Self-assemblies of plasmonic gold/layered double

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Nano Research
Nano Res
DOI 10.1007/s12274-015-0851-6
hydroxides with highly efficient antiviral effect against
hepatitis B virus
Gabriela Carja1 (), Elena Florentina Grosu1, Catalina Petrarean2, and Norica Nichita2
Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0851-6
http://www.thenanoresearch.com on July 4, 2015
© Tsinghua University Press 2015
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1
TABLE OF CONTENTS (TOC)
The highly efficient antiviral effect and good cytocompatibility of the self-assemblies of nanoparticles of
gold/layered double hydroxides are demonstrated for the first time, revealing the high potential of these
nanocomposites to be tailored as novel bioactive therapeutics for the treatment of hepatitis B.
hydroxides with highly efficient antiviral effect
against hepatitis B virus
Gabriela Carja1*, Elena Florentina Grosu1, Catalina
Petrarean2 and Norica Nichita
1
Departament of Chemical Engineering, Faculty of
Chemical Engineering and Environmental Protection,
Technical University “Gh. Asachi” of Iasi, Bd.
Mangeron No. 71, Iasi 700554, Romania
2
Department of Viral Glycoproteins, Institute of
Biochemistry of the Romanian Academy, Splaiul
Independentei 296, Bucharest 060031
Nano Research
DOI (automatically inserted by the publisher)
2
Nano Res.
Research Article
hepatitis B virus
Gabriela Carja1 (), Elena Florentina Grosu1, Catalina Petrarean2 and Norica Nichita2
Received: day month year
ABSTRACT
Revised: day month year
The design of complex nanocomposites engineered to interact with cells and to
Accepted: day month year
specifically act against hepatitis B virus (HBV) is a challenging approach in the
(automatically inserted by
the publisher)
© Tsinghua University Press
conditions that the current therapies against HBV infections have limited
efficacy and serious side effects. Herein, the antiviral effect of the
self-assemblies
of
plasmonic
gold
and
layered
double
hydroxides
and Springer-Verlag Berlin
(AuNPs/LDHs) is demonstrated, for the first time, using HBV as a model virus
Heidelberg 2014
and the hepatoma-derived HepG2.2.215 cells supporting viral replication,
assembly
and
secretion
of
infectious
virions
and
subviral
particles.
KEYWORDS
AuNPs/LDHs were obtained by a simple, cost-effective procedure in
plasmonic gold,
which small AuNPs (~ 3.5 nm) were directly obtained and organized on the
layered double
hydroxides; LDH,
surface of larger nanoparticles (~ 150 nm) of LDHs by exploiting the
antiviral effect,
“structural memory” in the aqueous solution of Au(O 2 CCH3 )3. The
HBV, hepatitis,
cytotoxicity
capability of MgLDH, ZnLDH and MgFeLDH to manifest their
self-assembly approach of AuNPs and LDHs was studied by transmission
electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), powder
X-ray diffraction (PXRD) and UV-Vis analysis (UV-Vis). All AuNPs/LDHs
reduced the amount of viral and subviral particles released from treated cells
by up to 80% and exhibited good cytocompatibility. AuNPs/MgFeLDH
showed the highest antiviral HBV response with more than 90%
inhibition of HBV secretion for the whole concentration range.
Preliminary investigations on the mechanism of HBV inhibition reveals that
in the presence of AuNPs/LDHs the HBV particles are sequestered within
treated cells. With these antiviral properties and low cytotoxicity plasmonic
gold/LDHs nanocomposites hold great potential to be tailored as novel efficient
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therapeutics for the treatment of hepatitis B.
1
platforms in anticancer therapies [8, 9]. Plasmonic
Introduction
NPs
show
specific
tunable
photo-responsive
Hepatitis B is a major health problem worldwide
behaviour upon illumination (e.g. surface plasmon
that is associated with as many as 1 milion death
resonance and electron storage/charging effects)
per year due to the development of hepatocellular
and extremely low quantum yield while the
carcinoma, cirrosis and other complications [1].
absorbed photon energy might be converted into
Hepatitis B is caused by the hepatitis B virus (HBV).
heat with high efficiency [10, 11 ]. To date it is still
HBV is a member of the Hepadanviridae family - of
challenging to join the functionalities of the
enveloped viruses with a DNA genome and is an
plasmonic
important human pathogen, currently affecting
nanocomposites in order to establish non-toxic and
over two billion people worldwide [1]. The viral
constructive interactions within the sophisticated
DNA genome is protected by a nucleocapsid
functions of the biological systems [12, 13]. It is
surrounded by the small (S), medium (M) and large
expected that coupling the plasmonic NPs and
(L) surface proteins which are able to self-assemble
nanostructured
into empty, non-infectious subviral particles (SVP),
cause a synergistic effect in their therapeutic
secreted in vast excess over the virions, both in
responses and achieved improved bioactivity and
infected patients and in vitro [2]. Significant
biospecificity [14]. One important aspect to be
prophylaxis against HBV infection is ensured
considered when NPs act on biological systems is
through vaccination; however, treatment of chronic
their cytotoxicity; this defines the NPs ability to
patients is problematic. Current therapies, relying
adversely affect the normal physiology of the cells,
on replication inhibitors and immune response
as well as, to directly alter the structure of the
stimulators have limited efficacy and serious side
normal cells [15]. Multiple studies have revealed
effects during the course of administration [3]. As a
that NPs aggregation process, their texture, size and
consequence, the number of chronically infected
stability are particularly important factors that can
patients has been alarmingly increasing over the
induce cytotoxicity [16]. Recent advances in NPs
last decades, urging the development of more
synthesis provide new tools to develop specific
efficient antiviral agents [4]. Due to the demand for
fabrication procedures in which the nano-texture
novel targets and mechanisms to eradicate HBV, the
might be controlled and nano-dimensions are
design of bioactive nanocomposites engineered
preserved by limiting aggregation processes of NPs
to interact with cells and to show efficient
[17]. Other aspects still remain key-points in
antiviral effect against hepatitis B virus (HBV)
establishing non-toxic effects of NPs, one of the
is a challenging approach [5, 6]. Recent research
most important being the toxicity of the organic
has demonstrated that AgNPs exhibit promising
surfactants that are used as structure-directing
cytoprotective activities towards HIV - infected
agents during NPs synthesis. [12]. For example,
T-cells and can inhibit in vitro production of HBV,
AuNPs are most often synthesized by using
reducing the amount of extracellular virions [7]
cetyltrimethylammonium
while AuNPs were found particularly attractive
cationic surfactant that tightly binds to AuNPs. It
NPs
in
complex
biocompatible
self-assembled
supports
bromide
might
(CTAB),
a
Address correspondence to [email protected]
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Research
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Nano Res.
has been shown that during NPs-cell interactions
through
clathrin-mediated
CTAB desorbs from AuNPs surface and induces
enhanced
cytotoxic effects and cells death [18, 19]. Another
manifesting toxic effects [30].
permeability and
endocytosis,
with
retention without
important aspect that might directly influence NPs
interactions with biological specimens is the use of
the
stabilizing
agents
to
prevent
nano-units
aggregation and to conserve their nano-features.
Such as AuNPs used for biological applications
might have coatings of the capping agents such as:
citric acid, polysaccharides, surfactants, proteins or
polymers [20]. The organic additives involved in
fabrication, stabilization and coatings of NPs can
strongly influence their direct interactions with
cellular and intracellular components resulting in
cytotoxic responses [21]. This motivates the needs
Figure 1 Illustration of synthesis process of AuNPs/LDHs
for establishing "greener procedures" to obtain
self-assemblies.
stable, non-toxic and bioactive nanostructures. In
order to address these issues we have recently
The
established a simple experimental procedure in
LDHsmatrices and the plasmonic NPs might be an
which small NPs might be directly obtained and
excellent motivation for testing cytotoxicity and
stabilized on the LDHs matrices without involving
antiviral effect of their self-assembly. Herein, we
any organic additives as structure-directing agents
report for the first time the antiviral properties of
and/or stabilizing agents [22-24]. It uses the
AuNPs/LDHs self-assemblies using HBV as a virus
capability of
NPs/LDHs
model and the hepatoma-derived HepG2.2.215 cells
self-assemblies while the freshly calcined LDHs
supporting viral replication, assembly and secretion
recover their original layered structure in the
of infectious virions and SVP. Three specific
aqueous solutions of metal salts Mey+Yx- by
compositions of the LDH clays were considered in
manifesting its “structural memory effect” [25, 26].
this study, denoted as MgLDH, ZnLDH and
More
MgFeLDH and both the cytotoxicity profiles and
the
precisely,
LDHs
in
the
to
form
active
environment
joined
bioactive
migration of Y from the salt aqueous solution to
self-assemblies were determined using quantitative
the clay inter-layers the LDH has also the capability
assays.
to take up the Me
cations, from the aqueous
significantly antiviral effect reducing the amount of
solution of Me Y and stabilize them on its surface
viral and subviral particles (SVPs) released from
as MeNPs and/or MexOyNPs [27]. This is schematic
treated cells by up to 80%, while no cellular toxicity
illustrated in Fig. 1. Toxicity studies that addressed
was observed.
y+
x-
LDHs clays have shown negligible cytotoxic effects
during LDHs-cells interactions while the influence
of LDHs nanoparticles on cells viability and
We
found
that
of
the
the
y+
activity
of
formed ,during the reconstruction process, by the
x-
antiviral
functionalities
AuNPs/LDHs
AuNPs/LDHs
have
2 Experimental
2.1 Preparation of the LDHs
proliferation was negligible [28, 29]. Moreover, it
was demonstrated that LDH large nanoparticles (50
Three different compositions of the LDHs were
to 200 nm) were selectively internalized into cells
prepared by a co-precipitation method similar to
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that described by Reichle [31]. MgLDH containing
voltage of 200 kV. X-ray photoelectron spectroscopy
Mg and Al (Mg /Al =2/1) as cations of the LDHs
(XPS) spectra were recorded using a Perkin-Elmer
layers was prepared by the slow addition of an
Model 5500-MT spectrometer equipped with Mg
aqueous
and
Kα radiation (1253.6 eV), operating at 15 kV and 20
Al(NO3)3.9H2O, 1M to a Na2CO3 (0.5 10 M) solution
mA; the binding energies (BE) were corrected by
under stirring, at 45°C. The pH value was kept
referencing the C1s peak to 284.8 eV. ICP analysis
constant at 9.5 by adding suitable amounts of 0.1 M
was also used to determine the gold content of the
NaOH aqueous solution. ZnLDH with Zn2+ and Al3+
samples. Structural characteristics, crystallinity and
in the LDH layers, (Zn2+/Al3+=2/1) was obtained by
purity
adding
of
diffraction (XRD) using a Shimadzu XRD 6100
Zn(NO ) 6H2O and Al(NO3)3.9H2O, 1M, to an
diffractometer with monochromatic light (λ=0.1541
aqueous solution of NaOH/Na2CO3 in such a way
nm), operating at 40 kV and 30 mA over a 2θ range
that the final pH remained at a value of 9. For
from 4 to 70°. UV-Vis absorption spectra were
MgFeLDH Mg , Fe , Al
recorded on a Jasco V550 spectrophotometer with
2+
3+
2+
3+
solution
of
Mg(NO3)2.6H2O
.
slowly
an
aqueous
-2
solution
3 2.
2+
3+
3+
were used as cations
layers in a molar ratio (Mg /Fe /Al =2/0.5/0.5)
2+
3+
3+
information
were
recorded
by
X-ray
integration sphere.
using a similar procedure based on the slow
addition of an aqueous solution of Mg(NO3)2.6H2O
and Al(NO3)3.9H2O, and Fe2(SO4)3 1M to a Na2CO3
2.4 Cell growth and treatment
(0.5.10-2 M) solution, under stirring, at 45°C. The
resulting slurries were aged about 20 h at 50°C. The
The
final products were recuperated by filtration,
two copies of the HBV genome, were grown in
washed several times with distilled water and dried
RPMI 1640 medium (Euroclone) containing 10%
under vacuum.
fetal bovine serum (FBS), 50 units/ml penicillin, 50
HepG2.2.2.15 cells, stably transfected with
μg/ml streptomycin, 2 mM Glutamax (Invitrogen)
2.2 Preparation of AuNPs/LDHs self-assemblies
and 200 μg/ml of G418 (Gibco). When indicated,
AuNPs/LDHs were added to the medium at
1g of the “freshly” calcined LDHs (at 500°C for 7 h)
different concentrations, to investigate their cellular
was added, under very vigorous stirring, at 150 ml
toxicity and potential antiviral properties against
of 0.1 M aqueous solutions of Au(O2CCH3)3 (see Fig.
HBV.
1). The obtained samples were aged at 30°C for 7 h,
washed, centrifuged, dried under vacuum and
denoted as AuNPs/LDHs, where LDHs is MgLDHs,
2.5 Cytotoxicity assay
ZnLDHs or MgFeLDH while the final pH value of
the reconstruction medium was 9.1, 8.3 and 8.7,
Cytotoxicity of AuNPs/LDHs was determined by
respectively.
MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth
oxyphenyl)-2-(4-sulfophenyl)-2H-te
assay, using the CellTiter 96®
2.3 Characterization
trazolium)
AQueous One
Solution Cell Proliferation Assay kit (Promega,
Madison, WI, USA).
HepG2.2.2.15 cells were
Transmission electron microscopy (TEM) imaging
treated with serial dilutions of NPs for 3 days,
was performed on a Hitachi H900 transmission
before addition of the MTS reagent. The 550 nm
electron microscope operating at an accelerating
absorbance was measured by using a microplate
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Nano Res.
reader
(Mitras
Berthold)
and
expressed
as
percentage from untreated (control) cells.
NanoDrop2000 spectrophotometer (Thermo). Equal
amounts of RNA were reverse-transcribed using the
SuperScript III First-Strand Synthesis kit (Invitrogen)
2.6 Quantification of HBV secretion by real-time
PCR
and the HBV-specific DNA was further quantified
by real-time PCR, as described above. Actin
amplification was employed as internal control. As
HepG2 2.2.15 cells were grown in the absence
the primers used for the HBV amplification are
(control) or presence of AuNPs/LDHs for 3 days.
specific for the S gene, the pregenomic (pg) RNA,
Encapsidated viral DNA was purified form cell
which overlaps the entire coding region of the viral
supernatants by phenol-chloroform extraction as
genome, will also be quantified using this assay, in
described [32]. The DNA was quantified by
addition to the envelope proteins transcripts.
real-time PCR using the Corbett Rotor Gene 6000
system and the Maxima SYBR Green qPCR Master
Mix (Fermentas). Primers were designed to amplify
2.9 Quantification of intracellular HBV
nucleocapsid DNA
a 279 bp HBV-specific fragment within the S gene:
HBV3575-3854_for,
5’-
HepG2.2.2.15 cells were grown in the absence
and
(control) or presence of AuNPs/LDHs for 3 days.
5’-
Cells were counted under the microscope and equal
TGGCCCCCAATACCACATCATCC-3’.
The
numbers were lysed in a buffer containing 1M
number
was
Tris-HCl (pH 8), 250 mM EDTA and 0.5 % NP-40.
determined using a calibration curve containing
Encapsidated viral DNA was further purified form
known amounts of HBV DNA and expressed as
cell lysates by phenol-chloroform extraction and
percentages from control (untreated) samples.
quantified by real-time PCR as described above.
2.7 Quantification of HBV SVP secretion by
2.10 Quantification of intracellular HBV envelope
TCCAGGATCCTCAACAACCAGCACG-3’
HBV3575-3854_rev,
of
viral
genome
equivalents
ELISA
proteins by sodium-dodecyl
sulfate-polyacrylamide gel electrophoresis
The HepG2.2.2.15 supernatants used for HBV
(SDS-PAGE) and Western Blotting.
quantification were also analysed for the amount of
secreted SVP, using the Monolisa HBsAg Ultra Kit
AuNPs/LDHs-treated and control HepG2.2.2.15
(Bio-Rad) and a Mitras Berthold microplate reader.
cells were lysed in a buffer containing 10 mM
The results were obtained as ratios of signal to
Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA,
cut-off value and were converted to percentages
0.5 % TritonX-100 and a mixture of protease
form control (untreated) sample.
inhibitors
(Sigma-Aldrich).
Cell
lysates
were
centrifuged at 14,000 x g for 10 min and the total
2.8 Quantification
transcripts
by
of
intracellular
reverse-transcription
HBV
protein
(RT)
supernatants using the BCA method (Pierce). Equal
real-time PCR.
amounts
content
of
SDS-PAGE
was
proteins
and
determined
were
transferred
loaded
to
in
onto
clear
the
nitrocellulose
Total RNA from control or AuNPs/LDHs-treated
membranes using a semi-dry blotter (BioRad). The
HepG2.2.2.15 cells was isolated by Trizol extraction
HBV proteins were detected by Western blotting
(Invitrogen)
following successive incubations of the membrane
and
quantified
using
the
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Nano Res.
with mouse anti-PreS1 (Santa Cruz, dilution 1:1000)
(200), (220), (311) and (222)
planes of fcc gold can
and HRP-labelled rabbit anti-mouse antibodies
be indexed [35, 36]. Hence, the small NPs identified
(Santa Cruz, dilution 1:10000). The proteins were
by the dark dots dispersed on the particles of LDHs
visualized using the ECL detection system (GE
might be identified as NPs of Au.
Healthcare).
3 Results and discussions
3.1 Physical_chemical characteristics of
AuNPs/LDHs self-assemblies
The
morphology
and
microstructure
of
AuNPs/LDHs assemblies are evidenced by TEM
and HRTEM results (see Fig. 2). A typical TEM
image it is shown in Fig. 2A from which it is clearly
evident the presence of aggregates of large and thin
platelets with a mean diameter of about 150 nm.
This morphology has been well consistent to that
typical reported for LDHs-like crystallites [33, 34]
and is almost similar for all AuNPs/LDHs. More
magnified
images
show
that
very
small
nanoparticles, easily observed as dark contrast dots,
are well-dispersed not only over the surface (Fig. 2B)
but also at the interfaces (Fig. 2D) of the LDHs
particles. The small NPs are almost spherical as
shape and their average size, obtained by statistical
analysis, were determined to be 3.2 nm, 3.7 nm and
2.9 nm, for AuNPs/MgLDH, AuNPs/MgFeLDH and
AuNPs/ZnLDH, respectively (see Table 1). No
evidence of small NPs unattached to the LDHs was
found confirming these were formed and stabilized
only on the LDHs surface. From the HRTEM image
Figure
2
TEM
image
of
A)
AuNPs/MgLDH;
B)
AuNPs/ZnLDH; C) HRTEM image of AuNPs/MgFeLDH; D)
AuNPs/MgLDH.
XPS results further confirm the formation of
AuNPs/LDHs assemblies and indicate that their
similar Au content (equal to~ 4%, as indicated in
Table 1). For AuNPs/MgFeLDH the survey XPS
spectra is presented in Fig. 3 while the XPS spectra
of AuNPs/MgLDHs and AuNPs/ZnLDHs are given
in Fig. S1.
Table 1 Physical-chemical characteristics of AuNPs/LDHs
self-assemblies.
(Fig. 2C) the lattice fringes with an interplanar
spacing of 0.24 nm are observed and assigned to the
{111} planes of face-centered cubic (fcc) Au (JCPDS
No. 04-0784). The HRTEM image confirmed the
crystalline character of the small NPs. Furthermore,
the selected area electron diffraction (SAED) pattern
in the inset of Fig. 2C reveals a set of bright
concentric rings in which the diffractions from (111),
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Figure 4 XRD patterns of _ AuNPs/ZnLDH; _ AuNPs/MgLDH;
Figure 3 The survey XPS spectrum of AuNPs/MgFeLDH.
_ AuNPs/MgFeLDH.
The XPS analysis detected peaks from Au, Mg, Fe,
also
Al and O for AuNPs/MgFeLDH while Au, Zn, Al,
reconstruction process of the LDHs has been
and O were identified in the surface elemental
completed for AuNPs/MgLDH while was not yet
composition
completed
of
AuNPs/ZnLDH.
For
identified.
This
for
indicates
that
AuNPs/ZnLDH
the
and
AuNPs/MgLDH Au, Mg, Al and O were identified
AuNPs/MgFeLDH. It can be also observed that all
from the binding energy (BE) values of Au4f, Mg2p,
(00l) reflections are characterized by high intensities
Al2p and O1s. Hence, XPS results indicate the
demonstrating
joined surface composition of AuNPs and specific
reconstructed LDH-like phase. The LDHs average
LDH clay matrix. The phase identity of the
crystallite sizes were calculated from the (003) and
AuNPs/LDHs was further confirmed by XRD
(006) reflections employing the Debye-Scherrer
analysis. The XRD patterns are depicted in Fig. 4.
equation [38]. The obtained values are around 150
For AuNPs/MgLDH the X-ray diffraction pattern
nm and are consistent with that given by TEM
reveals the presence of a single crystalline phase
analysis. Assuming a 3R stacking of the layers and
with reflections clearly assigned to the regular
from the positions of the peaks due to the planes
layered structure of hydrotalcite-like clay (ICDD file
(003) and (110) the interlayer space (where 0.48 nm
No. 22-700), in which a series of sharp and
is the width of the brucite-like layer) is calculated
symmetric basal reflections of the (00ℓ, ℓ = 3, 6, 9)
1.24±0.1 nm. The value is the consequence of the
planes and broad, less intense, reflections for the
expanded LDHs layers, due to the intercalation of
nonbasal (01ℓ, ℓ = 2, 5, 8) planes are easily
acetate anions into the inter-layer spaces of the
recognized [34, 37]. On the other hand, in the XRD
reconstructed LDHs and is consistent with that
patterns of AuNPs/ZnLDH and AuNPs/MgFeLDH
previously reported by Prevot et al. [39] for acetate
besides the reflections assigned to the regular
intercalated LDHs. No peaks characteristic of Au
layered structure of LDH some weaker reflections
phase
(marked by (*) in Fig. 4), with positions which
AuNPs/LDHs though it should be considered that
roughly coincide with those of mixed oxides that
such small and highly dispersed NPs might not be
define the structure of the calcined LDHs, can be
detected by XRD analysis [40]. In a previous study
can
be
the
high
noticed
crystallinity
in
the
of
patterns
the
of
9
Nano Res.
we showed that calcination at high temperatures of
during the calcination process. Fig. 6 compares the
NPs/LDHs self-assemblies gives rise to a high
UV-Vis spectra of AuNPs/LDHs. As expected from
increase (up to ten times) in the size of the
the change in colour of AuNPs/LDHs (the inset of
supported NPs due to their aggregation process [23].
Fig. 6) the strong and broad surface plasmon
As a consequence, after calcination, the larger NPs
resonance (SPR) band, a marker of well-dispersed
could be detected by XRD analysis [22, 23]. Hence,
AuNPs [41], can be clearly observed in all
we further analysed the XRD patterns of the
AuNPs/LDHs.
AuNPs/LDHs calcined at 750°C (see Fig. 5).
Figure 5 The XRD patterns of
AuNPs/MgLDH;
_
_
AuNPs/ZnLDH;
_
AuNPs/MgFeLDH after calcination at
750°C.
Figure 6 UV-Vis absorption spectra of
It shows that the LDHs layered structure is
_
AuNPs/ZnLDH;
_
AuNPs/MgLDH; _ AuNPs/MgFeLDH.
destroyed giving rise to mixtures of mixed oxides
with compositions determined by the specific
This provides a strong evidence for the presence of
compositions of the LDH precursors. Further, the
the nanosized-Au in AuNPs/LDHs assemblies. For
XRD spectra of the calcined samples show some
all the samples the absorption in the UV region is
common reflections defined by four well developed
defined by high intensities peaks that can be
reflections at 2θ = 38.1, 44.3, 64.5 and 77.4˚ that
assigned to the optical responses of the LDHs
matched to the diffraction lines of the (111), (200),
matrices and/or their derived mixed oxides, in the
(220) and (311) planes of face-centered cubic (fcc) of
conditions that he XRD results point out that the
Au
peak
structural reconstruction processes of ZnLDH and
corresponding to the (111) plane is more intense
MgFeLDH were not complete. Apart from the
than the other planes suggesting that (111) is the
absorption in the UV region the SPR band of
predominant orientation. The identification of the
AuNPs can be clearly identified by a peak centered
crystalline Au by XRD analysis, after calcination at
at 535 nm. This value indicates the presence of the
750°C, is due to the increase in the size of AuNPs
gold in its metallic state [42]. Hence, this confirms
(JCPDS
Card
No.
65-2870).
The
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10
Nano Res.
that the state of AuNPs was not influenced by the
different composition of the LDHs. On the other
hand, the SPR peak intensity increased from
AuNPs/MgLDH
to
AuNPs/ZnLDH
and
AuNPs/MgFeLDH, respectively, without any shift
in the position. It is known that the intensity of the
plasmon absorbance might be influenced by the
gold content, the particle size of gold and/or the
nano-gold surroundings environment [43, 44].
Considering that AuNPs/LDHs are defined by the
similar content of gold and similar dimensions of
AuNPs (see Table 1) the different compositions of
the LDHs might provide specific environmental
characteristics for AuNPs in gold-clay assemblies
[23]. Thus, the specific environment of the clay
matrix might give rise to the specific features of the
plasmonic responses of AuNPs in AuNPs/LDHs
assemblies.
Figure 7 Cytoxicity data for A) AuNPs/MgFeLDH; B)
AuNPs/ZnLDH; C) AuNPs/MgLDH.
3.2 Cytotoxicity of AuNPs/LDHs
Based on these results we considered the non-toxic
Cytotoxicity
of
AuNPs/LDHs
assemblies
was
concentration range [0-125] µ g/ml for all the
evaluated to rule out any interference with potential
samples to further evaluate their effect against
antiviral effects. The viability of HepG2.215 cells
HBV.
was determined relative to control, as a function of
the different AuNPs/LDHs concentrations (15, 30,
3.3 Antiviral activity of AuNPs/LDHs
60, 125 and 250 µ g/ml) after 3 days of incubation, by
MTS assay (Fig. 7). The results show that the cells
To determine the ability of AuNPs/LDH to inhibit
exposed to AuNPs/MgFeLDH are more than 94%
HBV, the virus particles load was quantified in
viable for the whole concentration range (panel A).
supernatants of HepG2.2.2.15 cells treated with a
Cells incubation with AuNPs/ZnLDH resulted in no
range of NPs concentrations. Remarkably, all
significant loss of viability for concentrations up
AuNPs/LDH formulations have shown a significant
to125 µ g/ml; however, when the AuNPs/ZnLDHs
antiviral effect (Fig. 8) AuNPs/MgFeLDH being
concentration was increased to 250 µ g/ml the cells
most potent of all (more than 90% inhibition of HBV
viability lowered by almost 20% (panel B). Further,
secretion) (Fig. 8, panel A).
AuNPs/MgLDH was revealed to be completely
inhibited HBV in a dose-dependent manner (Fig. 8,
non-toxic to the cells regardless the concentration
panel B), a peculiar behaviour was observed for
used (panel C).
AuNPs/MgFeLDH,
which
While AuNPs/ZnLDH
was
already
very
efficient at low concentrations (Fig. 8 panel A). A
similar effect was evidenced for AuNPs/MgLDH,
11
Nano Res.
which, however, appeared to lose some antiviral
AuNPs/LDHs nanostructured assemblies do not
potency with increasing concentrations (Fig. 8 C).
impair the conventional cell secretory pathway [45].
Figure 8 Inhibitory effect of A) AuNPs/MgFeLDH; B)
Figure 9 Inhibitory effect of A) AuNPs/MgFeLDH; B)
AuNPs/ZnLDH; C) AuNPs/MgLDH on the expression of
AuNPs/ZnLDH; C) AuNPs/MgLDH on the expression of SVP.
hepatitis B virus.
In contrast, the AuNPs/MgLDH inhibition of both
Considering the similar content of nano-gold in
viral and subviral HBV particles (Fig. 8 and Fig 9,
AuNPs/LDHs the specific antiviral behaviour might
panels C), might indicate an interference with either
be a consequence of the particular kinetics features
their assembly or secretion. It is interesting to note
of intracellular Au release from AuNPs/LDHs
that a previous report has shown little efficacy of 10
assemblies that could be a function of the different
nm gold nanoparticles prepared by sodium citrate
composition of the LDHs. Thus, we might assume
reduction, against HBV [46]. These differences in
that
the
HBV inhibition cannot be accounted for by the NPs
AuNPs/MgFeLDH and AuNPs/MgLDH than for
size and shape, as the AuNPs introduced here are
the AuNPs/ZnLDH. This hypothesis supposes that
also very small, ranging between 2.9 and 3.7 nm.
the threshold required for the Au-induced HBV
Rather, the environment provided by the clay
inhibition is rather low. Interestingly, unlike the
matrix structure, possibly enabling a controlled
AuNPs/MgLDH,
AuNPs
release of nano-gold and preventing premature
formulations did not significantly inhibit the
aggregation of NPs might be the key factor of the
secretion of non-infectious HBV particles (Fig. 9,
improved antiviral properties of these assemblies.
panels A and B). This suggests that the effect
Furthermore,
observed
as-synthesized LDHs showed a modest antiviral
this
process
in
Fig.
is
the
8
more
efficient
other
is
two
for
virus-specific
and
results
reveal
that
all
the
Research
12
Nano Res.
activity of about 20% inhibition of HBV (see Fig. 2S).
significantly higher in the presence of AuNPs/LDHs
It is also important to note that the cellular toxicity
(Fig. 10B).
of AuNPs/LDHs nanostructured assemblies is
significantly lower than that of the LDH matrices
(see Fig. 3S), revealing a cell protective effect
induced by the presence of nano-gold.
3.1 The
mechanism
of
AuNPs/LDHs
action
against HBV
The HBV life-cycle begins with virion attachment to
specific receptors at the hepatocyte surface, removal
of the envelope within the endocytic pathway and
the release of the circular, partially double stranded
DNA
(pdsDNA)
genome
from
the
naked
nucleocapsid [47]. The hepatocyte nucleus hosts the
pdsDNA convertion into covalently closed circular
DNA (cccDNA), which serves as template for
transcription of pregenomic (pg) and subgenomic
mRNAs. These are exported to the cytoplasm where
the pgRNA is reverse-transcribed into pdsDNA
within
the
viral
nucleocapsid
[47].
The
HepG2.2.2.15 cells are stably transfected with two
Figure 10 A) Intracellular analysis of HBV RNA --- control;
copies of the HBV genome and allow for full
AuNPs/MgFeLDH;
replication cycle, assembly and secretion of SVPs
Intracellular
and infectious virions; however, they are not
AuNPs/MgFeLDH; -- AuNPs/ZnLDH;
susceptible
for
infection
and
no
horizontal
---
AuNPs/ZnLDH;
analysis
of
HBV
---
AuNPs/MgLDH; B)
DNA
---
---
---
control;
---
AuNPs/MgLDH; for a
AuNPs/LDHs concentration of 125 μg/ml.
spreading of the virus occurs in these cells (or in
other in vitro cellular models). Therefore, to search
Corroborated with the drastic reduction of the virus
for the mechanism of HBV inhibition, we have
particles in cell supernatants (Fig. 8), this result
investigated in detail important steps of viral
strongly suggests that the HBV particles are
replication/assembly and protein synthesis in the
sequestered within treated cells. This hypothesis is
presence of the highest AuNPs/LDHs concentration
also supported by the analysis of the L envelope
(125 μg/ml), which has efficiently reduced the virus
protein, which plays crucial roles in nucleocapsid
load. Quantification of the HBV transcripts has
envelopment, while being less incorporated in SVPs.
shown no significant changes between control and
Similar to the effect on the viral DNA, the
treated cells (Fig. 10A), indicating that neither viral
AuNPs/LDHs induced a clear accumulation of both,
transcription nor the RNAs stability were affected
glycosylated (gp) and non-glycosylated (p) L
by AuNPs/LDHs. Next, the synthesis of HBV DNA
isoforms (Fig. 11). As the level of viral mRNAs was
was investigated in intact nucleocapsids extracted
similar in control and treated cells, the effect
form
observed indicates a strong intracellular retention of
control
and
AuNPs/LDHs-treated
cells.
Surprisingly, the amount of viral DNA was
these proteins in the presence of AuNPs/LDHs.
13
Nano Res.
cells. AuNPs/LDHs are similar regarding the gold
content, the texture of the gold nanoparticles hence
the
specific
antiviral
behaviour
might
be
a
consequence of the specific composition of the
LDHs that might establish particular features of the
gold release from AuNPs/LDHs composites and/or
give rise to specific synergetic effects between the
plasmonic
gold
environment.
and
The
the
particular
fabrication
LDHs
procedure
of
AuNPs/LDHs is simple taking the advantage of the
Figure 11 Western blotting revealing the glycosylated (gp) and
capability of the LDHs to form small NPs of Au (~
nonglycosylated (p) isoforms of the HBV large (L) envelope
3.5 nm) on the larger clay particles during the
protein in untreated, control cells (1) and cells treated with
structural reconstruction of the calcined LDHs in
AuNPs/MgFeLDH
or
the aqueous solution of Au(O2CCH3)3. With respect
AuNPs/MgLDH (4), at the concentrations indicated. The
to conventional antiviral HBV drugs, the prominent
numbers on the left represent the protein molecular weight in
advantages
of
kiloDaltons (kDa).
interesting
mechanism
(2),
AuNPs/ZnLDH
(3)
AuNPs/LDHs,
of
such
action
as
their
and
good
biocompatibility, open new ways towards the
These interesting effects suggest a direct interaction
development
between viral particles and AuNPs/LDHs, leading
nanocomposites.
of
highly
efficient
antiviral
to trapping of the formers inside cells. Future
studies will address the exact nature of these
interactions and whether the naked nucleocapsid,
Acknowledgments
enveloped particles or even the viral DNA are
involved. Inhibition of HBV secretion is a valuable
The authors are grateful for the financial supports
mechanism of action, especially in the context of a
from
complete HBV life cycle in vivo, preventing virus
Scientific
spreading and reinfection.
(PN-II-ID-PCE-75/2013)
the
Romanian
National
Research,
Authority
for
CNCS-UEFISCDI
and
the
Romanian
Academy.
4 Conclusions
Electronic Supplementary Material Supplementary
material (image characterization and supporting
Self-assemblies of plasmonic gold and layered
figures as referred to in the manuscript) is available
double hydroxides have been probed as novel
in
potential antiviral therapeutics against hepatitis B
http://dx.doi.org/10.1007/s12274-***-****-*
virus. AuNPs/MgFeLDH has shown the highest
antiviral HBV response with more than 90%
inhibition
of
HBV
secretion
at
very
low
concentrations, while for AuNPs/ZnLDH the HBV
inhibition is dose dependent. A direct interaction
between viral particles and AuNPs/LDHs was
the
online
version
of
this
article
at
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Electronic Supplementary Material
hepatitis B virus
Gabriela Carja1 (), Elena Florentina Grosu1, Catalina Petrarean2 and Norica Nichita2
Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)
Figure S1 The survey XPS spectrum of A) AuNPs/MgLDH; B) AuNPs/ZnLDH.
Research
Nano Res.
Figure S2 Inhibitory effect of A) MgFeLDH; B) ZnLDH; C) MgLDH on the expression of hepatitis B virus.
Figure S3 Cytoxicity data for A) MgFeLDH; B) ZnLDH; C) MgLDH.
Address correspondence to: [email protected]

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