Novel ginsenoside-based multifunctional liposomal delivery system for combination therapy of gastric cancer

Abstract

The clinical treatment of gastric cancer (GC) is hampered by the development of anticancer drug resistance and the unfavorable pharmacokinetics, off-target toxicity, and inadequate intratumoral accumulation of the current chemotherapy treatments. Ginsenosides combined with paclitaxel (PTX) have been shown to exert synergistic inhibition of human GC cell proliferation. In the present study, we developed a novel multifunctional liposome system, in which ginsenosides functioned as the chemotherapy adjuvant and membrane stabilizer. These had long blood circulation times and active targeting abilities, thus creating multifunctionality of the liposomes and facilitating drug administration to the GC cells. Methods: Three ginsenosides with different structures were used to formulate the unique nanocarrier, which was prepared using the thin-film hydration method. The stability of the ginsenoside liposomes was determined by particle size analysis using dynamic light scattering. The long circulation time of ginsenoside liposomes was compared with that of conventional liposome and polyethylene glycosylated liposomes in vivo. The active targeting effect of ginsenoside liposomes was examined with a GC xenograft model using an in vivo imaging system. To examine the antitumor activity of ginsenoside liposomes against GC, MTT, cell cycle, and apoptosis assays were performed on BGC-823 cells in vitro and PTX-loaded ginsenoside liposomes were prepared to evaluate the therapeutic efficacy on GC in vivo. Results: The ginsenosides stabilized the liposomes in a manner similar to cholesterol. We confirmed the successful delivery of the bioactive combination drugs and internalization into GC cells via analysis of the glucose-related transporter recognition and longer blood circulation time. PTX was encapsulated in different liposomal formulations for use as a combination therapy, in which ginsenosides were found to exert their inherent anticancer activity, as well as act synergistically with PTX. The combination therapy using these targeted liposomes significantly suppressed GC tumor growth and outperformed most reported PTX formulations, including Lipusu^® and Abraxane^®. Conclusion: We established novel ginsenoside-based liposomes as a tumor-targeting therapy, in which ginsenoside functioned not only as a chemotherapy adjuvant, but also as a functional membrane material. Ginsenoside-based liposomes offer a novel platform for anticancer drug delivery and may lead to a new era of nanocarrier treatments for cancer.

Keywords: Ginsenoside; combination therapy; gastric cancer; liposome; multifunction; paclitaxel..

Figure 1

Characterization of ginsenoside liposomes. (A) Chemical structure of cholesterol and the ginsenosides. (B) Transmission electron microscope images of ginsenoside liposomes and cholesterol liposome (C-lipo); scale bar = 50 nm. (C) Change in size and polydispersity index of different liposomal formulations stored at 4 °C (n = 3; mean ± standard deviation [SD]). (D) Blood circulation profiles of C-lipo, polyethylene glycolated C-lipo (PEG-C-lipo), and three ginsenoside liposomes (n = 3; mean ± SD).

Figure 2

Cellular uptake and internalization mechanism of liposomes in BGC-823 cells. Qualitative (A) and quantitative (B and C) cellular uptake of carboxyfluorescein (FAM)-labeled liposomes in BGC-823 cells. The cells were incubated with 500 ng/mL FAM-loaded liposomes at 37 °C for 4 h (n = 3; mean ± standard deviation [SD]); scale bar = 50 µm. (D-F) Quantitative cellular uptake of ginsenoside liposomes with different glucose transporter inhibitors. The cells were pre-incubated with 20 mM glucose, 0.3 mM phloridzin, or 0.2 mM quercetin for 60 min, respectively (n = 3; mean ± SD). ***P < 0.001; *P < 0.05.

Figure 3

In vivo targeting effect of ginsenoside liposomes. (A) In vivo imaging of DiR-labeled C-lipo and ginsenoside liposomes in BGC-823 tumor-bearing mice. (B) Ex vivo imaging of excised tumors at 24 h after injection of DiR-labeled liposomes. (C) The relative fluorescence intensity in tumors from different groups (n = 3; mean ± standard deviation). *P < 0.05; **P < 0.01.

Figure 4

In vitro anticancer activities of ginsenoside liposomes. (A-E) Cytotoxicity of different ginsenoside liposomes and free ginsenosides to BGC-823 cells (n = 6; mean ± standard deviation [SD]). (E-F) Cell-cycle progression of BGC-823 cells treated with different ginsenoside liposomes and free ginsenosides (n = 3; mean ± SD). (G-I) Apoptosis induced by different ginsenoside liposomes and free ginsenosides in BGC-823 cells (n = 3; mean ± SD). **P < 0.01; ***P < 0.001.

Figure 5

In vitro anticancer activities of paclitaxel (PTX)-loaded ginsenoside liposomes. Cytotoxicity (A) and IC₅₀ values (B) of free PTX and different types of PTX-loaded liposomes in BGC-823 cells (n = 6; mean ± standard deviation [SD]). (C) Cell-cycle analysis of BGC-823 cells treated with free PTX and different types of PTX-loaded liposomes (n = 3; mean ± SD). (D, F) Induction of apoptosis by free PTX and different types of PTX-loaded liposomes in BGC-823 cells (n = 3; mean ± SD). (E) Inverted fluorescence microscope images of cell apoptosis (arrows indicate apoptotic bodies; scale bar = 100 µm). **P < 0.01; ***P < 0.001.

Figure 6

In vivo anticancer activities of paclitaxel (PTX)-loaded ginsenoside liposomes. (A) In vivo tumor growth inhibition after intravenous injection of different PTX formulations at a dose of 10 mg/kg (n = 6; mean ± SD). (B) Excised tumors from BGC-823 tumor-bearing nude mice on the day after the last injection (n = 6; mean ± SD). (C) Tumor weights of excised tumors (n = 6; mean ± SD). (D) Body weight variation over the course of the treatment (n = 6; mean ± SD). (E) Representative hematoxylin and eosin stained sections of the heart, liver, spleen, lungs, and kidneys (Scale bar = 100 µm). ***P < 0.001.