Organic polydopamine nanoparticles ameliorate endothelial ischemia-reperfusion injury, increasing antioxidant defense and reducing inflammatory response

Background Protecting endothelial cells from oxidative stress is vital to alleviate ischemia/reperfusion injury (IRI). Polydopamine nanoparticles (PDA NPs), an organic biodegradable class of nanoparticles with excellent antioxidant properties, have shown great potential in treating myocardial IRI. Purpose To investigate the antioxidant and anti-inflammatory therapeutic properties of PDA NPs in a model of endothelial hypoxia-reperfusion (HR) injury. Methods Human umbilical vein endothelial cells (HUVEC) were treated with PDA NPs (25 µg/mL) after 24h of hypoxia followed by 6 or 24 h of reoxygenation. Cell uptake of PDA NPs was quantified by flow cytometry. Dichlorofluorescin diacetate and MitoSOX-based flow cytometric assay were performed to evaluate cell oxidative stress and mitochondrial oxidative stress, respectively. Real-time quantitative PCR (RT-qPCR) assessed mRNA levels of antioxidant gene enzymes. ELISA assay measured antioxidant enzyme activities and inflammatory protein expression. Results HUVECs subjected to 24 h of hypoxia followed by 6 or 24 hours of reoxygenation (H/R group) showed increased oxidative stress, inflammatory response and reduced expression of antioxidant gene enzymes compared to normoxic conditions. PDA NPs, internalized by HUVEC after 6h and 24 h of reoxygenation, reduced the H/R-induced ROS production (-76%±8% at 6 h, -83%±15 % at 24 h, p<0.01) and mitochondrial ROS generation (-35%±6% at 6 h, -51% ± 11%, p < 0.05). PDA NPs increased gene expression level of superoxide dismutase (2.2 and 4 fold increase at 6 and 24 h, respectively, p <0.01), catalase (1.5 and 2.5 fold increase at 6h and 24h, respectively, p <0.05) and glutathione peroxidase (2.8 and 3 fold increase, at 6h and 24h, respectively, p < 0.05). This upregulation is correlated to increased antioxidant activity of enzymes. Moreover, PDA NPs exhibited anti-inflammatory activity as demonstrated by reduced IL-6 (60±12 pg/mL vs H/R group 103±20 at 6h p < 0.01, 83 pg/mL vs H/R group 170±20 at 24 h, p < 0.01) and intercellular adhesion molecule 1-protein levels (108±17 pg/mL vs H/R group 157±15 at 6h, p < 0.05; 180±26 pg/mL vs H/R group 317±31 at 24 h, p < 0.01). Conclusions These findings demonstrate the antioxidant and anti-inflammatory activity of PDA NPs against hypoxia/reoxygenation injury, highlighting their potential as a promising therapeutic strategy for endothelial IRI.