**4.4 Encapsulation efficiency and** *in vitro* **drug release**

Drugs can be loaded onto nanosystems by incorporating them during the nanocapsule production, or either after the formation of the nanocapsules, incubating the carrier with the concentrated drug solution [51]. In both cases, the drug can be physically loaded onto the polymeric matrix or the oily core, or it can be adsorbed on the surface, in the function of the affinity and the physicochemical characteristics of both the drug and the components of the nanocapsules [52]. The total content of a drug in the nanocapsule suspension can be determined after dissolving or extracting the drug from the carrier, or calculated from the difference between the total and free drug concentrations after the separation of nanocapsules by centrifugation or ultrafiltration [23]. The determination of loaded or released drugs can be carried out by means of high-performance liquid chromatography (HPLC), fluorescence spectroscopy, UV-Vis spectroscopy, or another analytical technique.

Nanocapsule erosion or swelling can lead to drug release. *In vitro* drug delivery depends upon the localization of the drug within the particle, the physicochemical properties of both the drug and the nanocapsule constituents, size, morphology, and cross-linking, and also on release conditions, such as pH, temperature, polarity, and the presence of enzymes or an adjuvant. Regarding the release of the active principle, the process is governed by solubility, diffusion, and polymer biodegradation. In the case of nanospheres, where the drug is evenly distributed, drug release occurs through diffusion or matrix erosion. If diffusion is faster than erosion, the release mechanism is said to be controlled by diffusion. If the drug is weakly bound to the surface, a rapid initial release or "*burst*" will take place. If the drug has been

incorporated into the polymeric matrix (nanocapsule), it will present a relatively small "*burst*" effect and a sustained release profile instead. In this case, the release will be controlled by drug dissolution and diffusion through the polymeric membrane. To avoid the "*burst*" effect, compounds can be added to the matrix that reduces the drug solubility, or, as in the case of chitosan, ethylene oxide-propylene oxide block copolymer (PEO-PPO) can be added, which increases release rate because it diminishes drug-matrix interaction [1].

Determining the drug release mechanism from the particle system can give valuable information about the interactions between the drug and the nanocapsule. Drug release kinetics from nanocapsules may be obtained using ultracentrifugation, centrifugal ultrafiltration, dialysis techniques, or side-by-side diffusion cells with an artificial or biological membrane [23, 52]. Furthermore, the kinetic data can be fitted to mathematical models to determine the predominant release mechanism, which is very convenient in the design and evaluation of the utility of nanocapsules as drugdelivery systems in pharmaceutical applications [24].
