N32 - Targeted Modified Citrus Pectin-coated PLGA Nanoparticles for the Treatment of Pulmonary Fibrosis

Introduction: : Pulmonary fibrosis (PF) is a chronic interstitial disease with an increasing disease burden around the world, predominantly among the elderly and characterised by excess production of extracellular matrix (ECM) material and progressive formation of scar tissue throughout the lungs. Two drugs are currently available as PF-targeted therapies: pirfenidone and nintendanib (NIN), with few other prospects in sight and both carrying risks of off-target effects and limited efficacy. Our group has previously reported a highly promising citrus pectin-coated poly(lactic-co-glycolic) acid (PLGA)-based nanocarrier with inherent targeting ability and therapeutic action, both tied to citrus pectin’s ability to bind to and downregulate the production of galectin-3 (gal3). The current work sought to iterate upon this via the inclusion of modified citrus pectin (MCP) as a coating on the PLGA nanoparticle (NP) core, which itself encapsulated the anti-fibrotic agent NIN. Taken together, we hypothesized better batch-to-batch consistency, enhanced delivery to pulmonary fibroblasts (via MCP) and additive anti-fibrotic effects (via MCP+NIN, relative to MCP or NIN alone). We show that these particles can exert significant antifibrotic action not only on 2D in vitro models of pulmonary fibrosis but also penetrate and treat in vitro models of fibroblast foci: clusters of diseased fibroblasts with high ECM secretion activity observed in lungs of PF patients.

Materials and

Methods: : NPs were synthesized using amine-terminated PLGA (50:50) in an oil-in-water single-emulsion process (with NIN encapsulated by direct inclusion within the oil phase) and characterized via dynamic light scattering and S/TEM. Stability studies were carried out in various media including simulated lung fluid (Gambles fluid) over 7 days at 37°C. MCP coating was confirmed by way of FT-IR and TGA. NIN release kinetics were assessed in PBS (37°C, pH 7.4) over 21 days by means of UV-vis spectrophotometry. Cytocompatibility of both MCP and MCP-PLGA were assessed on MRC-5 human lung fibroblasts by means of the MTT assay. The targeting capabilities of our novel nanocarrier were evaluated by means of UV-vis spectrophotometry or confocal microscopy measuring the uptake of fluorescent MCP-PLGA or PLGA NPs by MRC-5 cells with or without TGFβ-induced fibrosis in both 2D- and 3D (spheroid) formats. Therapeutic response was evaluated as a function of the expression of α-smooth muscle actin (αSMA), gal3 and pSMAD3 in 2D- (ICC and Western blot) and 3D/spheroid (Western blot) fibroblast formats.

Results, Conclusions, and Discussions:: MCP-PLGA NPs of approx. 210 nm with a surface charge of -29 mV were synthesized successfully, with a NIN loading of 30 ug/mg NPs. Formulated NPs were found to be stable in suspension upto 6 days in all tested media, and cytocompatible (>80% viability) over 24 h upto 2 mg/mL. MCP-coated coumarin-6 (C6)-encapsulating NPs exhibited enhanced uptake by MRC-5 cells in general at rates significantly higher than non-coated controls, with diseased cells taking up coated NPs at significantly higher levels at 2 mg/mL. Uptake in models of fibroblast foci showed a similar trend, with high penetration/uptake at 1 mg/mL, and significantly higher uptake by diseased fibroblasts relative to all other groups at this concentration (see figure). ICC work on 2D cultures of fibroblasts (see figure) showed that MCP-PLGA NPs were able to exert a one-two punch effect on TGFβ-induced fibrosis: a rapid-onset (24 h) initial effect by virtue of the MCP coating, followed by a sustained antifibrotic effect at longer (72 h) timeframes. A similar effect was observed in Western blots carried out on both 2D and 3D cultures of fibroblasts.

Conclusions: Stable and cytocompatible MCP-PLGA NPs were synthesized with an average diameter of about 200 nm, a significant reduction from our previous iteration. Dose-dependent increase in NP uptake was observed in MRC-5 cells, with higher uptake by fibrotic cells noticeable even in in vitro models of fibroblast foci. NIN-containing NPs demonstrated more rapid antifibrotic activity than NPs without NIN or NIN or MCP alone, and showed clear evidence of a MCP+NIN additive effect over a 24-72 h period, with reductions of αSMA, pSMAD3 and gal3.

Acknowledgements and/or References (Optional):: This work was supported by the National Institute of Health grant R37 CA283937 to J.U.M.