2.13 Western blot analysis.
Harvested cells were resuspended in 20 mM RIPA buffer (pH 7.4) (Merk
Millipore, Vimodrone, MI, Italy) containing a cocktail of proteinase
inhibitors (Calbiochem, Merck, Darmstadt, Germany) and treated by
sonication 19 (Microson XL-2000, Minisonix, Farmingdale, NY, USA).
Aliquots of supernatants containing equal amounts of protein (30 µg) in
Laemmli buffer were separated on Bolt® Bis-Tris Plus gels 4-12% precast
polyacrylamide gels (Life Technologies, Monza, Italy) and then
transferred from the gel to a PVDF nitrocellulose membrane using iBlot 2
system (Life Technologies, Monza, Italy). Blots were stained with
Ponceau red to ensure equal loading and complete transfer of proteins.
Then, they were blocked for 1 hour, at room temperature, with 5% milk
in PBS 0.1% tween solution. Subsequently, the membranes were probed at
4°C overnight with the following primary antibodies: rabbit anti-uPAR
(1:500 FL 290, Santa Cruz Biotechnology, Cat# sc-10815); rabbit
anti-iNOS (1:1000, Cell signaling, Cat#2982) rabbit GAPDH antibody
(1:1000, Cell signaling Technology, Cat# 2118) was used to assess equal
amount of protein loaded in each lane. Anti-Rabbit IgG (whole
molecule)–Peroxidase antibody (Sigma, Cat#A0545) was used as the
secondary antibody; the ECL procedure was employed for development.
3. RESULTS AND DISCUSSION
After mixing, under continuous stirring, appropriate amounts of SS and
HAuCl4, the formation of AuNPs-SS was observed. More
specifically, by diluting the received SS with a dilution factor of 1:8,
fixing the HAuCl4 concentration at
4.0×10-4M, the colour of the reaction mixture changed
from light yellow (Figure 1A , picture 1) to light purple
(Figure 1A , picture 2). In particular, extending the contact
time to 48h, the colour of these solutions strikingly changed to dark
purple indicating the AuNPs-SS formation. As suggested by Yin et
al. ,[31] the optical properties of nanostructured gold depend
strongly on the size, shape, interactions between the nanoparticles and
the species adsorbed on their surface. The colour of the colloidal gold
is due to the surface plasmon resonance (SPR) absorption, a
characteristic signal in the visible absorption spectrum of AuNPs, in
general showed in the range from 500 to 600 nm in the UV–Vis absorption
spectrum. In this case, the mixture of HAuCl4 and SS
presents the AuNPs characteristic SPR band, that appeared slightly
broadened and centered at 550 nm (Figure 1B ). In particular, inFigure 1B , the AuNPs SPR band time evolution is reported. While
in Figures 1C and 1D , the wavelength and absorption
values at the maximum of the SPR are showed as function of time compared
with the Full Width at Half Maximum (FWHM). A shift towards higher
wavelength number values, along with an increase of the
Amax were observed by extending the contact time. At the
same time, the corresponding FWHM became larger than values observed at
the beginning of the reaction (Figures 1C, D ). Always according
Yin et al. ,[31] the variances in the SPR bands can be
attributed to a different distribution of the nanoparticles in the
solution and to their aggregation. Moreover, the SPR absorption
intensity of AuNPs depends on their size and shape as well as their
surrounding medium.[31]
Therefore, in agreement with the SPR band position and shape, the
spectral observations indicated polydispersed AuNPs that tend to
slightly increase their size with elapsing the contact time. Probably,
the observed increment in the Amax, passing from 4 to
48h, could be attributed to an increase in the AuNPs-SS concentration
due to the quite complete reaction of HAuCl4 with SS.
Further, it is worth mentioning that, incrementing the contact time
until 72h, the particles tended to aggregate to each other and the SPR
signal decreased, becoming barely evident in the UV-Vis spectrum
(data not shown ).
As a whole, the results indicated the reduction of Au(III) to Au(0),
forming nanoparticles thanks to the synergistic action, as reducing
agents, of main components of snail mucus. Indeed, the chemical
reduction of HAuCl4 aqueous solution is one of the most
widely used methods for the synthesis of colloidal gold.[32] Not
surprisingly, by looking at the AuNPs-SS spectrum shape line, in
particular below 400 nm, the contribution of chemical species, like
proteins/peptides and amino acids, mainly present in SS,[33]
absorbing in this region, was observed. The finding was confirmed by
comparing the UV-Vis spectrum of SS solutions with the AuNPs spectrum
(Figure S1) . The UV-Vis spectra of SS collected at different
dilutions (to better evidence the contribution of the absorbance values
in the reported wavelength region) clearly displayed the contribution of
the main SS components, indicated with arrows, on the AuNPs surface.
A Zeta potential value of approximately -35±2 mV was measured for
AuNPs-SS, indicating their good stability in water. Further,Figure S2 shows the comparison between the UV-Vis spectra of
AuNPs-SS collected soon after the synthesis (selecting 24h as contact
time) and after 2 months. It is clearly evident as AuNPs-SS are
characterized by a long-time stability, as indicated by the presence of
the AuNPs SRP signal.
3.1. FESEM Analysis
FESEM investigation revealed the formation of irregularly shaped AuNPs
(Figure 2A, B ), with minimum and maximum size of about 4 and 50
nm, respectively, and average size of 14±6 nm (particles size
distribution in Figure 2C ). FESEM images confirmed the hybrid
organic-inorganic nature of the AuNPs-SS and, in particular, evidenced
the presence of a thin organic layer covering the Au nanoparticles
(thickness ranging between 4 and 8 nm, Figure 2B ).
So, starting from these considerations and considering an AuNPs-SS
average size of 14±6 nm, a molar absorption coefficient of
3.7×108 M-1cm-1was calculated. As a result, each synthesis produced, adopting 24h as
contact time, AuNPs-SS stock solutions with a mean concentration of
5×10-9 M. It means 1.64×1012AuNPs/mL.
3.2 XPS analysis
XPS results showed that the surface atomic concentrations of carbon,
oxygen, nitrogen and gold are about 64, 18, 11 and 7%, respectively.Figure 3A reports the high-resolution Au 4f XPS spectrum
consisting of a doublet (i.e., Au 4f7/2 and Au
4f5/2 at 83.2 and 86.9 eV, respectively) due to
spin-orbit coupling, the position being in agreement with literature on
AuNPs.[18,34-37] The curve fitting of the Au 4f signal indicates
three Au 4f7/2 components, i.e., the main component at
83.2 eV (68%) attributed to metallic gold (Au(0)), and the minor peaks
at about 84.2 eV (23%) and 85.4 eV (9%) ascribed to Au(I) and Au(III),
respectively (the total area of the Au 4f7/2 is taken as
100%). [37-40] The high-resolution C1s XPS spectrum inFigure 3B is curve-fitted with four peaks and, specifically,
the hydrocarbon component at 285 eV (51%), the component at 286.4 eV
(31%) ascribed to both C-N and C-O groups, the peak at 288.0 eV
(14.5%) assigned to both C=O and O-C-O moieties and the weak peak at
289.2 eV (3.5%) due to carboxylic functionalities. The O1s spectrum inFigure 3C displays two components, i.e., the peak at 531.7 eV
(57%) that can be ascribed to O=C moieties and the component at 533.1
eV (43%) associated with O-C groups. The N1s signal (Figure
3D ) shows one main peak at about 400.1 eV, consistent with the presence
of amine and amide groups.[41-43] To unveil the nature of these
compounds on the AuNPs surface, ATR-FTIR and MALDI-MS analyses were
performed.
3.3 ATR-FTIR spectroscopic measurements
FTIR measurements were carried out to recognize the possible functional
groups responsible for efficient stabilization of gold nanoparticles
prepared by using SS. For this purpose, a comparison between the
ATR-FTIR spectra of SS before and after the formation of AuNPs, and the
FTIR spectrum of AuNPs-SS was performed (Figure 4 ).
Starting from as received SS, bands around 1700 cm-1and 1580 cm-1 were detected, and in accordance with
literature,[33,44,45] these peaks indicated the main presence of
amide bonds of peptides and proteins as glycosoaminoglycans and
proteoglycans. Further, the bands at about 1388 cm-1and 1700 cm-1 could suggest the presence of
carboxylate moieties associated with acid derivates of sugars and amino
acids chains. Indeed, the peaks at 1192 cm-1 and at
lower wavenumber values (below 900 cm-1) indicated the
O-glyosidic linkages.[33] In this region, the contribute of
sulphated, acylated and ester based compounds should be also taken into
account. The shoulder at 1360 cm-1 with a partial
contribute at 1380 cm-1 indicated the presence of
carbon chains of proteins and carbohydrates.[33,44,45] Accordingly,
there was evidence at 2850 cm-1 and 2930
cm-1 of CH2 and CH3arisen from protein skeleton and lipidic component of the slime.[30]
The observed bands between 2900 and 3500 cm-1 can be
ascribed to the presence of a large amount of OH and NH moieties. The
contributes of polyphenols, mainly in the range 1500-3500
cm-1, cannot be excluded.[18] Overtones of the
benzene rings of phenylaniline and tyrosine were detected above 1800
cm-1 and 2600 cm-1.[33]
Interestingly, after the contact with HAuCl4 solution,
and thus after the AuNPs formation, the SS spectrum appeared almost
unchanged as a whole in comparison with the one observed at the
beginning of the reaction. In particular, the signal at 1580
cm-1 disappeared, but others main vibrations at 1700
cm-1 and 1192 cm-1 were retained.
Then, if, on one hand, the disappearance of the of amide bond signals
from peptides and proteins features was indicative of their role in the
formation of AuNPs-SS, on the other hand, the counterpart of the
spectrum ascribed to sugar derivates did not show important changes. The
wavenumber region 2800-3500 cm-1 appeared also
affected, suggesting the contribute of biomolecules having OH and NH
functionalities. At the same time, the bands intensity at 2850
cm-1 and 2930 cm-1 was reduced
further suggesting the role of proteins in nanoparticles formation.
These results were confirmed when the FTIR-ATR of the AuNPs-SS was
collected. Indeed, the main signals at 1645 and 1545
cm-1, indicated the presence of a protein component
based on the Amide I (C=O stretching) (1645 cm-1) and
II (1545 cm-1) (N–H bending and C–N stretching)
bands.[44] Interestingly, if these bands were compared with signals
of SS and ascribed to the same vibration modes, the observed shift
towards lower wavenumber values could be attributed to their
coordination on the AuNPs surface. The characteristic absorption in the
region 1200–1300 cm-1, which represented the amide
III, due to the C–N stretching and N–H deformation bands, was also
detected. Accordingly, signals between 3300 and 3750
cm-1 arise also from the AuNPs-SS surface due to N–H
amine group stretching vibrations superimposed on the side of O–H
hydroxyl group stretching bands.[46] Once again, comparing the
position of these bands with those observed in the as received SS, a
clear shift towards higher wavenumbers was observed confirming that the
molecules in the SS could coordinate to AuNPs through formation of Au-N
or Au-O covalent bonds. In the spectral region from 750 to 1400
cm-1 only very weak signals can be observed. The
lacking of clear peaks at 1192, 1388 cm-1and 1700
cm-1 could suggest the absence O-glycosidic linkages
and carboxylate moieties associated with acid derivates of sugars and
amino acids chains. The signal at 2850 cm-1 and 2930
cm-1 observed in the ATR-FTIR of AuNPs-SS could
confirm the presence of protein skeleton. So, from the ATR-FTIR analyses
it has been possible to assess the main presence of peptides and
aminoacids on the surface of AuNPs-SS, acting as reductant agents during
the formation of AuNPs.
3.4 MALDI (+) FT-ICR MS Analysis.
The presence of amino acids and peptides was supported by MALDI (+)
FT-ICR MS experiments (Figure 5 ). The MALDI (+) FT-ICR MS
spectrum for as received SS sample (Figure 5A ) shows a huge
number of MS signals, thus revealing the complexity of the sample in
terms of its metabolic profile. Thanks to higher levels of accuracies
reached by HRMS technique,[47] it was possible to unequivocally
identify several protonated amino acids, i.e. threonine
([C4H10NO3]+,
m/z 120.12747), tryptophan
([C11H13N2O2]+,
m/z 205.23364), arginine
([C6H15N4O2]+,
m/z 175.20953), hystidine
([C6H10N3O2]+,
m/z 156.16258), asparagine
([C4H9N2O3]+,
m/z 133.12607), cysteine
([C3H8NO2S]+,
m/z 122.16670), glutamine
([C5H11N2O3]+,
m/z 147.15341), proline
([C5H10NO2]+,
m/z 116.13903) and serine
([C3H7NO3]+,
m/z 106.10083), already found in snail slime sample.[48] 6198
peptides could be identified showing the presence of at least one unit
of cysteine. The presence of peptides showing high contents of cysteine,
proline, serine and glycine was accessed too, these ones very well-known
in the literature for their high antimicrobial activity.[48]
Instead, analysis of AuNPs-SS sample (Figure 5B ) shows a very
different situation. In this case, MALDI (+) FT-ICR MS spectrum of the
sample shows a lower number of MS signals in comparison to the pure
sample (3158 and 209694 signals, respectively). No free amino acids were
found and only 3 free peptides could be identified, thus suggesting a
drastic shrinking of the range of free peptides present in the sample,
indicative of their role in the formation of AuNPs.
3.5 pH dependence of synthesized AuNPs.
The AuNP stability, after their synthesis, was investigated under
different pH values ranging from 2 to 12. Once again, by observing the
SPR of AuNPs, the wavelength and FWHM were monitored. Results, reported
in Figure 6A , indicated that both the position and the FWHM
were stable in the pH range from 4 to 10 and slight changes were
detected at the lowest and the highest investigated pH value,i.e. pH 2 and 12. More specifically, at pH 2 the SPR signal shift
towards higher wavelength values indicating the contribution of larger
AuNPs that under this condition tend to agglomerate.[31] Yinet al .[31] suggested that a large number of
H+ in the aqueous solution could be adsorbed onto the
surface of the AuNPs, modifying their surface and their charges.
Accordingly, at the same time, the FWHM increased. Then, AuNPs tended to
agglomerate easily and consequently the final particle size was larger.
Conversely, at pH 12 the contribute of smaller AuNPs occurred in
agreement with their stabilization, under this condition of work. The
FWHM in this case reduced its value, indicating that the particles were
more monodisperse. These findings were confirmed by performing Zeta
potential measurements as function of pH values, as reported inFigure 6B . It is worth mentioning that the Zeta potential gives
information about the AuNP suspension stability, since a higher electric
surface charge of the AuNPs prevents aggregation. Indeed, the AuNPs were
negatively charged in the range of pH from 4 to 12 and, in particular,
the charge decreased at the increasing of the pH value. On the other
hand, at pH 2 a positive Zeta potential value, of approximately +25mV,
was observed, affecting the AuNPs surface as previously described. The
presence of functional groups arisen from the main components of SS,
mainly the carboxylic moieties of proteins and aminoacids (and/or
polyphenols) present on the AuNP surface, produced a negative electrical
charge and the observed dependence on the pH. At pH values below 4, the
organic moieties were in the protonated form and the AuNPs slightly
aggregated.
3.6 AuNPs-SS enhance scratch wound closure of NCTC 2544 keratinocytes.
To determine whether AuNPs-SS could affect cell motility, scratch assays
were performed in a confluent monolayer of keratinocytes and evaluated
at 0h, 24h and 48h after treatment with increasing doses of AuNPs-SS. By
24h, even though the keratinocytes normally showed an active migratory
phenotype, scratches treated with AuNPs-SS displayed a significant
acceleration of wound closure as compared with the untreated (CTRL)
wound (Figures 7A, B ). By 48h the scratch treated with the
highest AuNPs dose was completely closed, thus showing a significant
increase in closure rate from 24h while no statistically significant
change in cell viability was observed on AuNPs-SS treatment as shown inFigure 7C . Moreover, western blot analysis displayed that
AuNPs-SS treatment induced a significant increase of the urokinase-type
plasminogen activator receptor (uPAR), which is essential for
keratinocyte adhesion, spreading and migration in vitro and in vivo
(Figure 8C ).[49,50]
3.7 AuNPs-SS reduce the inflammatory response of LPS-activated
macrophages.
To further assess possible effects of the AuNPs-SS on biological
inflammatory response, RAW 264.7 macrophages were pre-treated with LPS
and the day after loaded with two doses of AuNPs-SS. Morphological
changes and an inflammatory gene panel were evaluated at the end of the
sequential treatments. As shown in Figure 8A, the mouse
macrophage cells took up AuNP regardless of treatment with LPS. Cells
without LPS treatment were more homogeneous in size, and AuNPs appeared
to be dispersed throughout the cytoplasm. After LPS treatment,
macrophages became activated as seen by variations in size. The higher
the AuNP concentration, the more activated cells appeared, presenting
extended pseudopods. It was found there was no statistically significant
change in macrophage viability (Figure 8B ). Gene expression of
cytokines IL1-β, IL-6 and inducible nitric oxide synthase (iNOS), showed
that, in the absence of LPS treatment, there were moderate changes in
cytokines expression after AuNPs-SS treatment. LPS induced a massive
increase in cytokines and iNOS expression. Interestingly, the AuNPs-SS
synergistically reduced these elevated cytokine levels and completely
abrogated the synthesis of iNOS (Figure 8C ). The effect of
AuNPs-SS on iNOS levels before and after LPS stimulation was confirmed
also by western blot analysis (Figure 8D ). Altogether these
results suggest that AuNPss-SS may have modulatory effects that could be
beneficial in reducing inflammatory or pathogenic responses.
4. CONCLUSIONS
For the first time, during this work, the Snail Slime was used to induce
the formation of AuNPs, proposing in literature a novel and green
approach for their synthesis. A comprehensive investigation was
performed in our laboratory by adopting several complementary techniques
such as UV-Vis and ATR-FTIR spectroscopy, DLS, XPS, and high-resolution
MALDI-MS analyses. AuNPs 14±6 wide were observed through the FESEM
investigation that evidenced the formation of the organic layer Snail
Slime-based surrounding AuNPs. The latter was confirmed by ATR-FTIR, XPS
and MALDI-MS analyses. Indeed, the main presence of peptides and
aminoacids was observed at the surface of the AuNPs giving them a high
Zeta potential value in water (-35±2 mV), indicative of their great
stability. Indeed, the temporal and pH stability of the proposed
colloidal gold solution was investigated, which resulted in a suspension
stable for a long time and appeared only slightly affected under hard
acidic pH value (pH 2), far from biological conditions, in which
aggregation of AuNPs was observed. With the prospect of using the AuNPs
for biomedical devices, we studied the effect of AuNPs on wound healing
in human keratinocytes and evaluated their potential anti-inflammatory
properties in Murine Macrophages. Our findings demonstrated that the
accelerated wound closure promoted by AuNPs-SS was associated with an
increased expression of the receptor for urokinase (uPAR) which, besides
that canonical conversion of plasminogen to plasmin and extracellular
matrix (ECM) degradation, directly controls cell adhesion,
differentiation, proliferation and migration through non-proteolytic
mechanisms.[49,50]
It has been established that in the classic process following infection
or injury, macrophages play a key role as regulators of inflammation.
However, if the inflammatory response of macrophages is not properly
controlled, it may lead to severe diseases such as
atherosclerosis,[51] rheumatoid arthritis (RA), vascular injury and
cancer.[52] Accordingly, to moderate inflammation, the macrophages
have been targeted for specific ablation, inhibition of infiltration and
reduction of the pro-inflammatory cytokines release. In this work, we
demonstrated for the first time the AuNPs-SS were able to modulate the
inflammatory response induced by LPS in murine macrophages by
significantly reducing the IL1-β, IL-6 cytokines levels. It is known
that macrophages isolated from chronic wounds in diabetic patients
exhibit a proinflammatory phenotype with the release of IL-1β, MMP-9,
TNF-α, and IL-6. In a recent study Mirza and coworkers[53] showed
that IL-1β sustains the proinflammatory macrophage phenotype observed in
poorly healing wounds of humans and mice. The authors of the study
demonstrated that targeting the IL-1β pathway upregulated expression of
pro-healing factors in wounds of diabetic mice and improved healing of
these wounds. Indeed Sumbayev et al. reported that citrate‐stabilized
gold nanoparticles were able to down‐regulate the inflammatory process
by selectively targeting the IL‐1β dependent pathway[54].
We also observed a massive drop of the inducible nitric oxide synthase
(iNOS) levels, which is essential for the macrophage activation and thus
for cytokines release. Regulation of iNOS gene expression is, through
transcriptional regulation, particularly influenced by NF-κB activation.
Indeed, in mice, iNOS gene promoter contains two NF-κB binding sites,
both of which need to be bound in order to get full induction of iNOS by
LPS stimulation
Hence, a novel horizon in the field of biotechnology is opened with this
work, proposing an innovative nanoplatform that should have other
properties, under investigation in our research groups. The applications
in cosmetic formulations of AuNPs as an additional ingredient would give
the possibility to avoid, for example, the use of preservatives, due to
the potential antimicrobial activity of gold that, at least, should also
preserve the snail slime components.
In conclusion, our findings demonstrated that AuNPs-SS promote effective
wound healing and may pave the way to accelerate the regeneration of
difficult-to-heal wounds. A more in-depth understanding of the effects
of NPs on macrophage activation may lead to a more potent and efficient
control of inflammatory-related diseases.
Acknowledgements
We gratefully acknowledge Mr. Sergio Nuzzo for the skillful and
excellent technical assistance, and Francesco Paolo Perrotta and
Francesca D’Andretta, Società Agricola Dap di Francesco Paolo Perrotta
& C. S.a.s - via Diego Rapolla, - 85029 Venosa (Potenza, Basilicata,
Italy), for the snail slime production and Patrizia Iannece for her
technical support, provided for Mass Spectrometry analysis. This
research was supported by the Italian Ministry for Education, University
and Research (MIUR) under grant PONa3_00369 – “Laboratorio SISTEMA”.