Fig. 3

Thrombolysis and in vitro clot-formation assay. Thrombolytic effect: supplementation with a low dose (LD), moderate dose (MD), and high dose (HD) of dipyridamole (DIP)-fucoidan (FU)-polypyrrole (PPy) nanoparticles (NPs) to in vitro clots. The quantitative thrombolytic activity of formulations was evaluated using (a) a microscopic assay (DFC7000T, LEICA) and (b) a colorimetric assay to assess the release of red blood cells (RBCs) from blood clots via Drabkin’s method. (c) Inhibition of in vitro clot formation using colorimetric assays: the addition of LD, MD, and HD of DIP or FU to platelet-poor plasma (PPP)-platelet-rich plasma (PRP) (inhibition of in vitro clot formation) before CaCl₂ was added (ECHO CHEMICAL, 2.5 mM) and thrombin (Sigma-Aldrich, 1 U/mL). (d) Platelet aggregation using colorimetric assays: the addition of the MD of DIP or FU to PPP-PRP in the presence of CaCl₂ and thrombin. (e) Fluorescence microscopic (DFC7000T, LEICA) images of non-activated and activated platelets (induced by TRAP-6, 10 µM) treated with MD DIP and DIP-FU-PPy NPs (scale bar: 50 μm). (f) Cellular P-selectin levels assessed by microscope. Quantitative (g) P-selectin and (h) DIP levels of non-activated platelets or activated platelets using a microscopic assay. (i) Fluorescent microscopic data showing a fluorescence image of the nano-formulation interacting with or without LPS-induced bovine aortic endothelial cells treated with 4’,6-giamidino-2-phenylindole (DAPI, Biotium #40,043, 1:1000, 30 min, 23 °C), propidium iodide (PI, ThermoFisher, 1.5 µM, 30 min, 23 °C), or 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, 40 µM, 30 min, 37 °C) for ROS, and NB100-65392 (1:500, 1 h, 37 °C) as a P-selectin marker (scale bar: 100 μm). Control group: cells without LPS treatment. (* p < 0.05; NS, non-significant at p > 0.05)