cell death or destruction, including of tumor cells,
malignant cells, or
otherwise aberrantly proliferating cells.
[0013] One particular advantage of technologies described herein is that
mechanical
force is applied, and appropriate results achieved, without significant
generation of heat. Thus,
for example, in certain embodiments, the present disclosure provides
compositions and methods
that achieve selective cell destruction without significant generation of
heat. In particular,
provided technologies apply mechanical force, and in certain embodiments,
achieve selective
cell destruction, without generating heat sufficient to result in off-target
cell damage.
[0014] In one aspect, the invention is directed to an alternating current
superconductor
(ACSC) system for external control of the movement of magnetic particles
within the body of a
subject, the system comprising: an alternating current (AC) power source for
powering the
system; a controller for controlling a dynamic magnetic field produced by the
system; a cooling
unit for cooling the system; and an actuator comprising superconducting
windings, wherein the
actuator has a geometry operable to apply the dynamic magnetic field to a
working volume of
known geometry within the body of a subject, and wherein the dynamic magnetic
field is from
about 0.1 T to about 3 T to induce movement of particles located within the
working volume in
the body of the subject.
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[0015] In certain embodiments, the controller comprises an inverter. In
certain
embodiments, the cooling unit comprises a cryogenic cooling unit. In certain
embodiments, the
windings comprise a high temperature superconductor material coated conductor.
In certain
embodiments, the high temperature superconductor material comprises YBa2Cu30õ
(YBCO). In
certain embodiments, the induced movement includes rotation.
[0016] In certain embodiments, the particles comprise superparamagnetic
nanoparticles.
[0017] In certain embodiments, the working volume extends to a maximum
depth in a
range selected from the group consisting of greater than 1 cm, greater than 2
cm, greater than 3
cm, between 5 to 10 cm, between 10 to 20 cm, and between 20 to 30 cm from an
accessible
surface of the subject.
[0018] In certain embodiments, the surface of the subject comprises skin
of the subject or
the interior surface of an accessible cavity of the subject.
[0019] In certain embodiments, the system comprises particles for binding
to a target
structure. In certain embodiments, a mechanical force within the range of
about 1 fN to about 1
nN is applied to the target structure of the subject, but the particles are
not significantly heated.
In certain embodiments, a mechanical force within the range of about 1 pN to
about 1 fN is
applied to the target structure of the subject, but the particles are not
significantly heated.
[0020] In another aspect, the invention is directed to a method of
operating an alternating
current superconductor (ACSC) system for external control of the movement of
magnetic
particles within the body of a subject, the system comprising: an alternating
current (AC) power
source for powering the system; a controller for controlling a dynamic
magnetic field produced
by the system; a cooling unit for cooling the system; and an actuator
comprising superconducting
windings, wherein the actuator has a geometry operable to apply the dynamic
magnetic field to a
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working volume of known geometry within the body of a subject, and wherein the
dynamic
magnetic field is from about 0.1 T to about 3 T to induce movement of
particles located within
the working volume in the body of the subject, the method comprising: remotely
controlling
application of the dynamic field produced by the system to induce movement of
particles located
within the working volume in the body of the subject.
[0021] In certain embodiments, controlling is performed by Internet, by
WLAN, or
otherwise. In certain embodiments, the induced movement comprises rotation.
[0022] In certain embodiments, the particles comprise superparamagnetic
nanoparticles.
[0023] In certain embodiments, the working volume extends to a maximum
depth
selected from the range consisting of greater than 1 cm from an accessible
surface of the subject,
greater than 2 cm from an accessible surface of the subject, greater than 3 cm
from an accessible
surface of the subject, between 5 to 10 cm from an accessible surface of the
subject, between 10
to 20 cm from an accessible surface of the subject, and between 20 to 30 cm
from an accessible
surface of the subject.
[0024] In certain embodiments, the surface of the subject comprises skin
of the subject or
the interior surface of an accessible cavity of the subject.
[0025] In certain embodiments, the ACSC system is operated under 50 Hz.
In certain
embodiments, the ACSC system is operated under 30 Hz. In certain embodiments,
the ACSC
system is operated from about 10 Hz to 30 Hz. In certain embodiments, the ACSC
system is
operated at about 20 Hz.
[0026] In certain embodiments, a mechanical force within the range of
about 1 fN to
about 1 nN is applied to a target structure of the subject. In certain
embodiments, a mechanical
force within the range of about 1 pN to 1 fN is applied to a target structure
of the subject.
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[0027] In certain embodiments, the particles are or comprise
nanoparticles. In certain
embodiments, the particles are or comprise superparamagnetic nanoparticles. In
certain
embodiments, the particles are or comprise iron oxide nanoparticles
("SPIONs"). In certain
embodiments, the particles are or comprise superparamagnetic nanoparticles
characterized by an
iron oxide core. In certain embodiments, the particles lose their magnetism
when not exposed to
an external magnetic field. In certain embodiments, the particles are
superparamagnetic
nanoparticles associated with a targeting agent that specifically binds to the
target structure. In
certain embodiments, the particles are bound to a target structure in the body
of the subject. In
certain embodiments, the particles are bound to a target structure in the body
of the subject, and a
lysosomal membrane and the superparamagnetic nanoparticles are associated with
a targeting
agent that specifically binds to the target structure. In certain embodiments,
the particles are not
significantly heated such that no tissue damage is caused by generated heat.
[0028] In certain embodiments, the targeting agent is covalently linked
to the magnetic
nanoparticles. In certain embodiments, the targeting agent is or comprises a
member selected
from the group consisting of an antibody agent, a polypeptide, a small
molecule, a glycan, a lipid,
and a nucleic acid that specifically binds to a target moiety in or on the
target structure. In
certain embodiments, the target structure is or comprises a member selected
from the group
consisting of a cell membrane, a tumor-associated entity, a tumor-associated
marker, an ion
channel, an intracellular membrane, a lysosomal membrane, an intracellular
entity such as an
organelle such as the endoplasmic reticulum (ER), the golgi apparatus, the
mitochondria, a
component of transcription machinery, a splicosome, a ribosome, and
combinations thereof. In
certain embodiments, the targeting agent is covalently linked to the magnetic
nanoparticles. In
certain embodiments, the targeting agent specifically binds to a target moiety
on the surface of
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the lysosomal membrane. In certain embodiments, the target moiety is or
comprises LAMP-1
(CD107a), LAMP-2 (CD107b), or LAMP-3 (CD63).
[0029] In certain embodiments, the method comprises exposing a target
structure to the
magnetic nanoparticles so that the nanoparticles bind to the target structure
with a density
sufficient to apply a desired force across a relevant area. In certain
embodiments, the exposed
target structure is coincident with the working volume or is within the
working volume.
[0030] In certain embodiments, the method comprises exposing the target
structure to the
magnetic nanoparticles so that, on average, from about 1 to about 60 magnetic
nanoparticles
become bound to each lysosome. In certain embodiments, the step of exposing
comprises
exposing the target structure to the magnetic nanoparticles so that, on
average, from about 10 to
about 50 magnetic nanoparticles become bound to each lysosome. In certain
embodiments, the
step of exposing comprises exposing the target structure to the magnetic
nanoparticles so that, on
average, about 30 magnetic nanoparticles become bound to each lysosome.
[0031] In certain embodiments, the method comprises applying a DMF field
with a
strength within the range of about 1 mT to 1 T.
[0032] In another aspect, the invention is directed to a method of
applying mechanical
force to a target structure, the method comprising: exposing a target
structure to magnetic
particles so that the particles bind to the target structure; and applying a
dynamic magnetic field
(DMF) to the nanoparticles sufficient to induce movement of the particles.
[0033] In certain embodiments, the induced movement comprises rotation.
[0034] In certain embodiments, a mechanical force within a range of from
about 1 fN to
about 1 nN is applied to the target structure without the particles being
significantly heated.
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[0035] In certain embodiments, the particles are or comprise
nanoparticles. In certain
embodiments, the particles are or comprise magnetic nanoparticles. In certain
embodiments, the
particles are or comprise iron oxide nanoparticles ("SPIONs"). In certain
embodiments, the
magnetic nanoparticles are characterized by an iron oxide core. In certain
embodiments, the
particles are characterized in losing their magnetism when not exposed to an
external magnetic
field. In certain embodiments, the particles are characterized in maintaining
their magnetism
when not exposed to an external magnetic field. In certain embodiments, the
magnetic
nanoparticles are associated with a targeting agent that specifically binds to
the target structure.
[0036] In certain embodiments, the targeting agent is covalently linked
to the magnetic
nanoparticles. In certain embodiments, the targeting agent is or comprises an
antibody agent. In
certain embodiments, the targeting agent is or comprises a member selected
from the group
consisting of a polypeptide, a small molecule, a glycan, a lipid, a nucleic
acid that specifically
binds to a target moiety in or on the target structure, and combinations
thereof In certain
embodiments, the target moiety is or comprises a polypeptide. In certain
embodiments, the
target moiety is or comprises a glycan. In certain embodiments, the target
moiety is or comprises
a nucleic acid.
[0037] In certain embodiments, the target structure is or comprises a
cell membrane. In
certain embodiments, the target structure is or comprises a tumor-associated
marker. In certain
embodiments, the target structure is or comprises an ion channel. In certain
embodiments, the
target structure is or comprises an intracellular membrane. In certain
embodiments, the target
structure is or comprises a lysosomal membrane. In certain embodiments, the
target structure is
or comprises an intracellular entity.
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[0038] In certain embodiments, the intracellular entity is or comprises
an organelle. In
certain embodiments, the organelle is selected from the group consisting of
the endoplasmic
reticulum (ER), the golgi apparatus, the mitochondria, and combinations
thereof. In certain
embodiments, the intracellular entity is or comprises a component of
transcription machinery, a
splicosome, or a ribosome.
[0039] In certain embodiments, the target structure is or comprises a
lysosomal
membrane and the magnetic nanoparticles are associated with a targeting agent
that specifically
binds to the target structure.
[0040] In certain embodiments, the targeting agent is covalently linked
to the magnetic
nanoparticles. In certain embodiments, the targeting agent specifically binds
to a target moiety
on the surface of the lysosomal membrane. In certain embodiments, the target
moiety is or
comprises LAMP-1 (CD107a), LAMP-2 (CD107b), or LAMP-3 (CD63).
[0041] In certain embodiments, the step of exposing comprises exposing
the target
structure to the magnetic nanoparticles so that the nanoparticles bind to the
target structure with a
density sufficient to apply a desired force across a relevant area. In certain
embodiments, the
step of exposing comprises exposing the target structure to the magnetic
nanoparticles so that, on
average, about 1 to about 60 magnetic nanoparticles become bound to each
lysosome. In certain
embodiments, the step of exposing comprises exposing the target structure to
the magnetic
nanoparticles so that, on average, about 10 to about 50 magnetic nanoparticles
become bound to
each lysosome. In certain embodiments, the step of exposing comprises exposing
the target
structure to the magnetic nanoparticles so that, on average, about 30 magnetic
nanoparticles
become bound to each lysosome.
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[0042] In certain embodiments, the step of applying DMF treatment
comprises applying
a DMF field with a strength within the range of about 1 mT to 5 T. In certain
embodiments, the
step of applying DMF treatment comprises applying a DMF field with a reach
within the range
of about mm to about cm. In certain embodiments, the step of applying DMF
treatment
comprises applying a DMF field with a reach of at least 1 cm. In certain
embodiments, the step
of applying DMF treatment comprises applying a DMF field with a reach of at
least 2 cm to 5
cm. In certain embodiments, the step of applying DMF treatment comprises
applying a DMF
field with a reach of at least 10 cm (e.g., at least 15 cm, at least 18 cm, at
least 20 cm, at least 23
cm, at least 25 cm, at least 28 cm, or at least 30 cm). In certain
embodiments, the step of
applying DMF treatment comprises applying a DMF field with a reach of at least
50 cm.
[0043] In certain embodiments, the target structure is or comprises a
tumor-associated
entity. In certain embodiments, the tumor-associated entity is or comprises a
cell-surface entity.
In certain embodiments, the tumor-associated entity is or comprises and
intracellular entity.
[0044] In another aspect, the invention is directed to a composition for
use with an
alternating current superconductor (AC SC) system for external control of the
movement of
magnetic particles within the body of a subject, the composition comprising
superparamagnetic
nanoparticles.
[0045] In certain embodiments, the composition is or comprises iron oxide
nanoparticles
("SPIONs"). In certain embodiments, the nanoparticles are or comprise
superparamagnetic
nanoparticles characterized by an iron oxide core.
[0046] In certain embodiments, the composition further comprises
superparamagnetic
nanoparticles associated with a targeting agent that specifically binds to a
target structure in the
body of the subject. In certain embodiments, the nanoparticles are bound to a
target structure in
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the body of the subject, and a lysosomal membrane and the superparamagnetic
nanoparticles are
associated with a targeting agent that specifically binds to the target
structure. In certain
embodiments, the nanoparticles comprise a targeting agent covalently linked to
the
superparamagnetic nanoparticles.
[0047] In certain embodiments, the targeting agent is or comprises a
member selected
from the group consisting of an antibody agent, a polypeptide, a small
molecule, a glycan, a lipid,
and a nucleic acid that specifically binds to a target moiety in or on the
target structure.
[0048] Elements of embodiments involving one aspect of the invention
(e.g., methods)
can be applied in embodiments involving other aspects of the invention, and
vice versa.
Description of the Drawings
[0049] FIG. 1 presents a schematic depiction of exemplified use of LAMP1-
SPIONs with
DMF to disrupt lysosomal membranes and induce apoptosis. Additional supporting
information,
including Movie 1: Illustrating the rotational control of magnetic
microparticles achieved with a
DMF device; Movie 2: Illustrating the rotational control of magnetic
nanoparticles (300 nm
diameter) achieved with a DMF device; and Movie 3: Dynamic confocal microscopy
study
illustrating the real-time destruction of lysosomes via DMF and LAMP1-SPIONs,
is available
free of charge via the Internet at http://pubs.acs.org.
[0050] FIGS. 2A-2B illustrate DMF controlled rotation of magnetic
nanoparticles.
[0051] FIG. 2A shows schematic representation of a DMF generator utilized
in the
present Examples. The device controls rotation and movement of magnetic
nanoparticles (e.g.,
SPIONs) with a low frequency (10-40 Hz) field. In contrast to reported
alternating magnetic
field generators, the DMF generator causes the nanoparticles to rotate around
their own axis.
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[0052] FIG. 2B illustrates principle of use of the DMF generator and
remote induction of
apoptosis. As depicted in FIG. 2B, when targeted nanoparticles (LAMP1-SPIONs)
first come
into contact with cell membranes, their internalization can be enhanced by
activation of slow
nanoparticle rotation. This causes rotational motion (rolling) of the
nanoparticles across the cell
membrane and eventually internalization. Once internalized, the LAMP1-SPIONs
enter
lysosomes and bind to the lysosomal membrane. When the DMF is activated at
this point, the
nanoparticles start to rotate and the resulting shear forces cause injury to
the lysosomal
membrane. This in turn causes leakage of the lysosomal contents into the
cytoplasm, leading to
a decrease in its pH and subsequently apoptosis.
[0053] FIGS. 3A-3D demonstrates DMF-induced rotation of magnetic
particles. In order
to enable better visualization of the effect of the DMF on magnetic particles
under the
microscope, larger micrometer sized magnetic beads (diameter 5.8 m) were
used. A dish
containing beads in a physiologic salt solution (Krebs buffer) was placed in
the vicinity of the
DMF device. Once the DMF was switched on, the beads started to rotate around
their own axis,
which also caused a slow directional movement of the beads across the floor of
the dish. The
beads completed a rotation of 360 in seconds (time depends on the viscosity
of the liquid)
between the FIG. 3A and FIG. 3D. The speed of rotation can be controlled by
varying the
frequency setting on the DMF device and in this experiment was varied between
5-15 Hz.
Movie 1, noted above, depicts this rolling.
[0054] FIGS. 4A-4D demonstrates the loading of magnetic nanoparticle into
lysosomes
in INS-1 cells.
[0055] FIG. 4A shows confocal imaging of SPIONs location in INS-1 cells.
The
SPIONs conjugated with the fluorescent dye TRITC (upper left image) were
incubated with
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living cells in a static magnetic field for 5 minutes. Thereafter the cells
were treated by DMF
with 20 Hz, for 20 min. and then confocal microscopy images obtained (upper
middle image).
Plasma membrane and early endosomes were stained with CellMask (upper right
image); nuclei
and lysosomes were stained with Hoechst 32580 (lower left image) and
LysoTracker Green
(green, lower middle image), respectively. The squares in the merge stack
(lower right image)
indicate SPIONs located in the lysosomes. Scale bars = 2 p.m.
[0056] FIG. 4B shows statistical analysis of SPION co-localization with
LysoTracker
Green and CellMask under same conditions as in FIG. 4A.
[0057] FIG. 4C shows co-localization analysis of lysosomes with SPION and
CellMask
under same conditions as FIG. 4A.
[0058] FIG. 4D demonstrates loading efficiency of LAMP1 antibody
conjugated SPIONs
(LAMP1-SPION) increased under condition with the DMF treatment. The loading
efficiency is
calculated by the ratio of TRITC fluorescence intensity (lighter greyscale
values shown in the
merged image) over nuclear intensity. The data was collected from three
independent
experiments. The "*" symbol indicates p<0.05, and "***" symbol indicates
p<0.001.
[0059] FIGS. 5A-5D demonstrates that DMF treatment decreases
intracellular lysosomes
and the pH in LAMP1-SPION loaded INS-1 cells.
[0060] FIG. 5A, shows cells (in the lower images) treated by DNIF (20 Hz,
20 min.), and
lysosomes (in all four images) stained with LysoTracker Green. Scale bars = 5
p.m.
[0061] FIG. 5B shows the mean intensity of fluorescence measured under
the various
different conditions shown in FIG. 5A.
[0062] FIG. 5C shows representative confocal images indicating the
intracellular pH
value using an acidotropic probe, LysoSensor Green DND 189 in INS-1 cells.
Scale bars = 5 p.m.
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[0063] FIG. 5D shows the mean intensity of fluorescence measured under
the various
different conditions shown in FIG. 5C. The data were collected from five
experiments with at
least 6 cells under each condition. The "**" symbol indicates p<0.01, and
"***" symbol
indicates p<0.001.
[0064] FIG. 6 is a temperature plot demonstrating that DMF-field induced
SPION
rotation does not increase temperature. A dish with 100 nm SPIONs (10 mg/ml)
in water and a
control dish containing water only, each containing a temperature probe, were
placed
simultaneously on the DMF device. The dishes were then subjected to the DMF
field for 20 min.
at 20 Hz. No significant change in temperature was observed in either the dish
with the SPIONs
(thicker, darker curve) or the control dish (thinner, lighter curve).
[0065] FIGS. 7A-7F demonstrates that DMF treatment disrupts lysosomes in
human
pancreatic beta cells after loading with LAMP1-SPIONs.
[0066] FIG. 7A shows immunostaining of human islet beta cells with or
without DMF
treatment. Lysosomes were stained with the anti-LAMP1 antibody (two left
images), SPIONs
with TRITC (two left-center images) and islet beta-cells with an anti-insulin
antibody (two right-
center images), respectively. Scale bars = 2 p.m.
[0067] FIG. 7B shows the SPIONs located in the membrane of a lysosome in
which an
intensity profile (right plot) was derived along the horizontal line shown in
the stained image
(left image).
[0068] FIG. 7C shows the SPIONs located in the membrane of a lysosome,
after
treatment with DMF. An intensity profile (right plot) was also derived based
on the horizontal
line shown in the corresponding stained image (left).
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[0069] FIG. 7D illustrates differences in size distribution of lysosomes
before (grey bars)
and after (black bars) DMF treatment.
[0070] FIG. 7E illustrates average sizes of lysosomes without and with
DMF treatment.
The "***" symbol indicates p<0.001.
[0071] FIG. 7F shows transmission electron microscopy (TEM) images of the
intracellular distribution of SPIONs in INS-1 cells. Images on the bottom are
magnified versions
of the areas indicated with white boxes (shown in the top images). While
without DMF
treatment the LAMP1-SPIONs are clustered in vesicular structures, their
distribution is scattered
throughout the cytosol after DMF treatment.
[0072] FIGS. 8A-8D demonstrate DMF treatment-induced apoptosis in LAMP1-
SPION
loaded cells.
[0073] FIG. 8A shows INS-1 cells treated with DMF for 20 min. at 20 Hz
and stained
with the nuclear marker Hoechst (shown as grey values in the left image and
center-left image),
the apoptosis marker annexin V (lighter grey values in the right image) and 7-
AAD (lighter grey
values in the right image and center-right image). Scale bars = 5 p.m.
[0074] FIGS. 8B and 8C show that after 6 hours of incubation (5% CO2, 37
C), early
i
(FIG. 8B) and late (FIG. 8C) stage apoptosis were detected by percentage of
FAnnexn V(7AAD) - xioo
'Hoechst 34580
number of annexin V and 7-AAD positive cells to the number of Hoechst stained
cells. As
demonstrated, DMF caused significant increase in apoptosis in LAMP1-SPION-
loaded cells
compared to when loading was done using conventional SPIONs. Each treatment
was conducted
with 28 cells. The "*" symbol indicates p<0.05.
[0075] FIG. 8D shows decrease of the rate of cell growth in LAMP1-SPION
loaded INS-
1 cells. Cells were treated with DMF (20 Hz, 20 minutes) once/day. Data are
from 3
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independent experiments and represent mean values S.E.M. The "***" symbol
indicates
p<0.001.
[0076] FIG. 9 shows an illustrative ACSC system as described herein.
[0077] FIG. 10 shows illustrative actuator geometries as described
herein.
[0078] FIG. 11 shows a block diagram of an example network environment
for use in the
methods and systems described herein, according to an illustrative embodiment
of the present
disclosure.
[0079] FIG. 12 shows a block diagram of an example computing device and
an example
mobile computing device, for use in illustrative embodiments of the present
disclosure.
[0080] FIG. 13 is a schematic depicting components that can be included
in an ACSC
system, according to an illustrative embodiment of the present disclosure.
Components can
include, for example, a transformer, a grid connection, a converter, a
controller, a field unit (e.g.,
actuator), and cooler.
[0081] FIG. 14 is a schematic depicting an exemplary system in which an
actuator
component of an ACSC system is located in proximity to (e.g., underneath) a
subject, according
to an illustrative embodiment of the present disclosure.
[0082] FIG. 15 shows a map profile of the magnetic field (B) as a
function of distance
(mm) produced by a field generator system as described herein, according to an
illustrative
embodiment of the present disclosure.
Definitions
[0083] Administration: As used herein, the term "administration" refers
to the
administration of a composition to a subject or system. Administration to an
animal subject
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(e.g., to a human) may be by any appropriate route. For example, in certain
embodiments,
administration may be bronchial (including by bronchial instillation), buccal,
enteral,
interdermal, intra-arterial, intradermal, intragastric, intramedullary,
intramuscular, intranasal,
intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal,
oral, rectal,
subcutaneous, sublingual, topical, tracheal (including by intratracheal
instillation), transdermal,
vaginal and vitreal.
[0084]
Affinity: As is known in the art, "affinity" is a measure of the tightness
with a
particular ligand binds to its partner. Affinities can be measured in
different ways. In certain
embodiments, affinity is measured by a quantitative assay. In some such
embodiments, binding
partner concentration may be fixed to be in excess of ligand concentration so
as to mimic
physiological conditions. Alternatively or additionally, in certain
embodiments, binding partner
concentration and/or ligand concentration may be varied. In some such
embodiments, affinity
may be compared to a reference under comparable conditions (e.g.,
concentrations).
[0085]
Agent: The term "agent" as used herein may refer to a compound or entity of
any chemical class including, for example, polypeptides, nucleic acids,
saccharides, lipids, small
molecules, metals, or combinations thereof. In certain embodiments, an agent
is or comprises a
natural product in that it is found in and/or is obtained from nature. In
certain embodiments, an
agent is or comprises one or more entities that is man-made in that it is
designed, engineered,
and/or produced through action of the hand of man and/or is not found in
nature. In certain
embodiments, an agent may be utilized in isolated or pure form; in certain
embodiments, an
agent may be utilized in crude form. In certain embodiments, potential agents
are provided as
collections or libraries, for example that may be screened to identify or
characterize active agents
within them. Some particular embodiments of agents that may be utilized in
accordance with the
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present disclosure include small molecules, antibodies, antibody fragments,
aptamers, nucleic
acids (e.g., siRNAs, shRNAs, DNA/RNA hybrids, antisense oligonucleotides,
ribozymes),
peptides, peptide mimetics, etc. In certain embodiments, an agent is or
comprises a polymer. In
certain embodiments, an agent is not a polymer and/or is substantially free of
any polymer. In
certain embodiments, an agent contains at least one polymeric moiety. In
certain embodiments,
an agent lacks or is substantially free of any polymeric moiety.
[0086] Animal: As used herein, the term "animal" refers to any member of
the animal
kingdom. In certain embodiments, "animal" refers to humans, at any stage of
development. In
certain embodiments, "animal" refers to non-human animals, at any stage of
development. In
certain embodiments, the non-human animal is a mammal (e.g., a rodent, a
mouse, a rat, a rabbit,
a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In certain
embodiments, animals
include, but are not limited to, mammals, birds, reptiles, amphibians, fish,
and/or worms. In
certain embodiments, an animal may be a transgenic animal, genetically-
engineered animal,
and/or a clone.
[0087] Antibody agent: As used herein, the term "antibody agent" refers to
an agent that
specifically binds to a particular antigen. In certain embodiments, the term
encompasses any
polypeptide with immunoglobulin structural elements sufficient to confer
specific binding.
Suitable antibody agents include, but are not limited to, human antibodies,
primatized antibodies,
chimeric antibodies, bi-specific antibodies, humanized antibodies, conjugated
antibodies (i.e.,
antibodies conjugated or fused to other proteins, radiolabels, cytotoxins),
Small Modular
ImmunoPharmaceuticals ("SMIPsTm"), single chain antibodies, cameloid
antibodies, and
antibody fragments. As used herein, the term "antibody agent" also includes
intact monoclonal
antibodies, polyclonal antibodies, single domain antibodies (e.g., shark
single domain antibodies
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(e.g., IgNAR or fragments thereof)), multispecific antibodies (e.g. bi-
specific antibodies) formed
from at least two intact antibodies, and antibody fragments so long as they
exhibit the desired
biological activity. In certain embodiments, the term encompasses stapled
peptides. In certain
embodiments, the term encompasses one or more antibody-like binding
peptidomimetics. In
certain embodiments, the term encompasses one or more antibody-like binding
scaffold proteins.
In come embodiments, the term encompasses monobodies or adnectins. In many
embodiments,
an antibody agent is or comprises a polypeptide whose amino acid sequence
includes one or
more structural elements recognized by those skilled in the art as a
complementarity determining
region (CDR); in certain embodiments an antibody agent is or comprises a
polypeptide whose
amino acid sequence includes at least one CDR (e.g., at least one heavy chain
CDR and/or at
least one light chain CDR) that is substantially identical to one found in a
reference antibody. In
certain embodiments an included CDR is substantially identical to a reference
CDR in that it is
either identical in sequence or contains between 1-5 amino acid substitutions
as compared with
the reference CDR. In certain embodiments an included CDR is substantially
identical to a
reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In
certain
embodiments an included CDR is substantially identical to a reference CDR in
that it shows at
least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference
CDR. In certain
embodiments an included CDR is substantially identical to a reference CDR in
that at least one
amino acid within the included CDR is deleted, added, or substituted as
compared with the
reference CDR but the included CDR has an amino acid sequence that is
otherwise identical with
that of the reference CDR. In certain embodiments an included CDR is
substantially identical to
a reference CDR in that 1-5 amino acids within the included CDR are deleted,
added, or
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substituted as compared with the reference CDR but the included CDR has an
amino acid
sequence that is otherwise identical to the reference CDR. In certain
embodiments an included
CDR is substantially identical to a reference CDR in that at least one amino
acid within the
included CDR is substituted as compared with the reference CDR but the
included CDR has an
amino acid sequence that is otherwise identical with that of the reference
CDR. In certain
embodiments an included CDR is substantially identical to a reference CDR in
that 1-5 amino
acids within the included CDR are deleted, added, or substituted as compared
with the reference
CDR but the included CDR has an amino acid sequence that is otherwise
identical to the
reference CDR. In certain embodiments, an antibody agent is or comprises a
polypeptide whose
amino acid sequence includes structural elements recognized by those skilled
in the art as an
immunoglobulin variable domain. In certain embodiments, an antibody agent is a
polypeptide
protein having a binding domain which is homologous or largely homologous to
an
immunoglobulin-binding domain.
[0088] Antibody: As is known in the art, an "antibody" is an
immunoglobulin that binds
specifically to a particular antigen. The term encompasses immunoglobulins
that are naturally
produced in that they are generated by an organism reacting to the antigen,
and also those that
are synthetically produced or engineered. An antibody may be monoclonal or
polyclonal. An
antibody may be a member of any immunoglobulin class, including any of the
human
classes: IgG, IgM, IgA, and IgD. A typical immunoglobulin (antibody)
structural unit as
understood in the art, is known to comprise a tetramer. Each tetramer is
composed of two
identical pairs of polypeptide chains, each pair having one "light"
(approximately 25 kD) and
one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain
defines a variable
region of about 100 to 110 or more amino acids primarily responsible for
antigen recognition.
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The terms "variable light chain"(VL) and "variable heavy chain" (VH) refer to
these light and
heavy chains respectively. Each variable region is further subdivided into
hypervariable (HV)
and framework (FR) regions. The hypervariable regions comprise three areas of
hypervariability
sequence called complementarity determining regions (CDR 1, CDR 2 and CDR 3),
separated by
four framework regions (FR1, FR2, FR2, and FR4) which form a beta-sheet
structure and serve
as a scaffold to hold the HV regions in position. The C-terminus of each heavy
and light chain
defines a constant region consisting of one domain for the light chain (CL)
and three for the
heavy chain (CH1, CH2 and CH3). In certain embodiments, the term "full length"
is used in
reference to an antibody to mean that it contains two heavy chains and two
light chains,
optionally associated by disulfide bonds as occurs with naturally-produced
antibodies. In certain
embodiments, an antibody is produced by a cell. In certain embodiments, an
antibody is
produced by chemical synthesis. In certain embodiments, an antibody is derived
from a
mammal. In certain embodiments, an antibody is derived from an animal such as,
but not limited
to, mouse, rat, horse, pig, or goat. In certain embodiments, an antibody is
produced using a
recombinant cell culture system. In certain embodiments, an antibody may be a
purified
antibody (for example, by immune-affinity chromatography). In certain
embodiments, an
antibody may be a human antibody. In certain embodiments, an antibody may be a
humanized
antibody (antibody from non-human species whose protein sequences have been
modified to
increase their similarity to antibody variants produced naturally in humans).
In certain
embodiments, an antibody may be a chimeric antibody (antibody made by
combining genetic
material from a non-human source, e.g., mouse, rat, horse, or pig, with
genetic material from
humans).
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[0089] Antibody fragment: As used herein, an "antibody fragment" includes
a portion of
an intact antibody, such as, for example, the antigen-binding or variable
region of an antibody.
Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments;
triabodies;
tetrabodies; linear antibodies; single-chain antibody molecules; and multi
specific antibodies
formed from antibody fragments. For example, antibody fragments include
isolated fragments,
"Fv" fragments, consisting of the variable regions of the heavy and light
chains, recombinant
single chain polypeptide molecules in which light and heavy chain variable
regions are
connected by a peptide linker ("ScFv proteins"), and minimal recognition units
consisting of the
amino acid residues that mimic the hypervariable region. In many embodiments,
an antibody
fragment contains sufficient sequence of the parent antibody of which it is a
fragment that it
binds to the same antigen as does the parent antibody; in certain embodiments,
a fragment binds
to the antigen with a comparable affinity to that of the parent antibody
and/or competes with the
parent antibody for binding to the antigen. Examples of antigen binding
fragments of an
antibody include, but are not limited to, Fab fragment, Fab' fragment, F(ab')2
fragment, scFv
fragment, Fv fragment, dsFy diabody, dAb fragment, Fd' fragment, Fd fragment,
and an isolated
complementarity determining region (CDR) region. An antigen binding fragment
of an antibody
may be produced by any means. For example, an antigen binding fragment of an
antibody may
be enzymatically or chemically produced by fragmentation of an intact antibody
and/or it may be
recombinantly produced from a gene encoding the partial antibody sequence.
Alternatively or
additionally, antigen binding fragment of an antibody may be wholly or
partially synthetically
produced. An antigen binding fragment of an antibody may optionally comprise a
single chain
antibody fragment. Alternatively or additionally, an antigen binding fragment
of an antibody
may comprise multiple chains which are linked together, for example, by
disulfide linkages. An
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antigen binding fragment of an antibody may optionally comprise a
multimolecular complex. A
functional antibody fragment typically comprises at least about 50 amino acids
and more
typically comprises at least about 200 amino acids.
[0090] Antigen: An "antigen" is a molecule or entity that i) elicits an
immune response;
and/or (ii) is specifically bound by a T cell receptor (e.g., when presented
by an MHC molecule)
or an antibody (e.g., produced by a B cell), for example when exposed or
administered to an
organism. In certain embodiments, an antigen elicits a humoral response (e.g.,
including
production of antigen-specific antibodies) in an organism; alternatively or
additionally, in certain
embodiments, an antigen elicits a cellular response (e.g., involving T-cells
whose receptors
specifically interact with the antigen) in an organism. It will be appreciated
by those skilled in
the art that a particular antigen may elicit an immune response in one or
several members of a
target organism (e.g., mice, rabbits, primates, humans), but not in all
members of the target
organism species. In certain embodiments, an antigen elicits an immune
response in at least
about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of a target organism
species. In
certain embodiments, an antigen binds to an antibody and/or T cell receptor,
and may or may not
induce a particular physiological response in an organism. In certain
embodiments, for example,
an antigen may bind to an antibody and/or to a T cell receptor in vitro,
whether or not such an
interaction occurs in vivo. In general, an antigen may be or include any
chemical entity such as,
for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate,
a lipid, a polymer
[in certain embodiments other than a biologic polymer (e.g., other than a
nucleic acid or amino
acid polymer)] etc. In certain embodiments, an antigen is or comprises a
polypeptide. In certain
embodiments, an antigen is or comprises a glycan. Those of ordinary skill in
the art will
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appreciate that, in general, an antigen may be provided or utilized in
isolated or pure form, or
alternatively may be provided in crude form (e.g., together with other
materials, for example in
an extract such as a cellular extract or other relatively crude preparation of
an antigen-containing
source). In certain embodiments. In certain embodiments, an antigen is or
comprises a
recombinant antigen. In certain embodiments, an antigen is or comprises a
polypeptide or portion
thereof. In certain embodiments, an antigen is associated with (e.g.,
expressed by) an infectious
agent. In certain embodiments, an antigen is associated with cancer (e.g.,
with tumor cells and/or
metastases).
[0091] Approximately: As used herein, the terms "approximately" and
"about" are each
intended to encompass normal statistical variation as would be understood by
those of ordinary
skill in the art as appropriate to the relevant context. In certain
embodiments, the terms
"approximately" or "about" each refer to a range of values that fall within
25%, 20%, 19%, 18%,
17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or
less in
either direction (greater than or less than) of a stated value, unless
otherwise stated or otherwise
evident from the context (e.g., where such number would exceed 100% of a
possible value).
[0092] Aptamer: As used herein, the term "aptamer" refers to a
macromolecule
composed of nucleic acid (e.g., RNA, DNA) that binds tightly to a specific
molecular target (e.g.,
an umbrella topology glycan). A particular aptamer may be described by a
linear nucleotide
sequence and is typically about 15-60 nucleotides in length. Without wishing
to be bound by
any theory, it is contemplated that the chain of nucleotides in an aptamer
form intramolecular
interactions that fold the molecule into a complex three-dimensional shape,
and this three-
dimensional shape allows the aptamer to bind tightly to the surface of its
target molecule. Given
the extraordinary diversity of molecular shapes that exist within the universe
of all possible
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nucleotide sequences, aptamers may be obtained for a wide array of molecular
targets, including
proteins and small molecules. In addition to high specificity, aptamers
typically have very high
affinities for their targets (e.g., affinities in the picomolar to low
nanomolar range for proteins).
In many embodiments, aptamers are chemically stable and can be boiled or
frozen without loss
of activity. Because they are synthetic molecules, aptamers are amenable to a
variety of
modifications, which can optimize their function for particular applications.
For example,
aptamers can be modified to dramatically reduce their sensitivity to
degradation by enzymes in
the blood for use in in vivo applications. In addition, aptamers can be
modified to alter their
biodistribution or plasma residence time.
[0093] Associated with: Two events or entities are "associated" with one
another, as that
term is used herein, if the presence, level and/or form of one is correlated
with that of the other.
For example, a particular entity (e.g., polypeptide) is considered to be
associated with a
particular disease, disorder, or condition, if its presence, level and/or form
correlates with
incidence of and/or susceptibility of the disease, disorder, or condition
(e.g., across a relevant
population). In certain embodiments, two or more entities are physically
"associated" with one
another if they interact, directly or indirectly, so that they are and remain
in physical proximity
with one another. In certain embodiments, two or more entities that are
physically associated
with one another are covalently linked to one another; in certain embodiments,
two or more
entities that are physically associated with one another are not covalently
linked to one another
but are non-covalently associated, for example by means of hydrogen bonds, van
der Waals
interaction, hydrophobic interactions, magnetism, and combinations thereof.
[0094] Binding: It will be understood that the term "binding", as used
herein, typically
refers to a non-covalent association between or among two or more entities.
"Direct" binding
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involves physical contact between entities or moieties; indirect binding
involves physical
interaction by way of physical contact with one or more intermediate entities.
Binding between
two or more entities can typically be assessed in any of a variety of contexts
¨ including where
interacting entities or moieties are studied in isolation or in the context of
more complex systems
(e.g., while covalently or otherwise associated with a carrier entity and/or
in a biological system
or cell).
[0095] Binding agent: In general, the term "binding agent" is used herein
to refer to any
entity that binds to a target of interest as described herein. In many
embodiments, a binding
agent of interest is one that binds specifically with its target in that it
discriminates its target from
other potential binding partners in a particular interaction contact. In
general, a binding agent
may be or comprise an entity of any chemical class (e.g., polymer, non-
polymer, small molecule,
polypeptide, carbohydrate, lipid, nucleic acid, etc.). In certain embodiments,
a binding agent is a
single chemical entity. In certain embodiments, a binding agent is a complex
of two or more
discrete chemical entities associated with one another under relevant
conditions by non-covalent
interactions. For example, those skilled in the art will appreciate that in
certain embodiments, a
binding agent may comprise a "generic" binding moiety (e.g., one of
biotin/avidin/streptaviding
and/or a class-specific antibody) and a "specific" binding moiety (e.g., an
antibody or aptamers
with a particular molecular target) that is linked to the partner of the
generic biding moiety. In
certain embodiments, such an approach can permit modular assembly of multiple
binding agents
through linkage of different specific binding moieties with the same generic
binding poiety
partner. In certain embodiments, binding agents are or comprise polypeptides
(including, e.g.,
antibodies or antibody fragments). In certain embodiments, binding agents are
or comprise small
molecules. In certain embodiments, binding agents are or comprise nucleic
acids. In certain
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embodiments, binding agents are aptamers. In certain embodiments, binding
agents are
polymers; in certain embodiments, binding agents are not polymers. In certain
embodiments,
binding agents are non-polymeric in that they lack polymeric moieties. In
certain embodiments,
binding agents are or comprise carbohydrates. In certain embodiments, binding
agents are or
comprise lectins. In certain embodiments, binding agents are or comprise
peptidomimetics. In
certain embodiments, binding agents are or comprise scaffold proteins. In
certain embodiments,
binding agents are or comprise mimotopes. In certain embodiments, binding
agents are or
comprise stapled peptides. In certain embodiments, binding agents are or
comprise nucleic
acids, such as DNA or RNA.
[0096] Characteristic portion: As used herein, the term "characteristic
portion" is used,
in the broadest sense, to refer to a portion of a substance whose presence (or
absence) correlates
with presence (or absence) of a particular feature, attribute, or activity of
the substance. In
certain embodiments, a characteristic portion of a substance is a portion that
is found in the
substance and in related substances that share the particular feature,
attribute or activity, but not
in those that do not share the particular feature, attribute or activity. In
certain embodiments, a
characteristic portion shares at least one functional characteristic with the
intact substance. For
example, in certain embodiments, a "characteristic portion" of a protein or
polypeptide is one
that contains a continuous stretch of amino acids, or a collection of
continuous stretches of amino
acids, that together are characteristic of a protein or polypeptide. In
certain embodiments, each
such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or
more amino acids. In
general, a characteristic portion of a substance (e.g., of a protein,
antibody, etc.) is one that, in
addition to the sequence and/or structural identity specified above, shares at
least one functional
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characteristic with the relevant intact substance. In certain embodiments, a
characteristic portion
may be biologically active.
[0097] Combination therapy: As used herein, the term "combination
therapy" refers to
those situations in which a subject is simultaneously exposed to two or more
therapeutic
regimens (e.g., two or more therapeutic agents). In certain embodiments, two
or more agents
may be administered simultaneously; in certain embodiments, such agents may be
administered
sequentially; in certain embodiments, such agents are administered in
overlapping dosing
regimens.
[0098] Comparable: The term "comparable", as used herein, refers to two
or more
agents, entities, situations, sets of conditions, etc. that may not be
identical to one another but
that are sufficiently similar to permit comparison therebetween so that
conclusions may
reasonably be drawn based on differences or similarities observed. In certain
embodiments,
comparable sets of conditions, circumstances, individuals, or populations are
characterized by a
plurality of substantially identical features and one or a small number of
varied features. Those
of ordinary skill in the art will understand, in context, what degree of
identity is required in any
given circumstance for two or more such agents, entities, situations, sets of
conditions, etc. to be
considered comparable. For example, those of ordinary skill in the art will
appreciate that sets of
circumstances, individuals, or populations are comparable to one another when
characterized by
a sufficient number and type of substantially identical features to warrant a
reasonable
conclusion that differences in results obtained or phenomena observed under or
with different
sets of circumstances, individuals, or populations are caused by or indicative
of the variation in
those features that are varied.
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[0099] Corresponding to: As used herein, the term "corresponding to" is
often used to
designate a structural element or moiety in an agent of interest that shares a
position (e.g., in
three-dimensional space or relative to another element or moiety) with one
present in an
appropriate reference agent. For example, in certain embodiments, the term is
used to refer to
position/identity of a residue in a polymer, such as an amino acid residue in
a polypeptide or a
nucleotide residue in a nucleic acid. Those of ordinary skill will appreciate
that, for purposes of
simplicity, residues in such a polymer are often designated using a canonical
numbering system
based on a reference related polymer, so that a residue in a first polymer
"corresponding to" a
residue at position 190 in the reference polymer, for example, need not
actually be the 190th
residue in the first polymer but rather corresponds to the residue found at
the 190th position in
the reference polymer; those of ordinary skill in the art readily appreciate
how to identify
"corresponding" amino acids, including through use of one or more commercially-
available
algorithms specifically designed for polymer sequence comparisons.
[00100] Designed: As used herein, the term "designed" refers to an agent
(i) whose
structure is or was selected by the hand of man; (ii) that is produced by a
process requiring the
hand of man; and/or (iii) that is distinct from natural substances and other
known agents.
[00101] Detection entity The term "detection entity" as used herein refers
to any
element, molecule, functional group, compound, fragment or moiety that is
detectable. In certain
embodiments, a detection entity is provided or utilized alone. In certain
embodiments, a
detection entity is provided and/or utilized in association with (e.g., joined
to) another agent.
Examples of detection entities include, but are not limited to: various
ligands, radionuclides (e.g.,
3H, '4C,
18 19 32 35 135 125 123 64 187 111 90 99m 177 89
H, C, F, F, P, S, I, I, I, Cu, Re, In, Y, Tc, Lu, Zr,
etc.),
fluorescent dyes (for specific exemplary fluorescent dyes, see below),
chemiluminescent agents
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(such as, for example, acridinum esters, stabilized dioxetanes, and the like),
bioluminescent
agents, spectrally resolvable inorganic fluorescent semiconductors
nanocrystals (i.e., quantum
dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.)
nanoclusters, paramagnetic
metal ions, enzymes (for specific examples of enzymes, see below),
colorimetric labels (such as,
for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens,
and proteins for
which antisera or monoclonal antibodies are available.
[00102] Determine: Many methodologies described herein include a step of
"determining". Those of ordinary skill in the art, reading the present
specification, will
appreciate that such "determining" can utilize or be accomplished through use
of any of a variety
of techniques available to those skilled in the art, including for example
specific techniques
explicitly referred to herein. In certain embodiments, determining involves
manipulation of a
physical sample. In certain embodiments, determining involves consideration
and/or
manipulation of data or information, for example utilizing a computer or other
processing unit
adapted to perform a relevant analysis. In certain embodiments, determining
involves receiving
relevant information and/or materials from a source. In certain embodiments,
determining
involves comparing one or more features of a sample or entity to a comparable
reference.
[00103] Diagnostic information: As used herein, "diagnostic information" or
"information for use in diagnosis" is information that is useful in
determining whether a patient
has a disease, disorder or condition and/or in classifying a disease, disorder
or condition into a
phenotypic category or any category having significance with regard to
prognosis of a disease,
disorder or condition, or likely response to treatment (either treatment in
general or any
particular treatment) of a disease, disorder or condition. Similarly,
"diagnosis" refers to
providing any type of diagnostic information, including, but not limited to,
whether a subject is
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likely to have or develop a disease, disorder or condition (such as cancer),
state, staging or
characteristic of a disease, disorder or condition as manifested in the
subject, information related
to the nature or classification of a tumor, information related to prognosis
and/or information
useful in selecting an appropriate treatment. Selection of treatment may
include the choice of a
particular therapeutic (e.g., chemotherapeutic) agent or other treatment
modality such as surgery,
radiation, etc., a choice about whether to withhold or deliver therapy, a
choice relating to dosing
regimen (e.g., frequency or level of one or more doses of a particular
therapeutic agent or
combination of therapeutic agents), etc.
[00104] Dosage form: As used herein, the term "dosage form" refers to a
physically
discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for
administration to a
subject. Each unit contains a predetermined quantity of active agent. In
certain embodiments,
such quantity is a unit dosage amount (or a whole fraction thereof)
appropriate for administration
in accordance with a dosing regimen that has been determined to correlate with
a desired or
beneficial outcome when administered to a relevant population (i.e., with a
therapeutic dosing
regimen). Those of ordinary skill in the art appreciate that the total amount
of a therapeutic
composition or agent administered to a particular subject is determined by one
or more attending
physicians and may involve administration of multiple dosage forms.
[00105] Dosing regimen: As used herein, the term "dosing regimen" refers to
a set of unit
doses (typically more than one) that are administered individually to a
subject, typically
separated by periods of time. In certain embodiments, a given therapeutic
agent has a
recommended dosing regimen, which may involve one or more doses. In certain
embodiments, a
dosing regimen comprises a plurality of doses each of which are separated from
one another by a
time period of the same length; in certain embodiments, a dosing regimen
comprises a plurality
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of doses and at least two different time periods separating individual doses.
In certain
embodiments, all doses within a dosing regimen are of the same unit dose
amount. In certain
embodiments, different doses within a dosing regimen are of different amounts.
In certain
embodiments, a dosing regimen comprises a first dose in a first dose amount,
followed by one or
more additional doses in a second dose amount different from the first dose
amount. In certain
embodiments, a dosing regimen comprises a first dose in a first dose amount,
followed by one or
more additional doses in a second dose amount same as the first dose amount.
In certain
embodiments, a dosing regimen is correlated with a desired or beneficial
outcome when
administered across a relevant population (i.e., is a therapeutic dosing
regimen).
[00106] Engineered: In general, the term "engineered" refers to the aspect
of having been
manipulated by the hand of man. For example, a polynucleotide is considered to
be
"engineered" when two or more sequences, that are not linked together in that
order in nature,
are manipulated by the hand of man to be directly linked to one another in the
engineered
polynucleotide. For example, in certain embodiments, an engineered
polynucleotide comprises a
regulatory sequence that is found in nature in operative association with a
first coding sequence
but not in operative association with a second coding sequence, is linked by
the hand of man so
that it is operatively associated with the second coding sequence. Comparably,
a cell or
organism is considered to be "engineered" if it has been manipulated so that
its genetic
information is altered (e.g., new genetic material not previously present has
been introduced, for
example by transformation, mating, somatic hybridization, transfection,
transduction, or other
mechanism, or previously present genetic material is altered or removed, for
example by
substitution or deletion mutation, or by mating protocols). As is common
practice and is
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understood by those in the art, progeny of an engineered polynucleotide or
cell are typically still
referred to as "engineered" even though the actual manipulation was performed
on a prior entity.
[00107] High affinity binding: The term "high affinity binding", as used
herein refers to a
high degree of tightness with which a particular ligand binds to its partner.
Affinities can be
measured by any available method, including those known in the art. Those
skilled in the art
will be aware of affinities that are appropriately considered to be "high" in
a particular context.
In certain embodiments, high affinity binding may be characterized by
preferentially binding to a
particular target when in the presence of alternative potential (e.g.,
competitive) targets,
particularly when such competitive targets are present in excess relative to
the target of interest.
In certain embodiments, high affinity binding may be characterized by
relatively rapid on-rate
and/or slow off-rate.
[00108] Isolated: As used herein, the term "isolated" refers to a substance
and/or entity
that has been (1) separated from at least some of the components with which it
was associated
when initially produced (whether in nature and/or in an experimental setting),
and/or (2)
designed, produced, prepared, and/or manufactured by the hand of man. Isolated
substances
and/or entities may be separated from about 10%, about 20%, about 30%, about
40%, about
50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about
93%, about
94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about
99% of the
other components with which they were initially associated. In certain
embodiments, isolated
agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,
about 94%,
about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99%
pure. As
used herein, a substance is "pure" if it is substantially free of other
components. In certain
embodiments, as will be understood by those skilled in the art, a substance
may still be
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considered "isolated" or even "pure", after having been combined with certain
other components
such as, for example, one or more carriers or excipients (e.g., buffer,
solvent, water, etc.); in such
embodiments, percent isolation or purity of the substance is calculated
without including such
carriers or excipients. In certain embodiments, isolation involves or requires
disruption of
covalent bonds (e.g., to isolate a polypeptide domain from a longer
polypeptide and/or to isolate
a nucleotide sequence element from a longer oligonucleotide or nucleic acid).
[00109] Low affinity binding: The term "low affinity binding", as used
herein refers to a
low degree of tightness with which a particular ligand binds to its partner.
As described herein,
affinities can be measured by any available method, including methods known in
the art. Those
skilled in the art will be aware of affinities that are appropriately
considered to be "low" in a
particular context. In certain embodiments, low affinity binding may be
characterized by failure
to discriminate among potential (e.g., competitive) targets, particularly when
they are present at
comparable levels. In certain embodiments, low affinity binding may be
characterized by
relatively slow on-rate and/or rapid off-rate.
[00110] Marker: A marker, as used herein, refers to an entity or moiety
whose presence
or level is a characteristic of a particular state or event. In certain
embodiments, presence or
level of a particular marker may be characteristic of presence or stage of a
disease, disorder, or
condition. To give but one example, in certain embodiments, the term refers to
a gene
expression product that is characteristic of a particular tumor, tumor
subclass, stage of tumor, etc.
Alternatively or additionally, in certain embodiments, a presence or level of
a particular marker
correlates with activity (or activity level) of a particular signaling
pathway, for example that may
be characteristic of a particular class of tumors. The statistical
significance of the presence or
absence of a marker may vary depending upon the particular marker. In certain
embodiments,
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detection of a marker is highly specific in that it reflects a high
probability that the tumor is of a
particular subclass. Such specificity may come at the cost of sensitivity
(i.e., a negative result
may occur even if the tumor is a tumor that would be expected to express the
marker).
Conversely, markers with a high degree of sensitivity may be less specific
that those with lower
sensitivity. According to the present disclosure, a useful marker need not
distinguish tumors of a
particular subclass with 100% accuracy.
[00111] Nucleic acid: As used herein, the term "nucleic acid," in its
broadest sense, refers
to any compound and/or substance that is or can be incorporated into an
oligonucleotide chain.
In certain embodiments, a nucleic acid is a compound and/or substance that is
or can be
incorporated into an oligonucleotide chain via a phosphodiester linkage. As
will be clear from
context, in certain embodiments, "nucleic acid" refers to individual nucleic
acid residues (e.g.,
nucleotides and/or nucleosides); in certain embodiments, "nucleic acid" refers
to an
oligonucleotide chain comprising individual nucleic acid residues. In certain
embodiments, a
"nucleic acid" is or comprises RNA; in certain embodiments, a "nucleic acid"
is or comprises
DNA. In certain embodiments, a nucleic acid is, comprises, or consists of one
or more natural
nucleic acid residues. In certain embodiments, a nucleic acid is, comprises,
or consists of one or
more nucleic acid analogs. In certain embodiments, a nucleic acid analog
differs from a nucleic
acid in that it does not utilize a phosphodiester backbone. For example, in
certain embodiments,
a nucleic acid is, comprises, or consists of one or more "peptide nucleic
acids", which are known
in the art and have peptide bonds instead of phosphodiester bonds in the
backbone, are
considered within the scope of the present disclosure. Alternatively or
additionally, in certain
embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-
phosphoramidite
linkages rather than phosphodiester bonds. In certain embodiments, a nucleic
acid is, comprises,
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or consists of one or more natural nucleosides (e.g., adenosine, thymidine,
guanosine, cytidine,
uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).
In certain
embodiments, a nucleic acid is, comprises, or consists of one or more
nucleoside analogs (e.g., 2-
aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl
adenosine, 5-
methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,
C5-
bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-
propynyl-cytidine,
C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-
oxoadenosine, 8-
oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases,
intercalated bases, and
combinations thereof). In certain embodiments, a nucleic acid comprises one or
more modified
sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose)
as compared with
those in natural nucleic acids. In certain embodiments, a nucleic acid has a
nucleotide sequence
that encodes a functional gene product such as an RNA or protein. In certain
embodiments, a
nucleic acid includes one or more introns. In certain embodiments, nucleic
acids are prepared by
one or more of isolation from a natural source, enzymatic synthesis by
polymerization based on a
complementary template (in vivo or in vitro), reproduction in a recombinant
cell or system, and
chemical synthesis. In certain embodiments, a nucleic acid is at least 3, 4,
5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,
130, 140, 150, 160,
170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,
600, 700, 800,
900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues
long.
[00112] Pharmaceutical composition: As used herein, the term
"pharmaceutical
composition" refers to an active agent, formulated together with one or more
pharmaceutically
acceptable carriers. In certain embodiments, active agent is present in unit
dose amount
appropriate for administration in a therapeutic regimen that shows a
statistically significant
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probability of achieving a predetermined therapeutic effect when administered
to a relevant
population. In certain embodiments, pharmaceutical compositions may be
specially formulated
for administration in solid or liquid form, including those adapted for the
following: oral
administration, for example, drenches (aqueous or non-aqueous solutions or
suspensions),
tablets, e.g., those targeted for buccal, sublingual, and systemic absorption,
boluses, powders,
granules, pastes for application to the tongue; parenteral administration, for
example, by
subcutaneous, intramuscular, intravenous or epidural injection as, for
example, a sterile solution
or suspension, or sustained-release formulation; topical application, for
example, as a cream,
ointment, or a controlled-release patch or spray applied to the skin, lungs,
or oral cavity;
intravaginally or intrarectally, for example, as a pessary, cream, or foam;
sublingually; ocularly;
transdermally; or nasally, pulmonary, and to other mucosal surfaces.
[00113] Pharmaceutically acceptable: The term "pharmaceutically acceptable"
as used
herein, refers to agents that, within the scope of sound medical judgment, are
suitable for use in
contact with tissues of human beings and/or animals without excessive
toxicity, irritation,
allergic response, or other problem or complication, commensurate with a
reasonable benefit/risk
ratio.
[00114] Pharmaceutically acceptable carrier: As used herein, the term
"pharmaceutically
acceptable carrier" means a pharmaceutically-acceptable material, composition
or vehicle, such
as a liquid or solid filler, diluent, excipient, or solvent encapsulating
material, involved in
carrying or transporting the subject compound from one organ, or portion of
the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in the sense
of being
compatible with the other ingredients of the formulation and not injurious to
the patient. Some
examples of materials which can serve as pharmaceutically-acceptable carriers
include: sugars,
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such as lactose, glucose and sucrose; starches, such as corn starch and potato
starch; cellulose,
and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose
and cellulose
acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and
soybean oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar;
buffering agents, such as
magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic
saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,
polycarbonates and/or
polyanhydrides; and other non-toxic compatible substances employed in
pharmaceutical
formulations.
[00115] Polypeptide: The term "polypeptide", as used herein, generally has
its art-
recognized meaning of a polymer of at least three amino acids, linked to one
another by peptide
bonds. In certain embodiments, a polypeptide may include at least 3-5 amino
acids, each of
which is attached to others by way of at least one peptide bond. Those of
ordinary skill in the art
will appreciate that polypeptides sometimes include "non-natural" amino acids
or other entities
that nonetheless are capable of integrating into a polypeptide chain. Those of
ordinary skill in
the art understand that protein sequences generally tolerate some substitution
without destroying
activity. Thus, in certain embodiments, any polypeptide that retains activity
and shares at least
about 30-40% overall sequence identity, often greater than about 50%, 60%,
70%, or 80%, and
further usually including at least one region of much higher identity, often
greater than 90% or
even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions,
usually
encompassing at least 3-4 and often up to 20 or more amino acids, with another
polypeptide
(e.g., a reference polypeptide) is considered to be of the same class as that
other polypeptide. In
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certain embodiments, polypeptides of a particular class all show significant
sequence homology
or identity with a relevant reference polypeptide. In many embodiments, such
member also
shares significant activity with the reference polypeptide. For example, in
certain embodiments,
a member polypeptide shows an overall degree of sequence homology or identity
with a
reference polypeptide that is at least about 30-40%, and is often greater than
about 50%, 60%,
70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or
includes at
least one region (i.e., a conserved region, often including a characteristic
sequence element) that
shows very high sequence identity, often greater than 90% or even 95%, 96%,
97%, 98%, or
99%. Such a conserved region usually encompasses at least 3-4 and often up to
20 or more
amino acids; in certain embodiments, a conserved region encompasses at least
one stretch of at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino
acids. In certain
embodiments, a polypeptide may contain L-amino acids, D-amino acids, or both.
In certain
embodiments, a polypeptide may contain any of a variety of amino acid
modifications or analogs
known in the art. For example, in certain embodiments, a polypeptide may
include one more
modifications such as, e.g., terminal acetylation, amidation, methylation,
etc. In certain
embodiments, a polypeptide may comprise natural amino acids, non-natural amino
acids,
synthetic amino acids, and/or combinations thereof. The term "peptide" is
generally used to
refer to a polypeptide having a length of less than about 100 amino acids,
less than about 50
amino acids, less than 20 amino acids, or less than 10 amino acids. In certain
embodiments,
proteins are antibodies, antibody fragments, biologically active portions
thereof, and/or
characteristic portions thereof.
[00116] Prevention: The term "prevention", as used herein, refers to a
delay of onset,
and/or reduction in frequency and/or severity of one or more symptoms of a
particular disease,
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disorder or condition. In certain embodiments, prevention is assessed on a
population basis such
that an agent is considered to "prevent" a particular disease, disorder or
condition if a statistically
significant decrease in the development, frequency, and/or intensity of one or
more symptoms of
the disease, disorder or condition is observed in a population susceptible to
the disease, disorder,
or condition
[00117] Prognostic and predictive information: As used herein, the terms
"prognostic
information" and "predictive information" are used to refer to any information
that may be used
to indicate any aspect of the course of a disease or condition either in the
absence or presence of
treatment. Such information may include, but is not limited to, the average
life expectancy of a
patient, the likelihood that a patient will survive for a given amount of time
(e.g., 6 months, 1
year, 5 years, etc.), the likelihood that a patient will be cured of a
disease, the likelihood that a
patient's disease will respond to a particular therapy (wherein response may
be defined in any of
a variety of ways). Prognostic and predictive information are included within
the broad category
of diagnostic information.
[00118] Protein: As used herein, the term "protein" refers to a polypeptide
(i.e., a string
of at least two amino acids linked to one another by peptide bonds). Proteins
may include
moieties other than amino acids (e.g., may be glycoproteins, proteoglycans,
etc.) and/or may be
otherwise processed or modified. Those of ordinary skill in the art will
appreciate that a
"protein" can be a complete polypeptide chain as produced by a cell (with or
without a signal
sequence), or can be a characteristic portion thereof Those of ordinary skill
will appreciate that
a protein can sometimes include more than one polypeptide chain, for example
linked by one or
more disulfide bonds or associated by other means. Polypeptides may contain L-
amino acids, D-
amino acids, or both and may contain any of a variety of amino acid
modifications or analogs
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known in the art. Useful modifications include, e.g., terminal acetylation,
amidation,
methylation, etc. In certain embodiments, proteins may comprise natural amino
acids, non-
natural amino acids, synthetic amino acids, and combinations thereof. The term
"peptide" is
generally used to refer to a polypeptide having a length of less than about
100 amino acids, less
than about 50 amino acids, less than 20 amino acids, or less than 10 amino
acids. In certain
embodiments, proteins are antibodies, antibody fragments, biologically active
portions thereof,
and/or characteristic portions thereof
[00119]
Reference: The term "reference" is often used herein to describe a standard or
control agent, individual, population, sample, sequence or value against which
an agent,
individual, population, sample, sequence or value of interest is compared. In
certain
embodiments, a reference agent, individual, population, sample, sequence or
value is tested
and/or determined substantially simultaneously with the testing or
determination of the agent,
individual, population, sample, sequence or value of interest. In certain
embodiments, a
reference agent, individual, population, sample, sequence or value is a
historical reference,
optionally embodied in a tangible medium. Typically, as would be understood by
those skilled
in the art, a reference agent, individual, population, sample, sequence or
value is determined or
characterized under conditions comparable to those utilized to determine or
characterize the
agent, individual, population, sample, sequence or value of interest.
[00120]
Response: As used herein, a response to treatment may refer to any beneficial
alteration in a subject's condition that occurs as a result of or correlates
with treatment. Such
alteration may include stabilization of the condition (e.g., prevention of
deterioration that would
have taken place in the absence of the treatment), amelioration of symptoms of
the condition,
and/or improvement in the prospects for cure of the condition, etc. It may
refer to a subject's
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response or to a tumor's response. Tumor or subject response may be measured
according to a
wide variety of criteria, including clinical criteria and objective criteria.
Techniques for
assessing response include, but are not limited to, clinical examination,
positron emission
tomatography, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy,
presence or level
of tumor markers in a sample obtained from a subject, cytology, and/or
histology. Many of these
techniques attempt to determine the size of a tumor or otherwise determine the
total tumor
burden. Methods and guidelines for assessing response to treatment are
discussed in Therasse et.
al., "New guidelines to evaluate the response to treatment in solid tumors",
European
Organization for Research and Treatment of Cancer, National Cancer Institute
of the United
States, National Cancer Institute of Canada, I Natl. Cancer Inst., 2000,
92(3):205-216. The
exact response criteria can be selected in any appropriate manner, provided
that when comparing
groups of tumors and/or patients, the groups to be compared are assessed based
on the same or
comparable criteria for determining response rate. One of ordinary skill in
the art will be able to
select appropriate criteria.
[00121] Risk: As will be understood from context, a "risk" of a disease,
disorder or
condition is a degree of likelihood that a particular individual will develop
the disease, disorder,
or condition. In certain embodiments, risk is expressed as a percentage. In
certain embodiments,
risk is from 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 100%. In certain
embodiments risk is expressed as
a risk relative to a risk associated with a reference sample or group of
reference samples. In
certain embodiments, a reference sample or group of reference samples have a
known risk of a
disease, disorder, or condition. In certain embodiments a reference sample or
group of reference
samples are from individuals comparable to a particular individual. In certain
embodiments,
relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
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[00122] Sample: As used herein, the term "sample" typically refers to a
biological sample
obtained or derived from a source of interest, as described herein. In certain
embodiments, a
source of interest comprises an organism, such as an animal or human. In
certain embodiments,
a biological sample is or comprises biological tissue or fluid. In certain
embodiments, a
biological sample may be or comprise bone marrow; blood; blood cells; ascites;
tissue or fine
needle biopsy samples; cell-containing body fluids; free floating nucleic
acids; sputum; saliva;
urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph;
gynecological fluids; skin
swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a
ductal lavages or
broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue
biopsy
specimens; surgical specimens; feces, other body fluids, secretions, and/or
excretions; and/or
cells therefrom, etc. In certain embodiments, a biological sample is or
comprises cells obtained
from an individual. In certain embodiments, obtained cells are or include
cells from an
individual from whom the sample is obtained. In certain embodiments, a sample
is a "primary
sample" obtained directly from a source of interest by any appropriate means.
For example, in
certain embodiments, a primary biological sample is obtained by methods
selected from the
group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy),
surgery, collection of
body fluid (e.g., blood, lymph, feces etc.), etc. In certain embodiments, as
will be clear from
context, the term "sample" refers to a preparation that is obtained by
processing (e.g., by
removing one or more components of and/or by adding one or more agents to) a
primary sample.
For example, filtering using a semi-permeable membrane. Such a "processed
sample" may
comprise, for example nucleic acids or proteins extracted from a sample or
obtained by
subjecting a primary sample to techniques such as amplification or reverse
transcription of
mRNA, isolation and/or purification of certain components, etc.
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[00123] Small molecule: As used herein, the term "small molecule" means a
low
molecular weight organic and/or inorganic compound. In general, a "small
molecule" is a
molecule that is less than about 5 kilodaltons (kD) in size. In certain
embodiments, a small
molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In certain
embodiments, the
small molecule is less than about 800 daltons (D), about 600 D, about 500 D,
about 400 D, about
300 D, about 200 D, or about 100 D. In certain embodiments, a small molecule
is less than
about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less
than about 800
g/mol, or less than about 500 g/mol. In certain embodiments, a small molecule
is not a polymer.
In certain embodiments, a small molecule does not include a polymeric moiety.
In certain
embodiments, a small molecule is not a protein or polypeptide (e.g., is not an
oligopeptide or
peptide). In certain embodiments, a small molecule is not a polynucleotide
(e.g., is not an
oligonucleotide). In certain embodiments, a small molecule is not a
polysaccharide. In certain
embodiments, a small molecule does not comprise a polysaccharide (e.g., is not
a glycoprotein,
proteoglycan, glycolipid, etc.). In certain embodiments, a small molecule is
not a lipid. In
certain embodiments, a small molecule is a modulating agent. In certain
embodiments, a small
molecule is biologically active. In certain embodiments, a small molecule is
detectable (e.g.,
comprises at least one detectable moiety). In certain embodiments, a small
molecule is a
therapeutic.
[00124] Specific binding: As used herein, the terms "specific binding" or
"specific for"
or "specific to" refer to an interaction (typically non-covalent) between a
target entity (e.g., a
target protein or polypeptide) and a binding agent (e.g., an antibody, such as
a provided
antibody). As will be understood by those of ordinary skill, an interaction is
considered to be
"specific" if it is favored in the presence of alternative interactions. In
many embodiments, an
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interaction is typically dependent upon the presence of a particular
structural feature of the target
molecule such as an antigenic determinant or epitope recognized by the binding
molecule. For
example, if an antibody is specific for epitope A, the presence of a
polypeptide containing
epitope A or the presence of free unlabeled A in a reaction containing both
free labeled A and the
antibody thereto, will reduce the amount of labeled A that binds to the
antibody. It is to be
understood that specificity need not be absolute. For example, it is well
known in the art that
numerous antibodies cross-react with other epitopes in addition to those
present in the target
molecule. Such cross-reactivity may be acceptable depending upon the
application for which the
antibody is to be used. In particular embodiments, an antibody specific for
receptor tyrosine
kinases has less than 10% cross-reactivity with receptor tyrosine kinase bound
to protease
inhibitors (e.g., ACT). One of ordinary skill in the art will be able to
select antibodies having a
sufficient degree of specificity to perform appropriately in any given
application (e.g., for
detection of a target molecule, for therapeutic purposes, etc.). Specificity
may be evaluated in
the context of additional factors such as the affinity of the binding molecule
for the target
molecule versus the affinity of the binding molecule for other targets (e.g.,
competitors). If a
binding molecule exhibits a high affinity for a target molecule that it is
desired to detect and low
affinity for non-target molecules, the antibody will likely be an acceptable
reagent for
immunodiagnostic purposes. Once the specificity of a binding molecule is
established in one or
more contexts, it may be employed in other, preferably similar, contexts
without necessarily re-
evaluating its specificity.
[00125]
Specific: The term "specific", when used herein with reference to an agent or
entity having an activity, is understood by those skilled in the art to mean
that the agent or entity
discriminates between potential targets or states. For example, an agent is
said to bind
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"specifically" to its target if it binds preferentially with that target in
the presence of competing
alternative targets. In certain embodiments, the agent or entity does not
detectably bind to the
competing alternative target under conditions of binding to its target. In
certain embodiments,
the agent or entity binds with higher on-rate, lower off-rate, increased
affinity, decreased
dissociation, and/or increased stability to its target as compared with the
competing alternative
target(s).
[00126] Stage of cancer: As used herein, the term "stage of cancer" refers
to a qualitative
or quantitative assessment of the level of advancement of a cancer. Criteria
used to determine
the stage of a cancer include, but are not limited to, the size of the tumor
and the extent of
metastases (e.g., localized or distant).
[00127] Subject: By "subject" is meant a mammal (e.g., a human, in certain
embodiments
including prenatal human forms). In certain embodiments, a subject is
suffering from a relevant
disease, disorder or condition. In certain embodiments, a subject is
susceptible to a disease,
disorder, or condition. In certain embodiments, a subject displays one or more
symptoms or
characteristics of a disease, disorder or condition. In certain embodiments, a
subject does not
display any symptom or characteristic of a disease, disorder, or condition. In
certain
embodiments, a subject is someone with one or more features characteristic of
susceptibility to
or risk of a disease, disorder, or condition. A subject can be a patient,
which refers to a human
presenting to a medical provider for diagnosis or treatment of a disease. In
certain embodiments,
a subject is an individual to whom therapy is administered.
[00128] Substantially: As used herein, the term "substantially" refers to
the qualitative
condition of exhibiting total or near-total extent or degree of a
characteristic or property of
interest. One of ordinary skill in the biological arts will understand that
biological and chemical
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phenomena rarely, if ever, go to completion and/or proceed to completeness or
achieve or avoid
an absolute result. The term "substantially" is therefore used herein to
capture the potential lack
of completeness inherent in many biological and chemical phenomena.
[00129] Suffering from: An individual who is "suffering from" a disease,
disorder, or
condition has been diagnosed with and/or exhibits or has exhibited one or more
symptoms or
characteristics of the disease, disorder, or condition.
[00130] Susceptible to: An individual who is "susceptible to" a disease,
disorder, or
condition (e.g., influenza) is at risk for developing the disease, disorder,
or condition. In certain
embodiments, an individual who is susceptible to a disease, disorder, or
condition does not
display any symptoms of the disease, disorder, or condition. In certain
embodiments, an
individual who is susceptible to a disease, disorder, or condition has not
been diagnosed with the
disease, disorder, and/or condition. In certain embodiments, an individual who
is susceptible to a
disease, disorder, or condition is an individual who has been exposed to
conditions associated
with development of the disease, disorder, or condition. In certain
embodiments, a risk of
developing a disease, disorder, and/or condition is a population-based risk
(e.g., family members
of individuals suffering from the disease, disorder, or condition).
[00131] Symptoms are reduced: According to the present disclosure,
"symptoms are
reduced" when one or more symptoms of a particular disease, disorder or
condition is reduced in
magnitude (e.g., intensity, severity, etc.) or frequency. For purposes of
clarity, a delay in the
onset of a particular symptom is considered one form of reducing the frequency
of that symptom.
[00132] Therapeutic agent: As used herein, the phrase "therapeutic agent"
refers to any
agent that elicits a desired pharmacological effect when administered to an
organism. In certain
embodiments, an agent is considered to be a therapeutic agent if it
demonstrates a statistically
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significant effect across an appropriate population. In certain embodiments,
the appropriate
population may be a population of model organisms. In certain embodiments, an
appropriate
population may be defined by various criteria, such as a certain age group,
gender, genetic
background, preexisting clinical conditions, etc. In certain embodiments, a
therapeutic agent is
any substance that can be used to alleviate, ameliorate, relieve, inhibit,
prevent, delay onset of,
reduce severity of, and/or reduce incidence of one or more symptoms or
features of a disease,
disorder, and/or condition.
[00133] Therapeutic regimen: A "therapeutic regimen", as that term is used
herein, refers
to a dosing regimen whose administration across a relevant population is
correlated with a
desired or beneficial therapeutic outcome.
[00134] Therapeutically effective amount: As used herein, the term
"therapeutically
effective amount" refers to an amount of a therapeutic protein (e.g., receptor
tyrosine kinases
antibody) which confers a therapeutic effect on the treated subject, at a
reasonable benefit/risk
ratio applicable to any medical treatment. The therapeutic effect may be
objective (i.e.,
measurable by some test or marker) or subjective (i.e., subject gives an
indication of or feels an
effect). In particular, the "therapeutically effective amount" refers to an
amount of a therapeutic
protein or composition effective to treat, ameliorate, or prevent a desired
disease or condition, or
to exhibit a detectable therapeutic or preventative effect, such as by
ameliorating symptoms
associated with the disease, preventing or delaying the onset of the disease,
and/or also lessening
the severity or frequency of symptoms of the disease. A therapeutically
effective amount is
commonly administered in a dosing regimen that may comprise multiple unit
doses. For any
particular therapeutic protein, a therapeutically effective amount (and/or an
appropriate unit dose
within an effective dosing regimen) may vary, for example, depending on route
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administration, on combination with other pharmaceutical agents. Also, the
specific
therapeutically effective amount (and/or unit dose) for any particular patient
may depend upon a
variety of factors including the disorder being treated and the severity of
the disorder; the activity
of the specific pharmaceutical agent employed; the specific composition
employed; the age,
body weight, general health, sex and diet of the patient; the time of
administration, route of
administration, and/or rate of excretion or metabolism of the specific fusion
protein employed;
the duration of the treatment; and like factors as is well known in the
medical arts.
[00135] Treatment: As used herein, the term "treatment" (also "treat" or
"treating") refers
to any administration of a substance (e.g., provided compositions) that
partially or completely
alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity
of, and/or reduces
incidence of one or more symptoms, features, and/or causes of a particular
disease, disorder,
and/or condition (e.g., influenza). Such treatment may be of a subject who
does not exhibit signs
of the relevant disease, disorder and/or condition and/or of a subject who
exhibits only early
signs of the disease, disorder, and/or condition. Alternatively or
additionally, such treatment
may be of a subject who exhibits one or more established signs of the relevant
disease, disorder
and/or condition. In certain embodiments, treatment may be of a subject who
has been
diagnosed as suffering from the relevant disease, disorder, and/or condition.
In certain
embodiments, treatment may be of a subject known to have one or more
susceptibility factors
that are statistically correlated with increased risk of development of the
relevant disease,
disorder, and/or condition.
[00136] Unit dose: The expression "unit dose" as used herein refers to an
amount
administered as a single dose and/or in a physically discrete unit of a
pharmaceutical
composition. In many embodiments, a unit dose contains a predetermined
quantity of an active
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agent. In certain embodiments, a unit dose contains an entire single dose of
the agent. In certain
embodiments, more than one unit dose is administered to achieve a total single
dose. In certain
embodiments, administration of multiple unit doses is required, or expected to
be required, in
order to achieve an intended effect. A unit dose may be, for example, a volume
of liquid (e.g.,
an acceptable carrier) containing a predetermined quantity of one or more
therapeutic agents, a
predetermined amount of one or more therapeutic agents in solid form, a
sustained release
formulation or drug delivery device containing a predetermined amount of one
or more
therapeutic agents, etc. It will be appreciated that a unit dose may be
present in a formulation
that includes any of a variety of components in addition to the therapeutic
agent(s). For example,
acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents,
stabilizers, buffers,
preservatives, etc., may be included as described infra. It will be
appreciated by those skilled in
the art, in many embodiments, a total appropriate daily dosage of a particular
therapeutic agent
may comprise a portion, or a plurality, of unit doses, and may be decided, for
example, by the
attending physician within the scope of sound medical judgment. In certain
embodiments, the
specific effective dose level for any particular subject or organism may
depend upon a variety of
factors including the disorder being treated and the severity of the disorder;
activity of specific
active compound employed; specific composition employed; age, body weight,
general health,
sex and diet of the subject; time of administration, and rate of excretion of
the specific active
compound employed; duration of the treatment; drugs and/or additional
therapies used in
combination or coincidental with specific compound(s) employed, and like
factors well known in
the medical arts.
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Detailed Description
[00137] As described herein, the present disclosure provides technologies
that utilize
certain magnetic particles, and particularly utilize targeted such particles,
to apply mechanical
force to a target structure, through application of dynamic magnetic field
(DMF) treatment. In
particular embodiments, the present disclosure provides technologies for
applying mechanical
force to cellular membranes and/or to intracellular membranes. In certain
embodiments, the
present disclosure provides technologies for disrupting or otherwise damaging
or injuring (e.g.,
permeabilizing) cellular membranes and/or intracellular membranes. In certain
embodiments,
the present disclosure provides technologies for inducing or promoting
apoptosis and/or cell
death or destruction. In certain embodiments, the present d provides
technologies for inducing
and/or stimulating apoptosis and/or cell death or destruction in malignant
(e.g., cancer, tumor,
and/or metastatic) cells.
[00138] Among other advantages, provided technologies can achieve
application of
mechanical force, and particularly can achieve specific application of
mechanical force (i.e., to a
particular, e.g., preselected, site of interest) without generation of heat.
Provided technologies
show a variety of advantages as compared with other strategies for inducing
and/or stimulating
apoptosis and/or cell death or destruction, including those that utilize
magnetic particles, many of
which generate heat, with undesirable effect(s).
Dynamic Magnetic Field Treatment
[00139] Provided technologies utilize magnetic particles (e.g.,
superparamagnetic,
ferromagnetic, or ferrimagnetic particles, particularly nanoparticles) that
respond to application
of dynamic magnetic field (DMF) treatment.
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[00140] Certain such particles, superparamagnetic iron oxide nanoparticles
("SPIONs"),
have found wide-spread applications in the biomedical field spanning in vitro
diagnostic tests
such as nanosensors, in vivo imaging and therapies such as magnetic fluid
hyperthermia or drug
delivery. Recent investigations have also explored the capability of
controlling the position or
temperature of magnetic nanoparticles (e.g., SPIONs) within cells and tissues
by remote
application of magnetic fields. So far, this possibility has been investigated
using permanent
magnets that set nanoparticles in a longitudinal motion, using alternating
magnetic fields, or
through rotating permanent magnets outside of the tissues of interest. In the
latter scenario, the
nanoparticles describe circular motions, but do not individually rotate around
their own axis.
The combination of alternating magnetic fields and magnetic nanoparticles
allows one to
transform energy into forces or heat. Hyperthermia is used as an adjunctive
treatment in cancer
therapy; here, high-frequency alternating (but not moving) magnetic fields in
the KHz/MHz
range have been used to kill cancer cells loaded with magnetic nanoparticles
through thermal
induction. However, such treatment is not without risks particularly near
thermally sensitive
structures such as the gut or gallbladder if nanoparticles are injected
systemically, as the heat
induction cannot be controlled spatially with high precision and could cause
tissue necrosis.
Therefore, in contrast to thermal ablation systems, ambient temperature
increases greater than
46 C are not desirable for purposes of remote controlling apoptosis with
magnetic fields.
[00141] Fundamentally different from prior studies using high frequency
alternating
magnetic fields that cause apoptosis via heat induction, the present
disclosure describes a
principle of controlling particle rotation and inducing apoptosis via
mechanical forces exerted on
membranes by targeted particles. Specifically, the present disclosure utilizes
dynamic magnetic
field (DMF) treatment to induce and precisely control the rotation of magnetic
nanoparticles
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around their own axis. DMF treatment creates a dynamic force field, which is
converted inside
the particle into a magnetic flux field B, which operates on a particle with a
magnetic Moment M
and a moment of inertia I. The field generates a torque i equal to = x .
This approach
enables for the first time the ability to induce rotation of individual
magnetic particles around
their own axis, and furthermore allows precise control of the rotation speed.
The present
disclosure therefore provides technologies that permit specific application of
mechanical force
via remote triggering of individual magnetic particle rotation about its own
axis. Among other
advantages, the present disclosure achieves such rotation, and therefore such
application of
mechanical force, without generating heat (e.g., without heating the
particle).
[00142] The present disclosure specifically exemplifies induction of this
kind of rotation
in certain magnetic particles (e.g., superparamagnetic nanoparticles) bound to
a target structure,
and demonstrates that it can be used to remotely apply mechanical force to a
target structure.
The present disclosure particularly demonstrates that such magnetic particles
can be targeted to
an intracellular site, internalized into cells, and bound to the targeted
site. Furthermore, the
present disclosure demonstrates that subsequent remote activation of a dynamic
magnetic field
causes the bound particles to exert mechanical force on the target structure.
The particular
context exemplified here represents a specific biological application; those
skilled in the art will
appreciate that the same principle should enable many other new applications
in the fields of
nanotechnology and nanomedicine.
[00143] In certain embodiments, provided technologies do not utilize high-
frequency (e.g.,
above the Hz range, for example in the kHz [sometimes considered to be medium-
frequency],
MHz, or GHz range) magnetic field oscillations. In many embodiments, provided
technologies
utilize low-frequency (e.g., within the Hz range) oscillations.
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[00144] In many embodiments, the present disclosure applies a DMF
treatment (e.g.,
utilizes a DMF device, for example such as that described in German patent no.
DE 10 2005 030
986) that does not heat the particles. In certain embodiments, the present
disclosure applies a
DMF treatment that does not aggregate the particles. One feature of the
present disclosure is
that, in many embodiments, it applies a DMF treatment that rotates individual
particles about
their axes, without also necessarily moving the particles in a direction
(e.g., in a circulatory
motion) or moving them in bulk. Such individual rotation about a particle's
own axis, as
provided herein, is particularly and surprisingly useful in the application of
mechanical force,
especially to structures such as cell membranes or intracellular membranes,
for example as
exemplified herein.
Particles
[00145] Particles for use in accordance with the present disclosure
include any particles
responsive to DMF as described herein so that their movement, and particularly
their rotation
about their axis, can be specifically controlled.
[00146] In certain embodiments, the present disclosure utilizes magnetic
particles with a
high, positive magnetic susceptibility, including for example,
superparamagnetic, paramagnetic,
ferromagnetic, or ferrimagnetic particles.
[00147] In certain embodiments, the present disclosure utilizes
nanoparticles (i.e.,
particles whose longest dimension is below 1 um). In certain embodiments, the
present
disclosure utilizes nanoparticles whose longest dimension is less than about
300 nm; on some
embodiments such nanoparticles are particularly useful for delivery into or
onto cells. In certain
embodiments, the present disclosure utilizes nanoparticles whose longest
dimension is less than
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about 200 nm. In certain embodiments, the present disclosure utilizes
nanoparticles whose
longest dimension is less than about 100 nm. In certain embodiments, the
present disclosure
utilizes nanoparticles whose size is within a range of about 10 - about 500
nm, or within a range
of about 10, about 20, about 30, about 40 or about 50 nm to about 100 nm,
about 200 nm, or
about 300 nm.
[00148] Those of ordinary skill in the art, reading the present
specification, would
immediately be aware of a variety of appropriate particles, many of which are
commercially
available, for use in accordance with the present disclosure. Exemplified
herein are
superparamagnetic iron oxide nanoparticles (SPIONs); those of ordinary skill
in the art will
readily appreciate the extent to which exemplified results can be expected to
be generalizable to
other particular nanoparticle formats. Relevant considerations include, for
example, that larger
magnetic moments can be generated with larger particles, but such can also
generate unwanted
heat. Additionally, some particles (e.g., certain ferromagnetic particles) may
tend to aggregate,
which could be undesirable particularly in certain biological contexts. Those
skilled in the art
will appreciate that certain available coating or other technologies may be
utilized in certain
embodiments to adjust particle characteristics (e.g., to reduce accumulation)
if desired.
[00149] In many embodiments, useful magnetic particles have a metal or
metal oxide core
(e.g., iron oxide), and particularly a ferromagnetic metal oxide core,
optionally associated or
coated with one or more inorganic (e.g., silica, gold) or organic (e.g.,
polypeptides, small
molecule, glycan, lipid and/or nucleic acid) components.
[00150] In certain embodiments, the nanoparticles are crystalline. In
certain
embodiments, the nanoparticles (e.g., crystalline iron oxide) amplify incoming
magnetic fields.
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[00151] In certain embodiments, useful magnetic particles are
characterized in that they
lose their magnetism when not exposed to an external magnetic field.
[00152] As noted above, the present specification specifically exemplifies
use of
superparamagnetic nanoparticles, and particularly superparamegnetic iron oxide
nanoparticles
(e.g., SPIONs; see Hofmann-Amtenbrink et al "Superparamagnetic Nanoparticles
for Biomedical
Applications" in Nanostructured Materials for Biomedical Applications, 2009).
In certain
embodiments, utilized SPIONs comprise a single ferrimagnetic unit (elementary
cell). In certain
embodiments one elementary cell includes 8Fe2+ ions. Use of such particles
permits precise
computation of parameters such as magnetic energy, torque, and/or mechanical
forces, as may
not be readily feasible with certain larger particles, for example with
changing internal structures
(e.g., Bloch walls).
[00153] Among other things, the present disclosure establishes that use of
specifically
targeted DMF-responsive magnetic particles (i.e., magnetic particles
associated with a targeting
agent that specifically binds to a target structure - e.g., to a target entity
or moiety in or on a
target structure of interest) shows surprising advantages, even as compared
with use of otherwise
identical magnetic particles not associated with the targeting agent. Those
skilled in the art will
be aware of a wide variety of appropriate targeting agents for use in
accordance with the present
disclosure, as appropriate for particular target entities or moieties of
interest.
[00154] In certain embodiments, a targeting agent is or comprises a
polypeptide, a small
molecule, a glycan, a lipid, and/or a nucleic acid. In some particular
embodiments, a targeting
agent is or comprises an antibody agent. In certain embodiments, a targeting
agent is or
comprises an antibody or antigen-binding portion thereof.
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Target Structures
[00155] Those skilled in the art, reading the present disclosure, will
immediately
appreciate that provided technologies are useful to direct appropriate
magnetic particles to any
of a variety of target structures, for application of mechanical force
thereto. For many
biomedical applications, target structures of particular interest are cells or
cell structures. In
some particular embodiments, target structures are cell membranes or
intracellular membranes.
In certain embodiments, target structures are organelles, for example selected
from the group
consisting of lysosomes, the endoplasmic reticulum (ER), the golgi apparatus,
the mitochondria.
[00156] In certain embodiments, a target structure, entity, or moiety is
or comprises a
surface marker. In certain embodiments, a surface marker is or comprises a
receptor, an enzyme,
a channel, etc.
[00157] In certain embodiments, a target structure, entity or moiety is or
comprises an
intracellular target. In certain embodiments, a target structure, entity or
moiety is or comprises a
marker specifically associated with a disease, disorder, or condition (e.g.,
with an infectious
disease, with cancer, etc.). In certain embodiments, a target structure,
entity, or moiety is or
comprises a surface marker is or comprises component of transcription
machinery, a splicosome,
or a ribosome.
[00158] The present disclosure particularly exemplifies targeting of
superparamagnetic
nanoparticles to lysosomal membranes, and application of DMF treatment so that
individual
superparamagnetic particles rotate about their axes and exert mechanical force
on the lysosomal
membrane. In certain embodiments, superparamagnetic nanoparticles are targeted
to one or
more lysosomal membrane components such as, for example, LAMP-1 (CD107a), LAMP-
2
(CD107b), or LAMP-3 (CD63).
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Applied Forces
[00159] In certain embodiments, application of DMF to magnetic particles
as described
herein exerts a force on the target structure that is within the range of
about 10-9 (i.e., nN range)
to about 10-12 (i.e., pN range); in certain embodiments within the range of
about 10-10 to 10-12
Newtons.
[00160] In certain embodiments, application of DMF to magnetic particles
as described
herein exerts torque on the target structure within the range of about 10-18
Nm ¨ 10-20 Nm. In
certain embodiments, the torque is about 10-18 Nm.
[00161] In certain embodiments, magnetic particles are delivered to a
target structure in
accordance with the present disclosure so that they interact with the target
structure at a density
sufficient to apply a desired force across an appropriate area.
[00162] As described herein, application of DMF treatment to appropriate
magnetic
particles bound to lysosomes at a density within the range of about 1-60
nanoparticles, on
average, per lysosome, is sufficient to achieve lysosomal disruption and/or
permeabilization. In
certain embodiments, magnetic particles are bound to lysosomes at an average
density of about
30 nanoparticles/lysosome.
Applications
[00163] In certain embodiments, application of DMF to magnetic particles
as described
herein is utilized to disrupt or otherwise damage or injure (e.g.,
permeabilize) the target structure
(e.g., a biological membrane). In some particular embodiments, the technology
described herein
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is utilized to induce apoptosis in cells. Advantageously, such disruption
(e.g., permeabilization)
and/or induction can be achieved without heating.
[00164] In particular embodiments, the inventive technology is utilized to
kill cancer cells.
The cytosol of normal human cells has a neutral pH value (pH about 7). The
surrounding of
normal human cells in the body has a pH about 7.1, also approximately neutral.
Lysosomes, by
contrast, have an acidic pH (pH about 5); lysosomal enzymes are typically only
active at such
low pH. The cytosol of cancerous cells, like that of normal human cells, has a
neutral pH value,
but their surroundings have an acidic pH. In certain embodiments, the present
disclosure
provides technologies to apply mechanical force to lysosomal membranes within
cancerous cells,
so that lysosomal enzymes are released into the cytosol, from which they are
pumped out of the
cells. In accordance with certain embodiments of the present disclosure, such
released lysosomal
enzymes actively become activated in the low-pH extracellular milieu that
surrounds cancerous
cells. They therefore digest materials, including cancer cells, within that
surrounding milieu.
Without wishing to be bound by any particular theory, it is contemplated that
such released
lysosomal enzymes will become inactivated once cancerous tissue is destroyed,
as normal tissues
have a neutral pH milieu.
Alternating current superconductor (ACSC) systems
[00165] In certain embodiments, the system used for DMF treatment includes
classical
aperture, e.g., copper windings, an iron chore with or without integrated
cooling. Such systems
may have limited penetration depth ¨ e.g., they may be able to produce a
magnetic field for
control of nanoparticles (internal to the subject's body) no greater than
about 1 cm away from the
field generator, located external to the subject's body. This limitation may
be due to the
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maximum current density in classical copper windings, e.g., about 2.5 A/mm2,
above which the
wire can burn. Thus, field amplitude is limited.
[00166] It is found that use of a superconducting wire, e.g., with maximum
current density
at least about 150 A/mm2 allows stronger magnetic fields (greater flux
density) and deeper
penetration depth for control of nanoparticles that are greater than 1 cm from
the field generator
at the surface of the subject's body, e.g., from 5 to 10 cm, or from 10 to 20
cm, or from 20 to 30
cm.
[00167] In vitro tests with INS-1 cells and a standard device showed that
an incoming
magnetic field density of 35 mT was strong enough to control the particles at
a 2 cm distance
from the device. For a standard device with an optimized cooling system, flux-
densities of 200
mT can be achieved at this distance. In contrast, a superconducting system
(such as the systems
described herein) produces larger flux-densities due to, for instance, higher
current densities. For
example, using the systems provided herein, Tesla values in the low Tesla
range (e.g., less than 3
T, e.g., less than 2 T, e.g., less than 1 T, e.g., about 200 mT) can be
achieved at larger (e.g., up to
20 cm) distances compared to the Tesla values and penetration depths of
standard lab-devices.
[00168] FIG. 13 depicts components that can be included in an ACSC system.
Components can include, not are not limited to, a transformer, a grid
connection, a converter, a
controller, a field unit (e.g., actuator), and cooler. In certain embodiments,
a field unit activates
magnetic particles (e.g., activates movement of the particles) due to its
magnetic field. In certain
embodiments, the system includes an actuator featuring a superconducting wire
(e.g., made of
high temperature superconductor material, HTS, e.g., a YBa2Cu3Ox (YBCO) coated
conductor).
In certain embodiments, superconducting systems establish field-density values
in the low Tesla
range. With HTS materials, the magnetic field strength achievable in an
alternating current (AC)
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system is from about 1 to 3 T for low frequencies, which is sufficient for
deep penetration depths
and applicable for a variety of working volumes. FIG. 15 shows a map profile
of the magnetic
field (B, T) as a function of distance (mm) produced by a field generator
system as described
herein. Improvements in cooling technologies and materials permit higher flux
densities.
[00169] The system also includes an alternating current (AC) power source,
a controller
(e.g., inverter and controlling system), and a cooling apparatus (e.g.,
cryogenic cooling system),
as illustrated in the schematic below.
[00170] The geometry of the actuator can be adapted for a given working
volume needed
for a particular patient and/or for a particular kind of treatment (e.g.,
particular type and/or
location of tissue being treated). Various geometries of the actuator ¨ e.g.,
flat, arc, and
toroid/cylinder/sphere/hemisphere are shown in the schematic below, together
with the working
volume applicable for the particular geometry. The working volume is the
volume in which
rotatable nanoparticles, as described herein, are located in vivo in a subject
and whose movement
(e.g., rotation) can be controlled by the externally-applied magnetic field
for the desired
treatment. For example, FIG. 14 depicts a schematic where an actuator
component of an ACSC
system is located near (e.g., underneath) a patient for a particular kind of
treatment. In certain
embodiments, the externally-applied magnetic field causes rotation of the
nanoparticles located
in the subject at a distance of up to 30 cm from the surface of the field
generator (which is
located outside the subject). For example, the magnetic field is effective to
rotate nanoparticles
located in the subject at a distance of 0 to 30 cm from the surface of the
field generator, or at a
distance of 0 to 25 cm, or 0 to 20 cm, or 0 to 15 cm, e.g., effective at a
distance no less than 10
cm, no less than 15, no less than 20 cm, no less than 25 cm, or no less than
30 cm from the
surface of the nearest field generator. In certain embodiments, multiple field
generators can be
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used, e.g., to provide for increased effective working volume of rotation of
nanoparticles. One or
more field generators can be shaped (e.g., as described above, including flat
beds, tubes, half-
tubes, etc.) and/or arranged to provide an effective working volume of
nanoparticle rotation over
a desired region of the subject, e.g., during treatment. In certain
embodiments, one or more
components are hand-held, e.g., such that the magnetic field can be moved over
the subject
during treatment.
[00171] With a flat actuator, there is a large working volume but a
smaller penetration
depth. The more circular (rounder) the actuator, the smaller the working
volume gets, but the
greater the penetration that is possible. The geometry of the actuator can be
adapted for a desired
penetration depth and location of in vivo tissue to be treated. Moreover, in
certain embodiments,
the actuator moves in relation to the subject (or the subject is moved in
relation to the actuator)
so that the working volume is adjusted over time, e.g., to treat a larger
volume of tissue at the
necessary depth.
[00172] Furthermore, in addition to adjusting actuator geometry, the
magnetic flux
distribution can be optimized for a desired working volume by adapting the
winding system for
the applied field, e.g. by varying the number of turns, the number of phases,
or the distribution of
the windings within the actuator, for example. The actuator is generally non-
homogeneous, with
ferromagnetic parts (iron teeth and yoke), paramagnetic parts (air, isolation)
and diamagnetic
parts (copper, superconductor). This may lead to ripples in the flux
distribution and saturation
effects. The voltage and/or current of the inverter can be controlled
according to the geometry of
the actuator to minimize the influence of these effects. Furthermore, the
efficiency of the
superconducting windings can be increased by deformation of the standard
sinusoidal current.
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[00173] In certain embodiments, the system is operated remotely, e.g., the
magnetic field
generator may be controlled remotely. Operation of the system may be performed
by a
practitioner (e.g., nurse, or doctor) from a location remote from the subject.
In certain
embodiments, components of the system (e.g., the field generator) are portable
and/or wearable.
Therapy by controlled movement of nanoparticles in in vivo tissue may require
treatment over
multiple sessions, or over one or more sessions that are lengthy (e.g., more
than an hour, two
hours, or longer). The size, remote operability, and/or portability of the
system may allow more
convenient treatment (e.g., cancer treatment), e.g., treatment in a clinical
as opposed to an
operating room, or even home treatment.
[00174] In certain embodiments, the magnetic field and consequently the
magnetic field
density depends on the field-producing current (e.g., where field ranges can
be calculated via the
"Maxwell Equations"). Values of field amplification inside nanoparticles
(e.g., crystalline iron
nanoparticles) and field-particle interactions (e.g., forces, e.g., moments)
can be computed for
anisotropic material (e.g., magnetic particles) to design systems and methods
for various
treatment applications.
Illustrative network environment
[00175] FIG. 11 shows an illustrative network environment 1100 for use in
the methods
and systems described herein. In brief overview, referring now to FIG. 11, a
block diagram of an
exemplary cloud computing environment 1100 is shown and described. The cloud
computing
environment 1100 may include one or more resource providers 1102a, 1102b,
1102c
(collectively, 1102). Each resource provider 1102 may include computing
resources. In some
implementations, computing resources may include any hardware and/or software
used to
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process data. For example, computing resources may include hardware and/or
software capable
of executing algorithms, computer programs, and/or computer applications. In
some
implementations, exemplary computing resources may include application servers
and/or
databases with storage and retrieval capabilities. Each resource provider 1102
may be connected
to any other resource provider 1102 in the cloud computing environment 1100.
In some
implementations, the resource providers 1102 may be connected over a computer
network 1108.
Each resource provider 1102 may be connected to one or more computing device
1104a, 1104b,
1104c (collectively, 1104), over the computer network 1108.
[00176] The cloud computing environment 1100 may include a resource
manager 1106.
The resource manager 1106 may be connected to the resource providers 1102 and
the computing
devices 1104 over the computer network 1108. In some implementations, the
resource manager
1106 may facilitate the provision of computing resources by one or more
resource providers
1102 to one or more computing devices 1104. The resource manager 1106 may
receive a request
for a computing resource from a particular computing device 1104. The resource
manager 1106
may identify one or more resource providers 1102 capable of providing the
computing resource
requested by the computing device 1104. The resource manager 1106 may select a
resource
provider 1102 to provide the computing resource. The resource manager 1106 may
facilitate a
connection between the resource provider 1102 and a particular computing
device 1104. In
some implementations, the resource manager 1106 may establish a connection
between a
particular resource provider 1102 and a particular computing device 1104. In
some
implementations, the resource manager 1106 may redirect a particular computing
device 1104 to
a particular resource provider 1102 with the requested computing resource.
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[00177] FIG. 12 shows an example of a computing device 1200 and a mobile
computing
device 1250 that can be used in the methods and systems described in this
disclosure. The
computing device 1200 is intended to represent various forms of digital
computers, such as
laptops, desktops, workstations, personal digital assistants, servers, blade
servers, mainframes,
and other appropriate computers. The mobile computing device 1250 is intended
to represent
various forms of mobile devices, such as personal digital assistants, cellular
telephones, smart-
phones, and other similar computing devices. The components shown here, their
connections
and relationships, and their functions, are meant to be examples only, and are
not meant to be
limiting.
[00178] The computing device 1200 includes a processor 1202, a memory
1204, a storage
device 1206, a high-speed interface 1208 connecting to the memory 1204 and
multiple high-
speed expansion ports 1210, and a low-speed interface 1212 connecting to a low-
speed
expansion port 1214 and the storage device 1206. Each of the processor 1202,
the memory
1204, the storage device 1206, the high-speed interface 1208, the high-speed
expansion ports
1210, and the low-speed interface 1212, are interconnected using various
busses, and may be
mounted on a common motherboard or in other manners as appropriate. The
processor 1202 can
process instructions for execution within the computing device 1200, including
instructions
stored in the memory 1204 or on the storage device 1206 to display graphical
information for a
GUI on an external input/output device, such as a display 1216 coupled to the
high-speed
interface 1208. In other implementations, multiple processors and/or multiple
buses may be
used, as appropriate, along with multiple memories and types of memory. Also,
multiple
computing devices may be connected, with each device providing portions of the
necessary
operations (e.g., as a server bank, a group of blade servers, or a multi-
processor system).
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[00179] The memory 1204 stores information within the computing device
1200. In some
implementations, the memory 1204 is a volatile memory unit or units. In some
implementations,
the memory 1204 is a non-volatile memory unit or units. The memory 1204 may
also be another
form of computer-readable medium, such as a magnetic or optical disk.
[00180] The storage device 1206 is capable of providing mass storage for
the computing
device 1200. In some implementations, the storage device 1206 may be or
contain a computer-
readable medium, such as a floppy disk device, a hard disk device, an optical
disk device, or a
tape device, a flash memory or other similar solid state memory device, or an
array of devices,
including devices in a storage area network or other configurations.
Instructions can be stored in
an information carrier. The instructions, when executed by one or more
processing devices (for
example, processor 1202), perform one or more methods, such as those described
above. The
instructions can also be stored by one or more storage devices such as
computer- or machine-
readable mediums (for example, the memory 1204, the storage device 1206, or
memory on the
processor 1202).
[00181] The high-speed interface 1208 manages bandwidth-intensive
operations for the
computing device 1200, while the low-speed interface 1212 manages lower
bandwidth-intensive
operations. Such allocation of functions is an example only. In some
implementations, the high-
speed interface 1208 is coupled to the memory 1204, the display 1216 (e.g.,
through a graphics
processor or accelerator), and to the high-speed expansion ports 1210, which
may accept various
expansion cards (not shown). In the implementation, the low-speed interface
1212 is coupled to
the storage device 1206 and the low-speed expansion port 1214. The low-speed
expansion port
1214, which may include various communication ports (e.g., USB, Bluetoothg,
Ethernet,
wireless Ethernet) may be coupled to one or more input/output devices, such as
a keyboard, a
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pointing device, a scanner, or a networking device such as a switch or router,
e.g., through a
network adapter.
[00182] The computing device 1200 may be implemented in a number of
different forms,
as shown in the figure. For example, it may be implemented as a standard
server 1220, or
multiple times in a group of such servers. In addition, it may be implemented
in a personal
computer such as a laptop computer 1222. It may also be implemented as part of
a rack server
system 1224. Alternatively, components from the computing device 1200 may be
combined
with other components in a mobile device (not shown), such as a mobile
computing device 1250.
Each of such devices may contain one or more of the computing device 1200 and
the mobile
computing device 1250, and an entire system may be made up of multiple
computing devices
communicating with each other.
[00183] The mobile computing device 1250 includes a processor 1252, a
memory 1264,
an input/output device such as a display 1254, a communication interface 1266,
and a transceiver
1268, among other components. The mobile computing device 1250 may also be
provided with
a storage device, such as a micro-drive or other device, to provide additional
storage. Each of
the processor 1252, the memory 1264, the display 1254, the communication
interface 1266, and
the transceiver 1268, are interconnected using various buses, and several of
the components may
be mounted on a common motherboard or in other manners as appropriate.
[00184] The processor 1252 can execute instructions within the mobile
computing device
1250, including instructions stored in the memory 1264. The processor 1252 may
be
implemented as a chipset of chips that include separate and multiple analog
and digital
processors. The processor 1252 may provide, for example, for coordination of
the other
components of the mobile computing device 1250, such as control of user
interfaces,
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applications run by the mobile computing device 1250, and wireless
communication by the
mobile computing device 1250.
[00185] The processor 1252 may communicate with a user through a control
interface
1258 and a display interface 1256 coupled to the display 1254. The display
1254 may be, for
example, a TFT (Thin-Film-Transistor Liquid Crystal Display) display or an
OLED (Organic
Light Emitting Diode) display, or other appropriate display technology. The
display interface
1256 may comprise appropriate circuitry for driving the display 1254 to
present graphical and
other information to a user. The control interface 1258 may receive commands
from a user and
convert them for submission to the processor 1252. In addition, an external
interface 1262 may
provide communication with the processor 1252, so as to enable near area
communication of the
mobile computing device 1250 with other devices. The external interface 1262
may provide, for
example, for wired communication in some implementations, or for wireless
communication in
other implementations, and multiple interfaces may also be used.
[00186] The memory 1264 stores information within the mobile computing
device 1250.
The memory 1264 can be implemented as one or more of a computer-readable
medium or media,
a volatile memory unit or units, or a non-volatile memory unit or units. An
expansion memory
1274 may also be provided and connected to the mobile computing device 1250
through an
expansion interface 1272, which may include, for example, a SIMM (Single In
Line Memory
Module) card interface. The expansion memory 1274 may provide extra storage
space for the
mobile computing device 1250, or may also store applications or other
information for the
mobile computing device 1250. Specifically, the expansion memory 1274 may
include
instructions to carry out or supplement the processes described above, and may
include secure
information also. Thus, for example, the expansion memory 1274 may be provided
as a security
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module for the mobile computing device 1250, and may be programmed with
instructions that
permit secure use of the mobile computing device 1250. In addition, secure
applications may be
provided via the SIMM cards, along with additional information, such as
placing identifying
information on the SIMM card in a non-hackable manner.
[00187] The memory may include, for example, flash memory and/or NVRAM
memory
(non-volatile random access memory), as discussed below. In some
implementations,
instructions are stored in an information carrier and, when executed by one or
more processing
devices (for example, processor 1252), perform one or more methods, such as
those described
above. The instructions can also be stored by one or more storage devices,
such as one or more
computer- or machine-readable mediums (for example, the memory 1264, the
expansion
memory 1274, or memory on the processor 1252). In some implementations, the
instructions
can be received in a propagated signal, for example, over the transceiver 1268
or the external
interface 1262.
[00188] The mobile computing device 1250 may communicate wirelessly
through the
communication interface 1266, which may include digital signal processing
circuitry where
necessary. The communication interface 1266 may provide for communications
under various
modes or protocols, such as GSM voice calls (Global System for Mobile
communications), SMS
(Short Message Service), EMS (Enhanced Messaging Service), or MMS messaging
(Multimedia
Messaging Service), CDMA (code division multiple access), TDMA (time division
multiple
access), PDC (Personal Digital Cellular), WCDMA (Wideband Code Division
Multiple Access),
CDMA2000, or GPRS (General Packet Radio Service), among others. Such
communication
may occur, for example, through the transceiver 1268 using a radio-frequency.
In addition,
short-range communication may occur, such as using a Bluetoothg, Wi-FiTM, or
other such
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transceiver (not shown). In addition, a GPS (Global Positioning System)
receiver module 1270
may provide additional navigation- and location-related wireless data to the
mobile computing
device 1250, which may be used as appropriate by applications running on the
mobile computing
device 1250.
[00189] The mobile computing device 1250 may also communicate audibly
using an
audio codec 1260, which may receive spoken information from a user and convert
it to usable
digital information. The audio codec 1260 may likewise generate audible sound
for a user, such
as through a speaker, e.g., in a handset of the mobile computing device 1250.
Such sound may
include sound from voice telephone calls, may include recorded sound (e.g.,
voice messages,
music files, etc.) and may also include sound generated by applications
operating on the mobile
computing device 1250.
[00190] The mobile computing device 1250 may be implemented in a number of
different
forms, as shown in the figure. For example, it may be implemented as a
cellular telephone 1280.
It may also be implemented as part of a smart-phone 1282, personal digital
assistant, or other
similar mobile device.
[00191] Various implementations of the systems and techniques described
here can be
realized in digital electronic circuitry, integrated circuitry, specially
designed ASICs (application
specific integrated circuits), computer hardware, firmware, software, and/or
combinations
thereof. These various implementations can include implementation in one or
more computer
programs that are executable and/or interpretable on a programmable system
including at least
one programmable processor, which may be special or general purpose, coupled
to receive data
and instructions from, and to transmit data and instructions to, a storage
system, at least one
input device, and at least one output device.
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[00192] These computer programs (also known as programs, software,
software
applications or code) include machine instructions for a programmable
processor, and can be
implemented in a high-level procedural and/or object-oriented programming
language, and/or in
assembly/machine language. As used herein, the terms machine-readable medium
and
computer-readable medium refer to any computer program product, apparatus
and/or device
(e.g., magnetic discs, optical disks, memory, Programmable Logic Devices
(PLDs)) used to
provide machine instructions and/or data to a programmable processor,
including a machine-
readable medium that receives machine instructions as a machine-readable
signal. The term
machine-readable signal refers to any signal used to provide machine
instructions and/or data to
a programmable processor.
[00193] To provide for interaction with a user, the systems and techniques
described here
can be implemented on a computer having a display device (e.g., a CRT (cathode
ray tube) or
LCD (liquid crystal display) monitor) for displaying information to the user
and a keyboard and
a pointing device (e.g., a mouse or a trackball) by which the user can provide
input to the
computer. Other kinds of devices can be used to provide for interaction with a
user as well; for
example, feedback provided to the user can be any form of sensory feedback
(e.g., visual
feedback, auditory feedback, or tactile feedback); and input from the user can
be received in any
form, including acoustic, speech, or tactile input.
[00194] The systems and techniques described here can be implemented in a
computing
system that includes a back end component (e.g., as a data server), or that
includes a middleware
component (e.g., an application server), or that includes a front end
component (e.g., a client
computer having a graphical user interface or a Web browser through which a
user can interact
with an implementation of the systems and techniques described here), or any
combination of
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such back end, middleware, or front end components. The components of the
system can be
interconnected by any form or medium of digital data communication (e.g., a
communication
network). Examples of communication networks include a local area network
(LAN), a wide
area network (WAN), and the Internet. In certain embodiments, the operating
unit is connected
to a magnetic field system (such as those described herein) via WLAN.
[00195] The computing system can include clients and servers. A client and
server are
generally remote from each other and typically interact through a
communication network. The
relationship of client and server arises by virtue of computer programs
running on the respective
computers and having a client-server relationship to each other.
[00196] In certain embodiments, the system (e.g., control unit) includes a
webserver
program that enables operation of the system. In certain embodiments, the
webserver is
managed with a standard web browser (e.g., tablet, smartphone, computer,
personal computer)
that is connected via a standard computer network.
Examples
Example 1:
[00197] The present Example demonstrates that use of DMF to induce
rotation as
described herein in targeted superparamagnetic iron oxide nanoparticles
(SPIONs) can be used to
remotely activate apoptosis. Specifically, the present Example demonstrates
that SPIONs
conjugated with LAMP1 (Lysosomal-associated membrane protein 1) antibodies
(LAMP1-
SPION) internalize into cells and bind to lysosomal membranes. This Example
further
demonstrates that subsequent remote activation of the dynamic magnetic field
causes mechanical
disruption and/or permeabilization of lysosomes, which leads to apoptosis via
extravasation of
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lysosomal contents into the cytoplasm and a decrease of intracellular pH. As
those skilled in the
art will appreciate, certain examples of SPIONs conjugated with LAMP1
antibodies are
described in Gruttner et al, "Synthesis and Antibody Conjugation of Magnetic
Nanoparticles
with Improved Specific Power Absorption Rates for Alternating Magnetic Field
Cancer
Therapy," Journal Magnetism and Magnetic Materials 2007.
RESULTS
Dynamic magnetic field stimulation results in rotation of individual
nanoparticles
[00198] A DNIF generator as described in German Patent No: DE 10 2005 030
986 was
utilized to control directional movement and self-centered rolling. FIG. 2A
shows a schematic
representation of a DNIF generator utilized in the present Exemplification.
The device controls
rotation and movement of magnetic nanoparticles (e.g., SPIONs) with a low
frequency (10-40
Hz) field. The DNIF generator causes the nanoparticles to rotate around their
own axis. To
demonstrate the pattern of the particle movement, the rotation of larger
magnetic beads of
different sizes (5.8, 1, 0.5 and 0.3 i_tm diameter) was first monitored by
filming them in a cell
culture dish under a microscope. Once the DMF is switched on, the beads start
to rotate around
their own axis, which also causes a slow directional movement of the beads
across the floor of
the dish, as shown in FIGS. 3A-3D. The present study clearly demonstrated that
the applied
DMF treatment enables a self-turning of magnetic particles. The speed of
rotation can be
controlled by varying the frequency setting on the DNIF device. This
observation suggested that
the DNIF could be used to contrive a virus-like interaction between the SPIONs
and the cell
surface, which in turn could enhance internalization of the SPIONs into the
cytosol. Once the
SPIONs have internalized into the intracellular compartments, e.g. endosomes
or lysosomes, the
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loaded SPIONs can be operated non-invasively by DMF to regulate the cellular
compartmental
activities and further cell functions, as for example, shown in FIG. 2B.
Dynamic magnetic field stimulation enhances uptake of superparamagnetic
nanoparticles
[00199] Internalization of SPIONs into living cells was reported
previously. In the present
study, the internalization efficiency in the absence or presence of DMF
stimulation is first
evaluated. To monitor the process of SPION internalization, the present study
used fluorescently
labeled (TRITC) 100-nm SPIONs and incubated them with rat insulinoma cells
(INS-1). 300-nm
SPIONs were also tested, but they exhibited markedly lower loading efficiency.
The present
study then applied a DMF field for 20 min. at a frequency of 20 Hz, before
staining the cells
using the plasma membrane marker CellMask, the lysosomal marker LysoTracker
Green and the
nuclear marker Hoechst 34580. The cells were then imaged by live confocal
microscopy, which
demonstrated that the majority of the SPIONs were loaded into the lysosomes
after 20 min. of
DMF treatment, as shown in FIG. 4A. FIG. 4B shows 71.2 3.8% of SPIONs
colocalized with
the lysosomal marker LysoTracker Green, while only 18.2 2.2% of SPIONs
colocalized with the
plasma membrane and early endosome probe CellMask. Conversely, FIG. 4C shows
91 8.7%
of the LysoTracker Green fluorescence appeared in conjunction with SPIONs,
which means that
nearly all lysosomes contained several loaded SPIONs.
[00200] Next the present study conjugated SPIONs with an antibody against
the lysosomal
membrane protein, LAMP1 (LAMP1-SPION). The loading efficiencies between SPION
and
LAMP1-SPION were compared in order to evaluate if the conjugation of the LAMP1
antibody
enhanced internalization and loading into the lysosomes. LAMP1-SPION
nanoparticles were
more efficiently loaded into the lysosomes than the unconjugated SPIONs, and
the loading
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efficiency after 20 min. FIG. 4D shows the loading efficiencies of DMF
treatment averaged
13.3 2.3% and 21.2 2.4% for SPIONs and LAMP1-SPIONs, respectively.
Dynamic magnetic field stimulation can injure lysosomes via antibody-
conjugated
superparamagnetic nanoparticles
[00201] To evaluate whether DMF treatment has the potential to injure
lysosomes in
LAMP1-SPION-loaded cells, the present study visualized the lysosome
compartment with the
marker LysoTracker Green. After DMF-facilitated loading of the LAMP1-SPION
nanoparticles,
the cells were cultured at 37 C for 40 min to allow binding of the antibody
paratope on the
nanoparticles to LAMP1 in the lysosomal membrane. After that culture period,
any remaining
LAMP1-SPION nanoparticles outside the cells were removed by washing, before
subjecting the
cells to DNIF treatment (20 Hz) for 20 minutes.
[00202] The capability of the DMF treatment to disrupt and/or permeabilize
the
compartments of the lysosomes was evaluated by assessing changes in
LysoTracker Green
fluorescence intensity. Indeed, in LAMP1-SPION-loaded cells, the DNIF
treatment significantly
decreased LysoTracker Green fluorescence by 75% as compared to cells loaded
with
conventional SPIONs without the LAMP1 antibody (483.6 84.2 A.U. vs. 120.3 20.9
A.U. for
SPION- and LAMP1-SPION-treated cells, respectively, as shown in FIGS. 5A and
5B. To
confirm these findings, the present study next used the acidotropic probe
(pKa=5.2) LysoSensor
Green DND 189 as described in Eto et al., "Glucose Metabolism and Glutamate
Analog Acutely
Alkalinize Ph of Insulin Secretory Vesicles of Pancreatic Beta-Cells,"
American Journal of
Physiology Endocrinology and Metabolism 2003. The rationale for this
experiment was that
disruption of lysosomes would reduce the volume of the very acidic
compartments in the cell and
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lead to a decrease in LysoSensor Green fluorescence. The results are shown in
FIGS. 5C and
5D.
[00203] Prior to DMF treatment, the present study found no difference in
fluorescence
intensity between SPION- and LAMP1-SPION-loaded cells. DNIF treatment had no
effect in
SPION-loaded cells. In contrast, in LAMP1-SPION-treated cells fluorescence
intensity dropped
(769.5 82.5 A.U./cell vs. 368.4 69.6 A.U./cell in SPION- vs. LAMP1-SPION
treated cells,
respectively; P<0.001). In light of the low frequency of the alternating
magnetic fields utilized
herein, heat induction, which is common if not ubiquitous when high frequency
alternating
magnetic fields are utilized, was not expected to occur. The present study
confirmed the absence
of heat induction by monitoring temperature runs in phantoms. These
experiments showed that
no significant change in temperature was caused by the DNIF induced magnetic
field, as shown
in the temperature plot of FIG. 6. Taken together, these results suggest that
the remote
application of the DNIF treatment causes permeabilization of lysosomal
compartments, induced
via the torque of the membrane-bound LAMP1-SPIONs.
Effects of dynamic magnetic field stimulation of superparamagnetic
nanoparticles in human
primary cells
[00204] To monitor the LAMP1-SPION loaded lysosomes, SPIONs are covalently
attached to both LAMP1 antibodies and the fluorescence marker TRITC (TRITC-
LAMP1-
SPION). To further validate the possibility to translate the present findings
to clinical settings,
the present study used isolated primary cells from human pancreatic islets and
seeded on glass
bottom petri dishes. Then TRITC-LAMP1-SPIONs were added to cell culture
medium, followed
by DNIF treatment or no treatment, and finally the cells were fixed for
confocal microscopy.
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[00205] Human primary islet cells contained larger lysosomes than INS-1
cells as assessed
by TRITC fluorescence (as shown in FIG. 7A, upper images). To assess co-
localization of
LAMP1 and TRITC-LAMP1-SPION, LAMP1 was detected by indirect
immunocytochemistry
using a Cy2-tagged secondary antibody. In cells not exposed to DMF treatment,
LAMP1 and
TRITC-LAMP1-SPION co-localized by 49.9 9.2%, which was not significantly
affected by
DMF treatment (54.8 9.5%) (Data not shown). These results indicate that the
bonds between
LAMP1 and TRITC remain stable during DMF treatment. Interestingly, the TRITC-
LAMP1-
SPIONs loaded into the cells mainly appeared around the boundaries of
structures, which are
likely to represent a location in the lysosomal membrane, as for example shown
in FIG. 7B.
However, after a second round of DMF treatment, most of the LAMP1 was
apparently separated
from the TRITC-LAMP1-SPIONs (as shown in FIG. 7A, bottom images) and the
SPIONs
aggregated tightly (as shown in FIG. 7C).
[00206] DMF treatment also led to a marked downward shift in the
distribution of
lysosomal sizes, as for example shown in FIG. 7D, and accordingly the average
size of
lysosomes decreased from 1.77 0.06 i_tm (n=141) to 0.79 0.05 i_tm (n=105)
after DMF
treatment, as shown in FIG. 7E. These results suggest that the second round of
DMF treatment
in cells loaded with TRITC-LAMP1-SPIONs results in a certain degree of damage
to the
lysosome membrane. However, a potential alternative explanation could be that
the detachment
of TRITC-LAMP1-SPIONs occurs from the lysosome membrane without disruption
which can
lead to the particles aggregating in the center of the still intact lysosomes.
[00207] To address this possibility, the subcellular locations of the
LAMP1-SPIONs were
further identified by transmission electrical microscopy (TEM). The TEM images
clearly
showed that LAMP1-SPION particles had accumulated within intracellular
compartments after
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loading into cells (as shown in FIG. 7F, left). In contrast, after the second
round of DMF
treatment, the LAMP1-SPIONs were scattered throughout the cells (as shown in
FIG. 7F, right).
These results demonstrate that DMF-induced rotational movement of LAMP1-SPIONs
are also
capable of disrupting lysosomal membranes in human primary cells.
Consequences of DMF-mediated disruption of lysosomes
[00208] Disruption of lysosomes has previously been reported to activate
apoptotic
reactions. To determine whether DMF treatment can elicit apoptosis in LAMP1-
SPION-loaded
cells, the present study measured the extent of apoptosis in INS-1 cells with
and without DMF.
Annexin V and 7-AAD were used to indicate early and late stage apoptosis,
respectively (as
shown in FIG. 8A). After DNIF treatment, early and late apoptosis in LAMP1-
SPION loaded
INS-1 cells significantly increased from 4.56 0.55% to 12.45 1.6% and from
0.73 0.17% to
1.31 0.16%, as evidenced by positive staining for Annexin V or 7-AAD,
respectively (shown in
FIGS. 8B and 8C). Furthermore, the elevated rates of apoptosis also had
consequences on cell
proliferation during culture. A single 20-minute DNIF treatment (20 Hz) in
SPION-loaded cells
had no significant effect on cell number during a 6-days culture period when
compared to control
cells. In contrast, the number of LAMP1-SPION loaded cells after DNIF
treatment was
significantly (p < 0.001) lower from day 2 and onwards (as shown in FIG. 8D).
These results
indicate that the attack on lysosomes via DMF-activated lysosomal membrane-
targeted SPIONs
prompts apoptotic cell death and affects the growth of the cell population.
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DISCUSSION
[00209] The present Example describes a novel biomedical platform based on
a unique
dynamic magnetic field generator, which in combination with superparamagnetic
nanoparticles
can be utilized for various new applications.
[00210] Several prior studies have investigated the effect of 'alternating
magnetic fields'
on magnetic nanoparticles. These studies generally used high-frequency
(typically MHz range),
or at least medium-frequency (e.g., kHz range), alternations in the magnetic
field polarity, and
observed that targeted iron oxide nanoparticles caused damage to cellular
membranes leading to
permeabilization. In most such studies, energy dissipated locally as heat by
the iron oxide
nanoparticles was thought to lead to (and therefore presumably to be required
for) disruption of
lipid bilayers. Huang et at., "Remote Control of Ion Channels and Neurons
through Magnetic-
Field Heating of Nanoparticles," Nature Nanotechnology 2010 demonstrated a
temperature-
induced change in fluorescence when a fluorochrome was attached to a magnetic
nanoparticle
and exposed to an alternating magnetic field, whereas no change was observed
in free
fluorochromes.
[00211] In contrast to these reports using high-frequency alternating (but
not dynamic)
magnetic fields, the present DMF approach uses low-frequency (¨ 10-20 Hz),
dynamic (i.e.
moving) magnetic fields of ¨30 mT that uniquely induce rotation of every
individual particle in
the field around their own axis (as illustrated in FIG. 2B). The speed of this
rotation (and its
direction) can be controlled by varying the frequency setting on the device.
For example, at
lower frequencies nanoparticles can be prompted to roll over cell membranes,
which may mimic
the movement of viruses along cell membrane surfaces, as for example,
described in Burckhardt
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et al., "Virus Movements on the Plasma Membrane Support Infection and
Transmission between
Cells,". PLoS Pathogen 2009, and increase the efficiency with which
nanoparticles internalize
into cells. Higher rotational speeds can be used in order to destroy
particular targets via
rotational shear forces without inducing unwanted thermal effects. Molecular
simulations of
lipid bilayers have shown that both incremental shear and tension can
destabilize cell
membranes, and that the energy that is required to cause such membrane damage
can be
achieved with rotating magnetic nanoparticles.
[00212] The present study has applied the DMF on LAMP1 antibody-conjugated
superparamagnetic iron oxide nanoparticles to facilitate nanoparticle uptake
into cells and to
disrupt the lysosomal membrane as a means to induce cell apoptosis. The DMF
approach
described herein has at least two major advantages: 1) Nanoparticles can be
rotated around their
own axis, and 2) No significant heat is created.
[00213] Heat creation is the presumed mechanism of how high-frequency
alternating
magnetic fields cause damage to cell membranes as suggested in Ivkov,
"Application of High
Amplitude Alternating Magnetic Fields for Heat Induction of Nanoparticles
Localized in
Cancer," Clinical Cancer Research 2005. This however can potentially cause
extensive and
unspecific cellular necrosis. By contrast, the DMF technology described herein
does not induce
heating; when applied in cellular systems or organisms, therefore, temperature
at the site can be
maintained within the physiological temperature range (e.g., typically
considered to be within a
range of about 37 C to about 42 C). Technologies described herein achieve
induction of
apoptosis specifically in nanoparticle-loaded cells only. Apoptotic cells are
removed in vivo by
endogenous scavenger systems e.g. the innate immune system and macrophages.
Tissue damage
is thus limited to only the targeted cells, in contrast to procedures leading
to supraphysiological
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temperatures and resultant necrosis, potentially sparing wide-spread acute
inflammatory
reactions.
[00214] In certain embodiments, provided methodologies achieve a
particularly high
degree of specificity of nanoparticle-mediated intervention, by targeting of
nanoparticles to a
particular cell type and/or into a desired subcellular compartment is
important. Among other
things, in certain embodiments, the provided technology platform represents an
example of
unique utilization of DMF s to target magnetic nanoparticles to specific
intracellular
compartments.
[00215] Previous reports have demonstrated the usefulness of magnetic
nanoparticles for
controlling activity of plasma membrane receptors or ion channels. Upon
protracted stimulation
of receptors or ion channels there is solid evidence for down-regulation of
their activities. One
important mechanism of such desensitization is internalization of receptors or
ion channels to
intracellular sites where they reside in an inactive standby pool. In the
context of nanoparticle-
mediated activation of receptors or ion channels this means that after
activation, the number of
the receptors/ion channels in plasma membrane decreases and the desired
cellular signals and
responses are blunted. Moreover, the magnetic nanoparticles themselves are
internalized already
after short incubation with live cells via the endocytotic pathway, as for
example described in
Gupta et al., "Cytotoxicity Suppression and Cellular Uptake Enhancement of
Surface Modified
Magnetic Nanoparticles," Biomaterials 2005 and Bogart et al., "Photothermal
Microscopy of the
Core of Dextran-Coated Iron Oxide Nanoparticles During Cell Uptake," ACS Nano
2012.
[00216] In certain embodiments, the present disclosure makes use of this
property of
internalization and facilitates the process by DMF treatment to accelerate
delivery of
nanoparticles (e.g., as exemplified, of LAMP1 antibody-attached nanoparticles)
to intracellular
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compartments, and specifically to the lysosome. Following down the endocytotic
pathway, the
targeted nanoparticles enter the early- and late-endosomes and afterwards they
should enter other
compartments, e.g. ER, via recycling endosomes, or being removed from the
lysosomes. In the
lysosomes, the targeting agent associated with its nanoparticle recognizes and
binds to its target
moiety (e.g., LAMP1, which is highly expressed in the lysosomal membrane). In
response to a
low frequency DMF, as employed in the disclosure herein, the now bound
nanoparticle generates
dynamic forces strong enough to tear the lysosomal membrane, leading to
destruction of the
lysosome integrity, leakage of lysosomal enzymes and finally induction of
apoptosis.
[00217] In INS-1 cells, the present study observed that the SPIONs mostly
loaded into
lysosomes after 20 minutes DMF treatment (FIG. 4A). This preferential
lysosomal localization
was also observed when SPIONs were located into primary human pancreatic islet
cells (as
shown in FIG. 7A). However, as the direction and extent of intracellular
membrane trafficking
may differ markedly between cell types, those of skill in the art will
appreciate that adjustments
to lysosome loading efficiency may be desirable when this technology is
applied to other cell
types. Those skilled in the art will appreciate that this technology can
provide tools to regulate
specific subcellular compartmental functions including not only the nuclei,
but also
compartments such as the ER, Golgi apparatus and different types of endosomes
along the
intracellular membrane trafficking system.
[00218] Those skilled in the art will also appreciate that adjustments
(e.g., stronger fields
etc.) may be desirable, when applying embodiments of provided technology to in
vivo contexts.
For example, the particular DMF field strength utilized in the present Example
was about 30 mT,
and may have a reach of up to about 1 cm, though such penetration was not
required (reach of
even 1 mm being sufficient in certain instances). Those skilled in the art
will appreciate that
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stronger fields (e.g., potentially within the range of mT to T) may be
required for certain in vivo
applications, for example in order to reach deep organs, which may well
involve penetration of
cm, 20 cm, 30 cm, 40 cm, or more. Regardless, ultimate clinical translation
using presently
disclosed low frequency DMF approach may be more straightforward than using
high-frequency
alternating fields, among other things because the DMF fields are not expected
to cause
nonspecific heating of tissues through induced eddy currents and should
therefore have a better
safety profile.
[00219] The achievement of rotational control on nanoparticles with the
DMF method as
described herein has many other potential applications beyond the model
systems used here, in
both biomedical and non-biological nanotechnology fields. To give but a few
examples,
magnetic actuation has been shown to control timing and drug release from
vesicles containing
iron oxide nanoparticles. An increase in permeability of lysosomal membranes
not only can be
used to promote apoptosis, such as through the release of proteolytic enzymes
and increase in
reactive oxygen species, but can also increase the efficacy of drugs trapped
in lysosomes.
Because sequestration of drugs in lysosomes is responsible for up to 40% of
whole tissue drug
uptake, lysosomal drug trapping plays an important role in the development of
tumor drug
resistance. Thus, is some embodiments, provided DMF-mediated lysosomal
membrane
permeabilization technologies could be used to treat cancer drug resistance.
CONCLUSIONS
[00220] In summary, using a unique dynamic magnetic field (DMF) generator
the present
exemplification can control rotational movements of superparamagnetic iron
oxide nanoparticles
(SPIONs) in solution. This rotational nanoparticle movement was applied for
remote induction
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of cell death by injuring lysosomal membrane structures. SPIONs are covalently
coated with
antibodies targeting the lysosomal protein marker LAMP1 (LAMP1-SPION). Remote
activation
of slow rotation of LAMP1-SPIONs with 20 Hz for 20 minutes, significantly
improved the
efficacy of cellular internalization of the nanoparticles. It is observed that
71.2 3.8% of
LAMP1-SPIONs accumulated along the membrane in lysosomes in rat insulinoma
tumor cells
due to binding of LAMP1-SPIONs to endogenous LAMP l. Further activation of
torque by the
LAMP1-SPIONs bound to lysosomes resulted in rapid decrease in size and number
of
lysosomes, attributable to tearing of the lysosomal membrane by the shear
force of the
rotationally activated LAMP1-SPIONs. This remote activation resulted in
increased cell
apoptosis and impaired cell growth. The findings of the present disclosure
suggest that DMF
treatment of lysosome-targeted nanoparticles offers a non-invasive tool to
induce apoptosis
remotely, providing an important platform technology for a wide range of
biomedical
applications.
METHODS
[00221]
Nanoparticle assembly. The protocol of conjugation of LAMP1 antibodies to
magnetic nanoparticles was described previously, for example, in Gruttner,
"Synthesis and
Antibody Conjugation of Magnetic Nanoparticles with Improved Specific Power
Absorption
Rates for Alternating Magnetic Field Cancer Therapy," Journal Magnetism and
Magnetic
Materials 2007. Briefly, SPION nanoparticles (Micromod, Germany) were amino-
functionalized and the density of amino groups per mg of particles was
determined. After
washing, the other parts of amino groups were reacted with sulfo-SMCC in PBS-
EDTA buffer to
introduce maleimide groups on the particle surface. The monoclonal LAMP1
antibody (Abcam,
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UK) was purified with a G-25 column containing PBS/EDTA buffer to remove the
glycerin and
sodium azide. After purification the LAMP1 antibody was treated with
imnothiolane solution to
introduce the SH groups. After washing the SH-modified antibody with PBS-EDTA
buffer in a
G-25 column, the maleimide-modified particles were added. The particles were
shaken for 1 h at
room temperature. Then cystein was added to quench the remaining reactive
sites. Finally the
particles were purified with PBS buffer in magnetic columns in a high gradient
magnetic field.
[00222] DMF device. The dynamic fields to control SPION rotation used in
this study
were created with a DMF generator (DM-01, Feldkraft and Stetter Elektronik,
Germany). The
device consists of an array of multiphase coil systems, where the coils are
displaced against each
other. The field can be altered in a highly dynamic fashion. A device-
integrated digital
controller regulates the frequency as well as the magnetic flux. The dynamic
flux produces an
electromagnetic gradient force F = (mV ) = B , where m are the magnetic
moments of the beads in
total and B is the dynamic flux density field vector. This vector is
established by the H field of
the present device. The magnetic field strength generated by the DMF device
used in this study
is approximately as large as 30 mT (rms).
[00223] Live cell imaging. Cells were seeded onto glass coverslips. After
DMF
treatment, live images were acquired using a Zeiss 510 Meta confocal system
with a x 40 water
immersion objective (NA=1.2). SPION-TRITC was visualized by excitation at 543
nm and
emitted light collected using a long-pass 560 nm filter. The pinhole was ¨1
airy unit and the
scanning frame was 512x512 pixels. The cellular location of SPION was stained
with the
plasma membrane marker CellMask (Invitrogen, USA), lysosome marker LysoTracker
Green
(Invitrogen, USA) and a marker for cell nuclei, Hoechst 34580 (Invitrogen,
USA). Image
analysis was performed with ZEN 2009 software (Zeiss, Germany). Co-
localization was
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analyzed by a Pearson's efficiency methods within ZEN 2009. The SPION-TRITC co-
localization of lysosomes was calculated by coefficient CSPION
colocalized¨ ; , X 100. Likewise,
n
,,Xe,total
PiXel
the LysoTracker-labeled lysosomes were counted by coefficient clysosome¨
colocalizedx 100.
pixeitotai
The coefficient is reported as a percentage from 0-100, with 0 meaning no co-
localization and
100 meaning all pixel-pairs are co-localized. Since TRITC labelled SPIONs were
loaded into all
the INS-1 cells after DMF treatment, the present study used the fluorescence
intensity of TRITC
to indicate the amount of SPIONs in a cell after 3 times washing. The total
SPION loading
nnexinV(7¨AAD)
efficiency was calculated by percentage of FA L.
xioo number of fluorescence intensity
Hoechst 34580
of TRITC to the intensity of Hoechst 34580 which reflects cell number in a
field.
[00224] Transmission electron microscopy (TEM) imaging. The preparation of
cells for
TEM is now discussed. Briefly, INS-1 insulinoma tumor cells were treated with
DMF, followed
by fixation in 2.5% glutaraldehyde for 1 hour at 4 C. The cells were then
treated with 1%
osmium tetroxide, dehydrated, and embedded in AGAR100 (Oxford instruments
Nordiska AB,
Stockholm) before being sliced in ultra-thin sections (70-90 nm). After
slicing, the samples were
placed on Cu grids and contrasted with uranyl acetate and lead citrate. The
TEM images were
obtained using a JEM 1230 electron microscope (Jeol-USA, Peabody, MA, USA).
[00225] Human islet cell Immunostaining. Human islets were provided by the
EXODIAB Human Tissue Lab and the Nordic Network of Clinical Islet
Transplantation
Programme (www.nordicislets.org). The islet cells were separated in a Ca2+-
free solution at 37
C for 10 minutes and treated with DMF after 12 hour culture on the cover slips
centered dish
(MatTek, Germany). Then the cells were fixed with 3% PFA-PIPES and 3% PFA-
Na2B04 for 5
min. and 10 min., respectively, followed by permeabilization with 0.1% Triton-
X 100 for 30
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min. and blocked with 5% normal donkey serum in PBS for 15 minutes. Guinea pig
sourced
antibody against insulin (EuroProxima, Netherlands) was diluted in 5% block
buffer and
incubated overnight at 4 C. Immunoreactivity was done using fluorescently
labeled secondary
antibodies: cy2 anti-guinea pig (1:400) and cy5 anti-mouse (1:400). The images
of SPION-
TRITC (EX: 543 nm), cy2-labeled insulin (EX: 488 nm) and cy5-labeled LAMP1
(EX: 633 nm)
were visualized through three or four channels by a confocal system (Zeiss,
Germany).
Lysosomal size (LAMP1 marked) was calculated using the profile function of the
ZEN 2009
software based on the shapes of the fluorescence areas.
[00226] Intracellular pH measurement. INS-1 cells were seeded on the glass
centered
dish (MatTek, Germany), loaded with SPION or SPION-LAMP1 and treated by DMF.
Then the
cells were incubated with 1 M acidic indicator, LysoSensor Green DND 189
(Invitrogen, USA)
for 30 min. in 37 C. After incubation, the cells were washed out and the
fluorescence images
were acquired by confocal microscopy. The average fluorescence intensity per
cell was
measured using the ZEN 2009 software and further applied for quantitative
analysis.
[00227] Cell apoptosis detection. FITC-Annexin V (51-65874X, BD, USA) and
7-AAD
(51-68981E, BD, USA) were used to assess early and late apoptosis of INS-1
cells. After DMF
treatment, the INS-1 cells were incubated with the dyes at 37 C for 30
minutes. Images were
acquired under identical conditions (using the same settings for pinhole (1
airy unit), exposure
time, gain and scanning speed). Fluorescence intensity was analyzed by the ZEN
2009 software
and early/late stage apoptosis was quantified by percentage of
FAnnexinV(7¨AAD) xioo number of
'Hoechst 34580
Annexin V/7-AAD positive cells to the number of Hoechst 34580 stained cells.
[00228] Proliferation. INS-1 cells were seeded on 24-well plates loaded
with SPION or
SPION-LAMP1 nanoparticles and treated by DNIF once/day. Then the cell number
was
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calculated by a plate cytometer after 6 hours of DMF treatment. Cells were
counted once per
day for 5 days.
[00229] Temperature measurements. A dish with 100 nm SPIONs (10mg/m1) in
water
and a dish without the SPIONs in water as a control, each containing a
temperature sensor
(Radial leaded glass-encapsulated NTC thermistor, EPCOS Inc., Germany), were
placed
simultaneously on the DMF device. The dishes were then subjected to the DMF
field for 20
minutes at 20 Hz and the temperature recorded in each dish.
[00230] Statistical analysis. The data was presented as average standard
error of the
mean (S.E.M.). Statistical comparison of paired-factors experiments was
performed by Student's
T-test and one-way analysis of variance (ANOVA) and the Friedman tests were
performed for
multi-factor experiments which have more than two group treatments in one
experiment.
Example 2:
[00231] In this example, a cooler is used to remove emerging losses
produced by
superconductors in AC fields with AC current transport. Losses in ACSC systems
depend on
moving vortexes, which are classical field enclosures in the superconductor.
Such vortexes may
be due to the standard frequencies (e.g., 50 Hz, e.g., 60 Hz) that are used in
existing systems;
however, the present devices operate at frequencies that are considerably
lower than standard
frequencies, as described below. Vortex losses are high at standard
frequencies; however, they
are far lower in the lower frequency range presented herein (e.g., 30Hz or
less, e.g., 20 Hz).
Such losses make it difficult to operate the superconductor at cryogenic
temperatures. Efforts to
reduce losses are not cost effective, and therefore, these efforts have not
been pursued heretofore.
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[00232] The present devices are operated at 20 Hz to establish a field
with magnetic
character (e.g., no material constants). As a result, vortex losses are
significantly reduced
compared to devices that are operated at standard frequencies (e.g., 50 Hz,
e.g., 60 Hz), thereby
minimizing heating inside the superconductor. Therefore, an inexpensive
cooling medium, for
example, liquid nitrogen can be used to maintain the conductors in the state
of superconductivity.
In contrast, devices that utilize standard frequencies and experience high
losses demand
expensive cooling media like hydrogen, neon, or helium..
[00233] In vitro tests with INS-1 cells showed that an incoming magnetic
field density of
35 mT was strong enough to control the particles. A standard lab-device can
establish this flux-
density in a 2 cm distance. For a standard available device with an optimized
cooling system,
flux-densities of 200 mT can be achieved. However, a superconducting system
(such as the
systems described herein) produces larger flux-densities due to, for example,
higher current
densities. For example, using the systems provided herein, Tesla values (e.g.,
less than 3 T, e.g.,
less than 2 T, e.g., less than 1 T, e.g., about 200 mT) can be achieved at
larger (e.g., up to 20 cm)
distances compared to the Tesla values and penetration depths of standard lab-
devices.
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