Application of Albumin-Embedded Magnetic Nanoheaters for Release
Total Page：16
File Type：pdf, Size：1020Kb
ANTICANCER RESEARCH 38 : 2683-2690 (2018) doi:10.21873/anticanres.12510 Application of Albumin-embedded Magnetic Nanoheaters for Release of Etoposide in Integrated Chemotherapy and Hyperthermia of U87-MG Glioma Cells MELÁNIA BABINCOVÁ 1, HANA VRBOVSKÁ 1, PAUL SOURIVONG 2, PETER BABINEC 1 and ŠTEFAN DURDÍK 3 1Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia; 2Oklahoma Cancer Specialists and Research Institute, Tulsa, OK, U.S.A.; 3Department of Surgical Oncology, Saint Elisabeth Cancer Institute and Faculty of Medicine, Comenius University, Bratislava, Slovakia Abstract. Background/Aim: Malignant gliomas remain high resistance to radiotherapy and chemotherapy (1, 2). The refractory to several therapeutic approaches and the main reasons for the poor prognosis of GBM are diagnosis requirement for novel treatment modalities is critical to at a late stage and lack of efficient therapies. The standard combat this disease. Etoposide is a topoisomerase-II therapeutic protocols for the treatment of GBM have only inhibitor, which promotes DNA damage and apoptosis of limited benefits and provide a median survival of patients of cancer cells. In this study, we prepared albumin with no longer than 15 months (3). embedded magnetic nanoparticles and etoposide for in vitro Etoposide (VP-16) is a semi-synthetic derivative of a evaluation of combined hyperthermia and chemotherapy. naturally occurring antibiotic, podophyllotoxin (Figure 1A), Material and Methods: Magnetic nanoparticles were introduced into cancer clinical trials in 1971, and U.S. Food prepared by a modified co-precipitation method in the and Drug Administration-approved since 1983 (4-6). It presence of human serum albumin and etoposide. A cellular inhibits topoisomerase II re-ligation of cleaved DNA proliferation assay was used to determine the effects of these molecules, resulting in the accumulation of double-strand nanostructures on the viability of U87 glioma cells in an DNA breaks. This leads to late S and G 2 cell-cycle arrest. alternating magnetic field. Results: The in vitro experiments Previous studies have reported that etoposide is effective showed that cell viability decreased to 59.4% after heat against glioma cell lines and it is currently widely used in treatment alone and to 53.8% on that with free etoposide, the treatment of lung and ovarian cancer, as well as recurrent while combined treatment resulted in 7.8% cell viability. childhood brain tumors. Effective against GBM at high Conclusion: Integrating hyperthermia and chemotherapy doses, etoposide leads to toxic side-effects such as nausea, using albumin co-embedded magnetic nanoheaters and weight loss, alopecia, myelosuppression with leucopenia, and etoposide may represent a promising therapeutic option for thrombocytopenia (7, 8). glioblastoma. Nanostructures are nowadays widely used in experimental and clinical medicine applications as diagnostic, imaging, Glioblastoma multiforme (GBM), an astrocytic glioma, is and therapeutic agents. Magnetic nanoparticles (MNPs) one of the most common malignant primary brain tumors, exposed to an external alternating magnetic field (AMF) are characterized by intense and aberrant vascularization and heated through either hysteresis loss or relaxation loss depending on their size and properties. In magnetic hyperthermia, MNPs act as nanoheaters through energy conversion from external AMF into heat. Because cancer Correspondence to: Melánia Babincová, Professor of Biophysics, cells are killed at a temperature of about 43˚C, whereas Department of Nuclear Physics and Biophysics, Faculty of normal cells survive at these higher temperatures, Mathematics, Physics and Informatics, Comenius University, magnetically mediated hyperthermia induced by AMF can be Mlynská dolina F1, 842 48 Bratislava, Slovakia. Tel: +42 1904180025, e-mail: [email protected] used to selectively destroy cancer cells in which magnetic particles have accumulated. Key Words: Glioblastoma, magnetic nanoparticles, human serum Thermotherapy involving the use of an AMF in albumin, hyperthermia, etoposide, chemotherapy. conjunction with MNPs has proven to be an effective method 2683 ANTICANCER RESEARCH 38 : 2683-2690 (2018) for treating patients with GBM. Initial tests have shown that in 96-well plates (Corning Inc., Corning, NY, USA) in a humidified MNPs have minimal toxicities for patients, although further atmosphere of 95% air and 5% CO 2 at 37˚C. The cells were seeded 3 testing must be performed to confirm these findings (9). at a density of 5×10 cells/well, 24 h before experiments. Much like other methods that are used to combat GBM, Cell viability assay. The in vitro cell viability was performed using a MNPs do not serve as a cure on their own; they have been modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide shown to be most effective when used as a combination with (MTT) assay (22). (Sigma, St. Louis, MO, USA) U87-MG cells were other treatment modalities, for example combining plated (5000 in 100 μl of medium) into each well of a 96-well plate fractionated radiotherapy with thermotherapy has been and incubated for 24 h. Fifty microliters of etoposide, MNPs, or MNP- shown to have a survival advantage in patients with relapsed HSA-ETO suspended in medium were added and incubated for 24 h. GBM (10). The supernatant was carefully removed and 10 μl of media and 20 μl of a 5 mg/ml MTT solution added and plates incubated for a further 3 Since the report by Zimmermann and Pilwat (11), MNPs h. As a control, 150 μl of PBS at pH 7.4 was added to cells in eight of have attracted attention not only as nanoheaters for cancer the wells. The supernatant in each well was aspirated and 150 μl of therapy (hyperthermia) but also because of their potential as dimethyl sulfoxide was added to solubilize the cells and MTT crystals. contrast agents for magnetic resonance imaging (MRI), and After 1 h of shaking on an Eppendorf Thermomixer at 37˚C and 400 magnetic drug targeting (12-18). rpm to dissolve all crystals, the blue color was read in a multiwell Our aim in this study was to demonstrate effectivity of scanning spectrophotometer at 540 nm using a microplate reader MNPs for integrated cancer therapy of the glioblastoma cell (Multiskan GO spectrophotometer; ThermoFisher Scientific). Cell viability was quantified by the relative absorbance of the drug-treated line U87. For these purposes, we prepared MNPs wells to the control wells without drug treatment. The cell viability was functionalized with human serum albumin (HSA) (19-21) calculated by comparing the sample absorption to the one of the control embedded with etoposide. cells, which was by definition 100%. Materials and Methods Setup for application of electromagnetic hyperthermia. AMF with a frequency of 3.5 MHz and an amplitude of 1.2 kA m −1 was generated using a 3.5 MHz radiofrequency generator (model GV6A; Preparation of HSA immobilized MNPs. MNPs were prepared by a ZEZ a.s., Rychnov nad Nisou, Czech Republic) with a power modified co-precipitation method (19) in the presence of HSA dissipation of 6 kW. The coil-shaped and water-cooled antenna with which facilitated the in situ immobilization of crystallized MNPs by a diameter of 15 cm was made of three copper windings, connected the protein. A volume of 180 ml of deoxygenated water with 1 g of to a water-cooled resonance circuit which produced the HSA (Sigma, St. Louis, MO, USA) and 3 ml of ammonium electromagnetic field. Magnetic field amplitude and frequency hydroxide (30%, w/v) under constant stirring and nitrogen flow, 20 produced in our equipment were within the safety range (23). For ml of iron salts (1.08 g of FeCl •6H O and 0.4 g of FeCl •4H O; 3 2 2 2 the evaluation of the effect of hyperthermia, U87-MG glioma cells Centralchem, Bratislava, Slovakia) was added dropwise at room were seeded on 35 mm Petri dishes (2×10 5 in 3 ml of growth temperature. The reaction mixture was then heated and processed at medium per dish). Prior to AMF heating, samples and controls were 70˚C for another 15 min. The resulting products were dialyzed in incubated at 37˚C for 15 min to stabilize temperature. During the phosphate-buffered saline (20 mM, pH 7.4) to remove excess experiments samples were placed in the center of the induction coil ammonium hydroxide and residual iron salts. Five milligrams of for the desired time. To measure the temperature changes over time etoposide (BioVision, Inc., San Francisco, CA, USA) was dissolved during exposure to the AMF, we used an optic fiber thermometer in 1 ml of dimethylsulfoxide (Centralchem) and the solution was FOB101 with automatic registration (Omega Engineering, Norwalk, dropwise added to the desired amount of MNP-HSA. The mixture CT, USA). After the heat treatment, the suspension of U87-MG was stirred at 900 rpm for 4 h. Unloaded etoposide was removed by glioma cells was diluted to 4.8 ml media and seeded in a 96-well ultrafiltration (MW cutoff: 10,000 Da; Millipore, Merck, Darmstadt, plate at a density of 5×10 3 cells/well. Germany) to obtain final stock of MNP-HSA-ETO (Figure 1B). Statistical analysis. The experiments were performed at least five Etoposide release under AMF. The percentage of etoposide released times. Data are expressed as the mean±standard deviation (SD). due to AMF heating was obtained from the total amount (WT) of Statistical correlation of data was checked for significance by added etoposide in a MNP-HSA-ETO sample and the amount of ANOVA and Student’s t-test. Values of p< 0.05 were considered to released etoposide (W ) in supernatant after centrifugation (10000× R indicate a statistically significant difference. g, 30 min) using the formula (W R/WT) ×100%. The concentration of etoposide was quantified using a calibration curve obtained from absorbance measurement using UV MINI 1240 UV-VIS Results and Discussion spectrophotometer (Shimadzu, Kyoto, Japan).