Highly Flexible Platform for Tuning Surface Properties of Silica

Subscriber access provided by RMIT University Library

Article

A highly flexible platform for tuning surface properties of silica nanoparticles and monitoring their biological interaction Isaac Ojea-Jiménez, Patricia Urbán, Francisco Barahona, Matteo Pedroni, Robin Capomaccio, Giacomo Ceccone, Agnieszka Kinsner-Ovaskainen, Francois Rossi, and Douglas Gilliland ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11216 • Publication Date (Web): 18 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

A highly flexible platform for tuning surface properties of silica nanoparticles and monitoring their biological interaction Isaac Ojea-Jiménez,1,* Patricia Urbán,1 Francisco Barahona,1 Matteo Pedroni,1 Robin Capomaccio,1,2 Giacomo Ceccone,1 Agnieszka Kinsner-Ovaskainen,1 François Rossi,1 Douglas Gilliland1,* 1

European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Via E. Fermi 2749, 21027 Ispra, VA, Italy.

2

Institut de Biologie et Chimie des Protéines, BMSSI-UMR 5086, Université Lyon 1, Université de Lyon, 69367 Lyon, France

Email: [email protected], [email protected] Keywords Silica nanoparticles, fluorescent nanoparticles, epoxide functionalization, surface charge, A549 cells, toxicity, uptake, cell membrane interaction

Abstract: The present work offers a simple, reliable and scalable seeding-growth methodology to prepare silica nanoparticles (SiO2 NPs) (20, 30, 50 and 80 nm) directly in aqueous phase, both as plain- as well as fluorescent-labelled silica. The amount of fluorescent label per particle remained constant regardless of size, which facilitates measurements in terms of number-based concentrations. SiO2 NPs in dispersion were functionalized with an epoxysilane, thus providing a flexible platform for the covalent linkage of wide variety of molecules under mild experimental conditions. This approach was validated with ethylenediamine, two different aminoacids and three akylamines to generate a variety of surface modifications. Accurate characterization of particle size, size distributions, morphology and surface chemistry is provided, both for assynthesized particles and after incubation in cell culture medium. The impact of physico-chemical properties of SiO2 NPs was investigated with human alveolar basal

1 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

epithelial cells (A549) such as the effect in cytotoxicity, cell internalization and membrane interaction.

Introduction The unique physico-chemical properties displayed by nanoparticles have raised their use in a number of commercial activities and emerging technologies. As part of our daily life, SiO2 NPs have found commercial application as additives in food and cosmetics,1,2 food contact materials, printer toners and industrial catalysis.3 Furthermore, biotechnology and biomedicine are fields of widespread application for SiO2 and SiO2coated NPs as drug delivery systems,4,5 in biosensing,6 as imaging contrast agents7 or gene carriers;8 which raises very strict requirements of purity, stability, and dispersibility. According to the specific needs of biological applications, diverse organic fluorophores such as fluorescein9,10 or rhodamine11 derivatives have also been successfully incorporated into SiO2 NPs, which provides them distinct properties such as high quantum yield, photostability, hydrophilicity and biocompatibility. Although the Stöber process,12 together with its multiple modifications,13,14 currently remains the dominant route for the synthesis of SiO2 NPs, the polydispersity obtained increases sharply when trying to prepare smaller NPs, typically 15-20 nm. Only recently, in an important breakthrough, Yokoi and co-workers have reported an alternative method to synthesise highly monodisperse SiO2 NPs of small sizes (12-23 nm),15,16 which has been later extended by Hartlen and co-workers to the preparation of monodisperse SiO2 NPs ranging from 15 nm to 200 nm by adopting a seeded regrowth approach.17 Such methodology makes use of aminoacids (i.e L-Lys or L-Arg) as a catalyst in place of ammonia for the silica condensation in aqueous medium, while TEOS is delivered heterogeneously using a top organic layer, thus ensuring a very low increase of the solution supersaturation.18 Compared to the Stöber method, the main advantage of a water-based synthesis is that it avoids particles to be transferred from ethanol into aqueous media if such particles are intended for biological applications. SiO2 NPs could undergo irreversible aggregation upon drying19 or even when the interparticle distance is drastically reduced during centrifugation or ultrafiltration,20 mostly occurring when alkoxysilanes are adsorbed at the surface of the particles.21 In addition, further purification steps such as dialysis could be applied in a more practical way (i.e. avoiding solvent resistant dialysis devices or large volumes of non-aqueous


Page 2 of 41

Page 3 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


solvent dialysates) when as-synthesized SiO2 NPs are readily dispersed in aqueous medium. Well-characterized monodisperse SiO2 NPs are needed to standardize and validate in vitro experimental procedures and to provide reliable data on their relative toxicity. Among the physicochemical properties of SiO2 NPs, their surface chemistry has been a subject widely investigated since small variations may lead to potential hazards to human health and to environmental risks. However, scientific insight into the toxicity of SiO2 NPs is still limited by the lack of detailed mechanistic information on the effect of the particle surface on their biological interactions. This is in part due to the fact that the surface modification of colloidal silica is still a challenging issue requiring careful consideration of reaction parameters (e.g. solvent, reagents, reaction time, and temperature). For example, agglomeration can severely hinder the outcome of the particle functionalization since the organic tethers employed, mainly alkoxysilanes, may condense with each other and crosslink particles if a non-aqueous solvent or high concentrations are employed.22,23 Therefore, a crucial factor in the design of surfacemodified SiO2 NPs for the subsequent immobilization of small molecules or biomolecules is the controlled covalent attachment of desired functional groups onto the particle surface while maintaining their dispersibility. Although surface modification with aminosilanes is by far the most common strategy adopted to obtain reactive groups on SiO2 NPs, only very limited number of studies describe for example their functionalization with carboxylate groups, which mainly implies the conversion of the pre-existing amino groups into carboxylic acid functionalities upon reaction with succinic anhydride.24 In this work, we report a highly robust, reliable and scalable seeding-growth method in aqueous phase to prepare a range of SiO2 NPs (nominal sizes of 20, 30, 50 and 80 nm) both as plain silica and as fluorescent-labelled silica (SiO2–Rubpy) employing a cationic inorganic dye, tris (2,2′bipyridyl) dichlororuthenium(II) hexahydrate (Ru(bpy)3). The main advantage of this seeding regrowth approach relies on a precise control over the particle size distribution while maintaining a constant amount of fluorescent label among all the different diameters of SiO2 NPs synthesized, which is highly desirable for comparative purposes in terms of particle concentrations for biological applications. Different particles prepared were subsequently functionalized with an epoxysilane coupling agent to produce epoxy-functionalized SiO2 NPs. These were later employed as reactive intermediates for the covalent immobilization under mild experimental conditions of 3 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 41

ethylenediamine, aminoacids (L-Serine and L-Glutamic acid) and alkylamines (methylamine, propylamine and butylamines), thus generating a myriad of surface characteristics (Figure 1). All materials were extensively characterized by an array of techniques including Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS),

Z-potential,

Differential Centrifugal

Sedimentation

(DCS),

Asymmetric Flow Field Flow Fractionation (AF4), Inductively Coupled Plasma Mass Spectrometry (ICP-MS), UV-vis and fluorescence spectroscopies, and the successful functionalization was demonstrated by a colorimetric assay, FT-IR spectroscopy and Xray Photoelectron Spectroscopy (XPS). The effect of particle size, functionalization and surface charge of SiO2 NPs were systematically determined by toxicity studies with human alveolar basal epithelial cells (A549) using an MTT assay, whereas cellular uptake and membrane interactions were evaluated by flow cytometry and TEM examination, respectively.

-Insert Figure 1 here-

Results and discussion Preparation and characterization of SiO2 NPs and SiO2–Rubpy NPs A robust and reliable seeding-growth method was achieved following optimization of the synthetic conditions described in previous literature references.17,18 This allowed the preparation of SiO2 NPs with highly tunable size control (20, 30, 50 and 80 nm) and high batch-to-batch reproducibility (Figure 2). Herein, we describe protocols regarding the highest volumes attempted in our laboratory (500 mg of SiO2 per batch), although scale up of this experimental procedure will be attempted in the future. Similarly, the full series of SiO2–Rubpy NPs of different diameters (20, 30, 50 and 80 nm) was obtained using the same seeding-growth approach (Figure 3). The most remarkable advantage of the process is that the four types of NPs obtained maintain exactly the same ratio of dye per NP, which allows rescaling direct fluorescence measurements in terms of particle concentrations for biological applications. This could be achieved since the synthesis of fluorescent SiO2–Rubpy NPs (20 nm) was carried out in the presence of the cationic inorganic dye Ru(bpy)3 in the initial reaction mixture, while the other diameters were obtained by a seeding-growth strategy using 20 nm particles as seeds. The amount of Ru(bpy)3 not incorporated into the 20 nm SiO2–Rubpy NPs was


Page 5 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


estimated to be approximately 0.1 % by measuring the absorbance of the filtrate solvent after centrifugation with filter units (10 KDa MWCO) with a UV-visible spectrophotometer (Molar extinction coefficient of 14600 M-1 cm-1 at 452 nm). Particle size distribution analysis, summarized in Table 1, was performed for all the particles by TEM, DLS, DCS and AF4 and the surface charge was analyzed by Z-potential measurements. The relative standard deviations (RSDs) of the TEM mean diameter varied between 4.5 % and 17.4 %, suggesting good monodispersity. These results are also in agreement with the hydrodynamic diameter measured by DLS and the particle size distribution calculated from the Stokes' law of sedimentation from DCS measurements. The corresponding polydispersity indexes (PdI) for both DLS and DCS measurements are also reported in Table 1. Initial DCS measurements were performed assuming a value of particle density of colloidal silica (2.2 g/cm3),25 media refractive index 1.344 and sample refractive index 1.45 (Figures 4A and 4B). Nevertheless, the apparent densities of the particles were also calculated based on the combination between the sedimentation time given by DCS and the hydrodynamic diameter found by DLS (Table 1).26,27 It could be observed that the apparent densities for 20 nm SiO2 NPs and SiO2–Rubpy NPs (1.58 and 1.62 g/cm3, respectively) were significantly lower than those of other sizes (between 1.71-1.84 g/cm3), which can be explained by the enhanced contribution of the hydrodynamic layer to the measured particle size for small volumes. As expected, when re-calculating the entire range of mean particle diameter values in DCS assuming their corresponding apparent densities, it could be observed that correlated well with the hydrodynamic diameters measured by DLS. The previously developed AF4–UV-vis–MALS method optimized for separation and sizing of SiO2NPs in the range between 20 nm and 150 nm, was applied to all SiO2 NPs and SiO2– Rubpy NPs (Supporting Information Table S1 and Figure S1).28 Fractograms showed well-defined single peaks that confirm the presence of monomodal size distributions. Mean particle sizes were in good agreement with the hydrodynamic diameter values obtained by off-line DLS measurements (Table 1).





1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

-Insert Table 1 here-

Both UV-vis absorption spectra and fluorescence spectra were determined for all samples containing Ru(bpy)3 and compared to that of a freshly prepared solution of the dye at the same concentration (Figures 4C and 4D). Appropriate dilutions of SiO2– Rubpy NPs solutions of 20, 30 and 50 nm in diameter were carried out in order to obtain the same concentration of dye as in the solution of 80 nm SiO2–Rubpy NPs (i.e. approximately 1.5 µg/mL Ru(Bpy)3). From the UV-vis spectra, one can observe a peak at 453 nm of the same absorbance maximum in all the cases except for particles of 80 nm in diameter, whose high contribution to the scattering component resulted in an increased optical density that hindered the peak. In the fluorescence spectra, all different sizes of SiO2–Rubpy NPs showed a maximum at the same wavelength (603 nm), which was slightly shifted from the value of free dye in solution (609 nm). A similar blue-shift has also been previously observed at low concentrations of Ru(bpy)3,29 which was attributed to the silica nanomatrix restricting the motion of dye molecules and reducing their aggregation by π-stacking. Furthermore, it was observed that the increasing width of the outer silica shell had an influence on the fluorescence intensity. For 30, 50 and 80 nm SiO2–Rubpy NPs, as the ratio of near surface dye molecules to the inner ones is decreased, the diffusion of atmospheric O2 has access to a lower proportion of dye molecules thus reducing a quenching effect and increasing their fluorescence intensity. The exact concentrations of silica in the SiO2 NP and SiO2–Rubpy NP dispersions (20, 30, 50 and 80 nm) were determined after measuring the Si-content by ICP-MS (Table 2). Measurements were carried out both by direct introduction of the particles into the equipment and also after digestion of the particles with hydrofluoric acid to assess the efficiency of the ionization. In both cases, the results were consistent with the expected values calculated from gravimetric determination, and the ratios between digested and undigested particles ranged between 88 and 115 % across the different sizes. Consequently, direct quantification of SiO2 NPs was possible and reliable in the size range considered here and is in good agreement with previous work.28 The Ru-content was also determined in samples of SiO2–Rubpy NPs by ICP-MS and the results were consistent with the expected values calculated from the added amount of Ru(bpy)3 dye, which confirms that most of the dye was embedded into the silica matrix.


Page 6 of 41

Page 7 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



-Insert Table 2 here-

Surface functionalization of SiO2 and SiO2–Rubpy NPs The highly versatile epoxy-functionalized SiO2 NPs and SiO2–Rubpy NPs were initially prepared by modification with (3-glycidyloxypropyl)trimethoxysilane (GPTMS) via a silanization reaction (Figure 1). The synthesis of this intermediate is very attractive since it presents reactive groups for covalent immobilization of a myriad of molecules. Epoxyde functional groups are reactive under mild experimental conditions toward many nucleophiles including amine, hydroxyl and thiol groups, thus leaving a very stable chemical bond under normal storage conditions. Although epoxysilanes have been extensively used for the modification and activation of glass slides, glass beads and other silica materials, there are just few number of reports on their use on SiO2 NPs.30-32 The aim of this strategy was to exploit epoxysilane chemistry as a convenient and versatile route for activation of SiO2 NPs. Hydrodynamic diameters measured by DLS show a uniform increase of the mean particle diameter (between 1.7 and 5.6 nm) while maintaining their size distributions (Figures 5A and 5B). The amount of epoxide groups incorporated into SiO2 50 nm NPs was estimated by a colorimetric assay upon reaction with fluorescein cadaverine (NH2-Fluo), a bifunctional molecule containing an amino and a fluorescein group (Supporting Information Figure S2). A significant decrease in absorbance of the filtered solutions confirmed the adsorption of the dye in epoxy-functionalized SiO2 NP dispersions. However, pristine SiO2 NPs also showed a drop in absorbance, presumably due to electrostatic adsorption of the dye onto the silica surface. From absorption differences measured by UV-vis spectroscopy, it could be estimated a total number of approximately 150 epoxide groups available per NP.33 These results confirmed that epoxy-functionalities on SiO2 NPs were reactive towards amine groups, and that these could be used directly for the covalent immobilization of molecules without compromising the stability.




1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 41

Initially, preparation of positively charged SiO2 NPs was investigated by the surface modification

epoxy-functionalized

SiO2

NP

solutions

with

ethylenediamine.

Crosslinking between particles was avoided by precise control on the reaction conditions (pH as well as concentrations of NPs and diamine molecule), and demonstrated since particle size distribution by DLS measurements showed no significant difference in the mean size compared with epoxy-SiO2 NPs (data not shown). A control experiment was performed in order to demonstrate the successful functionalization with ethylenediamine. Both pristine SiO2 NPs and epoxy-modified SiO2 NPs of 20 nm at the same concentrations (0.5 mg/mL) were reacted with ethylenediamine (2.24 mM) under the same reaction conditions as before. After 24 h the pH of the solutions was adjusted to 6-6.5 value by addition of 1M HCl. Z-potential measurements showed a large difference in surface charge depending on the initial particle (-19.6 mV for SiO2 NPs and 25.9 mV for epoxy-SiO2 NPs), while DLS measurements indicated no size changes for neither of the solutions. In a similar way, two different aminoacids (i.e. L-Ser and L-Glu) were reacted with the epoxy-modified SiO2 NPs. The evolution of the surface charge at different pH values of ethylenediamine, L-Ser and L-Glu-modified SiO2 NPs of 20 nm in diameter was evaluated by Z-potential measurements and are in good agreement with the expected values (Figure 5C). However, analysis of pristine SiO2 NPs and epoxy-SiO2 NPs unexpectedly resulted in relatively high negative values, which could be explained by the presence of L-Arg as a surfactant in pristine SiO2 NPs and the marked hydrophobic character of epoxy-SiO2 NPs.30 Measurements by FT-IR spectroscopy showed strong characteristic stretching bands for SiO2 NPs of -OH (3300-3700 cm-1) and Si-O-Si (~ 1100 cm-1) (Figure 5D). In addition, the peak observed at ~ 1634 cm-1 indicated the presence of adsorbed water molecules on the NPs surface, while the shoulder observed at ~ 1457 cm-1 (N-H bending) are characteristic of -NH2 groups on amine-SiO2 NPs. C-H stretching vibration bands characteristic of epoxydes were also observed around 2927-2944 cm-1, which confirmed the successful epoxysilane attachment onto the surface of SiO2 NPs.30,31 Surface modifications with ethylenediamine and L-Ser were successfully achieved with the complete range of sizes for SiO2 and SiO2–Rubpy NPs, always showing outstanding stability by DLS. Surface modification of SiO2 NPs of 20 nm in diameter was also


Page 9 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


analyzed by XPS measurements (Supporting Information Table S2 and Figure S3). XPS surface elemental analysis and carbon C1s core level spectra support the different types of functionalization. Finally, further covalent modifications of epoxy-functionalized particle surfaces were also approached with SiO2 NPs of 20 nm in diameter and a series of aliphatic amines such as methylamine, propylamine and butylamine. Successful functionalization was demonstrated by analyzing the relative hydrophobicity of the different aliphatic-functionalyzed SiO2 NPs by surface adsorption measurements of a hydrophobic probe molecule such as Rose Bengal, which showed a consistent larger adsorption to particle surface with increasing hydrophobicity (Supporting Information Figure S4).

Stability and dissolution of SiO2 NPs in cell culture medium In view of the fact that agglomeration can severely hinder determination of fundamental dose-response relatonships,34 the time-dependent stability of pristine SiO2 NPs, positively charged amine-SiO2 NPs and negatively charged L-Ser-SiO2 NPs in cell culture medium F12+GlutaMAXTM-I without serum was studied at a concentration of 200 µg/mL of silica for all the different size ranges. For evaluation of the size distribution and state of dispersion of SiO2 NPs in cell culture media, it has been shown that DLS in conjunction with TEM are suitable techniques.35 First of all, the evolution of the hydrodynamic diameters monitored by DLS over 48 h under these conditions indicates

insignificant agglomeration

among

all

particle

sizes

and

surface

functionalizations tested (Supporting Information Figure S5), which is in line with several previous stability studies performed at similar concentrations in serum free medium conditions.35-38 Clearly, TEM observation of NPs confirmed their dispersibility in culture medium after 48 h of incubation (Supporting Information Figures S6-S8), which was attributed to the relatively low concentrations of particles used and their high absolute Z-potential values at physiological pH. These experiments demonstrate that neither the ionic strength of physiological buffer nor chemical reactions with molecules derived from the medium cause agglomeration to this kind of particles. Moreover, the slight increase in hydrodynamic diameter observed by DLS for the charged amine-SiO2 NPs and L-Ser-SiO2 NPs of 80 nm in culture medium, as compared to pristine SiO2 NPs, can be attributed to the presence of thicker double layers (Debye length) for large sizes around the silica surface.36 Overall, good dispersibility in culture medium was also observed for the fluorescent-labeled SiO2–Rubpy NPs (data not shown). 9 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In the meanwhile, DLS measurements also show a gradual decrease in hydrodynamic diameter along the incubation period in culture medium, indicating dissolution of SiO2 NPs. TEM observation and image analysis were used to investigate the morphology and degradation. In comparison to pristine SiO2 NPs before incubation, dispersions of pristine SiO2 NPs, amine-SiO2 NPs and L-Ser-SiO2 NPs of 30, 50 and 80 nm in diameter present a much broader size distribution after 48 h incubation in culture medium (Supporting Information Table S3). Noticeably, the presence of smaller size populations and less dense non-spherical shapes could be found under TEM examination. Similar results could also be observed upon DCS analysis (data not shown). In the case of SiO2 NPs 20 nm in diameter, we could not detect significant dissolution or variation of morphology for any of the surface functionalizations compared to as-synthesized particles. Surprisingly, this observation contrasts with previous studies that found that dissolution rates on a per mass basis are almost independent of particle size at non-equilibrium conditions.39 Although we are aware of the limitations of DLS and TEM techniques to reliably size low electron density SiO2 NPs under 20 nm in diameter, a quantitative study of their dissolution rates is out of the scope of this work. Nevertheless, fluorescence spectroscopy of filtered solutions of SiO2–Rubpy NPs 20 nm in diameter after 48 h incubation in culture medium did not show in any signal coming from the dye, which discarded the dye escaping the particle matrix to any measurable extent. The dissolution process of silica in water has been well documented by a number of different works under various conditions such as temperature, pH or ionic strength.40,41 In particular, Vogelsberger et al. observed that the dissolution of nanodisperse silica shows a strong size effect, which leads to a significantly higher dissolution rate at first stages of the process but that slightly decreases at longer dissolution times.42,43 Concerning the toxicity of SiO2 NPs, solubility has been recognized to be a crucial factor in the clearance mechanism involved in the removal of amorphous silica from lung.44 The observed dissolution of SiO2 NPs is in agreement with its lack of agglomeration, since the formation of agglomerates would hinder dissolution (and thus increase persistence in the organism) by reducing the average equilibrium solubility of the particle system and by introducing kinetic hindrance to the diffusion process.

Nanoparticle-cell interaction studies with functionalized SiO2 NPs and SiO2– Rubpy NPs 10 Environment ACS Paragon Plus

Page 10 of 41

Page 11 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The respiratory system seems to be the most vulnerable barrier facing SiO2 NPs exposure and has received most attention in toxicological investigations. Consequently, a vast majority of the toxicity studies with SiO2 NPs involves exposure models of the respiratory tract such as adenocarcinomic human alveolar basal epithelial cells (A549).45 The main factors determining the toxicity of NPs are their large specific surface area making contact with cells, the chemical reactivity and intrinsic chemical toxicity of the material the NP is composed of, the particle morphology, the primary particle size influencing cell penetration and the dissolution ability determining persistence in the organism. In addition, surface charge, ionic strength and biological milieu may alter the properties of SiO2 NPs and potentially affect their biological effects. To systematically study the toxicological effect of the surface charge of SiO2 NPs in function of their size, A549 cells were exposed to pristine SiO2 NPs, positively charged amine-SiO2 NPs and negatively charged L-Ser-SiO2 NPs of different sizes (20, 30, 50 and 80 nm). Cell viability was evaluated using the well-established MTT assay, which measures the mitochondrial activity of the cells (Figure 6). A series of controls were carried out in order to verify that the influence of several steps of the experimental design did not affect to the final outcome of the experiments (see experimental details).46 Overall no corrections were applied in regard with the controls, as these readings were considered insignificant, i.e. less than five times higher than negative controls. After 24 h incubation time, in all the range of mass concentrations tested, pristine SiO2 NPs did not show a significant toxicity except for the case of 20 nm in diameter, which experienced a slight reduction to ~ 80 % of cell viability at 250 µg/mL (by far exceeding any realistic dose for unintentional exposure). Multiple examples in the literature can be found regarding studies with amorphous SiO2 NPs in A549 cells that show an overall low cytotoxicity below a concentration threshold over which it could be considered an unrealistic biological dose.47-52 By contrast, a toxic effect of positively charged amine-SiO2 NPs could clearly be observed, which was more noticeable for the highest particle sizes (50 and 80 nm) at concentrations > 0.15 µg/mL (Supporting Information Figure S9). Statistical data analysis applying a one-way ANOVA with a Bonferroni's multiple comparison test confirmed that there were significant differences only in the cases of 50 and 80 nm amine-SiO2 NPs when compared with the other functionalizations (pristine and L-Ser). These results were partly anticipated, as studies with airborne silica particles and the 11 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

examination of published evidences carried out by Bagchi and co-workers have suggested that the toxicity of these particles is strongly related with the high levels of positive charge they carry.53 Overall, biological environments appear to have low tolerance for positive charge or electron deficiency, not only since surface charge could play a part in membrane attachment or disruption but also because there is evidence that the final active forms of most chemical carcinogens are electron deficient, electrophilic, or positively charged reagents.54 However, and contrary to what some references have disclosed before with non-functionalized SiO2 NPs (i.e. a size-dependent toxicity with smaller sizes causing cell death to a greater extent),37,55 our results suggest that a high local dose of positive surface charge (i.e. for large particle sizes) could increase local membrane destabilization thus inducing higher toxicity. We are aware that these particles might have a different effect on cell metabolism or proliferation depending on the cell line tested. However, in contrast with other similar studies, the particles employed in these experiments were demonstrated to remain well dispersed under cell incubation conditions so that the toxicity observed could unarguably be attributed to the effects of size and surface characteristics of each type of NP. In regard to anionic L-SerSiO2 NPs, overall no effects on cell viability were observed, only showing a slight reduction to ~ 85 % at high mass concentrations (> 100 µg/mL) in the case of particles of 80 nm in diameter. However, statistical data analysis resulted in non-significant differences when compared with pristine SiO2 NPs.


We have recently demonstrated the efficiency of the fluorescent dye, Ru(bpy)3, embedded in SiO2 NPs as a marker to determine the cellular uptake in A549 cells, both quantitatively by flow cytometry and qualitatively by fluorescence microscopy, thus allowing visualization of NPs inside the cells.56 In the present work, flow cytometry experiments were first considered in order to quantify the uptake of pristine SiO2– Rubpy NPs, positively charged amine-SiO2–Rubpy NPs and negatively charged L-SerSiO2–Rubpy NPs of different sizes (20, 30, 50 and 80 nm) at equal mass concentrations (200 µg/mL) after 3 h incubation in serum free medium. The fact that the amount of Ru(bpy)3 dye per NP was maintained constant for all the different sizes facilitated a direct comparison among the full range of diameters. This was accomplished upon


Page 12 of 41

Page 13 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dividing the mean fluorescence intensities by the initial particle number concentrations incubated under each condition (Figure 7A).57 As expected, it was observed that the cells possessed a higher affinity for all the examples of cationic amine-SiO2–Rubpy NPs when compared with the negatively charged analogs. In particular, larger sizes showed an increased interaction. Such an increase of membrane adhesion in serum free conditions supported the overall higher cytotoxicity observed for cationic SiO2 NPs, and suggests possible disruption of the cell membrane. These observations agree with recent studies of cellular uptake with SiO2 NPs functionalized with different amounts of positive surface charge, which showed a correlation between surface charge and uptake efficiency.58 Similar work with Au NPs also disclosed an enhanced affinity for cell membranes and internalization of cationic NP-bioconjugates.59 Uptake of pristine SiO2–Rubpy NPs was moderate showing also a rising trend while increasing the particle diameter, thus suggesting that uptake was more efficient for larger sizes. Although flow cytometry is not able to distinguish between internalized and membrane associated particles, SiO2 NPs have been recurrently seen to be actively internalized after interacting with the membrane of A549 cells in the course of few hours.51 The dose metric for nanoscale particles poses an additional complexity to biological assays, as particle number and surface area may also play a significant role in addition to the traditional mass-based metric. For this reason, it was considered critical to reproduce flow cytometry experiments for equal particle number concentrations (3x1011 NP/mL) under the same reaction conditions (Figure 7B).57 Again, A549 cells possessed an overall higher affinity for amine-SiO2–Rubpy NPs followed by pristine SiO2–Rubpy NPs and L-Ser-SiO2–Rubpy NPs, which showed similar degrees of uptake. The most remarkable observation is that under a particle number dose-metric conditions the observed levels of uptake were not size-dependent for any of the different surface chemistries, i.e. the same number of NPs interact with the cell independently of their size. This observation has important bearings on the experimental design and the interpretation of biological assays concerning NPs, as disclosure of results could depend on the selection of dose metrics.38


TEM examination was used to visualize and localize the SiO2 NPs inside and/or around the cells after incubation (Figure 8). A549 cells were exposed to pristine SiO2 NPs, 13 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

positively charged amine-SiO2 NPs and negatively charged L-Ser-SiO2 NPs of different sizes (20, 30, 50 and 80 nm) for 3 h in serum free medium. Overall, it could be observed that NPs remained well-dispersed in the extracellular environment thus confirming the preliminary stability studies, and that the incubation with an equal mass concentration of particles (200 µg/mL) did not result in obvious structural damage of cells compared to untreated controls (Supporting Information Figures S10-S11). In agreement with flow cytometry experiments, cationic amine-SiO2 NPs were shown to have the highest affinity for adsorption to cell surfaces that eventually lead to internalization by endocytosis (Supporting Information Figure S12). The increased interaction, as compared to the pristine and L-Ser counterparts, can be explained in terms of the greater electrostatic attraction of cationic particles towards the negatively charged head-groups of the phospholipids bilayers forming the cell membrane. Moreover, it appears that NPs do not penetrate and cross the membrane but accumulate until the membrane is recycled and NPs are internalized inside endosomes. Experiments to determine the behaviour of SiO2 NPs in complete cell culture media are the subject of an ongoing study, in particular if the consequent opsonization with serum proteins (i.e. hardening of the protein corona, agglomeration or chemical modification of the surface) may deactivate these vectors for cellular uptake or toxicity.


Conclusions The present work offers a simple preparation procedure in order to precisely control the size of SiO2 NPs, by using a regrowth approach in water, and exploits epoxysilane chemistry as a convenient and versatile route for the activation and introduction of different functional groups on the NP surface while maintaining its dispersibility. In addition, the fluorescent-labelled version (i.e. SiO2–Rubpy NPs) can be obtained by incorporating a precise amount of inorganic dye Ru(bpy)3 while ensuring that the intensity of fluorescent label per particle remains constant among all the different sizes. Such an experimental design permits comparison of variations in both mass- and number-based concentration in biological studies, and highlights the importance of the selection of dose metrics. The impact of size and surface charge of SiO2 NPs was thoroughly investigated regarding their ability to induce harm in biological systems. As


Page 14 of 41

Page 15 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


expected, positively charged SiO2 NPs were not only more toxic to A549 cells but also had a greater level of cellular uptake and affinity for cell membranes, when compared to other functionalizations. Under a mass dose-metric condition, it could clearly be observed that cellular interaction increased as a function of increasing particle size, whereas particle number dose-metrics revealed no significant correlation with size. The presence of endocytic vesicles suggested that endocytosis was the predominant route of internalization. The dispersions of SiO2 NPs developed in this work offer a unique opportunity for standardization and validation of in vitro experimental procedures and provide reliable data on the toxicity and uptake of NPs in relation to their physico-chemical properties. For that reason, accurate characterization of particle size and size distributions, morphologic features (especially in the biological media used for experimental set-up), functionalization and surface chemistry has been performed, together with a complete description of the synthesis and assay conditions. As the scope of this study was merely to examine the authentic contribution of the surface chemistry of SiO2 NPs on biological interactions, all the results presented so far were obtained in serum free conditions. Realistic studies in order to investigate the effect of protein coating and modulation are underway.

Experimental Materials Tetraethylorthosilicate (TEOS, 99 %), L-Arginine (L-Arg, 98 %), tris (2,2′-bipyridyl) dichlororuthenium(II)

hexahydrate

(Ru(bpy)3,

99.95

%),

(3-

glycidyloxypropyl)trimethoxysilane (GPTMS, > 98 %) ethylenediamine (> 99 %), LSerine (L-Ser, > 99 %), L-Glutamic acid (L-Glu, > 99 %), methylamine (> 98 %), propylamine (> 99 %), butylamine (> 99 %) and cyclohexane (> 99 %) were purchased from Sigma-Aldrich and used as received without further purification. Fluorescein cadaverine dihydrobromide salt was purchased from Life Technologies. Water used in all the experiments was Milli-Q® ultrapure grade water. Cellulose dialysis membranes of 14 kDa MWCO (Sigma-Aldrich) were washed twice with boiling water before use. Centrifugal filter units of 10 kDa MWCO Amicon Ultra-15 (Millipore, Germany) were washed 3 x times with water and centrifuged (3500 rcf, 10 min, 4 ºC) before use.

Methods 15 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TEM (JEOL JEM 2100, Japan) at an accelerating voltage of 200 kV was used to visualize the nanoparticles. Samples were prepared by placing a drop (4 µL) onto ultrathin Formvar-coated 200-mesh copper grids (Tedpella Inc.) and leaving dry in air at 4 ºC. Digital images were analyzed with the ImageJ software and a custom macro performing smoothing (3x3 or 5x5 median filter), manual global threshold and automatic particle analysis provided by the ImageJ. The circularity filter of 0.8 was used to exclude agglomerates that occurred during drying. For each sample, the size of at least 500 particles was measured to obtain the average and the size distribution. DLS and Z-potential measurements were obtained using a Zetasizer Nano-ZS instrument (Malvern Instruments, UK). Hydrodynamic diameters were calculated using the internal software analysis from the DLS intensity-weighted particle size distribution. Z-potential was measured for independent samples just after adjusting the pH of the dispersions with either NaOH 1 M or HCl 1 M and at a total NaCl concentration of 1 mM. Particle size distribution was also determined by Disc Centrifuge Photosedimentometer model DC24000UHR (CPS Instruments Europe, The Netherlands). The instrument was operated in line-start mode at a disc rotation speeed of 22000 rpm using a sucrose gradient (8-24 % w/w) capped with dodecane to prevent solvent evaporation. Each reference was precedented by a calibration step done using an aqueous reference standard of 239 nm diameter polyvinyl chloride (PVC) spheres. All samples were vortexed for 30 seconds prior to injection. FT-IR spectra were adquired with a Confocheck Tensor27 spectrometer (Bruker, Germany). Samples were prepared by addition of NaCl (120 mg) to the different dispersions (2 mL) in order to destabilize the NPs, solutions were then centrifuged (15000 rcf, 1.5 hr, 4 ºC) and the resulting pellets dried in the oven at 60 ºC overnight. Pellets were prepared with KBr (25 mg) containing 1 % (w/w) of dry NPs. Elemental analysis was performed using a PerkinElmer NexIon 300D quadrupole ICP-MS, equipped with a SC Fast peristaltic pump, a Meinhard concentric nebulizer, a glass cyclonic spray chamber and a standard quartz torch (2.5 mm i.d). The system operated in standard mode monitoring isotopes m/z 28 and 29 for Si with dwell time of 5 ms and integration time of 1 s, as well as m/z 100.9 for Ru, under the same conditions. The quantification was performed by external calibration with silicon standards for ICP (TraceCert, Sigma-Aldrich) and ruthenium standards for ICP (Romil PrimAg® xtra, Romil Ltd., Cambridge, UK). SiO2 NP and SiO2–Rubpy NP samples were dissolved 100 x times in milliQ water. Elemental analysis of digested silica by ICP-MS was performed by mixing 1 mL of each silica suspension with 200 µL 16 Environment ACS Paragon Plus

Page 16 of 41

Page 17 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of concentrated hydrofluoric acid (HF) in 10 mL polypropylene tubes, which were subsequently filled up to 10 mL with water. The tubes were sealed and placed in an ultrasound bath thermostated at 60 °C during 1 h. The solutions were subjected to 1:10 dilution with water prior ICP-MS analysis. The content of silicon was determined using an ICP-MS HF-compatible configuration, which consisted on a PFA-ST nebuliser, a PFA cyclonic spray chamber, a 1.8 mm sapphire injector and a ceramic torch. The system operated with identical settings. The quantification was also performed by external calibration with ionic silicon standards for ICP-MS. UV-vis absorption spectra were recorded with an Evolution 300 UV-Vis Spectrophotometer (Thermo Scientific, USA) at room temperature. Fluorescence spectra were recorded with a Varian Cary Eclipse (Agilent Technologies, USA). The instrument was operated at an excitation wavelength of 460 nm using 5 nm slits for both excitation and emision and a medium scan control. Statistical analysis was performed with Prism 5 (GraphPad Software, Inc., USA).

Synthesis of SiO2 NPs First generation of SiO2 NPs 20 nm in diameter was obtained as described previously.17,18 Briefly, cyclohexane (16.25 mL) was mixed with a solution of L-Arg (330 mg, 1.9 mmol) in water (250 mL). The biphasic mixture was pre-heated at 50 ºC before TEOS (20 mL) was slowly added and the reaction was then stirred gently at a rate of approximately 300 rpm for 24 h at 50 ºC. Second generation of SiO2 NPs 30 nm in diameter was obtained by mixing 50 mL of as-synthesized SiO2 NPs of 20 nm with water (180 mL) and cyclohexane (25 mL), so that the total concentration of L-Arg was 1.5 mM. TEOS (18.5 mL) was slowly added and the biphasic mixture was kept under the same reaction conditions as before. Third generation of SiO2 NPs 50 nm in diameter was obtained by mixing 20 mL of as-synthesized SiO2 NPs of 30 nm with cyclohexane (10 mL) and a solution of L-Arg (12 mg, 0.07 mmol) in water (72 mL), so that the total concentration of L-Arg was 1 mM. TEOS (7.4 mL) was slowly added and the biphasic mixture was kept under the same reaction conditions as before. Fourth generation of SiO2 NPs 80 nm in diameter was obtained by mixing 20 mL of as-synthesized SiO2 NPs of 50 nm with cyclohexane (10 mL) and a solution of L-Arg (13.7 mg, 0.08 mmol) in water (72 mL), so that the total concentration of L-Arg was 1 mM. TEOS (7.4 mL) was slowly added and the biphasic mixture was kept under the same reaction conditions as



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

before. In all the cases, the reaction mixtures were allowed to cool down to room temperature and the aqueous phase was separated from the organic layer using a separating funnel. Particles were purified by dialysis against water for 24 h in order to eliminate the excess of non-conjugating molecules. The approximate concentration for all the samples was 2 mg/mL, calculated by gravimetric analysis upon destabilization with 300 mg NaCl, centrifugation (15000 rcf, 1.5 h, 4 ºC) and drying of samples (5 mL). Particle size distributions were determined by TEM, DLS and DCS, the surface charge by Z-potential measurements and elemental analysis of Si-content by ICP-MS.

Synthesis of SiO2–Rubpy NPs First generation of SiO2–Rubpy NPs 20 nm in diameter was obtained in a similar way as plain SiO2 NPs but in the presence the cationic inorganic dye Ru(bpy)3. In a typical experiment to obtain SiO2–Rubpy NPs 20 nm in diameter, cyclohexane (16.25 mL) was mixed with a solution of Arg (400 mg, 2.3 mmol) and Ru(bpy)3 (37.5 mg, 0.05 mmol) in water (250 mL). The mixture was kept at room temperature and at a constant stirring rate of approximately 300 rpm before TEOS (20 mL) was slowly added and the reaction was kept under these conditions for 48 h. The full series of SiO2–Rubpy NPs of different diameters (30, 50 and 80 nm) was obtained using the same seeding-growth strategy as for SiO2 NPs, but starting from as-synthesized SiO2–Rubpy NPs of 20 nm. Particles were purified using the same extraction and clean-up procedure as before and the approximate concentrations calculated by gravimetric analysis were 2 mg/mL in all the cases. Particle size distribution was determined by TEM, DLS and DCS, the surface charge by Z-potential measurements and elemental analysis of Si-and Ru-content by ICP-MS. UV-vis absorption and fluorescent spectra were obtained for 20, 30, 50 and 80 nm at concentrations of 0.025, 0.13, 0.43 and 2.3 mg/mL of SiO2 respectively, so that all samples had the same concentration of Ru(Bpy)3 (~ 1.5 µg/mL).

Epoxy-functionalization of SiO2 NPs and SiO2–Rubpy NPs A corresponding sample of either plain SiO2 NPs or SiO2–Rubpy NPs (120 mL) was diluted in water (360 mL) and the pH increased with the addition of 1M NaOH (240 µL). GPTMS (120 µL, 0.54 mmol) was immediately added and the reaction mixture was stirred at room temperature for 24 h. Particle size distributions were determined by DLS, which showed an increase of size of around 2-6 nm in all the cases. Surface charge of epoxy-functionalized SiO2 NPs of 20 nm was determined by Z-potential 18 Environment ACS Paragon Plus

Page 18 of 41

Page 19 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


measurements at different pH values after adjusting with 1M NaOH or HCl. The amount of epoxy groups incorporated into the SiO2 NPs protein was estimated upon reaction with NH2-Fluo. Both pristine SiO2 NPs and epoxy-modified SiO2 NPs of 50 nm in diameter were incubated for 16 h under dark conditions at a concentration of 0.6 mg/mL with NH2-Fluo (0.04 mg/mL) in a phosphate buffer (100 mM, pH 8). Particles were removed from the solutions using centrifugal filter units of 100 kDa MWCO Amicon Ultra-0.5 (15000 rcf, 15 min) and the absorbance of the filtrates was measured with a UV-visible spectrophotometer. Molar extinction coefficient of NH2-Fluo is 57 000 M-1 cm-1 at 494 nm.60 A control experiment was run without NPs under the same reaction conditions.

Surface modification of epoxy-functionalized SiO2 NPs and SiO2–Rubpy NPs All sizes (20, 30, 50 and 80 nm in diameter) of amine-functionalized SiO2 NPs and SiO2–Rubpy NPs were obtained from their corresponding aqueous solutions of epoxymodified SiO2 NPs (240 mL) at an approximate concentration of 0.5 mg/mL of silica. Ethylenediamine was directly added to a final concentration of 2.24 mM and at pH = 9, the reaction mixtures were stirred at room temperature for 24 h and finally 1M HCl (800 µL) was added. In a similar manner, stock aqueous solutions of L-Ser or L-Glu at 10 mg/mL (for L-Glu the pH was increased to 9 with 1M NaOH in order to ensure solubility) were added to epoxy-modified SiO2 NPs to a final concentration of 1.6 mM and stirred at room temperature for 24 h to obtain carboxylic acid-functionalized SiO2 NPs and SiO2–Rubpy NPs. Aliphatic modifications of epoxy-functionalized SiO2 NPs were obtained by addition of methylamine, propylamine or butylamine (final concentrations of 2.24 mM) at pH = 9 to a 20 nm particle solution (4 mL at 0.5 mg/mL of silica) and the reaction mixture left react for 24 h. All the samples were concentrated to their original stock concentrations (approx. 2 mg/mL of silica) using centrifugal filter units of 10 kDa MWCO Amicon Ultra-15 (3500 rcf, 5 min), and then purified through two additional washing steps with water in order to eliminate the excess of nonconjugating molecules. DLS measurements showed no major differences in the mean hydrodynamic size for any of the samples, which was a good indicator of the colloidal stability of these solutions. Surface charge of amine- and carboxylic acid-functionalized SiO2 NPs of 20 nm was determined by Z-potential measurements at different pH values after adjusting with 1 M NaOH or HCl, and their functional groups were analyzed by FT-IR. 19 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 41

Cell culture and characterization of particles in cell culture medium Adenocarcinomic human alveolar basal epithelial cell line (A 549) was obtained from American Tissue Type Culture Collection (ATCC). The A459 cells were cultured in 75 cm2 flasks (Corning) in HAM-F12+GlutamaxTM-I medium (Life Technologies) supplemented with 10 % heat-inactivated foetal bovine serum (Life Technologies), 10.000U Penicillin/Streptomycin (Life Technologies) and 0.5 % (v/v) Hepes (Life Technologies) under normal cell culture conditions (37 °C, 5 % CO2, 95 % humidity). Previously to the incubation with cells, pristine SiO2 NPs, amine-SiO2 NPs and L-SerSiO2

NPs,

both

plain

and

Ru(Bpy)3-modified,

were

incubated

in

HAM-

F12+GlutamaxTM-I medium without serum at 37 °C in a CO2 incubator at a concentration of 200 µg/mL. The stability of the particles was analyzed by DLS after 0, 5, 24 and 48 h and by TEM after 48 h of incubation.

Assessment of cytotoxicity Cells were trypsinized, seeded into 96-well plates (Corning) at a density of 1x104 cells/well and left in the incubator. After 48 h, the complete culture medium was replaced with medium without serum containing SiO2 NPs at the desired concentration or the corresponding solvent controls, and cells were incubated for 24 h. After the incubation time, the medium was removed, cells were washed with PBS and the MTT solution

(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

bromide,

0.5

mg/mL in culture medium) was added to each well and incubated for 2 h at 37 °C. After incubation of cells with the NPs, the medium was removed, the formazan crystals formed in each well were dissolved with DMSO and after shaking of the plate for 10 minutes, the absorbance of the samples was measured at 550 nm using an Omega Fluostar plate reader (BMG, Germany). Negative controls were performed with cells incubated with medium in the absence of particles. Blank controls were performed with particles incubated with medium in the absence of cells and not washed before performing the viability assay. To evaluate the intrinsic absorbance of the NPs, particle controls were performed with cells incubated with medium containing particles at the highest concentrations tested that did not undergo the viability assay (i.e. no incubation with MTT). Two independent experiments were performed with five replicates each. Percentages of mitochondrial activity or cell viability were calculated relative to absorbance values of the negative controls. Values corresponding to concentrations of 0 20 Environment ACS Paragon Plus

Page 21 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mg/mL represent the relative solvent controls, which were isolated from the NPs by centrifugal filter units of 10 kDa MWCO.

Flow cytometry A549 cells were trypsinized, seeded in 12-well plates at a density of 2x105 cells/well and left in the incubator for at least 96 h, replacing the medium after 48 h. When 80-90 % of confluency was reached, cells were exposed to SiO2–Rubpy NPs at a mass concentration of 200 µg/mL or to the corresponding controls in medium without serum for 3 h. Additionally, a series of experiments was repeated for an equal particle number concentration of SiO2–Rubpy NPs, upon a previous dilution of all the samples to the same particle concentration present in 80 nm SiO2–Rubpy NPs at 200 µg/mL (i.e. 3x1011 NP/mL). At the end of the exposure, cells were washed in PBS (3 x 1 mL), trypsinized (Trypsin-EDTA 0.05 %, 200 µL) and re-suspended in fresh medium to a final concentration of 1x106 cells/mL. After washing with PBS, the cell associated fluorescence, side scatter (SSC) and forward scatter (FSC) were measured using CyFlow space flow cytometer (Partec, Germany) and the data analysed using FCS Express 4 software (De Novo, USA). Laser excitation was 488 nm and emission band pass wavelength was 590/50 nm for SiO2–Rubpy NPs related fluorescence. A minimum of 15.000 cells per sample were analysed and all conditions were performed for three independent experiments with three replicates each. Cells debris, dead cells and NPs were excluded from the analysis by gating on the FSC versus SSC log graph and median fluorescence intensity of NPs treated cells was compared with the fluorescence of control cells.

TEM sample preparation and imaging For resin inclusion for TEM, A549 cells were trypsinized, seeded in 12-well plates at a density of 2x105 cells/well and left in the incubator for at least 96 h, replacing the medium after 48 h. When 80-90 % of confluency was reached, cells were exposed to SiO2 NPs at a mass concentration of 200 µg/mL or to the corresponding controls in medium without serum (1 mL) for 3 h. After incubation, cells were washed with PBS (3 x 1 mL), trypsinized (Trypsin-EDTA 0.05 %, 200 µL) and centrifuged (2000 rpm, 5 min). The resulting pellets were collected and fixed in a 0.1 mM PB solution (100 µL) containing 2.5 % glutaraldehyde at 4 ºC overnight. The cellular pellet was postfixed in a 1 % osmium tetroxide:cacodylate 0.1 M solution (1:2) (250 µL, 2 x times, 30 minutes 21 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

each), dehydrated in a graded series of ethanol (30, 50, 75, 95 and 100 %) and rinsed with propylene oxide (2 x times 10 minutes each). Finally warm epoxy resin was mixed with ethanol (1:1) for infiltration and resin only was used for cell embedding (60 ºC, 48 h). Ultrathin sections (50-70 nm) were cut with a Leica EM-UC7 ultramicrotome and stained with 5 % uranyl acetate in water and 1 % aqueous lead citrate solution. The sections obtained were imaged using a TEM (JEOL JEM-2100, Japan) at an accelerating voltage of 120 kV.

Acknowledgements The work leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° NMP4LA-2013-310451. The authors thank Dr. Luigi Calzolai and Dr. Pascal Colpo from Nanobiosciences Unit (IHCP-JRC) for support in the calculations of the apparent densities of particles and NMR analysis of GPTMS ligand. The authors also acknowledge Dr. Jessica Ponti from Nanobiosciences Unit (IHCP-JRC) for protocols for processing and contrasting cellular pellets.

Supporting Information Available. Separation and sizing of SiO2 NPs and SiO2– Rubpy NPs by AF4–UV-vis–MALS method; UV-vis absorption spectra of pristine SiO2 NPs and epoxy-SiO2 NPs solutions after incubation with fluorescein cadaverine; hydrophobicity measurements by Rose Bengal surface adsorption; XPS surface compositions and C1s core level spectra of SiO2 NPs; time evolution DLS measurements; TEM micrographs and particle size distributions of functionalized SiO2 NPs after incubation with serum free medium; cell viability tests at low mass concentrations of amine-SiO2 NPs of 50 nm and 80 nm; higher magnification TEM micrographs of A549 cells incubated with functionalized SiO2 NPs and TEM images of A549 cells incubated with solvent controls. This information is available free of charge via the Internet at http://pubs.acs.org.

References (1)

Peters, R.; Kramer, E.; Oomen, A. G.; Herrera Rivera, Z. E.; Oegema, G.;

Tromp, P. C.; Fokkink, R.; Rietveld, A.; Marvin, H. J. P.; Weigel, S.; Peijnenburg, A.


Page 22 of 41

Page 23 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A. C. M.; Bouwmeester, H. Presence of Nano-Sized Silica during In Vitro Digestion of Foods Containing Silica as a Food Additive ACS Nano 2012, 6, 2441-2451. (2)

Peters, R.; Puck Brandhoff, P.; Weigel, S.; Marvin, H.; Bouwmeester, H.;

Aschberger, K.; Rauscher, H.; Amenta, V.; Arena, M.; Botelho-Moniz, F.; Gottardo, S.; Mech, A. Inventory of Nanotechnology applications in the agricultural, feed and food sector; CFT/EFSA/FEED/2012/01; 2014. (3)

Banerjee, S.; Santra, S. Remarkable Catalytic Activity of Silica Nanoparticle in

the bis-Michael Addition of Active Methylene Compounds to Conjugated Alkenes Tetrahedron Lett. 2009, 50, 2037-2040. (4)

Rosenholm, J. M.; Sahlgren, C.; Linden, M. Towards Multifunctional, Targeted

Drug Delivery Systems Using Mesoporous Silica Nanoparticles - Opportunities & Challenges Nanoscale 2010, 2, 1870-1883. (5)

Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C.-W.; Lin, V. S. Y. Mesoporous Silica

Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers Adv. Drug Del. Rev. 2008, 60, 1278-1288. (6)

Tapec, R.; Zhao, X. J.; Tan, W. Development of Organic Dye-Doped Silica

Nanoparticles for Bioanalysis and Biosensors J. Nanosci. Nanotechnol. 2002, 2, 405409. (7)

Liberman, A.; Mendez, N.; Trogler, W. C.; Kummel, A. C. Synthesis and

Surface Functionalization of Silica Nanoparticles for Nanomedicine Surf. Sci. Rep. 2014, 69, 132-158. (8)

Kumar, M. N. V. R.; Sameti, M.; Mohapatra, S. S.; Kong, X.; Lockey, R. F.;

Bakowsky, U.; Lindenblatt, G.; Schmidt, C. H.; Lehr, C. M. Cationic Silica Nanoparticles as Gene Carriers: Synthesis, Characterization and Transfection Efficiency In vitro and In vivo J. Nanosci. Nanotechnol. 2004, 4, 876-881.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(9)

Nyffenegger, R.; Quellet, C.; Ricka, J. Synthesis of Fluorescent, Monodisperse,

Colloidal Silica Particles J. Colloid Interface Sci. 1993, 159, 150-157. (10)

Van Blaaderen, A.; Vrij, A. Synthesis and Characterization of Colloidal

Dispersions of Fluorescent, Monodisperse Silica Spheres Langmuir 1992, 8, 2921-2931. (11)

Verhaegh, N. A. M.; Blaaderen, A. v. Dispersions of Rhodamine-Labeled Silica

Spheres: Synthesis, Characterization, and Fluorescence Confocal Scanning Laser Microscopy Langmuir 1994, 10, 1427-1438. (12)

Stöber, W.; Fink, A.; Bohn, E. Controlled Growth of Monodisperse Silica

Spheres in the Micron Size Range J. Colloid Interface Sci. 1968, 26, 62-69. (13)

Giesche, H. Synthesis of Monodispersed Silica Powders II. Controlled Growth

Reaction and Continuous Production Process J. Eur. Ceram. Soc. 1994, 14, 205-214. (14)

Van Blaaderen, A.; Van Geest, J.; Vrij, A. Monodisperse Colloidal Silica

Spheres from Tetraalkoxysilanes: Particle Formation and Growth Mechanism J. Colloid Interface Sci. 1992, 154, 481-501. (15)

Yokoi, T.; Sakamoto, Y.; Terasaki, O.; Kubota, Y.; Okubo, T.; Tatsumi, T.

Periodic Arrangement of Silica Nanospheres Assisted by Amino Acids J. Am. Chem. Soc. 2006, 128, 13664-13665. (16)

Yokoi, T.; Wakabayashi, J.; Otsuka, Y.; Fan, W.; Iwama, M.; Watanabe, R.;

Aramaki, K.; Shimojima, A.; Tatsumi, T.; Okubo, T. Mechanism of Formation of Uniform-Sized Silica Nanospheres Catalyzed by Basic Amino Acids Chem. Mat. 2009, 21, 3719-3729. (17)

Hartlen, K. D.; Athanasopoulos, A. P. T.; Kitaev, V. Facile Preparation of

Highly Monodisperse Small Silica Spheres (15 to >200 nm) Suitable for Colloidal Templating and Formation of Ordered Arrays Langmuir 2008, 24, 1714-1720.


Page 24 of 41

Page 25 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18)

Fouilloux, S.; Daillant, J.; Thill, A. Single Step Synthesis of 5-30 nm

Monodisperse Silica Nanoparticles: Important Experimental Parameters and Modeling Colloids Surf., A 2012, 393, 122-127. (19)

Depasse, J.; Watillon, A. The Stability of Amorphous Colloidal Silica J. Colloid

Interface Sci. 1970, 33, 430-438. (20)

Izak-Nau, E.; Voetz, M.; Eiden, S.; Duschl, A.; Puntes, V. F. Altered

Characteristics of Silica Nanoparticles in Bovine and Human Serum: The Importance of Nanomaterial Characterization Prior to its Toxicological Evaluation Part. Fib. Toxicol. 2013, 10, 56-68. (21)

Pálmai, M.; Nagy, L. N.; Mihály, J.; Varga, Z.; Tárkányi, G.; Mizsei, R.;

Szigyártó, I. C.; Kiss, T.; Kremmer, T.; Bóta, A. Preparation, Purification, and Characterization of Aminopropyl-functionalized Silica Sol J. Colloid Interface Sci. 2013, 390, 34-40. (22)

Pham, K. N.; Fullston, D.; Sagoe-Crentsil, K. Surface Charge Modification of

Nano-Sized Silica Colloid Aust. J. Chem. 2007, 60, 662-666. (23)

Weichold, O.; Tigges, B.; Bertmer, M.; Möller, M. A Comparative Study on the

Dispersion Stability of Aminofunctionalised Silica Nanoparticles Made from Sodium Silicate J. Colloid Interface Sci. 2008, 324, 105-109. (24)

An, Y.; Chen, M.; Xue, Q.; Liu, W. Preparation and Self-assembly of

Carboxylic Acid-functionalized Silica J. Colloid Interface Sci. 2007, 311, 507-513. (25)

Ilker, R. K. The Chemistry of Silica: Solubility, Polymerization, Colloid and

Surface Properties and Biochemistry of Silica; Wiley: New York, 1979. (26)

Calculated from the sedimentation time t given by the Stokes’ law formula: D =

[(ln Rf/R0 x 18η)/(t x (ρp- ρf) x ω2]1/2, where D is the hydrodynamic diameter, R0 and Rf the start and the end of the gradient respectively, ρp and ρf the densities of the particle



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and the fluid respectively, η the dynamic viscosity of the fluid and ω the rotational speed in radians. (27)

Capomaccio, R.; Ojea-Jiménez, I.; Colpo, P.; Gilliland, D.; Ceccone, G.; Rossi,

F.; Calzolai, L. Determination of Structure and Morphology of Gold Nanoparticle-HSA Protein Complexes Nanoscale, 2015, 7, 17653-17657. (28)

Barahona, F.; Geiss, O.; Urbán, P.; Ojea-Jiménez, I.; Gilliland, D.; Barrero

Moreno, J. Simultaneous Determination of Size and Quantification of Silica Nanoparticles by Asymmetric Flow Field-Flow Fractionation Coupled to ICPMS Using Silica Nanoparticles Standards Anal. Chem. 2015, 87, 3039-3047. (29)

Liang, S.; Shephard, K.; Pierce, D. T.; Zhao, J. X. Effects of a Nanoscale Silica

Matrix on the Fluorescence Quantum Yield of Encapsulated Dye Molecules Nanoscale 2013, 5, 9365-9373. (30)

Sun, Y.; Zhang, Z.; Wong, C. P. Study on Mono-dispersed Nano-size Silica by

Surface Modification for Underfill Applications J. Colloid Interface Sci. 2005, 292, 436-444. (31)

Vejayakumaran, P.; Rahman, I. A.; Sipaut, C. S.; Ismail, J.; Chee, C. K.

Structural and Thermal Characterizations of Silica Nanoparticles Grafted with Pendant Maleimide and Epoxide Groups J. Colloid Interface Sci. 2008, 328, 81-91. (32)

Zhang, Q.; Huang, R.; Guo, L.-H. One-step and High-density Protein

Immobilization on Epoxysilane-modified Silica Nanoparticles Chin. Sci. Bull. 2009, 54, 2620-2626. (33)

The concentration of covalently bound NH2-Fluo was calculated from the

absorbance difference at λmax. = 494 nm between the filtered solutions of epoxy-SiO2 NPs and SiO2 NPs and divided by the number of particles in solution. The concentration of SiO2 NPs (approximately 1013 NP/mL) was calculated using the mean particle


Page 26 of 41

Page 27 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


diameter by TEM (56.8 nm), the density of colloidal silica (2.2 g/cm3) and the concentration of silica (2.29 mg/mL) by ICP-MS. (34)

Nel, A.; Xia, T.; Mädler, L.; Li, N. Toxic Potential of Materials at the Nanolevel

Science 2006, 311, 622-627. (35)

Murdock, R. C.; Braydich-Stolle, L.; Schrand, A. M.; Schlager, J. J.; Hussain, S.

M. Characterization of Nanomaterial Dispersion in Solution Prior to In Vitro Exposure Using Dynamic Light Scattering Technique Toxicol. Sci. 2008, 101, 239-253. (36)

Thomassen, L. C. J.; Aerts, A.; Rabolli, V.; Lison, D.; Gonzalez, L.; Kirsch-

Volders, M.; Napierska, D.; Hoet, P. H.; Kirschhock, C. E. A.; Martens, J. A. Synthesis and Characterization of Stable Monodisperse Silica Nanoparticle Sols for in Vitro Cytotoxicity Testing Langmuir 2009, 26, 328-335. (37)

Napierska, D.; Thomassen, L. C. J.; Rabolli, V.; Lison, D.; Gonzalez, L.; Kirsch-

Volders, M.; Martens, J. A.; Hoet, P. H. Size-Dependent Cytotoxicity of Monodisperse Silica Nanoparticles in Human Endothelial Cells Small 2009, 5, 846-853. (38)

Lison, D.; Thomassen, L. C. J.; Rabolli, V.; Gonzalez, L.; Napierska, D.; Seo, J.

W.; Kirsch-Volders, M.; Hoet, P.; Kirschhock, C. E. A.; Martens, J. A. Nominal and Effective Dosimetry of Silica Nanoparticles in Cytotoxicity Assays Toxicol. Sci. 2008, 104, 155-162. (39)

Diedrich, T.; Dybowska, A.; Schott, J.; Valsami-Jones, E.; Oelkers, E. H. The

Dissolution Rates of SiO2 Nanoparticles As a Function of Particle Size Environ. Sci. Technol. 2012, 46, 4909-4915. (40)

Cauda, V.; Schlossbauer, A.; Bein, T. Bio-degradation Study of Colloidal

Mesoporous Silica Nanoparticles: Effect of Surface Functionalization With Organosilanes and Poly(ethylene glycol) Microporous Mesoporous Mat. 2010, 132, 60-71.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(41)

Icenhower, J. P.; Dove, P. M. The Dissolution Kinetics of Amorphous Silica into

Sodium Chloride Solutions: Effects of Temperature and Ionic Strength Geochim. Cosmochim. Acta 2000, 64, 4193-4203. (42)

Roelofs, F.; Vogelsberger, W. Dissolution Kinetics of Synthetic Amorphous

Silica in Biological-Like Media and Its Theoretical Description J. Phys. Chem. B 2004, 108, 11308-11316. (43)

Vogelsberger, W.; Schmidt, J.; Roelofs, F. Dissolution Kinetics of Oxidic

Nanoparticles: The Observation of an Unusual Behaviour Col. Surf., A 2008, 324, 5157. (44)

Borm, P.; Klaessig, F. C.; Landry, T. D.; Moudgil, B.; Pauluhn, J.; Thomas, K.;

Trottier, R.; Wood, S. Research Strategies for Safety Evaluation of Nanomaterials, Part V: Role of Dissolution in Biological Fate and Effects of Nanoscale Particles Toxicol. Sci. 2006, 90, 23-32. (45)

Napierska, D.; Thomassen, L. C.; Lison, D.; Martens, J. A.; Hoet, P. H. The

Nanosilica Hazard: Another Variable Entity Part. Fibre Toxicol. 2010, 7, 1-32. (46)

Geys, J.; Nemery, B.; Hoet, P. H. M. Assay Conditions Can Influence the

Outcome of Cytotoxicity Tests of Nanomaterials: Better Assay Characterization is Needed to Compare Studies Toxicol. In vitro 2010, 24, 620-629. (47)

Jin, Y.; Kannan, S.; Wu, M.; Zhao, J. X. Toxicity of Luminescent Silica

Nanoparticles to Living Cells Chem. Res. Toxicol. 2007, 20, 1126-1133. (48)

Lin, W.; Huang, Y.-w.; Zhou, X.-D.; Ma, Y. In vitro Toxicity of Silica

Nanoparticles in Human Lung Cancer Cells Toxicol. Appl. Pharmacol. 2006, 217, 252– 259.


Page 28 of 41

Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(49)

Choi, S.-J.; Oh, J.-M.; Choy, J.-H. Toxicological Effects of Inorganic

Nanoparticles on Human Lung Cancer A549 Cells J. Inorg. Biochem. 2009, 103, 463471. (50)

Shi, Y.; Yadav, S.; Wang, F.; Wang, H. Endotoxin Promotes Adverse Effects of

Amorphous Silica Nanoparticles on Lung Epithelial Cells in Vitro J. Toxicol. Environ. Health, Part A 2010, 73, 748-756. (51)

Nowak, J. S.; Mehn, D.; Nativo, P.; García, C. P.; Gioria, S.; Ojea-Jiménez, I.;

Gilliland, D.; Rossi, F. Silica Nanoparticle Uptake Induces Survival Mechanism in A549 Cells by the Activation of Autophagy but not Apoptosis Toxicol. Lett. 2014, 224, 84-92. (52)

Izak-Nau, E.; Kenesei, K.; Murali, K.; Voetz, M.; Eiden, S.; Puntes, V. F.;

Duschl, A.; Madarász, E. Interaction of Differently Functionalized Fluorescent Silica Nanoparticles with Neural Stem- and Tissue-type Cells Nanotoxicology 2013, 8, 138148. (53)

Bagchi, N. What Makes Silica Toxic? Br. J. Ind. Med. 1992, 49, 163-166.

(54)

Miller, J. A.; Miller, E. C., Ultimate Chemical Carcinogens as Reactive

Mutagenic Electrophiles. In Origins of human cancer, Hiatt, H. H.; Watson, J. D.; Winsten, J. A., Eds. Cold Spring Harbor: New York, 1977; pp 605-627. (55)

Wottrich, R.; Diabaté, S.; Krug, H. F. Biological Effects of Ultrafine Model

Particles in Human Macrophages and Epithelial Cells in Mono- and co-Culture Int. J. Hyg. Environ. Health 2004, 207, 353–361. (56)

Halamoda-Kenzaoui, B.; Ceridono, M.; Colpo, P.; Valsesia, A.; Urbán, P.; Ojea-

Jiménez, I.; Gioria, S.; Gilliland, D.; Rossi, F.; Kinsner-Ovaskainen, A. Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake PLoS ONE 2015, 10, e0141593.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(57)

Concentrations of SiO2–Rubpy NPs were calculated using the mean particle

diameter by TEM, the density of colloidal silica (2.2 g/cm3) and the concentration of silica by ICP-MS. (58)

Chung, T.-H.; Wu, S.-H.; Yao, M.; Lu, C.-W.; Lin, Y.-S.; Hung, Y.; Mou, C.-

Y.; Chen, Y.-C.; Huang, D.-M. The Effect of Surface Charge on the Uptake and Biological Function of Mesoporous Silica Nanoparticles in 3T3-L1 Cells and Human Mesenchymal Stem Cells Biomaterials 2007, 28, 2959-2966. (59)

Ojea-Jiménez, I.; García-Fernández, L.; Lorenzo, J.; Puntes, V. F. Facile

Preparation of Cationic Gold Nanoparticle-Bioconjugates for Cell Penetration and Nuclear Targeting ACS Nano 2012, 6, 7692-7702. (60)

Taki, M.; Shiota, M.; Taira, K. Transglutaminase-mediated N- and C-terminal

Fluorescein Labeling of a Protein Can Support the Native Activity of the Modified Protein Protein Eng. Des. Sel. 2004, 17, 119-126.


Page 30 of 41

Page 31 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures and Tables

Figure 1. Schematic representation of the functionalization route employed, through epoxy-SiO2 NPs as intermediates, for the introduction of different functional groups onto the SiO2 NP surface and the interaction of positively charged amine-SiO2 NPs and negatively charged L-Ser-SiO2 NPs with A549 cells (drawn not at scale).



20.1 ± 3.9 nm

150

20 nm

100 50 0 0

20

40 60 80 Diameter (nm)

31.1 ± 2.5 nm

40

Number of NPs

Number of NPs

200

30 20 10 0 0

100

30 nm

20


100

200 nm

50 nm

56.8 ± 3.8 nm

60 40 20 0 0

20


100

40

Number of NPs

80 Number of NPs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 41

80 nm

86.3 ± 3.9 nm

30 20 10 0 0

20 40 60 80 Diameter (nm)

100

Figure 2. TEM morphological characterization and size distribution analysis of SiO2 NPs.


18.7 ± 3.6 nm

80 60

20 nm

40 20 0 0

30

20


47.4 ± 4.7 nm

50 nm

10

20


100

28.6 ± 3.7 nm

60

30 nm

40 20 0 0

100

20

0 0

80

20


100

50 Number of NPs

Number of NPs

100

Number of NPs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Number of NPs

Page 33 of 41

40

80 nm

79.6 ± 5.1 nm

30 20 10 0 0

20


100

100 nm

Figure 3. TEM morphological characterization and size distribution analysis of SiO2– Rubpy NPs.



C

1.0

53.9

82.1

SiO2 20nm SiO2 30nm SiO2 50nm SiO2 80nm

0.5

0.0 0

20

40

60 80 100 120 140 Diameter (nm)

0.6

Normalized weightDCS (a.u.)

B 18.2 32.3

D

Ru(Bpy)3 1.5 ug/mL SiO2-Rubpy 20 nm SiO2-Rubpy 30 nm SiO2-Rubpy 50 nm SiO2-Rubpy 80 nm

0.4

0.2

Intensity (a.u.)

Normalized weightDCS (a.u.)

A

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 41

1.0

18.1 30.4 45.0

71.2

SiO2-Rubpy 20nm SiO2-Rubpy 30nm SiO2-Rubpy 50nm SiO2-Rubpy 80nm

0.5

0.0 0

20

40

60 80 100 120 140 Diameter (nm)

300

Ru(Bpy)3 1.5 ug/mL SiO2-Rubpy 20 nm

603 nm

SiO2-Rubpy 30 nm SiO2-Rubpy 50 nm

200

SiO2-Rubpy 80 nm

609 nm

100

453 nm

0.0 300

400 500 Wavelength (nm)

600

0 500

600 700 Wavelength (nm)

800

Figure 4. Peak maximum provided by DCS analysis from particle size distribution in weight of SiO2 NPs (A) and SiO2–Rubpy NPs (B). Absorption (C) and fluorescence (D) spectra of SiO2–Rubpy NPs compared with a solution of inorganic dye Ru(bpy)3 at the same concentration.


Page 35 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


e

Size distribution Sample

SiO2 NPs SiO2 NPs SiO2 NPs SiO2 NPs SiO2-Rubpy NPs SiO2-Rubpy NPs SiO2-Rubpy NPs SiO2-Rubpy NPs

Size (nm) 20 30 50 80 20 30 50 80

a

TEM Mean (nm) 20.1 31.1 56.8 86.3 18.7 28.6 47.4 79.6

RSD (%) 19.4 8.0 6.7 4.5 19.3 12.9 9.9 6.4

DLS

Z-Ave 25.2 39.8 65.2 99.9 26.9 37.7 54.8 89.0

PdI 0.110 0.016 0.009 0.006 0.134 0.042 0.009 0.002

c

b

CLS

Mean (nm) 17.0 32.9 53.9 82.1 18.8 30.4 41.3 71.6

HW 4.8 6.2 6.4 6.7 8.4 12.2 13 12.1

PdI 1.157 1.163 1.169 1.026 1.104 1.116 1.099 1.048

AF 4

Mean (nm) 28.0 46.4 69.8 100.6 25.0 37.5 55.6 90.9

RSD (%) 0.8 0.4 0.2 1.0 1.5 n.a. n.a. 2.3

Apparent ρ 3 SiO2 (g/cm )

Mean (mV)

SD (mV)

1.58 1.84 1.84 1.84 1.62 1.80 1.71 1.80

-23.0 -28.9 -32.7 -32.4 -34.9 -34.0 -32.4 -33.5

45.9 30.1 15.7 12.0 11.3 12.6 16.7 20.7

Table 1. Particle size distributions determined by TEM, DLS, DCS and AF4, and surface charge measured by Z-potential of SiO2 NPs and SiO2–Rubpy NPs. aZ-average and polydispersity index (PdI) obtained by DLS cumulants analysis. bMean particle diameter measured by DCS assuming a value of particle density of colloidal silica (2.2 g/cm3); PdI defined as D(w)/D(n), where D(w) is the mean particle size expressed in a weight basis while D(n) is the mean particle size expressed in a number basis. cMean size distributions determined from the time of elution according to an external size calibration (plotting hydrodynamic diameter by DLS versus time of elution) using mixtures of SiO2 NPs size standards.28 dCalculated density from the combination of the sedimentation time given by DCS and correlated with the hydrodynamic diameter found by DLS using the Stokes’ law formula.26 eZ-potential measured at pH 8.5. n.a.: not applicable.


Z-potential

d


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Sample SiO2 NPs SiO2 NPs SiO2 NPs SiO2 NPs SiO2-Rubpy NPs SiO2-Rubpy NPs SiO2-Rubpy NPs SiO2-Rubpy NPs

Size (nm) 20 30 50 80 20 30 50 80

Measured by ICP-MS b Digested NPs Undigested NPs SiO2 (mg/mL) Ru(bpy) 3 SiO2 (mg/mL) Mean SD (µg/mL) 2.1 0.2 n.a. 2.4 2.6 0.1 n.a. 2.4 2.3 0.1 n.a. 2.1 2.5 0.1 n.a. 2.2 2.6 0.1 102.6 2.5 2.6 0.1 26.6 2.8 2.3 0.1 5.0 2.0 2.5 0.1 1.2 2.3 a

Page 36 of 41

Estimated c

d

Ratio (%)

SiO2 (mg/mL)

115 92 92 89 96 108 88 92

1.6 1.9 1.3 1.7 2.1 2.5 1.7 2.2

e

Ru(bpy) 3 (µg/mL) n.a. n.a. n.a. n.a. 150.0 33.0 7.1 1.5

Table 2. Elemental analysis performed by ICP-MS of SiO2 NPs and SiO2–Rubpy NPs. a

Direct analysis of Si-content (by triplicate) and Ru-content of undigested particles. bSi-

content analysis of particles digested in hydrofluoric acid. cRatio between digested and undigested methods. dFrom gravimetric determination. eFrom the added amount of Ru(bpy)3 dye in the synthesis of SiO2–Rubpy NPs 20 nm. n.a.: not applicable.


Page 37 of 41

A

B

30

30

SiO2 20 nm SiO2 30 nm

1.8 4.4

SiO2 50 nm SiO2 80 nm

20

1.7

epoxy-SiO2 20 nm epoxy-SiO2 30 nm epoxy-SiO2 50 nm epoxy-SiO2 80 nm

10

0 10

100

20

SiO2-Rubpy 20 nm SiO2-Rubpy 30 nm SiO2-Rubpy 50 nm SiO2-Rubpy 80 nm

2.1

epoxy-SiO2-Rubpy 20 nm epoxy-SiO2-Rubpy 30 nm epoxy-SiO2-Rubpy 50 nm epoxy-SiO2-Rubpy 80 nm

10

0 10

1000

Diameter (nm)

C

IntensityDLS (%)

3.8 5.6

100

1000

Diameter (nm)

D Pristine SiO2

40

Transmittance (a.u.)

IntensityDLS (%)

3.1 1.8

Z-Potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Epoxy-SiO2 Amine-SiO2

20

L-Ser-SiO2 L-Glu-SiO2

0 -20

Pristine SiO2

Epoxy-SiO2 OC-H

Amine-SiO2 OC-H SiO-H

-40

L-Ser-SiO2

N-H2

HO-H Si-O-Si

2

4

6

8

10

12

pH

4000

3000 2000 Wavenumber (cm-1)

1000

Figure 5. Particle size distributions as determined by intensity-mean DLS measurements of SiO2 NPs (A) and SiO2–Rubpy NPs (B) before and after epoxyfunctionalization with GPTMS. Increases in mean particle sizes are indicated. Evolution of Z-potential as a function of pH (C) and FT-IR spectra (D) of the different surface functionalized SiO2 NPs of 20 nm in diameter.



B

120

20 nm

80 60 40 Pristine SiO2 Amine-SiO2 L-Ser-SiO2

20 0

0

C 120

50 100 150 200 250 Concentration of SiO2 (µg/mL)


20 0

0


30 nm


20 0

0

D


120

50 nm

100

120 100

Cell viability (%)

Cell viability (%)

100

80 nm

100 Cell viability (%)

A

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 41


20 0

0


Figure 6. Cell viability MTT-test as mitochondrial activity of alveolar A549 cells incubated in cell culture medium without serum for 24 h with pristine SiO2 NPs, positively charged amine-SiO2 NPs or negatively charged L-Ser-SiO2 NPs of nominal sizes: 20 nm (A), 30 nm (B), 50 nm (C) and 80 nm (D). For conditions giving significant toxicity, further experiments were performed with lower concentrations until toxicity was considerably decreased.


Page 39 of 41

A Pristine SiO2-Rubpy Amine-SiO2-Rubpy L-Ser-SiO2-Rubpy

10 8 6 4 2 0

20 nm

30 nm

50 nm

80 nm

Mean Intensity (a.u.)

B

Mean Intensity 12 (x10 /conc. NPs0) (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8 7 6 5 4 3 2 1 0

Pristine SiO2-Rubpy Amine-SiO2-Rubpy L-Ser-SiO2-Rubpy

20 nm

30 nm

50 nm

80 nm

Figure 7. Flow cytometry measurements of alveolar A549 cells incubated in cell culture medium without serum for 3 h with pristine SiO2–Rubpy NP, positively charged amineSiO2–Rubpy NPs or negatively charged L-Ser-SiO2–Rubpy NPs of different nominal sizes. (A) Mean fluorescence intensity normalized by the initial particle number concentration in experiments performed at an equal mass concentration of silica (200 µg/mL). (B) Mean fluorescence intensity in experiments performed at an equal particle concentration (3x1011 NP/mL).



SiO2 NPs + SiO2 NPs

- SiO2 NPs

50 nm

30 nm

20 nm

pristine

80 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 41

Figure 8. TEM images of alveolar A549 cells incubated in cell culture medium without serum for 3 h with pristine SiO2 NPs, positively charged amine-SiO2 NPs or negatively charged L-Ser-SiO2 NPs of different nominal sizes. Representative images selected from duplicate experiments.


Page 41 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC

ethylenediamine

GPTMS

L-Serine


Highly Flexible Platform for Tuning Surface Properties of Silica

Recommend Documents