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Rabies Virus-Inspired Silica-Coated Gold Nanorods as

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a Photothermal Therapeutic Platform for Treating Brain
Tumors
Changkyu Lee, Ha Shin Hwang, Sungin Lee, Bomi Kim, Jong Oh Kim, Kyung Taek Oh,
Eun Seong Lee, Han-Gon Choi, and Yu Seok Youn*

The unique biological and surface properties of viruses have encephalomyelitis.[7] Delivery of drugs or particles into the brain
inspired the design of various nanosized delivery vehicles.[1] is very difficult because of the BBB. In general, drugs > 500 Da
Viruses vary widely in size (10 nm to >1 m) and shape (ico with low lipid solubility generally cannot cross the BBB due to
sahedrons, spheres, rods, and tubes).[2] Surface proteins of unique brain capillary endothelial cell structures such as tight
viruses or peptides derived from these proteins facilitate cel junctions and efflux transporters.[8] Thus, 98% of small mole
lular uptake of viruses, such as human immunodeficiency virus cules as well as colloidal-sized particles are restricted from
(HIV) or brome mosaic virus (BMV), which is useful for active entry into the brain, which is considered to be a crucial hurdle
targeting.[3] Mimicry of viral characters has yielded nanovehi for the treatment of brain tumors.[9]
cles that are better internalized into cells and that have a longer To overcome the BBB, strategies involving nanovehicles
systemic residence than conventional nanovehicles, improving related to the RVG ligand of RABV have been proposed.[10] It is
their therapeutic efficacy.[4] Viral-mimetic particles are therefore well documented that RVG interacts specifically with the nico
considered an attractive therapy delivery platform. tinic acetylcholine receptor (AchR) expressed on neuronal cells,
Rabies virus (RABV) is a prototypical neurotropic virus in which enables RABV to enter the central nervous systems.[11]
the genus Lyssavirus in the Rhabdoviridae family that causes In particular, a short (29-residue) peptide derived from RVG
hydrophobia accompanied by difficulty swallowing and panic (hereafter referred to as RVG29; Figure S1, Supporting Infor
in mammalian hosts.[5] RABV is enveloped by five proteins: a mation) also binds to nicotinic AchR-expressing neuronal cells
nucleoprotein, phosphoprotein, matrix protein, glycoprotein, such as Neuro 2a (N2a) cells and barely interacts with non-
and viral RNA polymerase.[6] Among these, the rabies virus neuronal cells.[11c] Furthermore, this peptide is able to competi
glycoprotein (RVG) enables rabies virus virions to travel to tively inhibit the binding of snake-venom toxin -bungarotoxin
the central nervous system (CNS) via neuronal pathways and (BTX), which is one of the most potent ligands of the nico
bypass the bloodbrain barrier (BBB), which can result in fatal tinic AchR, and vice versa in a dose-dependent manner.[11c,12]
Accordingly, the strategic use of RVG for drug delivery systems
may allow treatment of brain tumors.
C. Lee, H. S. Hwang, S. Lee, B. Kim As seen with viruses, several factors such as size, shape,
School of Pharmacy surface charge, and flexibility influence the in vivo biodistribu
Sungkyunkwan University tion and cellular uptake of nanoparticles (NPs). Among these,
Suwon 16419, Republic of Korea particle shape is considered to be one of the most powerful
Prof. J. O. Kim tools for enhancing nanoparticle internalization into cells in the
College of Pharmacy
Yeungnam University context of designing effective nanomedicines.[13] In particular,
Gyongsan 38541, Republic of Korea elongated nanoparticles exhibit greater affinity for cells than
Prof. K. T. Oh spherical nanoparticles because elongated NPs have a greater
College of Pharmacy chance of interacting with the cell surface because of their
Chung-Ang University greater surface area than spherical NPs.[13a,c] Nanorods were
Seoul 06974, Republic of Korea
shown to penetrate tumors more rapidly and accumulate to a
Prof. E. S. Lee
greater extent in tumors due to greater transmembrane trans
Division of Biotechnology
The Catholic University of Korea port and diffusion rates than nanospheres with the same effec
Bucheon 14662, Republic of Korea tive hydrodynamic diameter.[14] NPs with a high aspect ratio
Prof. H.-G. Choi (AR: length/width) tend to be taken up to a greater extent by
College of Pharmacy cells and at a faster rate than NPs with a low AR.[15] It has been
Hanyang University reported that particles with an AR of 3 can penetrate fourfold
Ansan 15588, Republic of Korea
more rapidly than those with an AR of 1.[1b]
Prof. Y. S. Youn
School of Pharmacy Herein, we introduce unique rabies virus-mimetic silica-
Biomedical Institute for Convergence at SKKU coated gold nanorods (RVG-PEG-AuNRs@SiO2) to treat brain
Sungkyunkwan University gliomas. These gold nanorods (AuNRs) were surface-modified
Suwon 16419, Republic of Korea with RVG29, enabling them to bypass the BBB and enter
E-mail: ysyoun@skku.edu
the brain by way of the central nervous system, presumably
DOI: 10.1002/adma.201605563 through the nicotinic AchR. Their rod shape increased their

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interaction with the nicotinic AchR and increased the local
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ized surface plasmon resonance (LSPR) signal in response
to near-infrared (NIR) light in the context of photothermal
therapy (PTT) (Figure 1A). We primarily focused on synthe
sizing AuNRs with a shape closest to that of RABV (Figure 1B).
RABV has a unique bullet-like shape with one rounded end
and one planar end. Although the size of RABV varies widely
in terms of diameter (45100 nm) and length (100430 nm), it
is 180 nm long and 75 nm wide on average, with an aspect
ratio of 2.4.[16] To fabricate a rabies virus-like nanoparticle plat
form, we utilized anisotropic (nonspherical) gold nanoparticle
(AuNP) chemistry because gold is a useful flexible inorganic
material that can be used to create a variety of nanoparticle
shapes including spheres, rods, shells, cages, and cubes.[17] Fur
thermore, gold is basically inert and nontoxic, and particles of
various shapes in the size range of 1150 nm can be facilely
and precisely synthesized.[18]
To create rabies virus-like AuNRs, we first synthesized longi
tudinally grown gold nanorods. For this purpose, we used the
well-established seeded growth synthesis approach.[19] HAuCl4
was associated by silver nitrate (AgNO3) in the presence of a
surfactant (cetyltrimethylammonium bromide: CTAB) and
a strong reducing agent (sodium borohydride: NaBH4). The
resulting gold seeds were 4.7 1.3 nm in size and spherical
(Figure 1C(a)). To increase the efficiency of longitudinal growth
of AuNRs, hydroquinone was added as a reducing agent instead
of ascorbic acid, because nanoparticle growth is highly sensitive
to the concentration of ascorbic acid added. Therefore, minute
change of ascorbic acid amount can generate little grown gold
nanorods or spherical particles. In contrast, hydroquinone
can be used to produce long gold nanorods with a high AR
in high purity and yield.[19] In actuality, longitudinally grown
AuNRs had a length and a width of 45.7 6.8 and 7.9 0.8 nm,
respectively, an AR of >5.0, and displayed LSPR at 870 nm
(Figure 1C(b),D).
Next, we induced transverse growth on the AuNRs to reduce
their AR to that of RABV (2.4). However, because transverse
overgrowth of AuNRs can result in partial or complete loss
of LSPR around 820 nm due to the low AR, striking a bal
ance between increasing the width and preserving the LSPR is
important. We considered the appropriate AR for AuNRs to be
4.0. In other words, the width of the AuNRs would have to
increase (transverse), but they would have to remain unchanged
longitudinally and thus be transformed from slender rods to
thick rods. Kou et al. established a transverse growth method
for AuNRs, referred to as the cysteine-induced method. Mol
ecules with thiol residues, such as cysteine and glutathione,
tend to bind preferentially to the ends of AuNRs at a low con
centration, which is believed to block longitudinal growth and
to predominantly induce transverse growth on the surface of
AuNRs.[20] As a result, the AR of the AuNRs can be precisely
tuned by modulating the amount of cysteine or glutathione
added. In our study, AuNRs with a length and a width of 79.9
7.3 to 20.1 1.4 nm, respectively, and an AR of 4.0 were effec
tively produced using cysteine; these AuNRs had an AR closer
to that of RABV and possessed a significant LSPR (820 nm)
for photothermal therapy (Figure 1C(c),D).
Next, we created an outmost material layer on the surface of
the AuNRs to (i) synchronize the aspect ratio with rabies virus
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and (ii) mimic the outermost glycoprotein layer on the envelope
of rabies virus virions. At the initial stage, an albumin layer
between AuNRs body and RVG29 peptide had been designed to
more closely mimic the matrix protein (lipoproteins) of RABV.
The lipoprotein matrix is known to play a functional role in not
only virus assembling or budding but also bridging between
the nuclease protein and outer virion membrane of RABV.[7]
However, this albumin layer showed insufficient thickness and
low conjugation yield.
In this regard, a silica material, tetraethyl orthosilicate
(TEOS), was coated on the surface of the AuNRs to complete
structural mimicry of RABV. Importantly, an extra 15 nm
thick shell layer of AuNRs was required to decrease their aspect
ratio from 4.0 to 2.4. It is well documented that the SiO2 shell
thickness of gold nanorods is tunable in the range of a few nm
to 20 nm by altering concentrations of CTAB and TEOS.[21]
Consistent with this, silica shell thickness was controlled to
13.8 1.2 nm by modulating the amounts of both CTAB and
TEOS added (Figure 1B,C). The newly formed mesoporous
SiO2 shell of AuNRs closely resembled the spike-like glyco
protein layer of RABV. RABV has two major structural com
ponents: a helical ribonucleoprotein core and a surrounding
envelope. The glycoprotein forms 400 trimeric spikes tightly
arranged on the surface of RABV virions.[16] Amine-function
alized AuNRs@SiO2 (AuNRs@SiO2-NH2) previously modified
with 3-aminopropyltriethoxysilane (APTES) via a 5 kDa polyeth
ylene glycol linker (MAL-PEG5k-NHS) were modified by cova
lent binding of the RVG29-cysteine peptide to allow efficient
binding to the nicotinic AchR and to stabilize the gold nano
structure (Figure S2, Supporting Information). Furthermore,
surface density of RVG peptide on gold nanorods is a critical
factor for cellular uptake.[22a] Also in our study, cellular uptake
of AuNRs@SiO2 increased sharply by increasing surface
modification of RVG29 in the range of 220 g (mg1 AuNRs)
(Figure S3, Supporting Information). This RVG29 density-
dependent uptake of AuNRs@SiO2 was highly significant in
N2a cells which express nicotinic AchR, not in HeLa cells (no
nicotinic AchR) (Figure S4, Supporting Information). In this
regard, we maximized the density of RVG29 on the surfaces
of AuNRs@SiO2-NH2 to mimic the glycoproteins on RABV.
The conjugation yield of bifunctional PEG (MAL-PEG5k-NHS)
at both ends has been reported to be 15%.[22b,c] Therefore, we
conjugated 20 g of RVG29 with 1 mg AuNRs@SiO2-NH2
or AuNPs@SiO2-NH2 (20 2.5 and 22 1.8 g RVG29 mg1
AuNRs@SiO2-NH2 g and 1 mg AuNPs@SiO2-NH2, respec
tively) (Figure S5, Supporting Information). After all of these
procedures, the length and width of RVG29-PEG-AuNRs@SiO2-
NH2 (hereafter referred to as RVG-PEG-AuNRs@SiO2) were
determined to be 117.7 7.3 and 50.3 3.1 nm, respectively,
and the AR value of these nanoparticles (2.34) as well as their
physical appearance were very similar to those of RABV, except
at one planar end (Figure 1B,C). Our RVG-PEG-AuNRs@SiO2
therefore mimicked the rabies virus in size, shape, and surface
function.[13] Also, the RVG-PEG-AuNRs@SiO2 maintained a
significant LSPR (maximum absorbance at 820 nm), showing
a dark brown color and a slightly positive zeta potential
(+14.2 2.5 mV), because they still had a gold structure with
an AR of 4.0, despite the reduced AR (2.34) of the entire struc
ture (RVG-PEG-AuNRs@SiO2) due to silica layers with RGV29
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Figure 1. A) A scheme showing brain delivery of the rabies virus-mimetic RVG-PEG-AuNRs@SiO2 via neuronal pathway and photothermal therapy
using NIR laser. B) A scheme of the synthesis strategy for fabricating rabies virus-mimetic silica-coated gold nanorods (RVG-PEG-AuNRs@SiO2):
(a) Au seed, (b) AuNRs (longitudinally grown), (c) AuNRs (after transverse growth), (d) silica-coated AuNRs, (e) RVG29 peptide-PEG5k-conjugated
AuNRs@SiO2 (hereafter termed as RVG-PEG-AuNRs@SiO2; 73 000 nm3); AR = aspect ratio (length/width). C) TEM images showing morphologies of
AuNRs at each step (a, b, c, and e) and gold nanoparticles (RVG-PEG-AuNPs@SiO2; 68 000 nm3; f). D) UVvisNIR spectra for RVG-PEG-AuNRs@
SiO2 at each synthesis step (0.25 103 m, DW). E) Photographs showing the representative colors for RVG-PEG-AuNRs@SiO2 at each synthesis step
(0.25 103 m, DW). F) Surface zeta potentials of RVG-PEG-AuNRs@SiO2 at each synthesis step (0.25 103 m, DW).

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(Figure 1C(e),D,E). Additionally, it was of our interest that sur
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face silica layers greatly decrease systemic toxicity derived from
plain gold nanoparticles.[22d]
As a comparator, spherical AuNPs were fabricated. Briefly,
AuNPs were grown by serial addition of HAuCl4, CTAB, and
ascorbic acid and were surface-decorated with mesoporous
silica layers and PEG-RGV29 (Figure S6, Supporting
Information).[20b] Importantly, the volumetric dimension of
RVG-PEG-AuNPs@SiO2 (68 000 nm3) was similar to that of
RVG-PEG-AuNRs@SiO2 (73 000 nm3), which allowed us to
investigate the influence of shape alone. The resulting RVG-
PEG-AuNPs@SiO2 were 73.9 1.4 nm in size and had an AR
of 1.0 (spheres; Figure 1C(f)). In vitro and in vivo characteris
tics such as cellular uptake and biodistribution of RVG-PEG-
AuNRs@SiO2 and RVG-PEG-AuNPs@SiO2 were compared in
N2a cells and in the brain and spinal cord of mice. As shown
in Figure 2A and Figure S7 (Supporting Information), RVG-
PEG-AuNRs@SiO2 were more efficiently internalized into
N2a cells, which express nicotinic AchRs, than were RVG-
PEG-AuNPs@SiO2, PEG-AuNPs@SiO2, and PEG-AuNRs@
SiO2 (without RVG29). This demonstrated that our rabies
virus-shaped mimetic gold nanorods were more rapidly inter
nalized into cells than plain spherical gold nanoparticles over
a limited duration of time (4 h). Furthermore, the improved
cellular uptake of RVG-PEG-AuNRs@SiO2 was due to the pres
ence of RVG29 on AuNRs@SiO2, which binds specifically to
the nicotinic AchR on N2a cells; internalization was obviously
inhibited by the addition of -bungarotoxin (Figure 2B). Fur
thermore, transmission electron microscopy (TEM) observa
tion of PEG-AuNRs@SiO2- or RVG-PEG-AuNRs@SiO2-treated
N2a cells directly demonstrated that a much greater amount of
RVG-PEG-AuNRs@SiO2 than PEG-AuNRs@SiO2 was local
ized inside the cells, emphasizing the importance of the surface
presence of RVG29 (Figure 2C). Cyanine dye-based fluores
cence visualization revealed a much greater amount of Cy5.5-
tagged RVG-PEG-AuNRs@SiO2 in the spinal cord of mice than
Cy5.5-tagged plain PEG-AuNRs@SiO2 (without RVG29) fol
lowing intravenous injections via the tail vein (Figure 2D). This
finding supported our hypothesis that RVG-PEG-AuNRs@SiO2,
as seen similar to the in vivo biological behavior of actual rabies
virus, were able to enter the central nervous system by way of
nicotinic AchRs expressed by particular neural cells in vivo.
Finally, as shown in Figure 2E, RVG-PEG-AuNRs@SiO2 were
most highly localized in the brain region of mice 224 h after
intravenous injections than were the other gold nanoparticle
groups. We believe that the superior in vivo brain targetability
of RVG-PEG-AuNRs@SiO2 is directly attributable to mimicry of
the rabies virus in terms of size, shape, and surface properties.
Nonetheless, significant fractions of RVG-PEG-AuNRs@SiO2
were located in other tissue organs, such as, livers, kidneys,
spleen, lungs, and heart of ICR mice (Figure S8, Supporting
Information). It is well-documented that biodistribution of
gold nanoparticles is affected by several factors, such as surface
RVG ligand, silica presence, and size or charge.[22a,23] Similarly,
RVG-PEG-AuNRs@SiO2 except brain organs were mainly dis
tributed in liver and kidneys, which might be due to nonspe
cific recognition of RVG29 peptide and silica materials.
Gold nanorods have attracted great interest in biomedical
applications because of their unique photothermal and optical
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properties.[24] Gold nanorods generate strong LSPR in the NIR
wavelength range of 700950 nm (maximally 1200 nm), which
is the best spectral region for imaging and therapy due to the
so-called water window of all aqueous tissues.[24a,25] In this
context, gold nanorods have a noticeable advantage over spher
ical gold nanoparticles that respond only to visible light in the
wavelength range from 500 to 550 nm (Figure S9, Supporting
Information). Moreover, the peak absorption band of gold
nanorods in the NIR region can be easily tuned by adjusting
their aspect ratios, which offers a wide spectrum of therapeutic
practicality.[24b,25] Additionally, the absorption coefficients of
gold nanorods are usually 104106 times higher than those of
conventional dyes; thus, their photothermal efficiency can be
very high.[26] The temperature of gold nanorods can increase in
the range from 10 to 1000 C upon NIR irradiation and opti
mization of irradiation conditions such as laser power, irra
diation time, and concentration of gold nanorods.[27] Tempera
ture changes caused by the LSPR of gold nanorods can result
in irreversible damage to or death of tumor cells, facilitating
hyperthermic treatment of cancers.[28] Also, some studies have
demonstrated that tumor cells are far more sensitive to damage
by elevated temperature than normal cells due to the hypoxic
environment of the tumor.[29] Our gold nanorods exhibited a
significant LSPR in response to the NIR wavelength of 808 nm.
Their surface temperature increased to 50 C depending upon
the concentration of AuNRs, whereas the temperature of plain
AuNPs hardly changed because they responded to light in the
wavelength range of 500550 nm (Figure 3A,B). Importantly,
RVG-PEG-AuNRs@SiO2 were highly localized in the tumors
of N2a cell-xenografted male BALB/c nude mice. When visu
alized by a photothermal camera, the local tumor temperature
of mice treated with RVG-PEG-AuNRs@SiO2 increased to
>50 C after 5 min of NIR exposure, which is sufficient for a
hyperthermal effect, whereas the local tumor temperature of
mice treated with plain PEG-AuNRs@SiO2 (without RVG) was
around 40 C (Figure 3C). These observations demonstrate that
our rabies virus mimetic RVG-PEG-AuNRs@SiO2 clearly tar
geted brain tumor cells and could respond to LSPR. This likely
facilitated their cytotoxic effects in N2a cells in contrast to PEG-
AuNRs@SiO2 and AuNRs groups without NIR laser irradiation
(Figure 3D,E). Additionally, increased cytotoxicity caused by
RVG-PEG-AuNRs@SiO2 (vs PEG-AuNRs@SiO2) is due to not
only extent but also rate of cellular uptake, on the basis of the
RVG29 presence. Due to high interaction between RVG29 and
nicotinic AchR, RVG-PEG-AuNRs@SiO2 can internalize much
faster than PEG-AuNRs@SiO2. This can be more significant in
an in vitro setting that removes gold nanoparticles outside cells
before NIR laser irradiation. Also, this fact can be more possible
in a dynamic in vivo environment that might rapidly eliminate
plain PEG-AuNRs@SiO2 (vs RVG-PEG-AuNRs@SiO2) from
neuronal path or blood stream near tumor tissues because of
weak nicotinic AchR interaction.
We next evaluated the feasibility of photothermal therapy
(PTT) using RVG-PEG-AuNRs@SiO2 in N2a tumor-bearing
mice. N2a cells were inoculated into the right dorsal flanks of
male BALB/c nu/nu mice as a conventional xenograft tumor
model; in a second setting, N2a cells were inoculated into the
striatum of male BALB/c nu/nu mice as an orthotopic brain
tumor model. Gold nanoparticles (2.5 103 m, 200 L) were
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Figure 2. A) In vitro cellular uptake of PEG-AuNRs@SiO2, RVG-PEG-AuNRs@SiO2, PEG-AuNPs@SiO2, and RVG-PEG-AuNPs@SiO2 (0.1 103 m)
in N2a cells after a 4 h incubation. B) Cellular uptake of RVG-PEG-AuNRs@SiO2 (0.1 103 m) by N2a cells with BTX (20 and 200 g) or without BTX
after a 4 h incubation. C) TEM images showing intracellular localization of RVG-PEG-AuNRs@SiO2 and PEG-AuNRs@SiO2 (0.1 103 m) in N2a cells
after a 4 h incubation. D) Ex vivo images of harvested spinal cords of mice intravenously treated with (a) Cy5.5-RVG-PEG-AuNRs@SiO2 (2.5 103 m,
200 L), (b) Cy5.5-PEG-AuNRs@SiO2 (2.5 103 m, 200 L), or (c) saline (200 L). E) In vivo fluorescence images of RVG-PEG-AuNRs@SiO2, RVG-
PEG-AuNPs@SiO2, PEG-AuNRs@SiO2, and PEG-AuNPs@SiO2 (2.5 103 m, 200 L) in orthotopic glioma-bearing mice at predetermined time points
(2, 4, 8, 24 h) after intravenous injections.

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Figure 3. A) Kinetic changes of surface temperature after NIR laser irradiation (808 nm, 1.5 W cm2) of different concentrations of AuNRs (after transverse
growth: 0, 0.01, 0.025, 0.05, 0.1 103 m) and AuNPs (0.1 103 m) for 5 min. B) Thermographic images of different concentrations of AuNRs (after trans-
verse growth: 0.01, 0.05, 0.1 103 m) and AuNPs (0.1 103 m) after NIR laser irradiation (808 nm, 1.5 W cm2) obtained using a thermal camera. C) In
vivo whole body thermal images of N2a cells-xenografted mice treated with RVG-PEG-AuNRs@SiO2, PEG-AuNRs@SiO2 (2.5 103 m, 200 L), or saline
(200 L) after NIR laser irradiation (808 nm, 1.5 W cm2) for 5 min; images were obtained using a thermal camera. D) Cell viability of cells treated with
different concentrations of RVG-PEG-AuNRs@SiO2 and PEG-AuNRs@SiO2 (0.020.1 103 m) after NIR laser irradiation (808 nm, 1.5 W cm2, 5 min) and
a 4 h incubation: *P < 0.001 over PEG-AuNRs@SiO2; **P < 0.01 over PEG-AuNRs@SiO2. E) Live-dead assay of N2a cells treated with RVG-PEG-AuNRs@
SiO2, PEG-AuNRs@SiO2 (each 0.1 103 m), or PBS buffer after NIR laser irradiation (808 nm, 1.5 W cm2, 5 min) and a 4 h incubation.

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Figure 4. (1) Side flank tumor xenograft model. A) Tumor volumes of BALB/c nu/nu mice treated with RVG-PEG-AuNRs@SiO2, PEG-AuNRs@SiO2
(2.5 103 m, 200 L), or saline (200 L) with or without NIR laser irradiation (808 nm, 1.5 W cm2, 5 min): *P < 0.001 over PEG-AuNRs@SiO2 (NIR+);
**P < 0.003 over PEG-AuNRs@SiO2 (NIR+); ***P < 0.004 over saline (NIR+). B) Representative photos of N2a cells-xenografted mice treated with PEG-
AuNRs@SiO2, RVG-PEG-AuNRs@SiO2 (each 2.5 103 m, 200 L), or saline (200 L) at predetermined days (0, 1, 3, 7, 10, and 13 d) after NIR laser
irradiation (808 nm, 1.5 W cm2, 5 min). C) Photographs of tumors excised from mice treated with PEG-AuNRs@SiO2, RVG-PEG-AuNRs@SiO2 (each
2.5 103 m, 200 L), or saline (200 L) with or without NIR laser irradiation (808 nm, 1.5 W cm2, 5 min). (2) Orthotopic brain tumor model. D) MRI
images of brains of orthotopic N2a glioma-bearing mice treated with (a) saline (200 L), (b) PEG-AuNRs@SiO2, or (c) RVG-PEG-AuNRs@SiO2 (each
2.5 103 m, 200 L) with NIR laser irradiation (808 nm, 1.5 W cm2, 5 min). E) Histological observations of brains of N2a glioma-bearing mice treated
with saline (200 L), PEG-AuNRs@SiO2 or RVG-PEG-AuNRs@SiO2 (each 2.5 103 m, 200 L) with NIR laser irradiation (808 nm, 1.5 W cm2, 5 min).

injected intravenously at predetermined times; after 4 h, the
respective tumor sites were irradiated by an 808 nm laser at
the low power density of 1.5 W cm2 for 5 min. The laser irra
diation alone (without gold nanoparticles) did not induce skin
damage (burn or charring spots) and tumor suppression. As
shown in Figure 4AC, RVG-PEG-AuNRs@SiO2 coordinated
by PTT suppressed growth of xenografted tumors in mice to
a remarkable extent in terms of tumor volume, whereas plain
PEG-AuNRs@SiO2 barely suppressed tumor growth. Tumor
volumes in mice treated with RVG-PEG-AuNRs@SiO2 and an
NIR laser were markedly smaller than those of mice treated
with PEG-AuNRs@SiO2 or control saline (124.8 147.5,
1067.4 295.4, and 2323.2 436.3 mm3, respectively) at 7 d
after treatment. Interestingly, the tumors of two mice treated
with RVG-PEG-AuNRs@SiO2 nearly vanished, and the slight
skin damage caused by 808 nm laser irradiation was healed
after 13 d. As shown in the magnetic resonance imaging (MRI)
and H&E staining photos (Figure 4D,E), orthotopic tumors of
mice treated with RVG-PEG-AuNRs@SiO2 were suppressed to
a greater extent by NIR laser irradiation than were the tumors
in the other two groups (Figure S10, Supporting Information).
These results indicate that our RVG-PEG-AuNRs@SiO2 have
good potential for treating brain tumors based on their superior
targetability and thermal response to NIR.
In summary, we successfully developed gold nanorods that
mimic the rabies virus in terms of size, shape, surface glycopro
tein property, and in vivo behavior. Our RVG-PEG-AuNRs@SiO2
had a length and a width of 120 and 50 nm, respectively, and
an aspect ratio of 2.34, which is very similar to that of the live
rabies virus (2.4). Considering our data, the rod shape of our
RVG-PEG-AuNRs@SiO2 appeared to play a crucial role in facili
tating cellular uptake into neuronal cells in vitro and inducing
a hyperthermal effect in response to NIR laser irradiation. The
surface-modified RVG29 peptide substantially improved the in
vivo distribution of the nanorods in the central nervous system.
Importantly, it is of great interest that these nanorods not only

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resembled the appearance of the live rabies virus but targeted
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the brain through the neuronal pathway bypassing the blood
brain barrier. Also, RVG-PEG-AuNRs@SiO2 were able to induce
a hyperthermal effect in response to NIR laser (808 nm) irra
diation, based on localized surface plasmon resonance, to effec
tively suppress brain tumors of mice. Together, these results
support that the rabies virus mimetic gold nanorods are a poten
tial prototype delivery platform for treating brain tumors.
Experimental Section
Materials, details on preparation and characterization of RVG-PEG-
AuNRs@SiO2, and procedures of cell experiments, imaging, and cancer
studies using animals are included in the Supporting Information. The
mice were cared for according to the guidelines issued by National
Institute of Health for the care and use of laboratory animals (NIH
publication 8023, revised in 1996).
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
This research was supported by a grant from the Korea Health Technology
R&D Project through the Korea Health Industry Development Institute
(KHIDI), funded by the Ministry of Health and Welfare, Republic of
Korea (No. HI15C2221).
Received: October 16, 2016
Revised: December 9, 2016
Published online:
[1] a) E. S.Lee, D.Kim, Y. S.Youn, K. T.Oh, Y. H.Bae, Angew. Chem.
Int. Ed. 2008, 47, 2418; b) R. A.Petros, J. M.DeSimone, Nat. Rev.
Drug Discovery 2010, 9, 615.
[2] a) Y. J.Ma, R. J. M.Nolte, J. J.Cornelissen, Adv. Drug Delivery Rev.
2012, 64, 811; b) Y.Geng, P.Dalhaimer, S. S.Cai, R.Tsai, M.Tewari,
T.Minko, D. E.Discher, Nat. Nanotechnol. 2007, 2, 249.
[3] a) S.Fawell, J.Seery, Y.Daikh, C.Moore, L. L.Chen, B.Pepinsky,
J.Barsoum, Proc. Natl. Acad. Sci. USA 1994, 91, 664;
b) X. P.Qi, T.Droste, C. C.Kao, Mol. Plant-Microbe Interact. 2011,
24, 25.
[4] a) W.Wu, S. C.Hsiao, Z. M.Carrico, M. B.Francis, Angew.
Chem. Int. Ed. 2009, 48, 9493; b) L.Xu, P.Frederik, K. F.Pirollo,
W. H.Tang, A.Rait, L. M.Xiang, W. Q.Huang, I.Cruz, Y. Z.Yin,
E. H.Chang, Hum. Gene Ther. 2002, 13, 469; c) X. B.Xiong,
H.Uludag, A.Lavasanifar, Biomaterials 2010, 31, 5886.
[5] a) Y.Liu, R. Q.Huang, L.Han, W. L.Ke, K.Shao, L. Y.Ye, J. N.Lou,
C.Jiang, Biomaterials 2009, 30, 4195; b) M. J.Warrell, D. A.Warrell,
Lancet 2004, 363, 959.
[6] a) Y. K.Gao, Z. Y.Wang, J. H.Zhang, Y. X.Zhang, H.Huo, [27] V. P.Zharov, K. E.Mercer, E. N.Galitovskaya, M. S.Smeltzer, Bio-
T. Y.Wang, T. Y.Jiang, S. L.Wang, Biomacromolecules 2014, 15,
1010; b) J. Y.Kim, W. I.Choi, Y. H.Kim, G.Tae, Biomaterials 2013,
34, 1170.
[7] M. J.Schnell, J. P.McGettigan, C.Wirblich, A.Papaneri, Nat. Rev.
Microbiol. 2010, 8, 51.
1605563 (8 of 8) wileyonlinelibrary.com 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
[8] H.Yang, Pharm. Res. 2010, 27, 1759.
[9] H. J.Byeon, L. Q.Thao, S.Lee, S. Y.Min, E. S.Lee, B. S.Shin,
H. G.Choi, Y. S.Youn, J. Controlled Release 2016, 225, 301.
[10] a) L.Biddlestone-Thorpe, N.Marchi, K.Guo, C.Ghosh, D.Janigro,
K.Valerie, H.Yang, Adv. Drug Delivery Rev. 2012, 64, 605; b) A. G.de
Boer, P. J.Gaillard, Annu. Rev. Pharmacol. Toxicol. 2007, 47, 323.
[11] a) T. L.Lentz, T. G.Burrage, A. L.Smith, J.Crick, G. H.Tignor,
Science 1982, 215, 182; b) M.Lafon, J. NeuroVirol. 2005, 11, 82;
c) P.Kumar, H. Q.Wu, J. L.McBride, K. E.Jung, M. H.Kim,
B. L.Davidson, S. K.Lee, P.Shankar, N.Manjunath, Nature 2007,
448, 39.
[12] T. L.Lentz, J. Mol. Recognit. 1990, 3, 82.
[13] a) P.Kolhar, A. C.Anselmo, V.Gupta, K.Pant, B.Prabhakarpandian,
E.Ruoslahti, S.Mitragotri, Proc. Natl. Acad. Sci. USA 2013, 110,
10753; b) S.Dasgupta, T.Auth, G.Gompper, Nano Lett. 2014, 14,
687; c) S.Salatin, S. M.Dizaj, A. Y.Khosroushahi, Cell Biol. Int.
2015, 39, 881.
[14] V. P.Chauhan, Z.Popovic, O.Chen, J.Cui, D.Fukumura,
M. G.Bawendi, R. K.Jain, Angew. Chem. Int. Ed. 2011, 50, 11417.
[15] a) X. L.Huang, X.Teng, D.Chen, F. Q.Tang, J. Q.He, Bioma-
terials 2010, 31, 438; b) R.Agarwal, V.Singh, P.Jurney, L.Shi,
S. V.Sreenivasan, K.Roy, Proc. Natl. Acad. Sci. USA 2013, 110,
17247.
[16] a) P.Guichard, T.Krell, M.Chevalier, C.Vaysse, O.Adam,
F.Ronzon, S.Marco, J. Struct. Biol. 2011, 176, 32; b) R. L.Guerrant,
in Tropical Infectious Diseases: Principles, Pathogens, and Practice,
Vol. 2 (Eds: R. L. Guerrant, D. H. Walker, P. F. Weller), Elsevier,
Philadelphia, PA, USA 2006, pp. 839851.
[17] E. C.Dreaden, A. M.Alkilany, X. H.Huang, C. J.Murphy,
M. A.El-Sayed, Chem. Soc. Rev. 2012, 41, 2740.
[18] a) E. E.Connor, J.Mwamuka, A.Gole, C. J.Murphy, M. D.Wyatt,
Small 2005, 1, 325; b) P.Ghosh, G.Han, M.De, C. K.Kim,
V. M.Rotello, Adv. Drug Delivery Rev. 2008, 60, 1307.
[19] L.Vigderman, E. R.Zubarev, Chem. Mater. 2013, 25, 1450.
[20] a) X. S.Kou, S. Z.Zhang, Z.Yang, C. K.Tsung, G. D.Stucky,
L. D.Sun, J. F.Wang, C. H.Yan, J. Am. Chem. Soc. 2007, 129, 6402;
b) R.Fenger, E.Fertitta, H.Kirmse, A. F.Thunemann, K.Rademann,
Phys. Chem. Chem. Phys. 2012, 14, 9343.
[21] W. C.Wu, J. B.Tracy, Chem. Mater. 2015, 27, 2888.
[22] a) J.Lee, E. J.Jeong, Y. K.Lee, K.Kim, I. C.Kwon, K. Y.Lee, Small
2016, 12, 1201; b) I.Kim, T. H.Kim, K.Ma, E. S.Lee, D.Kim,
K. T.Oh, D. H.Lee, K. C.Lee, Y. S.Youn, Bioconjugate Chem. 2010,
21, 1513; c) H. J.Byeon, S. Y.Min, I.Kim, E. S.Lee, K. T.Oh,
B. S.Shin, K. C.Lee, Y. S.Youn, Bioconjugate Chem. 2014, 25, 2212;
d) F.Tang, L.Li, D.Chen, Adv. Mater. 2012, 24, 1504.
[23] a) Q.He, Z.Zhang, F.Gao, Y.Li, J.Shi, Small 2011, 7, 271;
b) E.Blanco, H.Shen, M.Ferrari, Nat. Biotechnol. 2015, 33, 941;
c) Y.Gao, Z. Y.Wang, J.Zhang, Y.Zhang, H.Huo, T.Wang, T.Jiang,
S.Wang, Biomacromolecules 2014, 15, 1010.
[24] a) A. M.Alkilany, L. B.Thompson, S. P.Boulos, P. N.Sisco,
C. J.Murphy, Adv. Drug Delivery Rev. 2012, 64, 190; b) B.Jang,
J. Y.Park, C. H.Tung, I. H.Kim, Y.Choi, ACS Nano 2011, 5, 1086;
c) Z. J.Zhang, L. M.Wang, J.Wang, X. M.Jiang, X. H.Li, Z. J.Hu,
Y. H.Ji, X. C.Wu, C. Y.Chen, Adv. Mater. 2012, 24, 1418.
[25] H.Huang, P. K.Jain, I. H.El-Sayed, M. A.El-Sayed, Laser Med. Sci.
2008, 23, 217.
[26] E.Dulkeith, M.Ringler, T. A.Klar, J.Feldmann, A. M.Javier,
W. J.Parak, Nano Lett. 2005, 5, 585.
phys. J. 2006, 90, 619.
[28] E. B.Dickerson, E. C.Dreaden, X. H.Huang, I. H.El-Sayed,
H. H.Chu, S.Pushpanketh, J. F.McDonald, M. A.El-Sayed, Cancer
Lett. 2008, 269, 57.
[29] H. C.Huang, K.Rege, J. J.Heys, ACS Nano 2010, 4, 2892.
Adv. Mater. 2017, 1605563