ABSTRACT
Objective To prepare a new bone-targeted drug of epirubicin-oxidized dextran-alendronate compound,and explore the effect of compound on osteosarcoma cells.Methods (1) Based on Schiff's base theory,we synthesized the compound.(2) We investigated physicochemical character with ultraviolet (UV),infrared (IR),and 1H-nuclear magnetic resonance (1H-NMR),molecular weight and aldehyde group content in oxidized dextran,alendronate content in oxidized dextran-alendronate compound,epirubicin drug loading capacity in the compound,and rate of charge for three matters,and affinity to bone in vitro though Hydroxyapatite (HA) absorbing test.(3) We investigated the compound's cytotoxicity effect through methyl thiazolyl tetrazolium (MTT) test,apoptosis influence through flow cytometry (FCM).Results (1) There were typical physicochemical characters.(2) Oxidized dextran molecular weight (MW) was 4 251 ± 68,number of average molecular weight (MN) was 2 551 ± 42,and molecular weight distribution width was 1.78.Aldehyde group content of oxidized dextran was (10.41 ± 0.2)mmol/mg,epirubicin drug loading capacity was (5.28 ± 0.23) ％,rate of charge was 5,and 2 ～ 3 between aldehyde group content in oxidized dextran and alendronate (ALN).(3) In the MTT test,epirubicin (EPI)-Dex-ALN,EPI,ALN obviously restrained osteosarcoma cell proliferation,the minimum concentration(μg/ml) was 1,1,and 40; IC50 (μg/ml) of EPI-Dex-ALN,EPI,ALN was 0.142,0.505,0.219; 0.251,0.739,and 0.518; 45.21,27.97,and 18.96 after 24 h,48 h,72 h; in FCM (flow cytometry),apoptosis and necrosis rate of EPI-Dex-ALN,ALN,and Dex was 80.13 ±4.05,97.01 ±2.58,31.53 ±6.9,and 14.01 ±2.51.Conclusions We successfully synthesized a new bone-targeted drug delivery system,which showed better bone affinity in vivo,and kept biological activity with powerful targeted function.
ABSTRACT
Objective To investigate the effect of adiponectin on the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and the cell proliferation of osteosarcoma MG-63 cells.Methods The MG-63 cells were seeded in 6-cm board with a inoculum density of 1 × 105 cells/ml.When the cells grew up to about 80％,a volume (5 ml) of medium containing adiponectin (concentration 1 μg/ml) was added to each plate and incubated for 0 min,15 min,30 min,60 min,and 120 min,respectively ; and Western Blotting was used to detect the levels of AMPK phosphorylation,and the optimal time point processed by adiponectin was selected.The control and adiponectin groups were set (0,0.001,0.01,0.1,1 μg/ml) according to the optimal processing time of adiponectin,respectively; and Western blotting was used to detect the levels of AMPK phosphorylation and determine the optimal concentration of adiponectin.Based on the optimal processing time and optimal concentration,PBS was used as a control,the corresponding concentrations of adiponectin were used as the experimental group.Western blotting was used to detect the levels of AMPK phosphorylation to determine the effect of adiponectin on AMPK phosphorylation of osteosarcoma MG-63 cells.CCK-8 assay was used to investigate the effect of adiponectin on the cell proliferation of MG-63 cells.The MG-63 cells were seeded in the 96-well plates (5,000 cells/well) and cultured overnight.The experiment was set as blank control group and adiponectin-recombinant groups with 5 different concentration gradients (0.001,0.01,0.1,1,10 μg/ml).Five parallel wells were set for each group,and the cells were cultured for 24 h.During the last 4 h of culturing,a volume (10 μl) of CCK-8 reagent was added to each well,and the cells were cultured for another 2.5 h.The optical density (OD490) was obtained.Results When the concentration of adiponectin was greater than 0.01 μg/ml,the level of AMPK phosphorylation in MG-63 cells were significantly elevated (t =5.894,P =0.007).The short-time stimulation of adiponectin did not inhibit the proliferation of MG-63 cells (F =6.335,P =0.072).Conclusions Adiponectin can enhance the AMPK phosphorylation.The short-time stimulation of adiponectin did not inhibit the proliferation of MG-63 cells.