Glycopolymer nanomicelles: pH-responsive drug delivery, endocytosis pathway, autophagy behavior, and the effect of autophagy inhibitors

Abstract

Glycopolymer drug delivery nanosystems have attracted increasing attention in the field of sustainable biomaterials and clinical biomedicine, while few studies addressed their intracellular drug delivery properties, endocytosis pathways, intracellular trafficking, autophagy behaviors and the effect of autophagy inhibitors. Based on our previous study, in this work, a pH-responsive glycopolymer (PMAgala₁₈-b-P(MAA₂₄-co-MAChol₆)) was synthesized and used as a drug delivery carrier, to encapsulate antitumor drug doxorubicin (DOX) into nanomicelles, with high DOX loading efficiency and pH-responsive DOX release properties. The cytotoxicity, cell proliferation inhibition, endocytosis pathway, intracellular trafficking/localization, and autophagy behavior of the blank glycopolymer micelles and/or DOX-loaded micelles were studied in a Human Glioblastoma Carcinoma (H4) and green fluorescent protein-tagged H4-GFP-LC3 cell lines. The glycopolymer micelles could be taken up into the cells through favorable caveolae-mediated and clathrin-mediated endocytic pathways, and their intracellular trafficking/localization were associated with endosome-lysosome systems. Notably, after treating with DOX-loaded glycopolymer micelles (or free DOX) to the H4-GFP-LC3 cells, exogenous substances-induced autophagosome accumulation was observed. The autophagy inhibitors: 3-methyladenine (3-MA) and hydroxychloroquine (HCQ) were used to monitor the autophagy behavior of H4-GFP-LC3 cells incubated with the micelles. Interestingly, the autophagy inhibitors could significantly enhance the antitumor performance of the free DOX and/or DOX-loaded micelles, the drug combination effect of autophagy inhibitors and DOX was further studied by Bliss independent model analysis. Taken together, this work provided a preliminary understanding of the intracellular drug delivery properties of glycopolymer micelles and demonstrated the effect of different autophagy inhibitors, which might inspire future innovation of "autophagy regulator-combined nanotherapeutics" toward efficient cancer chemotherapy.

Scheme 1

Proposed cellular uptake, intracellular trafficking, and autophagy behaviors of DOX-loaded glycopolymer nanomicelles in Human Glioblastoma Carcinoma cells

Fig. 1

Top: Preparation of DOX-loaded glycopolymer micelles with pH-responsive manners; Bottom: (A) DLS characterization of the hydrodynamic particle size and distributions of the blank and DOX-loaded micelles (inset: TEM image of the DOX-loaded micelles, scale bar: 200 nm); (B) time- dependent colloidal stability of the DOX-loaded micelles in PBS buffer (pH 7.4, 10 mM) at 37 °C

Fig. 2

In vitro DOX release profiles of DOX-loaded micelles and free DOX at different release media: phosphate buffer (pH 7.4, 10 mM) and acetate buffer (pH 5.0, 10 mM)

Fig. 3

CCK-8 cytotoxicity assay results (A) and LDH release assay results (B) for the PMAgala₁₈-b- P(MAA₂₄-co-MAChol₆) nanoparticles in H4 cells incubated for 24 h and 4 h, respectively. Data is presented as the average with ± standard deviation (n = 6)

Fig. 4

Cytotoxicity assay results for the free DOX and DOX-loaded nanoparticles with H4 cells (A, B) and H4-GFP-LC3 cells (C, D). A, C Cell viability at different DOX dosage after 24 h incubation; (B, D) time-dependent cell viability with the DOX dosage of 0.5 μg/mL (n = 6)

Fig. 5

Intracellular properties for the DOX-loaded micelles in H4 cells. A Endocytosis pathways: relative DOX fluorescence intensity in the presence of different endocytic inhibitors (n = 3, **P < 0.01) evaluated by flow cytometry. B Intracellular localization: representative fluorescence images for the H4 cells treated with DOX-loaded micelles for 1 h and 6 h (DOX dosage: 10 μg/mL; Scale bar: 20 μm)

Fig. 6

A Exogenous substance-induced autophagy: Fluorescence images of the H4-GFP-LC3 cells treated with free DOX or DOX-loaded micelles for 4 h (DOX dosage: 10 μg/mL). B co-localization images of autophagosome accumulated H4-GFP-LC3 cells treated with free DOX or DOX-loaded micelles for 4 h (DOX dosage: 10 μg/mL; Scale bar: 20 μm)

Fig. 7

Fluorescence images for H4-GFP-LC3 cells treated with blank micelles (50 μg/mL) and 3-MA (0.2 mM) or HCQ (1.0 μM) for 1 h. The GFP-LC3 puncta (autophagosome accumulation) were highlighted by white color arrows (Scale bar: 20 μm)

Fig. 8

A H4-GFP-LC3 cell viability in the presence of the autophagy inhibitors 3-MA or HCQ (B) cell viability after the co-treatment of autophagy inhibitors and free DOX or DOX-loaded micelles for 24 h (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001)