Figure 4: Intracellular ATP-triggered Dox release.

(a) Dox released in MDA-MB-231 cells of Dox/NG and Dox/cNG obtained using the flow cytometry. The cells were incubated with Dox/NG or Dox/cNG at 37 °C for 2 h, and subsequently incubated with fresh culture medium at 37 °C for additional 2 h or 4 h after removal of the excess nanogels. Error bars indicate s.d. (n=3). **P<0.01 (two-tailed Student’s t-test). (b) The ATP content in MDA-MB-231 cells after different treatments. Error bars indicate s.d. (n=3). **P <0.01 compared with the control group (two-tailed Student’s t-test). (c) Dox released in MDA-MB-231 cells of Dox/NG obtained using the flow cytometry. The cells were incubated with Dox/NG at 37 °C for 2 h, and then incubated with the fresh culture medium at 4 °C or with IAA at 37 °C for additional 2 h or 4 h after removal of the excess nanogels. Error bars indicate s.d. (n=3). **P<0.01 (two-tailed Student’s t-test). (d) Intracellular delivery of Dox/NG on MDA-MB-231 cells at different time observed by CLSM. The cells were incubated with Dox/NG at 37 °C for 2 h, and further incubated with fresh culture medium at 37 °C for additional 4 h after removal of the excess nanogels. The late endosomes and lysosomes were stained by LysoTracker Green, and the nuclei were stained by Hoechst 33342. Scale bar is 10 μm. (e,f) In vitro cytotoxicity of Dox/cNG and Dox/NG on MDA-MB-231 (e) and MCF-7 cells (f) for 24 h. Error bars indicate s.d. (n=4). *P<0.05, **P<0.01 (two-tailed Student’s t-test). (g) Flow cytometric analysis of MDA-MB-231 cell apoptosis induced by Dox/cNG and Dox/NG for 12 h using the Annexin V-FITC/PI staining. In each panel, the lower-left (Annexin V-FITC⁻, PI⁻), lower-right (Annexin V-FITC⁺, PI⁻) and upper-right (Annexin V-FITC⁺, PI⁺) quadrants represent the populations of live cells, early apoptotic cells and late apoptotic cells, respectively. The average population (%) in each quadrant is indicated by the numbers at the corner of the panels. (h) MDA-MB-231 cell apoptosis induced by Dox/cNG and Dox/NG for 20 h using the APO-BrdU TUNEL assay. Alexa Fluor 488-stained nick end label showed green fluorescence, and PI-stained nuclei showed red fluorescence. Scale bar is 100 μm.