*3.1. Characterization of the Films*

The ATR-FTIR spectra of films at different preparation stages are shown in Figure 2. PU presented peaks originating from C=O and C–N at 1700 cm−<sup>1</sup> and 1530 cm−<sup>1</sup> , respectively (Figure 2a) [36]. A dominant absorption peak was observed at 2285 cm−<sup>1</sup> , which suggested that the –NCO group was successfully grafted on the PU surface (Figure 2b) [37,38]. However, Figure 2c shows that the peak of –NCO disappeared, and two symmetric and asymmetric –CH<sup>2</sup> stretching vibrations attributed to PHMG were noted at 2854 cm−<sup>1</sup> and 2924 cm−<sup>1</sup> [39], respectively, which confirmed that –NCO totally reacted with the –NH<sup>2</sup> of PHMG. Nevertheless, the spectra of PU-(PHMG/HA) (Figure 2d) showed no obvious change compared with that of PU-PHMG, suggesting that the first layer of HA might have little influence on the improvement of the surface properties. The wide peaks at 3324 cm−<sup>1</sup> assigned to the –OH group in HA increased (Figure 2e,f), indicating that the PHMG/HA bilayers were successfully assembled on the PU film [40]. Furthermore, the relatively broad peak at 1150 cm−<sup>1</sup> belonging to the ester group [41] in COOH– activated HA was found in the spectra of PU-(PHMG/HA)5/5-5 and PU-(PHMG/HA)10/5-5, while it did not appear in that of PU-PHMG and PU-(PHMG/HA)1/5-5. This phenomenon indicated that HA partially covalently bonded on the surface as expected. The reason might be due to the following: The activated –COOH provided by HA was not sufficient and completely reacted with the –NH<sup>2</sup> of PU-PHMG to –CO–NH during the preparation of the first bilayer PHMG/HA. Additionally, the –CO–NH was not able to be distinguished due to its original existence in any of the PU and modified PU films. With the increase in bilayer number, more HA provided more reactive ester groups, which could meet the demand in crosslinking of HA-PHMG. Nevertheless, the peak of the ester group was reduced with the increased bilayer, according to the spectra comparison of PU-(PHMG/HA)5/5-5 and PU-(PHMG/HA)10/5-5. This might be attributed to molecular rearrangement during the proceeding of assembly, which created more chances for the ester group to react with –NH2. In addition, the peak of –CH<sup>2</sup> weakened with the increased number of bilayers, but still existed on the surface of all assembled films in Figure 2d–f. It was supposed that the molecules of HA and PHMG were assembled in an entangled manner, which resulted in incomplete coverage of the HA chains on the surface [42].

The variation of the WCA was likewise related to the introduction of functional groups/molecules onto the surface. The WCA of the original PU was 90.1◦ due to its hydrophobicity [43,44]. The successful grafting of hydrophobic isocyanate on PU resulted in the WCA of PU-NCO increasing to 96.8◦ [37,45]. However, the succeeding PHMG onto the surface led to a low WCA (82.3◦ ) of PU-PHMG because of the introduction of the hydrophilic –NH<sup>2</sup> group. After HA was covalently bonded and electrostatic self-assembled onto PU-PHMG films, the surface became more hydrophilic. Subsequently, PHMG and HA alternately assembled onto the surface, which contributed to the WCA of corresponding films with a zig-zag effect (Figure 3). The HA (odd) layer achieved smaller WCA than that of the PHMG layer (even), suggesting higher hydrophilicity of HA than PHMG and proving that films with alternating deposition of polyelectrolyte were successfully obtained. In addition, the concentration of polyelectrolyte had an obvious effect on the WCA of the films. The increase or decrease in PHMG concentration both caused the WCA of the surface with relatively high value based on the comparison of preparation groups PHMG (10 mg/mL), PHMG (5 mg/mL), and PHMG (2 mg/mL) when HA was fixed at 5 mg/mL (Figure 3). One explanation might be less PHMG, leading to less HA loaded. The other might be the excessive PHMG providing more –CH<sup>2</sup> exposed on the surface when they entangled with HA. Therefore, it was found that the combination of HA (5 mg/mL)-PHMG

(5 mg/mL) achieved the greatest reduction in the WCA of the modified films among those prepared by other concentration combination of HA-PHMG. At this HA-PHMG concentration pair, HA and PHMG were well matched and reached a dynamic balance during the assembly process. Nevertheless, the WCA of PU-(PHMG/HA)n/5-5 had almost no apparent reduction, indicating that excessive assembled layers might have little impact on the function promotion of the surface. *Polymers* **2021**, *13*, x FOR PEER REVIEW 6 of 14

**Figure 2.** ATR−FTIR spectra of (**a**) PU; (**b**)PU-NCO; (**c**)PU-PHMG; (**d**) PU-(PHMG/HA)1/5-5; (**e**) PU-(PHMG/HA)5/5-5; (**f**) PU-(PHMG/HA)10/5-5. **Figure 2.** ATR–FTIR spectra of (**a**) PU; (**b**)PU-NCO; (**c**)PU-PHMG; (**d**) PU-(PHMG/HA)1/5-5; (**e**) PU-(PHMG/HA)5/5-5; (**f**) PU-(PHMG/HA)10/5-5. *Polymers* **2021**, *13*, x FOR PEER REVIEW 7 of 14

comparison of preparation groups PHMG (10 mg/mL), PHMG (5 mg/mL), and PHMG (2 mg/mL) when HA was fixed at 5 mg/mL (Figure 3). One explanation might be less **Figure 3.** Water contact angle (WCA) of modified PU films. n in PU-(PHMG/HA)n/5-5, PU-(PHMG/HA)n/5-2, PU-(PHMG/HA)n/2-5 and PU-(PHMG/HA)n/5-10 was the number of bi-**Figure 3.** Water contact angle (WCA) of modified PU films. n in PU-(PHMG/HA)n/5-5, PU- (PHMG/HA)n/5-2, PU-(PHMG/HA)n/2-5 and PU-(PHMG/HA)n/5-10 was the number of bilayer.

PHMG, leading to less HA loaded. The other might be the excessive PHMG providing more –CH2 exposed on the surface when they entangled with HA. Therefore, it was found that the combination of HA (5 mg/mL)-PHMG (5 mg/mL) achieved the greatest reduction in the WCA of the modified films among those prepared by other concentration combination of HA-PHMG. At this HA-PHMG concentration pair, HA and PHMG were well matched and reached a dynamic balance during the assembly process. Nevertheless, the WCA of PU-(PHMG/HA)n/5-5 had almost no apparent reduction, indicating that excessive assembled layers might have little impact on the function promotion of the surface. layer. The surface topography of the film was determined by AFM. The surface of the original PU was fairly flat and smooth with a root-mean-square (RMS) roughness of 36.4 ± 2.5 nm (Figure 4). However, the RMS of PU-PHMG surfaces increased significantly to 177.7 ± 2.3 nm (*p* < 0.001) compared to the PU films. One layer of HA assembled on PU-PHMG made little contribution to lower roughness of surface (173.9 ± 3.3 nm), which was consistent to the result of the ATR-FTIR spectra. However, after alternating PHMG and HA modification on PU films a few times, the surface roughness of PU-(PHMG/HA)n (e.g., PU-(PHMG/HA)5/5-5) decreased in comparison with that of The surface topography of the film was determined by AFM. The surface of the original PU was fairly flat and smooth with a root-mean-square (RMS) roughness of 36.4 ± 2.5 nm (Figure 4). However, the RMS of PU-PHMG surfaces increased significantly to 177.7 ± 2.3 nm (*p* < 0.001) compared to the PU films. One layer of HA assembled on PU-PHMG made little contribution to lower roughness of surface (173.9 ± 3.3 nm), which was consistent to the result of the ATR-FTIR spectra. However, after alternating PHMG and HA modification on PU films a few times, the surface roughness of PU-(PHMG/HA)<sup>n</sup> (e.g., PU-(PHMG/HA)5/5-5) decreased in comparison with that of PU-PHMG, but was still rougher than that of PU. Table 2 lists the surface roughness value for various samples. With the increase of HA concentration, the roughness of the films showed no obvious change

PU-PHMG, but was still rougher than that of PU. Table 2 lists the surface roughness value for various samples. With the increase of HA concentration, the roughness of the

PU-(PHMG/HA)5/5-2. With the increase in PHMG concentration, the roughness of the films decreased in comparison with PU-(PHMG/HA)5/5-2, PU-(PHMG/HA)5/5-5, and PU-(PHMG/HA)5/5-10. This influence might be related to the molecular weight of HA (>10 kDa) and PHMG (~600 Da). PHMG with far lower molecular weight than HA had relative flexibility and more PHMG was able to fill the void, which resulted in the lower roughness of the surface. Additionally, the number of assembled layers positively influenced the roughness of the modified films at the fixed preparation concentration based on the comparison of PU-(PHMG/HA)1/5-5, PU-(PHMG/HA)5/5-5, and PU-(PHMG/HA)10/5-5, whereas the increase in the bilayer number had a minor contribution to lower the roughness when the number of bilayers was more than five. PU-(PHMG/HA)10/5-5 possessed the smoothest surface with a RMS roughness value of 130.8 ± 2.6 nm, followed by PU-(PHMG/HA)5/5-10 and PU-(PHMG/HA)5/5-5. The roughness of the above three films had no remarkable differences. Therefore, PU-(PHMG/HA)5/5-5 was the optimum film when taking into account the preparation

**Figure 4.** Atomic force microscopy (AFM) images of (**a**) PU; (**b**) PU-PHMG; (**c**)

costs.

PU-(PHMG/HA)5/5-5.

layer.

based on the comparison of PU-(PHMG/HA)5/2-2 and PU-(PHMG/HA)5/5-2. With the increase in PHMG concentration, the roughness of the films decreased in comparison with PU-(PHMG/HA)5/5-2, PU-(PHMG/HA)5/5-5, and PU-(PHMG/HA)5/5-10. This influence might be related to the molecular weight of HA (>10 kDa) and PHMG (~600 Da). PHMG with far lower molecular weight than HA had relative flexibility and more PHMG was able to fill the void, which resulted in the lower roughness of the surface. Additionally, the number of assembled layers positively influenced the roughness of the modified films at the fixed preparation concentration based on the comparison of PU-(PHMG/HA)1/5-5, PU-(PHMG/HA)5/5-5, and PU-(PHMG/HA)10/5-5, whereas the increase in the bilayer number had a minor contribution to lower the roughness when the number of bilayers was more than five. PU-(PHMG/HA)10/5-5 possessed the smoothest surface with a RMS roughness value of 130.8 ± 2.6 nm, followed by PU-(PHMG/HA)5/5-10 and PU- (PHMG/HA)5/5-5. The roughness of the above three films had no remarkable differences. Therefore, PU-(PHMG/HA)5/5-5 was the optimum film when taking into account the preparation costs. PU-(PHMG/HA)5/5-2. With the increase in PHMG concentration, the roughness of the films decreased in comparison with PU-(PHMG/HA)5/5-2, PU-(PHMG/HA)5/5-5, and PU-(PHMG/HA)5/5-10. This influence might be related to the molecular weight of HA (>10 kDa) and PHMG (~600 Da). PHMG with far lower molecular weight than HA had relative flexibility and more PHMG was able to fill the void, which resulted in the lower roughness of the surface. Additionally, the number of assembled layers positively influenced the roughness of the modified films at the fixed preparation concentration based on the comparison of PU-(PHMG/HA)1/5-5, PU-(PHMG/HA)5/5-5, and PU-(PHMG/HA)10/5-5, whereas the increase in the bilayer number had a minor contribution to lower the roughness when the number of bilayers was more than five. PU-(PHMG/HA)10/5-5 possessed the smoothest surface with a RMS roughness value of 130.8 ± 2.6 nm, followed by PU-(PHMG/HA)5/5-10 and PU-(PHMG/HA)5/5-5. The roughness of the above three films had no remarkable differences. Therefore, PU-(PHMG/HA)5/5-5 was the optimum film when taking into account the preparation costs.

**Figure 3.** Water contact angle (WCA) of modified PU films. n in PU-(PHMG/HA)n/5-5,

PU-(PHMG/HA)n/5-2, PU-(PHMG/HA)n/2-5 and PU-(PHMG/HA)n/5-10 was the number of bi-

The surface topography of the film was determined by AFM. The surface of the original PU was fairly flat and smooth with a root-mean-square (RMS) roughness of 36.4 ± 2.5 nm (Figure 4). However, the RMS of PU-PHMG surfaces increased significantly to 177.7 ± 2.3 nm (*p* < 0.001) compared to the PU films. One layer of HA assembled on PU-PHMG made little contribution to lower roughness of surface (173.9 ± 3.3 nm), which was consistent to the result of the ATR-FTIR spectra. However, after alternating PHMG and HA modification on PU films a few times, the surface roughness of PU-(PHMG/HA)n (e.g., PU-(PHMG/HA)5/5-5) decreased in comparison with that of PU-PHMG, but was still rougher than that of PU. Table 2 lists the surface roughness value for various samples. With the increase of HA concentration, the roughness of the films showed no obvious change based on the comparison of PU-(PHMG/HA)5/2-2 and

**Figure 4.** Atomic force microscopy (AFM) images of (**a**) PU; (**b**) PU-PHMG; (**c**) PU-(PHMG/HA)5/5-5. **Figure 4.** Atomic force microscopy (AFM) images of (**a**) PU; (**b**) PU-PHMG; (**c**) PU- (PHMG/HA)5/5-5.


*Polymers* **2021**, *13*, x FOR PEER REVIEW 7 of 14

