Anales RANF

Juan Aparicio Blanco, Ignacio A. Romero, Jean P Benoit, Ana I. Torres Suárez @Real Academia Nacional de Farmacia. Spain 208 Figure 6: Quantitative analysis of the in vivo biodistribution of DiD-labeled LNCs in the brain of healthy mice expressed as percentage of the injected dose per gram of brain. The colours and fill patterns of the LNC formulations correspond to those of figure 2. *: p<0.05, **: p<0.01, ***: p<0.001. Altogether, the consistency between the in vitro and in vivo results served to validate our in vitro BBB model with the human brain endothelial cell line hCMEC/D3 as a versatile screening method to evaluate the passage of nanocarriers across the BBB that meets the high- throughput demands in the early stages of the drug discovery and lacks ethical constraints. Analogously, the glioma targeting ability has been evaluated through uptake experiments conducted with the human glioblastoma cell line U373MG. To this end, we have utilized the same LNCs than for the BBB-targeting ability (Figure 2). The quantitative analysis of the in vitro glioma- targeting ability is also shown in Figure 3. A decrease in particle size consistently yielded a higher in vitro uptake by human glioblastoma cells (3.0-fold for undecorated LNCs, p<0.05 and 3.5-fold for CBD-adsorbed LNCs, p<0.001). Given the effect of particle size on the cell uptake, the influence of CBD adsorption was then assessed from a comparison of equally-sized LNCs. The functionalization of LNCs with CBD enhanced by 3.4-fold (p < 0.001) the in vitro glioma targeting properties of their equally-sized undecorated counterparts. The 3D reconstruction from the Z-stacks of the images taken by confocal microscopy further support qualitatively the internalization of LNCs within the U373MG cells (Figure 7). The enhancement in glioma targeting achieved with the adsorption of CBD on LNCs equals that observed with other targeting moieties such as the aptamer AS1411 (57) or angiopep-2 (58) and outperformed that reported for transferrin (59), T7 peptide (60) or mannose (59). The exogenous and non-peptide nature of CBD makes it less prone to develop competitive phenomena with physiological ligands or cause immunogenicity. A comparison of the uptake studies with both cell lines reveals that the enhancements in the targeting effects achieved with the reduction in LNC size (p < 0.05 for undecorated LNCs and p< 0.001 for CBD-functionalized LNCs) and CBD adsorption (p < 0.001) were statistically more significant with the human glioblastoma cells (Figure 3).