Small molecule and protein-based drugs, while critically important therapies, cannot treat all diseases. As such, alternative treatment modalities must be developed to complement existing strategies. One potential alternative is small interfering RNA (siRNA) therapeutics, which are capable of specific inhibition of a wide range of intracellular, membrane, and extracellular proteins. siRNAs are hydrophilic due to their anionic backbone and do not readily diffuse across cellular membranes. During systemic delivery, naked siRNAs are rapidly filtered by the kidneys or degraded by serum nucleases and can often initiate an immune response. Thus, for siRNAs to be useful as therapeutics, they must be complexed with delivery vehicles for protection during extracellular transport and cellular internalization. Once delivered to the cytoplasm, siRNAs act through RNA interference (RNAi) to degrade messenger RNAs (mRNAs) in a sequence-specific manner, thereby reducing target protein expression. Despite the recent clinical success, development of siRNA therapeutics is limited due to the inefficiency, toxicity, and immunogenicity of current delivery vehicles. To overcome these hurdles, this research aimed to understand the role of delivery vehicle characteristics in influencing the cellular uptake and processing of siRNA-containing complexes. While many types of delivery vehicles have been developed for siRNAs, the characteristics that are essential for success are still not well understood. To address this issue, we synthesized a variety of silica nanoparticles (sNPs), and assessed their ability to effectively deliver siRNAs to human lung carcinoma cells (H1299). By varying the concentration of amines and dextran during sNP synthesis, we defined chemical/physical characteristics important for active siRNA delivery. Another roadblock in the development of siRNA therapeutics is a limited understanding of the intracellular processing of siRNA-containing complexes leading to initiation of RNAi. With recent evidence showing that the intracellular fate of endocytosed material was influenced by the endocytic pathway used for internalization, we developed a novel assay capable of differentiating uptake among the different endocytic pathways and assessing their functionality in initiating RNAi. Our results showed that Lipofectamine 2000 (LF2K) was internalized by Graf1-, Arf6-, or flotillin-mediated endocytosis for the initiation of RNAi, depending on cell type. Additionally, our study identified functional differences among endocytic pathways in a cell, indicating that uptake alone was not sufficient to initiate silencing. In a mixed cell population, we found that targeted inhibition of the non-functional pathways in some cells enhanced silencing in the uninhibited cells. These findings suggest that designing delivery vehicles for specific endocytic pathways may enhance the activity of the delivered siRNAs by directing them preferentially to the intended target cells.Finally, due to the limitations of current techniques, the intracellular pathways used in processing siRNA-containing complexes are not well defined. As a result, it is unclear how delivery vehicle characteristics affect the intracellular trafficking of siRNAs. To address this issue, we developed a novel microscopy-based assay that uses automated multi-well live-cell imaging to track the intracellular location of siRNAs over time. Through this assay we determined the intracellular pathways utilized in sNP-mediated siRNA delivery and identified how dextran functionalization of sNPs altered the intracellular trafficking of siRNAs. This assay provides a new analytical technique to assess intracellular pathways and could aid in the development of more efficient siRNA delivery vehicles.
Read
