Microprecision delivery of oligonucleotides in a 3D tissue model and its characterization using optical imaging

Despite significant potential of oligonucleotides (ONs) for therapeutic and diagnostic applications, rapid and widespread intracellular delivery of ONs in cells situated in tissues such as skin, head and neck cavity, and eye has not been achieved. This study was aimed at evaluating the synergistic combination of microneedle (MN) arrays and biochemical approaches for localized intratissue delivery of oligonucleotides in living cells in 3D tissue models. This synergistic combination was based on the ability of MNs to precisely deliver ONs into tissues to achieve widespread distribution, and the ability of biochemical agents (streptolysin O (SLO) and cholesterol conjugation to ONs) to enhance intracellular ON delivery. The results of this study demonstrate that ON probes were uniformly coated on microneedle arrays and were efficiently released from the microneedle surface upon insertion in tissue phantoms. Co-insertion of microneedles coated with ONs and SLO into 3D tissue models resulted in delivery of ONs into both the cytoplasm and nucleus of cells. Within a short incubation time (35 min), ONs were observed both laterally and along the depth of a 3D tissue up to a distance of 500 μm from the microneedle insertion point. Similar widespread intratissue distribution of ONs was achieved upon delivery of ON-cholesterol conjugates. Uniformity of ON delivery in tissues improved with longer incubation times (24 h) postinsertion. Using cholesterol-conjugated ONs, delivery of ON probes was limited to the cytoplasm of cells within a tissue. Finally, delivery of cholesterol-conjugated anti-GFP ON resulted in reduction of GFP expression in HeLa cells. In summary, the results of this study provide a novel approach for efficient intracellular delivery of ONs in tissues.