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. 2020 Apr 29;6(18):eaax1346. doi: 10.1126/sciadv.aax1346

Fig. 4. Evaluations on the antitumor effect of the nanoformulation in vitro.

Fig. 4

(A) Flow cytometric analysis on the apoptosis levels of 4T1 cells after incubation with PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV), ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V) and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (VI) for 12 and 24 hours. (B) CLSM observation apoptosis levels of 4T1 cells after incubation with PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV), ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V) and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (VI) for 24 hours. (C) Bright-field microscopy images of 4T1 cells after incubation with PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV), ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V) and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (VI) for 24 hours. (D) The proposed molecular mechanism for the nanoformulation-induced synergistic ferroptotic/apoptotic cell death. (E) Western blot analysis on the expression of key ferroptosis makers including BID, AIF, and EndoG, as well as apoptosis markers including NOX4, Caspase-3, BAX, and Bcl-2 in 4T1 cells after incubation with PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV), ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V) and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (VI). (F) DNA laddering assay on the DNA damage in 4T1 cells after incubation with PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV), ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V) and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (VI).