Such strategies have to be developed for the new two-step nanoprecipitation procedure as well. Having accomplished protein loaded nano-sized PLGA particles, we tested the development of the sustained release nanoparticles into an application platform. We selected Cyt-c as

model protein because it has been employed in experiments geared towards better cancer treatment options [24]. The size of our particles makes them potentially useful in passive and also active targeting of cancer tissues [37,38]. For example, Santra et al. [24] demonstrated recently the therapeutic potential of Cyt-c in nanoparticles by their capability to induce apoptosis in lung carcinoma cells after uptake by the cells by endocytosis. However, their vehicle consisted of a water-soluble hyperbranched GSI-IX research buy polyhydroxyl polymer not selleck screening library approved in medical applications. In contrast, our nanoparticles employ an already FDA approved and commercially available polymer (PLGA) and a straight forward encapsulation method. We hypothesized that encapsulation of Cyt-c via the two step nanoprecipitation method should work using the optimum conditions identified for lysozyme (Table 7) because both proteins have a similar molecular weight (12 and 14▒kDa, respectively) and are basic [39]. The encapsulation efficiency for Cyt-c was with 72% is similar to that obtained for lysozyme under identical conditions (Table 8). The peroxidase activity of

Cyt-c was comparable to values prior to precipitation and encapsulation and only few aggregates were formed indicating good preservation of structural integrity during the process. The size of the

particles obtained was 340▒nm and thus potentially useful to enable passive delivery to cancer tissues based on the EPR effect [37,38]. In vitro release of Cyt-c from the PLGA nanoparticles showed an initial “burst” release within 24▒h that was reasonably small with ca. 20% ( Fig. 2). Burst release values of >20% are frequently found for such PAK5 systems, in particular when nanosized systems are being used [ 40]. During a 100-day incubation period, Cyt c was released completely from the nanospheres. Since the release was slow, the amount of protein released per day was small and the residual activity during release could not be measured with accuracy. Future experiments using cell cultures and animal models will shed light into the bioactivity of the developed system. However, since 100% of the protein was released, we can exclude the formation of buffer-insoluble Cyt-c during the release period. Since there are some reports that PLGA nanoparticles could be internalized by cells, we investigated whether the Cyt-c-PLGA NPs would be toxic to cancer cells. We selected a human cervical cancer cell line (HeLa) as a model system and incubated the cells for 24, 48, 72, and 96▒h at 37▒°C under 5% CO2 with various concentrations of drug-loaded and empty PLGA nanoparticles and determined the cell viability (Fig. 3).