http://orcid.org/0000-0002-9853-913X
Figures & data
Figure 1. Schematic illustration of CD44-targeted delivery of HA-CH-NP/siRNA as a selective PLXDC1 siRNA delivery carrier for anti-angiogenesis tumor therapy. (A) Overview of this study. (B) Expression of PLXDC1 in normal tissues and in ovarian tissues from ovarian cancer patients. Scale bars indicate 50 µm.
Figure 2. Physical properties of CH-NP/PLXDC1 siRNA and HA-CH-NP/PLXDC1 siRNA. (A) Preparation of HA-CH-NP/PLXDC1 siRNA. (B) Particle size and (C) zeta potential of HA-CH-NP/PLXDC1 siRNA. The particle size and zeta potential were measured by dynamic light scattering with a particle size analyzer. (D) The efficiency of loading Cy5-labeled control siRNA into CH-NPs and HA-CH-NPs was determined by fluorescence spectrophotometry. (E) Binding efficiency of HA on the surface of HA-CH-NPs. (F) FITC-HA-labeled with HA-CH-NPs was examined by UV-visible spectrophotometry at 494 nm. (G) The complexation of the HA-CH-NPs was determined by FT-IR spectroscopy. The FT-IR spectra of the HA-CH-NP were confirmed by amide bonds for NH vibration (N-H bending at 1650 cm−1) of CH and -ROOH bonding of HA at 1550 cm−1. (H) The morphologies of the CH-NPs and HA-CH-NPs were determined by AFM. Scale bar: 250 nm. Error bars represent SEM; *p < .05.
Figure 3. The biological effect of PLXDC1 in HUVEC cells. (A) Expression of PLXDC1 in ovarian tumor cells or endothelial cells. (B) PLXDC1 silencing using PLXDC1 siRNA in endothelial cells. (C) Invasion, migration, and tube formation in HUVEC cells following PLXDC1 silencing. Error bars represent SEM; *p < .05.
Figure 4. CD44-targeted delivery of HA-CH-NPs. (A) CD44 expression in ovarian tumor cells or endothelial cells. (B) CD44 expression in tumor endothelial cells against A2780 tumor tissue. CD44 (green) and tumor neovasculature protein (CD31, red) were shown in colocalization (yellow) in tumor tissue. Scale bars indicate 20 µm. (C) CD44-mediated intracellular delivery of CH-NPs and HA-CH-NPs. Scale bars indicate 10 µm. Error bars represent SEM; *p < .05.
Figure 5. In vivo distribution of HA-CH-NPs/Cy5 siRNA. (A) In vivo distribution of HA-CH-NPs/Cy5 siRNA after i.v. injection in A2780 tumor-bearing mice. (B) HA-CH-NPs/Cy5 siRNA localization in tumor endothelial cells. The HA-CH-NPs/Cy5 siRNA was injected into mice through tail vein. The nuclei were stained with Hoechst 33258 (blue), vascular endothelial cells were stained with anti-CD31 (red), and CD44 receptors were stained with anti-CD44 (yellow). Scale bars indicate 20 µm. Error bars represent SEM; *p < .05.
Figure 6. Therapeutic efficacy of HA-CH-NPs in an orthotopic ovarian cancer model. Treatment with HA-CH-NPs was started 1 week after the intraperitoneal (i.p.) injection of mice with (A) A2780 (PLXDC1-negative) and (B) HeyA8 (PLXDC1-positive) tumor cells. HA-CH-NP/PLXDC1 siRNA was injected i.v. twice per week at doses of 150 µg/kg PLXDC1 siRNA-based on body weight. The fold change in levels of PLXDC1 mRNA represents the mean of triplicates evaluated by qRT-PCR. (C) Immunohistochemical analyses for markers of PLXDC1 expression in endothelial cells (PLXDC1 antibody), cell proliferation (Ki67), microvessel density (MVD, CD31), and TUNEL were performed on A2780 tumor tissues (scale bar: 50 µm). Results represent the mean ± standard deviation (SD). Statistical tests were two-sided and p values were evaluated by analysis of variance (ANOVA); *p < .05.
