U.S. patent application number 16/511605 was filed with the patent office on 2019-11-07 for non-invasive systems and methods for in-situ photobiomodulation.
This patent application is currently assigned to Immunolight, LLC.. The applicant listed for this patent is Duke University, Immunolight, LLC.. Invention is credited to Frederic A. BOURKE, JR., Tuan VO-DINH, Harold WALDER.
Application Number | 20190336786 16/511605 |
Document ID | / |
Family ID | 41135939 |
Filed Date | 2019-11-07 |
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United States Patent
Application |
20190336786 |
Kind Code |
A1 |
BOURKE, JR.; Frederic A. ;
et al. |
November 7, 2019 |
NON-INVASIVE SYSTEMS AND METHODS FOR IN-SITU PHOTOBIOMODULATION
Abstract
Products, compositions, systems, and methods for modifying a
target structure which mediates or is associated with a biological
activity, including treatment of conditions, disorders, or diseases
mediated by or associated with a target structure, such as a virus,
cell, subcellular structure or extracellular structure. The methods
may be performed in situ in a non-invasive manner by application of
an initiation energy to a subject thus producing an effect on or
change to the target structure directly or via a modulation agent.
The methods may further be performed by application of an
initiation energy to a subject in situ to activate a pharmaceutical
agent directly or via an energy modulation agent, optionally in the
presence of one or more plasmonics active agents, thus producing an
effect on or change to the target structure. Kits containing
products or compositions formulated or configured and systems for
use in practicing these methods.
Inventors: |
BOURKE, JR.; Frederic A.;
(Aspen, CO) ; VO-DINH; Tuan; (Durham, NC) ;
WALDER; Harold; (Oak Island, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunolight, LLC.
Duke University |
Detroit
Durham |
MI
NC |
US
US |
|
|
Assignee: |
Immunolight, LLC.
Detroit
MI
Duke University
Durham
NC
|
Family ID: |
41135939 |
Appl. No.: |
16/511605 |
Filed: |
July 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15151642 |
May 11, 2016 |
10391330 |
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16511605 |
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12417779 |
Apr 3, 2009 |
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15151642 |
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61042561 |
Apr 4, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
19/02 20180101; A61P 25/28 20180101; A61P 37/00 20180101; A61P 9/10
20180101; A61K 41/0057 20130101; A61P 19/08 20180101; A61P 29/00
20180101; A61N 2005/0642 20130101; A61P 25/00 20180101; A61K
47/6923 20170801; A61P 27/02 20180101; A61P 1/04 20180101; A61P
25/02 20180101; A61N 5/0618 20130101; A61N 2005/0661 20130101; A61N
5/10 20130101; A61N 2005/1098 20130101; A61N 5/022 20130101; A61P
43/00 20180101; A61P 25/04 20180101; A61N 2005/0659 20130101; A61P
13/12 20180101; A61P 31/00 20180101; A61P 35/02 20180101; A61N
5/062 20130101; A61P 25/08 20180101; A61P 37/04 20180101; A61P
13/08 20180101; A61P 25/16 20180101; A61K 41/0061 20130101; A61P
31/04 20180101; A61N 5/0622 20130101; A61N 2005/0662 20130101; A61P
37/06 20180101; A61P 31/12 20180101; A61P 17/02 20180101; A61P
35/00 20180101 |
International
Class: |
A61N 5/06 20060101
A61N005/06; A61K 41/00 20060101 A61K041/00; A61N 5/02 20060101
A61N005/02; A61N 5/10 20060101 A61N005/10; A61K 47/69 20060101
A61K047/69 |
Claims
1. (canceled)
2. A method for wound healing in a subject, comprising:
administering at least one energy modulation agent to a region of a
subject in need of wound healing, wherein the at least one energy
modulation agent is selected to have an emitted energy to be of a
wavelength sufficient to cause the wound healing upon application
of an applied initiation energy; and applying the initiation energy
from at least one source, wherein the initiation energy is
converted by the at least one energy modulation agent into the
emitted energy that directly causes wound healing in the region of
the subject.
3. The method of claim 2, wherein said initiation energy is capable
of penetrating completely through said subject.
4. The method of claim 2, wherein said initiation energy is applied
from a single source.
5. The method of claim 2, wherein said initiation energy is applied
from more than one source.
6. The method of claim 2, wherein said energy modulation agent
decreases the wavelength of the initiation energy.
7. The method of claim 2, wherein said energy modulation agent
increases the wavelength of the initiation energy.
8. The method of claim 2, wherein the initiation energy is UV
radiation, visible light, IR radiation, x-rays, gamma rays, an
electron beam, microwaves or radio waves.
9. The method of claim 2, wherein the initiation energy is
intensified by a nanoparticle or nanocluster of atoms and is
further absorbed by the energy modulation agent.
10. The method of claim 2, wherein a plasmonics-active agent is
further applied which enhances or modifies the applied initiation
energy, such that the enhanced initiation energy is absorbed,
intensified or modified by the energy modulation agent into the
energy that effects the predetermined change in said target
structure.
11. The method of claim 2, wherein the at least one energy
modulation agent is one or more members selected from a
biocompatible fluorescing metal nanoparticle, fluorescing metal
oxide nanoparticle, fluorescing metal coated metal oxide
nanoparticle, fluorescing dye molecule, gold nanoparticle, silver
nanoparticle, gold-coated silver nanoparticle, a water soluble
quantum dot encapsulated by polyamidoamine dendrimers, a
luciferase, a biocompatible phosphorescent molecule, a combined
electromagnetic energy harvester molecule, and a lanthanide chelate
exhibiting intense luminescence.
12. The method of claim 10, wherein the plasmonics-active agent is
a PEPST probe with multi plasmonics resonance mode.
13. The method of claim 10, wherein the plasmonics-active agent is
a PEPST probe comprising plasmonics-active metal
nanostructures.
14. The method of claim 13, wherein the metal nanostructures are
nanospheres, nanorods, nanocubes, nanopyramids, nanoshells,
multi-layer nanoshells and combinations thereof.
15. The method of claim 10, wherein the plasmonics-active agent is
a PEPST probe with multiple structures for different plasmonics
activation regimes.
16. The method of claim 15, wherein the plasmonics activation
regime is NIR and/or X rays.
17. The method of claim 10, wherein the plasmonics-active agent is
an exciton-induced phototherapy (EIP) probe possessing exciton
properties.
18. The method of claim 2, wherein the at least one energy
modulation agent is activated prior to administration and after
administration to the subject, the activated energy modulation
agent is triggered to emit an energy that induces the predetermined
change.
19. The method of claim 18, wherein the at least one energy
modulation agent is an infrared-triggered phosphor.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 15/151,642, filed May 11, 2016, now allowed, which is a
continuation of U.S. patent application Ser. No. 12/417,779, filed
Apr. 3, 2009, which claims priority to U.S. provisional patent
application 61/042,561, filed Apr. 4, 2008, and is also related to
U.S. patent application Ser. No. 11/935,655, filed Nov. 5, 2007;
U.S. patent application Ser. No. 12/059,484, filed Mar. 31, 2008;
U.S. patent application Ser. No. 12/389,946, filed Feb. 20, 2009;
the entire contents of each of which are hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
Field of Invention
[0002] The present invention relates to methods and systems for
treating a disorder or condition in a subject, that provide better
distinction between normal, healthy cells and those cells suffering
the disorder or condition (hereafter "target cells") and preferably
that can be performed using non-invasive or minimally invasive
techniques.
Discussion of the Background
Photobiomodulation
[0003] Photobiomodulation also known as low level laser therapy
(LLLT), cold laser therapy, and laser biostimulation, is an
emerging medical and veterinary technique in which exposure to
low-level laser light can stimulate or inhibit cellular function
leading to beneficial clinical effects. The "best" combination of
wavelength, intensity, duration and treatment interval is complex
and sometimes controversial with different diseases, injuries and
dysfunctions needing different treatment parameters and
techniques.
[0004] Certain wavelengths of light at certain intensities
(delivered by laser, LED or another monochromatic source) will, for
example, aid tissue regeneration, resolve inflammation, relieve
pain and boost the immune system. The exact mechanism is still
being explored and debated but it is agreed that the mechanism is
photochemical rather than heat-related. Observed biological and
physiological effects include changes in cell membrane
permeability, and up-regulation and down-regulation of adenosine
triphosphate and nitric oxide.
[0005] All light-induced biological effects depend on the
parameters of the irradiation (wavelength, dose, intensity,
irradiation time, depth of a target cell, and continuous wave or
pulsed mode, pulse parameters). (See, e.g., Karu I T, Low-Power
Laser Therapy", in Biomedical Photonics Handbook, Vo-Dinh T. Ed.,
CRC Press, Boca Raton, Fla., pp. 48-1 to 48-25, (2003)). Laser
average power is typically in the range of 1-500 mW; some high peak
power, short pulse width devices are in the range of 1-100 W with
typically 200 ns pulse widths. The average beam irradiance then is
typically 10 mW/cm.sup.2-5 W/cm.sup.2. The wavelength is typically
in the range 600-1000 nm. The red-to-near infrared (NIR) region is
preferred for photobiomodulation. Other wavelengths may be also
used, e.g., UV light for neurons and green light for prostate
tissue. Maximum biological responses are occurring when irradiated
at 620, 680, 760, and 820-830 nm (Karu T I, et al., (1998). The
Science of Low Power Laser Therapy. Gordon and Breach Sci. Publ.,
London). Large volumes and relatively deeper layers of tissues can
be successfully irradiated by laser only (e.g., inner and middle
ear diseases, injured siatic or optical nerves, inflammations). The
LEDs are used for irradiation of surface injuries.
[0006] A photoacceptor must first absorb the light used for the
irradiation. After promotion of electronically excited states,
primary molecule processes from these states can lead to a
measurable biological effect (via secondary biochemical reaction,
or photosignal transduction cascade, or cellular signaling) at the
cellular level. A photoacceptor for eukaryotic cells in red-to-NIR
region is believed to be the terminal enzyme of the respiratory
chain cytochrome c oxidase located in cell mitochondrion. In the
violet-to blue spectra region, flavoprotein (e.g.,
NADHdehydrogenase in the beginning of the respiratory chain) is
also among the photoacceptors.
[0007] Clinical applications of photobiomodulation include, for
example, treating soft tissue and bone injuries, chronic pain,
wound healing, nerve regeneration, sensory regeneration/restoration
and possibly even resolving viral and bacterial infections,
treating neurological and phychiatric diseases (e.g., epilepsy and
Parkinson's disease) (e.g., Zhang F., et al., Nature, 446:617-9
(Apr. 5, 2007; Han X., et al., PloS ONE, 2(3):e299 (Mar. 21, 2007);
Arany P R, et al., Wound Repair Regen., 15(6):866-74 (2007); Lopes
C B, et al., Photomed. Laser Surg., 25(2):96-101 (2007)). One
clinical application showing great promise is the treatment of
inflammation, where the anti-inflammatory effect of
location-and-dose-specific laser irradiation produces similar
outcomes as NSAIDs, but without the potentially harmful
side-effects (Bjordal J M, Couppe C, Chow R T, Tuner J, Ljunggren E
A (2003). "A systematic review of low level laser therapy with
location-specific doses for pain from chronic joint disorders". The
Australian journal of physiotherapy 49(2):107-16).
[0008] An NIR light treatment can prevent cell death (apoptosis) in
cultured neurons (brain) cells (Wong-Reiley M T, et al., JBC,
280(6):4761-71 (2005)). Specific wavelengths of light can promote
cellular proliferation to the activation of mitochondria, the
energy-producing organelles within the cell via cytochrome c
oxidase. An NIR treatment can augment mitochondrial function and
stimulate antioxidant protective pathways. The evidence that the
NIR treatment can augment mitochondrial function and stimulate
antioxidant protective pathways comes from photobiomodulation
experiments carried out using a laboratory model of Parkinson's
disease (PD) (cultures of human dopaminergic neuronal cells)
(Whelan H., et. al., SPIE, Newsroom, pages 1-3 (2008)).
[0009] It has also been shown that light has both inductive and
inhibitory effect on cell growth and division in a red tide
flagellate, Chattonella antique (Nemote Y., Plant and Cell
Physiol., 26(4):669-674 (1985)).
[0010] When the excitable cells (e.g., neurons, cardiomyocites) are
irradiated with monochromatic visible light, the photoacceptors are
also believed to be components of respiratory chain. It is clear
from experimental data (Karu, T. I., (2002). Low-power laser
therapy. In: CRC Biomedical Photonics Handbook, T. Vo-Dinh,
Editor-in-Chief, CRC Press, Boca Raton (USA)) that irradiation can
cause physiological and morphological changes in nonpigmental
excitable cells via absorption in mitochondria. Later, similar
irradiation experiments were performed with neurons in connection
with low-power laser therapy. It was shown in 80's that He--Ne
laser radiation alters the firing pattern of nerves; it was also
found that transcutaneous irradiation with HeNe laser mimicked the
effect of peripheral stimulation of a behavioral reflex. These
findings were found to be connected with pain therapy (Karu T I, et
al., (2002)).
[0011] When photoacceptors absorb photons, electronic excitation
followed by photochemical reactions occurring from lower excitation
states (first singlet and triplet) take place. It is also known
that electronic excitation of absorbing centers alters their redox
properties. Until yet, five primary reactions have been discussed
in literature (Karu T I, et al., (2002)). Two of them are connected
with alteration of redox properties and two mechanisms involve
generation of reactive oxygen species (ROE). Also, induction of
local transient (very short time) heating of absorbing chromophores
is possible. Details of these mechanisms can be found in (Karu T I,
et. al., (2002); Karu T I, et al., (1998). The Science of Low Power
Laser Therapy. Gordon and Breach Sci. Publ., London).
[0012] Photobiological action via activation of respiratory chain
is believed to be a general mechanism occurring in cells. Crucial
events of this type of cell metabolism activation are occurring due
to a shift of cellular redox potential into more oxidized direction
as well as due to ATP extrasynthesis. Susceptibility to irradiation
and capability for activation depend on physiological status of
irradiated cells: the cells, which overall redox potential is
shifted to more reduced state (example: some pathological
conditions) are more sensitive to the irradiation. The specificity
of final photobiological response is determined not at the level of
primary reactions in the respiratory chain but at the transcription
level during cellular signaling cascades. In some cells, only
partial activation of cell metabolism happens by this mechanism
(example: redox priming of lymphocytes).
[0013] Far red and NIR radiation have been shown to promote wound
healing, e.g., infected, ischemic, and hypoxic wounds (Wong-Reley,
W T T, JBC, 280(6):4761-4771 (2005)). Red-to-NIR radiation also
protects the retina against the toxic actions of methanol-derived
formic acid in a rodent model of methanol toxicity and may enhance
recovery from retinal injury and other ocular diseases in which
mitochondrial dysfunction is postulated to play a role (Eells J T.,
PNAS, 100(6):3439-44 (2003)). Other clinical applications of
photobiomodulation is repair of soft and bone tissues by IR laser
irradiation (Martinez Me., et al., Laser in Med. Sci., 2007).
Invasive laser assisted liposuction is a recently developed method,
wherein a laser fiber is introduced through a tube into the skin
and directly to the fat cells causing the cells to rapture and
drain away as liquid (Kim K H, Dermatol. Surg., 32(2):241-48
(2006)). Tissue around the area is coagulated. Yet, another
application of photobiomodulation is a non-surgical varicose vein
treatment (an endovenous laser therapy), wherein a laser is
threaded through an incision and the full length of the varicose
vein (Kim H S, J. Vasc. Interv. Radiol., 18(6):811 (2007)). When
the laser is slowly withdrawn, heat is applied to the vein walls,
causing the vein to permanently close and disappear.
[0014] Technological advances such as laser have redefined the
surgical treatment of enlarged prostate. The green light laser is a
laser that vaporizes and removes the enlarged prostate tissue
(Heinrich E., Eur. Urol., 52(6):1632-7 (2007)). The significance of
the color of the laser light (green) is that this results in
absorption by hemoglobin which is contained within red blood cells
and not absorbed by water. The procedure may also be known as laser
prostatectomy or laser Transurethral resection of the prostate
(TURP). The technique involves painting the enlarged prostate with
the laser until the capsule of the prostate is reached. By
relieving this portion of the prostate, patients are able to void
much easier through a wide-open channel in the prostate. The
procedure needs to be performed under general or spinal anesthesia.
An advantage of the procedure is that even patients taking blood
thinners (e.g., aspirin to prevent stroke) can be treated because
there is less bleeding compared to a traditional surgery.
[0015] Yet, another area of application of photobiomodulation is a
direct control of brain cell activity with light. The technique is
based upon NIR spectroscopy and is simpler to use and less
expensive than other methods such as functional magnetic resonance
imaging and positron emission tomography.
[0016] Whenever a region of the brain is activated that part of the
brain uses more oxygen. This technique works by measuring the blood
flow and oxygen consumption in the brain. The light emitted by NIR
laser diodes is carried through optical fibers to a person's head.
The light penetrates the skull where it assesses the brain's oxygen
level and blood volume. The scattered light is then collected by
optical fibers, sent to detectors and analyzed by a computer. By
examining how much of the light is scattered and how much is
absorbed, portions of the brain and extract information about brain
activity can be mapped. By measuring the scattering, it is
determined where the neurons are firing. This means that scientists
can simultaneously detect both blood profusion and neural activity.
The technique could be used in many diagnostic, prognostic and
clinical applications. For example, it could be used to find
hematomas in children, to study blood flow in the brain during
sleep apnea, and to monitor recovering stroke patients on a daily,
or even hourly, basis (that would be impractical to do with MRI).
To validate the technique, hemoglobin oxygen concentrations in the
brain obtained simultaneously by NIR spectroscopy and by functional
MRI, the current "gold standard" in brain studies, was compared.
Both methods were used to generate functional maps of the brain's
motor cortex during a periodic sequence of stimulation by finger
motion and rest. Spatial congruence between the hemoglobin signal
and the MRI signal in the motor cortex related to finger movement
was demonstrated. The researchers also demonstrated collocation
between hemoglobin oxygen levels and changes in scattering due to
brain activities. The changes in scattering associated with fast
neuron signals came from exactly the same locations.
[0017] A low-intensity laser light-oxygen cancer therapy is another
application of photobiomodulation. The light-oxygen effect (LOE),
which involves activation of or damage to biosystems by optical
radiation at low optical doses by direct photoexcitation of
molecular oxygen dissolved in a biosystem so that it is converted
to the singlet state, i.e., by photogeneration of molecular singlet
oxygen from O.sub.2 dissolved in cells, similar to photodynamic
effect (Zakharov S D, et al., Quantum Electronics, 29(12):1031-53
(1999)). It was shown that the He--Ne laser radiation destroys
tumor cells in the presence or absence of the photosensitiser. The
LOE can be activated by small optical doses, which are 4-5 orders
of magnitude lower that those found if a comparison is made with
the familiar analogue in the form of the photodynamic effect
(PDE).
[0018] Photobiostimulation using "caged" molecules and
light-sensitive proteins
[0019] This type of photobiomodulation methods fall into two
general categories: one set of methods uses light to uncage a
compound that then becomes biochemically active, binding to a
downstream effector. For example, this method involves applying
"caged" chemicals to a sample and then using light to open the cage
to invoke a reaction. Modified glutamate is useful for finding
excitatory connections between neurons, since the uncaged glutamate
mimics the natural synaptic activity of one neuron impinging upon
another. This method is used for elucidation of neuron functions
and imaging in brain slices using, for example, two-photon
glutamine uncageing (Harvey C D, et al., Nature, 450:1195-1202
(2007); Eder M, et al., Rev. Neurosci., 15:167-183 (2004)). Other
signaling molecules can be released by UV light stimulation, e.g.,
GABA, secondary messengers (e.g., Ca.sup.2+ and Mg.sup.2),
carbachol, capsaicin, and ATP (Zhang F., et al., 2006).
[0020] The other major photostimulation method is the use of light
to activate a light-sensitive protein such as rhodopsin (ChR2),
which can then excite the cell expressing the opsin.
[0021] It has been shown that channelrhodopsin-2, a monolithic
protein containing a light sensor and a cation channel, provides
electrical stimulation of appropriate speed and magnitude to
activate neuronal spike firing. Recently, photoinhibition, the
inhibition of neural activity with light, has become feasible with
the application of molecules such as the light-activated chloride
pump halorhodopsin to neural control. Together, blue-light
activated channelrhodopsin-2 and the yellow light-activated
chloride pump halorhodopsin enable multiple-color, optical
activation and silencing of neural activity.
[0022] ChR2 photostimulaiton involves genetic targeting ChR2 to
neurons and light pulsing the neurons expressing ChR2 protein. The
experiments have been conducted in vitro and in vivo in mice by in
vivo deep-brain photostimulaiton using optical fibers to deliver
light into the lateral hypothalamus (Adamantidis A R, et al.,
Nature 450:420-425 (2007)). Genetic targeting of ChR2 allows
exclusive stimulation of defined cellular subsets and avoids the
need for addition of the caged glutamate, facilitating
photostimulation in vivo (Wang H., et al., PNAS, 104(19):8143-48
(2007)). ChR2 photostimulation has been used for restoring visual
activity in mice with impaired vision, to evoke behavioral
responses in worms and flies (Wang H., et al., 2007). The robust
associative learning induced by ChR2-assisted photostimulaiton in
mice opens the door to study the circuit basis of perception and
cognition in vivo (Huber D., et al., 2007). This kind of neuronal
targeting and stimulation might have clinical application, e.g.,
deep brain stimulation to treat Parkinson's disease and other
disorders, controlling behavioral, perceptional and cognitive
characteristics, and for imaging and studying how the brain works
(Zhang F., et al., Nature Methods, 3(10):785-792 (2006); Wong-Riley
M T., et al., JBC, 280(6):4761-4771 (2005)).
[0023] Another gene, chloride pump (NpHR), which is borrowed from a
microbe called an archaebacterium, can make neurons less active in
the presence of yellow light. Combined, the two genes ChR2 and NpHR
can now make neurons obey pulses of light like drivers obey a
traffic signal: Blue means "go" (emit a signal), and yellow means
"stop" (don't emit).
[0024] Light-sensitive proteins can be introduced into cells or
live subjects via a number of techniques including electroporation,
DNA microinjection, viral delivery, liposomal transfection and
calcium-phosphate precipitation.
[0025] A third photostimulation technique is chemical modification
of ion channels and receptors to render them light-responsive. Some
of the most fundamental signaling mechanisms in a cell involve the
release and uptake of Ca.sup.2+ ions. Ca.sup.2+ is involved in
controlling fertilization, differentiation, proliferation,
apoptosis, synaptic plasticity, memory, and developing axons. It
has been shown that Ca.sup.2+ waves can be induced by UV
irradiation (single-photon absorption) and NIR irradiation
(two-photon absorption) by releasing caged Ca.sup.2+, an
extracellular purinergic messenger InsP3 (Braet K., et al., Cell
Calcium, 33:37-48 (2003)), or ion channel ligands (Zhang F., et
al., 2006).
[0026] Directly controlling a brain cell activity with light is a
novel means for experimenting with neural circuits and could lead
to therapies for some disorders. This accomplishment is a step
toward the goal of mapping neural circuit dynamics on a millisecond
timescale to see if impairments in these dynamics underlie severe
psychiatric symptoms. Knowing the effects that different neurons
have could ultimately help researchers figure out the workings of
healthy and unhealthy brain circuits. If use of the technique can
show that altered activity in a particular kind of neuron underlies
symptoms, for example, this insight will allow development of
targeted genetic or pharmaceutical treatments to fix those neurons.
Conceivably, direct control of neuronal activity with light could
someday become a therapy in itself.
[0027] In living organisms, scientists were able to cause worms, C.
elegans, to stop swimming while their genetically altered motor
neurons were exposed to pulses of yellow light intensified through
a microscope. In some experiments, exposure to blue light caused
the worms to wiggle in ways they weren't moving while unperturbed.
When the lights were turned off, the worms resumed their normal
behavior.
[0028] Meanwhile, in experiments in living brain tissues extracted
from mice, the researchers were able to use the technique to cause
neurons to signal or stop on the millisecond timescale, just as
they do naturally. Other experiments showed that cells appear to
suffer no ill effects from exposure to the light. They resume their
normal function once the exposure ends.
[0029] The most direct application of an optical neuron control is
experimenting with neural circuits to determine why unhealthy ones
fail and how healthy ones work.
[0030] In patients with Parkinson's disease, for example,
researchers have shown that electrical "deep brain stimulation" of
cells can help patients, but they don't know precisely why. By
allowing researchers to selectively stimulate or dampen different
neurons in the brain, the light stimulation techniques could help
in determining which particular neurons are benefiting from deep
brain stimulation. That could lead to making the electrical
treatment, which has some unwanted side effects, more targeted.
[0031] Another potential application is experimenting with
simulating neural communications. Because neurons communicate by
generating patterns of signals-sometimes on and sometimes off like
the 0s and 1s of binary computer code-flashing blue and yellow
lights in these patterns could compel neurons to emit messages that
correspond to real neural instructions. In the future, this could
allow researchers to test and tune sophisticated neuron behaviors.
Much farther down the road, the ability to artificially stimulate
neural signals, such as movement instructions, could allow doctors
to bridge blockages in damaged spinal columns, perhaps restoring
some function to the limbs of paralyzed patients.
[0032] Finally, the technique could be useful in teasing out the
largely unknown functioning of healthy brains.
[0033] Problems with LLLT, Cold Laser Therapy, and Laser
Biostimulation
[0034] The laser systems currently used for biostimulation do not
allow performing photobiomodulation in a region deep within thick
tissue without a surgical invasion. Laser therapy is mostly
conducted in surface or near surface target cells and tissue
because penetration of UV and red-to-N IR radiation used for
photobiomodulation and photobiostimulaiton is no more than a few
centimeters beneath the surface of the skin. In addition, imaging
and stimulation of brain cells is mainly possible in thin brain
slices, or a thin monolayer or suspension of cells. For deeper
tissue laser therapy in situ, a subject undergoes various invasive
surgical procedures, e.g., invasive insertion of a fiber via
incisions into a fat layer or veins, implanting a radiation source
in deep tissue, or implanting a glass window above the barrel
cortex (Huber D., et al., Nature, 451:61-66 (2007)). It is further
well recognized that another problem associated with the existing
methods of photobiomodulation is in differentiation of normal cells
from target cells.
Phototherapy
[0035] There are two main types of reactions in phototherapy:
[0036] (1) Type I reactions involve electrons and hydrogen atoms,
which are transferred between photo-active molecules (also called
photosensitizers) and substrates or solvent molecules. Oxygen may
participate in subsequent reactions: e.g., psoralens in
photopheresis and PUVA. [0037] (2) Type II reactions involve
singlet oxygen formation by energy transfer from PA molecules in
the lowest triplet state to oxygen in the ground state: e.g.,
photodynamic therapy (PDT) Photodynamic therapy (PDT) is a
treatment modality that uses a photosensitizing agent and laser
light to kill cells. PDT is a relatively new light-based treatment,
which has recently been approved by the United States Food &
Drug Administration (FDA) for the treatment of both early and
late-stage lung cancer. Other countries have approved PDT for
treatment of various cancers as well. Unlike chemotherapy,
radiation, and surgery, PDT is useful in treating all cell types,
whether small cell or non-small cell carcinoma. PDT involves
treatment of diseases such as cancer using light action on a
special photoactive class of drugs, by photodynamic action in vivo
to destroy or modify tissue [Dougherty T. J. and Levy J. G.,
"Photodynamic Therapy and Clinical Applications", in Biomedical
Photonics Handbook, Vo-Dinh T., Ed., CRC Press, Boca Raton Fla.
(2003)]. PDT, which was originally developed for treatment of
various cancers, has now been used to include treatment of
pre-cancerous conditions, e.g. actinic keratoses, high-grade
dysplasia in Barrett's esophagus, and non-cancerous conditions,
e.g. various eye diseases, e.g. age related macular degeneration
(AMD). Photodynamic therapy (PDT) is approved for commercialization
worldwide both for various cancers (lung, esophagus) and for
AMD.
[0038] The PDT process requires three elements: (1) a PA drug
(i.e., photosensitizer), (2) light that can excite the
photosensitizer and (3) endogenous oxygen. The putative cytotoxic
agent is singlet oxygen, an electronically excited state of ground
state triplet oxygen formed according to the Type II photochemical
process, as follows.
PA+h.nu..fwdarw..sup.1PA* (S) Excitation
[0039] .sup.1PA* (S).fwdarw..sup.3PA* (T) Intersystem crossing for
singlet to triplet state .sup.3PA*
(T)+O.sub.2.fwdarw..sup.1O*.sub.2+PA Energy transfer from the drug
to singlet oxygen where PA=photo-active drug at the ground state;
.sup.1PA*(S)=excited singlet state; .sup.3PA*(T)=excited triplet
state; .sup.1O*.sub.2=singlet excited state of oxygen
[0040] Because the triplet state has a relatively long lifetime
(msec to seconds) only photosensitizers that undergo efficient
intersystem crossing to the excited triplet state will have
sufficient time for collision with oxygen in order to produce
singlet oxygen. The energy difference between ground state and
singlet oxygen is 94.2 kJ/mol and corresponds to a transition in
the near-infrared at .about.1270 nm. Most PA photosensitizers in
clinical use have triplet quantum yields in the range of 40-60%
with the singlet oxygen yield being slightly lower. Competing
processes include loss of energy by deactivation to ground state by
fluorescence or internal conversion (loss of energy to the
environment or surrounding medium).
[0041] However, while a high yield of singlet oxygen is desirable
it is by no means sufficient for a photosensitizer to be clinically
useful. Pharmacokinetics, pharmacodynamics, stability in vivo and
acceptable toxicity play critical roles as well [Henderson B W,
Gollnick S O, "Mechanistic Principles of Photodynamic Therapy", in
Biomedical Photonics Handbook, Vo Dinh T, Ed., CRC Press, Boca
Raton Fla. (2003)]. For example, it is desirable to have relatively
selective uptake in the tumor or other tissue being treated
relative to the normal tissue that necessarily will be exposed to
the exciting light as well. Pharmacodynamic issues such as the
subcellular localization of the photosensitizer may be important as
certain organelles appear to be more sensitive to PDT damage than
others (e.g. the mitochondria). Toxicity can become an issue if
high doses of photosensitizer are necessary in order to obtain a
complete response to treatment. An important mechanism associated
with PDT drug activity involves apoptosis in cells. Upon absorption
of light, the photosensitiser (PS) initiates chemical reactions
that lead to the direct or indirect production of cytotoxic species
such as radicals and singlet oxygen. The reaction of the cytotoxic
species with subcellular organelles and macromolecules (proteins,
DNA, etc) lead to apoptosis and/or necrosis of the cells hosting
the PDT drug. The preferential accumulation of PDT drug molecules
in cancer cells combined with the localized delivery of light to
the tumor, results in the selective destruction of the cancerous
lesion. Compared to other traditional anticancer therapies, PDT
does not involve generalized destruction of healthy cells. In
addition to direct cell killing, PDT can also act on the
vasculature, reducing blood flow to the tumor causing its necrosis.
In particular cases it can be used as a less invasive alternative
to surgery.
[0042] There are several chemical species used for PDT including
porphyrin-based sensitizers. A purified hematoporphyrin derivative,
Photofrin.RTM., has received approval of the US Food and Drug
Administration. Porphyrins are generally used for tumors on or just
under the skin or on the lining of internal organs or cavities
because theses drug molecules absorbs light shorter than 640 nm in
wavelength. For tumors occurring deep in tissue, second generation
sensitizers, which have absorbance in the NIR region, such as
porphyrin-based systems [R. K. Pandey, "Synthetic Strategies in
designing Porphyrin-Based Photosensitizers`, in Biomedical
Photonics Handbook, Vo-Dinh T., Ed., CRC Press, Boca Raton Fla.
(2003)], chlorines, phthalocyanine, and naphthalocyanine have been
investigated.
[0043] PDT retains several photosensitizers in tumors for a longer
time than in normal tissues, thus offering potential improvement in
treatment selectivity. See corner C., "Determination of [3H]- and
[14C] hematoporphyrin derivative distribution in malignant and
normal tissue," Cancer Res 1979, 3 9: 146-15 1; Young S W, et al.,
"Lutetium texaphyrin (PCI-0123) a near-infrared, water-soluble
photosensitizer," Photochem Photobiol 1996, 63:892-897; and
Berenbaum M C, et al., "Meso-Tetra(hydroxyphenyl)porphyrins, a new
class of potent tumor photosensitizers with favorable selectivity,"
Br J Cancer 1986, 54:717-725. Photodynamic therapy uses light of a
specific wavelength to activate the photosensitizing agent. Various
light sources have been developed for PDT, which include dye lasers
and diode lasers. Light generated by lasers can be coupled to
optical fibers that allow the light to be transmitted to the
desired site. See Pass 1-11, "Photodynamic therapy in oncology:
mechanisms and clinical use," J Natl Cancer Inst 1993, 85:443-456.
According to researchers, the cytotoxic effect of PDT is the result
of photooxidation reactions, as disclosed in Foote C S, "Mechanisms
of photooxygenation," Proa Clin Biol Res 1984, 170:3-18. Light
causes excitation of the photosensitizer, in the presence of
oxygen, to produce various toxic species, such as singlet oxygen
and hydroxyl radicals. It is not clear that direct damage to DNA is
a major effect; therefore, this may indicate that photoactivation
of DNA crosslinking is not stimulated efficiently.
[0044] Furthermore, when laser light is administered via external
illumination of tissue surfaces, the treatment effect of PDT is
confined to a few millimeters (i.e. superficial). The reason for
this superficial limitation is mainly the limited penetration of
the visible light used to activate the photosensitizer. Thus, PDT
is used to treat the surfaces of critical organs, such as lungs or
intra-abdominal organs, without damage to the underlying
structures. However, even these treatments require significantly
invasive techniques to treat the surface of the affected organs.
Clinical situations use the procedure in conjunction with surgical
debulking to destroy remnants of microscopic or minimal gross
disease. It is possible that the laser light and small amount of
remaining microscopic and minimal gross disease results in too
little or highly damaged structures. Pre-clinical data show that
some immune response is generated, but clinical trials have
reported no auto vaccine effect similar to that produced by
extracorporeal photopheresis in clinical conditions. Instead, the
immune response appears to be vigorous only under limited
conditions and only for a limited duration. PDT retains several
photosensitizers in tumors for a longer time than in normal
tissues, thus offering potential improvement in treatment
selectivity. See Comer C., "Determination of [3H]- and [14C]
hematoporphyrin derivative distribution in malignant and normal
tissue," Cancer Res 1979, 3 9: 146-15 1; Young S W, et al.,
"Lutetium texaphyrin (PCI-0123) a near-infrared, water-soluble
photosensitizer," Photochem Photobiol 1996, 63:892-897; and
Berenbaum M C, et al., "Meso-Tetra(hydroxyphenyl)porphyrins, a new
class of potent tumor photosensitizers with favorable selectivity,"
Br J Cancer 1986, 54:717-725. Photodynamic therapy uses light of a
specific wavelength to activate the photosensitizing agent. Various
light sources have been developed for PDT that include dye lasers
and diode lasers. Light generated by lasers can be coupled to
optical fibers that allow the light to be transmitted to the
desired site. See Pass 1-11, "Photodynamic therapy in oncology:
mechanisms and clinical use," J Natl Cancer Inst 1993, 85:443-456.
According to researchers, the cytotoxic effect of PDT is the result
of photooxidation reactions, as disclosed in Foote C S, "Mechanisms
of photooxygenation," Proa Clin Biol Res 1984, 170:3-18. Light
causes excitation of the photosensitizer, in the presence of
oxygen, to produce various toxic species, such as singlet oxygen
and hydroxyl radicals. It is not clear that direct damage to DNA is
a major effect; therefore, this may indicate that photoactivation
of DNA crosslinking is not stimulated efficiently.
[0045] Photopheresis has been successfully used for treatment of
cell proliferation disorders. Exemplary cell proliferation
disorders may include, but are not limited to, cancer, bacterial
infection, immune rejection response of organ transplant, solid
tumors, viral infection, autoimmune disorders (such as arthritis,
lupus, inflammatory bowel disease, Sjogrens syndrome, multiple
sclerosis) or a combination thereof, as well as aplastic conditions
wherein cell proliferation is low relative to healthy cells, such
as aplastic anemia. Of these, cancer is perhaps the most well
known.
[0046] Other successful application of PDT is, for example, cardiac
ablasion therapy, e.g., treating cardiac arrhythmias and atrial
fibrillation which are believed to be a significant cause of
cerebral stroke.
[0047] U.S. Pat. No. 6,811,562 describes administering a
photoactivatable agent and subjecting cardiac tissue containing the
administered agent to laser irradiation having a wavelength from
350 to 700 nm using invasive techniques, e.g., a fiber optic
element.
[0048] Yet, another application of PDT is photoangioplasty for
arterial diseases including de novo atherosclerosis and restinosis
(Rockson A G, et al., Circulation, 102:591-596 (2000); Hsiang Y N.,
et al., J. Endovasc. Surg., 2:365-371 (1995)). In human clinical
applications, endovascular light (730 nm) is delivered through a
cylindrical fiber after intravenous administration of motexafin
lutetium. PDT is also used for preventing and treatment of intimal
hyperlpasia in blood vessels in vivo (see, e.g., U.S. Pat. No.
6,609,014).
[0049] Age-related macular degeneration (AMD) is a cause of new
blindness. Choroidal neovascularization leads to hemorrhage and
fibrosis in a number of ocular diseases. Conventional treatments
utilize the argon laser to occlude the leaking vessel by thermal
coagulation. However, the percentage of patients eligible for this
treatment is limited. PDT is used for treating AMD and involves
injecting verteporfin followed by the application of non-thermal
light at 692 nm.
[0050] Improvement of clinical appearance of psoriatic plaques and
palmopustular psoriasis using PUVA with hematopotphyrin was first
reported in 1937. Acne, apopecia areata, portwine stains and hair
removal also show promise with PDT treatment.
[0051] The choice of therapy usually depends on the location and
severity of the disorder, the stage of the disease, as well as the
patient's response to the treatment.
[0052] While some treatments may only seek to manage and alleviate
symptoms of the disorder, the ultimate goal of any effective
therapy is the complete removal or cure of all disordered cells
without damage to the rest of the body.
[0053] In one existing treatment known as extracorporeal
photopheresis (ECP), excellent results have been observed since its
initial approval by the FDA in 1988.
[0054] Extracorporeal photopheresis is a leukapheresis-based
immunomodulatory therapy that has been approved by the US Food and
Drug Administration for the treatment of cutaneous T-cell lymphoma
(CTCL). ECP, also known as extracorporeal photochemotherapy, is
performed at more than 150 centers worldwide for multiple
indications. Long-term follow-up data are available from many
investigators that indicate ECP produces disease remission and
improved survival for CTCL patients. In addition to CTCL, ECP has
been shown to have efficacy in the treatment of other T-cell
mediated disorders, including chronic graft versus host disease
(GVHD) and solid organ transplant rejection. ECP use for the
treatment of autoimmune disease, such as systemic sclerosis and
rheumatoid arthritis, is also being explored.
[0055] ECP is generally performed using the UVAR XTS Photopheresis
System developed by Therakos, Inc (Exton, Pa.). The process is
performed through one intravenous access port and has 3 basic
stages: (1) leukapheresis, (2) photoactivation, and (3) reinfusion,
and takes 3-4 hours to complete. A typical treatment session would
resemble the following sequence of events:
[0056] (1) One 16-gauge peripheral intravenous line or central
venous access is established in the patient;
[0057] (2) Blood (225 mL) is passed through 3 cycles of
leukapheresis, or 125 mL of blood is passed through 6 cycles,
depending on the patient's hematocrit value and body size. At the
end of each leukapheresis cycle, the red blood cells and plasma are
returned to the patient;
[0058] (3) The collected WBCs (including approximately 5% of the
peripheral blood mononuclear cells) are mixed with heparin, saline,
and 8-methoxypsoralen (8-MOP), which intercalates into the DNA of
the lymphocytes upon exposure to UVA light and makes them more
susceptible to apoptosis when exposed to UVA radiation;
[0059] (4) The mixture is passed as a 1-mm film through a sterile
cassette surrounded by UVA bulbs, resulting in an average UVA
exposure of 2 J/cm.sup.2; and
[0060] (5) The treated WBC mixture is returned to the patient.
[0061] Over the past 20 years, on-going research has explored the
mechanism of action of ECP. The combination of 8-MOP and UVA
radiation causes apoptosis of the treated T cells and may cause
preferential apoptosis of activated or abnormal T cells, thus
targeting the pathogenic cells of CTCL or GVHD. However, given that
only a small percentage of the body's lymphocytes are treated, this
seems unlikely to be the only mechanism of action.
[0062] Other evidence suggests that ECP also induces monocytes to
differentiate into dendritic cells capable of phagocytosing and
processing the apoptotic T-cell antigens. When these activated
dendritic cells are reinfused into the systemic circulation, they
may cause a systemic cytotoxic CD8.sup.+ T-lymphocyte-mediated
immune response to the processed apoptotic T-cell antigens.
[0063] Finally, animal studies indicate that photopheresis may
induce antigen-specific regulatory T cells, which may lead to
suppression of allograft rejection or GVHD.
[0064] However, there are still many limitations to ECP. For
example, ECP requires patient to be connected to a machine for
hours per treatment. It requires establishing peripheral
intravenous line or central venous access, which may be difficult
to do in certain disease states such as systemic sclerosis or
arthritis. There is also a risk of infection at the venous or
central line site, or in the central line catheter. Further, it
requires removing typically several hundred milliliters of whole
blood from the patient, hence, the treatment is limited to patients
who has sufficiently large initial volume of blood to be withdrawn.
The American Association of Blood Blanks recommend a limit of
extracorporeal volume to 15% of the patient's whole body blood
volume. Therefore, the size of the volume that can be treated
generally has to be at least 40 kg or more. Risk of contracting
blood-born pathogen (Hepatitis, HIV, etc.) due to exposure to
contaminated operating system is also a concern.
[0065] Alternatively, a patient can be treated in vivo with a
photosensitive agent followed by the withdrawal of a sample from
the patient, treatment with UV radiation in vitro (ex vivo), and
reinjecting the patient with the treated sample. This method is
known for producing an autovaccine. A method of treating a patient
with a photosensitive agent, exposing the patient to an energy
source and generating an autovaccine effect wherein all steps are
conducted in vivo has not been described. See WO 03/049801, U.S.
Pat. Nos. 6,569,467; 6,204,058; 5,980,954; 6,669,965; 4,838,852;
7,045,124, and 6,849,058. Moreover, the side effects of
extracorporeal photopheresis are well known and include nausea,
vomiting, cutaneous erythema, hypersensitivity to sunlight, and
secondary hematologic malignancy. Researchers are attempting to use
photopheresis in experimental treatments for patients with cardiac,
pulmonary and renal allograft rejection; autoimmune diseases, and
ulcerative colitis.
[0066] A survey of known treatment methods reveals that these
methods tend to face a primary difficulty of differentiating
between normal cells and target cells when delivering treatment,
often due to the production of singlet oxygen which is known to be
non-selective in its attack of cells, as well as the need to
perform the processes ex vivo, or through highly invasive
procedures, such as surgical procedures in order to reach tissues
more than a few centimeters deep within the subject.
[0067] U.S. Pat. No. 5,829,448 describes sequential and
simultaneous two photon excitation of photo-agents using
irradiation with low energy photons such as infrared or near
infrared light (NRI). A single photon and simultaneous two photon
excitation is compared for psoralen derivatives, wherein cells are
treated with the photo agent and are irradiated with NRI or UV
radiation. The patent suggests that treating with a low energy
irradiation is advantageous because it is absorbed and scattered to
a lesser extent than UV radiation. However, the use of NRI or UV
radiation is known to penetrate tissue to only a depth of a few
centimeters. Thus any treatment deep within the subject would
necessarily require the use of ex vivo methods or highly invasive
techniques to allow the irradiation source to reach the tissue of
interest. Also, this patent does not describe initiation energy
sources emitting energy other than UV, visible, and near infrared
energy; energy upgrading other than within the range corresponding
to UV and IR light, and downgrading from high to low energy.
[0068] Chen et al., J. Nanosci. and Nanotech., 6:1159-1166 (2006);
Kim et al., JACS, 129:2669-2675 (2007); U.S. 2002/0127224; and U.S.
Pat. No. 4,979,935 each describe methods for treatment using
various types of energy activation of agents within a subject.
However, each suffers from the drawback that the treatment is
dependent on the production of singlet oxygen to produce the
desired effect on the tissue being treated, and is thus largely
indiscriminate in affecting both healthy cells and the diseased
tissue desired to be treated.
[0069] U.S. Pat. No. 6,908,591 discloses methods for sterilizing
tissue with irradiation to reduce the level of one or more active
biological contaminants or pathogens, such as viruses, bacteria,
yeasts, molds, fungi, spores, prions or similar agents responsible,
alone or in combination, for transmissible spongiform
encephalopathies and/or single or multicellular parasites, such
that the tissue may subsequently be used in transplantation to
replace diseased and/or otherwise defective tissue in an animal.
The method may include the use of a sensitizer such as psoralen, a
psoralen-derivative or other photosensitizer in order to improve
the effectiveness of the irradiation or to reduce the exposure
necessary to sterilize the tissue. However, the method is not
suitable for treating a patient and does not teach any mechanisms
for stimulating the photosensitizers, indirectly.
[0070] U.S. Pat. No. 5,957,960 discloses a two-photon excitation
device for administering a photodynamic therapy to a treatment site
within a patient's body using light having an infrared or near
infrared waveband. However, the reference fails to disclose any
mechanism of photoactivation using energy modulation agent that
converts the initiation energy to an energy that activates the
activatable pharmaceutical agent and also use of other energy
wavebands, e.g., X-rays, gamma-rays, electron beam, microwaves or
radio waves.
[0071] U.S. Pat. No. 6,235,508 discloses antiviral applications for
psoralens and other photoactivatable molecules. It teaches a method
for inactivating viral and bacterial contaminants from a biological
solution. The method includes mixing blood with a photosensitizer
and a blocking agent and irradiating the mixture to stimulate the
photosensitizer, inactivating substantially all of the contaminants
in the blood, without destroying the red blood cells. The blocking
agent prevents or reduces deleterious side reactions of the
photosensitizer, which would occur if not in the presence of the
blocking agent. The mode of action of the blocking agent is not
predominantly in the quenching of any reactive oxygen species,
according to the reference.
[0072] Also, U.S. Pat. No. 6,235,508 suggests that halogenated
photosensitizers and blocking agents might be suitable for
replacing 8-methoxypsoralen (8-MOP) in photopheresis and in
treatment of certain proliferative cancers, especially solid
localized tumors accessible via a fiber optic light device or
superficial skin cancers. However, the reference fails to address
any specific molecules for use in treating lymphomas or any other
cancer. Instead, the reference suggests a process of photopheresis
for antiviral treatments of raw blood and plasma.
[0073] U.S. Pat. No. 6,235,508 teaches away from 8-MOP and
4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) and many other
photoactivatable molecules, which are taught to have certain
disadvantages. Fluorescing photosensitizers are said to be
preferred, but the reference does not teach how to select a system
of fluorescent stimulation or photoactivation using fluorescent
photosensitizers. Instead, the fluorescing photosensitizer is
limited to the intercalator that is binding to the DNA. The
reference suggests that fluorescence indicates that such an
intercalator is less likely to stimulate oxygen radicals.
[0074] U.S. published application 2002/0127224 discloses a method
for a photodynamic therapy comprising administering light-emitting
nanoparticles and a photoactivatable agent, which may be activated
by the light re-emitted from the nanoparticles via a two-photon
activation event. An initiation energy source is usually a light
emitting diode, laser, incandescent lamp, or halogen light, which
emits light having a wavelength ranging from 350 to 1100 nm. The
initiation energy is absorbed by the nanoparticles. The
nanoparticles, in turn, re-emit light having a wavelength from 500
to 1100 nm, preferably, UV-A light, wherein the re-emitted energy
activates the photoactivatable agent. Kim et al., (JACS,
129:2669-75, Feb. 9, 2007) discloses indirect excitation of a
photosensitizing unit (energy acceptor) through fluorescence
resonance energy transfer (FRET) from the two-photon absorbing dye
unit (energy donor) within an energy range corresponding to 300-850
nm. These references do not describe initiation energy sources
emitting energy other than UV, visible, and near infrared energy;
energy upgrading other than within the range corresponding to
wavelength of 350-1100 nm, and downgrading from high to low
energy.
[0075] These references fail to disclose any mechanism of
photoactivation of an photoactivatable molecules other than by
direct photoactivation by UV, visible, and near infrared
energy.
Psoralens and Related Compounds
[0076] U.S. Pat. No. 6,235,508 further teaches that psoralens are
naturally occurring compounds which have been used therapeutically
for millennia in Asia and Africa. The action of psoralens and light
has been used to treat vitiligo and psoriasis (PUVA therapy;
Psoralen Ultra Violet A). Psoralen is capable of binding to nucleic
acid double helices by intercalation between base pairs; adenine,
guanine, cytosine and thymine (DNA) or uracil (RNA). Upon
sequential absorption of two UV-A photons, psoralen in its excited
state reacts with a thymine or uracil double bond and covalently
attaches to both strands of a nucleic acid helix. The crosslinking
reaction appears to be specific for a thymine (DNA) or a uracil
(RNA) base. Binding proceeds only if psoralen is intercalated in a
site containing thymine or uracil, but an initial photoadduct must
absorb a second UVA photon to react with a second thymine or uracil
on the opposing strand of the double helix in order to crosslink
each of the two strands of the double helix, as shown below. This
is a sequential absorption of two single photons as shown, as
opposed to simultaneous absorption of two or more photons.
##STR00001##
[0077] In addition, the reference teaches that 8-MOP is unsuitable
for use as an antiviral, because it damages both cells and viruses.
Lethal damage to a cell or virus occurs when the psoralen is
intercalated into a nucleic acid duplex in sites containing two
thymines (or uracils) on opposing strands but only when it
sequentially absorbs 2 UVA photons and thymines (or uracils) are
present. U.S. Pat. No. 4,748,120 of Wiesehan is an example of the
use of certain substituted psoralens by a photochemical
decontamination process for the treatment of blood or blood
products.
[0078] Additives, such as antioxidants are sometimes used with
psoralens, such as 8-MOP, AMT and I-IMT, to scavenge singlet oxygen
and other highly reactive oxygen species formed during
photoactivation of the psoralens. It is well known that UV
activation creates such reactive oxygen species, which are capable
of seriously damaging otherwise healthy cells. Much of the viral
deactivation may be the result of these reactive oxygen species
rather than any effect of photoactivation of psoralens. Regardless,
it is believed that no auto vaccine effect has been observed.
[0079] The best known photoactivatable compounds are derivatives of
psoralen or coumarin, which are nucleic acid intercalators. The use
of psoralen and coumarin photosensitizers can give rise to
alternative chemical pathways for dissipation of the excited state
that are either not beneficial to the goal of viral inactivation,
or that are actually detrimental to the process. For psoralens and
coumarins, this chemical pathway is likely to lead to the formation
of a variety of ring-opened species, such as shown below for
coumarin:
##STR00002##
[0080] Research in this field over-simplifies mechanisms involved
in the photoactivating mechanism and formation of highly reactive
oxygen species, such as singlet oxygen. Both may lead to
inactivating damage of tumor cells, viruses and healthy cells.
However, neither, alone or combined, lead to an auto vaccine
effect. This requires an activation of the body's own immune system
to identify a malignant cell or virus as threat and to create an
immune response capable of lasting cytotoxic effects directed to
that threat. It is believed, without being limiting in any way,
that photoactivation and the resulting apoptosis of malignant cells
that occurs in extracorporeal photophoresis causes the activation
of an immune response with cytotoxic effects on untreated malignant
cells. While the complexity of the immune response and cytotoxic
effects is fully appreciated by researchers, a therapy that
harnesses the system to successfully stimulate an auto vaccine
effect against a targeted, malignant cell has been elusive, except
for extracorporeal photopheresis for treating lymphoma.
[0081] Midden (W. R. Midden, Psoralen DNA photobiology, Vol II (ed.
F. P. Gaspalloco) CRC press, pp. 1. (1988) has presented evidence
that psoralens photoreact with unsaturated lipids and photoreact
with molecular oxygen to produce active oxygen species such as
superoxide and singlet oxygen that cause lethal damage to
membranes. U.S. Pat. No. 6,235,508 teaches that 8-MOP and AMT are
unacceptable photosensitizers, because each indiscriminately
damages both cells and viruses. Studies of the effects of cationic
side chains on furocoumarins as photosensitizers are reviewed in
Psoralen DNA Photobiology, Vol. I, ed. F. Gaspano, CRC Press, Inc.,
Boca Raton, Fla., Chapter 2. U.S. Pat. No. 6,235,508 gleans the
following from this review: most of the amino compounds had a much
lower ability to both bind and form crosslinks to DNA compared to
8-MOP, suggesting that the primary amino functionality is the
preferred ionic species for both photobinding and crosslinking.
[0082] U.S. Pat. No. 5,216,176 of Heindel discloses a large number
of psoralens and coumarins that have some effectiveness as
photoactivated inhibitors of epidermal growth factor. Halogens and
amines are included among the vast functionalities that could be
included in the psoralen/coumarin backbone. This reference is
incorporated herein by reference.
[0083] U.S. Pat. No. 5,984,887 discloses using extracorporeal
photopheresis with 8-MOP to treat blood infected with CMV. The
treated cells as well as killed and/or attenuated virus, peptides,
native subunits of the virus itself (which are released upon cell
break-up and/or shed into the blood) and/or pathogenic
noninfectious viruses are then used to generate an immune response
against the virus, which was not present prior to the
treatment.
Problems with PDT
[0084] It is well recognized that a major problem associated with
the existing methods of diagnosis and treatment of cell
proliferation disorders is in differentiation of normal cells from
target cells. Radiation therapy works by irradiating cells with
high levels of high energy radiation such as high energy photon,
electron, or proton. These high energy beams ionize the atoms which
make up a DNA chain, which in turn leads to cell death. Unlike
surgery, radiation therapy does not require placing patients under
anesthesia and has the ability to treat disorders deep inside the
body with minimal invasion of the body. However, the high doses of
radiation needed for such therapies damages healthy cells just as
effectively as it does diseased cells. Thus, similar to surgery,
differentiation between healthy and diseased cells in radiation
therapy is only by way of location. There is no intrinsic means for
a radiation beam to differentiate between a healthy cell from a
diseased cell either. Another problem encountered in PDT therapy is
the inability to treat target areas that are more than a few
centimeters beneath the surface of the skin without significant
invasive techniques.
[0085] Therefore, there still exists a need for better and more
effective treatments that can more precisely target the diseased
cells without causing substantial side-effects or collateral
damages to healthy tissues, and which are capable of treating
disorders by non-invasive or minimum invasive techniques.
SUMMARY OF THE INVENTION
[0086] Accordingly, one object of the present invention is to
provide a method for the treatment of a condition, disorder or
disease in a subject that permits treatment of a subject in any
area of the body while being non-invasive and having high
selectivity for targeted cells relative to healthy cells.
[0087] A further object of the present invention is to provide a
method for treatment of a condition, disorder or disease in a
subject which can use any suitable energy source as the initiation
energy source to induce a predetermined change in a target
structure in a subject in situ to treat said condition, disorder or
disease.
[0088] A further object of the present invention is to provide a
method for treatment of a condition, disorder or disease using a
modulation agent which adsorbs, intensifies or modifies the
initiation energy into an energy that effects a predetermined
change in a target structure.
[0089] These and other objects of the present invention, which will
become more apparent in conjunction with the following detailed
description of the preferred embodiments, either alone or in
combinations thereof, have been satisfied by the discovery of a
method for treating a condition, disorder or disease in a subject,
comprising:
[0090] applying an initiation energy from at least one source to a
target structure in a subject in need of treatment, wherein the
initiation energy contacts the target structure and induces a
predetermined change in said target structure in situ, [0091] thus
treating said condition, disorder or disease.
[0092] Yet a further object of the invention is further administer
at least one energy modulation agent to said subject which adsorbs,
intensifies or modifies said initiation energy into an energy that
effects a predetermined change in said target structure.
[0093] A further object of the present invention is to provide a
method for treatment of a condition, disorder or disease which can
use any suitable energy source as the initiation energy source to
activate the activatable pharmaceutical agent and thereby cause a
predetermined change in a target structure to treat a condition,
disorder or disease.
[0094] A further object of the present invention is to provide a
method for treatment of a condition, disorder or disease using an
energy cascade to activate an activatable pharmaceutical agent that
then treats cells suffering from a condition, disorder or
disease.
[0095] A further object of the present invention is to provide a
method for generating an autovaccine effect in a subject, which can
be in vivo thus avoiding the need for ex vivo treatment of subject
tissues or cells, or can be ex vivo.
[0096] A further object of the present invention is to provide a
method for generating an autovaccine effect in a subject, which can
be in vivo thus avoiding the need for ex vivo treatment of subject
tissues or cells, or can be ex vivo.
[0097] A further object of the present invention is to provide a
computer implemented system for performing the methods of the
present invention.
[0098] A still further object of the present invention is to
provide a kit and a pharmaceutical composition for use in the
present invention methods.
[0099] These and other objects of the present invention, which will
become more apparent in conjunction with the following detailed
description of the preferred embodiments, either alone or in
combinations thereof, have been satisfied by the discovery of a
method for modifying a target structure which mediates or is
associated with a biological activity comprising:
[0100] applying an initiation energy from at least one source to a
target structure in a subject in need of treatment, wherein the
initiation energy contacts the target structure and induces a
predetermined change in said target structure in situ,
[0101] wherein said predetermined change modifies the target
structure and modulates the biological activity of the target
structure.
[0102] A further object of the present invention is to provide a
method for modifying a target structure which mediates or is
associated with a biological activity, comprising: [0103] (1)
contacting said target structure with at least one activatable
pharmaceutical agent (PA) that is capable of effecting a
predetermined change in a target structure when activated,
optionally in the presence of at least one member selected from the
group consisting of energy modulation agents, plasmonics-active
agents and combinations thereof; and [0104] (2) applying an
initiation energy from an initiation energy source to said target
structure, [0105] wherein the energy modulation agent, if present,
upgrades or downgrades the initiation energy to an activation
energy capable of activating the at least one activatable
pharmaceutical agent; [0106] wherein the plasmonics-active agent,
if present, enhances or modifies the applied initiation energy or
the activation energy generated by the energy modulation agent, or
both; and [0107] thus causing the predetermined change to the
target structure to occur, wherein said predetermined change
modifies the target structure and modulates the biological activity
of the target structure; [0108] and a kit for performing the
methods, pharmaceutical compositions, computer implemented systems
for performing the methods and a method and system for causing an
autovaccine effect in a subject.
[0109] A further object of the present invention is to provide such
methods which can use any suitable energy source as the initiation
energy source in combination with plasmonics materials to activate
the activatable pharmaceutical agent and thereby cause the
predetermined change.
[0110] A further object of the present invention is to provide such
methods using plasmonics in an energy cascade to activate an
activatable pharmaceutical agent that then cause the predetermined
change.
[0111] A further object of the present invention is to provide such
methods for in situ generation of energy which causes, either
directly or indirectly, the predetermined change.
[0112] A further object of the present invention is to provide a
method for the treatment of a cell proliferation disorder that
permits treatment of a subject in any area of the body while being
non-invasive and having high selectivity for targeted cells
relative to healthy cells through the use of exciton-plasmon
enhancement.
[0113] A further object of the present invention is to provide a
method for treatment of a condition, disorder or disease which can
use any suitable energy source as the initiation energy source in
combination with exciton-plasmon enhancement to activate the
activatable pharmaceutical agent and thereby cause a predetermined
change to treat cells suffering from a condition, disorder or
disease.
[0114] A further object of the present invention is to provide a
method for treatment of a condition, disorder or disease using
exciton-plasmon enhancement in an energy cascade to activate an
activatable pharmaceutical agent that then treats cells suffering
from a condition, disorder or disease.
[0115] Another object of the invention is a method for treating a
condition, disorder, or disease associated with a target structure
in a subject, comprising: [0116] (1) administering to the subject
at least one activatable pharmaceutical agent that is capable of
effecting a predetermined change in a target structure when
activated and at least one plasmonics-active agent; and [0117] (2)
applying an initiation energy from an initiation energy source to
the subject, [0118] wherein the plasmonics-active agent enhances or
modifies the applied initiation energy, such that the enhanced
initiation energy activates the activatable agent in situ,
[0119] thus causing the predetermined change to the target
structure to occur, wherein said predetermined change modifies the
target structure and treats said condition, disorder, or disease.
The condition, disorder, or disease may be mediated by abnormal
cellular proliferation and said predetermined change can ameliorate
the abnormal cellular proliferation. Abnormal cellular
proliferation may be higher than that of cells from a subject not
having said condition, disorder or disease or may be lower.
[0120] The treated condition, disorder, or disease may or may not
be significantly mediated by abnormal cellular proliferation and
said predetermined change does not have to substantially affect
cellular proliferation.
[0121] Yet another object of the invention is a method for
modifying a target structure which mediates or is associated with a
biological activity, comprising: [0122] (1) contacting said target
structure with at least one activatable pharmaceutical agent that
is capable of effecting a predetermined change in a target
structure when activated and at least one plasmonics-active agent;
and [0123] (2) applying an initiation energy from an initiation
energy source to target structure
[0124] wherein the plasmonics-active agent enhances or modifies the
applied initiation energy, such that the enhanced initiation energy
activates the activatable agent, [0125] thus causing the
predetermined change to the target structure to occur, wherein said
predetermined change modifies the target structure and modulates
the biological activity of the target structure. The target
structure need not be present inside an organism, but may be one in
vitro or ex vivo. The predetermined change may enhance the
expression of, promote the growth of, or increase the quantity of
the target structure; or the predetermined change can enhance,
inhibit or stabilize the usual biological activity of the target
structure compared to a similar untreated target structure. For
example, the predetermined change can alter the immunological or
chemical properties of the target structure which may be a cell,
cell membrane, internal cellular structure, polypeptide or
non-polypeptide compound which can be modified by said
predetermined change to be more or less antigenic or immunogenic.
In another embodiment, modifying the target structure can be done
without the need for a pharmaceutical agent, or a plasmonics-active
agent.
BRIEF DESCRIPTION OF THE FIGURES
[0126] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0127] FIG. 1 provides an exemplary electromagnetic spectrum in
meters (1 nm equals 10.sup.-9 meters).
[0128] FIG. 2A and FIG. 2B are graphical representations of the
depth of penetration of various wavelengths of energy into living
tissue
[0129] FIG. 3 illustrates a system according to one exemplary
embodiment of the present invention.
[0130] FIG. 4 illustrates an exemplary computer implemented system
according to an embodiment of the present invention.
[0131] FIG. 5 illustrates an exemplary computer system (1201) for
implementing various embodiments of the present invention.
[0132] FIGS. 6A and 6B are graphical representations of plasmonic
nanostructures and their theoretical electromagnetic enhancement at
different excitation wavelengths. FIG. 6A shows silver nanospheres
with different radii. FIG. 6B shows nanoshells with different
dielectric core/metallic shell radial ratios. [M. M. Kerker, Acc.
Chem. Res., 17, 370 (1984)]
[0133] FIG. 7 provides representative embodiments of plasmonics
photo-active probes useful in the present invention.
[0134] FIG. 8 is a graphical explanation of the plasmonics-enhanced
effect of photospectral therapy used in the present invention.
[0135] FIG. 9 provides representative embodiments of
plasmonics-active nanostructures.
[0136] FIG. 10 is a graphical representation of one embodiment of a
PEPST probe with remote drug release. Plasmonics excitation of
metal nanoparticles enhances photo-active probes with remote drug
release mechanism. Radiation of suitable wavelength (RF, MW, IR,
NIR, VIS, UV to X ray and y ray) is used to excited PA and or
nanoparticles.
[0137] FIG. 11 is a graphical representation of several embodiments
of PEPST probes with various linkers for remote drug release.
[0138] FIG. 12 is a graphical representation of several embodiments
of plasmonics photo-active probes with bioreceptors.
[0139] FIG. 13 is a graphical representation of the "therapeutic
window" in tissue and absorption spectra of biological
components.
[0140] FIG. 14 is a graphical representation of an embodiment of
the energy modulation agent (or excitation energy
converter/EEC)-photo activator (PA) system of the present
invention. X-ray is used to excite an "excitation energy converter"
(EEC) molecular system. The EEC absorbs the X-ray energy and emits
light that is absorbed by the "photo-activator" (PA) molecule. The
PA molecule becomes an activated drug for disease treatment.
[0141] FIGS. 15A-15F are graphical representations of several
embodiments of plasmonics photo-active energy modulation agent-PA
probes. FIG. 15A: PA molecules bound to EEC and to plasmonic metal
nanoparticles; FIG. 15B: Plasmonic metal nanoparticle with EEC
nanocap covered with PA molecules; FIG. 15C: PA-covered
nanoparticle with plasmonic metal nanoparticles; FIG. 15D:
EEC-containing nanoparticle covered with PA molecules and plasmonic
metal nanocap; FIG. 15E: Plasmonic metal nanoparticle core with EEC
nanoshell covered with PA molecules; FIG. 15F: PA molecule bound to
EEC (attached to plasmonics metal nanoparticle) nanoparticle by
detachable biochemical bond.
[0142] FIG. 16 shows structures of various preferred embodiments of
gold complexes exhibiting XEOL.
[0143] FIG. 17 shows the structure of a further embodiment of
compound exhibiting XEOL, namely a tris-8-hydroxyquinoline-aluminum
complex. [Taken from: S. J. Naftel, P. Zhang, P.-S. Kim, and T. K.
Sham, I. Coulthard, W. J. Antel, Jr., J. W. Freeland, and S. P.
Frigo, M.-K. Fung and S. T. Lee, Y. F. Hu and B. W. Yates, Soft
x-ray-excited luminescence and optical x-ray absorption fine
structures of tris 0.8-hydroxyquinoline. Aluminum, Appl. Phys.
Lett, 78, 1844, 2001]
[0144] FIG. 18 is a graphical representation of a
plasmonics-enhanced mechanism for a photo-active energy modulation
agent-PA probe of the present invention.
[0145] FIG. 19 is a graph showing excitation and emission
fluorescence spectra of psoralens
[0146] FIGS. 20A-20C are graphical representations of an embodiment
of a PEPST energy modulation agent-PA system with detachable bond.
FIG. 20A shows that an EEC nanoparticle improves delivery of PA
molecules (e.g., psoralen) into target disease cells. FIG. 20B
shows that, inside the cell, photon radiation releases PA which can
go into the nucleus. FIG. 20C shows that radiation of suitable
wavelength (NIR to X ray) induces plasmonic field to activate PA
intercalated into DNA.
[0147] FIG. 21 is a graphical representation of an embodiment of
PEPST probes for dual plasmonic excitation.
[0148] FIG. 22 is a graphical representation of an embodiment of a
use of encapsulated photoactive agents.
[0149] FIG. 23 is a simplified graphical representation of the use
of the present invention principle of non-invasive PEPST
modality.
[0150] FIG. 24 is a photomicrograph showing nanocaps
(half-nanoshells) comprising polystyrene nanospheres coated with
silver.
[0151] FIG. 25 shows various schematic embodiments of basic EIP
probes.
[0152] FIGS. 26A-E are graphical representations of fluorescence
spectra of PAH compounds. FIG. 26A shows fluorescence, excitation,
emission, and synchronous spectra of phenanthrene. FIG. 26B shows
fluorescence, excitation, emission, and synchronous spectra of
anthracene. FIG. 26C shows fluorescence, excitation, emission, and
synchronous spectra of perylene. FIG. 26D shows conventional
fluorescence spectrum of a mixture of naphthalene, phenanthrene,
anthracene, perylene, and tertracene. FIG. 26E shows synchronous
spectrum of the mixture. [Source: T. Vo-Dinh, Multicomponent
analysis by synchronous luminescence spectrometry, Anal. Chem.;
1978; 50(3) pp 396-401]
[0153] FIG. 27 is a graph showing the XEOL of Eu doped in BaFBr
matrix. [Source: N Subramanian et al, X-ray excited optical
luminescence, photoluminescence, photostimulated luminescence and
x-ray photoemission spectroscopy studies on BaFBr:Eu J. Phys.:
Condens. Matter 9 4769-4780, 1997]}
[0154] FIG. 28 provides further embodiments of schematic designs of
EIP probes.
[0155] FIG. 29 is a graphical representation of various embodiments
of basic EPEP probes.
[0156] FIG. 30 is a graphical representation of various embodiments
of basic EPEP probes.
[0157] FIG. 31 is a graphical representation of various embodiments
of EPEP probes having NPs, NWs and NRs.
[0158] FIG. 32 is a graphical representation of various embodiments
of EPEP probes having NPs, NWs, NRs and bioreceptors.
[0159] FIG. 33 is a graphical representation of an embodiment of
EPEP probes having NPs and multiple NWs.
[0160] FIG. 34A-34G show photo-active probes in which a
photo-active molecule is bound to plasmonics probes. FIG. 34A: PA
molecules bound to metal nanoparticle; FIG. 34B: X-ray converter
nanoparticle covered with metal nanoparticles; FIG. 34C: Metal
nanoparticle covered with X-ray converter nanocap; FIG. 34D: X-ray
converter nanoparticle covered with metal nanocap; FIG. 34E: Metal
nanoparticle covered with X-converter nanoshell; FIG. 34F: X-ray
converter nanoparticle covered with metal nanoshell; and FIG. 34G:
X-ray converter nanoparticle covered with metal nanoshell with
protective coating layer.
[0161] FIG. 35A-35G show plasmonics photo-active probes that have a
dielectric layer between the metal and the UC materials FIG. 35A:
PA molecules bound to metal nanoparticle; FIG. 35B: X-ray converter
nanoparticle covered with dielectric layer and metal nanoparticles;
FIG. 35C: Metal nanoparticle covered with dielectric layer and
X-ray converter nanocap; FIG. 35D: X-ray converter nanoparticle
covered with dielectric layer and metal nanocap; FIG. 35E: Metal
nanoparticle covered with dielectric layer and X-ray converter
nanoshell; FIG. 35F: X-ray converter nanoparticle covered with
dielectric layer and metal nanoshell; FIG. 35G: X-ray converter
nanoparticle covered with dielectric layer, metal nanoshell and
protective coating.
DETAILED DESCRIPTION OF THE INVENTION
[0162] The present invention sets forth a novel method of modifying
a target structure which mediates or is associated with a
biological activity, which includes treating a condition, disorder
or disease in a subject, that is effective, specific, and has few
side-effects. Those cells suffering from a condition, disorder or
disease are referred to herein as the target cells.
[0163] All methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, with suitable methods and materials being
described herein. All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including definitions, will control. Further, the materials,
methods, and examples are illustrative only and are not intended to
be limiting, unless otherwise specified.
[0164] Generally, the present invention provides method for
modifying a target structure which mediates or is associated with a
biological activity comprising:
[0165] applying an initiation energy from at least one source to a
target structure in a subject in need of treatment, wherein the
initiation energy contacts the target structure and induces a
predetermined change in said target structure in situ,
[0166] wherein said predetermined change modifies the target
structure and modulates the biological activity of the target
structure.
[0167] A further object of the present invention is to provide a
method for modifying a target structure which mediates or is
associated with a biological activity, comprising: [0168] (1)
contacting said target structure with at least one activatable
pharmaceutical agent (PA) that is capable of effecting a
predetermined change in a target structure when activated,
optionally in the presence of at least one member selected from the
group consisting of energy modulation agents, plasmonics-active
agents and combinations thereof; and [0169] (2) applying an
initiation energy from an initiation energy source to said target
structure, [0170] wherein the energy modulation agent, if present,
upgrades or downgrades the initiation energy to an activation
energy capable of activating the at least one activatable
pharmaceutical agent; [0171] wherein the plasmonics-active agent,
if present, enhances or modifies the applied initiation energy or
the activation energy generated by the energy modulation agent, or
both; and [0172] thus causing the predetermined change to the
target structure to occur, wherein said predetermined change
modifies the target structure and modulates the biological activity
of the target structure.
[0173] In a preferred embodiment, the present invention provides
methods for the treatment of a condition, disorder or disease, in
which an initiation energy source provides an initiation energy
that causes the predetermined cellular changes directly to treat
target cells within a subject. In one preferred embodiment, the
initiation energy source is applied indirectly via an energy
modulation agent, preferably in proximity to the target cells. The
present invention further provides methods for the treatment of a
condition, disorder or disease, in which an initiation energy
source provides an initiation energy that activates an activatable
pharmaceutical agent to treat target cells within the subject. In
one preferred embodiment, the initiation energy source is applied
indirectly via an energy modulation agent to the activatable
pharmaceutical agent, preferably in proximity to the target cells.
The present invention also provides methods for the treatment of a
condition, disorder or disease in which an initiation energy source
is enhanced or modified by a plasmonic-active agent, such that the
enhanced initiation energy activates the pharmaceutical agent in
situ.
[0174] As noted above, an object of the present invention is to
modify a target structure which mediates or is associated with a
biological activity, and in a preferred embodiment to treat a
condition, disorder or disease, in a subject using
photobiomodulation. Exemplary conditions, disorders or diseases may
include, but are not limited to, cancer, autoimmune diseases, soft
and bone tissue injury, chronic pain, wound healing, nerve
regeneration, viral and bacterial infections, fat deposits
(liposuction), varicose veins, enlarged prostate, retinal injuries
and other ocular diseases, Parkinson's disease, and behavioral,
perceptional and cognitive disorders. Exemplary conditions also may
include nerve (brain) imaging and stimulation, a direct control of
brain cell activity with light, control of cell death (apoptosis),
and alteration of cell growth and division.
[0175] Accordingly, in one embodiment, the present invention
provides methods that are capable of overcoming the shortcomings of
the existing methods. In general, a method in accordance with the
present invention utilizes an initiation energy from at least one
source applied to a target structure in a subject in need of
treatment, wherein the initiation energy contacts the target
structure and induces a predetermined change in said target
structure in situ, thus modifying a target structure which mediates
or is associated with a biological activity, preferably treating a
condition, disorder or disease. The initiation energy can
preferably penetrate completely through the subject and can be
applied from a single source or more than one source. Exemplary
initiation energy may be UV radiation, visible light, infrared
radiation (IR), x-rays, gamma rays, an electron beam, microwaves or
radio waves.
[0176] In one embodiment, a plasmonics-active agent enhances or
modifies the applied initiation energy, such that the enhanced
initiation energy causes the predetermined change in said target
structure. In a different embodiment, a plasmonics-active agent
enhances or modifies the applied initiation energy, such that the
enhanced initiation energy is absorbed, intensified or modified by
the modulation agent into the energy that effects the predetermined
change in said target structure.
[0177] In yet another preferred embodiment, a method in accordance
with the present invention utilizes the principle of energy
transfer to and among molecular agents to control delivery and
activation of cellular changes by irradiation such that delivery of
the desired effect is more intensified, precise, and effective than
the conventional techniques. At least one energy modulation agent
can be administered to the subject which adsorbs, intensifies or
modifies said initiation energy into an energy that effects a
predetermined cellular change in said target structure. The energy
modulation agent may be located around, on, or in said target
structure. Further, the energy modulation agent can transform a
photonic initiation energy into a photonic energy that effects a
predetermined change in said target structure. In one preferred
embodiment, the energy modulation agent decreases the wavelength of
the photonic initiation energy.
[0178] In another preferred embodiment, the energy modulation agent
can increase the wavelength of the photonic initiation energy. In a
different embodiment the modulation agent is one or more members
selected from a biocompatible fluorescing metal nanoparticle,
fluorescing metal oxide nanoparticle, fluorescing dye molecule,
gold nanoparticle, silver nanoparticle, gold-coated silver
nanoparticle, a water soluble quantum dot encapsulated by
polyamidoamine dendrimers, a luciferase, a biocompatible
phosphorescent molecule, a combined electromagnetic energy
harvester molecule, and a lanthanide chelate exhibiting intense
luminescence.
[0179] Another object of the present invention is to treat a
condition, disorder or disease in a subject using an activatable
pharmaceutical agent. Exemplary conditions, disorders or diseases
may include, but are not limited to, cancer, autoimmune diseases,
cardiac ablasion (e.g., cardiac arrhythmia and atrial
fibrillation), photoangioplastic conditions (e.g., de novo
atherosclerosis, restinosis), intimal hyperplasia, arteriovenous
fistula, macular degeneration, psoriasis, acne, hopecia areata,
portwine spots, hair removal, rheumatoid and inflammatory
arthrisis, joint conditions, lymph node conditions, and cognitive
and behavioral conditions.
[0180] Accordingly, in one embodiment, the present invention
provides methods utilizing the principle of energy transfer to and
among molecular agents to control delivery and activation of
pharmaceutically active agents such that delivery of the desired
pharmacological effect is more focused, precise, and effective than
the conventional techniques.
[0181] In yet another preferred embodiment, the initiation energy
source is applied directly or indirectly (via a modulation agent)
to the activatable pharmaceutical agent, preferably in proximity to
the target cells.
[0182] Within the context of the present invention, the phrase
"applied indirectly" (or variants of this phrase, such as "applying
indirectly", "indirectly applies", "indirectly applied",
"indirectly applying", etc.), when referring to the application of
the initiation energy, means the penetration by the initiation
energy into the subject beneath the surface of the subject and to
the modulation agent and/or activatable pharmaceutical agent within
a subject. In one embodiment, the initiation energy interacts with
a previously administered energy modulation agent which then
activates the predetermined cellular changes. In another
embodiment, the initiation energy interacts with a previously
administered energy modulation agent which then activates the
activatable pharmaceutical agent. In another embodiment, the
initiation energy itself activates the activatable pharmaceutical
agent. In either embodiment, the initiation energy source cannot be
within line-of-sight of the modulation agent and/or the activatable
pharmaceutical agent. By "cannot be within line-of-sight" is meant
that if a hypothetical observer were located at the location of the
modulation agent or the activatable pharmaceutical agent, that
observer would be unable to see the source of the initiation
energy.
[0183] Although not intending to be bound by any particular theory
or be otherwise limited in any way, the following theoretical
discussion of scientific principles and definitions are provided to
help the reader gain an understanding and appreciation of the
present invention.
[0184] As used herein, the term "subject" is not intended to be
limited to humans, but may also include animals, plants, or any
suitable biological organism.
[0185] As used herein, the phrase "a disease or condition" refers
to a condition, disorder or disease that may include, but are not
limited to, cancer, soft and bone tissue injury, chronic pain,
wound healing, nerve regeneration, viral and bacterial infections,
fat deposits (liposuction), varicose veins, enlarged prostate,
retinal injuries and other ocular diseases, Parkinson's disease,
and behavioral, perceptional and cognitive disorders. Exemplary
conditions also may include nerve (brain) imaging and stimulation,
a direct control of brain cell activity with light, control of cell
death (apoptosis), and alteration of cell growth and division. Yet
other exemplary a condition, disorder or disease may include, but
are not limited to, cardiac ablasion (e.g., cardiac arrhythmia and
atrial fibrillation), photoangioplastic conditions (e.g., de novo
atherosclerosis, restinosis), intimal hyperplasia, arteriovenous
fistula, macular degeneration, psoriasis, acne, hopecia areata,
portwine spots, hair removal, rheumatoid and inflammatory
arthritis, joint conditions, and lymph node conditions.
[0186] As used herein, the term "target structure" refers to an
eukaryotic cell, prokaryotic cell, a subcellular structure, such as
a cell membrane, a nuclear membrane, cell nucleus, nucleic acid,
mitochondria, ribosome, or other cellular organelle or component,
an extracellular structure, virus or prion, and combinations
thereof.
[0187] The nature of the predetermined cellular change will depend
on the desired pharmaceutical outcome. Exemplary cellular changes
may include, but are not limited to, apoptosis, necrosis,
up-regulation of certain genes, down-regulation of certain genes,
secretion of cytokines, alteration of cytokine receptor responses,
regulation of cytochrome c oxidase and flavoproteins, activation of
mitochondria, stimulation antioxidant protective pathway,
modulation of cell growth and division, alteration of firing
pattern of nerves, alteration of redox properties, generation of
reactive oxygen species, modulation of the activity, quantity, or
number of intracellular components in a cell, modulation of the
activity, quantity, or number of extracellular components produced
by, excreted by, or associated with a cell, or a combination
thereof. Predetermined cellular changes may or may not result in
destruction or inactivation of the target structure.
[0188] As used herein, an "energy modulation agent" refers to an
agent that is capable of receiving an energy input from a source
and then re-emitting a different energy to a receiving target.
Energy transfer among molecules may occur in a number of ways. The
form of energy may be electronic, thermal, electromagnetic,
kinetic, or chemical in nature. Energy may be transferred from one
molecule to another (intermolecular transfer) or from one part of a
molecule to another part of the same molecule (intramolecular
transfer). For example, a modulation agent may receive
electromagnetic energy and re-emit the energy in the form of
thermal energy. In preferred embodiments, the energy modulation
agent receives higher energy (e.g. x-ray) and re-emits in lower
energy (e.g. UV-A). Some modulation agents may have a very short
energy retention time (on the order of fs, e.g. fluorescent
molecules) whereas others may have a very long half-life (on the
order of minutes to hours, e.g. luminescent or phosphorescent
molecules). Suitable energy modulation agents include, but are not
limited to, a biocompatible fluorescing metal nanoparticle,
fluorescing dye molecule, gold nanoparticle, a water soluble
quantum dot encapsulated by polyamidoamine dendrimers, a
luciferase, a biocompatible phosphorescent molecule, a combined
electromagnetic energy harvester molecule, and a lanthanide chelate
capable of intense luminescence. Various exemplary uses of these
are described below in preferred embodiments.
[0189] The modulation agents may further be coupled to a carrier
for cellular targeting purposes. For example, a biocompatible
molecule, such as a fluorescing metal nanoparticle or fluorescing
dye molecule that emits in the UV-A band, may be selected as the
energy modulation agent.
[0190] The energy modulation agent may be preferably directed to
the desired site (e.g. a tumor) by systemic administration to a
subject. For example, a UV-A emitting energy modulation agent may
be concentrated in the tumor site by physical insertion or by
conjugating the UV-A emitting energy modulation agent with a tumor
specific carrier, such as a lipid, chitin or chitin-derivative, a
chelate or other functionalized carrier that is capable of
concentrating the UV-A emitting source in a specific target
tumor.
[0191] Additionally, the energy modulation agent can be used alone
or as a series of two or more energy modulation agents wherein the
energy modulation agents provide an energy cascade. Thus, the first
energy modulation agent in the cascade will absorb the activation
energy, convert it to a different energy which is then absorbed by
the second energy modulation in the cascade, and so forth until the
end of the cascade is reached with the final energy modulation
agent in the cascade emitting the energy necessary to activate the
activatable pharmaceutical agent.
[0192] Exemplary energy modulation agents may include, but are not
limited to, at least one energy modulation agent selected from the
group consisting of a biocompatible fluorescing metal nanoparticle,
fluorescing metal oxide nanoparticle, fluorescing dye molecule,
gold nanoparticle, silver nanoparticle, gold-caoted silver
nanoparticle, a water soluble quantum dot encapsulated by
polyamidoamine dendrimers, a luciferase, a biocompatible
phosphorescent molecule, a combined electromagnetic energy
harvester molecule, and a lanthanide chelate exhibiting intense
luminescence.
[0193] As used herein, an "activatable pharmaceutical agent" is an
agent that normally exists in an inactive state in the absence of
an activation signal. When the agent is activated by a matching
activation signal under activating conditions, it is capable of
effecting the desired pharmacological effect on a target cell (i.e.
preferably a predetermined cellular change).
[0194] Signals that may be used to activate a corresponding agent
may include, but are not limited to, photons of specific
wavelengths (e.g. x-rays, or visible light), electromagnetic energy
(e.g. radio or microwave), thermal energy, acoustic energy, or any
combination thereof.
[0195] Activation of the agent may be as simple as delivering the
signal to the agent or may further premise on a set of activation
conditions. For example, in the former case, an activatable
pharmaceutical agent, such as a photosensitizer, may be activated
by UV-A radiation. Once activated, the agent in its active-state
may then directly proceed to effect a cellular change.
[0196] Where activation may further premise upon other conditions,
mere delivery of the activation signal may not be sufficient to
bring about the desired cellular change. For example, a photoactive
compound that achieves its pharmaceutical effect by binding to
certain cellular structure in its active state may require physical
proximity to the target cellular structure when the activation
signal is delivered. For such activatable agents, delivery of the
activation signal under non-activating conditions will not result
in the desired pharmacologic effect. Some examples of activating
conditions may include, but are not limited to, temperature, pH,
location, state of the cell, presence or absence of co-factors.
[0197] Selection of an activatable pharmaceutical agent greatly
depends on a number of factors such as the desired cellular change,
the desired form of activation, as well as the physical and
biochemical constraints that may apply. Exemplary activatable
pharmaceutical agents may include, but are not limited to, agents
that may be activated by photonic energy, electromagnetic energy,
acoustic energy, chemical or enzymatic reactions, thermal energy,
or any other suitable activation mechanisms.
[0198] When activated, the activatable pharmaceutical agent may
effect cellular changes that include, but are not limited to,
apoptosis, redirection of metabolic pathways, up-regulation of
certain genes, down-regulation of certain genes, secretion of
cytokines, alteration of cytokine receptor responses, or
combinations thereof.
[0199] The mechanisms by which an activatable pharmaceutical agent
may achieve its desired effect are not particularly limited. Such
mechanisms may include direct action on a predetermined target as
well as indirect actions via alterations to the biochemical
pathways. A preferred direct action mechanism is by binding the
agent to a critical cellular structure such as nuclear DNA, mRNA,
rRNA, ribosome, mitochondrial DNA, or any other functionally
important structures. Indirect mechanisms may include releasing
metabolites upon activation to interfere with normal metabolic
pathways, releasing chemical signals (e.g. agonists or antagonists)
upon activation to alter the targeted cellular response, and other
suitable biochemical or metabolic alterations.
[0200] The treatment of the present invention can be by the unique
methods described in U.S. application Ser. No. 11/935,655, filed
Nov. 6, 2007 (incorporated by reference above), or by a modified
version of a conventional treatment such as PDT, but using a
plasmonics-active agent to enhance the treatment by modifying or
enhancing the applied energy or, in the case of using an energy
modulation agent, modifying either the applied energy, the emitted
energy from the energy modulation agent, or both.
[0201] In one preferred embodiment, the activatable pharmaceutical
agent is capable of chemically binding to the DNA or mitochondria
at a therapeutically effective amount. In this embodiment, the
activatable pharmaceutical agent, preferably a photoactivatable
agent, is exposed in situ to an activating energy emitted from an
energy modulation agent, which, in turn receives energy from an
initiation energy source.
[0202] Suitable activatable agents include, but are not limited to,
photoactive agents, sono-active agents, thermo-active agents, and
radio/microwave-active agents. An activatable agent may be a small
molecule; a biological molecule such as a protein, a nucleic acid
or lipid; a supramolecular assembly; a nanoparticle; or any other
molecular entity having a pharmaceutical activity once
activated.
[0203] The activatable agent may be derived from a natural or
synthetic origin. Any such molecular entity that may be activated
by a suitable activation signal source to effect a predetermined
cellular change may be advantageously employed in the present
invention.
[0204] Suitable photoactive agents include, but are not limited to:
psoralens and psoralen derivatives, pyrene cholesteryloleate,
acridine, porphyrin, fluorescein, rhodamine, 16-diazorcortisone,
ethidium, transition metal complexes of bleomycin, transition metal
complexes of deglycobleomycin, organoplatinum complexes,
alloxazines such as 7,8-dimethyl-10-ribityl isoalloxazine
(riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin),
7,8-dimethyl alloxazine (lumichrome), isoalloxazine-adenine
dinucleotide (flavine adenine dinucleotide [FAD]), alloxazine
mononucleotide (also known as flavine mononucleotide [FMN] and
riboflavine-5-phosphate), vitamin Ks, vitamin L, their metabolites
and precursors, and napththoquinones, naphthalenes, naphthols and
their derivatives having planar molecular conformations,
porphyrins, dyes such as neutral red, methylene blue, acridine,
toluidines, flavine (acriflavine hydrochloride) and phenothiazine
derivatives, coumarins, quinolones, quinones, and anthroquinones,
aluminum (111) phthalocyanine tetrasulfonate, hematoporphyrin, and
phthalocyanine, and compounds which preferentially adsorb to
nucleic acids with little or no effect on proteins. The term
"alloxazine" includes isoalloxazines.
[0205] Endogenously-based derivatives include synthetically derived
analogs and homologs of endogenous photoactivated molecules, which
may have or lack lower (1 to 5 carbons) alkyl or halogen
substituents of the photosensitizers from which they are derived,
and which preserve the function and substantial non-toxicity.
Endogenous molecules are inherently non-toxic and may not yield
toxic photoproducts after photoradiation.
[0206] Table 1 lists some photoactivatable molecules capable of
being photoactivated to induce an auto vaccine effect.
TABLE-US-00001 TABLE 1 SSET and TTET rate constants for
bichromophoric peptides K.sub.SSET (S.sup.-1) R.sub.model ( )
Compound .lamda..sub.ex (nm) E.sub.SSET K.sub.S of donor (S.sup.-1)
K.sub.SSET (S.sup.-1) (Average) R.sub.0 ( ) R ( ) (Average)
E.sub.TTET k.sub.TTET (S.sup.-1) 1B 224 96.3 9.5 .times. 10.sup.0
2.44 .times. 10.sup.8 1.87 .times. 10.sup.8 14.7 9 9.5 266 95 1.8
.times. 10.sup.8 2.5 5 .times. 10.sup.2 280 94 1.36 .times.
10.sup.8 1A 224 80 9.5 .times. 10.sup.0 3.8 .times. 10.sup.7 3.67
.times. 10.sup.7 14.7 11.8 14.1 266 79 3.6 .times. 10.sup.7 2 3.6
.times. 10.sup.2 280 79 3.6 .times. 10.sup.7 2B 224 77 9.5 .times.
10.sup.0 3.1 .times. 10.sup.7 3.9 .times. 10.sup.7 14.7 11.9 5.5
266 81 3.9 .times. 10.sup.7 32 9.4 .times. 10.sup.3 280 83 4.7
.times. 10.sup.7 2A 224 69 9.5 .times. 10.sup.0 2.1 .times.
10.sup.7 3 .times. 10.sup.7 14.7 12.2 8.1 74.3 5.7 .times. 10.sup.4
266 80 3.7 .times. 10.sup.7 280 77 3.2 .times. 10.sup.7
##STR00003## ##STR00004## ##STR00005## ##STR00006##
[0207] Table 2 lists some additional endogenous photoactivatable
molecules.
TABLE-US-00002 TABLE 2 Biocompatible, endogenous fluorophore
emitters. Excitation Max. Emission Max. Endogenous Fluorophores
(nm) (nm) Amino acids: Tryptophan 280 350 Tyrosine 275 300
Phenylalanine 260 280 Structural Proteins: Collagen 325, 360 400,
405 Elastin 290, 325 340, 400 Enzymes and Coenzymes: flavin adenine
dinucleotide 450 535 reduced nicotinamide dinucelotide 290, 351
440, 460 reduced nicotinamide dinucelotide phosphate 336 464
Vitamins: Vitamins A 327 510 Vitamins K 335 480 Vitamins D 390 480
Vitamins B.sub.6 compounds: Pyridoxine 332, 340 400 Pyridoxamine
335 400 Pyridoxal 330 385 Pyridoxic acid 315 425 Pyrictoxal
phosphate .sup. 5'-330 400 Vitamin B.sub.12 275 305 Lipids:
Phospholipids 436 540, 560 Lipofuscin 340-395 540, 430-460 Ceroid
340-395 430-460, 540 Porphyrins 400-450 630, 690
[0208] FIG. 1 provides an exemplary electromagnetic spectrum in
meters (1 nm equals meters).
[0209] Although the activatable pharmaceutical agent and the energy
modulation agent can be distinct and separate, it will be
understood that the two agents need not be independent and separate
entities. In fact, the two agents may be associated with each other
via a number of different configurations. Where the two agents are
independent and separately movable from each other, they generally
interact with each other via diffusion and chance encounters within
a common surrounding medium. Where the activatable pharmaceutical
agent and the energy modulation agent are not separate, they may be
combined into one single entity.
[0210] The initiation energy source can be any energy source
capable of providing energy at a level sufficient to cause cellular
changes directly or via a modulation agent which transfer the
initiation energy to energy capable of causing the predetermined
cellular changes. Also, the initiation energy source can be any
energy source capable of providing energy at a level sufficient
activate the activatable agent directly, or to provide the energy
to a modulation agent with the input needed to emit the activation
energy for the activatable agent (indirect activation). Preferable
initiation energy sources include, but are not limited to, UV-A
lamps or fiber optic lines, a light needle, an endoscope, and a
linear accelerator that generates x-ray, gamma-ray, or electron
beams. In a preferred embodiment the initiation energy capable of
penetrating completely through the subject. Within the context of
the present invention, the phrase "capable of penetrating
completely through the subject" is used to refer to energy that can
penetrate to any depth within the subject to activate the
activatable pharmaceutical agent. It is not required that the any
of the energy applied actually pass completely through the subject,
merely that it be capable of doing so in order to permit
penetration to any desired depth to activate the activatable
pharmaceutical agent. Exemplary initiation energy sources that are
capable of penetrating completely through the subject include, but
are not limited to, UV light, visible light, IR radiation, x-rays,
gamma rays, electron beams, microwaves and radio waves.
[0211] An additional embodiment of the present invention is to
provide a method for treatment of a condition, disease or disorder
by the in-situ generation of energy in a subject in need thereof,
where the energy generated can be used directly to effect a change
thereby treating the condition, disease or disorder, or the energy
can be used to activate an activatable pharmaceutical agent, which
upon activation effects a change thereby treating the condition,
disease or disorder. The energy can be generated in-situ by any
desired method, including, but not limited to, chemical reaction
such as chemiluminescence, or by conversion of an energy applied to
the subject externally, which is converted in-situ to a different
energy (of lower or higher energy than that applied), through the
use of one or more energy modulation agents.
[0212] A further embodiment of the present invention combines the
treatment of a condition, disease or disorder with the generation
of heat in the affected target structure in order to enhance the
effect of the treatment. For example, in the treatment of a cell
proliferation disorder using a photoactivatable pharmaceutical
agent (such as a psoralen or derivative thereof), one can activate
the photoactivatable pharmaceutical agent by applying an initiation
energy which, directly or indirectly, activates the pharmaceutical
agent. As noted elsewhere in the present application, this
initiation energy can be of any type, so long as it can be
converted to an energy suitable for activating the pharmaceutical
compound. In addition to applying this initiation energy, in this
embodiment of the present invention, an energy is applied that
causes heating of the target structure. In the case of a cell
proliferation disorder such as cancer, the heating would increase
the proliferation rate of the cancer cells. While this may seem
counterintuitive at first, when the cell proliferation disorder is
being treated using a DNA intercalation agent, such as psoralen or
a derivative thereof, this increase in cell proliferation can
actually assist the psoralen in causing apoptosis. In particular,
when psoralen becomes intercalated into DNA, apoptosis occurs when
the cell goes through its next division cycle. By increasing the
rate at which the cells divide, one can use the present invention
methods to enhance the onset of apoptosis.
[0213] For this embodiment, the heat can be generated in any
desired manner.
[0214] Preferably, the heat can be generated using the application
of microwaves or NIR energy to the target structure or by the use
of use of nanoparticles of metal or having metal shells. In the
nanoparticles embodiment, as is done in tumor thermotherapy,
magnetic metal nanoparticles can be targeted to cancer cells using
conventional techniques, then used to generate heat by application
of a magnetic field to the subject under controlled conditions.
(DeNardo S J, DeNardo G L, Natarajan A et al.: Thermal dosimetry
predictive of efficacy of 111In-ChL6 NPAMF-induced thermoablative
therapy for human breast cancer in mice. J. Nucl. Med. 48(3),
437-444 (2007).)
[0215] Alternatively, one can generate heat through the application
of NIR to nanoparticles having metal shells which is converted into
thermal energy. (Hirsch L R, Stafford R J, Bankson J et al
Nanoshell-mediated near-infrared thermal therapy of tumors under
magnetic resonance guidance. Proc. Natl Acad. Sci. USA 100(23),
13549-13554 (2003)).
[0216] In one embodiment, the source of the initiation energy can
be a radiowave emitting nanotube, such as those described by K.
Jensen, J. Weldon, H. Garcia, and A. Zettl in the Department of
Physics at the University of California at Berkeley (see
http://socrates.berkeley.edu/.about.argon/nanoradio/radio.html, the
entire contents of which are hereby incorporated by reference).
These nanotubes can be administered to the subject, and preferably
would be coupled to the activatable pharmaceutical agent or the
energy modulation agent, or both, or be located in proximity of a
target cell such that upon application of the initiation energy,
the nanotubes would accept the initiation energy (preferably
radiowaves), then emit radiowaves in close proximity to the
activatable pharmaceutical agent, or in close proximity to the
energy modulation agent, or to the target cell to then cause the
predetermined cellular changes or activation of the activatable
pharmaceutical agent. In such an embodiment, the nanotubes would
act essentially as a radiowave focusing or amplification device in
close proximity to the activatable pharmaceutical agent or energy
modulation agent or the target cell.
[0217] Alternatively, the energy emitting source may be an energy
modulation agent that emits energy in a form suitable for
absorption by the transfer agent or a target cell. For example, the
initiation energy source may be acoustic energy and one energy
modulation agent may be capable of receiving acoustic energy and
emitting photonic energy (e.g. sonoluminescent molecules) to be
received by another energy modulation agent that is capable of
receiving photonic energy. Other examples include transfer agents
that receive energy at x-ray wavelength and emit energy at UV
wavelength, preferably at UV-A wavelength. As noted above, a
plurality of such energy modulation agents may be used to form a
cascade to transfer energy from initiation energy source via a
series of energy modulation agents to activate the activatable
agent or the predetermined cellular change.
[0218] Signal transduction schemes as a drug delivery vehicle may
be advantageously developed by careful modeling of the cascade
events coupled with metabolic pathway knowledge to sequentially or
simultaneously cause the predetermined cellular change or activate
multiple activatable pharmaceutical agents to achieve
multiple-point alterations in cellular function.
[0219] Photoactivatable agents may be stimulated by an energy
source, such as irradiation, resonance energy transfer, exciton
migration, electron injection, or chemical reaction, to an
activated energy state that is capable of effecting the
predetermined cellular change desired. In a preferred embodiment,
the photoactivatable agent, upon activation, binds to DNA or RNA or
other structures in a cell. The activated energy state of the agent
is capable of causing damage to cells, inducing apoptosis.
[0220] One preferred method of treating a condition, disorder or
disease mediated by a target structure in a subject comprises:
[0221] (1) administering to the subject at least one activatable
pharmaceutical agent that is capable of effecting a predetermined
change to the target structure when activated; and [0222] (2)
applying an initiation energy from an initiation energy source to
the subject, [0223] wherein the applied initiation energy activates
the activatable agent in situ, [0224] thus causing the
predetermined change to the target structure to occur, wherein said
predetermined change treats the condition, disorder, or
disease.
[0225] Another preferred method for treating a condition, disorder
or disease mediated by a target structure in a subject, comprises:
[0226] (1) administering to the subject at least one activatable
pharmaceutical agent that is capable of activation by a multi
photon absorption event and of effecting a predetermined change in
said target when activated; and [0227] (2) applying an initiation
energy from an initiation energy source to the subject, [0228]
wherein the applied initiation energy activates the activatable
agent by the multi photon absorption event in situ, [0229] thus
causing the predetermined change to occur, wherein said
predetermined change treats the condition, disorder, or
disease.
[0230] The concept of multi-photon excitation is based on the idea
that two or more photons of low energy can excite a fluorophore in
a quantum event, resulting in the emission of a fluorescence
photon, typically at a higher energy than the two or more
excitatory photons. This concept was first described by Maria
Goppert-Mayer in her 1931 doctoral dissertation. However, the
probability of the near-simultaneous absorption of two or more
photons is extremely low. Therefore a high flux of excitation
photons is typically required, usually a femtosecond laser. This
had limited the range of practical applications for the
concept.
[0231] Perhaps the most well-known application of the multi-photon
excitation concept is the two-photon microscopy pioneered by
Winfried Denk in the lab of Watt W. Webb at Cornell University. He
combined the idea of two-photon absorption with the use of a laser
scanner.
[0232] There is an important difference between "sequential" and
"simultaneous" two-photon excitation. In sequential two-photon
excitation to a higher allowed energy level, the individual
energies of both the first photon and the second photon must be
appropriate to promote the molecule directly to the second allowed
electronic energy level and the third allowed electronic energy
level. In contrast, simultaneous two-photon excitation requires
only that the combined energy of the first of two photons and the
second of two photons be sufficient to promote the molecule to a
second allowed electronic energy level.
[0233] In two-photon excitation microscopy, an infrared laser beam
is focused through an objective lens. The Ti-sapphire laser
normally used has a pulse width of approximately 100 femtoseconds
and a repetition rate of about 80 MHz, allowing the high photon
density and flux required for two photons absorption and is tunable
across a wide range of wavelengths. Two-photon technology is
patented by Winfried Denk, James Strickler and Watt Webb at Cornell
University.
[0234] Two known applications are two-photon excited fluorescence
(TPEF) and non-linear transmission (NLT). The most commonly used
fluorophores have excitation spectra in the 400-500 nm range,
whereas the laser used to excite the fluorophores lies in the
.about.700-1000 nm (infrared) range. If the fluorophore absorbs two
infrared photons simultaneously, it will absorb enough energy to be
raised into the excited state. The fluorophore will then emit a
single photon with a wavelength that depends on the type of
fluorophore used (typically in the visible spectrum). Because two
photons need to be absorbed to excite a fluorophore, the
probability of emission is related to the intensity squared of the
excitation beam. Therefore, much more two-photon fluorescence is
generated where the laser beam is tightly focused than where it is
more diffuse. Effectively, fluorescence is observed in any
appreciable amount in the focal volume, resulting in a high degree
of rejection of out-of-focus objects. The fluorescence from the
sample is then collected by a high-sensitivity detector, such as a
photomultiplier tube. This observed light intensity becomes one
pixel in the eventual image; the focal point is scanned throughout
a desired region of the sample to form all the pixels of the image.
Two-photon absorption can be measured by several techniques.
[0235] Accordingly, in one aspect, the radiative signal may be of
the exact energy required to active the photoactive agent. In this
aspect, the radiative energy may be directly targeted at the
desired coordinate or region where the photoactive agent is
present. The initiation energy source in this embodiment may be,
for example, x-rays, gamma rays, an electron beam, microwaves or
radio waves.
[0236] In another aspect, the radiative signal may be of a lower
energy than the excitation energy of the photoactive agent. In this
aspect, the radiative signal does not have sufficient energy to
activate the photoactive agent in a conventional way. Activation of
the photoactive agent may be achieved via an "energy upgrade"
mechanism such as the multi-photon mechanism described above.
Activation of the photoactive agent may further be mediated by an
intermediary energy transformation agent. For example, the
radiative energy may first excite a fluorophore that emits a photon
at the right energy that excites the photoactive agent. The signal
is delivered to the target photoactive agent by way of this
intermediary agent. In this way, in addition to energy upgrading
(and downgrading, as described below), a signal relay mechanism is
also introduced. The initiation energy source may be x-rays, gamma
rays, an electron beam, microwaves or radio waves. Also, in one
embodiment, if the initiation energy is an infrared energy, the
energy activating the activatable agent is not UV or visible light
energy. Thus, another preferred method for treating a condition,
disease, or disorder mediated by a target structure in a subject,
comprises: [0237] (1) administering to the subject at least one
energy modulation agent and at least one activatable pharmaceutical
agent that is capable of activation by multi photon absorption and
of effecting a predetermined cellular change when activated; and
[0238] (2) applying an initiation energy from an initiation energy
source to the subject, [0239] wherein the energy modulation agent
upgrades the applied initiation energy to an energy, which then
activates the activatable agent by a multi photon absorption event
in situ, [0240] thus causing the predetermined cellular change to
occur, wherein said predetermined cellular change treats the
condition, disease or disorder.
[0241] In one embodiment, the energy upgrades are obtained via 2,
3, 4, or 5 simultaneous photon absorptions.
[0242] Yet another preferred method for treating a condition,
diseases, or disorder mediated by a target structure in a subject,
comprises: [0243] (1) administering to the subject at least one
energy modulation agent and at least one activatable pharmaceutical
agent that is capable effecting a predetermined cellular change
when activated; and [0244] (2) applying an initiation energy from
an initiation energy source to the subject, [0245] wherein the
energy modulation agent upgrades the applied initiation energy to
an energy, which then activates the activatable agent in situ,
[0246] thus causing the predetermined cellular change to occur,
wherein said predetermined cellular change treats the condition,
disease or disorder.
[0247] In yet another aspect, the radiative energy may be of a
higher energy than the excitation energy of the photoactive agent.
In this aspect, the photoactive agent may be activated via an
"energy downgrade" mechanism. In one scenario, via the multi-photon
mechanism, two lower energy photons having energy x may be absorbed
by an agent to excite the agent from ground state E0 to a higher
energy state E2. The agent may then relax down to an intermediate
energy state E1 by emitting a photon having an energy y that is
equal to the energy gap between E2 and E1, where y is less than x.
Other mechanisms of energy downgrade may be mediated by energy
transformation agents such as quantum dots, nanotubes, or other
agents having suitable photo-radiation properties. The initiation
energy source may be, for example, UV radiation, visible light,
infrared radiation, x-rays, gamma rays, an electron beam,
microwaves or radio waves. Thus, yet another preferred method for
treating a condition, disease, or disorder mediated by a target
structure in a subject, comprises: [0248] (1) administering to the
subject at least one energy modulation agent and at least one
activatable pharmaceutical agent that is capable of activation by
multi photon absorption and of effecting a predetermined cellular
change when activated; and [0249] (2) applying an initiation energy
from an initiation energy source to the subject, [0250] wherein the
energy modulation agent downgrades the applied initiation energy to
an energy, which then activates the activatable agent by a multi
photon absorption event in situ, [0251] thus causing the
predetermined cellular change to occur, wherein said predetermined
cellular change treats the condition, disease, or disorder.
[0252] Thus, yet another preferred method for treating a condition,
disease, or disorder mediated by a target structure in a subject,
comprises: [0253] (1) administering to the subject at least one
energy modulation agent and at least one activatable pharmaceutical
agent that is capable of effecting a predetermined cellular change
when activated; and [0254] (2) applying an initiation energy from
an initiation energy source to the subject, [0255] wherein the
energy modulation agent downgrades the applied initiation energy to
an energy, which then activates the activatable agent in situ,
[0256] thus causing the predetermined cellular change to occur,
wherein said predetermined cellular change treats the condition,
disorder or disease.
[0257] In a further preferred embodiment, the present invention
provides a method for treating a condition, disorder or disease
mediated by a target structure in a subject, comprising: [0258] (1)
administering to the subject an activatable pharmaceutical agent
that is capable of effecting a predetermined change in said target
structure when activated; and [0259] (2) applying an initiation
energy from an initiation energy source to the subject, [0260]
wherein the initiation energy applied and activatable
pharmaceutical agent upon activation produce insufficient singlet
oxygen in the subject to produce cell lysis, and wherein the
initiation energy activates the activatable pharmaceutical agent in
situ, [0261] thus causing the predetermined change to occur via
said target structure, wherein said predetermined change targets
the condition, disorder or disease.
[0262] In a different preferred embodiment, the present invention
provides a method for treating a condition, disorder or disease
mediated by a target structure in a subject, comprising: [0263] (1)
administering to the subject an activatable pharmaceutical agent
that is capable of activation by multi photon absorption and
effecting a predetermined change in said target structure when
activated; and [0264] (2) applying an initiation energy from an
initiation energy source to the subject, [0265] wherein the
initiation energy applied and activatable pharmaceutical agent upon
activation produce insufficient singlet oxygen in the subject to
produce cell lysis, and wherein the initiation energy activates the
activatable pharmaceutical agent by the multi photon absorption
event in situ, [0266] thus causing the predetermined change to
occur via said target structure, wherein said predetermined change
targets the condition, disorder or disease.
[0267] Work in the area of photodynamic therapy has shown that the
amount of singlet oxygen required to cause cell lysis, and thus
cell death, is 0.32.times.10.sup.-3 mol/liter or more, or 10.sup.9
singlet oxygen molecules/cell or more. In one preferred embodiment,
it is preferable to avoid production of an amount of singlet oxygen
that would cause cell lysis, due to its indiscriminate nature of
attack, lysing both target cells and healthy cells. Accordingly, it
is preferred in one preferred embodiment that the level of singlet
oxygen production caused by the initiation energy used or
activatable pharmaceutical agent upon activation be less than level
needed to cause cell lysis.
[0268] One advantage is that multiple wavelengths of emitted
radiation may be used to selectively stimulate one or more
photoactivatable agents or energy modulation agents capable of
stimulating the one or more photoactivatable agents. The energy
modulation agent is preferably stimulated at a wavelength and
energy that causes little or no damage to healthy cells, with the
energy from one or more energy modulation agents being transferred,
such as by Foerster Resonance Energy Transfer, to the
photoactivatable agents that damage the cell and cause the onset of
the desired cellular change, e.g., apoptosis of the cells.
[0269] Another advantage is that side effects can be greatly
reduced by limiting the production of free radicals, singlet
oxygen, hydroxides and other highly reactive groups that are known
to damage healthy cells. Furthermore, additional additives, such as
antioxidants, may be used to further reduce undesired effects of
irradiation.
[0270] Resonance Energy Transfer (RET) is an energy transfer
mechanism between to two molecules having overlapping emission and
absorption bands. Electromagnetic emitters are capable of
converting an arriving wavelength to a longer wavelength. For
example, UV-B energy absorbed by a first molecule may be
transferred by a dipole-dipole interaction to a UV-A-emitting
molecule in close proximity to the UV-B-absorbing molecule.
Alternatively, a material absorbing a shorter wavelength may be
chosen to provide RET to a non-emitting molecule that has an
overlapping absorption band with the transferring molecule's
emission band. Alternatively, phosphorescence, chemiluminescence,
or bioluminescence may be used to transfer energy to a
photoactivatable molecule.
[0271] Alternatively, one can administer the initiation energy
source to the subject. Within the context of the present invention,
the administering of the initiation energy source means the
administration of an agent, that itself produces the initiation
energy, in a manner that permits the agent to arrive at the target
cell within the subject without being surgically inserted into the
subject. The administration can take any form, including, but not
limited to, oral, intravenous, intraperitoneal, inhalation, etc.
Further, the initiation energy source in this embodiment can be in
any form, including, but not limited to, tablet, powder, liquid
solution, liquid suspension, liquid dispersion, gas or vapor, etc.
In this embodiment, the initiation energy source includes, but is
not limited to, chemical energy sources, nanoemitters, nanochips,
and other nanomachines that produce and emit energy of a desired
frequency. Recent advances in nanotechnology have provided examples
of various devices that are nanoscale and produce or emit energy,
such as the Molecular Switch (or Mol-Switch) work by Dr. Keith
Firman of the EC Research and Development Project, or the work of
Cornell et al. (1997) who describe the construction of nanomachines
based around ion-channel switches only 1.5 nm in size, which use
ion channels formed in an artificial membrane by two gramicidin
molecules: one in the lower layer of the membrane attached to a
gold electrode and one in the upper layer tethered to biological
receptors such as antibodies or nucleotides. When the receptor
captures a target molecule or cell, the ion channel is broken, its
conductivity drops, and the biochemical signal is converted into an
electrical signal. These nanodevices could also be coupled with the
present invention to provide targeting of the target cell, to
deliver the initiation energy source directly at the desired
site.
[0272] In another embodiment, the present invention includes the
administration of a source of chemical energy such as
chemiluminescence, phosphorescence or bioluminescence. The source
of chemical energy can be a chemical reaction between two or more
compounds, or can be induced by activating a chemiluminescent,
phosphorescent or bioluminescent compound with an appropriate
activation energy, either outside the subject or inside the
subject, with the chemiluminescence, phosphorescence or
bioluminescence being allowed to activate the activatable
pharmaceutical agent in vivo after administration. In one
embodiment, the activatable pharmaceutical agent and the source of
chemical energy can be administered. The administration can be
performed sequentially in any order or simultaneously. In the case
of certain sources of such chemical energy, the administration of
the chemical energy source can be performed after activation
outside the subject, with the lifetime of the emission of the
energy being up to several hours for certain types of
phosphorescent materials for example. There are no known previous
efforts to use resonance energy transfer of any kind to activate an
intercalator to bind DNA.
[0273] Yet another example is that nanoparticles or nanoclusters of
certain atoms may be introduced such that are capable of resonance
energy transfer over comparatively large distances, such as greater
than one nanometer, more preferably greater than five nanometers,
even more preferably at least 10 nanometers. Functionally,
resonance energy transfer may have a large enough "Foerster"
distance (R.sub.0), such that nanoparticles in one part of a cell
are capable of stimulating activation of photoactivatable agents
disposed in a distant portion of the cell, so long as the distance
does not greatly exceed R.sub.0. For example, gold nanospheres
having a size of 5 atoms of gold have been shown to have an
emission band in the ultraviolet range, recently.
[0274] In one embodiment, an aggressive cell proliferation disorder
has a much higher rate of mitosis, which leads to selective
destruction of a disproportionate share of the malignant cells
during even a systemically administered treatment. Stem cells and
healthy cells may be spared from wholesale programmed cell death,
even if exposed to photoactivated agents, provided that such
photoactivated agents degenerate from the excited state to a lower
energy state prior to binding, mitosis or other mechanisms for
creating damage to the cells of a substantial fraction of the
healthy stem cells. Thus, an auto-immune response may not be
induced.
[0275] Alternatively, a blocking agent may be used that prevents or
reduces damage to stem cells or healthy cells, selectively, which
would otherwise be impaired. The blocking agent is selected or is
administered such that the blocking agent does not impart a similar
benefit to malignant cells, for example.
[0276] In one embodiment, stem cells are targeted, specifically,
for destruction with the intention of replacing the stem cells with
a donor cell line or previously stored, healthy cells of the
patient. In this case, no blocking agent is used. Instead, a
carrier or photosensitizer is used that specifically targets the
stem cells.
[0277] Any of the photoactivatable agents may be exposed to an
excitation energy source implanted in a subject preferably near a
target site. The photoactive agent may be directed to a receptor
site by a carrier having a strong affinity for the receptor site.
Within the context of the present invention, a "strong affinity" is
preferably an affinity having an equilibrium dissociation constant,
at least in the nanomolar, nM, range or higher. Preferably, the
carrier may be a polypeptide and may form a covalent bond with a
photoactive agent, for example. The polypeptide may be an insulin,
interleukin, thymopoietin or transferrin, for example.
Alternatively, a photoactive agent may have a strong affinity for
the target cell without binding to a carrier.
[0278] A receptor site may be any of the following: nucleic acids
of nucleated blood cells, molecule receptor sites of nucleated
blood cells, the antigenic sites on nucleated blood cells,
epitopes, or other sites where photoactive agents are capable of
destroying a targeted cell.
[0279] In one embodiment, thin fiber optic lines are inserted in
the subject and laser light is used to photoactivate the agents. In
another embodiment, a plurality of sources for supplying
electromagnetic radiation energy or energy transfer are provided by
one or more molecules administered to a patient. The molecules may
emit stimulating radiation in the correct band of wavelength to
stimulate the target structure directly or to simulate the
photoactivatable agents, or the molecules may transfer energy by a
resonance energy transfer or other mechanism directly to the target
structure or the photoactivatable agent or indirectly by a cascade
effect via other molecular interactions.
[0280] The phenomenon of ultra weak emission from cellular systems
has been a topic of various inquiries since the 1900s. This topic
can be traced back to the early investigations of the Russian
biologist Gurwitsch Alexander G. Gurwitsch more than seventy years
ago, who speculated that ultraweak photon emission transmit
information in cells [A. G. Gurwitsch, S. S. Grabje, and S.
Salkind, "Die Natur des spezifischen Erregers der Zellteilung,"
Arch. Entwicklungsmech. Org. 100, 11-40, 1923].
[0281] In the 1970s, this area of research was investigated by a
number of investigators. The presence of biological radiation from
a variety of cells was later investigated by several research
groups in Europe and Japan using low-noise, sensitive
photon-counting detection systems [B. Ruth and F.-A. Popp,
"Experimentelle Untersuchungen zur ultraschwachen Photonenemission
biologischer Systeme," Z. Naturforsch., A: Phys. Sci. 31c, 741-745,
1976; T. I. Quickenden and S. S. Que-Hee, "The spectral
distribution of the luminescence emitted during growth of the yeast
Saccharomyces cerevisiae and its relationship to mitogenetic
radiation," Photochem. Photobiol. 23, 201-204, 1976; H. Inaba, Y.
Shimizu, Y. Tsuji, and A. Yamagishi, "Photon counting spectral
analysing system of extra-weak chemi- and bioluminescence for
biochemical applications," Photochem. Photobiol. 30, 169-175,
1979]. Popp and coworkers suggested the evidence of some
`informational character` associated with the ultra-weak photon
emission from biological systems, often referred by Popp as
"bio-photons". Other studies reported ultra-weak photon emission
from various species including plant, and animals cells [H. J.
Niggli, C. Scaletta, Y. Yan, F.-A. Popp, and L. A. Applegate,
"Ultraweak photon emission in assessing bone growth factor
efficiency using fibroblastic differentiation," J. Photochem.
Photobiol., B, 64, 62-68, 2001;]. Results of experiments of
UV-irradiated skin fibroblasts indicated that repair deficient
xeroderma pigmentosum cells show an efficient increase of ultraweak
photon emission in contrast to normal cells. [H. J. Niggli,
"Artificial sunlight irradiation induces ultraweak photon emission
in human skin fibroblasts," J. Photochem. Photobiol., B 18,281-285
(1993)].
[0282] A delayed luminescence emission was also observed in
biological systems [F.-A. Popp and Y. Yan, "Delayed luminescence of
biological systems in terms of coherent states," Phys. Lett. A 293,
93-97 (2002); A. Scordino, A. Triglia, F. Musumeci, F. Grasso, and
Z. Rajfur, "Influence of the presence of Atrazine in water on
in-vivo delayed luminescence of acetabularium acetabulum," J.
Photochem. Photobiol., B, 32, 11-17 (1996); This delayed
luminescence was used in quality control of vegetable products [A.
Triglia, G. La Malfa, F. Musumeci, C. Leonardi, and A. Scordino,
"Delayed luminescence as an indicator of tomato fruit quality," J.
Food Sci. 63, 512-515 (1998)] or for assessing the quality or
quality changes of biological tissues [Yu Yan, Fritz-Albert Popp *,
Sibylle Sigrist, Daniel Schlesinger, Andreas Dolf, Zhongchen Yan,
Sophie Cohen, Amodsen Chotia, "Further analysis of delayed
luminescence of plants", Journal of Photochemistry and Photobiology
B: Biology 78, 235-244 (2005)].
[0283] It was reported that UV excitation can further enhance the
ultra-weak emission and a method for detecting UV-A-laser-induced
ultra-weak photon emission was used to evaluate differences between
cancer and normal cells. [H. J. Niggli et al,
Laser-ultraviolet-A-induced ultraweak photon emission in mammalian
cells, Journal of Biomedical Optics 10(2), 024006 (2005)].
[0284] Accordingly, in one embodiment of the present invention,
upon applying an initiation energy from at least one source to a
target structure in a subject in need of treatment, the initiation
energy contacts the target structure and induces a predetermined
change in said target structure in situ,
[0285] wherein the predetermined change is the enhancement of
energy emission from the target, which then mediates, initiates or
enhances a biological activity of other target structures in the
subject, or of a second type of target structure (e.g., a different
cell type).
[0286] In another embodiment, the patient's own cells are removed
and genetically modified to provide photonic emissions. For
example, tumor or healthy cells may be removed, genetically
modified to induce bioluminescence and may be reinserted at the
site of the disease or condition to be treated. The modified,
bioluminescent cells may be further modified to prevent further
division of the cells or division of the cells only so long as a
regulating agent is present.
[0287] In a further embodiment, a biocompatible emitting source,
such as a fluorescing metal nanoparticle or fluorescing dye
molecule, is selected that emits in the UV-A band. The UV-A
emitting source is directed to the site of a disease or condition.
The UV-A emitting source may be directed to the site of the disease
or condition by systemically administering the UV-A emitting
source. Preferably, the UV-A emitting source is concentrated in the
target site, such as by physical insertion or by conjugating the
UV-A emitting molecule with a specific carrier that is capable of
concentrating the UV-A emitting source in a specific target
structure, as is known in the art.
[0288] In one preferred embodiment, the UV-A emitting source is a
gold nanoparticle comprising a cluster of 5 gold atoms, such as a
water soluble quantum dot encapsulated by polyamidoamine
dendrimers. The gold atom clusters may be produced through a slow
reduction of gold salts (e.g. HAuCl.sub.4 or AuBr.sub.3) or other
encapsulating amines, for example. One advantage of such a gold
nanoparticle is the increased Foerster distance (i.e. R.sub.0),
which may be greater than 100 angstroms. The equation for
determining the Foerster distance is substantially different from
that for molecular fluorescence, which is limited to use at
distances less than 100 angstroms. It is believed that the gold
nanoparticles are governed by nanoparticle surface to dipole
equations with a 1/R.sup.4 distance dependence rather than a
1/R.sup.6 distance dependence. For example, this permits
cytoplasmic to nuclear energy transfer between metal nanoparticles
and a photoactivatable molecule, such as a psoralen and more
preferably an 8-methoxypsoralen (8-MOP) administered orally to a
patient, which is known to be safe and effective at inducing an
apoptosis of leukocytes.
[0289] In another embodiment, a UV- or light-emitting luciferase is
selected as the emitting source for exciting a photoactivatable
agent. A luciferase may be combined with ATP or another molecule,
which may then be oxygenated with additional molecules to stimulate
light emission at a desired wavelength. Alternatively, a
phosphorescent emitting source may be used. One advantage of a
phosphorescent emitting source is that the phosphorescent emitting
molecules or other source may be electroactivated or photoactivated
prior to insertion into a target site either by systemic
administration or direct insertion into the region of the target
site. Alternatively, some of these materials can be activated, with
the energy being "stored" in the activated material, until emission
is stimulated by application of another energy. For example, see
the discussion of U.S. Pat. No. 4,705,952 below with respect to
infrared-triggered phosphors.
[0290] Phosphorescent materials may have longer relaxation times
than fluorescent materials, because relaxation of a triplet state
is subject to forbidden energy state transitions, storing the
energy in the excited triplet state with only a limited number of
quantum mechanical energy transfer processes available for
returning to the lower energy state. Energy emission is delayed or
prolonged from a fraction of a second to several hours. Otherwise,
the energy emitted during phosphorescent relaxation is not
otherwise different than fluorescence, and the range of wavelengths
may be selected by choosing a particular phosphor.
[0291] Among various materials, luminescent nanoparticles have
attracted increasing technological and industrial interest. In the
context of the present invention, nanoparticle refers to a particle
having a size less than one micron. While the description of the
invention describes specific examples using nanoparticles, the
present invention in many embodiments is not limited to particles
having a size less than one micron. However, in many of the
embodiments, the size range of having a size less than one micron,
and especially less than 100 nm produces properties of special
interest such as for example emission lifetime luminescence
quenching, luminescent quantum efficiency, and concentration
quenching and such as for example diffusion, penetration, and
dispersion into mediums where larger size particles would not
migrate.
[0292] U.S. Pat. No. 4,705,952 (the contents of which are hereby
incorporated herein by reference) describes an infrared-triggered
phosphor that stored energy in the form of visible light of a first
wavelength and released energy in the form of visible light of a
second wavelength when triggered by infrared light. In some cases,
U.S. Pat. No. 4,705,952 describes that "the upconversion continues
for as long as several days before a new short recharge is
required." The phosphors in U.S. Pat. No. 4,705,952 were
compositions of alkaline earth metal sulfides, rare earth dopants,
and fusible salts. The phosphors in U.S. Pat. No. 4,705,952 were
more specifically phosphors made from strontium sulfide, barium
sulfide and mixtures thereof; including a dopant from the rare
earth series and europium oxide, and mixtures thereof, and
including a fusible salt of fluorides, chlorides, bromides, and
iodides of lithium, sodium, potassium, cesium, magnesium, calcium,
strontium, and barium, and mixtures thereof. The materials
described in U.S. Pat. No. 4,705,952 are useful in various
embodiments of the invention.
[0293] In some cases, U.S. Pat. No. 4,705,952 describes that "the
storage times become extremely long, on the order of years." The
material is thus adapted to receive infrared photons and to emit
higher energy photons in a close to 1:1 relation. With storage
times this long, these infrared-triggered phosphors can be used in
various embodiments of the present invention as a viable mechanism
where commercial IR lasers are used to activate phosphorescence in
a medium, thereby in a patient generating visible or ultraviolet
light.
[0294] In another embodiment, a combined electromagnetic energy
harvester molecule is designed, such as the combined light
harvester disclosed in J. Am. Chem. Soc. 2005, 127, 9760-9768, the
entire contents of which are hereby incorporated by reference. By
combining a group of fluorescent molecules in a molecular
structure, a resonance energy transfer cascade may be used to
harvest a wide band of electromagnetic radiation resulting in
emission of a narrow band of fluorescent energy. By pairing a
combined energy harvester with a photoactivatable molecule, a
further energy resonance transfer excites the photoactivatable
molecule, when the photoactivatable molecule is nearby stimulated
combined energy harvester molecules. Another example of a harvester
molecule is disclosed in FIG. 4 of "Singlet-Singlet and
Triplet-Triplet Energy Transfer in Bichromophoric Cyclic Peptides,"
M.S. Thesis by M. O. Guler, Worcester Polytechnic Institute, May
18, 2002, which is incorporated herein by reference.
[0295] In another embodiment, a Stokes shift of an emitting source
or a series of emitting sources arranged in a cascade is selected
to convert a shorter wavelength energy, such as X-rays, to a longer
wavelength fluorescence emission such a optical or UV-A, which is
used to stimulate a photoactivatable molecule at the location of
the target structure. Preferably, the photoactivatable molecule is
selected to cause the predetermined change in target structure
without causing substantial harm to normal, healthy cells.
[0296] In an additional embodiment, the photoactivatable agent can
be a photocaged complex having an active agent contained within a
photocage. The active agent is bulked up with other molecules that
prevent it from binding to specific targets, thus masking its
activity. When the photocage complex is photoactivated, the bulk
falls off, exposing the active agent. In such a photocage complex,
the photocage molecules can be photoactive (i.e. when
photoactivated, they are caused to dissociate from the photocage
complex, thus exposing the active agent within), or the active
agent can be the photoactivatable agent (which when photoactivated
causes the photocage to fall off), or both the photocage and the
active agent are photoactivated, with the same or different
wavelengths. For example, a toxic chemotherapeutic agent can be
photocaged, which will reduce the systemic toxicity when delivered.
Once the agent is concentrated in the tumor, the agent is
irradiated with an activation energy. This causes the "cage" to
fall off, leaving a cytotoxic agent in the tumor cell. Suitable
photocages include those disclosed by Young and Deiters in
"Photochemical Control of Biological Processes", Org. Biomol.
Chem., 5, pp. 999-1005 (2007) and "Photochemical Hammerhead
Ribozyme Activation", Bioorganic & Medicinal Chemistry Letters,
16(10), pp. 2658-2661 (2006), the contents of which are hereby
incorporated by reference.
[0297] In one preferred embodiment, the use of light for uncaging a
compound or agent is used for elucidation of neuron functions and
imaging, for example, two-photon glutamine uncaging (Harvey C D, et
al., Nature, 450:1195-1202 (2007); Eder M, et al., Rev. Neurosci.,
15:167-183 (2004)). Other signaling molecules can be released by UV
light stimulation, e.g., GABA, secondary messengers (e.g.,
Ca.sup.2+ and Mg.sup.2+), carbachol, capsaicin, and ATP (Zhang F.,
et al., 2006). Chemical modifications of ion channels and receptors
may be carried out to render them light-responsive. Ca.sup.2+ is
involved in controlling fertilization, differentiation,
proliferation, apoptosis, synaptic plasticity, memory, and
developing axons. In yet another preferred embodiment, Ca.sup.2+
waves can be induced by UV irradiation (single-photon absorption)
and NIR irradiation (two-photon absorption) by releasing caged
Ca.sup.2+, an extracellular purinergic messenger InsP3 (Braet K.,
et al., Cell Calcium, 33:37-48 (2003)), or ion channel ligands
(Zhang F., et al., 2006).
[0298] Genetic targeting allows morphologically and
electrophysipologically characterization of genetically defined
cell populations. Accordingly, in an additional embodiment, a
light-sensitive protein is introduced into cells or live subjects
via a number of techniques including electroporation, DNA
microinjection, viral delivery, liposomal transfection, creation of
transgenic lines and calcium-phosphate precipitation. For example,
lentiviral technology provides a convenient combination a
conventional combination of stable long-term expression, ease of
high-titer vector production and low immunogenicity. The
light-sensitive protein may be, for example, channelrhodopsin-2
(ChR2) and chloride pump halorhodopsin (NpHR). The light protein
encoding gene(s) along with a cell-specific promoter can be
incorporated into the lentiviral vector or other vector providing
delivery of the light-sensitive protein encoding gene into a target
cell. ChR2 containing a light sensor and a cation channel, provides
electrical stimulation of appropriate speed and magnitude to
activate neuronal spike firing, when the cells harboring Ch2R are
pulsed with light.
[0299] In one embodiment, a lanthanide chelate capable of intense
luminescence is used. For example, a lanthanide chelator may be
covalently joined to a coumarin or coumarin derivative or a
quinolone or quinolone-derivative sensitizer. Sensitizers may be a
2- or 4-quinolone, a 2- or 4-coumarin, or derivatives or
combinations of these examples. A carbostyril 124
(7-amino-4-methyl-2-quinolone), a coumarin 120
(7-amino-4-methyl-2-coumarin), a coumarin 124
(7-amino-4-(trifluoromethyl)-2-coumarin),
aminoinethyltrimethylpsoralen or other similar sensitizer may be
used. Chelates may be selected to form high affinity complexes with
lanthanides, such as terbium or europium, through chelator groups,
such as DTPA. Such chelates may be coupled to any of a wide variety
of well known probes or carriers, and may be used for resonance
energy transfer to a psoralen or psoralen-derivative, such as
8-MOP, or other photoactive molecules capable of binding DNA. In
one alternative example, the lanthanide chelate is localized at the
site of the disease using an appropriate carrier molecule, particle
or polymer, and a source of electromagnetic energy is introduced by
minimally invasive procedures to irradiate the target structure,
after exposure to the lanthanide chelate and a photoactive
molecule.
[0300] In another embodiment, a biocompatible, endogenous
fluorophore emitter is selected to stimulate resonance energy
transfer to a photoactivatable molecule. A biocompatible emitter
with an emission maxima within the absorption range of the
biocompatible, endogenous fluorophore emitter may be selected to
stimulate an excited state in fluorophore emitter. One or more
halogen atoms may be added to any cyclic ring structure capable of
intercalation between the stacked nucleotide bases in a nucleic
acid (either DNA or RNA) to confer new photoactive properties to
the intercalator. Any intercalating molecule (psoralens, coumarins,
or other polycyclic ring structures) may be selectively modified by
halogenation or addition of non-hydrogen bonding ionic substituents
to impart advantages in its reaction photochemistry and its
competitive binding affinity for nucleic acids over cell membranes
or charged proteins, as is known in the art.
[0301] Skin photosensitivity is a major toxicity of
photosensitizers. Severe sunburn occurs if skin is exposed to
direct sunlight for even a few minutes. Early murine research
hinted at a vigorous and long term stimulation of immune response;
however, actual clinical testing has failed to achieve the early
promises of photodynamic therapies. The early photosensitizers for
photodynamic therapies targeted type II responses, which created
singlet oxygen when photoactivated in the presence of oxygen. The
singlet oxygen caused cellular necrosis and was associated with
inflammation and an immune response. Some additional
photosensitizers have been developed to induce type I responses,
directly damaging cellular structures.
[0302] Porfimer sodium (Photofrin; QLT Therapeutics, Vancouver, BC,
Canada), is a partially purified preparation of hematoporphyrin
derivative (HpD). Photofrin has been approved by the US Food and
Drug Administration for the treatment of obstructing esophageal
cancer, microinvasive endobronchial non-small cell lung cancer, and
obstructing endobronchial non-small cell lung cancer. Photofrin is
activated with 630 nm, which has a tissue penetration of
approximately 2 to 5 mm. Photofrin has a relatively long duration
of skin photosensitivity (approximately 4 to 6 weeks).
[0303] Tetra (m-hydroxyphenyl) chlorin (Foscan; Scotia
Pharmaceuticals, Stirling, UK), is a synthetic chlorine compound
that is activated by 652 nm light. Clinical studies have
demonstrated a tissue effect of up to 10 mm with Foscan and 652 nm
light. Foscan is more selectively a photosensitizer in tumors than
normal tissues, and requires a comparatively short light activation
time. A recommended dose of 0.1 mg/kg is comparatively low and
comparatively low doses of light may be used. Nevertheless,
duration of skin photosensitivity is reasonable (approximately 2
weeks). However, Foscan induces a comparatively high yield of
singlet oxygen, which may be the primary mechanism of DNA damage
for this molecule.
[0304] Motexafin lutetium (Lutetium texaphryin) is activated by
light in the near infared region (732 nm). Absorption at this
wavelength has the advantage of potentially deeper penetration into
tissues, compared with the amount of light used to activate other
photosensitizers (FIGS. 2A and 2B). Lutetium texaphryin also has
one of the greatest reported selectivities for tumors compared to
selectivities of normal tissues. Young S W, et al.: Lutetium
texaphyrin (PCI-0123) a near-infrared, water-soluble
photosensitizer. Photochem Photobiol 1996, 63:892-897. In addition,
its clinical use is associated with a shorter duration of skin
photosensitivity (24 to 48 hours). Lutetium texaphryin has been
evaluated for metastatic skin cancers. It is currently under
investigation for treatment of recurrent breast cancer and for
locally recurrent prostate cancer. The high selectivity for tumors
promises improved results in clinical trials.
[0305] In general, the approach may be used with any source for the
excitation of higher electronic energy states, such as electrical,
chemical and/or radiation, individually or combined into a system
for activating an activatable molecule. The process may be a
photopheresis process or may be similar to photophoresis. While
photophoresis is generally thought to be limited to photonic
excitation, such as by UV-light, other forms of radiation may be
used as a part of a system to activate an activatable molecule.
Radiation includes ionizing radiation which is high energy
radiation, such as an X-ray or a gamma ray, which interacts to
produce ion pairs in matter. Radiation also includes high linear
energy transfer irradiation, low linear energy transfer
irradiation, alpha rays, beta rays, neutron beams, accelerated
electron beams, and ultraviolet rays. Radiation also includes
proton, photon and fission-spectrum neutrons. Higher energy
ionizing radiation may be combined with chemical processes to
produce energy states favorable for resonance energy transfer, for
example. Other combinations and variations of these sources of
excitation energy may be combined as is known in the art, in order
to stimulate the activation of an activatable molecule, such as
8-MOP. In one example, ionizing radiation is directed at a solid
tumor and stimulates, directly or indirectly, activation of 8-MOP,
as well as directly damaging the DNA of malignant tumor cells. In
this example, either the effect of ionizing radiation or the
photophoresis-like activation of 8-MOP may be thought of as an
adjuvant therapy to the other.
[0306] In one embodiment, the present invention provides a method
for treating a condition, disease or disorder mediated by a target
structure in a subject, comprising: [0307] (1) administering to the
subject an activatable pharmaceutical agent that is capable of
effecting a predetermined change when activated; and [0308] (2)
applying an initiation energy from an initiation energy source to
the subject, [0309] wherein the initiation energy source is a
source of energy capable of penetrating completely through the
subject, and wherein the applying activates the activatable agent
in situ, [0310] thus causing the predetermined change to occur,
wherein occurrence of the predetermined change in the target
structure causes an increase in rate or decrease in rate of cell
division and/or growth to treat the condition, disease or
disorder.
[0311] In a further embodiment, the present invention provides a
method for treating a condition, disease or disorder mediated by a
target structure in a subject, comprising: [0312] (1) administering
to the subject one or more energy modulation agents and an
activatable pharmaceutical agent that is capable of effecting a
predetermined change in the target structure when activated; and
[0313] (2) applying an initiation energy from an initiation energy
source to the subject, [0314] wherein the one or more energy
modulation agents convert the initiation energy applied to UV-A or
visible energy, which then activates the activatable agent in situ,
[0315] thus causing the predetermined change to occur, wherein
occurrence of the predetermined change causes an increase in rate
or decrease in rate of cell division and/or growth to treat the
condition, disease or disorder.
[0316] In a different embodiment, the activatable pharmaceutical
agent can be activated by a single or multiphoton absorption
event.
[0317] Work in the area of photodynamic therapy has shown that the
amount of singlet oxygen required to cause cell lysis, and thus
cell death, is 0.32.times.10.sup.-3 mol/liter or more, or 10.sup.9
singlet oxygen molecules/cell or more. However, in the present
invention, it is most preferable to avoid production of an amount
of singlet oxygen that would cause cell lysis, due to its
indiscriminate nature of attack, lysing both target cells and
healthy cells. Accordingly, it is most preferred in the present
invention that the level of singlet oxygen production caused by the
initiation energy used or activatable pharmaceutical agent upon
activation be less than level needed to cause cell lysis.
[0318] In yet another embodiment, the activatable pharmaceutical
agent, preferably a photoactive agent, is directed to a receptor
site by a carrier having a strong affinity for the receptor site.
The carrier may be a polypeptide and may form a covalent bond with
a photo active agent, for example. The polypeptide may be an
insulin, interleukin, thymopoietin or transferrin, for example.
Alternatively, a photoactive pharmaceutical agent may have a strong
affinity for the target cell without a binding to a carrier.
[0319] For example, a treatment may be applied that acts to slow or
pause mitosis. Such a treatment is capable of slowing the division
of rapidly dividing healthy cells or stem cells without pausing
mitosis of cancerous cells. Thus, the difference in growth rate
between the non-target cells and target cells are further
differentiated to enhance the effectiveness of the methods of the
present invention.
[0320] In a further embodiment, methods in accordance with the
present invention may further include adding an additive to
alleviate treatment side-effects. Exemplary additives may include,
but are not limited to, antioxidants, adjuvant, or combinations
thereof. In one exemplary embodiment, psoralen is used as the
activatable pharmaceutical agent, UV-A is used as the activating
energy, and antioxidants are added to reduce the unwanted
side-effects of irradiation.
[0321] In another aspect, the present invention also provides
methods for producing an autovaccine, including: (1) providing a
population of targeted cells; (2) treating the cells ex vivo with a
psoralen or a derivative thereof; (3) activating the psoralen with
an initiation energy source to induce a predetermined change in a
target structure in the population of the target cells; and (4)
returning the treated cells back to the host to induce an
autovaccine effect against the targeted cell, wherein the treated
cells cause an autovaccine effect.
[0322] In a different embodiment, a method for generating an
autovaccine for a subject, comprises: [0323] (1) providing a
population of target cells; [0324] (2) treating the target cells ex
vivo in an environment separate and isolated from the subject with
an activatable pharmaceutical agent capable of activation by a
multi photon absorption event; [0325] (3) exposing the treated
target cells to an energy source; [0326] (4) activating the
activatable pharmaceutical agent with the energy source by the
multi photon absorption event to induce a predetermined change in
at least one target structure in the target cells; and [0327] (5)
returning the thus changed cells back to the subject to induce in
the subject an autovaccine effect against the target cell, wherein
the changed cells act as an autovaccine and the energy source is
x-rays, gamma rays, an electron beam, microwaves or radio
waves.
[0328] In a further embodiment, methods in accordance with the
present invention may further include a method for modifying a
target structure which mediates or is associated with a biological
activity, comprising: [0329] (1) contacting said target structure
with at least one activatable pharmaceutical agent that is capable
of effecting a predetermined change in a target structure when
activated and at least one plasmonics-active agent; and [0330] (2)
applying an initiation energy from an initiation energy source to
target structure [0331] wherein the plasmonics-active agent
enhances or modulates the applied initiation energy, such that the
enhanced initiation energy activates the activatable agent [0332]
thus causing the predetermined change to the target structure to
occur, wherein said predetermined change modifies the target
structure and modulates the biological activity of the target
structure.
[0333] In a different embodiment, the predetermined change enhances
the expression of, promotes the growth of, or increases the
quantity of said target structure; enhances, inhibits or stabilizes
the usual biological activity of said target structure compared to
a similar untreated target structure, and/or alters the
immunological or chemical properties of said target structure. In a
different embodiment, said target structure is a compound that is
modified by said predetermined change to be more or less antigenic
or immunogenic
[0334] The activatable pharmaceutical agent and derivatives thereof
as well as the energy modulation agent, can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the activatable pharmaceutical
agent and a pharmaceutically acceptable carrier. The pharmaceutical
composition also comprises at least one additive having a
complementary therapeutic or diagnostic effect, wherein the
additive is one selected from an antioxidant, an adjuvant, or a
combination thereof.
[0335] As used herein, "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions. Modifications can be made to the compound of the
present invention to affect solubility or clearance of the
compound. These molecules may also be synthesized with D-amino
acids to increase resistance to enzymatic degradation. If
necessary, the activatable pharmaceutical agent can be
co-administered with a solubilizing agent, such as
cyclodextran.
[0336] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, rectal administration, and
direct injection into the affected area, such as direct injection
into a tumor. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerin, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates, and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0337] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, or phosphate buffered saline (PBS). In all
cases, the composition must be sterile and should be fluid to the
extent that easy syringability exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0338] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, methods of preparation are vacuum
drying and freeze-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0339] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0340] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0341] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0342] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0343] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0344] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0345] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0346] Methods of administering agents according to the present
invention are not limited to the conventional means such as
injection or oral infusion, but include more advanced and complex
forms of energy transfer. For example, genetically engineered cells
that carry and express energy modulation agents may be used. Cells
from the host may be transfected with genetically engineered
vectors that express bioluminescent agents. Transfection may be
accomplished via in situ gene therapy techniques such as injection
of viral vectors or gene guns, or may be performed ex vivo by
removing a sample of the host's cells and then returning to the
host upon successful transfection.
[0347] Such transfected cells may be inserted or otherwise targeted
at the site where diseased cells are located. In this embodiment,
the initiation energy source may be a biochemical source as such
ATP, in which case the initiation energy source is considered to be
directly implanted in the transfected cell. Alternatively, a
conventional micro-emitter device capable of acting as an
initiation energy source may be transplanted at the site of the
diseased cells.
[0348] It will also be understood that the order of administering
the different agents is not particularly limited. Thus in some
embodiments the activatable pharmaceutical agent may be
administered before the energy modulation agent, while in other
embodiments the energy modulation agent may be administered prior
to the activatable pharmaceutical agent. It will be appreciated
that different combinations of ordering may be advantageously
employed depending on factors such as the absorption rate of the
agents, the localization and molecular trafficking properties of
the agents, and other pharmacokinetics or pharmacodynamics
considerations.
[0349] A further embodiment is the use of the present invention for
the treatment of skin cancer. In this example, a photoactivatable
agent, preferably psoralen, is given to the patient, and is
delivered to the skin lesion via the blood supply. An activation
source having limited penetration ability (such as UV or IR) is
shined directly on the skin--in the case of psoralen, it would be a
UV light, or an IR source. With the use of an IR source, the
irradiation would penetrate deeper and generate UV via two single
photon events with psoralen.
[0350] In a further embodiment, methods according to this aspect of
the present invention further include a step of separating the
components of the treated cells into fractions and testing each
fraction for autovaccine effect in a host. The components thus
isolated and identified may then serve as an effective autovaccine
to stimulate the host's immune system to suppress growth of the
targeted cells.
[0351] In another aspect, the present invention further provides
systems and kits for practicing the above described methods.
[0352] In one embodiment, a system for producing an auto-vaccine in
a subject, comprises: [0353] at least one activatable
pharmaceutical agent that is capable of activation by a multiphoton
absorption event and of inducing a predetermined cellular change
via at least one target structure in a target cell in said subject;
[0354] means for placing said at least one activatable
pharmaceutical agent in said subject; and [0355] an initiation
energy source to provide initiation energy capable of activating
the at least one activatable pharmaceutical agent in said target
cell by the multi photon absorption event, wherein activation is
either direct or indirect.
[0356] In a different embodiment, a system in accordance with the
present invention may include: (1) an initiation energy source; and
(2) one or more energy modulation agents. The system may further
comprise (3) one or more activatable pharmaceutical agents. In an
additional embodiment, the system may comprise only (1) the
initiation energy source. In yet another embodiment, the system may
comprise (1) an initiation energy source; and (3) one or more
activatable pharmaceutical agents. FIG. 3 illustrates a system
according to one exemplary embodiment of the present invention.
Referring to FIG. 3, an exemplary system according to one
embodiment of the present invention may have an initiation energy
source 1 directed at the subject 4. An activatable pharmaceutical
agent 2 and an energy modulation agent 3 are administered to the
subject 4. The initiation energy source may additionally be
controlled by a computer system 5 that is capable of directing the
delivery of the initiation energy.
[0357] In preferred embodiments, the initiation energy source may
be a linear accelerator equipped with image guided computer-control
capability to deliver a precisely calibrated beam of radiation to a
pre-selected coordinate. One example of such linear accelerators is
the SmartBeam.TM. IMRT (intensity modulated radiation therapy)
system from Varian medical systems (Varian Medical Systems, Inc.,
Palo Alto, Calif.).
[0358] In other embodiments, endoscopic or laproscopic devices
equipped with appropriate initiation energy emitter may be used as
the initiation energy source. In such systems, the initiation
energy may be navigated and positioned at the pre-selected
coordinate to deliver the desired amount of initiation energy to
the site.
[0359] In further embodiments, dose calculation and robotic
manipulation devices may also be included in the system.
[0360] In yet another embodiment, there is also provided a computer
implemented system for designing and selecting suitable
combinations of initiation energy source, energy transfer agent,
and activatable pharmaceutical agent, comprising:
[0361] a central processing unit (CPU) having a storage medium on
which is provided: [0362] a database of excitable compounds; [0363]
a first computation module for identifying and designing an
excitable compound that is capable of binding with a target
cellular structure or component; and [0364] a second computation
module predicting the resonance absorption energy of the excitable
compound,
[0365] wherein the system, upon selection of a target cellular
structure or component, computes an excitable compound that is
capable of binding with the target structure followed by a
computation to predict the resonance absorption energy of the
excitable compound.
[0366] FIG. 4 illustrates an exemplary computer implemented system
according to this embodiment of the present invention. Referring to
FIG. 4, an exemplary computer-implemented system according to one
embodiment of the present invention may have a central processing
unit (CPU) connected to a memory unit, configured such that the CPU
is capable of processing user inputs and selecting a combination of
initiation source, activatable pharmaceutical agent, and energy
transfer agent based on an energy spectrum comparison for use in a
method of the present invention.
[0367] FIG. 5 illustrates a computer system 1201 for implementing
various embodiments of the present invention. The computer system
1201 may be used as the controller 55 to perform any or all of the
functions of the CPU described above. The computer system 1201
includes a bus 1202 or other communication mechanism for
communicating information, and a processor 1203 coupled with the
bus 1202 for processing the information. The computer system 1201
also includes a main memory 1204, such as a random access memory
(RAM) or other dynamic storage device (e.g., dynamic RAM (DRAM),
static RAM (SRAM), and synchronous DRAM (SDRAM)), coupled to the
bus 1202 for storing information and instructions to be executed by
processor 1203. In addition, the main memory 1204 may be used for
storing temporary variables or other intermediate information
during the execution of instructions by the processor 1203. The
computer system 1201 further includes a read only memory (ROM) 1205
or other static storage device (e.g., programmable ROM (PROM),
erasable PROM (EPROM), and electrically erasable PROM (EEPROM))
coupled to the bus 1202 for storing static information and
instructions for the processor 1203.
[0368] The computer system 1201 also includes a disk controller
1206 coupled to the bus 1202 to control one or more storage devices
for storing information and instructions, such as a magnetic hard
disk 1207, and a removable media drive 1208 (e.g., floppy disk
drive, read-only compact disc drive, read/write compact disc drive,
compact disc jukebox, tape drive, and removable magneto-optical
drive). The storage devices may be added to the computer system
1201 using an appropriate device interface (e.g., small computer
system interface (SCSI), integrated device electronics (IDE),
enhanced-IDE (E-IDE), direct memory access (DMA), or
ultra-DMA).
[0369] The computer system 1201 may also include special purpose
logic devices (e.g., application specific integrated circuits
(ASICs)) or configurable logic devices (e.g., simple programmable
logic devices (SPLDs), complex programmable logic devices (CPLDs),
and field programmable gate arrays (FPGAs)).
[0370] The computer system 1201 may also include a display
controller 1209 coupled to the bus 1202 to control a display 1210,
such as a cathode ray tube (CRT), for displaying information to a
computer user. The computer system includes input devices, such as
a keyboard 1211 and a pointing device 1212, for interacting with a
computer user and providing information to the processor 1203. The
pointing device 1212, for example, may be a mouse, a trackball, or
a pointing stick for communicating direction information and
command selections to the processor 1203 and for controlling cursor
movement on the display 1210. In addition, a printer may provide
printed listings of data stored and/or generated by the computer
system 1201.
[0371] The computer system 1201 performs a portion or all of the
processing steps of the invention (such as for example those
described in relation to FIG. 5) in response to the processor 1203
executing one or more sequences of one or more instructions
contained in a memory, such as the main memory 1204. Such
instructions may be read into the main memory 1204 from another
computer readable medium, such as a hard disk 1207 or a removable
media drive 1208. One or more processors in a multi-processing
arrangement may also be employed to execute the sequences of
instructions contained in main memory 1204. In alternative
embodiments, hard-wired circuitry may be used in place of or in
combination with software instructions. Thus, embodiments are not
limited to any specific combination of hardware circuitry and
software.
[0372] As stated above, the computer system 1201 includes at least
one computer readable medium or memory for holding instructions
programmed according to the teachings of the invention and for
containing data structures, tables, records, or other data
described herein. Examples of computer readable media are compact
discs, hard disks, floppy disks, tape, magneto-optical disks, PROMs
(EPROM, EEPROM, flash EPROM), DRAM, SRAM, SDRAM, or any other
magnetic medium, compact discs (e.g., CD-ROM), or any other optical
medium, punch cards, paper tape, or other physical medium with
patterns of holes, a carrier wave (described below), or any other
medium from which a computer can read.
[0373] Stored on any one or on a combination of computer readable
media, the present invention includes software for controlling the
computer system 1201, for driving a device or devices for
implementing the invention, and for enabling the computer system
1201 to interact with a human user (e.g., print production
personnel). Such software may include, but is not limited to,
device drivers, operating systems, development tools, and
applications software. Such computer readable media further
includes the computer program product of the present invention for
performing all or a portion (if processing is distributed) of the
processing performed in implementing the invention.
[0374] The computer code devices of the present invention may be
any interpretable or executable code mechanism, including but not
limited to scripts, interpretable programs, dynamic link libraries
(DLLs), Java classes, and complete executable programs. Moreover,
parts of the processing of the present invention may be distributed
for better performance, reliability, and/or cost.
[0375] The term "computer readable medium" as used herein refers to
any medium that participates in providing instructions to the
processor 1203 for execution. A computer readable medium may take
many forms, including but not limited to, non-volatile media,
volatile media, and transmission media. Non-volatile media
includes, for example, optical, magnetic disks, and magneto-optical
disks, such as the hard disk 1207 or the removable media drive
1208. Volatile media includes dynamic memory, such as the main
memory 1204. Transmission media includes coaxial cables, copper
wire and fiber optics, including the wires that make up the bus
1202. Transmission media also may also take the form of acoustic or
light waves, such as those generated during radio wave and infrared
data communications.
[0376] Various forms of computer readable media may be involved in
carrying out one or more sequences of one or more instructions to
processor 1203 for execution. For example, the instructions may
initially be carried on a magnetic disk of a remote computer. The
remote computer can load the instructions for implementing all or a
portion of the present invention remotely into a dynamic memory and
send the instructions over a telephone line using a modem. A modem
local to the computer system 1201 may receive the data on the
telephone line and use an infrared transmitter to convert the data
to an infrared signal. An infrared detector coupled to the bus 1202
can receive the data carried in the infrared signal and place the
data on the bus 1202. The bus 1202 carries the data to the main
memory 1204, from which the processor 1203 retrieves and executes
the instructions. The instructions received by the main memory 1204
may optionally be stored on storage device 1207 or 1208 either
before or after execution by processor 1203.
[0377] The computer system 1201 also includes a communication
interface 1213 coupled to the bus 1202. The communication interface
1213 provides a two-way data communication coupling to a network
link 1214 that is connected to, for example, a local area network
(LAN) 1215, or to another communications network 1216 such as the
Internet. For example, the communication interface 1213 may be a
network interface card to attach to any packet switched LAN. As
another example, the communication interface 1213 may be an
asymmetrical digital subscriber line (ADSL) card, an integrated
services digital network (ISDN) card or a modem to provide a data
communication connection to a corresponding type of communications
line. Wireless links may also be implemented. In any such
implementation, the communication interface 1213 sends and receives
electrical, electromagnetic or optical signals that carry digital
data streams representing various types of information.
[0378] The network link 1214 typically provides data communication
through one or more networks to other data devices. For example,
the network link 1214 may provide a connection to another computer
through a local network 1215 (e.g., a LAN) or through equipment
operated by a service provider, which provides communication
services through a communications network 1216. The local network
1214 and the communications network 1216 use, for example,
electrical, electromagnetic, or optical signals that carry digital
data streams, and the associated physical layer (e.g., CAT 5 cable,
coaxial cable, optical fiber, etc). The signals through the various
networks and the signals on the network link 1214 and through the
communication interface 1213, which carry the digital data to and
from the computer system 1201 maybe implemented in baseband
signals, or carrier wave based signals. The baseband signals convey
the digital data as unmodulated electrical pulses that are
descriptive of a stream of digital data bits, where the term "bits"
is to be construed broadly to mean symbol, where each symbol
conveys at least one or more information bits. The digital data may
also be used to modulate a carrier wave, such as with amplitude,
phase and/or frequency shift keyed signals that are propagated over
a conductive media, or transmitted as electromagnetic waves through
a propagation medium. Thus, the digital data may be sent as
unmodulated baseband data through a "wired" communication channel
and/or sent within a predetermined frequency band, different than
baseband, by modulating a carrier wave. The computer system 1201
can transmit and receive data, including program code, through the
network(s) 1215 and 1216, the network link 1214, and the
communication interface 1213. Moreover, the network link 1214 may
provide a connection through a LAN 1215 to a mobile device 1217
such as a personal digital assistant (PDA) laptop computer, or
cellular telephone.
[0379] The exemplary energy spectrum previously noted in FIG. 1 may
also be used in this computer-implemented system.
[0380] The reagents and chemicals useful for methods and systems of
the present invention may be packaged in kits to facilitate
application of the present invention. In one exemplary embodiment,
a kit including a psoralen, and fractionating containers for easy
fractionation and isolation of autovaccines is contemplated. A
further embodiment of kit would comprise at least one activatable
pharmaceutical agent capable of causing a predetermined cellular
change, at least one energy modulation agent capable of activating
the at least one activatable agent when energized, and containers
suitable for storing the agents in stable form, and preferably
further comprising instructions for administering the at least one
activatable pharmaceutical agent and at least one energy modulation
agent to a subject, and for applying an initiation energy from an
initiation energy source to activate the activatable pharmaceutical
agent. The instructions could be in any desired form, including but
not limited to, printed on a kit insert, printed on one or more
containers, as well as electronically stored instructions provided
on an electronic storage medium, such as a computer readable
storage medium. Also optionally included is a software package on a
computer readable storage medium that permits the user to integrate
the information and calculate a control dose, to calculate and
control intensity of the irradiation source.
[0381] In different aspect of the invention, a kit for modifying a
target structure which mediates or is associated with a biological
activity, comprising: [0382] at least one agent selected from the
group consisting of energy modulation agents, plasmonics-active
agents and combinations thereof; [0383] wherein the energy
modulation agent, if present, upgrades or downgrades an initiation
energy to an activation energy capable of causing, either directly
or indirectly, a predetermined change in the target structure;
[0384] wherein the plasmonics-active agent, if present, enhances or
modifies the applied initiation energy or the activation energy
generated by the energy modulation agent, or both; and [0385] one
or more containers suitable for storing the agents in stable
forms.
[0386] In a different embodiment, a kit for performing a condition,
disorder or disease treatment, comprises:
[0387] at least one energy modulation agent capable of adsorbing,
intensifying or modifying an initiation energy into an energy that
is capable of causing a predetermined change in a target structure;
and
[0388] containers suitable for storing the agents in stable
form.
[0389] In yet another embodiment, the kit may further comprise
instructions for administering the at least one energy modulation
agent to a subject.
[0390] Plasmonics Enhanced Photospectral Therapy
[0391] In the PEPST embodiment of the present invention, the
present invention is significantly different from the phototherapy
technique often referred to Photothermal Therapy (PTT). To
illustrate the difference between the present invention PEPST, a
form of photospectral therapy (PST) and the PTT technique, the
photochemical processes involved in PST and PPT is discussed
below.
[0392] When drug molecules absorb excitation light, electrons
undergo transitions from the ground state to an excited electronic
state. The electronic excitation energy subsequently relaxes via
radiative emission (luminescence) and radiationless decay channels.
When a molecule absorbs excitation energy, it is elevated from
S.sub.o to some vibrational level of one of the excited singlet
states, S.sub.n, in the manifold S.sub.1, . . . , S.sub.n. In
condensed media (tissue), the molecules in the S.sub.n state
deactivate rapidly, within 10.sup.-13 to 10.sup.-11 s via
vibrational relaxation (VR) processes, ensuring that they are in
the lowest vibrational levels of S.sub.n possible. Since the VR
process is faster than electronic transitions, any excess
vibrational energy is rapidly lost as the molecules are deactivated
to lower vibronic levels of the corresponding excited electronic
state. This excess VR energy is released as thermal energy to the
surrounding medium. From the S.sub.n state, the molecule
deactivates rapidly to the isoenergetic vibrational level of a
lower electronic state such as S.sub.n-1 via an internal conversion
(IC) process. IC processes are transitions between states of the
same multiplicity. The molecule subsequently deactivates to the
lowest vibronic levels of S.sub.n-1 via a VR process. By a
succession of IC processes immediately followed by VR processes,
the molecule deactivates rapidly to the ground state S.sub.1. This
process results in excess VR and IC energy released as thermal
energy to the surrounding medium leading to the overheating of the
local environment surrounding the light absorbing drug molecules.
The heat produced results in local cell or tissue destruction. The
light absorbing species include natural chromophores in tissue or
exogenous dye compounds such as indocyanine green,
naphthalocyanines, and porphyrins coordinated with transition
metals and metallic nanoparticles and nanoshells of metals. Natural
chromophores, however, suffer from very low absorption. The choice
of the exogenous photothermal agents is made on the basis of their
strong absorption cross sections and highly efficient light-to-heat
conversion. This feature greatly minimizes the amount of laser
energy needed to induce local damage of the diseased cells, making
the therapy method less invasive. A problem associated with the use
of dye molecules is their photobleaching under laser irradiation.
Therefore, nanoparticles such as gold nanoparticles and nanoshells
have recently been used. The promising role of nanoshells in
photothermal therapy of tumors has been demonstrated [Hirsch, L.
R., Stafford, R. J Bankson, J. A., Sershen, S. R., Rivera, B.,
Price, R. E., Hazle, J. D., Halas, N. J., and West, J. L.,
Nanoshell-mediated near-infrared thermal therapy of tumors under
magnetic resonance guidance. PNAS, 2003. 100(23): p. 13549-13554].
The use of plasmonics-enhanced photothermal properties of metal
nanoparticles for photothermal therapy has also been reviewed
(Xiaohua Huang & Prashant K Jain & Ivan H. El-Sayed &
Mostafa A. El-Sayed, "Plasmonic photothermal therapy (PPTT) using
gold nanoparticles", Lasers in Medical Science, August 2007)
[0393] The PST method of the present invention, however, is based
on the radiative processes (fluorescence, phosphorescence,
luminescence, Raman, etc) whereas the PTT method is based on the
radiationless processes (IC, VR and heat conversion) in
molecules.
Basic Principle of Plasmonics and Enhanced Electromagnetic
Fields
[0394] Whereas the photothermal properties of plasmonics metal
nanoparticles have been used, the spectroscopic absorption and
emission of plasmonics-active nanoparticles in phototherapy have
not been reported.
[0395] In the present invention PEPST, the plasmonics-enhanced
spectroscopic properties (spectral absorption, emission,
scattering) are the major factors involved in the treatment.
[0396] The PEPST principle is based on the enhancement mechanisms
of the electromagnetic field effect. There are two main sources of
electromagnetic enhancement: (1) first, the laser electromagnetic
field is enhanced due to the addition of a field caused by the
polarization of the metal particle; (2) in addition to the
enhancement of the excitation laser field, there is also another
enhancement due to the molecule radiating an amplified emission
(luminescence, Raman, etc.) field, which further polarizes the
metal particle, thereby acting as an antenna to further amplify the
Raman/Luminescence signal.
[0397] Electromagnetic enhancements are divided into two main
classes: a) enhancements that occur only in the presence of a
radiation field, and b) enhancements that occur even without a
radiation field. The first class of enhancements is further divided
into several processes. Plasma resonances on the substrate
surfaces, also called surface plasmons, provide a major
contribution to electromagnetic enhancement. An effective type of
plasmonics-active substrate comprises nanostructured metal
particles, protrusions, or rough surfaces of metallic materials.
Incident light irradiating these surfaces excites conduction
electrons in the metal, and induces excitation of surface plasmons
leading to Raman/luminescence enhancement. At the plasmon
frequency, the metal nanoparticles (or nanostructured roughness)
become polarized, resulting in large field-induced polarizations
and thus large local fields on the surface. These local fields
increase the luminescence/Raman emission intensity, which is
proportional to the square of the applied field at the molecule. As
a result, the effective electromagnetic field experienced by the
analyte molecule on these surfaces is much larger than the actual
applied field. This field decreases as 1/r.sup.3 away from the
surface. Therefore, in the electromagnetic models, the
luminescence/Raman-active analyte molecule is not required to be in
contact with the metallic surface but can be located anywhere
within the range of the enhanced local field, which can polarize
this molecule. The dipole oscillating at the wavelength .lamda. of
Raman or luminescence can, in turn, polarize the metallic
nanostructures and, if .lamda. is in resonance with the localized
surface plasmons, the nanostructures can enhance the observed
emission light (Raman or luminescence).
[0398] There are two main sources of electromagnetic enhancement:
(1) first, the laser electromagnetic field is enhanced due to the
addition of a field caused by the polarization of the metal
particle; (2) in addition to the enhancement of the excitation
laser field, there is also another enhancement due to the molecule
radiating an amplified Raman/luminescence field, which further
polarizes the metal particle, thereby acting as an antenna to
further amplify the Raman/luminescence signal. Plasmonics-active
metal nanoparticles also exhibit strongly enhanced visible and
near-infrared light absorption, several orders of magnitude more
intense compared to conventional laser phototherapy agents. The use
of plasmonic nanoparticles as highly enhanced photoabsorbing agents
thus provides a selective and efficient phototherapy strategy. The
tunability of the spectral properties of the metal nanoparticles
and the biotargeting abilities of the plasmonic nanostructures make
the PEPST method promising.
[0399] The present invention PEPST is based on several important
mechanisms: [0400] Increased absorption of the excitation light by
the plasmonic metal nanoparticles, resulting in enhanced
photoactivation of drug molecules [0401] Increased absorption of
the excitation light by the plasmonic metal nanoparticles that
serve as more efficient energy modulation agent systems, yielding
more light for increased excitation of PA molecules [0402]
Increased absorption of the excitation light by the photoactive
drug system adsorbed on or near the plasmonic metal nanoparticles
[0403] Increased light absorption of the energy modulation agent
molecules adsorbed on or near the metal nanoparticles [0404]
Amplified light emission from the energy modulation agent molecules
adsorbed on or near the metal nanoparticles [0405] Increased
absorption of emission light emitted from the energy modulation
agent by the PA molecule
[0406] One of several phenomena that can enhance the efficiency of
light emitted (Raman or luminescence) from molecules adsorbed or
near a metal nanostructures Raman scatter is the surface-enhanced
Raman scattering (SERS) effect. In 1984, the general applicability
of SERS as an analytical technique was first reported by one of the
present inventors, and the possibility of SERS measurement for a
variety of chemicals including several homocyclic and heterocyclic
polyaromatic compounds [T. Vo-Dinh, M. Y. K. Hiromoto, G. M. Begun
and R. L. Moody, "Surface-enhanced Raman spectroscopy for trace
organic analysis," Anal. Chem., vol. 56, 1667, 1984]. Extensive
research has been devoted to understanding and modeling the Raman
enhancement in SERS since the mid 1980's. FIG. 6, for example,
shows the early work by Kerker modeling electromagnetic field
enhancements for spherical silver nanoparticles and metallic
nanoshells around dielectric cores as far back as 1984 [M M Kerker,
Acc. Chem. Res., 17, 370 (1984)]. This figure shows the result of
theoretical calculations of electromagnetic enhancements for
isolated spherical nanospheres and nanoshells at different
excitation wavelengths. The intensity of the normally weak Raman
scattering process is increased by factors as large as 10.sup.13 or
10.sup.15 for compounds adsorbed onto a SERS substrate, allowing
for single-molecule detection. As a result of the electromagnetic
field enhancements produced near nanostructured metal surfaces,
nanoparticles have found increased use as fluorescence and Raman
nanoprobes.
[0407] The theoretical models indicate that it is possible to tune
the size of the nanoparticles and the nanoshells to the excitation
wavelength. Experimental evidence suggests that the origin of the
10.sup.6- to 10.sup.15-fold Raman enhancement primarily arises from
two mechanisms: a) an electromagnetic "lightning rod" effect
occurring near metal surface structures associated with large local
fields caused by electromagnetic resonances, often referred to as
"surface plasmons"; and b) a chemical effect associated with direct
energy transfer between the molecule and the metal surface.
[0408] According to classical electromagnetic theory,
electromagnetic fields can be locally amplified when light is
incident on metal nanostructures. These field enhancements can be
quite large (typically 10.sup.6- to 10.sup.7-fold, but up to
10.sup.15-fold enhancement at "hot spots"). When a nanostructured
metallic surface is irradiated by an electromagnetic field (e.g., a
laser beam), electrons within the conduction band begin to
oscillate at a frequency equal to that of the incident light. These
oscillating electrons, called "surface plasmons," produce a
secondary electric field which adds to the incident field. If these
oscillating electrons are spatially confined, as is the case for
isolated metallic nanospheres or roughened metallic surfaces
(nanostructures), there is a characteristic frequency (the plasmon
frequency) at which there is a resonant response of the collective
oscillations to the incident field. This condition yields intense
localized field enhancements that can interact with molecules on or
near the metal surface. In an effect analogous to a "lightning
rod," secondary fields are typically most concentrated at points of
high curvature on the roughened metal surface.
Design, Fabrication and Operation of PEPST Probes
[0409] FIG. 7 shows a number of the various embodiments of PEPST
probes that can be designed: [0410] (A) probe comprising PA
molecules bound to a metal (gold) nanoparticle; [0411] (B)
PA-containing nanoparticle covered with metal nanoparticles; [0412]
(C) Metal nanoparticle covered with PA nanocap; [0413] (D)
PA-containing nanoparticle covered with metal nanocap; [0414] (E)
Metal nanoparticle covered with PA nanoshell; [0415] (F)
PA-containing nanoparticle covered with metal nanoshell; and [0416]
(G) PA-containing nanoparticle covered with metal nanoshell with
protective coating layer.
[0417] A basic embodiment of the PEPST probe is shown in FIG. 7A.
This probe comprises PA molecules bound to a metal (e.g., gold)
nanoparticle. FIG. 8 illustrates the plasmonics-enhancement effect
of the PEPST probe. The gold nanoparticles can serve as a drug
delivery platform. Gold nanoparticles have been described as a
novel technology in the field of particle-based tumor-targeted drug
delivery [Giulio F. Paciotti and Lonnie Myer, David Weinreich, Dan
Goia, Nicolae Pavel, Richard E. McLaughlin, Lawrence Tamarkin,
"Colloidal Gold: A Novel Nanoparticle Vector for Tumor Directed
Drug Delivery, Drug Delivery, 11:169-183, 2004]. Particle delivery
systems capable of escaping phagocytic clearance by the
reticuloendothelial system (RES) can facilitate targeting cancer
therapeutics to solid tumors. Such delivery systems could
preferentially accumulate within the tumor microenvironment under
ideal conditions. A particle delivery system capable of
sequestering a phototherapeutic drug selectively within a tumor may
also reduce the accumulation of the drug in healthy organs.
Consequently, these delivery systems may increase the relative
efficacy or safety of therapy (less radiation energy and
intensity), and therefore, will increase the drug's therapeutic
efficiency.
[0418] Radiation of suitable energy is used to excite the PA drug
molecules (e.g., aminolevulinic acid (ALA), porphyrins) and make
them photoactive. For example, with the PDT drug ALA, light of a
HeNe laser (632.8-nm excitation) can be used for excitation. In
this case the metal nanoparticles are designed to exhibit strong
plasmon resonance band around 632.8 nm. The surface plasmon
resonance effect amplifies the excitation light at the
nanoparticles, resulting in increased photoactivation of the PA
drug molecules and improved therapy efficiency. The
plasmonics-enhanced mechanism can also be used with the other PEPST
probes in FIGS. 7B, 7C, 7D, 7E, 7F and 7G.
[0419] FIG. 34 shows yet other embodiment of plasmonics
photo-active probes. FIG. 35 shows yet other embodiment of
plasmonics photo-active probes that have a dielectric layer between
the metal and the UC materials.
[0420] In one embodiment, a method for treating a condition,
disorder or disease in accordance with the present invention
comprises: [0421] (1) administering to the subject at least one
activatable pharmaceutical agent that is capable of effecting a
predetermined change in a target structure when activated and at
least one plasmonics-active agent; and [0422] (2) applying an
initiation energy from an initiation energy source to the subject,
[0423] wherein the plasmonics-active agent enhances or modifies the
applied initiation energy, such that the enhanced initiation energy
activates the activatable agent in situ, [0424] thus causing the
predetermined change to the target structure to occur, wherein said
predetermined change modifies the target structure and treats said
condition, disorder, or disease.
[0425] In a different embodiment, a method in accordance with the
present invention comprises: [0426] (1) contacting said target
structure with at least one activatable pharmaceutical agent that
is capable of effecting a predetermined change in a target
structure when activated and at least one plasmonics-active agent;
and [0427] (2) applying an initiation energy from an initiation
energy source to target structure [0428] wherein the
plasmonics-active agent enhances or modifies the applied initiation
energy, such that the enhanced initiation energy activates the
activatable agent [0429] thus causing the predetermined change to
the target structure to occur, wherein said predetermined change
modifies the target structure and modulates the biological activity
of the target structure.
[0430] In a different embodiment, at least one energy modulation
agent and/or excitation-generating energy modulation agent material
may be also added. In one embodiment, the energy modulation agent
or excitation-generating energy modulation agent material may
adsorb, intensify or modify the initiation energy which is then
enhanced by at least one plasmonic agent. In a different
embodiment, the energy modulation agent or excitation-generating
energy modulation agent material may adsorb, intensify or modify
energy enhanced by the at least plasmonics-active agent and emit an
energy that is capable to activate the pharmaceutical activatable
agent.
[0431] In another embodiment, the predetermined change enhances the
expression of, promotes the growth of, or increases the quantity of
said target structure. In yet, different embodiment, the
predetermined change enhances, inhibits or stabilizes the usual
biological activity of said target structure compared to a similar
untreated target structure. In a different embodiment, the
predetermined change alters the immunological or chemical
properties of said target structure. In a different embodiment, the
target structure is a compound that is modified by said
predetermined change to be more or less antigenic or
immunogenic.
Structures of Plasmonics-Active Metal Nanostructures
[0432] Plasmon resonances arise within a metallic nanoparticle from
the collective oscillation of free electrons driven by an incident
optical field. The plasmonic response of nanoparticles have played
a role in a growing number of applications, including
surface-enhanced Raman scattering (SERS), chemical sensing, drug
delivery, photothermal cancer therapy and new photonic devices. The
investigation and application of plasmonics nanosubstrates for SERS
detection has been used by one of the present inventors for over
two decades [T. Vo-Dinh, "Surface-Enhanced Raman Spectroscopy Using
Metallic Nanostructures," Trends in Anal. Chem., 17,557 (1998)].
The first report by one of the present inventors on the practical
analytical use of the SERS techniques for trace analysis of a
variety of chemicals including several homocyclic and heterocyclic
polyaromatic compounds was in 1984 [T. Vo-Dinh, M. Y. K. Hiromoto,
G. M Begun and R. L. Moody, "Surface-enhanced Raman spectroscopy
for trace organic analysis," Anal. Chem., vol. 56, 1667, 1984].
Since then, the development of SERS technologies for applications
in chemical sensing, biological analysis and medical diagnostics
has been ongoing. The substrates involve nanoparticles and
semi-nanoshells comprising a layer of nanoparticles coated by a
metal (such as silver) on one side (nanocaps or half-shells).
Several groups have shown that plasmon resonances of spherical
shells can be tuned by controlling the shell thickness and aspect
ratios of the nanoshell structures [M. M. Kerker, Acc. Chem. Res.,
17, 370 (1984); J. B. Jackson, S. L. Westcott, L. R. Hirsch, J. L.
West and N. H. Halas, "Controlling the surface enhanced Raman
effect via the nanoshell geometry," Appl. Phys. Lett., vol. 82,
257-259, 2003; S. 0.1. Norton and T Vo-Dinh, "Plasmonic Resonances
of nanoshells of Spheroidal Shape", IEEE Trans. Nanotechnology, 6,
627-638 (2007)]. These shells typically comprise a metallic layer
over a dielectric core. In one embodiment of the present invention,
these shells comprise spheroidal shells, since the plasmon
resonances (both longitudinal and transverse modes) are influenced
by both shell thickness and aspect ratio. A number of researchers
have examined the plasmonic response of the solid spheroidal
particle in their analysis of surface-enhanced Raman scattering,
although the spheroidal shell appears not to have been
investigated. The present invention also includes prolate and
oblate spheroidal shells, which show some interesting qualitative
features in their plasmon resonances. The spheroidal shell presents
two degrees of freedom for tuning: the shell thickness and the
shell aspect ratio [S. J. Norton and T Vo-Dinh, "Plasmonic
Resonances of Nanoshells of Spheroidal Shape", IEEE Trans.
Nanotechnology, 6, 627-638 (2007)].
[0433] FIG. 9 shows some of the various embodiments of
plasmonics-active nanostructures that can be designed, and are
preferred embodiments of the present invention: [0434] (A) Metal
nanoparticle; [0435] (B) Dielectric nanoparticle core covered with
metal nanocap; [0436] (C) Spherical metal nanoshell covering
dielectric spheroid core; [0437] (D) Oblate metal nanoshell
covering dielectric spheroid core; [0438] (E) Metal nanoparticle
core covered with dielectric nanoshell; [0439] (F) Metal nanoshell
with protective coating layer; [0440] (G) Multi layer metal
nanoshells covering dielectric spheroid core; [0441] (H)
Multi-nanoparticle structures; [0442] (I) Metal nanocube and
nanotriangle/nanoprism; and [0443] (J) Metal cylinder. PEPST Probes
with Remotely-Activated Drug Release
[0444] In a further embodiment of the present invention, the PA
drug molecules can be incorporated into a material (e.g.,
biocompatible polymer) that can form a nanocap onto the metal
(gold) nanoparticles. The material can be a gel or biocompatible
polymer that can have long-term continuous drug release properties.
Suitable gel or biocompatible polymers include, but are not limited
to poly(esters) based on polylactide (PLA), polyglycolide (PGA),
polycarpolactone (PCL), and their copolymers, as well as
poly(hydroxyalkanoate)s of the PHB-PHV class, additional
poly(ester)s, natural polymers, particularly, modified
poly(saccharide)s, e.g., starch, cellulose, and chitosan,
polyethylene oxides, poly(ether)(ester) block copolymers, and
ethylene vinyl acetate copolymers. The drug release mechanism can
also be triggered by non-invasive techniques, such as RF, MW,
ultrasound, photon (FIG. 10).
[0445] FIG. 11 shows other possible embodiments where the PA drug
molecule is bound to the metal nanoparticles via a linker that can
be cut by a photon radiation. Such a linker includes, but is not
limited to, a biochemical bond (FIG. 11A), a DNA bond (FIG. 11B),
or an antibody-antigen bond (FIG. 11C). In another embodiment, the
linker is a chemically labile bond that will be broken by the
chemical environment inside the cell. These types of probes are
useful for therapy modalities where the PA molecules have to enter
the nucleus (e.g., psoralen molecules need to enter the nucleus of
cells and intercalate onto DNA). Since it is more difficult for
metal nanoparticles to enter the cell nucleus than for smaller
molecules, it is desirable to PEPST probes that have releasable PA
molecules.
Disease-Targeted PEPST Probes
[0446] Aggregation of metal (such as silver or gold) nanoparticles
(nanopsheres, nanorods, etc) is often a problem, especially with
citrate-capped gold nanospheres, cetyl trimethylammonium bromide
(CTAB)-capped gold nanospheres and nanorods and nanoshells because
they have poor stability when they are dispersed in buffer solution
due to the aggregating effect of salt ions. The biocompatibility
can be improved and nanoparticle aggregation prevented by capping
the nanoparticles with polyethylene glycol (PEG) (by conjugation of
thiol-functionalized PEG with metal nanoparticles). Furthermore,
PEGylated nanoparticles are preferentially accumulated into tumor
tissues due to the enhanced permeability and retention effect,
known as the "EPR" effect [Maedaa H, Fanga J, Inutsukaa T, Kitamoto
Y (2003) Vascular permeability enhancement in solid tumor: various
factors, mechanisms involved and its implications. Int
Immunopharmacol 3:319-328; Paciotti G F, Myer L, Weinreich D, Goia
D, Pavel N, McLaughlin R E, Tamarkin L (2004) Colloidal gold: a
novel nanoparticles vector for tumor directed drug delivery. Drug
Deliv 11:169-183]. Blood vessels in tumor tissue are more "leaky"
than in normal tissue, and as a result, particles, or large
macromolecular species or polymeric species preferentially
extravasate into tumor tissue. Particles and large molecules tend
to stay a longer time in tumor tissue due to the decreased
lymphatic system, whereas they are rapidly cleared out in normal
tissue. This tumor targeting strategy is often referred to as
passive targeting whereas the antibody-targeting strategy is called
active targeting.
[0447] To specifically target diseased cells, specific genes or
protein markers, the drug systems of the present invention can be
bound to a bioreceptor (e.g., antibody, synthetic molecular imprint
systems, DNA, proteins, lipids, cell-surface receptors, aptamers,
etc.). Immunotargeting modalities to deliver PA agents selectively
to the diseased cells and tissue provide efficient strategies to
achieving specificity, minimizing nonspecific injury to healthy
cells, and reducing the radiation intensity used.
Biofunctionalization of metal nanoparticles (e.g., gold, silver)
can be performed using commonly developed and widely used
procedures. There are several targeting strategies that can be used
in the present invention: (a) nanoparticles conjugated to
antibodies that recognize biomarkers specific to the diseased
cells; (b) nanoparticles passivated by poly (ethylene) glycol
(PEG), which is used to increase the biocompatibility and
biostability of nanoparticles and impart them an increased blood
retention time.
PEPST Probes with Bioreceptors
[0448] Bioreceptors are the key to specificity for targeting
disease cells, mutated genes or specific biomarkers. They are
responsible for binding the biotarget of interest to the drug
system for therapy. These bioreceptors can take many forms and the
different bioreceptors that have been used are as numerous as the
different analytes that have been monitored using biosensors.
However, bioreceptors can generally be classified into five
different major categories. These categories include: 1)
antibody/antigen, 2) enzymes, 3) nucleic acids/DNA, 4) cellular
structures/cells and 5) biomimetic. FIG. 12 illustrates a number of
embodiments of the various PEPST probes with bioreceptors that can
be designed. The probes are similar to those in FIG. 2 but have
also a bioreceptor for tumor targeting.
[0449] Antibody Probes.
[0450] Antibody based targeting is highly active, specific and
efficient. The antibodies are selected to target a specific tumor
marker (e.g., anti-epidermal growth factor receptor (EGFR)
antibodies targeted against overexpressed EGFR on oral and cervical
cancer cells; anti-Her2 antibodies against overexpressed Her2 on
breast cancer cells) Antibodies are biological molecules that
exhibit very specific binding capabilities for specific structures.
This is very important due to the complex nature of most biological
systems. An antibody is a complex biomolecule, made up of hundreds
of individual amino acids arranged in a highly ordered sequence.
For an immune response to be produced against a particular
molecule, a certain molecular size and complexity are necessary:
proteins with molecular weights greater then 5000 Da are generally
immunogenic. The way in which an antigen and its antigen-specific
antibody interact may be understood as analogous to a lock and key
fit, by which specific geometrical configurations of a unique key
enables it to open a lock. In the same way, an antigen-specific
antibody "fits" its unique antigen in a highly specific manner.
This unique property of antibodies is the key to their usefulness
in immunosensors where only the specific analyte of interest, the
antigen, fits into the antibody binding site.
[0451] DNA Probes.
[0452] The operation of gene probes is based on the hybridization
process. Hybridization involves the joining of a single strand of
nucleic acid with a complementary probe sequence. Hybridization of
a nucleic acid probe to DNA biotargets (e.g., gene sequences of a
mutation, etc) offers a very high degree of accuracy for
identifying DNA sequences complementary to that of the probe.
Nucleic acid strands tend to be paired to their complements in the
corresponding double-stranded structure. Therefore, a
single-stranded DNA molecule will seek out its complement in a
complex mixture of DNA containing large numbers of other nucleic
acid molecules. Hence, nucleic acid probe (i.e., gene probe)
detection methods are very specific to DNA sequences. Factors
affecting the hybridization or reassociation of two complementary
DNA strands include temperature, contact time, salt concentration,
and the degree of mismatch between the base pairs, and the length
and concentration of the target and probe sequences.
[0453] Biologically active DNA probes can be directly or indirectly
immobilized onto a drug system, such as the energy modulation agent
system (e.g., gold nanoparticle, a semiconductor, quantum dot, a
glass/quartz nanoparticles, etc.) surface to ensure optimal contact
and maximum binding. When immobilized onto gold nanoparticles, the
gene probes are stabilized and, therefore, can be reused
repetitively. Several methods can be used to bind DNA to different
supports. The method commonly used for binding DNA to glass
involves silanization of the glass surface followed by activation
with carbodiimide or glutaraldehyde. The silanization methods have
been used for binding to glass surfaces using 3
glycidoxypropyltrimethoxysilane (GOP) or
aminopropyltrimethoxysilane (APTS), followed by covalently linking
DNA via amino linkers incorporated either at the 3' or 5' end of
the molecule during DNA synthesis.
[0454] Enzyme Probes.
[0455] Enzymes are often chosen as bioreceptors based on their
specific binding capabilities as well as their catalytic activity.
In biocatalytic recognition mechanisms, the detection is amplified
by a reaction catalyzed by macromolecules called biocatalysts. With
the exception of a small group of catalytic ribonucleic acid
molecules, all enzymes are proteins. Some enzymes require no
chemical groups other than their amino acid residues for activity.
Others require an additional chemical component called a cofactor,
which may be either one or more inorganic ions, such as Fe.sup.2+,
Mg.sup.2+, Mn.sup.2+, or Zn.sup.2+, or a more complex organic or
metalloorganic molecule called a coenzyme. The catalytic activity
provided by enzymes allows for much lower limits of detection than
would be obtained with common binding techniques. The catalytic
activity of enzymes depends upon the integrity of their native
protein conformation. If an enzyme is denatured, dissociated into
its subunits, or broken down into its component amino acids, its
catalytic activity is destroyed. Enzyme-coupled receptors can also
be used to modify the recognition mechanisms.
PEGylated-Vectors for PEPST Probes
[0456] The synthesis of these particles was first reported by
Michael Faraday, who, in 1857, described the chemical process for
the production of nanosized particles of Au0 from gold chloride and
sodium citrate (Faraday 1857). Initial formulations of the vector,
manufactured by binding only TNF to the particles, were less toxic
than native TNF and effective in reducing tumor burden in a murine
model. Subsequent studies revealed that the safety of this vector
was primarily due to its rapid uptake and clearance in the RES.
This vector was reformulated to include molecules of
thiol-derivatized polyethylene glycol (PEG-THIOL) that were bound
with molecules of TNF on the gold nanoparticles surface. The new
vector, PT-cAu-TNF, avoids detection and clearance by the RES, and
actively and specifically sequesters TNF within a solid tumor. The
altered biodistribution correlated to improvements. In the present
invention, a preferred embodiment includes the use of PEGylated-Au
nanoparticles-PA drug systems to avoid detection and clearance by
the RES.
Immobilization of Biomolecules to Metal Nanoparticles
[0457] The immobilization of biomolecules (PA molecules, drugs,
proteins, enzymes, antibodies, DNA, etc.) to a solid support can
use a wide variety of methods published in the literature. Binding
can be performed through covalent bonds taking advantage of
reactive groups such as amine (--NH.sub.2) or sulfide (--SH) that
naturally are present or can be incorporated into the biomolecule
structure. Amines can react with carboxylic acid or ester moieties
in high yield to form stable amide bonds. Thiols can participate in
maleimide coupling, yielding stable dialkylsulfides.
[0458] A solid support of interest in the present invention is the
metal (preferably gold or silver) nanoparticles. The majority of
immobilization schemes involving metal surfaces, such as gold or
silver, utilize a prior derivatization of the surface with
alkylthiols, forming stable linkages. Alkylthiols readily form
self-assembled monolayers (SAM) onto silver surfaces in micromolar
concentrations. The terminus of the alkylthiol chain can be used to
bind biomolecules, or can be easily modified to do so. The length
of the alkylthiol chain has been found to be an important
parameter, keeping the biomolecules away from the surface, with
lengths of the alkyl group from 4 to 20 carbons being preferred.
For example, in the case for DNA hybridization this has been shown
to displace nonspecifically adsorbed HS--(CH2)6-ss-DNA and reorient
chemically attached HS--(CH2)6-ss-DNA in such a way to make the
majority of surface bound probes accessible for hybridization (M.
Culha, D. L. Stokes, an dT. Vo-Dinh, "Surface-Enhanced Raman
Scattering for Cancer Diagnostics: Detection of the BLC2 Gene,"
Expert Rev. Mol. Diagnostics, 3, 669-675 (2003)). Furthermore, to
avoid direct, non-specific DNA adsorption onto the surface,
alkylthiols have been used to block further access to the surface,
allowing only covalent immobilization through the linker [Steel, A.
B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-7;
Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119,
8916-20]
[0459] There are many methods related to the preparation of stable
oligonucleotide conjugates with gold particles by using
thiol-functionalized biomolecules that had previously been shown to
form strong gold-thiol bonds. Oligonucleotides with 5'-terminal
alkanethiol functional groups as anchors can be bound to the
surface of gold nanoparticles, and the resulting labels were robust
and stable to both high and low temperature conditions [R.
Elghanian, J. J. Storhoff R. C. Mucic, R. L. Letsinger and C. A.
Mirkin, Selective colorimetric detection of polynucleotides based
on the distance-dependent optical properties of gold nanoparticles.
Science 277 (1997), pp. 1078-1081]. A cyclic
dithiane-epiandrosterone disulfide linker has been developed for
binding oligonucleotides to gold surfaces [R. Elghanian, J. J.
Storhoff R. C. Mucic, R. L. Letsinger and C. A. Mirkin, Selective
colorimetric detection of polynucleotides based on the
distance-dependent optical properties of gold nanoparticles.
Science 277 (1997), pp. 1078-1081]. Li et al. have reported a
trithiol-capped oligonucleotide that can stabilize gold metal
nanoparticles having diameters .gtoreq.100 nm, while retaining
hybridization properties that are comparable to acyclic or
dithiol-oligonucleotide modified particles [Z. Li, R. C. Jin, C. A.
Mirkin and R. L. Letsinger, Multiple thiol-anchor capped DNA gold
nanoparticle conjugates. Nucleic Acids Res. 30 (2002), pp. 1558
1562].
[0460] In general silver nanoparticles cannot be effectively
passivated by alkylthiol-modified oligonucleotides using the
established experimental protocols that were developed for gold
particles. A method of generating core-shell particles comprising a
core of silver and a thin shell of gold has allowed silver
nanoparticles to be readily functionalized with
alkylthiol-oligonucleotides using the proven methods used to
prepare pure gold particle-oligonucleotide conjugates. [Y. W. Cao,
R. Jin and C. A. Mirkin, DNA-modified core-shell Ag/Au
nanoparticles. J. Am. Chem. Soc. 123 (2001), pp. 7961-7962].
[0461] To facilitate the use of biomolecule-conjugated
plasmonics-active nanoprobes (PAN) it is important that the
recognition region of the biomolecule is fully accessible to the
biotarget. Commonly a polynucleotide extension sequence is
incorporated to serve as a spacer between the PAN and the
oligonucleotide recognition region. To achieve high sensitivity and
selectivity in assays based on DNA hybridization it is important
that the PAN label colloidal solution is stable. Recently, Storhoff
et al. [11 Storhoff, R. Elghanian, C. A. Mirkin and R. L.
Letsinger, Sequence-dependent stability of DNA-modified gold
nanoparticles. Langmuir 18 (2002), pp. 6666-6670] have shown that
the base composition of the oligonucleotide has a significant
effect on colloid stability and on oligonucleotide surface
coverage. Otsuka et al. have used a heterobifunctional thiol-PEG
(polyethylene glycol) derivative as a linker to stabilize gold PRPs
[H. Otsuka, Y. Akiyama, Y. Nagasaki and K. Kataoka, Quantitative
and reversible lectin-induced association of gold nanoparticles
modified with .alpha.-lactosyl-.omega.-mercapto-poly(ethylene
glycol). J. Am. Chem. Soc. 123 (2001), pp. 8226-8230].
[0462] Proteins are usually bound to PANs using non-covalent,
passive absorption. Alternatively, a mercapto-undecanoic acid
linker/spacer molecule can be used to attach NeutrAvidin covalently
to gold and silver segmented nanorods [I. D. Walton, S. M. Norton,
A. Balasingham, L. He, D. F. Oviso, D. Gupta, P. A. Raju, M. J.
Natan and R. G. Freeman, Particles for multiplexed analysis in
solution: detection and identification of striped metallic
particles using optical microscopy. Anal. Chem. 74 (2002), pp.
2240-2247]. The thiol groups bind to the metal surface, and the
carboxyl functional groups on the particle surface are activated
using EDC and s-NHS reagents and then cross-linked to the amino
groups in NeutrAvidin. The ability to fabricate core-shell
particles where the core is metal and the shell is composed of
latex, silica, polystyrene or other non-metal material provides a
promising alternative approach to immobilizing biomolecules and
engineering particle surfaces [T. K. Mandal, M. S. Fleming and D.
R. Walt, Preparation of polymer coated gold nanoparticles by
surface-confined living radical polymerization at ambient
temperature. Nano Letters 2 (2002), pp. 3-7; S. O. Obare, N. R.
Jana and C. J. Murphy, Preparation of polystyrene-and silica-coated
gold nanorods and their use as templates for the synthesis of
hollow nanotubes. Nano Letters 1 (2001), pp. 601-603; C. Radloff
and N. J. Halas, Enhanced thermal stability of silica-encapsulated
metal nanoshells. Appl. Phys. Lett. 79 (2001), pp. 674-676; L.
Quaroni and G. Chumanov, Preparation of polymer-coated
functionalized silver nanoparticles. J. Am. Chem. Soc. 121 (1999),
pp. 10642-10643p; F. Caruso, Nanoengineering of particle surfaces.
Adv. Mater. 13 (2001), pp. 11-22].
[0463] Silver surfaces have been found to exhibit controlled
self-assembly kinetics when exposed to dilute ethanolic solutions
of alkylthiols. The tilt angle formed between the surface and the
hydrocarbon tail ranges from 0 to 15.degree.. There is also a
larger thiol packing density on silver, when compared to gold
[Burges, J. D.; Hawkridge, F. M. Langmuir 1997, 13, 3781-6]. After
self-assembled monolayer (SAM) formation on gold/silver
nanoparticles, alkylthiols can be covalently coupled to
biomolecules. The majority of synthetic techniques for the covalent
immobilization of biomolecules utilize free amine groups of a
polypeptide (enzymes, antibodies, antigens, etc) or of
amino-labeled DNA strands, to react with a carboxylic acid moiety
forming amide bonds. As a general rule, a more active intermediate
(labile ester) is first formed with the carboxylic acid moiety and
in a later stage reacted with the free amine, increasing the
coupling yield. Successful coupling procedures include, but are not
limited to:
[0464] Binding Procedure Using N-Hydroxysuccinimide (NHS) and its
Derivatives
[0465] The coupling approach involves the esterification under mild
conditions of a carboxylic acid with a labile group, an
N-hydroxysuccinimide (NHS) derivative, and further reaction with
free amine groups in a polypeptide (enzymes, antibodies, antigens,
etc) or amine-labeled DNA, producing a stable amide [Boncheva, M.;
Scheibler, L.; Lincoln, P.; Vogel, H.; Akerman, B. Langmuir 1999,
15, 4317-20]. NHS reacts almost exclusively with primary amine
groups. Covalent immobilization can be achieved in as little as 30
minutes. Since H.sub.2O competes with --NH.sub.2 in reactions
involving these very labile esters, it is important to consider the
hydrolysis kinetics of the available esters used in this type of
coupling. The derivative of NHS,
O--(N-succinimidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate,
increases the coupling yield by utilizing a leaving group that is
converted to urea during the carboxylic acid activation, hence
favorably increasing the negative enthalpy of the reaction.
[0466] Binding Procedure Using Maleimide
[0467] Maleimide can be used to immobilize biomolecules through
available --SH moieties. Coupling schemes with maleimide have been
proven useful for the site-specific immobilization of antibodies,
Fab fragments, peptides, and SH-modified DNA strands. Sample
preparation for the maleimide coupling of a protein involves the
simple reduction of disulfide bonds between two cysteine residues
with a mild reducing agent, such as dithiothreitol,
2-mercaptoethanol or tris(2-carboxyethyl)phosphine hydrochloride.
However, disulfide reduction will usually lead to the protein
losing its natural conformation, and might impair enzymatic
activity or antibody recognition. The modification of primary amine
groups with 2-iminothiolane hydrochloride (Traut's reagent) to
introduce sulfydryl groups is an alternative for biomolecules
lacking them. Free sulfhydryls are immobilized to the maleimide
surface by an addition reaction to unsaturated carbon-carbon bonds
[Jordan, C. E., et al., 1997].
[0468] Binding Procedure Using Carbodiimide.
[0469] Surfaces modified with mercaptoalkyldiols can be activated
with 1,1'-carbonyldiimidazole (CDI) to form a carbonylimidazole
intermediate. A biomolecule with an available amine group displaces
the imidazole to form a carbamate linkage to the alkylthiol
tethered to the surface [Potyrailo, R. A., et al., 1998].
[0470] Other Experimental Procedures to Conjudate Biomolecules to
Metal (e.g., Silver, Gold) Nanoparticles.
[0471] In one preferred embodiment, nanoparticles of metal colloid
hydrosols are prepared by rapidly mixing a solution of AgNO.sub.3
with ice-cold NaBH.sub.4. For developing a SMP probes, a DNA
segment is bound to a nanoparticle of silver or gold. The
immobilization of biomolecules (e.g., DNA, antibodies, enzymes,
etc.) to a solid support through covalent bonds usually takes
advantage of reactive groups such as amine (--NH.sub.2) or sulfide
(--SH) that naturally are present or can be incorporated into the
biomolecule structure. Amines can react with carboxylic acid or
ester moieties in high yield to form stable amide bonds. Thiols can
participate in maleimide coupling yielding stable
dialkylsulfides.
[0472] In one preferred embodiment, silver nanoparticles are used.
In one preferred embodiment, the immobilization schemes involving
Ag surfaces utilize a prior derivatization of the surface with
alkylthiols, forming stable linkages are used. Alkylthiols readily
form self-assembled monolayers (SAM) onto silver surfaces in
micromolar concentrations. The terminus of the alkylthiol chain can
be directly used to bind biomolecules, or can be easily modified to
do so. The length of the alkylthiol chain was found to be an
important parameter, keeping the biomolecules away from the
surface. Furthermore, to avoid direct, non-specific DNA adsorption
onto the surface, alkylthiols were used to block further access to
the surface, allowing only covalent immobilization through the
linker.
[0473] Silver/gold surfaces have been found to exhibit controlled
self-assembly kinetics when exposed to dilute ethanolic solutions
of alkylthiols. The tilt angle formed between the surface and the
hydrocarbon tail ranges from 0 to 15.degree.. There is also a
larger thiol packing density on silver, when compared to gold.
[0474] After SAM formation on silver nanoparticles, alkylthiols can
be covalently coupled to biomolecules. The majority of synthetic
techniques for the covalent immobilization of biomolecules utilize
free amine groups of a polypeptide (enzymes, antibodies, antigens,
etc) or of amino-labeled DNA strands, to react with a carboxylic
acid moiety forming amide bonds. In one embodiment, more active
intermediate (labile ester) is first formed with the carboxylic
acid moiety and in a later stage reacted with the free amine,
increasing the coupling yield. Successful coupling procedures
include:
[0475] The coupling approach used to bind DNA to a silver
nanoparticle involves the esterification under mild conditions of a
carboxylic acid with a labile group, an N-hydroxysuccinimide (NHS)
derivative, and further reaction with free amine groups in a
polypeptide (enzymes, antibodies, antigens, etc) or amine-labeled
DNA, producing a stable amide [4]. NHS reacts almost exclusively
with primary amine groups. Covalent immobilization can be achieved
in as little as 30 minutes. Since H.sub.2O competes with --NH.sub.2
in reactions involving these very labile esters, it is important to
consider the hydrolysis kinetics of the available esters used in
this type of coupling. The derivative of NHS used in FIG. 101,
O--(N-succinimidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate,
increases the coupling yield by utilizing a leaving group that is
converted to urea during the carboxylic acid activation, hence
favorably increasing the negative enthalpy of the reaction.
Spectral Range of Light Used for PEPST
[0476] A plasmonics enhanced effect can occur throughout the
electromagnetic region provided the suitable nanostructures,
nanoscale dimensions, metal types are used. Therefore, the PEPST
concept is valid for the entire electromagnetic spectrum, i.e,
energy, ranging from gamma rays and X rays throughout ultraviolet,
visible, infrared, microwave and radio frequency energy. However,
for practical reasons, visible and NIR light are used for silver
and gold nanoparticles, since the plasmon resonances for silver and
gold occur in the visible and NIR region, respectively. Especially
for gold nanoparticles, the NIR region is very appropriate for
non-invasive therapy.
Photon Excitation in the Therapeutic Window of Tissue
[0477] There are several methods using light to excite
photoactivate compounds non-invasively. We can use light having
wavelengths within the so-called "therapeutic window" (700-1300
nm). The ability of light to penetrate tissues depends on
absorption. Within the spectral range known as the therapeutic
window (or diagnostic window), most tissues are sufficiently weak
absorbers to permit significant penetration of light. This window
extends from 600 to 1300 nm, from the orange/red region of the
visible spectrum into the NIR. At the short-wavelength end, the
window is bound by the absorption of hemoglobin, in both its
oxygenated and deoxygenated forms. The absorption of oxygenated
hemoglobin increases approximately two orders of magnitude as the
wavelength shortens in the region around 600 nm. At shorter
wavelengths many more absorbing biomolecules become important,
including DNA and the amino acids tryptophan and tyrosine. At the
infrared (IR) end of the window, penetration is limited by the
absorption properties of water. Within the therapeutic window,
scattering is dominant over absorption, and so the propagating
light becomes diffuse, although not necessarily entering into the
diffusion limit. FIG. 13 shows a diagram of the therapeutic window
of tissue. The following section discusses the use of one-photon
and multi-photon techniques for therapy.
Light Excitation Methods: Single-Photon and Multi-Photon
Excitation
[0478] Two methods can be used, one-photon or multi-photon
excitation. If the two-photon technique is used, one can excite the
PA molecules with light at 700-1000 nm, which can penetrate deep
inside tissue, in order to excite molecules that absorb in the
350-500 nm spectral region. This approach can excite the psoralen
compounds, which absorb in the 290-350 nm spectral region and emit
in the visible. With the one-photon method, the photo-activator
(PA) drug molecules can directly absorb excitation light at
600-1300 nm. In this case we can design a psoralen-related system
(e.g., psoralens having additional aromatic rings or other
conjugation to alter the ability to absorb at different
wavelengths) or use other PA systems: photodynamic therapy drugs,
ALA, etc.
PEPST Modality for Photopheresis Using X Ray Excitation
Need for X-Ray Excitation
[0479] Photopheresis has been demonstrated to be an effective
treatment for a number of diseases. However, there is a strong need
to develop non-invasive modalities where the excitation light can
directly irradiate the photoactive compounds without the need for
removal and reinfusion of blood from patients. One method for an
improved and practical modality for such therapy was described in
U.S. Ser. No. 11/935,655, filed Nov. 6, 2007, the entire contents
of which are hereby incorporated by reference.
[0480] Although X-ray can excite compounds in deep tissue
non-invasively, X-ray is not easily absorbed by organic drug
compounds. The present invention provides a solution to that
problem, by the providing of a molecular system that can absorb the
X-ray energy and change that energy into other energies that can be
used to activate drug molecules. More specifically, the molecular
system that can absorb and change the X-ray energy in the present
invention is the PEPST probes comprising nanoparticles.
[0481] In this embodiment, the present invention uses X-rays for
excitation. The advantage is the ability to excite molecules
non-invasively since X-ray can penetrate deep in tissue. However,
the limitation is the fact that X-ray does not interact with most
molecules. In one embodiment of the present invention, the drug
molecule (or PA) is bound to a molecular entity, referred to as an
"energy modulation agent" that can interact with the X-rays, and
then emit light that can be absorbed by the PA drug molecules.
(FIG. 14)
PEPST Probes for X Ray Excitation
[0482] In the previous sections, the advantage of gold
nanoparticles as plasmonics-active systems have been discussed.
Furthermore, gold nanoparticles are also good energy modulation
agent systems since they are biocompatible and have been shown to
be a possible candidate for contrast agents for X-ray [Hainfeld et
al, The British Journal of radiology, 79, 248, 2006]. The concept
of using high-Z materials for dose enhancement in cancer
radiotherapy was advanced over 20 years ago. The use of gold
nanoparticles as a dose enhancer seems more promising than the
earlier attempts using microspheres and other materials for two
primary reasons. First, gold has a higher Z number than iodine (I,
Z=53) or gadolinium (Gd, Z=64), while showing little toxicity, up
to at least 3% by weight, on either the rodent or human tumor
cells. The gold nanoparticles were non-toxic to mice and were
largely cleared from the body through the kidneys. This novel use
of small gold nanoparticles permitted achievement of the high metal
content in tumors necessary for significant high-Z radioenhancement
[James F Hainfeld, Daniel N Slatkin and Henry M Smilowitz, The use
of gold nanoparticles to enhance radiotherapy in mice, Phys. Med.
Biol. 49 (2004)]
[0483] Delivering a lethal dose of radiation to a tumor while
minimizing radiation exposure of nearby normal tissues remains the
greatest challenge in radiation therapy. The dose delivered to a
tumor during photon-based radiation therapy can be enhanced by
loading high atomic number (Z) materials such as gold (Au, Z=79)
into the tumor, resulting in greater photoelectric absorption
within the tumor than in surrounding tissues. Thus, gold clearly
leads to a higher tumor dose than either iodine or gadolinium.
Second, nanoparticles provide a better mechanism than microspheres,
in terms of delivering high-Z materials to the tumor, overcoming
some of the difficulties found during an earlier attempt using gold
microspheres [Sang Hyun Cho, Estimation of tumor dose enhancement
due to gold nanoparticles during typical radiation treatments: a
preliminary Monte Carlo study, Phys. Med. Biol. 50 (2005)]
[0484] Gold (or metal) complexes with PA ligands: Gold (or metal)
complexes with PA can preferably be used in the present invention.
The metal can be used as an energy modulation agent system. For
example, gold complexes with psoralen-related ligands can be used
as a hybrid energy modulation agent-PA system. The gold molecules
serve as the energy modulation agent system and the ligand
molecules serve as the PA drug system. Previous studies indicated
that gold(I) complexes with diphosphine and bipyridine ligands
exhibit X-ray excited luminescence [Ref 3: Kim et al, Inorg. Chem.,
46, 949, 2007].
[0485] FIG. 15 shows a number of the various embodiments of PEPST
probes that can be preferably used for X ray excitation of energy
modulation agent-PA system. These probes comprise: [0486] (A) PA
molecules bound to energy modulation agent and to plasmonic metal
nanoparticle; [0487] (B) Plasmonic metal nanoparticle with energy
modulation agent nanocap covered with PA molecules; [0488] (C)
PA-covered nanoparticle with plasmonic metal nanoparticles; [0489]
(D) Energy modulation agent-containing nanoparticle covered with PA
molecules and plasmonic metal nanocap; [0490] (E) Plasmonic metal
nanoparticle core with energy modulation agent nanoshell covered
with PA molecule; and [0491] (F) PA molecule bound to energy
modulation agent (attached to plasmonics metal nanoparticle)
nanoparticle by detachable biochemical bond.
Examples of PEPST System Based on Energy Modulation Agent-PA
[0492] For purposes of simplification, the following discussion is
centered on gold as the metal material and CdS as the energy
modulation agent material (which can also be used as DNA stabilized
CdS, see Ma et al, Langmuir, 23 (26), 12783-12787 (2007)) and
psoralen as the PA molecule. However, it is to be understood that
many other embodiments of metal material, energy modulation agent
and PA molecule are possible within the bounds of the present
invention, and the following discussion is for exemplary purposes
only. Suitable metals that can be used in plasmon resonating shells
or other plasmon resonating structures can be include, but are not
limited to, gold, silver, platinum, palladium, nickel, ruthenium,
rhenium, copper, and cobalt.
[0493] In the embodiment of FIG. 15A, the PEPST system comprises
gold nanoparticles, an energy modulation agent nanoparticle (e.g.,
CdS) linked to a PA drug molecule (e.g., psoralen). X ray is
irradiated to CdS, which absorbs X rays [Hua et al, Rev. Sci.
Instrum., 73, 1379, 2002] and emits CdS XEOL light (at 350-400 nm)
that is plasmonics-enhanced by the gold nanoparticle. This enhanced
XEOL light is used to photoactivate psoralen (PA molecule). In this
case the nanostructure of the gold nanoparticle is designed to
enhance the XEOL light at 350-400 nm.
[0494] In the embodiment of FIG. 15B, the PEPST system comprises a
plasmonics-active metal (gold) nanoparticle with energy modulation
agent nanocap (CdS) covered with PA molecules (e.g., psoralen). X
ray is irradiated to CdS, which absorbs X ray and emits XEOL light
that is plasmonics-enhanced by the gold nanoparticle. This enhanced
XEOL light is used to photoactivate psoralen (PA molecule).
[0495] In the embodiment of FIG. 15C, the PEPST system comprises a
PA (e.g., psoralen)-covered CdS nanoparticle with smaller plasmonic
metal (gold) nanoparticles. X ray is irradiated to CdS, which
absorbs X ray and emits XEOL light that is plasmonics-enhanced by
the gold nanoparticle. This enhanced XEOL light is used to
photoactivate psoralen (PA molecule).
[0496] In the embodiment of FIG. 15D, the energy modulation agent
core comprises CdS or CsCl nanoparticles covered with a nanocap of
gold. X ray is irradiated to CdS or CsCl, which absorbs X ray
[[Jaegle et al, J. Appl. Phys., 81, 2406, 1997] and emits XEOL
light that is plasmonics-enhanced by the gold nanocap structure.
This enhanced XEOL light is used to photoactivate psoralen (PA
molecule).
[0497] Similarly, the embodiment in FIG. 15E comprises a spherical
gold core covered by a shell of CdS or CsCl. X ray is irradiated to
CdS or CsCl material, which absorbs X ray [Jaegle et al, J. Appl.
Phys., 81, 2406, 1997] and emits XEOL light that is
plasmonics-enhanced by the gold nanosphere. This enhanced XEOL
light is used to photoactivate psoralen (PA molecule).
[0498] In the embodiment of FIG. 15F, the PEPST system comprises
gold nanoparticles, and an energy modulation agent nanoparticle
(e.g., CdS) linked to a PA drug molecule (e.g., psoralen) by a link
that can be detached by radiation. X ray is irradiated to CdS,
which absorbs X ray and emits CdS XEOL light (at 350-400 nm) that
is plasmonics-enhanced by the gold nanoparticle. This enhanced XEOL
light is used to photoactivate psoralen (PA molecule). In this case
the nanostructure of the gold nanoparticle is designed to enhance
the XEOL light at 350-400 nm.
[0499] In alternative embodiments, the metal nanoparticles or
single nanoshells are replaced by multi layers of nanoshells [Kun
Chen, Yang Liu, Guillermo Ameer, Vadim Backman, Optimal design of
structured nanospheres for ultrasharp light-scattering resonances
as molecular imaging multilabels, Journal of Biomedical Optics,
10(2), 024005 (March/April 2005)].
[0500] In other alternative embodiments the metal nanoparticles are
covered with a layer (1-30 nm) of dielectric material (e.g.
silica). The dielectric layer (or nanoshell) is designed to prevent
quenching of the luminescence light emitted by the energy
modulation agent (also referred to as EEC) molecule(s) due to
direct contact of the metal with the energy modulation agent
molecules. In yet other alternative embodiments, the energy
modulation agent molecules or materials are bound to (or in
proximity of) a metal nanoparticle via a spacer (linker). The
spacer is designed to prevent quenching of the luminescence light
emitted by the energy modulation agent molecules or materials.
Other Useable Materials
[0501] The energy modulation agent materials can include any
materials that can absorb X ray and emit light in order to excite
the PA molecule. The energy modulation agent materials include, but
are not limited to:
[0502] metals (gold, silver, etc);
[0503] quantum dots;
[0504] semiconductor materials;
[0505] scintillation and phosphor materials;
[0506] materials that exhibit X-ray excited luminescence
(XEOL);
[0507] organic solids, metal complexes, inorganic solids, crystals,
rare earth materials (lanthanides), polymers, scintillators,
phosphor materials, etc.; and
[0508] materials that exhibit excitonic properties.
[0509] Quantum dots, semiconductor nanostructures. Various
materials related to quantum dots, semiconductor materials, etc.
can be used as energy modulation agent systems. For example
CdS-related nanostructures have been shown to exhibit X-ray excited
luminescence in the UV-visible region [Hua et al, Rev. Sci.
Instrum., 73, 1379, 2002].
[0510] Scintillator Materials as energy modulation agent systems.
Various scintillator materials can be used as energy modulation
agents since they absorb X-ray and emit luminescence emission,
which can be used to excite the PA system. For example, single
crystals of molybdates can be excited by X-ray and emit
luminescence around 400 nm [Mirkhin et al, Nuclear Instrum. Meth.
In Physics Res. A, 486, 295 (2002].
[0511] Solid Materials as energy modulation agent systems: Various
solid materials can be used as energy modulation agents due to
their X-ray excited luminescence properties. For example CdS (or
CsCl) exhibit luminescence when excited by soft X-ray [Jaegle et
al, J. Appl. Phys., 81, 2406, 1997].
[0512] XEOL materials: lanthanides or rare earth materials [L.
Soderholm, G. K Liu, Mark R. Antonioc, F. W. Lytle, X-ray excited
optical luminescence .XEOL. detection of x-ray absorption fine
structure .XAFZ, J. Chem. Phys, 109, 6745, 19981 Masashi Ishiia,
Yoshihito Tanaka and Tetsuya Ishikawa, Shuji Komuro and Takitaro
Morikawa, Yoshinobu Aoyagi, Site-selective x-ray absorption fine
structure analysis of an optically active center in Er-doped
semiconductor thin film using x-ray-excited optical luminescence,
Appl. Phys. Lett, 78, 183, 2001]
[0513] Some examples of metal complexes exhibiting XEOL which can
be used as energy modulation agent systems are shown in FIGS. 16
and 17. Such structures can be modified by replacing the metal atom
with metal nanoparticles in order to fabricate a plasmonics-enhance
PEPST probe. In the present invention, the experimental parameters
including size, shape and metal type of the nano structure can be
selected based upon the excitation radiation (NIR or X ray
excitation), the photoactivation radiation (UVB), and/or the
emission process from the energy modulation agent system (visible
NIR).
[0514] U.S. Pat. No. 7,008,559 (the entire contents of which are
incorporated herein by reference) describes the upconversion
performance of ZnS where excitation at 767 nm produces emission in
the visible range. The materials described in U.S. Pat. No.
7,008,559 (including the ZnS as well as Er.sup.3+ doped BaTiO.sub.3
nanoparticles and Yb.sup.3+ doped CsMnCl.sub.3) are suitable in
various embodiments of the invention.
[0515] Further materials suitable as energy modulation agents
include, but are not limited to, CdTe, CdSe, ZnO, CdS,
Y.sub.2O.sub.3, MgS, CaS, SrS and BaS. Such materials may be any
semiconductor and more specifically, but not by way of limitation,
sulfide, telluride, selenide, and oxide semiconductors and their
nanoparticles, such as Zn.sub.1-xMn.sub.x S.sub.y,
Zn.sub.1-xMn.sub.xSe.sub.y, Zn.sub.1-xMn.sub.xTe.sub.y,
Cd.sub.1-xMnS.sub.y, Cd.sub.1-xMn.sub.xSe.sub.y,
Cd.sub.1-xMn.sub.xTe.sub.y, Pb.sub.1-xMn.sub.xS.sub.y,
Pb.sub.1-xMn.sub.xSe.sub.y, Pb.sub.1-xMn.sub.xTe.sub.y,
Mg.sub.1-xMnS.sub.y, Ca.sub.1-xMn.sub.xS.sub.y,
Ba.sub.1-xMn.sub.xS.sub.y and Sr.sub.1-x, etc. (wherein,
0<x.ltoreq.1, and 0<y.ltoreq.1). Complex compounds of the
above-described semiconductors are also contemplated for use in the
invention--e.g. (M.sub.1-zN.sub.z).sub.1-xMn.sub.xA.sub.1-yB.sub.y
(M=Zn, Cd, Pb, Ca, Ba, Sr, Mg; N=Zn, Cd, Pb, Ca, Ba, Sr, Mg; A=S,
Se, Te, O; B.dbd.S, Se, Te, 0; 0<x.ltoreq.1, 0<y.ltoreq.1,
0<z.ltoreq.1). Two examples of such complex compounds are
Zn.sub.0.4Cd.sub.0.4Mn.sub.0.2S and
Zn.sub.0.9Mn.sub.0.1S.sub.0.8Se.sub.0.2. Additional energy
modulation materials include insulating and nonconducting materials
such as BaF.sub.2, BaFBr, and BaTiO.sub.3, to name but a few
exemplary compounds. Transition and rare earth ion co-doped
semiconductors suitable for the invention include sulfide,
telluride, selenide and oxide semiconductors and their
nanoparticles, such as ZnS; Mn; Er; ZnSe; Mn, Er; MgS; Mn, Er; CaS;
Mn, Er; ZnS; Mn, Yb; ZnSe; Mn, Yb; MgS; Mn, Yb; CaS; Mn, Yb etc.,
and their complex compounds:
(M.sub.1-zN.sub.z).sub.1-x(Mn.sub.qR.sub.1-q).sub.xA.sub.1-yB.sub.y
(M=Zn, Cd, Pb, Ca, Ba, Sr, Mg; N=Zn, Cd, Pb, Ca, Ba, Sr, Mg; A=S,
Se, Te, O; B=S, . . . 0<z<1, o<q<1).
[0516] Some nanoparticles such as ZnS:Tb.sup.3+, Er.sup.3+;
ZnS:Tb.sup.3; Y.sub.2O.sub.3:Tb.sup.3+; Y.sub.2O.sub.3:Tb.sup.3+,
Er3.sup.-'; ZnS:Mn.sup.2+; ZnS:Mn,Er.sup.3+ are known in the art to
function for both down-conversion luminescence and upconversion
luminescence, and can thus be used in various embodiments of the
present invention.
Principle of Plasmonics-Enhancement Effect of the PEPST Probe Using
X-Ray Excitation
[0517] One embodiment of the basic PEPST probe embodiment comprises
PA molecules bound to an energy modulation agent and to plasmonic
metal (gold) nanoparticles. First the metal nanoparticle can serve
as a drug delivery platform (see previous discussion). Secondly,
the metal nanoparticle can play 2 roles: [0518] (1) Enhancement of
the X-ray electromagnetic field [0519] (2) Enhancement of the
emission signal of the energy modulation agent system.
[0520] The X ray radiation, used to excite the energy modulation
agent system, is amplified by the metal nanoparticle due to plasmon
resonance. As a result the energy modulation agent system exhibits
more emission light that is used to photoactivate the PA drug
molecules (e.g., psoralens) and make them photoactive. In this case
the metal nanoparticles are designed to exhibit strong plasmon
resonance at or near the X ray wavelengths. The surface plasmon
resonance effect amplifies the excitation light at the
nanoparticles, resulting in increased photoactivation of the PA
drug molecules and improved therapy efficiency. The
plasmonics-enhanced mechanism can also be used with the other PEPST
probes described above.
[0521] FIG. 18 illustrates the plasmonics-enhancement effect of the
PEPST probe.
[0522] X-ray used in medical diagnostic imaging has photon energies
from approximately 10 to 150 keV, which is equivalent to
wavelengths range from 1.2 to 0.0083 Angstroms. [2
(Angstrom)=12.4/E (keV)]. Soft X ray can go to 10 nm. The dimension
of plasmonics-active nanoparticles usually have dimensions on the
order or less than the wavelengths of the radiation used. Note that
the approximate atomic radius of gold is approximately 0.15
nanometers. At the limit, for gold the smallest "nanoparticle" size
is 0.14 nm (only 1 gold atom). A nanoparticle with size in the
hundreds of nm will have approximately 10.sup.6-10.sup.7 gold
atoms. Therefore, the range of gold nanoparticles discussed in this
invention can range from 1-10.sup.7 gold atoms.
[0523] The gold nanoparticles can also enhance the energy
modulation agent emission signal, which is use to excite the PA
molecule. For psoralens, this spectral range is in the UVB region
(320-400 nm). Silver or gold nanoparticles, nanoshell and nanocaps
have been fabricated to exhibit strong plasmon resonance in this
region. FIG. 19 shows excitation and emission fluorescence spectra
of a psoralen compound (8-methoxypsoralen).
PEPST Energy Modulation Agent-PA Probe with Detachable PA.
[0524] Some photoactive drugs require that the PA molecule to enter
the nucleus. FIG. 20 shows an embodiment of a PEPST probe where the
PA drug molecule is bound to the metal nanoparticles via a linker
(FIG. 20A) that can be cut by photon radiation (FIG. 20B). Such a
probe is useful for therapy modalities where the PA molecules have
to enter the nucleus, e.g., psoralen molecules need to enter the
nucleus of cells and intercalate onto DNA (FIG. 20C). Since it is
more difficult for metal nanoparticles to enter the cell nucleus
than for smaller molecules, it is preferable to use PEPST probes
that have releasable PA molecules.
[0525] Suitable linkers for linking the PA drug molecule to the
metal nanoparticles include, but are not limited to, labile
chemical bonds that can be broken by remote energy excitation (from
outside the body, e.g., MW, IR, photoacoustic energy, ultrasound
energy, etc.), labile chemical bonds that can be broken by the
chemical environment inside cells, antibody-antigen, nucleic acid
linkers, biotin-streptavidin, etc.
Nanoparticle Chain for Dual Plasmonics Effect
[0526] As discussed previously, there is the need to develop
nanoparticle systems that can have dual (or multi) plasmonics
resonance modes. FIG. 21 illustrates an embodiment of the present
invention PEPST probe having a chain of metal particles having
different sizes and coupled to each other, which could exhibit such
dual plasmonics-based enhancement. For example the parameters
(size, metal type, structure, etc) of the larger nanoparticle (FIG.
21, left) can be tuned to NIR, VIS or UV light while the smaller
particle (FIG. 21, right) can be tuned to X ray. There is also a
coupling effect between these particles.
[0527] These nanoparticle chains are useful in providing plasmonics
enhancement of both the incident radiation used (for example, x-ray
activation of CdS) as well as plasmonics enhancement of the emitted
radiation that will then activate the PA. Similar nanoparticles
systems have been used as nanolens [Self-Similar Chain of Metal
Nanospheres as an Efficient Nanolens, Kuiru Li, Mark I. Stockman,
and David J. Bergman, Physical Review Letter, VOLUME 91, NUMBER 22,
227402-1, 2003].
Drug Delivery Platforms
[0528] Liposome Delivery of Energy Modulation Agent-PA Systems
[0529] The field of particle-based drug delivery is currently
focused on two chemically distinct colloidal particles, liposomes
and biodegradable polymers. Both delivery systems encapsulate the
active drug. The drug is released from the particle as it lyses, in
the case of lipsomes, or disintegrates, as described for
biodegradable polymers. One embodiment of the present invention
uses liposomal delivery of energy modulation agent-PA systems
(e.g., gold nanoshells) for therapy. An exemplary embodiment is
described below, but is not intended to be limiting to the specific
lipids, nanoparticles or other components recited, but is merely
for exemplary purposes:
[0530] Preparation of Liposomes.
[0531] The liposome preparation method is adapted from Holig et. al
Holig, P., Bach, M., Volkel, T., Nande, T., Hoffmann, S., Muller,
R., and Kontermann, R. E., Novel RGD lipopeptides for the targeting
of liposomes to integrin-expressing endothelial and melanoma cells.
Protein Engineering Design and Selection, 2004. 17(5): p. 433-441].
Briefly, the lipids PEG-DPPE, PC, and Rh-DPPE are mixed in
chloroform in a round bottom flask and evaporated (Hieroglyph
Rotary Evaporator, Rose Scientific Ltd., Edmonton, Alberta, Canada)
to eliminate chloroform. The dry film is dehydrated into aqueous
phase with using PBS solution. A dry lipid film is prepared by
rotary evaporation from a mixture of PC, cholesterol, and PEG-DPPE
and then hydrated into aqueous phase using PBS. The mixture is
vigorously mixed by overtaxing and bath solicited (Instrument,
Company) and the suspension extruded through polycarbonate filter
using Liposofast apparatus (Avestin Inc., Ottawa, ON, Canada)
(pore-size 0.8 .mu.m). Preparation of liposomes is performed as
follows; 0.1 mmol of PC is dispersed in 8 ml of chloroform and
supplemented with 0.5 mol of PEG-DPPE in 20 ml of chloroform. 0.3
mmol rhodamine-labeled phosphatidylethanolamine (Rh-DPPE) is then
incorporated into the liposomes. The organic solvents are then
removed by rotary evaporation at 35.degree. C. for 2 h leaving a
dry lipid film. Gold nanoshells are encapsulated into liposomes by
adding them to the PBS hydration buffer and successively into the
dry lipid film. This mixture is emulsified in a temperature
controlled sonicator for 30 minutes at 35.degree. C. followed by
vortexing for 5 min. Encapsulated gold nanoshells are separated
from unencapsulated gold nanoshells by gentle centrifugation for 5
minutes at 2400 r.p.m (1200 g). The resulting multilamellar
vesicles suspension is extruded through polycarbonate filter using
Liposofast apparatus (Avestin Inc., Ottawa, ON, Canada) (pore-size
0.8 .mu.m). The aqueous mixture is obtained and stored at 4.degree.
C.
[0532] Fabrication of Gold Nanoparticles:
[0533] The Frens method [Frens, G., Controlled nucleation for the
regulation of the particle size in monodisperse gold solutions.
Nature (London) Phys Sci, 1973. 241: p. 20-22] can be used in the
present invention to synthesize a solution of gold nanoparticles
ranging in diameter from 8-10 nm. Briefly, 5.0.times.10.sup.-6 mol
of HAuCl.sub.4 is dissolved in 19 ml of deionized water producing a
faint yellowish solution. This solution is heated with vigorous
stirring in a rotary evaporator for 45 minutes. 1 ml of 0.5% sodium
citrate solution is added and the solution is stirred for an
additional 30 minutes. The color of the solution gradually changed
from the initial faint yellowish to clear, grey, purple and finally
a tantalizing wine-red color similar to merlot. The sodium citrate
used serves in a dual capacity, first acting as a reducing agent,
and second, producing negative citrate ions that are adsorbed onto
the gold nanoparticles introducing surface charge that repels the
particles and preventing nanocluster formation.
[0534] Preparation and Internalization of Liposome-Encapsulated
Gold Nanoshells:
[0535] Liposome-encapsulated gold nanoshells are incubated with
MCF-7 cells grown on partitioned cover-slips for intracellular
delivery. This is done by adding 10 .mu.l of liposome-encapsulated
gold nanoshells per 1 ml of cell culture medium. This is incubated
for 30 minutes in a humidified (86% RH) incubator at 37.degree. C.
and 5% CO.sub.2. This cell is used for localization studies; to
track the rhodamine-DPPE-labeled liposomes into the cytoplasm of
the MCF-7 cell. After incubation, the cells grown on cover-slips
are washed three times in cold PBS and fixed using 3.7%
formaldehyde in PBS. Rhodamine staining by rhodamine-DPPE-labeled
liposomes is analyzed using a Nikon Diaphot 300 inverted microscope
(Nikon, Inc., Melville, N.Y.).
Non-Invasive Cleavage of the Drug System In Vivo
[0536] After delivery of the drug system into the cell, there is
sometimes the need to have the PA system (e.g. psoralen) in the
nucleus in order to interact with DNA. If the PA is still linked to
the energy modulation agent, both of them have to be transported
into the nucleus. In the case with gold nanoparticles as the energy
modulation agent system, there are several methods to incubate
cells in vitro. For in vivo applications, one can link the PA to
the gold nanoparticles using a chemical linkage that can be
released (or cut) using non-invasive methods such as infrared,
microwave, or ultrasound waves. An example of linkage is through a
chemical bond or through a bioreceptor, such as an antibody. In
this case, the PA is the antigen molecule bound to the energy
modulation agent system that has an antibody targeted to the
PA.
[0537] When the energy modulation agent-Ab-PA enters the cell, the
PA molecules can be released from the energy modulation agent Ab
system. To release the PA molecule from the antibody, chemical
reagents can be used to cleave the binding between antibody and
antigen, thus regenerating the biosensor [Vo-Dinh et al, 1988].
This chemical procedure is simple but is not practical inside a
cell due to possible denaturation of the cell by the chemical. In
previous studies, it has been demonstrated that the gentle but
effective MHz-range ultrasound has the capability to release
antigen molecules from the antibody-energy modulation agent system
[Moreno-Bondi, M., Mobley, J., and Vo-Dinh, T., "Regenerable
Antibody-based Biosensor for Breast Cancer," J. Biomedical Optics,
5, 350-354 (2000)]. Thus, an alternative embodiment is to use
gentle ultrasonic radiation (non-invasively) to remove the PA
(antigen) from the antibody at the energy modulation agent
system.
[0538] In a preferred embodiment, the PA molecule is bound to the
energy modulation agent by a chemically labile bond [Jon A. Wolff,
and David B. Rozema, Breaking the Bonds: Non-viral Vectors Become
Chemically Dynamic, Molecular Therapy (2007) 16(1), 8-15]. A
promising method of improving the efficacy of this approach is to
create synthetic vehicles (SVs) that are chemically dynamic, so
that delivery is enabled by the cleavage of chemical bonds upon
exposure to various physiological environments or external stimuli.
An example of this approach is the use of masked endosomolytic
agents (MEAs) that improve the release of nucleic acids from
endosomes, a key step during transport. When the MEA enters the
acidic environment of the endosome, a pH-labile bond is broken,
releasing the agent's endosomolytic capability.
Use of Ferritin and Apoferritin as Targeted Drug Delivery
[0539] Another embodiment to deliver the energy modulation agent-PA
drugs involves the use of ferritin and apoferritin compounds. There
is increasing interest in ligand-receptor-mediated delivery systems
due to their non-immunogenic and site-specific targeting potential
to the ligand-specific bio-sites. Platinum anticancer drug have
been encapsulated in apoferritin [Zhen Yang, Xiaoyong Wang, Huajia
Diao, Junfeng Zhang, Hongyan Li, Hongzhe Sun and Zijian Guo,
Encapsulation of platinum anticancer drugs by apoferritin, Chem.
Commun. 33, 2007, 3453-3455]. Ferritin, the principal iron storage
molecule in a wide variety of organisms, can also be used as a
vehicle for targeted drug delivery. It contains a hollow protein
shell, apoferritin, which can contain up to its own weight of
hydrous ferric oxide-phosphate as a microcrystalline micelle. The
24 subunits of ferritin assemble automatically to form a hollow
protein cage with internal and external diameters of 8 and 12 nm,
respectively. Eight hydrophilic channels of about 0.4 nm, formed at
the intersections of subunits, penetrate the protein shell and lead
to the protein cavity. A variety of species such as gadolinium
(Gd.sup.3+) contrast agents, desferrioxamine B, metal ions, and
nanoparticles of iron salts can be accommodated in the cage of
apoferritin. Various metals such as iron, nickel, chromium and
other materials have been incorporated into apoferritin [Iron
incorporation into apoferritin. The role of apoferritin as a
ferroxidase, The Journal of Biological Chemistry [0021-9258] Bakker
yr: 1986 vol: 261 iss: 28 pg: 13182-5; Mitsuhiro Okuda.sup.1, Kenji
Iwahori.sup.2, Ichiro Yamashita.sup.2, Hideyuki Yoshimura.sup.1,
Fabrication of nickel and chromium nanoparticles using the protein
cage of apoferritin, Biotechnology Bioengineering, Volume 84, Issue
2, Pages 187-194]. Zinc selenide nanoparticles (ZnSe NPs) were
synthesized in the cavity of the cage-shaped protein apoferritin by
designing a slow chemical reaction system, which employs
tetraaminezinc ion and selenourea. The chemical synthesis of ZnSe
NPs was realized in a spatially selective manner from an aqueous
solution, and ZnSe cores were formed in almost all apoferritin
cavities with little bulk precipitation [Kenji Iwahori, Keiko
Yoshizawa, Masahiro Muraoka, and Ichiro Yamashita, Fabrication of
ZnSe Nanoparticles in the Apoferritin Cavity by Designing a Slow
Chemical Reaction System, Inorg. Chem., 44 (18), 6393-6400,
2005].
[0540] A simple method for synthesizing gold nanoparticles
stabilized by horse spleen apoferritin (HSAF) is reported using
NaBH.sub.4 or 3-(N-morpholino)propanesulfonic acid (MOPS) as the
reducing agent [Lei Zhang, Joe Swift, Christopher A. Butts, Vijay
Yerubandi and Ivan J. Dmochowski, Structure and activity of
apoferritin-stabilized gold nanoparticles, Journal of Inorganic
Biochemistry, Vol. 101, 1719-1729, 2007]. Gold sulfite (Au.sub.2S)
nanoparticles were prepared in the cavity of the cage-shaped
protein, apoferritin. Apoferritin has a cavity, 7 nm in diameter,
and the diameter of fabricated Au.sub.2S nanoparticles is about the
same size with the cavity and size dispersion was small. [Keiko
Yoshizawa, Kenji Iwahori, Kenji Sugimoto and Ichiro Yamashita,
Fabrication of Gold Sulfide Nanoparticles Using the Protein Cage of
Apoferritin, Chemistry Letters, Vol. 35 (2006), No. 10 p. 1192].
Thus, in a preferred embodiment, the PA or energy modulation
agent-PA compounds are encapsulated inside the apoferrtin
shells.
Use of Ferritin and Apoferritin as Enhanced Targeting Agents
[0541] It was reported that ferritin could be internalized by some
tumor tissues, and the internalization was associated with the
membrane-specific receptors [S. Fargion, P. Arosio, A. L.
Fracanzoni, V. Cislaghi, S. Levi, A. Cozzi, A Piperno and A. G.
Firelli, Blood, 1988, 71, 753-757; P. C. Adams, L. W. Powell and J.
W. Halliday, Hepatology, 1988, 8, 719-721]. Previous studies have
shown that ferritin-binding sites and the endocytosis of ferritin
have been identified in neoplastic cells [M. S. Bretscher and J. N.
Thomson, EMBO J., 1983, 2, 599-603]. Ferritin receptors have the
potential for use in the delivery of anticancer drugs into the
brain [S. W. Hulet, S. Powers and J. R. Connor, J. Neurol. Sci.,
1999, 165, 48-55]. In one embodiment, the present invention uses
ferritin or apoferritin to both encapsulate PA and energy
modulation agent-PA systems and also target tumor cells selectively
for enhanced drug delivery and subsequent phototherapy. In this
case no additional bioreactors are needed.
[0542] FIG. 22 schematically illustrates the use of encapsulated
photoactive agents (FIG. 22A) for delivery into tissue and
subsequent release of the photoactive drugs after the encapsulated
systems enter the cell. Note the encapsulated system can have a
bioreceptor for selective tumor targeting (FIG. 22B). Once inside
the cell, the capsule shell (e.g., liposomes, apoferritin, etc.)
can be broken (FIG. 22C) using non-invasive excitation (e.g.,
ultrasound, RF, microwave, IR, etc) in order to release the
photoactive molecules that can get into the nucleus and bind to DNA
(FIG. 22D).
Non-Invasive Phototherapy Using PEPST Modality
[0543] FIG. 23 illustrates the basic operating principle of the
PEPST modality. The PEPST photoactive drug molecules are given to a
patient by oral ingestion, skin application, or by intravenous
injection. The PEPST drugs travel through the blood stream inside
the body towards the targeted tumor (either via passive or active
targeting strategies). If the disease is systematic in nature a
photon radiation at suitable wavelengths is used to irradiate the
skin of the patient, the light being selected to penetrate deep
inside tissue (e.g., NIR or X ray). For solid tumors, the radiation
light source is directed at the tumor. Subsequently a treatment
procedure can be initiated using delivery of energy into the tumor
site. One or several light sources may be used as described in the
previous sections. One embodiment of therapy comprises sending NIR
radiation using an NIR laser through focusing optics. Focused beams
of other radiation types, including but not limited to X ray,
microwave, radio waves, etc. can also be used and will depend upon
the treatment modalities used.
Exciton-Plasmon Enhanced Phototherapy (EPEP)
[0544] Basic Principle of Exciton-Induced Phototherapy
[0545] Excitons in Solid Materials
[0546] Excitons are often defined as "quasiparticles" inside a
solid material. In solid materials, such as semiconductors,
molecular crystals and conjugated organic materials, light
excitation at suitable wavelength (such as X ray, UV and visible
radiation, etc) can excite electrons from the valence band to the
conduction band. Through the Coulomb interaction, this newly formed
conduction electron is attracted, to the positively charged hole it
left behind in the valence band. As a result, the electron and hole
together form a bound state called an exciton. (Note that this
neutral bound complex is a "quasiparticle" that can behave as a
boson--a particle with integer spin which obeys Bose--Einstein
statistics; when the temperature of a boson gas drops below a
certain value, a large number of bosons `condense` into a single
quantum state--this is a Bose--Einstein condensate (BEC). Exciton
production is involved in X-ray excitation of a solid material.
Wide band-gap materials are often employed for transformation of
the x-ray to ultraviolet/visible photons in the fabrication of
scintillators and phosphors [Martin Nikl, Scintillation detectors
for x-rays, Meas. Sci. Technol. 17 (2006) R37-R54]. The theory of
excitons is well known in materials research and in the fabrication
and applications of semiconductors and other materials. However, to
the present inventors' knowledge, the use of excitons and the
design of energy modulation agent materials based on exciton
tunability for phototherapy have not been reported.
[0547] During the initial conversion a multi-step interaction of a
high-energy X-ray photon with the lattice of the scintillator
material occurs through the photoelectric effect and Compton
scattering effect; for X-ray excitation below 100 keV photon energy
the photoelectric effect is the main process. Many excitons (i.e.,
electron-hole pairs) are produced and thermally distributed in the
conduction bands (electrons) and valence bands (holes). This first
process occurs within less than 1 ps. In the subsequent transport
process, the excitons migrate through the material where repeated
trapping at defects may occur, leading to energy losses due to
nonradiative recombination, etc. The final stage, luminescence,
consists in consecutive trapping of the electron-hole pairs at the
luminescent centers and their radiative recombination. The
electron-hole pairs can be trapped at the defects and recombine,
producing luminescent. Luminescent dopants can also be used as
traps for exciton.
[0548] Exciton Traps
[0549] Exciton traps can be produced using impurities in the
crystal host matrix. In impure crystals with dipolar guest
molecules the electron trap states may arise when electron is
localized on a neighbor of the impurity molecule. Such traps have
been observed in anthracene doped with carbazole [Kadshchuk, A. K.,
Ostapenko, N. L, Skryshevskii, Yu. A., Sugakov, V. I. and
Susokolova, T. O., Mol. Cryst. and Liq. Cryst., 201, 167 (1991)].
The formation of these traps is due to the interaction of the
dipole moment of the impurity with charge carrier. When the
concentration of the dopant (or impurities) is increased, spectra
exhibit additional structure of spectrum due to the trapping of
carriers on clusters of impurity molecules. Sometimes, impurities
and dopants are not required: the electron or exciton can also be
trapped on a structural defect in such crystals due to the
electrostatic interaction with reoriented dipole moment of
disturbed crystal molecules [S. V. Izvekov, V. I. Sugakov, Exciton
and Electron Traps on Structural Defects in Molecular Crystals with
Dipolar Molecules, Physica Scripta. Vol. T66, 255-257, 1996]. One
can design structural defects in molecular crystals that serve as
exiton traps. The development of GaAs/AlGaAs nanostructures and use
of nanofabrication technologies can design engineered exciton traps
with novel quantum mechanical properties in materials
Design, Fabrication and Operation of EIP Probes
[0550] FIG. 25 shows various embodiments of EIP probes that can be
designed: [0551] (A) probe comprising PA molecules bound (through a
linker, which can be fixed or detachable) to an energy modulation
agent particle that can produce excitons under radiative excitation
at a suitable wavelength (e.g., X-ray). In this preferred
embodiment, the energy modulation agent materials have structural
defects that serve as traps for excitons. [0552] (B) probe
comprising PA molecules bound (through a linker, which can be fixed
or detachable) to an energy modulation agent particle that can
produce excitons under radiative excitation at a suitable
wavelength (e.g., X-ray). In this preferred embodiment, the energy
modulation agent materials have impurities or dopant molecules that
serve as traps for excitons.
[0553] EIP Probes with Tunable Emission:
[0554] The embodiment in probes B provide the capability to tune
the energy conversion from an X ray excitation source into a
wavelength of interest to excite the PA molecules. In 1976, D'Silva
et al demonstrated that polynuclear aromatic hydrocarbons (PAH)
molecules doped in a frozen n-alkane solids could be excited by
X-ray and produce luminescence at visible wavelengths
characteristics of their luminescence spectra. [A. P. D'Silva, G.
J. Oestreich, and V. A. Fassel, X-ray excited optical luminescence
of polynuclear aromatic hydrocarbons, Anal. Chem.; 1976; 48(6) pp
915-917]. Tunable EIP probes can be designed to contain such
luminescent dopants such as highly luminescent PAHs exhibiting
luminescence emission in the range of 300-400 nm suitable to
activate psoralen. A preferred embodiment of the EIP with tunable
emission comprises a solid matrix (semiconductors, glass, quartz,
conjugated polymers, etc) doped with naphthalene, phenanthrene,
pyrene or other compounds exhibiting luminescence (fluorescence) in
the 300-400 nm range [T Vo-Dinh, Multicomponent analysis by
synchronous luminescence spectrometry, Anal. Chem.; 1978; 50(3) pp
396-401]. See FIG. 26. The EEC matrix could be a semiconductor
material, preferably transparent at optical wavelength of interest
(excitation and emission).
[0555] Other dopant species such as rare earth materials can also
be used as dopants. FIG. 27 shows the X ray excitation optical
luminescence (XEOL) of Europium doped in a matrix of BaFBr,
emitting at 370-420 nm. U.S. Patent Application Publication No.
2007/0063154 (hereby incorporated by reference) describes these and
other nanocomposite materials (and methods of making them) suitable
for XEOL.
[0556] FIG. 28 shows various embodiments of EIP probes that can be
designed: [0557] (A) probe comprising PA molecules bound around the
energy modulation agent particle or embedded in a shell around an
energy modulation agent particle that can produce excitons under
radiative excitation at a suitable wavelength (e.g., X-ray). In
this preferred embodiment, the energy modulation agent materials
has structural defects that serve as traps for excitons. [0558] (B)
probe comprising PA molecules bound around the energy modulation
agent particle or embedded in a shell around an energy modulation
agent particle that can produce excitons under radiative excitation
at a suitable wavelength (e.g., X-ray). In this preferred
embodiment, the energy modulation agent materials have impurities
or dopant molecules that serve as traps for excitons.
Principle of Exciton-Plasmon Enhanced Phototherapy (EPEP)
[0559] There is recent interest in an advanced photophysical
concept involving quantum optical coupling between electronic
states (excitons), photons and enhanced electromagnetic fields
(plasmons). Such a concept involving coupling between excitons and
plasmons can be used to enhance a phototherapy modality, referred
to as
Exciton-Plasmon Enhanced Phototherapy (EPEP).
[0560] A fundamental key concept in photophysics is the formation
of new quasiparticles from admixtures of strongly-coupled states.
Such mixed states can have unusual properties possessed by neither
original particle. The coupling between excitons and plasmons can
be either weak or strong. When the light-matter interaction cannot
be considered as a perturbation, the system is in the strong
coupling regime. Bellesa et al showed a strong coupling between a
surface plasmon (SP) mode and organic excitons occurs; the organic
semiconductor used is a concentrated cyanine dye in a polymer
matrix deposited on a silver film [Ref: J. Bellessa, * C. Bonnand,
and J. C. Plenet, J. Mugnier, Strong Coupling between Surface
Plasmons and Excitons in an Organic Semiconductor, Phys. Rev. Lett,
93 (3), 036404-1, 2004]. Govorov et al describe the photophysical
properties of excitons in hybrid complexes consisting of
semiconductor and metal nanoparticles. The interaction between
individual nanoparticles can produce an enhancement or suppression
of emission. Enhanced emission comes from electric field amplified
by the plasmon resonance, whereas emission suppression is a result
of energy transfer from semiconductor to metal nanoparticles.
[Alexander O. Govorov,*, .dagger. Garnett W Bryant, .dagger-dbl.
Wei Zhang, t Timur Skeini, .dagger. Jaebeom Lee, .sctn. Nicholas A.
Kotov, .sctn. Joseph M. Slocik, | and Rajesh R. Naik|,
Exciton-Plasmon Interaction and Hybrid Excitons in Semiconductor
Metal Nanoparticle Assemblies, Nano Lett., Vol. 6, No. 5, 984,
2006]. Bondarev et al also described a theory for the interactions
between excitonic states and surface electromagnetic modes in
small-diameter (<1 nm) semiconducting single-walled carbon
nanotubes (CNs). [I. V. Bondarev, K. Tatur and L. M. Woods, Strong
exciton-plasmon coupling in semiconducting carbon nanotubes].
[0561] Fedutik et al reported about the synthesis and optical
properties of a composite metal-insulator-semiconductor nanowire
system which consists of a wet-chemically grown silver wire core
surrounded by a SiO.sub.2 shell of controlled thickness, followed
by an outer shell of highly luminescent CdSe nanocrystals [Yuri
Fedutik, .dagger. Vasily Temnov, .dagger. Ulrike Woggon, .dagger.
Elena Ustinovich, .dagger-dbl. and Mikhail Artemyev*.dagger-dbl.,
Exciton-Plasmon Interaction in a Composite
Metal-Insulator-Semiconductor Nanowire System, J. Am. Chem. Soc.,
129 (48), 14939-14945, 2007]. For a SiO.sub.2 spacer thickness of
.about.15 nm, they observed an efficient excitation of surface
plasmons by excitonic emission of CdSe nanocrystals. For small d,
well below 10 nm, the emission is strongly suppressed (PL
quenching), in agreement with the expected dominance of the
dipole-dipole interaction with the damped mirror dipole [G. W. Ford
and W. H. Weber, Electromagnetic interactions of molecules with
metal surfaces," Phys. Rep. 113, 195-287 (1984)]. For nanowire
lengths up to .about.10 .mu.m, the composite
metal-insulator-semiconductor nanowires ((Ag)SiO.sub.2)CdSe act as
a waveguide for 1D-surface plasmons at optical frequencies with
efficient photon out coupling at the nanowire tips, which is
promising for efficient exciton-plasmon-photon conversion and
surface plasmon guiding on a submicron scale in the visible
spectral range.
[0562] Experiments on colloidal solutions of Ag nanoparticles
covered with J-aggregates demonstrated the possibility of using the
strong scattering cross section and the enhanced field associated
with surface plasmon to generate stimulated emission from
J-aggregate excitons with very low excitation powers. [Gregory A.
Wurtz, * Paul R. Evans, William Hendren, Ronald Atkinson, Wayne
Dickson, Robert J. Pollard, and Anatoly V. Zayals, Molecular
Plasmonics with Tunable Exciton-Plasmon Coupling Strength in
J-Aggregate Hybridized Au Nanorod Assemblies, Nano Lett., Vol. 7,
No. 5, 1297, 2007]. Their coupling to surface plasmons excitations
therefore provides a particularly attractive approach for creating
low-powered optical devices. This process can lead to efficient
X-ray coupling for phototherapy. In addition, the coupling of
J-aggregates with plasmonics structures presents genuine
fundamental interest in the creation of mixed plasmon-exciton
states.
Design, Fabrication and Operation of EPEP Probes
[0563] FIG. 29 shows various embodiments of EPEP probes of the
present invention showing the exciton-plasmon coupling: [0564] (A)
probe comprising a PA molecule or group of PA molecules bound
(through a linker, which can be fixed or detachable) to an energy
modulation agent particle that can produce excitons under radiative
excitation at a suitable wavelength (e.g., X-ray). The energy
modulation agent particle is bound to (or in proximity of) a metal
nanoparticle covered with a nanoshell of silica (or other
dielectric material). The silica layer (or nanoshell) (see FIG. 24A
and FIG. 24B; layer nanoshell in white between energy modulation
material and metal nanostructures) is designed to prevent quenching
of the luminescence light emitted by the energy modulation agent
particle excited by X-ray. The metal nanoparticle (Au, Ag, etc) is
designed to induce plasmons that enhance the X ray excitation that
subsequently leads to an increase in the energy modulation agent
light emission, ultimately enhancing the efficiency of
photoactivation, i.e. phototherapy. The structure of the
nanoparticle can also be designed such that the plasmonics effect
also enhances the energy modulation agent emission light. These
processes are due to strong coupling between excitons (in the
energy modulation agent materials and plasmons in the metal
nanoparticles; and [0565] (B) probe comprising a PA molecule or
group of PA molecules bound (through a linker, which can be fixed
or detachable) to an energy modulation agent particle that can
produce excitons under radiative excitation at a suitable
wavelength (e.g., X-ray). The energy modulation agent particle is
bound to (or in proximity of) a metal nanoparticle via a spacer
(linker). The spacer is designed to prevent quenching of the
luminescence light emitted by the energy modulation agent particle
excited by X-ray.
[0566] FIG. 30 shows yet further embodiments of EPEP probes of the
present invention:
[0567] (A) probe comprising a PA molecule or group of PA molecules
bound (through a linker, which can be fixed or detachable) to an
energy modulation agent particle that can produce excitons under
radiative excitation at a suitable wavelength (e.g., X-ray). The
energy modulation agent particle is covered with a nanoshell of
silica (or other dielectric material), which is covered by a layer
of separate nanostructures (nano islands, nanorods, nanocubes, etc.
. . . ) of metal (Au, Ag). The silica layer (or other dielectric
material) is designed to prevent quenching of the luminescence
light emitted by the EEC (also referred to as energy modulation
agent) particle excited by X-ray. The metal nanostructures (Au, Ag,
etc) are designed to induce plasmons that enhance the X ray
excitation that subsequently leads to an increase in the EEC light
emission, ultimately enhancing the efficiency of photoactivation,
i.e. phototherapy. The structure of the nanoparticle can also be
designed such that the plasmonics effect also enhance the energy
modulation agent emission light. These processes are due to strong
coupling between excitons (in the energy modulation agent materials
and plasmons in the metal nanostructures).
[0568] (B) probe comprising a group of PA molecules in a particle
bound (through a linker, which can be fixed or detachable) to an
energy modulation agent particle that can produce excitons under
radiative excitation at a suitable wavelength (e.g., X-ray). The
PA-containing particle is covered with a layer of metallic
nanostructures (Au, Ag). The metal nanostructures (Au, Ag, etc) are
designed to induce plasmons that enhance the energy modulation
agent light emission, ultimately enhancing the efficiency of
photoactivation, i.e. phototherapy.
[0569] (C) probe comprising a PA molecule or group of PA molecules
bound (through a linker, which can be fixed or detachable) to an
energy modulation agent particle that can produce excitons under
radiative excitation at a suitable wavelength (e.g., X-ray). The
energy modulation agent particle is covered with a nanoshell of
silica (or other dielectric material), which is covered by a layer
of metallic nanostructures (Au, Ag). The silica layer (or other
dielectric material) is
[0570] designed to prevent quenching of the luminescence light
emitted by the energy modulation agent particle excited by X-ray.
The metal nanostructures (Au, Ag, etc) are designed to induce
plasmons that enhance the X ray excitation that subsequently leads
to an increase in the energy modulation agent light emission,
ultimately enhancing the efficiency of photoactivation, i.e.
phototherapy. In addition. the PA-containing particle is covered
with a layer of metallic nanostructures (Au, Ag). The metal
nanostructures (Au, Ag, etc) are designed to induce plasmons that
enhance the EEC light emission, ultimately enhancing the efficiency
of photoactivation, i.e. phototherapy.
Hybrid EPEP Nano-Superstructures
[0571] EPEP probes can also comprise hybrid self-assembled
superstructures made of biological and abiotic nanoscale
components, which can offer versatile molecular constructs with a
spectrum of unique electronic, surface properties and photospectral
properties for use in phototherapy.
[0572] Biopolymers and nanoparticles can be integrated in
superstructures, which offer unique functionalities because the
physical properties of inorganic nanomaterials and the chemical
flexibility/specificity of polymers can be used. Noteworthy are
complex systems combining two types of excitations common in
nanomaterials, such as excitons and plasmons leading to coupled
excitations. Molecular constructs comprising building blocks
including metal, semiconductor nanoparticles (NPs), nanorods (NRs)
or nanowires (NWs) can produce EPEP probes with an assortment of
photonic properties and enhancement interactions that are
fundamentally important for the field of phototherapy. Some
examples of assemblies of some NW nanostructures and NPs have been
reported in biosensing. Nanoscale superstructures made from CdTe
nanowires (NWs) and metal nanoparticles (NPs) are prepared via
bioconjugation reactions. Prototypical biomolecules, such as
D-biotin and streptavidin pair, were utilized to connect NPs and
NWs in solution. It was found that Au NPs form a dense shell around
a CdTe NW. The superstructure demonstrated unusual optical effects
related to the long-distance interaction of the semiconductor and
noble metal nanocolloids. The NW .quadrature.NP complex showed
5-fold enhancement of luminescence intensity and a blue shift of
the emission peak as compared to unconjugated NW. [Jaebeom Lee,
.dagger. Alexander O. Govorov, .dagger-dbl. John Dulka,
.dagger-dbl. and Nicholas A. Kotov*, .dagger., Bioconjugates of
CdTe Nanowires and Au Nanoparticles: Plasmon-Exciton Interactions,
Luminescence Enhancement, and Collective Effects, Nano Lett., Vol.
4, No. 12, 2323, 2004].
[0573] To the present inventors' knowledge, these advanced concepts
have not been applied to phototherapy and EPEP probes comprising
superstructures from NPs, NRs and NWs are still a new unexplored
territory of phototherapy.
[0574] FIG. 31 shows various embodiments of EPEP probes of the
present invention comprising superstructures of NPs, NWs and
NRs.:
[0575] (A) probe comprising a PA molecule or group of PA molecules
bound (through a linker, which can be fixed or detachable) to an
energy modulation agent particle that can produce excitons under
radiative excitation at a suitable wavelength (e.g., X-ray). The
energy modulation agent particle is bound to (or in proximity of) a
metal nanowire (or nanorod) covered with a nanoshell cylinder of
silica (or other dielectric material). The silica nanoshells
cylinder is designed to prevent quenching of the luminescence light
emitted by the energy modulation agent particle excited by X-ray.
The metal nanoparticle (Au, Ag, etc) is designed to induce plasmons
that enhance the X ray excitation that subsequently leads to an
increase in the energy modulation agent light emission, ultimately
enhancing the efficiency of photoactivation, i.e. phototherapy. The
structure of the nanoparticle can also be designed such that the
plasmonics effect and/or the exciton-plasmon coupling (EPC) effect
also enhances the energy modulation agent emission light. These
processes are due to strong coupling between excitons (in the
energy modulation agent materials and plasmons in the metal
nanoparticles; and
[0576] (B) probe comprising a PA molecule or group of PA molecules
bound (through a linker, which can be fixed or detachable) to an
energy modulation agent particle that can produce excitons under
radiative excitation at a suitable wavelength (e.g., X-ray). The
energy modulation agent particle is bound to (or in proximity of) a
metal nanoparticles via a spacer (linker). The spacer is designed
to prevent quenching of the luminescence light emitted by the
energy modulation agent particle excited by X-ray. Same effect as
above in (A)
[0577] FIG. 32 shows another set of embodiments of EPEP probes of
the present invention comprising superstructures of NPs, NWs and
NRs and bioreceptors (antibodies, DNA, surface cell receptors,
etc.). The use of bioreceptors to target tumor cells has been
discussed previously above in relation to PEPST probes. Note that
in this embodiment the PA molecules are attached along the NW axis
in order to be excited by the emitting light form the NWs.
[0578] FIG. 33 shows another embodiment of EPEP probes of the
present invention comprising superstructures of NPs linked to
multiple NWs.
[0579] For some embodiments, by adding metal nanostructures
designed to interact specifically with the excitons in the energy
modulation agent system, there are significant improvements:
[0580] (1) an additional radiative pathway from exciton to photon
conversion is introduced
[0581] (2) the metal nanostructures can be designed to amplify (due
to the plasmonics effect) the excitation radiation (e.g., X-ray)
and/or the emission radiation (e.g, UV or visible) to excite the
photo-active (PA) molecule, thereby enhancing the PA
effectiveness.
[0582] Various metallic nanostructures that can be used in EPEP
probe embodiments of the present invention are the same as those
illustrated in FIG. 9 for the PEPST probes.
EPEP Probes with Microresonators
[0583] In a preferred embodiment the energy modulation agent system
can be designed to serve also as a microresonator having micron or
submicron size. Lipson et al described a resonant microcavity and,
more particularly, to a resonant microcavity which produces a
strong light-matter interaction [M Lipson; L. C. Kimerling; Lionel
C, Resonant microcavities, U.S. Pat. No. 6,627,923, 2000]. A
resonant microcavity, typically, is formed in a substrate, such as
silicon, and has dimensions that are on the order of microns or
fractions of microns. The resonant microcavity contains
optically-active matter (i.e., luminescent material) and reflectors
which confine light in the optically-active matter. The confined
light interacts with the optically-active matter to produce a
light-matter interaction. The light-matter interaction in a
microcavity can be characterized as strong or weak. Weak
interactions do not alter energy levels in the matter, whereas
strong interactions alter energy levels in the matter. In strong
light-matter interaction arrangements, the confined light can be
made to resonate with these energy level transitions to change
properties of the microcavity.
Experimental Methods
[0584] Preparation of Nanoparticles (Ag, Au)
[0585] There many methods to prepare metal nanoparticles for EPEP
or PEPST probes. Procedures for preparing gold and silver colloids
include electroexplosion, electrodeposition, gas phase
condensation, electrochemical methods, and solution-phase chemical
methods. Although the methodologies for preparing homogeneous-sized
spherical colloidal gold populations 2-40 nm in diameter are well
known [N. R. Jana, L. Gearheart and C. J. Murphy, Seeding growth
for size control of 5-40 nm diameter gold nanoparticles. Langmuir
17 (2001), pp. 6782-6786], and particles of this size are
commercially available. An effective chemical reduction method for
preparing populations of silver particles (with homogeneous optical
scattering properties) or gold particles (with improved control of
size and shape monodispersity) is based on the use of
small-diameter uniform-sized gold particles as nucleation centers
for the further growth of silver or gold layers.
[0586] A widely used approach involves citrate reduction of a gold
salt to produce 12-20 nm size gold particles with a relatively
narrow size distribution. The commonly used method for producing
smaller gold particles was developed by Brust et al [Brust, M.;
Walker, M; Bethell, D.; Schiffrin, D. J; Whyman, R. Chem. Commun.
1994, 801]. This method is based on borohydride reduction of gold
salt in the presence of an alkanethiol capping agent to produce 1-3
nm particles. Nanoparticle sizes can be controlled between 2 and 5
nm by varying the thiol concentration, [Hostetler, M. J.; Wingate,
J. E.; Zhong, C. J.; Harris, J. E.; Vachet, R. W.; Clark, M R.;
Londono, D.; Green, S. I; Stokes, J. J.; Wignall, G. D.; Glish, G.
L.; Porter, M D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14,
17]. Phosphine-stabilized gold clusters have also been produced and
subsequently converted to thiol-capped clusters by ligand exchange
in order to improve their stability [Schmid, G.; Pfeil, R.; Boese,
R.; Bandrmann, F.; Meyer, S.; Calis, G. H. M; van der Velden, J. W.
A. Chem. Ber. 1981, 114, 3634; Warner, M G.; Reed, S. M; Hutchison,
J. E. Chem. Mater. 2000, 12, 3316.] and phosphine-stabilized
monodispersed gold particles were prepared using a similar protocol
to the Brust method [Weare, W. W.; Reed, S. M; Warner, M G.;
Hutchison, J. E. J. Am. Chem. Soc. 2000, 122, 12890]. See also
recent review: Ziyi Zhong, Benoit.sup.1 Male, Keith B..sup.1 Luong,
John H. T., More Recent Progress in the Preparation of Au
Nanostructures, Properties, and Applications, Analytical Letters;
2003, Vol. 36 Issue 15, p 3097-3118]
[0587] Fabrication of Nanoparticle of Metal Coated with Nanoshells
of Dyes
[0588] The fabrication of metal nanoparticles coated with
nanoshells of dye molecules can be performed using the method
described by Masuhara et al [AKITO MASUHARA_, SATOSHI OHHASHIy,
HITOSHI KASAI; SHUJI OKADA, FABRICATION AND OPTICAL PROPERTIES OF
NANOCOMPLEXES COMPOSED OF METAL NANOPARTICLES AND ORGANIC DYES,
Journal of Nonlinear Optical Physics & Materials Vol. 13, Nos.
3 & 4 (2004) 587-592]. Nanocomplexes composed of Ag or Au as a
core and
3-carboxlymethyl-5-[2-(3-octadecyl-2-benzoselenazolinylidene)
ethylidene]rhodanine (MCSe) or copper (II) phthalocyanine (CuPc) as
a shell are prepared by the co-reprecipitation method. In the case
of Ag-MCSe nanocomplexes, 0.5 mM acetone solution of MCSe are
injected into 10 ml of Ag nanoparticle water dispersion, prepared
by the reduction of AgNO.sub.3 using NaBH.sub.4: Au-MCSe
nanocomplexes are also fabricated in a similar manner. A water
dispersion of Au nanoparticles was prepared by the reduction of
HAuCl.sub.4 using sodium citrate. Subsequently, 2 M NH.sub.4OH (50
.mu.l) was added and the mixture was thermally treated at
50.degree. C. This amine treatment often stimulates the J-aggregate
formation of MCSe.6 Ag-CuPc and Au-CuPc nanocomplexes were also
fabricated in the same manner: 1 mM 1-methyl-2-pyrrolidinone (NMP)
solution of CuPc (200 .mu.l) was injected into a water dispersion
(10 ml) of Ag or Au nanoparticles.
[0589] The present invention treatment may also be used for
inducing an auto vaccine effect for malignant cells, including
those in solid tumors. To the extent that any rapidly dividing
cells or stem cells may be damaged by a systemic treatment, then it
may be preferable to direct the stimulating energy directly toward
the tumor, preventing damage to most normal, healthy cells or stem
cells by avoiding photoactivation or resonant energy transfer of
the photoactivatable agent.
[0590] Alternatively, a treatment may be applied that slows or
pauses mitosis. Such a treatment is capable of slowing the division
of rapidly dividing healthy cells or stem cells during the
treatment, without pausing mitosis of cancerous cells.
Alternatively, a blocking agent is administered preferentially to
malignant cells prior to administering the treatment that slows
mitosis.
[0591] In one embodiment, an aggressive cell proliferation disorder
has a much higher rate of mitosis, which leads to selective
destruction of a disproportionate share of the malignant cells
during even a systemically administered treatment. Stem cells and
healthy cells may be spared from wholesale programmed cell death,
even if exposed to photoactivated agents, provided that such
photoactivated agents degenerate from the excited state to a lower
energy state prior to binding, mitosis or other mechanisms for
creating damage to the cells of a substantial fraction of the
healthy stem cells. Thus, an auto-immune response may not be
induced.
[0592] Alternatively, a blocking agent may be used that prevents or
reduces damage to stem cells or healthy cells, selectively, which
would otherwise be impaired. The blocking agent is selected or is
administered such that the blocking agent does not impart a similar
benefit to malignant cells, for example.
[0593] In one embodiment, stem cells are targeted, specifically,
for destruction with the intention of replacing the stem cells with
a donor cell line or previously stored, healthy cells of the
patient. In this case, no blocking agent is used. Instead, a
carrier or photosensitizer is used that specifically targets the
stem cells.
[0594] Work in the area of photodynamic therapy has shown that the
amount of singlet oxygen required to cause cell lysis, and thus
cell death, is 0.32.times.10.sup.-3 mol/liter or more, or 10.sup.9
singlet oxygen molecules/cell or more. However, in one embodiment
of the present invention, it is most preferable to avoid production
of an amount of singlet oxygen that would cause cell lysis, due to
its indiscriminate nature of attack, lysing both target cells and
healthy cells. Accordingly, it is most preferred in the present
invention that the level of singlet oxygen production caused by the
initiation energy used or activatable pharmaceutical agent upon
activation be less than level needed to cause cell lysis.
[0595] In a further embodiment, methods in accordance with the
present invention may further include adding an additive to
alleviate treatment side-effects. Exemplary additives may include,
but are not limited to, antioxidants, adjuvant, or combinations
thereof. In one exemplary embodiment, psoralen is used as the
activatable pharmaceutical agent, UV-A is used as the activating
energy, and antioxidants are added to reduce the unwanted
side-effects of irradiation.
[0596] An advantage of the methods of the present invention is that
by specifically targeting cells affected by a cell proliferation
disorder, such as rapidly dividing cells, and triggering a cellular
change, such as apoptosis, in these cells in situ, the immune
system of the host may be stimulated to have an immune response
against the diseased cells. Once the host's own immune system is
stimulated to have such a response, other diseased cells that are
not treated by the activatable pharmaceutical agent may be
recognized and be destroyed by the host's own immune system. Such
autovaccine effects may be obtained, for example, in treatments
using psoralen and UV-A.
[0597] The present invention methods can be used alone or in
combination with other therapies for treatment of cell
proliferation disorders. Additionally, the present invention
methods can be used, if desired, in conjunction with recent
advances in chronomedicine, such as that detailed in Giacchetti et
al, Journal of Clinical Oncology, Vol 24, No 22 (August 1), 2006:
pp. 3562-3569. In chronomedicine it has been found that cells
suffering from certain types of disorders, such as cancer, respond
better at certain times of the day than at others. Thus,
chronomedicine could be used in conjunction with the present
methods in order to augment the effect of the treatments of the
present invention.
[0598] In another aspect, the present invention further provides
systems and kits for practicing the above described methods.
[0599] In one embodiment, a system in accordance with the present
invention may include: (1) an initiation energy source; (2) one or
more energy modulation agents; and (3) one or more activatable
pharmaceutical agents.
[0600] In another embodiment, a system in accordance with the
present invention may include an initiation energy source and one
or more activatable pharmaceutical agents.
[0601] In preferred embodiments, the initiation energy source may
be a linear accelerator equipped with image guided computer-control
capability to deliver a precisely calibrated beam of radiation to a
pre-selected coordinate. One example of such linear accelerators is
the SmartBeam.TM. IMRT (intensity modulated radiation therapy)
system from Varian medical systems (Varian Medical Systems, Inc.,
Palo Alto, Calif.).
[0602] In other embodiments, endoscopic or laproscopic devices
equipped with appropriate initiation energy emitter may be used as
the initiation energy source. In such systems, the initiation
energy may be navigated and positioned at the pre-selected
coordinate to deliver the desired amount of initiation energy to
the site.
[0603] In further embodiments, dose calculation and robotic
manipulation devices may also be included in the system.
[0604] The reagents and chemicals useful for methods and systems of
the present invention may be packaged in kits to facilitate
application of the present invention. In one exemplary embodiment,
a kit including a psoralen, and fractionating containers for easy
fractionation and isolation of autovaccines is contemplated. A
further embodiment of kit would comprise at least one activatable
pharmaceutical agent capable of causing a predetermined cellular
change, at least one energy modulation agent capable of activating
the at least one activatable agent when energized, at least one
plasmonics agent and containers suitable for storing the agents in
stable form, and preferably further comprising instructions for
administering the at least one activatable pharmaceutical agent, at
least one plasmonics agent and at least one energy modulation agent
to a subject, and for applying an initiation energy from an
initiation energy source to activate the activatable pharmaceutical
agent. The instructions could be in any desired form, including but
not limited to, printed on a kit insert, printed on one or more
containers, as well as electronically stored instructions provided
on an electronic storage medium, such as a computer readable
storage medium. Also optionally included is a software package on a
computer readable storage medium that permits the user to integrate
the information and calculate a control dose, to calculate and
control intensity of the irradiation source.
[0605] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only and are not intended to be limiting unless otherwise
specified.
EXAMPLES
[0606] Preparation of Silver Nanoparticles
[0607] Silver (or gold) colloids were prepared according to the
standard Lee-Meisel method: 200 mL of 10.sup.-3 M AgNO.sub.3
aqueous solution was boiled under vigorous stirring, then 5 mL of
35-mM sodium citrate solution were added and the resulting mixture
was kept boiling for 1 h. This procedure was reported to yield
.about.10.sup.11 particles/mL of homogenously sized colloidal
particles with a diameter of .about.35-50 nm and an absorption
maximum at 390 nm. The colloidal solutions were stored at 4.degree.
C. and protected from room light. Further dilutions of the
colloidal solutions were carried out using distilled water.
[0608] Fabrication/Preparation of Metal Nanocaps
[0609] One approach has involved the use of nanospheres spin-coated
on a solid support in order to produce and control the desired
roughness. The nanostructured support is subsequently covered with
a layer of silver that provides the conduction electrons required
for the surface plasmon mechanisms. Among the techniques based on
solid substrates, the methods using simple nanomaterials, such as
Teflon or latex nanospheres, appear to be the simplest to prepare.
Teflon and latex nanospheres are commercially available in a wide
variety of sizes. The shapes of these materials are very regular
and their size can be selected for optimal enhancement. These
materials comprise isolated dielectric nanospheres (30-nm diameter)
coated with silver producing systems of half-nanoshells, referred
to as nanocaps.
[0610] FIG. 24 shows a scanning electron micrograph (SEM) of 300-nm
diameter polymer nanospheres covered by a 100-nm thick silver
nanocaps (half-nanoshell) coating. The nanoparticles can be
sonicated to release them from the underlying substrate. The effect
of the sphere size and metal layer thickness upon the SERS effect
can be easily investigated. The silver coated nanospheres were
found to be among the most plasmonics-active investigated. Gold can
also be used instead of silver to coat over nanoparticles
comprising PA drug molecules.
[0611] Fabrication of Gold Nanoshells
[0612] Gold nanoshells have been prepared using the method
described by Hirsch et al. [Hirsch L R, Stafford R J, Bankson J A,
Sershen S R, Price R E, Hazle J D, Halas N J, West J L (2003)
Nanoshell-mediated near infrared thermal therapy of tumors under MR
Guidance. Proc Natl Acad Sci 100:13549-13554] using a mechanism
involving nucleation and then successive growth of gold
nanoparticles around a silica dielectric core. Gold nanoparticles,
the seed, prepared as described above using the Frens method, were
used to grow the gold shell. Silica nanoparticles (100 nm) used for
the core of the nanoshells were monodispersed in solution of 1
APTES in EtOH. The gold "seed" colloid synthesized using the Frens
method were grown onto the surface of silica nanoparticles via
molecular linkage of amine groups. The "seed" covers the aminated
silica nanoparticle surface, first as a discontinuous gold metal
layer gradually growing forming a continuous gold shell. Gold
nanoparticles used as the "seed" were characterized using optical
transmission spectroscopy (UV-Vis Spectrophotometer, Beckman
Coulter, Fullerton, Calif.) and atomic force microscopy (Atomic
Force Microscope, Veeco Instruments, Woodbury, N.Y.) while gold
nanoshells were characterized using optical transmission
spectroscopy and scanning electron microscopy (Scanning Electron
Microscope, Hitachi S-4700, Hitachi High Technologies America, Inc.
Pleasanton, N.Y.).
[0613] Probe for Measurement of Apoptosis with the PDT Drug ALA
[0614] A method has been developed using nanosensors that can be
used to evaluate the effectiveness of PEPST probes. Although one
can use conventional methods (not requiring nanosensors), we
describe the nanosensor method previously developed [P. M. Kasili,
J. M Song, and T Vo-Dinh, "Optical Sensor for the Detection of
Caspase-9 Activity in a Single Cell", J. Am. Chem. Soc., 126
2799-2806 (2004)]. The method comprises measuring caspases
activated by apoptosis induced by the photoactive drugs. In this
experiment, we measure two sets of cells I and II. Set I is treated
with the drug ALA and set II is treated by the drug ALA conjugated
to a PEPST probe described in the previous section. By comparing
the results (amount of Caspases detected), one can evaluate the
efficiency of the PEPST-ALA drug compared to ALA alone.
[0615] In the classical model of apoptosis, caspases are divided
into initiator caspases and effector caspases according to their
function and their sequence of activation. Initiator caspases
include caspase-8, -9, while effector caspases include, caspases-3,
-6 and -7. The activation of caspases is one of the earliest
biomarkers of apoptosis making caspases an early and ideal target
for measuring apoptosis. Apoptosis, or programmed cell death, is a
mode of cell death characterized by specific morphological and
biochemical features. The results obtained in these experiments can
be used to evaluate the effectiveness of phototherapeutic drugs
that induce apoptosis (e.g. PDT drugs). Since caspases play a
central role in the induction of apoptosis, tetrapeptide-based
optical nanosensors were used to determine their role in response
to a photodynamic therapy (PDT) agent, .delta.-aminolevulinic acid
(ALA) in the well-characterized human breast carcinoma cell line,
MCF-7. MCF-7 cells were exposed to the photosensitizer ALA to
explore ALA-PDT induced apoptosis by monitoring caspase-9 and
caspase-7 activity. Caspase-9 and caspase-7 protease activity was
assessed in single living MCF-7 cells with the known caspase-9 and
caspase-7 substrates, Leucine-aspartic-histidine-glutamic acid
7-amino-4-methylcoumarin (LEHD-AMC) and aspartic-glutamic
acid-valine-aspartic acid 7-amino-4-methylcoumarin (DEVD-AMC)
respectively, covalently immobilized to the nanotips of optical
nanosensors. Upon the induction of apoptosis, activated target
caspases recognize the tetrapeptide sequence and specifically
cleaves it. The recognition of substrate by caspases is immediately
followed by a cleavage reaction yielding the fluorescent AMC which
can be excited with a Helium-Cadmium (HeCd) laser to generate a
measurable fluorescence signal. By comparing the fluorescence
signal generated from AMC within cells with activated caspases and
from those with inactive caspases, we are able to successfully
detect caspase activity within a single living MCF-7 cell.
[0616] Chemicals and Reagents
[0617] .delta.-aminolevulinic acid (ALA), phosphate buffered saline
(PBS), hydrochloric acid (HCl), nitric acid (HNO.sub.3),
Glycidoxypropyltrimethoxysilane (GOPS), 1,1'-Carbonyldiimidazole
(CDI), and anhydrous acetonitrile were purchased from
Sigma-Aldrich, St. Louis, Mo. Caspase-9 substrate,
LEHD-7-amino-4-methylcoumarin (AMC), Caspase-7 substrate,
DEVD-7-amino-4-methylcoumarin (AMC), 2.times. reaction buffer,
dithiothreitol (DTT), and dimethylsulfoxide (DMSO) were purchased
from BD Biosciences, Palo Alto. Calif.
[0618] Cell Lines
[0619] Human breast cancer cell line, MCF-7, was obtained from
American Type Culture Collection (Rockville, Md., USA, Cat-no.
HTB22). MCF-7 cells were grown in Dulbecco's Modified Eagle's
Medium ((DMEM) (Mediatech, Inc., Herndon, Va.)) supplemented with 1
mM L-glutamine (Gibco, Grand Island, N.Y.) and 10% fetal bovine
serum (Gibco, Grand Island, N.Y.). Cell culture was established in
growth medium (described above) in standard T25 tissue culture
flasks (Corning, Corning, N.Y.). The flasks were incubated in a
humidified incubator at 37.degree. C., 5% CO.sub.2 and 86%
humidity. Cell growth was monitored daily by microscopic
observation until a 60-70% state of confluence was achieved. The
growth conditions were chosen so that the cells would be in log
phase growth during photosensitizer treatment with ALA, but would
not be so close to confluence that a confluent monolayer would form
by the termination of the chemical exposure. In preparation for
experiments, cells were harvested from the T25 flasks and 0.1 ml
(10.sup.5 cells/ml) aliquots were seeded into 60 mm tissue culture
dishes (Corning Costar Corp., Corning, N.Y.) for overnight
attachment. The MCF-7 cells were studied as four separate groups
with the first group, Group I, being the experimental, exposed to
0.5 mM ALA for 3 h followed by photoactivation ([+]ALA[+]PDT). This
involved incubating the cells at 37.degree. C. in 5% CO.sub.2 for 3
h with 0.5 mM ALA. Following incubation the MCF-7 cells were
exposed to red light from a HeNe laser (.lamda. 632.8 nm, <15
mW, Melles Griot, Carlsbad, Calif.) positioned about 5.0 cm above
the cells for five minutes at a fluence of 5.0 mJ/cm.sup.2 to
photoactivate ALA and subsequently induce apoptosis. The second and
third groups, Group 11 and 111 respectively, served as the "treated
control" and were exposed to 0.5 mM ALA for 3 hours without
photoactivation ([+]ALA[-]PDT) and photoactivation without 0.5 mM
ALA ([-]ALA[+]PDT]) respectively. The fourth group, Group IV was
the "untreated control," which received neither ALA nor
photoactivation ([-]ALA[-]PDT
Experimental Protocol
[0620] Preparation of Enzyme Substrate-Based Optical
Nanosensors
[0621] Briefly, this process involved cutting and polishing plastic
clad silica (PCS) fibers with a 600-.mu.m-size core (Fiberguide
Industries, Stirling, N.J.). The fibers were pulled to a final tip
diameter of 50 nm and then coated with .about.100 nm of silver
metal (99.999% pure) using a thermal evaporation deposition system
(Cooke Vacuum Products, South Norwalk, Conn.) achieving a final
diameter of 150 nm. The fused silica nanotips were acid-cleaned
(HNO.sub.3) followed by several rinses with distilled water.
Finally, the optical nanofibers were allowed to air dry at room
temperature in a dust free environment. The nanotips were then
silanized and treated with an organic coupling agent, 10%
Glycidoxypropyltrimethoxysilane (GOPS) in distilled water. The
silanization agent covalently binds to the silica surface of the
nanotips modifying the hydroxyl group to a terminus that is
compatible with the organic cross-linking reagent, 1'1,
Carbonyldiimidazole (CDI). The use of CDI for activation
introducing an imidazole-terminal group was particularly attractive
since the protein to be immobilized could be used without chemical
modification. Proteins bound using this procedure remained securely
immobilized during washing or subsequent manipulations in
immunoassay procedures, as opposed to procedures that use
adsorption to attach proteins. The silanized and activated nanotips
for measuring caspase-9 activity were immersed in a solution
containing DMSO, 2.times. reaction buffer, PBS, and LEHD-AMC, and
allowed to incubate for 3 h at 37.degree. C., while those for
measuring caspase-7 activity were immersed in a solution containing
DMSO, 2.times. reaction buffer, PBS, and DEVD-AMC, and allowed to
incubate for 3 h at 37.degree. C.
[0622] Measurement System and Procedure
[0623] A schematic representation of the experimental setup used in
this work is described in a previous work [[P. M Kasili, J. M Song,
and T Vo-Dinh, "Optical Sensor for the Detection of Caspase-9
Activity in a Single Cell", J. Am. Chem. Soc., 126, 2799-2806
(2004)]. The components included a HeCd laser (Omnichrome, <5 mW
laser power) for excitation, an optical fiber for delivery of
excitation light to the optical nanosensor, a Nikon Diaphot 300
inverted fluorescence microscope (Nikon, Inc., Melville, N.Y.), a
photon counting photomultiplier tube (PMT) and a PC for data
acquisition and processing. This experimental set-up, used to probe
single cells, was adapted for this purpose from a standard
micromanipulation and microinjection apparatus. The Nikon Diaphot
300 inverted microscope was equipped with a Diaphot 300/Diaphot 200
Incubator to maintain the cell cultures at 37.degree. C. on the
microscope stage, during these experiments. The micromanipulation
equipment consisted of MN-2 (Narishige Co. Ltd., Tokyo, Japan)
Narishige three-dimensional manipulators for coarse adjustment, and
Narishige MMW-23 three-dimensional hydraulic micromanipulators for
fine adjustments. The optical nanosensor was mounted on a
micropipette holder (World Precision Instruments, Inc., Sarasota,
Fla.). The 325 nm laser line of a HeCd laser was focused onto a
600-.mu.m-delivery fiber that is terminated with a subminiature A
(SMA) connector. The enzyme substrate-based optical nanosensor was
coupled to the delivery fiber through the SMA connector and secured
to the Nikon inverted microscope with micromanipulators. To record
the fluorescence generated by AMC molecules at the nanotips, a
Hamamatsu PMT detector assembly (HC125-2) was mounted in the front
port of the Diaphot 300 microscope. The fluorescence emitted by AMC
from the measurement made using single live cells was collected by
the microscope objective and passed through a 330-380 nm filter set
and then focused onto a PMT for detection. The output from the PMT
was recorded using a universal counter interfaced to a personal
computer (PC) for data treatment and processing.
[0624] In Vitro Determination of Caspase Activity
[0625] After incubation using the following treatment groups, group
(I)-[+]ALA[+]PDT, group II--[+]ALA[-]PDT, group III--[-]ALA[+]PDT,
and group IV--[-]ALA[-]PDT, MCF-7 cells were washed with PBS
solution, pH 7.4, and then resuspended in lysis buffer (100 mM
HEPES, pH 7.4, 10% sucrose, 0.1%
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS),
1 mM EDTA, 10 mM dithiothreitol (DTT), 1 mM phenylmethylsulphonyl
fluoride (PMSF), 10 mg/ml pepstatin, 10 mg/ml leupeptin) and left
on ice for 45 minutes. The cells were then repeatedly passed
through a syringe with a 25-gauge needle until most of the cell
membrane was disrupted, and centrifuged at 1500 RPM for 10 min.
Activity of caspases was measured using the fluorogenic substrate
peptides; LEHD-AMC for caspase-9 and DEVD-AMC for caspase-7. The
release of AMC was measured after incubating optical nanosensors in
picofuge tubes containing the cell lysates from the various
treatment groups and using a HeCd laser (excitation 325 nm) to
excite AMC. Caspase activity was expressed as fluorescence
intensity of AMC as a function of equivalent nanomoles of LEHD-AMC
and DEVD-AMC respectively.
[0626] The results of the in vitro measurement of caspase-9 and
caspase-7 activity were plotted. The curves for each fluorescent
measurement of AMC were plotted for each as a function of AMC
concentration. Caspase-9 activity was determined by incubation of
optical nanosensors with the substrate
LEHD-7-amino-4-methylcoumarin (AMC) in cell lysate (.about.10.sup.5
cells) obtained from the following treatment groups; group I, II,
III and IV, described earlier in the article. The release of AMC
was measured after excitation using HeCd laser (325 nm) and
collecting the fluorescence signal using a 380 nm longpass filter.
The peak emission wavelength of AMC is about 440 nm. Likewise,
Caspase-7 activity was determined by incubation in cell lysate
(.about.10.sup.5 cells) obtained from the following treatment
groups I, II, III, and IV. The release of AMC was measured after
excitation using a HeCd laser (325 nm) and collecting the
fluorescence signal using a 380 nm longpass filter.
[0627] In this experiment, we measure two sets of cells 1 and 11:
(1) Set 1 is treated with the drug ALA and (2) set II is treated by
the drug ALA conjugated to a PEPST probe described in the previous
section. By comparing the results (amount of caspase detected), one
can evaluate the efficiency of the PEPST-ALA drug compared to ALA
alone.
[0628] Additional modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described herein.
* * * * *
References