Inhibiting pathogen surface adherence by multilayer polyelectrolyte

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Surfaces, Interfaces, and Applications

Inhibiting pathogen surface adherence by multilayer polyelectrolyte films functionalized with glucofuranose derivatives Valeria Villalobos, Angel Leiva, Hernan E. Ríos, Jorge Enrique Pavez, Carlos Silva, Mohammed Ahmar, Yves Queneau, Jenny M. Blamey, Francisco P. Chávez, and Marcela D. Urzúa ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03605 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 23, 2018

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ACS Applied Materials & Interfaces

Inhibiting pathogen surface adherence by multilayer polyelectrolyte films functionalized with glucofuranose derivatives Valeria Villalobos† ‡, Ángel Leiva#, Hernán E. Ríos†, Jorge Pavez§, Carlos P. Silva§, Mohammed Ahmar∥, Yves Queneau∥, JM. Blamey⊥, Francisco P. Chávez*¶ and Marcela D. Urzúa*† †

Departamento de Química, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425,

Ñuñoa, Chile ‡

Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de

Chile, El Llano Subercaseaux 2801, San Miguel, Santiago 8900000, Chile #

Departamento de Química-Física, Facultad de Química, Pontificia Universidad Católica de

Chile, Vicuña Mackenna 4860, Macul, Chile §

Departamento de Química de los Materiales, Facultad de Química y Biología, Universidad de

Santiago de Chile, Soft Matter Research-Technology Center, SMAT-C, Av. B. O’Higgins 3363, Casilla 40, Correo 33, Santiago, Chile ∥

Université de Lyon, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires,

ICBMS, UMR 5246, CNRS, UCBL, INSA Lyon, CPE Lyon, Bâtiment Lederer, 1 Rue Victor Grignard, 69622 Villeurbanne Cedex, France

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

⊥Fundación

¶

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Biociencia, José Domingo Cañas 2280, Ñuñoa, Santiago, Chile

Laboratorio de Microbiología de Sistemas. Departamento de Biología, Facultad de Ciencias,

Universidad de Chile, Las Palmeras 3425, Ñuñoa, Chile.

* Corresponding Authors

¶

Laboratorio de Microbiología de Sistemas. Departamento de Biología, Facultad de Ciencias,

Universidad de Chile, Las Palmeras 3425, Ñuñoa, Chile. E-mail: [email protected] †

Departamento de Química, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425,

Ñuñoa, Chile. E-mail: [email protected]

KEYWORDS. Carbohydrate Polyelectrolytes; P. aeruginosa; S. Typhimurium; Antibacterial Surfaces; Adherence Inhibition

ABSTRACT. Inhibiting pathogenic bacterial adherence on surfaces is an ongoing challenge to prevent the development of biofilms. Multilayer polyelectrolyte films are feasible antibacterial


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materials. Here we have designed new films made of carbohydrate polyelectrolytes to obtain antibacterial coatings that prevent biofilm formation. The polyelectrolyte films were constructed from poly(maleic anhydride-alt-styrene) functionalized with glucofuranose derivatives and quaternized poly(4-vinylpyridine) N-alkyl. These films prevent Pseudomonas aeruginosa and Salmonella Typhimurium, two important bacterial contaminants in clinical environments, from adhering to surfaces. When the film was composed of more than ten layers, the bacterial population was greatly reduced, while the bacteria remaining on the film were morphologically damaged, as atomic force microscopy revealed. The antibacterial capacity of the polyelectrolyte films was determined by the combination of thickness, wettability, surface energy, and most importantly, the conformation that polyelectrolyte adopt in function of nature of the carbohydrate group. This polyelectrolyte film constitutes the first green approach to preventing pathogenic bacterial surface adherence and proliferation without killing the bacterial pathogen.


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1. INTRODUCTION Human health is threatened by the proliferation of pathogenic bacteria on abiotic surfaces and the subsequent formation of biofilms, as well as increasing antibiotic resistance. Bacterial cells in biofilm are more resistant to antibiotics and common antibacterial agents than are their planktonic counterparts.1-2 Efforts have been made for many years to design antibacterial surfaces, because bacterial surface colonization of surfaces and resistance against antibiotics are extremely complex phenomena from a biochemical point of view.3-4 There are currently two main approaches to enhancing the antibacterial capacity of surfaces, one being to kill bacterial cells when they comes in contact with the surface, and the other being to prevent bacteria from reaching the surface.5-7 Ideally, these two approaches can be combined.5 The capacity of bacterial cells to mutate makes them increasingly resistant to existing antibacterial methods.8-10 This coupled with the capacity of bacteria to form biofilms, makes it imperative to create new surfaces with different properties that prevent bacterial infection and mutation.8, 11-12 The literature describes methods to produce antibacterial surfaces13-14 with polymers covalently bound to the surface,5 and polyelectrolyte multilayers15 resistant to bacterial colonization16-19 that are composed of several kinds of polymers, such as chitosan/heparin,20 poly(vinyl amine)/poly(acrylic acid),21 poly(L-arginine hydrochloride)/poly(hyaluronic acid),22 quaternized poly(4-vinyl pyridine) N-alkyl/carboxymethyl cellulose,23 and (poly(acrylic acid)gentamicin/poly(ethylenimine).24 Wang et al. designed and constructed a film using two layerby-layer (LBL) systems composed of (heparin/chitosan)10−(polyvinylpyrrolidone/poly(acrylic acid))10, based chemically on electrostatic interactions and hydrogen bonds. The degradation of


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(PVP/ PAA) 10 films can be controlled from the upper to the lower films, generating resistance to bacterial adhesion. Once the upper (PVP / PAA) film has degraded, the underlying multilayer (HEP/ CHI) films with antibacterial properties are exposed, killing microorganisms on contact.25 Junter et al. described polysaccharides as anti-adherence and bactericidal coatings that prevent biofilm formation on the surfaces of materials exposed to bacterial contamination. Hydrophilic coatings have been based on hialuronic acid, heparin and chitosan, and most recently, the algal polysaccharide Ulvan.26 However, there are no studies describing modification of surfaces coated with polyelectrolytes with carbohydrates in their side chain to inhibit biofilm formation. This represents a new strategy to alter the functionality, molecular orientation, topography, and other surface properties of polymeric films. The antimicrobial effect of properties like wettability, surface topography, surface charge, steric factor of polyelectrolytes, and surface energy of PEM films are increasingly being studied. 27-28

Correlations have been reported between bacterial attachment, hydrophobic character and

the rigidity of the pendant group in (meth)acrylates covering certain surfaces.29 However, there have few conclusive reports on the antibacterial effects of these parameters.16 Guo et al. summarized studies on controlling bacterial and cellular adhesion on surfaces with LbL assembles, including applications like tissue engineering and antimicrobial surfaces. One area of focus has been the physicochemical properties of the surface, which can critically influence bacterial adherence and control methods. They concluded that studies have generally focused on obtaining multifunctional films to prevent bacterial adhesion using a single LBL system as a versatile material platform to adopt and integrate strategies that limit viable bacterial adhesion.30 Consequently, polyelectrolytes with bulky carbohydrate groups in their side chain could allow for modifying the flexibility and hydrophobic character of polyelectrolytes. The presence of


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charged groups make it easier to form multilayers by electrostatic interactions. The use of polyelectrolytes to form multilayers can also improve physical and/or chemical properties as previously mentioned. This type of system is an optimal option to prevent bacterial adhesion by inhibiting the proliferation of microorganisms in the surface. The synthesis of polyelectrolytes containing carbohydrates and the subsequent modification of the surface with PEM films is interesting, given the high presence of hydroxyl groups belonging to carbohydrate molecules. Carbohydrate hydroxyl groups can be also modified with hydrophobic protector groups, thereby changing physicochemical properties and consequently surface polarity, which in turn affects the interaction with bacteria. The main goal of this work is to obtain new PEMs capable of preventing bacterial adherence without killing the bacterial pathogen. This dual approach aims to prevent the generation of new antibiotic resistant bacterial strains that currently represent a major health and environmental problem. In the present work, we have synthesized anionic polyelectrolytes from poly(maleic anhydridealt-styrene) and P(MA-alt-Sty), derivatives of different molecular weights that were functionalized with glucofuranose and poly(4-vinylpyridine) quaternized with ethyl or pentyl bromide. Both polyelectrolytes were adsorbed onto substrates to form polyelectrolyte multilayer LBL films. The multilayer films were characterized by ellipsometry, atomic force microscopy, and contact angle to determine thickness, morphology, wettability and surface energy. The antibacterial capacity of the polyelectrolyte films was assessed against Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium.


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These polyelectrolytes, which contain carbohydrates in their lateral chains, offer a new and green approach to protect in surfaces using the LBL method, combining anti-adherence mechanisms with killing bacterial to prevent biofilm formation.


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2. EXPERIMENTAL SECTION 2.1 Polymer Synthesis Poly(styrene-alt-maleic anhydride) copolymers (Mv~6.2x104 g/mol and Mv~3.2x104 g/mol) were synthesized according to a previously described method.31 The average molecular weight was determined by capillary viscometry in tetrahydrofuran at 30°C, with a=0.81 and K=5.07x105

as the Mark-Houwink-Sakurada constants. Poly(4-vinylpyridine) (Mn~6.2x104 g/mol) was fully

quaternized with ethyl or pentyl bromide to obtain cationic polyelectrolytes, as previously described (Scheme 1).23All reagents and solvents were purchased from Sigma-Aldrich®, and were of the highest purity available. 2.2 Carbohydrate synthesis Carbohydrate compounds were prepared with the hydroxyl-derivatives, 1,2-O-isopropylideneα-D-glucofuranose and 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose as starting materials. The synthesis consisted of three steps. First, the target –OH group of the protected starting sugar was transformed into a leaving group like tosylate (TsO-) or triflate (TfO-) to obtain the derivatives α32 and dα33, as described previously. Second, the azide derivatives α32 and 3-azide-3-deoxy1,2;5,6-di-O-isopropylidene-α-D-allofuranose34 were prepared by SN2 displacement reaction of the corresponding leaving group with sodium azide. Finally, the azide derivatives were treated with triphenylphosphine or Pd/C/H2 to obtain the carbohydrates with protected -OH groups following hydrogenation. All the solvents were previously distilled from appropriate drying agents. Solutions were concentrated under reduced pressure at 40-50ºC in a water bath. Reactions were monitored by


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thin layer chromatography on 0.2 mm silica gel 60 F254 (Merck) aluminum supported plates. Spots were evidenced with UV light, after charring with a 5% w:v H2SO4 ethanol solution. Column chromatography was performed with silica gel 60 (230–400 mesh, Merck). Nuclear magnetic resonance (1H and

13

C-NMR) spectra were recorded on a Bruker ALS 300 or Bruker

DRX 300 spectrometer. TMS in CDCl3 was used as an internal standard. All the signals obtained were compared with signals in the literature. XPS spectra were recorded on an XPS-Auger PerkinElmer electron spectrometer (Model PHI 1257), which consisted of a hemispherical electron energy analyzer and an X-ray source that provided unfiltered K radiation from its Mg anode (hv= 1253.6 eV). The pressure of the main spectrometer chamber during data acquisition was in the range of 10-6 Pa. The binding energy (BE) scale was calibrated using the peak of adventitious carbon, which was set at 284.8 eV [Handbook of XPS]. The samples were studied in normal angle emission at power source of 200 W. Synthesis of 6-Amino-6-deoxy-1,2-O-isopropylidene-α-D-glucofuranose A 1:1-mole base mixture of α−glucofuranose and triphenylphospyne in THF/H2O (4:1) was left overnight to react at room temperature. Solvent was evaporated under reduced pressure and the residue was dissolved in water and washed twice with ether. The aqueous layer was dried under vacuum to obtain the amino derivative as brown syrup. Synthesis of 3-Amino-3-deoxy-1,2;5,6-di-O-isopropylidene-α-D-allofuranose α-allofuranose (285 mg, 1mmol) was dissolved in absolute ethanol (2mL) under a nitrogen atmosphere. Palladium on charcoal (10% weight) was added and the reaction was hydrogenated


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at 1 atm H2. After several hours of stirring at room temperature and monitoring TLC, the reaction mixture was filtered using a celite pad. The solvent was removed at reduced pressure to yield the corresponding amine. 2.3 Functionalizing the copolymer poly(styrene-alt-maleic anhydride) with carbohydrates General Procedure P(MA-alt-Sty) with two different molecular weights (3.2x104 and 6.2x104 gmol-1) were functionalized with the carbohydrates 6-amino-6-desoxy-1,2-isopropylidene-α-D-glucofuranose, and 3-amino-3-deoxy-1,2;5,6-di-O-isopropylidene-α-D-allofuranose, to obtain the respective sodium salts (Scheme 1).

O

O Na n

O

O Na n

NH

O HO

O O

O

O HN

HO

O O

O O

O

P-Allo-f-(Iso)2

P-Glu-f-Iso(OH)2

n

N Br (CH2)n H3C

n = 1 or 4

QPVP-Cn

Scheme 1. Polyelectrolytes used in the construction of PEM films


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The copolymer P(MA-alt-St) was functionalized with carbohydrates in DMSO with 0.3% mole of triethylamine as a catalyst. The copolymer/carbohydrate stoichiometric ratio was 1:1. The solution was stirred for one week at 60°C. The product was neutralized with diluted NaHCO3, ultrafiltered to eliminate impurities and freeze-dried to obtain the anionic polyelectrolytes. The anionic polyelectrolytes were named P32-Glu-f-Iso(OH)2, P62-Glu-fIso(OH)2, P32-Allo-f-(Iso)2, P62-Allo-f-(Iso)2, and cationic polyelectrolytes were named QPVPC2 and QPVP-C5, as summarized in Table 1.

Table 1. Information Samples Polymer Mw

P(MA-alt-Sty)

QPVP

Polyelectrolyte

Side Group

3.2x104

P32-Glu-f-Iso(OH)2

Glucofuranose

6.2x104

P62-Glu-f-Iso(OH)2

Glucofuranose

3.2x104

P32-Allo-f-(Iso)2

Allofuranose

6.2x104

P62-Allo-f-(Iso)2

Allofuranose

6.0x104

QPVP-C2

Ethyl

6.0x104

QPVP-C5

Pentyl

2.4 Polyelectrolyte solutions Solutions of P-Glu-f-Iso(OH)2 and P-Allo-f-(Iso)2 polyelectrolytes and the quaternized poly(4vinylpyridine) N-alkyl were prepared at 1 mg/mL in 0.1M NaCl, at pH 4. MilliQ water was used in all solutions. 2.5 PEMs film construction The substrates used to construct the multilayer polyelectrolytes were 1x1cm2 silicon wafers that had previously been subjected to an oxidative wash (H2O, H2O2 and NH4OH (5: 1: 1)) and


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then immersed for 30 minutes each at room temperature, first in the solution of quaternized poly(4-vinylpyridine) N-alkyl, and then in a solution of polyelectrolyte functionalized with carbohydrates. The procedure was successively repeated until a total of twelve layers had been formed. The substrates coated with the films were kept dry for subsequent analysis. 2.6 Characterization of PEMs films 2.6.1. PEMs film thickness The thickness of PEM films was determined in a vertical computer-controlled ellipsometer (DRE-EL02 model, Ratzeburg, Germany). The incidence angle (Φ) was set at 70° and the wavelength of the laser at 632.8 nm. The measurements were made at 23 ± 1°C.31 2.6.2. Surface Analysis of PEMs films Surface analysis of the PEM films that included topographic and morphological images, RMS roughness, and Surface Potential Measurements (KFM-FM9 were obtained in an AFM/SPM Controller 9500 Series System, (Keysight Technologies, CA, USA) with a 7500 scanner. For topographic and morphological images the surfaces were scanned in the magnetic-AC mode (MAC-Mode® Keysight) with a scan rate of 0.3 Hz, using commercial AFM probes (Olympus). The images were collected under both air conditions and in solution (for bacterial samples) at room temperature (24-25ºC). Keysight PicoView software was used for image analysis and to determine the root mean square (RMS) roughness. The Surface Potential Measurements were made in Kelvin Force Microscopy-Frequency Modulation mode, using an µMasch HQ:XSC11/Pt(B) tip (Resonance frequency 80 KHz), with an oscillating bias voltage Vac=600 mV at a frequency, ωe= 7 KHz.


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2.6.3. PEMs film wettability and hysteresis surface angle The wettability of the multilayers was determined by measuring the advancing contact angle (θA). The contact angle was determined by the static drop method, which involved depositing 8 µL of water on the surface of the PEMs films. The receding angle (θR), which is obtained by extracting 4 µL of the previously deposited 8 µL, is used to determine the hysteresis surface angle (θH) with the equation: θH= θA - θR 2.6.4. Surface energy of PEM films The surface energy of the multilayers was calculated by measuring the contact angle of two liquids: one apolar and the other polar. In this case, we use diiodomethane and water, respectively. The total surface energy was calculated using Young’s equation and the geometric model theory.35 2.7 Antibacterial Behavior Studies Microbiological tests were performed to determine the antibacterial capacity of the PEM films. Pseudomonas aeruginosa PAO1 and Salmonella enterica serovar Typhimurium (Strain 14028s) were inoculated in 5 mL of LB medium. To visualize S. Typhimurium with fluorescence microscopy (Olympus MVX10, Japan), cells were previously transformed with pDiGC plasmid, which constitutively expressed green fluorescent protein (GFP). The strains were incubated at 180 rpm at 37°C for eighteen hours (overnight culture) and then diluted for use in the antimicrobial test. Prior to incubation with bacteria, the modified substrates with different PEMs were placed in a 48 multiwell plate system and sterilized with UV light for 10 minutes, after which 7µL of each


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bacterial suspension (0.1 optical density, OD) was deposited and dried under sterile conditions in a biosafety cabinet. The bacterial suspension was dried and then poured onto the modified substrate of 1mL of top LBA agar (LB + Ampicillin, 7.5 g of agar per liter of LB medium, ampicillin 50 mg/mL). The multi-well plate system was incubated at 37°C for 18 hours. All assays were performed at least in triplicate. To evaluate the antibacterial capacity of the PEM films, the bacterial population was estimated by fluorescence microscopy, either in the phase contrast mode (P. aeruginosa) or in the fluorescence mode (S. Typhimurium). The antimicrobial capacity of different PEM films was determined by the ratio of bacteria in the control substrate (with no PEM) relative to that in the modified substrate with PEM films after counting bacterial colony forming units using Image J software, which estimates colony forming units in films. The assays were carried out in triplicate. The statistical significance of the results was determined with an ANOVA (Bonferroni test) using GraphPad (Prism 6.0).


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3. RESULTS AND DISCUSSION 3.1 Characterization of P-Glu-f-Iso(OH)2 y P-Allo-p-(Iso)2 The IR spectrum of maleic anhydride (Figure 1-(A), red line) showed two characteristic bands at 1857 cm-1 and 1779 cm-1, which correspond to carbonyl maleic groups. In the IR spectra of PGlu-f-Iso(OH)2 (Figure 1-(A), black line), disappear and broad bands appear at approximately 3400 cm-1, corresponding to amide -NH and -OH groups. At 1693 cm-1, characteristic bands of the carboxylate and amide carbonyl groups are visible and may overlap. The P-Allo-p-(Iso)2 IR spectrum pattern is similar, while the signals of the

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C-NMR spectra of P-Glu-f-Iso(OH)2

(Figure 1-(B)) appear from 40 ppm to 120 ppm, which corresponds to the carbon atoms of the carbohydrate. The signal at 168 ppm corresponds to the carbon of the carboxylate group. Two additional signals appear at 173 ppm and 179 ppm, the first probably being a carbon of the carboxylate group and the second representing the carbon amide group that belongs to the same monomer unit. The degree of substitution was estimated by XPS. The atomic species detected on the surface of the sample were calculated based on these spectra. From this values it was possible to estimate the Na:N ratio as 5:1, which is the same as the ratio between these atomic species in the monomeric block. Based on this ratio, the degree of substitution degree estimated as close to 32% in both cases.


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(A)

a O

O Na a

b

n

b 1736

Absorption

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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NH

O HO

1857

HO

d O

O

1779

3372

4000

3500

1693

3000

2500

2000

1500

c O

P-Glu-f-Iso(OH)2 1000

500

-1

Wavenumber (cm ) (B)

Figure 1. (A) FT-IR Spectrum of P-Glu-f-Iso(OH)2 and (B)

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C-NMR Spectrum of P-Glu-f-

Iso(OH)2


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3.2 Construction and characterization of PEMs The PEMs films construction began with deposition of a primary layer of QPVP-Cn (cationic polyelectrolyte) onto hydroxyl-terminated silicon wafers. Petri et al. found that QPVP-Cn having a pyridinium group, interact with a hydroxyl-terminated substrate, thereby generating a first layer of QPVP-Cn chains, which remain strongly adsorbed onto hydroxyl-functionalized surface. Thus, the interaction is strong enough to prevent later desorption of the cationic polyelectrolyte layers, as well as the aggregation and conformational changes of QPVP-Cn once the anchoring takes place.23 Layers of P-Glu-f-Iso(OH)2 and P-Allo-f-(Iso)2 (anionic polyelectrolytes) were deposited alternately with layers of QPVP-Cn. This procedure was repeated to form a PEM films with a maximum of twelve layers. The thickness of the PEM films were determined by ellipsometry and ranged from 5 to 90 nm (Figure 2). These PEM films show a regular buildup regime, i.e., the amount of adsorbed polyelectrolyte regularly increases with the number of layers. Through the alternate deposit of anionic polyelectrolyte and cationic polyelectrolyte new layers are adsorbing on the surface. This regime implies that these new layers of polyelectrolytes will be adsorbed according to their opposite charges onto those previously adsorbed, in this way that there is no diffusion of the adsorbed polyelectrolyte to the solution interphase and the PEM films thickness would be stepped from these inner layers, consequently the outermost layers of the film would remain invariant. This growing process is mainly driven by electrostatic interactions that take place during the charge compensation processes within the films, due to the adsorption of oppositely charged polyelectrolytes. Similar behavior was observed in multilayers formed by sodium poly(styrenesulfonate) and poly(allylamine) hydrochloride.36-37 Here, the films thickness was not affected by the molecular weight of the polyelectrolyte, whereas the thickest multilayer were


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those formed by P-Glu-Iso(OH)2 with QPVP-C2 or QPVP-C5. The construction of the PEMs was also monitored by UV-visible spectroscopy, which corroborated the results obtained by ellipsometry (Figure S1). Absorbance increased proportionally with the number of layers, independent of the molecular weight of the P-Glu-f-Iso(OH)2 and P-Allo-f-(Iso)2 polyelectrolytes, indicating that the polyelectrolyte layers are absorbed onto the substrate and the layers already absorbed. Thickness was not affected by the molecular weight of the polyelectrolytes. The thickest multilayers were those composed of P-Glu-Iso(OH)2 with QPVP-C2 or QPVP-C5. However, there was an effect of molecular weight in the case of multilayers composed of P-Allo-f-(Iso)2, which could be due to the conformation that the polyelectrolytes take on in an aqueous solution as a result of hydrogen bond interactions, which causes the polyelectrolyte hypercoiling.31 P-GluIso(OH)2 has two -OH groups available that favor inter- and intramolecular interactions, yielding a more compact random coil form in an aqueous solution that is maintained when it is adsorbed onto the QPVP-C2 or QPVP-C5 layer during PEM construction (Figure 3). Other reports have shown that the construction of multilayers of carboxymethyl cellulose (CMC) QPVP-Cn with different hydrophobic characters are due to cooperative ion pair and hydrophobic interactions.23 P-Allo-f-(Iso)2 does not have –OH groups that favor cooperative interactions through a hydrogen bridge. As well, the size of the side chain can be a steric hindrance to absorption. The desorption of all the PEMs was also studied, which involved measuring layer thickness before and after that the PEMs were immersed for 72 hours in water and LB culture medium. Thickness was not affected, showing no PEMs desorption during the experimental period. Consequently, the PEMs were stable for at least this length of time. Previous studies have shown


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that cationic polyelectrolytes, the side chains of which contain aliphatic compounds, generate strong hydrophobic interactions, with consequent increased thickness of the absorbed layer.38-39 90

100

(A)

(B)

90 80

80 70

70

60

Γ(nm)

60

Γ(nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50 40

50 40 30

30 20

20

10

10 0

0 0

1

2

3

4

5

6

7

8

9

10 11 12

0

1

2

3

4

5

6

7

8

9

10 11 12

Number of Layers

Number of Layers

Figure 2. PEM thickness according to the number of deposited QPVP-C2 layers (■) and QPVPC5 (□) and layers of polyelectrolyte derivatives of P(MA-alt-St) Mv=6.2x104gmol-1 (green) and MV=3.2x104gmol-1 (red). (A) P-Glu-f-Iso(OH)2 (B) P-Allo-f-(Iso)2


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Figure 3. Schematic representation of polyelectrolyte adsorption behavior The surfaces were characterized by atomic force microscopy (AFM). After twelve layers of polyelectrolytes have been deposited, the PEM surfaces were observed with combination of both polyelectrolytes with homogeneous morphology and globular aggregates that depend on the substituent in the anionic polyelectrolyte side chain (Figure 4, 5).


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Figure 4. Atomic Force Microscopy images (sizes are 3 × 3 µm) of PEMs containing twelve layer. (A) P62-Glu-f-Iso(OH)2/QPVP-C5, (B) P62-Glu-f-Iso(OH)2/QPVP-C2, (C) P32-Glu-fIso(OH)2/QPVP-C5 and (D) P32-Glu-f-Iso(OH)2/QPVP-C2.

Figure 5. Atomic force microscopy images of PEMs containing twelve layer. (A) P62-Allo-f(Iso)2/QPVP-C5, (B)P62-Allo-f-(Iso)2/QPVP-C2, (C)P32-Allo-f-(Iso)2/QPVP-C5 and (D)P32-Allof-(Iso)2/QPVP-C2. Images A, B and D sizes are 3 × 3 µm; C size is 2 × 2 µm.


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The topography of some multilayers was rough. The P-Glu-f-Iso(OH)2 and QPVP-C2 films of both molecular weights had RMS values of 13.60 and 13.10 nm, respectively. P-Glu-f-Iso(OH)2 contains a glucofuranose derivative with hydroxyl groups available in its side chain that can interact with other hydroxyl groups to form aggregates in solution that can be adsorbed on the surface, thus increasing roughness. A smoother surface is obtained by the polyelectrolyte P62Allo-f-(Iso)2 with QPVP-C5 (RMS = 0.55 nm), which contains derivatives of glucofuranose completely protected in its side chain, thus yielding smoother surfaces (Table 2). The contact angle values of water droplets on PEM films were 10° higher than those of water droplets on the bare substrate. PEM films have varying hydrophilic/hydrophobic balances, reflected in contact angle values ranging between 36° and 68°. This suggests that the film surface in contact with the air is formed by functional hydrophobic and hydrophilic groups, in agreement with the high values of the hysteresis angle. The film composed of P62-Allo-f-(Iso)2/QPVP-C2 was the most hydrophilic, with a contact angle of 36°, while the film composed of P62-Glu-fIso(OH)2/ QPVP-C2 was the most hydrophobic, with a contact angle of 68° (Table 2). The film composed of P32-Allo-f-(Iso)2/QPVP-C5 had the highest hysteresis angle and the highest degree of chemical heterogeneity on its surface, while the PEM composed of by P62-Gluf-Iso(OH)2/QPVP-C5 had the lowest hysteresis angle and the lowest chemical heterogeneity on its surface (Table S1). The surface energy values of the PEM films ranged from 46 to 63 mJ.m-2, which are considered low (Table 2). The film with P32-Allo-f-(Iso)2/QPVP-C5 had the highest surface energy, while the film with P62-Glu-f-Iso(OH)2/ QPVP-C2 had the lowest surface energy (Table S2).


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Table 2. Percentage of P. aeruginosa cells and physicochemical properties of the multilayer Anionic Polyelectrolyte

MV (g/mol)

3.2x10

4

Remaining bacteria (%) 8.0

Γ (nm)

RMS (nm)

θA (°)

θH (°)

γ (mJ.m-2)

89.73

13.36

50±1

12±4

56±0

39.6

67.85

0.92

48±4

15±3

59±3

19.6

42.94

13.10

68±3

22±6

46±0

QPVP-C5

8.0

72.46

6.91

51±2

10±3

55±2

QPVP-C2

107.6

81.74

11.41

40±2

13±0

63±1

QPVP-C5

115.0

77.08

6.20

50±5

27±4

58±1

QPVP-C2

102.0

11.88

0.72

36±2

15±2

62±6

QPVP-C5

115.0

5.62

0.55

63±2

25±2

51±3

Cationic Polyelectrolyte QPVP-C2 QPVP-C5

P-Glu-f-Iso(OH)2 6.2 x10

3.2x10

4

QPVP-C2

4

P-Allo-f-(Iso)2 6.2 x10

4

MIC (minimal inhibitory concentration): P-Glu-f-Iso(OH)2, P-Allo-f-(Iso)2 ≥ 10.0 mg/L

3.3 Antibacterial capacity and adherence inhibition on PEM films To determine the antibacterial capacity of PEM films, P-Glu-f-Iso(OH)2 and P-Allo-f-(Iso)2 with QPVP-C5 and QPVP-C2 were tested with Pseudomonas aeruginosa (P. aeruginosa, PAO1), a multidrug-resistant pathogen that is known to form biofilm in different surfaces.40 For this, a novel multi-well plate system was designed for the antimicrobial tests. PEM films were incubated with bacteria for 5 hours, and then agar was added to measure the remaining cells on the films. Plates with PEM films were analyzed in triplicate after 18 hours of incubation at 37°C. The antibacterial capacity of the multilayer was determined by quantifying remaining bacterial cell colonies formed in the multi-well plates, based on images taken by phase contrast and/or fluorescence microscopy. The remaining cells were quantified and compared to the control unmodified substrate.


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The effect of the number of layers on PEM antibacterial capacity was studied using films composed of six, ten and twelve polyelectrolyte layers. As the number of deposited layers increased, the percentage of bacteria that remained attached decreased, indicating that the number of layers influences the capacity of bacteria to adhere to the surface (Figure 6).

D)

Figure 6. Phase contrast microscopy images showing PEM thicknesses according to the number of deposited QPVP-C2 and P32-Glu-f-Iso(OH)2 layers: (A) six layers (light blue), (B) ten layers (red), (C) twelve layers (yellow) and (D) PEM thickness according to the number of deposited layers.

The phase contrast images (Figure 7) indicate that the film composed of the polyelectrolytes PGlu-f-Iso(OH)2 was the most effective, with the lowest number of remaining bacterial cells attached. The remaining colonies observed were quantified and the percentages of bacteria capable of developing in each multilayer compared to the unmodified substrate were determined. Figure 7 shows that the PEM films composed of P32-Glu-f-Iso(OH)2/QPVP-C2 and P62-Glu-fIso(OH)2/QPVP-C5 were the most effective against P. aeruginosa adhesion, with the lowest number of attached bacteria (8% of bacteria remaining in both cases).


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CONTROL

P62-Allo-f-(Iso)2/QPVP-C5



20


40

P62-Glu-f-Iso(OH)2/QPVP-C5

60

Control

80

P32-Glu-f-Iso(OH)2 /QPVP-C2

100


120


140

Percentage of remaining bacteria

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

Multilayer

Figure 7. Percentage of P. aeruginosa cells remaining on PEM films, and images obtained by phase contrast microscopy of PEM films tested with P. aeruginosa. In order to deepen about the possible causes explaining the very low bacterial adhesion value found for the P32-Glu-f-Iso(OH)2/QPVP-C2 film. This effect could be explain by the local electrostatic properties of films by surface potential measurements for testing the electrostatic interactions between the charged surface of polyelectrolyte film and the conducting AFM tip. A high correlation between the surface potential and the anti-adhesion capacity of the films was found. The film composed by 12 layers of QPVP-C2 and P32-Glu-f-Iso(OH)2 polyelectrolytes, that proved to be the most repellent to bacteria, showed a very high surface potential profile value (Figure S2). Thus, the high surface charge density of the film acts in detriment of the bacterial adhesion. Some 19.6% and 39.6% of bacterial cells remain attached to the P62-Glu-fIso(OH)2/QPVP-C2 and P32-Glu-f-Iso(OH)2/QPVP-C5 films, respectively (Table 2). The number


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of bacterial cells on the PEM films composed of P-Allo-f-(Iso)2, with QPVP-C2 and QPVP-C5 was similar to that on the control film. The lower number of bacterial cells on the polyelectrolyte multilayer films mainly indicates that bacterial cells were excluded from the polymeric film surface during incubation. In addition, there is a clear difference between the antibacterial capacity of PEMs composed of P-Glu-f-Iso(OH)2 and those composed of P-Allo-f-(Iso)2, independent of the molecular weight or the QPVP-Cn side chain length. Most likely the conformation that P-Glu-fIso(OH)2 may adopt at the surface is quite different to that of P-Allo-f-(Iso)2 because the former having two -OH can be more compact than the second one. Thus, in the first case the pyridinium group may be more exposed to the bacterial cells. Finally, as shown in Table 2, other physicochemical properties are difficult to interpret because their trends do not correlate well with overall antibacterial capacity. To study in more detail the in vivo interaction between bacterial cells and PEMs, we have used atomic force microscopy (AFM). Unlike scanning electron microscopy and other ex vivo techniques, AFM does not require pretreated or modified samples. Consequently, bacterial samples can be observed in vivo on the surface of the PEM films. Furthermore, these studies allowed us to observe any damage or morphological changes to bacterial cell envelopes. We have compared live cell AFM images of P. aeruginosa PAO1 on the bare substrate (control) to those on the PEM films with twelve layers of polyelectrolytes (Figure 8). The live cell AFM images showed homogeneous distribution of P. aeruginosa cells (Figure 8A). Bacteria display clear flagellar structures, as well as homogenous distribution over the entire substrate. In contrast, live cell AFM images obtained from PEM films composed of P32-Glu-f-Iso(OH)2 and


26

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QPVP-C2 (Figure 8B) had considerably lower numbers of bacterial cells, with dissimilar distribution to that of the control substrate. PEM films showed numerous areas without bacterial cells or with only bacterial fragments. Isolated bacterial cells without flagella were observed in other areas (Figure 8B).

(A)

(B)

Figure 8. AFM images of live P. aeruginosa cells on (A) control and (B) PEM films. PEM films composed of P-Glu-f-Iso(OH)2 and QPVP-Cn. Image sizes: A are 100 × 100 µm (left), 15 × 15 µm (right); B are 100 × 100 µm (left), 15 × 15 µm (center), 15 × 15 µm (right). The absence of bacterial aggregations and flagellar structures in the AFM images indicates the antibacterial capacity of PEM films, which is consistent with a decrease in cellular cluster forming units in the agar-agar plate assays.


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Figure 9 shows an AFM image of cells on the surface of a PEM film. The surface phenotype of these cells is different from that of bacterial cells on the control substrate. The surface of the bacterial cell envelope changed from smooth to rough, suggesting some structural damage to the bacterial cells and its exopolymeric material. It is worth noting that the effect of monovalent halogens as antibacterial agents has been previously reported, and in the case of our PEMs, Brwas present in the structure of the cationic polyelectrolyte.41 Similar results were reported in Escherichia coli cells where AFM studies demonstrated that smooth envelopes (live bacteria) became corrugated (dead bacteria) after antibiotic treatment.42 Interestingly, carbohydrate polyelectrolytes in solution did not kill any bacterial pathogens even at high concentration of the polymer in solution (>10 µg/mL), suggesting that the damage of bacterial cells only occurs when bacteria are in contact with the surface of the PEM film (Figure 9). With this result we have demonstrated that some constructed PEMs prevent the adherence of the cells in the surface without killing the bacterial pathogens in solution.


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Figure 9. AFM image of live P. aeruginosa cells on PEM films. As shown in the electronic micrograph, the morphology of the P. aeruginosa cells on the surface was different from that of the bacterial cells on the control substrate (Figure 7A). To determine if the antimicrobial capacity of the PEM films is exclusive to P. aeruginosa cells, similar studies were carried out with green fluorescent protein (GFP)-tagged Salmonella enterica serovar Typhimurium cells. Salmonella cells can survive without a host and are often found in polluted water and contaminated food. In our study, fluorescent images were used to estimate the number of live bacterial cells on the films. PEM film images obtained by using fluorescence microscopy (Figure 10) showed that PEM films had a lower antibacterial capacity against Salmonella than against P. aeruginosa.

CONTROL

140

0




20


40


60


80


100


120

control

Percentage of remainig bacteria

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Multilayer

Figure 10. Percentage of S. Typhimurium cells remaining on PEM films, and images obtained by phase contrast microscopy of PEM films tested with S. Typhimurium


29


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The PEM film composed of polyelectrolytes P32-Glu-f-Iso(OH)2/QPVP-C had the strongest antibacterial capacity against the S. Typhimurium cells5 with 14.6% of bacteria remaining, followed by the PEM film composed of P62-Allo-f-(Iso)2/QPVP-C5 with 16% of bacteria remaining. Bacterial survival rates were significantly higher with the other PEM films, with 25% remaining with P32-Allo-f-(Iso)2/QPVP-C5 and over 50% remaining with P32-Glu-fIso(OH)2/QPVP-C2, P62-Glu-f-Iso(OH)2 with QPVP-C2 and QPVP-C5, and PEM films composed of P32-Allo-f-(Iso)2/QPVP-C2 and P62-Allo-f-(Iso)2/QPVP-C2. As occurs with the P. aeruginosa strain, the percentage of bacterial S. Typhimurium cells that remain on films is not influenced by any specific physical-chemical property of the PEM film surfaces, but rather by a combination of them. In fact, PEM films with similar thickness, wetting capacity, chemical heterogeneity and surface energy had different antibacterial capacities (Table S3). In summary, PEM films composed of P-Glu-f-Iso(OH)2, with QPVP-C2 and QPVP-C5, had the highest antibacterial capacity against the P. aeruginosa cells. AFM revealed that cell envelope of living cells in PEM films were morphologically damaged which could explain the inability of bacterial cells to form corrected biofilms in the surface. PEM films composed of P-Allo-f-(Iso)2 of two molecular weights, with QPVP-C2 and QPVP-C5, had the highest antibacterial capacity against the S. Typhimurium strain. To our knowledge, this is the first PEMs that prevent pathogenic bacterial surface adherence and proliferation without killing the bacterial pathogen.


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4. CONCLUSION The interest of the industry in developing new antibiotics is decreasing mainly by the spread of multidrug-resistant bacterial pathogens. Consequently, the discovery of novel antimicrobial surfaces that specifically inhibit bacterial surface adherence without killing the bacterial pathogen can be a promising strategy to prevent antibiotic resistance. Here we show that multilayer formed by P-Glu-f-Iso(OH)2 with QPVP-Cn inhibit surface pathogen adherence no matter the polyelectrolyte molecular weight. The antibacterial ability of PEM films was influenced by the nature of the carbohydrate group in the polyelectrolyte side chain which brings about a different conformation of the polyelectrolyte onto the PEMs surface. Finally, we modified the material surface with polymeric layered films that prevent bacterial pathogens from adhering, thus excluding bacteria from the PEM film surfaces, while damaging the bacterial cells that remain attached to the surface. To our knowledge, this is a novel approach in generating multilayer polyelectrolyte films that inhibit the surface adherence of two important clinical pathogens: P. aeruginosa and S. Typhimurium. The design of novel surfaces polymer that affect the capacity of bacteria to adhere to surfaces definitely without killing the bacterial cells can contribute to diminish current problems associated with antibiotic resistance.


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ASSOCIATED CONTENT (A)Absorption in the multilayer construction process using UV-Vis spectroscopy. Figure S1. PEM absorbance values according to the number of deposited layers of QPVP-C2 (■) and QPVP-C5 (□) and layers of polyelectrolyte derivatives of P(MA-alt-St) Mv=6.2x104gmol-1 (black) and MV=3.2x104 gmol-1 (red). (A) P-Glu-f-Iso(OH)2 (B) P-Allo-f-(Iso)2. (B)Advancing contact angle (θA), reverse (θR) and hysteresis (θH) and surface energy of PEMs. Table S1. Advancing contact angle (θA), reverse (θR) and hysteresis (θH) of PEMs. Advancing contact angle (θA), reverse (θR) and hysteresis (θH) of PEMs. Table S2. Contact angle (θ) and surface energy (γtotal) of PEMs. (C)Percentage of cells of S. Typhimurium and physicochemical properties of the multilayers PEMs Table S3. Percentage of cells of S. Typhimurium and physicochemical properties of the multilayers. (D) Surface Potential Measurements, made by Kelvin Force Microscopy-Frequency Modulated. Figure S2. AFM images of P32-Glu-f-Iso(OH)2 film: (A) Topography and (B) surface potential (KFM-FM) images. AFM images of P32-Glu-f-Iso(OH)2/ QPVP-C2 film: (C) Topography and (D) surface potential (KFM-FM) images.


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AUTHOR INFORMATION Corresponding Author *Dra. Marcela Urzúa, Departamento de Química, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile. E- mail: [email protected] *Dr. Francisco P.Chávez, Laboratorio de Microbiología de sistemas, Departamento de Biología Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile. Email: [email protected] Authors Contributions The manuscript was through contributions of all authors. All authors have given approval to the final version of the manuscript. Valeria Villalobos‡, Ángel Leiva# ‡, Hernán E. Ríos†, Jorge Pavez§, Carlos P. Silva§, Mohammed Ahmar∥, Yves Queneau∥, JM. Blamey⊥, Francisco P. Chávez*¶ and Marcela D. Urzúa*† contributed equally. ACKNOWLEDGMENT We are grateful for FONDECYT grants 1151221 and 1120209, ANILLO ACT Project 1412, FONDEQUIP EQM160036, and CONICYT scholarship grant 21120806. C.P.S. thanks to Proy. POSTDOC_DICYT-USACH (021740PI PostDoc). We also appreciate the collaboration of Marcos Flores in measuring XPS.


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Synthesis of polymers

Control film AFM

Screening for an8microbial surfaces PEMs film Carbohydrate

PEMs film

Control P. aeruginosa

film

S. Typhimurium PEMs film


Inhibiting pathogen surface adherence by multilayer polyelectrolyte

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