http://www.sciencedaily.com (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
"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 T.
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;
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 the invention.
 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 groups.
 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
 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
 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
 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 nanoplate.
 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
 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
 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
 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 nm.
 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, respectively.
 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 20 nm.
 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 nm.
 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
 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
 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 gold.
 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 reference herein.
 SEQ ID NO: 1 is a randomized control DNA sequence.
 SEQ ID NO: 2 is a DNA sequence including the AS1411
 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
 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 indicates otherwise.
 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 claims.
 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, 1997
 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 or Fab.
 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
 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
 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 determined.
 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 gold nanoparticles.
 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 700-2000 nm.
 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
 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
 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 particular cell.
 "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
 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, N.J.
 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 tumor.
 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 nanoparticle.
 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 adenocarcinoma.
 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 ligands.
 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 layer.
 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
 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 therapy.
 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 vinorelbine.
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
 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 the AuNFs.
 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 intracellular imaging.
 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.,
 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 x-ray imaging.
 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 locations.
 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
 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 effects.
 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
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
 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 scattered light.
 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 Imaris software.
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
 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 second.
 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
 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
 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
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
 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
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 the AuNFs.
 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
 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
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 was added.
 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 imaging.
 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
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 certain shapes.
 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
 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
 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 following conditions:
0.5 nM, 13 nm seed NH2OH (400 mM) 1%
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.
Uptake In Vitro
 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
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
(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
 Exemplary antibodies and small molecules that can be
conjugated to the disclosed nanoparticles are provided in Table
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 microscopy.
Photothermal Therapy with
 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
 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.
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 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.
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