Antimicrobial Activities of Hydrophobically Modiﬁed Poly(Acrylate) Films and Their Complexes with Di ﬀ erent Chain Length Cationic Surfactants

: Multilayer ﬁlms from hydrophobically modiﬁed poly(acrylic acid) (HMPA) and their complexes with cationic surfactants were successfully prepared using the layer-by-layer (LbL) method. Alkyl trimethylammonium bromide derivatives with various lengths of the hydrophobic chain (C 10 –C 18 ) were used to interact with the HMPA polymer, generating highly hydrophobic domains in the ﬁlms and contributing to the antimicrobial properties of the prepared coating. The antimicrobial e ﬃ ciency against common pathogens such as Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginosa , and Candida albicans was investigated in relationship with the morphology and composition of the thin ﬁlms. The wettability and roughness of the multilayered systems were evaluated using atomic force microscopy (AFM) and contact angle measurements. The e ﬀ ects of the microbial exposure on the surface properties of the prepared ﬁlms were investigated in order to assess the stability of the HMPA-deposited multilayers and the durability of the antimicrobial activity. The hydrophobically modiﬁed ﬁlms exhibited antimicrobial activity against the studied pathogens. The best e ﬃ ciency was registered in the case of S. aureus , which showed an inhibition of growth up to 100% after 2 h. to the e ﬀ ect of the multilayers deposited from HMPA–surfactant complexes. These results suggest that HMPA and HMPA–surfactant complex LbL multilayer ﬁlms can be used as promising materials in antimicrobial surface coatings with increased resistance to pathogens during exposure.


Introduction
Polymeric materials are widely used as antimicrobial nanocoatings to prevent biocontamination, which has a direct impact on human health. In recent years, polymeric thin films, membranes, The hydrophobically modified polymer PAC 18 Na used for the fabrication of multilayer films was prepared according to the synthesis previously reported, together with the characterization of the prepared polymers [22]. The polymer-surfactant complexes PAC 18 Na-C x TAB (x = 10, 12, 14 and 18) were prepared according to our previous study [20], and the aqueous PAC 18 Na solution was added to a C x TAB micellar solution under continuous stirring. The complexes were formed due to the electrostatic interaction between the negatively charged PAC 18 Na and the positively charged C x TAB. To ensure that the complexes were formed, measurements of zeta potential were carried out as in our previous paper [18]. The polymer-surfactant complexes (PAC 18 Na-C n TAB), (10 −2 M solution containing 0.01 M NaCl), PDADMAC (10 −2 M), and PEI (5 × 10 −2 M) were prepared and stored in a refrigerator for further use. The multilayers were deposited alternatively on a glass previously treated with PEI by using PDADMAC and PAC 18 Na solutions.
The LbL depositions were achieved using a programmable dipping machine (Dipping Robot DR-3, Riegler & Kirstein GmbH, Potsdam, Germany). The dipping procedure was reported elsewhere [20,21]. After six months of aging at room temperature and in the dark, the investigated films were exposed to a microbial environment. The composition of polyelectrolyte-surfactant films exposed to a microbial environment is presented in Table 1. Table 1. Hydrophobically modified poly(acrylate)-surfactant films investigated for antimicrobial properties.

Antimicrobial Activity Testing
The antimicrobial activity of the polyelectrolyte-surfactant LbL films was tested against S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853 and C. albicans ATCC 10231 according to the National Committee for Clinical Laboratory Standards [23]. All the experiments were performed in triplicate. Fresh microbial cultures obtained on nutrient agar were used to make 0.5 McFarland suspensions that were further diluted till a working solution of 1.5 × 10 6 colony forming units (CFUs). The films (initially sterilized by exposure for 30 min to UV on either side) were incubated in tubes with 9 mL of saline water and 1 mL of bacterial suspension at 37 • C under continuous shaking. After different periods of time (right after initial contact, 2, 3, 4, 5, 6 and 24 h, respectively), the number of CFUs was determined.

Characterization Methods
The contact angle measurements were collected using a Drop Shape Analysis System, model DSA1 (FM40 Easy Drop) from KRÜSS GmbH (Hamburg, Germany). The water drop volume was of 3 mL, and the measurements were done in static regime at room temperature in air. Zeta potential measurements were carried out on a MalvernNano ZS Zetasizer (Malvern Ltd., Malvern, UK) at room temperature to measure the electrokinetic potential. AFM measurements were carried out in noncontact mode with an XE-100 (Park Systems, Suwon, Korea) equipped with flexure-guided, cross-talked eliminated scanners, as recommended for soft materials. The morphological AFM images were taken at 2 × 2 µm 2 . The XEI program (v.1.8.0) was used for image processing and roughness evaluation. In order to have a better view of the surface morphology, the AFM images are presented in the so-called "enhanced color view mode".

Results and Discussion
From the LbL deposition method, thin films consisting of a mixture of hydrophobically modified polymers and surfactants were obtained. Various surfactants with hydrocarbon chains from C 10 to C 18 were used in order to improve the antimicrobial activity and to modify the morphology of the thin film. We observed that the contact angle increased suddenly after the deposition of five bilayers. Moreover, after aging six months, the contact angle and the rough surface increased significantly for the films containing 20 bilayers according to our study [20].
Also, our previous study evidenced that a PDADMAC outer layer leads to ball-like particles being randomly deposed [20]. Consequently, the obtained films presented higher roughness and contact angle compared with the films without a PDADMAC outer layer. In order to observe if one or more layers were removed from the polyelectrolyte multilayer films, zeta potential measurements were conducted. The zeta potential data proved to be negative for all of the samples. The results are presented in Table 2. The stability of the thin films in aqueous solution was investigated and the results are shown in Table 2. The samples were kept for 24 h at room temperature to observe if the electrokinetic potential changed during the experiment. After 2 h of stabilization, zeta potentials reached values that did not change significantly (~1 to 2 mV) during the rest of the 22 h of storage in aqueous solution. Results indicated that the surfaces did not seem to lose polymer even when the polyelectrolyte or polyelectrolyte-surfactant films were immersed for 1 day in liquid media. The results were similar with the behavior previously observed by Costerton et al. for a multilayered architecture immersed in water, MeOH, hexanes, acetone, dichloromethane, and ethyl acetate [24].

Evaluation of the Antimicrobial Activity of Multilayered Films
In order to evaluate the antimicrobial activity of multilayered films, S. aureus, E. coli, P. aeruginosa and C. albicans were selected as model microorganisms, as they are responsible for the most frequent hospital infections. The antimicrobial properties were investigated by testing the bacterial growth in liquid broth media. Figure 1 presents the bacterial growth of E. coli in liquid media on polymer-surfactant complex films of PAC 18 Na-C x TAB-NaCl/PDADMAC (x = 10, 12, 14, and 18) compared to the results of bacterial growth on PAC 18 Na-NaCl/PDADMAC and untreated glass. On the control material (untreated glass), the bacteria showed a constant growth rate after 24 h of exposure. The antimicrobial activity of all the tested materials (polyelectrolyte-surfactant films and pristine PAC 18 Na film) on E. coli proved to be very good after 24 h of incubation, as demonstrated by the complete bacterial growth inhibition. An unexpectedly high efficiency was recorded for the film prepared with the C 14 TAB surfactant (PAC 18 Na-C 14 TAB/PDAMAC) 20 , since no CFUs after 3 h of contact were present on the tested multilayers.
changed during the experiment. After 2 h of stabilization, zeta potentials reached values that did not change significantly (~1 to 2 mV) during the rest of the 22 h of storage in aqueous solution. Results indicated that the surfaces did not seem to lose polymer even when the polyelectrolyte or polyelectrolyte-surfactant films were immersed for 1 day in liquid media. The results were similar with the behavior previously observed by Costerton et al. for a multilayered architecture immersed in water, MeOH, hexanes, acetone, dichloromethane, and ethyl acetate [24].

Evaluation of the Antimicrobial Activity of Multilayered Films
In order to evaluate the antimicrobial activity of multilayered films, S. aureus, E. coli, P. aeruginosa and C. albicans were selected as model microorganisms, as they are responsible for the most frequent hospital infections. The antimicrobial properties were investigated by testing the bacterial growth in liquid broth media. Figure 1 presents the bacterial growth of E. coli in liquid media on polymersurfactant complex films of PAC18Na-CxTAB-NaCl/PDADMAC (x = 10, 12, 14, and 18) compared to the results of bacterial growth on PAC18Na-NaCl/PDADMAC and untreated glass. On the control material (untreated glass), the bacteria showed a constant growth rate after 24 h of exposure. The antimicrobial activity of all the tested materials (polyelectrolyte-surfactant films and pristine PAC18Na film) on E. coli proved to be very good after 24 h of incubation, as demonstrated by the complete bacterial growth inhibition. An unexpectedly high efficiency was recorded for the film prepared with the C14TAB surfactant (PAC18Na-C14TAB/PDAMAC)20, since no CFUs after 3 h of contact were present on the tested multilayers.  Even though Pseudomonas aeruginosa is also a Gram-negative bacteria just like E. coli, it was more resistant to the antimicrobial activity of the films. (PAC 18 Na-NaCl/PDADMAC) 20 and (PAC 18 Na-C 10 TAB-NaCl/PDAMAC) 20 inhibited the strain's development after 24 h and (PAC 18 Na-C 18 TAB-NaCl/PDAMAC) 20 film presence determined a colony forming unit (CFU) decrease after 5 and 6 h of incubation compared with the control sample ( Figure 2).
The multilayers prepared from mixtures of hydrophobically modified polymers and cation surfactants with longer alkyl chains (C 12 -C 18 ) exhibited no antibacterial activity. The films from polymer (PAC 18 Na/PDADMAC) 20 without a surfactant and with a short chain surfactant (C 10 ) exhibited the inhibition of bacterial growth after 24 h of exposure. In Figure 3, the development and survival of S. aureus strains in the presence of the films containing hydrophobically modified polyacrylate and the complexes with various alkyltrimethylammonium bromide at 20 bilayers are shown. All the multilayers exhibited a significant inhibition of bacterial growth after 2 or 3 h of incubation, while the film (PAC18Na/PDADMAC)20 without a surfactant proved to be the most effective.
The fungal strain C. albicans was less susceptible to the films' activity, as can be observed in Figure 4, and the number of CFUs was constant for all the tested samples. The exception was the film prepared with the C14 surfactant, which showed moderate activity: 24 h of exposure resulted in the inhibition of fungal growth.  In Figure 3, the development and survival of S. aureus strains in the presence of the films containing hydrophobically modified polyacrylate and the complexes with various alkyltrimethylammonium bromide at 20 bilayers are shown. All the multilayers exhibited a significant inhibition of bacterial growth after 2 or 3 h of incubation, while the film (PAC 18 Na/PDADMAC) 20 without a surfactant proved to be the most effective.
The fungal strain C. albicans was less susceptible to the films' activity, as can be observed in Figure 4, and the number of CFUs was constant for all the tested samples. The exception was the film prepared with the C 14 surfactant, which showed moderate activity: 24 h of exposure resulted in the inhibition of fungal growth. In Figure 3, the development and survival of S. aureus strains in the presence of the films containing hydrophobically modified polyacrylate and the complexes with various alkyltrimethylammonium bromide at 20 bilayers are shown. All the multilayers exhibited a significant inhibition of bacterial growth after 2 or 3 h of incubation, while the film (PAC18Na/PDADMAC)20 without a surfactant proved to be the most effective.
The fungal strain C. albicans was less susceptible to the films' activity, as can be observed in Figure 4, and the number of CFUs was constant for all the tested samples. The exception was the film prepared with the C14 surfactant, which showed moderate activity: 24 h of exposure resulted in the inhibition of fungal growth.

Effect of Bacterial Growth on the Multilayered Hydrophobic Film Properties
In order to observe if the bacterial media affected the wettability of the films, contact angle measurements were performed. In Table 3, the contact angle values for untreated multilayer films and those exposed to bacterial media are presented. As expected, the results show that the increase of the alkyl chain length of the surfactant in the composition of the multilayer films led to higher contact angles. The CA values determined on the films incubated with E. coli and S. aureus did not show significant changes, while the presence of P. aeruginosa produced a decrease of the hydrophobicity of the films prepared with all surfactants. Figure 5 presents the water contact angle profiles for hydrophobic layer-by-layer films based on modified poly(acrylate) and their complexes with cationic surfactants. As can be observed, the experimental data demonstrated that the contact angle increased with the alkyl chain length of the polymer graft and furthermore obeyed the trend previously observed for unaged or freshly prepared films [18,19]. In addition, the measured contact angle of the films subjected to a bacterial environment had almost the same values. For example, the contact angle of (PAC18Na-NaCl/PDADMAC)20 before bacterial exposure was 93°, while the contact angle for the polyacrylate films exposed to E. coli was 90°. For (PAC18Na-C12TABNaCl/PDADMAC)20 exposed to bacteria, the contact angle remained the same, namely, 103°.

Effect of Bacterial Growth on the Multilayered Hydrophobic Film Properties
In order to observe if the bacterial media affected the wettability of the films, contact angle measurements were performed. In Table 3, the contact angle values for untreated multilayer films and those exposed to bacterial media are presented. As expected, the results show that the increase of the alkyl chain length of the surfactant in the composition of the multilayer films led to higher contact angles. The CA values determined on the films incubated with E. coli and S. aureus did not show significant changes, while the presence of P. aeruginosa produced a decrease of the hydrophobicity of the films prepared with all surfactants. Figure 5 presents the water contact angle profiles for hydrophobic layer-by-layer films based on modified poly(acrylate) and their complexes with cationic surfactants. As can be observed, the experimental data demonstrated that the contact angle increased with the alkyl chain length of the polymer graft and furthermore obeyed the trend previously observed for unaged or freshly prepared films [18,19]. In addition, the measured contact angle of the films subjected to a bacterial environment had almost the same values. For example, the contact angle of (PAC 18 Na-NaCl/PDADMAC) 20 before bacterial exposure was 93 • , while the contact angle for the polyacrylate films exposed to E. coli was 90 • . For (PAC 18 Na-C 12 TABNaCl/PDADMAC) 20 exposed to bacteria, the contact angle remained the same, namely, 103 • . The wettability results of polyelectrolyte films exposed to S. aureus cells are presented in Figure  6. The contact angle of the untreated (PAC18Na/PDADMAC)20 was 93°, while for (PAC18Na/PDADMAC)20 exposed to S. aureus, the contact angle was calculated to be 97°. For (PAC18Na-C18TAB/PDADMAC)20 exposed to S. aureus, the contact angle was 122°, and for the uninfected one, CA = 124°. The wettability results of polyelectrolyte films exposed to S. aureus cells are presented in Figure 6. The contact angle of the untreated (PAC 18 Na/PDADMAC) 20 was 93 • , while for (PAC 18 Na/PDADMAC) 20 exposed to S. aureus, the contact angle was calculated to be 97 • . For (PAC 18 Na-C 18 TAB/PDADMAC) 20 exposed to S. aureus, the contact angle was 122 • , and for the uninfected one, CA = 124 • . The wettability results of polyelectrolyte films exposed to S. aureus cells are presented in Figure  6. The contact angle of the untreated (PAC18Na/PDADMAC)20 was 93°, while for (PAC18Na/PDADMAC)20 exposed to S. aureus, the contact angle was calculated to be 97°. For (PAC18Na-C18TAB/PDADMAC)20 exposed to S. aureus, the contact angle was 122°, and for the uninfected one, CA = 124°.

The Morphology of the Films after Exposure to Bacterial Media
AFM measurements were performed in order to demonstrate the possible film structure modifications occurring after exposure to bacterial media. Figure 7 presents the AFM images of multilayer films exposed to E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa and without bacteria. The morphological measurements for the untreated multilayer films of (PAC 18 Na/PDADMAC) 20 (Figure 7a 0 ), (PAC 18 Na-C 14 TAB/PDADMAC) 20 (Figure 7b 0 ), and (PAC 18 Na-C 14 TAB/PDADMAC) 20 (Figure 7c 0 ) reveal a rough morphology wherein air bubbles could be locked. The presence of the alkyl chain length indicated a higher root-mean-square (RMS) roughness and contact angle [19]. The AFM images of multilayer films exposed to E. coli ATCC 25922 indicated an irregular film structure, consisting of hills and valleys. The line scan was located within 0.9 nm for (PAC 18 Na/ PDADMAC) 20 (Figure 7a 1 ), 2 nm for (PAC 18 Na-C 14 TAB/PDADMAC) 20 (Figure 7b 1 ), and 6 nm for (PAC 18 Na-C 14 TAB/PDADMAC) 20 (Figure 7c 1 ). The RMS roughness for (PAC 18 Na/ PDADMAC) 20 was 0.290 nm, while for (PAC 18 Na-C 14 TAB/PDADMAC) 20 , it was 0.394 nm. For PAC 18 Na-C 18 TAB/PDADMAC at 20 bilayers, the RMS was 5.845 nm. When comparing the fresh multilayer film [19] with the ones incubated in a bacterial environment, we noticed that the bacterial growth did not change the surface morphology. These data confirmed our supposition that multilayer films are resistant to the bacterial attack of E. coli.
In order to observe the influence of S. aureus on multilayer films, AFM measurements were performed. Figure 7a 2 ,b 2 ,c 2 presents two-dimensional topographic AFM images at the scale of 2 × 2 µm 2 of the glass substrate coated with PAC 18 Na/PDADMAC, PAC 18 Na-C 14 TAB/PDADMAC, and PAC 18 Na-C 18 TAB/PDADMAC, respectively, together with characteristic surface profiles (line scans) along the horizontal direction. All films presented a uniform morphology consisting of hills and valleys, which, according to the line scans, were located within 1 nm (vertical direction) for the PAC 18 Na/PDADMAC film and 6 nm for the PAC 18 Na-C 18 TAB/PDADMAC film, respectively. Another important feature is that film exposure to bacteria led to a decrease in RMS roughness. For example, the RMS for PAC 18 20 polyelectrolyte-surfactant multilayers [18].
After AFM mapping, the data showed that the morphology of the investigated films in the presence of P. aeruginosa presented changes in film structure. All films presented a morphology consisting of hills and valleys, which, according to the line scans (vertical direction), were located within 4 nm for the PAC 18 20 (Figure 7c 3 ), the RMS was 0.864. The water contact angle data for hydrophobically modified poly(acrylate) and their complexes with cationic surfactants showed a decrease in contact angle values for the films treated with P. aeruginosa ATCC 2785. For example, the contact angle for untreated (PAC 18 Na-C 14 TAB/PDADMAC) 20 was 107 • , while for the exposed one, the contact angle was 98 • . For (PAC 18 Na-C 18 TAB/PDADMAC) 20 exposed to the bacteria with S. aureus, the contact angle was calculated to be 113 • , and for the uninfected one, the CA was 124 • . In Figure 8, two-dimensional topographic AFM images at the scale of 2 × 2 µm 2 and the water contact angle profiles of the polyelectrolyte or polyelectrolyte-surfactant multilayered films with C. albicans, together with the characteristic surface profiles (line scans) along the horizontal direction, are presented. In Figure 8, two-dimensional topographic AFM images at the scale of 2 × 2 µ m 2 and the water contact angle profiles of the polyelectrolyte or polyelectrolyte-surfactant multilayered films with C. albicans, together with the characteristic surface profiles (line scans) along the horizontal direction, are presented. The multilayered films presented a morphology consisting of hills and valleys, which, according to the line scans (vertical direction), were located within 1.2 nm for the PAC18Na/PDADMAC film and 13 nm for the PAC18Na-C14TAB/PDADMAC film, respectively. The RMS roughness was 3.649 for (PAC18Na/PDADMAC)20 and 2.731 for (PAC18Na-C14TAB/PDAMAC)20, while for (PAC18Na-C18TAB/PDAMAC)20, the RMS roughness was 10.407.

Conclusions
Hydrophobically modified polyacrylate and their complexes with cationic surfactants have demonstrated their abilities in multilayer film fabrication via electrostatic layer-by-layer assembly. Our study revealed that (PAC18Na/PDADMAC)20 and (PAC18Na-C14TAB/PDAMAC)20 systems inhibited the growth of the S. aureus by 100%. However, the mentioned films showed poor activity towards C. albicans. Future studies will develop complex films to further increase the antimicrobial nature of multilayered structured films. Thus, the use of hydrophobically modified PAA polymercationic surfactant complexes proved to be an effective way to construct thin films with remarkable stability, broad antibacterial activity, and low cytotoxicity. These films could find applications in selfcleaning and/or antimicrobial coatings for biomedical devices or decontamination of public areas.  The multilayered films presented a morphology consisting of hills and valleys, which, according to the line scans (vertical direction), were located within 1.2 nm for the PAC 18 Na/PDADMAC film and 13 nm for the PAC 18 Na-C 14 TAB/PDADMAC film, respectively. The RMS roughness was 3.649 for (PAC 18 Na/PDADMAC) 20 and 2.731 for (PAC 18 Na-C 14 TAB/PDAMAC) 20 , while for (PAC 18 Na-C 18 TAB/PDAMAC) 20 , the RMS roughness was 10.407.

Conclusions
Hydrophobically modified polyacrylate and their complexes with cationic surfactants have demonstrated their abilities in multilayer film fabrication via electrostatic layer-by-layer assembly. Our study revealed that (PAC 18 Na/PDADMAC) 20 and (PAC 18 Na-C 14 TAB/PDAMAC) 20 systems inhibited the growth of the S. aureus by 100%. However, the mentioned films showed poor activity towards C. albicans. Future studies will develop complex films to further increase the antimicrobial nature of multilayered structured films. Thus, the use of hydrophobically modified PAA polymer-cationic surfactant complexes proved to be an effective way to construct thin films with remarkable stability, broad antibacterial activity, and low cytotoxicity. These films could find applications in self-cleaning and/or antimicrobial coatings for biomedical devices or decontamination of public areas.