Pharmacokinetics of DOX-BSA/MnO2 Nanoparticles

SD female rats participated in the in vivo pharmacokinetic evaluation of DOX-BSA/MnO₂ NPs. Therefore, 12 rats were fasted with ad libitum water access and split into two groups (each n = 6) randomly, including 1) DOX solution (5 mg/kg); 2) DOX-BSA/MnO₂ NPs (5 mg/kg equivalent to DOX). Rats were given the DOX solution or the DOX-BSA/MnO₂ NPs by tail injection. Then, about 500 μL blood was gathered in a 1.5 mL heparinized centrifuge tube at designated time intervals (2, 5, 10, 15, 20, 30, 40, 60, 90, 120, 180, and 300 min). An identical amount of normal saline heated to body temperature was administered intraperitoneally to recover blood volume. Samples were centrifuged at 13,000 rpm for 5 min immediately to recover plasma, which was kept at −80°C for additional processing.

1) Determination of DOX by HPLC

High-pressure liquid chromatography (HPLC) (LC-20A, Shimadzu, Tokyo, Japan) using a fluorescence detector measured DOX concentration in plasma, organs, or tumors. Plasma samples were extracted by precipitation of proteins (acetonitrile: dichloromethane = 1:4) and using daunorubicin (DNR) as an internal standard. The excitation and emission wavelengths used to monitor DOX were 238 and 554 nm, respectively. The mobile phase comprised acetonitrile with 0.1% trifluoroacetic acid (25:75, v/v); online mixing and pumping were performed using a quaternary pump at a 1.0 mL/min flow rate. DOX was separated by a Phenomenex C18 column (250 × 4.6 mm, 5 μm) at 30°C with a 10 μL injection volume. DOX and DNR were eluted in around 3 and 7 min, respectively. The developed HPLC method was verified in the specificity, linearity, precision, accuracy, recovery, limit of detection (LOD), as well as limit of quantitation (LOQ).
A two-compartment model with Phoenix WinNonlin 10.0 program (Pharsight, Mountain View, CA, United States) calculated the pharmacokinetic metrics. The following parameters were estimated: maximum plasma concentration (C_max), area under the concentration-time curve from baseline to terminal time analyzed (AUC), mean residence time (MRT), clearance rate (Cl), volume of distribution V) and elimination half-life (t_1/2).