Synthesis, characterization, and application of sorbents for the mixed-mode extraction of antisense oligonucleotides from cerebrospinal fluid samples.

BACKGROUND: Antisense oligonucleotides are synthetic nucleic acid molecules capable of selectively modulating gene expression. As the number of approved antisense oligonucleotide therapies grows, reliable analytical procedures are needed to monitor their pharmacokinetics, metabolism, and safety. Current solid phase extraction strategies often suffer from limited recovery or poor reproducibility. Methods using non-polar, hydrophilic or anion-exchange sorbents, require application of ion-pair reagents, or high concentration of salts or long conditioning. On the other hand, the potential of sorbents with hydrophobic-hydrophilic properties used for mixed-mode extraction remains unexplored. Our study presents the first application of newly synthesized materials for the isolation of therapeutic antisense oligonucleotides from cerebrospinal fluid samples.

RESULTS: Five silica-based sorbents modified with aminopropyl, aliphatic, aromatic, and dicarboxylic groups were designed, synthesized, and characterized. The functionalization of sorbents enables various interactions with oligonucleotides, namely electrostatic, hydrophobic, π … π interactions, and hydrogen bonding. A new, mixed-mode dispersive solid phase extraction procedure was developed using a central composite design, enabling a systematic evaluation of the factors governing antisense oligonucleotide desorption. The effectiveness of the procedure was assessed for oligonucleotides varying in chemical modification and length. Recoveries depended on both factors and were the highest for shortmers with 2'-O-methyl modification. This effect is advantageous for the extraction of antisense oligonucleotide metabolites. The developed method was successfully used for the extraction of antisense oligonucleotides from cerebrospinal fluid, enabling reproducible recoveries (40-93 %). The matrix effects ranged from 89 % to 100 %. The highest recoveries were obtained for an oligonucleotide modified at two structural elements, making the method advantageous for the extraction of oligonucleotides used in therapy.

SIGNIFICANCE: The proposed mixed-mode dispersive solid-phase extraction procedure provides a simple, fast, and reproducible approach for isolating of antisense oligonucleotides from cerebrospinal fluid. The method significantly simplifies sample preparation, since the procedure may be straightforwardly applied without additional purification steps. These results for the first time demonstrate the suitability and high analytical potential of hydrophobic-hydrophilic sorbents for the extraction of antisense oligonucleotides from biological samples.