Ultraviolet spectrophotometry

DOX-CA was incubated with different pH values of phosphate-buffered saline (PBS) solution (pH 4.0, pH 5.0, pH 6.8 and pH 7.4). At designated time intervals, the solution was used for high performance liquid chromatography (HPLC) (Shimadzu 1630, Japan) measurements. HPLC analysis was performed on Agilent 1260 Infinity LC system (Agilent, Germany). Chromatographic separation was carried out at room temperature on a CAPCELL PAK C18 column (3.0 mm × 100 mm, 3 μm, SHISEIDO, Japan). The mobile phase consisted of solvent A (sodium dodecyl sulfate solution), solvent B (acetonitrile) and solvent C (methanol). The flow rate was 0.8 ml/min and the injection volume was 10 μL. The detection wavelength was set to 254 nm. Then, the release rate of DOX from the nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was investigated by a dialysis method. Typically, 1 mL of nanocomplex solution (2 mg/mL) was dialyzed (MWCO: 3.5 kDa) against 20 mL PBS solution (pH 7.4, 6.8 or 5.0) at 37 °C. At given intervals, 100 μL of external buffer was collected and replaced by the same volume PBS solution. The concentration of DOX released from polymeric micelles was quantified by ultraviolet spectrophotometry (Shimadzu, Japan) at 481 nm.