Letter

pubs.acs.org/NanoLett

A Peptide-Based Nanobrous Hydrogel as a Promising DNA

Nanovector for Optimizing the Ecacy of HIV Vaccine
Yue Tian,, Huaimin Wang, Ye Liu, Lina Mao, Wenwen Chen,, Zhening Zhu,, Wenwen Liu,,
Wenfu Zheng, Yuyun Zhao, Deling Kong, Zhimou Yang,*, Wei Zhang, Yiming Shao,*,
and Xingyu Jiang*,

Beijing Engineering Research Center for BioNanotechnology and CAS Key Lab for Biological Eects of Nanomaterials and
Nanosafety, National Center for NanoScience and Technology, No., 11 Zhongguancun Beiyitiao, Beijing 100190, China

University of Chinese Academy of Sciences, Beijing 100049, China

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, and College of
Life Sciences, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin
300071, China

State Key Laboratory for Infectious Disease Prevention and Control, National Center for AIDS/STD Control and Prevention,
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Chinese Center for Disease Control and
Prevention, 155 Changbai Road, Changping District, Beijing 102206, China
S Supporting Information
*

ABSTRACT: This report shows that a nanovector composed

of peptide-based nanobrous hydrogel can condense DNA to
result in strong immune responses against HIV. This
nanovector can strongly activate both humoral and cellular
immune responses to a balanced level rarely reported in
previous studies, which is crucial for HIV prevention and
therapy. In addition, this nanovector shows good biosafety in
vitro and in vivo. Detailed characterizations show that the
nanobrous structure of the hydrogel is critical for the
dramatically improved immune responses compared to existing materials. This peptide-based nanobrous hydrogel shows great
potential for ecacious HIV DNA vaccines and can be potentially used for delivering other vaccines and drugs.
KEYWORDS: Peptide-based nanobrous hydrogel, HIV DNA vaccine, DNA condensation, immune response, safety evaluation

activity of DNA during complex preparation.13,14 Therefore, it

is still challenging to prepare a proper delivery system that is
both safe and eective for broad application.
Nanobers provide an ideal platform to interact with
biomolecules or modify their biological functions. They can
guide the growth of neurons, improve the limit of detection in
disease diagnosis, and elongate DNA through contacts at
nanoscale.1517 The structure of nanobers can also modulate
immune responses of human immune cells and deliver cargos
to cells,18,19 indicating a great potential in promoting immune
responses of DNA vaccine. Another platform suited for DNA
delivery is based on hydrogel because of its high loading
capacity, mild working conditions, and good biocompatibility.20,21 Supramolecular hydrogel is a kind of hydrogel
composed of nanobers formed by the self-assembly of small
molecules (molecular weight usually <2000) in aqueous
solutions.22 They can rapidly respond to various external
stimuli. For example, the change of pH, ionic concentration,

IV infection is presently incurable and has led to more

than 30 million deaths.1 There is an urgent need to
develop ecacious vaccines that can prevent HIV infection.
Traditional vaccines such as live or attenuated viruses are
ineective and pose potential risks. As a consequence, safe
alternatives such as DNA vaccines have attracted great
attention. DNA vaccines can generate long-term humoral and
cellular immune responses which provide protective immunity.2,3 However, the major drawback of DNA vaccines is their
low immunogenicity in clinical tests.4 DNA vaccines are easily
degraded by DNases and lysosomes, and injected naked DNA
plasmids are poorly distributed and ineciently expressed;
thus, DNA vaccines can only induce modest humoral and
cellular immune responses.5 As such, DNA vaccines must be
injected with a delivery system to enhance the immune
responses. Dierent synthetic delivery systems have been
constructed to promote ecient delivery of DNA into
mammalian cells and protect them from degradation, including
polymers,68 liposomes,9 and nano- or microparticles.10
Although these delivery systems can be eective, some
disadvantages limit their further usage, such as toxicity,11
small amount of antigen loading,12 and decreased biological
2014 American Chemical Society

Received: December 9, 2013

Revised: February 16, 2014
Published: February 24, 2014
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Figure 1. Synthesis and characterization of peptide-based nanobrous hydrogels. (a) Chemical structures of all precursors used to form peptidebased nanobrous hydrogels and schematic illustration of enzymatic conversion. (b) CD spectra of three nanovectors. (c) TEM images of three
nanovectors. The insets show details of their helicity. Scale bar: 100 nm (black); 50 nm (white, left); 25 nm (white, middle); 50 nm (white, right).

Scheme 1. Process of Peptide-Based Nanobrous Hydrogel for Enhancing Immune Responses of HIV DNA Vaccines

strong antibody responses in mice without other adjuvant.34

Therefore, these eorts suggest that peptide-based nanobrous
hydrogel might be a good candidate for HIV DNA vaccine
nanovector for enhancing immune responses.
Nap-GFFY (Nap represents naphthalene acetic acid, G
represents glycine, F represents phenylalanine, Y represents
tyrosine) was demonstrated as a short peptide to construct
gelators of nanobrous hydrogel.35 The C-terminal residue of
the short peptide is very important for their self-assembly
property and applications.36 For example, amide-terminated
dipeptide of FF can self-assemble into nanospheres and deliver
ssDNA into cells.37 Our previous work designed Nap-GFFYOMe (G-OMe) and Nap-GFFY-OH (G-OH) structures, and
we found that Nap-GFFY-OMe could form peptide-based
nanobrous hydrogel with a minimum gelation concentration
(MGC) of 0.01 wt % after enzymatic conversion, which is the

temperature, or addition of enzymes could control the assembly

of supramolecular hydrogelators.2325 Recently, peptide-based
delivery system emerged as an alternative means to eciently
introduce genes or drugs into cells both in vitro and in vivo.26
Some enzyme-triggered peptide-based nanobrous hydrogel are
useful for the entrapment of drug molecules without reducing
their activity because that they can work in mild conditions.2730 They are also biocompatible and have welldened structures. Subcutaneous or intraperitoneal injection of
peptide-based nanobrous hydrogels with diameter 20 nm
cause little acute or long-term toxicity in vivo,31 and new tissues
could form as implanted supramolecular hydrogel biodegrades.32 Some peptide-based hydrogelators can assemble into
two-layered nanobrous structures with onion-like architecture and provide long-term sustained release.33 In addition,
assembly of peptides with T and B cell epitopes can elicit
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Figure 2. Immunological evaluation. (a) Anti-HIV antibody and IFN- secretion by mice immunized with HIV Env DNA as antigen, complexes of
antigen with G-NMe, G-OMe, G-OH, PEI, and CpG, and other pure gels. Animals were immunized intramuscularly at the 0th, 2nd, and 4th week
and sacriced at the 6th week. Results are presented as the mean SD of titers (log 10) or spots. a1, p = 0.001; b1, p = 0.7527; c1, p = 0.6177; a2, p
= 0.0003; b2, p = 0.3131; c2, p = 0.5497. (b) Anti-HIV antibody productions. Animals were injected i.m., i.d., and s.c. with G-NMe loaded DNA or
with naked DNA at weeks 0, 2, and 4. Mice were sacriced at week 6. Mice sera were collected at weeks 0 (preimmunization), 2 (1st immunization),
4 (2nd immunization), and 6 (3rd immunization). Antibody titers were determined by ELISA. Dierences were statistically signicant (p < 0.05). (c)
Cellular immune responses elicited by dierent immunization regimens, including antigen-specic IFN- or IL-4 producing splenocytes generated by
G-NMe complexed with DNA or DNA alone using ELISPOT assays.

Synthesis and Characterization of Peptide-Based

Nanobrous Hydrogels. The synthesis of G-NMe(p), GOMe(p), and G-OH(p) was straightforward (Supporting
Information Scheme S1). Three precursors dissolved well in
phosphate buered saline (PBS). After the addition of alkaline
phosphatase, the solution became slightly turbid immediately,
and the precursors turned into translucent hydrogels within 10
min at room temperature. Compared with other microemulsion
or nanoparticle-based delivery systems, which typically resort to
harsh conditions (such as high shear, an organic/aqueous
interface, localized high temperature, freeze-drying, surfactants,
and/or detergents),41,42 our system will not expose DNA to
these conditions that have the potential to cause damage or
degradation.
We characterized these supramolecular nanovectors using
circular dichroism (CD) and transmission electron microscopy
(TEM). All the nanovectors showed a positive band near 210
nm, a broad negative band at 260280 nm in CD, and two
negative peaks around 196 and 230 nm (Figure 1b), which
could be assigned to the * transition of naphthyl aromatics
and n* transition of local peptide backbones.4345 In
addition, G-NMe showed a negative CD peak at around 325
nm, while G-OMe and G-OH showed a positive CD peak at
around 300 and 350 nm. TEM images also show the distinct
structure of G-NMe, which is left-handed and in the width 12
nm, while the nanobers of G-OMe and G-OH are righthanded with widths between 4 and 6 nm (Figure 1c and Figure

lowest MGC value for peptide-based gelator reported up to

now.38 Gels with low MGCs are particularly attractive for DNA
carrier because the low gelation concentration can decrease the
toxicity of gelators and increase the drug loading of the
resulting hydrogels.39
In this report, based on the structure of Nap-GFFY-OMe, we
design a novel gelator (Nap-GFFY-NMe, G-NMe, whose
precursor is Nap-GFFpY-NMe, G-NMe(p)) modied with the
methylamino group, which has been proved to enhance
immune responses (Figure 1a).40 These peptide-based
precursors can be dissolved in an aqueous solution and
assemble into nanobrous hydrogels when triggered by an
enzyme at room temperature (Figure 1a). G-NMe nanovector
signicantly enhances both humoral and cellular immunity of
HIV Env DNA (DNA encoding the HIV-1 envelope protein gp
145) via the immunization by intramuscular (i.m.), intradermal
(i.d.), or subcutaneous (s.c.) injection. The enhanced immunity
by G-NMe nanovector probably results from the left-handed
structure of nanobers, which can condense DNA and protect
them from degradation, thus promoting the entry of DNA into
mammalian cells (Scheme 1). In addition, this nanovector can
be metabolized gradually in the body, and it is safe for use. This
is the rst report on the peptide-based nanobrous hydrogel
used as DNA vaccine nanovector. Our study could provide a
safe and eective DNA vaccine nanovector for the prevention
of the HIV infection. It may open the door for the vaccination
strategy based on peptide-based nanobrous hydrogels.
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cells.5254 Signicant humoral immune responses induced by

this nanovector/antigen complex encouraged us to investigate
cellular immune responses, which is also crucial for a successful
HIV vaccine.
To comprehensively evaluate the cellular immune responses,
we used ELISPOT to determine the levels of IL-4 and IFN-,
which are typical representative for cellular immunity. We
coadministered HIV DNA vaccine with the nanovector (GNMe) and found signicantly augmented numbers of
lymphocytes that secret IL-4 and IFN- compared with naked
DNA alone (Figure 2c). This nding was a pleasant surprise
because (i) injection of DNA alone even at large quantities
often fails to induce positive IL-4 response55 and (ii) of the
nanovectors or delivery systems we have worked with and
reported in the literature, little has induced a signicant increase
in the level of IL-4.5658 Furthermore, this positive cellular
immune response is achieved in all three routes of
administration (i.m, i.d., and s.c.). IL-4 is a cytokine crucial
for promoting the maturation of B cells, producing antigenspecic antibodies to neutralize the virus.59 This result
correlates very well with the increased amount of antibody
(Figure 2b). The augmented IFN- responses play a critical role
in the control of HIV replication by targeting the internal
epitopes which are not accessible to antibodies to kill infected
cells.60 The enhanced IL-4 and IFN- responses to HIV antigen
suggest that our nanovector has the potential to induce the
polyfunctionality of HIV vaccine, which is a hallmark in longterm nonprogress persons (individuals who are infected by HIV
but does not develop AIDS) and is able to eectively control
HIV replication to prevent the onset of AIDS.6164 Because the
levels of these cellular factors are critical parameters for clinical
trials of HIV vaccine, we strongly believe that this reported
nanovector shows great promise for clinical success.
Taken together, these data showed that G-NMe supramolecular nanovector could induce balanced immune response
(strong antigen specic antibody secretion, IFN- and IL-4
secreting lymphocytes production), which is rarely seen in
other delivery systems8,56,57 and is considered to be crucial for
protecting human beings from HIV infection. This nanovector
can induce immune responses via multiple routes of injection,
including i.m., i.d., and s.c. Compared with other delivery
system based on nanomaterials, such as gold nanorods that only
elicit enhanced immunogenicity by i.d. route,56 our system
allows more options in the administration of the vaccine for
clinical trials and thus may translate into an increased rate of
success for HIV vaccine.
Enhanced Transfection Eciency Depends on DNA
Condensation. To explore the mechanism of the enhanced
immune responses of G-NMe, we characterized the G-NMe/
DNA complexes by CD and TEM. Previous research shows
that the condensation status of DNA can help protect DNA
from degradation.65,66 Highly condensed DNA will result in
increased transfection eciency in mammalian cells. The
transfection eciency of 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP, cationic liposome)/L--dioleoylphosphatidylethanolamine (DOPE, neutral lipids) can be improved as
compared to DOTAP alone because DOTAP/DOPE induces
the condensation of DNA.67 Similarly, poly-L-lysine can
package DNA into a condensed structure and lead to a higher
transfection eciency than poly-D-lysine.68 In our experiments,
DNA alone exhibited short-wave negative and long-wave
positive CD bands of similar magnitudes, which is typical for
random coli-like DNA conformation (Figure 3a and Figure

S4). The dierent chirality of G-NMe is consistent with the CD

results, which may be induced by the substitution of an Nmethyl group.46
Enhanced Humoral and Cellular Immune Responses.
We evaluated the humoral and cellular immune responses of
three nanovectors in mice model. HIV Env DNA was chosen as
a model antigen in our in vivo research, considering that it had
been already used in human trials.47,48
HIV-specic enzyme-linked immunosorbent assay (ELISA)
and enzyme linked immunospot assay (ELISPOT) were used
to evaluate humoral and cellular immune responses. Both
humoral and cellular immune responses are indispensable for
protecting human beings from the attacks of HIV viruses.
Antibody titer is an important parameter for the evaluation of
humoral immune responses, and a high level of antigen specic
antibodies can neutralize the viruses, inhibit the progression of
HIV, and reduce HIV infection. IFN- determination by
ELISPOT is a typical assay for cellular immunity, which plays a
crucial role in eliminating infected cells and building pivotal
defensive barriers against HIV. We intramuscularly (i.m.)
immunized mice with three kinds of nanovectors or nanovetor/
antigen complexes at the zeroth, second, and fourth week and
detected the immune responses at the sixth week. Naked DNA,
commercial transfection agent polyethylenimine (PEI), and
CpG oligodeoxynucleotide (CpG-ODN) were used as
controls.4951 Signicantly enhanced humoral and cellular
immune responses were found in the G-NMe/antigen group,
the antibody titer, and numbers of IFN- secreting lymphocytes
increased 8- and 3-fold, respectively (Figure 2a). The
nanovectors themselves did not elicit detectable antigen-specic
immune responses (Figure 2a), and the complexes of DNA
with G-OMe and G-OH nanovectors induced similar humoral
and cellular immune responses to those in the naked DNA
group (Figure 2a). While the PEI/antigen complex showed
poor immune responses, even lower than those of the naked
DNA group (Figure 2a), which may be due to the toxicity of
PEI, because the large amount of positive charges of PEI often
damage cellular membranes or bind to important cellular
components to cause severe cellular damage.11 We next
systematically evaluated the humoral and cellular responses of
G-NMe as HIV DNA vaccine nanovector.
To evaluate the eects of nanovector (G-NMe) on DNA
vaccine in inducing humoral immune responses, we conducted
two additional immunization regimens (intradermal, i.d., and
subcutaneous, s.c.) and detected specic antibody titers at
weeks 0, 2, 4, and 6 (Figure 2b). Before immunization, mice in
all groups showed negative anti-HIV antibody responses. The
dierence between titers of anti-HIV antibodies obtained from
the HIV antigen (Env DNA) and G-NMe/antigen group by
i.m. injection was not statistically signicant initially (p > 0.05).
The antibody titers rose quickly after just two G-NMe/antigen
injections, with a signicantly (p = 0.0003) improved titer (14fold increase) compared with those induced by naked DNA
alone. After the third immunization, antibody titers induced by
G-NMe/antigen were from 1:512001:204800, signicantly
higher than that in naked DNA group (p < 0.05). The antibody
titers of mice injected with G-NMe/antigen by i.d. and s.c.
injections showed a similar trend to i.m. immunizations. These
data demonstrated that DNA vaccine administered with GNMe nanovector elicited high antibody titers in all three
immunization regimes. High levels of specic antibodies can
eciently inhibit both HIV-1 infection and HIV envelope
protein-mediated cell fusion of infected cells and uninfected
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Figure 3. Characterization of peptide-based nanobrous hydrogels formulated with DNA. (a) CD spectrum of DNA with dierent concentration of
supramolecular hydrogel. 1, 0.05 wt %; 2, 0.1 wt %; 3, 0.2 wt %. (b) TEM images of the nanovector/DNA complexes. The arrows show status of
DNA after complexation with each gel. Scale bar: 100 nm. (c) Fluorescence images of 293T cells transfected by nanovector/EGFP plasmid.
Concentration of precursor in each gel is 0.2 wt %. DNA with PEI and DNA alone were used as positive control and negative control, respectively.
Scale bar: 100 m.

S5a).67 Once combined with G-NMe nanovector, the positive

peak of DNA CD signature at 280 nm decreased and the
magnitude of the negative CD band further increased. PEI,
which condensed DNA eciently by positive charges, showed a
similar CD spectrum change of DNA. Such patterns typically
indicate a conversion of the randomly coiled DNA into a
condensed form.69 In comparison, DNA composited with GOH or G-OMe showed less conformational change, suggesting
that no condensation took place. The dierent modes of
condensation with DNA were also proved by TEM characterization. The DNA was condensed into spheres by G-NMe with
diameters 30 nm, which was similar to the morphology of
condensed DNA in PEI solution, while DNA in G-OMe or GOH exhibit random coil-like structures (Figure 3b and Figure
S5b).70,71 These results demonstrated that the left-handed
structure of G-NMe can eciently condense DNA, protect
them from degradation, and enhance the eciency of
transfection into cells.
To further prove our hypothesis that condensed DNA can
easily enter cells and induce protein expression, we evaluated
the transfection eciency of these gels in vitro by using a
plasmid for enhanced green uorescent protein (pEGFP) as a
reporter. GFP expression level is higher in G-NMe group than
that in G-OH and G-OMe treatment (Figure 3c). The maximal
transfection eciency of G-NMe is 30.38%36.60%, while the
transfection eciency of G-OH and G-OMe (<1%) is similar to
the naked DNA (<1%, Figure S5c,d). Although the transfection
eciency of G-NMe is lower than PEI (65.48%85.17%), GNMe has no cytotoxic eect even at a high concentration while
PEI displays a signicant toxicity on 293T cells (Figure S5e).
The LD50 (median lethal dose) value of PEI for cells is 10 g/
mL; these results are paralleled by the ndings from in vivo
immune assay of PEI.

Excellent Safety and Biocompatibility of Supramolecular Nanovector. To prepare for clinical trials, we
evaluated the key factors such as cellular toxicity, histological
change at the administration site, uctuation in body weight,
and metabolic property to qualitatively assess the toxicity of GNMe nanovector. The in vitro cytotoxicity of the G-NMe was
evaluated on mouse primary T cells, B cells, macrophages, and
human umbilical vein endothelial cells (HUVEC) using a cell
counting kit (CCK). The cell viabilities for G-NMe were higher
than 95% after incubation for 72 h, suggesting good
biocompatibility to all types of cells tested (Figure 4a). To
further investigate in vivo toxicity of G-NMe, a histological
analysis of muscle, epidermis, and dermis from the injection site
was performed. There was no evidence of inammatory cell
inltration or necrosis and no obvious aggregation of
macrophages and lymphocytes in all these samples (Figure
4b). The arrangement of the muscle bers and dermal glands
were not aected. Histological analysis shows no visible
dierence between the G-NMe-treated and control tissues
(Figure 4b). We next evaluated the increase of body weight of
mice over 30 days. Throughout this period, mice injected with
G-NMe did not show any adverse eects in their growth. In the
highest dose group, the gained body weight increased from 0.0
0.2 g (1st day) to 4.8 1.6 g (30th day), which is close to
that of the normal and PBS-treated group (Figure 4c). We used
125
I-labeled Nap-GFFY-NMe to evaluate the metabolism of this
nanovector. Signal intensity induced by injection of 125I-labeled
G-NMe in mice showed a concentration-dependent eect; the
representative radioactive intensity in mice was acquired at
dierent time points (Figure S6). Mice injected with G-NMe
exhibited a high level of radioactivity immediately after
administration, and the radioactivity decreased with time. At
day 21 there were 3.03%, 2.88%, and 1.44% of the radioactivity
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NMeDNA complexes. This material is available free of charge

via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*E-mail xingyujiang@nanoctr.cn (X.J.).

*E-mail yangzm@nankai.edu.cn (Z.Y.).
*E-mail yshao08@gmail.com (Y.S.).
Author Contributions

Y.T. and H.W. contributed equally.

Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
We acknowledge Ministry of Science and Technology
(2012AA022703), the Ministry of Health P. R. China
(2012ZX10001-008), the Chinese Academy of Science
(NNCAS-2010-5 and XDA01020304), the National Science
Foundation of China (21025520, 81361140345, and
51222303), and Beijing Municipal Science & Technology
Commission (Z131100002713024). We thank members of Dr.
Shao laboratory for helpful discussions and Ms. Qi X. Y. for
help with TEM.

Figure 4. Biosafety evaluation of G-NMe. (a) Cytotoxicity of G-NMe

containing 0.2 wt % precursors on primary T lymphocytes, B
lymphocytes, macrophages, and HUVECs. Values represent the
relative viability compared to untreated cells as mean SEM of one
representative experiment (n = 3). (b) Representative muscle,
epidermis, and dermis histology for the control and G-NMe injected
mice. These tissues did not exhibit signs of toxicity. Scale bar: 100 m.
(c) The weight increase of mice in dierent groups (normal: without
any treatments; PBS: with 50 L of PBS; and 0.3, 0.5, and 1.0 wt %:
with 50 L of gels from 0.3, 0.5, and 1.0 wt % of Nap-GFFpY-NMe,
respectively). (d) Radioactivity remaining (% of activity at day 0) at
the injection sites of G-NMe containing 0.3, 0.5, and 1 wt % precursor
at dierent time points.

remaining at the injection sites of 0.3, 0.5, and 1 wt % group,

respectively (Figure 4d). These results prove that the G-NMe is
safe to use as a vaccine nanovector.
In conclusion, the Nap-GFFY-NMe nanovector provides a
safe, straightforward, and eective approach for HIV DNA
vaccine. This nanovector can achieve optimized cellular and
humoral immune responses in mice through multiple
administration routes. The reason for this high eciency is
that this nanovector can condense DNA, promote DNA
transfection, and enhance gene expression in vitro. Furthermore, this peptide-based nanovector shows promising biocompatibility and no obvious toxicity. This is the rst report on
peptide-based nanobrous hydrogel used as a HIV DNA
nanovector. We expect that this nanovector can open the doors
for eective vaccination based on peptide-based nanobrous
hydrogels.

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ASSOCIATED CONTENT

S Supporting Information
*

Methods and experimental procedures; synthetic scheme of

peptide-based nanobrous hydrogels, the characterization of GNMe(p) with 1H NMR, 13C NMR, 31P NMR, and MS, the
characterization of nanovectorsDNA complexes with CD
spectrometer, TEM images for nanovectorsDNA complexes,
images of cell transfection, radioactive images of injected G1444

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(16) Yang, D. Y.; Niu, X.; Liu, Y. Y.; Wang, Y.; Gu, X.; Song, L. S.;
Zhao, R.; Ma, L. Y.; Shao, Y. M.; Jiang, X. Y. Adv. Mater. 2008, 20
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