Biofunctional Nanofibrous Substrate for Local TNF-Capturing as a Strategy to Control Inflammation in Arthritic Joints

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease that affects the synovial cavity of joints, and its pathogenesis is associated with an increased expression of pro-inflammatory cytokines, namely tumour necrosis factor-alpha (TNF-α). It has been clinically shown to have an adequate response to systemic administration of TNF-α inhibitors, although with many shortcomings. To overcome such limitations, the immobilization of a TNF-α antibody on a nanofibrous substrate to promote a localized action is herein proposed. By using this approach, the antibody has its maximum therapeutic efficacy and a prolonged therapeutic benefit, avoiding the systemic side-effects associated with conventional biological agents' therapies. To technically achieve such a purpose, the surface of electrospun nanofibers is initially activated and functionalized, allowing TNF-α antibody immobilization at a maximum concentration of 6 µg/mL. Experimental results evidence that the biofunctionalized nanofibrous substrate is effective in achieving a sustained capture of soluble TNF-α over time. Moreover, cell biology assays demonstrate that this system has no deleterious effect over human articular chondrocytes metabolism and activity. Therefore, the developed TNF-capturing system may represent a potential therapeutic approach for the local management of severely affected joints.

Keywords: TNF-α capture; antibody immobilization; electrospun nanofibers; human articular chondrocytes; rheumatoid arthritis.

Figure 1

Box plot of anti-TNF-α antibody immobilization at concentrations ranging from 0 to 12 µg/mL. Data were analysed by nonparametric way of a Kruskal-Wallis test followed by Tukey’s HSD test: (a) denotes significant differences compared to concentration 0, (b) denotes significant differences compared to concentration 1, (c) denotes significant differences compared to concentration 2, and (d) denotes significant differences compared to concentration 4.

Figure 2

SEM micrograph of a biofunctionalized NFM (A). Fluorescence micrograph of a NFM with the TNF-α antibody immobilized at the maximum concentration (6 µg/mL) (B).

Figure 3

TNF-α captured (median and interquartile range) over 72 h. Data were analysed by nonparametric way of a Kruskal-Wallis test, followed by Tukey’s HSD test: (a) denotes significant differences compared to immobilized anti-TNF-α at the surface of electrospun NFM (condition NFM + Ab).

Figure 4

TNF-α captured (median and interquartile range) over 15 days. Data were analyzed by nonparametric way of a Kruskal-Wallis test, followed by Tukey’s HSD test: (a) denotes significant differences compared to immobilized anti-TNF at the surface of electrospun NFM (condition NFM + Ab); and (b) denotes significant differences compared to soluble anti-TNF (condition sAb).

Figure 5

SEM micrographs of hACs cultures on tissue culture polystyrene (TCPs), UV-O treated electrospun PCL NFM (NFMs) and anti-TNF-α immobilized at the NFMs’ surface (NFM + Ab) for 14, 21 and 28 days.

Figure 6

Box plot of human articular chondrocytes proliferation (A) and protein synthesis (B) when cultured on tissue culture polystyrene (TCPs), UV-O treated electrospun PCL NFM (NFM) and anti-TNF-α immobilized at the NFMs’ surface (NFM + Ab). Data were analysed by nonparametric way of a Kruskal-Wallis test, followed by Tukey’s HSD test: (a) denotes significant differences compared to NFM + Ab and (b) denotes significant differences compared to NFM.

Figure 7

Blox plot of human articular chondrocytes GAGs accumulation when cultured on tissue culture polystyrene (TCPs), UV-O treated electrospun PCL NFM (NFM) and anti-TNF-α immobilized at the NFMs’ surface (NFM + Ab) (A). Data were analysed by nonparametric way of a Kruskal-Wallis test, followed by Tukey’s HSD test: (a) denotes significant differences compared to NFM + Ab and (b) denotes significant differences compared to NFM. Alcian blue staining evidencing sulphated proteoglycans deposition in samples from TCPs (B), NFMs (C), and NFM + Ab (D) on the 28^th day of the experiment.