Implementing Efficient Peptoid-Mediated Delivery of RNA-Based Therapeutics to the Vocal Folds.

OBJECTIVE: We 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. METHODS: In 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. RESULTS: In 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. CONCLUSIONS: In 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 EVIDENCE: NA.

Nanoparticle delivery of RNA-based therapeutics to alter the vocal fold tissue response to injury.

OBJECTIVES/HYPOTHESIS: Our laboratory and others hypothesized that Smad3 is a principle mediator of the fibrotic phenotype in the vocal folds (VFs), and we further posited that alteration of Smad3 expression through short interfering (si)RNA holds therapeutic promise, yet delivery remains challenging. To address this issue, we employed a novel synthetic oligomer, lipitoid, complexed with siRNA to improve stability and cellular uptake with the goal of increased efficiency of RNA-based therapeutics. STUDY DESIGN: In vitro study and in vivo animal model. METHODS: In vitro, lipitoid cytotoxicity was quantified via colorimetric and LIVE/DEAD assays in immortalized human VF fibroblasts and primary rabbit VF fibroblasts. In addition, optimal incubation interval and solution for binding siRNA to lipitoid for intracellular delivery were determined. In vivo, a rabbit model of VF injury was employed to evaluate Smad3 knockdown following locally injected lipitoid-complexed siRNA. RESULTS: In vitro, lipitoid did not confer additional toxicity compared to commercially available reagents. In addition, 20-minute incubation in 1× phosphate-buffered saline resulted in maximal Smad3 knockdown. In vivo, Smad3 expression increased following VF injury. This response was significantly reduced in injured VFs at 4 and 24 hours following injection (P = .035 and .034, respectively). CONCLUSIONS: The current study is the first to demonstrate targeted gene manipulation in the VFs as well as the potential utility of lipitoid for localized delivery of genetic material in vivo. Ideally, these data will serve as a platform for future investigation regarding the functional implications of therapeutic gene manipulation in the VFs. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E178-E183, 2018.

Stem Cell-Mediated Paracrine Signaling Alters Fibroplasia in Human Vocal Fold Fibroblasts in Vitro.

OBJECTIVES: Interactions between mesenchymal stem cells (MSCs) and native vocal fold fibroblasts (VFFs) have not been described in spite of promising preliminary data regarding the effects of MSCs on vocal fold repair in vivo. The current study employed a conditioned media (CM) model to investigate the paracrine effects of bone marrow-derived mesenchymal stem cells (BMSCs) on VFFs. METHODS: Human VFFs were treated with transforming growth factor-ß1 (TGF-ß1; 10 ng/mL), CM from human BMSCs following 48 hours of TGF-ß1 stimulation, or CM+TGF-ß1. Proliferation, immunocytochemistry for alpha smooth muscle actin (αSMA), migration, and collagen gel contraction were quantified as well as transcription of components of the TGF-ß signaling pathway. RESULTS: Transforming growth factor-ß1 accelerated proliferation and induced αSMA in VFFs; these effects were suppressed with CM ( P = .009, P < .001, respectively). The CM+TGF-ß1 condition increased cell migration ( P = .02) and decreased gel contraction; CM+TGF-ß1 also inhibited TGF-ß signaling via significant upregulation of NR4A1 as well as downregulation of S MAD3 and TGF-ß1 relative to TGF-ß1 stimulation in the absence of CM ( P = .002, P < .001, and P = .005, respectively). CONCLUSIONS: Conditioned media affected many profibrotic cell activities in TGF-ß1-stimulated VFFs, likely related to altered TGF-ß signaling. These data provide preliminary insight regarding the antifibrotic effects of MSCs and further support their progression to clinical utility.

NR4A1 is an endogenous inhibitor of vocal fold fibrosis.

OBJECTIVES/HYPOTHESIS: NR4A1 was recently identified as an endogenous inhibitor of transforming growth factor (TGF)-ß-induced fibrosis, and the role of this nuclear receptor has not been elucidated in tissue health or the response to injury in the vocal folds. Given the clinical implications of vocal fold fibrosis, we investigated NR4A1 expression during vocal fold wound healing in vivo and the regulatory roles of NR4A1 on vocal fold fibroblasts (VFFs) in vitro with the ultimate goal of developing targeted therapies for this challenging patient population. STUDY DESIGN: In vivo and in vitro. METHODS: In vivo, the temporal pattern of NR4A1 mRNA expression was quantified following rat vocal fold injury. In vitro, the role of NR4A1 on TGF-ß1-mediated transcription of genes underlying fibrosis as well as myofibroblast differentiation and collagen gel contraction was quantified in our human VFF line. Small interfering RNA was employed to alter NR4A1 expression to further elucidate this complex system. RESULTS: Nr4a1 mRNA increased 1 day after injury and peaked at 7 days. Knockdown of NR4A1 resulted in upregulation of COL1A1 and TGF-ß1, with TGF-ß1 stimulation (both P < .001) in VFFs. NR4A1 knockdown also resulted in increased α-smooth muscle actin-positive cells (P = .013) and contraction (P = .002) in response to TGF-ß1. CONCLUSIONS: NR4A1 has not been described in vocal fold health or disease. Upregulation of TGF-ß following vocal fold injury was concurrent with increased NR4A1 expression. These data provide a foundation for the development of therapeutic strategies given persistent TGF-ß signaling in vocal fold fibrosis. LEVEL OF EVIDENCE: N/A Laryngoscope, 127:E317-E323, 2017.

SMAD3 expression and regulation of fibroplasia in vocal fold injury.

OBJECTIVE: Recent reports highlight the efficacy of small interfering RNA (siRNA) targeting SMAD3 to regulate transforming growth factor ß (TGF-ß)-mediated fibroplasia in vocal fold fibroblasts. The current study sought to investigate SMAD3 expression during wound healing in vivo and quantify the downstream transcriptional events associated with SMAD3 knockdown in vitro. STUDY DESIGN: In vivo and in vitro. METHODS: Unilateral vocal fold injury was created in a rabbit model. SMAD3 and SMAD7 mRNA expression was quantified at 1 hour and 1, 3, 7, 14, 30, 60, and 90 days following injury. In vitro, multi-gene analysis technology was employed in our immortalized human vocal-fold fibroblast cell line following TGF-ß1 stimulation ± SMAD3 knockdown across time points. RESULTS: SMAD3 mRNA expression increased following injury; upregulation was significant at 3 and 7 days compared to control (both P < 0.001). SMAD7 mRNA was also upregulated at 3, 7, and 14 days (P = 0.02, P < 0.001, and P < 0.001, respectively). In vitro, SMAD3 knockdown reduced the expression of multiple profibrotic, TGF-ß signaling, and extracellular matrix metabolism genes at 6 and 24 hours following TGF-ß1 stimulation. CONCLUSION: Cumulatively, these data support SMAD3 as a potential master regulator of TGF-ß-mediated fibrosis. SMAD3 transcription peaked 7 days following injury. Multi-gene analysis indicated that the therapeutic effectiveness of SMAD3 knockdown may be related to regulation of downstream mediators of fibroplasia and altered TGF-ß signaling. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E308-E316, 2017.

Mesenchymal stem cells have antifibrotic effects on transforming growth factor-ß1-stimulated vocal fold fibroblasts.

OBJECTIVES/HYPOTHESIS: Mesenchymal stem cells (MSCs) hold therapeutic promise for vocal fold scar, yet the precise mechanism(s) underlying tissue level changes remain unclear. We hypothesize that MSCs interact with native fibroblasts to favorably affect healing. Furthermore, we hypothesize that these interactions vary based on MSC source. METHODS: Vocal fold fibroblasts (VFFs), adipose-derived stem cells, and bone marrow-derived stem cells (BMSCs) were extracted from Sprague-Dawley rats; and a coculture model was employed culturing VFFs ± transforming growth factor (TGF-ß1) (10 ng/mL) ± MSCs. Monoculture MSCs were also prepared as a control. Both extracellular matrix (ECM) and components of the TGF-ß signaling pathway were analyzed via polymerase chain reaction and western blotting. RESULTS: Significantly decreased TGF-ß1 mRNA and α-smooth muscle actin protein was observed in VFFs in response to TGF-ß1 in the coculture with both MSCs (P < 0.05, P < 0.01). BMSCs significantly downregulated collagen I (P < 0.05), collagen III (P < 0.05), Smad3 (P < 0.01), and TGF-ß1 receptor I (P < 0.01) mRNA in VFFs. Hyaluronic synthase-1 and 2 increased in cocultured BMSCs when compared with monocultured BMSCs at baseline and in response to TGF-ß1 (P < 0.01). CONCLUSION: MSCs had a favorable effect on ECM regulation as well as suppression of TGF-ß1 signaling in VFF. Bidirectional paracrine signaling was also observed as VFFs altered ECM regulation in MSCs. These data provide insight into the regenerative effects of MSCs and provide a foundation for clinical application. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E35-E41, 2017.

Preliminary study of a novel transfection modality for in vivo siRNA delivery to vocal fold fibroblasts.

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-ß 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% to 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 five-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 goal of clinical utility. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E231-E237, 2017.

The role of Smad3 in the fibrotic phenotype in human vocal fold fibroblasts.

OBJECTIVES/HYPOTHESIS: To investigate the role of Smad3 as a regulator of transforming growth factor (TGF)-ß1-mediated cell activities associated with fibrosis in normal human vocal fold fibroblasts. We also sought to confirm the temporal stability of Smad3 knockdown via small inhibitor ribonucleic acid (siRNA). Vocal fold fibroblasts were employed to determine the effects of Smad3 knockdown on TGF-ß1-mediated migration and contraction, as well as regulation of connective tissue growth factor (CTGF). We hypothesized that Smad3 is an ideal candidate for therapeutic manipulation in vivo based on its role in fibrosis. STUDY DESIGN: In vitro. METHODS: Knockdown of Smad3 via siRNA was performed in our normal human vocal fold cell line. Three-dimensional collagen gel contraction and scratch assays were employed to determine the role of Smad3 on TGF-ß1-mediated contraction and migration, respectively. The role Smad3 in the induction of CTGF was characterized via sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effects of Smad3 signaling on Smad7 messenger (m)RNA and protein were also quantified. RESULTS: Smad3 knockdown was temporally-stable up to 72 hours (P < 0.001), diminished TGF-ß1-mediated collagen gel contraction and migration, and blunted induction of CTGF, but it had no effect on TGF-ß1-mediated Smad7 mRNA or protein induction. CONCLUSION: Transforming growth factor-ß1 stimulated profibrotic cell activities in our cell line and these actions were largely reduced with Smad3 knockdown. These data provide continued support for therapeutic targeting of Smad3 for vocal fold fibrosis because it appears to regulate the fibrotic phenotype. LEVEL OF EVIDENCE: N/A. Laryngoscope, 126:1151-1156, 2016.