U.S. patent application number 15/088574 was filed with the patent office on 2016-09-22 for methods compositions and kits for imaging cells and tissues using nanoparticles and spatial frequency heterodyne imaging.
This patent application is currently assigned to Brown University. The applicant listed for this patent is Brown University, Rhode Island Hospital. Invention is credited to Zoltan Derdak, Vivian Ortiz, Danielle Rand, Christoph Rose-Petruck, Jack R. Wands.
Application Number | 20160274086 15/088574 |
Document ID | / |
Family ID | 48086236 |
Filed Date | 2016-09-22 |
United States Patent
Application |
20160274086 |
Kind Code |
A1 |
Rose-Petruck; Christoph ; et
al. |
September 22, 2016 |
Methods compositions and kits for imaging cells and tissues using
nanoparticles and spatial frequency heterodyne imaging
Abstract
Methods, compositions, systems, devices and kits are provided
herein for preparing and using a nanoparticle composition and
spatial frequency heterodyne imaging for visualizing cells or
tissues. In various embodiments, the nanoparticle composition
includes at least one of: a nanoparticle, a polymer layer, and a
binding agent, such that the polymer layer coats the nanoparticle
and is for example a polyethylene glycol, a polyelectrolyte, an
anionic polymer, or a cationic polymer, and such that the binding
agent that specifically binds the cells or the tissue. Methods,
compositions, systems, devices and kits are provided for
identifying potential therapeutic agents in a model using the
nanoparticle composition and spatial frequency heterodyne
imaging.
Inventors: |
Rose-Petruck; Christoph;
(Barrington, RI) ; Wands; Jack R.; (East
Greenwich, RI) ; Rand; Danielle; (Quincy, MA)
; Derdak; Zoltan; (Riverside, RI) ; Ortiz;
Vivian; (Nashville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Brown University
Rhode Island Hospital |
Providence
Providence |
RI
RI |
US
US |
|
|
Assignee: |
Brown University
Providence
RI
Rhode Island Hospital
Providence
RI
|
Family ID: |
48086236 |
Appl. No.: |
15/088574 |
Filed: |
April 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13645938 |
Oct 5, 2012 |
9316645 |
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15088574 |
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61546484 |
Oct 12, 2011 |
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61544419 |
Oct 7, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/56966 20130101;
B82Y 5/00 20130101; A61K 49/0428 20130101; G01N 2500/00 20130101;
G01N 33/57484 20130101; G01N 33/5011 20130101; G01N 33/574
20130101; G01N 33/57438 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 33/574 20060101 G01N033/574; A61K 49/04 20060101
A61K049/04 |
Goverment Interests
GOVERNMENT FUNDING
[0002] A portion of this work was supported by U.S. Department of
Energy grant DE-FG02-08ER15937, U.S. Department of Education GAANN
award P200A090076, and the National Institutes of Health grant
CA123544. The government has certain rights in this invention.
Claims
1-13. (canceled)
14. A method of identifying in a model system a potential
therapeutic agent for treating or preventing a disease condition,
the method comprising: contacting a first sample and a second
sample of cells or tissue having the disease condition with a
composition including: a nanoparticle and at least one of a polymer
layer coating the nanoparticle and a binding agent that
specifically binds the disease agent; contacting the second sample
with the potential therapeutic agent; and, measuring a presence or
an amount of a marker in the first sample and the second sample,
wherein the marker is characteristic of the disease condition,
wherein a greater amount of the marker in the first sample compared
to that in the second sample is a measure of treatment and
protection by the potential therapeutic agent, thereby identifying
the potential therapeutic agent for treating or preventing the
disease condition.
15. The method according to claim 14, wherein detecting the
presence of the marker in the first sample and the second sample
comprises measuring or detecting the nanoparticle using X-ray
scatter imaging or spatial frequency heterodyne imaging.
16. The method according to claim 14, wherein prior to contacting,
the method further comprises constructing the nanoparticle or a
plurality of nanoparticles comprising at least one material
selected from the group of: a metal, a metal oxide, a magnetic
resonance imaging agent, and a combination thereof.
17. The method according to claim 16, wherein constructing the
nanoparticle comprises forming a shell or core of the nanoparticle
with at least one from the group of: with at least one of: silver,
copper, gold, cadmium, zinc, nickel, palladium, platinum, rhodium,
platinum, manganese, gadolinium, dysprosium, tantalum, titanium,
and iron.
18. The method according to claim 14, wherein the binding agent
comprises at least one selected from the group of: a drug, a
protein, a carbohydrate, and a nucleotide sequence.
19. The method according to claim 14, wherein the disease condition
is associated with or produced by at least one from the group of: a
virus, a tumor, a cancer, a fungus, a bacterium, a parasite, a
pathogenic molecule, and a protein.
20. The method according to claim 14, wherein prior to contacting,
the method further comprises constructing the nanoparticle by
attaching or conjugating the binding agent to an external surface
of the nanoparticle, wherein the binding agent comprises at least
one selected from the group of: a drug, a protein, a carbohydrate,
and a nucleotide sequence.
21. The method according to claim 20, wherein the protein is an
antibody selected from the group of: a monoclonal antibody; a
polyclonal antibody; a single-chain antibody (scFv); a recombinant
heavy-chain-only antibody (VHH); an Fv; a Fab; a Fab'; and a
F(ab').sub.2.
22. The method according to claim 14, wherein measuring further
comprises observing a localization of the marker in the cells or
the tissue of the first sample and the second sample.
23. The method according to claim 14, further comprising spatial
frequency heterodyne imaging the nanoparticles in the cells or the
tissue using an absorption grid and a detector.
24. A kit for imaging cells or a tissue in a subject comprising: a
composition comprising a nanoparticle including at least one
selected from the group of: a polymer layer and a binding agent,
wherein the composition binds to and/or is phagocytosed by the
cells or the tissue; instructions for use, wherein the instructions
describe: contacting the cells or the tissue with the composition,
and imaging the cells and the tissue and detecting X-ray scattering
of the composition with a device; and a container.
25. The kit according to claim 24, wherein the nanoparticles
comprise at least one selected from the group of: a metal, a metal
oxide, an inorganic material, an organic material, a magnetic
resonance imaging agent, and a combination thereof.
26. A composition for imaging cells or a tissue comprising: a metal
nanoparticle having attached to an external surface of the
nanoparticle a polymer layer, and FB50 monoclonal antibody that
specifically binds an antigen of hepatocellular carcinoma, wherein
the polymer layer comprises at least one of: a polyethylene glycol,
a polyelectrolyte, an anionic polymer, and a cationic polymer.
Description
RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
provisional application Ser. No. 61/544,419 filed Oct. 7, 2011 and
61/546,484 filed Oct. 12, 2011 in the U.S. Patent and Trademark
Office, and which are incorporated herein by reference in their
entireties.
TECHNICAL FIELD
[0003] Systems, compositions, methods and kits are provided using
nanoparticle compositions and spatial frequency heterodyne imaging
for visualizing and detecting cells or a tissue such as a tumor,
for identifying a potential therapeutic agent for treating a
disease condition, and for preparing the nanoparticle
compositions.
BACKGROUND
[0004] Hepatocellular carcinoma (HCC) is the most common form of
liver cancer in adults, accounting for approximately three of every
four cancers in the liver (El-Sarag et al. 1999, New England
Journal of Medicine 340: 745-750). The American Cancer Society
estimates that more than 24,000 new cases of primary liver cancer
develop each year in the United States, of which approximately
19,000 result in death. HCC is common in developing countries,
particularly in sub-Saharan Africa and Southeast Asia (Trevisani et
al. 2008 Carcinogenesis 29: 1299-1305; and O'Brien 2004 Cancer
Journal 10: 67-73).
[0005] More than 500,000 people are diagnosed with HCC each year
worldwide (Trevisani et al. 2008 Carcinogenesis 29: 1299-1305; and
Bruix et al. 2006 Oncogene 25: 3848-3856). HCC is difficult to
diagnose in its earliest stages because there are currently no
screening tests available, and HCC generally becomes symptomatic
when the tumor is approximately 4.5 centimeters to eight
centimeters in diameter (Trevisani et al. 2008 Carcinogenesis 29:
1299-1305; and Colombo 1992 Hepatol 15: 225-236).
[0006] Detection by ultrasound and imaging by computed tomography
(CT) scans or magnetic resonance imaging (MRI) lack ability to
definitively and reproducibly diagnose early stage cancers,
particularly small HCC tumors (Bruix et al. 2006 Oncogene 25:
3848-3856; Hain et al. 2004 Cancer Journal 10: 121-127; Okuda 2000
J. Hepatol 32: 225-237; and Sheu et al. 1985 S. Cancer 56:
660-666). Misdiagnosis of HCC yielding false positive or false
negative results is common from these imaging techniques, and the
American Cancer Society estimates that HCC patients have a
five-year survival rate of just 10%.
[0007] New techniques for imaging and early diagnosis of HCC and
other cancers are needed to improve prognosis of cancers such as
HCC.
SUMMARY
[0008] An aspect of the invention provides a method of imaging
cells or a tissue, the method including: contacting a sample of the
cells or the tissue with a nanoparticle composition containing at
least one selected from the group of: a nanoparticle, a polymer
layer coating the nanoparticle, and a binding agent that
specifically binds a molecular species; irradiating the sample with
an X-ray beam; and, detecting by X-ray scatter imaging the
nanoparticle in the cells or the tissue. In various embodiments,
the polymer layer is composed of at least one of: a polyethylene
glycol, a polyelectrolyte, an anionic polymer, and a cationic
polymer. The polyelectrolyte in various embodiments includes a
protein, organic acid, or a polysaccharide; for example the
polyelectrolyte is a poly(acrylic acid) or a poly(allylamine
hydrochloride).
[0009] The method in various embodiments further includes prior to
contacting, constructing the nanoparticle with at least one of: a
metal, a metal oxide, an inorganic material, an alloy, and an
organic material. In a related embodiment, the method further
includes prior to contacting, constructing the nanoparticle with a
MRI agent, a positive contrast agent, or a negative contrast agent.
For example, the MRI agent includes an oil, a metal (e.g., iron and
magnesium) sulfate, a metal chloride, or a metal ammonium
citrate.
[0010] The method in various embodiments further includes prior to
contacting, constructing the nanoparticle with at least one of:
silver, copper, gold, cadmium, zinc, nickel, palladium, platinum,
rhodium, platinum, manganese, gadolinium, dysprosium, tantalum,
titanium, and iron. For example, the nanoparticle includes
gadolinium-diethylene triamine pentaacetic acid (DTPA).
[0011] In various embodiments, the nanoparticles comprise a
metallic core or a metallic shell. In various embodiments, the
shell electron density is greater than the core electron density.
Alternatively, in certain embodiments the core electron density is
greater than the shell electron density. In various embodiments,
the nanoparticles are non-toxic or biocompatible.
[0012] In an embodiment of the method, constructing the
nanoparticle involves producing the nanoparticle to have an average
diameter of at least about five nanometers (nm). In various
embodiments of the method, the nanoparticle has a diameter of at
least about: two nm, five nm, ten nm, 20 nm, 30 nm, 40 nm, 50 nm,
60 nm, 70 nm, 80 nm, 90 nm, or at least about 100 nm. In an
alternative embodiment of the method, the nanoparticle is less than
about 100 nm in diameter. In an embodiment of the method,
constructing the nanoparticle includes engineering a plurality of
nanoparticles having an average diameter greater than about five nm
and less than about 100 nm.
[0013] In various embodiments of the method, irradiating the sample
includes locating or inserting an absorption grid adjacent to the
sample between an X-ray source and a detector. In certain
embodiments, the method involves placing the absorption grid
millimeters, centimeters or meters in the vicinity of or adjacent
to the sample. The method in certain embodiments involves placing
the absorption grid between an x-ray source and the sample, or
alternatively in between the sample and the detector.
[0014] Detecting the presence of the nanoparticles in various
embodiments of the method involves spatial frequency heterodyne
imaging, spatial harmonic imaging. The terms, "spatial frequency
heterodyne imaging" and "spatial harmonic imaging" are used
interchangeably herein. In various embodiments, the spatial
frequency heterodyne imaging involves performing a Fourier
transformation of x-ray scatter images obtained by the detector.
For example, the detector includes a light sensor such as a camera
or a charge-coupled device.
[0015] In various embodiments of the method, the binding agent
attached or conjugated to the nanoparticle includes at least one
molecule selected from the group of: a drug, a protein, a
carbohydrate, and a nucleotide sequence. In various embodiments of
the method, the protein is an antibody selected from the group of:
a monoclonal antibody; a polyclonal antibody; a single-chain
antibody (scFv); a recombinant heavy-chain-only antibody (VHH); an
Fv; a Fab; a Fab'; and a F(ab').sub.2. In related embodiments of
the method, the antibody (e.g., a monoclonal antibody and a
polyclonal antibody) specifically binds a tumor antigen selected
from the group of: aspartyl (asparaginyl)-.beta.-hydroxylase,
alpha-fetoprotein, carcinoembryonic antigen (CA), CA-125, mucin 1,
epithelial tumor antigen, tyrosinase, melanoma-associated antigen,
tumor protein 53, human chorionic gonadotropin, vimentin, CD34,
desmin, prostate specific antigen, and glial fibrillary acidic
protein. For example, the monoclonal antibody used in the method
includes all or a portion of FB50 antibody (Wands et al. U.S. Pat.
No. 6,797,696 issued Sep. 28, 2004), or SF25 antibody (Wands et al.
U.S. Pat. No. 5,212,085 issued May 18, 1993).
[0016] In a related embodiment, the binding agent binds to or
targets genetic material (e.g., a nucleotide sequence) in the cell.
In various embodiments, the genetic material includes a DNA or an
RNA, such that the RNA is selected from: mRNA, tRNA, rRNA, siRNA,
RNAi, miRNA, and dsRNA, or a portion thereof. In certain
embodiments, the binding agent binds to a cell surface receptor or
to an intracellular receptor.
[0017] In various embodiments, the method images the tissue that
contains a plurality of cells selected from at least one of the
group of: cancerous, non-cancerous, epithelial, hematopoietic,
stem, spleen, kidney, pancreas, prostate, liver, neuron, breast,
glial, muscle, sperm, heart, lung, ocular, brain, bone marrow,
fetal, blood, leukocyte, and lymphocyte. For example, the tissue is
a sample obtained from a subject for example the tissue is a
portion of an organ (e.g., a liver, heart, brain, and stomach).
[0018] In certain embodiments of the method, the binding agent
attached or conjugated to the nanoparticle binds to an antigen, or
a nucleotide sequence that encodes the antigen. For example, the
antigen is a cancer antigen or a tumor antigen. In various
embodiments, the method further includes diagnosing or prognosing a
disease condition in the subject.
[0019] In various embodiments, the method further includes
detecting or imaging a tumor in the cells or the tissue, wherein
the tumor is selected from the group consisting of: melanoma; colon
carcinoma; pancreatic; lymphoma; glioma; lung; esophagus; mammary;
prostate; head; neck; ovarian; stomach; kidney; liver; and
hepatocellular carcinoma.
[0020] In various embodiments, the method further includes
administering a therapeutic agent to the cells, the tissue, or to
the subject. In various embodiments, the therapeutic agent is at
least one of: an antibiotic, an anti-viral, an anti-cancer, an
anti-tumor, an anti-proliferative, and an anti-inflammatory.
[0021] An aspect of the invention provides a method of identifying
in a model system a potential therapeutic agent for treating or
preventing a disease condition, the method including: contacting a
first sample and a second sample of cells or tissue having the
disease condition with a composition containing: a nanoparticle and
at least one of a polymer layer coating the nanoparticle and a
binding agent that specifically binds the disease agent; contacting
the second sample with the potential therapeutic agent; and,
measuring a presence or an amount of a marker in the first sample
and the second sample, such that the marker is characteristic of
the disease condition, such that a greater amount of the marker in
the first sample compared to that in the second sample is a measure
of treatment and protection by the potential therapeutic agent,
thereby identifying the potential therapeutic agent for treating or
preventing the disease condition. In various embodiments of the
method, the composition includes a plurality of nanoparticles.
[0022] Detecting the presence of the marker in the first sample and
the second sample includes in various embodiments of the method
measuring or detecting the nanoparticle using X-ray scatter imaging
or spatial frequency heterodyne imaging.
[0023] In various embodiments, prior to contacting, the method
further includes constructing the nanoparticle or a plurality of
nanoparticles comprising at least one material selected from the
group of: a metal, a metal oxide, a MRI agent, and a combination
thereof.
[0024] In various embodiments, constructing the nanoparticles
involves a layer-by-layer coating of one or more polymers on the
nanoparticles. For example, at least one of the polymers is
selected from: a polyethylene glycol, a polyelectrolyte, an anionic
polymer, and a cationic polymer.
[0025] In various embodiments of the method, constructing the
nanoparticle includes forming a shell or core of the nanoparticle
with at least one from the group of: silver, copper, gold, cadmium,
zinc, nickel, palladium, platinum, rhodium, platinum, manganese,
gadolinium, dysprosium, tantalum, titanium, and iron. In a related
embodiment of the method, constructing the nanoparticle involves
vapor-phase synthesis.
[0026] In certain embodiments of the method, the binding agent
includes at least one molecule selected from the group of: a drug,
a protein, a carbohydrate, and a nucleotide sequence.
[0027] In various embodiments of the method, the disease condition
is associated with or produced by at least one from the group of: a
virus, a tumor, a cancer, a fungus, a bacterium, a parasite, a
pathogenic molecule, and a protein. For example, the cancer is a
carcinoma such as a hepatoma or a melanoma.
[0028] Prior to contacting, the method in various embodiments
further includes constructing the nanoparticle by attaching or
conjugating the binding agent to an external surface of the
nanoparticle, such that the binding agent comprises at least one
selected from the group of: a drug, a protein, a carbohydrate, and
a nucleotide sequence. The protein in various embodiments is an
antibody selected from the group of: a monoclonal antibody; a
polyclonal antibody; a single-chain antibody (scFv); a recombinant
heavy-chain-only antibody (VHH); an Fv; a Fab; a Fab'; and a
F(ab').sub.2.
[0029] In various embodiments of the method, measuring further
includes observing a localization of the marker or the nanoparticle
in the cells or the tissue of the first sample and the second
sample. For example, the nanoparticle (or plurality of
nanoparticles) is located in the nucleus or is located in cytoplasm
of a cell in the first sample and the second sample.
[0030] The method in various embodiments further includes spatial
frequency heterodyne imaging the nanoparticles in the cells or the
tissue using an absorption grid and a detector.
[0031] An aspect of the invention provides a kit for imaging cells
or a tissue in a subject, the kit including: a composition
containing a nanoparticle including at least one selected from the
group of: a polymer layer and a binding agent, such that the
composition binds to and/or is phagocytosed by the cells or the
tissue; instructions for use, such that the instructions describe:
contacting the cells or the tissue with the composition, and
imaging the cells and the tissue and detecting X-ray scattering of
the composition with a device; and a container.
[0032] The nanoparticles in various embodiments of the kit include
at least one material selected from the group of: a metal, a metal
oxide, an inorganic material, an organic material, a MRI agent, and
a combination thereof.
[0033] An aspect of the invention provides a composition for
imaging cells or a tissue, the composition including: a metal
nanoparticle having attached to an external surface of the
nanoparticle a polymer layer, and FB50 monoclonal antibody that
specifically binds an antigen of hepatocellular carcinoma, such
that the polymer layer comprises at least one of: a polyethylene
glycol, a polyelectrolyte, an anionic polymer, and a cationic
polymer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a drawing of methods for layer-by-layer
polyelectrolyte coating of gold nanoparticles using an anionic
poly(acrylic acid) (PAA), and a cationic poly(allylamine
hydrochloride) (PAH). FIG. 1 Gold (Au) nanoparticles were contacted
with PAA to produce PAA-encapsulated nanoparticles (AU-PAA) having
carboxylic acid functional groups (--COOH) and de-protonated
carboxylic acid functional groups (--COO.sup.-) extending from the
nanoparticles. The PAA-encapsulated nanoparticles were contacted
with PAH to form PAA-PAH encapsulated nanoparticles (Au-PAA-PAH).
The PAH layer lies outside the PAA layer of PAA-PAH encapsulated
nanoparticle resulting in amine functional groups (--NH.sub.2) and
protonated amino functional groups (--NH.sub.3.sup.+) extending
from the nanoparticle.
[0035] FIG. 2 is a drawing of the X-ray imaging system used in
examples herein. An X-ray source directs electromagnetic radiation
to an absorption grid that is a periodic structure positioned in
the radiation propagation direction and positioned proximal to a
sample and a charged-coupled device (CCD). The grid scatters the
directed radiation to the sample positioned in a container or vial.
The X-ray radiation passes through the grid and the sample
respectively, and is then detected using the CCD.
[0036] FIG. 3 is a set of visible light images and X-ray scatter
images after Fourier transformation of scattered X-radiation of a
sample obtained by the imaging system shown in FIG. 2. A beam of
X-radiation was directed through an absorption grid and sample in a
vial, and was detected using an CCD. The original image (F) is
shown in the lower left of FIG. 3. A Fourier transformation was
performed resulting in an image in the spatial frequency domain.
Different peaks in the spatial frequency image contained different
information regarding scattering of incident x-radiation by the
sample. Selecting an area around a specific peak in the convolution
and Fourier back-transforming this area returns the logarithm of
the scattered intensities to real space and gives a processed image
that contains anisotropic information regarding scattering of the
incident X-rays by the sample. The area surrounding the central
0.sup.th-order peak (F.sup.-1; S0: 0.sup.th order; FIG. 3 bottom
right) corresponds to the original X-ray absorption image without
scatter and is used for normalization. The image from the central
0.sup.th-order peak is subtracted from the higher order images to
remove all absorption features. The area around the 1.sup.st-order
peak (F.sup.-1; S1: 1.sup.st order left; FIG. 3 center bottom)
corresponds to scattering in the x-direction, and therefore gives a
processed left 1.sup.st-order scatter image upon Fourier
back-transformation and normalization. Similarly, the area around
the 1.sup.st-order peak immediately above the 0.sup.th-order peak
(FIG. 3 top right box) corresponds to scattering in the
y-direction, and therefore gives a processed upper 1.sup.st-order
scatter image upon Fourier back-transfounation and normalization.
Each X-ray image yields at least two processed images; one image
results from X-radiation scattered horizontally (F.sup.-1; S1:
1.sup.st order left; FIG. 3 center bottom), and the other image
results from X-radiation scattered vertically (FIG. 3 top right
box). Both 1.sup.st order images measure identical scatter signals
because of the isotropic scattering of the spherical
nanoparticles.
[0037] FIG. 4 panels A-D are a set of images showing vials with
cell pellets containing approximately 10.sup.7 FOCUS cells
(Friendship of China and United States; a human hepatocellular
carcinoma cell line; He, L. et al. 1984 In Vitro 20(6): 493-504)
labeled with PAA-PAH coated gold nanoparticles having a diameter of
10 nm (right vial in each panel) or 50 nm (left vial in each
panel). Control cells were not contacted with gold nanoparticles
(center vial in each panel). Top boxes and bottom boxes in each of
FIG. 4 panels B-D outline areas selected for intensity profiles of
the supernatant and pellet, respectively. Data show improved
sensitivity and clarity for spatial harmonic images of coated
nanoparticles compared to absorbance images.
[0038] FIG. 4 panel A is a camera photograph of FOCUS cell pellets
under cell culture medium. Pellets of cells contacted with ten nm
PAA-PAH coated gold nanoparticles (FIG. 4 panel A left vial) and
with 50 nm PAA-PAH coated gold nanoparticles (FIG. 4 panel A right
vial) are visibly darker in appearance that control pellets of
cells not contacted with the gold nanoparticles (FIG. 4 panel A
center vial).
[0039] FIG. 4 panel B is an absorption image of the vials
containing FOCUS cell pellets contacted with ten nm PAA-PAH coated
gold particles (right image), contacted with 50 nm PAA-PAH coated
gold particles (left image), or control pellets of cells not
contacted with the gold nanoparticles (center image).
[0040] FIG. 4 panel C is an left 1st-order processed image of the
vials containing FOCUS cell pellets contacted with ten nm PAA-PAH
coated gold particles (right image), contacted with 50 nm PAA-PAH
coated gold particles (left image), or control pellets of cells not
contacted with the gold nanoparticles (center image).
[0041] FIG. 4 panel D is an upper 1st-order processed image of the
vials containing FOCUS cell pellets contacted with ten nm PAA-PAH
coated gold particles (right image), contacted with 50 nm PAA-PAH
coated gold particles (left image), or control pellets of cells not
contacted with the gold nanoparticles (center image).
[0042] FIG. 5 is a drawing showing methods for producing a
polyethylene glycol-coated FB50 antibody conjugated gold
nanoparticles and injecting the nanoparticle into a subject having
hepatocellular carcinoma (HCC). The FB50 antibody conjugated to the
nanoparticle specifically targets an antigen of HCC and allows for
imaging of a HCC tumor site using spatial harmonic imaging. The
drawing shows contacting a gold nanoparticle (AuNP) with a
bi-functional polyethylene glycol (PEG) having carboxylic acid
functional groups (--COOH). The PEG undergoes facile attachment to
the external surface of the nanoparticle (Au-PEG). The gold
nanoparticle having attached PEG was bio-conjugated to FB50
antibody using carbodiimide-amine (EDC/NHS) linking chemistry. The
polyethylene glycol coated, FB50 antibody conjugated gold
nanoparticles (Au-PEG-FB50) were injected into a mouse having HCC,
and the FB50 antibody component of the conjugated nanoparticle
specifically bound to an antigen of HCC. Spatial harmonic imaging
of the mouse resulted in imaging of the HCC tumor bound to the
polyethylene glycol-coated FB50 antibody-conjugated gold
nanoparticles.
[0043] FIG. 6 panels A-B are graphs of calculation of overall
diameter of coated or conjugated 50 nm gold nanoparticles, and
uptake of the coated or conjugated ten nm gold nanoparticles by
FOCUS cell pellets or by NIH/3T3 (a fibroblast cell line) cell
pellets. FB50 antibody (specific for an antigen of HCC) or a
control antibody specific for Murutucu tropical antibody (MUK) were
conjugated to the nanoparticles. Data show that the PEG-coated,
FB50 antibody conjugated nanoparticles produced by methods herein
were effectively and specifically bound to and transported into the
FOCUS cell pellets and not NIH/3T3 cell pellets.
[0044] FIG. 6 panel A is a line graph of the overall average
diameter in nm of: PEG-coated 50 nm gold nanoparticles (AU-PEG;
middle peak), PEG-coated FB50 antibody conjugated 50 nm gold
nanoparticles (AU-PEG-FB50; right most peak); and control 50 nm
nanoparticles that were neither coated with PEG nor conjugated with
FB50 (AU; left most peak). Data show that the diameter of the FB50
antibody conjugated 50 nm gold nanoparticles (93.9.+-.12.1 nm) was
larger than PEG-coated 50 nm gold nanoparticles (81.8.+-.12.1 nm)
and control gold nanoparticles (71.3.+-.10.0 nm).
[0045] FIG. 6 panel B is a bar graph showing average percent uptake
(Average % yield) of FOCUS cell pellets (checkered; three left
columns) and NIH/3T3 cell pellets (solid; two right columns)
contacted with conjugated ten nm gold nanoparticles. The FOCUS cell
pellets were contacted with one of: PEG-coated ten nm gold
nanoparticles (FOCUS+AU-PEG; left most column); PEG-coated FB50
antibody-conjugated ten nm gold nanoparticles (FOCUS+AU-PEG-FB50;
second column from left); or PEG-coated MUK antibody-conjugated 50
nm gold nanoparticles (AU-PEG-FB50; third from the left). The
NIH/3T3 cell pellets were contacted with either: PEG-conjugated ten
nm gold nanoparticles (FOCUS+AU-PEG; second column from the right);
or PEG-coated FB50 antibody-conjugated ten nm gold nanoparticles
(FOCUS+AU-PEG-FB50; right most column). Data show that at least
thirty fold more of the PEG-coated FB50 antibody conjugated ten nm
gold nanoparticles were transported into the FOCUS cell pellets
than into the NIH/3T3 cell pellets.
[0046] FIG. 7 panels A-B are a set of photographs, X-ray scatter
images, and absorption images of mice injected in vivo either with
PEG-coated 50 nm gold nanoparticles or with saline (negative
control). Mice were injected in the tail vein twice in 24 hours and
were sacrificed 48 hours after the first injection. Livers of
subjects injected with the PEG-coated 50 nm gold nanoparticles
showed a significant average signal enhancement (23.0.+-.14.1%)
compared livers from subjects injected with saline only.
[0047] FIG. 7 panel A is a set of photographs (left column) and
X-ray scatter images (right column) of livers of subjects injected
with either saline (top row) or with PEG-coated 50 nm gold
nanoparticles (AU-PEG; bottom row). The sizes of the livers for the
subjects injected with saline or with nanoparticles were
comparable. Improved X-ray scatter image clarity and sensitivity
were observed in livers of subjects injected with PEG-coated 50 nm
gold nanoparticles (FIG. 7 panel A right column bottom row)
compared to subjects injected with saline only (FIG. 7 panel A
right column top row).
[0048] FIG. 7 panel B is a set of total absorption images (left)
and a X-ray total scatter images (right) of excised livers from
subjects injected with either saline (top image) or PEG-coated 50
nm gold nanoparticles (AU-PEG; bottom image). The X-ray scatter
images (right) more clearly delineated the actual size of the
livers for the subjects compared to the absorption images (left),
and the nanoparticles enhanced the imaging of the livers compared
to the saline.
DETAILED DESCRIPTION
[0049] Nanoparticles are structures/particles that have an
approximate size of one nm to 100 nm and are used in applications,
including addition to surfaces or fluids for catalytic reactions,
self-cleaning and antibacterial products, glass dyeing, sunscreen
lotions and manufacturing of optical components (e.g., optical
fibers). See Raj ala et al. U.S. Pat. No. 8,231,369 issued Jul. 31,
2012. Nanoparticles are composed of a variety of materials
including metals, metal oxides, MRI agents, semiconductors, and
polymers, and possess unique characteristics because of their small
size.
[0050] Laboratory-scale and industrial-scale techniques are used to
manufacture nanoparticles having for example a specific size
distribution (mono-dispersivity), anti-agglomeration, and
homogeneity (see Davis et al. U.S. Pat. No. 8,263,035 issued Sep.
11, 2012; Magdassi et al. U.S. Pat. No. 8,227,022 issued Jul. 24,
2012; and Fiannaca et al. U.S. patent application number US
2008/0050592 A1 published Feb. 28, 2008). Nanoparticles are
produced for example by wet chemical processes and by vapor phase
processes (see Murphy et al. U.S. Pat. No. 8,241,922 issued Aug.
14, 2012; and Brooks et al. U.S. Pat. No. 7,985,398 issued Jul. 26,
2011). Vapor phase processes (also known as aerosol reactor
processes) use a number of different devices and techniques
including: flame reactors, hot-wall reactors, plasma reactors, gas
condensation methods, laser ablation and spray pyrolysis (see Jun
et al. U.S. Pat. No. 7,988,761 issued Aug. 2, 2011; and Yang, Y. et
al. 2006 Catalysis Communications 7:281-284) to synthesize
nanoparticles of controlled size and composition.
[0051] Nanoparticles composed of metals are used as imaging agents
that are imaged using a number of different techniques including
MRI, ultrasonography, and X-ray computer tomography (see Grinstaff
et al. U.S. Pat. No. 5,505,932 issued Apr. 9, 1996). Imaging of
nanoparticles containing for example gadolinium, dysoprium,
manganese, iron, and platinum involves specialized excitation and
data manipulation of detected radiofrequency signals from non-zero
spin nuclei which have a non-equilibrium nuclear spin state
distribution. (Weiler et al. U.S. Pat. No. 6,370,415 issued Apr. 9,
2002; and Weiler et al. U.S. Pat. No. 6,595,211 issued Jul. 22,
2003). In conventional MRI the nuclei responsible for the detected
signals are protons (e.g., protons of water), and the
non-equilibrium spin state distribution is achieved by placing the
subject in a strong magnetic field (to enhance the population
difference between the proton spin states at equilibrium), and then
exposing the subject to pulses of radiofrequency radiation at the
proton Larmor frequency, which excites spin state transitions and
creates a non-equilibrium spin state distribution (see Seri et al.
U.S. Pat. No. 5,811,077 issued Sep. 22, 1998).
[0052] Nanoparticles such as gold nanoparticles are potential
contrast agents for X-ray imaging because the nanoparticles are
constructed to be non-toxic and to have a higher atomic number and
X-ray absorption coefficient than typical iodine-based contrast
agents (Hainfeld, J. F. et al. 2006 Br. J. Radiol. 79: 248-253;
Kim, D. et al. 2007 J. Am. Chem. Soc. 129: 7661-7665; and Kojima,
C. et al. 2010 Nanotechnology 21: 245104). Biodistribution for
example of small gold nanoparticles injected intravenously is
detected by X-ray imaging (Hainfeld, J. F. et al. 2006 Br. J.
Radiol. 79: 248-253). Gold nanoparticles approximately 30 nm in
diameter have been injected intravenously and used for in vivo
computed tomography (CT) imaging of hepatoma in the liver (Kim, D.
et al. 2007 J. Am. Chem. Soc. 129: 7661-7665). However, successful
CT scan and MRI imaging of tissues required in vivo injection of
large quantities of gold nanoparticles, or creating a high density
of such particles at the image target for example, by linking the
nanoparticles to targeted delivery vehicles.
[0053] Without being limited by any particular theory or mechanism
of action, it is here envisioned that the spatial frequency
heterodyne imaging/spatial harmonic imaging techniques described in
Examples herein have the advantage of using a much reduced amount
of nanoparticles (e.g., gold nanoparticles) to produce a
visibly-enhanced contrast compared to typical absorption based
X-ray imaging. Additionally, the imaging described herein using
nanoparticles provides a nearly background-free image because
scattered x-radiation is very well separated from transmitted
radiation due to the different angles at which the scattered
radiation reaches a detector.
[0054] X-rays employed for medical diagnostic imaging are
electromagnetic radiation of approximately 0.01 nm to ten nm
wavelength and high energy, approximately 100 electronvolts to 100
kiloelectronvolts (keV) and typically about two keV to 50 keV. A
beam of X-rays directed to an atom is absorbed or deflected. The
deflected X-rays define extent of scatter. Compton scattering
describes an incident X-ray photon that is deflected from its
original path by an electron. Scatter traditionally serves little
purpose in imaging of tissues and patients, as scattering yields a
diffuse signal that reduces contrast and clarity of the image
(Feldmesser et al. U.S. Pat. No. 6,529,582 issued Mar. 4, 2003).
Tissue images are generally prepared using only X-rays passed
directly through the patient without colliding with atoms along the
path. At a given point of the image plane or detector, the quantity
of X-rays at that point indicates the degree of absorption of the
primary beam in the patient on the line from the X-ray source to
the X-ray receptor (e.g., the film). The scattered X-rays arrive at
the X-ray film from various angles and places in the body not
related to the path from the source to the receptor. Thus unwanted
scattered X-rays cause the image to be distorted and cloudy. These
distortions in the image from scattering result in reduced image
contrast, and obscure the small variations in X-ray absorption that
exist within the body of a subject, for which images should be
obtained for an accurate diagnosis or prognosis.
[0055] Spatial frequency heterodyne imaging (also known as spatial
hainionic imaging) was used in examples herein with an X-ray
scatter reducing grid for absorbing rays scattered when radiation
was transmitted through the subject, and to obtain a high quality
image to reduce or eliminate scatter radiation. The grid was placed
in the direction of propagation of an X-ray beam for example in
parallel and in the shape of a flat plate or box. Radiation was
transmitted through the grid and to a sample or a subject, such
that the scattered radiation traveled obliquely and was absorbed
and reduced by the radiation-absorbing portions. The primary
radiation was transmitted (substantially linearly) through a vial
containing the sample or through the subject. The primary radiation
transmitted through the radiation-transmitting portions (e.g.,
wood, a metal, a plastic, and voids) of the grid and then the
sample reached a detector that formed a radiation-transmitted
image. The radiation-absorbing portions in certain embodiments are
formed from an absorbing material or a dense shield material that
attenuates x-radiation such as lead or the like. The
radiation-transmitting portions and radiation-absorbing portions
were in certain embodiments alternately or closely arranged, e.g.,
symmetrical positions. The radiation-transmitting portions have a
high transmittance to avoid reducing transmission of the primary
radiation to the sample and the detector (see Ogawa U.S. Pat. No.
6,707,884 issued Mar. 16, 2004; Stein, A. F. et al. 2010 Opt.
Express 18: 13271-13278; Wen, H. et al. 2009 Radiology 251:
910-918; and Wen, H. et al. 2008 IEEE Trans Med Imaging 27:
997-1002, each of which is incorporated by reference herein in its
entirety). Fourier transformation was is used to process the image
produced by the detector (FIG. 1).
[0056] An imaging technique described herein involves in certain
embodiments use of surface-modified gold nanoparticles and spatial
frequency heterodyne imaging. The nanoparticles (containing at
least one of a metal or a metal oxide) are modified in certain
examples by attaching a binding agent and/or a polymer (e.g., a
polyelectrolyte) layer. The nanoparticles were observed to be
biocompatible and non-toxic and were taken up by cells.
[0057] Nanoparticles used in various embodiments of the invention
were constructed using layer-by-layer coatings of polymers such as
electrolytes. Layer-by-layer coating of polyelectrolytes is a
versatile method for modifying the surface chemistry of nanoscale
materials including gold nanoparticles and nanorods (Mayya, K. S.
et al. 2003 Adv. Funct. Mater. 13: 183-188; Gittins, D. I. et al.
2001 J. Phys. Chem. B. 105: 6846-6852; Gole, A. et al. 2005 Chem.
Mater. 17: 1325-1330; and Murphy, C. J. 2005 Chem. Mater. 17:
1325-1330). Layer-by-layer coatings of charged polyelectrolytes are
useful to stabilize colloidal suspensions.
[0058] Deposition of polyelectrolyte coatings (e.g., poly(acrylic
acid) and poly(allylamine hydrochloride) on surfaces of structures
under specific pH conditions result in the ability of the coated
surfaces to bind cells (Mendelsohn, J. D. et al. 2003
Biomacromolecules 4: 96-106). Poly(acrylic acid) and
poly(allylamine hydrochloride) are weak polyelectrolytes with
different degrees of ionization, and as a result each of these
electrolytes has a strength of electrostatic interaction that is
pH-dependent (Shiratori, S. S. et al. 2000 Macromolecules 33:
4213-4219). Poly(acrylic acid) stock solutions and poly(allylamine
hydrochloride) stock solutions used in certain embodiments herein
were adjusted to have a pH of 8.4 and 3.7, respectively, and under
these conditions poly(acrylic acid) has a pKa of about 4.5 and
poly(allylamine hydrochloride) has a pKa about 8.5. Stock solutions
of poly(acrylic acid) and poly(allylamine hydrochloride) described
herein were fully charged, i.e., anionically and cationically,
respectively.
[0059] The de-protonated carboxylic acid groups of anionic
poly(acrylic acid) (PAA) interacted strongly with protonated amine
groups of cationic poly(allylamine hydrochloride) (PAH), such that
the oppositely charged layers produced thin, flat coatings on the
nanoparticle surface. Water is unable to penetrate the surface
layer of the PAA-PAH coated nanoparticle because the PAA-PAH
polyelectrolyte layers were so tightly cross-linked by the anionic
and cationic (i.e., ionic) interactions. The electrolyte coated
nanoparticles produced by this method therefore resulted in a very
hydrophobic polymer barrier to a surrounding aqueous environment.
The hydrophobicity of the electrolyte coating resulted in
nanoparticle surfaces that were cytophilic (Lee, J. H. et al. 1995
Prog. Polym. Sci. 20: 1043-1079; Alexis, F. et al. 1998 Mol. Pharm.
5: 505-515; Brandenberger, C. et al. 2010 Small 6: 1669-1678).
Furthermore, the thickness and specificity of the polyelectrolyte
coating is controlled by varying and optimizing the number of
layers deposited on the surface of the nanoparticles (Mayya, K. S.
et al. 2003 Adv. Funct. Mater. 13: 183-188; and Gittins, D. I. et
al. 2001 J. Phys. Chem. B. 105: 6846-6852). Without being limited
by any particular theory or mechanism of action, it is here
envisioned that the polyelectrolyte-coated nanoparticles described
herein were effective vehicles for cellular uptake by mechanisms
such as phagocytosis and endocytosis.
[0060] Specificity of the nanoparticles was further enhanced by
conjugating a variety of antibody binding agents to the
polyelectrolyte coatings. Layer-by-layer coating and antibody
binding agent conjugation of nanoparticle composition
functionalized the nanoparticle compositions and increased the
specific binding of the nanoparticles to different types of cells
and tissues (Murphy, C. J. 2005 Chem. Mater. 17: 1325-1330).
Phagocytosis of the polymer coated gold nanoparticles in living
cells was measured in a model system using FOCUS cells, a human
hepatocellular carcinoma (HCC) cell line (He, L. et al. 1984 In
Vitro 20: 493-504). FOCUS cells are a model for the study of
targeted imaging of cells because these cells express specific HCC
antigens that are recognized and bound by binding agents such as
monoclonal antibodies (Hurwitz, E. et al. 1990 Bioconjugate Chem.
1: 285-290; Takahashi, H. et al. 1989 Gastroenterology 96:
1317-1329; Mohr, L. et al. 2004 Gastroenterology 127: S225-S231;
and Luu, M. et al. 2009 Hum. Pathol. 40: 639-644). A strong
antibody-antigen interaction exists between FB50 antibody and
aspartyl (asparaginyl)-.beta.-hydroxylase, a protein over-expressed
in liver cancer cells such as HCC (Luu, M. et al. 2009 Hum. Pathol.
40: 639-644).
[0061] Examples herein used antibody-conjugated nanoparticles and
spatial frequency heterodyne imaging to directly target and image
tumors in an animal model (Mohr, L. et al. 2004 Gastroenterology
127: S225-S231; and Luu, M. et al. 2009 Hum. Pathol. 40: 639-644).
The N-terminal (amino terminus) of FB50 antibody was conjugated to
the outer surface of a PEG coated nanoparticle by
1-ethyl-3-3-dimethylaminopropyl]carbodiimide and amine (EDC/NHS)
cross-linking chemistry (FIG. 5). In certain embodiments, the
polymer (e.g., PEG, PAA, and PAH) coatings are mixed with and bound
to the antibody binding agent prior to being contacted with the
nanoparticle to produced a polymer-coated antibody-conjugated
nanoparticle, which can be used for example to binding normal
tissues or cancer tissues in vivo (FIGS. 6-7). In certain
embodiments, the binding agent (e.g., protein) or plurality of
binding agents is conjugated or attached to the nanoparticles at a
carboxy-terminus or an amino-terminus.
[0062] Nanoparticles are coated with a polyethylene glycol (PEG) to
prevent nonspecific protein adsorption, to reduce nonspecific
cellular uptake, and to increase the circulation times of
nanoparticles in the bloodstream (Alexis, F. et al. 1998 Mol.
Pharm. 5: 505-515; Brandenberger, C. 2010 Small 6: 1669-1678;
Hucknall, A. et al. 2009 Adv. Maert. 21: 2441-2446; and Lipka, J.
et al. 2010 Biomaterials 31: 6574-6581). In certain embodiments,
the PEG polymer layer was coated to gold nanoparticles using
layer-by-layer coating techniques and then used to conjugate
binding agents to the nanoparticlesthat target specific cancers in
vivo (FIG. 5). The gold nanoparticles were then used to deliver
gold to the HCC tumors, and the cells and the nanoparticles were
imaged and diagnosed.
[0063] Cancer cells such as FOCUS cells were targeted in vivo by
contacting the cells with nanoparticles conjugated with an FB50
antibody binding agent that binds to HCC antigens. The binding
agent-conjugated nanoparticles contact the cells and/or or deliver
therapeutic agents to the targeted cells, and avoid being taken up
by healthy cells. Spatial frequency heterodyne imaging was
performed on samples of pellets of FOCUS cells incubated with
different amount of PAA-PAH coated gold nanoparticles (ten nm or 50
nm diameters). Control samples were incubated in absence of gold
nanoparticles. Each set of pellets was imaged multiple times to
exclude false signals due to possible non-uniformities of the
sensitivity of the imaging system. A vial holder was designed and
used to keep vials containing cell samples and nanoparticles in a
position that could be reproduced. Apertures of the same size were
drilled through a thin block of aluminum so that the vials, placed
into these apertures, were positioned at exactly the same position
and height.
[0064] Examples herein in certain embodiments used cell pellets
each containing approximately 10.sup.7 FOCUS cells. The pellet size
of FOCUS cells corresponded to the approximate size of a small
tumor (several millimeters in diameter). The FOCUS cells were
cultured and incubated with gold nanoparticles (ten nm and 50 nm
diameter). Beams of X-rays were directed through an absorption grid
and a vial containing the sample of cells, and the scatter X-ray
radiation was detected using a charge-coupled camera (CCD). Fourier
transformation of the X-ray scatter data produced by the spatial
frequency heterodyne imaging yielded clear and sensitive images of
the cells. Data herein show that spatial frequency heterodyne
imaging of cells and tissues contacted with the nanoparticles was
more sensitive than typical absorption-based imaging techniques,
for example, tissues imaged with CT scans and MRI imaging.
[0065] Data show enhanced FOCUS cell phagocytosis of the gold
nanoparticles having a bi-layer of PAA and the cationic PAH
compared to phagocytosis of gold nanoparticles having no coating
(FIG. 4). Presence of the polymer layers of polyelectrolytes on the
nanoparticle surface resulted in nearly double amount of uptake of
the nanoparticles by the FOCUS cells (see Table 1). The mass of
gold taken up by each cell corresponds to several hundred 50 nm
gold nanoparticles and tens of thousands of ten nm gold
nanoparticles (Table 2).
TABLE-US-00001 TABLE 1 Cellular uptake of gold nanoparticles.
uncoated 10 nm 50 nm 10 nm PAA-PAH PAA-PAH nanoparticles
nanoparticles nanoparticles mass of gold taken up per cell 1.2 .+-.
0.5 2.8 .+-. 0.4 2.2 .+-. 0.3 (picograms) approximate number of
108,000 275,000 1730 nanoparticles per cell approximate volume
fraction 0.00025% 0.00063% 0.00049% of nanoparticles in each
cell
TABLE-US-00002 TABLE 2 Average signal enhancements of cells due to
gold labeling incubation with PAA-PAH gold nanoparticles. replicate
1 replicate 2 mass of gold taken up per cell (pg) 0.45 .+-. 0.09
0.76 .+-. 0.11 average number of 10 nm 44,600 75,200 nanoparticles
per cell 50 nm 356 602 change in signal per original image 1.3 .+-.
4.4 1.1 .+-. 3.0 pellet (%) .sup.a processed image 1.6 .+-. 0.3 4.4
.+-. 0.8 signal enhancement original image 2.9 .+-. 9.8 1.4 .+-.
3.9 per 1 pg of gold taken processed image 3.6 .+-. 0.7 5.7 .+-.
1.1 up per cell (%) approximate potential signal enhancement 11
.+-. 2 17 .+-. 3 for a pellet of 10.sup.7 cells (%) .sup.a All
enhancements are reported in logarithm scale.
[0066] A set of representative X-ray scatter imaging photographs of
the pellets under cell culture medium shows gold labeling by the
nanoparticles (see FIG. 4). Processed X-ray scatter images using
spatial frequency heterodyne imaging showed significant image
enhancement due to gold labeling by the polymer modified
nanoparticles that was almost five-fold greater than enhancements
identified in the absorption images (Table 2). Further, examples
herein using polymer coated antibody-conjugated gold nanoparticle
compositions produced data showing increased cellular uptake of
gold nanoparticles into cells and tissues compared to results for
cells contacted with only polymer coated gold nanoparticles (FIG.
6). For example, data herein showed that less than 0.001% of each
cell volume is occupied by polymer coated gold nanoparticles,
allowing for significantly increased amounts of gold nanoparticles
to be further introduced into each cell, thereby increasing scatter
signal and, ultimately, enhancing visibility in scatter images. The
sensitivity and potential specificity of the nanoparticle-based
imaging techniques described herein show that these are effective
methods, compositions and kits for early detection and diagnosis of
cancers such as HCC.
[0067] Examples herein show that cell pellet samples of FOCUS cells
phagocytosed gold nanoparticles of different sizes, and that the
gold nanoparticle-containing cell pellets of FOCUS cells were
distinguishable from cell pellets of FOCUS devoid of gold
nanoparticles using spatial frequency heterodyne imaging. Spatial
frequency heterodyne images of FOCUS cell pellets labeled with gold
nanoparticles showed that greater than 85% of the images indicate a
signal enhancement compared to X-ray scatter images of FOCUS
pellets containing no gold. Data herein showed that each FOCUS cell
phagocytosed approximately three picograms of gold nanoparticles
per cell (Table 1), and that X-ray scatter imaging enhances
visibility by up to 5.7% for every picogram of gold in the cells
(Table 2). Without being limited by any particular theory or
mechanism of action, it is here envisioned that that spatial
frequency heterodyne imaging enhanced visibility of gold
nanoparticle-labeled cells by more than 17% on a logarithmic
scale.
[0068] Examples herein obtained images of cell pellets that were
several millimeters in diameter, which corresponds to the
approximate size of a small tumor. Spatial frequency heterodyne
imaging of nanoparticles was used in Examples to detect tumor-sized
cell samples that are significantly below the detection limits of
current imaging techniques for HCC and many other cancers.
Currently cancers such as HCC go undiagnosed until the tumors are
several centimeters in size, viz., the cancers are detected only
upon reaching a size an order of magnitude greater than the sizes
detected using the methods described herein for X-ray scatter
imaging of nanoparticles. Thus, methods, compositions, and kits
described herein were effective for imaging and for differentiating
cancerous tissues and normal tissues.
[0069] Spatial frequency heterodyne imaging was performed on cell
pellet samples that were placed under water in an in vivo model,
because water and tissues (e.g., liver tissues) share a similar
radiological density. Data showed that in vitro X-ray scatter
imaging of the submerged cells and nanoparticles was effective in
visualizing the cells. Without being limited by any particular
theory or mechanism of action, it is here envisioned that spatial
frequency heterodyne imaging of nanoparticles containing a metal as
described herein would detect small in situ tumors (less than a few
millimeters in size) in liver and in other internal organs and
tissues.
[0070] Metal nanomaterials (containing for example gold) have been
used for cancer therapy applications (e.g., introduction of
bioactive agents) as well as imaging applications (see Sung et al.
U.S. Pat. No. 7,985,426 issued Jul. 26, 2011). For example, gold
nanoshells have been developed for use in photothermal cancer
therapy (Hirsch, L. R. et al. 2003 Proc. Natl. Acad. Sci. U.S.A.
100: 13549-13544; Choi, M. et al. 2007 S. Nano Lett. 7: 3759-3765;
Lal, S.; Clare, S. E. et al. 2008 Ace. Chem. Res. 41: 1842-1851).
These nanoshells composed of metals and metal oxides have highly
tunable plasmon resonances, allowing strong absorption of light
even under the circumstances of the frequency of the incoming light
matching plasmon oscillation frequencies of the nanoshells. The
nanoshells convert energy absorbed from directed light into heat,
killing cells containing the nanoshells and leaving unharmed the
surrounding unlabeled cells and tissues. Clinical trials of
therapies using nanoshells and nanostructures are being
investigated (Clare, S. E. et al. 2008 Acc. Chem. Res. 41:
1842-1851).
[0071] Gold nanoparticles have also been used to provide dose
enhancement in cancer radioablation therapy. Small gold
nanoparticles have for example been injected intravenously such
that the nanoparticles accumulated in tumors and improved X-ray
therapy at the tumor site (Hainfeld, J. F. 2004 Physics in Medicine
and Biology 49: N309-N315). As both of the therapeutic applications
discussed above use electron-dense nanomaterials, it is envisioned
herein that the X-ray scatter imaging methods and compositions
described herein would be effective in combination with these and
similar applications for the dual imaging and therapy of cells and
tissues.
[0072] An aspect of the invention provides a composition for
enhanced imaging and/or diagnosing a cells or a tissue for example
a tumor, the composition including a nanoparticle having at least
one polymer layer coating the nanoparticle, such that the
nanoparticle binds to and/or is phagocytosed by the cells or the
tissue to enhance visibility of the tumor by X-ray scatter
imaging.
[0073] In an embodiment of the composition, the nanoparticle
includes a metal, for example the metal is at least one of: silver,
copper, gold, mercury, cadmium, zinc, nickel, palladium, platinum,
rhodium, mercury. In an embodiment of the composition, the metal is
a transition metal for example titanium or iron.
[0074] In an embodiment of the composition, the nanoparticle
includes a shell of a material for example a metal or a carbon,
surrounding a core of a material with an electron density lower
than that of the shell. For example, a carbon core or a silica core
is surrounded by a layer of gold or another suitable metal, metal
oxide, MRI agent, inorganic material, or organic material.
[0075] In an embodiment of the composition, the nanoparticle is at
least about five nm in diameter. In various embodiments of the
composition, the nanoparticle has a diameter of at least about: two
nm, five nm, ten nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80
nm, 90 nm, and 100 nm. In an embodiment, the nanoparticle includes
a plurality of nanoparticles having an average diameter greater
than about five nm.
[0076] In an embodiment of the composition, the nanoparticle is
less than about 100 nm in diameter. In various embodiments of the
composition, the nanoparticle includes a diameter less than about:
100 nm, 90 nm, 80 nm, 75 nm, 65 nm, 60 nm, 50 nm, 45 nm, 40 nm, 30
nm, 25 nm, 20 nm, 15 nm, and ten nm. In an embodiment, the
nanoparticle includes a plurality of nanoparticles having an
average diameter less than about 100 nm.
[0077] In an embodiment of the composition, the polymer layer
includes at least one of: an anionic polyelectrolyte, a cationic
polyelectrolyte, and a polyethylene glycol. In an embodiment of the
composition, the polyethylene glycol polymer is about 10,000
average molecular weight in size. In other embodiments, the
polyethylene glycol polymer includes an average molecule weight in
size of at least one of: about 1,000; about 2,000; about 4,000;
about 6,000; about 8,000; about 12,000; about 15,000; about 20,000;
and about 30,000.
[0078] In an embodiment of the composition, the polymer layer
includes an anionic polyelectrolyte. In various embodiments of the
composition, the anionic polyelectrolyte is at least one selected
from: a poly(acrylic acid), a 4-Styrenesulfonic acid,
poly(2-acrylamido-2-methyl-1-propanesulfonic acid), a
poly(2-acrylamido-2-methyl-1-propanesulfonic acid, a
poly(4-styrenesulfonic acid), a poly(4-styrenesulfonic
acid-co-maleic acid), a polyanetholesulfonic acid, a
poly(vinylsulfonic acid), and a salt thereof. In various
embodiments of the composition, the polymer layer is a
bio-compatible layer or a non-toxic-layer.
[0079] In an embodiment of the composition, the polymer layer
includes a cationic polyelectrolyte. In various embodiments of the
composition, the cationic polyelectrolyte is at least one selected
from: a diallyldimethylammonium, a
poly(acrylamide-co-diallyldimethylammonium chloride), a
poly(allylamine hydrochloride, a poly(diallyldimethylammonium
chloride), and a salt thereof.
[0080] In an embodiment of the composition, the anionic
polyelectrolyte and/or the cationic polyelectrolyte include an
anionic poly(acrylic acid) and an cationic poly(allylamine
hydrochloride), respectively.
[0081] The composition further includes for example at least one
binding agent that selectively binds the nanoparticle to the
tissue, for example, the binding agent includes at least one
selected from the group of: a drug, a protein such as an antibody
or a binding protein, a carbohydrate such as a sugar, and a
nucleotide sequence. For example, the antibody is a polyclonal
antibody, a monoclonal antibody, or a portion thereof for example a
Fv; a Fab; a Fab'; or a F(ab').sub.2. In an embodiment of the
composition, the antibody includes a fusion protein or a chimeric
protein.
[0082] The tumor in various embodiments of the composition is
located or associated with a site or cancer selected from the group
of: melanoma; sarcoma; carcinoma (e.g., colon and hepatocellular);
pancreatic; lymphoma; glioma; lung; esophagus; mammary; prostate;
head; neck; ovarian; kidney; and liver.
[0083] An aspect of the invention provides a composition including:
a gold nanoparticle; a polymer layer coating the nanoparticle
comprising for example a polyethylene glycol or a polyelectrolyte
such as an anionic poly(acrylic acid), and a cationic
poly(allylamine hydrochloride); and, a monoclonal antibody or
portion thereof that specifically binds a tumor antigen and is
bound to the polymer layer.
[0084] In an embodiment of the composition, the nanoparticle is at
least about five nm in diameter. In an embodiment of the
composition, the nanoparticle is less than about 100 nm in
diameter. For example, the nanoparticle is about ten nm or about 50
nm in diameter.
[0085] In an embodiment of the composition, the monoclonal antibody
is FB50 and the tumor antigen is aspartyl
(asparaginyl)-.beta.-hydroxylase. In an embodiment of the
composition, the tumor antigen is associated with hepatocellular
carcinoma.
[0086] An aspect of the invention provides a method of diagnosing a
presence of a tumor in a subject including: contacting a tissue
with a composition having: a gold nanoparticle; a polymer layer
coating the nanoparticle, such that the polymer layer comprises for
example a polyethylene glycol or a polyelectrolyte such as an
anionic poly(acrylic acid) or a cationic poly(allylamine
hydrochloride); and, a binding agent that specifically binds a
tumor antigen, such that the binding agent is bound to the polymer
layer; and, detecting the presence or absence of the tumor attached
to the nanoparticle using an imaging device, such that detecting
comprises identifying presence or accumulation the nanoparticles in
the tissue by X-ray scatter imaging to detect the tumor. In an
embodiment of the method, the tissue is in situ or in vivo.
Alternatively, the tissue is in vitro, for example the method in an
embodiment includes, prior to contacting the tissue with the
composition, collecting or obtaining the tissue from the
subject.
[0087] In various embodiments of the method, the imaging device
includes a X-ray device or a MRI device. In various embodiments of
the method, detecting using the X-device involves generating X-rays
using an X-ray tube or laser or accelerator generated X-rays.
[0088] In an embodiment of the method, detecting the presence of
the tumor includes spatial frequency heterodyne imaging or spatial
harmonic imaging, for example the spatial harmonic imaging involves
irradiating with an X-ray source and detecting with an absorption
grid, and/or a detector. In an embodiment of the method, detecting
the presence of the tumor involves identifying in situ the presence
of the tumor. Alternatively, detecting involves identifying in
vitro the presence of the tumor.
[0089] In various embodiments of the method, the nanoparticle
includes at least one of: silver, copper, gold, mercury, cadmium,
zinc, nickel, palladium, platinum, rhodium, mercury, and a
combination thereof.
[0090] In various embodiments of the method, the anionic
poly(acrylic acid) is directly contacting the nanoparticle, and the
cationic poly(allylamine hydrochloride) and the polyethylene glycol
coat the anionic poly(acrylic acid).
[0091] The method in an embodiment further includes prior to
contacting the tissue, preparing the nanoparticle by coating the
nanoparticle with the polymer layer and attaching the binding
agent.
[0092] In various embodiments of the method, the tissue includes a
plurality of cells selected from the group of: epithelial cells,
hematopoietic cells, stem cells, spleen cells, kidney cells,
pancreas cells, liver cells, neuron cells, glial cells, smooth or
striated muscle cells, sperm cells, heart cells, lung cells, ocular
cells, bone marrow cells, fetal cells, peripheral blood mononuclear
cells, leukocyte cells, lymphocyte cells, and living postmitotic
cells.
[0093] In an embodiment of the method, the binding agent includes
at least one selected from the group of: a drug, a protein, a
carbohydrate, and a nucleotide sequence. In an embodiment of the
method, the binding agent includes a polyclonal antibody or a
portion thereof. In an embodiment of the method, the binding agent
includes a monoclonal antibody or a portion thereof. In an
embodiment of the method, the antibody is specific for the tumor
antigen selected from the group of: aspartyl
(asparaginyl)-.beta.-hydroxylase, alphafetoprotein,
carcinoembryonic antigen (CA), CA-125, mucin 1, epithelial tumor
antigen, tyrosinase, melanoma-associated antigen, tumor protein 53,
human chorionic gonadotropin, vimentin, CD34, desmin, and glial
fibrillary acidic protein. For example, the binding agent is
monoclonal antibody FB50 or SF25. In various embodiments of the
method, the antibody is an immunoglobulin selected from the group
consisting of: IgA, IgD, IgE, and IgG. In an embodiment of the
method, the antibody is from at least one origin selected from:
human, murine, ovine, bovine, feline, canine, hircine, and
equine.
[0094] In various embodiments of the method, the tumor is selected
from the group of: melanoma; colon carcinoma; pancreatic; lymphoma;
glioma; lung; esophagus; mammary; prostate; head and neck; ovarian;
kidney; liver, and hepatocellular carcinoma.
[0095] The method in an embodiment further includes therapeutically
treating the tumor, for example to surgically excising the tumor or
to reduce size of the tumor. In various embodiments, the method
further includes administering a therapeutic agent to the tumor or
to the subject. In various embodiments, the therapeutic agent is at
least one of: an anti-cancer, anti-tumor, an anti-proliferative,
and an anti-inflammatory.
[0096] An aspect of the invention provides a method of
manufacturing a composition for imaging and/or diagnosing a tumor
involving: coating a nanoparticle with a polymer layer, such that
the polymer layer coats the nanoparticle and includes for example a
polyethylene glycol and/or a polyelectrolyte such as an anionic
poly(acrylic acid) or a cationic poly(allylamine hydrochloride), to
obtain a resulting polymer-coated nanoparticle; and contacting the
resulting polymer-coated nanoparticle with at least one binding
agent that selectively attaches to the polymer layer and binds the
tumor, thereby producing the composition for imaging and/or
diagnosing the tumor. In an embodiment, the polymer layer includes
both the polyethylene glycol and the polyelectrolyte layer.
[0097] In an embodiment of the method, the binding agent that binds
the tumor includes at least one selected from the group of: a drug,
a protein, a carbohydrate, and a nucleotide sequence.
[0098] In an embodiment of the method, the binding agent includes a
monoclonal antibody that specifically targets the tumor or a
surface protein or peptide located on the tumor. For example, the
binding agent is monoclonal antibody FB50 or monoclonal antibody
SF25.
[0099] In an embodiment of the method, the antibody includes a
polyclonal antibody that specifically targets the tumor or a
surface protein or peptide located on the tumor.
[0100] In various embodiments of the method, the binding agent
binds a tumor antigen that is at least one selected from the group
of: aspartyl (asparaginyl)-.beta.-hydroxylase, alphafetoprotein,
carcinoembryonic antigen (CA), CA-125, mucin 1, epithelial tumor
antigen, tyrosinase, melanoma-associated antigen, tumor protein 53,
vimentin, CD34, desmin, and glial fibrillary acidic protein.
[0101] In various embodiments of the method, the tumor is selected
from the group of melanoma; colon carcinoma; pancreatic; lymphoma;
leukemia; glioma; lung; esophagus; mammary; prostate; head and
neck; ovarian; kidney; liver, and hepatocellular carcinoma.
[0102] The method in an embodiment includes prior to contacting the
nanoparticle with at least one polymer layer, constructing the
nanoparticle to have a diameter greater than about five nm in
diameter, or to have a diameter less than about 100 nm in
diameter.
[0103] An aspect of the invention provides a kit for diagnosing
presence of a tumor in a subject with a composition, the kit
including: a nanoparticle; a polymer layer coating the
nanoparticle, having: a polyethylene glycol or a polyelectrolyte
such as an anionic poly(acrylic acid) and/or a cationic
poly(allylamine hydrochloride); and, a binding agent that
specifically binds a tumor antigen, wherein the antibody is bound
to the polymer layer; the kit further including instructions for
use and a container.
[0104] In an embodiment of the kit, the nanoparticle comprises a
metal, for example a transition metal. In various embodiments of
the kit, the metal includes at least one of: silver, copper, gold,
mercury, cadmium, zinc, nickel, palladium, platinum, rhodium,
mercury, and a combination thereof.
[0105] In an embodiment of the kit, the anionic poly(acrylic acid)
contacts the nanoparticle, and the cationic poly(allylamine
hydrochloride) contacts the anionic poly acrylic acid.
[0106] In an embodiment of the kit, the binding agent includes at
least one selected from the group of: a drug, a protein, a
carbohydrate, and a nucleotide sequence. In an embodiment of the
kit, the binding agent that selectively attaches the composition to
the tumor is an antibody or a portion thereof, for example the
antibody includes a monoclonal antibody or a portion thereof. In an
embodiment of the invention, the antibody includes a polyclonal
antibody or a portion thereof. In an embodiment of the kit, the
binding agent is monoclonal antibody FB50 or monoclonal antibody
SF25, or a portion thereof.
[0107] In an embodiment of the kit, the tumor is selected from the
group of: melanoma; colon carcinoma; pancreatic; lymphoma; glioma;
lung; esophagus; mammary; prostate; head and neck; ovarian; kidney;
liver, and hepatocellular carcinoma. In certain embodiments, the
kit includes any pharmaceutical composition described herein.
[0108] In various embodiment of the kit, the tumor antigen is at
least one selected from the group of: aspartyl
(asparaginyl)-.beta.-hydroxylase, alphafetoprotein,
carcinoembryonic antigen (CA), CA-125, mucin 1, epithelial tumor
antigen, tyrosinase, melanoma-associated antigen, tumor protein 53,
vimentin, CD34, desmin, and glial fibrillary acidic protein.
[0109] An aspect of the invention herein provides a method of
identifying in a model system a potential therapeutic agent for
treating or preventing a tumor, the method including: contacting a
first sample of cells or tissue having a tumor with a composition
including: a gold nanoparticle; a polymer layer coating the
nanoparticle comprising for example a polyethylene glycol or a
polyelectrolyte, for example the polyelectrolyte includes an
anionic poly(acrylic acid) or a cationic poly(allylamine
hydrochloride); and, a monoclonal antibody that specifically binds
a tumor and is bound to the polymer layer, contacting a second
sample of the cells or tissue having the tumor with the composition
and a potential therapeutic agent; and measuring in the first
sample and the second sample, an amount of the marker, such that
the marker is characteristic of the tumor, such that the amount of
the marker in the second sample compared to that in the first
sample is a measure of treatment and protection by the potential
therapeutic agent, such that a decreased amount of the marker in
the second sample compared to the first sample is an indication
that the potential therapeutic agent is therapeutic, thereby
identifying the potential therapeutic agent for treating or
preventing the tumor. In an embodiment of the method, measuring
further comprises detecting the presence of the tumor in first
sample and the second sample using X-ray scatter imaging, for
example spatial harmonic imaging.
[0110] An aspect of the invention provides a composition for
imaging and/or diagnosing a tumor including: nanoparticles having
at least one binding agent that binds to and/or is suitable for
phagocytosis by the tumor, such that the nanoparticles are viewed
by X-ray scattering imaging, for example spatial harmonic imaging,
to detect, image, or diagnose the tumor.
[0111] In an embodiment of the composition, the nanoparticles
include a metal for example at least one of: silver, copper, gold,
mercury, cadmium, zinc, nickel, palladium, platinum, rhodium,
mercury, or a combination thereof, and the nanoparticles are
determined to be at least about five nm in diameter and less than
about 100 nm in diameter.
[0112] In an embodiment of the composition, the nanoparticles
include a core surrounded by a metal shell with a higher electron
density than the core. For example, the core includes carbon or
silica.
[0113] In an embodiment of the composition, the binding agent is
specifically attached to a portion of a surface of the
nanoparticles, for example the binding agent is attached using a
linker such as an amino acid, a polymer, or a nucleotide, and such
that the nanoparticles effectively bind to the tumor and scatters
X-rays once irradiated.
[0114] In an embodiment of the composition, the nanoparticles
include a fullerene or C.sub.60 Bucky ball, and the binding agent
is linked for example to the Bucky ball. In various embodiments of
the composition, the binding agent includes at least one selected
from the group of: a drug, a protein such as an antibody or a
binding protein, a carbohydrate such as a sugar, and a nucleotide
sequence. For example, the binding agent includes a monoclonal
antibody, a polyclonal, or a portion thereof.
[0115] An aspect of the invention provides a method of diagnosing a
presence of a tumor in a subject comprising: contacting a tissue
with a composition comprising a nanoparticle having at least one
binding agent that binds to and/or is suitable for phagocytosis by
the tumor; and, detecting the presence of the tumor attached to the
nanoparticle using X-ray scatter imaging for example spatial
harmonic imaging. In various embodiments of the method, the
composition further comprises any of compositions described
herein.
[0116] In an embodiment of the method, detecting the presence of
the tumor includes applying a mathematical operation such as a
Fourier transform to detect X-ray scatter measurements or data. In
an embodiment of the method, detecting the presence of the tumor is
performed using an absorption grid. In an embodiment of the method,
the detector includes a camera such as a charge coupled device that
detects the presence of the composition and/or nanoparticle.
[0117] An aspect of the invention provides a kit for diagnosing a
presence of a tumor in a subject comprising: a composition
including a nanoparticle having at least one binding agent that
binds to and/or is suitable for phagocytosis by the tumor;
instructions for use; and a container. In various embodiments of
the kit, the composition comprises any of the compositions
described herein for detecting or diagnosing the tumor by X-ray
scattering imaging including for example at least one of:
nanoparticles having at least one binding agent that binds to
and/or is suitable for phagocytosis by the tumor; a composition
having: a gold nanoparticle, a polymer layer coating the
nanoparticle, such that the polymer layer includes for example a
polyethylene glycol or a polyelectrolyte such as an anionic
poly(acrylic acid) or a cationic poly(allylamine hydrochloride);
and, a binding agent that specifically binds a tumor antigen, such
that the binding agent is bound to the polymer layer. The kit in an
embodiment further includes a receptacle.
[0118] The compositions, methods, kits and devices herein show an
imaging technique for visualizing cells and/or for the early
diagnosis of cancers and tumors (e.g., hepatocellular carcinoma)
using surface-modified nanoparticles (having an attached binding
agent) and X-ray imaging. The binding agent selectively binds to
for example a tumor or cancer cell, and in certain embodiments is
an antibody such as a monoclonal antibody, a polyclonal antibody,
or a portion thereof. In certain embodiments the monoclonal
antibody is a FB50 antibody (Rand et al. 2011 Nano Lett.
11:2678-2683), or a SF25 antibody. See also Takahashi et al., 1988
Cancer Res 48: 6573 and Takahashi et al. U.S. Pat. No. 5,212,085
issued May 18, 1993. Cancerous issues labeled with these
electron-dense particles and imaged using spatial harmonic imaging
show enhanced X-ray scattering over normal tissues, allowing for
effectively differentiation of the cancerous cells containing the
nanoparticles from normal non-cancerous cells not containing the
nanoparticles. Data show both in vitro and in vivo detection of
tumors as small as a few millimeters in size.
Binding Agents
[0119] The methods of the present invention use nanoparticles and
spatial frequency heterodyne imaging in certain embodiments to
image cells or a tissue, and to diagnose or prognose presence or
progression of a type of cell or tissue (e.g., cancerous). The
nanoparticles in embodiments herein include a binding agent such as
least one selected from the group of: a drug, a protein, a
carbohydrate, and a nucleotide sequence
[0120] In certain embodiments, the binding agent is an antibody
that is attached or conjugated to the nanoparticle and selectively
binds the cells or the tissue, such as a tumor. The term "antibody"
as used herein includes whole antibodies and any antigen binding
fragment (i.e., "antigen-binding portion") or single chains of
these. A naturally occurring "whole" antibody is a glycoprotein
including at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds.
[0121] As used herein, an antibody that "specifically binds to a
tumor" refers to an antibody that binds to tumor or tumor antigen.
As used herein, an antibody that "specifically binds to a cell"
refers to an antibody that binds to a cellular antigen. For example
the antibody has a K.sub.D of 5.times.10.sup.-9 M or less,
2.times.10.sup.-9 M or less, or 1.times.10.sup.-10 M or less. For
example, the antibody is a monoclonal antibody or a polyclonal
antibody. The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a target such as cells or a particular cellular epitope. The
antibody is for example an IgM, IgE, IgG such as IgG1 or IgG4.
[0122] Also useful for systems, method and kits herein is an
antibody that is a recombinant antibody. The term "recombinant
human antibody", as used herein, includes all antibodies that are
prepared, expressed, created or isolated by recombinant means, such
as antibodies isolated from an animal (e.g., a mouse). Mammalian
host cells for expressing the recombinant antibodies used in the
methods herein include for example Chinese Hamster Ovary (CHO
cells) including dhff CHO cells, described Urlaub and Chasin, Proc.
Natl. Acad. Sci. USA 77:4216-4220, 1980 used with a DHFR selectable
marker, e.g., as described in R. J. Kaufman and P. A. Sharp, 1982
Mol. Biol. 159:601-621, NSO myeloma cells, COS cells and SP2 cells.
In particular, for use with NSO myeloma cells, another expression
system is the GS gene expression system shown in WO 87/04462, WO
89/01036 and EP 338,841. To produce antibodies, expression vectors
encoding antibody genes are introduced into mammalian host cells,
and the host cells are cultured for a period of time sufficient to
allow expression of the antibody in the host cells or secretion of
the antibody into the culture medium in which the host cells are
grown. Antibodies are recovered from the culture medium using
standard protein purification methods.
[0123] Standard assays to evaluate the binding ability of the
antibodies toward the target of various species are known in the
art, including for example, ELISAs, western blots and RIAs. The
binding kinetics (e.g., binding affinity) of the antibodies also
can be assessed by standard assays known in the art, such as by
Biacore analysis.
[0124] General methodologies for antibody production, including
criteria to be considered when choosing an animal for the
production of antisera, are described in Harlow et al. (Antibodies,
Cold Spring Harbor Laboratory, pp. 93-117, 1988). For example, an
animal of suitable size such as goats, dogs, sheep, mice, or camels
are immunized by administration of an amount of immunogen, such as
the intact protein or a portion thereof containing an epitope
(e.g., HCC or CD44), effective to produce an immune response. An
exemplary protocol is as follows. The animal is subcutaneously
injected in the back with 100 micrograms to 100 milligrams of
antigen, dependent on the size of the animal, followed three weeks
later with an intraperitoneal injection of 100 micrograms to 100
milligrams of immunogen with adjuvant dependent on the size of the
animal, for example Freund's complete adjuvant. Additional
intraperitoneal injections every two weeks with adjuvant, for
example Freund's incomplete adjuvant, are administered until a
suitable titer of antibody in the animal's blood is achieved.
Exemplary titers include a titer of at least about 1:5000 or a
titer of 1:100,000 or more, i.e., the dilution having a detectable
activity. The antibodies are purified, for example, by affinity
purification on columns containing a cellular antigen used for
immunization.
[0125] The technique of in vitro immunization of human lymphocytes
is used to generate monoclonal antibodies. Techniques for in vitro
immunization of human lymphocytes are well known to those skilled
in the art. See, e.g., Inai, et al., Histochemistry, 99(5):335 362,
May 1993; Mulder, et al., Hum. Immunol., 36(3):186 192, 1993;
Harada, et al., J. Oral Pathol. Med., 22(4):145 152, 1993; Stauber,
et al., J. Immunol. Methods, 161(2):157 168, 1993; and
Venkateswaran, et al., Hybridoma, 11(6) 729 739, 1992. These
techniques can be used to produce antigen-reactive monoclonal
antibodies, including antigen-specific IgG, and IgM monoclonal
antibodies.
Pharmaceutical Compositions
[0126] An aspect of the present invention provides pharmaceutical
compositions that include a nanoparticle having at least one of a
polymer layer and a binding agent. In certain embodiments, the
composition comprises a plurality of nanoparticles that are
administered to cells, a tissue, or a subject. The nanoparticles in
certain embodiments undergo cellular uptake by for example
phagocytosis and endocytosis. In various embodiments, the
nanoparticles are conjugated to a binding agent that binds to a
molecular antigen of cell or a tissue. In related embodiments, the
pharmaceutical composition is formulated sufficiently pure for
administration to a subject, e.g., a human, a mouse, a rat, a dog,
a cat, and a cow. The pharmaceutical composition is administered
for example to an abdomen or a vascular system.
[0127] In certain embodiments, the pharmaceutical composition
further includes at least one therapeutic agent selected from the
group consisting of growth factors, anti-inflammatory agents,
vasopressor agents including but not limited to nitric oxide and
calcium channel blockers, collagenase inhibitors, topical steroids,
matrix metalloproteinase inhibitors, ascorbates, angiotensin II,
angiotensin III, calreticulin, tetracyclines, fibronectin,
collagen, thrombospondin, transforming growth factors (TGF),
keratinocyte growth factor (KGF), fibroblast growth factor (FGF),
insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs),
epidermal growth factor (EGF), platelet derived growth factor
(PDGF), neu differentiation factor (NDF), hepatocyte growth factor
(HGF), vascular endothelial growth factor (VEGF), heparin-binding
EGF (HBEGF), thrombospondins, von Willebrand Factor-C, heparin and
heparin sulfates, and hyaluronic acid. See Toole et al. U.S. Pat.
No. 5,902,795 issued May 11, 1999, which is incorporated by
reference herein in its entirety.
[0128] The therapeutic agent in various embodiments includes an
anti-cancer or anti-tumor agent selected from the group of:
alkylating agents, such as mechlorethamine, cyclophosphamide,
melphalan, uracil mustard, chlorambucil, busulfan, carmustine,
lomustine, semustine, streptozoticin, and decrabazine;
antimetabolites, such as methotrexate, fluorouracil,
fluorodeoxyuridine, cytarabine, azarabine, idoxuridine,
mercaptopurine, azathioprine, thioguanine, and adenine arabinoside;
natural product derivatives, such as irinotecan hydrochloride,
vinblastine, vincristine, dactinomycin, daunorubicin, doxorubicin,
mithramycin, taxanes (e.g., paclitaxel) bleomycin, etoposide,
teniposide, and mitomycin C; and miscellaneous agents, such as
hydroxyurea, procarbezine, mititane, and cisplatinum. See Brown et
al. U.S. publication number 20050267069 published Dec. 1, 2005,
which is incorporated by reference herein in its entirety.
[0129] In other embodiments, the therapeutic agent is a compound,
composition, biological or the like that potentiates, stabilizes or
synergizes the effects of another molecule or compound on a cell or
tissue. In some embodiments, the drug includes without limitation
anti-tumor, antiviral, antibacterial, anti-mycobacterial,
anti-fungal, anti-proliferative or anti-apoptotic agents. Drugs
that are included in the compositions of the invention are well
known in the art. See for example, Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman, et al.,
eds., McGraw-Hill, 1996; and Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 12th Ed., Hardman, et al.,
eds., McGraw-Hill, 2010, the contents of which are herein
incorporated by reference herein.
[0130] As used herein, the term "pharmaceutically acceptable
carrier" includes any and all solvents, diluents, or other liquid
vehicle, dispersion or suspension aids, surface active agents,
isotonic agents, thickening or emulsifying agents, preservatives,
solid binders, lubricants and the like, as suited to the particular
dosage form desired. Remington's Pharmaceutical Sciences 20.sup.th
Edition by Gennaro, Mack Publishing, Easton, Pa., 2003 provides
various carriers used in formulating pharmaceutical compositions
and known techniques for the preparation thereof. Some examples of
materials which can serve as pharmaceutically acceptable carriers
include, but are not limited to, sugars such as glucose and
sucrose; excipients such as cocoa butter and suppository waxes;
oils such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil, and soybean oil; glycols such a propylene
glycol; esters such as ethyl oleate and ethyl laurate; agar;
buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen-free water; isotonic saline;
Ringer's solution; ethyl alcohol; and phosphate buffer solutions,
as well as other non-toxic compatible lubricants such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
releasing agents, coating agents, preservatives and antioxidants
can also be present in the composition, the choice of agents and
non-irritating concentrations to be detelmined according to the
judgment of the formulator.
Therapeutically Effective Dose
[0131] Methods provided herein involves administering a
pharmaceutical composition to cells or to a subject, for example,
administering a therapeutically effective amount of a
pharmaceutical composition having nanoparticles having at least one
of a polymer layer and a binding agent. The pharmaceutical
composition in certain embodiments optionally further includes a
therapeutic agent in such amounts and for such time as is necessary
to achieve the desired result.
[0132] The compositions, according to the method of the present
invention, are administered using any amount and any route of
administration effective for contacting cells or a subject. The
exact dosage is chosen by the individual physician in view of the
patient to be treated. Dosage and administration are adjusted to
provide sufficient levels of the active agent(s) or to maintain the
desired effect. Additional factors which may be taken into account
include the severity of the disease state, e.g., intermediate or
advanced stage of a disease condition; age, weight and gender of
the patient; diet, time and frequency of administration; route of
administration; drug combinations; reaction sensitivities; and
tolerance/response to therapy. Long acting pharmaceutical
compositions might be administered hourly, twice hourly, every
three to four hours, daily, twice daily, every three to four days,
every week, or once every two weeks depending on half-life and
clearance rate of the particular composition.
[0133] The active agents of the invention are preferably formulated
in dosage unit form for ease of administration and uniformity of
dosage. The expression "dosage unit form" as used herein refers to
a physically discrete unit of active agent appropriate for the
patient to be diagnosed or to be treated. It will be understood,
however, that the total daily usage of the compositions of the
present invention will be decided by the attending physician within
the scope of sound medical judgment. For any active agent, the
therapeutically effective dose can be estimated initially either in
cell culture assays or in animal models, as provided herein,
usually mice, but also potentially from rats, rabbits, dogs, or
pigs. The animal cell model provided herein is also used to achieve
a desirable concentration and total dosing range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans.
[0134] A therapeutically effective dose refers to that amount of
active agent that can be clearly imaged for diagnosis of cells or
tissues (e.g., tumors) at a very early stage, or that ameliorates
the symptoms or prevents progression of a pathology or condition.
Therapeutic efficacy and toxicity of active agents can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED50 (the dose is therapeutically
effective in 50% of the population) and LD50 (the dose is lethal to
50% of the population). The dose ratio of toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
ratio, LD50/ED50. Pharmaceutical compositions which exhibit large
therapeutic indices are preferred. The data obtained from cell
culture assays and animal studies are used in formulating a range
of dosage for human use.
Administration of Pharmaceutical Compositions
[0135] As formulated with an appropriate pharmaceutically
acceptable carrier in a desired dosage, the pharmaceutical
composition provided herein is administered to humans and other
mammals topically such as ocularly (as by solutions, ointments, or
drops), nasally, bucally, orally, rectally, topically,
parenterally, intracisternally, intravaginally, or
intraperitoneally.
[0136] Injections include intravenous injection, direct or parental
injection into the tissues (e.g., cancerous and non-cancerous), or
injection into the external layers of the tissue or adjacent
tissues, such as for example injection into the peritoneal cavity,
stomach, liver, breast, leg, or lung.
[0137] The pharmaceutical composition in various embodiments is
administered with inert diluents commonly used in the art such as,
for example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the delivered compositions can also include
adjuvants such as wetting agents, and emulsifying and suspending
agents.
[0138] Dosage forms for topical or transdermal administration of an
inventive pharmaceutical composition include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants, or
patches. The active agent is admixed under sterile conditions with
a pharmaceutically acceptable carrier and any needed preservatives
or buffers as may be required. For example, ocular or cutaneous
routes of administration are achieved with aqueous drops, a mist,
an emulsion, or a cream. Administration may be diagnostic,
prognostic, therapeutic or it may be prophylactic. The invention
includes delivery devices, surgical devices, audiological devices
or products which contain disclosed compositions (e.g., gauze
bandages or strips), and methods of making or using such devices or
products. These devices may be coated with, impregnated with,
bonded to or otherwise treated with a composition as described
herein.
[0139] Transdermal patches have the added advantage of providing
controlled delivery of the active ingredients to the body. Such
dosage forms can be made by dissolving or dispensing the compound
in the proper medium. Absorption enhancers can also be used to
increase the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the compound in a polymer matrix or gel.
[0140] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For the purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. The
injectable formulations can be sterilized, for example, by
filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use. In order to
prolong the effect of an active agent, it is often desirable to
slow the absorption of the agent from subcutaneous or intramuscular
injection. Delayed absorption of a parenterally administered active
agent may be accomplished by dissolving or suspending the agent in
an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the agent in biodegradable polymers such
as polylactide-polyglycolide. Depending upon the ratio of active
agent to polymer and the nature of the particular polymer employed,
the rate of active agent release can be controlled. Examples of
other biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the agent in liposomes or microemulsions which are
compatible with body tissues.
[0141] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the active
agent(s) of the invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active agent(s).
[0142] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active agent is mixed with at least one inert, pharmaceutically
acceptable excipient or carrier such as sodium citrate or dicalcium
phosphate and/or a) fillers or extenders such as starches, sucrose,
glucose, mannitol, and silicic acid, b) binders such as, for
example, carboxymethylcellulose, alginates, gelatin,
polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as
glycerol, d) disintegrating agents such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, 0 absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof.
[0143] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as milk sugar as well as high molecular weight
polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active agent(s) may be admixed with at least one inert diluent such
as sucrose or starch. Such dosage forms may also comprise, as is
normal practice, additional substances other than inert diluents,
e.g., tableting lubricants and other tableting aids such a
magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active agent(s)
only, or preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
which can be used include polymeric substances and waxes.
[0144] A portion of the invention is provided in a publication in
the Journal Nano Letters and is entitled "Nanomaterials for X-ray
Imaging: Gold Nanoparticle-Enhancement of X-ray Scatter Imaging of
Hepatocellular Carcinoma" by Danielle Rand, Vivian Ortiz, Yanan
Liu, Zoltan Derdak, Jack R. Wands, Milan Tati{hacek over (c)}ek,
and Christoph Rose-Petruck (Rand et al. 2011 Nano Lett. 11:
2678-2683), which is hereby incorporated by reference in its
entirety.
[0145] The following examples and claims are illustrative only and
not intended to be further limiting. Those skilled in the art will
recognize or be able to ascertain using no more than routine
experimentation, numerous equivalents to the specific procedures
described herein. Such equivalents are within the scope of the
present invention and claims. The contents of all references
including issued patents and published patent applications cited in
this application are hereby incorporated by reference in their
entireties. A skilled person will recognize that many suitable
variations of the methods may be substituted for or used in
addition to those described above and in the claims. It should be
understood that the implementation of other variations and
modifications of the embodiments of the invention and its various
aspects will be apparent to one skilled in the art, and that the
invention is not limited by the specific embodiments described
herein and in the claims. Therefore, it is contemplated to cover
the present embodiments of the invention and any and all
modifications, variations, or equivalents that fall within the true
spirit and scope of the basic underlying principles disclosed and
claimed herein.
EXAMPLES
Example 1
Materials
[0146] Chemicals were purchased from Sigma-Aldrich Inc. (St. Louis,
Mo.) unless otherwise specified. Uncoated gold nanoparticles (ten
nm and 50 nm) stabilized in citrate buffer were purchased from
British Biocell International (Cardiff, UK): 10 nm gold
nanoparticles were BBI catalog #EMGC10, batch #15683; and 50 nm
gold nanoparticles were BBI catalog #EMGC50, batch #15693.
Example 2
Layer-by-Layer Coating of Nanoparticles
[0147] Stock solutions of poly(acrylic acid) (PAA) and
poly(allylamine hydrochloride) (PAH) were prepared (10 mg/mL PAA or
PAH in one millimolar (mM) aqueous sodium chloride solution).
Unpurified one milliliter (mL) aliquots of ten nm gold
nanoparticles or of 50 nm gold nanoparticles were mixed with 100
microliters (.mu.L) of 1 mM sodium chloride and 200 .mu.L of PAA
stock solution. The mixture was incubated for 30 minutes. Excess
polymer in the supernatant was removed by centrifugation. Anionic
poly(acrylic acid) (PAA) was deposited on the gold nanoparticles,
resulting in a gold nanoparticle being coated with carboxylic acid
groups located on the nanoparticle surface (FIG. 1; AU-PAA
nanoparticle).
[0148] The PAA-coated nanoparticle were re-suspended in phosphate
buffered saline (PBS). The PAA encapsulated gold nanoparticles (one
mL aliquots) were then mixed with 100 .mu.L of 1 mM sodium chloride
and 200 .mu.L of cationic poly(allylamine hydrochloride) stock
solution for 30 minutes. The electrostatic interaction between the
oppositely charged PAA and PAH polymer layers yielded a
layer-by-layer coating of the PAA and PAH polyelectrolytes on the
surface of the nanoparticles.
[0149] The mixture was then centrifuged to remove excess polymer in
the supernatant. The resulting gold nanoparticles were coated with
an inner layer of PAA polymer and an outer layer of PAH polymer.
Solutions containing the PAA-PAH coated gold nanoparticles were
stored at room temperature.
Example 3
Preparation of FOCUS Cells
[0150] FOCUS cells were cultured and maintained at 37.degree. C.
(5% CO.sub.2) in Eagle's Minimum Essential Medium (EMEM)
supplemented with 10% fetal bovine serum, 1%
penicillin/streptomycin and 1% L-glutamine. Cells were grown to
confluence and formed a monolayer. Trypsin was added to detach the
FOCUS cells, which were re-suspended in serum-free EMEM.
Example 4
Incubation of FOCUS Cells with Coated Nanoparticles
[0151] To prepare samples for X-ray scatter imaging, cell pellet
samples containing approximately 10.sup.7 FOCUS cells were
incubated in vials with nanoparticle solutions containing either
ten nm PAA-PAH coated gold nanoparticles, or 50 nm PAA-PAH coated
gold nanoparticles. Control cell pellet samples were incubated with
either no gold, or with ten nm uncoated gold nanoparticles only,
viz., no PAA-PAH coating. The number of FOCUS cells in the cell
pellet samples was chosen to model the size of a small tumor having
a diameter of a few millimeters.
[0152] After each incubation, the cells were collected, washed and
imaged. The approximate amount of gold nanoparticles contained in
each incubation with the cell pellet samples was measured in
Example 8 by spectrometric methods.
Example 5
Spatial Frequency Heterodyne Imagine of the FOCUS Cells and
Nanoparticles
[0153] Spatial frequency heterodyne imaging was used to image
samples of FOCUS cell pellets contacted with gold nanoparticles.
The spatial frequency heterodyne imaging system shown in FIG. 2
directed X-ray radiation to an absorption grid and a sample, and
then detected the X-rays scattered by the sample to using a
detector and Fourier transformation (Stein, A. F. et al. 2010 Opt.
Express 18: 13271-13278; Wen, H. et al. 2009 Radiology 251:
910-918; and Wen, H. et al. 2008 IEEE Trans Med Imaging 27:
997-1002). A vial holder positioned the vials containing the cell
pellets-gold nanoparticles.
[0154] The X-ray scatter measurements were obtained with a
micro-focus X-ray tube (Trufocus Corporation, model: TFX-3110EW)
with a tungsten anode. The tube was operated at an electrical power
of 20 watts (W), producing a maximum voltage of 95.6 kilovolts
(kV). High voltages reduced the exposure times, and were observed
to be better suited for in vivo applications, as imaging a tissue
in a subject required large penetration depths. The distance
between the source and camera was 1.6 meters (m), and the sample
was placed at a distance halfway (0.8 m) between the source and
camera to enhance resolution (Wen, H. et al. 2009 Radiology 251:
910-918).
[0155] The absorption grid was stainless steel with a pitch of 50
micrometer (.mu.m), and was purchased from Small Parts, Inc.
(Seattle, Wash.). The grid was positioned directly in front of the
vials containing the cell pellet samples and the gold
nanoparticles. The images were acquired with an X-ray CCD camera
(Princeton Instruments, Model Quad-RO 4096). The total exposure
time for each image was 180 seconds.
Example 6
Fourier Transformation of Images
[0156] Fourier transformation was then performed on the original
images (FIG. 3 bottom left image; F, Original). The Fourier
transformation converted the product of X-ray scatter
transmittances of the sample and the grid into a convolution in the
spatial frequency domain (see FIG. 3). The grid, a periodic
structure, produced a series of peaks in this convolution. Each
peak produced by the grid was "surrounded" by the spatial frequency
spectrum of the sample.
[0157] Selecting an area around a specific peak in the convolution
and Fourier back-transforming this area yielded a logarithm of the
scattered intensities to real space and provided a processed image
that contained anisotropic information about the scattering of the
incident X-rays by the sample. The area surrounding the central
0.sup.th-order peak (FIG. 3 bottom right image; F.sup.-1, S.sub.0:
0.sup.th order) corresponded to the original X-ray absorption image
without scatter, which was then used for normalizing the images.
The X-ray absorption image without scatter was subtracted from the
higher order images to remove absorption features that distort
clear visualization of the cell pellet samples.
[0158] The area around the 1.sup.st-order peak to the immediate
left of the 0.sup.th-order peak (FIG. 3 bottom center image;
F.sup.-1, S.sub.1: 1.sup.st order, left) corresponded to scattering
in the x-direction, and therefore produced a processed "left
1.sup.st-order" scatter image upon Fourier back-transformation and
normalization. Further, the area around the 1.sup.st-order peak
immediately above the 0.sup.th-order peak (FIG. 3 top image top
right box) corresponded to scattering in the y-direction, and
therefore producing a processed "upper 1.sup.st-order" scatter
image upon Fourier back-transformation and normalization. Thus, the
original X-ray image obtained yielded two Fourier transformation
processed images. A processed image resulting from X-radiation
scattered horizontally (FIG. 3 bottom center image; F.sup.-1,
S.sub.1: 1.sup.st order), and the processed image from x-radiation
scattered vertically (FIG. 3 top image top right box). Both of
these 1.sup.st order images produced identical scatter signals
because of the isotropic scattering of the spherical
nanoparticles.
Example 7
Calculation of Scatter Signals and Enhancement Factors
[0159] The scattering signal (S) for each processed image was
calculated according to the following equation:
S = - log ( I 1 / G 1 I 0 / G 0 ) ##EQU00001##
such that I.sub.1 and I.sub.0 were the detected X-ray signals with
sample in the 1.sup.st and 0.sup.th order, respectively. In the
equation above, G.sub.1 and G.sub.0 were the detected X-ray signals
without sample in the 1.sup.st and 0.sup.th order, respectively.
The signal S was calculated for each vial in the area that contains
the cell pellet (S.sub.cells) and the area of the supernatant
(S.sub.super). Precautions were taken to avoid detecting any signal
from the walls of the vials. The normalized scattering signal
(S.sub.norm) for each cell pellet was calculated by dividing the
signal measured in the area containing the cell pellet
(S.sub.cells) by the signal measured in the area of the supernatant
(S.sub.super) as shown in the equation below:
S norm = S cells S super ##EQU00002##
The normalized scattering signal was then compared to the
absorbance measured in the original absorption X-ray images. The
absorbance was calculated using the following equation:
A = - log ( I sample I flat field ) ##EQU00003##
such that, I.sub.sample was the detected X-ray intensity with a
sample vial and I.sub.flat field was the detected X-ray intensity
without a sample vial. The absorbance was calculated for each vial
in the area containing the cell pellet (A.sub.cells) and in the
area of the supernatant (A.sub.super), respectively. The normalized
absorbance signal (A.sub.norm) measured for each pellet in the
absorption images was calculated by dividing the signal measured in
the area containing the cell pellet (A.sub.cells) by the signal
measured in the area of the supernatant (A.sub.super) using the
equation shown below:
A norm = A cells A super ##EQU00004##
The scattering signal (S.sub.norm) was compared to the absorption
signal (A.sub.norm) measured in the absorption images by
calculating an enhancement factor as shown in an equation
below:
Enhancement factor = S norm A norm ##EQU00005##
Example 8
ICP-AES Analysis of Gold in Cell Pellet Samples
[0160] The signal enhancement in the processed X-ray images due to
increased scattering by the gold nanoparticles was normalized by
spectrophotometric techniques for gold content (mass) in the cells.
After the pellets were imaged using X-ray scattering, the amount of
gold taken up by the FOCUS cell pellets during incubation was
determined using inductively-coupled plasma atomic emission
spectroscopy (ICP-AES).
[0161] Samples were prepared for ICP-AES analysis by digesting gold
and organics with aqua regia (1:3 mixture of HNO.sub.3:HCl)
followed by dilution in 2% nitric acid. Data from ICP-AES analysis
showed that the samples contained four micrograms to eight
micrograms (.mu.g), corresponding to several hundred 50 nm
nanoparticles per cell and tens of thousands of ten nm
nanoparticles per cell (shown in Tables 1 and 2).
Example 9
Images Analysis
[0162] FIG. 4 panels A-D are representative photographs (FIG. 4
panel A), absorption images (FIG. 4 panel B) and X-ray scatter
images (FIG. 4 panels C-D) of vials containing FOCUS cell pellets
incubated with either 50 nm PAA-PAH coated gold nanoparticles (FIG.
4 left image in each panel), or ten nm. PAA-PAH coated gold
nanoparticles (FIG. 4 right image in each panel). Control samples
of FOCUS cell pellets were incubated with no gold nanoparticles
(FIG. 4 center image in each panel).
[0163] Visual analysis of photographs clearly indicated that
samples incubated with PAA-PAH coated gold nanoparticles were
labeled with gold, and that control samples incubated with no gold
nanoparticles were not labeled (FIG. 4 panel A). Cell pellets
containing gold nanoparticles were stained a distinctive brown
color. It was observed that for each of the samples the EMEM
supernatant located above the cell pellet was naturally pink and
showed no presence of gold nanoparticles. No clear labeling of
FOCUS cell pellets was observed in images obtained using
conventional absorption techniques. For the absorption images,
FOCUS cell pellets incubated with gold nanoparticles and the cell
pellet samples incubated with no gold nanoparticles were
indistinguishable from each other (FIG. 4 panel B). Most important,
images obtained by spatial frequency heterodyne imaging showed a
signal enhancement in samples incubated with the PAA-PAH coated
gold nanoparticles compared to control cells having no
nanoparticles because of uptake of the gold nanoparticles into the
cell pellets (FIG. 4 panels C-D).
[0164] The signal enhancement for spatial frequency heterodyne
imaging compared to absorbance imaging was calculated by first
obtaining an average intensity profile of the absorbance image at
the pellet height (A.sub.cells), such that the average absorbance
intensity varied depending on presence of gold nanoparticle
concentration contacting the cells. The absorbance value was
normalized using the intensity of the supernatant (A.sub.super)
which was approximately the same for each of the samples
irrespective of whether samples contained gold nanoparticles or
not. The upper right boxes and two lower boxes shown in FIG. 4
panels B-D outline the areas selected for calculating intensity
profiles of the supernatant and pellet, respectively. The intensity
values were averaged using the optical data from the outlined
areas. Examples herein used a vial holder for positioning the vials
containing the cell pellets-gold nanoparticles described above
ensured that the average signal intensities were standardized for
each image obtained, as absorbance and signal comparisons between
the samples were obtained from the same relative positions (having
the same thickness) in the vials containing the samples.
[0165] Data show that gold nanoparticles not coated with PAA and
PAH polyelectrolytes were not effectively uptaken by the FOCUS cell
pellets. Data show that less than 25% of the uncoated nanoparticles
used for incubation were phagocytosed by the FOCUS cells after the
one hour contact with the nanoparticles. Clearly these control cell
pellets were observed to have reduced intensity due to the
relatively low gold concentrations in each cell (see Table 1).
[0166] Coating gold nanoparticles with the PAA-PAH polymer coatings
enhanced FOCUS cell pellets cellular uptake (e.g., phagocytosis) of
the nanoparticles compared using non-coated control nanoparticles.
Layer-by-layer coating of the nanoparticles resulted in increased
amount of gold in each cell, and therefore the increased scattering
signal observed in the X-ray images obtained (Table 1). Data in
Table 1 further show that the FOCUS cells phagocytosed more than
twice as many ten nm PAA-PAH coated gold nanoparticles (275,000
nanoparticles) as ten nm uncoated gold nanoparticles (108,000
nanoparticles).
[0167] Most important, FOCUS cells phagocytosed twice as much mass
of gold per cell of 10 nm PAA-PAH coated gold nanoparticles
(2.8.+-.0.4 picograms per cell) and 50 nm PAA-PAH coated gold
nanoparticles (2.2.+-.0.3 picograms per cell) compared to uncoated
gold nanoparticles (1.2.+-.0.5 picograms per cell). The uptake of
the nanoparticles was enhanced by the PAA-PAH coating, as data
showed that FOCUS cells phagocytosed an average of approximately
275,000 polyelectrolyte-coated ten nm PAA-PAH gold nanoparticles
per cell, compared to approximately 108,000 uncoated ten nm gold
nanoparticles per cell (Table 1).
[0168] It was observed that FOCUS cell pellets phagocytosed
approximately the same amount of PAA-PAH coated nanoparticles
irrespective if the nanoparticles were ten nm (2.8.+-.0.4
picograms) or 50 nm (2.2.+-.0.3) in size (Table 1). Without being
limited by any particular theory or mechanism of action, it is here
envisioned that the total volume of gold nanoparticles incubated
with the cells that is an important factor in determining the
extent of cellular uptake. Calculation of the percentage volume of
the nanoparticles in the cell indicated than only a very small
portion of each cell is actually occupied by the coated gold
nanoparticles. Data show that the FOCUS cells phagocytosed a cell
volume of much less than 0.001% of PAA-PAH polyelectrolyte-coated
nm gold nanoparticles: 10 nm PAA-PAH coated gold nanoparticles
(0.00063%), 50 nm PAA-PAH coated gold nanoparticles (0.00049%), and
uncoated 10 nm gold nanoparticles (0.00025%).
[0169] Analysis of the absorption X-ray images further shows that
the signal measured for FOCUS cell pellets incubated with PAA-PAH
coated gold nanoparticles was 1.2% greater than the signal measured
for FOCUS cell pellets incubated without gold nanoparticles (see
Table 2). However, of the total number (12) of absorption images
taken of FOCUS cell pellets labeled with gold nanoparticles, only
58% (seven) were observed to have exhibit any enhancement due to
gold labeling by the nanoparticles. The large standard deviation
calculated for absorption techniques illustrates the extent that
absorption images are unreliable for imaging cells in vitro, let
alone in vivo.
[0170] The spatial frequency heterodyne imaging described herein
visualized and differentiated between FOCUS cell pellets incubated
with PAA-PAH coated gold nanoparticles and control FOCUS cell
pellets incubated with no gold nanoparticles. The left
1.sup.st-order scatter images (FIG. 4 panel C) and upper
1.sup.st-order scatter images (FIG. 4 panel D) produced using
spatial frequency heterodyne imaging were analyzed and used to
produce normalized intensity profiles for the cell pellet samples.
These calculated normalized profiles corresponded to the logarithm
of the intensity of the scattered x-radiation. Without being
limited by any particular theory or mechanism of action, it is
envisioned that the signal differences between the 1.sup.st order
images were due to the side interfaces produced by either air in
the vials (FIG. 4 panel C) or the bottom interface of the vials
(FIG. 4 panel D). Thus the signal differences in the 1.sup.st-order
spatial frequency heterodyne imaging were from anisotropic X-ray
scattering at smooth material interfaces.
[0171] Analysis of processed spatial frequency heterodyne (X-ray
scatter) images and the ICP-AES data in Examples herein shows an
average signal enhancement due to gold labeling that ranges from
approximately 1.6% to 4.4% (Table 2). Of the X-ray scatter images
(24 total images) taken of FOCUS cell pellets labeled with gold
nanoparticles, 88% (21 images) showed a signal enhancement when
compared to X-ray scatter images of FOCUS pellets containing no
gold. Overall, the data herein of processed spatial frequency
heterodyne images and ICP-AES show an at least three or at least a
five enhancement factor per picogram of gold uptaken by the cell
(Table 2).
[0172] These data show that for every picogram of gold taken up by
a cell, the signal observed in the processed scatter images was
enhanced by an average of 3.6% and 5.7% (Table 2; replicate 1 and
replicate 2, respectively). It was observed that each cell
contained an average of about three picograms of
polyelectrolyte-coated gold nanoparticles (Table 1), resulting in a
potential signal enhancement due to gold labeling of approximately
11% to approximately 17% in logarithm scale (Table 2).
Example 10
Model of In Vivo Imaging Using Nanoparticles and Spatial Frequency
Heterodyne Imaging
[0173] Examples herein show development of an X-ray scatter imaging
system for in vivo imaging of cells and tissues. The in vivo model
utilized signal enhancement produced by gold nanoparticle labeling
such that the signal enhancement would correspond to visualizing
cells and structures beneath many layers of tissue.
[0174] Spatial frequency heterodyne imaging was performed as
described in FIG. 2 for a cell pellet sample incubated with 50 nm
PAA-PAH coated gold nanoparticles, in which X-ray radiation
progressed through one centimeter of water prior to reaching the
cell pellets previously contacted with nanoparticles. X-ray
radiation traversing one centimeter of water positioned above the
cell pellet-gold nanoparticles was chosen to imitate closely a
thickness of tissue which X-ray radiation delivered to a patient's
skin might traverse at the point of entry into the patient.
[0175] Water and liver tissues have similar radiological densities,
in fact the electron densities of water and liver tissues differ by
less than 5% (Yang, M. et al. 2010 Physics in Medicine and Biology
55: 1343-1362). X-ray scatter imaging an additional traversal
through one centimeter of water resulted in the detection of
gold-labeled FOCUS cell pellets. Data show that normalized signal
intensities in the scatter images increased by 1.8.+-.0.5% for
cells incubated with gold nanoparticles compared to a control cell
pellet incubated with no gold nanoparticles.
Example 11
Imaging Cells Using Antibody-Conjugated Nanoparticles
[0176] Nanoparticles linked to HCC-specific antibodies were
prepared to determine whether such compositions would be useful as
X-ray visible immunolabels, for in vivo detection and X-ray imaging
of HCC tumors in a murine mouse model.
[0177] The FB50 monoclonal antibody was generated by cellular
immunization of mice with FOCUS HCC cells. The FB50 antibody
specifically binds and targets FOCUS cells, and the antibody is
localized intracellularly. To reduce nonspecific uptake of the gold
nanoparticles by the other types of tissue, the nanoparticle
surface was coated with a polyethylene glycol (PEG). Without being
limited by any particular theory or mechanism of action, it is here
envisioned that PEG prevented the non-specific adsorption of the
proteins onto the nanoparticle surface, allowing for longer
circulation times in vivo and enhanced accumulation of the
nanoparticle specifically in the liver of the subject.
[0178] Bi-functional PEG (HS-PEG-COOH) was added to the
nanoparticle, for facile attachment to the nanoparticle surface.
Bio-conjugation of the gold nanoparticles to the FB50 antibody was
performed using techniques involving EDC/NHS cross-linking
chemistry. The bio-conjugation involved linking the carboxylic acid
groups on the PEG coating around the nanoparticles to amine groups
present on the FB50 antibody (See FIG. 5). Dynamic light scattering
techniques showed that each of the steps of producing the PEG
coated the gold nanoparticles and the conjugated FB50 antibody
resulted in increases to the diameter of the 50 nm gold
nanoparticles (FIG. 6 panel A). Specifically data showed that the
uncoated 50 nm gold particles had a diameter of 71.3.+-.10.0 nm,
that the PEG-coated 50 nm gold nanoparticles had a diameter of
81.8.+-.12.1 nm, and that the PEG-coated FB50 antibody conjugated
50 nm gold nanoparticles had a diameter of 93.9.+-.18.3 nm (FIG. 6
panel A). Without being limited by any particular theory or
mechanism of action, it is here envisioned that PEG coated on the
nanoparticle surface prevented the non-specific adsorption of the
antibody-conjugated nanoparticles to cells not expressing HCC,
allowing for longer circulation times in vivo and enhanced
accumulation of the nanoparticle specifically in the liver of the
subject.
[0179] Cellular uptake by FOCUS cells pellets and by NIH/3T3 cell
pellets of the PEG coated gold nanoparticles or the PEG coated FB50
conjugated ten nm gold nanoparticles was measured (FIG. 6 panel B).
The specificity of FB50 antibody was evaluated by using as a
negative control the NIH/3T3 fibroblasts, which do not express HCC.
An anti-Murutucu tropical virus (MUK) antibody was also used as an
additional negative control antibody for the FOCUS cells, as the
MUK antibody does not specifically bind HCC (FIG. 6 panel B).
[0180] Data show that at least thirty fold more of the PEG-coated
FB50 antibody conjugated ten nm gold nanoparticles were taken into
the FOCUS cell pellets compared to the NIH/3T3 cell pellets.
Further, little or no PEG-coated MUK antibody conjugated gold
nanoparticles were transported into the FOCUS cell or the NIH/3T3
cells. Thus, the PEG-coated FB50 antibody conjugated ten nm gold
nanoparticles specifically bound to the HCC antigen and enhanced
cellular uptake of the nanoparticles into the FOCUS cells.
Example 12
In Vivo Imaging of Livers
[0181] Examples herein used the PEG/FB50 antibody conjugated
nanoparticles were tested in an in vivo a murine mouse model. Mice
were injected twice over a 24 hour period into the tail vein with
50 nm PEG/FB50 antibody conjugated gold nanoparticles, or saline as
a negative control. Subjects were sacrificed 48 hours after the
first injection, and were fixed in formaldehyde and imaged in the
spatial frequency heterodyne imaging system (FIG. 2) and imaging
was performed in situ. Without being limited by any particular
theory or mechanism of action, it is envisioned that injected
nanoparticles that were not conjugated to the targeting/binding
agent FB50 antibody such as the Au-PEG nanoparticles (shown in FIG.
4) concentrated mainly in the liver due to the high phagocytic
activity of the Kuppfer cells located in that tissue. In vivo
spatial frequency heterodyne imaging was performed in the area of
the liver of each subject.
[0182] In situ imaging showed that 80% of livers in subjects
injected with PEG/FB50 antibody conjugated gold nanoparticles (FIG.
7 panel A right column bottom row) exhibited a brighter normalized
scatter signal than livers from subjects injected with the saline
control (FIG. 7 panel A right column top row).
[0183] Livers were excised from subjects and spatial frequency
heterodyne imaging system was performed. It was observed that
subjects injected with PEG/FB50 antibody conjugated gold
nanoparticles also clearly showed enhanced signal enhancement of
the liver compared to spatial frequency heterodyne images of livers
from control subjects injected with saline only (FIG. 7 panel B).
The spatial frequency heterodyne imaging was also performed in situ
for spleens, kidneys and lungs from subjects, and the liver was the
primary organ to show significant signal enhancement with an
average enhancement of 23.0.+-.14.1%. Enhanced signal enhancements
were observed in both absorption images (FIG. 7 panel B left
images) and spatial frequency heterodyne imaging (FIG. 7 panel B
right images). A greater than a factor of ten signal enhancement
was calculated in the scatter images compared to the absorption
images. Thus, spatial frequency heterodyne imaging effectively
visualized in vivo the size and contours of in vivo organs and
greatly outperformed the results obtained by absorption
imaging.
Example 13
In Vivo Imaging of Tissues in the Body
[0184] Nanoparticles (metal nanoparticles, metal oxide
nanoparticles, and MRI agent nanoparticles) are prepared for in
vivo administering and imaging of tissues including muscle, bone,
cartilage, skin, and blood vessels.
[0185] The nanoparticles are contacted with a polymer such that a
polymer layer coated the nanoparticles. The polymer layer provides
the nanoparticles with a hydrophobic barrier that enhances
non-specific cellular uptake. Alternatively, nanoparticles are
constructed with both a polymer and a binding agent such that the
binding agent extends from the nanoparticle to specifically bind to
molecular antigens present on and in cells and tissues.
[0186] The nanoparticles described herein are stable at a range of
storage temperatures (e.g., room temperature and below freezing)
and are non-toxic to subjects. The nanoparticles are formulated
into compositions and injected into subjects for imaging.
[0187] Absorption imaging and spatial frequency heterodyne imaging
are performed in vivo by directing X-ray radiation to tissues of
subjects injected with nanoparticles. Deflection of incident X-rays
from the primary beam direction are detected in Examples herein by
placing an absorption grid between the sample and the X-ray source.
Fourier transformations are performed on the original X-ray scatter
images to obtain processed images that analyzed by a blind panel of
doctors to allow for histological information of the tissues.
[0188] The samples of the tissues are then excised, fixed and/or
decalcified. Histopathology slides for each tissue are prepared,
and multiple images are acquired using a standard microscope and an
image analysis software. The panel of doctors then uses the images
obtained from the histological slides to identify the type and
histological condition of each of the tissues. Data analyses are
performed (e.g., sensitivity and specificity) to compare the
results obtained from each of the absorption imaging, spatial
frequency heterodyne imaging, and the actual histological analysis.
Data show that spatial frequency heterodyne imaging using metal
nanoparticles, metal oxide nanoparticles, and MRI agent
nanoparticles is much more sensitive in imaging and differentiating
tissue than absorption imaging. Most important, the spatial
frequency heterodyne imaging of tissues injected with the
nanoparticles is as effective in diagnosing tissue conditions as
actual histological analysis.
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