Polymorphs of arsenic sulfide and their promising anti-cancer effects

 

A. Zorkovská1, Z. Bujňáková1, P. Baláž1, J. Sedlák2

 

1Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, Košice, Slovakia

2Cancer Research Institute, Slovak Academy of Sciences, 833 91 Bratislava, Slovakia

zorkovska@saske.sk

 

Keywords: realgar, pararealgar, milling, nanoparticles, anti-cancer effect

 

Abstract

Nanosuspensions of arsenic sulfide (As4S4) polymorphs (realgar and pararealgar) were prepared by circulation mill, with average particle size below 150 nm. The nanosuspensions were stable up to six weeks. Their anti-cancer effects were tested and compared on human lung cancer H460 cell line. Induction of DNA damage and increase of apoptotic cells was observed. The arsenic dissolution from the nanosuspensions in simulated gastric and intestinal fluids reached 12–13.5%.

Introduction

Arsenic sulfides have been utilized for a long time in the manufacture of cosmetics, foods, glass, insecticides, pigments, and in medicine as well [1]. In Western medicine, approximately 60 different arsenic preparations have been developed and used in pharmacological history. In traditional Chinese medicines different forms of mineral arsenicals are used, and realgar alone is included in 22 oral remedies, recognized by the Chinese Pharmacopeia Committee (2005). In the recent years its potential anticancer effects have been studied [2,3]. Production of nanocrystals is an approach to increase the drug solubility and its bioavailability. Here, the arsenic sulfides were prepared as nanosuspensions in circulation mill.

The As4S4 has at least three distinct polymorphs: i) the α-As4S4 phase, with monoclinic crystal structure (space group P21/n), structurally identical to the mineral realgar, which is stable at room temperature, ii) the high temperature phase, β-As4S4, with base-centered monoclinic crystal structure (space group C2/c) stable above 260°C, which slowly reconverts to the α-phase upon cooling, iii) and the monoclinic pararealgar (space group P21/c) bright yellow product formed upon exposure to visible light of both red α and β-phases [4]. There are two As–As covalent bonds and four As–S–As covalent bridges in the As4S4 subunits of both realgar (α or β-phase) and pararealgar. Transformation from realgar to pararealgar needs rearrangement of the molecular subunits involving As–S and As–As bond breaking.

Experimental

The investigation was carried out with mineral realgar – sample A, collected from Allchar locality (R. Macedonia) and pararealgar. The pararealgar was prepared innovately by milling – sample B (planetary mill Pulverisette 6, Fritzsch, Germany, sample weight 5 g, revolutions of the milling shaft 400 min-1, milling time 60 min) or classically, by exposure of realgar to sunlight for 1 month – sample C. The preparation of the nanosuspensions was performed in a laboratory circulation mill MiniCer (Netzsch, Germany) in the presence of 300 mL of 0.5% polyvinylpyrrolidone (PVP) solution as nonionic stabilizer. PVP is one of the most used carriers for the preparation of nanosuspensions for medical application. The mill was loaded with yttrium stabilized ZrO2 milling balls. After milling, the resulting nanoparticle suspensions were filtrated through a 0.22 μm sterile filter.

X-ray diffraction measurements were carried out using a D8 Advance diffractometer (Bruker, Germany) equipped with a Q/Q goniometer, Cu Ka radiation (40 kV, 40 mA), secondary graphite monochromator, and scintillation detector. The diffraction data were collected over an angular range 10 < 2Q< 100° with steps 0.03° and a counting time 20 s/step. The commercial Diffracplus Eva software has been used for phase analysis according to the ICDD - PDF2 database.

The particle size distribution was measured by photon cross-correlation spectroscopy using a Nanophox particle sizer (Sympatec, Germany).

Dissolution tests were conducted in simulated gastric fluid (SGF) composed of 0.2% NaCl in 0.7% HCl (pH = 1.3) and in a simulated intestinal fluid (SIF) composed of 0.042% NaOH, 0.4% NaH2PO4.9H2O and 0.6% NaCl with pH 6.5 at 36.5°C over a period of 240 minutes. The cytotoxicity on human lung cancer H460 was determined by colony forming assay. H460 cells were seeded onto 6-well plates with a density of 60 cells per well and incubated overnight. The cells were then treated with samples at various concentrations (0.156, 0.625, 1, 4, 16 and 64 μg/ml). After incubation for 10 days the values of 50% inhibition concentration (IC50) were determined. Cell cycle progression was monitored using DNA flow cytometry.

Results

Characterization of the materials

Realgar – sample A.

High-purity mineral, realgar, crystallizing in monoclinic crystal structure, space group P21/n, JCPDS 01-076-9449 (Fig. 1a).

Pararealgar – sample B and C, prepared by two alternative pathways:

B - Milling of realgar in a planetary mill Pulverisette 6. The XRD pattern of the sample after milling is shown on Fig.1c, the pararealgar-b phase, crystallizing in the base centered monoclinic system, space group C2/c, JCPDS 01-075-8666 was confirmed (Fig.1b). Line broadening and relative intensity decrease indicate the decrease of crystallite size and amorphization.

C - Irradiation of realgar by sunlight during one month. Absorption of visible photons with energies in the range 1.85–2.48 eV leads to irreversible isomerization of realgar to pararealgar, whereby the positions of one arsenic atom and one sulfur atom in the As4S4 cluster become exchanged [5], the process is accompanied by visible color change of the mineral from red to yellow. The transformation, realized by structural rearrangement and bond-breakings, leads also to considerable amorphization, as it can be seen from the XRD pattern on Fig. 1c. The product is a structurally non-homogeneous, multiphase system, with the main component pararealgar (monoclinic P21/c phase, JCPDS 01-083-1013) and b - phase. Non-transformed realgar can be also detected.

The nanosuspensions were prepared from samples A and C. The estimated average particle size x50 was 137 nm (142 nm) for the realgar (pararealgar) nanosuspensions, respectively, and 99% of particles were confirmed to be smaller than 200 nm. Interestingly, the main pararealgar component, present in the sample C, can not be detected in the obtained nanosuspension, which is composed of the majority pararealgar-b phase and of some non-transformed realgar (Fig.2).

Dissolution in simulated gastric and intestinal fluids

Great rise in the solubility of arsenic was achieved by nanomilling, the amount of dissolved arsenic after 240 minutes of leaching in SGF + SIF increased from 2% to 12% (13.5%) for the nanomilled realgar (pararealgar), respectively. These results are very promising with respect to the published literature results. For comparison, only 0.6% of arsenic of the total realgar content was finally released into simulated gastric juice in [6], whereas some authors reported that 4% of arsenic from realgar were traced in gastric and intestinal fluids [7].

Anti-cancer effects

The nanomilled samples showed increased cytotoxicity. The values of 50% inhibition concentration (IC50) of milled samples to H460 cells were 0.033 (0.031) μg/mL for the nanomilled realgar (pararealgar). For comparison, this concentration for the used anti-cancer agent, cisplatin, is 0.01 μg/mL. In general, the results imply that the lung cancer cells are susceptible to the treatment with these samples.

Cell cycle progression and induction of apoptotic cells

The H460 cells were treated with various concentrations of arsenic sulfides for 24, 48 and 72 h. The cell cycle distribution was determined, monitoring the G1 (growth phase), S (DNA replication phase) and G2/M (growth phase immediately preceding cell mitosis) phases.

 

Figure 1. XRD patterns showing the phase composition of the source samples for preparation of nanosuspensions.

 

Figure 2. XRD pattern of the nanomilled sample C (light-irradiated realgar).

The treatment of H460 cells with nanomilled A and C samples for 24 and 48 h resulted in reduction of G1 phase, accumulation of G2/M phase, and appearance of SubG1 cells (indicating apoptotic cells). Similarly to cisplatin, significantly increased number of SubG1 cells was observed after 72 h, indicating the cell cycle interference which may trigger the apoptotic pathways.

 

Figure 3. Cell cycle perturbation and apoptotic cell death induced by nanosuspensions prepared from samples A and C, and cisplatin for comparison.

Summary

Nanosuspensions of realgar and light irradiated realgar (composed of pararealgar, b-phase and realgar) with average particle size below 150 nm were prepared in a circulation mill.  The nanosuspensions were stable for more then one month. They have shown increased cytotoxicity and DNA damage activity on H460 lung cancer cells, with accumulation of G2/M phase inducing apoptotic cells.

 

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Acknowledgements.

The Slovak Grant Agency VEGA (2/0064/14 and 2/0027/14), the Agency for Science and Development (project APVV-0189-10) and the European Regional Development Fund (nanoCEXmat I and II - ITMS 26220120019 and 26220120035) are gratefully acknowledged.