Research ArticleMATERIALS SCIENCE

New power of self-assembling carbonic anhydrase inhibitor: Short peptide–constructed nanofibers inspire hypoxic cancer therapy

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Science Advances  06 Sep 2019:
Vol. 5, no. 9, eaax0937
DOI: 10.1126/sciadv.aax0937
  • Fig. 1 Molecular design of self-assembled CA IX inhibitors and their hypoxic cancer cell–targeted self-assembly.

    (A) Depending on overexpression of CA IX enzymes of hypoxic cancer cells, small molecules of N-pepABS achieve CA IX–targeted self-assembly, which concentrate CA IX inhibitors on hypoxic cancer cell membrane and subsequently interrupt the normal activities of hypoxic cancer cells. In addition, these N-pepABS–based nanofibers undergo CA IX–regulated endocytosis, promoting intracellular uptakes of the self-assembled nanofibers under hypoxia. During the process of internalization (stages I to IV), N-pepABS–based nanofibers may convert to much bigger bunches of nanofiber that pierce intracellular acid vesicles, therefore introducing highly selective toxicities for hypoxic cancer cells. (B) The chemical structures of self-assembled CA IX inhibitors. (C) In vitro gelation performance of N-pepABS, N-pep, pepABS, and ABS at pH 6.5 or pH 5.5. (D) Transmission electron microscope (TEM) images of hydrogels formed by 0.75 wt % of N-pepABS and N-pep at pH 6.5. (E) Environment scanning electron microscope images of MDA-MB-231 cells after 24-hour incubation with 500 μM medium control, N-pepABS, or N-pep under hypoxia condition (1.0% of O2) (Photo credit: Chunying Chen, The National Center for Nanoscience and Technology of China).

  • Fig. 2 Nanofibers inhibit CA IX–associated cancer cell behaviors.

    (AW) Self-assembled nanofibers may concentrate CA inhibitors on the hypoxic cancer cell membrane, enhancing their inhibitory effect. (A) The alterations of extracellular culture medium after 48-hour treatment of 500 μM N-pepABS or N-pep for MDA-MB-231 cells under hypoxia, with the appearance of gel-like materials (black arrows) on the bottom of culture plate. (B) Transwell data of MDA-MB-231 cells treated with 200 μM N-pepABS, ABS, and solvent control for 24 hours under hypoxia, followed by another 10-hour migration in the upper chamber. (C) The mRNA and protein expression level of CA IX after cells incubated for 48 hours. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Cell proliferation of MDA-MB-231 with treatment of N-pepABS, N-pep, pepABS, or ABS, respectively, for 72 hours under both hypoxia (sky blue) and normoxia (black). The bar image was represented as means ± SD, while *P < 0.05 was thought as significant difference (Photo credit: Chunying Chen, The National Center for Nanoscience and Technology of China).

  • Fig. 3 Nanofibers promote CA IX–regulated endocytosis under hypoxia.

    (AW) Artwork here illuminates the whole process of CA IX-regulated endocytosis for nanofibers under hypoxia. (A) CA IX–regulated endocytosis of N-pepABS nanofibers has appeared under hypoxia after 24-hour treatment of 500 μM N-pepABS (+) or medium control (−). Scale bar, 20 μm. We further observe subsequent (B) endolysosomal swelling and (C and D) intracellular acid vesicles injuries after 48-hour treatment of 500 μM N-pepABS under hypoxia. Scale bars, 20 μm. Then, blockage of protective autophagy has been detected after 48-hour treatment of 500 μM N-pepABS. (E) Ratio of mRNA levels of Atg5/GADPH, with the bar image represented as means ± SD, while *P < 0.05 was thought as significant difference. (F) Fluorescence images of autophagosome accumulation in the cytoplasm of hypoxic cancer cells. Scale bars, 20 μm. LC3B, light chain 3B. (G) Western blot assays of autophagy-related signals in MDA-MB-231 cells and their (H) autophagy flux study with 1-hour pretreatment of 10 nM bafilomycin A1 (Baf). cPARP, cleaved poly(adenosine diphosphate–ribose) polymerase.

  • Fig. 4 CA IX–induced nanofiber internalizations in hypoxic cancer cells.

    (AW) Hypothesis of structure upgrades during CA IX-induced nanofiber internalization. TEM images of nanofibers formed by 0.75 wt % of N-pepABS at (A) pH 6.5 or (B to D) pH 5.5. TEM images of nanofibers (E) dissociatively appearing in the cytoplasm (black arrows) and (F) destroying monolayer vesicles (white arrows) of hypoxic MDA-MB-231 cells, after 48-hour treatment of 500 mM N-pepABS. Scale bars, 5 μm (E) and 500 nm (F).

  • Fig. 5 Antihypoxic and antimetastasis performance of N-pepABS treatment in tumor models.

    Immunofluorescence images of expression alterations on (A) CA IX, (B) HIF-1α, (C) LC3B, and (D) Ki67 after treatments of phosphate-buffered saline (PBS) control, N-pep, pepABS, ABS, and N-pepABS. Inhibition of tumor growth and metastasis in 4T1 tumor model: (E) N-pepABS performs in vitro inhibitory effects on hypoxic 4T1 cell growth; (F) N-pepABS treatment apparently inhibits tumor growth and decreases tumor weight of 4T1 cancer. The bar graph was represented as means ± SD, while *P < 0.05 was thought as significant difference (n = 5), using two-tailed t tests. (G) Statistical analysis of flow cytometry for CA IX expression in tumor tissues after N-pepABS treatment. The bar graph was represented as means ± SD, while *P < 0.05 was thought as significant difference (n = 3), using two-tailed t tests. (H) Hematoxylin and eosin staining images of lung tissues with metastasis of 4T1 tumor cells from six different samples and (I) its statistical analysis of the number of tumor lesions per lobi pulmonis. The bar graph was represented as means ± SD, *P < 0.05 was thought as significant difference (n = 5), using two-tailed t tests. (J) Immunofluorescence images of endothelial marker CD31, indicating blood vessel variation after N-pepABS treatment. (Photo credit: Chunying Chen, The National Center for Nanoscience and Technology of China).

  • Fig. 6 The combined therapeutic efficacy of N-pepABS and low dose of Dox on MDA-MB-231 xenografts.

    (A to D)Tumor growth was apparently inhibited after the combination use of N-pepABS and Dox. Their volumes and weights were compared and statistically analyzed with different treatment exposures. The bar graph was represented as means ± SD, while *P < 0.05 was thought as significant difference (n = 6), performing nonparametric tests. (E) Necrotic regions were observed by hematoxylin and eosin staining experiments with different treatment exposures. (F) Western blots assays of the expression of CA IX in tumor tissues. Immunofluorescence images of (G) Ki67 illuminated cell proliferation attenuation, and (H) CD31 indicated blood vessel variation with different treatment exposures.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax0937/DC1

    Fig. S1. Hydrogel performances and characterizations of N-pepABS.

    Fig. S2. CA IX–related cell behaviors in MDA-MB-231 and HeLa cells.

    Fig. S3. CA IX–related endocytosis performances in MDA-MB-231.

    Fig. S4. CA IX down-regulation inhibits nanofiber endocytosis under hypoxia.

    Fig. S5. Acid vesicle injuries blocked protective autophagy in MDA-MB-231 cells.

    Fig. S6. The antihypoxic cancer cells effects of N-pepABS treatment in vivo.

    Fig. S7. Inhibition of tumor growth and metastasis on 4T1 tumor model.

    Fig. S8. In vivo toxicity evaluation after different treatments.

    Fig. S9. The synthesis and characterization of samples.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Hydrogel performances and characterizations of N-pepABS.
    • Fig. S2. CA IX–related cell behaviors in MDA-MB-231 and HeLa cells.
    • Fig. S3. CA IX–related endocytosis performances in MDA-MB-231.
    • Fig. S4. CA IX down-regulation inhibits nanofiber endocytosis under hypoxia.
    • Fig. S5. Acid vesicle injuries blocked protective autophagy in MDA-MB-231 cells.
    • Fig. S6. The antihypoxic cancer cells effects of N-pepABS treatment in vivo.
    • Fig. S7. Inhibition of tumor growth and metastasis on 4T1 tumor model.
    • Fig. S8. In vivo toxicity evaluation after different treatments.
    • Fig. S9. The synthesis and characterization of samples.

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