*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 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/cm<sup>2</sup> for *E. coli*, 14.0 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/cm<sup>2</sup> for *P. aeruginosa*, and 24.3 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/cm<sup>2</sup> 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 <sup>×</sup> <sup>10</sup><sup>5</sup> , 0.0153 <sup>×</sup> <sup>10</sup><sup>5</sup> , and 0.160 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/cm<sup>2</sup> 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.

**Table 3.** Antibacterial test of films against *E. coli*, *P. aeruginosa*, and *S. aureus*.


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 property but nearly no antifouling performance due to electrostatic adsorption and hydrophobic interaction [55]. Therefore, PU-PHMG merely maintained the antibacterial properties at the initial stage, but was gradually covered by dead bacteria and lost its function during long-term incubation with bacteria. To our delight, bacteria were barely observed on the surface of PU-(PHMG/HA)5/5-5 (Figure 6c), indicating no biofilm had formed. The high bactericidal efficiency was attributed to two aspects. On one hand, PHMG can kill bacteria temporarily adhered to the surface. On the other hand, the size of almost all of the bacteria was larger than 500 nm, which made the bacteria unable to be entrapped in the rough area. Therefore, the killed bacteria, gently adsorbed on the surface, can be easily stripped by simple hydraulic turbulence [46] due to the hydrophilicity of HA. The result of the antifouling property of three test films indicated that the antifouling property of the surface was important for an antibacterial effect. PU-(PHMG/HA)5/5-5 with good hydrophilicity containing PHMG and HA exhibited excellent antibacterial and antifouling properties, suggesting that it had an ideal antibacterial surface for future biomedical usage.

**Samples** 

**Colonies (×105, CFU/cm2)** 

**Table 3.** Antibacterial test of films against *E. coli*, *P. aeruginosa*, and *S. aureus*.

**Colonies (×105, CFU/cm2)** 

PU 29.2 ± 8.77 / 14.0 ± 0.283 / 24.3 ± 0.778 /

5-5 0 99.99 0.00514 ± 0.000247 99.96 0 99.99

*E. coli P. aeruginosa S. aureus* 

**Inhibition (%)** 

**Colonies (×105, CFU/cm2)** 

**Inhibition (%)** 

rial surface for future biomedical usage.

**Inhibition (%)** 

**Figure 6.** Confocal laser scanning microscopy (CLSM) images of *E. coli* adherent onto (**a**) PU, (**b**) PU-PHMG, and (**c**) PU-(PHMG/HA)5/5-5 film. The live bacteria appear as green fluorescence and dead bacteria appear as red fluorescence. Scale bars are 50 μm. **Figure 6.** Confocal laser scanning microscopy (CLSM) images of *E. coli* adherent onto (**a**) PU, (**b**) PU-PHMG, and (**c**) PU-(PHMG/HA)5/5-5 film. The live bacteria appear as green fluorescence and dead bacteria appear as red fluorescence. Scale bars are 50 µm.

#### *3.4. Cytotoxicity Test 3.4. Cytotoxicity Test*

cell viability.

Biocompatibility is an essential requirement in bio-materials for their potential biomedical application [56]. Cytotoxicity testing can generally be performed in two ways—contact (direct) and extraction (indirect) [35]. The extraction method was applied due to the anti-adhesion property of the film surface, which was difficult for cells to adhere on. The conditioned cell culture medium mimicked the effect of the film on the physiological environment. The results of the cytotoxicity of L929 cultured in leaching solution of films are shown in Figure 7. PU and PU-PHMG films had high cell viability (over 88%) and the PU-(PHMG/HA)5/5-5 film had no cytotoxicity against L929 cell compared to the control, which indicated that the final surface modification was favorable to Biocompatibility is an essential requirement in bio-materials for their potential biomedical application [56]. Cytotoxicity testing can generally be performed in two ways—contact (direct) and extraction (indirect) [35]. The extraction method was applied due to the antiadhesion property of the film surface, which was difficult for cells to adhere on. The conditioned cell culture medium mimicked the effect of the film on the physiological environment. The results of the cytotoxicity of L929 cultured in leaching solution of films are shown in Figure 7. PU and PU-PHMG films had high cell viability (over 88%) and the PU-(PHMG/HA)5/5-5 film had no cytotoxicity against L929 cell compared to the control, which indicated that the final surface modification was favorable to cell viability. *Polymers* **2021**, *13*, x FOR PEER REVIEW 11 of 14

property but nearly no antifouling performance due to electrostatic adsorption and hydrophobic interaction [55]. Therefore, PU-PHMG merely maintained the antibacterial properties at the initial stage, but was gradually covered by dead bacteria and lost its function during long-term incubation with bacteria. To our delight, bacteria were barely observed on the surface of PU-(PHMG/HA)5/5-5 (Figure 6c), indicating no biofilm had formed. The high bactericidal efficiency was attributed to two aspects. On one hand, PHMG can kill bacteria temporarily adhered to the surface. On the other hand, the size of almost all of the bacteria was larger than 500 nm, which made the bacteria unable to be entrapped in the rough area. Therefore, the killed bacteria, gently adsorbed on the surface, can be easily stripped by simple hydraulic turbulence [46] due to the hydrophilicity of HA. The result of the antifouling property of three test films indicated that the antifouling property of the surface was important for an antibacterial effect. PU-(PHMG/HA)5/5-5 with good hydrophilicity containing PHMG and HA exhibited excellent antibacterial and antifouling properties, suggesting that it had an ideal antibacte-

#### extracts. **4. Conclusions**

version of the manuscript.

Scientist Joint Research Center.

(50321102117022).

*C-Mater. Biol. Appl.* **2017**, *71*, 1166–1174, doi:10.1016/j.msec.2016.11.125.

**References** 

**4. Conclusions**  In this study, we focused on surface modification with hydrophilic material and an antibacterial agent to simultaneously improve the antifouling and antibacterial properties of the PU film. We successfully created PHMG/HA multilayer films on PU by using layer-by-layer self-assembly with COOH-activated HA and PHMG as polyanions and polycations. An optimal film named as PU-(PHMG/HA)5/5-5 with the lowest WCA and medium roughness was obtained, which possessed excellent protein repelling performance. The adsorption levels of BFG, HSA, and LYS reduced 67.85%, 85.33% and 80.31%, respectively, compared with that on the PU film. In addition, the high bacteriostatic rate of over 99.9% against the three tested bacteria and excellent antibacterial ad-In this study, we focused on surface modification with hydrophilic material and an antibacterial agent to simultaneously improve the antifouling and antibacterial properties of the PU film. We successfully created PHMG/HA multilayer films on PU by using layer-bylayer self-assembly with COOH-activated HA and PHMG as polyanions and polycations. An optimal film named as PU-(PHMG/HA)5/5-5 with the lowest WCA and medium roughness was obtained, which possessed excellent protein repelling performance. The adsorption levels of BFG, HSA, and LYS reduced 67.85%, 85.33% and 80.31%, respectively, compared with that on the PU film. In addition, the high bacteriostatic rate of over 99.9% against the three tested bacteria and excellent antibacterial adhesion property showed that PU-(PHMG/HA)5/5-5 possessed high antimicrobial and anti-biofouling performance.

hesion property showed that PU-(PHMG/HA)5/5-5 possessed high antimicrobial and anti-biofouling performance. Furthermore, the film had nearly no cytotoxicity against L929

**Author Contributions:** Conceptualization, H.Y. and M.L.; Funding acquisition M.L.; Methodology, H.Y., C.X., and J.Z.; Resources C.X. and Z.Y.; Software, C.X.; Validation, C.X. and J.Z.; Investigation, C.X.; Resources, C.X.; Writing—original draft preparation, H.Y., C.X., Z.Y., and M.L.; Writing—review and editing, H.Y., C.X. and M.L. All authors have read and agreed to the published

**Funding:** This research was funded by the Science and Technology Commission of Shanghai Municipality (STCSM, 20520712500) and the Fundamental Research Funds for the Central Universities

**Acknowledgments:** This work was supported by the Science and Technology Commission of Shanghai Municipality (STCSM, 20520712500) and the Fundamental Research Funds for the Central Universities (50321102117022). Additional support was provided by the Feringa Nobel Prize

**Conflicts of Interest:** The authors declare no conflict of interest.

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2. Gorbunova, M.A.; Anokhin, D.V.; Badamshina, E.R. Recent Advances in the Synthesis and Application of Thermoplastic Sem-

3. Yuan, H.H.; Qian, B.; Chen, H.Y.; Lan, M.B. The influence of conditioning film on antifouling properties of the polyurethane

icrystalline Shape Memory Polyurethanes. *Polym. Sci. Ser. B* **2020**, *62*, 427–450, doi:10.1134/s1560090420050073.

film modified by chondroitin sulfate in urine. *Appl. Surf. Sci.* **2017**, *426*, 587–596, doi:10.1016/j.apsusc.2017.06.314.

Furthermore, the film had nearly no cytotoxicity against L929 cells, which made it possible for biomedical applications in the future.

**Author Contributions:** Conceptualization, H.Y. and M.L.; Funding acquisition M.L.; Methodology, H.Y., C.X., and J.Z.; Resources C.X. and Z.Y.; Software, C.X.; Validation, C.X. and J.Z.; Investigation, C.X.; Resources, C.X.; Writing—original draft preparation, H.Y., C.X., Z.Y., and M.L.; Writing—review and editing, H.Y., C.X. and M.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Science and Technology Commission of Shanghai Municipality (STCSM, 20520712500) and the Fundamental Research Funds for the Central Universities (50321102117022).

**Acknowledgments:** This work was supported by the Science and Technology Commission of Shanghai Municipality (STCSM, 20520712500) and the Fundamental Research Funds for the Central Universities (50321102117022). Additional support was provided by the Feringa Nobel Prize Scientist Joint Research Center.

**Conflicts of Interest:** The authors declare no conflict of interest.
