Assessing Binding Capacity of Melanin-Based Nanoparticles

Brimonidine, HR97-biotin, and HR97-brimonidine at a range of concentrations (3.125, 6.25, 12.5, 25, 50, 100 µg/mL) were dissolved in pH 6.5 PBS solution. The solutions (400 µL) were then mixed thoroughly with 400 µL of 1 mg/mL mNPs in pH 6.5 PBS solution and transferred to the inner reservoir of the rapid equilibrium dialysis (RED) device inserts (8 K MWCO). The outer reservoir was filled with 800 µL of pH 6.5 PBS solution. The samples were incubated on an orbital shaker with temperature control at 37 °C and 300 rpm for 48 h (n = 3). The solutions from outer reservoir (free drug) were than collected and transferred to an autosampler vial for HPLC analysis (Prominence LC2030, Shimadzu, Columbia, MD) with photodiode-array detection (PDA) system. Separation was achieved with a Luna® 5 µm C18(2) 100 Å LC column 250 × 4.6 mm (Phenomenex, Torrance, CA) at 40 °C using isocratic flow. The amount of bound drug was used to calculate the binding capacity (mol drug/mg melanin) and the dissociation constant (K_d) as previously described^{20 (link),34 (link)}.