U.S. patent application number 13/136939 was filed with the patent office on 2012-02-23 for laser activated nanothermolysis of cells.
Invention is credited to Dmitri Lapotko, Alexander Oraevsky.
Application Number | 20120046593 13/136939 |
Document ID | / |
Family ID | 36692962 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120046593 |
Kind Code |
A1 |
Oraevsky; Alexander ; et
al. |
February 23, 2012 |
Laser activated nanothermolysis of cells
Abstract
Provided herein are methods and systems to increase selective
thermomechanical damage to a biological body, such as a cancer cell
or cell associated with a pathophysiological condition. The
biological body or cancer cell is specifically targeted with
nanoparticulates comprising one or more targeting moieties which
form nanoparticulate clusters thereon or therewithin. Pulsed
electromagnetic radiation, e.g., optical radiation, having a
wavelength spectrum selected for a peak wavelength near to or
matching a peak absorption wavelength of the nanoparticulates
selectively heats the nanoparticulates thereby generating vapor
microbubbles around the clusters causing damage to the targets
without affecting any surrounding medium or normal cells or
tissues. Also provided are methods for treating leukemia and for
selectively and thermomechanically causing damage to cells
associated with a pathophysiological condition using the methods
and system described herein.
Inventors: |
Oraevsky; Alexander;
(Houston, TX) ; Lapotko; Dmitri; (Minsk,
BY) |
Family ID: |
36692962 |
Appl. No.: |
13/136939 |
Filed: |
August 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11795856 |
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PCT/US2006/002186 |
Jan 22, 2006 |
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13136939 |
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60646018 |
Jan 22, 2005 |
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Current U.S.
Class: |
604/6.01 ;
435/173.1; 435/173.3; 435/283.1; 977/773; 977/810; 977/904 |
Current CPC
Class: |
A61K 47/6929 20170801;
A61K 41/0052 20130101; A61P 35/02 20180101; A61P 43/00 20180101;
A61K 47/6923 20170801; A61P 31/12 20180101; A61P 35/00 20180101;
Y10S 977/70 20130101; A61K 47/6873 20170801; A61K 47/6891 20170801;
Y10S 977/702 20130101; A61P 31/04 20180101; B82Y 5/00 20130101 |
Class at
Publication: |
604/6.01 ;
435/173.1; 435/173.3; 435/283.1; 977/773; 977/810; 977/904 |
International
Class: |
A61M 1/36 20060101
A61M001/36; C12M 1/42 20060101 C12M001/42; C12N 13/00 20060101
C12N013/00 |
Claims
1. A method for increasing selective therapeutic thermomechanically
induced damage to a biological body, comprising: specifically
targeting a biological body comprising a medium with a plurality of
nanoparticulates each conjugated to at least one targeting moiety,
said nanoparticulates effective to form one or more nanoparticulate
clusters on or in the biological body upon targeting thereto;
irradiating the biological body with at least one pulse of
electromagnetic radiation having a spectrum of wavelengths selected
to have a peak wavelength that is near to or that matches a peak
absorption wavelength of the nanoparticulates; and generating vapor
microbubbles from heat produced via absorption of the
electromagnetic radiation into the nanoparticulate cluster(s),
wherein the vapor microbubbles cause selective and increased
thermomechanical damage to the targeted biological body.
2. The method of claim 1, further comprising: filtering the
products of the thermomechanical damage from the medium.
3. The method of claim 1, further comprising: receiving a
photothermal signal or generating an optical image of
thermomechanical effects to monitor and to guide selective
thermomechanical damage to the biological body.
4. The method of claim 1, wherein the nanoparticulate has a
dimension of about 1 nm to about 1000 nm.
5. The method of claim 1, wherein the nanoparticulate cluster has a
total volume about 2 to about 200 times greater than a volume of
the nanoparticulate comprising the same.
6. The method of claim 1, wherein the nanoparticulate is a
spherical nanoparticle, a nanorod or a nanoshell at least partially
comprising gold or silver or is a carbon nanotube.
7. The method of claim 1, wherein the targeting moiety is a
monoclonal antibody or a peptide specifically targeted to a
receptor site on the biological body.
8. The method of claim 7, wherein the receptor site further
comprises another monoclonal antibody or peptide attached thereto
specific for the targeted monoclonal antibody.
9. The method of claim 7, wherein the nanoparticulate further
comprises complementary strands of a nucleic acid conjugated
thereto or a combination thereof.
10. The method of claim 7, wherein the nanoparticulate further
comprises PEG molecules.
11. The method of claim 1, wherein the wavelength spectrum is a
range of wavelengths of about 300 nm to about 300 mm.
12. The method of claim 1, wherein the pulse of electromagnetic
radiation is optical radiation.
13. The method of claim 12, wherein the pulse of optical radiation
has wavelength in the range from 500 nm to 1150 nm.
14. The methods of claim 1, wherein the pulse of electromagnetic
radiation is about 1 ns to about 100 ns in duration.
15. The method of claim 1, wherein the biological body is an
abnormal cell, a bacterium or a virus.
16. A system for increasing selective therapeutic thermomechanical
damage to abnormal cells, comprising: a chamber containing the
abnormal cells in a medium; a source of nanoparticulates modified
to specifically target the abnormal cells fluidly connected to the
cell chamber; an optical chamber adapted to contain the targeted
abnormal cells fluidly connected to the cell chamber; a pulsed
source of electromagnetic radiation directed against the targeted
cancer cells in the optical chamber, said source configured to emit
a spectrum of wavelengths selected to have a peak wavelength that
is near to or that matches a peak absorption wavelength of said
nanoparticulates; and means for filtering out cells damaged by
thermomechanical effects resulting from absorption of the
electromagnetic radiation emitted at the peak wavelength, said
filtering means fluidly connected to the cell chamber.
17. The system of claim 16, further comprising: means for receiving
a photothermal signal or for generating an optical image of the
thermomechanical effects.
18. The system of claim 16, wherein the nanoparticulates each
comprise at least one targeting moiety specifically targeted to a
receptor site on the cancer cell.
19. The system of claim 18, wherein the receptor site further
comprises another targeting moiety attached thereto specific for
said targeting moiety on the nanoparticulates.
20. The system of claim 18, wherein the targeting moiety is a
monoclonal antibody or a peptide attached thereto specific for the
targeted monoclonal antibody.
21. The system of claim 18, wherein the nanoparticulate further
comprises complementary strands of a nucleic acid conjugated
thereto or a combination thereof.
22. The method of claim 21, wherein the nanoparticulate further
comprises PEG molecules.
23. The system of claim 16, wherein the nanoparticulate has a
dimension of about 1 nm to about 1000 nm.
24. The system of claim 16, wherein the nanoparticulate is a
spherical nanoparticle, a nanorod or a nanoshell at least partially
comprising gold or silver or is a carbon nanotube.
25. The system of claim 16, wherein the wavelength spectrum is a
range of wavelengths of about 300 nm to about 300 mm.
26. The system of claim 16, wherein the pulse of electromagnetic
radiation is optical radiation having a wavelength in the range
from 500 nm to 1150 nm.
27. The system of claim 16, wherein the pulse of electromagnetic
radiation is about 1 ns to about 100 ns in duration.
28. The system of claim 16, wherein the abnormal cells are leukemic
cancer cells.
29. A method for treating a leukemia in an individual, comprising:
a) obtaining a sample comprising normal and leukemic cells from the
individual; b) placing the sample in the cell chamber of the system
of claim 16; c) targeting the cancer cells in the sample with the
modified nanoparticulates, said modified nanoparticulates forming
one or more clusters on or in the targeted cancer cell; d)
irradiating the targeted leukemic cells with electromagnetic
radiation emitted from the pulsed source, wherein the
electromagnetic radiation absorbed by the nanoparticulates causes
selective and increased thermomechanical effects damaging to the
targeted cancer cells, but not to the normal cells comprising the
sample; e) filtering out the damaged cells from the sample; f)
returning the normal cells remaining in the sample to the
individual thereby treating the leukemia; and. g) repeating said
method steps a) to f) zero or more times, thereby treating the
leukemia.
30. The method of claim 29, further comprising: receiving a
photothermal signal or generating an optical image of the
thermomechanical effects to monitor and to guide selective
thermomechanical damage to the cancer cells.
31. The method of claim 29, wherein the thermomechanical effects
are caused by heat generated within the nanoparticulates from
absorbed electromagnetic radiation sufficient to form vapor
microbubbles around the nanoparticulate clusters.
32. The method of claim 29, wherein the nanoparticulate cluster has
a total volume about 2 to about 200 times greater than a volume of
the nanoparticulate comprising the same.
33. A method for selectively and thermomechanically damaging cells
associated with a pathophysiological condition, comprising:
targeting the cells with a first monoclonal antibody specific
thereto; targeting the cells with gold nanoparticulates modified
with a second monoclonal antibody specific to the first monoclonal
antibody whereupon one or more clusters of the nanoparticulates
form on or in the targeted cells; heating one or more clusters of
gold nanoparticulates formed on or in the targeted cells; and
generating vapor bubbles around the heated clusters sufficient to
thermomechanically damage the cells.
34. The method of claim 33, further comprising photothermally or
optically monitoring the thermomechanical damage.
35. The method of claim 33, wherein the gold nanoparticulates are
spherical nanoparticles, nanorods or nanoshells.
36. The method of claim 33, wherein the clustered gold
nanoparticulates are heated with optical radiation having a
wavelength in a range from 500 nm to 1150 nm.
37. The method of claim 33, wherein the optical radiation is pulsed
for a duration of about 1 ns to about 100 ns.
38. The method of claim 33, wherein the nanoparticulate has a
dimension of about 1 nm to about 1000 nm.
39. The method of claim 33, wherein the nanoparticulate cluster(s)
has a total volume about 2 to about 200 times greater than a volume
of the nanoparticulate comprising the same.
40. The method of claim 33, wherein the cell is a cancer cell, a
bacterial cell or a virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part application under 35 U.S.C.
.sctn.120 of pending U.S. Ser. No. 11/795,856, filed Jul. 23, 2007,
which is a national stage application under 35 U.S.C. 371 of
international application PCT/US2006/002186, filed Jan. 22, 2006,
now abandoned, which claims benefit of priority under 35 U.S.C.
119(e) of provisional U.S. Ser. No. 60/646,018, filed Jan. 22,
2005, now abandoned, the entirety of all of which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the fields of medical
therapies employing electromagnetic radiation and of nanoparticles.
More specifically, the present invention relates to a method and a
system for electromagnetic radiation induced selective destruction
of abnormal biological bodies or structures utilizing bioconjugated
nanoparticles.
[0004] 2. Description of the Related Art
[0005] In a variety of medical applications, it is desirable to
inactivate, ablate or otherwise achieve the destruction and
elimination of abnormal cells, while preserving normal and healthy
cells. Recent advances in biomedical optics and nanotechnology has
created a solid basis for development of effective therapies for
human diseases, such as cancer or atherosclerosis. The major
breakthrough in this area is the possibility for a therapeutic
agent to target selectively certain types of cells with molecular
specificity. Furthermore, new imaging modalities are being
developed to visualize not only abnormal cells and tissues, but
also to monitor and guide therapeutic procedures, making them more
effective and safe. The prior art discloses laser (optical) methods
of therapy, which can be, in principle, guided by imaging based on
optical contrast or otherwise enabled by optical activation.
[0006] Selective and specific inactivation, that is, damage or
destruction, of cells with optical means requires substantial
optical contrast between target cells and all non-target normal
cells. General approaches to achieve cell inactivation include
thermomechanical damage through high-intensity pulsed laser
interaction with cells, thermal damage through continuous wave high
power interaction, such as used for hyperthermia and coagulation,
and biochemical damage through relatively low power interactions of
photons with molecules resulting in production of chemically active
species, such as ions, radicals and metastable excited states (1).
Similarly, these types of interactions can be the result of
interactions of electromagnetic radiation of various wavelengths,
e.g., X-rays, UV, visible light, near-infrared, infrared photons,
microwave, and radiofrequency quanta, and also high intensity
acoustic waves.
[0007] Laser ablation is based on thermal and mechanical effects
caused in cells by absorption of high-intensity laser pulses (2).
One of the advanced concepts of laser ablation, selective
photothermolysis, was introduced more than 20 years ago (3).
Selective photothermolysis employs short laser pulsed interaction
with absorbing tissue microstructures to induce localized physical
damage by avoiding thermal diffusion of the deposited laser energy.
Other advanced concepts of precise laser ablation with limited
thermal collateral damage to surrounding tissue employs conditions
of pressure confinement upon short pulse laser irradiation to
produce tensile wave causing cavitation bubbles at temperatures
below 100.degree. C. (4) or ultrashort laser pulses to cause
nonlinear absorption and rapid micro-explosion that occurs prior to
thermal diffusion (5). Slow heating with long pulses of
electromagnetic radiation can also be used for selective damage of
cells, however, due to thermal diffusion requires much stronger
optical contrast to achieve specificity and to not damage adjacent
cells or tissues (6). As an alternative to instantaneous
thermomechanical destruction of target cells, photodynamic therapy
employs low-intensity laser irradiation to produce necrosis and
apoptosis through delayed damage mechanisms caused by
photochemically produced toxic species, for example, radicals and
singlet oxygen (7).
[0008] Both high and low intensity interactions of laser radiation
with cells can be made selective and specific to target cells. Such
selectivity requires utilization of either endogenous or exogenous
chromophores, which strongly absorb specific colors of laser
spectrum in the visible and near-infrared, i.e. in the range of
wavelengths where majority of tissue constituents do not absorb.
Since endogenous tissue chromophores strongly absorbing in the red
and near-infrared are limited to hemoglobin and melanin, the
medical applications of natural contrast agents are limited to
blood vessels and retinal pigmented epithelium (8,9). Selective
damage of leukemia and other cancerous cells demands exogenous
contrast agents. Optical contrast agents that possess very strong
absorption of near-infrared radiation attract attention of
researchers because normal cells and tissues are transparent in
this spectral range, so there is a great potential to achieve an
exceptional selectivity of cell damage through the contrast agent.
Chromophore-assisted laser inactivation (CALI) is a term to
describe selective inactivation of certain proteins in cellular
membranes using laser irradiation of cells stained with molecular
dyes strongly absorbing red laser pulses (10).
[0009] It was recently revealed that gold nanoparticles can be
designed to absorb any desirable color of near-infrared radiation
by either changing the thickness gold shells on the silica core
(11) or changing the aspect ratio of gold nanorods, i.e.,
ellipsoids or other prisms with one elongated axis (12). Gold
nanoparticles and especially silver nanoparticles absorb
near-infrared light much stronger than nanoparticles of organic
dyes, which makes them superior contrast agents for imaging a small
cluster of cancer cells in the depth of tissue (13). Nanoparticle
assisted selective laser thermolysis of cells was recently
demonstrated by targeting optically absorbing nanoparticles to cell
surface receptors and superheating them with laser pulses (14).
Based on the experimental results obtained with microparticles, the
prior art speculated that the cavitation bubble generation that
results in cell inactivation after laser irradiation with certain
optical fluence may depend on particle size. On the other hand, the
prior art neither provides an explanation of the underlying
physical phenomena, nor provides a solution for achieving highly
effective cell damage using low threshold fluence of
electromagnetic radiation.
[0010] U.S. Pat. No. 6,530,944 teaches optically active
nanoparticles that can be used in therapeutic and diagnostic
methods. However, therapeutic applications disclosed by West et al.
are limited to methods of hyperthermia, i.e. slow heating usually
with continuous wave lasers and other optical sources.
Nanoparticles will extravasate preferentially at locations where
the blood vessel walls have increased porosity or have
microvascular surface changes, especially at tumor sites. O'Neal et
al. demonstrated that intravenous injection of gold nanoshells,
which are nanoparticles strongly absorbing in the near-infrared
spectral range, into a mouse, resulted in nonspecific, but
effective targeting of an implanted tumor. Further, it enabled
hyperthermic damage of the tumor through heating the nanoshells to
temperatures several degrees higher than that in surrounding tissue
via continuous wave laser illumination of the tumor area (15). U.S.
Pat. No. 6,165,440 taught that a combination of radiation and
nanoparticles or microparticles could be used to temporarily open
pores in cancer cell membranes and blood vessels to allow better
penetration of drugs into solid tumors.
[0011] U.S. Pat. Nos. 6,530,944 and 6,699,724 disclosed optical
diagnostic uses of nanoparticles that emit near-infrared light,
i.e., nanodots or absorb near-infrared red light, i.e., nanoshells.
Sokolov et al. utilized the capability of gold nanoparticles to
reflect light strongly and thereby enhance contrast of abnormal or
cancerous tissue specifically targeted with nanoparticles
conjugated with monoclonal antibody (16). Oraevsky et al. (17,18)
predicted that various nanoparticles can enhance optical absorption
in tissue and emit thermoacoustic waves, which in turn can be
utilized in optoacoustic imaging of tissue. U.S. Patent Pub. No.
20050175540 described non-spherical nanoparticles and a method by
which to optoacoustically detect the presence of objects as small
as 1-mm in a body, which can be penetrated by electromagnetic
radiation. It was discovered that at least partially metallic
nanoparticulates fabricated or manipulated to be non-spherical not
only will shift the optical absorption spectrum into the
near-infrared range for deeper penetration of radiation into a
body, but also will both narrow the absorption band and
simultaneously increase the effective absorbance, in certain
instances by more than an order of magnitude. This greatly
increases the optoacoustic efficacy of the nanoparticulate, making
the manipulated nanoparticulate a very high contrast optoacoustic
imaging agent.
[0012] U.S. Pat. Nos. 5,213,788, 5,411,730, 5,427,767, 5,521,289,
6,048515, 6,068,857, 6,165,440, 6,180,415, 6,344,272, 6,423,056,
and 6,428,811 disclose various electromagnetically active
nanoparticles for use as therapeutic or imaging contrast agents. It
was established by many groups that specific targeting using
antibodies increases efficacy of anticancer therapies (19). In
addition to IgG type antibodies, short peptides can be used as
targeting vectors (20).
[0013] It is well recognized that any minimally invasive therapy
may benefit from imaging methods that can precisely guide the
therapeutic procedure. Lapotko et al. have developed methods of
photothermal detection and imaging that enable one to visualize
individual cells and thermomechanical processes that occur in cells
upon pulsed laser irradiation (21-22). U.S. Pat. Nos. 5,840,023 and
6,309,352 taught a method and a system of optoacoustic imaging that
helps detection, localization and real-time monitoring of abnormal
tissue in the depth of normal tissue.
[0014] Chemotherapy and radiotherapy are often ineffective in
treating human cancer, including hematological malignancies, such
as leukemia. The state of the art therapy methods and systems have
serious limitations, associated with significant treatment toxicity
and generation of drug-resistant tumor cells (24-27). The residual
cells, therefore, must be eliminated from blood or bone marrow
grafts by methods generally called "purging". Available purging
methods employ pharmacological and photochemical (PDT) treatment,
magnetic and fluorescence based sorting and elimination in cuvette
through adsorption to mab attached to its bottom. These methods
provide help and relief to cancer patients, but do not provide
sufficient efficacy, i.e. 100% elimination, or adequate speed of
cell elimination (28). New treatment strategies that are more
effective, faster and less expensive are therefore necessary to
overcome these problems.
[0015] However, there is a recognized need in the art for an
effective and safe method and system that provides selective lysis,
i.e., destruction, of targeted abnormal cells and other
microstructures or microbodies while leaving all normal cells
intact, using electromagnetic radiation. Specifically, the prior
art is deficient in therapeutic laser methods and systems utilizing
nanoparticulate contrast agents. More specifically, the prior art
is deficient in methods and systems of Laser Activated
Nano-Thermolysis Cell Elimination Technology (LANTCET) that utilize
metal nanoparticles for laser therapy of cancer. The present
invention fulfils this longstanding need in the art.
SUMMARY OF THE INVENTION
[0016] The present invention is directed to a method for increasing
selective therapeutic thermomechanically induced damage to a
biological body. The method comprises specifically targeting a
biological body comprising a medium with a plurality of
nanoparticulates each conjugated to at least one targeting moiety.
The nanoparticulates are effective to form one or more
nanoparticulate clusters on or in the biological body upon
targeting thereto. The biological body is irradiated with at least
one pulse of electromagnetic radiation having a spectrum of
wavelengths selected to have a peak wavelength that is near to or
that matches a peak absorption wavelength of the nanoparticulates.
Subsequently, vapor microbubbles are generated from heat produced
via absorption of the electromagnetic radiation into the
nanoparticulates such that the microbubbles cause selective and
increased thermomechanical damage to the targeted biological body.
In a related invention the method further may comprise filtering
the products of the thermomechanical damage from the medium. In
another related invention the method further may comprise receiving
a photothermal signal or generating an optical image of
thermomechanical effects to monitor and to guide selective
thermomechanical damage to the biological body.
[0017] The present invention also is directed to a system for
increasing selective therapeutic thermomechanical damage to
abnormal cells. The system comprises a chamber containing the
abnormal cells in a medium, a source of nanoparticulates adapted to
specifically target the abnormal cells which is fluidly connected
to the cell chamber, an optical chamber adapted to contain the
targeted abnormal cells which is fluidly connected to the cell
chamber, and a means for filtering out cells damaged by
thermomechanical effects resulting from absorption of the
electromagnetic radiation emitted at the peak wavelength which is
fluidly connected to the cell chamber. A pulsed source of
electromagnetic radiation is directed against the targeted cancer
cells in the optical chamber, where the source is configured to
emit a spectrum of wavelengths selected to have a peak wavelength
that is near to or that matches a peak absorption wavelength of the
nanoparticulates. In a related invention the system further
comprises a means for receiving a photothermal signal or for
generating an optical image of the thermomechanical effects.
[0018] The present invention is directed further to a method for
treating a leukemia in an individual. The method comprises
obtaining a sample comprising normal and leukemic cells from the
individual and placing the sample in the cell chamber of the system
described herein. The cancer cells in the sample are targeted with
the nanoparticulates described herein and the targeted cancer cells
are irradiated with electromagnetic radiation emitted from the
pulsed source comprising the system. The electromagnetic radiation
is absorbed by the nanoparticulates thereby causing selective and
increased thermomechanical effects damaging to the targeted cancer
cells, but not to the normal cells comprising the sample. The
damaged cells are filtered out from the sample and the normal cells
remaining in the sample are returned to the individual thereby
treating the leukemia. The method steps may be repeated zero or
more times. In a related invention the method further may comprise
receiving a photothermal signal or generating an optical image of
thermomechanical effects to monitor and guide selective
thermomechanical damage to the biological body.
[0019] The present invention is directed further still to a method
for selectively and thermomechanically damaging cells associated
with a pathophysiological condition. The method comprises targeting
the cells with a first monoclonal antibody specific thereto and
targeting the cells with gold nanoparticulates described herein
that are modified with a second monoclonal antibody specific to the
first monoclonal antibody whereupon one or more clusters of the
nanoparticulates form on or in the targeted cells. One or more of
the clusters of gold nanoparticulates formed on or in the targeted
cells are heated and vapor microbubbles are generated around the
heated clusters sufficient to thermomechanically damage the cells.
In a related invention, the method further comprises photothermally
or optically monitoring the thermomechanical damage.
[0020] Other and further aspects, features, benefits, and
advantages of the present invention will be apparent from the
following description of the presently preferred embodiments of the
invention given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The appended drawings have been included herein so that the
above-recited features, advantages and objects of the invention
will become clear and can be understood in detail. These drawings
form a part of the specification. It is to be noted, however, that
the appended drawings illustrate preferred embodiments of the
invention and should not be considered to limit the scope of the
invention.
[0022] FIGS. 1A-1B show the calculated optical absorption spectra
of gold (FIG. 1A) and of silver (FIG. 1B) nanorods. With an
increasing aspect ratio, the peak of plasmon resonance absorption
gradually shifts to longer wavelengths in the near-infrared. Aspect
ratios left to right in graphs: 2a=20 nm, 2c=60 nm; 2a=20 nm,
2c=100 nm; 2a=20 nm, 2c=140 nm.
[0023] FIGS. 2A-2C show electron microphotographs (FIGS. 2A-2B) of
gold nanorods with diameter of 15 nm and 2 different aspect ratios,
3.2 and 6.9, placed on the surface of a glass slide, and an
experimentally measured optical absorption spectra (FIG. 2C) of
these gold nanorods, conjugated with PEG and suspended in
water.
[0024] FIG. 3 shows optoacoustic signal as a function of laser
fluence incident upon suspension of spherical gold nanoparticles
with 100 nm diameter. Absorption cross-section of these
nanoparticles is .sigma..sub..alpha.=1.4.times.10.sup.-9
cm.sup.2.
[0025] FIG. 4 shows threshold fluence for the laser damage of tumor
cells targeted with gold nanoparticles that formed clusters inside
the target cells.
[0026] FIG. 5 is a sketch of a LANTCET system modified from a blood
dialysis system.
[0027] FIGS. 6A-6B comprise a schematic diagram of LANTCET
depicting simultaneous monitoring and guidance by photothermal
microscopy.
[0028] FIGS. 7A-7B are electron microscopy images of K562 cells.
FIG. 7A is a control cell without nanoparticles and FIG. 7B is a
cell specifically targeted with primary MABs (CD 15 and glycophorin
A) and 30 nm diameter spherical gold nanoparticles conjugated to
secondary MAB. Small black dots are single nanoparticles, larger
black spots are clusters of nanoparticles.
[0029] FIGS. 8A-8C illustrate PT responses obtained after a single
laser pulse at 532-nm wavelength and duration of 10-ns. FIG. 3A
shows a single K562 cell targeted with bare nanoparticles--no
bubble, no damage at optical fluence of 35 J/cm2. FIG. 8B shows a
suspension of 30-nm diameter single gold nanoparticles; optical
fluence of 35 J/cm.sup.2. FIG. 8C shows a single K562 specifically
targeted cell with clusters of NP, optical fluence of 5 J/cm2.
[0030] FIG. 9 illustrates the cell damage probabilities, DP,
experimentally obtained for different pump laser pulse (532 nm, 10
ns) fluence levels for specifically targeted common B acute
lymphoblasts (lymphoblasts) and normal stem cells, for specifically
targeted K562 cells (test), for non-specifically targeted K562
cells (control #1), for K562 cells targeted with bare NP (control
#2) and for untargeted K562 cells (control #3).
[0031] FIGS. 10A-10B are optical microscopic images of normal stem
cells (FIG. 10A) and common B acute lymphoblasts (FIG. 10B) in the
cuvette after irradiation of cell suspension with a single broad
laser pulse with optical fluence of 1.7 J/cm2. Cells were prepared
using specific targeting protocol.
[0032] FIGS. 11A-11B are schematic diagrams of cell irradiation and
laser photothermal microscopy for a single cell (FIG. 11A) and for
a cell suspension (FIG. 11B) of ALL tumor cells and normal
cells.
[0033] FIGS. 12A-12F are optical (FIGS. 12A-12B) and fluorescent
(FIGS. 12C-12D) images of human leukemia cells (B-lymphoblasts) and
corresponding fluorescence signal profiles (FIGS. 12E-12F) for
images obtained after the stage 1, incubation at 48 C (FIGS. 12A,
12C, 12E) and after the stage 2, incubation at 378 C (FIGS. 12B,
12D, 12F). White lines on the cell images show the cross-section
for amplitude profile and cell boundaries (nuclei and of outer
membranes).
[0034] FIGS. 13A-13B are histograms of fluorescent image parameters
of tumor cells: the maximal values of pixel amplitude in peaks Max
(FIG. 13A) and radial distribution Mir of fluorescent peaks (FIG.
13B).
[0035] FIGS. 14A-14B illustrate the kinetics of the clusterization
of NPs during cell incubation at 48.degree. C. (stage 1) and
37.degree. C. (stage 2) for normal BM cells (FIG. 14A) and tumor
cells (FIG. 14B) (O-37.degree. C., peaks at membrane, -37.degree.
C., peaks inside cell, .DELTA.-4.degree. C., peaks at membrane,
.tangle-solidup.4.degree. C., peaks inside cell).
[0036] FIGS. 15A-15C illustrate a typical bubble-specific PT
response (FIG. 15A), PT-image (FIG. 15B), and optical image of same
cell before irradiation (FIG. 15C) obtained for a tumor cell after
single laser pulse for the incubation conditions being 37.degree.
C., 2 hours.
[0037] FIGS. 16A-16B illustrate the influence of incubation
parameters on cell damage. FIG. 16A shows the level of survived
live tumor cells LLC experimentally obtained after a single laser
pulse (532 nm, 0.6 J/cm2) for primary MABs CD19, CD20 and CD 22
applied during the first stage of cell targeting with spherical
nanoparticles. FIG. 16B shows the level of survived live cells LLC
experimentally obtained for tumor CD10+ cells and normal BM cells
after their irradiation with single laser pulse (532 nm, 0.6 J/cm2)
at different temperatures during the second stage of
incubation.
[0038] FIGS. 17A-17C are images and spectra of K562 myeloid culture
cells obtained with an optical scattering microscope. FIG. 17A
shows a cell without nanorods. FIG. 17B shows a cell with
cytoplasm-located clusters of gold nanorods. FIG. 17C is an optical
scattering spectra for intact cells (red) and for nanorod clusters
in the cell (black).
[0039] FIGS. 18A-18D are optical microscopic images of an AML cell
before (FIG. 18A) and 5 sec after (FIG. 18D) cell treatment with
single laser pulse (10 ns, 780 nm and a photothermal image (FIG.
18B) and response (FIG. 18C) of laser-induce PTB in the cell shown
in FIG. 18B.
[0040] FIGS. 19A-19B illustrate bubble generation probability
spectra (FIG. 19A) and bubble lifetime spectra (FIG. 19B) for
single gold nanorods and human bone marrow tumor cells.
[0041] FIGS. 20A-20D are optical microscopic images of Hep-2C cell
before (FIG. 20A) and 5 sec after (FIG. 20D) cell treatment with
single laser pulse (10 ns, 720 nm) and a photothermal image (FIG.
20B) and response (FIG. 20C) of laser-induce PTB in the cell shown
in FIG. 20B.
[0042] FIGS. 21A-21B are vertical cross sections of the tumor that
was treated with spherical nanoparticles and laser pulse (FIG. 21A)
and the control tumor which was not treated with spherical
nanoparticles but was treated with the laser (FIG. 21B). Blue is an
intact area and white is a necrotic area, scale bar in mm.
DETAILED DESCRIPTION OF THE INVENTION
[0043] In one embodiment of the present invention there is provided
a method for increasing selective therapeutic thermomechanically
induced damage to a biological body, comprising specifically
targeting a biological body comprising a medium with a plurality of
nanoparticulates each conjugated to at least one targeting moiety,
where the nanoparticulates are effective to form one or more
nanoparticulate clusters on or in the biological body upon
targeting thereto; irradiating the biological body with at least
one pulse of electromagnetic radiation having a spectrum of
wavelengths selected to have a peak wavelength that is near to or
that matches a peak absorption wavelength of the nanoparticulates;
and generating vapor microbubbles from heat produced via absorption
of the electromagnetic radiation into the nanoparticulates where
the vapor microbubbles cause selective and increased
thermomechanical damage to the targeted biological body.
[0044] Further to this embodiment the method may comprise filtering
the products of the thermomechanical damage from the medium. In
another further embodiment the method may comprise receiving a
photothermal signal or generating an optical image of
thermomechanical effects to monitor and guide selective
thermomechanical damage to the biological body. In all embodiments
the biological body may be an abnormal cell, a bacterium or a
virus.
[0045] In these embodiments the nanoparticulates may have a
dimension of about 1 nm to about 1000 nm. Also, the nanoparticulate
cluster may have a total volume about 2 to about 200 times greater
than a volume of the nanoparticulate comprising the same. In
addition, the nanoparticulate may be a spherical nanoparticle, a
nanorod or a nanoshell at least partially comprising gold or silver
or is a carbon nanotube.
[0046] Furthermore, in all embodiments the nanoparticulates
comprise a targeting moiety that is a monoclonal antibody or a
peptide specifically targeted to a receptor site on the biological
body. In an aspect, the receptor site further comprises another
monoclonal antibody or peptide attached thereto specific for the
targeted monoclonal antibody. Further to all embodiments the
nanoparticulates may comprise complementary strands of a nucleic
acid conjugated thereto or a combination thereof. Further still the
nanoparticulate may comprise PEG molecules.
[0047] In all embodiments the wavelength spectrum of the pulse of
electromagnetic radiation may have a range of wavelengths of about
300 nm to about 300 mm. In an aspect the pulse of
electricalmagnetic radiation is optical radiation. This optical
radiation may have a wavelength in the range from 500 nm to 1150
nm. Also, in all embodiments the pulse of electromagnetic radiation
is about 1 ns to about 100 ns.
[0048] In another embodiment of the present invention there is
provided a system for increasing selective therapeutic
thermomechanical damage to abnormal cells, comprising a chamber
containing the abnormal cells in a medium; a source of
nanoparticulates modified to specifically target the abnormal cells
fluidly connected to the cell chamber; an optical chamber adapted
to contain the targeted cancer cells fluidly connected to the cell
chamber; a pulsed source of electromagnetic radiation directed
against the targeted cancer cells in the optical chamber, where the
source is configured to emit a spectrum of wavelengths selected to
have a peak wavelength that is near to or that matches a peak
absorption wavelength of the nanoparticulates; and means for
filtering out cells damaged by thermomechanical effects resulting
from absorption of the electromagnetic radiation emitted at the
peak wavelength, where the filtering means is fluidly connected to
the cell chamber. In a further embodiment the system comprises a
means for receiving a photothermal signal or for generating an
optical image of the thermomechanical effects.
[0049] In these embodiments the nanoparticulates each comprise at
least one targeting moiety specifically targeted to a receptor site
on the cancer cell. Furthermore, the receptor site may comprise
another targeting moiety attached thereto which is specific for the
targeting moiety on the nanoparticulates. Examples of a targeting
moiety are a monoclonal antibody or a peptide. In another further
embodiment the nanoparticulates further may comprise complementary
strands of a nucleic acid conjugated thereto or a combination
thereof. Further still the nanoparticulate may comprise PEG
molecules.
[0050] In all embodiments the abnormal cells may be leukemic cancer
cells. Also, in all embodiments the dimensions, shapes, or metal or
carbon compositions of the nanoparticulates or nanoparticulate
clusters are as described supra. Furthermore, the spectrum
wavelengths and types of electromagnetic radiation and time of
pulse duration are as described supra.
[0051] In yet another embodiment of the present invention there is
provided a method for treating a leukemia in an individual,
comprising a) obtaining a sample comprising normal and leukemic
cells from the individual; b) placing the sample in the cell
chamber of the system described supra; c) targeting the leukemic
cells in the sample with the modified nanoparticulates comprising
the system; d) irradiating the targeted leukemic cells with
electromagnetic radiation emitted from the pulsed source comprising
the system, where the electromagnetic radiation absorbed by the
nanoparticulates causes selective and increased thermomechanical
effects damaging to the targeted cancer cells, but not to the
normal cells comprising the sample; e) filtering out the damaged
cells from the sample; f) returning the normal cells remaining in
the sample to the individual; and g) repeating the method steps a)
to f) zero or more times, thereby treating the leukemia.
[0052] Further to this embodiment the method comprises receiving a
photothermal signal or generating an optical image of the
thermomechanical effects to monitor and guide selective
thermomechanical damage to the cancer cells. In both embodiments
the thermomechanical effects are caused by heat generated within
the nanoparticulates from absorbed electromagnetic radiation
sufficient to form vapor microbubbles around the clusters.
[0053] In yet another embodiment of the present invention there is
provided a method for selectively and thermomechanically damaging
cells associated with a pathophysiological condition, comprising
targeting the cells with a first monoclonal antibody specific
thereto; targeting the cells with gold nanoparticulates modified
with a second monoclonal antibody specific to the first monoclonal
antibody whereupon one or more clusters of the nanoparticulates
form on or in the targeted cells; heating one or more clusters of
gold nanoparticulates formed on or in the targeted cells; and
generating vapor bubbles around the heated clusters sufficient to
thermomechanically damage the cells. Further to this embodiment,
the method may comprise photothermally or optically monitoring the
thermomechanical damage. In both embodiments, the gold
nanoparticulates and nanoparticulate clusters and the optical
radiation are as described supra. Also, in both embodiments, the
cells associated with a pathophysiological condition may be a
cancer cell, such as a leukemic or solid tumor cancer cell, a
bacterial cell or a virus.
[0054] As used herein, the term "a" or "an", when used in
conjunction with the term "comprising" in the claims and/or the
specification, may refer to "one," but it is also consistent with
the meaning of "one or more," "at least one," and "one or more than
one." Some embodiments of the invention may consist of or consist
essentially of one or more elements, method steps, and/or methods
of the invention. It is contemplated that any method or composition
described herein can be implemented with respect to any other
method or composition described herein.
[0055] As used herein, the term "or" in the claims refers to
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0056] As used herein, the term "nanothermolysis" refers to damage,
ablation, destruction of biological target cells assisted and
enabled by nanoparticles.
[0057] As used herein, the phrase "Laser Activated
Nano-Thermolysis" refers to selective damage of cells using
nanoparticles (nanoparticles) targeted to specific receptors
expressed in cancer cells, but not in normal cells.
[0058] As used herein, the phrase "cell elimination" refers to a
successful treatment procedure. Thus, as used herein, the phrase
"Laser Activated NanoThermolysis Cell Elimination Technology" or
"LANTCET" refers to a method and a system for electromagnetic
radiation induced selective destruction of abnormal biological
structures utilizing bioconjugated nanoparticles, such as tissues,
cells, bacteria and viruses. Because of their selectivity for
abnormal biological structures, LANTCET may be applied repeatedly
to insure or achieve a desired level of cell elimination, up to and
including complete cell elimination, although a single LANTCET
application with zero repeats is well within the scope of the
invention.
[0059] As used herein, the term "nanoparticulate" refers to a
single nanoparticle, a collection of nanoparticles, or a
nanoparticle aggregate. As used herein, the term "nanoparticulate
cluster" describes specific aggregation of nanoparticles in which
nanoparticles may touch each other or be in proximity to each
other, so that when irradiated with laser pulses or other pulses of
electromagnetic energy, they present themselves as a single
unresolved source of thermal energy.
[0060] As used herein, the phrase "at least partially metallic"
refers to a preferred nanoparticulate effective to absorb
electromagnetic radiation by the plasmon resonance mechanism, which
is known to yield very strong absorption coefficient.
[0061] Provided herein are methods and systems using Laser
Activated Nano-Thermolysis Cell Elimination Technology or LANTCET
for the electromagnetic radiation induced selective destruction and
purging of abnormal biological structures, such as tissues, cells,
bacteria and viruses. LANTCET has advantages over current methods
of purging as shown in Table 1.
TABLE-US-00001 TABLE 1 Magnetic Flow Method separators Cytometry
PDT LANTCET Rate, cell/s 10.sup.7 10.sup.4 10.sup.7 10.sup.7
Volume, ml 50-100 50 >1000 >1000 per one cycle Max amount of
10.sup.9 10.sup.6 10.sup.10 10.sup.10 treated cells Efficacy, % of
90 99.9 95 99.9 labeled cells Safety Additional Potential Poten-
Potential chemical cell tially laser treatment of the damage toxic
for damage to cells for removing at high normal 0.1-3% of magnetic
particles rate cells normal cells may damage normal cells
[0062] Although magnetic elimination is not damaging to normal
cells, its main disadvantage is that efficacy of this method is not
sufficient for purging residual cancer cells due to a lack of
strong magnetic force that separates small ferrous beads coupled to
the target cells from normal untargeted cells. The main advantage
of flow cytometry is its high efficiency of cell sorting, however,
the low throughput of this method is a limiting factor.
Photodynamic therapy lacks sufficient specificity to target cells
due to low contrast provided by organic dyes. Overall, these
methods and systems lack means for high through-put that is
effective, specific for the target cells and safe.
[0063] In a LANTCET method, strongly absorbing nanoparticles
provide contrast relative to untargeted cells that is unmatched by
any other contrast agent. Furthermore, formation of clusters of
nanoparticles in cells makes laser thermolysis of the target cells
by microbubbles a very effective, low threshold process that is
harmless to normal untargeted cells. Selectivity is conferred by
targeting an abnormal biological structure with bioconjugated
nanoparticles. These nanoparticles are targeted to molecular
receptors on the surface of the target cells using antibodies, such
as immunoglobulin type proteins, or short peptides, which help to
achieve selectivity of nanoparticle accumulation in cells.
[0064] The targeting protocol is designed to produce clusters of
nanoparticles on the surface of cells and/or inside the abnormal
cells in order to enhance efficacy of thermomechanical interactions
of electromagnetic radiation with the nanoparticles. Such
enhancement results in a substantially lower threshold of the
fluence or the power required to achieve the abnormal cell damage.
This in turn increases specificity and probability of damage to the
targeted cell damage and of safety to normal cells and tissues. The
methods and systems described herein have a variety of applications
including, but not limited to, minimally invasive therapy of
cancer, such as eliminating early subsurface tumors from tissue and
purging leukemia cells from bone marrow graft transplants or
blood.
[0065] Generally, the methods and systems described herein are
effective to substantially increase the thermomechanical damage to
an abnormal cell, biological body or microbody or microstructure,
such as, but not limited to, a biological cell, e.g., a cancer or
tumor cell, a bacterium or other microorganism, a virus, or
atherosclerotic plaques, resulting from a pulse of electromagnetic
radiation absorbed by the nanoparticles and to decrease the damage
to the surrounding normal body. The abnormal cell, biologic body,
cell or virus may be associated with a pathophysiological
condition, such as, but not limited to a cancer or other
malignancy, a bacterial or viral infection or atherosclerosis. More
particularly, the methods and systems described herein include the
following components and steps.
Nanoparticles
[0066] The nanoparticulates provided herein are designed with the
maximum capability to absorb electromagnetic radiation of a
wavelength effective to penetrate the body, but not to be absorbed
by molecular content of the body. That is, the nanoparticulates
must absorb electromagnetic radiation very strongly, so that such
absorption is sufficient to superheat them well above the boiling
point of the surrounding biological medium, e.g., water or a water
like medium, with a fluence of energy that produces insignificant
direct heating of the surrounding medium. Nanoparticulates that
strongly absorb electromagnetic radiation in the near-infrared
spectral range, the range of wavelengths where molecular
constituents of biological cells and tissues possess no or minimum
absorption are used. Preferably, the nanoparticles may be at least
partially metal nanoshells, metal nanorods or carbon nanotubes.
[0067] One skilled in the art can predict that many combinations
and complex structures can be created based on basic properties of
nanoparticles known in the art. It is contemplated that clusters of
other nanoparticulates, such as carbon nanotubes and liposomes
filled with organic dyes, can be utilized for LANTCET since
clusters of these particles can strongly absorb near-infrared
radiation. Although, the near-infrared spectral range seems to be
the preferred range based on what is known in the art,
nanoparticles may be designed to absorb x-rays, microwave (RF)
radiation or visible radiation. The absolute value of the
absorption coefficient for a given wavelength of electromagnetic
radiation is not as important as the contrast, that is, difference,
ratio between the absorption coefficient in the nanoparticulate and
the background surrounding medium or body that was not targeted
with said nanoparticulate.
[0068] The most preferred materials for nanoparticulate composition
are gold and silver and the most preferred shape of the
nanoparticulate is an elongated asymmetric shape, such as nanorods
or more complex structures involving nanorods, such as nanostars
and nanourchins. However, a symmetric composition, such as
spherical, particles are not excluded. For example, nanorods with
aspect ratio close to one are spheres, which have peak optical
absorption in the green spectral range. Formed nanoparticulate
clusters may be spherical or aspherical in three-dimensional space,
the formed shape may be fractal or chaotic and may be a combination
of various aggregate shapes and structures.
[0069] The most optimal nanoparticles in terms of maximum
absorption in the near-infrared spectral range are the silver
nanorods. Also, silver nanorods or more complex elongated silver
nanostructures can be designed to absorb near-infrared very
strongly, i.e., with an absorption cross-section up to 100 times
the physical cross-section. However, silver is not a completely
inert metal and can be toxic to normal cells, if used in large
concentration. The most optimal nanoparticles in terms of minimal
toxicity in the absence of radiation are gold nanoparticles.
Furthermore, gold nanorods or more complex structures encompassing
gold nanorods, such as gold nanostars or nanourchins, can be
designed to absorb near-infrared very strongly such that the
absorption cross-section exceeds the physical cross-section several
times over, next only to silver nanostructures.
[0070] The dimensions of the nanoparticulate are determined from
its size which must be sufficiently small to be suspended in a
water solution of an appropriate surfactant, e.g., PEG, as compared
to pores in biological tissues and blood vessels, so that the
nanoparticulate can diffuse through tissue and to be effectively
endocytosed by cells, and so that effective targeting of
nanoparticles to cell receptors can be accomplished. The size of
the nanoparticle must effective for absorbing electromagnetic
radiation of chosen wavelength due to plasmon resonance, which
requires that the maximum characteristic dimension of the
nanoparticulate will be smaller than the wavelength. Some
nonmetallic nanoparticles, such as semiconductor carbon nanotubes
do not have limitation of size for absorption, but any one skilled
in the art of radiation absorption can conclude that there is an
optimal size of any particle beyond which absorption of radiation
will be less effective and less homogeneous inside the particle.
Thus, the nanoparticulate must be no smaller than 1-2 nm and no
larger than 1000 nanometers. Nanoparticulate clusters, also
provided by the instant invention, may be larger than a single
nanoparticulate, i.e. have dimensions of up to several microns.
[0071] A preferred nanoparticulate is a partially metallic
nanoparticulate with an elongated shape, i.e. with an aspect ratio
greater than 1, which may be a collection of nanoparticles. A
non-spherical nanoparticulate comprising a nanoparticle aggregate
does not require that the nanoparticles of the aggregate be
non-spherical. The nanoparticles of the aggregate may comprise
spherical nanoparticles ordered in a structure to have the
properties of the nanoparticulate disclosed herein. Examples of
nanoparticulates are spherical nanoparticles or nanospheres,
nanorods or nanoshells.
[0072] Particularly, a nanoparticulate aggregate is so ordered and
the nanoparticles are at least partially coated with an organic
material suitably comprising complementary molecules with high
affinity to each other to ordain such order. For example, a
collection of spherical nanoparticles may be aggregated as an
elongated nanoparticulate, which shift their optical absorption as
a function of the aspect ratio, i.e. ratio of small axis length to
long axis length. One example of elongated nanoparticulate is gold
or silver nanorods. One skilled in the art can appreciate that
these types of nanoparticulates have tunable absorption in the
near-infrared spectrum of electromagnetic radiation and that their
absorption peaks are narrow and very strong, i.e., much stronger
than those of biological molecules. These properties are beneficial
for application in the Laser Activated Nano-Thermolysis Cell
Elimination Technology.
[0073] A nanoparticulate used in this invention may be combinations
of nanoparticles of one shape with nanoparticles of another shape
to form nanoparticulate geometries effective to absorb a selected
specific wavelength or range of wavelengths and further form
nanoparticulate clusters, which help to produce microbubbles within
target cells, which in turn produce maximum thermomechanical damage
by laser or other electromagnetic pulses. Thus, for medical or
biological applications the details of both dimension and shape are
important to LANTCET, since these parameters enable efficient
accumulation of nanoparticulate clusters in the target body, such
as abnormal biological cell.
[0074] The nanoparticulate may be at least partially metallic" and
be effective to absorb electromagnetic radiation by the plasmon
resonance mechanism. Alternatively, the present invention
encompasses nanoparticles, such as carbon nanotubes, that possess
properties of semiconductors and yet have very strong optical
absorption at various wavelengths of electromagnetic radiation.
Either are effective in the LANTCET methods and systems described
herein.
Bioconjugation
[0075] The present invention encompasses the use of nanoparticles
or aggregates of nanoparticles that are conjugated with biological,
i.e., organic material. The purpose of such bioconjugation is to
(i) produce nanoparticulates that are well suspended in water, (ii)
to target specific receptors in abnormal cells and (iii) to form
clusters of nanoparticles inside cells or on the cell surface, but
not outside the cells in suspension. It is desirable that the
nanoparticles have multiple molecules conjugated to their surfaces
to optimize the biological and chemical properties of the particles
and to maximize the desired formation of clusters inside target
cells, but not to form aggregates outside the cells. Coated metal,
partially metal and nonmetal nanoparticles or aggregates of these
nanoparticles as contrast agents for laser activated
nanothermolysis, i.e. selective thermomechanical damage to target
abnormal cells, may be used.
[0076] Conjugated nanoparticles may have coatings that are
covalently bound to the surface of the particles and/or coatings
that physically adhere to the surface of the particles. One bond
used most frequently in conjugation of gold and other metals to
biological molecules is a dative S.dbd. bond provided by the thiol
--SH group or sulfhydryl group. U.S. Pat. Nos. 6,821,730, 6,689,338
and 6,315,978 and others (29-34) teach methods of nanoparticle
bioconjugation for a variety of biomedical applications. One
skilled in the art may predict that numerous techniques exist to
conjugate nanoparticles with biological molecules so that such
conjugation is chemically stable upon administration of said
nanoparticulates in vitro and in vivo. Such conjugated
nanoparticulate also must be nontoxic in the absence of radiation
and be unrecogniseable as foreign by the human (or animal) immune
system to protect them from being scavenged by the immune system
before they reach the target body.
[0077] In addition proteins or other biological molecules may be
used as surfactants. Particularly desirable surfactants are block
copolymers, especially block copolymers in which one block is
polyethyleneglycol (PEG) (35). PEG can be labeled bi-functionally
on opposite sides of the polymer. One side is usually labeled with
a thiol or SH group to have strong affinity to metals, especially
gold. The opposite side is usually labeled with an NH.sub.2 group,
which permits convenient conjugation of proteins, such as a
monoclonal antibody. PEG, in having hydrophilic groups on the
outside of the polymer, prevents nanoparticles from being
recognized by neutrophils, macrophages and other scavengers in the
circulation or in the mixture of blood cells. The invention further
encompasses the use of a nanoparticulate comprising nanoparticles
that are stabilized against uptake by the reticuloendothelial
system using appropriate surfactants or other particle
coatings.
[0078] Another purpose of the surfactants or other substances used
to coat the particles is to prevent particle aggregation outside
the target cells. Aggregation of the individual particles would
lead to particle growth and to precipitation of the particles from
the suspension that would shorten the shelf life of any conjugate
formulation. The instant invention encompasses the use of
bioconjugates that are stabilized against particle aggregation and
precipitation through the use of surfactants or other particle
coatings.
[0079] Optionally, the surfactant may serve as a platform for the
attachment of other chemical species with desirable biological or
chemical properties, which may help to form clusters of
nanoparticles inside the target cells. For this purpose, a
surfactant or other surface-active agent with reactive functional
groups is desirable. As a result, both the surfactant and the
attachment site should have reactive functional groups. An optional
spacer or linker also should have a pair of reactive functional
groups.
[0080] An enabling component of nanoparticulate bioconjugates is a
targeting vector or moiety. The targeting vectors or moieties may
be antibody protein, protein fragments, short peptides or other
molecules with a strong or high affinity to target receptors and no
or little affinity to target other biological molecules on or in
the surrounding medium or body. Regardless of whether the targeting
vectors adhere directly to the surface of the nanoparticle or is
attached indirectly through the surfactant, the specific receptors
for the targeting vectors may be chemical groups, proteins, or
other species that are overexpressed by abnormal target tissue or
cells. Generally, the receptors may be any chemical or biochemical
feature of tissue or cell type to be treated and eliminated. In
addition the nanoparticles may be conjugated to a secondary vector
or moiety, for example, an antibody or peptide having high and
specific affinity to the primary vectors. Such secondary antibody
may help to produce aggregates of nanoparticles around the primary
targeted nanoparticles on the cell surface.
[0081] Specific antibodies for targeting leukemia or other tumor
cells for elimination of these tumor cells from the body, such as
bone marrow transplants), depend on the type of cells being
targeted. Examples include, but are not limited to CD33 and CD123
for acute myeloid leukemia (AML), CD20 for chronic lymphocytic
leukemia (CLL), or CD19, CD20 and CD22 for acute lymphoblastic
leukemia (ALL).
[0082] In addition to successful targeting, clusterization of
accumulated nanoparticles in cells of interest must occur.
Preferred clusters are two- and three-dimensional structures
comprising even numbers of elongated nanoparticles, e.g., two- and
three-dimensional stars pyramids or other such structures. Methods
for organizing metal nanoparticles into stabilized clusters of
aggregates are well-known. For example, covering the surface of
different gold particles with complementary strands of DNA favors
the self-assembly of the particles into ordered aggregates (36-38).
Aggregate formation results from the favorable interaction between
the complementary strands of DNA. The instant invention encompasses
the use of contrast agents for optoacoustic imaging comprising
aggregates of particles coated with complementary strands of
artificial or natural DNA, RNA or analogs of RNA or DNA.
[0083] Preferably, the nanoparticulate comprises aggregates of
metal particles. Such aggregates exhibit a collective plasmon
resonance that enhances the total accumulated thermal energy over
that expected for single particles in proportion to the total
volume of a cluster. Furthermore, clusters of metal nanoparticles
can exhibit collective resonance absorption, which is stronger than
a simple additive of the optical absorption by single particles
(39). For example, a cubic stack of 16 nanorods of 4 layers with 4
nanorods in each layer will absorb more optical radiation than 16
separate nanorods. The presence of a collective plasmon resonance
for a collection of nanoparticles is evidenced experimentally by a
non-linear increase in the intensity of the optoacoustic signal as
the particle concentration increases and aggregates are formed.
[0084] A useful biochemical means of promoting controlled particle
aggregation is to coat different particles with complementary
sequences of nucleic acids, referred to as nucleotides, oligo- and
polynucleotides, including deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). Physical means also may be used to promote
aggregation, e.g. heating of the nanoparticulates with infrared
light (40). Elevated temperature also promotes endocytosis. The
endocytosis may be enhanced using an internalizing mab (40).
Stimulated Clusterization
[0085] A cluster of strongly absorbing nanoparticles must be formed
in order to produce effective local thermomechanical effect, such
as generation of an expanding vapor bubble. The cluster has a
characteristic dimension, D, comprising small particles with a
characteristic dimension, d. A single large particle with a
characteristic dimension, D, is not effective as the
laser-activated target because of several limitations.
[0086] First, large single metal particles do not absorb laser
radiation as strongly as a group of smaller nanoparticles because
Plasmon resonance interactions are limited to nanoparticles much
smaller than the wavelength of electromagnetic radiation. The
near-infrared radiation 650-1150 nm penetrates cells with minimum
absorption, thus being the most beneficial for selective laser
treatment. Nanoparticles that are much smaller than 650-1150 nm are
in the range of 10-250 nm, will possess maximum absorption per
particle, but are too small to generate vapor bubbles at the
temperature of .DELTA.T=100.degree. C., i.e., the temperature of
adiabatic vaporization.
[0087] A large particle made of organic dyes, polymers or other
absorbing materials, can be used to generate vapor bubble at the
temperature of 100.degree. C.:
.DELTA.T=.mu..sub..alpha.F/.rho.C=100.degree. C. (1),
where .mu..sub.a is the optical absorption coefficient, F is the
laser fluence, .rho. is the nanoparticle density, and C is the heat
capacity of the nanoparticle material. However, due to relatively
low absorption of these particles, a very high laser fluence, F, is
required to reach 100.degree. C. in these particles. Such high
laser fluence is not safe for normal cells and selectivity of the
treatment would be lost.
[0088] Secondly, large and strongly absorbing particles also are
not effective as a contrast agent, because these particles cannot
be effectively targeted to cells. The efficiency of targeting is
roughly inversely proportional to the particle size. This is
explained easily because a large particle cannot be held strongly
by a single chemical bond formed between the antibody and the
receptor. Also, large particles can not be effectively conjugated
to vector molecules, such as monoclonal antibodies or peptides,
specific to receptors of abnormal cells. Large particles also are
very hard to keep in water suspension with no sedimentation at the
bottom of the cuvette. Large particles also will not penetrate
through tissues in case their target is not on the very
surface.
[0089] Clusterization of nanoparticles on and in target cells
occurs due to specially designed targeting procedure, which
utilizes complementary molecules and high affinity molecular
reactions to increase probability of nanoparticle-nanoparticle
interaction in cells. The strength of nanoparticle-cell receptor
interactions is also maximized by choice of monoclonal antibody. In
addition conditions of time duration of targeting, temperature of
the body to be targeted, concentration of nanoparticles, and
conditions of cellular internalization process or endocytosis must
be optimized in order to achieve maximum rate of nanoparticle
clusterization. The efficacy of clusterization can be represented
as a bell-shaped function of each of these quantitative conditions.
One of ordinary skill in the art of cell biology can appreciate
that the specific optimal conditions depend on specific medical
application.
[0090] Various general methods known in the art (33-41) may be used
to stimulate clusterization of nanoparticulates in the body. A site
of interest, e.g., a tumor cell or tissue comprising the same, may
be pretreated with a monoclonal antibody having multiple binding
sites is targeted with nanoparticles conjugated to a secondary
monoclonal antibody specific for the first monoclonal antibody.
Alternatively, nanoparticles conjugated with a primary antibody are
targeted to the site of interest followed by targeting
nanoparticles conjugated to a second monoclonal antibody to the
first monoclonal antibody.
[0091] In another alternative method, nanoparticles conjugated to a
primary monoclonal antibody and further conjugated to a first
aggregating molecule, such as biotin, are targeted to a site of
interest. Subsequently, nanoparticles conjugated with the primary
monoclonal antibody and further conjugated to a second aggregating
molecule, such as streptavidin, are targeted to the avidin-linked
nanoparticles. The use of the first and the second aggregating
molecules that have high affinity to each other is not limited to
biotin-streptavidin linking. One of ordinary skill in the art would
be familiar with a variety of complementary chemical or biochemical
compounds or compositions that have a very strong affinity to each
other, for example, but not limited to, adenine-thymine and
guanine-cytosine nucleotides, protein A or immunoglobulin.
Furthermore, stimulation of clusterization inside target cells may
be accomplished by using an internalizing monoclonal antibody.
[0092] A two-stage targeting method, which provides delivery and
clusterization of the nanoparticles inside the target cell also is
provided. At the first stage, a high concentration of nanoparticles
is provided at the outer cell membrane using monoclonal antibodies
and specific staining conditions to prevent endocytosis of
nanoparticles inside the cell, which is being maintained at a low
temperature of 4.degree. C. At the second stage, after washing out
unbound nanoparticles from the cell suspension the temperature is
raised to 37.degree. C. for an optimal time interval of about 30
min to stimulate the process of endocytosis. The nanoparticles are
delivered thereby from the cell outer membrane to inside the cell
by endocytosis, including formation of vesicles at the cell
membrane. Several nanoparticles in proximity to each other will
then be captured at the cell membrane by emerging vesicles, which
then deliver nanoparticles inside the cell. As a result, the
nanoparticles are concentrated spatially within the vesicles and
their spatial distribution inside the cell represents the clusters.
Vesicles may exist in the cells for a long time, which to allows
further stages of LANTCET to be performed. Also vesicles may
deliver nanoparticles to other specific cell compartments where
further concentration of nanoparticles may occur.
[0093] This protocol can be applied to any type of cell because
endocytosis is a universal transport mechanism and vesicles will
emerge in any type of cell. The occurrence of the nanoparticulate
clusters in the target cells as well as single nanoparticles can be
visualized by electron microscopy (44). To avoid nonspecific
targeting of normal bodies or microstructures, such as cells), the
temperature in the targeting chamber is preferably reduced to
4.degree. C. It is demonstrated herein that minimal or no
accumulation of nanoparticles occurred in cells having no specific
receptors for targeting vectors used.
Administration of Nanoparticles
[0094] The methods and system provided herein are applicable to
animal or non-animal bodies, such as cancer cells or bacteria.
Thus, without limitation, in terms of medical significance, for
example, the body may be an in vivo or in vitro specimen and the
object may be a molecule or a virus or bacterium. Alternatively,
the body may be an ex vivo specimen, such as a disseminated cancer
cell, for example a leukemic cell. The body animate may be an
animate human or non human and the object may be biological and
comprise a specific tissue, cell or microorganism. For example, the
object detected may be a tumor in an animate human or a specific
cell, bacteria or virus harmful to the human.
[0095] The abnormal body or a cell, which is the subject of LANTCET
treatment with the goal of elimination, may be pretreated by
specific primary monoclonal antibodies or other vectors that can be
further used as receptors for secondary monoclonal antibodies or
other vectors in order to form clusters of nanoparticulates in the
abnormal body. Those skilled in the art can recognize that a
targeting vector against cancer receptor may be used not only for
selective delivery of nanoparticles to the target body, but also
for direct therapeutic purposes. Such conclusion comes with
understanding that targeting vectors, such as monoclonal
antibodies, attached to protein receptors on the surface of cancer
cells may disable vital functions of those receptors and thereby
kill those cancer cells.
[0096] An example of such therapeutic action is the monoclonal
antibody, trastuzumab, commercially known as HERCEPTIN, raised
against receptors associated with HER2/neu gene overexpressed in
breast cancer cells and other types of cancer. HERCEPTIN has been
successfully used for treatment of metastatic breast cancer (42).
As disclosed herein, Laser Activated Nanothermolysis of abnormal
cells may be enhanced by pretreatment of the target cells with
primary monoclonal antibodies or primary vectors raised against
vital receptors on target cells. In association with previously
disclosed therapeutic effect of targeting vectors, it is
contemplated that the nanoparticulates disclosed in this invention
also can be used as an anticancer therapeutic agent. In addition,
the designed nanoparticulate can contain an agent molecule to
enhance toxicity of such nanoparticulate to tumor cells. Such
addition is most desirable if the targeting protocol permits
absence of the nanoparticulate accumulation in normal cells.
[0097] For imaging a human or a non-human body, many modes of
application are possible, depending on the therapeutic
requirements. Administration of the therapeutic agent can be
systemic or local. Administration can be made intravenously,
orally, topically or through direct application of the agent to
human or non-human tissue or cells. Local administration of the
nanoparticulate agent may be by topical application, by means of a
catheter, with a suppository, or by means of an implant or by
mixing the nanoparticulate with target bodies (cells) in vitro.
Other means of local application will be apparent to those skilled
in the art.
[0098] Furthermore, the contrast agents may be administered in
conjunction with a hyperthermic application, that is, the
artificial elevation of the local temperature of an organ or
another body part. Hyperthermia accelerates the passage of
nanoparticles through the capillaries of the vascular system of
growing tumors (43). Hyperthermia also will enhance the uptake of
the nanoparticulate by other types of diseased tissue and cells
through widening pores and channels in cell membranes.
[0099] Any of the many different means of elevating temperature are
possible. These include, but are not limited to, the application of
thermostatic chambers, the use of focused ultrasound, microwave or
RF irradiation. Any or all of these heating procedures can be
actively applied while the contrast agent is applied, or,
alternatively, the heating can take place up to 24 hours prior to
the administration of the agent.
Electromagnetic Radiation
[0100] A nanoparticulate is administered to a medium surrounding
the body for treatment of the body. Preferably, the nanoparticulate
is at least partially metallic, has a formed non-spherical shape
having a minimal characteristic dimension in the range from about 1
to about 1000 nanometers and has a formed composition capable of
absorbing the electromagnetic radiation and of accumulating thermal
energy either in the nanoparticulate or in the body greater than
the irradiated body could produce in the absence of the
nanoparticulate.
[0101] In accordance with the invention, electromagnetic radiation
is directed onto the body. The electromagnetic radiation has a
specific wavelength or spectrum of wavelengths in the range of
about 1 nm to about 1 m which encompasses X-rays to radiofrequency.
More preferably, the range is about 300 nm to about 300 mm selected
so that the wavelength or wavelength spectrum is longer by a factor
of at least 3 than the minimum characteristic dimension of the
nanoparticulate. Even more preferably, the spectral range is from
green (520 nm) to infrared (1120 nm) wavelengths which can be
produced by commercially available lasers. Most preferably, the
range of wavelengths is in the near-infrared from about 650 nm to
about 900 nm, where tissue and cells have absolutely minimal
optical absorption and scattering within which the most preferred
gold and silver nanoparticles possess maximum absorption
cross-section.
[0102] The nanoparticulate made of elongated nanoparticles absorbs
the electromagnetic radiation more than would one or more
non-aggregated spherically shaped particles of the same total
volume with a composition identical to the nanoparticulate. The
nanoparticulate by such absorption produces an enhanced
thermomechanical effect resulting from the absorption.
[0103] The most effective and safe LANTCET procedure can be
performed with electromagnetic irradiation of a wavelength that
generates microbubbles around nanoparticulate clusters with minimal
fluence, i.e., energy per irradiated area, in the targeted body.
The irradiation can be generated with a laser, but the invention
encompasses the use of any radiation source, regardless of its
source. Examples of alternate radiation sources include, but are
not limited to, flash lamps, incandescent sources, magnetrons,
radioactive substances, or x-ray tubes. The invention encompasses
the use in LANTCET of nanoparticulates comprising metal particles
or aggregates of metal with electromagnetic irradiation having a
wavelength matching the wavelength of peak absorption in the
nanoparticulates.
[0104] Advantageously, the nanoparticles comprise gold or silver
and the wavelength for irradiation is about 520 nanometers to about
1120 nanometers. For example, the wavelength for irradiation is
about 520 nanometers to about 1120 nanometers and the nanoparticles
in a collection are at least partially gold or silver, are
elongated in at least one dimension and have an aspect ratio of at
least 2.0. Alternatively, the wavelength for irradiation is about
520 nanometers to about 1120 nanometers, the nanoparticles in a
collection are at least partially gold or silver, are elongated,
and have a bimodal distribution of aspect ratios. Particularly, one
local maximum in the distribution of aspect ratios is about 4 and
the other local maximum in the distribution of aspect ratios is
about 7. In a multimodal distribution of aspect ratios, the
electromagnetic radiation comprises two or more wavelength spreads.
In an example for a bimodal distribution of aspect ratios of
elongate at least partially gold nanoparticles, one wavelength band
is about 690 nanometers to about 800 nanometers and another
wavelength band is about 800 nanometers to about 1120 nanometers.
Alternatively, the same wavelength range is used and the
nanoparticulate is a carbon nanotube, preferably a single wall
carbon nanotube.
[0105] In a partially metallic nanoparticulate, heating thereof is
produced preferably through plasmon derived resonance absorption by
conductive electrons in the nanoparticulates. Suitably, the
electromagnetic radiation used is pulsed and is emitted from a
pulsing laser operating in the near-infrared spectral range.
Interaction of nanoparticles with the body being detected produces
a shift of the absorption maximum by the nanoparticles for the
selected wavelength or spread of wavelengths.
Interaction of Electromagnetic Pulses with Nanoparticles of Various
Sizes
[0106] Preferably, LANTCET utilizes elongated gold or silver
nanoparticles with an absorption peak in the near-infrared, i.e.,
620-1120 nm, the most suitable spectral range for deep tissue
imaging due to the relative transparency of tissues to
near-infrared light. Gold nanoellipsoids or elongated nanoprisms,
nanoshells, and other metal nanoparticles absorbing in the NIR can
be produced in limited quantities in the laboratory (45-47).
Therefore, commercially available spherical nanoparticles as a
nanoparticulate are used to demonstrate the feasibility of
selective ablation of tumor cells using laser activated
nano-thermolysis. The disclosure provided herein will allow one of
ordinary skill in the art to predict changes in the results of
LANTCET for various nanoparticles based on their optical and
thermal properties, shape and dimensions.
[0107] FIGS. 1A-1B show the absorption cross-section for gold and
silver nanoellipsoids (nanorods) as a function of their diameter
employing formulas described in detail elsewhere (39). One of
ordinary skill in the art can appreciate that the total optical
energy absorbed by a nanoellipsoid increases initially as a cube of
the diameter, that is, proportional to the volume of these
nanoparticles, and then increases as a square of the diameter, that
is, proportional to the area of the nanoparticles.
[0108] Superheating of the nanoparticle, which evaporates a layer
of surrounding water, may produce a microbubble. Surprisingly, it
was discovered herein that microbubbles cannot be produced around
small nanoparticles, such as about 10 nm to about 100 nm, using
optical fluence that heats these nanoparticles up to 100.degree.
C., the boiling point of water, and even up to 374.degree. C., the
critical temperature of water. Even with extremely high fluence of
pulsed electromagnetic radiation, only nanobubbles invisible by
optical microscopy can be produced. With a further increase of
absorbed energy, the small nanoparticles evaporate and disappear
from aqueous suspension. Even for larger nanoparticles (>100 nm)
it is statistically difficult to produce visible microbubbles.
[0109] The reason for such phenomenon is that a nanoparticle with
small volume can accumulate only a limited amount of thermal
energy, not sufficient to evaporate a volume of water required to
produce a microbubble that can sustain in suspension for a
measurable time. There are two major physical reasons for such
effect. First, strong surface tension that is inversely
proportional to the bubble radius makes the surface tension force
very strong for bubbles smaller than certain radius. Secondly,
viscosity of water provides an extremely strong force on small
nanobubbles thereby preventing their growth to microbubbles. Thus,
only when the nanoparticulate cluster has sufficient size and mass
to accumulate the thermal energy from electromagnetic radiation, is
it possible to generate microbubbles of vapor using energy fluence
that heats the nanoparticulate cluster to a temperature between
100.degree. C. and 374.degree. C. This energy fluence under optimal
experimental conditions can be safe for normal cells. In the
absence of nanoparticle clusters, the required energy fluence will
be much higher than 1 J/cm.sup.2, which is not safe for normal
cells.
[0110] Using the known absolute values for gold nanoparticle
absorbance along with the thermal diffusion models for different
shapes of heated objects (48), an estimate can be made of the
minimum laser fluence required to heat a nanoparticle or a
nanoparticulate cluster up to a boiling point of water at
100.degree. C. or a critical point of water around 374.degree. C.
Upon irradiation with a laser pulse, heat diffusion within a gold
nanoparticle occurs on the scale of picoseconds. Therefore, gold
nanoparticles will be homogeneously heated with a 5 ns to 50 ns
pulse width of a typical Q-switched laser suitable for LANTCET.
[0111] Heat diffusion time is the time required for the transfer of
about 2/3 of the thermal energy stored in a nanoparticle to the
surrounding medium. The heat diffusion time from a nanoparticle or
a cluster of nanoparticles to the surrounding water occurs on the
scale of sub-nanoseconds to tens of nanoseconds, depending on the
size of a nanoparticle, d, being heated by radiation, and its shape
(48):
.tau. HD = d 2 24 .chi. , if the shape is spherical ( eq . 2 a )
.tau. HD = d 2 16 .chi. , if the shape is cylindrical ( eq . 2 b )
.tau. HD = d 2 4 .chi. , if the nanoparticulate cluster is shaped
as disk ( eq . 2 c ) ##EQU00001##
[0112] In the formulas (2a-2b-2c), .chi.=1.310.sup.-3 cm.sup.2/s is
the thermal diffusivity of water at room temperature. The
expression describing the increase of temperature of the
nanoparticle during laser pulse, i.e. when the particle
simultaneously absorbs light and diffuses heat) can be presented in
the following fashion (13):
.DELTA. T NP = F .sigma. a NP V NP .rho. NP C NP .times. ( .tau. HD
.tau. L ) .times. [ 1 - exp ( - .tau. L .tau. HD ) ] ( eq . 3 )
##EQU00002##
[0113] where F[mJ/cm.sup.2] is the incident (upon the nanoparticle)
laser fluence and .sigma..sub..alpha..sup.NP is the plasmon-derived
absorption by the spherical gold nanoparticle at the wavelength of
laser irradiation, V.sub.NP is the volume of nanoparticle being
irradiated, .rho..sub.NP is the density of nanoparticle material
(for gold .rho..sub.g=19.3 g/ml), C.sub.NP is the heat capacity of
nanoparticle material (for gold C.sub.g=0.128 J/g.sup.0K),
.tau..sub.HD is the effective heat diffusion time from gold into
water (surrounding medium). As formula (3) indicates, for effective
utilization of the electromagnetic pulse (laser pulse) energy, the
pulse duration must be shorter than the heat diffusion time,
.tau..sub.HD. A typical heat diffusion time for preferred
nanoparticles with dimensions of 10-100 nm is in the range of
nanoseconds. Therefore, an electromagnetic pulse of near-infrared
radiation from a q-switched laser with a duration of a 3-10 ns may
be an example of a preferred pulse duration.
[0114] Sometimes, however, for the sake of cost reduction, pulsed
optical sources are being replaced with continuous wave sources
(11,16). In case of significant contrast between tumor cells and
normal cells, one of ordinary skill in the art can design a
successful treatment procedure even using continuous wave (over 1
sec long pulses). Nevertheless, short pulses having duration equal
or shorter than the time of thermal diffusion from the heat source
to surrounding tissue, will result in much more effective
thermomechanical interaction and much better spatial confinement of
the damage effects (2-4).
[0115] Formula (eq. 3) is true until the temperature reaches
100.degree. C. Then, any additional absorbed energy may contribute
to the evaporation of the water around the particle, as well as
heating the particle above 100.degree. C. Employing
.DELTA.T=80.degree. K, the difference between room temperature and
boiling temperature of water in formula (eq. 1), minimal optical
fluence required for generation of vapor bubble around absorbing
nanoparticles can be calculated.
[0116] For example, assume the diameter of a spherical gold
nanoparticle is d=200 nm. The heat diffusion time from this
particle to water equals 12.8 ns. Then for a typical laser pulse of
12.8 ns in duration, formula (eq. 3) will yield F=0.6 mJ/cm.sup.2,
which is the critical fluence needed to heat up the spherical gold
nanoparticles to 100.degree. C. It can be predicted that any
fluence larger than 0.6 mJ/cm.sup.2, will result in superheating
and possibly evaporation of water around the nanoparticle. Assuming
that evaporation, at least in the stage of a thin nanobubble around
nanoparticle, does not prevent the nanoparticle from being further
heated, the optical fluence that corresponds to a temperature
increase in a 200-nm diameter nanoparticle to 374.degree. C. equals
2.6 mJ/cm.sup.2. This is a temperature at which conversion of water
into vapor occurs instantly.
[0117] Surprisingly, however, no bubbles were detected at this
level of fluence of laser irradiation. FIG. 3 depicts a magnitude
of optoacoustic signal as a function of laser fluence. No deviation
occurs at 2.6 mJ/cm.sup.2 from a typical linear curve describing
the thermoelastic expansion of water. This means that at this level
of optical fluence microbubbles do not occur. If any nanobubbles
occur, they cannot contribute to the optoacoustic signal or to
thermomechanical damage to surrounding the body.
.sigma..sub..alpha.=1.4.times.10.sup.-9 cm.sup.2. A sharp increase
in the signal occurs at about 1 J/cm.sup.2 indicating contribution
of vapor bubbles to the optoacoustic signal. Note that the
threshold of deviation of the optoacoustic signal from the linear
curve of thermoelastic expansion occurs at a fluence much greater
than the fluence, F=0.0026 J/cm.sup.2, corresponding to the
critical temperature of water, 374.degree. C.
[0118] Results presented in FIG. 3 and the calculations above show
that the threshold of microbubble generation should decrease with
increase of the size of nanoparticles. On the other hand, a very
strong absorption of electromagnetic radiation due to plasmon
resonance may be lost by a large nanoparticle, e.g., microparticle,
since plasmon resonance theory requires that the size of the
nanoparticles must be smaller than the electromagnetic wavelength.
Furthermore, microparticles are not practical from the clinical
prospective, since these particles are too large (heavy) to be
suspended in water and can not propagate through biological cells
and tissues, which makes targeting of these types of particles to a
cell or other biological body difficult or impossible. Based on
these considerations, clusters of nanoparticles can be used to
effectively target cells and then to effectively generate vapor
microbubbles by a low fluence laser radiation. If an ideal size
cluster can be formed selectively in target cells, the laser
fluence required for cell damage will be in the range of only a few
mJ/cm.sup.2, which is absolutely safe for normal cells and
tissues.
[0119] FIG. 4 demonstrates the threshold of microbubble formation
from laser irradiated clusters of gold nanoparticles. A targeting
protocol is designed, which resulted in the accumulation of
clusters of about ten 30-nm diameter nanoparticles in the target
tumor cells, human B-lymphoblasts. The threshold fluence of the
cell damage, confirmed with observation of the microbubbles by
photothermal detection, was found to be about 100 mJ/cm.sup.2,
which is 50-60 times lower than that observed for individual gold
nanoparticles with diameter 200 nm and 30 times lower than the cell
damage threshold for cells nonspecifically targeted with the same
30-nm diameter nanoparticles. Both the microbubble generation
threshold and the threshold of cell damage was always significantly
higher in cases when no clusters in cells was observed.
[0120] FIGS. 1A-1B showing absorption coefficients of gold and
silver nanorods and expressions (eq. 1), (eq. 2b) and (eq. 3)
permit estimation of a nanoparticle temperature for a nanoellipsoid
of revolution. Assume a gold nanorod with diameter of 20-nm and
length of 100-nm. Its absorption cross-section is
8.5.times.10.sup.-10 cm.sup.2, i.e. almost equal to that of a gold
nanosphere with diameter of 200 nm, while its volume is only 200
times smaller than that of the nanosphere. Significantly stronger
optical absorption of gold nanorods compared with gold nanospheres
of equal volume results in a dramatically reduced minimum laser
fluence required to heat up nanorods to a temperature required for
vapor bubble formation. Thus, clusters of elongated metal
nanoparticles are preferred over clusters of spherical metal
nanoparticles for producing near-infrared radiation induced
microbubbles. FIGS. 2A-2C show that experimentally measured optical
properties of actual gold nanorods are very close to those
calculated theoretically. A controlled fabrication of relatively
monodisperse nanorods with an aspect ratio not deviating from the
maximum can be achieved. Furthermore, conjugation with PEG slightly
changes spectral width and position of the peak absorption.
[0121] Recent developments in nanotechnology allow for engineering
nanoparticles with various shape and dimensions thereby
facilitating the development of new imaging and therapeutic
nanoparticulates. Colloidal gold is especially attractive since it
is inert material that has been used for therapeutic applications
(49-50). It is noteworthy to mention that upon intravenous
injection of gold nanoparticles, unattached nanoparticles that
could be a source of background noise for LANTCET can be rapidly
removed from the circulating blood pool by liver and other organs
of the reticuloendothelial system (51).
[0122] In summary total absorbed energy of electromagnetic
radiation is proportional to the volume of a nanoparticulate. Thus,
the total thermal energy stored in said nanoparticulate is also
proportional to its volume. Heat diffusion rate decreases with
nanoparticle dimension to the second power. Probability of
microbubble generation is inversely proportional to dimension,
i.e., surface tension, and viscosity that strongly affects the
bubble generation is proportional to the nanoparticle dimension to
the second power. These factors require that a nanoparticulate
cluster is formed to reduce the threshold of microbubble formation,
that can be used for LANTCET.
Real-Time Imaging and Monitoring
[0123] Lapotko et al (21-23) teach a method and apparatus for
obtaining an image of a body, e.g. a cell, to allow examination of
a number of submicron heterogeneities simultaneously, such as
microbubbles or heated areas, including a method for detecting
size. A relatively large sample surface, bigger than the cell
diameter, is exposed to the pump laser radiation. The size of the
surface exceeds the wavelength of the pump laser beam used. In
fact, a surface of any size could be irradiated, but, logically,
the size could not exceed the size of the sample itself because the
chosen probe laser beam diameter is not smaller nor comparable with
the pump laser beam diameter and is not larger than the maximum
overall dimensions of the sample.
[0124] Spatial distribution of absorbing heterogeneities, e.g.
bubbles, in the irradiation zone is determined by the synchronous
measurement of the diffraction limited phase distribution through
the whole cross-section of the probe laser beam which is
transformed into an amplitude image by a phase contrast method. The
size of separate microheterogeneities larger than the pump laser
beam wavelength is determined by analyzing the amplitude image
structure. The amplitude image corresponds to the refraction index
change distribution induced by the pump laser in the object
observed.
[0125] The average size of microheterogeneities smaller than the
wavelength is measured indirectly by the characteristic time of
cooling which is dependent on the size. That measurement is based
on the speed measurement of the phase change of diffraction-limited
images of those microheterogeneities at various points of the probe
laser beam cross-section at different moments in time. Measurement
begins right after pump laser irradiation has taken place as the
chosen irradiation period is much shorter than the characteristic
time of cooling of the microheterogeneity observed.
[0126] A short-time irradiation can be performed by two methods.
The first method uses a single laser pulse. It is the duration of
the pulse that determines the period of effect. A pulse-periodic
mode, usually with a porosity greater than 1, also could provide
this effect. The second method uses continuous laser pump radiation
that is intensity-modulated with a relatively high modulation
frequency ranged from a few kHz to hundreds of MHz. In this case
duration of a single effect is determined by a modulation
semi-period. This effect repeats with the frequency determined by
the laser modulation frequency. Information about the time of
cooling would be carried by the probe laser beam time phase related
to the pump laser beam time phase.
[0127] A number of versions of the probe beam realization could be
used. For example, a part of the pump laser beam could be used as a
probe beam. Propagating the probe beam through an additional
optical delay line regulates its delay time as related to the main
beam. The chosen probe beam intensity should be considerably (at
least 5-10 times) lower than the main beam intensity, so as to have
minimal effect on the measurement results.
[0128] The phase distribution of the probe laser beam in the
function of the pump laser wavelength should be measured, so as to
obtain information about spectral properties of separate
microheterogeneities simultaneously with their sizes. It is
suggested that said measurement is to be accomplished by at least
two time delays as related to the pump beam pulse at every pump
laser wavelength. Dynamic change of the microheterogeneities or
microbubbles, induced by the pump beam, is examined by changing at
least two phase images, one of which is obtained immediately before
the pump pulse operation and the other obtained simultaneously with
the pulse operation or with a delay, with their subsequent
subtraction. This is particularly important in case there are
insignificant alterations of the image structure which are
difficult to identify using only one image, as the measurement
precision is low.
[0129] For the LANTCET method, an additional optical system of
phase contrast is used as an optical transformation unit to
transform phase distribution in the probe beam cross-section to an
amplitude image. A registration unit is a high-speed multi-channel
photodetector, for example CCD-matrix, in the pulse mode to
register the amplitude image of the probe beam at various moments
of time as related to the moment of the pump laser pulse operation.
Another version of the registration unit is a number of one-channel
photodetectors used to register time amplitude changes for one or
several zones in the amplitude image of the probe beam. The probe
beam falls simultaneously on all the detectors due to a
semi-transparent system of mirrors placed in the way of probe beam
behind the phase-contrast system. Another solution is a consecutive
spatial shift of said detector using an additional switch unit.
[0130] For examination of the microheterogeneities considerably
smaller than the wavelength, the fundamental solution is to
introduce a synchronizing unit and a time delay regulating unit
connected with each other, with the pump laser units, with the
probe laser forming unit, and with the registration unit. A
gradually regulated delay provides, when using a probe laser and
pump laser pulse regimens, a precise measurement of the cooling
time for the absorbing heterogeneities heated by the pump pulse to
estimate the average size of the heterogeneities. If the continuous
mode of the probe laser is used, a synchronizing unit that switches
the probe beam phase monitoring at the moment of the pump laser
pulse operation is used.
[0131] In the continuous mode of the pump laser with intensity
modulation, the device contains an additional intensity-modulating
unit placed in the path of the pump beam distribution. Registration
of the continuous probe beam modulation caused by pump radiation,
via refraction index modulation, is provided by the synchronous
integrating unit connected to a photodetector or multi-channel
photodetectors of the probe beam. The unit also receives a signal
from the pump beam modulator. The necessary information about the
pump laser (time) phase is carried by the signal.
[0132] Also provided is a one-channel mode, where pump radiation
accomplishes the probe beam functions simultaneously. In this case
phase distribution in the pump beam cross-section itself is
registered. The filter cutting the pump beam in front of the
photodetector should be removed to follow this scheme. The system
of splitting the pump laser beam into a main beam and an additional
beam is introduced into the path of the pump beam, coming from the
pump laser to use part of the pump laser as a probe beam, the
additional beam accomplishing the probe beam function. The optical
delay line connected with the time delay unit is introduced in the
path of the probe beam.
[0133] A probe beam-forming unit can be realized both as a
continuous laser connected with the synchronizing unit and as a
pulse laser connected with a time delay unit. A probe beam turning
unit should be introduced as related to the sample observed to
obtain a three-dimensional tomographic image. Another version is to
introduce the turning unit of the sample itself, where the turning
unit is connected with the synchronizing unit. The device can also
be equipped with an image processing unit connected with the
photodetectors, the synchronizing unit, and the time delay unit.
Its functions include image comparison at various moments of time,
and another one is comparison of photothermal and regular optical
images.
[0134] The device is additionally equipped with a pump beam
wavelength-changing unit connected with the pump laser unit.
Various methods can be used to provide the laser wavelength change.
These methods include temperature and pressure influence on the
active element; using spectral elements within a pump laser
resonator in the form of a prism, diffraction grid, interference
filters, or other applicable elements.
[0135] LANTCET begins when the object containing absorbing
microheterogeneities is irradiated with a probe laser beam where
the chosen probe beam diameter is not smaller than the pump beam
diameter and is not larger than the maximum overall dimensions of
the sample. Intensity of the probe beam should be considerably,
i.e., at least 5-10 times, smaller than the pump beam intensity to
cause minimal effect on the measurement results. The
diffraction-limited distribution of the probe laser beam phase over
the whole cross-section is then transformed to an amplitude image
using the phase contrast method. The obtained values of the probe
beam phase .phi..sub.0(x, y) and the phase-corresponding amplitude
I.sub.0(x', y') of the image are basic for further analysis.
[0136] The next step is irradiation of the object containing
absorbing microheterogeneities by a focused pump laser beam having
a short pulse width and a wavelength coinciding with the absorption
line of the microheterogeneities. The pump pulse immediately
irradiates a relatively large sample surface, where the size of the
surface is larger than the wavelength of the pump laser used. In
fact, the surface could be of any size, but logically, it couldn't
be larger than the sample itself. If such an effect occurs, light
energy absorption in the sample is not uniform:
microheterogeneities absorb light most actively. Thus, a live cell
has various absorbing structures, e.g., cytochromes, organelles,
and mitochondria, the size of which vary from a few nm to hundreds
of nm, i.e., considerably smaller than the average size of a cell
(5-20 microns). However, their ability to absorb light is so high,
that it causes thermal effects causing a temperature rise 10-1000
times higher than the temperature of the cell's environment.
Cooling of the structure that has absorbed light energy begins by
heat diffusion after the end of the pump pulse operation.
[0137] The time of cooling for a single sphere-looking object
is:
.tau. T = R 2 6.75 K , ( eq . 4 ) ##EQU00003##
where
[0138] t.sub.T is the time of cooling of the object, sec;
[0139] R is the radius of the object, m; and
[0140] K is the temperature conductivity coefficient
(m.sup.2/c).
This time equals 10.sup.-5-10.sup.-4 sec for the majority of blood
cells.
[0141] A primary thermal response could be presented as the
distribution of temperatures over the x-axis and the y-axis:
.DELTA. T ( x y ) = .alpha. ( x y .lamda. ) ( x y ) .rho. C , ( eq
. 5 ) ##EQU00004##
where
[0142] .DELTA.T is the distribution of temperatures over the x- and
y-axes,
[0143] .alpha. is the light energy absorption coefficient with the
wavelength,
[0144] .rho. is density, kg/m,
[0145] .epsilon. is energy density in the pump beam, J/m.sup.2,
[0146] C is thermal capacity, J/kg.degree. C.
[0147] Expressions (eq. 4) and (eq. 5) help estimate the
temperature effect both for an object (a cell) as a whole and for
its structural elements, i.e., absorbing heterogeneities. The
intensity of the effect depends on a specific absorption
coefficient and the heterogeneity's size. t.sub.T would be about
10.sup.-8 s and less for the submicron structures having the sizes
smaller than the wavelength (10.sup.-7-8 m). It means that
significant rise of the local temperature could be achieved only
providing the pump pulse width or the modulation period T=1/f
(where f is a modulation frequency) are smaller or at least
comparable with t.sub.T. Otherwise the local temperature effect,
being the very source of the local heat variations of the
refraction index, would not be achieved. The local heat variations
of the refraction index .DELTA.n(x, y) induced by the pump pulse
absorption could be presented as follows:
.DELTA. n ( x , y ) = .alpha. ( x , y , .lamda. ) ( x , y ) .rho. C
( n T ) p , ( eq . 6 ) ##EQU00005##
where
[0148] .alpha. is the light energy absorption coefficient with the
wavelength,
[0149] .rho. is density, kg/m,
[0150] .epsilon. is energy density in the pump beam, J/m.sup.2,
[0151] C is thermal capacity, J/kg.degree. C.; and
[0152] T is temperature.
[0153] A further step comprises irradiating the object containing
heated absorbing microheterogeneities by the probe laser beam where
the chosen probe beam diameter is not smaller than the pump beam
diameter and not larger than the maximum overall dimensions of the
sample. Intensity of the probe beam should be considerably, i.e.,
at least 5-10 times, smaller than the pump beam intensity to cause
minimal effect on the measurement results. The phase of the probe
beam wave front will be distorted from the local heat variations of
the refraction index when the probe beam would propagate through
the sample. The phase deviations .phi..DELTA.(x, y) could be
described as follows:
.PHI..DELTA. ( x , y ) = L .alpha. ( x , y , .lamda. ) 2 .pi. ( x ,
y ) .lamda. 0 .rho. C ( n T ) p , ( eq . 7 ) ##EQU00006##
where
[0154] L is the geometrical length of the probe beam way in a
heterogeneity,
[0155] n is the refraction index,
[0156] .DELTA.n is the refraction index variations on the x- and
y-axes,
[0157] .alpha. is the light energy absorption coefficient with the
wavelength,
[0158] .rho. is the density, kg/m,
[0159] .epsilon. is the energy density in the pump beam,
J/m.sup.2,
[0160] C is thermal capacity, J/kg.degree. C., and
[0161] T is the temperature.
[0162] In a further step the diffraction limited phase distribution
of the probe laser beam over the whole cross-section is transformed
to an amplitude image using the phase contrast method. Taking into
account the values .phi..sub.0(x,y) and I.sub.0(x',y') previously
obtained in an unexcited state, parameters of the probe beam
propagated through the exited sample at the moment of time t.sub.0
could be presented as follows:
.phi.(x,y)=.phi..sub.0(x,y)+.DELTA..phi.(x,y) (eq. 8),
where
[0163] .phi..sub.0(x, y) is the probe beam phase in the absorbing
zone of an unexcited object
[0164] .DELTA..phi.(x, y) is alteration of the probe beam in the
absorbing zone of an unexcited body,
I(x',y')=I.sub.0(x',y')+S(x',y') (eq. 9),
where
[0165] I.sub.0(x', y') is the amplitude of the photothermal signal
in an unexcited state,
[0166] S (x', y') is the required photothermal signal being subject
to registration and analysis.
[0167] The size of separate microheterogeneities larger than the
pump laser wavelength is determined using structural analysis of
the amplitude image measured immediately at the moment of the pump
laser operation and corresponding to the refraction index change
distribution induced by the pump laser in the object observed. To
determine the average size of the microheterogeneities smaller than
the wavelength, the phase alteration speed of the
diffraction-limited images of said microheterogeneities in various
points of the probe beam cross-section should be measured.
Measuring begins immediately after the pump pulse effect has taken
place. To achieve this, the probe beam irradiation and the phase
distortion analysis of the object are performed a number of times,
e.g., at two moments of time.
[0168] The theoretical limit of this method is conditioned by the
terminal time of transformation of optical energy to thermal energy
which is 10.sup.-13 seconds for condensed mediums. This time
corresponds to 1 .ANG. and could be achieved using femtosecond
lasers. The photothermal signal amplitude S is proportional to the
temperature change in the absorbing zone and decreases due to
thermal conductivity.
[0169] U.S. Pat. No. 5,840,023 teaches the acquisition of
optoacoustic images with contrast agents. In this method, a short
pulse of irradiation is followed by detection of the induced
pressure wave, which is then used for generation of an image. In
the practice of the present invention, the pulses of
electromagnetic radiation preferably have a duration of about 10 ns
to about 1000 ns. When the tissue to be imaged is simulated by a
solid slab tissue, the radiation fluence on the surface of the slab
is about 10 mJ/cm.sup.2. For other configurations of the test
sample or for living human or non-human bodies, the surface fluence
will vary, but will always be in the range of about 1 to about 100
mJ/cm.sup.2, which generally is considered safe.
[0170] In addition both photothermal and optoacoustic imaging,
either simultaneously or sequentially, can be utilized to monitor
pulsed laser interactions with nanoparticulates and their clusters
in the course of LANTCET procedure. The monitoring method may
include photothermal imaging and microscopy, optical and
optoacoustic detection of thermal field, thermal lens and bubbles
generated around nanoparticulates. The imaging can occur during
administration of the nanoparticulate, immediately after
administration, or at some later time to allow for accumulation of
the agent in the target cell or other body.
LANTCET System
[0171] FIG. 5 shows a designed system that utilizes the LANTCET
method of laser activated nanothermolysis for purging tumor cells.
For example such system may be a modification of a blood dialysis
system where cells containing or accumulating bioconjugated
nanoparticles can be irradiated extracorporally, i.e. outside human
or animal body. The main components of the system 10 are the cell
chamber 1, a source of targeting moieties 2, e.g., monoclonal
antibodies or peptides, and nanoparticulates 3, an optical chamber
4, such as an optical flow cuvette, a pulsed source of
electromagnetic radiation 5, and a means for filtering 6 and
collecting 7 the products of damaging thermomechanical effects,
e.g., a hemosorption system or similar system. The cell chamber 1
is independently fluidly connected to the source of targeting
nanoparticulates 2,3, to the optical chamber 4 and to the filtering
and collecting means 6,7. The pulsed source of electromagnetic
radiation 5 is positioned to irradiate the cells in the optical
chamber 4. Optionally, an imager 8 is positioned to receive a
photothermal signal or for generating an optical image of
thermomechanical effects affecting the cells in the optical chamber
4.
[0172] The system is configured so that bioconjugated nanoparticles
can selectively target the cells in the cell chamber whereby
nanoparticulate clusters accumulate in the cells. The targeted
cells containing the nanoparticulate clusters flow to the optical
cuvette where they are irradiated with laser or other
electromagnetic pulses. The laser pulse heats the nanoparticulates
which heat subsequently induces microbubble formation. The
formation of microbubbles causes selective and increased levels of
thermomechanical damage. Optionally, the imager may monitor the
process of cell nanothermolysis by laser-induced microbubbles
and/or direct the damaging thermomechanical effect against the
targeted cells.
[0173] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
Example 1
In Vitro Laser Activated Nanothermolysis of Tumor Cells Using
Nanospheres
[0174] An in vitro model with myeloid K562 cell line, cryopreserved
human cells, tumor cells (patient-derived acute B lymphoblast
leukemia), both referred to as tumor cells, and normal stem cells
are used. Well-defined specific MABs are used as primary MAB for
targeting, i.e., CD15 and Glycophorin-A for K562 cells and CD19 for
acute B-lymphoblast leukemia cells. Selection of MABs was realized
by using flow cytometry. MABs CD 15 and Glycophorin A were selected
for K562 cells based on their superior level of expression on
surface of those cells. CD 19 was selected in the same way for
acute B lymphoblast leukemia (ALL) cells. For controls for the
single-cell model, untreated K562 cells are used and for the
suspension model, normal stem cells are used.
Specific Targeting Protocol
[0175] Cells at a concentration of 800,000/ml in phosphate buffer
solution (PBS) with 1% fetal bovine serum (FBS) were preincubated
with two primary MABs CD15 and Glycophorin-A (20 ml/ml of each)
(K562 cells in single-cell model) and CD19 (acute B lymphoblast
leukemia cells and normal stem cells in suspension model) for 30
min with shaking in the dark at 4.degree. C. After incubation, the
cells were centrifuged at 300 g for 4 min and washed twice. Then
the cells were incubated again for 30 min with 2.times.10.sup.10/ml
of 30 nm gold NPs conjugated to secondary MAB, Goat anti-Mouse IgG
(British Biocell International, Cardiff, UK via Ted Pella Inc.,
Redding, Calif.). Cells were separated from free uncoupled NPs by
centrifugation at 300 g for 4 min. The bottom pellet of cells was
resuspended in PBS-FBS solution and immediately used. The same
cells were used for preparation of cell samples with modified
protocols.
Non-Specific Targeting Protocol
[0176] The same procedure as described above was applied, however,
without primary MAB, only with secondary MAB-NP complex. As a
simple control, bare gold NPs without any MAB were incubated with
cells under the same conditions. To determine cell viability,
trypan blue dye was used according to a routine protocol, on an
aliquot of cells from the stock suspension before the laser
treatment and on every treated sample after the laser treatment.
Four K562 cell samples were prepared as for the single-cell model.
Two cell samples (leukemic and normal stem cells) were prepared for
the suspension model using the specific targeting protocol.
Additional control sample of nanoparticles in solution without
cells was also prepared.
Laser Treatment Procedure and Cell Damage Measurements
[0177] LANTCET was experimentally studied in the two models, single
cell and suspension of cells. FIGS. 6A-6B depict a schematic
diagram of LANTCET with simultaneous monitoring and guidance by
photothermal microscopy. In the diagram a mixture of cells is
stained with MAB-conjugated gold nanoparticles that selectively
attach to target receptors and form clusters (FIG. 6A, right),
cells are irradiated by a laser pulse that induces the bubbles
around clusters of nanoparticles (FIG. 6B), the bubbles destroy
only cells targeted with nanoparticles and non-targeted cells (FIG.
6C, left) remain intact; and a photothermal image shows tumor cells
loaded with nanoparticles prior to pulsed laser irradiation and
fragments of cells destroyed by the laser pulse (FIG. 6D). In the
first model, single cells were irradiated one by one and any
possible damage was detected immediately. After the incubation with
gold nanoparticles, the samples of K562 cells were immediately
placed in the sample chamber (S-24737, Molecular Probes, OR)
mounted on a microscopic slide to produce a monolayer of cells.
Individual cells (total of 150) were irradiated within 7 min with
10-ns long single focused laser pulse at 532 nm. The green light at
532-nm is strongly absorbed by nanoparticles, but not by the cells.
All samples were exposed to the same laser irradiation conditions,
i.e. a specific optical fluence.
[0178] A photothermal (PT) microscope was used for visualization of
vapor bubbles around the laser irradiated NP [30]. Photothermal
microscopy may be used not only for detection of microbubbles
around the nanoparticles, but also for the real-time monitoring of
the laser-induced damage of individual cells with nanosecond
temporal resolution. Single cells were irradiated with two
collinear focused laser beams, i.e., a pump pulsed beam for bubble
generation and a probe low intensity continuous-wave beam for
detecting transient thermomechanical changes in the irradiated
cell. Any change in the optical refraction index caused by a
thermal field or a bubble produces a thermal lens effect that
influences the probe-beam intensity at the input of a
photodetector. Any bubble-specific photothermal response signal
detected from a cell at the same time indicates damage of the
irradiated cell [22].
[0179] Cells were irradiated one by one with single focused laser
pulse at 532 nm (10-ns duration) and the photothermal response from
each cell was recorded with a photodetector measuring changes in
the power of the probe laser at 633-nm (temporal profile of probe
laser signal) [21,22]. The probability PRB of bubble generation at
a specific laser fluence was measured as:
PRB=N.sub.2/N (eq. 10)
where N is the amount of irradiated cells and N.sub.2 is the amount
of cells with bubble-specific photothermal responses. The bubble
generation threshold fluence for a specific pump laser wavelength
was defined as the fluence that corresponds to a PRB level of
0.5.
[0180] In the second model, feasibility of LANTCET for purging was
evaluated using suspension of cells. Suspensions of test and
control cells were injected into rectangular glass chambers and
then were irradiated with single laser pulses. A laser beam with
diameter of 1 mm was scanned along a cuvette with dimensions of
0.4.times.0.1.times.10.0 mm for about one minute for each sample.
After the irradiation, the laser-induced cell damage was assessed
within 5 min in the same cuvette using the standard trypan blue
dye, optical microscope and digital camera for visualization and
counting the level of positively stained cells. The damage
probability, DP, was then calculated.
Electron Microscopy of Leukemic Cells
[0181] Binding of IgG-conjugated gold nanoparticles to primary MABs
attached to CD15 and Glycophorin-A receptors on the surface of K562
cells causes internalization of the complex. Whether phagocytosis
took the NPs into the cells was examined using transmission
electron microscopy. The targeted cells were fixed in situ in a
mixture of 2.5% glutaraldehyde and 1.5% formaldehyde in 0.05 M
cacodylate buffer pH 7.2 to which 0.03% trinitrophenol and 0.03%
CaCl2. After washing in 0.1 M cacodylate buffer cells were pelleted
and processed further as a pellet. They were post-fixed in 1% OsO4
in 0.1 M cacodylate buffer, en bloc stained with 2% aqueous uranyl
acetate, dehydrated in ethanol and embedded in Poly/Bed 812
(Polysciences, Warrington, Pa.). Ultrathin sections were cut on
Reichert-Leica Ultracut-S ultramicrotome, stained with lead citrate
and examined by a transmission electron microscope JEM 100 CX II
(JEOL, Japan). Amount of nanoparticles as per cell was estimated
through counting particles in four Emimages each visualizing a thin
(60 nm) slice of a cell, averaging counts in 10 different cells
(total of 40 microscopic slides were processed) followed by
extrapolation of obtained numbers to the total volume of the
cell.
Evaluation of Add Nanoparticle Uptake by Cells with Electron
Microscopy
[0182] Electron microscopy (EM) images of K562 cells (FIGS. 7A-7B)
revealed that nanoparticles fill the entire volume of the cell and
do not concentrate only on its outer membrane where target
receptors are located. Clusters of closely packed 5-20 particles
were found in cells that were selectively targeted using the
primary MAB and bioconjugates of nanoparticles with the secondary
monoclonal antibodies (FIG. 7B). The total average number of
nanoparticles per cell estimated by extrapolating the number of
nanoparticles counted in 60-nm thin slices corresponding to EM
images was about 31650.+-.4810 in specifically targeted cells. In
contrast, a much smaller number of nanoparticles was found in
non-specifically targeted cells, i.e., about 1500.+-.350
nanoparticles per cell and only in about 200.+-.90 nanoparticles
per cell in control cells incubated with bare nanoparticles. No
clusters were observed in non-specifically targeted and control
sample (FIG. 7A). High nanoparticle contrast (ratio of NP level for
specific targeting to that of non-specific targeting) was 21. This
ratio further increased to 158 for specific targeting relative to
the control of bare nanoparticles. The selectivity achieved by
specific targeting of tumor cells indicates a high degree of
LANTCET safety in that only cells with clusters of nanoparticle may
be destroyed while leaving other cells not damaged by laser
pulse.
Laser Elimination of Tumor Cells-Single Cell Model
[0183] Using the single cell model, PT responses from individual
K562 cells to pulsed laser radiation (532-nm, 10-ns) were studied.
PT responses from the free space between the cells (background),
from individual cells targeted with nanoparticles and from
suspension of single nanoparticles without cells were obtained. The
microbubble generation and cell damage were monitored
simultaneously with the probe laser pulse by analyzing profiles and
amplitudes of PT responses as depicted in FIGS. 8A-8C. Among all
studied samples, only the cells selectively targeted with gold
spherical nanoparticles showed prominent bubble-specific PT
response (FIG. 8C). These cells also frequently exhibited apparent
visual signs of the damage, such as fragmentation after irradiation
with a single laser pulse. Microbubbles with a duration 1-3.5 .mu.s
(FIG. 8C) were detected only in specifically targeted cells, while
in other test samples such long microbubbles were never observed
even at optical fluences 10 times exceeding the threshold of bubble
generation. Long bubbles were detected only among those cells that
manifested the clusters of nanoparticle in their EM images.
[0184] The explanation for this observation is found in the
previously discussed models of vapor bubbles generated around
superheated nanoparticles. The temperature of a laser-heated
nanoparticle is proportional to the ratio .tau..sub.HD/.tau..sub.L
of heat diffusion time and the laser pulse duration, where the heat
diffusion time from a spherical nanoparticle into aqueous medium
can be found as the ratio of nanoparticle diameter squared and the
heat diffusion coefficient (.chi.=0.0013 cm.sup.2/s) in water
.tau..sub.HD=d.sup.2/24.chi.[54]. Heat diffusion time equals 0.3 ns
for a single gold nanoparticle with diameter of 30-nm and this time
is much less than laser pulse length (10 ns). Such rapid heat
diffusion distributes the laser pulse energy over a larger volume,
so that only a 0.033 fraction of laser energy is used to heat the
nanoparticle. More importantly, for generation of vapor bubbles
around nanoparticles, the surface tension and the dynamic viscosity
of water prevent formation of expanding bubbles around
nanoparticles superheated substantially above the critical point
temperature of water (647.degree. K).
[0185] The situation changes for NP clusters with effective
diameter of more than 300 nm consisting of about 10-20
nanoparticles, so that the initial surface tension is 10 times
lower and the full thermal energy of a laser pulse is utilized to
heat the cluster of gold nanoparticles. Therefore, the threshold of
pulsed laser interaction with clusters of nanoparticles is
significantly lower than that for a single nanoparticle.
Superheating of the nanoparticle clusters generates a much bigger
vapor microbubble capable of damaging even large cells.
Nanoparticle clusters were destroyed by single laser pulses with
optical fluence of 5 J/cm2, which could superheat NP to a
temperature significantly above the boiling temperature of gold
(287.degree. K). Laser pulses that followed the first
microbubble-generating pulse produced no bubbles, and no
bubble-specific PT response was detected for the second pulse (FIG.
8A).
[0186] It is important to note the difference in PT response
amplitude and lifetime, the parameters dependent on the bubble
diameter, between the control suspension of nanoparticles (FIG. 8B)
and the cells specifically targeted with gold NPs (FIG. 8C). Small
amplitude and a short 0.3 ms duration of the PT response signal
detected in the suspension of gold nanoparticles (FIG. 8B)
indicates that only a very short-lived nanobubble was generated
even with optical fluence of 35 J/cm2. In contrast, cells
specifically targeted with nanoparticles (test sample) produced a
much stronger PT response (FIG. 8C), which indicates that the
bubble was almost one order of magnitude larger and, therefore,
lasted longer. Such increase in the bubble lifetime and size may be
explained only by formation of clusters of NPs in tumor cells.
[0187] Cell damage probability DP for test and control samples was
analyzed at several different levels of laser fluence: 5, 35 and 90
J/cm2 (FIG. 9). Both the DP level and the laser fluence threshold
for bubble generation depend on the presence of NP clusters in
cells. The DP increases and the damage threshold decreases for
target cells that may have clusters of nanoparticles compared with
control samples having for which no clusters were found during EM
examination. For example, the damage probability, DP, for
specifically targeted cells, reached its absolute maximum of 100%
at a fluence of 5 J/cm2, while for nonspecifically targeted cells
it was only 0.07 at the same fluence. For the control sample of
cells incubated with bare nanoparticles, the DP level was only 0.09
at a much higher fluence of 35 J/cm2. The damage threshold for the
specifically targeted K562 cells is estimated at about 1-2 J/cm2,
which is 30-50 times lower than the threshold fluence level for
destruction of the same cells without particles (control). Thus,
the specific targeting protocol provided a significant decrease of
the laser damage threshold compared to intact untargeted cells and
more than 10 times decrease compared with control K562 cells
incubated with NPs not conjugated to a MAB. The length of PT
response signal in targeted cells statistically varied from 0.2 to
3.5 ms. Maximal micro-bubble diameter reached 20 mm, as calculated
using previously developed model of cell damage based on the
experimentally measured lifetime of a microbubble [22].
Laser Elimination of Tumor Cells-Cell Suspension Model
[0188] In addition to laser irradiation of individual cells and
their real-time monitoring with the PT microscope, suspensions of
the human leukemic and normal stem cells were irradiated in the
cuvettes, simulating the first approximation to the bone marrow
purging procedure. In this case many cells (200-400) were
irradiated simultaneously with a broad laser beam. Damaging effects
of a single laser pulse was monitored by microscopic examination of
trypan blue uptake by cells after the laser treatment. Cells with
visual signs of destruction as well as positively stained were
considered damaged. Laser fluence in the range of 0.5-2 J/cm2 was
used in this experiment. At the level of 1.7 J/cm.sup.2, 100% of
specifically targeted tumor cells were damaged (FIG. 10B) and 16%
of normal stem cells were damaged (FIG. 9), while 84% of normal
cells survived the laser pulses (FIG. 10A). The bubble generation
threshold for tumor cells was in the range 0.1-0.3 J/cm2. This is
100-300 times lower than the bubble generation threshold of optical
fluence (30-70 J/cm2) determined for intact untreated cells such as
lymphocytes, K562 and lymphoblasts.
Example 2
[0189] Ex Vivo LANTCET of Human Normal and ALL Leukemia Cells Using
Spherical Nanoparticles
[0190] Cryopreserved samples of human bone marrow (BM) taken either
from normal donors or patients with the diagnosis of acute
B-lymphoblast leukemia (ALL) were used. Suspensions of normal and
tumor cells were not mixed. Normal BM samples had no tumor cells
and ALL tumor samples consisted mainly of tumor cells where the
level of B-lymphoblasts was 94% to 98% in samples from different
patients. Three normal and three tumor samples were all obtained
from different patients. Normal and tumor samples were prepared and
analyzed as separate samples though the same treatment protocol was
used for each. Leukemia cells express diagnosis-specific genes that
were determined individually for each ALL patient by using standard
clinical protocols [53]. Specific MAB, raised against cell membrane
receptors corresponding to specific genes, were used for each
sample of tumor cells. Phenotyping was performed with flow
cytometry.
[0191] As a result diagnosis-specific (primary) monoclonal
antibodies were determined (MAB1). For different patients different
MAB yielded maximal levels of expression. It was determined that
for each of the three patients an optimal MAB1 was different, i.e.,
CD10 for patient no. 1, CD19 for the patient no. 2 and CD20 for
patient no. 3. These MABs were used and each patient-specific
sample was treated with an MAB1 that corresponds to that
sample.
LANTCET Method
[0192] High selectivity of formation of clusters of nanoparticles
in the target tumor cells is achieved through a two stage
incubation procedure. At the first stage the samples (normal and
tumor) were separately incubated for 30 minutes at 4.degree. C.
with diagnosis-specific MAB1 (mouse anti-human CD10, CD19 or CD20
for each tumor sample) that selectively attach mainly to blast
cells. Then both samples were separately incubated for 30 minutes
at 4.degree. C. with 30 nm gold spherical NP that were conjugated
with secondary MAB2 (#15754, Ted Pella, Inc., Redding, Calif.).
NP-MAB2 comes as a factory-made complex and MAB2 (goat anti-mouse
IgG(H+L)(AH)) has high coupling efficiency for MAB1. The gold
nanoparticles were attached to cell membranes through the chain of
bonds, i.e., cell receptor-MAB1-MAD2-NP. All operations during
stage 1 were performed at 4.degree. C. which minimizes any
physiological processes and allows efficient MAB-receptor or
MAB-MAB interactions. Selectivity of the targeting after stage 1 is
good, however, not sufficient from the clinical point of view.
First, MAB1-specific receptors also may be expressed at minor
levels in some normal cells. Second, MAB2 may directly couple
through different uncontrollable mechanisms directly to the
membranes of normal, i.e., non-target cells. Thus, small amounts of
NPs may attach to membranes of normal cells causing death during
exposure to laser radiation.
[0193] Therefore, selectivity and LANTCET safety is improved via a
second incubation stage to create maximal clusters of NP inside the
target cells. A temperature of 37.degree. C. and incubation time of
30-120 minutes stimulates internalization of NPs through
endocytosis into the cell endosomes. At the end of stage 2 large NP
clusters were formed only in those cells, i.e., the target cells,
with a high initial level of membrane-bound NP. Because of a much
lower level of NP at the membranes of normal cells, the clusters
either were not formed at all or had much smaller dimensions. The
bigger the cluster size, the lower the laser fluence threshold of
bubble generation. An explanation for such reduction of the laser
fluence threshold is found in the combination parameters
responsible for vapor bubble formation, i.e., larger NP volume
increases heat accumulated in the vapor, longer heat diffusions
time increases effectiveness of laser interaction with
light-absorbing clusters, and a reduced surface tension and dynamic
viscosity allows expansion of vapor nuclei into microbubbles
[54].
[0194] Next, normal and tumor cell suspensions were injected into
separate sample chambers with a diameter of 2.5 mm (S-24737,
Molecular Probes, OR), and exposed to single laser pulses at a 532
nm wavelength matching a peak of light absorbance for spherical 30
nm gold NP. The laser pulse duration was 10 ns, which approximately
matches the heat diffusion time from a 200 nm cluster of gold NPs
into water. The laser energy(fluence) was set at the minimal level
that provides bubble generation only around the biggest NP clusters
(>200 nm) and does not induce any bubbles around smaller
clusters or around single NPs which have a higher threshold for
bubble generation. This fluence level provides high selectivity of
cell killing because no bubbles can be generated in normal cells
even if they accumulated some NP. Two experimental modes, i.e.,
single cell irradiation and simultaneous irradiation of many cells
in suspension, were studied. Tumor and normal samples were exposed
to laser pulses with equal fluence. In a single-cell mode (FIG.
11A) each individual cell (total of 150) was irradiated one by one
with single focused laser pulse at 532 nm (LS 2132, Lotis TII,
Minsk, Belarus). This mode was used for investigation of
laser-induced bubbles in cells. Biological damage to irradiated
cells was studied in the suspension mode (FIG. 11B) through
simultaneous irradiation of 4,000-20,000 cells in a sample chamber
with a wide laser beam (3 mm diameter, 532 nm, 10 nanoseconds).
After exposure to laser pulses the samples were collected from the
chambers and analyzed for cell viability.
Imaging and Measuring Nanoparticles in Individual Cells
[0195] CCD camera (model U2C-14S415, Ormins Ltd, Minsk, Belarus)
with a 12-bit dynamic range and a sensor size of 1300.times.1000
pixels was used with a fluorescent light microscope (Leica DML,
Leica Microsystems, Wetzlar, Germany) with
100.times.-microobjective. Standard "green" fluorescent excitation
mode was applied. Fluorescent signal amplitude was acquired and
measured in counts (0-12000) of camera digitizer. R-phycoerythirin
(PE) fluorescent dye (#P9787), Ted Pella, Inc., Redding, Calif.)
factory-conjugated with MAB3 (anti-goat IgG), was used as a marker
for NP. PE was coupled to NP through MAB3-MAB2 bonds and by
incubating the tumor and normal samples with PE-MAB3 after stage 1
and prior to stage 2 treatment.
[0196] Optical concentration of PE was determined by flow
cytometry. Tumor cells were divided into two samples. Sample 1,
NP-free cells, was incubated only with PE-MAB3 for 30 min at
4.degree. C. and was used as a reference source of non-specific
fluorescence of PE. Sample 2 was treated according to stage 1
protocol (cells with NP) and then was incubated with PE-MAB3 for 30
min at 4.degree. C. Sample 2 was used as the source of Np-specific
fluorescence. Several concentrations of PE were used. Concentration
of PE 1:2,5000 provided maximal ratio of 18 for the mean amplitudes
of flow cytometer output signals of the sample 2 (51.9.+-.29.5 au)
to the sample 1 (2.85.+-.2.58 au). This PE concentration was used
in all experiments for detecting and imaging NP in cells and the
level of non-specific fluorescence was less than 6%.
[0197] To allow quantitative estimation of NP concentration in
individual cells, the CCD camera was calibrated. The images of
homogenous NP solutions (without cells) in water at several
different concentrations of NP were obtained. The mean pixel
amplitude S of fluorescent signal was found to be almost linearly
proportional to the concentration of NP, which allowed one to
estimate the NP local concentration C through fluorescent amplitude
S in the corresponding point of the image by:
C=0.022.times.10.sup.11.times.(S-S.sub.bc-S.sub.ns) (11)
where S.sub.bc is a background signal of image detector (459
counts), S.sub.ns is a level of non-specific signal (6%).
[0198] For each cell, optical and fluorescent images were obtained
prior to its exposure to a laser pulse. Optical image was used for
determining cell diameter and location of outer membrane and
nuclear membrane. The fluorescent image was analyzed with special
software that was previously developed as part of the photothermal
microscope (21). Several image parameters were used to analyze such
as NP-related properties as the mean level of NP in an individual
cell, which is measured as the cell-averaged pixel amplitude, the
amount of NP in clusters which characterizes cluster size and is
measured as the maximal amplitude in the center of cluster-related
image, i.e., Max, the spatial radial distribution of NP and their
clusters inside the cell which are measured as the mean relative
radius Mir (normalized by cell radius) corresponding to location of
maximal amplitude, and the heterogeneity of spatial distribution of
the Np. Image parameters were calculated for each cell and then
were analyzed as histograms for each sample.
[0199] For the images with size close to the diffraction limit of
the microscope (300-350 nm), an actual size of the source of
fluorescent signal can not be determined because it can be much
less than that. Thus, the peak image amplitude was treated as the
measure of total number of NP in one cluster. Conventional
(non-confocal) microscopic image is the two-dimensional projection
of three-dimensional distribution of fluorescent signals in the
depth of focus of the micro-objective. Still, membrane-type
fluorescence could be differentiated from cytoplasm-type
fluorescence and the uniform fluorescence from cluster-type
fluorescence.
Detection of Laser-Induced Microbubbles in Cells
[0200] The photothermal microscope was used for detecting vapor
bubbles in individual cells. Each cell was illuminated with three
collinear focused laser beams, i.e., a pulsed beam for bubble
generation and two low-intensity continuous (633 nm, 0.1 mW) and
pulsed (640 nm, 8 ns) probe beams for detecting the bubble in a
cell in real time. Any bubble-related change of refractive index
causes the shift of the phase of the probe beam that influences
beam intensity in the input of the photodetector. Output of the
photodetector is measured as PT response by a high speed digitizer
(Bordo-211, Auria Ltd., Belarus) in case the bubble PT response has
a specific shape so that bubble generation can be detected during
irradiation of individual cells with a laser pulse.
[0201] Each individual cell (total of 150) was irradiated one by
one with a single focused laser pulse at 532 nm (Lotis TII, Minsk,
Belarus) and the PT response from each cell was simultaneously
recorded as the time-response of the probe laser intensity [22].
This mode allowed both bubbles detection and lifetime measurement
that characterizes maximal bubble diameter. After sequentially
irradiating 150 cells with a single pulse and registering the PT
response from each one, the probability PRB of bubble generation at
a specific laser fluence was as described in Example 1.
[0202] For time-resolved imaging of the bubbles in individual
cells, a dye pulsed probe laser beam at the wavelength of minimal
light absorption by the cell (640 nm, 8 ns) was used and which was
synchronized and delayed for 120 ns against the pump pulse [55].
Bubble location and diameter were analyzed from probe beam images
captured with a CCD camera so that only events occurring after this
delay and during probe pulse duration were visualized.
Cell Damage Detection
[0203] Laser-induced damage was measured by microscopy
(concentration counts in hemocytometer) and flow cytometry (outer
membrane damage detection as propidium iodide, PI, positive stains)
after each stage of sample preparation and 2 hours after laser
treatment. Microscopy detected the destruction of cells and flow
cytometry detected necrotic death of those cells that were damaged,
but not destroyed. Cells with compromised membranes that included
propidium iodide (PI) were considered as damaged. The cells that
were counted as PI-negative were considered as survived live cells.
The level of survived live cells LLC was used as a measure of
LANTCET efficacy and was analyzed experimentally for each normal
and tumor sample as a function of laser pulse fluence, number of
laser pulses, types of MAB1, and incubation stage.
[0204] This parameter describes the relative change of the level of
living cells in population due to laser treatment and counts cell
losses due to destruction and cell death:
LLC=(C.sub.al/C.sub.bl)*C.sub.PI-*100% (eq. 12)
where C.sub.bl is the initial concentration of all cells in the
sample (counted in hemocytometer) before laser treatment, C.sub.al
is the concentration of all cells in the sample after laser
treatment (both counted in hemocytometer), C.sub.PI- is the level
of PI-negative (live) cells obtained with a flow cytometer for
MAB1-positive cells in the tumor sample and for all cells in the
normal sample. Delayed cell death was not considered because the
bubble-related mechanism of cell damage acts very fast.
Fluorescent Imaging of Nanoparticles in Cells
[0205] Fluorescent images of tumor cells showed the two types of
spatial distribution of the signals: local peaks and uniform areas
(FIGS. 12A-12F). Local and relatively strong fluorescent peaks were
found in most of the images of the tumor cells that were
pre-incubated with nanoparticles. These peaks were associated
either with cell membrane (FIGS. 12C, 12E) or with cell cytoplasm
(FIGS. 12D, 12F). The amplitude in the peak (2000-10000 counts;
FIGS. 12E-12F) was 5-20 times higher than that for cell areas with
uniform signal (200-500 counts). All peak-related images had a
similar shape, a round spot with the diameter 0.4-0.8 .mu.m, that
is close to the diffraction limit of the microscope. The
fluorescent signals of such shape and amplitude were never observed
in cell-free space. Such peaks are considered as evidence for the
clusters of nanoparticles.
[0206] Using calibration data, the concentration of single NP in
tumor cells was estimated as
11.2.times.10.sup.11.+-.4.7.times.10.sup.11 for the uniform areas,
while in the clusters it varied from
57.times.10.sup.11.+-.21.times.10.sup.11 (48 C, 0.5 hours) to
122.times.10.sup.11.+-.55.times.10.sup.11 (37.degree. C., 2 hours).
Concentration of NP in tumor cells was found to be approximately 10
times (uniform areas) and 100 times (clusters) higher than the
concentration of free unbound NP during the first stage of cell
incubation. The actual size of NP clusters could not be measured
because it may be below the optical diffraction limit of the
microscope and, therefore, the actual concentration of NP in the
clusters may be even higher. Nevertheless the maximal peak
amplitude characterizes the total number of NP in the cluster. Some
of the normal BM cells also exhibited fluorescent images (not
shown) with cluster-related peaks that were similar to those
obtained for the tumor cells (FIG. 12C). Although the total level
of NP-positive normal BM cells was within 6%, the rest of normal
cells did not yield NP-specific fluorescence in their images.
Nanoparticle Accumulation in Cells after Stage 1 and Stage 2
[0207] The peak amplitudes and the homogeneous fluorescence were
measured in tumor and normal cells as a function of the incubation
time after stage 1 (4.degree. C., membrane coupling) and stage 2
(37.degree. C., internalization). All fluorescent images were
analyzed in two ways. First, the image parameters were calculated
for each cell and were plotted as the histograms for each stage of
cell incubation (FIG. 13A). Second, cell counts were performed for
three categories of fluorescent images, i.e., images without NP
clusters, images with peripheral NP clusters only that are located
at cell outer membrane and images with peripheral and intracellular
(located inside cell) clusters of NP (FIG. 13B). Image parameters
and cell counts were used to analyze the influence of incubation
temperature and time on clusterization of NP.
[0208] Distinct intracellular clusters of NP were found only for
the tumor cells that were incubated at 37.degree. C. (after stage
2, see FIG. 11D, 11F) with fluorescent peaks having maximal
amplitudes of 8,000-11,000 counts. To compare NP cluster size after
stage 2 and stage 1 the histograms were analyzed for the maximal
values of fluorescent amplitudes in peaks (FIG. 13A). This image
parameter is important because the largest clusters of NP in the
cell would produce the biggest bubbles and thus such clusters are
the main cell-killing nanostructures. Maximal peak amplitudes after
stage 1 were 2,000-4,000 counts for all incubation times, which is
two to four times lower than those after stage 2 (2 hours of
incubation). This difference may be interpreted as the difference
in NP cluster size because the amplitude is proportional to the
total number of NP in one cluster and the size of the cluster
correlates to the number of NP in it. Therefore the stage 2
(incubation at 37.degree. C., 2 hours) provided the largest
clusters of NP with their localization inside cells. Fluorescent
peaks within cell nuclei were not observed. Thus, the
internalization of NP was confined by the cytoplasm; NP did not
penetrate into cell nuclei that are quite large for this type of
cells (FIG. 11A-11D). Spatial distribution of fluorescent peaks in
cells was quantified through corresponding image parameter Mir and
as function of the time and temperature of cell incubation (FIG.
13B). Histogram analysis of this image parameter showed a clear
trend in re-localization of NP clusters from the periphery (Mir
value 0.6-0.8) after stage 1 to the inner site of the cell (Mir
value 0.3-0.6) after stage 2 of incubation. Such spatial
distribution of the fluorescent peaks confirms endocytotic
mechanism of NP clusterization.
[0209] Additional measurements were made to understand the kinetics
of the internalization process. The cells with images were counted
as in the three categories defined above categories, i.e., the
number of cells with intracellular peaks in their images and the
number of cells with membrane-located peaks in their images, as
function of cell incubation time and temperature (FIGS. 14A-14B).
No changes were found in those categories for normal BM cells (FIG.
14A) regardless of the time and the temperature of cell incubation.
The counts of cluster-related cells were within 6.0%.+-.1.1% for
any incubation conditions. So stage 2 did not add any NP or NP
clusters to normal cell after stage 1. For tumor cells the
situation was totally different (FIG. 14B). Incubation at
37.degree. C. (stage 2) caused steady increase of the number of
cells with intracellular clusters from 12.0%.+-.2.0% at the
beginning of stage 2 to 66.0%.+-.4.0% after 2 hours. Also during
stage 2 tumor cells yielded the decrease of cell counts for
membrane-located clusters from 84.0%.+-.4.2% at the beginning to
30.0%.+-.2.8% after 2 hours. Tumor cells that were incubated at
4.degree. C. (stage 1) showed no changes at all in the counts of
membrane-located and intracellular clusters (FIG. 14B). The level
of intracellular NP clusters after stage 1 was under 8.0%.+-.0.9%
and did not increase for 2 hours. Obtained experimental results
demonstrated that the formation of clusters of NP occurred at the
cellular membrane and inside the cells and that the largest NP
clusters emerge inside in the cytoplasm of the tumor cell during
the second stage of incubation from internalization. The difference
in NP levels in normal and tumor cells indicated significant
increase in selectivity of NP clusterization in tumor cells in
comparison with normal cells after an additional second stage of
incubation.
Laser-Induced Bubbles in Cells
[0210] Photothermal (PT) responses and images were obtained for
individual tumor cells (single cell mode) at different laser
fluencies. Typical bubble-specific PT responses and images that
were obtained after a single laser pulse at 0.6 J/cm.sup.2, 532 nm
for incubation conditions of 37.degree. C., 2 hrs are shown in
FIGS. 15A-15C. The duration of the PT response allows measurement
of the bubble lifetime and thus its maximal diameter can be
estimated. Location of the bubble-specific signals in the PT image
of the tumor cell (FIG. 15A) shows that the bubbles were generated
in the cytoplasm and probably not in the area of the nucleus. The
bubble generation sites spatially coincide with location of
nanoparticle clusters as shown in the fluorescent images. Also it
was found that the number of bubble-related signals in one cell,
from 1 to 4, was lower than the number of cluster-related peaks,
4-20, in those cells. Therefore, under a given laser fluence of 0.6
J/cm.sup.2, not every cluster produced the bubbles. It is likely
that only biggest clusters of nanoparticles are cell damaging
agents while the rest of smaller clusters and single nanoparticle
may not produce the bubbles and therefore do not contribute to
nanothermolysis of the cell.
[0211] The PT response of the bubble (FIG. 15A) was obtained at the
fluence of 0.6 J/cm.sup.2. No bubble-specific response was detected
at the same fluence when water suspension of individual
nanoparticles at a concentration of 8.times.10.sup.11/ml was
irradiated with a single laser pulse. Also, such PT responses were
not detected for normal bone marrow cells. This result may be
considered as additional evidence that only clusters of
nanoparticles produced the bubbles at this fluence level. It also
was discovered that the cells with nanoparticle clusters, shown in
their fluorescent images, may generate the bubbles during exposure
of the cell to several (up to 60) laser pulses. Single NPs that
produced bubbles at much higher fluencies exhibited first-pulse
bubble generation though never produced the bubbles during the
second and following pulses because they were destroyed, i.e.,
melted or evaporated, during the first laser pulse. Thus, the
cluster of nanoparticles are a photostable structure, unlike the
single nanoparticles that are destroyed after the first pulse, and
can be irradiated with more than one laser pulse. Using the model
of a laser-activated bubble, maximal bubble diameter is estimated
as 13 .mu.m for the PT signal (FIG. 15C). Such a bubble size is
comparable with the cell diameter, that is, 8-9 .mu.m (FIGS.
11A-11B) and therefore may rupture the cell outer membrane. This
causes lysis of any tumor an normal cell.
Laser-Induced Damage to Cells
[0212] Cell samples were irradiated in round 2.5 mm cuvettes with
single or several laser pulses at 532 nm. Cell damage was analyzed
through LLC dependence upon laser parameters (pulse fluence and
number of pulses) and incubation parameters (nanoparticle
concentration, incubation temperature and MAB1 types). Influence of
the laser parameters on the cell damage was studied for the same
incubation conditions such as one type of MAB1 (specific for each
patient), incubation temperature of 4.degree. C. and a nanoparticle
concentration during the first stage of incubation of 15000
nanoparticle/cell.
[0213] Increase of laser fluence from 0.2 to 2 J/cm.sup.2 caused a
gradual decrease of LLC for tumor cells from 3.9%.+-.0.6% at 0.2
J/cm.sup.2 to less than 1% at the fluencies above 0.6 J/cm.sup.2.
The samples obtained from 3 different patients with an ALL
diagnosis showed different degrees of damage of tumor cells. At the
fluence of 0.6 J/cm.sup.2 and single-pulse irradiation, the LLC was
found to be 1.5%.+-.0.3%, less than 0.1%, and 5%.+-.0.6%
respectively for the patients 1, 2 and 3. These samples were
treated with different MAB1 and the difference detected in LLC
values may be caused by the variations in nanoparticle targeting
efficacy. The change in LLC of the tumor cells after laser
treatment was due mainly to the decrease in the concentration of
cells, i.e., up to 10 times in comparison with initial
concentration, and, to a lesser extent, to cell membrane damage.
The LLC for normal bone marrow cells under the same conditions was
found to be 77%.+-.6.2% to 84%.+-.4.2%. Irradiation of tumor cells
with 10 laser pulses instead of 1 did not produce a significant
effect. The LLC decreased from 1.5%.+-.0.3% to 1.2%.+-.0.2%. This
means that the first laser-induced bubbles produce the damage and
unlike the damage through heating the bubble-related damage does
not have accumulative nature and occurs immediately after expansion
of the first bubble.
[0214] The influence of incubation parameters on cell damage was
studied. Under a fixed concentration of nanoparticles during the
first stage of incubation, the application of the different primary
MABs resulted in a variation of LLC from 0 to 67% (FIG. 16A). The
strongest effect, that is when LLC is 0, was reached by using the
combination of several primary monoclonal antibodies. The influence
of another incubation parameter, the temperature of the second
stage of incubation, is shown in FIG. 16B. Increase of the
incubation temperature from 4.degree. C. to 37.degree. C. caused a
2.6.times. decrease of LLC for tumor cells from 3.9%.+-.0.6% at
4.degree. C. to 1.5%.+-.0.3% at 37.degree. C. and a 1.6.times.
decrease of LLC for normal cells from 77%.+-.6.2% at 4.degree. C.
to 49%.+-.5% at 37.degree. C. Therefore in terms of the efficacy of
cell killing the two-stage incubation at 37.degree. C. has produced
better results than the standard incubation scheme at 4.degree. C.
Although "hot" incubation was less safe for normal BM cells. No
significant variation of LLC for tumor cells was discovered when
the concentration of nanoparticles varied from 6000 to 150000
nanoparticles/cell during the first stage of incubation. The LLC of
tumor cells decreased from 15% (6000 nanoparticle/cell) to 10%
(150,000 nanoparticle/cell). It is assumed that the concentration
from 15000 to 30000 nanoparticles/cell is quite saturating, because
further increase of nanoparticle concentration had no effect on
cell killing efficacy.
Example 3
LANTCET of Human Normal and Tumor Cells Using Nanorods or
Nanoshells
[0215] Cells and Antibody Conjugates
[0216] K562 cells that have high level of expression of CD33
antigens were used. Primary samples of human bone marrow (BM) taken
either from normal donors or patients with the diagnosis of acute
myeloid leukemia (AML) were used in experiments. All AML (tumor)
samples consisted mainly of tumor cells where the level of
B-lymphoblasts was 94 to 98% in samples from different patients.
AML cells expressing diagnosis-specific CD-33 genes were determined
with flow cytometry.
[0217] Conjugates of gold nanorods (NR) with and without antibodies
to CD33 were used. AML and K562 cells were incubated for 30 min at
37.degree. C. with the gold nanorods with dimensions 45.times.14 nm
and absorption maximum at 780 nm. Then living and fixed cells were
used as suspensions for bubble generation and detection.
[0218] Solid tumor cells were prepared as the monolayers of living
EGF-positive carcinoma cells (Hep-2C) that were grown on glass
surface and human lymphocytes were used as normal cells. Both
Hep-2C and normal lymphocytes were incubated 35 min at 37.degree.
C. with the conjugates of 40 nm gold nanoshells-C225 antibody that
was grown against EGFR. Cells were irradiated with single laser
pulse at the wavelength of 720 nm close to the peak of maximal
optical absorbance of the nanoshells.
Generation and Detection of Photothermal Bubbles (PTB) and
Imaging
[0219] The photothermal microscope described herein is used for
generating and detecting PTB in individual cells. Each cell was
illuminated with 3 collinear focused laser beams: pulsed beam for
bubble generation (532 nm or 720-840 nm), and low-intensity
continuous (633 nm, 0.1 mW) and pulsed probe beams for detecting
the bubble in a cell in real time. Each individual cell (total of
90 for each sample) was irradiated one by one with single focused
laser pulse of the same fluence at 532 nm (gold nanospheres) or
720-840 nm (gold nanoshells and nanorods) and PT image and response
from each cell were simultaneously recorded as time-resolved image
of pulsed probe laser and time-response of c.w. probe laser
intensity. After sequential irradiating of all cells in the sample
and registering PT images and responses from each cell we have
measured the probability PRB of bubble generation at specific laser
fluence is measured as described herein. A CCD-camera as described
herein is used for time-resolved imaging of the bubbles in
individual cells irradiated with a pulsed pump laser beam.
Optical Scattering Images and Spectra of Nanorod Clusters in
Cells
[0220] Formation of nanorod (NR) clusters was verified directly
with optical scattering microscopy and microspectroscopy. As the
source of light we used A white light continuous source was used.
Light was directed with an optical fiber at the glass slide with
the sample at an angle of 70-80.degree.. NR-treated and control
(untreated) K562 cells were imaged with light scatter photothermal
microscopy. Images of individual cells are shown in FIGS. 17A-17C.
Those images revealed two common features both for the AML and K562
cells. NP-related signals were detected in 90-95% of targeted cells
and spatial distribution of NP-related signals within the cell was
highly heterogeneous with apparent local peaks. Also no NP-related
signals were found in the images of control cells. Thus, regardless
of the targeting method used the nanorods aggregate into clusters
during interaction with living cells.
[0221] Amplitudes of scattering signals are 1230.+-.580 for intact
control cells and 3980.+-.1170 for NR-treated K562 and AML cells.
Scattering images were registered with a continuous white light
source and no photobleaching effects occurred, so they are more
suitable for NP imaging and quantitative studies. Untreated
(control) cells also produced some optical scattering though with
much lower amplitudes and without strong local peaks (FIG. 17A).
Optical scattering sensitivity may be improved by using a
monochromatic light source at the wavelength that matches plasmon
resonance wavelength for NR clusters. Spectral studies of the cells
were performed with a microspectrometer. Scattering spectra of a
local zone with diameter not bigger than 1 .mu.m were measured for
control and treated cells (FIG. 17C). For treated cells the spectra
were measured for the regions occupied by the clusters. The
cluster-related spectrum demonstrated a resonant nature of
scattering and is very close to that obtained for the water
suspension of nanorods. Their maximums almost coincide (680 nm for
NR water suspension and 670 nm for NR clusters in cells). The
conclusion is that the local scattering signal represent the
clusters of gold nanorods.
Photothermal Bubbles Around Single NP and in AML Cells Around Gold
Nanorods
[0222] A typical bubble-specific PT response (PTB) and images are
shown in FIGS. 18A-18C that were obtained for individual AML cells
with nanorod clusters at different laser fluencies. Peripheral
location of the bubble-specific signals in the PT image of the
tumor cell shows that the bubbles were generated in the cytoplasm
and probably not in the area of the nucleus. For all types of
studied cells, including cultured and primary tumor cells, a single
laser pulse accompanied by PTB caused cell lysis through mechanical
damage of the cytoplasmic membrane. Cell membrane damage can be
seen in its optical image (FIG. 18D). Even the smallest bubbles
with lifetimes of 20-40 ns caused cell damage. Table 2 gives bubble
generation thresholds for AML cells (780 nm, 10 ns).
TABLE-US-00002 TABLE 2 Bubble generation Samples thresholds,
J/cm.sup.2 Intact cells >50 Cells with NR (4.degree. C.)
.apprxeq.1.5 Cells with NR (37.degree. C.) 0.32
[0223] Analysis of the thresholds of PTB as shown in the Table 2
indicates that PRB increases gradually up to 100% with increase of
pulse fluence and this means that ALL the cells in population
accumulated nanorods. Also this dependence allows the bubble
generation threshold to be determined as one value for all cells.
Increasing the temperature during the incubation of the cells with
nanorods from 4.degree. C. (only chemical reactions are allowed) to
37.degree. C. (all physiological reactions including endocytosis
are allowed) caused significant increase of PRB and bubble size and
the decrease of bubble generation threshold by 5 times. Also in the
both cases PTB threshold levels are much lower than the threshold
obtained for suspension of single nanorods in water. The conclusion
in both cases is that clusters of nanorods were formed and that
nanorod clusters formed at 37.degree. C. are larger.
[0224] Spectral dependence of PTB lifetime and probability of PTB
generation around single NR in their water suspension and after
their clusterization in human bone marrow CD33+ AML cells was
measured (FIGS. 19A-19B). Bubble generation probability (PRB) was
measured at several NIR wavelengths at fixed laser fluence levels
that was 7.5 J/cm2 for single nanorods in water and 0.75 J/cm2 for
the cells treated with the same nanorods. The PRB spectrum for
single nanorods closely matches its absorption spectrum, while
formation of nanorod clusters has broadened the spectrum of PRB
obtained for the cells.
[0225] The PRB spectra for nanorods in water matches optical
absorption spectra of the same nanorod suspension with peak PRB and
OD at 780 nm. This means that bubbles are generated due to
absorption of laser energy. The cell spectrum for PRB also has peak
around 750-780 nm and matches the spectrum of PRB for NR
suspension. Significant broadening of PRB spectrum was not found as
it was found for optical absorption spectrum of aggregated NR in
water. Notably, the absorbance and PRB spectra of the nanorod
suspension in water match each other in terms of width of peak, but
in the case of cells there is no such match. Absorbance spectrum of
suspension of nanorod clusters is very broad, but PRB spectrum of
cells that apparently have nanorod clusters is not so broad and the
width of its peak is closer to the width of the peak of the PRB
spectrum obtained for individual nanorods in water. Comparing
bubble generation thresholds obtained for single spherical
nanoparticles and nanoshells in water, for normal cells and tumor
cells indicates that target tumor cells form big clusters resulting
in the decrease of bubble generation/cell damage laser fluence
threshold in tumor cells by almost 100 times relative to untreated
cells. Table 3 gives laser fluence thresholds (J/cm.sup.2) for
generation of PTB in normal and ALL tumor cells.
TABLE-US-00003 TABLE 3 Gold red Single NP NP type lymphocytes blood
cells K562 AML In water Spherical NP 9.4 27.0 43 (bare, 30 nm) (532
nm) (532 nm) (532 nm) NP-IgG-CD33 0.3 0.42 (conjugates) (532 nm)
(532 nm) NR-PEG 0.43 0.32 17 45 .times. 14 nm (780 nm) 780 nm) 780
nm) No particles 10.0 2.6 >45 >35 -- Intact cells (532 nm)
(532 nm) (532 nm) (532 nm) />40 (780 nm) />50 />50 />50
(780 nm) (780 nm) (780 nm)
[0226] Photothermal Bubbles Formed in Solid Tumor Cells Around Gold
Nanoshells
[0227] A solid tumor application was studied with Hep-2C model
cells and conjugates of 40 nm gold nanoshells with EGF-specific
antibody C225. Bubble images and responses obtained from carcinoma
cells Hep-2C are shown in FIGS. 20A-20D. Comparing bubble
generation thresholds obtained for single nanoshells in water, for
normal cells, and for tumor (Hep-2C) cells showed that normal cells
uptake some single nanoshells that do not form clusters and that
target tumor cells form large nanoshell clusters. These large
clusters cause a decrease in the bubble generation/cell damage
laser fluence threshold in Hep-2C cells by almost 100 times,
relative to untreated cells, and by 20 times, relative to normal
cells that also were treated with NP-C225 conjugates and laser
pulsed under the same conditions. Table 4 gives the laser fluence
thresholds (J/cm.sup.2) for generation of PTB (laser pulse 720 nm,
10 ns) in normal and solid Hep-2C tumor cells.
TABLE-US-00004 TABLE 4 red Single NP NP type lymphocytes blood
cells Hep-2C in water NS (bare) -- 11.0 -- NS-C225 12.0 -- 0.5 11.0
(conjugate) No particles >40 >50 >40 -- Intact cells
Example 4
LANTCET of a Sarcoma in a Rat Model Using Nanoparticles
[0228] Rats were used to grow polymorphic sarcoma 1 (tumor type M-1
obtained from the bank of Russian Oncological Research Center) to a
diameter of 0-15 mm. The skin layer was removed at the tumor site.
A drop of a nanoparticle suspension (5 .mu.l) was administered onto
the tumor surface and left for 40 min to be absorbed by the tumor.
Gold spherical 30 nm particles conjugated to goat anti-mouse IgG
(#15754, Ted Pella, Inc, Redding, Calif.) were then applied
topically onto the tumor surface. The skin layer was removed at the
tumor site. The drop of spherical nanoparticles suspension (5
.mu.l, with concentration of NP 8.times.10.sup.11/ml) was
administered onto the tumor surface and left for 40 min to be
absorbed by the tumor at room temperature. The tumor site was then
treated with the laser. A single laser pulse at 532 nm (maximum of
light absorbance by NPs), 10 ns duration, fluency of 0.75
J/cm.sup.2, and diameter of 3-4 mm was directed to the central area
of the tumor. After 24 hours following the laser treatment the
solid tumor was extracted from the animal and the degree of tumor
necrosis was measured according to the uptake of trypan blue.
[0229] The tumor that was treated with a single laser pulse showed
a necrotic (FIG. 21A, white area) area with diameter close to the
laser beam diameter (3-4 mm) and the depth of 1-2 mm that indicates
the spherical nanoparticle diffusion depth. The tumor that
underwent laser treatment without pretreatment with NPs showed no
signs of necrosis (FIG. 21B). The conditions of spherical NP-cell
interaction at physiological temperature and 40 min of interaction
time were sufficient to allow NP cluster formation inside tumor
cells.
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[0286] Any publications or patents mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually
incorporated by reference.
[0287] One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those objects,
ends and advantages inherent herein. Changes therein and other uses
which are encompassed within the spirit of the invention as defined
by the scope of the claims will occur to those skilled in the
art.
* * * * *