Study On The Antitumor Mechanism Of Realgar Nanoparticles

At present, it is urgent to improve the bioavailability and decline the potent toxicity of realgar since the increasing attention was put on its clinical application for tumor therapy. In the present study, the notion of nano-processed of realgar was introduced. The processes of milling, detoxification and solid dispersion were investigated, respectively. And raw realgar particles and realgar nanoparticles with the same background As2O3 concentrations were prepared. The effects of the two drug-carrier systems on cell proliferation, apoptosis, oxidative stress and membrane toxicity in promyelocytic leukemia HL-60 cells were compared. The main results are as follows: (1) The processes of milling, detoxification and solid dispersion were investigated, respectively. Mechanical milling was carried out using a high-energy planetary ball mill at 400 r/min in the presence of milling-supporting media. The average diameter of the product was about 327.4 nm with polydispersion index of 0.312 only after 6 h of milling. The resulting nanoparticles were then processed through acid washing, alkali washing or water refinement followed by either air drying or alcohol drying. It was concluded that the process of water refinement followed by alcohol drying could eliminate the As2O3 and prevent reuniting of nanoparticles effectively. The concentrations of As2O3 and the uniformity of paiticles were used as the indexes to determine the process of solid dispertion. The relative ideal process of solid dispersion was as the follows: the PEG6000/realgar powder ratio was 5/1, the stirring time was 30 min and the stirring temperature was 70±5℃. It was found that realgar nanoparticles could keep relative stability within 30 d of exposure period according to the results from the stability assay. (2) In order to further clarify the size effects of realgar nanoparticles, the raw realgar particles (marked as Raw-SD) and realgar nanoparticles (marked as Nano-SD) with the same concentration of As2O3 were prepared, and in the meantime, As₂O₃ particles at the same concentration (marked as As₂O₃-SD) were set up in order to further exclude the possibly synergetic effects of As2O3 in realgar particles. The effects on proliferation in HL-60 cells were investigated by trypan blue exclusion and MTT assays. And realgar nanoparticles-induced apoptosis was assayed by fluorescence microscopy, agrose gel electrophoresis and flow cytometric analysis. Results revealed that treatment with realgar nanoparticles resulted in considerably low cell viability and produced characteristic apoptotic events in HL-60 cells including morphorlogical changes, DNA ladder formation and increased number of cells with sub-G1 phase, while raw realgar particles with the same As2O3 concentration failed to induce apoptosis. The loss of mitochondrial membrane potential, demonstrated by diminished fluorescence intensity of rhodamine 123, a mitochondria-specific and voltage-dependent dye, examined by flow cytometry, suggested that treatment with realgar nanoparticles led to a significant decline of mitochondria membrane potential in HL-60 cells. Furthermore, reverse transcription and polymerase chain reaction (RT-PCR) analysis revealed that the mRNA level of Bcl-2 was significantly decreased in realgar nanoparticles-treated HL-60 cells. According to the data from the oxidative kinetics of raw realgar particles and realgar nanoparticles (in PBS buffer, 37℃, 5% CO2), it was found that the oxidative ratio of the latter was much higher. These results indicated that realgar nanoparticles led to enhanced growth inhibition and apoptotic induction in which they might exert their cytotoxicity through releasing the soluble arsenic. (3) The effects of realgar nanoparticles on the intracellular level of ROS, oxidative damage to lipid and protein and the antioxidant defense system were investigated. It was found that realgar nanoparticles promoted the generations of ROS accompanied by the potentiation of lipid peroxidation and protein oxidation, especially the loss of its free thiols. Moreover, the imbalance of the antioxidant enzymes system induced by realgar nanoparticles contributed to oxidative stress. Treatment with realgar nanoparticles for 36 h inhibited greatly the activity of catalase (CAT), stimulated the activities of total superoxide dismutase (T-SOD), manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPx) and had little effect on the activities of glutathione reductase (GR),glutathione-S-transferase (GST) and thioredoxin reductase (TR) in HL-60 cells. Furthermore, reverse transcription and polymerase chain reaction (RT-PCR) analysis revealed that the mRNA levels of MnSOD and GPx remained steady in HL-60 cells exposed to realgar nanoparticles, suggesting the absence of transcriptional control and the enhancement in the activities of both enzymes might be mediated post-transcriptionally or translationally. These results indicated that realgar nanoparticles could initiate oxidative stress that might be involved in apoptotic induction. (4) The effects of realgar nanoparticles on glutathione redox level were examined as a function of time. At the initial 12 h of exposure period, realgar nanoparticles resulted in the depletion of GSH coupled with the accumulation of GSSG. In the subsequent exposure, both GSH and GSSG remained relative higher level than those of the other groups. The results suggested that realgar nanoparticles might exert their cytotoxicity through destroying the redox status of glutathione. (5) The effects of realgar nanoparticles on cell membrane including membrane permeability, membrane mobility and the configuration of membrane protein were examined. Realgar nanoparticles had acute toxicity to cell membrane, increasing lactate dehydrogenase (LDH) release, reducing membrane fluidity and destroying the structures of membrane protein especially the surface protein. In addition, the information of infrared spectra in realgar nanoparticles-treated HL-60 cells provided a consistent proof for apoptotic induction.