Synthesis, antitumor activity and molecular mechanism of doxorubicin conjugated trimethyl-chitosan polymeric micelle loading Beclin1 siRNA for drug-resisted bladder cancer therapy

Abstract

Herein, we describe a convenient approach for the preparation of a polymeric micelle using doxorubicin (DOX) conjugated trimethyl-chitosan (TMC) with Beclin-1 siRNA (Si-Beclin1/DOX-TMC). This micelle displayed a potent capacity for autophagy inhibition and reversed drug-resistance to DOX in BIU-87/ADR cell lines. The Si-Beclin1/DOX-TMC micelle was highly cytotoxic to both drug-sensitive BIU-87 and drug-resistant BIU-87/ADR cells. Its capacity to reverse drug-resistance was dependent upon upregulation of autophagy levels in BIU-87/ADR cells. DOX was conjugated to TMC via a pH-sensitive Schiff base, which responded to the acidic lysosome microenvironment and resulted in the cytoplasmic release of DOX. The structure of DOX conjugation to the TMC polymeric micelle was characterized by NMR, GPC, TEM and DLS. DOX release profiles in different pH environment were determined by HPLC. Cellular uptake, changes to nuclei morphology and formation of autophagosomes were observed using a fluorescence microscope. Finally, in vivo antitumor activity of systemic Si-Beclin1/DOX-TMC micelle administration was evaluated in BIU-87/ADR xenograft models and Si-Beclin1/DOX-TMC micelles showed significantly suppressed tumor growth.

Fig. 1. Synthesis route of DOX-TMC.

Fig. 2. ¹H NMR spectra of TMC, O-TMC, DOX and DOX-TMC.

Fig. 3. Characterization of siRNA loaded DOX-TMC micelles. (a) Agarose gel electrophoresis of siRNA loaded by DOX-TMC; (b) the size distribution of siRNA loaded DOX-TMC micelles; (c) stability of siRNA loaded DOX-TMC micelles in 10% serum; (d) transmission electron microscopy images of siRNA loaded DOX-TMC micelles (scale bar indicates 100 nM); (e) hemolysis test results different concentrations of siRNA loaded DOX-TMC micelles after incubation with red blood cells suspension for 3 h.

Fig. 4. (a) The accumulative release profiles of doxorubicin from DOX-TMC and si-Beclin1/DOX-TMC in different pH environment; (b) cell viability curves of sensitive BIU87 cells and resistant BIU87/ADR cells incubated with siNC/TMC, si-Beclin1/TMC, free DOX, DOX-TMC and si-Beclin1/DOX-TMC, respectively.

Fig. 5. (a) The cellular uptake of doxorubicin of free DOX or DOX-TMC by incubated with BIU87/ADR cells for 24 h (scale bar indicates 200 μM); (b) cellular uptake of Cy3-labeled siRNA of free siRNA, complexed by TMC or complexed by DOX-TMC after incubation with BIU87/ADR cells for 24 h (scale bar indicates 20 μM); (c) observation of GFP-LC3 puncta stimulated by free DOX, DOX-TMC and si-Beclin1/DOX-TMC in BIU87 or BIU87/ADR cells (scale bar indicates 6 μM).

Fig. 6. The apoptosis assays of BIU87/ADR cells treated by free DOX, DOX-TMC, si-Beclin1/TMC and si-Beclin1/DOX-TMC, respectively; (a) flow cytometry assay stained by Annexin V/PI dual-staining; (b) the nuclei morphological changes stained by Hochest33258 (scale bar indicates 50 μM; ** p < 0.01).

Fig. 7. (a) The changes on total apoptosis levels following si-Beclin1/DOX-TMC combined with autophagy inhibitor 3-MA or apoptosis inhibitor Z-VAD-FMK; (b) the changes on autophagy or apoptosis related proteins following si-Beclin1/DOX-TMC combined with 3-MA or Z-VAD-FMK; (c) the changes on autophagy levels followed by si-Beclin1/DOX-TMC or free DOX combined with 3-MA or Z-VAD-FMK (scale bar indicates 6 μM; ** p < 0.01).

Fig. 8. Antitumor effects of si-Beclin1/DOX-TMC in vivo, compared to effects observed with NS, si-Beclin1/TMC and DOX-TMC. (a) curves of tumor volumes in each group; (b) statistics of tumor weight in each group; (c) curves of body weight changes; (d) immunohistological staining against TUNEL (scale bar indicates 50 μM), Ki67, Beclin1, SQSTM1 and cleaved caspase3 (scale bar indicates 20 μM); (e) quantification of each marker in panel C (*p < 0.05 to NS group, **p < 0.01 to NS group, respectively).