2.7. Determination of encapsulation efficiency and drug loading capacity

Encapsulation efficiency and drug loading capacity of the nanoparticles were analyzed by HPLC. Five-milliliter PTNs solution was placed in the dialysis bag and the bag was placed in 50 mL PBS (pH 7.4). Then it was dialyzed under magnetic stirring at 37 °C for 4 h. After the dialysate filtered with 0.22 μm microporous membrane, 40 μL filtrate was automatically injected into HPLC system and analyzed with Agilent SB-C18 (50 mm × 4.6 mm, 5 μm). The effluent was detected at 280 nm. The mobile phase was methanol/acetonitrile/0.003 mol/L KH₂PO₄ aqueous solution/triethylamine (50:30:20:0.05) with the flow rate at 1.0 mL/min. TET showed good linearity in the range of 0.4–20 μg/mL, and the encapsulation efficiency and drug loading capacity of TET were calculated by the following formula.

W₁ is the amount of free TET, W₂ is the amount of initial added TET, and W₃ is the total amount of the gathered PTNs.