The produced insulin-loaded PLGA-NP were freeze-dried with and without cryoprotectants added, and its physical-chemical properties was assessed after freeze-drying (Table 3). The presence of cryoprotectants in formulation SB203580 HCC is important also to avoid aggregation after redispersion of the lyophilizate.14 Trehalose for instance showed to facilitate the resuspension of polylactic acid (PLA)-polyethylene oxide (PEO) nanoparticles after freeze-drying.6 Table 3. Physical-chemical properties of insulin-loaded PLGA-NP after freeze-drying with and without cryoprotectants (n = 3, mean �� SD) The morphology of the obtained nanoparticles may be evaluated through the visualization of its microscopic appearance by TEM and SEM. On one hand, TEM may show us information essentially about the shape of PLGA-NP and SEM may show information about the surface of nanoparticles.

However, using SEM microscopy to visualize the surface of nanoparticles with a good definition is very difficult, and focusing the electron beam on such a small area may also damage the nanoparticles. To avoid these drawbacks, during the visualization by SEM particles with the higher particle size that often occur in such formulations were chosen. Therefore, it is possible to visualize the larger particles and infer its morphology and surface features to the produced nanoparticles. TEM allows the observation of the freeze-dried nanoparticles after their dilution, however the visualization of nanoparticles by SEM is very difficult when the cryoprotectant concentration is more than 5%, since a continuous matrix covering all the nanoparticles may be observed.

15 Therefore, the purification of the performed freeze-dried nanoparticles, in order to remove the cryoprotectant is crucial to be possible to properly visualize the particles by SEM. Insulin-loaded PLGA-NP was visualized after formulation and after freeze-drying with no cryoprotectants added, by TEM (Fig. 1) and by SEM (Fig. 2). They were also visualized after freeze-drying with the cryoprotectants used by TEM and SEM and the results are shown in Figure 3 and Figure 4, respectively. Figure 1. TEM microphotographs of insulin-loaded PLGA-NP after production (A) and after freeze-drying with no cryoprotectant added (B). (A) bar shows 200 nm and (B) bar shows 100 nm. Figure 2.

SEM microphotographs of insulin-loaded PLGA-NP after production (A) and after freeze-drying with no cryoprotectant added (B) (bar shows 5 ��m). Figure GSK-3 3. TEM microphotographs of insulin-loaded PLGA-NP after freeze-drying with: 10% (w/w) trehalose (A); 10% (w/w) sucrose (B); 10% (w/w) glucose (C); 10% (w/w) fructose (D) and 10% (w/w) sorbitol (E). (A and B) bar shows 100 nm, (C) bar shows … Figure 4. SEM microphotographs of insulin-loaded PLGA-NP after freeze-drying with: 10% (w/w) trehalose (A); 10% (w/w) sucrose (B); 10% (w/w) glucose (C); 10% (w/w) fructose (D) and 10% (w/w) sorbitol (E) (bar shows 30 ��m).