(Aug. 8, 2012)
Oh, My Stars and Hexagons! DNA Code Shapes Gold
DNA holds the genetic code for all sorts of biological molecules
and traits. But University of Illinois researchers have found that
DNA's code can similarly shape metallic structures.
The team found that DNA segments can direct the shape of gold
nanoparticles -- tiny gold crystals that have many applications in
medicine, electronics and catalysis. Led by Yi Lu, the Schenck
Professor of Chemistry at the U. of I., the team published its
surprising findings in the journal Angewandte Chemie.
"DNA-encoded nanoparticle synthesis can provide us a facile but
novel way to produce nanoparticles with predictable shape and
properties," Lu said. "Such a discovery has potential impacts in
bio-nanotechnology and applications in our everyday lives such as
catalysis, sensing, imaging and medicine."
Gold nanoparticles have wide applications in both biology and
materials science thanks to their unique physicochemical
properties. Properties of a gold nanoparticle are largely
determined by its shape and size, so it is critical to be able to
tailor the properties of a nanoparticle for a specific
"We wondered whether different combinations of DNA sequences could
constitute 'genetic codes' to direct the nanomaterial synthesis in
a way similar to their direction of protein synthesis," said
Zidong Wang, a recent graduate of Lu's group and the first author
of the paper.
Gold nanoparticles are made by sewing tiny gold seeds in a
solution of gold salt. Particles grow as gold in the salt solution
deposits onto the seeds. Lu's group incubated the gold seeds with
short segments of DNA before adding the salt solution, causing the
particles to grow into various shapes determined by the genetic
code of the DNA.
The DNA alphabet comprises four letters: A, T, G and C. The term
genetic code refers to the sequence of these letters, called
bases. The four bases and their combinations can bind differently
with facets of gold nanoseeds and direct the nanoseeds' growth
pathways, resulting in different shapes.
In their experiments, the researchers found that strands of
repeating A's produced rough, round gold particles; T's, stars;
C's, round, flat discs; G's, hexagons. Then the group tested DNA
strands that were a combination of two bases, for example, 10 T's
and 20 A's. They found that many of the bases compete with each
other resulting in intermediate shapes, although A dominates over
Next, the researchers plan to investigate exactly how DNA codes
direct nanoparticle growth. They also plan to apply their method
to synthesize other types of nanomaterials with novel
The National Science Foundation supported this work.
Zidong Wang, Longhua Tang, Li Huey Tan, Jinghong Li, Yi Lu.
"Discovery of the DNA “Genetic Code” for Abiological Gold
Angewandte Chemie International Edition
NUCLEIC ACID-MEDIATED SHAPE CONTROL OF NANOPARTICLES FOR
Inventor(s): WANG ZIDONG [US]; LU YI [US];
ZHANG JIEQIAN [US]; KENIS PAUL J A [US]; WONG NGO YIN [US]
'':'none')">+ (WANG ZIDONG, ; LU YI, ; ZHANG JIEQIAN, ; KENIS
PAUL J. A, ; WONG NGO YIN)
Applicant(s): UNIV ILLINOIS + (THE BOARD OF
TRUSTEES OF THE UNIVERSITY OF ILLINOIS)
Classification: - international:
A61K31/7088; A61K49/00; A61K9/51; A61M37/00; B05D7/00;
B32B5/16; C12N5/071; C12Q1/02; B82Y5/00
- European: A61K31/7088; A61K41/00U;
-- Embodiments of
a method for nucleic acid-mediated control of a nanoparticle shape
are disclosed. In some embodiments, one or more nucleic acid
oligomers are adsorbed to a metal nanoseed, and additional metal
is deposited onto the nanoseed to produce a shaped nanoparticle.
In certain embodiments, the nanoseed is gold and the oligomers are
5-100 nucleotides in length. The nanoparticle shape is determined
at least in part by the nucleic acid sequence of the oligomer(s).
Shaped nanoparticles produced by embodiments of the method include
nanoflowers, nanospheres, nanostars, and nanoplates. Embodiments
for using the shaped nanoparticles also are disclosed.
CROSS REFERENCE TO RELATED
 This application claims the benefit of the earlier filing
date of U.S. Provisional Patent Application No. 61/404,410, filed
Sep. 30, 2010, which application is incorporated herein by
reference in its entirety.
ACKNOWLEDGMENT OF GOVERNMENT
 This invention was made with government support under Grant
Nos. CMMI0749028, CTS0120978, and DMR0117792 awarded by the
National Science Foundation. The government has certain rights in
 Embodiments of a method for using nucleic acid molecules to
control the growth and shape of nanoparticles are disclosed, as
well nanoparticles and methods of using such nanoparticles.
 Metal nanoparticles have unique physicochemical properties
leading to potential applications in selective catalysis,
sensitive sensing, enhanced imaging, and medical
treatment.<1-9, 53, 54 >The properties of a metal
nanoparticle typically are affected by its size, shape, and
crystal structure, and therefore it is possible to tune the
properties of the particle by controlling its growth process.
Molecular capping agents such as organic surfactants and polymers
have been used to direct nanocrystal growth in a face-selective
fashion to produce shape-controlled nanoparticle synthesis.<8,9
>Despite tremendous progress made, the mechanism of the shape
control is not well understood, in part due to the difficulty in
defining structures and conformations of these surfactants and
polymers in solution and in systematic variation of functional
 DNA is a biopolymer with more defined structure and
conformation in solution and unique programmable nature to tune
its functional properties.<10-13 >Because of these
advantages, DNA has been used as a template to position
nanoparticles through DNA metallization,<14,15 >or
nanoparticle attachment,<16-21 >or to control the sizes
and/or the photo-luminescent properties of quantum dots.<22-28
>However, in contrast to proteins or peptides,<29-32 >DNA
has been much less explored to control the shape or morphology of
metal nanoparticles, and, therefore the promise of this field
remains to be fully realized. Such an investigation may result in
new nanoparticles with new shapes and offer deeper insights into
mechanisms of shape control.
 Embodiments of a method to use DNA and/or RNA for
modulating the shape and thus the optical properties of
nanoparticles are disclosed. Systematic variations of the nucleic
acid sequences offer mechanistic insights into the morphology
control. Nucleic acid molecules in such nanoparticles maintain
their bioactivity, allowing programmable assembly of new
nanostructures. In addition, the cell uptake ability and light
scattering property of the flower-shaped nanoparticles are also
demonstrated. In some embodiments, the nucleic acid-mediated
nanoparticle synthesis method is applied to synthesize
non-spherical gold nanoparticles with new shapes by using other
nanoseeds such as nanoprisms or nanorods.
 Embodiments of a method for controlling the shape of a
nanoparticle using nucleic acid (DNA and/or RNA) oligomers are
disclosed. In some embodiments, the method includes providing a
metal nanoseed, adsorbing a plurality of nucleic acid oligomers to
the metal nanoseed, wherein each nucleic acid oligomer has a
nucleic acid sequence, and depositing metal onto the metal
nanoseed to produce a shaped nanoparticle, wherein the shaped
nanoparticle has a shape determined at least in part by the
nucleic acid sequence of the oligomer. In some embodiments,
inorganic nanoseeds such as silica or metal oxide nanoseeds are
used. Following adsorption of the nucleic acid oligomers to the
inorganic nanoseed, additional inorganic material is deposited
onto the nanoseed to produce a shaped nanoparticle.
 In some embodiments, the metal nanoseed is gold. In certain
embodiments, the metal nanoseed is coated with citrate before
adsorbing the oligomer. In some embodiments, the metal nanoseed is
a nanosphere, a nanorod, or a nanoprism. In particular
embodiments, the metal nanoseed has a largest dimension ranging
from 1 nm to 1000 nm, such as from 1 nm to 25 nm, 1 nm to 50 nm, 1
nm to 100 nm, 1 nm to 250 nm, 1 nm to 500 nm, 5 nm to 20 nm, 5 nm
to 50 nm, 5 nm to 100 nm, 5 nm to 150 nm, 10 nm to 50 nm, 10 nm to
100 nm, 10 nm to 500 nm, 10 nm to 1000 nm.
 In some embodiments, each nucleic acid oligomer has a DNA
sequence selected from poly A, poly C, poly G, poly T, or a
sequence with mixed nucleotide of A, C, G, and/or T. In other
embodiments, the oligomer is an RNA oligomer, and the RNA sequence
is poly A, poly C, poly G, poly U, or a sequence with mixed
nucleotides of A, C, G, and/or U. In some embodiments, the
oligomer is an aptamer. In certain embodiments, the oligomer has
at least 5 nucleotides, such as at least 10, at least 50, or at
least 100 nucleotides, such as 5 to 100 nucleotides. In certain
embodiments, the oligomer is labeled with a detectable label. In
some embodiments, a plurality of oligomers is adsorbed to the
metal nanoseed. In particular embodiments, the sequence of each of
the plurality of oligomers is the same.
 In some embodiments, the metal nanoseed is a gold
nanosphere, a plurality of DNA oligomers is adsorbed to the gold
nanosphere, wherein each of the plurality of DNA oligomers has a
DNA sequence consisting of poly A, poly C, or a mixture of A and
C, and depositing gold onto the gold nanosphere produces a
nanoflower. In other embodiments, each of the plurality of DNA
oligomers has a DNA sequence consisting of poly T, and depositing
gold onto the gold nanosphere produces a spherical nanoparticle.
 In some embodiments, the metal nanoseed is a gold
nanoprism, a plurality of DNA oligomers are adsorbed to the gold
nanoprism, wherein each of the plurality of DNA oligomers has a
DNA sequence consisting of poly T or a mixture of T in majority
and C in minority, and depositing gold onto the gold nanoprism
produces a six-angled nanostar. In some embodiments, each of the
plurality of DNA oligomers has a DNA sequence consisting of poly
G, or a mixture of G in majority and T in minority, and depositing
gold onto the gold nanoprism produces a nanostar with multiple
tips. In other embodiments, each of the plurality of DNA oligomers
has a DNA sequence consisting of poly A, poly C, or a mixture of A
and C, and depositing gold onto the gold nanoprism produces a
 Also disclosed are embodiments of shaped nanoparticles
including a metal nanoparticle and a plurality of oligomers
extending from the metal nanoparticle, wherein at a least a
portion of each of the plurality of oligomers is embedded within
the metal nanoparticle. In some embodiments, the oligomers are at
least 5 nucleotides, such as at least 10, at least 50, or at least
100 nucleotides, such as 5 to 100 nucleotides in length. In
particular embodiments, the metal nanoparticle is gold.
 In some embodiments, the metal nanoparticle is gold, the
oligomers are DNA oligomers that are at least 5 nucleotides, such
as at least 10, at least 50, or at least 100 nucleotides, such as
5 to 100 nucleotides in length, each of the DNA oligomers has a
DNA sequence consisting of poly A, poly C, or a mixture of A and
C, and the shaped nanoparticle is a nanoflower or a nanoplate. In
other embodiments, each of the DNA oligomers has a DNA sequence
consisting of poly T, poly G or a mixture of T and G, and the
shaped nanoparticle is a nanosphere or a nanostar.
 In some embodiments, the oligomers are RNA oligomers that
are at least 5 nucleotides, such as at least 10, at least 50, or
at least 100 nucleotides, such as 5 to 100 nucleotides in length,
and each of the RNA oligomers has an RNA sequence consisting of
poly A, poly C, poly G, poly U, or a mixture of A, C, G, and/or U.
 Embodiments of methods of using the shaped nanoparticles
also are disclosed. In some embodiments, the shaped nanoparticle
is delivered to a target cell by contacting the shaped
nanoparticle with a target cell under conditions that allow the
shaped nanoparticle to enter or bind to the cell. In certain
embodiments, the shaped nanoparticle is conjugated to an antibody
specific for a protein on the surface of the target cell, thereby
delivering the shaped nanoparticle to the target cell. In
particular embodiments, the shaped nanoparticle comprises
oligomers including an aptamer sequence extending from the shaped
nanoparticle, wherein the aptamer sequence is capable of binding
to the target cell (e.g., to a protein on the surface of the
target cell), thereby delivering the shaped nanoparticle to the
target cell. In certain embodiments, the target cell is in a
subject, and contacting comprises administering the shaped
nanoparticle to the subject.
 Embodiments of methods of using the shaped nanoparticles
also are disclosed. In some embodiments, the shaped nanoparticle
is delivered to a target cell by contacting the shaped
nanoparticle with a target cell under conditions that allow the
shaped nanoparticle to bind to and/or enter the cell, wherein the
shaped nanoparticle comprises DNA or RNA aptamers specific for the
target cell, thereby delivering the shaped nanoparticle to a
target cell. In certain embodiments, the target cell is in a
subject, and contacting comprises administering the shaped
nanoparticle to the subject.
 In some embodiments, the shaped nanoparticle is imaged
after delivery to the target cell. In other embodiments, after the
shaped nanoparticle is delivered to the target cell in the
subject, near-infrared radiation is administered to the subject,
wherein the shaped nanoparticle absorbs at least a portion of the
near-infrared radiation, thereby producing a temperature increase
within the shaped nanoparticle.
 In some embodiments, a drug is delivered within a cell by
contacting an embodiment of a shaped nanoparticle with the cell,
wherein the shaped nanoparticle comprises a drug molecule
conjugated to the shaped nanoparticle to produce a drug-shaped
nanoparticle conjugate, and wherein the drug-shaped nanoparticle
conjugate is contacted with the cell under conditions sufficient
to allow the cell to bind to and/or internalize the drug-shaped
nanoparticle conjugate. In certain embodiments, the cell is in a
subject, and contacting comprises administering a therapeutic
amount of the drug-shaped nanoparticle to the subject.
 The foregoing and other objects and features of the
disclosure will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
BRIEF DESCRIPTION OF THE DRAWINGS
 The patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee.
 FIG. 1a depicts UV-visible
spectra of gold nanoparticle solutions prepared with A30
oligomers (AuNF_A30, dark blue line), C30 oligomers (AuNF_C30,
blue line), T30 oligomers (AuNF_T30, red line), in the absence
of DNA (AuNF_No DNA, pink line), or before reduction (AuNS/No
reduction, light pink line); FIG. 1b is a series of color
photographs of the corresponding gold nanoparticles.
 FIGS. 2a-d are a series of
transmission electron microscopy (TEM) images of gold
nanoparticles prepared with (a) A30 oligomers, (b), C30
oligomers, (c) T30 oligomers, (d) in the absence of DNA. The
scale bar indicates 20 nm.
 FIG. 3 is a TEM image of
gold nanoparticles prepared with G10 oligomers. The scale bar
indicates 20 nm.
 FIG. 4a is a TEM image of
200-nm gold nanoseeds (AuNS).
 FIG. 4b is a TEM image of
gold nanoparticles prepared in the absence of DNA but with the
addition of 20 mM NaCl. It is noted that aggregation of the gold
nanoparticles occurred during synthesis.
 FIGS. 5a-5d are color
photographs of AuNS solutions incubated with (a) A30 oligomers,
(b) C30 oligomers, (c) T30 oligomers, and (d) in the absence of
DNA before (left image of each pair) and after (right image of
each pair) the addition of 0.1 M NaCl.
 FIG. 5e is a series of
UV-visible spectra of the corresponding nanoparticle solutions
with and without the presence of 0.1 M NaCl.
 FIGS. 6a-f are TEM images
of gold nanoparticles prepared by reducing (a) 0.05 [mu]L, (b)
0.1 [mu]L, (c) 0.4 [mu]L, (d) 0.6 [mu]L, (e) 1.2 [mu]L, and (f)
2.0 [mu]L of 1% HAuCl4 aqueous solution with an excess amount of
NH2OH (20 mM). Before the reduction reaction, 100 [mu]L of 0.5
nM AuNS solution was incubated with 1 [mu]M poly A30. The scale
bar indicates 20 nm.
 FIGS. 7a-f are TEM images
of gold nanoparticles prepared by incubating AuNS solutions with
poly A30 at different molar ratios: AuNS:DNA=(a) 1:20, (b)
1:100, (c) 1:500, (d) 1:1000, (e) 1:2000, (f) 1:4000. The AuNS
solutions (0.5 nM) were incubated with DNA for 30 minutes,
followed by addition of 20 mM NH2OH and 167 [mu]M HAuCl4 to
complete the nanoparticle synthesis. The scale bar indicates 20
 FIGS. 8a-b are TEM images
of gold nanoparticles prepared with (a) adenosine monophosphate
(AMP), and (b) random 30-mer DNA. A similar synthesis procedure
was followed except that 0.5 nM AuNS was incubated with 30 [mu]M
AMP or 1 [mu]M random DNA with the sequence 5'-AGT CAC GTA TAC
AGC TCA TGA TCA GTC AGT-3' (SEQ ID NO: 3). The scale bar
indicates 20 nm.
 FIG. 9 depicts the
time-dependent evolution of the UV-visible spectra of gold
nanoflowers (AuNF) grown in the presence of A30 oligomers. From
bottom to top, the spectra illustrate the absorbance of the
growth solution after initiation of the reaction for 0 s, 3 s, 5
s, 10 s, 30 s, 60 s, 120 s, 240 s, 480 s, 720 s, and 840 s,
 FIGS. 10a-r are TEM images
of the nanoparticle intermediates prepared by stopping the
nanoparticle growth with mercaptopropionic acid (1.5 mM) after
0.5 s (a, g, m), 2 s (b, h, n), 5 s (c, i, o), 30 s (d, j, p), 5
min. (e, k, q) and 15 min. (f, l, r) of the reaction. The images
in the top row (a-f) represent the intermediates synthesized in
the presence of poly A30 oligomers; the images in the second row
(g-l) represent the intermediates synthesized in the presence of
poly T30 oligomers; the images in the last row (m-r) represent
the intermediates synthesized in the absence of DNA. Before
initiation of the reduction reaction, 100 [mu]L of 0.5 nM AuNS
solution was incubated with 1 [mu]M DNA. The scale bar indicates
 FIG. 11 is a TEM image of
small gold nanoparticles produced from the conversion of
Au(I)-mercaptopropionic acid complexes into metal particles on
the TEM grid upon electron-beam irradiation during TEM imaging.
HAuCl4 (167 [mu]M) was mixed with mercaptopropionic acid (1.5
mM), and the mixture was dropped on the TEM grid. The TEM image
was taken after the sample was dried. The scale bar indicates 20
 FIG. 12 is a schematic
illustration of one embodiment of a method for DNA-mediated
shape control of gold nanoparticles. Poly A (SEQ ID NO: 4); Poly
T (SEQ ID NO: 5); Poly C (SEQ ID NO: 6).
 FIG. 13 depicts melting
curves of the DNA on AuNFs (circles) and free DNA in solution
(squares). Both melting curves were obtained using buffer
containing 10 mM HEPES buffer (pH 7.1) and 50 mM NaCl.
 FIGS. 14a-d are TEM images
of nanoassemblies: (a) AuNF_A30 with AuNS5nm-S_T30; (b) AuNF_A30
with non-complementary AuNS5nm-S_A30; (c) AuNS_T30 with
AuNS5nm-S_A30; (d) AuNS_T30 with non-complementary
AuNS5nm-S_T30. The scale bar indicates 20 nm.
 FIGS. 15a-d are TEM images
of nanoassemblies: (a, b) AuNF_A30 with AuNS5nm-S_T30; (c, d)
AuNF_A30 with non-complementary AuNS5nm-S_A30. The scale bar
indicates 100 nm.
 FIG. 16 depicts Raman
spectra of the Raman tag (Trama) from AuNFs (upper line) and
AuNSs (lower line). The samples were excited with 603 nm laser.
 FIG. 17 is a dark-field
light-scattering image of gold nanoflowers. The scale bar
indicates 2 [mu]m.
 FIGS. 18a-b are dark-field
images of Chinese hamster ovary (CHO) cells (a) treated with
AuNF particles, (b) without nanoparticle treatment. The scale
bar indicates 10 [mu]m.
 FIGS. 19a-h are optical
and confocal fluorescence images of CHO cells treated with AuNF
nanoparticles synthesized with FAM-A30 (a-d) or without
nanoparticle treatment (e-h). FIG. 19a is a brightfield image of
the AuNF treated cells; FIGS. 19b-d are corresponding 3-D
reconstructed confocal fluorescence images of the AuNF treated
cells (b: top view; c, d: side views; unit scale: 1 [mu]m); FIG.
19e is a brightfield image of the control cells; FIGS. 19f-h are
corresponding 3-D reconstructed confocal fluorescence images of
the control cells (f: top view; g, h: side views; unit scale: 1
[mu]m). The scale bars in FIGS. 19a and 19e indicate 10 [mu]m.
The AuNFs (1 nM) were incubated with CHO cells for 20 hours
before imaging. The fluorescence arises from the incomplete
quenching of fluorophore by the gold nanoparticles. It was shown
that the fluorescent dots were distributed inside the cells,
indicating that the AuNFs were taken up by the cells after
incubation. As a comparison, the control cells without
nanoparticle treatment showed little fluorescence.
 FIGS. 20a-d are TEM images
of nanoparticles synthesized with A30 oligomers (a), T30
oligomers (b), C30 oligomers (c) and G10 oligomers (d) by using
gold nanoprisms as seeds.
 FIGS. 21a-c are TEM images
of nanorod seeds before reaction (a), and nanoparticles
synthesized with A30 oligomers (b), and T30 oligomers (c) using
the gold nanorod seeds.
 FIGS. 22a-d are TEM images
of nanoflowers synthesized with increasing concentrations of
 FIGS. 23a-b are graphs of
size versus gold salt concentration, demonstrating a linear
relationship between gold salt concentration and nanoflower
size. The nanoflowers were synthesized with a randomized DNA
construct (a) or an AS1411 aptamer (b); 50 particles were
counted to determine size.
 FIGS. 24a-24c are TEM
images of gold nanoflowers grown from 15-nm, 30-nm, and 50-nm
gold nanoparticles, respectively.
 FIG. 25 is a graph
illustrating the absorption spectra of gold nanoflowers grown
from 15-nm, 30-nm, and 50-nm gold nanoparticles.
 FIGS. 26a-26b are
dark-field optical images of MCF-7 cells incubated with
nanoflowers comprising control DNA (a) or nanoflowers comprising
the AS1411 aptamer (b). The images were obtained under identical
conditions and microscope settings.
 The nucleic acid sequences provided herein are shown using
standard letter abbreviations for nucleotide bases as defined in
37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is
shown, but the complementary strand is understood as included by
any reference to the displayed strand. The sequence listing is
submitted as an ASCII text file, named 7950-85921-02_ST25.txt,"
created on Sep. 27, 2011, 2011, 1.83 KB, which is incorporated by
 SEQ ID NO: 1 is a randomized control DNA sequence.
 SEQ ID NO: 2 is a DNA sequence including the AS1411 aptamer
 SEQ ID NO: 3 is a randomized DNA sequence.
 SEQ ID NO: 4 is a Poly A sequence.
 SEQ ID NO: 5 is a Poly T sequence.
 SEQ ID NO: 6 is a Poly C sequence.
 Embodiments of a method for using nucleic acids to control
nanoparticle shape are disclosed. The nucleic acids may be DNA or
RNA. Single strand DNA (ssDNA) has been found to adsorb on
citrate-coated gold nanospheres (AuNSs) in a sequence-dependent
manner.<33 >Deoxynucleosides dA, dC, dG have shown much
higher binding affinity to gold surfaces than deoxynucleoside
dT.<34 >To investigate the effect of different DNA sequences
on nanoparticle morphology during crystal growth, various DNA
oligomers were bound to gold nanoseeds, additional metal was
deposited onto the DNA-nanoseed constructs, and the resulting
nanoparticle morphology was determined.
 Nanoparticles made by some embodiments of the disclosed
method can be taken up by cells. Because metallic nanoparticles
can be visualized by, e.g., darkfield microscopy, such
nanoparticles may be useful for intracellular imaging.
Additionally, nanoparticles that can be taken up by cells may be
useful carriers for delivering drugs, contrast agents, genes, and
other molecules into cells.
I. TERMS AND ABBREVIATIONS
 The following explanations of terms and abbreviations are
provided to better describe the present disclosure and to guide
those of ordinary skill in the art in the practice of the present
disclosure. As used herein, "comprising" means "including" and the
singular forms "a" or "an" or "the" include plural references
unless the context clearly dictates otherwise. The term "or"
refers to a single element of stated alternative elements or a
combination of two or more elements, unless the context clearly
 Unless explained otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only
and not intended to be limiting. Other features of the disclosure
are apparent from the following detailed description and the
 Unless otherwise indicated, all numbers expressing
quantities of components, molecular weights, percentages,
temperatures, times, and so forth, as used in the specification or
claims are to be understood as being modified by the term
"about."Accordingly, unless otherwise indicated, implicitly or
explicitly, the numerical parameters set forth are approximations
that may depend on the desired properties sought and/or limits of
detection under standard test conditions/methods. When directly
and explicitly distinguishing embodiments from discussed prior
art, the embodiment numbers are not approximates unless the word
"about" is recited.
 Definitions of common terms in chemistry may be found in
Richard J. Lewis, Sr. (ed.), Hawley's Condensed Chemical
Dictionary, published by John Wiley & Sons, Inc., 1997 (ISBN
0-471-29205-2). All references herein are incorporated by
reference. In order to facilitate review of the various
embodiments of the disclosure, the following explanations of
specific terms are provided:
 Administration: To provide or give a subject an agent, such
as a nanoparticle preparation described herein, by any effective
route. Exemplary routes of administration include, but are not
limited to, topical, injection (such as subcutaneous,
intramuscular, intradermal, intraperitoneal, intratumoral, and
intravenous), oral, sublingual, rectal, transdermal, intranasal,
vaginal and inhalation routes.
 Adsorption: The physical adherence or bonding of ions and
molecules onto the surface of another molecule or substrate. An
ion or molecule that adsorbs is referred to as an adsorbate.
Adsorption can be characterized as chemisorption or physisorption,
depending on the character and strength of the bond between the
adsorbate and the substrate surface. Chemisorption is
characterized by a strong interaction between an adsorbate and a
substrate, e.g., formation of covalent and/or ionic bonds.
Physisorption is characterized by weaker bonding between an
adsorbate and a substrate. The weaker bond typically results from
van der Waals forces, i.e., an induced dipole moment between the
adsorbate and the substrate.
 Antibody: A polypeptide ligand comprising at least a light
chain or heavy chain immunoglobulin variable region which
specifically recognizes and binds an epitope of an antigen, such
as a tumor-specific protein. Antibodies are composed of a heavy
and a light chain, each of which has a variable region, termed the
variable heavy (VH) region and the variable light (VL) region.
Together, the VH region and the VL region are responsible for
binding the antigen recognized by the antibody.
 Antibodies include intact immunoglobulins and the variants
and portions of antibodies well known in the art, such as Fab
fragments, Fab' fragments, F(ab)'2 fragments, single chain Fv
proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv").
A scFv protein is a fusion protein in which a light chain variable
region of an immunoglobulin and a heavy chain variable region of
an immunoglobulin are bound by a linker, while in dsFvs, the
chains have been mutated to introduce a disulfide bond to
stabilize the association of the chains. The term also includes
genetically engineered forms such as chimeric antibodies (for
example, humanized murine antibodies), heteroconjugate antibodies
(such as, bispecific antibodies). See also, Pierce Catalog and
Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby,
J., Immunology, 3<rd >Ed., W.H. Freeman & Co., New York,
 Typically, a naturally occurring immunoglobulin has heavy
(H) chains and light (L) chains interconnected by disulfide bonds.
There are two types of light chain, lambda ([lambda]) and kappa
(k). There are five main heavy chain classes (or isotypes) which
determine the functional activity of an antibody molecule: IgM,
IgD, IgG, IgA and IgE.
 Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). In
combination, the heavy and the light chain variable regions
specifically bind the antigen. Light and heavy chain variable
regions contain a "framework" region interrupted by three
hypervariable regions, also called "complementarity-determining
regions" or "CDRs." The extent of the framework region and CDRs
have been defined (see, Kabat et al., Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human
Services, 1991, which is hereby incorporated by reference). The
Kabat database is now maintained online. The sequences of the
framework regions of different light or heavy chains are
relatively conserved within a species, such as humans. The
framework region of an antibody, that is the combined framework
regions of the constituent light and heavy chains, serves to
position and align the CDRs in three-dimensional space. The CDRs
are primarily responsible for binding to an epitope of an antigen.
The CDRs of each chain are typically referred to as CDR1, CDR2,
and CDR3, numbered sequentially starting from the N-terminus, and
are also typically identified by the chain in which the particular
CDR is located.
 References to "VH" or "VH" refer to the variable region of
an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv
or Fab. References to "VL" or "VL" refer to the variable region of
an immunoglobulin light chain, including that of an Fv, scFv, dsFv
 A "monoclonal antibody" is an antibody produced by a single
clone of B lymphocytes or by a cell into which the light and heavy
chain genes of a single antibody have been transfected. Monoclonal
antibodies are produced by methods known to those of skill in the
art, for instance by making hybrid antibody-forming cells from a
fusion of myeloma cells with immune spleen cells. Monoclonal
antibodies include humanized monoclonal antibodies.
 Aptamer: An oligonucleic acid that binds to a specific
target. Nucleic acid aptamers are capable of binding to various
molecular targets such as small molecules, proteins, nucleic
acids, or cells. DNA or RNA aptamers recognize target effector
molecules with high affinity and specificity (Ellington and
Szostak, Nature 346(6287):818-822, 1990; Tuerk and Gold, Science,
249:505-510, 1990). Aptamers have several unique properties.
First, aptamers for a given target can be obtained by routine
experimentation. For instance, in vitro selection methods can be
used (called systematic evolution of ligands by exponential
enrichment (SELEX)) to obtain aptamers for a wide range of target
effector molecules with exceptionally high affinity, having
dissociation constants in the picomolar range (Brody and Gold,
Reviews in Molecular Biotechnology, 74(1)5-13, 2000, Jayasena,
Clinical Chemistry, 45(9):1628-1650, 1999, Wilson and Szostak,
Ann. Rev. Biochem., 68:611-647, 1999, Ellington et al., Nature
1990, 346, 818-822; Tuerk and Gold Science 1990, 249, 505-510; Liu
et al., Chem. Rev. 2009, 109, 1948-1998; Shamah et al., Acc. Chem.
Res. 2008, 41, 130-138; Famulok, et al., Chem. Rev. 2007, 107,
3715-3743; Manimala et al., Recent Dev. Nucleic Acids Res. 2004,
1, 207-231; Famulok et al., Acc. Chem. Res. 2000, 33, 591-599;
Hesselberth, et al., Rev. Mol. Biotech. 2000, 74, 15-25; Morris et
al., Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2902-2907). Second,
aptamers are easier to obtain and less expensive to produce than
antibodies, because aptamers can be generated in vitro in short
time periods (for example, within days) and at economical cost.
Third, aptamers display remarkable structural durability and can
be denatured and renatured many times without losing their ability
to recognize their targets. The mononucleotides of an aptamer may
adopt a particular conformation upon binding to its target.
Aptamers that are specific to a wide range of targets from small
organic molecules such as adenosine, to proteins such as thrombin,
and even viruses and cells, have been identified (Chou et al.,
Trends in Biochem Sci. 2005, 30(5), 231-234; Liu et al., Chem.
Rev. 2009, 109, 1948-1998; Lee et al., Nucleic Acids Res. 2004,
32, D95-D100; Navani and Li, Curr. Opin. Chem. Biol. 2006, 10,
272-281; Song et al., TrAC, Trends Anal. Chem. 2008, 27, 108-117;
Tombelli et al., Bioelectrochemistry, 2005, 67(2), 135-141). In
one example the aptamer is specific for HIV (such as HIV-tat).
 Contacting: Placement in direct physical association,
including both a solid and liquid form. Contacting can occur in
vitro, for example, with isolated cells, such as tumor cells, or
in vivo by administering to a subject (such as a subject with a
tumor). Thus, the nanoparticles disclosed herein can be contacted
with cells in vivo or in vitro, under conditions that permit the
nanoparticle to be endocytosed into the cell.
 DNA melting temperature: The temperature at which a DNA
double helix dissociates into single strands, specifically the
temperature at which 50% of the DNA, or oligonucleotide, is in the
form of a double helix and 50% has dissociated into single
strands. The most reliable and accurate determination of melting
temperature is determined empirically. Methods for determining the
melting temperature of DNA are known to those with ordinary skill
in the art of DNA characterization. For single-stranded oligomers,
a complementary oligonucleotide is hybridized to the oligomer, and
the melting temperature of the double-stranded complex is
 Nanoflower (NF): A nanoparticle with a morphology in
microscopic view that resembles a flower.
 Nanoparticle (NP): A nanoscale particle with a size that is
measured in nanometers, for example, a particle that has at least
one dimension of less than about 100 nm. Nanoparticles may have
different shapes, e.g., nanofibers, nanoflowers, nanohorns,
nano-onions, nanopeanuts, nanoplates, nanoprisms, nanorods,
nanoropes, nanospheres, nanostars, nanotubes, etc.
 Nanoplate: A nanoparticle with a morphology in microscopic
view that resembles a substantially flat plate.
 Nanoseed (NS): A small nanoparticle used as a starting
material for larger nanoparticle synthesis. For example, gold ions
may be reduced and deposited onto gold nanoseeds to produce larger
 Nanostar: A nanoparticle with a morphology in microscopic
view that resembles a star.
 Near-infrared (NIR): The infrared spectrum is typically
divided into three sections, with near-infrared including the
shortest wavelengths. Although the region is not rigidly defined,
NIR typically encompasses light with wavelengths ranging from
 An oligomer is a general term for a polymeric molecule
consisting of relatively few monomers, e.g., 5-100 monomers. In
one example, the monomers are nucleotides.
 Pharmaceutically acceptable vehicles: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W.
Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995),
describes compositions and formulations suitable for
pharmaceutical delivery of the nanoparticles disclosed herein.
 In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids
such as water, physiological saline, balanced salt solutions,
aqueous dextrose, glycerol or the like as a vehicle. For solid
compositions (for example, powder, pill, tablet, or capsule
forms), conventional non-toxic solid carriers can include, for
example, pharmaceutical grades of mannitol, lactose, starch, or
magnesium stearate. In addition to biologically-neutral carriers,
pharmaceutical compositions to be administered can contain minor
amounts of non-toxic auxiliary substances, such as wetting or
emulsifying agents, preservatives, and pH buffering agents and the
like, for example sodium acetate or sorbitan monolaurate.
 A polymer is a molecule of repeating structural units
(e.g., monomers) formed via a chemical reaction, e.g.,
 "Specifically binds" refers to the ability of a molecule to
bind with specificity to a particular target. For example,
"specifically binds" refers to the ability of an individual
aptamer to specifically bind to a molecular target such as a small
molecule, a protein, a particular nucleic acid sequence, or a
 "Specifically binds" also refers to the ability of
individual antibodies to specifically immunoreact with an antigen,
such as a tumor-specific antigen, relative to binding to unrelated
proteins, such as non-tumor proteins, for example [beta]-actin.
For example, a HER2-specific binding agent binds substantially
only the HER-2 protein in vitro or in vivo. As used herein, the
term "tumor-specific binding agent" includes tumor-specific
antibodies and other agents that bind substantially only to a
tumor-specific protein in that preparation.
 The binding is a non-random binding reaction between an
antibody molecule and an antigenic determinant of the T cell
surface molecule. The desired binding specificity is typically
determined from the reference point of the ability of the antibody
to differentially bind the T cell surface molecule and an
unrelated antigen, and therefore distinguish between two different
antigens, particularly where the two antigens have unique
epitopes. An antibody that specifically binds to a particular
epitope is referred to as a "specific antibody".
 In some examples, an antibody (such as an antibody
conjugated to a nanoparticle of the present disclosure)
specifically binds to a target (such as a cell surface protein)
with a binding constant that is at least 10<3 >M<-1
>greater, 10<4>M<-1 >greater or 10<5 >M<-1
>greater than a binding constant for other molecules in a
sample or subject. In some examples, an antibody (e.g., monoclonal
antibody) or fragments thereof, has an equilibrium constant (Kd)
of 1 nM or less. For example, an antibody binds to a target, such
as tumor-specific protein with a binding affinity of at least
about 0.1*10<-8 >M, at least about 0.3*10<-8 >M, at
least about 0.5*10<-8 >M, at least about 0.75*10<-8
>M, at least about 1.0*10<-8 >M, at least about
1.3*10<-8 >M at least about 1.5*10<-8>M, or at least
about 2.0*10<-8 >M. Kd values can, for example, be
determined by competitive ELISA (enzyme-linked immunosorbent
assay) or using a surface-plasmon resonance device such as the
Biacore T100, which is available from Biacore, Inc., Piscataway,
 Subject or patient: A term that includes human and
non-human mammals. In one example, the subject is a human or
veterinary subject, such as a mouse.
 Therapeutically effective amount: An amount of a
composition that alone, or together with an additional therapeutic
agent(s) (such as a chemotherapeutic agent) sufficient to achieve
a desired effect in a subject, or in a cell, being treated with
the agent. The effective amount of the agent (such as the
nanoparticles disclosed herein) can be dependent on several
factors, including, but not limited to the subject or cells being
treated, the particular therapeutic agent, and the manner of
administration of the therapeutic composition. In one example, a
therapeutically effective amount or concentration is one that is
sufficient to prevent advancement, delay progression, or to cause
regression of a disease, or which is capable of reducing symptoms
caused by the disease, such as cancer. In one example, a
therapeutically effective amount or concentration is one that is
sufficient to increase the survival time of a patient with a
 In one example, a desired response is to reduce or inhibit
one or more symptoms associated with cancer. The one or more
symptoms do not have to be completely eliminated for the
composition to be effective. For example, administration of a
composition containing a nanoparticle disclosed herein, which in
some examples is followed by photothermal therapy can decrease the
size of a tumor (such as the volume or weight of a tumor, or
metastasis of a tumor) by a desired amount, for example by at
least 20%, at least 50%, at least 80%, at least 90%, at least 95%,
at least 98%, or even at least 100%, as compared to the tumor size
in the absence of the nanoparticle. In one particular example, a
desired response is to kill a population of cells by a desired
amount, for example by killing at least 20%, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 98%, or even at least 100% of the cells, as compared to
the cell killing in the absence of the nanoparticle. In one
particular example, a desired response is to increase the survival
time of a patient with a tumor (or who has had a tumor recently
removed) by a desired amount, for example increase survival by at
least 20%, at least 50%, at least 60%, at least 70%, at least 80%,
at least 90%, at least 95%, at least 98%, or even at least 100%,
as compared to the survival time in the absence of the
 The effective amount of the disclosed nanoparticles that is
administered to a human or veterinary subject will vary
significantly depending upon a number of factors associated with
that subject, for example the overall health of the subject. An
effective amount of an agent can be determined by varying the
dosage of the product and measuring the resulting therapeutic
response, such as the regression of a tumor. Effective amounts
also can be determined through various in vitro, in vivo or in
situ immunoassays. The disclosed agents can be administered in a
single dose, or in several doses, as needed to obtain the desired
response. However, the effective amount of the disclosed
nanoparticles can be dependent on the source applied, the subject
being treated, the severity and type of the condition being
treated, and the manner of administration. In certain examples, a
therapeutically effective dose of the disclosed nanoparticles is
at least 20 mg per kg body weight, at least 200 mg per kg, at
least 2,000 mg per kg, or at least 20 g per kg, for example when
administered intravenously (iv).
 In particular examples, a therapeutically effective dose of
an antibody conjugated to a nanoparticle of the present disclosure
is at least 0.5 milligram per 60 kilogram (mg/kg), at least 5
mg/60 kg, at least 10 mg/60 kg, at least 20 mg/60 kg, at least 30
mg/60 kg, at least 50 mg/60 kg, for example 0.5 to 50 mg/60 kg,
such as a dose of 1 mg/60 kg, 2 mg/60 kg, 5 mg/60 kg, 20 mg/60 kg,
or 50 mg/60 kg, for example when administered iv. However, one
skilled in the art will recognize that higher or lower dosages
also could be used, for example depending on the particular
nanoparticle. In particular examples, such daily dosages are
administered in one or more divided doses (such as 2, 3, or 4
doses) or in a single formulation. The disclosed nanoparticle can
be administered alone, in the presence of a pharmaceutically
acceptable carrier, in the presence of other therapeutic agents
(such as other anti-neoplastic agents).
 Treating: A term when used to refer to the treatment of a
cell or tissue with a therapeutic agent, includes contacting or
incubating an agent (such as a nanoparticle disclosed herein) with
the cell or tissue. A treated cell is a cell that has been
contacted with a desired composition in an amount and under
conditions sufficient for the desired response. In one example, a
treated cell is a cell that has been exposed to a nanoparticle
under conditions sufficient for the nanoparticle to enter the
cell, which is in some examples followed by phototherapy, until
sufficient cell killing is achieved.
 Tumor, neoplasia, malignancy or cancer: A neoplasm is an
abnormal growth of tissue or cells which results from excessive
cell division. Neoplastic growth can produce a tumor. The amount
of a tumor in an individual is the "tumor burden" which can be
measured as the number, volume, or weight of the tumor. A tumor
that does not metastasize is referred to as "benign." A tumor that
invades the surrounding tissue and/or can metastasize is referred
to as "malignant." A "non-cancerous tissue" is a tissue from the
same organ wherein the malignant neoplasm formed, but does not
have the characteristic pathology of the neoplasm. Generally,
noncancerous tissue appears histologically normal. A "normal
tissue" is tissue from an organ, wherein the organ is not affected
by cancer or another disease or disorder of that organ. A
"cancer-free" subject has not been diagnosed with a cancer of that
organ and does not have detectable cancer.
 Exemplary tumors, such as cancers, that can be treated with
the claimed nanoparticles include solid tumors, such as breast
carcinomas (e.g. lobular and duct carcinomas), sarcomas,
carcinomas of the lung (e.g., non-small cell carcinoma, large cell
carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma
of the lung, colorectal adenocarcinoma, stomach carcinoma,
prostatic adenocarcinoma, ovarian carcinoma (such as serous
cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ
cell tumors, testicular carcinomas and germ cell tumors,
pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular
carcinoma, bladder carcinoma (including, for instance,
transitional cell carcinoma, adenocarcinoma, and squamous
carcinoma), renal cell adenocarcinoma, endometrial carcinomas
(including, e.g., adenocarcinomas and mixed Mullerian tumors
(carcinosarcomas)), carcinomas of the endocervix, ectocervix, and
vagina (such as adenocarcinoma and squamous carcinoma of each of
same), tumors of the skin (e.g., squamous cell carcinoma, basal
cell carcinoma, malignant melanoma, skin appendage tumors, Kaposi
sarcoma, cutaneous lymphoma, skin adnexal tumors and various types
of sarcomas and Merkel cell carcinoma), esophageal carcinoma,
carcinomas of the nasopharynx and oropharynx (including squamous
carcinoma and adenocarcinomas of same), salivary gland carcinomas,
brain and central nervous system tumors (including, for example,
tumors of glial, neuronal, and meningeal origin), tumors of
peripheral nerve, soft tissue sarcomas and sarcomas of bone and
cartilage, and lymphatic tumors (including B-cell and T-cell
malignant lymphoma). In one example, the tumor is an
 The disclosed nanoparticles can also be used to treat
liquid tumors, such as a lymphatic, white blood cell, or other
type of leukemia.
 Under conditions sufficient for: A phrase that is used to
describe any environment that permits the desired activity. In one
example, "under conditions sufficient for" includes administering
a nanoparticle to a subject sufficient to allow the nanoparticle
to enter the cell. In particular examples, the desired activity is
killing the cells into which the nanoparticles entered, for
example following phototherapy of the cells. In another example,
"under conditions sufficient for" includes contacting DNA
oligomers with a nanoseed sufficient to allow the oligomers to
bind to the nanoseed, to form a nanoparticle of the desired shape.
II. NANOPARTICLE PREPARATION AND
NUCLEIC ACID-MEDIATED SHAPE CONTROL
 The disclosure provides nanoparticles having attached
thereto nucleic acid oligomers, wherein the DNA or RNA oligomer
can be used to control the shape of the nanoparticle. Also
provided are methods of making such shaped nanoparticles.
 In some embodiments, nanospheres (NSs) are used as
nanoseeds, or starting materials, for nanoparticle growth. In
other embodiments, the nanoseeds are nanoprisms, or nanorods. A
person of ordinary skill in the art of nanoparticle technology
will understand that nanoseeds of any shape may be used; however,
the final nanoparticle's morphology may depend at least in part
upon the shape of the nanoseed. Nanoseeds may comprise any
material to which nucleic acid oligomers can be attached. If the
nanoparticles will be administered to living subjects, it is
advantageous to use nanoseeds that do not have significant
cellular toxicity. In particular embodiments, gold nanoparticles
are produced from gold nanoseeds. Gold has very low cellular
toxicity, making gold nanoparticles (NPs) advantageous for
applications in living subjects. Other suitable materials include
other metals, such as silver and platinum, as well as inorganic
compounds (e.g., silica, metal oxide).
 Typically, the nanoseeds have a largest dimension, or
diameter, between 1 nm to 1000 nm, such as from 1 nm to 25 nm, 1
nm to 50 nm, 1 nm to 100 nm, 1 nm to 250 nm, 1 nm to 500 nm, 5 nm
to 20 nm, 5 nm to 50 nm, 5 nm to 100 nm, 5 nm to 150 nm, 10 nm to
50 nm, 10 nm to 100 nm, 10 nm to 500 nm, 10 nm to 1000 nm. In some
embodiments, AuNSs with a diameter of 5-20 nm were used.
 In some embodiments, nucleic acid oligomers comprising a
single type of nucleotide are used (e.g., poly A). In other
embodiments, the oligomers may include more than one type of
nucleotide (such as an oligomer containing a mixture of A and C).
Oligomers containing five or more nucleotides are suitable for use
in the disclosed embodiments. Oligomers with fewer than 5
nucleotides are too short to significantly influence the
nanoparticle morphology. The oligomers disclosed herein can be at
least 5 nucleotides in length, such as at least 10, at least 20,
at least 30, or at least 60 nucleotides in length, such as 5 to
100 nucleotides in length, 5 to 60 nucleotides, or 10 to 30
nucleotides in length. In some embodiments, all of the oligomers
bound to the NS have the same sequence and the same length. In
other embodiments, oligomers of differing sequences and/or
differing lengths may be used. In a working embodiment, 30-mer
DNAs consisting of either poly A, poly C, or poly T (designated as
A30, C30, and T30, respectively) were bound to AuNSs.
 In some embodiments, the oligomers may be modified or
labeled with a detectable label. Suitable detectable labels may
include, but are not limited to, fluorophores (e.g., fluorescein
dyes, Alexa Fluor(R) dyes, etc.), radioisotopes, biotin,
photo-sensitive linkers, and chemical functional groups (e.g.,
alkynyl, azide, carboxyl, etc.).
 In some embodiments, gold nanoseeds are coated with citrate
during nanoseed synthesis. Oligomers adsorb to the citrate-coated
AuNS surface via physisorption.
 After nucleic acid (NA) oligomers are adhered to the NS
surface, additional material is deposited onto the nucleic
acid-nanoseed (NA-NS) construct to produce nanoparticle growth. In
some embodiments, the nanoseed is gold, and nanoparticle growth is
achieved through gold ion reduction and deposition onto the
NA-functionalized AuNS surface. In working embodiments, hydrogen
tetrachloroaurate(III) (HAuCl4) was used as the gold ion source.
However, other soluble gold salts also may be used. Hydroxylamine
(NH2OH) is a suitable reducing agent for reducing HAuCl4 catalyzed
by the gold surface.<35 >Other reducing agents also may be
used, e.g., ascorbic acid, amines (poly(allylamine)
hydrochloride<51>, sodium diphenylamine
 Nanoparticle size is controlled by varying the size of the
nanoseed and/or varying the growth conditions. In some
embodiments, a set of growth conditions is selected to minimize
the amount of gold deposited onto the nanoseed. For example, the
amount of HAuCl4 can be limited and controlled to precisely
control the size of the resulting nanoparticle. In certain
embodiments, the nanoparticle has a largest dimension, or diameter
of 5-1,000 nm, such as 10-500 nm, 10-250 nm, or 20-200 nm.
Typically, particles with a largest dimension between 20 nm and
200 nm are suitable for in vivo applications. Nanoparticle size is
a result-effective variable that may influence uptake activity for
nanoparticles having a particular shape, surface
functionalization, and/or environment.
 The oligomer sequence affects the morphology of the
nanoparticle. In a working embodiment, gold nanoparticles were
synthesized in the presence of A30, C30, or T30 oligomers. DNA
oligomers were adsorbed onto small gold nanospheres. Gold ions in
solution subsequently were reduced and deposited onto the
DNA-nanosphere constructs to cause nanoparticle growth. Using
transmission electron microscopy to determine the nanoparticles'
morphology, the inventors unexpectedly discovered that the
nanoparticles synthesized in the presence of A30 and C30 were
flower shaped (FIGS. 2a-2b), while nanoparticles synthesized in
the presence of T30 were spherical (FIG. 2c). Nanoparticles
synthesized in the absence of DNA also were spherical (FIG. 2d),
as were nanoparticles synthesized in the presence of a 10-mer of
poly G (FIG. 3). Thus, it is apparent that the nucleic acid
sequence mediated the nanoparticle growth and controlled the
resulting shape of the nanoparticle.
 The length and number of oligomers adsorbed to the nanoseed
also significantly affect the nanoparticle shape. As previously
discussed, shorter oligomers (e.g., those with fewer than 5
nucleotides) have a lesser influence on the nanoparticle shape.
Furthermore, the number of oligomers adsorbed to the nanoseed
significantly affects the nanoparticle morphology. As shown in
FIGS. 7a-f, shape control becomes increasingly evident as the
number of oligomers increases.
 Thiol chemistry can be used to conjugate DNA and RNA to
gold surfaces. However, when thiolated (i.e., thiol-modified) DNA
is adsorbed onto gold nanospheres, all of the thiolated DNA can be
displaced by mercaptoethanol. In contrast, embodiments of AuNFs
produced with unmodified poly A oligonucleotides by the methods
disclosed herein are resistant to mercaptoethanol displacement,
and incubation with mercaptoethanol overnight displaces less than
one-third of the DNA strands. Thus, some embodiments of the
disclosed in situ synthesis and controlled reduction methods
advantageously can be used to prepare stable DNA-functionalized
gold surfaces with unmodified DNA. Certain embodiments of gold
nanoflowers produced by the disclosed methods are very stable in
aqueous solution, even in the presence of 0.3 M salt,
demonstrating that unmodified DNA oligomers can be attached to the
nanoparticles during their synthesis, and act as stabilizing
 Considering the remarkably high binding affinity of DNA to
the AuNFs (higher than thiol-gold binding), it was hypothesized
that the DNA in situ attached to AuNFs during reduction could be
partially buried in the AuNFs. As additional gold is deposited
onto the DNA-functionalized nanoseed, a portion of the DNA strand
becomes buried in the deposited gold, thereby firmly attaching the
DNA oligomer to the nanoparticle during nanoparticle growth.
Because the melting point of a DNA oligonucleotide bound to a
complementary oligonucleotide increases with the length of the
oligonucleotide, an attached DNA oligonucleotide may have a lower
melting point than that of a free oligonucleotide if a portion of
the attached oligonucleotide is buried within the gold
nanoparticle. In some embodiments, the attached oligonucleotides
have a melting point that is at least 10% or at least 20% (such as
10-20%) lower than that of the corresponding free
oligonucleotides, substantiating the hypothesis that a portion of
the DNA strand is embedded within the gold nanoparticle during
nanoparticle growth. In certain embodiments, it is preferable to
control nanoparticle size by varying the nanoseed size rather than
by varying the thickness of the deposited gold. Varying the
nanoseed size while minimizing the thickness of the deposited gold
allows minimal "trapping" of the DNA sequence by the growing gold
 To produce flower-shaped gold nanoparticles, the DNA
oligomer has at least 5 nucleotides. DNA oligomers with fewer
nucleotides are not long enough to significantly influence the
nanoparticle morphology. As discussed above, DNA oligomers
comprising poly A and poly C produced flower-shaped nanoparticles,
while DNA oligomers comprising poly T and poly G produced
spherical nanoparticles. It was observed that poly G oligomers
longer than 10 nucleotides had secondary structure due to internal
folding, thereby forming a compact structure that is hydrophobic,
and making poly G more difficult to use for nanoparticle
synthesis. In an oligomer containing a mixture of nucleotides, as
the percentage of A and C increases (such as a DNA oligomer
containing at least 75% A and C nucleotides, at least 80%, at
least 90%, or at least 95% A and C nucleotides, the flower
morphology becomes more pronounced. However, if a large majority
(e.g., at least 90%, at least 95%, at least 97% or at least 98%)
of the nucleotides of the DNA oligomer are T, the nanoparticle
will be spherical.
 In some embodiments, it is beneficial to maximize the
flower-like morphology of the nanoflower while minimizing the
thickness of the deposited gold. Nanoflower growth can be
monitored by the nanoparticle's UV absorbance. Gold spherical
nanoparticles exhibit specific UV absorbance in the 500-600 nm
range, and the absorption at this wavelength is a good indicator
of the size and the polydispersity of the nanoparticles. As
spherical nanoseeds grow into nanoflowers, the absorption peak
will blue shift (increase in wavelength) and the absorbance at the
original wavelength will decrease. By monitoring the subsequent
shifted peak that corresponds to the formation of the nanoflower
structure as well as the original peak of the nanosphere, it is
possible to assign a quality factor to track the growth of
nanoflowers that is expressed as:
 The optimum gold concentration that maximizes nanoflower
morphology with minimum gold growth can be determined by plotting
this quality factor vs. the amount of gold salt added.
 The sequence of the nucleic acid also mediates growth and
morphology of nanoparticles synthesized from non-spherical seeds.
In a working embodiment, when gold nanoprisms were functionalized
with A30 or C30 DNA oligomers and additional gold was deposited,
flat nanoplates were formed (FIGS. 20a, 20c). Thus, DNA oligomers
of poly A or poly C, or a mixture of A and C (such as a DNA
oligomer of at least 75% A and C), can be attached to gold
nanoprisms to make flat nanoplates. In contrast, gold nanoprisms
functionalized with T30 or G10 DNA oligomers formed multi-pointed
nanostars (FIG. 20b, 20d). Thus, DNA oligomers of poly T or poly
G, or a mixture of T and G (such as a DNA oligomer of at least 75%
T and G), can be attached to gold nanoprisms to make multi-pointed
nanostars. In another working embodiment, gold nanorods
functionalized with A30 DNA oligomers produced bone-shaped, or
dumbbell-shaped, nanoparticles (FIG. 21b), whereas nanorods
functionalized with T30 oligomers produced nanoparticles
resembling peanuts (FIG. 21c). Thus, DNA oligomers of poly A, or a
mixture of A with other nucleotides (such as a DNA oligomer of at
least 75% A), can be attached to gold nanorods to make
dumbbell-shaped, nanoparticles, while DNA oligomers of poly T, or
a mixture of T with other nucleotides (such as a DNA oligomer of
at least 75% T), can be attached to gold nanorods to make
nanoparticles resembling peanuts.
 In certain embodiments, a nucleic acid sequence is selected
based at least in part on its ability to bind to a target, e.g., a
target protein. In such embodiments, it is desirable to control
nanoflower size by selecting an appropriately sized nanoseed and
then depositing a thin layer of gold so that only a minimal
portion of the oligomer is buried in the deposited gold. For
example, aptamer AS1411 (SEQ ID NO: 2) recognizes and binds to
nucleolin, a eurkaryotic nucleolar phosphoprotein involved in the
synthesis and maturation of ribosomes. In order to facilitate
binding to its target, the entire AS1411 sequence preferably is
fully exposed. Thus, in some embodiments, the nanoseed is
functionalized with a plurality of oligomers comprising the
aptamer plus an additional "tail" of nucleotides, e.g., a poly C
tail, such that a portion of the tail is embedded in the deposited
metal while the aptamer sequence remains fully exposed. Based on
this teaching, one can select an appropriate aptamer based on the
target, and incorporate the selected aptamer into the disclosed
 In some examples, the disclosed nanoparticles further
include other molecules. In one example, the disclosed
nanoparticles further include antibodies or fragments thereof that
can be used to target a nanoparticle to a target cell. In one
example, the antibody is specific for a cell surface receptor,
such as a receptor on a cancer cell. Such nanoparticles can be
used for example to image or treat (e.g., kill) the cancer cell.
In another example, the disclosed nanoparticles further include a
therapeutic molecule that can be used to treat a target cell. For
example, the therapeutic molecule can be a drug that is used to
treat a disease, such as a chemotherapeutic agent (e.g.,
cisplatin, doxorubicin, fluorouracil). In another example, the
therapeutic molecule is a nucleic acid molecule used for gene
 Chemotherapeutic agents are known in the art (see for
example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86
in Harrison's Principles of Internal Medicine, 14th edition; Perry
et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd
ed., 2000 Churchill Livingstone, Inc; Baltzer and Berkery. (eds):
Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis,
Mosby-Year Book, 1995; Fischer Knobf, and Durivage (eds): The
Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book,
1993). Exemplary chemotherapeutic agents that can be conjugated to
a nanoparticle provided herein include but are not limited to,
carboplatin, cisplatin, paclitaxel, docetaxel, doxorubicin,
epirubicin, topotecan, irinotecan, gemcitabine, iazofurine,
gemcitabine, etoposide, vinorelbine, tamoxifen, valspodar,
cyclophosphamide, methotrexate, fluorouracil, mitoxantrone and
III. NANOPARTICLE USES
 Bio-functionalization of nanomaterials can provide the
nanomaterials with target recognition ability, and can enable
their controlled assembly.<41 >This functionalization step
typically involves chemical modifications of the nanoparticles or
the biomolecules to allow conjugation. For example, some
embodiments of the disclosed shaped nanoparticles (e.g.,
nanoflowers, nanoplates, nanospheres, and/or nanostars), are
capable of binding to and/or entering a target cell. In one
embodiment, a nucleic-acid functionalized nanoparticle comprises
an aptamer capable of binding to an antigen of interest. In
another embodiment, a molecule of interest (e.g., an antibody,
antibody fragment, peptide, protein, or drug molecule) is
conjugated to a nucleic acid-functionalized nanoparticle. The
molecule of interest may be conjugated to the nucleic acid
oligomer extending from the nanoparticle, or the molecule of
interest may be conjugated directly to the nanoparticle surface.
Certain embodiments of the disclosed shaped nanoparticles are
capable of forming larger nano-assemblies comprising a plurality
of shaped nanoparticles. Additionally, some embodiments of the
disclosed shaped nanoparticles have unique optical and/or
electrical properties that may provide utility for imaging and/or
biosensing applications, e.g., surface-enhanced Raman
 Nanoflowers have several advantages over nanospheres. For
example, nanoflowers have a much higher surface area than
nanospheres of a similar size. Therefore, more biomolecules or
drug can be loaded on each nanoflower. In addition, the tips of
the nanoscale protrusions and the nanocavities on the surface of
gold nanoflowers have strong localized near-field enhancement
effects, and they give a much stronger Raman signal enhancement
effect than the gold nanospheres. Furthermore, preliminary studies
indicate that AuNFs are more easily taken up and internalized by
cells via endocytosis than non-functionalized gold nanospheres.
 In some embodiments, nanoflowers also have different
optical properties than nanospheres. For example, AuNFs have a
peak absorbance at longer wavelengths (e.g., 600-630 nm) than gold
nanospheres (unfunctionalized or functionalized with T30), which
have a maximum absorbance at 520-530 nm (FIG. 1a). The absorbance
shift allows visualization of AuNFs with near-infrared radiation,
and also may make AuNFs suitable candidates for photothermal
therapies since near-IR absorption increases the temperature of
 Nucleic acid-functionalized nanoflowers may be used as
imaging agents and nano-carriers in a cellular environment. For
example, some embodiments of DNA-functionalized AuNFs can be taken
up by cells. Without being bound by any particular theory, it is
believed that this cellular uptake ability might be due to high
DNA loading on the AuNF surface and/or the morphology of the AuNF.
Intracellular AuNFs scatter light and can be visualized using
dark-field microscopy. The cellular uptake ability and light
scattering property make the AuNFs promising nano-carriers for
drug or gene delivery and promising contrast agents for
 In certain embodiments, a nucleic-acid functionalized
nanoflower comprises an aptamer capable of binding to an antigen
of interest. DNA aptamers have been shown to be a useful targeting
ligand for many biologically and medically relevant targets, and
have shown potential for in vivo targeting applications.<57,58
>Thus, an aptamer-functionalized nanoflower can be used to
deliver the nanoflower to a desired target (such as a particular
cell type). In one embodiment, a gold nanoflower comprises an
AS1411 aptamer, which binds specifically to nucleolin, a protein
that is over expressed ~20-fold on the surface of certain cancer
cells and is an exemplary binding target for human breast cancer
cells, e.g., MCF-7.
 Molecules of interest (e.g., antibodies, peptides,
proteins, drug molecules) may be attached to nucleic
acid-functionalized gold nanoflowers by conventional coupling
techniques. For example, molecules of interest can be attached to
DNA-functionalized gold nanoflowers by conventional gold or DNA
coupling techniques. In some embodiments, the nucleic acid
oligomers may be chemically modified to facilitate
functionalization with, e.g., antibodies, peptides, proteins,
and/or drug molecules. In other embodiments, the molecules of
interest may be attached directly to the nanoflower surface.
 Nanoflower-antibody conjugates may be used to deliver NFs
to desired targets. For example, an antibody that recognizes a
particular target antigen on a cell surface may be conjugated to
the NF. Alternatively, the NF may be conjugated to an antibody
that recognizes, e.g., mouse monoclonal antibodies. In such an
embodiment, a mouse monoclonal antibody specific for a target
antigen may be administered to a subject where it binds to the
target antigen, followed by administration of the anti-mouse
 In one embodiment, an antibody-NF conjugate may be used for
imaging target cells. For example, antibodies to an antigen found
on the surface of cancer cells may be conjugated to NFs. The
antibody-NFs may be administered to a subject, with the antibody
then recognizing and binding to the cancer cell antigens. The
cancer cells may be imaged by any suitable method, such as CT or
 In one embodiment, an antibody-NF conjugate may be used in
photothermal and/or radiotherapy, e.g., for treatment of cancer.
Photothermal therapy is a technique that converts electromagnetic
radiation (usually in the form of infrared) into thermal energy as
a therapeutic technique for medical conditions, such as cancer.
Gold and silver nanoparticles have emerged as powerful platforms
for in vitro and in vivo biomedical applications, due to their
high stability, low toxicity, and ability to be taken up by
cells.<59 >As the dimensions decrease in metals, the
properties of the surface become dominant and give nanoparticles
new properties. As the dimensions decrease in metals, the
properties of the surface become dominant and give nanoparticles
new properties. In noble metals, the coherent collective
oscillation of electrons in the conduction band induces large
surface electric fields which greatly enhance the radiative
properties of gold and silver nanoparticles when they interact
with resonant electromagnetic radiation. This makes the absorption
cross section of these nanoparticles orders of magnitude stronger
than that of the most strongly absorbing molecules and the light
scattering cross section orders of magnitude more intense than
that of organic dyes. It was realized that this intense absorption
provided a path to efficiently convert IR light to an intense
local heating around the nanoparticle.<60 >Photothermal
therapy places these metal nanoparticles only in and around
diseased and/or cancerous cells to create localized heating that
would selectively kill the targeted cells without damaging the
surrounding area.<61 >
 In order to be considered applicable for in vivo
applications, nanoparticles should absorb EM radiation most
efficiently from 700 nm to 900 nm, also known as the near IR
window where skin, tissues, and hemoglobin have minimum absorption
and scattering, allowing the radiation to penetrate deep into the
tissue. The efficiency with which a nanoparticle can convert near
IR radiation to thermal energy is partly determined by electric
fields that arise from the oscillations of surface electrons.
Sharp, pointed features, such as the morphological features of
nanoflowers, behave as focusing points for such oscillations and
can dramatically increase the radiative properties at these
 Gold nanoflowers have been shown to absorb energy in the
near-infrared region. Absorption of NIR energy will increase the
temperature of the AuNF. Thus, an antibody-AuNF conjugate bound to
a cancer cell may be irradiated with NIR radiation, thereby
heating the AuNF and destroying the cancer cell. Alternatively,
the AuNFs may be used to increase the dose of x-ray radiation
received by the cancer cells relative to the dose received by
normal tissue. The absorption characteristics of AuNFs may allow
effective treatment (e.g., cancer cell destruction) with less
radiation than conventional gold nanospheres.
 In other embodiments, nanoflowers may be used to deliver
molecules of interest to a cell. For example, a drug molecule may
be conjugated to the NF surface or to the nucleic oligomers
protruding from the NF surface. Because cells can take up
DNA-functionalized AuNFs (see Example 6), the AuNF may be used to
deliver a drug molecule to the cell interior. Alternatively,
coupling a drug molecule to an NF-antibody conjugate may be used
to deliver the drug to the immediate environment, or vicinity, of
a targeted cell. Thus, an anti-cancer drug, for example, could be
delivered specifically to a tumor site rather than disseminated
throughout the body. Such methods can be used in combination with
other therapies, such as other anti-neoplastic therapies, such as
radiation therapy, chemotherapy immunosuppressants (such as
Rituximab, steroids), and cytokines (such as GM-CSF).
 Nanoparticles prepared by embodiments of the disclosed
method also can be used to make nano-assemblies. Nucleic
acid-directed nano-assemblies may be used for biosensing and
nanoscale photonic device applications. A nanoflower
functionalized with oligomers of a given sequence can be prepared.
The oligomers can act as ligands to bind and attach additional
nanoparticles to which complementary oligomers are attached. For
example, nanoparticles functionalized with poly T oligomers can
bind to a gold nanoflower functionalized with poly A oligomers via
the interaction between the poly A and poly T oligomers. (See,
e.g., FIG. 14a.) However, if the added nanoparticles include
non-complementary oligomers, then little or no binding occurs. For
example, nanoparticles with bound poly A oligomers will not bind
to a poly A-nanoflower. Thus, formation of the nano-assemblies is
sequence specific. Additionally, the number of oligomers on the
"central" nanoparticle, or nanoflower, determines in part how many
"peripheral" nanoparticles including complementary oligomers can
be attached to form the nano-assembly. As the number of oligomers
on the central nanoparticle increases, so does the number of
peripheral nanoparticles that can assemble onto it. One of
ordinary skill in the art will understand that the number of
nanoparticles in the nano-assembly also depends at least in part
upon space constraints and the relative sizes of the
nanoparticles. A larger central nanoparticle can accommodate more
peripheral nanoparticles than a smaller central nanoparticle.
Similarly, using smaller peripheral nanoparticles allows more
nanoparticles to assemble onto the central nanoparticle.
 These flower-like nanoparticles may also have promising
applications in SERS (Surface Enhanced Raman Spectroscopy) based
biosensing. Raman spectroscopy is a useful technique that detects
and identifies molecules based on their vibrational energy levels
and corresponding Raman fingerprints. However, Raman scattering
from the molecules themselves without enhancement is very weak.
Colloidal Au nanospheres have been used to increase the scattering
efficiencies of Raman-active molecules by as much as
10<14>-10<15>-fold.<44 >Compared to these AuNSs
with smooth surfaces, AuNFs may be a better candidate for
fabricating SERS-active tags for a number of reasons: (i) the tips
of the nanoscale bumps and the nanocavities on the AuNF surface
have strong localized near-field enhancement effects<45,46>;
(ii) AuNFs have a larger total surface area due to the roughness
of the AuNF surface; and/or (iii) the surface plasmon resonance
peaks of the AuNFs (e.g., 630 nm for AuNFs) are nearer to the
excitation wavelength, which provides stronger enhancement
 Nanoparticles with different shapes have different
physiochemical properties. Thus, new nanoparticle shapes such as
nanoplates, nanostars, etc., have unique optical and/or electrical
properties that are significantly different from nanospheres or
nanoflowers. These new nanoparticles may have an improved
performance in SERS sensing, and imaging and drug delivery in
comparison with nanospheres. Furthermore, these nanoparticles with
different light scattering properties may also be used
collectively for multiplex sensing or imaging by encoding each
target with a different type of nanomaterial. For example, a
nanoplate may be functionalized (e.g., with an antibody or an
oligonucleotide probe) to couple to one target, while a nanostar
may be functionalized to couple to a different target.
Chemicals and Materials
 All oligonucleotides used herein were purchased from
Integrated DNA Technologies Inc. (Coralville, Iowa). Solutions of
20-nm and 5-nm gold nanospheres (AuNSs) were purchased from Ted
Pella (Redding, Calif.) and purified using a centrifuge before
use. Hydrogen tetrachloroaurate(III) hydrate (HAuCl4.3H2O,
99.999%; Sigma-Aldrich), hydroxylamine hydrochloride (NH2OH.HCl,
99.9999%; Sigma-Aldrich), sodium hydroxide (NaOH, 98%;
Sigma-Aldrich), adenosine 5'-monophosphate sodium salt (AMP, 99%;
Sigma-Aldrich), tris(2-carboxyethyl)phosphine hydrochloride (TCEP,
C9H15O6P.HCl; Sigma-Aldrich), 2-mercaptoethanol (ME, 98%;
Sigma-Aldrich) and mPEG thiol (CH2O-(CH2CH2O)6-CH2CH2SH, Mw=356.5;
Polypure) were used without further purification.
 Shapes and sizes of gold nanoparticles as well as the
nano-assemblies were analyzed using a JEOL 2010LaB6 transmission
electron microscope (TEM) operated at 200 kV. Samples were
prepared by putting a drop of a nanoparticle solution onto a
carbon-coated copper TEM grid (Ted pella).
 Absorbance of the nanoparticle solutions was characterized
using UV-Vis spectrophotometry (Hewlett-Packard 8453).
 Darkfield light-scattering images were acquired using a
Zeiss Axiovert 200M inverted microscope coupled with a CCD digital
camera. The individual nanoparticles on a glass coverslip were
imaged using an EC Epiplan 50* HD objective (NA=0.7), and the
Chinese hamster ovary (CHO) cells were imaged with a Plan-Neofluar
10* objective (NA=0.3). Prior to acquisition, the digital camera
was white-balanced using Zeiss Axiovision software so that colors
observed in the digital images represented the true color of the
 Z-stacks of fluorescence images of the cells were acquired
using Andor Technology Revolution System Spinning Disk Confocal
Microscope at 100* objective (oil immersion, excitation wavelength
488 nm). The collected z-stacks of images were then deconvoluted
and assembled into a 3D image using Autoquant X software and
Nanoparticle Synthesis and
 The concentration of purified 20-nm citrate-coated gold
nanospheres (AuNSs) was calculated based on the Beer-Lambert law
(extinction coefficient of 20-nm AuNS at 520 nm is 9.406*10<8
>M<-1 >cm<-1>) and then adjusted to 0.5 nM and
resuspended in pure water. A 300 [mu]L aliquot of 0.5 nM 20-nm
AuNS solution was first incubated with 1 [mu]M of DNA (poly A30,
poly C30 or poly T30) for 15 min to let DNA adsorb onto the AuNS
surface. This step was followed by addition of 15 [mu]L of 400 mM
NH2OH (adjusted to pH 5 with NaOH) to produce a final
concentration of 20 mM NH2OH. Three types of 30-mer DNAs
consisting of poly A, poly C, or poly T (designated as A30, C30,
and T30, respectively) were used. After vortexing, 2.1 [mu]L 1%
(wt/wt) HAuCl4 was introduced to AuNS mixture solution (final
concentration of HAuCl4 was 167 [mu]M), and the mixture was
rigorously vortexed to facilitate the reduction. A color change
was observed in seconds. The mixture solution was constantly
vortexed for another 15 min until the reaction was complete. Based
on the DNA sequences used and their shape, the synthesized gold
nanoparticles were called AuNF_A30, AuNF_C30 or AuNS_T30
respectively. Surprisingly, nanoparticle solutions synthesized in
the presence of A30 or C30 were blue colored, while the
nanoparticle solution synthesized with T30 was red colored (FIG.
1b). The resultant solutions were stable for days without showing
any nanoparticle aggregation or color change.
 To determine the morphology of the nanoparticles prepared
with different DNA sequences, transmission electron microscopy
(TEM) was employed to investigate each of the resulted
nanoparticle solutions. Surprisingly, those particles synthesized
with A30 or C30 were flower shaped (designated as AuNF_A30 and
AuNF_C30) (FIGS. 2a, 2b), while particles synthesized with T30
were spherical (AuNP_T30, FIG. 2c). The flower-shaped gold
nanoparticles had a broad surface plasmon absorbance that peaked
at 600 nm (for AuNF_C30) or 630 nm (for AuNF_A30) (FIG. 1a), which
is consistent with the absorbance of gold nanoflowers prepared by
other reported methods.<36 >
 Poly G30 was not tested due to synthetic difficulties
caused by the formation of a guanine tetraplex structure.<37
>Instead, a shorter DNA consisting of 10-mer poly G was tested,
and the resulting nanoparticles were nearly spherical (FIG. 3). In
contrast, only spherical nanoparticles were formed in the absence
of DNA (FIG. 2d) or in the presence of salt only (FIGS. 4a and b).
 No metal nanoparticles were formed upon mixing DNA, NH2OH
and HAuCl4 together, without the addition of AuNS as seeds. These
results demonstrated that the DNA mediates the morphology of the
gold nanoparticles, and the nanoparticle shape is sequence
 To understand the DNA sequence-dependent nanoparticle
formation and to determine the stability of DNA-adsorbed AuNs, the
adsorption step of single-stranded DNA (ssDNA) on AuNS was
investigated. Unmodified ssDNA is able to adsorb onto AuNS, and
enhances the electrostatic repulsion between AuNSs, thereby
reducing or preventing salt-induced aggregation.<38 >First,
100 [mu]L of 1 nM, 20 nm AuNS solutions were incubated with 1
[mu]M DNA (either poly A30, poly C30, or poly T30, respectively).
After 15 min incubation, 0.1 M NaCl was introduced to each of the
solutions. UV-vis spectroscopy was used to record the absorbance
of each solution before and after the addition of NaCl.
 As shown in FIGS. 5a-e, aggregation of AuNS happened
immediately when the T30 DNA sequence was used for incubation with
the AuNS, while AuNS incubated with A30 or C30 sequences remained
stable. Since the stability of the AuNS at the same salt
concentration is determined by the number of DNA adsorbed on its
surface,39 it was concluded that many fewer T30 molecules were
adsorbed onto the AuNS surface compared to A30 or C30, which is
consistent with the lower binding affinity of T30 towards the gold
nanoparticle surface. This result explains the differences in
shaping the gold nanoparticle by the T30 sequence in comparison
with A30 or C30.
 To further evaluate the mechanism of shape control process
of the flower-shaped nanoparticle directed by DNA, varying amounts
of HAuCl4 were added to A30, which was incubated with AuNS and 20
mM NH2OH to initiate the reduction. Since NH2OH was in large
excess, it was expected that the HAuCl4 would be completely
reduced to gold metal in the presence of AuNS seeds.<35 >As
shown in FIGS. 6a-f, with the addition of increasing amount of
HAuCl4, the resultant nanoparticle evolved from sphere shape to a
bud sphere and then into the flower-like shape. Upon further
increase of the HAuCl4 amount, the flower shaped nanoparticle
would grow even bigger.
 In order to investigate how the nanoparticle morphology was
affected by the number of DNA oligomers adsorbed on AuNS, varying
amounts of A30 were incubated with AuNS and followed by reduction
of equal amounts of HAuCl4. FIGS. 7a-f shows that the nanoparticle
shape changed from spherical to flower-like with increasing
numbers of DNA oligomers adsorbed on AuNS, while the size of the
gold nanoparticle remained the same. From the above observations,
it was determined that DNA of chain-like structure was able to
direct the deposition of the reduced gold metal on the AuNS and
guide the nanoparticle growth from a spherical into a flower-like
shape. This conclusion was further supported by the control
experiments, which showed that when the single deoxynucleotide,
adenosine monophosphate (AMP) was incubated with AuNS instead of a
DNA chain, the nanoparticles obtained were nearly spherical, while
a random 30-mer DNA sequence of mixed A, T, G, C caused the
formation of flower-shaped nanoparticles (FIGS. 8a-b).
 To further probe this DNA mediated AuNF growing process,
the absorbance of AuNF growth solution was monitored using
UV-visible spectroscopy. As shown in FIG. 9, after initiation of
the reaction for 3 seconds, the intensity of the nanoparticle
absorbance increased significantly, and the peak of the AuNSs at
520 nm broadened and red-shifted. With growth of the AuNS, a new
absorbance peak at 630 nm from the resultant AuNFs appeared, and
the reaction completed in about 15 minutes.
 This time-dependent AuNF growth process was further studied
using TEM by stopping the reaction at the early stages of NP
growth with excess mercaptopropionic acid (MPA). MPA has been
shown to quench the NP growth effectively by forming the less
reactive Au(I)-MPA complex with gold ion.<40 >As shown in
FIGS. 10a-r, both the 20-nm AuNSs and 1-3 nm small nanoparticles
(SNPs) could be observed after initiation of the reaction at 0.5
 A further control experiment showed that formation of the
SNPs could be due to the conversion of Au(I)-MPA complexes into
metal particles on the TEM grid upon electron-beam irradiation
during TEM imaging (FIG. 11). Flower-like nanoparticle
intermediates were observed after 2 seconds of reaction in both
A30 and T30 mediated syntheses. Interestingly, the flower-like
intermediates prepared with T30 grew further into nanospheres
within 30 s while the intermediates prepared with A30 maintained
their flower-like structure and stable AuNFs were produced. In the
absence of DNA, the AuNSs grew into bigger nanospheres and no
flower-like intermediate was observed. These results suggest that
DNA adsorbed on the AuNS surface acts as a template to mediate the
formation of flower-like gold nanoparticles. The formation of the
AuNF results from either selective deposition of the reduced gold
metal on AuNS templated by surface-bound DNA or from uneven growth
of the AuNS due to the binding of DNA to the surface.
 As depicted in FIG. 12, due to the strong binding affinity
of poly A (SEQ ID NO: 4) or poly C (SEQ ID NO: 6) to AuNS, a
number of A30 or C30 bind tightly to AuNS and induce the
inhomogeneous growth of AuNS, producing the flower-like
nanoparticles. In contrast, fewer poly T molecules bind weakly and
loosely to AuNS. The weakly bound poly T molecules produce the
flower-like intermediates at a very initial stage. However, they
are not able to stabilize the flower-like structures, and the
spherical particles are eventually formed.
Determination of the Number and
Stability of Thiolated and Unmodified Oligonucleotides on Gold
Preparation of Thiolated DNA-Gold
 Functionalization of thiolated DNA (HS-A30 or HS-T30) on
5-nm gold nanospheres was carried out by following a published
protocol<55 >with slight alterations. Briefly, 9 [mu]L of 1
mM thiolated DNA was first mixed with 1.5 [mu]L of 10 mM TCEP
(tris(2-carboxyethyl)phosphine) solution and 1 [mu]L of 500 mM
acetate buffer (pH 5.2) to activate the thiolated DNA. After a
30-minute reaction, the mixture was transferred into 3 mL of 5-nm
AuNS solution (82 nM, in pure water) followed by addition of 10 mM
Tris-HCl buffer (Tris=2-amino-2-hydroxymethyl-1,3-propanediol, pH
8.2). The nanoparticle solution was incubated overnight, and the
NaCl concentration was then increased to 0.1 M. The functionalized
5-nm AuNS solutions (designated as AuNS5nm-S_A30 or AuNS5nm-S_T30)
were incubated for another 12 h before usage. To purify the
nanospheres from the unreacted DNA, a Microcon(R) centrifugal
filter (Ultracel YM-100, MWCO=100K; Millipore, Billerica, Mass.)
was used by following the instructions from the manufacturer.
Preparation of Unmodified DNA
 Fluorophore (FAM) labeled poly A30 was used for AuNF
synthesis. The AuNFs were synthesized by incubating 1 [mu]M of
Fluorophore (FAM) labeled poly A30 (FAM-A30) with 300 [mu]L of 0.5
nM 20 nm AuNS solution for 15 min. 15 [mu]L of 400 mM NH2OH (pH 5)
and 2.1 [mu]L 1% (wt/wt) HAuCl4 were added to the nanoparticle
solution to initiate the AuNF formation (three samples were
prepared separately). Meanwhile, 300 [mu]L 1 [mu]M FAM-A30
solutions were prepared with the addition of 15 [mu]L of 400 mM
NH2OH (pH 5) and 2.1 [mu]L pure water and these solutions were
used as control solutions. After AuNF synthesis, the supernatants
were collected by removing the nanoparticles with centrifugation.
The oligonucleotide concentrations in both the collected
supernatants and the control solutions were quantified and
compared by using UV absorbances at 260 nm. The DNA concentration
in the supernatants was 825.6 nM, so the DNA attached to the AuNFs
during synthesis were 174.4 nM. Dividing this number by the AuNS
concentration (0.5 nM), it was estimated that the average number
of attached oligonucleotides on each AuNF was ~349.
Stability of Attached
 To probe the stability of the DNA attached to AuNFs, the
number of oligonucleotides on AuNFs after treatment with
mercaptoethanol was quantified using a fluorescence-based
method.<56 >The AuNF solutions (0.5 nM) were treated with
mercaptoethanol (ME) to a final concentration of 14 mM overnight.
The solutions containing the displaced oligonucleotides were
separated from AuNFs by centrifugation. Each supernatant (100
[mu]L) was added to 400 [mu]L 62.5 mM phosphate buffer (pH 7.2).
The pH and ionic strength of the sample and calibration standard
solutions were kept the same for all measurements due to the
sensitivity of the fluorescent properties of FAM to these
conditions. The fluorescence maximums (520 nm) were measured and
then converted to molar concentrations of the FAM labeled
oligonucleotides by using a standard linear calibration curve.
Standard curves were carried out with known concentrations of
fluorophore-labeled oligonucleotides under same buffer pH, salt,
and mercaptoethanol concentrations.
 The average number of displaced oligonucleotides for each
AuNF was obtained by dividing the calculated oligonucleotide molar
concentration by the original AuNF concentration. The results
demonstrated only ~110 strands were displaced by mercaptoethanol
(ME), and the majority (~240 strands) was still bound to the AuNF
after the treatment. Thiol-gold chemistry is the most used method
to conjugate DNA to gold surface. Under the same ME (14 mM)
treatment, however, all of the thiolated DNA oligonucleotides were
displaced by ME from the gold surface.<42 >
Melting Point Determination of
DNA-Functionalized Gold Nanoflowers
 Considering the remarkably high binding affinity of DNA to
the AuNFs (higher than thiol-gold binding), it was hypothesized
that the DNA in situ attached to AuNFs during reduction could be
partially buried in the AuNFs. To test this hypothesis and also
the functionality of the DNA on the AuNFs, experiments were
performed to test the melting point of the DNA in-situ attached on
 AuNFs were first treated with thiolated PEG (polyethylene
glycol, 6 [mu]M) molecules overnight to displace any weakly bound
DNA on AuNF surfaces.<43 >Purified AuNF_A30 (2 nM) was
hybridized with fluorophore (FAM) labeled Poly T30 (FAM-T30) (1
[mu]M) in a buffer solution containing 10 mM HEPES buffer (pH 7.1)
and 50 mM NaCl. The mixture solution was heated up to 65[deg.] C.
and cooled down to room temperature in about two hours. The
unhybridized fluorophore strands were removed by centrifugation,
and the AuNFs (2 nM) were redispersed in the same buffer solution.
 A fluorimeter (FluoroMax-P; Horiba Jobin Yvon, Edison,
N.J.) coupled with a temperature controller was used to obtain the
melting curve of the DNA hybridization on AuNFs. Since a gold
nanoparticle can effectively quench the fluorescence from its
surrounding fluorophores, the release of the fluorophore labeled
DNA from AuNFs due to DNA melting will result in a fluorescence
increase of the nanoparticle solution. The sample was kept at
target temperatures for 72 seconds after the temperature was
reached to ensure that the sample was at the stated temperature
during data collection at each temperature. As a comparison, free
A30 labeled with an organic quencher (Blank Hole Quencher-1, 200
nM)) was hybridized with FAM-T30 (200 nM) in the same buffer under
identical conditions, and its melting curve was collected as well.
 As shown in FIG. 13, the melting temperature of the DNA in
situ attached to AuNFs (around 42[deg.] C.) was significantly
lower than the free DNA (around 50[deg.] C.). This result
indicated that a small segment of DNA might be buried in the AuNFs
during the nanoparticle growth, while the majority part of DNA
exposed outside was still functional for DNA hybridization.
DNA-Functionalized Gold Nanoparticles
 The synthesized AuNF_A30 solution was first purified by
centrifugation (9000*g, 5 min.) twice and then redispersed in
water. The AuNF_A30 particles were then treated with 6 [mu]M mPEG
thiol for 2 hours and purified. After purification, AuNF_A30 (0.5
nM) was mixed with purified AuNS (50 nM) modified with thiolated
complementary DNA (AuNS5nm-S_A30 or AuNS5nm-S_T30 respectively) in
the presence of 10 mM phosphate buffer (pH 8) and 0.1 M NaCl. The
mixture solution was incubated overnight to allow nano-assembly.
The same procedure was used to assemble AuNS_T30 with
AuNS5nm-S_A30 or AuNS5nm-S_T30. After incubation, the nanoparticle
mixture solution was centrifuged at (9000*g, 2 min.) to remove
free 5-nm gold nanoparticles in the supernatant, and the pellet
was redispersed in buffer solution for TEM sample preparation.
 TEM was then employed to assess the assembly of the
nanoparticles. As shown in FIG. 14a, AuNF_A30 was surrounded by a
number of AuNS5nm_S_T30, forming the satellite structure. As a
comparison, when 5-nm AuNS functionalized with non-complementary
DNA A30 (AuNS5nm_S_A30) were used to incubate with AuNF_A30, no
assembly was observed (FIG. 14b). Additional large-area TEM images
containing multiple satellite assembled nanostructures are shown
in FIGS. 15a-d. These results further confirmed that the DNA
molecules were not only densely functionalized to AuNFs in a large
number, but also retained their molecule recognition properties.
Interestingly, when AuNS_T30 were incubated with AuNS5nm_S_A30
under similar conditions, only a few 5-nm particles were assembled
on AuNS_T30, while little assembly was observed with
non-complementary AuNS5nm_S_T30 (FIGS. 14c, 14d). This observation
indicates that fewer numbers of T30 oligonucleotides were attached
during synthesis, consistent with the fact that fewer T30
oligonucleotides were adsorbed on AuNS compared to A30 or C30.
Surface Enhanced Raman
Spectroscopy of DNA-Functionalized Gold Nanoflowers
 SERS enhancement from DNA functionalized AuNFs was compared
with AuNSs. Raman tag labeled DNA (Trama-A30) was used to grow
AuNFs and then the Raman signal was collected. As shown in FIG.
16, under the same conditions (excitation (603 nm), nanoparticle
concentration (0.5 nM), etc.), the Raman signal from the Raman tag
with the AuNFs was clearly observed while the signal from the
Raman tag with AuNS was too low to distinguish. These results
indicated that AuNFs provide a much stronger SERS effect over the
AuNS. The Raman spectrometer was a home-made instrument located at
Materials Research Lab at University of Illinois.
Cellular Uptake of Gold
 AuNFs were synthesized with 1 [mu]M of fluorophore (FAM)
labeled poly A30 (FAM-A30) by following the procedure in Example
2. The AuNFs were purified by centrifugation.
 CHO (Chinese hamster ovary) cells were cultured in
Dulbecco's modified eagle medium (DMEM; Cell Media Facility,
University of Illinois at Urbana-Champaign, Urbana, Ill.)
supplemented with 10% fetal bovine serum (FBS), penicillin (50
U/ml), and streptomycin (50 [mu]g/ml), at 37[deg.] C. in a
humidified atmosphere of 5% CO2. Cells were seeded at a density of
1*10<5 >cells/cm<2 >on 4 well Lab-Tek chambered #1
Borosilicate coverglass system (Fisher Scientific), and the cells
were grown for 24 hours before treatment with nanoparticles. After
18 hours, the cells were washed with 1*PBS buffer and fresh media
 To investigate the cell uptake of the AuNFs, nanoparticles
(0.5 nM or 1 nM) synthesized with fluorophore (FAM) labeled A30
were added to the cells and incubated for 18 hours. Excess AuNFs
were removed by washing the cells with 1*PBS five times prior to
 Dark-field light-scattering images were taken to visualize
the AuNF uptake by the cells.<47 >The light scattering
property of the AuNFs was first investigated using a dark-field
microscope coupled to a CCD digital camera. The digital camera was
white-balanced so that the observed colors represented the true
color of the scattered light. The AuNFs showed bright orange color
in the dark field image (FIG. 17). As shown in FIG. 18a, the
orange dots representing the AuNFs were observed in the
intracellular region of the cells while the untreated control
cells appeared dim yellow to green color due to the intrinsic
cellular scattering (FIG. 18b). This nanoparticle cellular uptake
was further confirmed by the 3-D reconstructed confocal microscope
images of the AuNF treated cells, showing that the AuNFs were
distributed inside the cells (FIGS. 19a-h). The results
demonstrated that AuNFs entered into cells during the incubation.
It is believed that this ability of the AuNF to be taken up by the
cell might be due to the high DNA loading on the AuNF
surface<48 >and/or the shape effect.<49 >The cellular
uptake ability and light scattering property make the AuNFs
promising nanocarriers for drug or gene delivery and contrast
agents for intracellular imaging.
Synthesis of Non-Spherical
 Gold nanoprisms were synthesized in the presence of
surfactants and iodine by following a previously reported
method.<50 >After removing the free surfactant with
centrifugation, these purified nanoseeds were incubated with DNA
of different sequences (A30, T30, C30) respectively for 15
minutes. NH2OH and HAuCl4 were then added to the nanoparticle
solution to initiate the particle growth.
 The morphologies of the prepared nanoparticles were studied
using scanning electron microscopy (SEM). Surprisingly, the
nanoprisms incubated with A30 or C30 grew into thicker round
nanoplates, while nanoprisms incubated with T30 grew into 2-D
six-angled nanostars (FIGS. 20a-c). Nanoprisms incubated with G10
also produced 2-D multiple angled nanostars were produced (FIG.
20d). These results demonstrated that DNA of different sequences
could direct the growth of the nanoprism into different shapes,
and each sequence encodes the formation of nanoparticles with
 Nanoparticle growth was also tested using gold nanorods as
seeds. Remarkably, the nanorods (FIG. 21a) were converted into
dogbone-like nanoparticles in the presence of A30 after growth
(FIG. 21b), while the nanorods were converted into peanut-like
nanoparticles in the presence of T30 (FIG. 21c).
 These results indicate that embodiments of the DNA-mediated
shape-control method can be readily adapted to synthesize other
non-spherical nanoparticles. This method can be used as a general
methodology to control growth of metal nanoparticles, and holds
great promise to produce a series of novel nanoparticles with
different shapes and unique properties.
Nanoflower Size and Quality
 Nanoflower size can be precisely controlled by controlling
the growth conditions for nanoflowers, e.g., by varying the amount
of gold available and/or by varying the nanoseed size.
 In one example, nanoflowers were synthesized using 300
[mu]L of a 0.5 nM solution of 13-nm gold nanoseeds (synthesized
according to available protocols) with increasing amounts of a 1%
w/v solution of HAuCl4, and the resulting nanoflowers were
analyzed by TEM. The nanoseeds were incubated with an AS1411
aptamer (1 [mu]M; SEQ ID NO: 2) or a randomized control construct
(1 [mu]M; SEQ ID NO: 1) prior to gold salt reduction. The protocol
described above in Example 1 was followed during synthesis.
 As shown in FIGS. 22a-d, increasing gold salt concentration
under identical conditions leads to increasing nanoflower size
with good uniformity. Using additional gold resulted in
non-uniform structures (not shown). FIGS. 22a-d are TEM images of
nanoflowers synthesized with the AS1411 aptamer under the
0.5 nM, 13 nm seed NH2OH (400 mM) 1% w/v
FIG. 22a 300 [mu]L 15 [mu]L 0.7 [mu]L
FIG. 22b 300 [mu]L 15 [mu]L 0.9 [mu]L
FIG. 22c 300 [mu]L 15 [mu]L 1.3 [mu]L
FIG. 22d 300 [mu]L 15 [mu]L 1.5 [mu]L
 The relationship between gold salt concentration and
nanoflower size was determined to be linear (FIGS. 23a-b). The
nanoflowers in FIG. 23a were synthesized with the randomized DNA
construct, and the nanoflowers in FIG. 23b were synthesized with
the AS1411 aptamer.
 In another example, nanoflower size was controlled by
varying the size of the nanoseed. Nanoflowers were synthesized
using 1 [mu]M AS1411 aptamer and 1% w/v HAuCl4 with 15-nm, 30-nm,
and 50-nm gold nanoparticles as nanoseeds. FIGS. 24a-c are TEM
images of the nanoflowers grown from 15-nm, 30-nm, and 50-nm gold
nanoparticle seeds synthesized with the AS1411 aptamer under the
conditions shown in Table 2. The protocol described above in
Example 1 was followed during synthesis. As seen in FIGS. 24a-c,
the nanoflower size increased with increasing nanoseed size.
200 [mu]L AuNP NH2OH (400 mM) 1% w/v
FIG. 24a 15 nm, 0.5 nM 15 [mu]L 3 [mu]L
FIG. 24b 30 nm, 0.31 nM 15 [mu]L 3 [mu]L
FIG. 24c 50 nm, 0.06 nM 15 [mu]L 4 [mu]L
 The nanoflower structure is ideally suited for photothermal
applications, and embodiments of the synthesized nanoflowers can
be tuned to absorb strongly within the near-IR window (i.e., from
700 nm to 900 nm). As shown in FIG. 25, the nanoflowers grown from
50-nm gold nanoparticle seeds are candidates for photothermal
applications with an absorption peak at 800 nm.
Cancer-Selective Targeted Uptake
 Two types of nanoflowers were synthesized. The first
nanoflower included the AS1411 apatmer (SEQ ID NO: 2); the second
construct was identical except the aptamer sequence was randomized
(SEQ ID NO: 1). The DNA sequences are shown in Table 3 below. Both
types of nanoflowers were grown from 15 nm gold seeds and
incubated with MCF-7 cells (human breast cancer cells).
Nanoflowers were synthesized following the protocol described
above in Example 1.
 Cells were incubated and grown according to standard
procedures and plated on glass cover slips inside a 6-well plate
(~100,000 cells per well). Cells were incubated for 12 hours in
cell medium (10% FBS) and washed with PBS buffer. After washing,
the cells were incubated with 100 [mu]L of nanoflower solution (10
nM suspended in deionized water) diluted with 900 [mu]L of
Opti-MEM for 2 hours at 37[deg.] C. and 5% CO2. After incubation,
the cells were washed 3* with PBS to remove excess nanoflowers,
and the glass slides were processed for imaging under fluorescence
microscope and dark-field optical microscope.
Control- SEQ ID NO: 1 5'-/56-FAM/TTG GTA GTA GTG ATT
GTA ATG GTA GTG
DNA A TTTTT TTTTT TTTTT CCCCC CCCCC CCCCC CCCCC
Aptamer- SEQ ID NO: 2 5'-/56-FAM/TTG GTG GTG GTG
GTT GTG GTG GTG GTG
DNA G TTTTT TTTTT TTTTT CCCCC CCCCC CCCCC CCCCC
(AS1411 aptamer sequence in bold)
 As shown in FIGS. 26a and 26b, nanoflowers functionalized
with the AS1411 aptamer (FIG. 26b) exhibited superior binding to
the MCF-7 cells compared to nanoflowers comprising control DNA
Diagnostic Imaging with Shaped
 Embodiments of the disclosed shaped nanoparticles (e.g.,
nanoflowers, nanoplates, nanostars, nanopeanuts, etc.) may be used
for diagnostic imaging, such as to visualize the location and/or
size of a tumor. For example, gold nanoflowers can be synthesized
as described in Example 1. An antibody that recognizes an antigen
on a tumor cell may be conjugated to the AuNFs by any suitable
method. Tumor-specific antibodies are well known in the art.
Alternatively, small molecules that specifically bind to tumor
antigens can be used instead of antibodies. In one example, an
aptamer specific for cancer cells is used.
 Exemplary antibodies and small molecules that can be
conjugated to the disclosed nanoparticles are provided in Table 4.
Antigen Exemplary Tumors Molecules
HER1 adenocarcinoma Cetuximab,
gefitinib, erlotinib, and
lapatinib can also be used.
HER2 breast cancer, ovarian Trastuzumab
cancer, stomach cancer, (Herceptin (R)), pertuzumab
CD25 T-cell lymphoma Daclizumab (Zenapax)
CEA colorectal cancer, some CEA-scan (Fab fragment,
gastric cancers, biliary approved by FDA),
Cancer antigen ovarian cancer, OC125 monoclonal
125 (CA125) mesothelioma, breast antibody
Alpha- hepatocellular carcinoma ab75705 (available
fetoprotein (AFP) Abcam) and other
Lewis Y colorectal cancer, biliary B3 (Humanized)
TAG72 adenocarcinomas B72.3 (FDA-approved
including colorectal, monoclonal antibody)
mammary, and non-small
cell lung cancer
 The antibody (or small molecule) may be conjugated to the
gold surface or to a DNA oligomer. The antibody-AuNF conjugates
may then be administered to a subject using routine methods, for
example by injection (for example intratumorally or i.v.). After
waiting for a period of time sufficient to allow the conjugates to
travel to and bind to the tumor cell antigens, the conjugates may
be visualized by CT or x-ray imaging, thus permitting
visualization of the tumor.
 Alternatively, an antibody that recognizes a tumor cell
antigen may be prepared. A second antibody that recognizes the
anti-antigen antibody may be conjugated to the AuNFs. The
anti-antigen antibody and the antibody-AuNF conjugates may be
administered sequentially or simultaneously to the subject. After
waiting for a period of time sufficient to allow the anti-antigen
antibody to bind to the tumor cell antigen, and the antibody-AuNF
conjugates to bind to the anti-antigen antibody, the conjugates
may be visualized by CT or x-ray imaging.
 In some examples, the antibody-AuNF conjugates are used to
image tumor cells ex vivo. For example, tumor cells from a subject
can be obtained (for example during a biopsy), and then incubated
with the antibody-AuNF conjugates under conditions that permit the
antibody to bind to its target protein. IN some examples live
cells are incubated with the antibody-AuNF conjugates, while in
other examples killed or fixed cells are incubated with the
antibody-AuNF conjugates. The cells can be processed for imaging
(for example fixed and embedded), for example using electron
Photothermal Therapy with Shaped
 Embodiments of the disclosed shaped nanoparticles (e.g.,
nanoflowers, nanoplates, nanostars, nanopeanuts, etc.) may be
delivered to a target cell of interest for use in photothermal
therapy. A shaped nanoparticle of a particular size and shape may
be selected based on its absorbance of energy within a given
wavelength range, e.g., near-infrared radiation. In certain
embodiments, the shaped nanoparticle is conjugated to a moiety
capable of recognizing and binding to the target cell. Suitable
moieties include but are not limited to antibodies and fragments
thereof, drug molecules, proteins, peptides, and aptamers.
 In one example, AuNF conjugates may be delivered to tumor
cells by the methods outlined in Example 10. Suitable AuNF doses
may range from 20 mg per kg body weight to 20 g per kg body
weight. Because AuNF conjugates are capable of absorbing
near-infrared (NIR) radiation, the tumor site may be irradiated
with NIR radiation (700 nm-1500 nm), such as from an NIR laser.
For example, a red laser that emits light with a wavelength of 790
to 820 nm or 800 nm to 810 nm (such as 800 nm or 810 nm) may be
used. In one example, the tumor is irradiated at a dose of at
least 0.5 W/cm<2 >for 2 to 60 minutes, for example 5 to 30
minutes or 3 to 10 minutes, such as at least 2 W/cm<2 >for 2
to 60 minutes, for example 5 to 30 minutes or 3 to 10 minutes, at
least 10 W/cm<2 >for 2 to 60 minutes, for example 5 to 30
minutes or 3 to 10 minutes, or 0.5 to 50 W/cm<2 >for 2 to 60
minutes, for example 5 to 30 minutes or 3 to 10 minutes. The tumor
cells may be destroyed via photothermal heating caused when the
AuNFs absorb energy from the laser.
Drug Delivery with Shaped
 Embodiments of the disclosed shaped nanoparticles (e.g.,
nanoflowers, nanoplates, nanostars, nanopeanuts, etc.) may be
utilized to deliver a therapeutic drug molecule to a subject. For
example, AuNFs can be synthesized as described in Example 1.
Therapeutic drug molecules may be conjugated to the AuNFs by any
suitable means. The drug molecule may be conjugated to the gold
surface or to a DNA oligomer. The drug-AuNF conjugate may then be
administered to a subject as described above at a therapeutically
effective dose. The drug-AuNF conjugates may be taken up by cells
(e.g., by endocytosis or receptor-mediated endocytosis), thereby
delivering drug to the cell interior. In one example, the drug is
a chemotherapeutic agent, and is administered to a subject in
order to treat a tumor in the subject.
 Alternatively, the drug-AuNF conjugate may further be
conjugated to an antibody that recognizes an antigen on a target
cell. The drug-AuNF-antibody conjugate may be administered to a
subject. The antibody may then bind to the target cell antigen,
thereby delivering the drug to the immediate vicinity of the
target cell while minimizing drug delivery to non-target cells.
(1) Murray, C. B.; Sun, S. H.; Doyle, H.; Betley, T. Mrs Bulletin
2001, 26, 985-991.
(2) Jin, R. C.; Cao, Y. W.; Mirkin, C. A.; Kelly, K. L.; Schatz,
G. C.; Zheng, J. G. Science 2001, 294, 1901-1903.
(3) Jana, N. R.; Gearheart, L.; Murphy, C. J. J. Phys. Chem. B
2001, 105, 4065-4067.
(4) Sun, Y. G.; Xia, Y. N. Science 2002, 298, 2176-2179.
(5) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293-346.
(6) Sonnichsen, C.; Reinhard, B. M.; Liphardt, J.; Alivisatos, A.
P. Nat. Biotechnol. 2005, 23, 741-745.
(7) Huang, X. H.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J.
Am. Chem. Soc. 2006, 128, 2115-2120.
(8) Tao, A. R.; Habas, S.; Yang, P. D. Small 2008, 4, 310-325.
(9) Xia, Y.; Xiong, Y.; Lim, B.; Skrabalak, S. E. Angew. Chem.,
Int. Ed. 2009, 48, 60-103.
(10) Seeman, N. C.; Nature 2003, 421, 427-431.
(11) Rothemund, P. W. K.; Nature 2006, 440, 297-302;
(12) Lu, J; Liu. J; Acc. Chem. Res. 2007, 40, 315-323.
(13) Wang, Z; Y. Lu, J. Mater. Chem. 2009, 19, 1788-1798.
(14) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998,
(15) Gu, Q.; Cheng, C. D.; Gonela, R.; Suryanarayanan, S.;
Anabathula, S.; Dai, K.; Haynie, D. T. Nanotechnology 2006, 17,
(16) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.;
Loweth, C. J.; Bruchez Jr, M. P.; and Schultz, P. G; Nature 1996,
(17) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J.
J. Nature 1996, 382, 607-609.
(18) Warner, M. G.; Hutchison, J. E. Nat. Mater. 2003, 2, 272-277.
(19) Le, J. D.; Pinto, Y.; Seeman, N. C.; Musier-Forsyth, K.;
Taton, T. A.; Kiehl, R. A. Nano Lett. 2004, 4, 2343-2347;
(20) Zhang, J.; Liu, Y.; Ke, Y.; Yan, H.; Nano Lett. 2006, 6,
(21) Lee, J. H.; Wernette, D. P.; Yigit, M. V.; Liu, J.; Wang, Z;
Lu, Y. Angew. Chem., Int. Ed. 2007, 46, 9006.
(22) Bigham, S. R.; Coffer, J. L. J. Phys. Chem. 1992, 96,
(23) Ma, N.; Dooley, C. J.; Kelley, S. O. J. Am. Chem. Soc. 2006,
(24) Kumar, A.; Jakhmola, A. Langmuir 2007, 23, 2915-2918.
(25) Ma, N.; Yang, J.; Stewart, K. M.; Kelley, S. O. Langmuir
2007, 23, 12783-12787.
(26) Berti, L.; Burley, G. A. Nat. Nanotechnol. 2008, 3, 81-87.
(27) Wang, Q. B.; Liu, Y.; Ke, Y. G.; Yan, H. Angew. Chem., Int.
Ed. 2008, 47, 316-319.
(28) Ma, N.; Sargent, E. H.; Kelley, S. O, Nat. Nanotechnol. 2009,
(29) Brown, S.; Sarikaya, M.; Johnson, E. J. Mol. Biol. 2000, 299,
(30) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.;
Stone, M. O, Nat. Mater. 2002, 1, 169-172.
(31) Banerjee, I. A.; Yu, L. T.; Matsui, H. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 14678-14682.
(32) Dickerson, M. B.; Sandhage, K. H.; Naik, R. R. Chem. Rev.
2008, 108, 4935-4978.
(33) Storhofff, J. J.; Elghanian, R.; Mirkin, C. A.; Letsinger, R.
L. Langmuir 2002, 18, 6666-6670.
(34) Ostblom, M.; Liedberg, B.; Demers, L. M.; Mirkin, C. A. J.
Phys. Chem. B 2005, 109, 15150-15160.
(35) Brown, K. R.; Natan, M. J. Langmuir 1998, 14, 726-728.
(36) Bakr, O. M.; Wunsch, B. H.; Stellacci, F. Chem. Mater. 2006,
(37) Poon, K.; Macgregor, R. B. Biopolymers 1998, 45, 427-434.
(38) Li, H. X.; Rothberg, L. J. J. Am. Chem. Soc. 2004, 126,
(39) Li, H. X.; Rothberg, L. Proc. Natl. Acad. Sci. U.S.A. 2004,
(40) Zhao, L.; Ji, X.; Sun, X.; Li, J.; Yang, W.; Peng, X. J.
Phys. Chem. C. 2009, 113, 16645-16651.
(41) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43,
(42) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.;
Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000,
(43) Kannan, B.; Kulkarni, R. P.; Majumdar, A. Nano Lett. 2004, 4,
(44) Nie, S.; Emory, S. R., Science 1997, 275, 1102-1106.
(45) Hao, E. C.; Bailey, R. C.; Schatz, G. G.; Hupp, J. T.; Li, S.
Y. Nano Lett. 2004, 4, 327-330.
(46) Bakr, O. M.; Wunsch, B. H.; Stellacci, F. Chem. Mater. 2006,
(47) Wax, A.; Sokolov, K. Laser & Photon. Rev. 2009, 3,
(48) Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J.
E.; Rosi, N. L.; Mirkin, C. A. Nano Lett. 2007, 7, 3818-3821.
(49) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Nano Lett.
2006, 6, 662-668.
(50) Millstone, J. E.; Wei, W.; Jones, M. R.; Yoo, Y; Mirkin, C.
A.; Nano. Lett. 2008, 8, 2526-2569.
(51) Newman, J. D. S.; Blanchard G. J. J. Nanoparticle Res. 2007,
(52) Liu Q.; et al., Mater. Sci. 2006, 41, 3657-3662.
(53) Popovtzer, R., et al., Nano. Lett. 2008, 8, 4593-4596.
(54) Hainfeld, J. et al., J. Pharmacy and Pharmacology 2008, 60,
(55) Liu, J.; Lu, Y., Nat. Protoc. 2006, 1, 246.
(56) Demers, L. M., et al., Anal. Chem. 2000, 72, 5535.
(57) Farokhzad et al., Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
(58) Giljohann et al., Nano Lett. 2007, 7, 3818.
(59) Hauck, T. S. et al., Small 2008, 4, 153.
(60) Jain et al., J. Phys. Chem. B 2006, 110, 7238.
(61) O'Neal et al., Cancer Letters 2004, 209, 171.
 In view of the many possible embodiments to which the
principles of the disclosure may be applied, it should be
recognized that the illustrated embodiments are only examples of
the disclosure and should not be taken as limiting the scope of
the invention. Rather, the scope of the disclosure is defined by
the following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
Your Support Maintains this
The Rex Research Civilization Kit
... It's Your Best Bet & Investment in Sustainable
Humanity on Earth ...
Ensure & Enhance Your Survival & Genome
Everything @ rexresearch.com on a Data DVD !