Objective An obstacle to clinical use of RNA-based gene suppression is instability and inefficiency of current delivery modalities. Nanoparticle delivery likely holds great promise, but the kinetics and transfection conditions must be optimized prior to in vivo utility. We investigated a RNA nanoparticle complex incorporating a “lipitoid” transfection reagent in comparison to a commercially-available reagent. Study Design In vitro Methods We investigated which variables influence transfection efficiency of lipitoid oligomers and a commercially-available reagent across species, in vitro. These variables included duration, dose, and number of administrations, as well as serum and media conditions. The target gene was Smad3, a signaling protein in the Transforming Growth Factor (TGF)-β cascade implicated in fibroplasia in the vocal folds and other tissues. Results The two reagents suppressed Smad3 mRNA for up to 96 hours; Lipitoid performed favorably and comparably. Both compounds yielded 60–80% mRNA knockdown in rat, rabbit, and human vocal fold fibroblasts (p< 0.05 relative to control). Dose and number of administrations played a significant role in gene suppression (p<0.05). Suppression was more dose-sensitive with Lipitoid; at a constant siRNA concentration, a 50% decrease in gene expression was observed in response to a 5-fold increase in Lipitoid concentration. Increased number of administrations enhanced gene suppression; ~45% decrease between one and four administrations. Neither serum nor media type altered efficiency. Conclusion Lipitoid effectively knocked down Smad3 expression across multiple transfection conditions. These preliminary data are encouraging and Lipitoid warrants further investigation with the ultimate goal of clinical utility. Level of Evidence N/A

N-Substituted glycine peptoid oligomers have recently attracted attention for their metal binding capabilities. Due to their efficient synthesis on solid phase, peptoids are well suited for generation of compound libraries, followed by screening for molecular recognition and other diverse functional attributes. Ideally, peptoids could be simultaneously screened for binding to a number of metal species. Here, we demonstrate the use of bench-top X-ray fluorescence (XRF) instrumentation to screen rapidly, on solid support, a library of peptoid oligomers incorporating metal-binding functionalities. A subset of the peptoid sequences exhibited significant metal binding capabilities, including a peptoid pentamer and a nonamer that were shown to selectively bind nickel. The binding capabilities were validated by colorimetric assay and by depletion of Ni(2+) ion concentration from solution, establishing bench-top XRF as a rapid, practicable high-throughput screening technique for peptoid oligomers. This protocol will facilitate discovery of metallopeptoids with unique material properties.

NA. Laryngoscope, 128:E178-E183, 2018.

Injectable collagen-based hydrogels offer great promise for tissue engineering and regeneration, but their use is limited by poor mechanical strength. Herein, we incorporate tannic acid (TA) to tailor the rheology of the corresponding hydrogels while simultaneously adding the therapeutic benefits inherent to this polyphenolic component. TA in the solution form and needle-shaped TA microparticles are combined with collagen and the respective systems studied for their time-dependent sol–gel transitions (from storage to body temperatures, 4–37 °C) as a function of TA concentration. Compared to systems incorporating TA microparticles, those with dissolved TA, applied at a similar concentration, generate a less significant enhancement of the elastic modulus. Premature gelation at a low temperature and associated colloidal arrest of the system are proposed as a main factor explaining this limited performance. A higher yield stress (elastic stress method) is determined for systems loaded with TA microparticles compared to the system with dissolved TA. These results are interpreted in terms of the underlying interactions of TA with collagen, as probed by spectroscopy and isothermal titration calorimetry. Importantly, hydrogels containing TA microparticles show high cell viability (human dermal fibroblasts) and comparative cellular activity relative to the collagen-only hydrogel. Overall, composite hydrogels incorporating TA microparticles demonstrate a new, simple, and better-performance alternative to cell culturing and difficult implantation scenarios.

ObjectiveWe hypothesize that Smad3 is a master regulator of fibrosis in the vocal folds (VFs) and RNA‐based therapeutics targeting Smad3 hold therapeutic promise. Delivery remains challenging. We previously described a novel synthetic peptoid oligomer, lipitoid L0, complexed with siRNA to improve stability and cellular uptake. An advantage of these peptoids, however, is tremendous structural and chemical malleability to optimize transfection efficiency. Modifications of L0 were assayed to optimize siRNA‐mediated alteration of gene expression.MethodsIn vitro, Smad3 knockdown by various lipitoid variants was evaluated via quantitative real‐time polymerase chain reaction in human VF fibroblasts. Cytotoxicity was quantified via colorimetric assays. In vivo, a rabbit model of VF injury was employed to evaluate the temporal dynamics of Smad3 knockdown following injection of the L0‐siRNA complex.ResultsIn vitro, similar reductions in Smad3 expression were established by all lipitoid variants, with one exception. Sequence variants also exhibited similar nontoxic characteristics; no statistically significant differences in cell proliferation were observed. In vivo, Smad3 expression was significantly reduced in injured VFs following injection of L0‐complexed Smad3 siRNA at 1 day postinjection. Qualitative suppression of Smad3 expression persisted to 3 days following injury, but did not achieve statistical significance.ConclusionsIn spite of the chemical diversity of these peptoid transfection reagents, the sequence variants generally provided consistently efficient reductions in Smad3 expression. L0 yielded effective, yet temporally limited knockdown of Smad3 in vivo. Peptoids may provide a versatile platform for the discovery of siRNA delivery vehicles optimized for clinical application.Level of EvidenceNA