*3.5.6* In vitro*,* ex vivo*, and* in vivo *drug release*

*In vitro* and *in vivo* studies provide extrapolated data to reflect the bioavailability of drug formulations. The *in vitro* drug release profile of nanoparticles can be determined with a Franz diffusion cell. The donor compartment has a cellophane membrane as a barrier, comprising the encapsulated drug in the nanoparticles. The receptor compartment is filled with buffer solution, usually phosphate-buffered saline (pH 7.4), and the system is stirred with a magnetic stirrer at 100 rpm and 37°C. At predetermined time intervals, samples of the dispersion are withdrawn from the medium of the receptor compartment and an equal amount of medium is returned to the system at this same time. The withdrawn sample is filtered using a 0.22–50 μm filter (e.g., Millipore, USA) and analyzed for drug release using UV-visible spectrophotometry at the specific wavelength that shows peak absorption of the drug [39, 43, 44].

An *ex vivo* drug release study profile of nanoparticles can be performed with a diffusion cell. Suitable skin and the underlying cartilage removed from ears can be cut up and placed on the diffusion cell filled with receptor solution. Vesicular process samples are placed on the dorsal surface of the skin. At predetermined time intervals, samples of the dispersion are withdrawn from the medium of the receptor compartment and an equivalent amount of the medium is returned at the same time. The withdrawn sample is then analyzed for the amount of drug released using high-performance liquid chromatography (HPLC) or UV-visible spectrophotometry at a specific wavelength [44].

*In vivo***,** drug release study profiles of nanoparticles can be carried out by administering the nanoparticles to living animals. At predetermined time intervals, blood samples are withdrawn, followed by centrifugation and analysis of drug release using HPLC.
