Novel pH-sensitive nanoformulated docetaxel as a potential therapeutic strategy for the treatment of cholangiocarcinoma

Abstract

Background: Cholangiocarcinoma (CC) is one of the fatal malignant neoplasms with poor prognosis. The traditional chemotherapy has been resistant to CC and does not improve the quality of life. The aim of the present study is to investigate the potential of chondroitin sulphate (CS)-histamine (HS) block copolymer micelles to improve the chemotherapeutic efficacy of docetaxel (DTX).

Results: pH-responsive property of CS-HS micelles was utilized to achieve maximum therapeutic efficacy in CC. In the present study, docetaxel-loaded CS-HS micelles (CSH-DTX) controlled the release of drug in the basic pH while rapidly released its cargo in the tumor pH (pH 5 and 6.8) possibly due to the breakdown of polymeric micelles. A nanosize of <150 nm will allow its accumulation in the tumor interstitial spaces via EPR effect. CSH-DTX effectively killed the cancer kills in a time- and concentration-dependent manner and showed pronounced therapeutic action than that of free drug at all-time points. CSH-DTX resulted in higher apoptosis of cancer cells with ~30% and ~50 of cells in early apoptosis quadrant when treated with 100 and 1000 ng/ml of equivalent drug. The micellar formulations showed remarkable effect in controlling the tumor growth and reduced the overall tumor volume to 1/5(th) to that of control and half to that of free drug treated group with no sign of drug-related adverse effects. Immunohistochemical analysis of tumor sections showed that fewer number of Ki-67 cells were present in CSH-DTX treated group comparing to that of free DTX treated group.

Conclusion: Our data suggests that nanoformulation of DTX could potentially improve the chemotherapy treatment in cholangiocarcinoma as well as in other malignancies.

Figure 1

Schematic representation of conjugation of chondroitin sulphate (CS)-histidine (HS) via chemical reactions. Schematic illustration of self-assembly of docetaxel (DTX) and CS-HS conjugate into polymeric micelles.

Figure 2

(a) Typical size distribution analysis of CSH-DTX by dynamic light scattering technique (b) transmission electron microscope (TEM) imaging of CSH-DTX (c) scanning electron microscope (SEM) imaging of CSH-DTX.

Figure 3

Release profile of DTX from CSH-DTX micelles incubated at phosphate buffered saline (pH 7.4) and acetate buffered saline (pH 6.8 and 5.0). The samples were incubated at 37°C in a rotary shaker (100 rpm). The data are presented as mean ± SD (n = 3). *p < 0.05, *p < 0.01 is the statistical difference between drug release at pH 5.0, pH 6.5, and pH 7.4.

Figure 4

(a) In vitro cytotoxicity of blank polymeric micelles at various concentrations against QBC939 cells (b-d in vitro cytotoxicity of free DTX and CSH-DTX against QBC939 cells incubated at 24, 48, and 72 h. The cytotoxicity of formulations was evaluated by MTT assay. The data are presented as mean ± SD (n = 6).

Figure 5

(a) Cellular morphology of QBC939 cells following incubation with free DTX and CSH-DTX (b) fluorescence microscopy images of the cell apoptosis induced by free DTX and CSH-DTX. The apoptosis of cells was analysed by Hoechst staining.

Figure 6

Flow cytometer analysis of cell apoptosis using annexinV-FITC and PI staining. The cells were exposed with free DTX and CSH-DTX at a concentration of 100 ng/ml and 1000 ng/ml and incubated for 24 h. **p < 0.01 is the statistical difference CSH-DTX and free DTX.

Figure 7

In vivo antitumor efficacy study (a) changes in tumor volume (b) changes in mice body weight (c) images of tumor sections. The antitumor study was carried out in QBC939 cells -bearing xenograft model and administered thrice at a fixed dose of 5 mg/kg. *p < 0.05, ***p < 0.001 is the statistical difference in the tumor volume between CSH-DTX and free DTX or CSH-DTX and control group.

Figure 8

(a) histopathology of tumor sections (b) immunohistochemical analysis of tumor cell proliferation (Ki-67) (c) immunohistochemical analysis of cleaved PARP (apoptosis marker).