U.S. patent application number 12/593359 was filed with the patent office on 2011-07-07 for in vivo tumor targeting and spectroscopic detection with surface enhanced raman nanoparticle tags.
Invention is credited to Dominic Ansari, Shuming Nie, Ximei Qian.
Application Number | 20110165077 12/593359 |
Document ID | / |
Family ID | 39808725 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110165077 |
Kind Code |
A1 |
Qian; Ximei ; et
al. |
July 7, 2011 |
IN VIVO TUMOR TARGETING AND SPECTROSCOPIC DETECTION WITH SURFACE
ENHANCED RAMAN NANOPARTICLE TAGS
Abstract
Nanostructures, methods of preparing nanostructures, methods of
detecting targets in subjects, and methods of treating diseases in
subjects, are disclosed. An embodiment, among others, of the
nanostructure includes a metallic gold surface-enhanced Raman
scattering nanoparticle, a Raman reporter and a protection
structure. The protection structure may include a
thiol-polyethylene glycol to which may be attached a
target-specific probe.
Inventors: |
Qian; Ximei; (Decatur,
GA) ; Ansari; Dominic; (Decatur, GA) ; Nie;
Shuming; (Atlanta, GA) |
Family ID: |
39808725 |
Appl. No.: |
12/593359 |
Filed: |
April 2, 2008 |
PCT Filed: |
April 2, 2008 |
PCT NO: |
PCT/US08/59117 |
371 Date: |
November 10, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60909656 |
Apr 2, 2007 |
|
|
|
Current U.S.
Class: |
424/9.1 |
Current CPC
Class: |
A61B 2503/40 20130101;
A61K 49/0089 20130101; Y10S 977/81 20130101; Y10S 977/773 20130101;
A61K 49/0041 20130101; B82Y 5/00 20130101; A61B 5/416 20130101;
A61K 49/0032 20130101; G01N 21/658 20130101; A61P 35/00 20180101;
A61K 49/0023 20130101; A61K 49/0065 20130101; A61B 5/0059 20130101;
A61K 49/0093 20130101 |
Class at
Publication: |
424/9.1 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under NIH
Grant No. R01 CA108468 awarded by the U.S. National Institutes of
Health of the United States government. The government has certain
rights in the invention.
Claims
1. A surface-enhanced Raman spectroscopic active composite
nanostructure comprising: a core metallic nanoparticle; a Raman
reporter molecule disposed on the surface of the core; and an
encapsulating protective layer disposed on the surface of the core
and the reporter molecule, wherein the encapsulated reporter
molecule has a measurable surface-enhanced Raman spectroscopic
signature.
2. The nanostructure of claim 1, wherein the Raman reporter
molecule is selected from an isothiocyanate dye, a multi-sulfur
organic dye, a multi-heterosulfur organic dye, a benzotriazole dye,
or combinations thereof.
3. The nanostructure of claim 1, wherein the reporter molecule is
selected from a thiacyanine dye, a dithiacyanine dye, a
thiacarbocyanine dye, or a dithiacarbocyanine dye.
4. The nanostructure of claim 1, wherein the reporter molecule is
selected from malachite green isothiocyanate,
tetramethylrhodamine-5-isothiocyante, X-rhodamine-5-isothiocyanate,
X-rhodamine-6-isothiocyanate, or 3,3'-diethylthiadicarbocyanine
iodide.
5. The nanostructure of claim 3, wherein the core metallic
nanoparticle is gold.
6. The nanostructure of claim 1, wherein the core has a diameter
less than about 200 nanometers.
7. The nanostructure of claim 1, wherein the encapsulating material
is a thiol-polyethylene glycol.
8. The nanostructure of claim 1, further comprising a
target-specific probe selectively binding a target on a cell.
9. The nanostructure of claim 8 wherein the target-specific probe
is selected from the group consisting of an antibody, a
polypeptide, a polynucleotide, a drug molecule, an inhibitor
compound, and a combination thereof, and wherein the targeting
probe has an affinity for a marker on the surface of a target
cell.
10. The nanostructure of claim 9 wherein the target-specific probe
is an immunoglobulin, or a fragment thereof.
11. The nanostructure of claim 8, wherein the probe is disposed on
the hydrophobic protection structure.
12. The nanostructure of claim 8, wherein the probe is a
tumor-targeting ligand.
13. A method of preparing a nanostructure, comprising: providing a
metallic nanoparticle; introducing the metallic nanoparticle to a
Raman reporter, whereupon the Raman reporter is disposed on the
surface of the nanoparticle to form a nanoparticle-reporter
complex; and disposing a protection structure layer on the surface
of the nanoparticle-reporter complex, wherein the reporter molecule
has a measurable surface-enhanced Raman spectroscopic
signature.
14. The method of claim 13, further comprising depositing a cell
target-specific probe to the protection structure layer, wherein
the probe is selected from an antibody, a polypeptide, a
polynucleotide, a drug molecule, an inhibitor compound, or a
combination thereof.
15. The method of claim 13, wherein the core metallic nanoparticles
are a colloid.
16. The method of claim 13, wherein the core metallic nanoparticles
is gold
17. The method of claim 13, wherein the Raman reporter molecule is
selected from an isothiocyanate dye, a multi-sulfur organic dye, a
multi-heterosulfur organic dye, a benzotriazole dye, or
combinations thereof.
18. The method of claim 13, wherein the reporter molecule is
selected from a thiacyanine dye, a dithiacyanine dye, a
thiacarbocyanine dye, or a dithiacarbocyanine dye.
19. The method of claim 13, wherein the reporter molecule is
selected from malachite green isothiocyanate,
tetramethylrhodamine-5-isothiocyante, X-rhodamine-5-isothiocyanate,
X-rhodamine-6-isothiocyanate, or 3,3'-diethylthiadicarbocyanine
iodide.
20. The method of claim 13, wherein the encapsulating material is a
thiol-polyethylene glycol.
21. A method of imaging a biological sample, comprising: delivering
at least one nanostructure to a cultured cell or to an animal or
human subject, wherein the nanostructure comprises a core gold
nanoparticle, a Raman reporter molecule disposed on the surface of
the core, and an encapsulating protective layer disposed over the
core and the reporter molecule, and wherein the encapsulated
reporter molecule has a measurable surface-enhanced Raman
spectroscopic signature; allowing the nanostructure to contact a
targeted biological cell or tissue; exciting the reporter molecule
with a source of radiation; and measuring the surface enhanced
Raman spectroscopy spectrum of the nanostructure corresponding to
the reporter molecule, thereby detecting the presence of the
nanostructure in the targeted cell or tissue.
22. The method of claim 21, wherein the nanostructure further
comprises a target-specific probe, wherein the targeting probe
selectively binds the nanoparticle to a targeted cell, thereby
allowing detection of the targeted cell.
23. The method of claim 22, wherein the target cell is in a tissue
of an animal or human subject.
24. The method of claim 21, wherein the target cell is a cancerous
cell of an animal or human subject.
25. The method of claim 21, wherein the target-specific probe is
selected from the group consisting of an antibody, a polypeptide, a
polynucleotide, a drug molecule, an inhibitor compound, or a
combination thereof, and wherein the targeting probe has an
affinity for a marker on the surface of a target cell.
26. The method of claim 21 wherein the target-specific probe is a
tumor-targeting ligand.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/909,656, entitled "A New Class of
nanoparticle Tags Based on Surface Enhanced Raman Scattering for In
Vitro and In Vivo Detection of Cancer Biomarkers" filed on Apr. 2,
2007, the entirety of which is hereby incorporated by
reference.
TECHNICAL FIELD
[0003] The present disclosure is generally related to
surface-enhanced Raman spectroscopy nanoparticles, and cell
detection uses thereof.
BACKGROUND
[0004] The development of biocompatible nanoparticles for in vivo
molecular imaging and targeted therapy is an area of considerable
current interest across a number of science, engineering and
biomedical disciplines. The basic rationale is that nanometer-sized
particles have functional and structural properties that are not
available from either discrete molecules or bulk materials. When
conjugated with biomolecular targeting ligands such as monoclonal
antibodies, peptides or small molecules, these nanoparticles can be
used to target malignant tumors with high specificity and affinity.
In the `mesoscopic` size range of 10- to 100-nm diameter,
nanoparticles also have large surface areas for conjugating to
multiple diagnostic (e.g., optical, radioisotopic or magnetic) and
therapeutic (e.g., anticancer) agents. Recent advances have led to
the development of biodegradable nanostructures for drug delivery,
iron oxide nanocrystals for magnetic resonance imaging, quantum
dots for multiplexed molecular diagnosis and in vivo imaging, and
nanoscale carriers for short interfering RNA (siRNA) delivery.
[0005] Colloidal gold has been safely used to treat rheumatoid
arthritis for half a century, and recent work indicates the
pegylated gold nanoparticles (colloidal gold coated with a
protective layer of polyethylene glycol or PEG) exhibit excellent
in vivo biodistribution and pharmacokinetic properties upon
systemic injection. In contrast to cadmium-containing quantum dots
and other toxic or immunogenic nanoparticles, gold colloids have
little or no long-term toxicity or other adverse effects in vivo.
The discovery of single-molecule and single-nanoparticle
surface-enhanced Raman scattering (SERS) has attracted considerable
interest, both for fundamental studies of enhancement mechanisms
and for potential applications in ultrasensitive optical detection
and spectroscopy. A number of researchers have shown that the
enhancement factors are as large as 10.sup.14-10.sup.15, leading to
Raman scattering cross sections that are comparable to or even
larger than those of fluorescent organic dyes. This enormous
enhancement allows spectroscopic detection and identification of
single molecules located on the surface of single nanoparticles or
at the junction of two particles at room temperature. Progress has
been made concerning both the structural and mechanistic aspects of
single-molecule SERS, but it is still unclear how this large
enhancement effect might be exploited for applications in
analytical chemistry, molecular biology, or medical diagnostics.
One major problem is the intrinsic interfacial nature of SERS,
which requires the molecules to adsorb on roughened metal surfaces.
For biological molecules such as peptides, proteins, and nucleic
acids, surface-enhanced Raman data are especially difficult to
obtain, hard to interpret, and nearly impossible to reproduce.
SUMMARY
[0006] Embodiments of a new cellular imaging technology based on
ultra-sensitive surface enhanced Raman scattering (SERS)
spectroscopy has been developed as a diagnostic and therapeutic
tool. Embodiments of the present disclosure relates to
spontaneously assembled SERS nanotags with a durable and versatile
protective coat for in vitro and in vivo applications. The image
brightness measurements can show that the SERS nanotags are at
least two orders of magnitude greater than a quantum dot tag.
Bifunctional polyethylene glycol polymers serve as a linker between
the gold nanoparticle core and the targeting or therapeutic agents
attached to the nanostructures.
[0007] Nanoparticles, methods of preparation thereof, and methods
of detecting a target molecule using embodiments of the
nanoparticle, are disclosed. One embodiment of an exemplary
nanoparticle, among others, includes a surface-enhanced Raman
spectroscopic active composite nanostructure. The surface-enhanced
Raman spectroscopic active composite nanostructure includes a core,
a reporter molecule, and an encapsulating material. The reporter
molecule is bonded to the core. The reporter molecule may be
selected from, but is not limited to, an isothiocyanate dye, a
multi-sulfur organic dye, a multi-heterosulfur organic dye, a
benzotriazole dye, and combinations thereof. The encapsulating
material is disposed over the core and the reporter molecule. The
encapsulated reporter molecule has a measurable surface-enhanced
Raman spectroscopic signature.
[0008] Briefly described, embodiments of this disclosure, among
others, encompass nanostructures, methods of preparing
nanostructures, methods of imaging by delivering a nanostructure of
the present disclosure to a specific target on or within a cell,
tissue or whole animal or human. The disclosure encompasses
nanostructures that comprise a metallic nanoparticle core, a Raman
reporter and a protective layer disposed thereon.
[0009] One aspect, therefore, of the disclosure encompasses
surface-enhanced Raman spectroscopic active composite
nanostructures comprising a core metallic nanoparticle, a Raman
reporter molecule disposed on the surface of the core, and an
encapsulating protective layer disposed on the surface of the core
and the reporter molecule, wherein the encapsulated reporter
molecule has a measurable surface-enhanced Raman spectroscopic
signature.
[0010] In embodiments of the disclosure, the Raman reporter
molecule may be selected from an isothiocyanate dye, a multi-sulfur
organic dye, a multi-heterosulfur organic dye, a benzotriazole dye,
and combinations thereof.
[0011] In embodiments of the disclosure, the reporter molecule is
selected from a thiacyanine dye, a dithiacyanine dye, a
thiacarbocyanine dye, and a dithiacarbocyanine dye. In other
embodiments, the reporter molecule is selected from malachite green
isothiocyanate, tetramethylrhodamine-5-isothiocyante,
X-rhodamine-5-isothiocyanate, X-rhodamine-6-isothiocyanate, and
3,3'-diethylthiadicarbocyanine iodide.
[0012] In one embodiment of the disclosure, the core is gold, and
may have a diameter less than about 200 nanometers.
[0013] In embodiments of the nanostructures of the disclosure, the
encapsulating material can be a thiol-polyethylene glycol.
[0014] In other embodiments of the disclosure the nanostructures
may further comprise a target-specific probe capable of selectively
binding a target on a cell.
[0015] In these embodiments, the target-specific probe may be
selected from the group consisting of: an antibody, a polypeptide,
a polynucleotide, a drug molecule, an inhibitor compound, and a
combination thereof, and wherein the targeting probe has an
affinity for at least one marker on the surface of a target
cell.
[0016] In one embodiment, the target-specific probe is an
immunoglobulin, or a fragment thereof and in the embodiments of the
disclosure the probe may be disposed on the hydrophobic protection
structure. In one embodiment, the probe is a tumor-targeting
ligand.
[0017] Another aspect of the disclosure encompasses methods of
preparing a nanostructure according to the disclosure, comprising
providing a gold nanoparticle, introducing the gold nanoparticle to
a Raman reporter, whereupon the Raman reporter is disposed on the
surface of the nanoparticle to form a nanoparticle-reporter
complex, and disposing a protection structure layer on the surface
of the nanoparticle-reporter complex, wherein the reporter molecule
has a measurable surface-enhanced Raman spectroscopic
signature.
[0018] In one embodiment of this aspect of the disclosure, the
methods may further comprise depositing a cell target-specific
probe onto the protection structure layer, wherein the probe is
selected from an antibody, a polypeptide, a polynucleotide, a drug
molecule, an inhibitor compound, or a combination thereof.
[0019] Yet another aspect of the disclosure encompasses methods of
imaging a biological sample, comprising delivering at least one
nanostructure to a cultured cell or to an animal or human subject,
wherein the nanostructure comprises a core metallic, and gold,
nanoparticle, a Raman reporter molecule disposed on the surface of
the core, and an encapsulating protective layer disposed over the
core and the reporter molecule, and wherein the encapsulated
reporter molecule has a measurable surface-enhanced Raman
spectroscopic signature, allowing the nanostructure to contact a
targeted biological cell or tissue, exciting the reporter molecule
with a source of radiation, and measuring the surface enhanced
Raman spectroscopy spectrum of the nanostructure corresponding to
the reporter molecule, thereby detecting the presence of the
nanostructure in the targeted cell or tissue.
[0020] In one embodiment of this aspect of the disclosure, the
nanostructure may further comprise a target-specific probe, wherein
the targeting probe selectively binds the nanoparticle to a
targeted cell, thereby allowing detection of the targeted cell.
[0021] In another embodiment of the disclosure, the target cell is
in a tissue of an animal or human subject.
[0022] In the embodiments of this aspect of the disclosure, the
target cell may be a cancerous cell of an animal or human subject
and the target-specific probe may selected from the group
consisting of an antibody, a polypeptide, a polynucleotide, a drug
molecule, an inhibitor compound, and a combination thereof, and
wherein the targeting probe has an affinity for a marker on the
surface of a target cell.
[0023] In one embodiment of the disclosure, the target-specific
probe is a tumor-targeting ligand.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Further aspects of the present disclosure will be more
readily appreciated upon review of the detailed description of its
various embodiments, described below, when taken in conjunction
with the accompanying drawings.
[0025] FIG. 1A illustrates the order of preparation and schematic
structures of the original gold colloid, a particle encoded with a
Raman reporter, and a particle stabilized with a layer of
thiol-polyethylene glycol (thiol-PEG). Approximately
1.4-1.5.times.10.sup.4 reporter molecules (e.g., malachite green)
are adsorbed on each 60-nm gold particle, which is further
stabilized with 3.0.times.10.sup.4 thiol-PEG molecules
[0026] FIG. 1B illustrates the optical absorptions obtained from
the original, Raman-encoded, and PEG-stabilized gold nanoparticles
shown in FIG. 1A.
[0027] FIG. 1C illustrates the transmission electron microscopy
(TEM) obtained from the original, Raman-encoded, and PEG-stabilized
gold nanoparticles shown in FIG. 1A.
[0028] FIG. 1D illustrates the dynamic light scattering size data
obtained from the original, Raman-encoded, and PEG-stabilized gold
nanoparticles shown in FIG. 1A.
[0029] FIGS. 2A-2F illustrate comparisons of pegylated SERS
nanoparticles and near-infrared-emitting quantum dots in the
spectral region of 650-750 nm.
[0030] FIGS. 2A and 2B show optical absorption and emission spectra
of SERS nanoparticles (FIG. 2A) and QD705 (FIG. 2B) under identical
experimental conditions.
[0031] FIGS. 2C and FIG. 2D show SERS and fluorescence images of
single gold nanoparticles (FIG. 2C) and single quantum dots (FIG.
2D) dispersed on glass slides and acquired under the same
conditions (EM-CCD camera, 633.+-.3 nm excitation, and 655 nm
long-pass emission). The speckles shown in FIG. 2D are optical
interference fringes, which become visible at low light levels.
[0032] FIG. 2E and FIG. 2F show line plots (FIG. 2E) and
statistical analysis (FIG. 2F) of the brightness differences
between SERS nanoparticles and quantum dots. S.D. in the Raman and
quantum dot signals are indicated by error bars.
[0033] FIGS. 3A and 3B illustrate cancer cell targeting and
spectroscopic detection by using antibody-conjugated SERS
nanoparticles.
[0034] FIG. 3A shows the preparation of targeted SERS nanoparticles
by using a mixture of SH-PEG and a hetero-functional PEG
(SH-PEG-COOH). Covalent conjugation of an anti-EGFR-specific scFv
antibody fragment occurs at the exposed terminal of the
hetero-functional PEG.
[0035] FIG. 3B shows SERS spectra obtained from EGFR-positive
cancer cells (Tu686) and from EGFR-negative cancer cells (human
non-small cell lung carcinoma NCI-H520), together with control data
and the standard tag spectrum. All spectra were taken in cell
suspensions with 785-nm laser excitation and were corrected by
subtracting the spectra of nanotag-stained cells by the spectra of
unprocessed cells. The Raman reporter molecule is
diethylthiatricarbocyanine (DTTC), and its distinct spectral
signatures are indicated by wave numbers (cm.sup.-1).
[0036] FIG. 4 illustrates in vivo SERS spectra obtained from
pegylated gold nanoparticles injected into subcutaneous and deep
muscular sites in live animals. The injection sites and laser beam
positions are indicated by circles on the animal.
[0037] FIGS. 5A-5C illustrate in vivo cancer targeting and
surface-enhanced Raman detection by using scFv-antibody conjugated
gold nanoparticles that recognize the tumor biomarker EGFR.
[0038] FIGS. 5A and 5B show SERS spectra obtained from the tumor
and the liver locations by using targeted (FIG. 5A) and nontargeted
(FIG. 5B) nanoparticles. Two nude mice bearing human head-and-neck
squamous cell carcinoma (Tu686) xenograft tumor (3-mm diameter)
received 90 .mu.l of scFv EGFR-conjugated SERS tags or pegylated
SERS tags (460 pM). The particles were administered via tail vein
single injection. SERS spectra were taken 5 hrs after
injection.
[0039] FIG. 5C shows photographs showing a laser beam focusing on
the tumor site or on the anatomical location of liver. In vivo SERS
spectra were obtained from the tumor site and the liver site with
2-s signal integration and at 785 nm excitation. The spectra were
background subtracted and shifted for better visualization. The
Raman reporter molecule is malachite green, with distinct spectral
signatures as labeled in FIGS. 5A and 5B. The laser power is about
20 mW.
[0040] FIG. 6 illustrates biodistribution data of targeted and
nontargeted gold nanoparticles in major organs at 5 hrs after
injection as measured by inductively coupled plasma-mass
spectrometry (ICP-MS). Note the difference in tumor accumulation
between the targeted and nontargeted nanoparticles. The s.d. error
bars were calculated based on four animals (n=4) in each study
group.
[0041] FIGS. 7A and 7B illustrate a stability comparison of
uncoated (left column) and PEG-SH coated (right column) Au-MGITC
complexes. Top panels are UV-vis absorption spectra of uncoated
(left) and coated (right) Au-MGITC in water (solid curves) and PBS
(dashed curves); middle panels are TEM images of uncoated (left)
and coated (right) Au-MGITC in PBS; bottom panels are the DLS size
distributions of uncoated (left) and coated (right) Au-MGITC in
PBS. MGITC is the abbreviation for malachite green isothiocyanate
(ITC).
[0042] FIG. 8 illustrates SERS spectra and correlated surface
plasmon imaging of single cancer cells. Upper panels: Reflective
mode dark-field images of live Tu686 cells (EGFR positive) and H520
6 cells (EGFR negative) tagged with scFv-conjugated gold
nanoparticles. The images were acquired with Olympus Q-Color 5 CCD
camera at an exposure time of 250 milliseconds. Lower panels: SERS
spectra obtained from single cells as indicated by arrows. The
Raman reporter dye was diethylthiatricarbocyanine (DTTC).
[0043] FIG. 9 illustrates a comparison of in-vivo distribution and
tumor uptake data for plain PEG-coated nanoparticles and
PEG-nanotags that are conjugated with a size-matched nonspecific
protein (27-KD recombinant GFP). The data were obtained at 5 hours
post injection by inductively coupled plasma-mass spectrometric
(ICP-MS) analysis of elemental gold.
[0044] FIG. 10 illustrates transmission electron micrographs
showing tumor uptake of EGFR-targeted gold nanoparticles, their
clustering and localization in intracellular organelles such as
endosomes. The inset is an expanded view of gold nanoparticles in
an organelle. Nu refers to cell nucleus.
[0045] FIG. 11 illustrates transmission electron micrographs
showing nonspecific uptake of gold nanoparticles by liver Kuffper
cells showing primarily single gold nanoparticles localized in
early- and late-stage endosomes (indicated by arrows).
[0046] FIG. 12 shows a schematic diagram of pegylated SERS
nanoparticles involved in active and passive tumor targeting. Both
the control and targeted nanoparticles can accumulate in tumors
through the EPR effect (enhanced permeability and retention
effect), but only the targeted nanoparticles can recognize
EGFR-positive cancer cells and rapidly enter these cells by
receptor-mediated endocytosis.
[0047] FIG. 13 compares the photostability of an SERS nanostructure
of the disclosure and the quantum dot QD705.
[0048] FIG. 14 illustrates the intensity of the SERS signal versus
the number of thiol-polyethylene glycols attached to a 60 nm gold
surface.
[0049] FIG. 15 illustrates the `lock-out effect` of encapsulating
the gold nanoparticle with a PEG-SH layer. (i) without PEG-SH
coating; (ii) 30,000 PEG-SH per nanoparticle; (iii) 300,000 PEG-SH
per nanoparticle; and (iv) PEG-SH attached before adding
reporter-dye locked out from nanoparticle.
[0050] FIG. 16 illustrates that a PEG coating prevents cross-talk
between a reporter molecule attached to the nanoparticle and a dye
on the outer surface of the PEG layer. (a) Au-MGITC alone; (b)
Au-RBITC alone; (c) RBITC locked out; and (d) 2 dyes co-absorbed on
the nanoparticle.
[0051] FIG. 17 illustrates the long-term stability of PEG coated
particles.
[0052] FIG. 18 illustrates SERS spectra of Au-MGITC-PEG-SH
redispersed in (panel a) pure water, (panel b) 10.times.PBS, (panel
c) pH 12 aqueous solution, (panel d) pH 2 aqueous solution, (panel
e) ethanol, (panel f) methanol, (panel g) DMSO, then transferred
back to water. The reporter dye is malachite green isothiocyanate
(MGITC), with distinct spectral signatures as labeled. Excitation
wavelength: 633 nm; laser power: 5 mW.
[0053] The details of some exemplary embodiments of the methods and
systems of the present disclosure are set forth in the description
below. Other features, objects, and advantages of the disclosure
will be apparent to one of skill in the art upon examination of the
following description, drawings, examples and claims. It is
intended that all such additional systems, methods, features, and
advantages be included within this description, be within the scope
of the present disclosure, and be protected by the accompanying
claims.
DETAILED DESCRIPTION
[0054] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, and as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
disclosure will be limited only by the appended claims.
[0055] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the disclosure.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the disclosure, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the disclosure.
[0056] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0057] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present disclosure
is not entitled to antedate such publication by virtue of prior
disclosure. Further, the dates of publication provided could be
different from the actual publication dates that may need to be
independently confirmed.
[0058] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0059] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of medicine, organic chemistry,
biochemistry, molecular biology, pharmacology, and the like, which
are within the skill of the art. Such techniques are explained
fully in the literature.
[0060] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a support" includes a plurality of
supports. In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings unless a contrary intention is
apparent.
[0061] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise. In this disclosure,
"comprises," "comprising," "containing" and "having" and the like
can have the meaning ascribed to them in U.S. patent law and can
mean "includes," "including," and the like; "consisting essentially
of" or "consists essentially" or the like, when applied to methods
and compositions encompassed by the present disclosure refers to
compositions like those disclosed herein, but which may contain
additional structural groups, composition components or method
steps (or analogs or derivatives thereof as discussed above). Such
additional structural groups, composition components or method
steps, etc., however, do not materially affect the basic and novel
characteristic(s) of the compositions or methods, compared to those
of the corresponding compositions or methods disclosed herein.
"Consisting essentially of" or "consists essentially" or the like,
when applied to methods and compositions encompassed by the present
disclosure have the meaning ascribed in U.S. patent law and the
term is open-ended, allowing for the presence of more than that
which is recited so long as basic or novel characteristics of that
which is recited is not changed by the presence of more than that
which is recited, but excludes prior art embodiments.
[0062] Prior to describing the various embodiments, the following
definitions are provided and should be used unless otherwise
indicated.
Definitions
[0063] The term "Raman light scattering" as used herein refers to
when certain molecules are illuminated, a small percentage of the
molecules which have retained a photon do not return to their
original vibrational level after remitting the retained photon, but
drop to a different vibrational level of the ground electronic
state. The radiation emitted from these molecules will therefore be
at a different energy and hence a different wavelength. This is
referred to as Raman scattering.
[0064] If the molecule drops to a higher vibrational level of the
ground electronic state, the photon emitted is at a lower energy or
longer wavelength than that absorbed. This is referred to as
Stokes-shifted Raman scattering. If a molecule is already at a
higher vibrational state before it absorbs a photon, it can impart
this extra energy to the remitted photon thereby returning to the
ground state. In this case, the radiation emitted is of higher
energy (and shorter wavelength) and is called anti-Stokes-shifted
Raman scattering. In any set of molecules under normal conditions,
the number of molecules at ground state is always much greater than
those at an excited state, so the odds of an incident photon
interacting with an excited molecule and being scattered with more
energy than it carried upon collision is very small. Therefore,
photon scattering at frequencies higher than that of the incident
photons (anti-Stokes frequencies) is minor relative to that at
frequencies lower than that of the incident photons (Stokes
frequencies). Consequently, it is the Stokes frequencies that are
usually analyzed.
[0065] The term "surface enhanced Raman scattering (SERS)" as used
herein refers to a significant increase in the intensity of Raman
light scattering that can be observed when molecules are brought
into close proximity to (but not necessarily in contact with)
certain metal surfaces. The metal surfaces need to be "roughened"
or coated with minute metal particles.
[0066] Metal colloids also show this signal enhancement effect. The
increase in intensity can be on the order of several million-fold
or more. The cause of the SERS effect is not completely understood;
however, current thinking envisions at least two separate factors
contributing to SERS. First, the metal surface contains minute
irregularities. These irregularities can be thought of as spheres
(in a colloid, they are spheroidal or nearly so). Those particles
with diameters of approximately 1/10th the wavelength of the
incident light were considered to contribute most to the effect.
The incident photons induce a field across the particles which,
being metal, have very mobile electrons.
[0067] In certain configurations of metal surfaces or particles,
groups of surface electrons can be made to oscillate in a
collective fashion in response to an applied oscillating
electromagnetic field. Such a group of collectively oscillating
electrons is called a "plasmon." The incident photons supply this
oscillating electromagnetic field. The induction of an oscillating
dipole moment in a molecule by incident light is the source of the
Raman scattering. The effect of the resonant oscillation of the
surface plasmons is to cause a large increase in the
electromagnetic field strength in the vicinity of the metal
surface. This results in an enhancement of the oscillating dipole
induced in the scattering molecule and hence increases the
intensity of the Raman scattered light. The effect is to increase
the apparent intensity of the incident light in the vicinity of the
particles.
[0068] A second factor considered to contribute to the SERS effect
is molecular imaging. A molecule with a dipole moment, which is in
close proximity to a metallic surface, will induce an image of
itself on that surface of opposite polarity (i.e., a "shadow"
dipole on the plasmon). The proximity of that image is thought to
enhance the power of the molecules to scatter light. This coupling
of a molecule may have an induced or distorted dipole moment to the
surface plasmons greatly enhances the excitation probability. The
result is a very large increase in the efficiency of Raman light
scattered by the surface-absorbed molecules.
[0069] The SERS effect can be enhanced through combination with the
resonance Raman effect. The surface-enhanced Raman scattering
effect is even more intense if the frequency of the excitation
light is in resonance with a major absorption band of the molecule
being illuminated. The resultant Surface Enhanced Resonance Raman
Scattering (SERRS) effect can result in an enhancement in the
intensity of the Raman scattering signal of seven orders of
magnitude or more.
[0070] The term "Raman reporter" as used herein can refer to small
organic compounds such as thiophenol, mercaptobenzoic acid, and
bispyridine previously used as Raman spectroscopic reporters. These
molecules give rise to simple Raman spectra, but it has been
difficult or impossible to achieve resonance Raman enhancement at
visible excitation wavelengths. As a result, the reported SERS
intensities are relatively low, even at high (millimolar) reporter
concentrations. Organic dyes with an isothiocyanate
(--N.dbd.C.dbd.S) group or with multiple sulfur atoms adsorb
strongly on the core particles and may be compatible with
encapsulation. For example, intense SERS spectra have been obtained
from (b) malachite green isothiocyanate (MGITC), (c)
tetramethylrhodamine-5-isothiocyanate TRITC), (d)
X-rhodamine-5-(and-6)-isothiocyanate (XRITC), and (a)
3,3'-diethylthiadicarbocyanine iodide (DTDC). Three of these
molecules contain an isothiocyanate group, while the fourth has two
sulfur atoms in ring structures.
[0071] The isothiocyanate group or sulfur atoms provide an
"affinity tag" for binding to gold surfaces, yielding a sulfur-gold
bond that is stable. For molecules without such an affinity tag
such as crystal violet and rhodamine 6G, intense SERS spectra may
be observed, but the signals disappeared after, for example, silica
coating. In addition, most of these dyes have strong electronic
transitions in the visible spectrum, so resonance Raman enhancement
can be used to further increase the signal intensities. In a strict
sense, these molecules should be called "resonant Raman reporters,"
to distinguish them from thiophenol and other nonresonant Raman
reporters. In most cases, resonance Raman provides about 2-3 orders
of magnitude of additional enhancement relative to surface
enhancement alone. Both fluorescent and nonfluorescent dyes can be
used as resonant Raman reporters because fluorescence emission is
efficiently quenched by the gold particles, not interfering with
Raman measurement. A series of benzotriazole dyes are excellent for
surface-enhanced resonance Raman scattering; due to the presence of
multiple nitrogen atoms, these molecules could provide a new class
of resonant Raman reporters for spectroscopic encoding and
multiplexing applications.
[0072] The term "protective layer" as used herein refers to a layer
that may totally or partially encapsulate a nanoparticle, thereby
preventing aggregation of the particles. The biocompatible layer
may comprise, but is not limited to, a thiol-polyethylene glycol
polymer, wherein the thiol group links the polymer to the
underlying nanoparticle. The distal end of the polymer may have a
reactive group to which a target-specific ligand may be coupled.
The protective layer may be disposed, i.e., located or deposited on
or around, in whole or in part, the surface of the metallic
nanoparticle and reporter nanostructure.
[0073] The term "quantum dot" (QDs) as used herein refers to
semiconductor nanocrystals or artificial atoms, which are
semiconductor crystals that contain anywhere between 100 to 1,000
electrons and range from about 2-10 nm. Some QDs can be between
about 10-20 nm in diameter. QDs have high quantum yields, which
makes them particularly useful for optical applications. QDs are
fluorophores that fluoresce by forming excitons, which can be
thought of the excited state of traditional fluorophores, but have
much longer lifetimes of up to 200 nanoseconds. This property
provides QDs with low photobleaching.
[0074] The terms "polypeptide" or "protein" as used herein are
intended to encompass a protein, a glycoprotein, a polypeptide, a
peptide, and the like, whether isolated from nature, of viral,
bacterial, plant, or animal (e.g., mammalian, such as human)
origin, or synthetic, and fragments thereof. A preferred protein or
fragment thereof includes, but is not limited to, an antigen, an
epitope of an antigen, an antibody, or an antigenically reactive
fragment of an antibody.
[0075] The term "nucleic acid" as used herein refers to DNA and
RNA, whether isolated from nature, of viral, bacterial, plant or
animal (e.g., mammalian, such as human) origin, synthetic,
single-stranded, double-stranded, comprising naturally or
non-naturally occurring nucleotides, or chemically modified.
[0076] The term "cancer", as used herein shall be given its
ordinary meaning and is a general term for diseases in which
abnormal cells divide without control. Cancer cells can invade
nearby tissues and can spread through the bloodstream and lymphatic
system to other parts of the body.
[0077] There are several main types of cancer, for example,
carcinoma is cancer that begins in the skin or in tissues that line
or cover internal organs. Sarcoma is cancer that begins in bone,
cartilage, fat, muscle, blood vessels, or other connective or
supportive tissue. Leukemia is cancer that starts in blood-forming
tissue such as the bone marrow, and causes large numbers of
abnormal blood cells to be produced and enter the bloodstream.
Lymphoma is cancer that begins in the cells of the immune
system.
[0078] When normal cells lose their ability to behave as a
specified, controlled and coordinated unit, a tumor is formed.
Generally, a solid tumor is an abnormal mass of tissue that usually
does not contain cysts or liquid areas (some brain tumors do have
cysts and central necrotic areas filled with liquid). A single
tumor may even have different populations of cells within it with
differing processes that have gone awry. Solid tumors may be benign
(not cancerous), or malignant (cancerous). Different types of solid
tumors are named for the type of cells that form them. Examples of
solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias
(cancers of the blood) generally do not form solid tumors.
[0079] Representative cancers include, but are not limited to,
bladder cancer, breast cancer, colorectal cancer, endometrial
cancer, head & neck cancer, leukemia, lung cancer, lymphoma,
melanoma, non-small-cell lung cancer, ovarian cancer, prostate
cancer, testicular cancer, uterine cancer, cervical cancer.
[0080] Cardiovascular disease, as used herein, shall be given its
ordinary meaning, and includes, but is not limited to, high blood
pressure, diabetes, coronary artery disease, valvular heart
disease, congenital heart disease, arrthymia, cardiomyopathy, CHF,
atherosclerosis, inflamed or unstable plaque associated conditions,
restinosis, infarction, thromboses, post-operative coagulative
disorders, and stroke.
[0081] Inflammatory disease, as used herein, shall be given its
ordinary meaning, and can include, but is not limited to,
autoimmune diseases such as arthritis, rheumatoid arthritis,
multiple sclerosis, systemic lupus erythematosus, other diseases
such as asthma, psoriasis, inflammatory bowel syndrome,
neurological degenerative diseases such as Alzheimer's disease,
Parkinson's disease, Huntington's disease, vascular dementia, and
other pathological conditions such as epilepsy, migraines, stroke
and trauma.
DESCRIPTION
[0082] In accordance with the purpose(s) of the present disclosure,
as embodied and broadly described herein, embodiments of the
present disclosure encompass surface-enhanced Raman spectroscopic
(SERS) active composite nanostructures, methods of fabricating
these nanostructures, and methods of using these nanostructures.
The SERS active composite nanostructures are distinguishable and
can be individually detected. In this regard, the SERS active
composite nanostructures can be modified so that the SERS active
composite nanostructures interact with certain target molecules,
which allow detection of the target molecules. In addition, the
SERS active composite nanostructures can be used in encoding
systems as well as in multiplexing systems. The SERS active
composite nanostructures can be used in many areas such as, but not
limited to, flow cytometry, chemical array systems, biomolecule
array systems, biosensing, biolabeling, high-speed screening, gene
expression studies, protein studies, medical diagnostics,
diagnostic libraries, and microfluidic systems.
[0083] The SERS active composite nanostructures provided by the
present disclosure include, but are not limited to, a core, a
reporter molecule disposed thereon, and an encapsulant protective
material or layer. In an embodiment, the core material is a metal.
In an embodiment the core is gold or silver. In an embodiment the
core is gold. The reporter molecules may be disposed (bonded) onto
the core, while the encapsulant material covers and protects the
core and reporter molecules. On the hydrophilic protective surface
of SERS nanostructures according to the present disclosure, there
may be a large number of functional groups that may be derivatized
and may allow the attachment of both diagnostic and therapeutic
agents or target-specific probes. With small-molecule ligands such
as synthetic organic molecules, short oligonucleotides and
peptides, many copies of the same ligand can be linked to single
nanoparticles, leading to multivalent SERS-nanoparticle-target
binding.
[0084] Such nanoparticles may each comprise a SES-active metal
nanoparticle, a submonolayer, monolayer, or multilayer of
spectroscopy-active species in close proximity to the metal
surface, and an encapsulating protective shell. This places the
spectroscopy-active molecule (the "reporter") at the interface
between the metal nanoparticle and the encapsulant. In a typical
and advantageous embodiment, a SERS nanostructure comprises (i) a
metal nanoparticle core (e.g., gold or silver), (ii) a Raman-active
reporter, that gives a unique vibrational signature, and (iii)
protective encapsulant that "locks" the reporter molecules in place
while also providing a highly biocompatible surface. The protective
coating, which is essentially SERS-inactive, also stabilizes the
particles against aggregation and prevents competitive adsorption
of unwanted species.
[0085] Although not intending to be bound by theory, the core
optically enhances the SERS spectrum, while the reporter molecule
provides a distinct spectroscopic SERS signature. Disposing the
encapsulant material over the core and reporter molecule does not
substantially impact the spectroscopic SERS signature of the
reporter molecule, while protecting the core and reporter
molecules. Unlike other SERS particles, the SERS active composite
nanostructure described in the present disclosure have strong SERS
intensities (more than about 10,000 counts with 1 mW laser power in
about a second). In some embodiments, the SERS active composite
nanostructure have measurable surface-enhanced resonance Raman
spectroscopic signatures.
[0086] The class of core-shell colloidal nanoparticles (e.g., SERS
active composite nanostructures) that are highly efficient for SERS
and herein disclosed are suitable for multiplexed detection and
spectroscopy at the single-particle level. With nearly optimized
gold cores and protective shells, the SERS active composite
nanostructures of this disclosure are stable in both aqueous
electrolytes and organic solvents, and yield intense
single-particle SERS spectra. Blinking or intensity fluctuation is
still observed, indicating that the SERS signals could arise from
single molecules at the interface between the core and the shell. A
surprising finding is that organic dyes with an isothiocyanate
(--N.dbd.C.dbd.S) group or multiple sulfur atoms are compatible
with the encapsulation process, and are an excellent group of Raman
reporters due to their rich vibrational spectra and the possibility
of combined surface enhancement and resonance enhancement.
[0087] In contrast to most previous SERS studies, the surface
enhanced Raman signals reported here do not come from the target
molecules, but from a reporter dye that is embedded in the SERS
active composite nanostructures. This design avoids the problems
of, among other things, surface adsorption, substrate variations,
and poor data reproducibility. This development has opened new
possibilities in using SERS for spectroscopic labeling of multiple
biomarkers in single cells and tissue specimens, including
Raman-activated flow cytometry and cell sorting. In comparison with
other biolabels such as fluorescent dyes and semiconductor quantum
dots, SERS active composite nanostructures contain a built-in
mechanism for signal enhancement and provide rich spectroscopic
information in ambient conditions. Furthermore, the extremely short
lifetimes of Raman scattering prevent photobleaching, energy
transfer, or quenching in the excited state.
[0088] The nanoparticle core may be a metallic nanoparticle known
in the art. As used herein, the term "nanoparticle",
"nanostructure", "nanocrystal", "nanotag," and "nanocomponent" are
used interchangeably to refer to a metallic particle with or
without additional layers such as an encapsulating protective
layer, having one dimension from about 1 nm to 1000 nm, including
any integer value between about 1 nm and 1000 nm. In some
embodiments, the metal nanoparticle core can be a spherical or
nearly spherical particle of about 20-200 nm in diameter. In some
embodiments the range is about 2 nm to 50 nm, in some embodiments
in the range of about 20 nm to 50 nm. Anisotropic nanoparticles may
have a length and a width. In some embodiments, the length of an
anisotropic nanoparticle is the dimension parallel to the aperture
in which the nanoparticle was produced. In the case of anisotropic
nanoparticles, in some embodiments, the nanoparticle can have a
diameter (width) of about 350 nm or less. In other embodiments, the
nanoparticle can have a diameter of about 250 nm or less and in
some embodiments, a diameter of about 100 nm or less. In some
embodiments, the width can be about 15 nm to 300 nm. In some
embodiments, the nanoparticle can have a length of about 10-350
nm.
[0089] Nanoparticles may be isotropic or anisotropic. Nanoparticles
include colloidal metal hollow or filled nanobars, magnetic,
paramagnetic, conductive or insulating nanoparticles, synthetic
particles, hydrogels (colloids or bars), and the like. It will be
appreciated by one of ordinary skill in the art that nanoparticles
can exist in a variety of shapes, including, but not limited to,
spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes,
nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped
nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped
nanoparticles, prism-shaped nanoparticles, and a plurality of other
geometric and non-geometric shapes.
[0090] The reporter molecule can include molecules such as, but not
limited to, organic dye molecules having an isothiocyanate group
(hereinafter "isothiocyanate dyes"), organic dye molecules having
two or more sulfur atoms (hereinafter "multi-sulfur organic dyes"),
organic dye molecules having two or more heterocyclic rings each
incorporating sulfur atoms (hereinafter "multi-heterosulfur organic
dyes"), and benzotriazole dyes. In addition, the reporter molecule
may include resonant Raman reporters, which have strong electronic
transitions in the visible spectrum, so that resonance Raman
enhancement can be used to further amplify the signal intensities.
The resonant Raman reporters include, but are not limited to,
organic dyes, biomolecules, porphyrins, and metalloporphyrins. In
particular, the resonant Raman reporters can include, but are not
limited to, malachite green isothiocyanate,
tetramethylrhodamine-5-isothiocyante, X-rhodamine-5-isothiocyanate,
X-rhodamine-6-isothiocyanate, 3,3'-diethylthiadicarbocyanine
iodide, and combinations thereof. A particularly advantageous
reporter molecule is malachite green.
[0091] Further, the reporter molecule can include, but is not
limited to, thiacyanine dyes, dithiacyanine dyes, thiacarbocyanine
dyes (e.g., thiacarbocyanine dyes, thiadicarbocyanine dyes, and
thiatricarbocyanine dyes), and dithiacarbocyanine dyes (e.g.,
dithiacarbocyanine dyes, dithiadicarbocyanine dyes, and
dithiatricarbocyanine dyes), and combinations thereof.
[0092] Furthermore, the reporter molecule can include:
3,3'-diethyl-9-methylthiacarbocyanine iodide; 1,1'-diethyl-2,2'
quinotricarbocyanine iodide; 3,3'-diethylthiacyanine iodide;
4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid,
disodium salt; benzophenone-4-isothiocyanate;
4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid, disodium
salt; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, disodium
salt; N-(4-(6-dimethylamino-2-benzofuranyl)phenylisothiocyanate;
7-dimethylamino-4-methylcoumarin-3-isothiocyanate;
eosin-5-isothiocyanate; erythrosin-5-isothiocyanate;
fluorescein-5-isothiocyanate;
(S)-1-p-isothiocyanatobenzyldiethylenetriaminepentaacetic acid;
Oregon Green.RTM. 488 isothiocyanate;
tetramethylrhodamine-5-isothiocyanate;
tetramethylrhodamine-6-isothiocyanate;
tetramethylrhodamine-5-(and-6)-isothiocyanate;
X-rhodamine-5-(and-6)-isothiocyanate, and combinations thereof.
[0093] The benzotriazole dyes can include, but are not limited to,
azobenzotriazoyl-3,5-dimethoxyphenylamine, and
dimethoxy-4-(6'-azobenzotriazolyl)phenol.
[0094] As mentioned above, the reporter molecules can have an
isothiocyanate group or two or more sulfur atoms (e.g.,
isothiocyanate dyes, multi-sulfur organic dyes, and
multi-heterosulfur organic dyes) that are capable of forming
sulfur-gold bonds that are stable against deposition of the
coupling agent and the encapsulant material. In addition, these
reporter molecules have strong electronic transitions in the
visible and near-infrared spectra (about 400-850 nm), so that
resonance Raman enhancement can be used to increase signal
intensity.
[0095] The SERS active composite nanostructure advantageously may
have a spherical diameter or substantially spherical diameter of
less than about 250 nanometers (nm), about 10 to 150 nm, and about
30 to 90 nm. The core diameter can be about 10 to 200 nm, about 20
to 100 nm, and about 40 to 80 nm. The encapsulant thickness can be
about 1 to 50 nm, about 2 to 50 nm, and about 5 to 10 nm. In
general, the greater the encapsulant diameter, the better the
protection that is provided. With increased diameter, however, the
overall size of the SERS active composite nanostructure increases.
Selection of the appropriate dimensions can be determined based on
the particular application.
[0096] In general, the reporter molecule can cover about 1 to 75%
of the surface of the core (e.g., the reporter molecule adsorbs
onto about 1 to 75% of the core particle surface), about 15 to 50%
of the surface of the core 12, about 15 to 30% of the surface of
the core 12, and about 20 to 25% of the surface of the core 12.
[0097] In embodiments including coupling agents, the coupling agent
can cover about 1 to 100% of the surface of the core, about 40 to
60% of the surface of the core 12, and about 45 to 50% of the
surface of the core. In an embodiment the reporter molecule can
cover about 1 to 75% of the surface of the core, about 15 to 50% of
the surface of the core 12, about 15 to 30% of the surface of the
core 12, and about 20 to 25% of the surface of the core.
[0098] The SERS active composite nanostructure can be prepared in
one or more ways. For example, the SERS active composite
nanostructure can be prepared by mixing the core with the reporter
molecule under conditions such that the reporter molecule bonds to
the core. In particular, the core may be mixed with reporter
molecules having a concentration from about 2.5.times.10.sup.-8 M
to 1.25.times.10.sup.-7 M and about 7.5.times.10.sup.-8 M for about
1 to 30 minutes. Then, in one embodiment, a coupling agent is mixed
with the core having reporter molecules disposed thereon. In
particular, the coupling agent may be added to a final
concentration of about 2.5.times.10.sup.-7 M for about 1 to 30
minutes. Subsequently, the core having reporter molecules disposed
thereon (and in some embodiments having coupling agents disposed
thereon) may be mixed with the encapsulating material at a pH of
about 9 to 11 for about 24 to 96 hours. Additional details
regarding the preparation of the SERS active composite
nanostructure are described in the examples presented herein.
[0099] The present disclosure encompasses the use of a protective
capsule disposed on the surface of the core-Raman reporter complex.
It is contemplated that a variety of materials may be used to
encapsulate the core-reporter. Most advantageously, the protective
layer comprises a thiol-polyethylene glycol, whereby the polymer is
coupled to the underlying core by means of the thiol group. The
distal end of the polymer may comprise an active group such as, but
not limited to, a carboxyl or amine group that may form a coupling
to a target-specific entity such as, but not limited to an
immunoglobulin or a fragment thereof.
[0100] The SERS active composite nanostructure can be attached to a
probe molecule. The SERS active composite nanostructure can also be
attached to a structure (e.g., in an assay) or float freely (e.g.,
in a microfluidic system or in flow cytometry). The probe molecule
can be any molecule capable of being linked to the SERS active
composite nanostructure either directly, or indirectly via a
linker. For example, the target-specific probe may be attached to
the protective encapsulating material such as thiol-polyethylene
glycol. The probe molecule can be attached to the SERS active
composite nanostructure by a stable physical and/or chemical
association.
[0101] The advantageous target-specific probe molecules
contemplated for use in the embodiments of the present disclosure
may have an affinity for one or more target molecules for which
detection is desired. If, for example, the target molecule is a
nucleic acid sequence, the probe molecule should be chosen so as to
be substantially complementary to the target molecule sequence,
such that the hybridization of the target and the probe occurs. The
term "substantially complementary," means that the probe molecules
are sufficiently complementary to the target sequences to hybridize
under the selected reaction conditions.
[0102] In one embodiment, the probe molecule has an affinity for
one or more target molecules (e.g., cancer cell) for which
detection (e.g., determining the presence of and/or proximal
position within the vessel (body)) is desired. If, for example, the
target molecule is a nucleic acid sequence, the probe molecule
should be chosen so as to be substantially complementary to the
target molecule sequence, such that the hybridization of the target
and the probe occurs. The term "substantially complementary," means
that the probe molecules are sufficiently complementary to the
target sequences to hybridize under the selected reaction
conditions.
[0103] The probe molecule and the target molecule can include, but
are not limited to, polypeptides (e.g., protein such as, but not
limited to, an antibody (monoclonal or polyclonal)), nucleic acids
(both monomeric and oligomeric), polysaccharides, sugars, fatty
acids, steroids, purines, pyrimidines, drugs (e.g., small compound
drugs), ligands, or combinations thereof. Advantageously, the probe
may be an antibody or a ligand compatible with, and capable of
binding to, a target molecule on the surface of a cell such as, but
not limited to, a cancer cell.
[0104] The nanostructures of the disclosure can include at least
two different types of probes, each being, for example, a targeting
probe that targets certain cells.
[0105] The present disclosure provides methods of targeting one or
more target cells in a sample or a subject (e.g., mammal, human,
cat, dog, horse, etc.). For example, the nanostructure can be used
to detect tumor cells in an animal using the nanostructures
according to the present disclosure.
[0106] It should also be noted that nanostructures could be used
for the detection of, as part of treatment of (e.g., drug
delivery), as an indication of delivery to one or more targets
(e.g., cancers), or combinations thereof, conditions and/or
diseases such as, but not limited to, cancers, tumors, neoplastic
diseases, autoimmune diseases, inflammatory diseases, metabolic
conditions, neurological and neurodegenerative diseases, viral
diseases, dermatological diseases, cardiovascular diseases, an
infectious disease, and combinations thereof.
[0107] It should be noted that a cell can be pre-labeled (e.g., in
vitro and in vivo) with nanostructures and/or microstructures. For
example, cells can be labeled with nanoparticle-block copolymer
microstructures in vitro through immunostaining, adsorption,
microinjection, cell uptake, and the like. The cells then can be
monitored in vitro, or traced in vivo with the nanoparticles as a
tracer, fluorescence, magnetic, combinations thereof, and the like,
while the expression of a gene may be modified by a probe attached
to the outer surface of the SERS nanostructures.
[0108] The present disclosure provides a method of detecting one or
more target molecules in a sample. The method includes attaching a
target molecule (e.g., via a target-specific probe molecule) to the
nanostructure and measuring the SERS spectrum of the nanostructure,
where the detection of SERS spectrum specific for the reporter
molecule indicates the presence of the target molecule specific for
the probe molecule. The SERS active composite nanostructure can be
used to detect the presence of one or more target molecules in
chemical array systems and biomolecular array systems. In addition,
SERS active composite nanostructures can be used to enhance
encoding and multiplexing capabilities in various types of
systems.
[0109] In one embodiment, a flow cytometer can be used in
multiplexed assay procedures for detecting one or more target
molecules using one or more SERS active composite nanostructure.
Flow cytometry is an optical technique that analyzes particular
particles (e.g., SERS active composite nanostructures) in a fluid
mixture based on the particles' optical characteristics. Flow
cytometers hydrodynamically focus a fluid suspension of SERS active
composite nanostructures into a thin stream so that the SERS active
composite nanostructures flow down the stream in substantially
single file and pass through an examination zone. A focused light
beam, such as a laser beam, illuminates the SERS active composite
nanostructures as they flow through the examination zone. Optical
detectors within the flow cytometer measure certain characteristics
of the light as it interacts with the SERS active composite
nanostructures. Commonly used flow cytometers can measure SERS
active composite nanostructure emission at one or more
wavelengths.
[0110] One or more target molecules can be detected using a SERS
active composite nanostructure and one or more probes having an
affinity for one or more of the target molecules. Each SERS active
composite nanostructure has a reporter molecule that corresponds to
the probe. Prior to being introduced to the flow cytometer, the
SERS active composite nanostructures specific for certain target
molecules are mixed with a sample that may include one or more
target molecules. The SERS active composite nanostructures interact
with (e.g., bond or hybridize) the corresponding target molecules
for which the probe has an affinity.
[0111] Next, the SERS active composite nanostructures are
introduced to the flow cytometer. As discussed above, the flow
cytometer is capable of detecting the SERS active composite
nanostructure after exposure to a first energy. Detection of a
certain Raman spectrum corresponding to a certain reporter molecule
indicates that a target molecule is present in the sample.
[0112] Images of cells containing Raman spectral information can be
obtained by a number of methods. A microscope can be coupled to a
CCD camera such that complete images of the object may be obtained.
Then, between the sample and the camera, a wavenumber filtering
device such as a monochromator or liquid crystal tunable filter is
inserted. The filtering device only allows a narrow bandwidth of
the scattered radiation to reach the camera at any one time.
Multiple images are collected, each covering a small spectral range
of the scattered radiation. The spectra from each point in the
image are assembled in software. At the other extreme, light from a
single point of an image may be dispersed through a monochromator
and the complete spectrum of that point can be acquired on an array
detector. The object is then scanned such that each point in the
image is acquired separately. The Raman image is then assembled in
software. In another approach, a line scan instrument can be
constructed that excites the sample with a line of radiation. The
line is imaged spatially along one axis of a CCD camera while
simultaneously being spectrally dispersed along the orthogonal
axis. Each readout of the camera acquires the complete spectrum of
each spatial pixel in the line. To complete the image the line is
scanned across the sample.
[0113] Thus, according to this disclosure, cells or cell
populations may be identified by using an antibody-conjugated SERS
nanostructure prepared with an antibody that may bind a cell
surface antigenic receptor expressed on a cell subpopulation.
[0114] SERS nanostructures according to the present disclosure may
also be used to detect intracellular targets. SERS nanostructures
may be introduced into cells via microinjection, electroporation,
endocytosis-mediated approaches including the use of amphipathic
peptides such as PEP-1, the use of cationic lipid-based reagents,
such as Lipofectamine (Invitrogen), and the use of micelles and
transfection reagents such as transferrin, mannose, galactose, and
Arg-Gly-Asp (RGD), and other reagents such as the dendrimer-based
reagent SuperFect (Qiagen).
[0115] Intracellular indirect methods can be used to prove that the
particles are bound to the desired targets. The simplest method to
demonstrate the specificity of the probes is to use
immunofluorescence to verify the location of the SERS
nanostructures. There are a number of commercially available
fluorescent probes that are useful for labeling cellular structures
(such as the mitochondria, Golgi apparatus and endoplasmic
reticulum) in living cells. By conjugating an antibody that targets
the same structure, what fraction of particles is actively labeling
their target can be determined; and what percentage are
non-specifically bound. Another approach to verifying the location
of the SERS nanostructures is to use fluorescent protein fusions,
such as GFP and its analogs.
[0116] The present disclosure, therefore, encompasses
nanostructures directed to imaging agents displaying important
properties in medical diagnosis. More particularly, the present
disclosure is directed to imaging agents comprising SERS
nanostructures. The imaging agents of the present disclosure are
useful in imaging a patient generally, and/or in specifically
diagnosing the presence of diseased tissue in a patient. By choice
of composition, the excitation and emission of SERS nanostructures
can be tuned to occur between about 633 nm and 1000 nm, in the
minimum region for absorption and scattering by tissues. The
imaging process may be carried out by administering an imaging
agent of the disclosure to a patient, and then scanning the patient
using any system that can perform spectral imaging, such as spot
scanning confocal microscopes, line scanning systems, and Optical
Coherence tomographic systems. SERS nanostructures of the present
disclosure can also be seen by any imaging system that detects only
over a single wavelength band, the list above as well as any
fluorescence imaging system that has an excitation light source and
filtered image detection. Also included are time domain methods,
such as dynamic light scattering spectroscopy and tomography,
time-of-flight imaging, quasi-elastic light scattering
spectroscopy, photon-correlation spectroscopy, Doppler
spectroscopy, and diffusion wave spectroscopy. All these techniques
allow differentiation between photons and where they have been
based on their time signatures. Since SERS nanostructures will have
different time signatures than fluorescent substances, etc., they
can be discriminated against tissues and other labels with these
methods. Useful instrument parameters are a modulated light source
and time sensitive detector. Modulation can be pulsed or
continuous.
[0117] The scanning results in spectra or images of an internal
region of a patient and/or of any-diseased tissue in that region.
By region of a patient, it is meant the whole patient, or a
particular area or portion of the patient. The imaging agent may be
employed to provide images of the vasculature, heart, liver, and
spleen, and in imaging the gastrointestinal region or other body
cavities, or in other ways as will be readily apparent to those
skilled in the art, such as in tissue characterization, blood pool
imaging, etc.
[0118] This disclosure also provides a method of diagnosing
abnormal pathology in vivo comprising, introducing a plurality of
SERS nanostructures targeted to a molecule involved in the abnormal
pathology into a bodily fluid contacting the abnormal pathology,
wherein the SERS nanostructures become associated to a molecule
involved in the abnormal pathology, and imaging the associated SERS
nanostructures in vivo. The method is generally applicable to any
organ accessible by the probes: gastro-intestinal tract, heart,
lung, liver cervix, breast, etc. In some embodiments, the SERS
nanostructures can be introduced via an endoscope, as in the case
of a colonoscopy, or a needle, or used with a disposable tip or
sleeve. In other embodiments, the SERS nanostructures may be
introduced by directly by the imaging probe itself. For example,
individual optical fibers, or bundles of optical fibers, can be
introduced into live organisms for imaging, and has been
demonstrated for imaging of nerves, brain, microvessels, cells, as
well as for characterizing biodistribution. Gel-coated optical
fibers are very well known in the sensor literature. SERS
nanostructures can be non-covalently bound to the gel, diffusing
into the relevant tissue upon introduction. A variety of other
methods to immobilize SERS nanostructures onto the outer surface of
fibers such that they diffuse into liquid phases to which they are
contacted can be envisioned.
[0119] The present disclosure also provides method for labeling an
animal with SERS nanostructures, comprising introducing SERS
nanostructures into an animal. SERS nanostructures can be
introduced into animals by any suitable means, such as by
subcutaneous implantation or intravenously, and detected using
appropriate equipment. The present disclosure also provides an
identification system and related methods for animals such as
livestock or house pets by utilizing SERS nanostructures implanted
under the hide or skin to identify the animal.
[0120] Under in vivo conditions, nanostructures according to the
disclosure can be delivered to tumors by both a passive targeting
mechanism and an active targeting mechanism. In the passive mode,
macromolecules and nanometer-sized particles are accumulated
preferentially at tumor sites through an enhanced permeability and
retention (EPR) effect. This effect is believed to arise from two
factors: (a) angiogenic tumors that produce vascular endothelial
growth factors (VEGF) that hyperpermeabilize the tumor-associated
neovasculatures and cause the leakage of circulating macromolecules
and small particles; and (b) tumors lack an effective lymphatic
drainage system, which leads to subsequent macromolecule or
nanoparticle accumulation.
[0121] One aspect, therefore, of the disclosure encompasses
surface-enhanced Raman spectroscopic active composite
nanostructures comprising a core metallic, advantageously gold,
nanoparticle, a Raman reporter molecule disposed on the surface of
the core, and an encapsulating protective layer disposed on the
surface of the core and the reporter molecule, wherein the
encapsulated reporter molecule has a measurable surface-enhanced
Raman spectroscopic signature.
[0122] In embodiments of the disclosure, the Raman reporter
molecule may be selected from an isothiocyanate dye, a multi-sulfur
organic dye, a multi-heterosulfur organic dye, a benzotriazole dye,
or combinations thereof.
[0123] In embodiments of the disclosure, the reporter molecule is
selected from a thiacyanine dye, a dithiacyanine dye, a
thiacarbocyanine dye, or a dithiacarbocyanine dye. In other
embodiments, the reporter molecule is selected from malachite green
isothiocyanate, tetramethylrhodamine-5-isothiocyante,
X-rhodamine-5-isothiocyanate, X-rhodamine-6-isothiocyanate, or
3,3'-diethylthiadicarbocyanine iodide.
[0124] In one embodiment of the disclosure, the core is gold, and
may have a diameter less than about 200 nanometers.
[0125] In the embodiments of the nanostructures of the disclosure,
the encapsulating material is a thiol-polyethylene glycol.
[0126] In other embodiments of the disclosure the nanostructures
may further comprise a target-specific probe selectively binding a
target on a cell.
[0127] In these embodiments, the target-specific probe may be
selected from the group consisting of an antibody, a polypeptide, a
polynucleotide, a drug molecule, an inhibitor compound, and a
combination thereof, and wherein the targeting probe has an
affinity for a marker on the surface of a target cell.
[0128] In one embodiment, the target-specific probe is an
immunoglobulin, or a fragment thereof and in the embodiments of the
disclosure the probe may be disposed on the hydrophobic protection
structure. In one embodiment, the probe is a tumor-targeting
ligand.
[0129] Another aspect of the disclosure encompasses methods of
preparing a nanostructure according to the disclosure, comprising
providing a gold nanoparticle, introducing the gold nanoparticle to
a Raman reporter, whereupon the Raman reporter is disposed on the
surface of the nanoparticle to form a nanoparticle-reporter
complex, and disposing a protection structure layer on the surface
of the nanoparticle-reporter complex, wherein the reporter molecule
has a measurable surface-enhanced Raman spectroscopic
signature.
[0130] In one embodiment of this aspect of the invention, the
methods may further comprise depositing a cell target-specific
probe to the protection structure layer, wherein the probe is
selected from an antibody, a polypeptide, a polynucleotide, a drug
molecule, an inhibitor compound, or a combination thereof.
[0131] In one embodiment of the method of this aspect of the
disclosure, the core metallic nanoparticles are a colloid. In an
advantageous embodiment, the core metallic nanoparticles is
gold.
[0132] In embodiments of this aspect of the disclosure, the Raman
reporter molecule may be selected from an isothiocyanate dye, a
multi-sulfur organic dye, a multi-heterosulfur organic dye, a
benzotriazole dye, or combinations thereof. In other embodiments of
the disclosure, the reporter molecule is selected from a
thiacyanine dye, a dithiacyanine dye, a thiacarbocyanine dye, or a
dithiacarbocyanine dye. In yet other embodiments of this method of
the disclosure, reporter molecule is selected from malachite green
isothiocyanate, tetramethylrhodamine-5-isothiocyante,
X-rhodamine-5-isothiocyanate, X-rhodamine-6-isothiocyanate, or
3,3'-diethylthiadicarbocyanine iodide.
[0133] In one embodiment of the disclosure, the encapsulating
material is a thiol-polyethylene glycol.
[0134] Yet another aspect of the disclosure encompasses methods of
imaging a biological sample, comprising delivering at least one
nanostructure to a cultured cell or to an animal or human subject,
wherein the nanostructure comprises a core gold nanoparticle, a
Raman reporter molecule disposed on the surface of the core, and an
encapsulating protective layer disposed over the core and the
reporter molecule, and wherein the encapsulated reporter molecule
has a measurable surface-enhanced Raman spectroscopic signature,
allowing the nanostructure to contact a targeted biological cell or
tissue, exciting the reporter molecule with a source of radiation,
and measuring the surface enhanced Raman spectroscopy spectrum of
the nanostructure corresponding to the reporter molecule, thereby
detecting the presence of the nanostructure in the targeted cell or
tissue.
[0135] In one embodiment of this aspect of the disclosure, the
nanostructure may further comprise a target-specific probe, wherein
the targeting probe selectively binds the nanoparticle to a
targeted cell, thereby allowing detection of the targeted cell.
[0136] In another embodiment of the disclosure, the target cell is
in a tissue of an animal or human subject.
[0137] In the embodiments of this aspect of the disclosure, the
target cell may be a cancerous cell of an animal or human subject
and the target-specific probe may selected from the group
consisting of an antibody, a polypeptide, a polynucleotide, a drug
molecule, an inhibitor compound, and a combination thereof, and
wherein the targeting probe has an affinity for a marker on the
surface of a target cell.
[0138] In one embodiment of the disclosure, the target-specific
probe is a tumor-targeting ligand.
[0139] The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present disclosure to its fullest extent. All
publications recited herein are hereby incorporated by reference in
their entirety.
[0140] It should be emphasized that the embodiments of the present
disclosure, particularly, any "preferred" embodiments, are merely
possible examples of the implementations, merely set forth for a
clear understanding of the principles of the disclosure. Many
variations and modifications may be made to the above-described
embodiment(s) of the disclosure without departing substantially
from the spirit and principles of the disclosure. All such
modifications and variations are intended to be included herein
within the scope of this disclosure, and the present disclosure and
protected by the following claims.
[0141] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
and compounds disclosed and claimed herein. Efforts have been made
to ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.), but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C., and pressure is at or near
atmospheric. Standard temperature and pressure are defined as
20.degree. C. and 1 atmosphere.
EXAMPLES
Example 1
[0142] Reagents: Ultrapure water (18 M.OMEGA. cm.sup.-1) was used
throughout the work. The following chemicals were obtained from
commercial sources and were used without further purification:
60-nm citrate-stabilized gold particles at a concentration of
2.6.times.1010 particles per milliliter (Ted Pella Inc.),
near-infrared-emitting quantum dots (QD705, Invitrogen), malachite
green isothiocyanate (MGITC) (Invitrogen),
diethylthiatricarbocyanine iodide (DTTC) (Exciton), mPEG-SH (MW
approximately 5 kDa) (Nektar Therapeutics), HS-PEG-COOH (MW
approximately 3 kDa) (Rapp Polymers). The human carcinoma cells
line Tu686 was established from a primary tumor in base of tongue.
Human carcinoma cell line NCI-H520 was purchased from the American
Type Culture Collection (ATCC). Cell culture media, fetal bovine
serum, hemocytometer, and cell culture supplies were purchased from
Fisher Scientific. All other reagents were obtained from
Sigma-Aldrich at the highest purity available.
Example 2
[0143] Synthesis: Gold colloids with a target diameter of about 60
nm were synthesized according to literature procedures. All
glassware was cleaned rigorously and rinsed with water prior to
use. In a 50 mL glass flask, 30 mL of a 0.01% aqueous solution of
HAuCl.sub.4 was brought to a boil under magnetic stirring. Upon
boiling, 180 .mu.L of 1% sodium citrate was rapidly injected.
Within minutes, the pale yellow solution turned deep purple and
quickly progressed to red. The colloid was boiled for approximately
15 minutes to ensure complete reduction, was allowed to cool to
room temperature, and was reconstituted to 30 mL before use.
[0144] To prepare SERS active composite nanostructures with an
embedded Raman reporter (i.e., a reporter molecule), about 0.1 g
mixed bed ion-exchange resin was stirred with the freshly prepared
gold colloid to remove excess ions. The resin was removed either by
filtration or careful decanting, and the colloid was diluted with
an equal amount of water. A Raman reporter was added under rapid
stirring to a concentration not exceeding about 7.5.times.10.sup.-8
M and was allowed to equilibrate for about 15 minutes.
[0145] Measurements: A scanning spectrophotometer (Shimadzu,
Columbia, Md.) was used to acquire UV-visible absorption spectra.
High-magnification transmission electron micrographs were taken
using a Phillips CM200 electron microscope and were recorded on a
TVIPS 2 k by 2 k CCD. Bulk Raman spectra were recorded using a
dispersive Raman spectroscopy system (Solution 633, Detection
Limit, Laramie, Wyo.). Single-particle spectra were obtained with
an inverted optical microscope (Diaphot 200, Nikon, Melville,
N.Y.), equipped with a mixed gas argon/krypton ion laser (Lexel
3500, Fremont, Calif.) for 647 nm excitation.
[0146] Regions of interest were first screened with wide-field
illumination, and Raman-active particles were located with a
video-rate intensified CCD (ICCD, PTI, Inc., Lawrenceville, N.J.)
mounted to the front microscope port. Confocal optics was then used
to focus on an individual SERS active composite nanostructures, and
back-scattered Raman signals were collected through a microscope
objective (Plan 100.times., oil immersion, NA=1.25). A
triple-bandpass filter (Chroma Tech, Brattleboro, Vt.) was used to
block the laser line and extraneous signals. Spectroscopic
signatures were obtained with a CCD detector (TKB512, Princeton
Instruments, Trenton, N.J.) mounted on a single-stage spectrometer
(Model 270M, Spex, Edison, N.J.).
Example 3
[0147] Preparation of pegylated SERS nanoparticles: A freshly
prepared reporter solution (3-4 .mu.M) was added dropwise to a
rapidly mixing gold colloid at a 1:6 reporter solution/colloid
volume ratio, which facilitated even distributions of the reporter
molecules on the gold particle surface. The molar ratio of reporter
molecules to gold particles was optimized for maximal SERS
intensities and minimal colloid aggregation. For example, the
optimized surface coverage values were 14,000 malachite green
isothiocyanate molecules per 60 nm gold particle, and about 15,300
crystal violet molecules per gold particle of the same size. It
should be noted that the above parameters (that is, stock reporter
concentration, volume ratio of stock reporter solution to gold
nanoparticle solution, and the rate of reporter addition to gold)
all affected the aggregation state of the resulting tags. When
reporter solution was added to gold colloid, we observed higher
SERS signals than when adding gold to reporter.
[0148] After 10 mins, a thiol-PEG solution (10 .mu.M) was added
dropwise to the Raman-encoded colloids, with a minimum ratio of
30,000 PEG-SH molecules per 60-nm gold particle. This surface
coverage corresponded to a complete PEG monolayer on the gold
particle surface, and was necessary to stabilize gold colloids
against aggregation under various conditions. Simple geometric
calculations showed that each thiol-PEG molecule occupied a
footprint area of 0.35 nm.sup.2 on the gold surface, consistent
with the literature data reported for PEG-SH in a brush
conformation. Importantly, addition of 10- to 20-fold excess PEG-SH
did not result in any changes in colloid stability or in the
thickness of the polymer coating layer.
Example 4
[0149] Nanoparticle characterization: UV-Vis absorption spectra
were recorded on a Shimadzu (UV-2401) spectrometer using disposable
polyacrylic cuvettes. Transmission electron micrographs (TEM) were
taken by using a Hitachi H7500 high-magnification electron
microscope. The nanoparticle sample (5 .mu.l) was dropped onto
copper 200-mesh grids that were pretreated with UV light to reduce
static electricity. After 30 min, the solvent was drained with a
filter paper and a phosphotungstic acid stain solution (1% by
weight, adjusted to pH 6) was applied for 30 secs Fresh tumor
tissue specimens were fixed in 0.1 M cacodylate buffer (pH 7.4)
containing 2.5% glutaraldehyde at 4.degree. C. The tissue was
rinsed three times in 0.1 M cacodylate buffer for 15 min,
post-fixed with 1% OsO.sub.4 buffer, and then dehydrated and
embedded in a resin (Epon). Ultrathin sections (approximately 60
nm) were produced with an ultratome machine, and were placed on
copper grids for TEM imaging.
[0150] DLS data were obtained by using a Brookhaven 90Plus particle
size analyzer instrument. Each sample was measured three times
consecutively. SERS spectra were recorded on a compact Raman system
using 633 nm (3 mW) or 785 nm (40 mW) excitation (Advantage Raman
Series, DeltaNu). In vivo SERS spectra were collected using 785-nm
laser excitation on a handheld Raman system (Inspector Series,
DeltaNu). The laser beam diameter was 35 .mu.m at the focal point,
so the probe volume was estimated to be about 23 nl at 633 nm
excitation and about 19 nl at 785 nm excitation. SERS intensities
were normalized to the Raman spectra of cyclohexane and polystyrene
to correct for variations in optical alignment and instrument
response. The spectral resolution was about 5 cm-1 for both the
Advantage and the Inspector Raman systems.
[0151] For imaging of single SERS nanoparticles and quantum dots, a
narrow bandwidth laser excitation filter (633.+-.3 nm) and a
long-pass emission filter (655LP, Chroma Tech) were employed with
an Olympus IX71 inverted microscope. The images were taken with 750
ms exposure time and were the average of 50 images by using an
electron-multiplying (EM) CCD camera (Hamamatsu, Model C9100-12)
attached to the microscope. The use of long exposure times and
image averaging cancelled out any signal fluctuations of single
nanoparticles. For quantitative comparison of SERS and quantum dot
signal intensities, the wavelength dependence factor was corrected
by using the CCD camera response curve.
Example 5
[0152] Conjugation with scFv ligands: scFv B10, an antibody
fragment specific for human EGFR, was isolated from the YUAN-FCCC
human naive phage display library by using established solid phase
biopanning methods. Large quantities of scFv were purified from
bacterial extracts under native conditions using a Ni.sup.2+
NTA-agarose column (Qiagen). Protein purity greater than 95% was
determined by using sodium dodecyl sulfate (SDS)-PAGE. The
heterofunctional linker HS-PEG-COOH (430 .mu.l and 1 .mu.M) was
added dropwise to 2.2 ml Au-MGITC (or Au-DTTCI) solution in a
polypropylene tube under rapid mixing. The number of carboxy groups
per gold particle was controlled to be approximately 5,000 by
changing the amount of linker molecules used. After 15 min of
mixing, the gold nanoparticles were exposed to a large volume of
PEG-SH (1.6 ml at 10 .mu.M) to fill the areas not covered by the
heterofunctional PEG, yielding well-shielded and stable particle
surfaces. Before covalent ligand conjugation at the carboxylic acid
functional groups, the gold particles were purified by three rounds
of centrifugation (1,000 g) and resuspension in PBS.
[0153] To activate the --COOH groups on the particle surface for
covalent conjugation, freshly prepared ethyl dimethylaminopropyl
carbodiimide (EDC) solution (5 .mu.l) at a concentration of 40
mg/ml) and sulfo-NHS (5 .mu.l at 110 mg/ml) were mixed vigorously
at 25.degree. C. for 15 min. Excess EDC and sulfo-NHS were
separated from the activated nanoparticles by three rounds of
centrifugation (1,000 g) and resuspension in PBS using Nanosep 10K
MWCO OMEGA membrane (Pall Life Sciences). The purified gold
particles with activated carboxyl groups were then reacted with the
scFv antibody (11.2 nmol) at 25.degree. C. for 2 h, and the
reaction mixture was stored at 4.degree. C. for overnight. Excess
scFv ligand was removed by three rounds of centrifugation and
resuspension in PBS using 100K MWCO OMEGA membranes. Based on
protein absorption measurement at 280 nm, we estimated that there
were about 600 scFv molecules per gold particle. This value was
further confirmed by using a fluorescently labeled scFv ligand to
determine the conjugation ratio at higher sensitivity. The fully
functionalized nanoparticles were characterized by UV-Vis, TEM and
DLS, and their colloidal stability and optical properties were
essentially the same as that of control nanoparticle tags.
Example 6
[0154] Cellular SERS studies.: Tu686 and H520 cells were cultured
in DMEM/Ham's F-12 (1:1) and RPMI-1640 supplemented with 10%
heat-inactivated fetal bovine serum and antibiotics (streptomycin,
penicillin G and amphotericin B), respectively, and were maintained
in a humidified incubator at 37.degree. C., 5% CO.sup.2. The cells
were grown to confluence in 35-mm dishes. Cell staining procedures
were performed under sterile conditions on a tabletop binding
incubator at 25.degree. C. Live cells were gently mixed with the
scFv-conjugated SERS nanoparticles (15 pM in PBS) for 30 min, and
then were harvested by gentle scraping. The cells were subjected to
four rounds of washing with ice-cold PBS, and were resuspended in
500 .mu.l PBS before SERS measurement. A portion of the cells were
incubated with pegylated control SERS tags to assess nonspecific
binding and internalization. An additional portion of the cells
received neither control SERS tags nor EGFR-SERS tags, and were
used as controls to assess background cell scattering. SERS spectra
were normalized to cell numbers as determined with a Coulter
counter.
[0155] For quantitative comparison, we subtracted the pure cell
scattering spectra to generate difference spectra in FIG. 3. All
spectra were taken in cell suspensions. Based on a cell density of
1.times.10.sup.6 cells per ml, we estimated that the laser
detection volume contained approximately 20 to 30 labeled cells. We
did not observe changes in either spectral signatures or
intensities upon repeated examination of the unfixed cell samples
over a period of 3 days or upon cell fixation in formaldehyde
solution. These cell-suspension measurements avoided the problems
of nanoparticle tagging and cellular heterogeneities and were found
to be highly reproducible.
Example 7
[0156] Tumor xenografts and in vivo SERS: A healthy nude mouse
received 50 femtomoles of pegylated SERS nanoparticles administered
at two locations: (i) subcutaneous injection (1-2 mm under skin);
and (ii) deep muscular injection (1 cm under the skin). Different
locations were examined by using an NIR Raman spectrometer
(Inspector Series, DeltaNu). The subcutaneous SERS spectrum was
obtained in 3 secs, the muscular spectrum in 21 secs, and the
control spectrum (obtained in an area away from the injection site)
also in 21 secs.
[0157] Tu686 cells (5.times.10.sup.6) were injected subcutaneously
into the back flank area of approximately 6- to 8-week-old female
nude mice (NC rathymic, nu/nu). The mice were divided into two
groups for passive and active targeting studies. When the tumor
size reached 3 mm diameter, the nude mice received 45 femtomoles of
scFv EGFR-conjugated SERS tags and pegylated control SERS tags,
respectively, by tail vein injection. After 5 hrs, the mice were
placed under anesthesia by injection of 70 .mu.l of ketamine and
xylazine mixture solution and were examined by using a Raman
spectrometer with 20 mW laser power at 785 nm. The laser beam was
focused to the tumor or the liver anatomical region for both the
targeted and nontargeted SERS nanoparticles. With a focal length of
approximately 9 mm, SERS spectra were obtained in a completely
noncontact and noninvasive manner. Results are shown in FIGS.
5A-5C. After spectroscopic data acquisition, the mice were killed
to collect major organs for ICP-MS biodistribution analysis. A
small portion of each fresh tissue sample was also fixed
immediately in 0.1 M cacodylate buffer to prepare TEM thin sections
(FIGS. 10 and 11).
[0158] Briefly, major organ tissues were rinsed with ethanol three
times and then lyophilized and weighed into clean vials for acid
digestion. After 2 days of strong acid digestion, the samples were
purified and diluted 35-fold for analysis by ICP-MS (inductively
coupled plasma-mass spectrometry). The experiments were carried out
in five independent runs for statistical analysis. Each run had two
mice with freshly prepared SERS tags, one with active targeting and
the other with passive targeting. One group of the animals was used
for longer term toxicity studies.
Example 8
[0159] Design and characterization of pegylated SERS nanotags:
FIGS. 1A-1D show the design and preparation of pegylated gold
nanoparticles with embedded spectroscopic tags and their schematic
structures. Also shown are their optical absorption spectra (FIG.
1B), transmission electron microscopy (TEM) structures (FIG. 1C),
and hydrodynamic size data (FIG. 1D). The original gold particles
(60-nm diameter) were encoded with a Raman reporter and stabilized
with a layer of thiol-PEG. Previous experimentation had shown that
gold nanoparticles with a core size of approximately 60-80 nm were
most efficient for SERS at red (630-650 nm) and near-infrared (785
nm) excitations (Krug et al., (1999) J. Am. Chem. Soc. 121:
9208-9214).
[0160] This spectral region is known as a `clear window` for
optical imaging because the hemoglobin (blood) and water absorption
spectra are minimal. Beyond the SERS effect, we also achieved
resonance Raman enhancement by using reporter molecules with
electronic transitions at 633 nm or 785 nm. The gold plasmonic
resonance spectra remained essentially unchanged (<1-nm red
shifts), even when the gold particles were coated with a large
number of molecules (about 1.4-1.5.times.104) and stabilized with a
layer of PEG molecules (FIG. 1B). We note that single-molecule SERS
occurs only at special active sites or junctions, and it is not
required for tumor detection. In fact, with a large number of
reporter molecules adsorbed on the particle surface, the achieved
total signal intensities exceeded that of single-molecule SERS. The
PEG coating was clearly observed as a thin white layer of
approximately 5 nm by TEM negative staining, whereas the particle's
`wet` hydrodynamic diameter increased by 20 nm after pegylation, as
measured by hydrodynamic light scattering (DLS) in buffered saline.
At a core particle size of 60 nm, a minimum of 30,000 thiol-PEG
molecules (MW=5 kDa) per gold nanoparticle was necessary to achieve
complete protection against salt-induced colloid aggregation. This
surface coverage corresponded to a footprint area of approximately
0.35 nm.sup.2 per PEG molecule, in agreement with that reported by
another group for thiol-PEG adsorbed on colloidal gold in a brush
conformation. After this shielding layer was completed, the use of
additional thiol-PEG up to 10- to 20-fold excess had little effect
on the coating thickness, as measured by both TEM and DLS.
Example 9
[0161] The stability of pegylated gold nanoparticles was studied by
measuring their SERS signals (both frequency and intensity) under a
wide range of conditions including concentrated salts (1-2 M),
strong acids (0.1 M HCl), strong bases (1 M NaOH) and organic
solvents (methanol, ethanol and dimethyl sulfoxide or DMSO) FIGS. A
and B. In the absence of PEG protection, the gold nanoparticles
rapidly `crash` (that is, aggregate and precipitate) under these
harsh conditions. With PEG protection, the gold particles and their
SERS spectra are completely stable, with only minor relative
intensity changes at pH 1-2 (due to protonation and relative
orientation changes of the reporter molecule on the gold
surface).
[0162] The observation of intense SERS signals with a thiol-PEG
coating is counterintuitive because the reporter molecules on the
particle surface are expected to be displaced by thiol compounds
(which are known to spontaneously form a monolayer on gold). Also
surprising is that a range of Raman reporters such as crystal
violet, Nile blue, basic fuchsin and cresyl violet were not
displaced by thiol-PEG, even without an anchoring isothiocyanate
(--N.dbd.C.dbd.S) group. In fact, the SERS signals of crystal
violet and other dyes were strongly protected by thiol-PEG, and
were stable for >11 months at 25.degree. C. A common feature for
these reporter dyes is that they are positively charged and contain
delocalized pi-electrons. In contrast, organic dyes with negative
charges such as sodium fluorescein gave only weak and unstable SERS
signals on the citrate-stabilized gold particles (also negatively
charged) used in this work. Thus, we believe that both
electrostatic interactions and delocalized pi-electrons are
important for strong dye adsorption, likely at gold surface sites
that do not compete with thiol-PEG adsorption. It is also possible
that the thiol-PEG layer protected and stabilized the adsorbed
reporter dyes by steric shielding and electronic interactions.
[0163] For cellular and in vivo imaging applications, we compared
the excitation and emission spectral properties of pegylated gold
nanoparticles and near-infrared quantum dots. The gold
nanoparticles provided much richer spectroscopic information, and
their emission peaks (full width at half maximum FWHM=1-2 nm) were
20-30 times narrower than those of quantum dots (FWHM=40-60 nm)
(FIGS. 2A and 2B). Under identical experimental conditions, the
pegylated gold particles were >200 times brighter (on a
particle-to-particle basis) than near-infrared-emitting quantum
dots in the spectral range of 650-750 nm (see single particle
images in FIGS. 2C and 2D, and statistical data in FIGS. 2E and
2F). The pegylated gold nanoparticles had hydrodynamic sizes of
about 80 nm (diameter) and were completely nontoxic to cultured
cells when tested over 3-6 days. In the absence of surface-enhanced
Raman signals, near-infrared gold nanoshells have recently been
used as a contrast enhancement agent for optical coherence
tomography as well as for photothermal tumor ablation, but this
approach does not provide molecular signatures for spectral
encoding or multiplexing.
Example 10
[0164] Spectroscopic detection of cancer cells: For cancer cell
detection, targeted gold nanoparticles were prepared by using a
mixture of thiol-PEG (about 85%) and a heterofunctional PEG
(SH-PEG-COOH) (about 15%). The heterofunctional PEG was covalently
conjugated to an scFv antibody (MW=25 kDa), a ligand that binds to
the EGFR with high specificity and affinity as schematically shown
in FIG. 3A. UV-Vis absorption and fluorescence data indicated that
about 600 copies of the scFv ligand were conjugated to each gold
nanoparticle. FIG. 3B shows cellular binding and SERS spectra
obtained by incubating the scFv-conjugated gold nanoparticles with
human carcinoma cells. The human head-and-neck carcinoma cells
(Tu686) were EGFR positive (10.sup.4-10.sup.5 receptors per cell),
and were detected by strong SERS signals. In contrast, the human
non-small cell lung carcinoma (NCI-H520) did not express EGFR,
showing little or no SERS signals. To confirm targeting
specificity, we preincubated Tu686 cancer cells in a tenfold excess
of free scFv EGFR antibody, and then added EGFR-labeled SERS
nanoparticles for competitive binding studies. After three rounds
of washing, the cells showed only negligible SERS signals. Also
tested and confirmed were the binding specificity of SERS
nanoparticles conjugated to secondary antibodies in a two-site
sandwich format. For control cancer cells (EGFR negative) and
control nanoparticles (plain PEG-coated nanotags and PEG-nanotags
functionalized with a nonspecific IgG antibody), the spectra showed
a weak but reproducible background as shown in FIG. 3B. The low
background was probably caused by residual SERS nanoparticles in
the mixing solution that were not completely removed during cell
isolation, but there could also have been contributions from
nonspecific binding or nanoparticle internalization. An infrared
dye (diethylthiatricarbocyanine or DTTC) was used as a
spectroscopic reporter, and achieved surface-enhanced resonance
Raman scattering (SERRS) at 785-nm excitation. This resonance
condition did not lead to photobleaching because the adsorbed dyes
were protected from photo-degradation by efficient energy transfer
to the metal particle. The resonance effect can further increase
the surface-enhanced Raman signals by 10- to 100-fold, sensitive
enough for Raman molecular profiling studies of single cancer cells
(FIG. 8). This sensitivity is important for investigating the
heterogeneous nature of cancer tissue specimens removed by surgery,
and circulating tumor, cells captured from peripheral blood
samples. Single-cell profiling studies are of great clinical
significance because EGFR is a validated protein target for
monoclonal antibody and protein-kinase-based therapies
Example 11
[0165] In vivo tumor targeting and detection: A major challenge for
in vivo optical imaging and spectroscopy is the limited penetration
depth, due to light scattering and absorption in animal tissues. To
determine whether SERS spectra can be acquired from pegylated gold
nanoparticles buried in animal tissues, we injected small dosages
of nanoparticles into subcutaneous and deep muscular sites in live
animals. Highly resolved SERS signals were obtained from
subcutaneous as well as muscular injections as shown in FIG. 4.
[0166] A healthy nude mouse received 50 .mu.l of the SERS
nanoparticles tags (1 nM) by subcutaneous (1-2 mm under the skin)
or muscular (approximately 1 cm under the skin) injection. The
subcutaneous spectrum was obtained in 3 secs, the muscular spectrum
in 21 sec, and the control spectrum (obtained in an area away from
the injection site) also in 21 sec. The reference spectrum was
obtained from the SERS nanoparticles in PBS solution in 0.1 secs
The spectral intensities are adjusted for comparison by a factor
(.times.1, .times.30 or .times.210) as indicated. The Raman
reporter molecule is malachite green, with spectral signatures at
427, 525, 727, 798, 913, 1,169, 1,362, 1,581 and 1,613 cm-1. These
features are distinct from the animal skin Raman signals (see the
skin spectrum). Excitation wavelength, 785 nm; laser power, 20
mW.
[0167] The in vivo SERS spectra were identical to that obtained in
vitro (saline solution), although the absolute intensities were
attenuated by 1-2 orders of magnitude. Based on the high
signal-to-noise ratios, we estimated that the achievable
penetration depth was about 1-2 cm for in vivo SERS tumor detection
(also confirmed by deep tissue injection studies).
[0168] For in vivo tumor targeting and spectroscopy, the gold
nanoparticles conjugated with the scFv antibody were injected
systemically (through tail veins) into nude mice bearing a human
head-and-neck tumor (Tu686). FIGS. 5A and 5B shows SERS spectra
obtained 5 hrs after nanoparticle injection by focusing a
near-infrared, 785-nm laser beam on the tumor site or on other
anatomical locations (e.g., the liver or a leg). Substantial
differences were observed between the targeted and nontargeted
nanoparticles in the tumor signal intensities, whereas the SERS
signals from nonspecific liver uptake were similar. This result
indicates that the scFv-conjugated gold nanoparticles were able to
target EGFR-positive tumors in vivo. Time-dependent SERS data
further indicate that nanoparticles were gradually accumulated in
the tumor for 4-6 hrs, and that most of the accumulated particles
stayed in the tumor for >24-48 hrs.
Example 12
[0169] In vivo nanoparticle distribution and intracellular
localization: Quantitative biodistribution studies using
inductively coupled plasma-mass spectrometry (ICP-MS) revealed that
the targeted gold nanoparticles were accumulated in the tumor 10
times more efficiently than the nontargeted particles as shown in
FIG. 6. The ICP-MS data also confirmed nonspecific particle uptake
by the liver and the spleen, but little or no accumulation in the
brain, muscle or other major organs, similar to the biodistribution
data reported for gold nanoshells injected into healthy mice31.
Ultrastructural TEM studies further revealed that the SERS
nanoparticles were taken up by the EGFR-positive tumor cells, and
were localized in intracellular organelles such as endosomes and
lysosomes as shown in FIGS. 10 and 11. The in vivo endocytosed
nanoparticles had crystalline and faceted structures, in agreement
with the finding that nearly identical SERS spectra were obtained
from the encoded gold nanoparticles in vitro and in vivo. The
pegylated gold particles appeared to be intact and stable in
systemic circulation as well as after being taken up into
intracellular organelles. No toxicity or other physiological
complications were observed for the animals after 2-3 months of
gold particle injection.
Example 13
[0170] Stability of Pegylated SERS Nanoparticles under Harsh
Conditions: Four independent techniques verified the high degree of
stability of Au-MGITC-PEG-SH in concentrated PBS solution. PEG-SH
coated and uncoated Au-MGITC complexes were examined by UV-vis
absorption spectroscopy, TEM, DLS, and visual observation, as shown
in FIGS. 7A and 7B) PBS addition to uncoated Au-MGITC immediately
aggregated and precipitated the colloid as evidenced by dramatic
spectral changes in UV-vis absorption spectrum, large aggregates in
TEM, and the appearance of a distinct population of particles of
600-1000 nm hydrodynamic diameter, and an obvious color change from
pink to clear. In contrast, PEG-SH coated Au-MGITC treated with PBS
showed a preservation of the characteristic plasmon resonance peak
of 60 nm gold, a majority of single particles by TEM (with a small
population of clusters due to solvent evaporation), a unimodal,
narrow size distribution of particles in DLS, and the pink color.
The effects of a wide range of conditions encountered in
bioconjugation and cell labeling procedures were investigated for
their effects on the spectral signatures of PEG-SH coated SERS
tags.
[0171] Au-MGITC-PEG-SH was pelleted by centrifugation, redispersed
in new solvents, and examined by SERS spectroscopy. There was no
significant spectral changes when Au-MGITC-PEG-SH was redispersed
in 10-fold concentrated PBS (1.37 M NaCl), basic water (pH 12),
acidic water (pH 2), ethanol, and methanol comparing with reference
spectrum of Au-MGITC in water (FIG. 18). A slight change in
relative peak intensities of the Raman bands at 1615, 1365, and
1172 cm-1 at pH 2 was noticed, possibly due to relative orientation
changes of MGITC on the Au surface, but no shift in vibrational
frequencies was observed within the instrument resolution of 5
cm-1.
[0172] Redispersion of Au-MGITC-PEG-SH in dimethylsulfoxide (DMSO)
masked the spectral features of the reporter due to the strong
Raman cross section of DMSO. Interestingly, the original MGITC
spectral signature was recovered after the DMSO solvated tag was
stored under ambient conditions for 60 days and then redispersed in
water (FIG. 18, panel `g`). Although uncoated Au-MGITC coalesced
upon 5 centrifugations, PEG-SH coated SERS tags did not form
aggregates under any of the above conditions tested.
Example 14
[0173] SERS Spectra and Correlated Plasmonic Imaging of Single
Cancer Cells: Tu686 and H520 cells were grown to confluence in an
8-chamber glass slide. scFv-conjugated SERS tags at a concentration
of 15 pM were introduced to 200 uL cell culture medium, and were
then gently mixed for 30 min. After the incubation period, cells
were washed thoroughly with PBS six times to remove free gold
nanoparticles before imaging. The reflective mode darkfield images
were obtained with an ExamineR microscope (DeltaNu, Laramie, Wyo.)
using 20.times. objective. A dark field condenser was used to
deliver a narrow beam of white light from a tungsten lamp to the
sample. In this mode, cells stained with SERS nano-tags on the cell
membrane displayed bright golden color due to the highly scattering
property of gold nanoparticles. EGFR-negative H520 cells showed a
mostly dark background. The Tu686 EGFR-positive cells exhibited a
high level of EGFR receptor binding while the H520 EGFR-negative
cells had only limited EGFR expression. Single-cell SERS spectra
were obtained by switching the microscope to the Raman mode with
785 nm laser excitation. The laser spot size using 20.times.
objective was 5.times.10 .mu.m at the focal plane. FIG. 8 showed
the SERS spectra recorded from the areas as indicated by the arrows
for EGFR-positive and EGFR-negative cells, respectively. Each
spectrum was acquired with an exposure time of 10 seconds.
Example 15
[0174] Biodistribution Studies of Nontargeted (Control) SERS
Nanoparticles: To investigate the behavior of SERS nanostructure
target-specific antibody conjugated probes in living animals, the
following were examined: their specific uptake and retention,
background or nonspecific uptake, blood clearance, and organ
distribution. Nonspecific nanostructure uptake and retention took
place primarily in the liver and the spleen, with little or no SERS
nanostructure accumulation in the brain, the heart, the kidney, or
the lung, as shown in FIG. 9. This pattern of in vivo organ uptake
and distribution was similar to that of dextran-coated magnetic
iron oxide nanoparticles. For polymer-encapsulated SERS
nanoparticles with excess COOH groups, no tumor targeting was
observed, indicating nonspecific organ uptake and rapid blood
clearance. For polymer-encapsulated SERS nanoparticles with surface
PEG groups, the rate of organ uptake was reduced and the length of
blood circulation was improved, leading to slow accumulation of the
nanoparticles in the tumors. For nanoparticles encapsulated by PEG
and bioconjugated with an anti-EGFR antibody, the nanoparticles
were delivered and retained by the tumor xenografts, but
nonspecific liver and spleen uptake was still apparent, as shown in
FIG. 6.
Example 16
[0175] Intracellular Localization Studies by Transmission Electron
Microscopy (TEM): Tumor, liver, spleen and kidney were examined
with TEM to determine where the gold nanoparticles are deposited
after cellular and tissue uptake. FIG. 10 shows a representative
TEM image of tumor tissue sections when EGFR targeted gold
nanoparticles were injected systemically for in-vivo tumor
targeting. The data clearly show that the gold nanoparticles are
internalized into tumor cells (most likely via receptor-mediated
endocytosis) and are located in intracellular organelles such as
endosomes and lysosomes.
[0176] To examine liver uptake of the nanoparticles, FIG. 11 shows
a Kupffer cell (macrophages lining the liver sinudoidal surface)
with gold nanoparticles captured in early- and late-stage
endosomes. Note that the nanoparticles nonspecifically taken up by
Kupffer cells are often isolated structures, in contrast to the
clustered structures inside tumor cells. A significant number of
gold nanoparticles are also identified inside spleen macrophage
cells. In all other organs, gold particles are only found at very
low densities. Overall, high-magnification TEM studies reveal that
pegylated gold nanoparticles are taken up into intracellular
organelles under in-vivo conditions, but their shape and morphology
remain intact.
Example 17
[0177] Passive Accumulation versus Active Targeting of SERS
Nanoparticle Tags to Tumors: See FIG. 12.
[0178] It should be emphasized that the above-described embodiments
of the present disclosure are merely possible examples of
implementations, and are merely set forth for a clear understanding
of the principles of this disclosure. Many variations and
modifications may be made to the above-described embodiment(s) of
the disclosure without departing substantially from the spirit and
principles of the disclosure. All such modifications and variations
are intended to be included herein within the scope of this
disclosure and protected by the following claims.
* * * * *