Augmented Anticancer Effects of Cantharidin with Liposomal Encapsulation: In Vitro and In Vivo Evaluation

Abstract

PEGylated liposomes have received much attention as pharmaceutical carriers to deliver chemotherapeutic agents for therapeutic purpose. The aim of this study was to prepare and characterize PEGylated liposome of cantharidin and investigate its therapeutic effect on human hepatocellular carcinoma treatment in vitro and in vivo. Liposomal cantharidin was evaluated for their anticancer effects in vitro using human hepatocellular carcinoma HepG2 cells and in vivo using HepG2-bearing nude mice compared to free drug. PEGylated liposome of cantharidin had a particle size of 129.9 nm and a high encapsulation efficacy of approximately 88.9%. The liposomal cantharidin had a higher anti-proliferative effect vis-à-vis free cantharidin in inducing G2/M cell cycle arrest and apoptosis. Liposomal cantharidin killed more HepG2 cancer cells at the same concentration equivalent to free cantharidin. Further study in vivo also showed that liposomal cantharidin achieved a higher tumor growth inhibition efficacy than free drug on hepatocellular carcinoma. As our study exhibited enhanced cytotoxicity against HepG2 cells and augmented tumor inhibitory effects in vivo, the results validate the potential value of cantharidin-liposome in improving the therapeutic efficacy of cantharidin for liver cancer.

Keywords: HepG2; PEGylated liposome; anti-proliferative effect; cantharidin; drug delivery system; hepatocellular carcinoma; xenograft tumor.

Figure 1

Cantharidin chemical structure and it possible anticancer mechanism. Cantharidin-induced apoptosis is probably via six pathways according to the literature: (a) tumor suppressors p53 and p21; (b) the mitochondrial Bax and Bcl-2 proteins; (c) the JAK/STAT pathway; (d) the transcription factor nuclear factor-κB (NF-κB); (e) Wnt-β catenin; and (f) down-regulated. FAS ligand gene (FASLG). Abbreviations: PP2A, protein phosphatase 2A; IKK, IκB kinase; IκB, inhibitor of NF-κB; TNF-α, tumor necrosis factor α; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; FasL, Fas ligand; Cyto C, cytochrome c; ROS, reactive oxygen species; JAK/STAT, janus tyrosine kinase/signal transducers and activators of transcription; Wnt, wingless-type MMTV integration site family; DVL1L1, dishevelled; GSK3, glycogen synthase kinase 3; APC, adenomatous polyposis coli; CK1, casein kinase 1;

Figure 2

(A) Schematic illustration of cantharidin encapsulated into PEGylated liposome; and (B) size distribution of liposomes. Particle size of liposomal cantharidin before extrusion, after extrusion, and the blank liposome after extrusion was determined by a Delsa Nano equipment.

Figure 3

(A) Anti-proliferative effects of cantharidin and liposomal cantharidin on HepG2 cells. HepG2 cells were treated with different concentrations of cantharidin and liposomal cantharidin for 24, 48, and 72 h. Results obtained from MTT assay are expressed as percentage of cell growth relative to controls. Results are an average of triplicate experiments and the SD is shown in a bar. ** p < 0.01 when compared to the free cantharidin group at the same concentration. (B) Morphological observation of HepG2 cells in different treatment groups. HepG2 cells were treated with different concentrations of free cantharidin or liposomal cantharidin for 24 h and stained with Hoechst 33342 followed observation with a fluorescence microscope. The morphological changes of the nuclei of HepG2 cells including chromatin condensation or fragmentation could be seen after treatment with cantharidin and liposomal cantharidin. Scale bar = 100 μm.

Figure 4

(A) Effects of cantharidin and liposomal cantharidin on cell-cycle distribution in HepG2 cells. HepG2 cells were treated with different concentrations of cantharidin and liposomal cantharidin for 24 h and stained with PI. Cellular DNA contents were monitored by flow cytometry. The cell cycle profiles of HepG2 cells under different treatment. (B) The percentages of each cell cycle are presented as the mean ± SD of three independent experiments. ** p < 0.01 vs. the control. (C) Analysis of apoptotic cell death by flow cytometry. HepG2 cells were treated with PBS, 50 µM of free cantharidin, and liposomal cantharidin for 24 h. The apoptotic cell death of HepG2 cells was analyzed with an Annexin V–FITC apoptosis detection kit by flow cytometry. (D) The quantitative data for later apoptotic cells(Q2 (Annexin V+ and PI+)) and early apoptotic cells (Q4 (Annexin V+ and PI-)) obtained by flow cytometry (n = 3, mean ± SD). ** p < 0.01 vs. the control and free cantharidin.

Figure 5

Anticancer efficacy of cantharidin and liposomal cantharidin on HepG2-tumor bearing nude mice in vivo. (A) Tumor volume profiles of nude mice in different treatment groups (n = 6, mean ± SEM). Mice were administrated with saline, free cantharidin and liposomal cantharidin at a cantharidin dose of 0.35 mg/kg for six intravenous injections in total at three-day intervals. (B) Body weights of mice were monitored during the whole experiment periods (n = 6, mean ± SEM). (C) After 42 days, tumors in different groups were excised and photographed. (D) The tumor weight was recorded at the end of the experiments on 42 days. Liposomal cantharidin showed significant lower tumor weights compared to control group (n = 6, means ± SEM). * p < 0.05 compared to the control.

Figure 6

Proposed mechanisms of better anticancer effect of liposomal cantharidin.