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  • Staurosporine br Intracellular localization br At a


    3.2.3. Intracellular localization
    At a very early time point (1 h), despite the different internalization kinetics recorded through flow cytometry, chitosan/HA and Nanocin/ HA NP did not showed a markedly different behavior: both of them were internalized in similar amounts with some, possibly initial signs of colocalization with lysosomal compartments (Fig. 4a). This snapshot evidenced initial NP localization in lysosomes (yellow from con-temporaneous emission of Lysotracker (green, 488 nm) and siRNA (red, 561 nm), but also signs of possible escape (red siRNA signal around or flanking the yellow or green organelles). At 24 h, the two HA-decorated L3-DY547-NP are internalized/accumulated in HCT-116 in similar amounts (Fig. 4b; n = 5 fields of view, average of 150 cells observed for each treatment), which is broadly in line with the flow cytometry re-sults. Of note, the siRNA cytosolic distribution seemed to be con-sistently more homogeneous with chitosan/HA NP, and more com-partmented (visibly brighter spots) with Nanocin/HA NP. This phenomenon may indicate a possibly lower availability of siRNA in the Nanocin/HA at this stage, which would stem from a tighter com-plexation of the nucleic Staurosporine with the polycation. This tight interaction
    Fig. 1. Physico-chemical characterization of nanoparticles. Z-average size (histogram) and ζ-potential (red dots) of as-prepared Nanocin- (a) and chitosan-based (b) nanoparticles. The effect of storage is respectively shown in (c) and (d) for particles without HA and a 1:4 polycation:HA ratio. A typical PAGE gel analysis (e) shows payload protection for 25% wt. siRNA-loaded nanoparticles after incubation with different concentrations of RNase I (for each column, from left to right: 0, 0.01, 0.1, 1 U/μL). Recovered siRNA is expressed as percentage with respect to its control, each value is reported as average ± of at least three independent samples. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    would be possibly caused by: a) the higher charge density on Nanocin and b) its lower size. The higher charge density of the polycation may cause a higher avidity; a more compact siRNA/polycation complex is possibly obtained with Nanocin (smaller Mw) that has an easier com-plexation compared to the larger polyelectrolytes (chitosan, entropic penalty for surface siRNA/polycation coupling) However, we should also consider that any factor increasing the complexation strength, such as a high charge density, is not necessarily detrimental: for example, using chitosan we have recently demonstrated that high density of amines increased chitosan binding to RNAs, but this effect was most likely overcome by higher endosomolytic efficiency (Lallana et al., 2017).
    3.3. Silencing efficacy: siRNA release from nanoparticles and mRNA-KRAS knock-down
    We have first investigated the silencing efficiency of a panel of 
    different siRNA, employing simple Nanocin/siRNA polyplexes, since their high positive charge (see Table 1) was supposed to stimulate the highest transfection. The silencing efficiency was evaluated with qRT-PCR, rather than immunostaining methods, due to its superior sensi-tivity; this is critical in KRAS analysis as the level of gene expression is low and therefore difficult to detect. We eventually selected the GGA CUCUGAAGAUGUACCUAGGUACAUCUUCAGAGUCCs sequence as a promising and more reproducible candidate for further experiments, after comparison of the silencing efficiencies of 14 siRNAs targeting different regions within the mRNA sequences (see Supplementary Information, section SI.2 and Fig. 1SI).
    Chitosan/HA NP resulted in KRAS silencing to a level comparable to the gold standard transfecting agent Lipofectamine (no statistical dif-ference between the two treatments at 48 h, Fig. 5a); more importantly, the mRNA-KRAS reduction was approximately 2-fold higher than with Nanocin/HA. As anticipated in Section 3.2.3, the higher charge density
    of Nanocin may be a limiting factor for siRNA intracellular availability, which is a possible explanation for its lower silencing activity. Of in-terest, the HA-decorated NP caused levels of silencing similar to those of the binary polyplexes, i.e. siRNA/polycation only (see Supplementary Information, Fig. 2SI); the latter are cationic species, and as such are characterized by an efficient cell adhesion and pene-tration, therefore it is remarkable that HA, most likely through its in-teractions with CD44, allows to overcome the effect of charge inversion.
    3.3.3. Effect of storage on silencing efficacy
    NP were also stored for 1 week at 4 °C in deionized water before using them in silencing experiments; this storage period caused no significant alteration in the silencing activity of both NP (Fig. 5b). In-terestingly, we observed that without HA (direct complexation between