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

Abstract

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.

Cellular viability and osteogenic differentiation potential of human gingiva‑derived stem cells in 2D culture following treatment with anionic, cationic, and neutral liposomes containing doxorubicin

“…The 12 groups of the present study were as follows: i) Unloaded control group (D0); ii) doxorubicin, 1 µg/ml (D1); iii) doxorubicin, 10 µg/ml (D10); iv) unloaded anionic group (A0); v) anionic group loaded with 1 µg/ml doxorubicin (A1); vi) anionic group loaded with doxorubicin at 10 µg/ml (A10); vii) unloaded cationic group (C0); viii) cationic group loaded with doxorubicin at 1 µg/ml (C1); ix) cationic group loaded with doxorubicin at 10 µg/ml (C10); x) unloaded neutral group (N0); xi) neutral group loaded with doxorubicin at 1 µg/ml (N1); and xii) neutral group loaded with doxorubicin at 10 µg/ml (N10) ( 7 , 9 ).…”

“…However, liposomal doxorubicin formulations are associated with lower reported odds ratios with regard to myelosuppression, cardiotoxicity and alopecia than conventional doxorubicin on its own ( 6 ). The effects of doxorubicin, particularly doxorubicin liposomes, on stem cells have remained to be fully elucidated ( 7 ). The aim of the present study was to evaluate the effects of anionic, cationic and neutral liposomes loaded with doxorubicin on the viability and osteogenic differentiation potential of human gingiva-derived stem cells in two-dimensional culture.…”

Osteogenic potential of cell spheroids composed of varying ratios of gingiva‑derived and bone marrow stem cells using concave microwells

“…In intact cells intracellular esterases produce green fluorescence by reacting with the nonfluorescent, cell-permeant calcein acetoxymethyl ester ( 20 ). The ethidium homodimer can enter damaged cells and produce red fluorescence ( 21 ). Following staining, the spheroids were observed under a fluorescence microscope (magnification, ×100; Axiovert 200; Zeiss GmbH, Jena, Germany).…”

Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression

“…In addition, the number of viable cells was quantitatively examined using a commercially available kit (Cell Counting Kit-8; Dojindo Molecular Technologies, Inc.) on days 1, 3, 5 and 7 according to the manufacturer's instructions. The specific time-points were selected for analysis according to a previous study (19). Experiments were carried out in triplicate.…”