Nucleolin-Targeting AS1411 Aptamer-Modified Micelle for the Co-Delivery of Doxorubicin and miR-519c to Improve the Therapeutic Efficacy in Hepatocellular Carcinoma Treatment

Abstract

Background: Multidrug resistance (MDR) has emerged to be a major hindrance in cancer therapy, which contributes to the reduced sensitivity of cancer cells toward chemotherapeutic drugs mainly owing to the over-expression of drug efflux transporters. The combination of gene therapy and chemotherapy has been considered as a potential approach to improve the anti-cancer efficacy by reversing the MDR effect.

Materials and methods: The AS1411 aptamer-functionalized micelles were constructed through an emulsion/solvent evaporation strategy for the simultaneous co-delivery of doxorubicin and miR-519c. The therapeutic efficacy and related mechanism of micelles were explored based on the in vitro and in vivo active targeting ability and the suppression of MDR, using hepatocellular carcinoma cell line HepG2 as a model.

Results: The micelle was demonstrated to possess favorable cellular uptake and tumor penetration ability by specifically recognizing the nucleolin in an AS1411 aptamer-dependent manner. Further, the intracellular accumulation of doxorubicin was significantly improved due to the suppression of ABCG2-mediated drug efflux by miR-519c, resulting in the efficient inhibition of tumor growth.

Conclusion: The micelle-mediated co-delivery of doxorubicin and miR-519c provided a promising strategy to obtain ideal anti-cancer efficacy through the active targeting function and the reversion of MDR.

Keywords: aptamer; micelle; multidrug resistance; nucleolin; tumor targeting.

Scheme 1

The illustration of micelle preparation.

Figure 1

Characterization and DOX release profile of MDPAS micelles. (A) The TEM images of M (a), MDP (b) and MDPAS micelles (c). The hydrodynamic size (B) and zeta potential (C) change of micelles stored at 4 °C for 7 days. (D) The cumulative release of DOX from MDPAS micelles under different pH conditions. Data were presented as mean ± SD of triplicate experiments.

Figure 2

Cellular uptake and intracellular distribution of MDPAS micelles. The cellular uptake efficiency of different formulations in HepG2 (A) and L02 cells (B) through FACS analysis (**p < 0.01). (C) The CLSM images for the intracellular distribution of MDPAS micelles in HepG2 cells after the incubation for 2, 6 and 12 h, respectively. Blue (DAPI, nucleus), green (LysoTracker Green, lysosome) and red (DOX). Scale bar: 50 μm.

Figure 3

Expression level of ABCG2 and apoptosis-related proteins. (A) The ABCG2 expression level in HepG2 cells after the treatment with different formulations through qPCR analysis (**p < 0.01, vs control group). (B) Western blotting analysis and the quantitative results (C) of procaspase-3, −8 and −9 and ABCG2 expression in HepG2 cells after the treatment with different formulations. (D) The intracellular DOX concentration in HepG2 cells after the treatment with micelles for different time by LC-MS/MS analysis.

Figure 4

Anti-proliferative effect of MDPAS micelles. (A) The cell viability of HepG2 cells after the treatment with different micelles by MTT assay (**p < 0.01, vs control group). (B) Live/Dead cell staining assay of HepG2 cells treated with control (a), free DOX (b), MDP (c) and MDPAS micelles (d), respectively. Scale bar: 200 μm. (C and D) FACS analysis of the cell apoptosis and cell cycle arrest of HepG2 cells treated with control (a), M (b), free DOX (c), MDP (d) and MDPAS micelles (e).

Figure 5

Distribution and cell apoptosis in 3D tumor spheroids. (A) The CLSM images of 3D tumor spheroids treated with different micelles by scanning along the Z-axis. Scale bar: 50 μm. (B) The 3D reconstruction of spheroids treated with free DOX (a), MDP (b) and MDPAS micelles (c), respectively. (C) FACS analysis for the cell apoptosis in 3D spheroids after the treatment with different formulations: control (a), M (b), free DOX (c), MDP (d) and MDPAS micelles (e).

Figure 6

Biodistribution analysis of MDPAS micelles. (A) In vivo biodistribution of micelles in different organs and tumor tissues (n=3 per group). (B–D) The mean fluorescence intensity of different organs and tumor tissues at 1, 4 and 12 h post-injection, respectively.

Figure 7

In vivo anti-cancer efficacy and biocompatibility of MDPAS micelles. (A) The tumor volume profile of HepG2-xenografted nude mice after the treatment with different formulations (n=6 per group, *p < 0.05 and **p < 0.01, vs control group). (B) Representative images of excised tumor tissues from tumor-bearing mice received different treatments. Scale bar: 1 cm. (C) Representative images of H&E and immunohistochemical staining of tumor tissues. Scale bar: 100 nm. (D) Representative images of H&E staining of heart, liver, spleen, lung and kidney at 28 days post-administration. Scale bar: 100 nm.