(560b) Synergistic Combination of Coated Microneedles and Biochemical Approaches for Oligonucleotide Delivery to Cells in 3D Tissues

Oligonucleotides (ONs) have the potential to provide significant clinical impact by enabling novel therapeutic and disease diagnosis strategies. For example, molecular beacons could be used for disease detection, while anti-RNA ON probes could be used to knockdown protein expression in cells to provide a therapeutic action. However, intracellular delivery of ONs, especially in 3D tissues is challenging. Two major barriers limit successful intracellular delivery of ONs in an intact 3D tissue. The first challenge is related to the inability to achieve wide-spread uniform delivery of ONs in the tissue such that all cells in the tissue can be exposed to a sufficient therapeutic dose of ONs. The second challenge is related to the poor intracellular delivery of ONs. The objective of this study was to synergistically combine (i) microneedles as devices to precisely deliver ONs into tissues to achieve extensive intra-tissue distribution of ON probes, with (ii) biochemical approaches including use of streptolysin O (SLO) and cholesterol-conjugation to enhance penetration of these widely dispersed ONs into cells of the 3D tissue.

Microneedles were uniformly coated with ONs. 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 wide-spread intra-tissue distribution of ONs was achieved upon delivery of ON-cholesterol conjugates. We also demonstrate that delivery of a cholesterol-conjugated anti-green fluorescent protein (GFP)-ON using coated microneedles resulted in reduction of GFP expression in HeLa cells in 3D culture. In conclusion, the results of this study provide a novel approach for efficient intracellular delivery of ONs in tissues.