Inhibitory Effects of Hybrid Liposomes on the Overgrowth Of Human Synovial Sarcoma Cells By Induction Of Apoptosis

Abstract

Copyright: © 2017 Matsumoto, et al. accurate colorimetric determination method based on the amino group present in the gentamicin molecule [24,25].This may prove to be an inexpensive alternative for quantitative gentamicin detection compared to the existing analytical methods, like HPLC, ELISA or fluorometry [26]. Materials and Methods Materials The chemicals were purchased from Sigma, with the exception of gentamicin sulphate, which was purchased from Molekula. The bone blocks were provided by the West-Hungarian Regional Tissue Bank, the blocks originated from the femoral head of human cadavers. Freeze-dried femoral head blocks were cut into 50±5 mg cube-shaped pieces under sterile conditions. MIC measurement Minimal inhibitory concentration (MIC) was determined on Escherichia coli (ATCC 11303) cells according to Andrews [27]. Briefly, serial dilution of antibiotic from 0 to 25 µg/ml was prepared in molten (50 °C) agar, which were mixed and poured onto Petri dishes. E. coli bacteria from overnight culture were inoculated to the plates so that approximately 104 cfu/spot was applied. Plates were incubated at 37°C for 18 hours. The MIC was defined as the lowest concentration of antibiotic at which there was no visible growth of organism. Antibiotic coating For the short-term antibiotic release, gentamicin (10 mg/ml) was used in aqueous solution. The bone graft was placed in 1 ml of antibiotic solution and was incubated at 25 °C for 24 hours. Subsequently, the soaked graft, submerged in the antibiotic solution, was frozen at -80 °C followed by lyophilization for 24 hours using a Labconco Freezone 2.5 freeze-drying machine in order to maximize drug content. The alginate coating for long-term release was prepared in two steps. First, the bone grafts were coated with gentamicin as described above; then the alginate film coating was created over the antibiotic layer as follows: Na-Alg solution (1 ml, 4 %) was applied on the surface of the antibiotic coated freeze-dried bone. The graft was dried afterwards at 37 °C for 4 hours on teflon plates. The film coating process was repeated with the dried coated grafts turned upside down, thus a double layer Na-Alg film was formed. Na-Alg was then converted into water insoluble Ca-Alg using CaCl2.To do so, the Na-Alg coated bone grafts were placed in 10 % CaCl2 solution for 60 seconds. In the final step, the grafts were washed with distilled water and dried at 37 °C.