He suggested that, the results obtained using agitation during lipoplex preparation may have implications for designing more efficient and successful siRNA delivery systems [31]. Silvander et al. prepared small unilamellar vesicles by ultrasonic irradiation of samples containing about 25mg of lecithin in 5mL pH 7.4 buffer (10mM Tris-HCl containing 150mM NaCl). The samples were sonicated for 1 hour and thereafter diluted with buffer to the desired concentration [32]. In

our work, we employ additional stirring by a high-shear mixer previous to sonication, and buffers of the same but also lower pH. Silvander Inhibitors,research,lifescience,medical using different characterization methods concluded that vesicles were formed. After that they added different anionic tensioactives and demonstrated the transition from vesicles to find more micelles through different intermediate states. As revealed by cryo-TEM micrographs, micelles of various types Inhibitors,research,lifescience,medical and shapes may form during solubilization of lecithin vesicles by alkyl sulfate surfactants [32]. All the evaluated

systems have shown globular micelles at high surfactant concentration, and for instance, we found this shape for the siRNA-loaded nanoparticles prepared at pH 5.0. Therefore, we cannot discard this transition to micelles, or at least the feasibility of coexistence between vesicles Inhibitors,research,lifescience,medical and micelles. The phosphocholine polar head is zwitterionic at pH between 3 and 11; this means that in this pH range the phosphate group of Inhibitors,research,lifescience,medical the polar head has a net negative charge of electrons, and the choline group has an equal positive charge with a spatial separation. In aqueous solution, 3–5 water molecules are bound to the phosphate group, while none is bound to the choline group. When salts are added to the solution, anions are attracted by the choline group, and cations are bound to the phosphate group [30, 33]. It can be supposed, then,

that nanometric spherical particles are formed Inhibitors,research,lifescience,medical at pH 5.0 because of the interaction between siRNA and PC, Oxygenase more specifically because of the interaction between the positively charged amine group of phosphatidylcholine and the phosphate groups of siRNA. Meanwhile, at pH 7.0, these interactions could be less relevant as a result of the decrease in the proportion of the positively charged forms of the zwitterionic phosphocholine polar head of the amphiphile, which is in agreement with the z potential values obtained. As a consequence, different conformational organization of molecules is acquired. Proper internalization of the delivered siRNA was tested on MCF-7 cells transfected with the vehicle. However, it must be taken into account that the final silencing effect depends also on the endolysosomal escape and the efficient incorporation of siRNA to the RNA-induced silencing machinery.