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”.