Materials Letters 104 (2013) 84–86

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Materials Letters

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Properties and bioaccessibility of arsenic sulphide nanosuspensions

Peter Baláž a, Zdenka Bujňáková a,n, Olga Kartachova b,c, Martin Fabián a, Bernhardt Stalder b

a Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, 04353 Košice, Slovakiab Bühler AG, CH-9240, Uzwil, Switzerlandc Deakin University, Locked Bag 20000, Geelong, Victoria 3220, Australia

a r t i c l e i n f o

Article history:Received 19 October 2012Accepted 2 April 2013Available online 10 April 2013

Keywords:Arsenic sulphideNanomillingDissolutionNanoparticle

7X/$ - see front matter & 2013 Published by Ex.doi.org/10.1016/j.matlet.2013.04.003

esponding author. Tel.: +42 1557922607; fax:ail address: bujnakova@saske.sk (Z. Bujňáková

a b s t r a c t

Dissolution tests of the nanosized realgar particles prepared by the novel wet milling technique aredescribed in this paper. For nanomilling of arsenic sulphide As4S4, laboratory Perl Mill PML2 (Bühler) wasused. The specific surface areas increased from 0.2 to 34.2 m2/g during milling. A nanosized, monomodaldistribution with a mean particle size of 146–198 nm was obtained. The ξ-potential values from −52 to−38 mV were determined. Dissolution experiments in simulated gastric and intestinal fluids demon-strate that the soluble arsenic from As4S4 available for absorption into the bloodstream is greaterthan 10%.

& 2013 Published by Elsevier B.V.

1. Introduction

Arsenic compounds had a long Janus-type interaction withhumanity. On one hand, they have been extensively utilised, buton the other hand, their poisonous properties have causedmisery and many deaths. Arsenic substances have also beenshown to have a therapeutic effect [1–3], and several reviewshave been published on this topic [4,5]. Realgar (Xiong Huang)is listed in the modern Chinese Pharmacopoeia, where itscurative application can be traced for a very long time. Realgar,chemically arsenic sulphide with As4S4 formula, has beenstudied as a drug for cancer treatment [6–12]. However, themain drawback to using this compound is its solubility [13] and,consequently, its bioavailability. It was reported that 70% of thepotential drug candidates were discarded due to low bioavail-ability related with poor solubility in water before they everreached the pharmaceutical department [14]. Because of thisproperty, some sort of pre-treatment is needed to modify thepotential drug.

In this paper, we report on the use of a novel mechanochemicalmethod for the preparation of arsenic sulphide nanosuspensionsby wet nanomilling. The changes in the solid state properties andthe enhancement of dissolution in fluids that simulate the humangastrointestinal tract are presented.

lsevier B.V.

+42 1557922604.).

2. Experimental

The investigation was performed using arsenic sulphide (98%As4S4 in purity, Sigma-Aldrich, USA). An XRD analysis confirmedthe presence of arsenic sulphide As4S4 (JCPDS 48-1247). A smallamount of zinc was estimated for the sample using EDX and XPSmethods [10]. The sample was purified by the washing proceduredescribed in [10].

The milling process was performed in laboratory Perl Mill PML2(Bühler, Switzerland) in the presence of 3 L of 0.075% sodiumdodecyl sulphate solution as an ionic stabiliser. The mill wasloaded to 80% with yttrium stabilised ZrO2 milling balls withdiameters of 1.5 mm. Two modes of milling were applied: (1)n¼1500–2500 min−1, tM¼10–200 min and m¼5 g (experimentno. 1) and (2) n¼2500 min−1, tM¼10–120 min and m¼15 g(experiment no. 2), where n refers to the revolutions of the millingshaft, tM is milling time and m is weight of the sample. Followingmilling, the resulting As4S4 nanoparticle suspensions were fil-trated through a 0.22 μm filter. The liquid and the solid phaseswere used for further investigations.

The specific surface area was determined by the low-temperature nitrogen absorption method using a Gemini 2360sorption apparatus (Micromeritics, USA).

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

The zeta-potential was measured using Zetasizer version 6.12(Malvern, Great Britain).

Dissolution tests were conducted in simulated gastric fluid(SGF) that was composed of 0.2% NaCl in 0.7% HCl (pH¼1.3) and insimulated intestinal fluid (SIF) that was composed of 0.042%

P. Baláž et al. / Materials Letters 104 (2013) 84–86 85

NaOH, 0.4% NaH2PO4∙9H2O and 0.6% NaCl (pH 7.4) at 36.5 1C over aperiod of 30 min [15].

3. Results and discussion

PropertiesSpecific surface area changes: The high-energy milling of the

As4S4 particles leads to an increased number of particles throughthe generation of fresh surfaces. The time-dependence of theincrease in the specific surface area SA is represented for twomodes of milling in Fig. 1. An increase in the SA from 0.24 m2/g to16.65 m2/g (2) and to 34.16 m2/g (1) can be observed for the two

Fig. 1. Specific surface area, SA vs. milling time, tM for the milled As4S4.

Fig. 2. The particle size distribution of the milled As4S4. 1—experiment

experiments. More dilute slurry was used in experiment no. 1,which resulted in higher values of SA in comparison with experi-ment no. 2. In both cases, the samples were milled in an ionicliquid stabiliser (0.075% sodium dodecyl sulphate), which sup-presses the aggregation of small particles and decreases theirspecific surface area. In this method, the stabilisation of the As4S4nanoparticles is achieved by electrostatic repulsion.

Nanoparticle size distribution: The size distribution of the As4S4nanoparticles, given in Fig. 2, can offer a deeper look into themilling process. In the two milling experiments, the nanosizeddispersion has a narrow, monomodal distribution profile withparticle sizes of 146–198 nm (experiment no. 1) and 224–281 nm(experiment no. 2). Broader profiles with large populations of the

no. 1, 2—experiment no. 2, tM—milling time, and D—particle size.

Fig. 3. Specific surface area, SA vs. zeta potential, ξ for the milled As4S4in experiment no. 2.

Fig. 4. Dissolution of As from milled As4S4. A—experiment no. 1, B—experiment no.2, and tD—dissolution time.

P. Baláž et al. / Materials Letters 104 (2013) 84–8686

finer particles (o100 nm) were observed for experiment no. 2,where the more dense suspensions were processed (see Table inFig. 1). Typically, suspensions of nanoparticles with a diameter ofless than 200 nm, with most of the particles being less than400 nm in diameter, can be obtained by milling [16,17].Nanoparticle-based drugs show improved solubility and bioavail-ability when used in this application [18]. However, the stabilityagainst aggregation also plays an important role in this type ofnanosuspension. In our case the stability performance of thesamples remained unchanged over 31 days. As can be seen inthe figure, the nanoparticles exhibit excellent physical stability(minimal change in particle size for more than 1 month).

The ξ-potential was measured to evaluate the surface charge ofthe nanoparticles and was used as a key parameter to predict thenanoparticles' stability. The addition of an ionic stabiliser, such assodium dodecyl sulphate (SDS), leads to a negative ξ-potentialvalue [16,17]. The measured ξ-potential values and the data inFig. 3 indicate that the As4S4 nanoparticles are stabilised. The shiftto less negative ξ-values with increasing SA can be explained bythe increased capture of SDS on the more extended surface of theAs4S4 nanoparticles. In spite of this, dependence on pH values wasnot monitored; we hypothetized that this phenomenon is con-nected with decrease of pH values. It was demonstrated that thepolar headgroup of the phospholipid in the cell membrane is themain site of interaction for the realgar nanoparticles. The nega-tively charged realgar nanoparticles may have an electrostaticinteraction with these phospholipides. The interactions betweenthe nanoparticles and the phospholipids resulted in structuralchanges in the lipid bilayers and in disruption of the membraneintegrity [19].

Dissolution tests: Because many drug nanoparticles aredesigned for use as tablets to disperse in the gastrointestinal tract(bioaccessibility), dissolution in a simulated gastric fluid (SGF)and/or a simulated intestinal fluid (SIF) should provide an initialestimate of the dissolution rate enhancement [16].

The results of the dissolution of the As4S4 nanoparticles in SGFand SIF media are given in Fig. 4. The arsenic concentration in

solution increases as the dissolution time increases; also, adependance on the milling mode is well-documented. In theliterature [20], an investigation of the bioaccessibility of arsenicsulphide has revealed that a maximum of 0.6% As is solubilised inSGF experiments. In our case (experiment no. 2), a value of 0.2% Aswas obtained after dissolution for 30 min. However, when theother mode of milling was applied (experiment no. 1), a value of6.3% As was attained, which is higher than the 4% As that wasreported recently in [21]. An additional 4.5% of dissolved arseniccan be gained in SIF experiments. Altogether, the amount ofsoluble arsenic available for absorption into the bloodstream isgreater than 10%. Such a value produces further challenges for theoral treatment of various cancers that are sensitive to soluble andbioaccessible arsenic.

4. Conclusions

Changes in the specific surface area (0.21–34.16 m2/g), thenanosized distribution (146–198 nm) and the ξ-potential values(−52 to −38 mV) of As4S4 samples were detected as consequencesof high-energy milling. Dissolution experiments in SGF and SIFwith more than 10% As in leach demonstrated the possibleapplication of As4S4 nanoparticles as an oral dose in future cancertreatments using arsenic-based drugs.

Acknowledgement

This work was supported by the Agency for Science and Devel-opment (Projects LPP-0107-09 and APVV-0189-10), Slovak GrantAgency (Project VEGA 2/0009/11) and the European Regional Devel-opment Fund (NANOCEXMAT 1 (ITMS 26220120019) and NANOCEX-MAT 2 (ITMS 26220120035)).

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