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Stabilization of TiO2 nanoparticles in complex medium through a pH adjustment protocol Olivier Spalla, and Camille Guiot Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es3040736 • Publication Date (Web): 14 Dec 2012 Downloaded from http://pubs.acs.org on December 20, 2012

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Environmental Science & Technology

Stabilization of TiO2 nanoparticles in complex medium through a pH adjustment protocol Camille Guiot, Olivier Spalla*. CEA Saclay, DSM/IRAMIS/SIS2M/LIONS UMR3299, 91191 Gif-sur Yvette, France. [email protected] ABSTRACT

Preparing TiO2 nanoparticle (NP) suspensions displaying well defined and reproducible dispersion state is a key feature to perform relevant toxicity experiments for environmental, animal or human concerns. Relying on the evolution of surface charge with pH, and interactions between nanoparticles in their medium, we developed an optimized dispersion protocol involving a pH adjustment before addition of bovine serum albumin (BSA). It yielded highly dispersed and stable concentrated stock suspensions of TiO2 NP at pH 7. It was designed for 4 kinds of manufactured TiO2 nanomaterials and can be extended to a wide range of TiO2 NP. The suspensions studied here were characterized by small-angle X-ray scattering (SAXS), using a model quantitatively describing fractal aggregates. Results were correlated with dynamic light scattering (DLS) measurements. Moreover, the stability in a typical biological medium was assessed by diluting stock suspensions in Luria Bertani (LB) medium. It resulted in highly dispersed and stable working suspensions. No sedimentation, followed by in situ DLS, was observed over 17 h for both the concentrated stock suspensions prepared according to the pH adjusted –BSA protocol and their dilution into LB medium.

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KEYWORDS Dispersion protocol, bovine serum albumin, BSA, nanoparticles, toxicity, TiO2, titanium dioxide, P25, small-angle X-ray scattering, SAXS, dynamic light scattering, DLS.


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Introduction

With the development of nanotechnology that occurred lately came a rapid increase in the amount and variety of engineered nanoparticles produced. This induces a risk of release in the environment and human exposure. Therefore, assessing the potential toxicity of nanoparticles has become a great matter of concern for researchers and industrials.1-3 When studying environmental, animal or human toxicity of nanoparticles (NP), one is faced to the major issue of preparing finely dispersed and stable suspensions. Indeed, when suspended in complex media used for toxicity studies, such as buffers, cell or bacteria culture media and biological fluids, NP are strongly aggregated4-6 and then sediment rapidly after preparation. The difficulty to control the aggregation state leads to a lack of reproducibility and many discrepancies in the results. Achieving stable suspensions in terms of aggregation state is tremendous to ensure a specific dose added to biological media, in mass, volume and specific surface area. A complete and accurate characterization of NP used must be performed.7,8 Among those characterizations, the dispersion state; i.e. finite size of aggregates, and any stabilizing properties (surface charge, adsorbed species, etc) are of prime interest. In that context our objective was to optimize a dispersion protocol based on physico-chemical and interaction considerations and applicable to a wide range of manufactured TiO2 NP. The suspensions were designed to serve as stock material for further dilution into a relevant medium yielding an accurate dose in toxicological tests. For that order, effective characterization of dispersion state was essential. Indeed, the classical dynamic light scattering does not yield a complete description of multiscale systems such as aggregates of finite size. In different protocols found in literature the Bovine Serum Albumin (BSA) is often used as the stabilizer because it is the most abundant protein in serum and it is present at high concentration in


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many media used for toxicity studies.4,6,9-11 It is usually added in pure water or in complement of a buffer whatever the surface charge of the particles. TiO2 nanoparticles are almost uncharged in water since their isoelectric point is around 6. At this pH, BSA adsorbs onto the surface but can generate a strong aggregation12-15 if one works at a too low concentration of BSA. To avoid this adverse effect, in the present protocol, the stabilization of TiO2 NP was achieved in 3 steps, involving first ultrasonication in acidic media to obtain the best dispersed state achievable, then addition of bovine serum albumin (BSA) to prevent the NP from aggregating strongly when adjusting the pH to physiological values in the third step. The design of the protocol is detailed first. The characterization by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) of the dispersion state of such suspensions are presented thereafter. In particular, the fitting of SAXS results by a unified aggregate scattering model helps describing finely the aggregates in terms of radius of gyration of primary particles, radius of gyration and fractal dimension of aggregates. The stability over time of as-prepared suspensions is also assessed by DLS through kinetics studies of sedimentation. To demonstrate the applicability in toxicity studies, we finally quantify the stability of such suspensions diluted into the phosphate-rich cell culture medium LB (Luria Bertani).

Materials and Methods Materials The four TiO2 nanomaterials used were provided by the Joint Research Center (Ispra, Italy) in the framework of NANOGENOTOX joint action, and are named according to their generic codes also used within OECD projects, i.e. NM102 (anatase, primary particle size 21 nm, specific surface area measured by SAXS on powder16 65 m²/g), NM103 (hydrophobic, rutile, 26 nm, 51 m²/g), NM104 (hydrophylic, rutile, 26 nm, 52 m²/g) and NM105 (approx. 86% anatase - 14% rutile, 21 nm, 47 m²/g). NM103 and NM104 are suspected to present Al2O3 surface coating. NM103 is also covered with a layer of


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polymethylsiloxane (dimethicone) to render it hydrophobic meanwhile NM104 is covered with a layer of glycerine to render it hydrophilic. According to OECD guidelines for testing manufactured nanomaterials,17 extensive physico-chemical characterization (size, specific surface area, morphology, crystallography, impurities, etc) is being performed within the framework of NANOGENOTOX project and will be released in forthcoming scientific papers and reports to be accessible from the NANOGENOTOX website. Data are gathered in table SI1 in the supporting information. Bovine serum albumin was purchased from Sigma-aldrich (A9418, Cohn fraction V) and used as received. LB medium was purchased from Sigma-Aldrich and prepared following the manufacturer instructions.

Physico-chemical characterizations Small-angle (SAXS) and ultra small-angle (USAXS) X-ray scattering measurements were performed with a Rigaku generator RUH3000 with copper rotating anode (λ= 1.54 Å) with respectively a 2D image plate detector MAR300 and a 1D high count rate CyberStar X200. SAXS and USAXS raw data are gathered on a common scale in the following. Dispersion state and surface charge of TiO2 suspensions were determined by dynamic light scattering (DLS) and zeta potential measurements performed on NanoSizer ZS (Malvern Instrument). For study of sedimentation of suspensions over time, 500 µL of sample were poured into DLS disposable low volume cell (optical path 1 mm) and left at rest in the apparatus during successive measurements performed at fixed attenuator and laser position. All characterization techniques, equipments and procedures are further described in supporting information.

pH adjusted-BSA optimized dispersion protocol

For studies implying nanoparticles in biological media, suspensions of nanoparticles are usually


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processed in pH-neutral and significant ionic strength media. However, preparing such suspensions with sufficient stability is not straightforward. The optimized dispersion protocol proposed here relies on a reflection over physico-chemical properties of NP in suspension and interactions responsible for either aggregation or stability. It has been developed to cover a wide range of TiO2 nanoparticles in terms of surface properties, specific surface area and aggregate size. Regarding TiO2, the evolution of zeta potential with pH for a constant ionic strength was studied for each NM and results are displayed in figure 1. Experimental method is detailed in supporting information. Isoelectric points (IEP) thereby obtained (table insert in figure 1) for NM102 and NM105, namely 6 and 6.6, are consistent with literature data for TiO2 nanoparticles.18,19 However, NM103 and NM104 exhibit a much higher IEP of 8.2, which is explained by the presence of an alumina coating at the surface of the nanoparticles. Indeed, typical IEP for alumina materials is found around 8.5-9.19,20 This preliminary study demonstrates that for a wide range of TiO2 nanoparticles, suspensions are not stable in the pH range 5-9, where the weak surface charge results in aggregation and subsequent sedimentation of nanoparticles. On the other hand, suspensions below pH 5 display zeta potentials above 30 mV, accounting for a high surface charge and therefore highly stable and finely dispersed suspensions. For typical suspensions at pH 2, additional results of DLS size measurements (figure SI1) and in situ sedimentation measurements (figure SI2) are reported in supporting information. On the other hand, BSA possesses an IEP around 512,21,22 and is thus negatively charged at the target pH (7-8). Therefore, as seen in figure 1, adding BSA to TiO2 nanoparticles at neutral pH would lead to fast aggregation of big flocs because of strong interactions between near-neutral or positively charged NP and negatively charged BSA. The basic idea of the protocol is then to adsorb BSA onto NP in a pH range where the two objects (BSA, and any type of TiO2 NP) are of identical sign, and form a stable and well dispersed suspension, i.e. at acidic pH. Based on those considerations, the optimized protocol is designed in 3 steps (typical zeta-pH values are reported on figure 1 for each step and numbered 1, 2 and 3). First, stable suspensions in their best dispersed state are achieved by ultrasonication in pH 2 medium.


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Titration of the Ti in solution after the US treatment yields a very low concentration of this metal under its ionic form in solution (

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