Controlled delivery of decorin and decorin-derived peptides using methacrylated hyaluronic acid microgels for the prevention of vocal fold fibrosis

Introduction:: Vocal fold (VF) scarring affects over 4% of children and is the primary cause of vocal impairment in pediatric patients. This can have devastating social, psychological, and physical effects. Current therapies for treating VF fibrosis include anti-inflammatory and hyaluronic acid (HA) laryngoplasty injections. However, these treatments have limited efficacy and fail to prevent and address the causes of fibrosis. One of the primary markers of fibrotic disease is the transition of native VF fibroblasts to disease-associated myofibroblasts. Myofibroblasts are contractile cells with abundant alpha-smooth muscle actin (α-SMA) that secrete and remodel key extracellular matrix components such as collagen. This results in severe scarring and subsequent fibrosis. The transition from fibroblasts to myofibroblasts can be mediated by TGF-β1; a potent cytokine that can stimulate the transcription of pro-fibrotic and immunomodulatory genes such as α-SMA. Our data suggests that decorin, a proteoglycan found in the extracellular matrix, reverses markers of fibrotic disease in VF fibroblasts in 2D and 3D. Decorin is a natural candidate for a therapeutic. However, it is costly and may have off-target effects as it binds to numerous receptors that activate different pathways. Thus, we investigated the use of decorin-derived peptides to reverse fibrotic gene expression while reducing off-target effects and cost. We have conducted preliminary screening studies and have discovered that the decorin-derived peptide LRR6 significantly lowers pro-fibrotic gene expression. Furthermore, we have engineered methacrylated hyaluronic acid (HAMA) microgels as an injectable drug delivery system for delivering DCN and DCN-derived peptides to the VF.

Materials and Methods:: Six established human VF fibroblast cell lines were used for all in vitro experiments.

Anti-fibrotic effect of DCN and DCN-derived peptides. We compared the response of (i) VF fibroblasts and (ii) TGFβ1-primed VF myofibroblasts to DCN (100ng/mL) and 10µM, 1µM, 100nM, and 10nM of multiple DCN-derived peptides after 1 day by RT-qPCR and immunostaining. For 3D studies, a solution of methacrylated type I collagen and fibroblasts was polymerized to form cell-laden hydrogels and used to test the effects of the mentioned anti-fibrotic treatment conditions. Elastic modulus was determined by unconfined compression testing. Cell viability was confirmed with calcein AM and ethidium homodimer staining.

HAMA Microgels (MG) and Microspheres (MS). HAMA MG and MS were fabricated using extrusion fragmentation and batch emulsion respectively and loaded with DCN or DCN peptides. For extrusion fragmentation, MG were passed through 4 needles of decreasing diameter. For batch emulsions, HAMA precursor solution was added dropwise to a solution of mineral oil in a round bottom flask stirring at 300rpm, exposed to UV light for 10 minutes, and then washed. The size was determined via optical and scanning electron microscopy with ImageJ analysis. Release profiles and loading were quantified using ELISA and HPLC.

In Vivo VF fibrosis. Sprague Dawley rats were selected for their tri-layered VF lamina propria, which is similar to humans. Fibrosis was induced by brushing with a rigid wire brush under endoscopy. This model can be used to assess in vivo efficacy of DCN and/or DCN-derived peptide delivery on VF fibrosis.

Results, Conclusions, and Discussions:: Results: Following the extensive screening of multiple DCN-derived peptides, the peptide LRR6 was found to have the most potent anti-fibrotic effects. DCN and DCN-derived peptide LRR6 significantly downregulated pro-fibrotic and immunomodulatory genes such as α-SMA, IL-6, IL-8, and CTGF. Both DCN and LRR6 also demonstrated a decrease in the rate of collagen fibrillogenesis as compared to a control. A concentration of 10nM of LRR6 and 100 ng/uL of DCN was sufficient to induce significant downregulation. DCN MG were fabricated with a mean diameter of approximately 110µM to minimize cellular uptake and ensure localization in the VF extracellular matrix after injection. MG release was quantified using an ELISA. Release of HAMA MG loaded with DCN was studied in both 1xPBS and 1xPBS with 5U of hyaluronidase to mimic the physiological concentration of hyaluronidase in the vocal fold. DCN was released at a faster rate in HAMA gels incubated in hyaluronidase and released DCN at therapeutically relevant concentrations above 100ng per day. The release profiles of MG and MS were also performed with both DCN and DCN-derived peptides. Furthermore, we established an in vivo model of VF fibrosis to test our delivery system.

Discussion: Previous treatments for VF fibrosis, such as anti-inflammatory drugs and laryngoplasty injections, suffer from rapid drug clearance, high off-target effects, and do not prevent VF fibrosis. We discovered novel drug candidates for preventing vocal fold fibrosis and other fibrotic diseases. Both the downregulation of pro-fibrotic and immunomodulatory genes and the decrease in the rate of collagen fibrillogenesis suggest the potent effects of both DCN and DCN-derived peptides. The successful release of DCN from HAMA MG indicates the potential for utilizing HAMA MG and MS as a delivery system for both DCN and DCN-derived peptides to the vocal fold. Furthermore, a drug delivery system optimized for either DCN-derived peptide or DCN release may be useful for treating fibrosis and scarring in other parts of the body.

Conclusion: DCN and DCN-derived peptides have potent anti-fibrotic effects on VF fibroblasts and myofibroblasts and can be easily integrated into hydrogel microgels for controlled, local delivery to treat VF fibrosis.

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