Figures & data
Figure 1. Properties of PLGA@Icaritin. (A) TEM image of PLGA without PEG, PLGA and PLGA@Icaritin. Scale bar, 100 nm. (B) The particle size and surface charge of PLGA without PEG, PLGA and PLGA @Icaritin. (C) The Zate potential of PLGA without PEG, PLGA and PLGA@Icaritin. (D) The release curve of PLGA@Icaritin at pH = 6 and pH = 7.4 solutions. Data are shown as the mean ± SD, n = 3. ***Indicates p < 0.001.
Figure 2. Cell uptake of PLGA@Icaritin. (A) CLSM image of MFC cells treated with PLGA@Icaritin for 0–12 h, and corresponding quantification of CLSM images using the green mean fluorescence intensity of FITC. Scale bar, 20 μm. (B) Flow cytometry images of MFC cells treated with PLGA@Icaritin for 0–12 h, and corresponding quantification of green mean fluorescence intensity of FITC inside cell. Data are shown as the mean ± SD, n = 3. ***Indicates p < 0.001.
Figure 3. In vitro anti-tumor effect of PLGA@Icaritin. (A) Cell Vabilities of MFC cells after incubation with PLGA, icaritin and PLGA@Icaritin. (B) LDH leakage of MFC cells after incubation with PLGA, icaritin and PLGA@Icaritin. (C) Cell cycle distribution images. (D) Statistical graph of G2/M phase rates. (E,F) The effect of PLGA, icaritin and PLGA@Icaritin on migration and invasion was assessed with transwell migration assay and tranwell invasion assay, respectively. 200×. Data are shown as the mean ± SD, n = 3. ***Indicates p < 0.001.
Figure 4. Mitochondrial oxidative damage induced by PLGA@Icaritin. (A) The generation of ROS in MFC cells treated with PLGA, icaritin and PLGA@Icaritin. (B) Corresponding quantification of green mean fluorescence intensity of FITC inside cells. (C,D) Flow cytometric analysis of untreated, PLGA-treated, icaritin-treated and PLGA@Icaritin-treated MFC cells stained with JC-1 after 72 h of exposure for mitochondrial transmembrane potential (MMP) evaluation. (E,F) Ox-mtDNA and TOM20 images of MFC cells, detected by CLSM. Scale bar, 20 μm. Data are shown as the mean ± SD, n = 3. ***Indicates p < 0.001.
Figure 5. ICD induced by PLGA@Icaritin in vitro. (A) CRT image of MFC cells after the incubation with PLGA, icaritin and PLGA@Icaritin. Scale bar, 30 μm. (B) HMGB1 image of MFC cells after the incubation with PLGA, icaritin and PLGA@Icaritin. Scale bar, 30 μm. (C) Relative expression of CRT in MFC cells after the incubation with PLGA, icaritin and PLGA@Icaritin. (D) Relative expression of HMGB1 in MFC cells after the incubation with PLGA, icaritin and PLGA@Icaritin. (E) The ATP secretion of MFC cell after incubation with PLGA, icaritin and PLGA@Icaritin. (F) Secreted HMGB1 of MFC cell after incubation with PLGA, icaritin and PLGA@Icaritin. Data are shown as the mean ± SD, n = 3. **Indicates p < 0.01; ***indicates p < 0.001.
Figure 6. In vivo anti-tumor effect of PLGA@Icaritin. (A) Image of tumors derived from mice treated with saline, PLGA, icaritin and PLGA@Icaritin. (B) Tumor volume of MFC tumors isolated from mice. (C) Tumor weight of MFC tumors isolated from mice. (D) Body weight of MFC tumor-bearing mice. Data are shown as the mean ± SD, n = 3. **Indicates p < 0.01; ***indicates p < 0.001.
Figure 7. Immune response induced by PLGA@Icaritin. (A) Production of CD3 + CD4+ and CD3 + CD8+ T cells in splenic lymphocytes harvested from immunized mice. (B,C) The proportions of CD3 + CD4+ and CD3 + CD8+ T cells in splenic lymphocytes. (D) Quantitative analysis of IFN-γ in serum samples of MFC tumor-bearing mice after different treatments. (E) Quantitative analysis of TNF-α in serum samples of MFC tumor-bearing mice after different treatments. (F) Quantitative analysis of IL-6 in serum samples of MFC tumor-bearing mice after different treatments. Data are shown as the mean ± SD, n = 3. **Indicates p < 0.01; ***indicates p < 0.001.
Scheme 1. Schematic diagram of the proposed therapeutic mechanisms of PLGA@Icaritin NPs.
