Sinonasal Stent Coated with Sustained-Release Varnish of Mometasone Furoate Inhibits Pro-Inflammatory Cytokine Release from Macrophages: An In Vitro Study

Abstract

The aim of the study was to develop a sustained-release varnish (SRV) containing mometasone furoate (MMF) for sinonasal stents (SNS) to reduce mucosa inflammation in the sinonasal cavity. The SNS' segments coated with SRV-MMF or an SRV-placebo were incubated daily in a fresh DMEM at 37 °C for 20 days. The immunosuppressive activity of the collected DMEM supernatants was tested on the ability of mouse RAW 264.7 macrophages to secrete the cytokines' tumor necrosis factor α (TNFα) and interleukin (IL)-10 and IL-6 in response to lipopolysaccharide (LPS). The cytokine levels were determined by respective Enzyme-Linked Immunosorbent Assays (ELISAs). We found that the daily amount of MMF released from the coated SNS was sufficient to significantly inhibit LPS-induced IL-6 and IL-10 secretion from the macrophages up to days 14 and 17, respectively. SRV-MMF had, however, only a mild inhibitory effect on LPS-induced TNFα secretion as compared to the SRV-placebo-coated SNS. In conclusion, the coating of SNS with SRV-MMF provides a sustained delivery of MMF for at least 2 weeks, maintaining a level sufficient for inhibiting pro-inflammatory cytokine release. This technological platform is, therefore, expected to provide anti-inflammatory benefits during the postoperative healing period and may play a significant role in the future treatment of chronic rhinosinusitis.

Keywords: chronic rhinosinusitis; cytokine; local drug delivery; nasal stent; steroids; sustained-release varnish.

Figure 1

(A) SNS without varnish. (B) SRV-MMF-coated stent. The coating was retained on both the polyurethane plastic material and the nitinol wires.

Figure 2

(A) DMF-differentiated PLB-985 cells were exposed to different concentrations of MMF (0.1, 1.0, and 10 µg/mL) and/or 10 ng/mL TNFα, and the ROS production was followed by monitoring the luminescence each minute for 60 min. MMF only slightly affected the TNFα-induced ROS production. (B) The same setup experiment as in (A) but with 100 ng/mL TNFα. At this TNFα concentration, the presence of 1.0 and 10 µg/mL MMF did not interfere with the TNFα-induced ROS production, and 0.1 µg/mL MMF even increased the TNFα-induced ROS production. RLU = Relative luminescence units. The values are the average of triplicates, and the standard deviation is presented.

Figure 3

A549 lung carcinoma cells were exposed to different concentrations of MMF (0.1, 1 µg/mL and 10 µg/mL) and 2.5 ng/mL IL-1β for 24 h. The GM-CSF levels in the supernatant were determined by an ELISA. MMF suppressed the IL-1β-induced GM-CSF production in A549 lung cancer cells. The lowest concentrations of mometasone (0.1–1 µg/mL) caused strong and similar inhibition of GM-CSF production, while the higher concentration of 10 µg/mL MMF had no effect. The values are the average of triplicates, and the standard deviation is presented. * p < 0.05.

Figure 4

A cellular model of inflammation. RAW264.7 macrophages were exposed to different concentrations of MMF (0.1, 1 µg/mL and 10 µg/mL) in the presence of 10 ng/mL LPS for 24 h. The levels of TNFα (A) and IL-6 (B) in the macrophage supernatants were determined by an ELISA. Mometasone suppressed both TNFα and IL-6 secretion from LPS-exposed macrophages. The macrophages produced a low amount of TNFα and IL-6 in the absence of LPS, which was strongly induced in its presence. The lower tested concentrations of MMF (0.1–1 µg/mL) caused stronger inhibition of TNFα than the higher one (10 µg/mL). The values are the average of triplicates, and the standard deviation is presented. * p < 0.05 compared to LPS alone.

Figure 5

A cellular model of inflammation: SRV-MMF and SRV-placebo-coated nasal stents were incubated in 2 mL DMEM for 24 h and daily transferred to a fresh DMEM for 20 days. Then, the RAW 264.7 macrophages were incubated with the supernatant in the presence of LPS (10 ng/mL) for 24 h, and the secreted IL-6 (A), TNFα (B), and IL-10 (C) levels were analyzed by an ELISA. The red line presents the IL-6, IL-10, and TNFα levels secreted by the macrophages in the presence of 10 ng/mL LPS, while the green line presents their levels in the macrophage samples exposed to LPS and 100 ng/mL MMF. The black lines are the SRV-placebo-coated samples of two individual samples, while the blue lines are the SRV-MMF-coated samples of three individual samples. Figure 6A,C shows the IL-6, TNFα, and IL-10 levels, respectively, secreted from the macrophages in the same experiment in the absence or presence of 10 ng/mL LPS and/or MMF. The inhibitory effect of the SRV-MMF-coated stents on the secretion of all three cytokines was statistically significant (p < 0.05), while the SRV-placebo had only a significant inhibition on IL-10 secretion (p < 0.05).

Figure 5

A cellular model of inflammation: SRV-MMF and SRV-placebo-coated nasal stents were incubated in 2 mL DMEM for 24 h and daily transferred to a fresh DMEM for 20 days. Then, the RAW 264.7 macrophages were incubated with the supernatant in the presence of LPS (10 ng/mL) for 24 h, and the secreted IL-6 (A), TNFα (B), and IL-10 (C) levels were analyzed by an ELISA. The red line presents the IL-6, IL-10, and TNFα levels secreted by the macrophages in the presence of 10 ng/mL LPS, while the green line presents their levels in the macrophage samples exposed to LPS and 100 ng/mL MMF. The black lines are the SRV-placebo-coated samples of two individual samples, while the blue lines are the SRV-MMF-coated samples of three individual samples. Figure 6A,C shows the IL-6, TNFα, and IL-10 levels, respectively, secreted from the macrophages in the same experiment in the absence or presence of 10 ng/mL LPS and/or MMF. The inhibitory effect of the SRV-MMF-coated stents on the secretion of all three cytokines was statistically significant (p < 0.05), while the SRV-placebo had only a significant inhibition on IL-10 secretion (p < 0.05).

Figure 6

The control samples of data presented in Figure 5. The same macrophages that were used for measuring the anti-inflammatory action of supernatants that have been exposed to either the SRV-placebo or SRV-MMF-coated SNS were exposed to 10 ng/mL LPS alone or in the presence of 100 ng/mL MMF. The IL-6 (A), TNFα (B), and IL-10 (C) levels were measured simultaneously with the samples in Figure 5 by an ELISA. MMF significantly prevented LPS-induced IL-6 secretion (A) and IL-10 (C), with only limited inhibition of TNFα secretion (B). The values are the average of triplicates, and the standard deviation is presented. * p < 0.05. The IL-6, IL-10, and TNFα levels of untreated (“control”) macrophages are also shown.