nc an Bra ocr nd rio gh- ions were characterized by XRD method, by measurement of specific surface area in nanosized region. In biological tests the sensitivity of human cancer cell lines s h icondu re stab aversa tive ag d in di 2,3]. An te 180 planetary mill Pulverisette 6 (Fritsch, Germany). The following milling Materials Letters 63 (2009) 1542–1544 Contents lists available at ScienceDirect Materials j ourna l homepage: www.e ls As2O3 which in comparison with As4S4 is fairly good soluble in water is applied in treatment of acute formsof leukemia [4,5]. However, its toxicity is high and a drug form possesses severe side effects. Realgar As4S4 was used very frequently in Chinese traditional medicine for treatment of various diseases. However, this sulphide is not soluble in water and a pretreatment is needed to enhance its solubility and/or to prepare particles in nanometer size with the better curing effect. Recently, the effect of cryo-grinding on production of realgar nanoparticles (176– 287nm)with the effects onviability of humanovarian and cervical cancer cell lines was investigated [6]. In this connection mechanochemistry can serve as a suitable tool conditions were used: loading of the mill with 10 mm balls, material of the milling vial and balls: tungsten carbide, ball charge 360 g, weight charge 3 g and rotational speed of the mill 200 rpm. Milling was performed in a dry mode (argon atmosphere) as well as in a wet mode (water+SDS) for milling time 15 min. The X-ray diffraction measurements were carried out using a diffractometer X Perth MPD (Philips, Netherlands) working in the 2 Θ geometry with CuKα radiation. The specific surface area was determined by the low temperature nitrogen adsorption method. Gemini 2360 sorption apparatus (Micro- because it offers a possibility to prepare n enhanced solubility by simple solid state app ⁎ Corresponding author. E-mail address:
[email protected] (P. Baláž). 0167-577X/$ – see front matter © 2009 Elsevier B.V. A doi:10.1016/j.matlet.2009.04.008 0. In 1878, a report from ler's solution (1% solution blood cell counts. Today Sigma-Aldrich, USA). Sodium dodecylsulphate SDS (Junsei, Japan) has been applied in milling tests as surfactant. The high-energy milling of realgar samples was performed in a Boston City hospital described the effect of Fow of As2O3 in K2CO3) on the reduction of white 1. Introduction Arsenic sulphide As4S4 is a red sem crystalline forms. The room temperatu identical tomineral realgar [1]. Realgar is an advanced material as well as cura optical properties have been describe applications inoptoelectronicmaterials [ compounds was first reported in the la ctor that exists in several le α-As4S4 is structurally tile substance classified as ent. Recently, interesting sordered As4S4 with the ticanceractivityof arsenic realgar prepared by high-energy milling under ambient temperature and to test its anticancer effect on selected cancer cell lines. 2. Experimental The investigation was carried out with realgar As4S4 (98% in purity, It is the aim of this paper to examine the properties of nanosized Anticancer effect Mechanochemical preparation and antica As4S4 nanoparticles Peter Baláž a,⁎, Martin Fabián a, Michal Pastorek b,c, D a Institute of Geotechnics, Slovak Academy of Sciences, 043 53 Košice, Slovakia b Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia c Institute of Molecular Biology, Faculty of Natural Sciences, Commenius University, 842 15 a b s t r a c ta r t i c l e i n f o Article history: Received 17 November 2008 Accepted 6 April 2009 Available online 8 April 2009 Keywords: Realgar Mechanochemistry Nanomaterial In the past two decades nan their intriguing properties a as therapeutic effects for va have been prepared by a hi various experimental condit and particle size distribution for the realgar nanoparticle anosized particles with roach [7–10]. ll rights reserved. as been clearly demonstrated. © 2009 Elsevier B.V. All rights reserved. er effect of realgar ka Cholujová b, Ján Sedlák b tislava, Slovakia ystalline sulphide semiconductors have attracted considerable interest due to structure diversities. Arsenic show interesting solid state phenomena as well us diseases. In this study, the nanosized realgar As4S4 particles (d–144 nm) energy milling. The solid state properties of the nanoparticles milled under Letters ev ie r.com/ locate /mat le t meritics, USA) has been applied. Particle size distribution was measured on NANOPHOX (Sympatec GmbH, Germany) particle size analyser based on photon cross correlation spectroscopy (PCCS). Anticancer effect of nanosized realgar particles has been tested by standard methods applied in cancer research [11–12]. For biological testing several human cancer cell lines have been selected: HL 60, K 562, ARH 77 and U 266which represent various forms of leukemia and myeloma cancer. 3. Results and discussion 3.1. Solid state properties The disintegration of realgar As4S4 particles by high-energymilling is accompanied by an increase of their number and by generation of fresh, previously unexposed surface. The dependence of specific adsorption surface and percentage of particles less than 10 μm on the time of mechanical activation is represented in Fig. 1. The traces for drymilling show that the rate of newsurface area and small particle formation are similar and limited by time of mechanical activation. After the stage (I) of mechanical activation where the increase of surface area is almost proportional to the milling time, the stage (II) is manifested. Besides the effect of disintegration resulting in a greater proportion of the fine particles in stage I, we can also observe the effect of surface area stagnation in stage II which is in close relation with the particles agglomeration. This is a common phenomenon for many solids activated in high-energymills [9]. Bymilling in a drymode for 15 min the value 1.1 m2g−1 of specific surface area was obtained. The authors would like to thank Mrs. Repčáková for milling and chemical analysis and Dr. Ďurišin for performing XRD analysis. The Fig. 2. Particle size distribution of realgar As4S4: A—non-treated, B—milled with SDS for Fig. 3. XRD patterns of realgar As4S4: A—non-treated sample, B—milled with SDS for 1543P. Baláž et al. / Materials Letters 63 (2009) 1542–1544 When the same milling was performed for 15 min in a wet mode (water+SDS) more than the 3-fold increase in the specific surface area value (SA=3.8 m2g−1) was obtained. This sample after filtration through 0.23 μm filter (marked as RK 10) has been applied for further biological testing. The particle size distribution of realgar is given in Fig. 2. Non- treated As4S4 (A) shows broad polymodal distribution of particles in the range 70–4000 nm. After milling (B), the distribution is changed to three-modal form where the main particle population has particle size less than 250 nm. However, the existence of larger particles with the size over 700 nm is here also manifested. After filtration a mono- modal distribution of realgar nanoparticles with the average hydro- dynamic parameter 144 nm was obtained (C). XRD patterns of realgar particles have been shown in Fig. 3. The patterns show many overlapping peaks with a tendency of the higher background, and a small amorphous fraction was probably present in the sample as a consequence of high-energy milling. All peaks belong to α-As4S4 as identified with JCPD card file No. 09-0441. No characteristic peaks of impurities are detected in the XRD patterns. Furthermore, the sharp diffraction peaks indicate the well crystal- lization of the as-prepared As4S4 nanocrystals. There is a mark which labels the peak in both patterns. This peak is a little bit enhanced in milled sample when comparing both traces. We speculate that some phase transition in realgar can start here because a possible occurrence of several As4S4 polymorphs have been reported to date [13–16] and milling creates favourite conditions for such transitions [9,10]. Fig. 1. Specific surface area, SA and percentage of realgar As4S4 particles less than 10 μm, Q10 vs. milling time, tM. Dry milling (without surfactant). 3.2. Anticancer effect In this study realgar nanoparticles were produced by milling with SDS for 15 min. SDS (sodium dodecyl sulphate) as a biocompatible surfactant has been applied. Cytotoxicity tests were performed with the selected human cancer cell lines (see Experimental). The results have been shown in Fig. 4. This Figure shows the concentration dependent decrease of cancer cells survival viability where the most sensitive cells for nanosized realgar effect are HL 60 and K 562 (respectively) followed by the more resistant cell lines ARH 77 and U 266. On Fig. 4 the calculated values of IC50 parameter are given. IC50 represents the concentration of arsenic that is required for 50% inhibition in comparison to control sample. 4. Conclusions Changes in surface area, nanosize distribution and solid state properties of realgar As4S4 particles were detected as a consequence of high-energy milling. The treatment of several cancer cell lines with the mechanochemically activated realgar was manifested in the decrease of their viability. This finding broadens the possibilities for application of the realgar nanocrystalline particles. Acknowledgements 15 min, C—milled with SDS for 15 min and filtrated through filter—0.23 μm. 15 min. support through the Agency for Science and Development (Project APVV 0347-06), the Slovak Grant Agency (Project VEGA 2/0035/08) and Center of Excellence of Slovak Academy of Sciences (NANOS- MART) is gratefully acknowledged. References [1] Mullen DJE, Nowacki V. Zetschrift für Kristallographie 1972;136:48. [2] Popescu M. Journal of Optoelectronic Advanced Materials 2005;7:2189. [3] Popescu M. Journal of Non-crystalline Solids 2006;352:887. [4] Antman KM. The Oncologist 2001;6(Suppl 2):1. [5] Wang ZY. Cancer Chemotherapy and Pharmacology 2001;48(Suppl 1):72. [6] Wu JZ, Ho PC. European Journal of Pharmaceutical Sciences 2006;29:35. [7] Baláž P. Extractive Metallurgy of Activated Minerals. Amsterdam: Elsevier; 2000. [8] Baláž P. Journal of Materials Science 2004;39:5097. [9] Baláž P, Choi WS, Dutková E. Journal of Physics and Chemistry of Solids 2007;68:1178. [10] Baláž P. Mechanochemistry in Nanoscience and Minerals Engineering. Berlin Heidelberg: Springer; 2008. [11] Mosmann T. Journal of Immunological Methods 1983;65:55. [12] Jakubíková J, Bao Y, Bodo J, Sedlák J. Neoplasma 2006;53:463. [13] Douglas DL, Shing Ch, Wang GE. American Mineralogist 1992;77:1266. [14] Bonazzi P, Menchetti S, Pratesi G. American Mineralogist 1995;80:400. [15] Bonazzi P, Menchetti S, Pratesi G, Muniz–Miranda M, Sbrana G. American Mineralogist 1996;81:874. [16] Bonazzi P, Bindi G. Zeitschrift für Kristallographie 2008;223:132. Fig. 4. Relative viability of the human cancer cell lines vs. arsenic concentration in nanosized realgar (sample RK 10) (■) HL 60, (□) K 562, (●) ARH 77, (○) U 266. 1544 P. Baláž et al. / Materials Letters 63 (2009) 1542–1544 Mechanochemical preparation and anticancer effect of realgar �As4S4 nanoparticles Introduction Experimental Results and discussion Solid state properties Anticancer effect Conclusions Acknowledgements References