Modeling the Delivery and Release of Tocopherol-Modified Oligos
Although the direct incorporation of tocopherol into a therapeutic oligonucleotide should efficiently mediate its delivery into a cell, its hydrophobic nature might inhibit the final function of the oligonucleotide once inside the cell. To this end, we synthesized oligonucleotides conjugated to tocopherol via a cleavable disulphide-bridge.
After delivery to the cell, the tocopherol could be easily removed from the oligonucleotide by breaking the disulphide bridge, leaving the oligonucleotide free to carry out its therapeutic function. To demonstrate the feasibility of this approach, we constructed a cell delivery model system in which a hydrophobic cartridge takes the place of the cell membrane.
Oligonucleotides were synthesized using standard phosphoramidite chemistry, introducing a disulphide bridge linker followed by tocopherol incorporation at the 5´ end. The resulting crude oligonucleotide has the general structure tocopherol-S-S-oligo. After synthesis, the oligonucleotide was cleaved and deprotected, then immediately loaded onto a standard purification cartridge (Figure 1).
The purification method mimics what we would expect to happen in cell delivery, in that the modified oligonucleotide can be “delivered” to the cartridge and then the active molecule released via cleavage of the S-S bond, retaining the delivery agent (tocopherol) on the cartridge.
Using thiol-modified oligonucleotides conjugated to tocopherol as an example, we have generated data indicating that tocopherol successfully mediates attachment of the conjugate to the cartridge, and that the oligonucleotide can be released at a high level of purity following cleavage of the disulphide bridge.
This data indicates that the oligonucleotide can be easily released from the tocopherol delivery agent with sufficient efficiency and purity to accurately carry out its therapeutic function.