2.2.4. Determination of Critical Micelle Concentration

The critical micelle concentrations (CMCs) of the blank and complex-loaded micellar pre-concentrates were determined using a previously described fluorometric technique [15]. Pyrene—acting as a fluorescent probe partition between the aqueous phase and the micellar hydrophobic core—was dissolved at 6 × 10⁻⁷ M in 0.1 M phosphate buffer (pH 6.8.) Next, the micellar pre-concentrates were diluted across a range of 100–1000 µg/mL in pyrene solution, vortexed, thermomixed at 60 °C for 2 h, and cooled under shaking overnight. The fluorescence excitation of the samples at λ_em = 390 nm was measured using a Tecan microplate reader (Tecan infinite 200, Tecan Austria GmbH, Salzburg, Austria). A decimal logarithmic concentration-versus-I₃₃₈/I₃₃₃ plot was generated to determine the CMC. Finally, the corresponding dilutions of the micelles and Kolliphor surfactants were analyzed individually for droplet size using 0.0001–0.1 dilutions in the buffer.