*3.2. Protein Adsorption*

Adsorption of protein on the surface works as the initial step of biofouling when implanted in vivo and further compromises the surface properties, promotes cell attachment, and initiates the final foreign body response [46,47]. Thus, the resistance to protein adsorption of PU films is first taken into consideration.

Due to the inherent hydrophobic and electrostatic interactions between the surface and proteins, generally, the adsorption of proteins on the hydrophobic surface of PU films has no obvious inhibitory effect on the adhesion of any kind of contaminant [48,49]. Therefore, improving hydrophilicity is a valid approach to enhance the antifouling property of film to some extent.

The adsorption amount of BFG, HSA, and LYS on different films is listed in Figure 5. The PU-(PHMG/HA)5/5-5 surface exhibited the best resistance to protein adsorption, on which the adsorption levels of BFG, HSA and LYS were 2.43, 0.49, 0.16 µg/cm<sup>2</sup> , respectively. The adsorption amount followed the order of the molecular weight of proteins, that is BFG > HSA > LYS, as the high molecular weight resulted in the high amount of protein adsorbed on to patch at the same adsorption sites. The corresponding protein adsorption level was reduced 67.85%, 85.33%, and 80.31% compared with that on PU film. This is attributed to the higher hydrophilicity and lower surface roughness of PU-(PHMG/HA)5/5-5.

charges, and nanotopography structure of the surface as well as the size, shape, and charges of the proteins were the important factors for the protein adsorption property. Among these facts, surface hydrophilicity was a dominating one for protein adsorption.

**Figure 5.** Protein adsorption on films. **Figure 5.** Protein adsorption on films.

*3.3. Bacteria Adhesion*  Bacterial infection is the main complication after stent implantation, and the adhesion and colonization of bacteria on the stent play an essential part in scaling. Therefore, antibacterial functionalization becomes the key target for surface modification. Three typical uropathogens were selected to evaluate the broad-spectrum antimicrobial properties of PU, PU-PHMG, and PU-(PHMG/HA)5/5-5 (best resistance to protein adsorption). The results are shown in Table 3. The amount of bacteria adhered to naked PU was 29.2 × 105 CFU/cm2 for *E. coli*, 14.0 × 105 CFU/cm2 for *P. aeruginosa*, and 24.3 × 105 CFU/cm2 for *S. aureus*. Compared with PU, PU-PHMG showed excellent antibacterial effect against the three strains, and the adherence levels of the corresponding bacteria were as low as 0.0345 × 105, 0.0153 × 105, and 0.160 × 105 CFU/cm2 with inhibitory rates of 99.88%, 99.89%, and 99.34%, respectively. The effective antibacterial activity of PU-PHMG was attributed to the bactericidal capacity of PHMG. The interaction between PHMG and the anionic components of bacterial cell wall compromises membrane integrity, further causing cell membrane rupture and leads to microbial death [53,54]. The inhibition rates of PU-(PHMG/HA)5/5-5 on *E. coli*, *P. aeruginosa*, and *S. aureus* were 99.99%, 99.96%, and 99.99%, respectively, indicating that the film had outstanding antibacterial activity. The inhibition rate was slightly higher than that of PU-PHMG, indicating that the improvement of surface hydrophilicity and roughness also affect the antibacterial effect. Aside from its antimicrobial activities, biofouling resistance is another crucial element affecting the long-term property of the films. Generally, bacteria will both adhere to the film to form colonies and participate in the formation of subsequent biofilms, covering up the function of antibacterial substances, and subsequently causing inevitable biological contamination. After incubation with bacteria for one day, the antifouling ability of the films was assessed by imaging bacterial adhesion on the surface. Figure 6 illustrates the bacterial adhesion on PU, PU-PHMG, and PU-(PHMG/HA)5/5-5, respectively. As observed, most of the live bacteria and few dead bacteria accumulated on the PU surface (Figure 6a) because of its hydrophobic property. PU-PHMG, in contrast, adhered to most of the dead bacteria (Figure 6b), showing that it had efficient antibacterial Besides hydrophilicity and surface roughness, the nanotopography structure also influenced the amount of protein adsorbed on these modified PU films. In Figure 5, BFG adsorption level on PU-PHMG films was markedly lower than that on PU-(PHMG/HA)5/2-2, PU-(PHMG/HA)5/5-2, and PU-(PHMG/HA)1/5-5, though the hydrophilicity and roughness of PU-PHMG were higher than that of the three films. The possible reason might be the brush structure of PHMG on PU films repulsing parts of the proteins. The surface entropy of PU-PHMG increased when PHMG brushes were compressed by BFG, which was disadvantageous to thermodynamic stability and led to repelling BFG adsorption on the surface [50,51]. Similarly, the amount of HSA and LYS adsorbed on PU-PHMG were comparatively low in comparison with that on PU-(PHMG/HA)10/5-5 and PU- (PHMG/HA)5/5-10, respectively. Thus, the brush structure dominated the protein adsorption on PU-PHMG. Likewise, the anomalous observation was the relatively low level of BFG adsorption on PU in contrast with that on films with high roughness and medium hydrophilicity. This result might embody the importance of the protein sharp in adsorption. Fibrinogen is known as a cylinder (diameter = 6 nm, length = 45 nm) [52], where the side-on orientations were difficult to adsorb stably on PU with a roughness of 36.4 nm because of stereohindrance. In addition, more assembled layers negatively affected the protein repelling property (typical case illustrated in PU-(PHMG/HA)10/5-5), which provided more internal space to capture smaller sized proteins such as HSA into the swelling inner films through the microstructure [52]. Among the three model proteins, LYS possessed the smallest size, and was a positively-charged (isoelectric point at 11.0) and 'hard' one. The electrostatic repulsion between LYS and PHMG was noticeable, which reflected in lower LYS adsorption quantity on the PU-(PHMG/HA)1/5-5 with high roughness and medium hydrophilicity compared with other PU-(PHMG/HA)<sup>n</sup> films. After the formation of the first bilayer of PHMG and HA, the two molecules might be coiled and the positive charge of PHMG was not well covered by HA, as explained in th ATR-FTIR spectra, which resulted in a surface with good LYS repelling performance. As seen in the results above, the hydrophilicity, roughness, charges, and nanotopography structure of the surface as well as the size, shape, and charges of the proteins were the important factors for the protein adsorption property. Among these facts, surface hydrophilicity was a dominating one for protein adsorption.
