Vortemp 56 evc

The diffusion of nanoparticles in pig intestinal mucus was performed as previously described [27 (link), 28 (link)]. Briefly, 4 mg Lumogen® Red-labeled nanoparticles (4 mg/mL) was dispersed in 0.5 g mucus and incubated for 2 h at 37 °C at 60 rpm (Labnet VorTemp 56 EVC, Labnet International, Inc., Edison, NJ). The mucus was obtained following the procedure previously described [27 (link), 28 (link)]. The movement of nanoparticles was recorded in a two-dimensional plane at 30 frames/s during 10 s by a high-speed camera (Allied Vision Technologies, Stadtroda, Germany) attached to a wide-field epifluorescence microscope used at 63 × magnification oil immersion lens (Leica DM IRB, Wetzlar, Germany). A minimum of 100 trajectories were captured and later tracked and analyzed using an image processing software (Fiji ImageJ).
The diffusion coefficient of the nanoparticles in water (D°) was obtained from the Stokes–Einstein equation [28 (link)], whereas the “Effective Diffusion Coefficient” (< Deff >) was calculated as follows: $$Deff=\frac{<MSD>}{4·\mathrm{\Delta }t}$$ in which < MSD > is the mean square displacement of 100 individual trajectories, 4 is a constant related to the 2-dimensional mode of video capture, and Δt is the selected time interval. All the formulations were expressed as the ratio (%) between their Deff and their D° (diffusions in mucus and in water, respectively).

Release experiments were conducted at 37 °C using simulated fluids for gastric (SGF; pH 1.2) and intestinal (SIF; pH 6.8) conditions prepared according to European Pharmacopoeia (EMA, European Pharmacopoeia 6.0, chapter 2.9.3 Dissolution Test for Solid Dosage Forms, 2008).
At different intervals, samples were collected and centrifuged at 10,000 rpm for 10 min (Centrifuge MIKRO 220, Hettich, Germany). For each specific time interval, 79 μg of P2Et, either free or entrapped into nanoparticles, was resuspended in 2 mL of the corresponding simulated fluid in polyvininyl chloride tubes and maintain under constant shaking at 1500 rpm at 37 °C (Labnet VorTemp 56 EVC, Labnet International, Inc.). The different formulations were kept in the SGF for 2 h before being transferred to SIF for 20 h. The amount of P2Et released was quantified using HPLC from the supernatants described above.