The effects of doxorubicin-loaded liposomes on viability, stem cell surface marker expression and secretion of vascular endothelial growth factor of three-dimensional stem cell spheroids

The aim of the present study was to evaluate the effects of anionic, cationic and neutral liposomes containing doxorubicin on the cellular viability and osteogenic differentiation of three-dimensional stem cell spheroids. Doxorubicin-loaded liposomes were prepared using the traditional thin-lipid-film-hydration method and were characterized using transmission electron microscopy and a zeta potential analyzer. The doxorubicin release profile from these liposomes was also analyzed in vitro. Three-dimensional cell spheroids were fabricated using silicon elastomer-based concave microwells. Qualitative results of cellular viability were observed using a confocal microscope and quantitative cellular viability was evaluated using a Cell-Counting Kit-8 (CCK-8) assay. Furthermore, the secretion of vascular endothelial growth factor was evaluated. Western blot analysis was performed to assess the expression of collagen I and glyceraldehyde 3-phosphate. Results indicated that the spheroids were well formed in silicon elastomer-based concave microwells on day 1. In general, the shapes of the cells in the in the doxorubicin-loaded anionic, cationic and neutral liposome groups were similar to the control group except for the 10 µg/ml groups on days 3, 5, and 7. No significant changes in cellular viability were noted with the addition of doxorubicin at day 1 but significant decreases in cellular viability were noted with application of doxorubicin at day 5. Notably, higher concentrations of doxorubicin reduced the secretion of vascular endothelial growth factor and stem cell marker expression. To conclude, the present study indicated that doxorubicin-loaded anionic liposomes produced the most sustained release profile and cationic liposomes produced the highest uptake of the stem cell spheroids. These findings suggested that higher concentrations of doxorubicin-loaded liposomes affected cellular viability, the secretion of vascular endothelial growth factor and stem cell marker expression.