(222a) Development of a Novel Method for Investigation of Polymer/siRNA Intracellular Trafficking

Despite substantial investment in RNA interference (RNAi)-based therapies, few clinical trials have shown promising results. This is due largely to a lack of methods and materials for the efficient delivery of small-interfering RNA (siRNA). Many new materials have been designed specifically for this purpose. Investigators commonly design such materials based on reasonable assumptions about the limiting barriers to efficient delivery and structure-activity relationships. Such an approach is often unsuccessful, however, because the intracellular mechanisms of siRNA delivery (e.g., cell surface binding, cellular uptake, endocytic escape, unpackaging of the nucleic acid, etc.) are unclear. Increased understanding of intracellular trafficking mechanisms and kinetics, and how these processes are affected by material properties, is required for rational design of siRNA delivery agents.

Here we report investigation of the kinetics of cellular internalization and endocytic processing of siRNA complexed with 25-kDa polyethylenimine (PEI) in HeLa cells. Conventional techniques including microscopy and cellular fractionation did not provide sufficient precision, prompting us to develop new methods for quantifying intracellular trafficking. GFP fusions with endocytic markers of early endosomes (Rab5), late endosomes (Rab7) and lysosomes (LAMP-1) were expressed individually in HeLa cells. Confocal fluorescence microscopy was used to quantify colocalization of fluorescently labeled siRNA with these markers. We observed high colocalization of siRNA with Rab5-GFP and Rab7-GFP at short times, which decreased linearly over the next 4 h. In contrast, little colocalization with LAMP-1-GFP was initially observed, but the siRNA was strongly localized in lysosomes from 30 minutes to 4 h post-transfection. However, significant overlap of the marker proteins in different organelles limited the precision of conventional microscopy to assess intracellular localization. In addition, laser-scanning confocal microscopy inherently favors observation of large particles (i.e., aggregates) over single polyplexes. Thus, we developed a unique technique for resolving the location of siRNA within the endocytic pathway. The method relies on the known endocytic trafficking kinetics of horseradish peroxidase (HRP) and the enzyme’s ability to catalyze polymerization of 3,3’-diaminobenzidine (DAB). In the presence of H2O2 and DAB, HRP-containing vesicles are cross-linked, making them easy to isolate for analysis of siRNA content. Using this new method, we observed ~60% of siRNA within early endosomes within 5 min, a rapid decrease to 15-20% by 30 min, and a slower decrease to <5% at 3 h post-transfection. siRNA localized in lysosomes was first detected (<5%) 30 min post-transfection, increased to ~20% by 30 min (due to apparent transfer of siRNA from the early endosomes) and further increased to only 16% by 3 h. Finally, this assay allowed us to observe a steady increase of siRNA in cytosol, presumably due to release from endocytic vesicles.

This method allows for more precise determination of intracellular localization and analysis of intracellular trafficking kinetics of siRNA/PEI polyplexes (or any nanoparticle delivery system) compared to more commonly employed methods. We are employing this method to compare intracellular trafficking of various siRNA delivery vectors and construct a quantitative model of the transfection process.