Ceramide lipid-based nanosuspension for enhanced delivery of docetaxel with synergistic antitumor efficiency

Abstract

Ceramide (CE), a bioactive lipid with tumor suppression, has been widely used as a drug carrier and enhancer for cancer therapy. CE-based combination therapy was prone to be attractive in cancer therapy. In our previous study, the combination of CE and docetaxel (DTX) was proved to be an effective strategy for cancer therapy. To further improve the antitumor efficiency of DTX, the CE lipid-based nanosuspensions (LNS) was prepared for the delivery of DTX to exhibit synergistic therapeutic effect. The enhanced delivery and synergistic therapeutic effect of DTX-loaded CE-LNS (CE + DTX-LNS) were evaluated. CE + DTX-LNS exhibited spherical or ellipsoidal shape, uniform particle size distribution (108.1 ± 3.8 nm), sustained release characteristics and good stability in vitro. Notably, CE + DTX-LNS could effectively co-localize CE and DTX into same tumor cell and subsequently play synergistic cell damage effect compared with CE-LNS + DTX-LNS (p < 0.05). The in vivo fluorescence imaging results showed that CE + DTX-LNS could effectively prolong the in vivo circulation time and enhance the accumulation in tumor sites. Moreover, the antitumor efficacy of CE + DTX-LNS observed in B16 murine melanoma model was 93.94 ± 2.77%, significantly higher than that of CE-LNS, DTX-LNS, Duopafei® (p < 0.01) and CE-LNS + DTX-LNS (p < 0.05), respectively, demonstrating that co-delivery of CE and DTX into same tumor cell was the basis for enhanced synergistic therapeutic effect. Furthermore, histological examination of Blank-LNS showed no visible tissue toxicity compared to normal saline. Consequently, CE-LNS could effectively delivery DTX and CE + DTX-LNS exhibit synergistic inhibition of tumor growth due to the co-localization of CE and DTX. CE-LNS hold great potential to be an appropriate carrier for CE-based combination chemotherapy.

Keywords: Ceramide; co-delivery; combination therapy; docetaxel; lipid-based nanosuspension.

Figure 1.

Scheme of the synergistic mechanisms of nanosized CE + DTX-LNS. After intravenous administration, CE + DTX-LNS was accumulated at tumor site through EPR effect and internalized into same cell through vesicles and caveolae mediated endocytosis. Subsequently, CE and DTX with different anti-cancer mechanisms were released independently from CE + DTX-LNS inside the same cancer cell and killed the cancer cell in an enhanced synergistic manner.

Figure 2.

Characteristics of CE + DTX-LNS in vitro. (A) Particle size (a), zeta potential (b), TEM images (c) and photographs (d) of freshly prepared CE+DTX-LNS; Photographs (e) and particle size (f) of lyophilized CE + DTX-LNS. (B) In vitro drug release of CE+DTX-LNS (n = 3). (a) The release profile of DTX from CE + DTX-LNS, DTX-LNS and Duopafei® (b) The release profile of NBD-CE from NBD-CE + DTX-LNS, NBD-CE-LNS and NBD-CE-Solution. *p < 0.05, **p < 0.01, statistically significant difference between NBD-CE+DTX-LNS and NBD-CE-Solution; # p < 0.05, ##p < 0.01, statistically significant difference between CE+DTX-LNS and Duopafei®. (C) Stability of CE + DTX-LNS was evaluated by determination of particle size alteration overtime in different media: PBS, complete cell culture media and PBS containing 10% plasma (n = 3).

Figure 3.

Co-delivery of CE and DTX via CE + DTX-LNS in vitro. (A) Co-delivery of CE and DTX via CE + DTX-LNS by fluorescence microscope (n = 3). B16 cells were incubated with NBD-CE and Rho-DTX labeled CE + DTX-LNS for 4 h while cells treated with NBD-CE-LNS + Rho-DTX-LNS were served as control (× 20). (B) Co-delivery of CE and DTX via CE + DTX-LNS by FACS analysis (n = 3). B16 and MCF-7 cells were incubated with NBD-CE and Rho-DTX labeled CE + DTX-LNS for 0.5, 2 and 4 h, respectively. Cells incubated with NBD-CE-LNS + Rho-DTX-LNS were served as control.

Figure 4.

The anti-proliferation effect and apoptosis of CE + DTX-LNS in vitro.(A) Effects of different treatments on cell viabilities of B16 and MCF-7 cells, (n = 3). Cells were treated with CE-LNS (No DTX-LNS), DTX-LNS (No CE-LNS), CE-LNS + DTX-LNS (with DTX-LNS 20 μM or with CE-LNS 10 μM) at different molar ratio and CE + DTX-LNS. After 48 h incubation, cell viabilities were evaluated by MTT assay. * p < 0.05, ** p < 0.01, statistically significant difference between CE-LNS and CE + DTX-LNS; # p < 0.05, ## p < 0.01, statistically significant difference between DTX-LNS and CE + DTX-LNS. (B) Caspase-3 activity after different treatments on B16 and MCF-7 cells. ** p < 0.01, statistically significant difference between CE-LNS and CE + DTX-LNS; ## p < 0.01, statistically significant difference between DTX-LNS and CE + DTX-LNS; & p < 0.05, && p < 0.01, statistically significant difference between CE + DTX-solution and CE + DTX-LNS.

Figure 5.

The biodistribution of CE + DTX-LNS by live imaging system in vivo and ex vivo. (A) Fluorescence images of B16 tumor bearing mice at different time intervals after intravenous administration of CE + DTX-LNS (n = 3). Mice administered with NBD-CE and Rho-DTX dual labeled CE + DTX-solution (NBD-CE and Rho-DTX dissolved in DMSO and diluted with PBS) and N.S were served as control. (B) Ex vivo fluorescence images of tissues and tumor after intravenous administration of CE + DTX-LNS and CE + DTX-solution. (C) Quantitative analysis for the ex vivo tissues and tumor (n = 3). # p < 0.05, ## p < 0.01.

Figure 6.

The antitumor effect of CE + DTX-LNS in B16 tumor bearing mice in vivo and the histological evaluation. Mice treated with CE-LNS (2.5/5 mg/kg), DTX-LNS (10/20 mg/kg), Duopafei® (10/20 mg/kg), physical mixture of CE-LNS + DTX-LNS, Blank-LNS and N.S were served as controls (n = 5). (A) and (B) Tumor volume; (C) Tumor weight; (D) Body weight change. (E) Representative microscopy images of H&E-stained histological sections treated with saline and Blank-LNS (n = 3). The magnification was 100. Note: ** p < 0.01, statistically significant difference between CE-LNS and CE + DTX-LNS; # p < 0.05, ## p < 0.01, statistically significant difference between DTX-LNS and CE + DTX-LNS; & p < 0.05, && p < 0.01, statistically significant difference between Duopafei® and CE + DTX-LNS; $ p < 0.05, statistically significant difference between DTX-LNS and Duopafei®.