2.6. Flocculation and Precipitation of Chondroitin Sulfate, Heparin, Non-Anticoagulant Heparin Carrying Polystyrene (NAC-HCPS) and Low-Molecular-Weight Heparin/Protamine Nanoparticles (LMWH/P NPs) with BiSCaO and BiSCa(OH)2 Colloidal Dispersions

Various amounts of 1 N HCl were added to 0.5 mL of 0.4 wt.% BiSCaO or BiSCa(OH)₂ colloidal dispersions containing 75 wt.% PP compared with BiSCaO or BiSCa(OH)₂ to adjust the pH, from strongly alkaline (>12) to weakly acidic (≈5), then 0.5 mL of 0.1 wt.% 0.1 mg/mL (0.01 wt.%) chondroitin sulfate, heparin, NAC-HCPS or LMWH/P NPs was added and each mixture was vortexed for 10 sec. Each generated flocculent/precipitate was separated by centrifugation (1000 rpm, 190× g) for 5 min. Chondroitin sulfate, heparin, and NAC-HCPS remaining in the supernatant were measured using a Blyscan Glycosaminoglycan Assay Kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and the optical densities of the supernatants at 656 nm were measured using a spectrophotometer.