Design and Cellular Fate of Bioinspired AuâAg Nanoshells@Hybrid

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Design and Cellular Fate of Bio-inspired AuAg Nanoshells@Hybrid Silica Nanoparticles. Samantha Soulé, Anne-Laure Bulteau, Stéphane Faucher, Bernard Haye, Carole Aimé, Joachim Allouche, Jean-Charles Dupin, Gaëtane Lespes, Thibaud Coradin, and Herve Martinez Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02810 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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Design and Cellular Fate of Bio-inspired Au-Ag Nanoshells@Hybrid Silica Nanoparticles. Samantha Soulé,† Anne-Laure Bulteau,ǂ Stéphane Faucher,ǂ Bernard Haye,§ Carole Aimé,§ Joachim Allouche,†* Jean-Charles Dupin, † Gaëtane Lespes,ǂ Thibaud Coradin§ and Hervé Martinez.† †

Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les Matériaux

(IPREM)-UMR CNRS/UPPA 5254, Equipe de Chimie Physique (ECP), Université de Pau et des Pays de l'Adour (UPPA), Technopôle Hélioparc Pau Pyrénées, 2, Avenue du Président Pierre Angot, 64053 PAU Cedex 09 France. ǂ

Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les Matériaux

(IPREM)-UMR CNRS/UPPA 5254, Laboratoire de Chimie Analytique Bio-inorganique et Environnement (LCABIE), Université de Pau et des Pays de l'Adour (UPPA), Technopôle Hélioparc Pau Pyrénées, 2, Avenue du Président Pierre Angot, 64053 PAU Cedex 09 France. §

Sorbonne Universités, UPMC Univ Paris 06, CNRS, Collège de France, Laboratoire de Chimie

de la Matière Condensée de Paris, 4 Place Jussieu, 75005 Paris, France

ACS Paragon Plus Environment

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ABSTRACT: Silica-coated gold-silver alloy nanoshells (NS) were obtained via a bioinspired approach using gelatin and poly-L-lysine (PLL) as biotemplates for the interfacial condensation of sodium silicate solutions. X-ray photoelectron spectroscopy was used as an efficient tool to the in-depth and complete characterization of the chemical features of nanoparticles along the whole synthetic process. Cytotoxicity assays using towards HaCaT cells evidenced the detrimental effect of the gelatin nanocoating and a significant induction of late apoptosis after silicification. In contrast PLL-modified nanoparticles had a lower biological impact that was further improved by the silica layer and uptake rates up to 50 % of the initial particles could be achieved. These results are discussed considering the effect of nanosurface confinement of the biopolymers on their chemical and biological reactivity.


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INTRODUCTION Hybrid metal- or metal oxide-polymer nanocomposites have been widely studied in the past decades due to their high potential in many applications, from catalysis to biomedical applications.(1-3) Considering the latter domain, some of the most important efforts in the past ten years have been devoted to the design and development of multifunctional core-shell nanomaterials in order to provide several functions in one single platform.(4-6) For instance, theranostic agents combining therapeutic and diagnostic functions have recently emerged as promising nanosystems for Nanomedicine.(7, 8) Besides, noble metal-based hybrid materials have attracted much attention due to their optical properties originating from the Plasmon Resonance phenomenon.(9) In particular, gold or gold-silver nanoshells are now considered as promising tools for cancer photothermal therapy since their optical absorption can be tuned to the near-infrared (NIR) “biological window”.(10, 11) In this context, our group has recently developed multifunctional hybrid gold nanoshell/mesoporous silica nanocomposites (12, 13) providing photothermally responsive platforms for drug delivery applications.(14) In these systems, thermoresponsive organic nanovalves allowed for pore opening under laser irradiation. In the present work, another type of hybrid silica shell is proposed, composed of a biopolymer/silica layer grown on gold-silver nanoshells. Indeed, we and others have already shown the possibility to design bioinspired nanocomposites through interfacial activation of silicates condensation by electrostatic interactions with amine-bearing polymers.(15–18). Here gelatin is selected as a first biotemplating polymer due to its high thermal sensitivity, that could respond to the photoinduced heating of the nanoshell. Poly-L-lysine (PLL) is also studied, not


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only because its interaction with silica sources is very-well documented (19, 20) but also for its potential in gene delivery.(21) The successful preparation of such nanocomposites is demonstrated, especially via a complete and in-depth characterization by X-ray photoelectron spectroscopy used as a unique and efficient tool for interfaces characterization. Foreseeing their biomedical application, the cytotoxicity and cellular uptake of these nanoparticles is evaluated using HaCaT keratinocyte cells. These experiments emphasize the influence of the biotemplating polymer on the biological behaviour of the particles and demonstrate the ability of the silica coating to modulate apoptosis induction.

EXPERIMENTAL Chemicals. Silver nitrate (AgNO3, Sigma, >99%), tetrachloroauric acid trihydrate (HAuCl4.3H2O, Sigma, >99.9%), tri-sodium citrate (Fisher, >99%), Polyvinylpyrrolidone (PVP, 10,000 g.mol-1, Sigma), sodium hydroxide (Fisher, >98%), Gelatin (from porcine skin, gel strength 300, type A, Sigma), Poly-L-lysine hydrobromide (PLL, 30,000-70,000 g.mol-1, Sigma), sodium silicate (≥10% NaOH, ≥27% SiO2, Sigma), hydrochloric acid (HCl, Sigma, 37%). Concerning total elemental analysis, a standard solution of gold at 1000 mg.L−1 (Au, >99.5%, SCP Science, France) was used for calibrations. A standard solution of Indium at 1000 mg.L−1 (In, >99.5%, SCP Science, France) was used as an internal standard. Solutions of nitric acid (HNO3, 70%, Atlantic laboratory, Bruges, Belgium), hydrofluoric acid (HF, 60%, Atlantic laboratory, Bruges, Belgium) and hydrochloric acid (HCl, 37%, Atlantic laboratory, Bruges, Belgium) were used for sample mineralization. All products for TEM sample preparation were purchased from LFB distribution and used without further purification.


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Synthesis of silver-gold nanoshells (NS) The synthesis of silver-gold alloy nanoshell (NS) was performed following our published procedure based on a galvanic replacement process on silver nanoparticles.(13) First 250 mL of a 0.002 M silver nitrate solution (1) were prepared and heated to 100°C. As soon as the solution started to boil, 12.5 mL of a 1 wt. % aqueous solution of tri-sodium citrate were quickly added to the silver solution and left under magnetic agitation for 1 hour during which its color turned yellow-brown indicating the formation of nanoparticles. The reaction was stopped by quickly cooling down the solution. The as-prepared silver nanoparticles dispersion was used for nanoshell preparation without further purification. A 50 mL gold precursor HAuCl4-xOHx stock solution (2) was prepared by hydroxide substitution of the HAuCl4 complex. Typically, a 0.2 wt. % aqueous solution of HAuCl4 was prepared and the pH was raised to 5 by the slow addition of a few drops of NaOH (2M) during 1 hour. This solution was kept 24 hours at 5 °C before use. For NS preparation, a solution of stabilized silver nanoparticles (3) was prepared by mixing 250 mL of 1 with 66.7 mL of a 1 wt. % PVP solution under sonication for a few seconds. Then, 31.3 mL of 2 diluted to 250 mL with deionized water were added dropwise to 3 under mild magnetic stirring. The color of the solution immediately changed indicating the occurrence of the redox reaction. Synthesis of NS@polymer nanoparticles. Before polymer adsorption, 10 mL of the asprepared NS solution were centrifuged and washed 3 times in order to remove the excess of PVP. Then, 10 mg of the polymer were added to the nanoparticles suspension. In the case of gelatin, the suspension was stirred 30 min at 45 °C and after three centrifugation/washing cycles at 40°C, the NS@Gel suspension was obtained. For poly-L-lysine, after the polymer addition, the suspension was kept under stirring at room temperature during two hours. Then the solution was centrifuged and washed three times to obtain the NS@PLL suspension.


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Silica shell growth (NS@polymer-SiO2 nanoparticles). First, 2mL of the NS@polymer suspension were diluted to 10 mL with milliQ water. In parallel, a clear silicate solution (4) ([SiO2] = 60 mM) was obtained by dilution of the starting sodium silicate solution in water followed by neutralization with 2M HCl to pH 7.0 ± 0.2. Finally, 1 mL or 2 mL of 4 were added dropwise to the NS@polymer diluted suspension to obtain a silicate concentration of 5.5.10-3 M or 1.10-2 M, respectively. The solution was stirred overnight at room temperature and three centrifugation/washing cycles were performed to obtain the NS@polymer-SiO2 nanoparticles suspension. Note that for in vitro assays, sterile water was used during centrifugation/washing cycles and also during the redispersion step to avoid bacterial contamination.

Scheme 1. The different steps of the synthesis of NS@polymer-SiO2 nanoparticles

Morphological and structural characterization. Dynamic Light Scattering measurements were performed with a DynaPro Nanostar system (Wyatt Technology, Santa Barbara, USA)


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equipped with a laser at wavelength λ0 = 658 nm and a photodiode detector at a detection angle of 90°. All size measurements were carried out on diluted particles suspension at 25°C using plastic cuvette of 1 cm path length and 2 mL active volume as sample holder. DYNAMICS software (v.7.1.7.16) developed by Wyatt Technology was used to analyze the data. Morphology of nanoparticles was studied by transmission electron microscopy (TEM). The images were recorded with a Philips CM 200 (200 KV) instrument equipped with a LaB6 source. The samples dispersed in water were dropped onto a carbon copper grid and dried before analysis. X-ray Photoelectron Spectroscopy (XPS). XPS measurements were performed on a Thermo K-alpha spectrometer with a hemispherical analyzer and a microfocused (400 µm diameter microspot) monochromated radiation (Al Kα, 1486.6 eV) operating at 72 W under a residual pressure of 1.10−9 mbar. The pass energy was set to 20 eV. Charge effects, mainly important for hybrid samples, were compensated by the use of a dual beam charge neutralization system (low energy electrons and Ar+ ions) which had the unique ability to provide consistent charge compensation. All spectra were energy-calibrated by using the hydrocarbon peak at a binding energy of 285.0 eV. Spectra were mathematically fitted with Casa XPS software© using a least squares algorithm and a nonlinear Shirley-type background.(22) The fitting peaks of the experimental curves were defined by a combination of Gaussian (70%) and Lorentzian (30%) distributions. Quantification was performed on the basis of Scofield’s relative sensitivity factors.(23) In vitro studies. HaCaT keratinocyte cells were obtained from Thermofischer (Saint Aubin, France) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were incubated for 24 hours with the nanoparticles at gold concentrations of 10


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µM and 100 µM. Then, the culture medium was removed, the cells were washed and replaced in fresh medium. Three replicates were performed for each gold concentration. To investigate the cellular impact of the nanoparticles, an Annexin V-FITC/PI apoptosis detection kit was used, as described by the manufacturer (Thermofischer, Saint Aubin, France). Flow cytometric analysis of apoptotic populations, was carried out using a BD Accuri™ C6 flow cytometer (BD Biosciences, Le Pont de Claix, France). For each gold concentration, three replicates were analysed and the presented results are the average of these three analyses. To quantify the amount of internalized particles, the intracellular gold amount was determined using ICP-MS. The samples were digested using a mixture of concentrated HNO3 (2 mL), HCl (6 mL) and HF (1 mL). Blank solutions were prepared identically but without sample. The samples and blanks were heated at 65oC for 4 h on digestion blocks (Digiprep MS, SCP Science, France). Because of the presence of hydrofluoric acid in the analyzed solutions, and in order to improve the cleaning of the system (gold removal) between two analyses, the samples were diluted about 1000 times in (HNO3 + HCl) 5% to reach a concentration range from 0.5 to 2 µg (Au) L-1. Analyses were performed with an Agilent 7900ce model ICP-MS instrument, equipped with a concentric nebulizer (MicroMist MM) and a cooled Scott-type spray. The operating parameters are summarized in Table T1 in Supporting Information. These parameters were optimized using a solution of 1 mg.L−1 Li, Y, Tl, Ce in 2 wt. % HNO3. The quantification was performed by external calibration. Indium was used as internal standard in order to eliminate the fluctuations due to the temporal signal variations. The monitored isotopes were 197Au and 115In. In these conditions, the limits of detection and quantification for gold (12 and 40 ng.L−1, respectively) were calculated according to the IUPAC recommendations.


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Recovered cells were fixed for 1 h at 4 °C with a 2.5 % glutaraldehyde solution in a 0.05 M cacodylate buffer. After washing with a 0.3 M saccharose solution in the same buffer, samples were reacted for 1 h at 4°C with a 2 wt % osmium tetraoxide solution in the 0.3 M saccharose/0.05 M cacodylate buffer. After washing, the samples were gradually dehydrated in baths of ethanol 50%, 70% and 95% during 5 minutes each, and 2 baths of 10 minutes in ethanol 100%. Ethanol was then replaced by propylene oxide in 2 baths of 10 minutes, first in 1/1 ethanol/propylene oxide mixture and then in pure propylene oxide. The dehydrated samples were then impregnated in a solution of Araldite (20 mL Araldite CY212, 22 mL DDSA (Dodecenyl Succinic Anhydride) and 1.1 mL BDMA (Benzyl Dimethyl Amine)) in three successive steps (2/1 propylene oxide/araldite for 1 h, 1/2 propylene oxide/araldite overnight and pure araldite). The hardening of araldite was left to occur at 60°C for 3 days. The resulting blocks were cut in ultrathin sections of 70 nm with an ultramicrotome (Ultracut Reichert, France). The cells were finally imaged by Transmission Electron Microscopy (TEM) using a JEOL 1011 electron microscope operating at 100 kV. The images were recorded on a Gatan Orius CCD camera.

RESULTS AND DISCUSSION Morphological characterization of the nanoparticles. The polymer adsorption on NS nanoshells was studied by dynamic light scattering, measuring the average hydrodynamic diameter in aqueous solution at low concentration to avoid multiple scattering phenomena. Figure 1b shows the particle size distribution of NS, NS@Gel and NS@PLL nanoparticles.


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Figure 1. a) SEM image of NS nanoparticles and b) DLS size distribution of nanoshells (NS), nanoshells coated with gelatin (NS@Gel) and poly-L-lysine (NS@PLL) For nanoshells stabilized by PVP, the particle size distribution is nearly monomodal and centered around 110 nm in agreement with SEM image (Figure 1a). For NS@Gel and NS@PLL nanoparticles, the particle size increases to respectively 160 nm and 130 nm reflecting polymers adsorption on nanoparticle surface. Such an adsorption is in agreement with the ability of cationic polymers to interact strongly with metallic nanoparticles.(24–27) Besides, the increase of the hydrodynamic diameter is more significant for NS@Gel than for NS@PLL suggesting the formation of a gel-like coating that can swell in cold water (T < 30°C).(27) Gelatin and poly-L-lysine are two cationic polymers that are known to activate silica formation in solution or after deposition on surfaces.(28–31) The ammonium groups of polyamine or peptide chains can act as adsorption sites for silicates, bringing them closer one to another and favoring their condensation. Here for both polymer-coated samples, the shell formation in the presence of silicates is clearly evidenced by TEM (Figure 2). For silicates concentration of 5.5 10-3 M, the coating is relatively thin and uneven, particularly in the case of gelatin. For 1.0.10-2 M silicate concentration, the shell becomes thicker (around 5-6 nm) and the silica deposit seems


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quite dense. Similar attempts performed with native (i.e. PVP-coated, Figure S1a, Supporting Information) NS lead to the deposition of an ill-defined layer on a small fraction of the particles (Figure S1b, Supporting Information). These observations suggest that the positive charge of the polymer is the key parameter driving silica deposition while the detailed polymer structure does not influence significantly the coating morphology. Previous reports have shown that depending on the density and localization of the ammonium groups on the polymer chain, silicate condensation can be either extended and form dense gels in the case of polyamines such as PLL or spatially constrained and form silica nanoparticles embedded within polymer fibers in the case of self-assembling proteins such as gelatin.(32) However, deposition of silica on gelatin nanoparticles results in a dense coating due to the reduced extension of protein brushes at the particle/water interface.(17) The same mechanism could explain the thin and dense silica coating obtained for our gelatin-templated materials. Moreover, the reduced gelatin brushes extension could be enhanced by the gelatin/gold interactions. In the next sections, silicate concentration was fixed to 1.10-2 M corresponding to thicker silica coatings.


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Figure 2. TEM images of nanoparticles NS@Gel-SiO2 (a, b) and NS@PLL-SiO2 (c, d) synthesized with two different concentrations of sodium silicates: 5.5.10-3 M (a, c) and 1.10-2 M (b, d). Chemical characterization by X-ray photo-electron spectroscopy. The chemical composition of the various coatings was studied by X-ray photoelectron spectroscopy. A preliminary study of several references, such as gelatin, poly-L-lysine and nanoshells, was performed to provide a chemical data set of binding energies associated with their corresponding chemical environments.


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Figure 3. C1s, N1s and O1s XPS spectra of gelatin (a, b, c) and poly-L-lysine (d, e, f) The whole XPS results of reference materials are gathered in Table 1. The C1s, N1s and O1s core peaks of gelatin and poly-L-lysine are presented in Figure 3. In the case of gelatin, the C1s spectrum (Figure 3a) evidences four environments. The component at 285.0 eV (35.9 at. %) corresponds to the aliphatic carbon atoms (C-C/C-H). The other one at 286.4 eV reveals the presence of C-(N, O) groups. The peak at 288.2 eV (13.1 at. %) is attributed to amide O=C-N groups which are characteristic of the peptide bond. The last one, at 289.2 eV (1.6 at. %) is assigned to both carbon atoms of guanidinium group (N=C-(NR)2) and carbonyl group (COOH). The N1s core peak (Figure 3b) shows a major component at 400.2 eV which mainly corresponds to the peptide bond (O=C-N). This component is associated with an atomic percentage of 12.0 at. % in agreement with the corresponding C1s component (13.1 at. % for the O=C-N component). The two other peaks, located at 398.5 eV and 402.1 eV, are attributed to imine (N=C-NH) and ammonium (C-NHx+) environments, respectively. On the O1s spectrum of gelatin (Figure 3c), the component located at 531.8 eV corresponds to the contribution of both O=C-N and O=C-O groups. The other one at 533.0 eV is attributed to O=C-O and C-O environments. The


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asymmetry observed on the high binding energy side of the peak has been reported in literature (33) and could be due to water traces.

Table 1. XPS binding energies and atomic percentages of C1s, N1s, O1s and Br3d core peaks of Gelatin and Poly-L-lysine. Gelatin

Poly-L-lysine

Assignments

BE (fwhm) at. % (eV)

BE (fwhm) at. % (eV)

285.0 (1.5)

35.9

285.0 (1.3)

42.4

C-(C/H)

286.4 (1.5)

20.4

286.4 (1.3)

18.4

C-(N/O)

288.2 (1.5)

13.1

288.1 (1.3)

8.2

O=C-N

289.2 (1.5)

1.6

C1s

Total

N 1s

71.0

Total

69.0

398.5 (1.5)

0.4

-

400.2 (1.5)

12.0

400.0 (1.5)

7.3

O=C-N/CN3

402.1 (1.5)

0.6

401.7 (1.5)

5.9

C-NHx+

Total

O 1s

COOH/CN3

13.0

N=C-NH

13.2

531.8 (1.7

11.9

531.7 (1.4)

6.9

O=C-(N/O)

533.0 (1.7)

3.3

532.9 (1.5)

3.1

O=C-O/C-O

534.3 (1.8)

0.8

534.1 (1.5)

0.7

H2O

16.0

10.7 68.1 (1.3)

4.2

69.2 (1.3)

2.9

Br 3d Total

7.1


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For poly-L-lysine, the C1s spectrum (Figure 3d) can be fitted with three components: at 285.0 eV (42.4 at. %) for the aliphatic carbon atoms (C-C/C-H), at 286.4 eV (18.4 at. %) for the C-(N, O) groups and at 288.1 eV (8.2 at. %) for the peptide bonds (O=C-N). The N1s core peak (Figure 3e) indicates two environments: at 400.0 eV corresponding to amide (O=C-N) and to non-protonated amine (C-NH2) groups; at 401.7 eV evidencing the presence of protonated amine groups. The O1s spectrum (Figure 3f) exhibits the same three components as gelatin (at 531.7 eV (O=C-N), 532.9 eV (C-O) and 534.1 eV (H2O)). Bromide is also detected in agreement with the initial composition of the polymer (poly-L-lysine hydrobromide) (Table 1). The nanoshells were also characterized by XPS before polymer adsorption. The analysis clearly shows the bimetallic composition Ag/Au of nanoparticles with the Ag3d core peak at 367.9-373.9 eV (Ag0) (Figure 4a) and the Au4f doublet at 83.9-87.6 eV (Au0) (Figure 4b). Moreover, carbon, nitrogen and oxygen are also detected in agreement with the presence of the PVP ligands on the nanoparticles surface (Figure S2, Supporting Information).

Figure 4. Ag3d (a) and Au4f (b) XPS spectra of NS

The main concern for XPS characterization of NS@polymer nanoparticles is that PVP initially present on the AuAg nanoshells includes an amide group that is also present in gelatin and PLL.


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In order to ascertain the presence of these polymers at the surface of the NS core nanoparticles, the estimation of the specific Ctot at. % / Ntot at. % ratio (r1) and C (O=C-N) at.% / Ntot at.% ratio (r2) can be useful (Table 2).

Table 2. Values of r1 = Ctot at.% / Ntot at.% and r2 = C (O=C-N) at.% / Ntot at.% ratios deduced from XPS analysis of Gelatin, Poly-L-lysine, NS, NS@Gel, NS@PLL, NS@Gel-SiO2 and NS@PLL-SiO2 nanoparticles r1

r2

Ctot at.% / Ntot at.%

C(O=C-N) at.% / Ntot at.%

Gelatin

5.5

1.0

Poly-L-lysine

5.1

0.6

NS

12.8

1.5

NS@Gel

5.1

1.0

NS@PLL

7.7

1.0

NS@Gel-SiO2

5.1

1.5

NS@PLL-SiO2

9.4

1.0

After gelatin adsorption (Figure 5), the C1s core peak is very similar to the one of the gelatin reference. The same four components at 285.0 eV (C-C/C-H), 286.2 eV (C-(N, O)), 287.9 eV (O=C-N) and 288.8 eV (COOH) are identified. The N1s and O1s spectra are reminiscent of both gelatin and PVP. However, both r1 and r2 ratios change with gelatin adsorption from 12.8 and 1.5 for PVP-coated NS to 5.1 and 1.0 for NS@Gel, respectively, both values being very close to those calculated for the protein reference.


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Figure 5. C1s (a), N1s (b) and O1s (c) XPS core peaks of NS@Gel nanoparticles Upon poly-L-lysine adsorption, the C1s and O1s spectra do not significantly change compared to initial NS systems (Figure S3, Supporting Information). Nevertheless, the N1s spectrum displays an additional component at 401.5 eV assigned to protonated amine groups confirming PLL adsorption on the NS surface. Note that the relative intensity associated with the ammonium groups is weak indicating that poly-L-lysine is predominantly adsorbed in the neutral form. In this case, r1 and r2 give intermediate values between those calculated for NS and PLL, which would suggest the presence of both PVP and PLL on the NS surface. After reaction with silicates, XPS analyses of the hybrid nanoparticles reveal the presence of Silicon in both NS@Gel-SiO2 and NS@PLL-SiO2. Indeed, the Si2p3/2 and the O1s components located at 103.1 eV and 532.7 eV for NS@Gel-SiO2 (Figure 6a, b) and 103.2 eV and 532.6 eV for NS@PLL-SiO2 (Figure S4a, S4b, Supporting Information) correspond to a SiO2 environment.(34) In the case of gelatin-based nanoparticles, the C1s component at 288.1 eV (Figure 6c) and the N1s component at 400.0 eV (Figure 6d) assigned to the peptide bonds are still detected while r1 and r2 are not significantly modified, confirming that no significant polymer desorption has occurred allowing the formation of the hybrid gelatin/silica shell.


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Figure 6. Si2p (a), O1s (b), C1s (c) and N1s (d) XPS core peaks of NS@Gel-SiO2 For NS@PLL-SiO2, the C1s core peak (Figure S4c, Supporting Information) displays the same components as before silica deposition. On the N1s spectrum (Figure S4d, Supporting Information), the component associated with the protonated amine groups is no longer detected. However, the presence of PLL is confirmed by the same r2 value as before silica coating. Note that the r1 value significantly changes from 7.7 for NS@PLL to 9.4 for NS@PLL-SiO2 suggesting the presence of carbon contamination. Silver and gold at metal oxidation state are also detected in NS@polymer-SiO2 nanoparticles (Figure S5, Supporting Information). Considering that the depth analysis of XPS is about 5 nm, this observation confirms that the silica shell is not much thicker than this value. It may also reflect a thickness heterogeneity of the polymer-silica shell.


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Cytotoxicity and cellular uptake. The interactions of the NS, polymer-coated NS and silicacoated NS particles with HaCaT keratinocyte cells were then studied. First the cytotoxicity of the different particles after 24 h of contact at 10 and 100 µM gold concentrations was investigated. It is well-known that nanomaterials can induce cell death by several mechanisms, that can be distinguished between apoptosis and necrosis.(35) The former corresponds to a cellularcontrolled process as a response to a stress whereas the latter is a catastrophic event leading to rapid cell lysis. However, it is important to point out that apoptosis can evolve into death or late apoptosis in a second stage of the cellular fate. (35)

Figure 7. Cytotoxicity after 24h incubation of (a) NS, (b) NS@Gel, (c) NS@Gel-SiO2, (d) NS@PLL and (e) NS@PLL-SiO2 nanoparticles evaluated by flow cytometry at 10 µM and 100 µM gold concentrations. As shown in Figure 7, for the lowest particle dose (10 µM), the overall cell death rate remains below 20 % for all particles, except for NS@Gel-SiO2 and NS@PLL for which more than 40% of the cells are dead. The apoptotic vs. dead or late apoptotic cell ratio is almost 1 for NS, NS@PLL and NS@PLL-SiO2. The former prevails for NS@Gel-SiO2 while the latter was more significant for NS@Gel. At a higher dose, cell death reaches ca. 60 % for NS, NS@PLL and NS@PLL-SiO2, 80 % for NS@Gel and the whole population of HaCaT cells are dead in the


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presence of 100 µM NS@Gel-SiO2. The apoptotic vs. dead or late apoptotic cell ratios are very similar to those obtained at 10 µM, indicating that the enhanced cell death reflects a dose effect and not a different mechanism of cytotoxicity. Overall these data suggest that (i) the two cationic polymers favor apoptosis and (ii) the presence of gelatin is more detrimental to cell survival than PLL. The first observation is in good agreement with the literature indicating that high MW PLL and porcine skin gelatin can induce apoptosis.(36, 37) The latter observation about the higher toxicity of gelatin-coated NS compared to PLL-coated is more surprising as this protein is widely considered as a safe biocompatible polymer.(38)

Figure 8. Evaluation of Au % in cells by ICP-MS after incubation with NS, NS@Gel, NS@GelSiO2, NS@PLL and NS@PLL-SiO2 at two gold concentrations (10 µM, 100 µM) To clarify this point, cell uptake experiments were also performed through TEM observations and measurements of intracellular gold amount determined by ICP-MS after 24 h incubation. As displayed in Figure 8, PLL-based materials exhibit higher capacities to be uptaken by cells with gold percentage values 3-5 times higher than those of the gelatin-based particles and ca. 10 times


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that of the native gold nanoshells. In addition, it is worth noting that the presence of silica does not significantly modify the uptake level. TEM images at a gold concentration of 100 µM on Figure 9 confirm the higher uptake of PLL-based materials (Figures 9d, 9f) compared to NS (Figure 9b), NS@Gel (Figure 9c) and NS@Gel-SiO2 (Figure 9e). At higher magnification (Figures 9g, 9h), intracellular metal nanoparticle aggregates are clearly evidenced.

Figure 9. TEM images of HaCaT cells after 24 h of incubation : (a) control, (b) cells treated with NS, (c) NS@Gel, (d) NS@PLL, (e) NS@Gel-SiO2 and (f, g, h) NS@PLL-SiO2 nanoparticles (gold concentration : 100 µM) It is usually considered that cationic nanoparticles are more easily uptaken than anionic ones due to their easiest adsorption on the negatively-charged cell membrane, favoring further internalization.(39) On this basis, it could be expected that both NS@PLL and NS@Gel would be easily uptaken whereas silica-coated particles would be less prone to be internalized.


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However, it is also well-known that surface adsorption of proteins present in the culture medium can modify the apparent charge of the nanoparticles.(40–43) Moreover, the culture medium can also change the state of dispersion of the particles, affecting the pathways of cellular uptake.(44) Considering PLL-based nanoparticles, both intracellular Au titration and TEM imaging demonstrate their facile incorporation. Noticeably the presence of the silica coating seems to favor this process and also leads to a decrease in cytotoxicity compared to NS@PLL. Such differences suggest that the silica coating is still present during the course of the experiments, although it is difficult to draw any clear conclusion about the intracellular fate of this coating from TEM images. Nevertheless it has been previously shown that internalized silica nanoparticles could undergo surface erosion inside intracellular compartments.(45) In the case of gelatin, the situation is strikingly different as the Au content remains low and only some dispersed nanoparticles are found within the cells on the TEM images. As a matter of fact, the high rate of cell death induced by NS@Gel and NS@Gel-SiO2 may even suggest that these correspond to particles that have passively diffused through a damaged cell membrane. This assumption is strengthened by the observation that for less cytotoxic NS particles, the Au cellular content is even lower than NS@Gel and colloids are mainly visualized on the outer part of the intact cell membrane. Thus, the most plausible explanation for the observed cytotoxicity of NS@Gel particles is that they can adhere to the cell surface, thanks to their positive surface charge, but cannot be actively internalized. The longer residence time of the particles on the cell membrane would allow for detrimental electrostatic interactions of gelatin with membrane lipids, as recently suggested.(46) Noticeably, NS@Gel-SiO2 particles are more toxic than NS@Gel, especially due to a larger relative contribution of the apoptosis death mechanism. Such an influence of silicification was not observed for NS@PLL particles, suggesting that this effect is


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specific of the gelatin/silica layer. It must be pointed out that the disruption of the gelatin/silica coating is very likely to occur in the course of the experiment due to the gel-to-sol transition of the protein at 37°C. This would release silica oligomers that have been reported to be toxic for some mammalian cells.(47)

CONCLUSION These results illustrate the versatility of the bioinspired polyamine-mediated interfacial deposition of silica that could be successfully applied to metallic nanoparticles. Our detailed XPS study enlightens the analytical challenges associated with the characterization of such multilayered hybrid nano-coatings. Accordingly the recorded cellular responses are highly complex as indicated by the variations in the relative contribution of apoptosis to cell death as a function of surface composition. While the observed cytotoxicity of gelatin nanosurfaces confirms the recent literature, the high uptake of PLL-coated particles is an interesting phenomenon that deserves further investigation, together with the role and fate of the silica layer during the internalization process.

ASSOCIATED CONTENT Supporting Information. Instrument parameters for ICP-MS analyses; TEM images of NS@PVP and NS@PVP-silica nanoparticles synthesized at 1.10-2 M silicates concentration; C1s, N1s and O1s XPS core peaks of Ag-Au nanoshells and NS@PLL; Si2p, O1s, C1s and N1s XPS core peaks of NS@PLL-SiO2; Ag3d and Au4f XPS core peaks of NS@gel-SiO2 and NS@PLL-SiO2. This material is available free of charge via the Internet at http://pubs.acs.org


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AUTHOR INFORMATION Corresponding Author *Joachim Allouche. [email protected]. Author Contributions. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript Funding Sources. CNRS, Region Aquitaine, Université de Pau et des Pays de l’Adour (UPPA).

ACKNOWLEDGMENT We thank Arkema for their assistance in TEM analyses and CNRS, Region Aquitaine and UPPA for their financial supports.

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Graphical abstract


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Scheme 1. The different steps of the synthesis of NS@polymer-SiO2 nanoparticles 427x160mm (150 x 150 DPI)


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Figure 1. a) SEM image of NS nanoparticles and b) DLS size distribution of nanoshells (NS), nanoshells coated with gelatin (NS@Gel) and poly-L-lysine (NS@PLL) 425x198mm (90 x 90 DPI)


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Figure 2. TEM images of nanoparticles NS@Gel-SiO2 (a, b) and NS@PLL-SiO2 (c, d) synthesized with two different concentrations of sodium silicates: 5.5.10-3 M (a, c) and 1.10-2 M (b, d). 337x330mm (116 x 116 DPI)


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Figure 3. C1s, N1s and O1s XPS spectra of gelatin (a, b, c) and poly-L-lysine (d, e, f) 456x282mm (150 x 150 DPI)


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Figure 4. Ag3d (a) and Au4f (b) XPS spectra of NS 380x186mm (111 x 112 DPI)


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Figure 5. C1s (a), N1s (b) and O1s (c) XPS core peaks of NS@Gel nanoparticles 489x154mm (106 x 106 DPI)


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Figure 6. Si2p (a), O1s (b), C1s (c) and N1s (d) XPS core peaks of NS@Gel-SiO2 309x280mm (118 x 118 DPI)


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Figure 7. Cytotoxicity after 24h incubation of (a) NS, (b) NS@Gel, (c) NS@Gel-SiO2, (d) NS@PLL and (e) NS@PLL-SiO2 nanoparticles evaluated by flow cytometry at 10 µM and 100 µM gold concentrations. 517x159mm (78 x 78 DPI)


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Figure 8. Evaluation of Au % in cells by ICP-MS after incubation with NS, NS@Gel, NS@Gel-SiO2, NS@PLL and NS@PLL-SiO2 at two gold concentrations (10 µM, 100 µM) 317x207mm (122 x 122 DPI)


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Figure 9. TEM images of HaCaT cells after 24 h of incubation : (a) control, (b) cells treated with NS, (c) NS@Gel, (d) NS@PLL, (e) NS@Gel-SiO2 and (f, g, h) NS@PLL-SiO2 nanoparticles (gold concentration : 100 µM) 615x341mm (107 x 107 DPI)


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