pubs.acs.org/Langmuir © 2009 American Chemical Society
Synthesis and Characterization of Stable Monodisperse Silica Nanoparticle Sols for in Vitro Cytotoxicity Testing )
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Leen C. J. Thomassen,† Alexander Aerts,† Virginie Rabolli,‡ Dominique Lison,‡ Laetitia Gonzalez,§ Micheline Kirsch-Volders,§ Dorota Napierska, Peter H. Hoet, Christine E. A. Kirschhock,† and Johan A. Martens*,†
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† Center for Surface Chemistry & Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, 3001 Heverlee, Belgium, ‡Industrial Toxicology and Occupational Medicine unit, Universit e catholique de Louvain, Avenue E. Mounier, 53.02, 1200 Brussels, Belgium, §Laboratory of Cell Genetics, Vrije Universiteit Brussel, Pleinlaan, 2, 1050 Brussels, Belgium, and Laboratory of Lung Toxicology, Katholieke Universiteit, Herestraat 49, 3000 Leuven, Belgium
Received June 8, 2009. Revised Manuscript Received July 22, 2009 For the investigation of the interaction of nanoparticles with biomolecules, cells, organs, and animal models there is a need for well-characterized nanoparticle suspensions. In this paper we report the preparation of monodisperse dense amorphous silica nanoparticles (SNP) suspended in physiological media that are sterile and sufficiently stable against aggregation. SNP sols with various particle sizes (2-335 nm) were prepared via base-catalyzed hydrolysis and polymerization of tetraethyl orthosilicate under sterile conditions using either ammonia (St€ober process1) or lysine catalyst (Lys-Sil process2). The series was complemented with commercial silica sols (Ludox). Silica nanoparticle suspensions were purified by dialysis and dispersed without using any dispersing agent into cell culture media (Dulbecco’s Modified Eagle’s medium) containing antibiotics. Particle sizes were determined by dynamic light scattering. SNP morphology, surface area, and porosity were characterized using electron microscopy and nitrogen adsorption. The SNP sols in cell culture medium were stable for several days. The catalytic activity of the SNP in the conversion of hydrogen peroxide into hydroxyl radicals was investigated using electron paramagnetic resonance. The catalytic activity per square meter of exposed silica surface area was found to be independent of particle size and preparation method. Using this unique series of nanoparticle suspensions, the relationship between cytotoxicity and particle size was investigated using human endothelial and mouse monocyte-macrophage cells. The cytotoxicity of the SNP was strongly dependent on particle size and cell type. This unique methodology and the collection of well-characterized SNP will be useful for further in vitro studies exploring the physicochemical determinants of nanoparticle toxicity.
Introduction One of the concerns about the introduction of nanotechnology is the potential adverse effect of nanoparticles on human health. Scientific insight into the toxicity of nanoparticles to be applied in nanomaterials and nanotechnological devices is still limited. The lung is the most probable portal of entry of nanoparticles and, therefore, has received most attention in toxicological investigations. Uptake via skin or digestive system is, however, possible as well.3 Evidence is accumulating that three main factors determine the toxicity of nanoparticles:4 (1) the chemical reactivity and intrinsic chemical toxicity of the material the nanoparticle is composed of, (2) the large specific surface area of nanoparticles making contact with cells, and (3) the particle morphology. Toxicological investigations of nanoparticles are hampered by a number of technical difficulties such as the difficulty to avoid aggregation of the nanoparticles. In aqueous media, nanoparticles tend to agglomerate, and this process is a variable and difficult to control phenomenon. The nanoparticle concentration, zeta potential, morphology, hydrophobicity, fluid characteristics, *Corresponding author: Fax (þ32) 16-321998; e-mail johan.martens@biw. kuleuven.be. (1) St€ober, W.; Fink, W.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62–69. (2) Davis, T. M.; Snyder, M. A.; Krohn, J. E.; Tsapatsis, M. Chem. Mater. 2006, 18, 5814–16. (3) Nel, A.; Xia, T.; Madler, L.; Li, N. Science 2006, 311, 622–27. (4) Donaldson, K.; Stone, V.; Tran, C. L.; Kreyling, W.; Borm, P. J. Occup. Environ. Med. 2004, 61, 727–28.
328 DOI: 10.1021/la902050k
and the method of dispersing, among other factors, influence the rate and degree of nanoparticle aggregation.5,6 The culture medium for in vitro studies, with its fixed pH and ionic strength, and addition of proteins or other surface-active molecules has an impact on nanoparticle aggregation behavior.7 Toxicity testing of nanoparticles often has been performed without precise control over the aggregation state of the tested nanomaterial. Agglomeration can, however, severely hinder determination of fundamental dose-response relationships.3 Nanotoxicologists strongly advice to characterize nanomaterials in their original form as well as in suspended state.8,9 Well-characterized materials are needed to standardize and validate in vitro experimental procedures and to provide reliable data on the relative toxicity of nanoparticles.10 In this respect, sols of amorphous silica nanoparticles (SNP) offer a unique opportunity to develop useful (5) Teeguarden, J. G.; Hinderliter, P. M.; Orr, G.; Thrall, B. D.; Pounds, J. G. Toxicol. Sci. 2007, 97, 614. (6) Allouni, Z. E.; Cimpan, M. R.; Hol, P. J.; Skodvin, T.; Gjerdet, N. R. Colloids Surf., B 2009, 68, 83–87. (7) Buford, M. C.; Hamilton, R. F., Jr.; Holian, A. Part. Fibre Toxicol. 2007, 4, 6. (8) Oberdorster, G.; Maynard, A.; Donaldson, K.; Castranova, V.; Fitzpatrick, J.; Ausman, K.; Carter, J.; Karn, B.; Kreyling, W.; Lai, D.; Olin, S.; Monteiro-Riviere, N.; Warheit, D.; Yang, H. Part. Fibre Toxicol. 2005, 2, 8. (9) Borm, P. J.; Robbins, D.; Haubold, S.; Kuhlbusch, T.; Fissan, H.; Donaldson, K.; Schins, R.; Stone, V.; Kreyling, W.; Lademann, J.; Krutmann, J.; Warheit, D.; Oberdorster, E. Part. Fibre Toxicol. 2006, 3, 11. (10) Sayes, C. M.; Reed, K. L.; Warheit, D. B. Toxicol. Sci. 2007, 97, 163–80.
Published on Web 08/21/2009
Langmuir 2010, 26(1), 328–335
Thomassen et al.
Article
model materials. They are conveniently synthesized over a broad size range (20-500 nm) using the St€ober method.1 More recently, a lysine-catalyzed synthesis of stable silica sol was reported by Davis et al.2,11 These Lys-Sil particles were used recently in a cellular environment. Particle stability against aggregation in cell culture medium and after dialyses was demonstrated by Snyder et al.12 Long time Ludox sols are commercially available representing highly concentrated silica sols with sizes in the 7-21 nm range.13 Recently, Brown et al.14 published toxicological data on St€ober silica particles measuring 100 and 200 nm. Those silica particles were dispersed in deionized water prior to dilution in cell culture medium. Agglomerates with substantially larger dimensions than the elementary St€ober silica nanospheres were formed in that medium. Those particles showed little toxicity to mesothelial cells. In a previous publication from our team, a monodisperse preparation of St€ober SNP measuring 29 ( 4 nm was assessed in cytotoxicity studies.15 Half maximal effective concentrations (EC50) of 37, 50, and 150 μg/mL were obtained in mouse monocyte-macrophage, human type II lung epithelium, and human endothelium cell lines, respectively, revealing the toxic potential of these dispersed amorphous nanoparticles. The sizedependent toxicity of a similar series of sols of amorphous St€ober and Ludox SNP toward a human endothelial cell line (EAHY926) was reported by Napierska et al.16 Here we report on the preparation, purification, and characterization of an extended series of stable monodisperse amorphous silica sols with a controlled variation of particle size in the range 2-335 nm. These SNP suspensions were found to be stable for several days in physiological media, which enables in vitro cytotoxicity studies without aggregation in the absence of any dispersing agent.
Acros. Ultrapure (Milli-Q) water was steam sterilized before use. The silica particles were synthesized according to the method of Davis et al.2 TEOS was added to lysine solution while stirring at 400 rpm. The Lys-Sil-2 synthesis took 5 days at room temperature. Lys-Sil-26, Lys-Sil-34, and Lys-Sil-36 were synthesized by dropwise addition of TEOS to lysine solution preheated at 60 °C under stirring. The synthesis took 3 days. All manipulations were performed under sterile conditions. The dialysis procedure was the same as explained for the Ludox samples. St€ ober silica was synthesized using ethanol (absolute) from VWR, ammonium hydroxide solution (25 wt %) from Fluka, and tetraethyl orthosilicate (TEOS, 98 wt %) from Acros. Ultrapure (Milli-Q) water was steam sterilized before use. TEOS was added dropwise to the solution under stirring at 400 rpm. The sol was stirred for 48 h at room temperature. Ethanol and ammonia were removed by dialysis using the procedure described for the Ludox samples. The effectiveness of the purification was verified using attenuated total reflection infrared spectroscopy (ATR-IR) (Bruker IFS 66v/S ATR-IR instrument; aperture 6 mm, 128 scans, resolution 4 cm-1). The samples were injected in a horizontal attenuated total reflectance flow cell from Pine Technologies. The background signal of ultrapure water was subtracted from all spectra. The silicon dioxide content of Ludox-Sil-14, Ludox-Sil-15, St€ ober-Sil-60, St€ ober-Sil-104, and St€ ober-Sil-335 SNP samples was quantified by atom absorption spectroscopy (AAS, Solaar 32 AA, TJA Solutions; acetylene-nitrogen oxide flame and silicon hollow-cathode lamp). The particles were dissolved in a 2 M sodium hydroxide solution and subsequently diluted with water to an appropriate concentration. The sodium hydroxide solution served as reference sample to assess the background intensity. The silicon dioxide content of Lys-Sil-2, Ludox-Sil-11, St€ ober-Sil-18, Lys-Sil-26, Lys-Sil 34, and Lys-Sil-36 was determined using inductive coupled plasma-atomic emission spectroscopy (ICPAES, Perkin-Elmer, 4300 dual view).
Materials and Methods
2. Physicochemical Characterization of Silica Nanopowober-Sil-104, ders. Ludox-Sil-14, Ludox-Sil-15, St€ober-Sil-60, St€
1. Preparation of Silica Sols. The code name for the silica sols was Lys-Sil-x, St€ ober-Sil-x, or Ludox-Sil-x with x representing the mean particle size according to transmission or scanning electron microscopy. All syntheses were performed under sterile conditions in a laminar flow hood. The composition of the synthesis mixtures is given in Table 1. Ludox-Sil-11, Ludox-Sil-14, and Ludox-Sil-15 were commercial Ludox SM-30, LS-30, and HS-40, respectively, obtained from Sigma. These commercial sols are electrostatically stabilized by sodium hydroxide (pH 9-10). The Ludox samples were diluted with steam sterilized (15 min at 121 °C) ultrapure water (Milli-Q) to a 10 wt % SiO2 concentration. 200 mL of the particle suspension was dialyzed against 5 L of distilled water to remove sodium from the suspension. Steam sterilized Nadir dialysis membranes were used (diameter 38 mm, pore diameter 2.5-3.0 nm from Carl Roth). The dialysate was refreshed four times with a minimum time interval of 2 h. The total dialysis time was 24 h. During dialyses, the dialysate was agitated with a magnetic stir bar. Lys-Sil sols were synthesized using L-(þ)-lysine monohydrate, 99% (Aldrich), and tetraethyl orthosilicate (TEOS, 98 wt %) from (11) Snyder, M. A.; Lee, J. A.; Davis, T. M.; Scriven, L. E.; Tsapatsis, M. Langmuir 2007, 23, 9924–28. (12) Snyder, M. A.; Demirgoz, D.; Kokkoli, E.; Tsapatsis, M. Microporous Mesoporous Mater. 2009, 118, 387–95. (13) Iler, R. K. The Chemistry of Silica: Solubility, Polymerization, Colloid and Surface properties and Biochemistry; Wiley: New York, 1979. (14) Brown, S. C.; Kamal, M.; Nasreen, N.; Baumuratov, A.; Sharma, P.; Antony, V. B.; Moudgil, B. M. Adv. Powder Technol. 2007, 18, 69–79. (15) 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. Toxicol. Sci. 2008, 104, 155–62. (16) Napierska, D.; Thomassen, L. C. J.; Rabolli, V.; Lison, D.; Gonzalez, L.; Kirsch-Volders, M.; Martens, J. A.; Hoet, P. H. Small 2009, 5, 846–53.
Langmuir 2010, 26(1), 328–335
and St€ ober-Sil-335 sols were dried at room temperature to obtain a powder. Ludox-Sil-11, St€ ober-Sil-18, Lys-Sil-2, Lys-Sil-26, LysSil 34, and Lys-Sil-36 were lyophilized (pressure
