Nanoparticles Characterization

Entrapment Efficiency:

%EE = [(Total amount of drug - Free drug in the supernatant)/Total amount of drug]
*100

Total amount of drug = mass of Ibu or Df initially applied

Free drug in the supernatant = the drug mass accumulated in the supernatant

1. Prepare the Ch/ γ-PGA, Ch/Ibu/γ-PGA and Ch/Df/γ-PGA NPs.

2. Remove and store 100 ul of each solution.
3. Centrifugated the NPs solution (the one that was not stored on step 2.) at
20.000 g for 30 minutes.
4. After that, the supernatant of the NPs was collected (Remove all the
supernatant and resuspend with the buffer).
NOTA: No meu caso, como tenho 2 ml da solução de NPs, removo os 2 ml e
ressuspendo em 200 ul do buffer.
5. Analyze the solution stored on step 2. and the supernatant of the NPs by
UV/Vis spectroscopy at 230 nm (Ibu) and 275 nm (Df) (Synergy Mx, BioTek),
using several concentrations of each free drug in a buffer as a calibration curve.

Release Assay:

1. Prepare the Ch/ γ-PGA, Ch/Ibu/γ-PGA and Ch/Df/γ-PGA NPs.

2. Incubate the NPs solution with PBS at pH 7.4.
3. At different timepoints (0h,1h,2h,4h,24h, and 48h) the PBS solution was
collected and replaced.
NOTA: No meu caso para ter deteção dos fármacos, necessitei de usar 6 ml de solução
das partículas para 12 ml de PBS e o que fazia era a diferentes timepoints retirar 500 ul
e colocar outros 500 ul de PBS.
4. The solutions obtained were centrifuged at 20000 g for 30 minutes.
5. In the end, the Ibu and Df concentration in the supernatant was measured at
230 nm (Ibu) or 275 nm (Df) (Synergy Mx, BioTek) and quantified through this
formula: % Dfreleased = [Df]supernatant × 100/[Df]initial. The absorbance levels of Ch/γ-PGA
NPs without drug were used as a control.