## 4 Discussion
Considering that similar behavior has been observed for our non-fouling coatings (i.e., PEOA of Figure 9) where almost no cells were observed on them, the delamination of the coatings could be likely due to components of the cell culture medium (i.e., ions, proteins) that may change the interfacial liquid and coating interactions and promote its detachment from the underlying substrate. Similar behavior was observed for conventional PEO films deposited by spin coating [39; 40]. In literature, it is reported that PEO-like films swell in PBS of approximately 20%, as revealed by in situ liquid-cell ellipsometry measurements at a time scale of several minutes [41]. Owing to the repetition of CH\({}_{2}\)CH\({}_{2}\)O ether groups, it is known that PEO forms complexes with various metal cations in organic solvents [42]. Complex formation between conventional PEO and such metal cations in water have been reported in the literature [40], demonstrating that PEO coatings show unique swelling behavior in aqueous salt systems. As noted earlier, since the chemical nature of oxygen in PEO is different concerning other hydrocarbon polymers, like polyvinyl alcohol, the interaction with metal cations present in the cell culture medium results in different swelling behavior. These findings on conventional PEO could explain also the behavior observed on plasma deposited PEO-like coatings [40]. The multi-step approach proposed in this paper for AP plasma processes was revealed to be a successful strategy to improve the resistance of the coating to delamination during in vitro cell-adhesion tests. AR-XPS analysis (Figure 12) performed on three-layer PEOA coatings shows that also in the last layer (\(\sim\)200 nm thick, Figure 10) there is a gradual enrichment of the O-containing moieties toward the topmost layer. The topmost layer shows a composition very similar to the PEOA deposited in a single step and to that deposited at LP. It is important to notice that the last deposition step, step 3, was carried out in-continuous, without interrupting the discharge, after closing the on-off valve of C\({}_{2}\)H\({}_{4}\) flow to the reactor. This process produces a vertical chemical gradient in the last layer due to the progressive consumption of C\({}_{2}\)H\({}_{4}\) after the closure of the on-off valve that connects the plasma chamber to the C\({}_{2}\)H\({}_{4}\) line. In fact, it is supposed that 5 min of discharge, is not enough for purging or consuming unreacted ethylene and its active species produced in the previous steps. Thus, probably ethylene contributes actively in a co-deposition with TEGDME in the last step going toward a topmost layer richer of COC motifs when C\({}_{2}\)H\({}_{4}\) is completely consumed. Almost all AP coatings resulting from the multilayer approach are stable in water and during cell culture experiments, except PEOA deposited at 16 kHz and 6.5 kV\({}_{\text{P-P}}\) that, similarly to AP coatings deposited in a single step approach, delaminates during cell culture experiments (Figure 13c). The improvement in film adhesion is due to a hydrophobic interlayer, like the substrate below, well attached to it, thanks to a plasma polymerization process that involves ethylene which is a highly reactive precursor. If soft conditions are used (i.e., low frequency and low peak-to-peak voltage) this strategy fails, probably due to a reduced crosslinking in the film.