Heparin coated tubes

Pharmacokinetics and biodistribution (see below) were evaluated in healthy Balb/C mice (Charles River) (n=3). Animals were maintained on an alfalfa-free diet (Teklad) for two weeks prior to the administration of fluorescently labeled PVX filaments to reduce tissue autofluorescence. A-PVX, A-PVX-P5B, or A-PVX-P20 formulations were injected via the tail vein (at 100 µg in 100 µL sterile PBS) and blood was collected into heparin-coated tubes (Fisher) using retro-orbital bleeding; time course studies were conducted and samples were collected up to 36 hours post-administration. Serum was isolated from samples by centrifuging at 14,500 g for 10 minutes. Fluorescence was read (λ_Ex = 600 nm, λ_Em = 660 nm) using a Tecan microplate reader, and the fluorescence reading was correlated to a standard curve normalized for each particle formulation to determine the amount of particle (in µg) at each time point. Percent injected dose was determined based on the fluorescence reading and amounts of PVX per 50 µL of serum from each time point and assuming a total blood volume of 1.5 mL.

Six to eight week old Balb/C mice (Charles River) were placed on an alfalfa free diet (Teklad) for three weeks before tail vein injection of 10 mg/kg A647-TMV-PEG and A647-TMV-GPRPP formulations. Blood samples were collected by orbital sinus bleeds at 3, 5, 10, 15, 20, 30, 60, 100, 140, and 180 min (n = 3) in heparin-coated tubes (Fisher). Serum was separated and collected by centrifugation at 7000 rpm in a tabletop centrifuge for 10 minutes. 50 μL of serum from each time point were added to a black 384-well plate and fluorescence read using a Tecan Infinite 200 microplate reader (λ_Ex = 600 nm, λ_Em = 665 nm). The results were analyzed using Prism software.