Preparation and Evaluation of Liposomes Co-Loaded with Doxorubicin, Phospholipase D Inhibitor 5-Fluoro-2-Indolyl Deschlorohalopemide (FIPI) and D-Alpha Tocopheryl Acid Succinate (α-TOS) for Anti-Metastasis

Abstract

Tumor metastasis has become a key obstacle to cancer treatment, which causes high mortality. Nowadays, it involves multiple complex pathways, and conventional treatments are not effective due to fewer targets. The aims of the present study were to construct a novel liposome delivery system co-loading a specific PLD inhibitor 5-fluoro-2-indolyldes-chlorohalopemide (FIPI) in combination with antitumor drug doxorubicin (DOX) and functional excipient D-alpha tocopheryl acid succinate (α-TOS) for anti-metastasis. In this study, the liposomes containing three components (DFT-Lip) with different physicochemical properties were successfully prepared by film dispersion method combined with pH-gradient method. Physicochemical parameters such as particles size, potential, encapsulation efficiency, stability, and release profiles were investigated. In vitro and in vivo anti-metastasis effectiveness against highly metastatic breast cancer MDA-MB-231 cell line was evaluated. The liposomes showed uniform particle size (approximately 119 nm), high drug encapsulation efficiency (> 90%), slow release characteristics and stability. In vitro anti-tumor cell metastasis study demonstrated DFT-Lip could greatly inhibit motility, migration and invasion of MDA-MB-231 cells compared to other liposomes, predicting a synergistic anti-tumor metastasis effect between FIPI with α-TOS in liposomes. In vivo anti-metastasis study showed that DFT-Lip prevented the initiation and the progression of metastasis of high metastatic breast cancer. These results suggested that the liposomes containing DOX, FIPI, and α-TOS might be a promising strategy for metastatic tumor therapy in clinics.

Keywords: Anti-metastasis; DOX; FIPI; Liposomes; α-TOS.

Fig. 1

Structural formulas of 5-fluoro-2-indolyldes-chlorohalopemide (FIPI), D-α-tocopheryl acid succinate (α-TOS), and doxorubicin hydrochloride

Fig. 2

In vitro release profiles of FIPI and DOX from DFT-lip

Fig. 3

Stability of DFT-lip at 4 °C and 25 °C in PBS for 15 days determined by particle size and polydispersity index

Fig. 4

Flow cytometric measurement of cellular uptake by breast cancer MDA-MB-231 cells after incubation with free DOX, DOX-lip, and DFT-lip at a concentration of 5 μM DOX for 4 h at 37 °C. The auto-fluorescence of cells was applied as the control. All the data presented here were calculated as mean ± SD (n = 3). Notes: ns, p > 0.05; ***p < 0.001

Fig 5

Cell viability of MDA-MB-231 cells incubated with Blank-lip, FIPI-lip, DF-lip, DT-lip, and DFT-lip for 7 h (a) or 24 h (b) at a concentration of 2 μM FIPI, respectively. All the data presented here were calculated as mean ± SD (n = 6). Notes: ns, p > 0.05 versus blank liposomes

Fig. 6

In vitro anti-metastasis study of the liposomes. a Inhibition effects on the cell motility in vitro. Images of MDA-MB-231 cells wound edge after incubation with control, Blank-lip, FIPI-lip, DF-lip, DT-lip, and DFT-lip for 48 h, respectively (magnification, × 100). b Inhibition effects on cell migration. Images of MDA-MB-231 cell migration after incubation with control, Blank-lip, FIPI-lip, DF-lip, DT-lip, and DFT-lip for 7 h at a concentration of 2 μM FIPI (magnification, × 100). c Inhibition effects on the cell invasion. Images of MDA-MB-231 cell invasion after incubation with control, Blank-lip, FIPI-lip, DF-lip, DT-lip, and DFT-lip for 24 h a concentration of 2 μM FIPI (magnification, × 100)

Fig. 7

In vivo anti-metastasis study of the liposomes. a Bioluminescent images of the nude mice model of lung metastasis of breast cancer, which was constructed with the injection of MDA-MB-231/Luc cells via tail vein. b Representative in vivo bioluminescent images of mice in a preventive protocol treated with Blank-lip and DFT-lip (n = 8). c The extent of tumor metastasis burden was quantified (n = 8). Notes: ***p < 0.001 versus Blank-Lip. d Body weight changes for the tumor-bearing mice

Fig. 8

Schematic diagram of action mechanism for DFT-liposome