High-efficiency , Low-toxicity Nanopieces Designed for siRNA Delivery in Extracellular Matrix-rich Musculoskeletal Tissues

Introduction: Since the discovery of RNA interference (RNAi), the use of small interfering RNA (siRNA) to silence diseaseinducing genes has shown great therapeutic potential due to its high specificity and efficiency in silencing target gene expression. However, a major challenge in this approach is that siRNA must infiltrate tissue and enter target cells to be effective. This is especially challenging for extracellular matrix-rich musculoskeletal systems. The second challenge is cytotoxicity of the current transfection vehicles. Lipid-based or lipidcomposited commercial transfection reagents, such as Lipofectamine 2000, X-tremeGENE and Nanojuice, show high levels of cytotoxicity upon transfection, which can induce a deleterious pro-inflammatory response and even apoptosis due to their lipid nature. Moreover, they are large in size, forming spheres of several hundred nanometers in diameter (after loading with RNAs), very difficult to penetrate matrix-rich musculoskeletal tissues. Therefore, we have developed a novel delivery vehicle, Nanopieces, consisting of co-assembled rosette nanotubes (RNTs) and siRNA via completely non-covalent bindings (Figure 1). Thus, Nanopieces showed superior biodegradability. Once Nanopieces delivered their siRNA cargo, their degradation products are highly biocompatible due to the biomimetic G^C base motif of the RNT. The ability of Nanopiece to deliver cargo effectively and degrade safely allows minimal levels of cytotoxicity, a prerequisite for in vivo therapeutic applications. Furthermore, Nanopieces have a nano-rod-like shape, 20-30 nm in diameter. This is more than 2000 times smaller in volume than Lipofectamine spherical particles, allowing the Nanopiece to transfect cells that are shielded by dense extracellular matrix. In this study, we tested two generations of Nanopieces. One is K1-RNT Nanopiece followed by the second generation TBL (twin base linker)-RNT Nanopiece that has two G^C basepair motifs per functional unit as opposed to a single G^C motif seen in the K1-RNT (Figure 1). The TBL-RNT has a stronger positive surface charge and larger diameter due to the second G^C motif allowing more RNAs to load than K1-RNT. In this study, we showed that both Nanopieces transfect matrix-rich musculoskeletal cells in vitro with higher transfection efficiency and lower cytotoxicity than commercial delivery vehicles.