Egm 2

Confluent HUVEC grown on a well plate coated with 8 µg/mL collagen (Gibco, Gaithersburg, MD, USA) were exposed to NPs at different concentrations (10, 20, 30, 40, 50, 100, 300 and 500 μg/mL) for 24 h. Cell Counting Kit-8 (CCK- 8 kit, Sigma, St. Louis, MO, USA) was used to measure the viability of cells. CCK-8 allows sensitive colorimetric determination of the number of viable cells using a formazan dye. After incubation, absorbance of the formazan dye was recorded at 450 nm using a microplate reader (Biotek Synergy H1, Winooski, VT, USA). Tested samples included ZnO NPs, Dye 847 alone, as well as ZnO particles conjugated to dye 847. Stock solutions of the NPs were prepared by sonicating and vortexing the NPs in water at a concentration of 1 mg/mL. Once the stock solution was prepared, particles were sonicated again for the time determined in the previous test and diluted to working concentrations in endothelial growth medium-2 (EGM-2, Lonza, Walkersville, MD, USA). The concentrations of NPs represent physiologically relevant doses of NPs, based on previous studies [41 (link),43 (link),44 (link),46 (link),96 (link),97 (link),98 (link)]. Cells in EGM-2 without NPs served as the positive control, and cells exposed to 2% Triton X-100 (Sigma- Aldrich, St. Louis, MO, USA) in EGM-2 served as the negative control.

To analyze cell viability in the HS fibrin gels, passage 3–5 AFC were dissociated and resuspended in EGM-2 (Lonza) at 4x10⁵ cells/mL. 75 uL HS (250 mM NaCl) and PS (145 mM NaCl) fibrin gels were fabricated using the 4X AFC suspension as drops in wells of a 6-well tissue culture-treated plate (three gels per well). After gelation, 2 mL of EGM-2 was added to each well and the gels were incubated at 37°C and 5% CO₂. After 1, 24, and 96 hours, EGM-2 was aspirated and cells were stained using the fluorescent LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen) according to kit instructions. Three images were captured from each gel using a Zeiss Observer.Z1 and long-distance objective (LD Plan-NEOFLUAR 20X/0,4 Ph2) and were used to count living and dead cells.
To analyze the cell-mediated degradation kinetics of HS and PS fibrin formulations, gels were fabricated with a final concentration of 1x10⁵ AFC/mL (P3-5) and incubated in EGM-2 +/- 1 mg/mL of the plasmin inhibitor 6-aminocaproic acid (Sigma, A2504) at 37°C and 5% CO₂. After 0, 7, and 14 days, gels were imaged using phase contrast (Zeiss ObserverZ.1) and wet weights were recorded.

The CSE was acquired as described previously^{14 (link)}. Briefly, we burned one cigarette (carbon monoxide 14 mg/cigarette, nicotine 1 mg, tar 13 mg; Tobacco Hunan Industrial, China), collected the smoke in 20 mL of EGM-2 by a vacuum pump, removed particles and bacteria by 0.22 μM pores, then diluted the solution to 1% CSE with EGM-2. In 2 mL of EGM-2, we dissolved 5 g of Dec (Sigma, California, USA), then diluted the solution to 2 μmol/L with EGM-2 and stored it at -80°C until the experiments.