Cellular Penetration and Intracellular Dynamics of Perfluorocarbon-Conjugated DNA/RNA as a Potential Means of Conditional Nucleic Acid Delivery.

Nucleic acid-based therapeutics represent a novel approach for controlling gene expression. However, a practical delivery system is required that overcomes the poor cellular permeability and intercellular instability of nucleic acids. Perfluorocarbons (PFCs) are highly stable structures that can readily traverse the lipid membrane of cells. Thus, PFC-DNA/RNA conjugates have properties that offer a potential means of delivering nucleic acid therapeutics, although the cellular dynamics of the conjugates remain unknown. Here, we performed systematic analysis of the cellular permeability of sequence-controlled PFC-DNA conjugates (N[PFC]n-DNA, n = 1,2,3,4,5) that can be synthesized by conventional phosphoramidite chemistry. We showed that DNA conjugates with two or more PFC-containing units (N[PFC]n≥2-DNA) penetrated HeLa cells without causing cellular damage. Imaging analysis along with quantitative flow cytometry analysis revealed that N[PFC]2-DNA rapidly passes through the cell membrane and is evenly distributed within the cytoplasm. Moreover, N[PFC]2-modified cyclin B1-targeting siRNA promoted gene knockdown efficacy of 30% compared with naked siRNA. A similar cell penetration without associated toxicity was consistent among the seven different human cell lines tested. These unique cellular environmental properties make N[PFC]2-DNA/RNA a potential nucleic acid delivery platform that can meet a wide range of applications.

Floxuridine Oligomers Activated under Hypoxic Environment.

Floxuridine oligomers are anticancer oligonucleotide drugs composed of a number of floxuridine residues. They show enhanced cytotoxicity per floxuridine monomer because the nuclease degradation of floxuridine oligomers directly releases highly active floxuridine monophosphate in cells. However, their clinical use is limited by the low selectivity against cancer cells. To address this limitation, we herein report floxuridine oligomer prodrugs that are active under hypoxia conditions, which is one of the distinguishing features of the microenvironment of all solid tumors. We designed and synthesized two types of floxuridine oligomer prodrugs that possess hypoxia-responsive moieties on nucleobases. The floxuridine oligomer prodrugs showed lower cytotoxicity under normoxia conditions (O2 = 20%), while the parent floxuridine oligomer showed similar anticancer effects under hypoxia conditions (O2 = 1%). The floxuridine oligomer prodrug enabled tumor growth suppression in live mice. This would be the first example demonstrating the conditional control of the medicinal efficacy of oligomerized nucleoside anticancer drugs.