2.3. Critical Micellar Concentration with Nile Red Assay

Critical Micellar Concentration (CMC) using nile red probe was determined as previously described [24]. Briefly, nile red was dissolved in solvent to 300 nM with or without the addition of micellar triblock copolymers at concentrations ranging from 0–10% (w/v). Using a microplate spectrophotometer (Biotek, Winooski, VT, USA), nile red fluorescence was measured between 575 and 700 nm at room temperature in phosphate buffer saline pH 7.4. The shift that occurs in maximum emission wavelength peak between nile red in solvent versus nile red in micelles was calculated and represented as change in maximum emission at different concentrations of micellar triblock copolymer. For the data that formed two distinct lines, the CMC was calculated as the intersect of two linear fits. For the data in which transition was too steep to provide distinct lines, a sigmoidal fit was applied, and the second derivative was used to calculate the CMC.