Effect of Size and Processing Method on the Cytotoxicity of Realgar Nanoparticles in Cancer Cell Lines
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International Journal of Nanomedicine Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Effect of size and processing method on the cytotoxicity of realgar nanoparticles in cancer cell lines Weizhong Zhao1 Abstract: In this study, the effects of the size and Chinese traditional processing (including Xun Lu3 elutriation, water cleaning, acid cleaning, alkali cleaning) on realgar nanoparticles (RN)-induced Yuan Yuan1 antitumor activity in human osteosarcoma cell lines (MG-63) and hepatoma carcinoma cell lines Changsheng Liu1 (HepG-2) were investigated. The human normal liver cell line (L-02) was used as control. RN Baican Yang3 was prepared by high-energy ball milling technology. The results showed that with the assistance Hua Hong1 of sodium dodecyl sulfate, the size of realgar could be reduced to 127 nm after 12 hours’ ball milling. The surface charge was decreased from 0.83 eV to -17.85 eV and the content of As O Guoying Wang3 2 3 clearly increased. Except for elutriation, the processing methods did not clearly change the size Fanyan Zeng2 of the RN, but the content of As2O3 was reduced dramatically. In vitro MTT tests indicated that 1The State Key Laboratory in the two cancer cell lines, RN cytotoxicity was more intense than that of the coarse realgar of Bioreactor Engineering, 2Key Laboratory for Ultrafine nanoparticles, and cytotoxicity was typically time- and concentration-dependent. Also, RN Materials of Ministry of Education cytotoxicities in the HepG-2 and L-02 cells all increased with increasing milling time. Due to and Engineering Research Center the reduction of the As O content, water cleaning, acid cleaning, and alkali cleaning decreased for Biomedical Materials of Ministry 2 3 of Education, East China University RN cytotoxicity in HepG-2, but RN after elutriation, with the lowest As2O3 (3.5 mg/g) and the of Science and Technology, 3Pharmacy smallest size (109.3 nm), showed comparable cytotoxicity in HepG-2 to RN without treatment. Department of Traditional Chinese Meanwhile, RN-induced cytotoxicity in L-02 cells was clearly reduced. Therefore, it can be Medicine, Shanghai University of Traditional Chinese Medicine, concluded that RN may provide a strong antiproliferation effect in the MG-63 and HepG-2 cells. Shanghai, People’s Republic of China Elutriation processing is a suitable approach to limit the dangerous side-effects of As2O3, while maintaining the effectiveness of RN. Keywords: realgar nanoparticles, cytotoxicity, size, processing, apoptosis Introduction Realgar (Xiong-Huang in Chinese), an arsenic-containing Chinese traditional mineral drug (its major component is As2S2 or As4S4), has been used for treatment of various diseases, such as carbuncles, scalds and burns, insect-bites, abdominal pains, infantile convulsions, and psoriasis, for more than 1500 years.1–4 Especially in recent years, with the confirmation of its clinical effectiveness for the treatment of some varieties of refractory or relapsed tumor or cancer, realgar has attracted increasing attention Correspondence: Yuan Yuan, in the biomedical field.5 In particular, previous investigations showed that realgar is Changsheng Liu 6 East China University of Science absorbed rapidly and excreted mostly within the first 24 hours’ urine. Unfortunately, and Technology, Box 112, No 130, poor natural water solubility and thus poor bioavailability greatly hampers wider Meilong Road, Shanghai 200237, clinical applications of realgar. It has been reported that only 0.10% and 0.40% of the People’s Republic of China Tel +86 21 6425 1308 coarse realgar (CR) powder dissolves in the stomach and intestinal fluids, and is finally Fax +86 21 6425 1358 absorbed.4 Therefore, there is an urgent need for methods to safely and effectively Email [email protected], [email protected] deliver poorly water-soluble realgar. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2011:6 1569–1577 1569 Dovepress © 2011 Zhao et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article http://dx.doi.org/10.2147/IJN.S21373 which permits unrestricted noncommercial use, provided the original work is properly cited. Zhao et al Dovepress Due to its inherent nonionizable property, there is Materials and methods a limited number of chemical approaches available to Materials overcome the disadvantages of realgar. Therefore, realgar Realgar (As2S2, 98% in purity) was purchased from Hubei nanoparticles (,100 nm in diameter) were proposed and Province. Arsenic trioxide (As2O3) was kindly provided by several strategies have been used to reduce the size of realgar Zhongshan Hospital. SDS (Junsei, Japan) has been used in the past few years. It has been proved that the physical previously in milling tests as a surfactant. MG-63, HepG-2, and chemical properties of realgar and other nanoparticles and L-02 cell lines were purchased from the American Type can be significantly improved or radically changed as their Culture Collection (ATCC). size is scaled down to small clusters of atoms, especially in clinical applications.6 The ensuing in vitro and in vivo experiments revealed that the size indeed had dramatic Methods effect on the antitumor activity of realgar. Compared with Preparation of RN by high-energy ball milling The ball milling equipment (Nanjing University Instrument CR, realgar nanoparticles (RN) exhibited greater toxicity Plant, Nanjing, China) was used to reduce the particle size in a cervical cancer cell line (HeLa), three human ovarian of realgar powder. There were four grinding vials in the cancer cell lines (CI80-13S, OVCAR, OVCAR-3),6 and nor- equipment and pure realgar powder (5.0 g) was also added mal ECV-304 cells.7 Accordingly, in our previous study, our with SDS aqueous solution (saturated concentration) to group also endeavored to prepare RN by high-energy milling each vial. The rate of milling ball and realgar was W(balls)/ technology. The results revealed that the disintegration of W(realgar) 40/1 and rate of rotation was 400 rpm. These realgar particles was accompanied by an obvious increase in = four realgar preparations were ground simultaneously to arsenic trioxide (As O ) from 2.82 to 9.75 mg/g. Research has 2 3 produce particles under the same operating conditions. In shown that inorganic As O is the most toxic arsenic species 2 3 order to study how the process altered RN size, three ball- and can induce complete remission in a high percentage of milling times were chosen: 4, 8, and 12 hours. patients with refractory acute promyelocytic leukemia.8–11 However, the high toxicity and severe side effects, especially in long-term use, make it unacceptable in clinic settings.12 Processing methods of the as-prepared RN That is to say, reduction of the As2O3 content of RN should The as-prepared RN was processed by the following four be considered prior to clinical use. methods: elutriation, water cleaning, acid cleaning, and alkali Processing is a widely used method to reduce the toxicity cleaning. The corresponding RN obtained were named E-RN, of traditional Chinese medicines. The most commonly used WC-RN, ACI-RN, and ALK-RN, respectively. processing methods include elutriation, water cleaning, acid 1. Elutriation: 10 g RN powder and 5 mL distilled water cleaning, and alkali cleaning. To date, the effects of these were first added into a mortar. After 5 minutes’ grind- processing methods on the As2O3 content and the change in ing, 100 mL distilled water was added and then agitated resulting antitumor activity have never been investigated. for 1 minute. The solution stood for 10 minutes, then In this study, our first objective was to prepare RN and the clear supernatant liquid was collected and the coarse investigate the cytotoxicity of the prepared RN in human powder remaining was ground again. This procedure was osteosarcoma cell lines (MG-63) and human hepatoma repeated eleven times to obtain E-RN. carcinoma cell lines (HepG-2), which has not been reported 2. Water cleaning: 10 g RN powder and 100 mL distilled previously. Our second objective was to study the effect water were added to a beaker and agitated for 30 minutes. of processing method (elutriation, water cleaning, acid Sucking filtration was then carried out and the processed cleaning, and alkali cleaning) on the physicochemical RN were collected. This procedure was repeated eleven properties and cytotoxicity of RN in MG-63 and HepG-2 times to obtain WC-RN. cell lines. The cytotoxicity of RN in human normal liver cell 3. Acid cleaning: 10 g RN powder and 100 mL dilute hydro- line was also investigated for comparison, and the potential chloric acid solution were added to a beaker and agitated apoptosis induction of RN particles after processing was for 30 minutes. Sucking filtration was then carried out investigated, too. RN were prepared by high-energy milling and the RN processed was collected. This procedure was technology with the help of sodium dodecyl sulfate (SDS) repeated eleven times to obtain ACI-RN. at room temperature. The cytotoxicity was evaluated by 4. Alkali cleaning: 10 g RN powder and 100 mL dilute MTT assay. caustic soda solution (3%) were added to a beaker and 1570 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2011:6 Dovepress Dovepress Cytotoxicity of realgar nanoparticles agitated for 30 minutes. Sucking filtration was then carried ethylenediamine tetraacetic acid (EDTA) and the cell density out and the RN processed was collected. This procedure was calculated. was repeated eleven times to obtain ALK-RN. These cells were preincubated in 96-well plates at a density of 5000 cells/well. After 24 hours, the cells were Characterization treated with different concentrations of RN for a determined X-ray diffraction measurement (XRD, D/max 2550 VB/PC; time (24, 48, and 72 hours). Then, the 3-(4,5-dimethylthiazol- Rigaku, Tokyo, Japan) and Fourier transform infrared 2-yl)-2,5-diphenyltetrazolium bromide (MTT) labeling spectrometer (Nicolet-5700 FTIR; Nicolet, Waltham, MA) solution was added onto the plates containing the RN-treated were used to study the changes in phase and chemical MG-63, HepG-2, and L-02 cells.