Aminoglucose-functionalized, redox-responsive polymer nanomicelles for overcoming chemoresistance in lung cancer cells

Abstract

Background: Chemotherapeutic drugs used for cancer therapy frequently encounter multiple-drug resistance (MDR). Nanoscale carriers that can target tumors to accumulate and release drugs intracellularly have the greatest potential for overcoming MDR. Glucose transporter-1 (GLUT-1) and glutathione (GSH) overexpression in cancer cells was exploited to assemble aminoglucose (AG)-conjugated, redox-responsive nanomicelles from a single disulfide bond-bridged block polymer of polyethylene glycol and polylactic acid (AG-PEG-SS-PLA). However, whether this dual functional vector can overcome MDR in lung cancer is unknown.

Results: In this experiment, AG-PEG-SS-PLA was synthetized successfully, and paclitaxel (PTX)-loaded AG-PEG-SS-PLA (AG-PEG-SS-PLA/PTX) nanomicelles exhibited excellent physical properties. These nanomicelles show enhanced tumor targeting as well as drug accumulation and retention in MDR cancer cells. Caveolin-dependent endocytosis is mainly responsible for nanomicelle internalization. After internalization, the disulfide bond of AG-PEG-SS-PLA is cleaved in the presence of high intracellular glutathione levels, causing the hydrophobic core to become a polar aqueous solution, which subsequently results in nanomicelle disassembly and the rapid release of encapsulated PTX. Reduced drug resistance was observed in cancer cells in vitro. The caspase-9 and caspase-3 cascade was activated by the AG-PEG-SS-PLA/PTX nanomicelles through upregulation of the pro-apoptotic proteins Bax and Bid and suppression of the anti-apoptotic protein Bcl-2, thereby increasing apoptosis. Furthermore, significantly enhanced tumor growth inhibition was observed in nude mice bearing A549/ADR xenograft tumors after the administration of AG-PEG-SS-PLA/PTX nanomicelles via tail injection.

Conclusions: These promising results indicate that AG-PEG-SS-PLA/PTX nanomicelles could provide the foundation for a paradigm shift in MDR cancer therapy.

Keywords: Cancer therapy; Gult-1; Multidrug resistance; Nanomicelles; Redox-responsive polymer.

Scheme 1

Schematic representation of the assembly, tumor targeting, and cellular internalization of AG-PEG-SS-PLA nanomicelles leading to reversal of MDR in lung cancer therapy

Scheme 2

Synthetic scheme showing the various steps required to prepare AG-PEG-SS-PLA

Fig. 1

¹H NMR of P-P, P-SS-P, and AG-P-SS-P

Fig. 2

a Excitation spectra of pyrene in an AG-P-SS-P solution after incubation with GSH for different time periods (λ_em = 373 nm). b I340-to-I336 ratio of AG-P-SS-P and P-SS-P after incubation with GSH for different time periods (λ_em = 373 nm). DLS measurement (c) and TEM images (d) of AG-P-SS-P/PTX, P-SS-P/PTX, and P-P/PTX. Scale bar = 100 nm. e In vitro PTX release profiles of AG-P-SS-P/PTX, P-SS-P/PTX, and P-P/PTX in PBS (pH 7.4) without GSH at 37 °C; release profile of AG-P-SS-P/PTX in the presence of 5 mM GSH; and release profiles of AG-P-SS-P/PTX and P-SS-P/PTX in the presence of 10 mM GSH. f Retention of PTX in A549/ADR cells after preincubation with AG-P-SS-P/PTX, P-SS-P/PTX, P-P/PTX, and Taxol for different time periods. All data are presented as the means ± standard deviations (n = 3); *P < 0.05, compared with P-P/PTX without GSH, P-SS-P/PTX without GSH, AG-P-SS-P/PTX without GSH and AG-P-SS-P/PTX with GSH (5 mM) nanomicelles. *P < 0.05, compared with Taxol, P-P/PTX and P-SS-P/PTX nanomicelles, respectively

Fig. 3

a The cellular uptake of AG-P-SS-P/Rh123 with 0 and 10 mM GSH by A549/ADR cells using laser scanning confocal microscopy (×400). b1 CLSM images of A549/ADR cells after 30 min of incubation with P-SS-P/Rh123 (non-targeted) and AG-P-SS-P/Rh123 (targeted) (×200). Free AG (10 mM) was used to block GLUT1 binding before adding AG-P-SS-P/Rh123. b2 Fluorescence intensity of Rh123-loaded P-SS-P (non-targeted) and AG-P-SS-P (targeted) in A549/ADR cells. Quantitative analysis was performed with ImageJ. *P < 0.05, compared with 10 mM GSH. *P < 0.05, compared with AG-P-SS-P/Rh123 nanomicelles, respectively. (1) Image of CLSM; (2) Fluorescence intensity of image

Fig. 4

Flow cytometry analyses of A549 (a) and A549/ADR (b) cells following a 2-h incubation with free Rh123, P-P/Rh123, P-SS-P/Rh123, or AG-P-SS-P/Rh123. c Laser scanning confocal microscopy-based comparison of the cellular uptake of Rh123 from different formulations by A549 and A549/ADR cells (scale bar, 30 mm). d Analysis of the pathway responsible for the uptake of AG-P-SS-P/Rh123 nanomicelles by A549 cells. This analysis was performed 30 min after incubation. The cells were blocked with different inhibitors: 2 mM AG, 10 mM AG, 1 μg/mL colchicine, 0.4 μg/mL PhAsO, or 0.5 μg/mL filipin. Green, Rh123 (scale bar, 50 mm). *P < 0.05, compared with Free Rh123 in A549/PTX cells, respectively. *P < 0.05, compared with PBS, respectively. (1) Image of CLSM; (2) fluorescence intensity of image

Fig. 5

Flow cytometry analyses of A549/ADR cells after incubation with AG-P-SS-P/Rh123. The A549/ADR cells were precultured with BSO or GSH-OEt. *P < 0.05, compared with Free Rh123 in A549/PTX cells, respectively. *P < 0.05, compared with PBS, respectively. 1, image of CLSM; 2, Fluorescence intensity of image

Fig. 6

a Viability of A549 (a1) and A549/ADR (a2) cells cultured with PTX-loaded nanomicelles in comparison with that of Taxol at the same PTX dose for 48 h. b Cell apoptosis rate detected by flow cytometry. A549 and A549/ADR cells were treated with different formulations that contained a total PTX concentration of 10 µM for 24 h. c Proteins involved in the apoptosis signaling pathways in A549 and A549/ADR cells as determined by Western blotting (c1). (1) Control (PBS); (2) Taxol; (3) P-P/PTX; (4) P-SS-P/PTX; and (5) AG-P-SS-P/PTX nanomicelles. Activity ratios of caspase-3 and caspase-9 and expression ratios of the pro-apoptotic proteins Bax and Bid and the anti-apoptotic proteins Bcl-2 and Bcl-xl in A549 and A549/ADR cells after incubation with the various formulations. β-actin was also assessed by Western blotting. All protein levels were quantified densitometrically and normalized to β-actin (c2). All data are presented as the means ± standard deviations (n = 3); (1) image of western blot; (2) grey level of western blot. *P < 0.05, compared with AG-P-SS-P/PTX nanomicelles. ^#P < 0.05, compared with A549 cells

Fig. 7

Tumor images (a1) and tumor growth inhibition graph (a2) for a murine model with A549/ADR xenografts after intravenous injection with the different formulations. b Body weight changes of the tumor-bearing mice after treatment with the various formulations. The arrow indicates the day of drug administration. The data are presented as the means ± standard deviations (n = 5); ^aP < 0.05 compared with the control; ^bP < 0.05 compared with Taxol; ^cP < 0.05 compared with P-P/PTX; and ^dP < 0.05 compared with P-SS-P/PTX nanomicelles