Enhanced biological activity of liposomal methylated resveratrol analog 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214) in 3D patient-derived ovarian cancer model

Abstract

3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214) belongs to methoxystilbenes family and is an active metabolite of 3,4,5,4'-tetramethoxystilbene (DMU-212). In several of our previous studies, the anti-apoptotic activity of DMU-214 was significantly higher than that of the parent compound, especially in ovarian cancer cells. Due to increased lipophilicity and limited solubility, methoxystilbenes require a solubilization strategy enabling DMU-214 administration to the aqueous environment. In this study, DMU-214-loaded liposomes were developed for the first time, and its antitumor activity was tested in the ovarian cancer model.First, several liposomal formulations of DMU-214 were obtained by the thin lipid film hydration method followed by extrusion and then characterized. The diameter of the resulting vesicles was in the range of 118.0-155.5 nm, and samples presented monodisperse size distribution. The release of DMU-214 from the studied liposomes was governed by the contribution of two mechanisms, Fickian diffusion and liposome relaxation.Subsequently, in vitro activity of DMU-214 in the form of a free compound or liposome-bound was studied, including commercial cell line SK-OV-3 and patient-derived ovarian cancer cells in monolayer and spheroid cell culture models. DMU-214 liposomal formulations were found to be more potent (had lower IC₅₀ values) than the free DMU-214 both in the monolayer and, more significantly, in both examined spheroid models. The above results, with particular emphasis on the patient-derived ovarian cancer model, indicate the importance of further development of liposomal DMU-214 as a potential anticancer formulation for ovarian cancer treatment.

Keywords: DMU-214; Resveratrol; liposomes; methylated resveratrol analogs; ovarian cancer; tumor spheroids.

Graphical abstract

Figure 1.

Characteristics of DMU-214 loaded liposomes and non-loaded analog formulations: a) liposome size expressed as z-average (nm) and polydispersity index (PDI), b) DMU-214 encapsulation efficiency (%) and concentration in liposomal formulations (µM), c) zeta potential (mV), d) in vitro release profiles of DMU-214-loaded formulations at 0.3 D/L ratio – open circles represent experimentally determined values, lines – correspond to data predicted from Peppas-Sahlin mathematical model. Standard deviations were obtained from measurements (a, b, c) or experiments (d) repeated three times.

Figure 2.

Effect of free DMU-214 and DMU-214 loaded into three liposomal formulations DMPC (A), DPPC (B) and POPC (C) on the SK-OV-3 monolayer cultured cells. DMPC/DPPC/POPC blank: liposomes with DMPC/DPPC/POPC but without DMU-214, diluted by the same dilution factor as the liposomal formulations. The viability was measured using the MTT test after 72 h of drug exposure. IC₅₀ values were calculated following a normalized dose-response inhibition curve fitting. Data represent mean values from three independent experiments ± SD.

Figure 3.

Effect of free DMU-214 and DMU-214 loaded into three liposomal formulations DMPC (A, D), DPPC (B, E), POPC (C, F) on the SK-OV-3 and P-3 patient-derived spheroids. DMPC/DPPC/POPC blank: liposomes with DMPC/DPPC/POPC but without DMU-214, diluted by the same dilution factor as the liposomal formulations. Spheroid viability was measured using CellTiter Glo 3 D after 72 h exposure to the drugs. IC₅₀ values were calculated following a normalized dose-response inhibition curve fitting. Data represent mean values from three independent experiments ± SD.

Figure 4.

Spheroids growth and morphology upon treatment with DMU-214 liposomal formulations (DMPC/DPPC/POPC). Spheroids were pre-formed for 7 days with 5 000 SK-OV-3 cells and treated with the indicated concentrations of DMU-214 formulations. The images were acquired 72 hours after administration of the formulation with the Olympus ix73 inverted microscope.