CALCIUM-SIRNA NANOCOMPLEXES: MECHANISM OF CELLULAR UPTAKE AND ENDOSOMAL RELEASE

Small interfering RNA (siRNA) represents a promising type of therapeutics exploiting the mechanism of RNA interference for silencing target genes. Yet, the clinical translation of siRNA has been limited due to delivery challenges. We recently described a novel Ca²⁺- siRNA nanocomplex capable of strong but reversible complexation, siRNA protection, cellular uptake, and cytoplasmatic unloading of its cargo. Here, we investigated the importance of Ca²⁺compared to other bi- or tri-valent cations in creating these nanocomplexes, and the cytocompatibility of the various nanocomplexes. Further, we elucidated cellular entry and endosome release mechanisms of Ca²⁺-siRNA nanocomplexes. The nanocomplexes were prepared by incubating siRNA (50 nM final) with either Ca²⁺, Mg²⁺, Zn²⁺, Ba²⁺, Mn²⁺, Fe²⁺ or Fe³⁺ ions (5 mM for divalent and 3.33mM for Fe³⁺) Of these nanocomplexes, only those prepared with Ca²⁺, Mg²⁺, Ba²⁺ andFe³⁺ were cytocompatible as judged by PrestoBlue® for cell viability. Effective eGFP silencing (~80%) in GFP expressing mouse colon carcinoma CT26 cells was achieved only with Ca²⁺- siRNA nanocomplexes. Cell uptake studies (using confocal microscopy) and silencing experiments (using flow-cytometry), were performed using different inhibitors of several possible entry mechanisms: Dynasore, Pitstop2®, EIPA, Nifedipine, Cadmium and Genistein. We revealed that the major endocytic pathways involved in the entry of Ca²⁺-siRNA nanocomplexes are clathrin and dynamin-dependent. Treatment with Bafilomycin, which inhibits endosome acidification after Ca²⁺ entry to endosomes, completely abolished siRNA-mediated silencing indicating that Ca²⁺ is critical for the endosomal unloading through a “proton sponge” effect. In conclusion, Ca²⁺ is a critical component for particle assembly, particle uptake and endosomal escape.