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Tumor microenvironment-activatable Fe-doxorubicin preloaded amorphous CaCO3 nanoformulation triggers ferroptosis in target tumor cells

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Science Advances  29 Apr 2020:
Vol. 6, no. 18, eaax1346
DOI: 10.1126/sciadv.aax1346
  • Fig. 1 Synthesis scheme and structural/compositional characterizations of the ACC-based nanoformulation.

    (A) Synthesis scheme of ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG and its complementary ferroptosis/apoptosis-based therapeutic action. After intravenous injection, ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG would accumulate in tumor tissues via the enhanced permeability and retention (EPR) effect and activated by the abnormally high MMP-2 levels at the tumor site, by which the exposed folic acid ligands could facilitate the tumor-specific uptake of the nanoformulation. (B to G) Scanning electron microscopy and TEM images of ACC@DOX.Fe2+ (B and E), ACC@DOX.Fe2+-CaSi (C and F), and ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (D and G). (H) Scanning transmission electron microscopy image of ACC-DOX.Fe2+ and the corresponding energy-dispersive spectroscopy (EDS) analysis results. (I) High-resolution transmission electron microscopy images of ACC@DOX.Fe2+. (J) Selected area electron diffraction pattern of ACC@DOX.Fe2+, indicating the amorphous nature of ACC@DOX.Fe2+ nanoparticles. (K) Fe2p x-ray photoelectron spectroscopy (XPS) spectra of ACC@DOX.Fe2+. a.u., arbitrary units.

  • Fig. 2 DOX loading and the pH-triggered drug release features.

    (A) Changes in DOX fluorescence after mixing DOX with Fe2+ at varied mix ratios in ethanol. It was observed that the intensity of DOX fluorescence reached the minimum when the DOX/Fe2+ ratio was 1:3. (B) Fluorescence spectroscopic analysis shows the dissociation of the DOX-Fe2+ complex in the buffer solution at pH 5.5, the color changes of the samples from 0 to 8 hours of incubation are indicated by the inserted images. Photo credit: Chen-Cheng Xue, Chongqing University. (C) The DOX release profiles from ACC@DOX- CaSi-PAMAM-FA/mPEG under pH 7.4 and pH 5.5. (D and E) Impact of pH on the release rate of DOX-Fe2+ complex from the ACC substrate, in which ACC@DOX.Fe2+-PAMAM-FA/mPEG was incubated in buffer solutions at different pH values for 24 hours. D, pH 7.4; E, pH 5.5.

  • Fig. 3 In vitro characterizations on the uptake and lysosomal release capabilities of the ACC-based nanoformulation.

    (A) CLSM images of 4T1 cells incubated with PBS (I), ACC@FITC-CaSi-PEG (II), ACC@FITC-CaSi-PAMAM-FA/mPEG (III) and MMP-2–treated ACC@FITC-CaSi-PAMAM-FA/mPEG (IV) for 12 and 24 hours. The blue, red, and green colors indicate cell nucleus, cell membrane, and the FITC-labeled nanoparticles, respectively. (B) CLSM evaluation on the lysosomal escape of nanoparticles where the 4T1 cells were incubated with PBS (I), ACC@FITC-CaSi-PEG (II), ACC@FITC-CaSi-PAMAM-FA/mPEG (III) and MMP-2–treated ACC@FITC-CaSi-PAMAM-FA/mPEG (IV) for 12 hours. The blue, red, and green colors indicate cell nucleus, lysosome, and the FITC-labeled nanoparticles, respectively. (C) ICP results on the intracellular iron levels of 4T1 cells after incubation with PBS, ACC@DOX.Fe2+-CaSi-PEG, ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG, and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG for 12 or 24 hours. The double-asterisk symbol indicates significance at P < 0.01. (D) Flow cytometric analysis on the intracellular lipoperoxide levels in 4T1 cells incubated 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. The lipid ROS indicator was BODIPY-C11. (E) CLSM observation on the intracellular distribution of lipoperoxides in 4T1 cells after incubation with PBS, ACC-CaSi-PAMAM-FA/mPEG, DOX, ACC@DOX-CaSi-PAMAM-FA/mPEG, ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG, and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG for 24 hours. The green fluorescence is the lipid ROS after the staining with BODIPY-C11. (F) CLSM observation on the changes in the mitochondrial membrane potential of 4T1 cells after incubation with PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV) and MMP-2–treated ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V) for 24 hours.

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

    (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).

  • Fig. 5 Fluorescence investigation on the distribution patterns of the nanoformulation in 4T1 and A375 tumor–bearing nude mice.

    (A and D) Fluorescence images of the 4T1 and A375 tumor–bearing nude mice after the intravenous injection of Cy5 (I), ACC@Cy5-CaSi (II), and ACC@Cy5-CaSi-PAMAM-FA/mPEG (III) at different time points. (B and E) Ex vivo fluorescence images of organs and tumors harvested at 24 hours for mice bearing 4T1 or A375 tumors, respectively. (C and F) Quantitative analysis on the MFI (mean fluorescence intensity) of major organs and tumors in mice bearing 4T1 or A375 tumors, respectively. Data were collected 24 hours after intravenous injection.

  • Fig. 6 Therapeutic efficacy of the nanoformulation in vivo.

    (A) Photographs of 4T1 and A375 tumor–bearing mice through the 21-day treatment period, PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV) and ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V). (B) Comparison of tumor tissues extracted from 4T1 and A375 tumor–bearing mice after the 21-day treatment period, PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV) and ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V). (C) Changes in the tumor volumes of the 4T1 tumor–bearing mice (six mice in each group) plotted against time, the tumor volume was measured every 2 days. (D) Final weight of tumor tissues extracted from 4T1 and A375 tumor–bearing mice after the 21-day treatment period. PBS (I), ACC-CaSi-PAMAM-FA/mPEG (II), DOX (III), ACC@DOX-CaSi-PAMAM-FA/mPEG (IV) and ACC@DOX.Fe2+-CaSi-PAMAM-FA/mPEG (V). (E) Survival rate of 4T1 tumor–bearing mice in 60 days (six mice in each group). (F) Survival rate of A375 tumor–bearing mice in 60 days (six mice in each group). Photo credit: Chen-Cheng Xue, Chongqing University.

Supplementary Materials

  • Supplementary Materials

    Tumor microenvironment-activatable Fe-doxorubicin preloaded amorphous CaCO3 nanoformulation triggers ferroptosis in target tumor cells

    Chen-Cheng Xue, Meng-Huan Li, Yang Zhao, Jun Zhou, Yan Hu, Kai-Yong Cai, Yanli Zhao, Shu-Hong Yu, Zhong Luo

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