Exceptional Nuclease Resistance of DNA and RNA with the Addition of Small-Molecule Nucleobase Mimics.

Nucleases present a formidable barrier to the application of nucleic acids in biology, significantly reducing the lifetime of nucleic acid-based drugs. Here, we develop a novel methodology to protect DNA and RNA from nucleases by reconfiguring their supramolecular structure through the addition of a nucleobase mimic, cyanuric acid. In the presence of cyanuric acid, polyadenine strands assemble into triple helical fibers known as the polyA/CA motif. We report that this motif is exceptionally resistant to nucleases, with the constituent strands surviving for up to 1 month in the presence of serum. The conferred stability extends to adjacent non-polyA sequences, albeit with diminishing returns relative to their polyA sections due to hypothesized steric clashes. We introduce a strategy to regenerate stability through the introduction of free polyA strands or positively charged amino side chains, enhancing the stability of sequences of varied lengths. The proposed protection mechanism involves enzyme failure to recognize the unnatural polyA/CA motif, coupled with the motif's propensity to form long, bundling supramolecular fibers. The methodology provides a fundamentally new mechanism to protect nucleic acids from degradation using a supramolecular approach and increases lifetime in serum to days, weeks, or months.

Polymeric Microspheres Containing Human Vocal Fold Fibroblasts for Vocal Fold Regeneration.

OBJECTIVE: Most acellular injectable biomaterials for vocal fold (VF) wound treatment have limited regenerative potential due to their fast enzymatic degradation and limited recruitment of native cells postinjection. The injection of cells as therapeutic treatment often results in apoptosis due to stresses within the needle and the immune response of the host. Degradable microspheres may improve treatment effectiveness by increasing cell residence time, shielding cells during injection, and offering early protection against the immune system response. The objective of the present study was to investigate the potential of human VF fibroblasts encapsulated in polymeric microspheres as an injectable therapeutic treatment in vitro. METHODS: Alginate, alginate-poly-L-lysine, and alginate-chitosan microspheres were fabricated using electrospraying and characterized in terms of biocompatibility, swelling, and mechanical properties as well as cytokine production. RESULTS: Alginate microspheres were found to have the most desirable properties for VF regeneration. They were resistant to mechanical challenges. They were found to have a stiffness similar to that reported for native VF-lamina propria. They were found to be biocompatible and increased the proliferation of fibroblasts. Human VF fibroblasts encapsulated in alginate microspheres induced the production of interleukin (IL)-8 and IL-4 at 24 hours. CONCLUSION: The alginate microspheres fabricated in this study were found to offer potential advantages, as cell delivery tool. This study highlights the importance of combining biomaterials and cells to expedite the wound-healing process through cytokine production. Future work is aimed to further analysis of the wound-healing properties the microspheres. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1828-1834, 2021.