*3.4. Quantification of Captured TNF-α*

The ultimate goal of this work was to engineer a system that could capture TNF-α. Accordingly, after the primary antibody immobilization optimization, the TNF-α capturing capacity of the biofunctionalized substrate was assessed. A TNF-α rich conditioned culture medium from stimulated monocyte-derived macrophages was used over two distinct time intervals: 3 days representing the half-life of the anti-TNF-α antibody and 15 days representing the time interval of anti-TNF-α antibody administration in the clinic. Four conditions were tested and compared: (1) the biofunctionalized NFM (with primary antibody); (2) a positive control with the soluble form of the anti-TNF-α; (3) a negative control with UV-O activated NFM; and (4) the conditioned culture medium alone. The quantification of TNF-α levels from the conditioned culture medium (condition 4) was used as a baseline, eliminating the quantification of degraded TNF-α but not captured. Therefore, to guarantee the values' accuracy and reproducibility between experiments, the values herein presented do not correspond to the ones quantified by ELISA, but just to the concentration of the TNF-α captured.

#### 3.4.1. TNF-α Capturing during 3 days

The proposed system comprising anti-TNF-α immobilized at the surface of NFMs (NFM + Ab) was found to capture soluble TNF-α in significant amounts after 8 hours of incubation when compared to the UV-O activated NFM (NFM) (*p* < 0.01) (Figure 3). This observation was also valid when the biofunctionalized substrate was compared to the soluble antibody (sAb), as the NFM + Ab exhibited significantly higher cytokine clearance for time points above the 10th hour of incubation (*p* < 0.01). In addition, no significant differences were found between the sAb and the control condition UV-O activated NFM (NFM).

**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).

The initial efficiency of the activated NFM over the TNF-α levels at early time points might be justified by the presence of free binding sites (such as the available amine groups quantified in Section 2.4) that unspecifically immobilize/adsorb proteins present in the conditioned medium, including TNF-α. Nevertheless, the capturing levels in this condition stabilize around zero for time points above 6 hours, unlike the biofunctionalized NFM + Ab, which maintains the capturing ability until the last time point. Likewise, the positive control sAb has a similar biological effect to the NFM + Ab testing condition during the first 10 hours. The sAb capturing ability decreases after this time point and becomes comparable to the NFM negative control condition. These results show that the immobilized form of anti-TNF-α is more stable and has longer action times, when compared to the non-immobilized one (i.e., sAb). However, this decrease might be partially justified by the gradual clearance of the soluble anti-TNF-α from the conditioned medium along the samples collection. It is envisioned that this decrease of sAb mimics its clearance in a living system, since the circulating antibody has higher propensity to be cleared by biological means [41]. Even if the sAb was locally administered, part of it would escape the articular cavity and enter into circulation, suffering also the clearance phenomena.
