Fig. 4: Neutralization of histones by HNPs after their binding to the cell surface. | Nature Communications

Fig. 4: Neutralization of histones by HNPs after their binding to the cell surface.

From: Synthetic hydrogel nanoparticles for sepsis therapy

Fig. 4

a Neutralization of the cell surface-bound histones by PEGHNPs. 2H-11 cells were treated with histones for 60 s. Then, PEGHNPs were added after the PBS washing to remove free histones in the medium. At 24 h after HNP addition, viable cells were determined by a WST-8 assay. Data represent the means ± s.d. n = 4. b Cellular uptake of histones. 2H-11 cells were incubated with Cy5-histones and PEGHNPs for 24 h. Then, the cells were lysed and the amount of Cy5-histones was measured. Significant difference; ***p < 0.0001 vs. Histones alone, p = 0.0012 vs. Naked HNP + His, p = 0.062 vs. PEGHNP8 + His, and p = 0.0001 vs. and PEGHNP11 + His. Data represent the means ± s.d. n = 3. Differences within a group were evaluated by one-way analysis of variance (ANOVA) with the Tukey post hoc test using Kaleidagraph (Version 4.5.3). c Cellular uptake of HNPs. 2H-11 cells were incubated with FITC-HNPs and/or histones for 24 h. Then, the cells were lysed and the amount of FITC-HNPs was measured. Significant difference; ***p < 0.0001 vs. Naked HNP(+), p = 0.0001 vs. PEGHNP8 (+), and p < 0.00012 vs. PEGHNP11 (+). Data represent the means ± s.d. n = 4. The data were analyzed by two-tailed Student’s t tests. d Removal of the cell surface-bound histones by PEGHNP washing. 2H-11 cells were treated with Cy5-labeled histones for 10 s. The cells were washed with PEGHNPs 1 or 3 times. Then, the cells were lysed and the fluorescent intensity of Cy5-histones was measured. Significant difference; p = 0.046 Histone alone vs. Naked HNP, p = 0.046 Histone alone vs. PEGHNP8, p = 0.046 Histone alone vs. PEGHNP11, and p = 0.026 Histone alone vs. PEGHNP12. Data represent the means ± s.d. n = 3. The data were analyzed by two-tailed Student’s t tests. e, f Localization of Cy5-histones and FITC-PEGHNPs. 2H-11 cells were treated with Cy5-histones for 10 s. Then, the cells were washed with FITC-PEGHNPs. At 30 min (e) and 24 h (f) after the washing, the localization of Cy5-histones and FITC-HNPs was observed laser scanning confocal microscopy. Red: histones; Green: HNPs; Bar: 50 µm. The experiment was repeated two times and three pictures were taken in each experiment.

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