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Fluoroalkylation promotes cytosolic peptide delivery

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Science Advances  12 Aug 2020:
Vol. 6, no. 33, eaaz1774
DOI: 10.1126/sciadv.aaz1774
  • Fig. 1 Fluoroalkylation of peptides.

    (A) Fluoroalkylation leads to self-assembly of peptides into nanostructures. (B) Sequences, molecular weight, hydrophobicity, and net charge of cargo peptides in this study. The hydrophobicity of peptides was calculated according to Kyte-Doolittle hydrophobicity values (40).

  • Fig. 2 Cytosolic delivery of fluoroalkylated peptides.

    (A) Transmission electron microscope (TEM) images of assembled F-P1 and F-P2. Scale bars, 500 nm. (B) The viability of HeLa cells treated with fluorous-tagged or unmodified peptides. Data are presented as means ± SD (n = 5). (C) Confocal images of HeLa cells treated with peptides for 6 hours. Scale bar, 40 μm. Unmodified, TAT-modified, and R8-modified peptides were tested as controls. Peptides (10 μM) were tested for each sample. (D) Relative fluorescence intensity of cells treated with the peptides in (C). The fluorescence intensity of cells treated with unmodified peptides was defined as 1. Data are presented as means ± SD (n = 3). ***P < 0.001 analyzed by one-factor analysis of variance (ANOVA). (E) Confocal images of HeLa cells treated with peptides modified with tag 2 or tag 3 for 6 hours (10 μM peptide). Scale bar, 40 μm.

  • Fig. 3 Endocytosis pathways of fluorous-tagged peptides.

    Confocal images of HeLa cells treated with different doses of P1 or F-P1 for 6 hours (A) or with 10 μM P1 or F-P1 for 1 to 8 hours (B). Scale bar, 40 μm. The fluorescence intensities of cells in (A) and (B) analyzed by flow cytometry are shown in (C) and (D), respectively. Data are presented as the means ± SD (n = 3). ***P < 0.001 analyzed by Student’s t test. (E) Confocal images of HeLa cells treated with 10 μM F-P1 for 6 hours. The cells were pretreated with different inhibitors before peptide incubation. Scale bar, 40 μm. (F) Proposed endocytosis pathways for F-P1.

  • Fig. 4 Proteolytic stability of fluorous-tagged peptides.

    (A) Degradation behavior of P1, TAT-P1, 2-P1, and F-P1 after incubation with chymotrypsin for 0 to 12 hours. Data are presented as the means ± SD (n = 3). (B) Fluorescence images of HeLa cells treated with F-P1 or TAT-P1 for 6 hours. The peptides were added with chymotrypsin before cytosolic delivery to prove the proteolytic stability of F-P1. A representative result from three independent experiments. Scale bar, 200 μm. (C) Flow cytometric analysis of peptide-incubated cells in (B). (D) Degradation behavior of P3, TAT-P3, 2-P3, and F-P3 after incubation with chymotrypsin for 0 to 12 hours. Data are presented as the means ± SD (n = 3). (E) Flow cytometric analysis of HeLa cells treated with F-P3 or TAT-P3 for 6 hours. The cells were added without or with chymotrypsin during cytosolic delivery. (F) Cytosolic delivery of F-P1 into 293T, NIH3T3, MCF7, and RAW264.7 cells, respectively, for 6 hours. The concentration of F-P1 in each well is 10 μM. Scale bar, 40 μm.

  • Fig. 5 Cytosolic delivery of fluorous-tagged KLA.

    (A) Cytosolic delivery of F-P6 into cancer cells induces cell apoptosis and death. (B) Confocal images of HeLa cells treated with 4 μM FITC-labeled peptides P6, F-P6, or TAT-P6 for 6 hours. The mitochondria in cells were stained by MitoTracker Red CMXRos. Scale bar, 40 μm. (C) TEM image and dynamic light scattering (DLS) of assembled F-P6a. Scale bar, 500 nm. (D) Confocal images of calcein-treated HeLa cells. The cells were incubated with 10 μM P6a, F-P6a, or TAT-P6a for 6 hours before calcein treatment. Scale bar, 40 μm. (E) LDH release from peptide-treated cells. Data are presented as means ± SD (n = 3). ***P < 0.001 analyzed by one-factor ANOVA. (F) Apoptosis of peptide-treated HeLa cells analyzed by an annexin V/propidium iodide (PI) staining assay. The cells were incubated with 10 μM P6a, F-P6a, or TAT-P6a for 24 hours.

  • Fig. 6 Therapeutic effect of fluorous-tagged proapoptotic peptide KLA in vivo.

    (A) Tumor volume of mice treated with PBS, P6a, 2-P6a, F-sP6a, TAT-P6a, and F-P6a, respectively. Data are presented as means ± SD (n = 5). **P < 0.01 and ***P < 0.001 analyzed by one-factor ANOVA. (B) Image of excised tumors from the therapeutic groups. Scale bar, 1 cm. (C) Tumor weight in different groups. Data are presented as means ± SD (n = 5). *P < 0.05 and ***P < 0.001 analyzed by one-factor ANOVA. (D) Body weight of mice during the therapeutic period. Data are presented as means ± SD (n = 5).

Supplementary Materials

  • Supplementary Materials

    Fluoroalkylation promotes cytosolic peptide delivery

    Guangyu Rong, Changping Wang, Lijie Chen, Yang Yan, Yiyun Cheng

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