1525 a binary pump

100 μl of supernatant was diluted with 1 mL of acetonitrile and vortexed vigorously. 200 μl of the resulting mixture was further diluted 10 times using acetonitrile and vortexed again. The amount of tacrolimus in final mixture was determined using reversed phase high performance liquid chromatography (HPLC). The HPLC system (Waters™ 1500 series controller, USA) was equipped with wavelength detector (Waters™ 2489 a Dual™ Absorbance detector, USA), pump (Waters™ 1525 a Binary pump, USA), and an automated sampling system (Waters™ 2707 Plus Autosampler, USA). The HPLC system was monitored by “Breeze (Waters™)” software. Tacrolimus was analyzed by injecting 100 μL to a C₁₈ column (Macherey-Nagel, 4.6 × 150 mm, 10 μm particle size). Acetonitrile and milli Q water (pH adjusted to 3 by orthophosphoric acid) at a ratio of 75:25 was used as a mobile phase. Flow rate of the mobile phase was 1 mL/min and detection was carried out at 215 nm. Column temperature was kept at 60 °C. Each sample was assayed for at least three times. Drug loading and encapsulation efficiency of drug loaded micelles were calculated using the following equations $\mathit{DL}(\%)=\frac{\mathit{Amount}\mathit{of}\mathit{Tacrolimus}}{\mathit{Amount}\mathit{of}\mathit{Tacrolimus}+\mathit{Polymer}}\times 100$ $\mathit{EE}(\%)=\frac{\mathit{Amount}\mathit{of}\mathit{drug}\mathit{loaded}}{\mathit{Amount}\mathit{of}\mathit{drug}\mathit{added}}\times 100$