br siRNA and polycations the
siRNA and polycations) the chitosan-based NP experienced a significant reduction in silencing performance (from > 60% to about 40%, Supplementary Information Fig. SI2), which is likely to ascribe to the low degree of protonation of chitosan amines, which eventually allows self-aggregation of the polymer and physical destabilization of the particles.
3.3.4. Effect of RNase I exposure on silencing performance
As a last step in the assessment of NP for in vivo administration, we tested whether siRNA loaded in Nanocin- and chitosan-NP could pre-serve its silencing functionality when the NP are exposed to potentially RNA-degrading condition, such as the presence of RNAse. To support our previous demonstration that the apparent integrity of the loaded siRNA (Fig. 1e) ensures the preservation of its silencing capacity, we
Fig. 3. Kinetics of internalization of HA-coated L3-DY547-NP in CD44-expressing cell lines: cancer (HCT-116, CD44+, CD44v6high) and fibroblasts (HDF, CD44low). Results are expressed as mean ± st.dev. of 10,000 events (n = 3 independent samples) at each time point. In mono-culture, similar kinetics of uptake are observed in both cell lines and for both NP. Whereas, a more tumor-like environment (co-culture of HCT-116 and HDFa) promotes a higher internalization of HA-coated NP, with increased uptake in cancer T-5224 rather than fibroblasts at each time point (fold increase of 2–3 at the endpoint).
Fig. 4. Nanoparticle and siRNA intracellular localization: a) lysosomal escape after 1 h incubation with NP, the arrows indicate lysosomes (green), the co-localization of nanoparticles in late endosomes/lysosomes (yellow) and released siRNA (red); b) internalization of siRNA in HCT-116 cells after 24 h incubation with NP, notably a similar percentage of siRNA was delivered in HCT-116, being 3.35% and 3.55% respectively for Nanocin/HA and chitosan/HA NP; a more ‘cloudy’ siRNA signal was observed in HCT-116 cells incubated with chitosan/HA NP, compared to a more localized siRNA signal for the Nanocin/HA counterparts. Scale bars: 25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
further tested the silencing efficiency of both NP after exposure to different concentration of RNase I (i.e. 0.01, 0.1, 1 U/μL) and compared the results to non-exposed NP (incubated in nuclease free water, con-trol). In these experiments, concentrations of RNAse up to 1 U/μL did not alter the performance of Nanocin/HA NP, whereas that of chitosan/ HA NP was in part decreased. This can be ascribed to the higher complexation strength of Nanocin (higher charge density, lower mole-cular weight), which makes more difficult for an enzyme to reach its target in the bulk of a particle. Indeed, similar effects can be seen also
for the polycomplexes formed without HA (see Supplementary in-formation, Fig. 3SI).
We compared two HA-based polyplex systems for the delivery of
siRNA in CD44+ cell lines (HCT-116 and HDFa), for a perspective KRAS-targeted tumor therapy. Our main findings are that: a) CD44high/ CD44v+ cancer cells (HCT-116) are more active for the internalization
Fig. 5. KRAS silencing efficacy of HA-coated NP. Relative KRAS expression and mRNA silencing was measured after 48 h treatment in HCT-116 cells with: a) as
prepared NP: results show no statistical difference of chitosan-nanoparticles with respect to lipofectamine and a significant difference between Nanocin NP and lipofectamine (n = 5, p < 0.0001); b) NP after one-week storage at 4 °C generally showed a silencing efficacy similar to that of as prepared NP. Data are expressed as percentage with respect to untreated cells and are the average on n = 3 independent experiments. Lipofectamine was used as control (data not reported); c) NP exposed (incubation prior use) to RNase I at different concentrations (0, 0.01, 0.1, 1 U/μL). Data are expressed as percentage with respect to untreated cells and are the average on n = 3 independent experiments. Lipofectamine was used as control (data not reported).
of HA-coated NP than stromal standard CD44+ cells (HDFa), and that this difference amplifies when cells are co-cultured. This is a very en-couraging finding that supports the use of HA for tumor targeting, with potential low off-target effects; b) the strength of the polyelectrolyte complexation is an important parameter that carries a delicate balance of favorable and detrimental effects. The stronger interactions between Nanocin and siRNA (and HA) appear to negatively affect the silencing efficacy of such NP, possibly due to the lower availability of the nucleic acid. On the other hand, they most likely provide a higher stability against nucleases (and other harmful agents for the nucleic acid). Remarkably, we found that chitosan NP presented the right compro-mise between polyplexes stability and avidity/efficiency of siRNA re-lease, which conferred them a high silencing efficacy comparable to the gold standard transfecting agent (i.e. Lipofectamine). With this rational, further in vivo studies will need to demonstrate whether the different complexation also affects the stability of the NP in the blood stream, and whether the higher silencing capacity of chitosan-based systems can be confirmed in real tumors.