Indomethacin-based stimuli-responsive micelles combined with paclitaxel to overcome multidrug resistance

Abstract

Development of multidrug resistance against antitumor agents is a major limiting factor for the successful chemotherapy. Currently, both amphiphilic polymeric micelles and chemosensitizers have been proposed to overcome MDR during chemotherapy. Herein, the redox-responsive polymeric micelles composed of dextran and indomethacin (as chemosensitizer) using a disulfide bond as the linker are prepared (DEX-SS-IND) for delivery of antitumor agent paclitaxel (PTX). The high level of glutathione in tumor cells selectively breaks the disulfide bond, leading to the rapid breakdown and deformation of redox-responsive polymeric micelles. The data show that DEX-SS-IND can spontaneously form the stable micelles with high loading content (9.48 ± 0.41%), a favorable size of 45 nm with a narrow polydispersity (0.157), good stability, and glutathione-triggered drug release behavior due to the rapid breakdown of disulfide bond between DEX and IND. In vitro antitumor assay shows DEX-SS-IND/PTX micelles effectively inhibit the proliferation of PTX-resistant breast cancer (MCF-7/PTX) cells. More impressively, DEX-SS-IND/PTX micelles possess the improved plasma pharmacokinetics, enhanced antitumor efficacy on tumor growth in the xenograft models of MCF-7/PTX cells, and better in vivo safety. Overall, DEX-SS-IND/PTX micelles display a great potential for cancer treatment, especially for multidrug resistance tumors.

Keywords: breast cancer; drug delivery system; indomethacin; multidrug resistance; paclitaxel.

Figure 1. Preparation and characterization of DEX-SS-IND/PTX micelles

(A) Schematic formation of DEX-SS-IND/PTX micelles. (B) ¹H NMR spectra of dextran, indomethacin and DEX-SS-IND (a and c, -CH-, at 3∼4 ppm; b and d, -CH-, 7∼8 ppm) (C) Negative-stain transmission electron microscopy of DEX-SS-IND blank micelles and DEX-SS-IND/PTX micelles. Scale bar = 100 nm.

Figure 2. In vitro release behaviors and stability of DEX-SS-IND/PTX micelles

(A) and (B) In vitro stability of DEX-SS-IND/PTX micelles at 4°C, including size and PDI. (C) The size changes of DEX-SS-IND/PTX micelles in the different reduction environments. ^* P < 0.05. (D) PTX release of DEX-IND/PTX and DEX-SS-IND/PTX micelles in PBS 7.4 with or without 10 mM GSH. (E) PTX release of DEX-SS-IND/PTX micelles in PBS 7.4 with various concentrations of GSH. (F) and (G) NR fluorescent images and quantification of DEX-SS-IND/NR or DEX-IND/NR micelles after incubation with different concentrations of GSH for 10, 20 and 40 min.

Figure 3

(A) Intracellular GSH levels of MCF-7/PTX cells and BSO-pretreated MCF-7/PTX cells, ^* P < 0.05. (B) In vitro GSH-triggered NR release from DEX-IND/NR micelles and DEX-SS-IND/NR micelles (red) in MCF-7/PTX and BSO-pretreated MCF-7/PTX cells, Scale bar = 25 nm. (C) The semi-quantitative values of fluorescence intensity of (B). (D) Quantitative determination of GSH-triggered NR release via flow cytometry. (E) The intracellular concentration of PTX in the DEX-SS-IND/PTX micelles at different time points (6 and 24 h). Data were presented as mean ± SD (n = 3), ^* P < 0.05. (F) Representative western blot analysis of MRP1 in MCF-7/PTX cells treated with DEX-SS-IND,β-actin was used as control. (G) Densitometric analysis for detecting thelevels of MRP1. Values were normalized against β-actin. Data were presented as mean ± SD (n = 3), ^*P < 0.05.

Figure 4. In vitro anti-tumor activity of DEX-SS-IND/PTX micelles

(A) and (B) MTT assay of blank DEX-SS-IND and DEX-IND micelles against MCF-7 and MCF-7/PTX cells after 48 h incubation (n = 3). (C) MTT assay of PTX-based formulations against MCF-7 and MCF-7/PTX cells after 48 h incubation (n = 3), ^* P < 0.05. (D) and (E) Imaging and quantitative analysis of live (green) cell assay for exposure of MCF-7/PTX cells to different PTX-based formulations, Scale bar = 100 nm.

Figure 5

(A) Maximum tolerated dose of DEX-SS-IND/PTX (n = 3). (B) Plasma concentration-time curves after intravenous administration of Taxol, DEX-IND/PTX and DEX-SS-IND/PTX micelles (equivalent 10 mg/kg PTX). (C) Biodistribution in tissue in mice after intravenous administration of Taxol, DEX-IND/PTX and DEX-SS-IND/PTX micelles at equivalent 10 mg/kg PTX for different times (6 and 12 h). Data were presented as mean ± SD (n = 4), ^* P < 0.05.

Figure 6. In vivo antitumor efficacy of DEX-SS-IND/PTX micelles

(A) and (B) Tumor growth curves of mice after administrated with different PTX-based formulations (n = 6). (C) MCF-7/PTX xenograft tumor sections stained with H&E for the histological examination, with Ki67 for the tumor proliferation analyses and with TUNEL for the apoptosis analyses (200 ×).

Figure 7. Reduced systemic toxicity of PTX by DEX-SS-IND/PTX micelles

(A) and (B) Body weight changes of nude mice bearing MCF-7 and MCF-7/PTX xenograft tumors, respectively (n = 6). (C) H&E staining microphotos of heart and liver tissues of mice bearing MCF-7/PTX xenograft tumors at the end of antitumor inhibition test (200×).