Evaluation of siRNA Stability and Interaction with Serum Components Using an Agarose Gel-Based Single-Molecule FRET Labeling Method.

Small interfering RNAs (siRNAs) are RNA molecules with promising therapeutic potential as a result of their selective mRNA cleavage. However, despite recent progress, low stability in the bloodstream is an impediment to successful administration in vivo. Thus, the availability of flexible and rapid methods for studying siRNA stability and vehicles is crucial for future novel siRNA-based therapeutics. Herein, we report a fast Förster resonance energy transfer (FRET) method based on agarose gel electrophoresis to evaluate the stability of siRNA in serum as well as siRNA interaction with serum proteins and enzymes.

An improved protocol for ICP-MS-based assessment of the cellular uptake of metal-based nanoparticles.

Along with a growing interest in biomedical applications of metal-based nanoparticles, there is a compelling need in systematic information on their behavior in human body systems, preferably at the cellular level. However, in most of the in-vitro uptake experiments, the nanomaterial was applied in its native form that in reality can hardly reach the cell. In this work, we developed an improved procedure in which prior to addition to the cells the particles are converted into the protein conjugates by incubation in human serum. The procedure was tested for gold nanoparticles of different size, chosen as a representative nanomaterial on multifunctional medicinal use, and MCF-7 cell line. Using ICP-MS to measure intracellular metal concentration, it was shown that an original state has significant effect on particle internalization. The protein corona significantly inhibits the uptake amount by MCF-7 cells, with the greatest influence (a 15-fold decrease compared to uncoated particles) being exerted over the smallest, 5-nm particles (3 pg Au/cell). Conjugates of larger particles (20 and 50 nm) are taken up more effectively (45 and 34 pg Au/cell, respectively). The advanced protocol makes the uptake results more reliable and its implementation may accelerate the preclinical development of metal-based nanoparticles as a viable theranostic implement.