Nanoparticle siRNA Transfection
Sigma-Aldrich’s N-TER™ nanoparticle siRNA transfection peptide-based system was developed to circumvent a number of drawbacks associated with lipid-based siRNA transfection agents. These include limited ability to transfect a variety of cell lines such as primary, neuronal, differentiated and nondividing cells; toxicity; and relatively longer times required for reagent uptake and subsequent target knockdown to occur.
Designed for use in transient knockdown of eukaryotic gene expression, the N-TER peptide binds siRNAs noncovalently, forming a nanoparticle that can interact directly with lipids on the surface of the plasma membrane, allowing the nanoparticle to cross the cell membrane and deliver the siRNA directly to the cytoplasm.
According to Steven Suchyta, product manager, functional genomics, this siRNA delivery system enters the cell via an as-yet undetermined pathway. The delivered siRNA becomes available to the RNAi interference machinery soon after entry into the cell.
“N-TER/siRNA complexes are generally more stable than a liposome,” Suchyta says, “and we have seen faster uptake and relatively long persistence within the cell.” N-TER is based on an amphipathic peptide that interacts directly with the siRNA to form a nanoparticle, which can efficiently deliver and release the siRNA into the cell.
In pointing out advantages that distinguish peptide-based nanoparticle delivery, Suchyta comments that this reagent exhibits reduced toxicity compared to lipid-based reagents. Particularly, he says, it is effective in extremely difficult-to-transfect cells. Recent experiments with the undifferentiated mouse skeletal myoblast cell line C2C12 could be successfully transfected with slight protocol optimization, for example, by varying cell densities.
Typically N-TER can work over a wide range of confluencies and works well even for cell types that are typically highly confluent such as HUVEC cells, he adds. Sigma Aldrich is now “exploring the potential in vivo use of N-TER,” he reports. “We have seen some promising initial data in mouse models. Allowing scientists to take the next step in their RNAi research using animal models is one of Sigma’s highest priorities, and we will continue to actively pursue technologies that facilitate in vivo RNAi.”
Through its Dharmacon products, Thermo Fisher Scientific offers siRNA delivery “without a transfection reagent,” says Devin Leake, Ph.D., director of research and development for genomics products. Dr. Leake and his colleagues have developed methods for siRNA design and identified chemical-modification patterns to enhance potency, stability, and specificity of siRNA transfection. Dr. Leake says that while lipid reagents were designed to get siRNA into “as many cell types as possible, certain cells including stem cells, immune cells, and primary cells remain challenging.”
According to the company, its Accell siRNA promotes cellular uptake in many cell types without the toxic and inflammatory responses typically associated with conventional lipid transfection agents. Further, it claims, the “passive nature” of these reagents reduces adverse off-target gene knockdown.
Dr. Leake explains that these chemically modified siRNAs enter almost any cell type directly from the culture medium. While transfection with the modified RNA requires no serum, or reduced serum conditions, “with assay optimization, a window can be found that allows transfection to occur, then cells can be returned to their normal serum-containing medium.”
According to Dr. Leake, researchers have reported successful stem cell, T-cell, Jurkat, and cadiomyocyte transfection with Accell siRNAs. The combination of chemical modifications and sequence selection provides silencing tools in these relevant cell types.