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

Abstract

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 (C₁₀–C₁₈) 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 efficiency 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 effects 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 efficiency was registered in the case of S. aureus, which showed an inhibition of growth up to 100% after 2 h. C. albicans proved to be less sensitive to the effect 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.

1. 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, and scaffolds have proved their usefulness in medical device fabrication, drug delivery, and health care products, along with other major applications in textiles, food packaging, water decontamination, and information technologies applied to diagnostic methods [1,2,3,4,5,6,7]. Many strategies and materials have been developed to obtain efficient self-cleaning and/or antimicrobial coatings [8]. The antimicrobial efficiency of a thin film is mainly related to the chemical composition, while the self-cleaning effect is due to its morphology. Thus, both the material and the preparation method influence the biological behavior of polymeric nanostructured films.

The layer-by-layer (LbL) self-assembly technique is a useful method to prepare highly hydrophobic and/or antimicrobial multilayer films due to the simplicity of the experimental conditions. It also allows extended variation of properties by evaluating molecular interactions between the different types of materials used and tuning the composition [9,10]. The low cost of preparation and the opportunity to control the composition and morphology, leading to improved structural, electrical, optical, and antimicrobial properties, make the self-assembly method the most attractive approach for the fabrication of films in biomedical applications [1,2,3,4,5,6,7,8,9,10,11]. When a multilayer film is obtained, the existing molecular interaction will generate new properties which allow applications on different materials. Moreover, it is possible to combine different properties of certain materials to create new synergetic systems, such as superhydrophobic architectures with antibacterial or antifungal properties.

Among various materials of interest used for the fabrication of self-assembled films, poly(acrylate) (PANa) is an intrinsically conducting polymer which possesses electrical, optical, and anticorrosive properties [12]. In addition, hydrophobically modified poly(acrylate) (HMPA) films exhibit high chemical and environmental stability; ease of manipulation, polymerization, and doping; and low-cost production [13]. Gratzl et al. found that the presence of acid-form of polyacrylic acid (PAA) in the polymer and slightly acidic conditions are important for good antimicrobial activities of the material, while counterions reduce its potency dramatically [14].

Cationic surfactants, consisting of a hydrophilic quaternary ammonium group and a hydrophobic alkyl chain, have strong bactericidal potential and are frequently used for disinfection in a variety of fields, such as medicine and food manufacturing. Moreover, cationic surfactants such as alkyltrimethylammonium bromide possess the same activity for both Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative (Staphylococcus aureus) bacteria, according to Campanha et al. [15]. Surfactants with quaternary ammonium groups were considered relatively safe antimicrobial agents, but their increased usage in the last decades as disinfection and sanitation agents led to the emergence of the pathogens resistant to cationic surfactants.

Extensive studies have been performed to address the challenges associated with the application of cationic compounds against resistant bacterial infections. Recent papers report the synthesis of other antimicrobial compounds bearing ammonium groups, such as gemini cationic surfactants [16] or modified polymers [17,18].

Our approach in the present research was based on the advantage of using the very good antimicrobial properties of quaternary ammonium surfactants while increasing their stability and efficiency by incorporating them into modified hydrophobic polymer films.

Among other polymeric compounds used in the fabrication of LbL films, poly(diallyldimethylammonium chloride) (PDADMAC) exhibits only a weak antimicrobial effect due to the quaternary nitrogen which is pendant away from the backbone chain. It is reported that PDADMAC requires the presence of hydrophobic groups and a high charge density for efficient biocidal effect [19].

In our previous work, we obtained LbL films by electrostatic interaction between hydrophobically modified sodium poly(acrylates) (PAC_nNa) with and without cationic surfactants and PDADMAC [20,21]. Our present study is focused on the antimicrobial activity of hydrophobically modified HMPA and HMPA–surfactant complex LbL multilayer films. To the best of our knowledge, this is the first work to investigate the antimicrobial activity of HMPA–surfactant architectures obtained using the LbL technique. The antimicrobial activity of films based on PAC₁₈Na-C_xTAB (x = 10, 12, 14, and 18) were tested against S. aureus, E. coli, P. aeruginosa and Candida albicans as model microbial strains.

2. Materials and Methods

2.1. Materials

Decyl-(C₁₀TAB), dodecyl-(C₁₂TAB), tetradecyl-(C₁₄TAB), and octadecyltrimethylammonium bromide (C₁₈TAB) from Fluka (Waltham, MA, USA). Chemicals products were used as purchased (98%, 98%, 99%, and 97% purity).

Poly(ethylenimine) (PEI) aqueous solution (50 wt.%) with M_W = 75,000, and poly(diallyldimethylammonium chloride) (PDADMAC) aqueous solution (23 wt.%) with M_W 100,000–200,000 were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Hydrophobically modified sodium polyacrylates, PAC_nNa (n = 18), were obtained in our laboratory, according to the procedure reported in a previous study [22].

2.2. Preparation of Hydrophobically Modified Poly(Acrylate)–Surfactant Films

The hydrophobically modified polymer PAC₁₈Na 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 PAC₁₈Na-C_xTAB (x = 10, 12, 14 and 18) were prepared according to our previous study [20], and the aqueous PAC₁₈Na solution was added to a C_xTAB micellar solution under continuous stirring. The complexes were formed due to the electrostatic interaction between the negatively charged PAC₁₈Na and the positively charged C_xTAB. To ensure that the complexes were formed, measurements of zeta potential were carried out as in our previous paper [18]. The polymer–surfactant complexes (PAC₁₈Na-C_nTAB), (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 PAC₁₈Na 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. Hydrophobically modified poly(acrylate)–surfactant films investigated for antimicrobial properties.

No.	Sample	Surfactant Concentration (M)
1	PAC18Na/PDADMAC	0
2	PAC18Na-C10TAB-NaCl/PDADMAC	2 × 10−2
3	PAC18Na-C12TAB-NaCl/PDADMAC	7 × 10−3
4	PAC18Na-C14TAB-NaCl/PDADMAC	2 × 10−3
5	PAC18Na-C16TAB-NaCl/PDADMAC	1 × 10−3

.

The multilayer films consisted of 20 bilayers of alternatively deposited polyelectrolyte–surfactant complexes and PDADMAC. In our previous study, we observed that a sudden increase of contact angle (CA) occurred up to five bilayers. In order to save time and materials, we decided to use films containing 20 bilayers. These films have a good contact angle and also a rough surface, as the atomic force microscopy (AFM) results suggest after six months of aging [21].

2.3. 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 × 10⁶ 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.

2.4. 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 µm². 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”.

3. 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 C₁₀ to C₁₈ 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. Zeta potential data for several deposed films.

Time (h)	Zeta Potential (mV)
	PAC18Na-NaCl/PDADMAC	PAC18Na-C10TAB-NaCl/PDADMAC	PAC18Na-C12TAB-NaCl/PDADMAC	PAC18Na-C14TAB-NaCl/PDADMAC	PAC18Na-C18TAB-NaCl/PDADMAC
0	−48.1	−10.4	−19.39	−15.63	−15.51
2	−55.8	−17.27	−19.9	−18.8	−20.2
4	−41.4	−16.6	−26.6	−16.7	−26.4
6	−41.4	−18.5	−29.8	−19.4	−25.3
24	−39.8	−17.8	−27.6	−18.42	−25.1

.

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].

3.1. 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 PAC₁₈Na-C_xTAB-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. (PAC₁₈Na-NaCl/PDADMAC)₂₀ and (PAC₁₈Na-C₁₀TAB-NaCl/PDAMAC)₂₀ inhibited the strain’s development after 24 h and (PAC₁₈Na-C₁₈TAB-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 (C₁₂–C₁₈) exhibited no antibacterial activity. The films from polymer (PAC₁₈Na/PDADMAC)₂₀ without a surfactant and with a short chain surfactant (C₁₀) 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.

3.2. 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 (CA) values for untreated films and films subjected to microbial attack with positive and negative bacteria.

Sample	CA for Untreated Films (°)	CA for Films Treated with E. coli (°)	CA for Films Treated with S. aureus (°)	CA for Films Treated with P. aeruginosa (°)
Clear glass	26	25	23	24
(PAC18Na/PDADMAC)20	93	90	97	85
(PAC18Na-C10TAB/PDAMAC)20	101	98	108	90
(PAC18Na-C12TAB/PDAMAC)20	103	103	115	96
(PAC18Na-C14TAB/PDAMAC)20	107	105	119	98
(PAC18Na-C18TAB/PDAMAC)20	124	126	122	113

, 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°.

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°.

3.3. 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 (PAC₁₈Na/PDADMAC)₂₀ (Figure 7a₀), (PAC₁₈Na-C₁₄TAB/PDADMAC)₂₀ (Figure 7b₀), and (PAC₁₈Na-C₁₄TAB/PDADMAC)₂₀ (Figure 7c₀) 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 (PAC₁₈Na/PDADMAC)₂₀ (Figure 7a₁), 2 nm for (PAC₁₈Na-C₁₄TAB/PDADMAC)₂₀ (Figure 7b₁), and 6 nm for (PAC₁₈Na-C₁₄TAB/PDADMAC)₂₀ (Figure 7c₁). The RMS roughness for (PAC₁₈Na/PDADMAC)₂₀ was 0.290 nm, while for (PAC₁₈Na-C₁₄TAB/PDADMAC)₂₀, it was 0.394 nm. For PAC₁₈Na-C₁₈TAB/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 7a₂,b₂,c₂ presents two-dimensional topographic AFM images at the scale of 2 × 2 µm² of the glass substrate coated with PAC₁₈Na/PDADMAC, PAC₁₈Na-C₁₄TAB/PDADMAC, and PAC₁₈Na-C₁₈TAB/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 PAC₁₈Na/PDADMAC film and 6 nm for the PAC₁₈Na-C₁₈TAB/PDADMAC film, respectively. Another important feature is that film exposure to bacteria led to a decrease in RMS roughness. For example, the RMS for PAC₁₈Na/PDADMAC decreased from 1.42 (Figure 7a₀) to 0.975 nm (Figure 7a₂), which further resulted in a similar contact angle. The increase of the alkyl chain of the cationic surfactant led to the formation of random superficial particles (ball-like) on top of the film for (PAC₁₈Na-C₁₄TAB-NaCl/PDAMAC)₂₀ and (PAC₁₈Na-C₁₈TAB-NaCl/PDAMAC)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 PAC₁₈Na/PDADMAC (Figure 7a₃) film and 11 nm for the PAC₁₈Na-C₁₄TAB/PDADMAC (Figure 7b₂) film, respectively. Moreover, the presence of P. aeruginosa ATCC 27853 in the aqueous media caused holes in the surface for (PAC₁₈Na/PDADMAC)₂₀ and (PAC₁₈Na-C₁₄TAB/PDAMAC)₂₀ films. The RMS roughness was 0.914 for (PAC₁₈Na/PDADMAC)₂₀ (Figure 7a₃) and 1.275 for (PAC₁₈Na-C₁₄TAB/PDAMAC)₂₀ (Figure 7b₃), while for (PAC₁₈Na-C₁₈TAB/PDAMAC)₂₀ (Figure 7c₃), 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 (PAC₁₈Na-C₁₄TAB/PDADMAC)₂₀ was 107°, while for the exposed one, the contact angle was 98°. For (PAC₁₈Na-C₁₈TAB/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 µm² 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.

4. 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 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.