A previous report about polypeptide-Dox nanoparticles was referred (40). To measure pharmacokinetics of Dox, 5 mg/kg Dox equivalent of aldoxorubicin, Dox-SA, or Dox-CBD-SA was injected intravenously into female FVB mice. Blood samples were collected in EDTA-coated tubes at 5 min, 30 min, 1 hour, 4 hours, 12 hours, 25 hours, 50 hours, and 75 hours after injection. Blood samples were stored at 4°C until the end of sample collection. The samples were centrifuged (2000g, 5 min), and plasma was collected. Plasma samples diluted in acidified isopropanol (75 mM HCl, 10% water, 90% isopropanol) were loaded onto a 96-well black plate (100 μl/well). Fluorescence was measured as described above. Plasma samples were also collected from mice, which received no injections, diluted in acidified isopropanol, and measured to create the standard curve of background fluorescence. Exponential two-phase decay (Y = Ae−αt + Be−βt) fitting was used to calculate the plasma half-life. Fast clearance half-life, t1/2,α; slow clearance half-life, t1/2,β. Data were analyzed using Prism software (v7, GraphPad).

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