U.S. patent application number 12/863163 was filed with the patent office on 2011-03-03 for treatments of disease or disorders using nanoparticles for focused hyperthermia to increase therapy efficacy.
Invention is credited to Parmeswaran Diagaradjane, Sunil Krishnan, Jon Alexander Schwartz, James Chunjay Wang.
Application Number | 20110052672 12/863163 |
Document ID | / |
Family ID | 40885860 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110052672 |
Kind Code |
A1 |
Krishnan; Sunil ; et
al. |
March 3, 2011 |
TREATMENTS OF DISEASE OR DISORDERS USING NANOPARTICLES FOR FOCUSED
HYPERTHERMIA TO INCREASE THERAPY EFFICACY
Abstract
Methods are provided for the treatment of diseases and disorders
using systematically-introduced nanoparticles to create a focused
localized hyperthermia in a target area to enhance the effect of
additional treatment therapies such as ionizing radiation.
Advantages include an enhancement of the therapeutic effect of
other therapies by increasing perfusion or reducing hypoxia in the
treatment area, further, the methods herein may also result in the
disruption of the vasculature, which provide further impetus for
such treatments, singly and in combination with conventional
therapies such as chemotherapy and radiation therapy. Methods for
treating a target area may comprise systemically introducing
nanoparticles into an organism; allowing the nanoparticles to
preferentially accumulate in the target area, applying an external
energy where the nanoparticles are adapted to transduce at least a
portion of the external energy into a heal energy so as to create a
focused localized hyperthermia; and applying a subsequent
additional therapy.
Inventors: |
Krishnan; Sunil; (Houston,
TX) ; Diagaradjane; Parmeswaran; (Houston, TX)
; Schwartz; Jon Alexander; (Sugar Land, TX) ;
Wang; James Chunjay; (Arlington, TX) |
Family ID: |
40885860 |
Appl. No.: |
12/863163 |
Filed: |
January 16, 2009 |
PCT Filed: |
January 16, 2009 |
PCT NO: |
PCT/US09/00315 |
371 Date: |
July 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61011266 |
Jan 16, 2008 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/649; 424/93.4; 977/915 |
Current CPC
Class: |
A61N 2005/1088 20130101;
A61P 35/00 20180101; A61N 2005/0659 20130101; A61N 2005/1098
20130101; A61N 1/406 20130101; A61N 5/1077 20130101; A61N 5/0625
20130101; A61K 41/0052 20130101; A61N 7/00 20130101; A61K 47/60
20170801; A61N 2005/067 20130101; A61N 5/062 20130101; A61N 2/002
20130101 |
Class at
Publication: |
424/450 ;
424/93.4; 424/649; 977/915 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 35/74 20060101 A61K035/74; A61P 35/00 20060101
A61P035/00; A61K 33/24 20060101 A61K033/24 |
Claims
1. A method for the treatment of cancer comprising the systemic
delivery of energy-absorbing particles to a tumor, the application
of electromagnetic or mechanical energy to the area resulting in an
elevated temperature in the tumor, and the application of ionizing
radiation to the tumor.
2. The method of claim 1 wherein the localized hyperthermia reduces
the hypoxia of the tumor.
3. The method of claim 1 wherein the tumor vasculature is
disrupted.
4. The method of claim 1 wherein the localized hyperthermia
initially reduces the hypoxia of the tumor and the combined
hyperthermia and radiation results in a disruption of the tumor
vasculature.
5. A method for the disruption of vasculature of a target area
comprising the delivery of energy-absorbing particles to the target
area, the application of electromagnetic or mechanical energy to
the area resulting in an elevated temperature in the vasculature of
the target area, and the application of ionizing radiation to the
target area.
6. A method for the disruption of vasculature of a target area
comprising the application of electromagnetic energy in a
wavelength absorbed by the blood component of the target area
resulting in an elevated temperature in the vasculature of the
target area, and the application of ionizing radiation to the
target area.
7. A method for the treatment of tumors comprising: increasing the
perfusion of tumors by the delivery of energy-absorbing particles
to the target area followed by the application of electromagnetic
or mechanical energy resulting in an elevated temperature of the
target area and the delivery of a therapeutic agent to the tumor
wherein the efficacy is enhanced by the increased perfusion.
8. The method of claim 7 wherein the therapeutic agent is a
chemotherapeutic drug.
9. The method of claim 7 wherein the therapeutic agent is a gene
therapy vector.
10. The method of claim 7 wherein the therapeutic agent is a drug
delivery vector.
11. The method of claim 10. wherein the drug delivery vector is
selected from among liposomes or micelles or hollow nanoparticles
or drug eluting nanoparticles.
12. The method of claim 7 wherein the therapeutic agent is an
immunotherapeutic agent.
13. The method of claim 7 wherein the therapeutic agent is
vascular-targeted therapy.
14. A method for the treatment of tumors comprising: increasing the
hypoxia of tumors by the systemic delivery of energy-absorbing
particles to the target area followed by the application of
electromagnetic or mechanical energy resulting in an elevated
temperature of the target area, followed by the application of
ionizing radiation to the tumor, resulting in the disruption of the
vasculature of the tumor and the delivery of a hypoxia-targeted
therapy to the tumor.
15. The method of claim 14 wherein the hypoxia-targeted therapy is
an anerobic bacterial spore.
16. The method of claim 14 wherein the hypoxia-targeted therapy is
an inhibitor of HIF1 Alpha or thioredoxin.
17. The method of claim 1 wherein the energy-absorbing particles
are selected from among, or are a combination of, nanoshells,
nanorods, carbon nanotubes, fullerenes, paramagnetic particles,
metallic nanoparticles, and other absorbers of electromagnetic
energy or absorbers of acoustic energy.
18. The method of claim 5 wherein the energy-absorbing particles
are selected from among, or are a combination of, nanoshells,
nanorods, carbon nanotubes, fullerenes, paramagnetic particles,
metallic nanoparticles, and other absorbers of electromagnetic
energy or absorbers of acoustic energy.
19. The method of claim 7 wherein the energy-absorbing particles
are selected from among, or are a combination of, nanoshells,
nanorods, carbon nanotubes, fullerenes, paramagnetic particles,
metallic nanoparticles, and other absorbers of electromagnetic
energy or absorbers of acoustic energy.
20. The method of claim 14 wherein the energy-absorbing particles
are selected from among, or are a combination of, nanoshells,
nanorods, carbon nanotubes, fullerenes, paramagnetic particles,
metallic nanoparticles, and other absorbers of electromagnetic
energy or absorbers of acoustic energy.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The patent application claims priority to and the benefit of
U.S. Provisional Patent Application Ser. No. 61/011,266, titled,
"The Treatment of Disease or Disorders Using Energy-Absorbing
Particles for Focused Hyperthermia to Increase the Efficacy of
Other Therapies," the entire specification of which is hereby
incorporated by reference.
BACKGROUND
[0002] The present invention generally relates to methods for the
treatment of a variety of diseases and disorders utilizing
systemically-introduced nanoparticles to create focused
hyperthermia of a target area so as to enhance the efficacy of
additional therapies.
[0003] Radiation therapy is often a component of the
multidisciplinary approach to the treatment of many tumors.
However, as a single modality, radiation therapy is unable to
eradicate all locoregional recurrences and/or cure localized
cancers. This ineffectiveness is largely related to the intrinsic
resistance of some cancer cells to ionizing radiation. Moeller, B.
J.; Richardson, R. A.; Dewhirst, M., W. Cancer Metastasis Rev.
2007, 26, (2), 241-48.
[0004] Intratumoral hypoxia is believed to be a key mediator of
this resistance to radiation therapy and is exacerbated by
inadequate oxygenation via mutated, chaotic and incomplete blood
vessels in tumors. McDonald, D. M.; Choyke, P. L. Nat Med 2003, 9,
(6), 713-25; Baluk, P.; Morikawa, S.; Haskell, A.; Mancuso, M.;
McDonald, D. M., Am J Pathol 2003, 163, (5), 1801-15. Hypoxia is
known to induce the expression of a spectrum of genes involved in
metabolism, proliferation, apoptosis, and angiogenesis. Harris, A.
L., Nat Rev Cancer 2002, 2, (1), 38-47; Hockel, M.; Vaupel, P., J
Natl Cancer Inst 2001, 93, (4), 266-76; These hypoxia-induced tumor
cellular and microenvironmental changes contribute to tumor
aggressiveness and resistance to radiation therapy. Zhang, Y.; Li,
M.; Yao, Q.; Chen, C. Med Sci Monit 2007, 13, (10), RA175-80.
Consequently, any therapeutic strategy that alleviates tissue
hypoxia could potentially overcome a major mechanism of
radioresistance and enhance the effects of radiation therapy.
[0005] One such highly effective therapeutic adjunct to radiation
therapy is mild temperature hyperthermia, which has direct
anti-tumor effects and tumor microenvironment effects mediated, in
part, through mitigation of hypoxia that contribute to the observed
radio-insensitization. Roti Roti, J. L. Int J Hyperthermia 2004,
20, (2), 109-14; Kampinga, H. H.; Dikomey, E. Int J Radical Biol
2001, 77, (4), 399-408; Moros, E. G.; Corry, P. M.; Orton, C. G.
Med Phys 2007, 34, (1), 1-4. Mild temperature hyperthermia mediates
its anti-tumor effects via subtle influences on the tumor
microenvironment, activation of immunological processes, induction
of gene expression and induction of protein synthesis. While these
effects do not independently cause tumor cell cytotoxicity, they
lead to greater effectiveness of other conventional treatment
modalities such as radiation therapy, chemotherapy and
immunotherapy. In particular, in its role as an adjunct to
radiation therapy, hyperthermia serves as a dose-modifying agent
that increases the therapeutic ratio of radiation therapy, i.e.
enhanced effectiveness without additional toxicity.
[0006] Various methods have been used to combine hyperthermia and
radiotherapy. One example included applying interstitial radiation
with interstitial hyperthermia in brain tumors. Another example
used magnetic particles directly injected into a tumor and external
beam radiation. Recently, iron oxide particles have been directly
injected into a tumor and an alternating magnetic field applied for
hyperthermia followed by ionizing radiation.
[0007] As another example, in U.S. Pat. No. 5,620,479, Diederich
describes a method and apparatus for thermal therapy of tumors
using piezoceramic tubular transducers for the delivery of
interstitial thermal therapy in conjunction with simultaneous
brachytherapy or radiotherapy from within the applicator. In yet
another example, in U.S. Pat. No. 6,957,108, Turner et al. describe
a microwave hyperthermia apparatus that can be inserted into the
body that includes a hollow central tube for the insertion of
radioactive therapy sources for hyperthermia and brachytherapy.
[0008] Several randomized trials have demonstrated improved
response rates and survival when patients with locally advanced
malignancies are treated with locoregional hyperthermia and
radiotherapy compared to radiotherapy alone. Despite convincing
evidence for hyperthermic radiosensitization, it is underutilized
in routine clinical practice for the following reasons: (a) the
invasive means of achieving and maintaining hyperthermia, (b) the
time commitment involved in a treatment, which can often last about
an hour, (c) the lack of good thermal dosimetry and (d) the
inability to achieve localized hyperthermic temperatures. Thus,
conventional methods for utilizing hyperthermia to enhance other
treatment therapies suffer from a variety of disadvantages.
[0009] A localized dose enhancement of ionizing radiation can also
result from the presence of certain elements in the tumor. For
example, in U.S. Pat. No. 7,367,934. Hainfeld et al. describe the
use of heavy metal particles delivered to a tissue or cells to
achieve a concentration within the tissue of at least 0.1% metal by
weight, applying ionizing radiation of specified energy and
achieving a localized radiation dose enhancement. The radiation
enhancement achieved from the interaction of the metal and the
radiation, requiring a minimum metal content for efficacy.
[0010] As an alternative approach to cancer therapy, vascular
disruptive agents ("VDA") are being developed in an attempt to
treat cancer through the elimination or disruption of the blood
supply. These agents may also be used in conjunction with ionizing
radiation. Vascular disruption of a single established blood
vessel, be it via subtle structural changes of dysmorphic
endothelial cells or induction of intravascular coagulation, could
potentially eliminate hundreds or thousands of tumor cells
downstream. Vascular disrupting agents in preclinical and early
clinical development include combretastatin A4 phosphate (CA4P),
ZD6126, TZT-1027, AVE8062, ABT-751, and MN-029, which target the
tubulin cytoskeletal network of endothelial cells;
5,6-dimethylxanthenone-4-acetic acid (DMXAA), which targets
autocrine endothelial regulatory cascades; and exherin (AFH-1),
which targets cell adhesion. While these agents have shown promise
in early trials, there is concern that more than just tumor vessels
may be targeted by systemic exposure to these agents. In
particular, damage to vascular compartments outside the tumor may
contribute to acute coronary syndromes and thromboembolic events.
Consequently, conventional treatment methods lack the ability to
focus such treatments on specific target areas.
[0011] While a principal use of ionizing radiation is in the
treatment of cancer, other diseases and disorders may benefit from
radiotherapy if the ionizing effects could be confined to the
target area or, alternatively, if the target area could be
sensitized to the effects of ionizing radiation by a non-invasive
method. For example, there are other medical conditions in which
disruption of the vasculature is desired, such as arteriovenous
malformations (AVMs), which can result in hemorrhage or other
deleterious effects depending on their location (in case of brain
AVMs, seizures and aberrant vascular perfusion of adjacent normal
brain).
[0012] Accordingly, improved treatment methods are needed to
address one or more of the disadvantages of the prior art.
SUMMARY
[0013] The present invention generally relates to methods for the
treatment of a variety of diseases and disorders utilizing
systemically-introduced nanoparticles to create focused
hyperthermia of a target area so as to enhance the efficacy of
additional therapies.
[0014] An example of a method for the treatment of a tumor residing
in a target area of an organism comprises the steps of:
systemically introducing a plurality of nanoparticles into a
circulating blood of an organism; allowing the nanoparticles to
preferentially accumulate in the target area; allowing application
of an external energy to the target area wherein the nanoparticles
are adapted to transduce at least a portion of the external energy
into heat energy wherein the external energy is an electromagnetic
or a mechanical energy; allowing the temperature of the area of
accumulation of the nanoparticles to elevate to a localized
elevated temperature by way of a transduction of the external
energy into heat energy by the nanoparticles; and applying ionizing
radiation to the target area.
[0015] An example of a method for the disruption of a vasculature
of a target area comprises the steps of: systemically introducing a
plurality of nanoparticles into a circulating blood of an organism;
allowing the nanoparticles to preferentially accumulate in the
target area; allowing application of an external energy to the
target area wherein the nanoparticles are adapted to transduce at
least a portion of the external energy into heat energy wherein the
external energy is an electromagnetic or a mechanical energy;
allowing a temperature of the target area to elevate to a localized
elevated temperature by way of a transduction of the external
energy into heat energy by the nanoparticles; and applying ionizing
radiation to the target area so as to disrupt the vasculature of
the target area.
[0016] An example of a method for the disruption of a vasculature
of a target area comprises the steps of: allowing application of an
external energy to the target area wherein the target area
transduces at least a portion of the external energy into heat
energy wherein the external energy is an electromagnetic or a
mechanical energy; allowing a temperature of the target area to
elevate to a localized elevated temperature by way of a
transduction of the external energy into heat energy; and applying
ionizing radiation to the target area so as to disrupt the
vasculature of the target area.
[0017] An example of a method for the disruption of a vasculature
of a target area comprises the steps of: applying electromagnetic
energy to the target area wherein the electromagnetic energy is in
a wavelength absorbed by a blood component of the target area;
allowing the applied electromagnetic energy to result in an
elevated temperature of the vasculature of the target area; and
applying ionizing radiation to the target area so as to disrupt the
vasculature of the target area.
[0018] The features and advantages of the present invention will be
apparent to those skilled in the art. While numerous changes may be
made by those skilled in the art, such changes are within the
spirit of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] A more complete understanding of the present disclosure and
advantages thereof may be acquired by referring to the following
description taken in conjunction with the accompanying figures,
wherein:
[0020] FIG. 0 illustrates a schematic view illustrating certain
elements of the present invention.
[0021] FIG. 1a illustrates absorption spectra of gold nanoshells
(silica core diam: 120.+-.12 nm; gold shell diam: 12.+-.3 nm).
[0022] FIG. 1b illustrates a temperature profile of tumor tissue
measured by thermocouples.
[0023] FIG. 1c illustrates MRTI images of tumor tissues at various
time periods.
[0024] FIG. 1d illustrates temperature profile in tumor tissue
estimated from the MRTI at various time points during laser
illumination at .quadrature.24 h after gold nanoshell
injection.
[0025] FIG. 2a illustrates normalized tumor volume plot of control,
hyperthermia, radiation, and thermoradiotherapy groups showing the
mean.+-.SE values at different time periods after the initiation of
each treatment.
[0026] FIG. 2b illustrates the corresponding tumor doubling time
after each treatment.
[0027] FIGS. 3a-h illustrate T1-weighted (a) precontrast, (b)
prehyperthermia DCE-MRI, (c) posthyperthermia DCE-MRI images of
tumor, and (d-f) the corresponding 3D pixel intensity distribution
profile. Enhanced contrast (bright tumor center) observed in
posthyperthermia DCE-MRI when compared to prehyperthermia shows
increased perfusion after gold nanoshell-mediated hyperthermia.
Pre- and posthyperthermia contrast uptake estimated from the Region
of Interest (ROI) encompassing the tumor core and whole tumor is
illustrated in (g) and (h), respectively.
[0028] FIGS. 4a-h illustrate H and E staining of tumor (a-d)
periphery and (e-h) core, tissues from control, hyperthermia,
radiation, and thermoradiotherapy treated groups, with the arrows
in (e-h) representing the regions of necrosis in the tumor center
and the arrow in (d) representing the depth of necrosis from the
tumor periphery. A representative scale bar is shown in the bottom
image of each column.
[0029] FIGS. 5a-l illustrate immunofluorescence staining of
control, hyperthermia, radiation, and thermoradiotherapy treated
tumors showing hypoxia, cell proliferation (a-d), and hypoxia,
perfusion in tumor periphery (e-h), and tumor core (i-l),
respectively. The white and grey colors on these images represents
cell proliferation, perfusion, and hypoxic regions in tumors.
Patchy hypoxic region seen in (l) is attributed to the vascular
disruption effect induced by gold nanoshell-mediated
thermoradiotherapy. Scale bars are represented in the bottom image
of each column.
[0030] FIGS. 6a-c illustrate (a) Immunofluorescence staining for
the microvessel density in control, hyperthermia, radiation, and
thermoradiotherapy treated tumors showing the vessel distribution
in tumor periphery (column 1) and tumor center (column 2). (b) Bar
chart representing mean.+-.SE of blood vessels in tumor periphery
and tumor core for different treatment groups. (c) SEM images
showing the low and high magnification images of gold nanoshell
distribution near the perivascular regions in tumors before (row 1:
low magnification: 1000.times.; high magnification: 7000.times.)
and after (row 2: low magnification: 2000.times.; high
magnification: 5000.times.) gold nanoshell-mediated hyperthermia.
The indicating arrows show the gold nanoshell distribution.
[0031] While the present invention is susceptible to various
modifications and alternative forms, specific exemplary embodiments
thereof have been shown by way of example in the drawings and are
herein described in detail. It should be understood, however, that
the description herein of specific embodiments is not intended to
limit the invention to the particular forms disclosed, but on the
contrary, the intention is to cover all modifications, equivalents,
and alternatives falling within the spirit and scope of the
invention as defined by the appended claims.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0032] The present invention generally relates to methods for the
treatment of a variety of diseases and disorders utilizing
systemically-introduced nanoparticles to create focused
hyperthermia of a target area so as to enhance the efficacy of
additional therapies.
[0033] Generally, the present invention describes a method for
treating tumors or target tissues using localized hyperthermia to
enhance the effect of other therapies such as drug treatments and
radiation treatments.
[0034] In certain embodiments, methods are provided for the
treatment of diseases and disorders using systemically-introduced
nanoparticles to create a focused localized hyperthermia in a
target area so to enhance the effect of an additional treatment
therapy such as ionizing radiation. Advantages of the methods
herein include an increase of the therapeutic effect of other
therapies by increasing perfusion or reducing hypoxia in the
treatment area. Further, in certain embodiments, the methods herein
may also result in the disruption of the vasculature, which provide
further impetus for such treatments, singly and in combination with
conventional therapies such as anti-hypoxic agents. Although the
examples provided herein relate in large part to the treatment of
tumors, it is explicitly recognized that the methods herein may be
used to treat any disease or disorder that would be enhanced by the
creation of a localized hyperthermia, such as, for example,
vascular conditions.
[0035] To facilitate a better understanding of the present
invention, the following examples of certain embodiments are given.
In no way should the following examples be read to limit, or
define, the scope of the invention.
[0036] In certain embodiments, methods for treating a tumor
residing in a target area of an organism comprise in part:
[0037] (a) systemically introducing a plurality of nanoparticles
into a circulating blood of the organism;
[0038] (b) allowing the nanoparticles to preferentially accumulate
in the target area of the organism;
[0039] (c) applying an external energy to the target area wherein
the nanoparticles are adapted to transduce at least a portion of
the external energy into a heat energy;
[0040] (d) allowing the heat energy to elevate the temperature of
the target area so as to create a focused localized hyperthermia;
and
[0041] (e) applying a subsequent additional therapy.
[0042] Each of these steps will now be discussed with reference to
FIG. 0. Organism 110 has a disease or disorder, such as a tumor,
residing, at least partially, in target area 120. In step (a), a
plurality of nanoparticles is systemically introduced into the
circulating blood of organism 110 via injection 130. In step (b),
the nanoparticles are allowed to preferentially accumulate in
target area 120 of the organism. This accumulation of nanoparticles
in the target area may occur passively through the enhanced
permeability and retention ("EPR") effect or through active
targeting mechanisms, both of which are described below in more
detail.
[0043] In step (c), an external energy 140 is applied to target
area 120 via external energy source 140. The external energy may be
any electromagnetic energy or a mechanical energy. Suitable forms
of electromagnetic energy include, but are not limited to,
ultraviolet radiation, visible light, infrared light, microwave
radiation, radiowaves, alternating magnetic fields, or any
combination thereof. Suitable forms of mechanical energy include,
but are not limited to, acoustic energy such as ultrasonic
waves.
[0044] External energy 140 is then transduced to heat at least
partially by the nanoparticles that have accumulated in target area
120. In this way, the transduction of energy by the nanoparticles
induces a localized temperature elevation or hyperthermia confined
to target area 120 and the region immediately adjacent thereto.
Accordingly, target area 120 is sensitized to subsequent therapies,
and effects on surrounding healthy tissue are minimized.
[0045] Upon achieving a localized focused hyperthermia in target
area 120, a subsequent additional therapy may be applied to the
target area. As described in more detail below, suitable additional
therapies include, but are not limited to, ionizing radiation, a
hypoxia-targeted therapy, a therapeutic agent, or any combination
thereof. In certain embodiments, ionizing radiation may optionally
be applied via external energy source 145. In certain embodiments,
the additional therapy may be a hypoxia-targeted therapy or a
therapeutic agent 150 delivered via systemic introduction 152 or
direct injection 154 to target area 120.
Suitable Nanoparticles
[0046] Nanoparticles suitable for use in conjunction with the
present invention include any nanoparticle that is adapted to at
least partially transduce an external energy into heat energy for
elevating the temperature of a target area. Suitable examples of
such materials and their methods of production and
functionalization are known in the art. See e.g., U.S. Pat. Nos.
6,344,272 and 6,685,986. These transducing nanoparticles include,
among others: nanoshells (including gold-shell silica core
nanoshells, gold-gold sulfide nanoshells and other variants), metal
nanorods, nanostars, hollow nanoparticles, nanocages, elliptical
"nanorice," carbon particles, fullerenes, carbon fullerenes,
metallic nanoparticles, metal colloids, carbon particles, carbon
nanotubes, buckyballs, and any combination thereof
[0047] In certain embodiments, the nanoparticles may be a magnetic
or paramagnetic (e.g., iron oxide particles) particularly when the
energy source is an alternating magnetic field. In another
embodiment, the nanoparticles may be a conducting material (e.g.,
gold or other metal colloids, nanoshells, nanorods, buckeyballs and
carbon nanotubes), particularly when the energy source is
radiowaves. Carbon fullerenes, nanocubes, nanostars, and
indocyanine green encapsulated in nanoparticles may also be used as
suitable nanoparticles.
[0048] In certain embodiments, the nanoparticles may be designed or
selected to absorb near-infrared energy, light in the visible
spectra, radiowaves, microwaves, magnetic energy, other forms of
electromagnetic radiation, or any combination thereof.
Alternatively, these particles can be designed to absorb mechanical
energy such as acoustic waves, for example, ultrasound waves.
[0049] In certain embodiments, more than one type of nanoparticle
may be simultaneously used. Each type of nanoparticle may be
designed or tuned to preferentially transduce a different type of
external energy.
[0050] In more particular embodiments, nanoparticles may be
designed to absorb electromagnetic radiation in the near-infrared
region (e.g. between 670 nm and 1200 nm), wavelengths that allow
the maximum penetration of this energy through normal tissue. Upon
the application of a laser emitting within these wavelengths, the
nanoparticles absorb and convert this energy into heat to elevate
the temperature of the tumor to a non-ablative level, increasing
the sensitivity of the tumor to a subsequent therapy, such as
ionizing radiation. The effect of the nanoshell-induced
hyperthermia is to create a temperature elevation confined to the
tumor and the region immediately adjacent thereto, localizing the
area of increased sensitivity to ionizing radiation to minimize the
effect on surrounding healthy tissue.
[0051] These particles can be delivered to the tumor by injection
or by systemic delivery, with or without targeting mechanisms. As
described further below, these particles may optionally be targeted
to the vasculature associated with the tumor. As used herein, the
term "nanoparticle" also includes particles of a size that may be
systemically be delivered to the target area through the blood
stream or lymphatic channels. In certain embodiments, a
nanoparticle will have a largest dimension of less than 1 micron,
and in other embodiments, preferably less than 200 nanometers.
Systemic Introduction and Accumulation of Nanoparticles in the
Target Area
[0052] The nanoparticles may be systemically introduced into the
organism to be treated. As used herein, the term "systemic
introduction" refers to any introduction of nanoparticles that
pertains to or affects the organism as a whole such as an
introduction of nanoparticles into the circulating blood of an
organism. As previously described, the mechanism by which the
nanoparticles accumulate in the target area may be by a passive
mechanism, an active mechanism, or a combination thereof.
[0053] In the passive mechanism, nanoparticles may be injected or
infused into the blood stream and accumulate at the target area or
tumor site through the enhanced permeability and retention ("EPR")
effect. Through this mechanism, passively targeted particles
accumulate in the tumor in a region near the disrupted blood
vessels. In certain embodiments, gold nanoshells measuring about
150-160 nm, when injected intravenously, accumulate preferentially
in tumors by the enhanced permeability and retention (EPR) effect,
where the leaky tumor vasculature containing wide interendothelial
junctions, abundant transendothelial channels, incomplete or absent
basement membranes, and dysfunctional lymphatics contribute to
passive extravasation of systemically injected macromolecules and
nanoparticles into tumors.
[0054] Active mechanisms for targeting the tumor site include
conjugating nanoparticles with an antibody to a cell surface
molecule, such as an anti-EGFr antibody, preferentially expressed
by a target cell. These particles may be inserted into the blood,
allowed to selectively accumulate in the target area, and
selectively bind to cells in the target area which have such
molecules present on their cell surface. Additionally, vascular
targeting agents (e.g., ligands for the integrin alpha.v beta.3,
VEGFr or phosphatidylserine) may be used to actively target the
target site. Similarly, particles actively targeted to the tumor
endothelial cells will accumulate at an endothelial surface.
[0055] A variety of ligands may be selected for use to
preferentially associate the exogenous material with the target
cells. The attachment of these ligands to exogenous materials has
been extensively described in the scientific literature. The choice
of ligand is dependent on the target cells. For example, if the
target is a tumor cell that expresses the HER2 receptor, molecules
that selectively bind to the HER2 receptor may be used.
Alternatively, the ligand may be selected for affinity to the, the
EGF receptor, an integrin, a hormonal receptor, or a variety of
other surface molecules. One of ordinary skill in the art, with the
benefit of this disclosure, will appreciate, that the ligand may be
selected from a variety of proteins, peptides, antibodies, antibody
fragments, aptamers or other compounds that has a preferential
affinity for the target over other circulating blood components.
The ligand(s) selected need not be specific for only the
target.
Application of External Energy to the Target Area
[0056] The external energy may be applied from a position external
to the body or from an applicator placed within or near the target
area. The external energy may comprise electromagnetic radiation,
mechanical energy, or any combination thereof. Suitable forms of
electromagnetic energy include, but are not limited to, ultraviolet
radiation, visible light, infrared light, microwave radiation,
radiowaves, alternating magnetic fields, or any combination
thereof. Suitable forms of mechanical energy include, but are not
limited to, acoustic energy such as ultrasonic waves.
[0057] Typically, the external energy will be selected to
correspond to the type of energy to which the nanoparticles
preferentially transduce. In certain embodiments, more than one
type of external energy may be used simultaneously or in sequence,
particularly where more than one type of nanoparticle is present in
the target area.
[0058] Generally, the external energy will be limited to a
non-ablative level that generates a focused localized hyperthermia.
Nevertheless, in certain embodiments, the external energy is
applied in a manner which both generates a focused localized
hyperthermia and causes the nanoparticles to ablate adjacent or
targeted cells.
Focused Localized Hyperthermia
[0059] The application of energy to these passively or actively
targeted nanoparticles will result in hyperthermia localized within
the target area, but more specifically in the area proximate to the
nanoparticle accumulations in the target area. These accumulations
may occur near the related vasculature or at the surface f target
cells. This non-invasive method of producing a focused localized
hyperthermia overcomes a number of the disadvantages previously
noted in the prior art.
[0060] Indeed, this type of non-invasive method to generate focused
localized hyperthermia in a target area is especially beneficial
for such treatments as hyperthermic sensitization of tumors to
chemotherapy (via increased vascular perfusion of areas of the
tumor that are otherwise shielded from exposure to chemotherapy
drugs due to inadequate blood supply) and hyperthermia-mediated
delivery of chemotherapy drug encapsulated in a
temperature-sensitive liposome.
[0061] Thus, by increasing perfusion or reducing hypoxia in the
treatment or target area, the efficacies of subsequent therapies
may be enhanced. Because of this enhanced efficacy of the
subsequent therapies, dose reductions of the subsequent therapies
may be realized. These dose reductions may especially beneficial in
subsequent therapies such as ionizing radiation where the ionizing
radiation poses increased independent risks of adverse effects.
Thus, by minimizing the effective dose of a subsequent therapy, any
adverse effects of a subsequent therapy can be minimized.
Applying a Subsequent Therapy
[0062] Upon generating focused localized hyperthermia in the target
area, a subsequent therapy may be applied to the target area.
Suitable additional therapies include, but are not limited to,
ionizing radiation, a hypoxia-targeted therapy, a therapeutic
agent, or any combination thereof. Where the additional therapy is
a hypoxia-targeted therapy or a therapeutic agent, it may be
delivered via systemic introduction into the circulating blood of
the organism or by direct injection to target area to be
treated.
[0063] Where ionizing radiation is the additional therapy applied,
it may be applied from an external source, an internal source (as
in Systemic Targeted Radionuclide Therapy), or alternatively from a
localized source such as brachytherapy seeds. The ionizing
radiation may be applied in a single dose or in multiple doses over
time. This technique of using ionizing radiation following a
focused localized hyperthermia is referred to herein as
thermoradiotherapy.
[0064] The subsequent application of ionizing radiation may have a
pronounced effect resulting in the disruption of the cells situated
in the area of preferential nanoparticle accumulation. Where such
nanoparticle accumulation is concentrated near the tumor
vasculature The subsequent application of ionizing radiation may
have a pronounced effect resulting in the disruption of the tumor
vasculature. This effect, although in some cases similar to that
achieved by vascular disrupting agents currently being
investigated, is localized to the tumor that is preferentially
exposed to hyperthermia and radiation so as to eliminate the side
effects normally associated with conventional systemically active
VDAs.
[0065] Alternatively, the use of particle-based localized
hyperthermia and ionizing radiation may be used to treat other
diseases and disorders, including vascular disorders, such as
arteriovenous malformations.
[0066] Alternatively, the energy-absorbing properties of the target
area may be used to deliver a localized hyperthermia to the
vasculature. In such an embodiment, the absorption properties of
the target, such as a high level of hemoglobin related to the
higher blood content of the target, may be used in conjunction with
a localized near-infrared laser to deliver a localized hyperthermia
to the vasculature of a target area in conjunction with ionizing
radiation to create a disruption of the vasculature of the
area.
[0067] The localized hyperthermia may be used to initially reduce
the hypoxia or increase the perfusion of the target area. This
increased perfusion may enhance the effect of other therapeutic
agents. When applied in conjunction with ionizing radiation, the
subsequent vascular disruption can result in a level of necrosis
through reduction of vessel density. Accordingly, this method of
localized hyperthermia and related vasculature disruption or
necrosis may result in increased hypoxia/anoxia in the target area
subsequent to this initial therapy, which may also be used with
agents or methods that use or require hypoxia for therapy.
[0068] It is explicitly recognized that any of the elements and
features of each of the devices described herein are capable of use
with any of the other devices described herein with no limitation.
Furthermore, it is explicitly recognized that the steps of the
methods herein may be performed in any order except unless
explicitly stated otherwise or inherently required otherwise by the
particular method.
EXAMPLES
[0069] To facilitate a better understanding of the present
invention, the following examples of certain aspects of some
embodiments are given. In no way should the following examples be
read to limit, or define, the scope of the invention.
[0070] The following examples demonstrate modulation of in vivo
tumor radiation response using gold nanoshell-mediated hyperthermia
via a dual vascular-focused mechanism: (a) an early increase in
perfusion that reduces the radioresistant hypoxic fraction of
tumors and (b) a subsequent induction of vascular
disruption/collapse and extensive necrosis that complements
radiation-induced cell death. These tumor vasculature focused
effects characterize a novel agent/device (gold nanoshells) that
serves as an integrated anti-hypoxic and localized vascular
disrupting agent.
[0071] Production of nanoparticles. Gold nanoshells were fabricated
as follows. Colloidal silica (120 nm.+-.12 nm diameter) was used as
the core material (Precision Colloids, LLC). Gold colloids of
.about.1-3 nm in diameter were grown by using the method of Duff
(Duff, D. G.; Baiker, A.; Edwards, P. P. Langumir 1993, 9, (9),
2301-2309) and aged for 2 weeks at 4.degree. C. and the aged gold
colloid suspension was mixed with aminated silica particles. Gold
colloid adsorbs to the amine groups on the surface of the silica
core to form nucleating sites, which were further reacted with
HAuCl.sub.4 in the presence of formaldehyde. This process reduces
additional gold onto the adsorbed colloid, which acts as a
nucleation site, causing the surface colloid to grow and coalesce
with neighboring gold colloid, forming a complete metal shell.
Particles were designed to have a 120 nm core diameter and a 12-15
nm-thick shell resulting in an absorption peak between 780 and 800
nm (FIG. 1-a), assessed by a UV-VIS spectrophotometer. For passive
targeting, a thiolated polyethylene glycol SH-PEG (Laysan Bio,
Huntsville, Ala.) was assembled onto nanoshell surfaces by
combining 5 .mu.M SH-PEG and nanoshells in DI H.sub.2O
(3.2.times.10.sup.5 SH-PEG molecules/particle) for 12 hrs, followed
by diafiltration to remove the excess SH-PEG. Resulting particles
were coated with an average of 3.2.times.10.sup.5 SH-PEG molecules
and suspended in 10% trehalose solution to create an iso-osmotic
solution for injection.
[0072] Localized mild-temperature hyperthermia can be induced
non-invasively by optically activated gold nanoshells and measured
non-invasively by magnetic resonance thermal imaging (MRTI). To
optimize the laser settings for generation of mild-temperature
hyperthermia over a short period of time without significant
over-heating of both tumor and/or surrounding normal tissues,
temperature increases were initially recorded under different laser
illumination conditions in an in vivo tumor model using
tumor-implanted thermocouples. Six to eight-week-old
immunocompromised male nude (Swiss nu/nu) mice weighing 20-25 g
each were subcutaneously inoculated with human colorectal cancer
cells (HCT 116; .about.2.times.10.sup.6 cells per 50 .mu.l of
sterile phosphate buffered saline) in the right thigh. When the
tumors attained a size of .about.7-8 mm in diameter,
.about.8.times.10.sup.8 nanoshells/g body weight (or .about.14
.mu.g/g body weight) were injected intravenously via the tail vein.
Localized hyperthermia was carried out at 20-24 hrs post-injection,
needle thermocouples (model HYP1-30-1/2-T-G-60-SMP-M, Omega
Engineering) were positioned in the tumor core and tumor base
(adjacent to muscular fascia), and core body temperature was
measured using a rectal probe (model RET-3, Braintree Scientific,
Inc.). The three different laser settings evaluated were 0.8, 0.6
and 0.4 W/cm.sup.2 in cohorts of 2-3 mice each. Prior to laser
illumination PEG diacrylate (M.sub.x 600, Sartomer, West Chester,
Pa.) was applied over the surface of the tumor as an index-matching
agent. An 808 nm NIR diode laser (Diomed-plus 15, Diomed corp, UK)
was used to illuminate the tumor surface (10 mm diameter spot size)
via a fiber optic cable with a collimating lens. Average baseline
tumor temperature was .about.30.+-.1.degree. C. Upon illumination
with a laser power of 0.8 W/cm.sup.2, a steep rise in tumor
temperature was observed within the first 5 min, followed by a
steady temperature plateau (.DELTA.T of .about.13-15.degree. C.)
for the remaining 15 min of laser illumination. While this was
still below the typical hyperthermia temperature threshold
(<45.degree. C.), because the intent was to generate
mild-temperature hyperthermia without inducing tissue damage by the
hyperthermia itself, and a lower power setting was chosen.
Illumination with a laser power of 0.4 W/cm.sup.2 achieved a
.DELTA.T of .about.4-5.degree. C. in the tumor core. Using an
intermediate laser power of 0.6 W/cm.sup.2, yielded .DELTA.Ts of
.about.10.+-.1.5.degree. C. and .about.8.+-.0.5.degree. C. in the
tumor core and base, respectively (FIG. 1-b). These illumination
parameters (808 nm NIR laser beam, power setting of 0.6 W/cm.sup.2,
75% duty cycle, final optical power output of .about.350
mW/cm.sup.2 at a spot size of 10 mm) were verified for
reproducibility in an additional cohort of mice and then chosen for
all subsequent hyperthermia experiments. The observed differences
in temperature between the tumor core and base were attributable to
(a) the limitation in depth of penetration of the NIR laser beam
within tumor tissues, (b) the absorption of NIR light by the gold
nanoshells in the superficial layers of the tissues and (c) the
preferential accumulation of and absorption of NIR light by gold
nanoshells within the perivascular space of blood vessels lining
the periphery of the tumor. To further confirm that the induced
temperature rise was mediated by gold nanoshells, temperature
measurements were undertaken using identical laser settings in a
cohort of control animals without nanoshells. .DELTA.Ts of
.about.2.5-3.5.degree. C. were observed in the tumor core, the base
of the tumor and in irradiated muscle of the contralateral
thigh.
[0073] The thermocouple measurements were subsequently validated by
non-invasive in vivo MRTI in an additional cohort of mice (see
supplementary information below for additional details). Upon laser
illumination with a power of 0.6 W/cm.sup.2, real-time MRTI
measurements demonstrated a temperature difference (.DELTA.T) of
.about.11.0.degree. C. consistent with the thermocouple
measurements. The T1-weighted images overlaid with the temperature
distribution images at 0, 5, 10 and 20 minutes after laser
illumination are illustrated in FIG. 1c. The .DELTA.T of
.about.15.0.degree. C. observed in the tumor periphery (MRTI) may
be attributable to the high concentration of nanoshells within the
highly vascularized tumor periphery. Regions of Interest (ROIs)
encompassing the whole tumor, tumor periphery and tumor core were
created and averaged to generate temperature plots at various time
points. A similar ROI from the background region was selected to
compensate for background temperature changes. The temperature rise
calculated from the real-time MRTI measurements is illustrated in
FIG. 1-d. Similar to the thermocouple measurements, an initial
steep rise in temperature was followed by a relatively sustained
plateau region for the remaining period of laser illumination.
However, the slope of the initial temperature. rise was different
from that of the thermocouple measurements, possibly attributable
to the averaging of temperature across a larger ROI in the MRTI
measurements as opposed to point measurements using the
thermocouple.
[0074] Gold nanoshell-mediated hyperthermia enhances the efficacy
of radiation therapy. In a separate experiment employing the tumor
re-growth delay assay, 36 mice bearing .about.7-8 mm diameter
tumors were evenly randomized to one of four treatment conditions:
(a) control treatment with gold nanoshells alone without
hyperthermia or radiation, (b) 20 min of laser illumination as
specified above for generation of mild-temperature hyperthermia,
(c) a single 10 Gy dose of radiation therapy using 125 kV X-rays
(Phillips RT-250 orthovoltage X-ray unit operated at 20 mA and
using a 2 mm Aluminum filter and a skin cone of 1 cm diameter to
collimate the beam to the tumor surface with the target to surface
distance of 22.4 cm; see supplementary information for experimental
setup), and (d) hyperthermia followed by 10 Gy radiation therapy
(thermoradiotherapy) .about.3-5 min later. Tumor growth was
followed by serially measuring tumor dimensions in two orthogonal
directions (long axis: a.sub.1; short axis: a.sub.2) twice weekly.
Tumor volume was calculated using the expression
(.pi./6)(a.sub.1)(a.sub.2).sup.2 and plotted over time as
represented in FIG. 2-a. The tumor volume of individual mice in
each treatment group was normalized with respect to the initial
tumor volume prior to treatment. Tumor re-growth delays, calculated
as the time to doubling of tumor volume, were observed to be
approximately 4, 9, 17, and 29 days for the control, hyperthermia,
radiation and thermoradiotherapy groups, respectively. As
illustrated in FIG. 2-b, there was a statistically significant
(p<0.005) difference in tumor doubling time between the
radiation and thermoradiotherapy groups.
[0075] Gold nanoshell-mediated hyperthermia enhances perfusion.
Because one of the purported mechanisms of radiosensitization by
mild-temperature hyperthermia is an acute increase in tumor
vascular perfusion, dynamic contrast magnetic resonance imaging
(DCE-MRI) was used to evaluate contrast uptake in tumors following
treatment. Approximately 3-5 min after the completion of laser
illumination at standard settings, DCE-MRI was performed in a
separate cohort of mice (see supplementary information below for
additional details). Representative pre-contrast MRI images and
pre- and post-hyperthermia T1-weighted DCE-MRI images are
illustrated in FIG. 3-(a-c). The baseline pre-hyperthermia DCE-MRI
image (FIG. 3-b) revealed contrast enhancement at the periphery of
the tumor with relative paucity of contrast in the tumor core.
After laser illumination, a significant increase in contrast was
observed within the tumor core (FIG. 3-c). This increase in
contrast within the tumor core is further illustrated by a 3D pixel
intensity representation of a region of interest (ROI) (34.times.37
pixels) encompassing the entire tumor in FIG. 3-(d-t).
Pre-hyperthermia contrast-enhanced images demonstrated higher pixel
intensity values near the tumor periphery whereas the immediate
post-hyperthermia contrast-enhanced images demonstrated an
approximately 50% increase in pixel intensity value in the tumor
core (FIG. 3-f) as compared to the prehyperthermia images (FIG.
3-e). The contrast uptake before and after gold nanoshell-mediated
hyperthermia in the tumor center and in the whole tumor is further
illustrated in FIGS. 3-g&h. The slopes (mean.+-.SE) of the pre-
and post-hyperthermia contrast uptake in the tumor core were
estimated as 2.49.+-.0.51 and 4.38.+-.0.60 arbitrary units
(a.u.)/sec, respectively and the corresponding values for the whole
tumor were 4.42.+-.0.22 and 8.99.+-.0.69 a.u./sec,
respectively.
[0076] Ex vivo analysis of tumor tissue. Two mice from each group
were euthanized 90 min after all treatment and tumors were
extracted for ex vivo analyses. Hematoxylin and eosin (H&E)
staining of the peripheral and core regions of tumors from all four
treatment groups is illustrated in FIG. 4-(a-h) with arrows
representing the necrotic regions. No necrotic regions were
observed in the tumor periphery of control, hyperthermia and
radiation groups (FIG. 4(a-c)), respectively. However, necrotic
regions were observed at a distance of .about.1.4 mm from the tumor
periphery in the thermoradiotherapy group (FIG. 4-d). The tumor
core of control, hyperthermia and radiation groups demonstrated
small necrotic regions in the range of .about.0.17-0.4 mm (FIG.
4-(e-g)), respectively. In contrast to these small necrotic
regions, large necrotic regions in the range of .about.0.6-1.2 mm
were observed in the tumor center of the thermoradiotherapy group
(FIG. 4-h).
[0077] Markers for tissue hypoxia, blood flow and proliferation. To
further understand the mechanism of extensive necrosis noted on
H&E staining, these tumor specimens from all four groups were
evaluated for changes in tumor cell proliferation as well as
changes in the tumor microenvironment, specifically with assessment
of hypoxia and blood flow (see supplementary information below for
marker injection and immunofluorescence staining procedure). The
hypoxic (white) and proliferative (grey) regions in control,
hyperthermia, radiation and thermoradiotherapy groups are
represented in FIGS. 5(a-d), respectively. In the control and
radiation groups, the hypoxic regions were predominantly in the
tumor core while the proliferative regions were largely confined to
the periphery of the tumor. However, in the hyperthermia group
(FIG. 5-b), the proliferative regions extended to the tumor core
with a corresponding decrease in the hypoxic area in the tumor
core. This may be attributable to the increased vascular perfusion
induced by gold nanoshell-mediated hyperthermia. Higher
magnification images of the periphery and tumor core of different
treatment groups demonstrating blood perfusion and tissue hypoxia
are shown in FIGS. 5-(e-h) & (i-l), respectively. Regions of
blood flow (grey) are associated with scant tissue hypoxia (white).
At higher magnification, more distinct differences in the patterns
of the hypoxic region were observed between the thermoradiotherapy
and other groups. The control, hyperthermia and radiation groups
demonstrated a structured pattern of a hypoxic region in the tumor
core with regions of perfusion between the hypoxic regions (FIG.
5-(i-k)). In contrast to this, the thermoradiotherapy group
demonstrated a distortion of this architecture characterized by
patchy hypoxic regions with no distinct regions of blood flow in
the tumor core (FIG. 5-i). We hypothesized as a reflection of a
hindrance to perfusion that also explains the massive necrosis
observed on H&E staining.
[0078] Assessment of tumor microvessels. To evaluate the pattern of
tumor vasculature in all four groups, CD31-immunofluorescence
staining was performed on all tumors extracted 90 min following
treatments (FIG. 6-a). No significant difference in the microvessel
density was observed between the control, hyperthermia and
radiation groups. However, the microvessel density was
significantly lower (p<0.05) in the thermoradiotherapy group
compared to other groups. The differences in microvessel density in
the tumor periphery and the tumor core of all groups are
illustrated in FIG. 6-b, which confirm the relative scarcity of
microvessels in tumors treated with thermoradiotherapy. Therefore,
these results indicate that thermoradiotherapy leads to an acute
increase in perfusion of tumors immediately following hyperthermia
(as demonstrated by DCE-MRI 3-5 min following hyperthermia) and a
subsequent distortion of vascular patterns (as demonstrated by the
decrease in microvessel density 90 min following radiation). These
observations are best explained by the nature of hyperthermia
induced by gold nanoshells wherein focal temperature rises are much
more pronounced adjacent to the nanoshells as compared to further
away from them, thereby creating heterogeneity in the distribution
of temperature within the entire tumor. Because this heterogeneity,
with sharp temperature gradients around the nanoshells, may
contribute to the observed vascular pattern disruption, the precise
location of gold nanoshells within tumors in the control and
hyperthermia groups was determined.
[0079] Localization of gold nanoshells within the tumor
microenvironment. The microscopic distribution of gold nanoshells
in tumors was determined by scanning electron microscopy (SEM). SEM
was performed using a JSM-5910 scanning electron microscope
operating at an accelerating voltage of 15 kV (JEOL, USA, Inc.,
Peabody, Mass.). In control tumors, SEM images revealed predominant
accumulation/distribution of nanoshells in perivascular regions
(.about.50-70 .mu.m from the vascular endothelium, represented by
white arrows) in the tumor tissues (FIG. 6c row 1). Similarly, in
the hyperthermia group, SEM images (FIG. 6c row 2) revealed intact
blood vessels with nanoshells distributed in the perivascular
region. Taken together, this perivascular sequestration of gold
nanoshells leads to a focal temperature rise near the blood vessels
after hyperthermia, which, in turn, leads to (i) an acute increase
in perfusion (FIG. 3) and (ii) a greater focal sensitivity to
subsequent radiation that results in vascular disruption (FIG.
5-i).
Supplementary Information
[0080] MRTI
[0081] MRTI was performed using a 1.5 T MR scanner (Signa
Echospeed, General Electric Medical Systems, Milwaukee, Wis.)
equipped with high performance gradients (23 mT/m maximum amplitude
and 120 T/m/sec maximum slew rate) and fast receiver hardware
(bandwidth, +/-500 MHz). A receive-only surface coil (3 inch
diameter) specially designed for small animal imaging was
positioned over the tumor for the MR imaging. T1-weighted (TR/TE
600/10.9 Bandwidth 25 KHz NEX 6 FOV 5.times.2.5 cm matrix
256.times.192) and T2-weighted (TR/TE 4500/15.7 Bandwidth 25 KHz
NEX 6 FOV 5.times.2.5 cm matrix 256.times.192) images were used to
localize the position for the MRTI by verifying the tumor location
and the laser axis. MRTI was performed by a complex
phase-difference technique (Ishihara, Y.; Calderon, A.; Watanabe,
H.; Okamoto, K.; Suzuki, Y.; Kuroda, K.; Suzuki, Y. Magn Reson Med
1995, 34, (6), 814-23) with a fast 2-D radiofrequency-spoiled
gradient-recalled echo sequence (TR/TE=360 ms/10 ms, flip
angle=30.degree., bandwidth=8.1 kHz; NEX 1; FOV 5.times.3 cm;
matrix 256.times.128). Real-time temperature was monitored with a
fast spoiled gradient-recalled (FSPGR) sequence. Temperature maps
and contrast uptake graphs were generated off-line, with the use of
MATLAB software (Mathworks, Natick, Mass.).
[0082] DCMRI
[0083] Dynamic Contrast Magnetic Resonant Imaging was performed
using Gd-DTPA (2:5 Gd concentration in saline, 1 .mu.l/gram body
weight). An FSPGR sequence was used for the dynamic contrast
enhanced (DCE) imaging (TR/TE 8.8/2.2 bandwidth 19.2 KHz NEX 12 FOV
5.times.3 cm matrix 256.times.128).
[0084] Markers Injection
[0085] Immediately after each treatment, the animals were injected
intravenously with 2.5 mg pimonidazole (Chemicon, Temecula, Calif.)
in 0.2 ml sterile saline to target the hypoxic cells. 15 mg of
bromodeoxyuridine (Sigma-Aldrich, St. Louis, Mo.) dissolved in 3.0
ml of sterile saline were injected intraperitoneally, 20 and 40 min
after the pimonidazole injection. The final injection of 0.4 mg of
Hoechst 33342 (Sigma-Aldrich, St. Louis, Mo.) in 0.1 ml of sterile
saline was administered intravenously 60 min after pimonidazole
injection and 1 min before euthanasia. Animals were euthanized by
cervical dislocation and the tumor tissues were immediately
dissected exactly into two halves along the central axis of the
tumor. One half of the tumor tissue was frozen in dry ice with
optimum cutting temperature (OCT) medium and stored at -80.degree.
C. and sequential sections of .about.6 to 8 .mu.m thickness were
made for immunofluorescence staining. The other half of the tumor
tissue was divided into two pieces and fixed in formalin for
hematoxylin and eosin (H&E) staining.
[0086] Immunofluorescence Staining
[0087] Three sequential sections from each animal were used for
immunofluorescence staining. Hypoxic cells were stained overnight
(at 4.degree. C.) with primary mouse anti-pimonidazole antibody
(Chemicon, Temecula, Calif.) diluted (1:50) in antibody diluent
(Dakocytomation) followed by 1 hr incubation (at 20.degree. C.).
with Alexa 488 (.lamda..sub.ex=488 nm, .lamda..sub.ex=520 nm)
labeled anti-mouse secondary antibody (Molecular Probes, Eugene,
Oreg.) diluted (1:100) in antibody diluent. Proliferating cells
were identified by incubating the tissue sections with 2N HCl for
10 min (at 20.degree. C.) for DNA denaturation (Zymed) followed by
10 min incubation with 0.1M Borax (at 20.degree. C.). Sections were
incubated overnight (at 4.degree. C.) with biotinylated
anti-bromodeoxyuridine antibody (Molecular Probes, Eugene, Oreg.)
diluted (1:50) in antibody diluent followed by 1 hr incubation (at
20.degree. C.) with Alexa 568 (.lamda..sub.ex=568 nm,
.lamda..sub.ex=603 nm) streptavidin conjugate (Molecular Probes,
Eugene, Oreg.) diluted (1:200) in antibody diluent. Microvessel
staining was performed by overnight incubation (at 4.degree. C.)
with 1:200 (diluted in antibody diluent) primary rat monoclonal
anti-CD31 antibody (Pharmingen) followed by 1 hr incubation (at
20.degree. C.) with TexasRed (.lamda..sub.ex=596 nm,
.lamda..sub.ex=615 nm) labeled secondary anti-rat IgG (1:100)
(Jackson Scientific) to stain the vasculature. Stained tissues were
covered with a cover slip using an anti-fade fluorescence-mounting
medium and observed under a Leica DM4000B fluorescence microscope
(Leica Microsystems, Wetzlar GmbH, Germany) equipped with a CCD
camera (RT KE/SE, Diagnostic Instruments Inc., Sterling Heights,
Mich., USA). The fluorescence from the Hoechst, Alexa-488,
Alexa-568 and TexasRed were collected using filter cubes with
suitable excitation and emission filters. The excitation and
emission bandpass-filter combinations of .lamda..sub.ex-BP360/40
nm, .lamda..sub.em-BP 470/40 nm and .lamda..sub.ex-BP480/40 nm,
.lamda..sub.em-BP 527/30 nm were used to detect the fluorescence
signal from Hoechst and Alexa-488, respectively. A combination of
BP515-560 nm excitation bandpass filter and LP590 nm longpass
emission filter was used to detect the fluorescence emission signal
from Alexa-568 and TexasRed. The acquired fluorescence images were
processed and analyzed using NIH ImageJ software.
[0088] Conclusions Relating to Examples
[0089] These examples indicate that hyperthermia mediated by gold
nanoparticles uniquely improves the efficacy of radiation therapy
by two mechanisms: (a) an early increase in perfusion which reduces
the fraction of hypoxic cells that contribute to radiation
resistance and (b) a subsequent (.about.90 min after treatment)
induction of vascular disruption/collapse and extensive necrosis
that complements radiation-induced cell death. This unique dual
effect of gold nanoparticle mediated hyperthermia is a consequence
of the focal temperature rise generated adjacent to tumor
vasculature where nanoshells have sequestered preferentially. The
first mechanism of improvement in the efficacy of radiation therapy
observed was due to the increased vascular perfusion of the tumor.
Tumors are typically perfused by a network of morphologically and
functionally abnormal vessels recruited by an orchestrated series
of dynamic events (angiogenesis) driven by the increasing demand
for oxygen and nutrients by proliferating tumor cells. Jain, P. K.;
Lee, K. S.; El-Sayed, I. H.; El-Sayed, M. A. J Phys Chem B 2006,
110, (14), 7238-48; and Jain, R. K. Cancer Res 1988, 48, (10),
2641-58. The resulting temporal and spatial heterogeneity of blood
flow leads to distinct regions where cells located distal to a
functional feeding blood vessel receive insufficient oxygen due to
its consumption by the cells closer to the blood vessel. These
hypoxic cells maintain their clonogenicity but their sensitivity to
radiation is up to three times less than that of normally
oxygenated cells. Brown, J. M. Methods Enzymol 2007, 435, 295-321.
These radioresistant cells not only contribute to tumor progression
but also their location distant from blood flow inevitably means
that blood-borne therapeutics has limited access to them.
Increasing perfusion via gold nanoshell-mediated hyperthermia,
therefore, reduces the fraction of cells within a tumor that are
hypoxic and thereby enhances radiation sensitivity.
[0090] The second mechanism of improvement of efficacy of radiation
therapy observed was due to vascular disruption. Vascular
disruption has been proposed as a viable anti-tumor strategy
because damaging a single established blood vessel, be it via
subtle structural changes of dysmorphic endothelial cells or
induction of intravascular coagulation, could potentially eliminate
hundreds or thousands of tumor cells downstream. Vascular
disrupting agents in preclinical and early clinical development
include combretastatin A4 phosphate (CA4P), ZD6126, TZT-1027,
AVE8062, ABT-751, and MN-029, which target the tubulin cytoskeletal
network of endothelial cells; 5,6-dimethylxanthenone-4-acetic acid
(DMXAA), which targets autocrine endothelial regulatory cascades;
and exherin (AFH-1), which targets cell adhesion. Patterson, D. M.;
Rustin, G. J. Clin Oncol (R Coll Radiol) 2007, 19, (6), 443-56;
Hinnen, P.; Eskens, F. A. Br J Cancer 2007, 96, (8), 1159-65;
Jameson, M. B.; Baguley, B. C.; Kestell, P.; Zhao, L.; Paxton, J.
W.; Thompson, P. I.; Waller, S. Cancer Chemother Pharmacol 2007,
59, (5), 681-7; O'Hanlon, L. H. J Natl Cancer Inst 2005, 97, (17),
1244-5; O'Hanlon, L. H., J. Nat'l Cancer Inst. 2005, 97, 17,
1244-45; Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nat Rev Cancer
2005, 5, (6), 423-35; Siemann, D. W.; Rojiani, A. M. Int J Radiat
Oncol Biol Phys 2005, 62, (3), 846-53; and Siemann, D. W.; Horsman,
M. R. Expert Rev Anticancer Ther 2004, 4, (2), 321-7. While these
agents have shown promise in early trials, there is concern that
more than just tumor vessels may be targeted by systemic exposure
to these agents. In particular, damage to vascular compartments
outside the tumor may contribute to acute coronary syndromes and
thromboembolic events. Van Heeckeren, W. J.; Bhakta, S.; Ortiz, J.;
Duerk, J.; Cooney, M. M.; Dowlati, A.; McCrae, K.; Remick, S. C. J
Clin Oncol 2006, 24, (10), 1485-8; and Van Heeckeren, W. J.;
Sanborn, S. L.; Narayan, A.; Cooney, M. M.; McCrae, K. R.;
Schmaier, A. H.; Remick, S. C. Curr Opin Hematol 2007, 14, (5),
468-80. In contrast to these agents, gold nanoshell-mediated
hyperthemia causes vascular disruption that is localized to the
tumor, permitting further increase in therapeutic ratio.
[0091] The observed radiation dose modification by gold nanoshells
is similar to that observed with other forms of hyperthermia. The
degree of necrosis observed, however, far exceeds that noted with
traditional means of achieving hyperthermia. Notably, this model
only evaluates the effects of this combination on single-fraction
high dose radiation therapy, similar to that used in clinical
stereotactic radiation therapy applications. This therapeutic
challenge could be converted to an opportunity for targeted therapy
using drugs targeting the hypoxia-inducible factor-1 transcription
factor, prodrugs activated by hypoxia, hypoxia-specific gene
therapy, and recombinant anaerobic bacteria. Alternate sequencing
strategies and alterations of the degree of temporal separation
between hyperthermia and radiation may be employed to maximize the
likelihood that radiation is administered during periods of peak
radiosensitivity defined by an optimal tumor oxygenation window or
a vascular normalization window. Dings, R. P.; Loren, M.; Heun, H.;
McNiel, E.; Griffioen, A. W.; Mayo, K. H.; Griffin, R. J. Clin
Cancer Res 2007, 13, (11), 3395-402; and Winkler, F.; Kozin, S. V.;
Tong, R. T.; Chae, S. S.; Booth, M. F.; Garkavtsev, I.; Xu, L.;
Hicklin, D. J.; Fukumura, D.; di Tomaso, E.; Munn, L. L.; Jain, R.
K. Cancer Cell 2004, 6, (6), 553-63. Lastly, targeting gold
nanoshells to tumor vasculature may further localize and
concentrate nanoshells along tumor vasculature to facilitate
tumor-directed therapy beyond image-guided physical collimation of
the laser beam to the tumor alone. Additional clinical benefit
could be realized if substances with a high atomic number (Z)
accumulate in tissues leading to greater radiation dose deposition,
particularly while utilizing kilovolt radiation therapy where
highly Z-dependent photoelectric interactions are the predominant
form of interaction. However, preliminary calculations, Monte Carlo
estimations and in vitro experiments do not bear this out to be a
sufficient source of dose escalation to be clinically significant
(data not shown). This is likely due to the insufficient tumor
parenchymal penetration beyond the immediate perivascular space by
the 160 nm nanoshells and the relatively low gold content of each
nanoshell in contrast to the significant dose enhancement
achievable with smaller nanogold particles. Hainfeld, J. F.;
Slatkin, D. N.; Smilowitz, H. M. Phys Med Biol 2004, 49, (18),
N309-15.
[0092] While the examples illustrate the use of gold nanoshells to
transducer near-infrared light, there are numerous other examples
of nanoparticles that may be used to transducer this or other
energy. See e.g., Liu Z, Cai W, He L, Nakayama N, Chen K, Sun X,
Chen X, Dai H "In vivo biodistribution and highly efficient tumour
targeting of carbon nanotubes in mice," Nat Nanotechnol. 2007 Jan;
2(1):47-52; Gannon C J, Patra C R, Bhattacharya R, Mukherjee P,
Curley S A. "Intracellular gold nanoparticles enhance non-invasive
radiofrequency thermal destruction of human gastrointestinal cancer
cells." J Nanobiotechnology, 2008 Jan. 30; 6:2. Additionally, while
the examples describe the passive accumulation of nanoparticles in
the tumor, one of ordinary skill will recognize that the
nanopaticles may be actively targeted to cell surfaces. See e.g.,
Lowery A R, Gobin A M, Day E S, Halas N J, West J L.,
"Immunonanoshells for targeted photothermal ablation of tumor
cells," Int J Nanomedicine, 2006, 1(2), 149-54.
[0093] Therefore, the present invention is well adapted to attain
the ends and advantages mentioned as well as those that are
inherent therein. The particular embodiments disclosed above are
illustrative only, as the present invention may be modified and
practiced in different but equivalent manners apparent to those
skilled in the art having the benefit of the teachings herein.
Furthermore, no limitations are intended to the details of
construction or design herein shown, other than as described in the
claims below. It is therefore evident that the particular
illustrative embodiments disclosed above may be altered or modified
and all such variations are considered within the scope and spirit
of the present invention. Also, the terms in the claims have their
plain, ordinary meaning unless otherwise explicitly and clearly
defined by the patentee.
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