B. Fibronectin adsorption on the grafted surfaces

1. Effect of fibronectin adsorption on surface energy

FN was adsorbed on the surface of nongrafted and grafted PET and PCL for 1 h at 37 °C in PBS. They were characterized by IR spectroscopy and contact angles. Interest bands at 1538 and 1550 cm−1 corresponding to amide-II on fibronectin2626. D. Usoltsev, V. Sitnikova, A. Kajava, and M. Uspenskaya, Biomolecules 9, 359 (2019). https://doi.org/10.3390/biom9080359 were found on both nongrafted and grafted PET [Fig. 3(a)] and PCL [Fig. 3(c)].FN covered the surface and changed the surface tension. The surface tension change depends on the organization of FN that was influenced by FN-surface interaction. Indeed, the water contact angle (WCA) decreased on the nongrafted surface after FN adsorption [Fig. 3(b)]. In contrast, WCA increased on grafted PET and PCL (∼10%) [Fig. 3(d)].

2. Conformation and nanomechanical properties of fibronectin on the grafted surface by AFM

The adsorbed FN was characterized by AFM using PF-QNM and FV in the air. FN fibrillogenesis was observed on nongrafted samples with 10–15 nm thickness of fibril [Figs. 4(a), 4(b), and S4 in the supplementary material].3838. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001165 for more information on contact angle and AFM analysis. Determination of the thickness of FN on PCL was a challenge due to the surface roughness. Therefore, the thickness of fibrils was estimated by roughness sections [Fig. 4(b)]. The conformation of FN was confirmed by adhesion maps [Figs. 4(a) and 4(b)—adhesion], which showed the contrast between FN fibrils and the substrate based on the interaction of tip-protein (dark) and tip-surface (bright).The particles appeared on the PNaSS-grafted surface of PET [Fig. 4(c)] that was confirmed by adhesion maps (dark dots) after FN adsorption. Similarly, particles were covered completely on the grafted surface [Fig. 4(d)] and they concealed the lamellar structure of PCL [Fig. 2(e)]. The particles were found on both grafted PET and PCL. This conformation of FN was reported on charged surfaces, e.g., sulfonation polystyrene2727. H. M. Kowalczyńska, R. Kołos, M. Nowak-Wyrzykowska, J. Dobkowski, D. Elbaum, A. Szczepankiewicz, and J. Kamiński, J. Biomed. Mater. Res. A 91, 1239 (2009). https://doi.org/10.1002/jbm.a.32473 or oxygen plasma-treated polyurethane surfaces.2828. W. Guo et al., Macromolecules 50, 8670 (2017). https://doi.org/10.1021/acs.macromol.7b01888 Unfortunately, determining the height of the FN particles on grafted PET and PCL was not possible due to the imprecision on defining the substrate level due to the roughness of the surface of PCL and possibly multiple-layer adsorption of FN.The morphology of FN on nongrafted and grafted surfaces was different as a result of the protein-surface interaction.29,3029. M. Bergkvist, J. Carlsson, and S. Oscarsson, J. Biomed. Mater. Res. A 64, 349 (2003). https://doi.org/10.1002/jbm.a.1042330. M. D. Heath, B. Henderson, and S. Perkin, Langmuir 26, 5304 (2010). https://doi.org/10.1021/la100678n Based on the interaction between the tip-protein by a nonspecific interaction measured by contact mode AFM (FV), we recorded the force-distance curves on the PCL surface with fibronectin adsorption [Figs. 4(b)–4(d)] to determine the organization of proteins (Fig. 5). The force curve response of fibronectin on the nongrafted and grafted surfaces was significantly different [Figs. 5(a), 5(b), and S2 in the supplementary material].3838. See the supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001165 for more information on contact angle and AFM analysis. Indeed, an extension force was observed in Fig. 5(b) for FN adsorption on the grafted PCL surface that showed at (3) on the withdraw curve [Fig. 5(d)]. The extension force was from the interaction of the tip and single fibronectin31–3331. Z. Sun, L. A. Martinez-Lemus, A. Trache, J. P. Trzeciakowski, G. E. Davis, U. Pohl, and G. A. Meininger, Am. J. Physiol. Hear. Circ. Physiol. 289, 2526 (2005). https://doi.org/10.1152/ajpheart.00658.200432. F. S. Ruggeri, F. Benedetti, T. P. J. Knowles, H. A. Lashuel, S. Sekatskii, and G. Dietler, Proc. Natl. Acad. Sci. U.S.A. 115, 7230 (2018). https://doi.org/10.1073/pnas.172122011533. S. S. Nair, C. Wang, and K. J. Wynne, Prog. Org. Coat. 126, 119 (2019). https://doi.org/10.1016/j.porgcoat.2018.10.008 [Fig. 5(g)]. There was no extension force observed on the withdraw curve on the surface of nongrafted PCL with FN adsorption [Figs. 5(a)–5(c)].The “pulling-off” fibronectin by an AFM tip was described in Fig. 5(g). Each step in Fig. 5(g) corresponds to the position in Fig. 5(b). (1) The tip approaches the surface and interacts with the protein on the surface. Then, it withdraws and pulls the protein (2). As a result, the protein is stretched until the contact was lost (3). The pull-off force (ΔF∼0.1nN) was measured in step (3), and the plateau length was determined from (2) to (3).23,3423. A. F. Oberhauser, C. Badilla-Fernandez, M. Carrion-Vazquez, and J. M. Fernandez, J. Mol. Biol. 319, 433 (2002). https://doi.org/10.1016/S0022-2836(02)00306-634. S. M. Früh, I. Schoen, J. Ries, and V. Vogel, Nat. Commun. 6, 7275 (2015). https://doi.org/10.1038/ncomms8275

We used the FV program for collected force-distance curves with a total of 1024 points. The pull-off was evident in >90% of the force-distance curves obtained on the PNaSS-grafted surface. The length of adsorbed FN on the grafted surface was estimated to be ∼130 nm.

In this work, albumin was used for passivation of the surfaces and for stabilizing the FN solution, and we focus on the FN adsorption and conformation changes on specific sites of the surface. The fibronectin adsorbed on the nongrafted surface folded [Fig. 5(f)] and formed the net of fibronectin [Figs. 4(a) and 4(b)]. However, fibronectin adsorbed can unfold on the charged surface of PNaSS grafted.1616. N. Pernodet, M. Rafailovich, J. Sokolov, D. Xu, N. L. Yang, and K. McLeod, J. Biomed. Mater. Res. A 64, 684 (2003). https://doi.org/10.1002/jbm.a.10394 As a result, FN unfolded partially: the charged domains of proteins interacted with the grafted surface by electrostatic interaction, and the uncharged domains formed a coil [Figs. 4(c) and 4(d)]. The extension force was produced due to the nonspecific interaction of the tip-protein.The results showed that the electrostatic interaction between FN and the grafted surface decreased the Gibbs free energy of folded FN and strategically conditioned to undergo conformational changes. This change revealed specific binding (α5β1), which confirmed that there is a strong interaction between the cell membrane and protein.3535. K. J. Johnson, H. Sage, G. Briscoe, and H. P. Erickson, J. Biol. Chem. 274, 15473 (1999). https://doi.org/10.1074/jbc.274.22.15473