Therapeutic polymeric nanomedicine: GSH-responsive release promotes drug release for cancer synergistic chemotherapy

Abstract

To obtain an efficient dual-drug release and enhance therapeutic efficiency for combination chemotherapy, a glutathione (GSH)-responsive therapeutic amphiphilic polyprodrug copolymer (mPEG-b-PCPT) is synthesized to load doxorubicin (DOX) via hydrophobic and π-π stacking interaction. In this nanomedicine system (mPEG-b-PCPT/DOX), the ratio of the two drugs can be easily modulated by changing the loading content of DOX. The in vitro drug release curves and laser confocal images suggested that the release of CPT and DOX is induced through a "release promotes release strategy": after internalization into tumor cells, the disulfide bonds in the nanomedicine are cleaved by glutathione (GSH) in the cytoplasm and then lead to the release of CPT. Meanwhile, the disassembly of nanomedicine immediately promotes the co-release of DOX. The optimum dose ratio of CPT and DOX is evaluated via the combination index (CI) value using HepG-2 cells. The results of cell apoptosis and cell viability prove the better synergistic efficiency of the nanomedicine than free drugs at the optimum dose ratio of 1. Consequently, this stimuli-responsive synergistic chemotherapy system provides a direction for the fabrication of nanomedicines possessing promising potential in clinical trials.

Scheme 1. Schematic illustration of the preparation of mPEG-b-PCPT/DOX nanomedicine and the “drug release promotes release strategy” of mPEG-b-PCPT/DOX for cancer synergistic chemotherapy.

Scheme 2. Synthetic route of mPEG-b-PCPT.

Fig. 1. Characterization of mPEG-b-PCPT and mPEG-b-PCPT/DOX. TEM images of (a) mPEG-b-PCPT and (b) mPEG-b-PCPT/DOX. TEM images of mPEG-b-PCPT/DOX after the treatment of GSH (10 mM) for (c) 2 h, (d) 6 h and (e) 24 h. Scale bar = 200 nm. Drug release curves of (f) CPT and (g) DOX from mPEG-b-PCPT/DOX NPs under different conditions. Data represent as mean ± SD (n = 3).

Fig. 2. CLSM images of HepG-2 cells treated with the mixture of free CPT and DOX, mPEG-b-PCPT/DOX NPs for 2 h, 6 h and 12 h, respectively. The cells for GSH pretreated group were pretreated with glutathione reduced ethyl ester (GSH-OEt) (concentration of 10 mM) for 2 h. Scale bar = 25 μm.

Fig. 3. (a) Viability of HepG-2 cells treated with the mixtures of CPT and DOX at indicated concentrations for 24 h. Data represent as mean ± SD (n = 4). (b) Calculated CI values for the CPT and DOX combinations for HepG-2 cells at indicated concentrations.

Fig. 4. Apoptosis analysis of HepG-2 cells treated with medium (a), mixture of free CPT and DOX (b) and mPEG-b-PCPT/DOX NPs (c) at the concentration of 0.1 μg mL⁻¹ CPT and DOX for 24 h. (d) Frequencies of necrotic cells (Q1), late apoptotic cells (Q2), early apoptotic cells (Q3), and living cells (Q4) analyzed by flow cytometry after treatments of indicated formulations. Data represent mean ± SD (n = 3). *p < 0.05, Student's t-test. (e) Viability of HepG-2 cells treated with various concentrations of the mixture of free CPT and DOX and mPEG-b-PCPT/DOX NPs at a drug mass ratio of 1 for 24 h. Data represent mean ± SD (n = 4). **p < 0.01, *p < 0.05, Student's t-test.