On the Infectivity of Bacteriophages in Polyelectrolyte Multilayer Films

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On the infectivity of bacteriophages in polyelectrolyte multilayer films: inhibition or preservation of their bacteriolytic activity? Jalal Bacharouche, Ozge Erdemli, Romain Rivet, Balla Doucouré, Céline Caillet, Angela Mutschler, Philippe Lavalle, Jerome F.L. Duval, Christophe Gantzer, and Grégory Francius ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10424 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 11, 2018

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ACS Applied Materials & Interfaces

On the infectivity of bacteriophages in polyelectrolyte multilayer films: inhibition or preservation of their bacteriolytic activity?

Jalal Bacharouche1,2, Ozge Erdemli2,3,4, Romain Rivet1,2, Balla Doucouré1,2, Céline Caillet5,6, Angela Mutschler3,4, Philippe Lavalle3,4, Jérôme F.L. Duval5,6, Christophe Gantzer1,2, Grégory Francius1,2*

1

Université de Lorraine, Laboratoire de Chimie Physique et Microbiologie pour

l’Environnement, LCPME, UMR 7564, Villers-lès-Nancy, F-54600, France. 2

CNRS, Laboratoire de Chimie Physique et Microbiologie pour l’Environnement, LCPME,

UMR 7564, Villers-lès-Nancy, F-54600, France. 3

Institut National de la Santé et de la Recherche Médicale, INSERM Unité 1121, 11 rue

Humann, 67085 Strasbourg Cedex, France. 4

Université de Strasbourg, Faculté de Chirurgie Dentaire, 8 rue Sainte Elisabeth, 67000

Strasbourg, France. 5

Université de Lorraine, Laboratoire Interdisciplinaire des Environnements Continentaux,

UMR 7360, 54501 Vandœuvre-lès-Nancy, France. 6

CNRS, Laboratoire Interdisciplinaire des Environnements Continentaux, UMR 7360, 54501

Vandœuvre-lès-Nancy, France.

* Corresponding authors: [email protected]

Keywords: Polyelectrolyte multilayer films, phages, antibacterial biomaterials, infectivity, qPCR, plaque bioassays, Atomic Force Microscopy, Dynamic Light Scattering.

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Abstract Antibiotic resistance in bacterial cells has motivated the scientific community to design new and efficient (bio)materials with targeted bacteriostatic and/or bactericide properties. In this work, a series of polyelectrolyte multilayer films differing in terms of polycation-polyanion combinations are constructed according to the layer-by-layer deposition method. Their capacities to host T4 and φx174 phage particles, maintain their infectivity and bacteriolytic activity are thoroughly examined. It is found that the macroscopic physicochemical properties of the films, which includes film thickness, swelling ratio or mechanical stiffness (as derived by atomic force microscopy and spectroscopy measurements), do not predominantly control the selectivity of the films for hosting infective phages. Instead, it is evidenced that the intimate electrostatic interactions locally operational between the loaded phages and the polycationic and polyanionic PEM components may lead to phage activity reduction and preservation/enhancement, respectively. It is argued that the underlying mechanism involves the screening of the phage capsid receptors (operational in cell recognition/infection processes) due to the formation of either polymer-phage heteroassemblies or polymer coating surrounding the bioactive phage surface.


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1.

Introduction The increased resistance of various pathogenic bacteria against antibiotics commonly

used by humans has prompted the scientific community to elaborate alternative and efficient strategies for preventing and controlling microbial-related infections and diseases. Bacteriophages are natural predators of bacteria and, as such, they offer a promising perspective in terms of sustainable therapeutic solutions against bacterial pathogens actions and/or proliferation.1-3 Bacteriophages belong to diverse groups of viruses,2 some of them being easily cultivable4 with potential applications in biotechnology,5-6 nanomedicine3, 7 and nanotherapeutics.1,

8

In addition, some of them are very specific in terms of cell targeting,

which provides an original basis for the design of novel nano-vectors for selective delivery of vaccines and genes, and for the elaboration of a new class of antimicrobial agents.3, 9-10 Over the last decade, due attention was drawn to the development of tuneable phagestorage/delivery systems, especially in the fields of agriculture, biotechnology, pharmaceutics, farming and food industries.7,

11-13

In this context, spray drying, extrusion dripping,

emulsification and polymerisation are among the methods that are commonly adopted for phage storage and encapsulation in organic matrices.14-17 For the sake of illustration, the emulsification technique applied to hydrogels composed of chitosan, alginate or pectin18-20 offers a proper control of phage storage and release.17-18,

20-21

However, all aforementioned

phage encapsulation processes suffer from a loss and/or reduction of bacteriophages activity once incorporated within a given 3D storing polymer-based matrix.17 Physico-chemical features of the internal matrix environment, e.g. temperature, hydration, salinity or pH, are known to unfavourably mediate bacteriophage infectivity and stability, i.e. their ability to efficiently infect and kill bacteria.22-24 Recent studies have further evidenced that the constitutive elements of the matrix itself and the nature of the phage encapsulation processes may impact on the infectivity of the bacteriophages.17, 19 The underlying mechanisms remain



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poorly understood and they possibly originate from irreversible alterations of the phage capsid proteins as a result of strong interactions with constitutive components of the phage-hosting matrix.25-26 Polyelectrolyte multilayer films (PEM) have been employed for almost two decades in the design and development of versatile and tailorable drug delivery systems.27-28 PEM films consist of cationic and anionic polymer layers alternatively deposited at the surface of any types of supporting substrates with virtually any shape. Beyond their now recognized potential in terms of biomedical applications, PEMs may further exhibit antimicrobial, antiadhesive and/or reservoir properties, depending on the nature of the PEM-constituting polyelectrolytes and on the physicochemical medium composition.29-32 In turn, PEM films are considered as interesting material candidates for embedding bioactive molecules, e.g. peptides, drugs, or DNA.33-35 The latter can be incorporated into PEM films following different strategies: adsorption of the bioactive molecules at various stages of the film construction, or upon immersion of a pre-formed film into a solution containing these biomolecules, the so-called post-diffusion method.36 PEM films are currently used as reservoir/storage and delivery systems for nanoparticles and various active drugs.29,

36-38

However, to the best of our knowledge, there is a lack of studies documenting the suitability of PEMs for hosting bacteriophages while concomitantly preserving their infectivity.39-41 So far, the focus has essentially been given to the elaboration of protocols in order to improve the detection of bacteriophages, regardless of their infective potential.42-43 In this context, the objective of the current work is to investigate the ability of PEMs differing in terms of chemical composition and nanomechanical characteristics, for storing two bacteriophages termed φx174 and T4, and maintaining their infection activity toward Escherichia coli. φx174 is an icosahedral phage with ca. 30 nm diameter, it hosts a circular 5386 nucleotides single-stranded DNA and belongs to the Microviridae family. T4 is a 200


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nm head-tailed phage of the Myoviridae family containing 168903 nucleotides doublestranded DNA. Both phages are classically employed as surrogates to describe the fate and behaviour of pathogenic viruses in natural aquatic media.44 Topography and mechanical features of the different polymeric matrices tested in this work are addressed by spatiallyresolved atomic force microscopy (AFM). The way physicochemical properties of the PEM materials determine (or not) their capacities to store phages and alter or maintain phages infectivity is systematically explored by considering PEM compositions that differ in terms of combinations of polyanionic and polycationic film-building blocks. The phage-storage performance of each polyelectrolyte multilayer film was evaluated by both quantitative polymerase chain reaction (qPCR) technique and plaque bioassays after 18 hours incubation of the PEMs in phage-containing media. qPCR allows estimation of total number of particles whereas plaque bioassays only evaluate the number of phages able to infect host bacteria. Phage infectivity was evaluated by bioassay measurements after completion of the phageloading process in the PEMs. The results are discussed in conjunction with bacteriophage size measurements performed by dynamic light scattering in relevant polycation/polyanion environments. This strategy makes it possible the identification of the contribution of each polyelectrolyte component to phage stability against aggregation and, therewith, to reduction in phage infectivity.

2.

Materials and methods

2.1.

Chemicals and preparation of polyelectrolyte multilayer films. Tris(hydroxymethyl)aminomethane (TRIS, ref. T4661), sodium chloride (NaCl, ref.

1.06406.0500), poly(allylamine hydrochloride) (PAH, Mw = 50,000 g/mol, ref. 283223), poly(L-lysine

hydrochloride)

(PLL,

Mw

>

30,000

g/mol,

ref.

P9404),

poly(dimethyldiallylammonium chloride) (PDADMAC, Mw = 400,000-500,000 g/mol, ref.



409030) and poly(acrylic acid sodium salt) (PAA, Mw ~ 50,000 g/mol, ref. 00627) and chitosan (CHI, Mw=100,000-300,000 g/mol, ref. 00281-100) were purchased from SigmaAldrich (France) and Polysciences (Biovalley, France). Sodium hyaluronate (HA, 301-450 kDa, ref. HA500K-1) was purchased from LifeCore Biomedical (Chaska, USA). Each polyelectrolyte was dissolved in buffer solution consisting of 150 mM NaCl and 10 mM TRIS (1 mg/mL) with pH adjusted to 7.4. For the sake of clarity, Table 1 reports the nature of the various polycationic and polyanionic polymers used for construction of the PEM films tested in this work. The automated sequential build-up of the multilayer films at room temperature (22°C) was performed using borosilicate slides as supporting substrate in order to facilitate subsequent film analysis by atomic force microscopy (AFM). Evaluation of the amount of phages in the film was carried out by reverse-transcription polymerase chain reaction (RTPCR). PEM films were constructed using a dipping robot (Riegler & Kirstein GmbH, Berlin, Germany). The first deposited layer consisted of the polycation (i.e. either PLL, PDADMAC, PAH or CHI, see Table 1), and the sample was then rinsed by immersion for 5 min in the buffer solution (150 mM NaCl, 10 mM TRIS at pH 7.4). The polyanion component of the PEM film (i.e. HA or PAA) was subsequently deposited with following the procedure invoked above for the deposition of polycations. This PEM build up process was iterated via alternate deposition of the polycationic and of the polyanionic polymer until the PEM film consisted of 30 bilayers. If not used immediately after construction, the PEM films, hereafter denoted as (polycation-polyanion) for short, were stored at 4°C in buffer solution (150 mM NaCl, 10 mM TRIS, pH 7.4). The polyelectrolyte multilayer films were loaded with phages via passive diffusion of the latter from the inoculum solution to the polymer matrices until equilibrium was reached (after 18 hours incubation). Production and purification of the here adopted T4 and φx174 phages are detailed below.


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2.2.

Production and purification of bacteriophages. The production of the bacteriophages of interest in this work requires host bacteria: E.

coli B for T4 (ATCC 11303) and E. coli C for φx174 (ATCC 13706). T4 and φx174 phages were replicated according to the ISO 10705-2 procedures, respectively, without the chloroform lysis step. The latter was not considered in order to (i) limit the presence of incomplete viral particles originating from chloroform-induced lysis of bacteria, and (ii) to avoid the selection of the only polar viruses, recalling that most apolar viruses may interact with chloroform and they may thus be eliminated together with this solvent. After replication, phage suspensions were centrifugated (Beck man [Fullerton, CA] model J2-22; 10,000 g; 10 min; 4°C) and the supernatant was filtered using a 0.22 µm membrane (Millex®-GP Millipore). Then 30 mL of this suspension was concentrated in 5.5 mL by Amicon Ultra-15 (PLHK, membrane Ultracel-PL, 100 kDa). The phage suspensions were purified by cesium chloride gradient after addition of 3.8g CsCl and centrifugation at 29000 g for 18h at 15°C. About 1.5 mL of phage suspension was extracted from central strip and then purified by dialysis (100 kDa molecular weight cutoff, Spectrum [Gadena, CA Spectrum Labs]), first against de-ionized water for 14 h, and then against 1 mM NaCl at pH 7 ± 0.3 for 14 h. The concentrations of phages obtained following the above protocol were in the range 109 to 1011 PFU/mL.

2.3. Quantifying the total amount of phages and the amount of infective phages in the films. The total amount of phages (infective and non-infective) corresponds to the amount of phage’s genome copies detected in the sample. Viral DNA was extracted from 100 µL of phage suspension from each sample (either the inoculum solution or the dissolved PEM, see details below) using the automated nucleic acid extractor EasyMag from Biomerieux and



according to the manufacturer’s instructions. DNA was eluted in 100 µL elution buffer and stored immediately at -80°C. The phage’s genomes from each sample were quantified by droplet digital PCR (ddPCR) using the QX100 system by BioRad and according to the manufacturer’s instruction. In brief, the amplification mix was composed of 10 µL of ddPCR™ Supermix for Probes (1863023 – Biorad), 600 nM of forward and reverse primers, 300 nM of fluorescent hydrolysis probe tagged with 6-FAM and BHQ-1, 5µL of DNA extract and DNase-free water up to 20 µL. The sequences of the primers and probes are listed in Table 2. The droplets generation was performed with a DG8 Cartridge on the QX100 Droplet Generator and following manufacturer’s instructions. Amplification was done with a Veriti Thermal Cycler (Applied Biosystem), the thermal ramping was fixed at 70% of the maximum (around 2.5°C/sec). The amplification program was 10min at 95°C followed by 50 cycles of 30 s at 94°C and 90 s at 60°C and a final step at 98°C for 10 min. Immediately after amplification, positive and negative droplets were counted on the QX100 Droplet Reader, calculation of concentration’s samples was then performed with help of the QuantaSoft™ Software. As the thickness of all constructed PEMs (Table 1) is -within experimental erroridentical for 4 out of the 5 materials tested (ca. 5 µm, see Table 3), the results were expressed in PFU units. It is stressed that ddPCR method probes the total amount of phages (infective and non-infective) in a given environment (solution or dissolved PEM volume) via detection of their DNA copies. The evaluation of the amount of the only infective phages was performed via plaque bioassays, which consists in counting plaques upon spotting the phages originating from inoculum or dissolved PEMs on a lawn of E. coli cells after 18 hours incubation as describe in the ISO 10705-2 procedure. More specifically, the amount of infective phages stored within PEMs was quantified upon application of bioassays method for originally phage-containing PEMs dissolved by osmotic shock in a 2 M NaCl buffer solution.


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The minimum inhibitory concentration of the tested polyelectrolytes, denoted as MIC100, was determined by broth microdilution. In detail, an overnight culture of E. coli C was diluted in order to reach an optical density (OD620) of 10-3, and microorganisms were subsequently plated in 96-well plates in the presence of cationic or anionic polyelectrolyte (Table 1) at 0.01, 0.10 and 1.00 mg/mL concentrations. After 24 hours incubation at 37°C, the microorganism growth was assessed by optical density OD620 measurements using a Multiskan™ EX microplate spectrophotometer (Thermo Fisher Scientific). The MIC100, defined as the lowest inoculum concentration of a given component (here the polyelectrolyte) leading to 100% growth inhibition, was determined on the basis of a modified Gompertz model as detailed by Lambert et al.45 For each polyelectrolyte tested, the normalized pathogen growth (NPG for short) was calculated on the basis of the following relationship (Figure S1 in Supporting Information).

NPG =

OD620 sample OD620 (control+ ) OD620 control- OD620 (control+ )

×100

(1)

, where control+ corresponds to the situation where bacterial suspensions contain efficient antibiotics (tetracycline at 10 µg/mL and cefotaxime at 0.1 µg/mL) for bacterial killing, control- refers to the case where bacterial suspension are free of these antibiotics and ‘sample’ pertains to the medium containing the tested anionic or cationic polymers (those used for the PEM constructions). All experiments were performed in biological triplicates.

2.4.

Dynamic Light Scattering measurements Prior to virus size measurements, phage suspensions were purified under cesium-

chloride gradient conditions to eliminate the possible presence of bacterial membrane residues. Dynamic light scattering (DLS) experiments were then performed on the soobtained suspensions with a Malvern Zetasizer NanoZS instrument using a U-shaped measurement cell (DTS1070) commonly dedicated to electrophoretic mobility determination, 9 Environment ACS Paragon Plus


which in turn allowed the use of only c.a. 500 µL phage suspensions. The sensitivity of the apparatus used for the phage size-measurements requires working with a minimum concentration of viruses of ca. 109 PFU/mL. The size of T4 and φx174 phages in buffer solution (150 mM NaCl, 10 mM TRIS at pH 7.4) containing c.a. 109 PFU/mL phages was evaluated in the presence of dispersed anionic or cationic polyelectrolytes (those used for PEM construction, Table 1). Control measurements were additionally performed in the absence of the polyelectrolytes. Measurements in the presence of polyelectrolytes (one polyanion, HA and two polycations, PAH and PDADMAC were tested here) were performed as follows. A mixture of 55 µL polyelectrolyte at 1 mg/mL and 500 µL phage suspension was homogenized manually to reach a final 0.1 mg/mL polyelectrolyte concentration. Then, a period of 5 minutes was considered prior to filling the measurement cell with the phagepolyelectrolyte suspension and setting that cell in the apparatus. Measurements were performed at 25°C. For each condition tested, 3 successive measurements were carried out on the same aliquot. Phage size was measured in the absence of polyelectrolyte following the protocol detailed elsewhere.46 As a first approximation, phage sizes were obtained from DLS measurements on the basis of Stokes-Einstein relationship.44

2.5.

Atomic Force Microscopy (AFM) AFM images and force spectroscopy measurements were performed using a Bioscope

Resolve (Bruker Nano Surface, Bruker France SAS, Palaiseau, France) and a MFP3D-BIO instrument (Asylum Research Technology, Oxford Instruments Company, Wiesbaden, Germany), respectively. Topography of the PEM films was imaged by AFM operating in contact mode. Silicon nitride cantilevers of conical shape purchased from Bruker (MLCT, Bruker France SAS, Palaiseau, France) with spring constant of about 9-14 pN/nm were used for both imaging and PEM nanomechanical measurements. All images were recorded with a


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resolution of 512 × 512 pixels and a scan rate of 1 Hz. Nanomechanical properties of the PEM films were addressed in PBS buffer solution (pH 7.4) by recording at least 3 Force-Volume Images (FVI) at different locations of the films. Each FVI consisted of a grid of 32 × 32 force curves measured with adopting a 2 µm.s-1 approach rate of the tip toward the sample. The Young modulus E was evaluated by analysing the force-indentation curves within the framework of the Sneddon model.47-48 In this model, the Young modulus is related to the applied force according to the equation given below:

F=

2 E ⋅ Tan (α ) π (1 − ν 2 )

R1/2 δ 2 ⋅ f BECC

(2)

, where δ is the indentation depth, ν the Poisson coefficient, α the semi-top angle of the conical tip and fBECC is the bottom effect cone-correction function that takes into account the stiffness of the glass substrate that supports the PEM material. All FVI were analysed using an automatic Matlab algorithm detailed elsewhere,49 and average Young moduli values given in this work were derived from at least 3072 force curves.

3.

Results and discussion

3.1.

Phage-storage performance of polyelectrolyte multilayer reservoirs Five different PEM matrices of 30 bilayers were built in a buffer solution and

subsequently analyzed by AFM before bacteriophage entrapment/storage. The analysis of the matrices topography, which includes PEM thickness evaluation (and corresponding PEM volume), determination of swelling ratio, surface roughness and elasticity (Young Modulus), are reported in Table 3. AFM imaging in aqueous medium evidences homogeneous surfaces for all PEM systems considered in this work (Figure S2 in Supporting Information). Average roughness values are in the 18-117 nm (Table 3) range with the lowest values obtained for PLL-HA and PAH-HA films, and the largest ones for CHI-HA and PDADMAC-PAA films



that exhibit globular types of surface structures (Figure S2). All PEM materials are relatively soft with Young moduli in the range 7-55 kPa except PAH-PAA that is the stiffest system adopted in this work with a Young modulus of ca. 6000 kPa. In line with this marked stiffness, PAH-PAA is the material defined by the lowest swelling ratio (1.6, see Table 3), the lowest thickness (2.5 µm) as compared to the other PEM materials (swelling ratio in the 4 to 12 range). The PLL-HA polyelectrolytes combination leads to the largest swelling ratio (12, Table 3) and the lowest Young modulus (7 kPa) as compared to the other PAH-HA, CHI-HA, PDADMAC-PAA systems (swelling ratio of ca. 4-5, Young Modulus in the range 12 to 55 kPa) while the thickness of all these materials is similar (ca. 5 µm). Basically, the softest films are those defined by an exponential growth phase, in agreement with literature,50-51 whereas the layer-by-layer construction of PAH-PAA material is defined by a regime intermediate between linear and exponential growth mechanism depending on medium composition.29, 52-53 Following the evaluation of the above physicochemical PEM attributes (Figure S2 and Table 3), PEM matrices were incubated 18 hours in a buffer solution containing φx174 or T4 bacteriophages in order to address their respective phage-entrapment capacities. Quantitative determination of the amount of infective phages was performed by plaque bioassay method at different stages of the phages entrapment procedure. In detail, after the incubation period of 18 hours, the supernatant was collected and analyzed by plaque bioassays (blue bars in Figure 1). In addition, the last rinsing bath (the last of the three baths used for PEM construction) collecting the non-entrapped phages and the phages that are poorly attached to the PEM surfaces were also titrated by plaque bioassays (orange bars in Figure 1). Finally, the PEM matrices were dissolved by osmotic shock in a 2 M NaCl buffer solution and so dissolved PEM solutions were titrated in order to evaluate the number of infective phages originally entrapped within the PEM body volume (green bars in Figure 1).


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All results are reported in the form of histograms for φx174 (Figure 1a) and T4 (Figure 1b) phages. Concerning φx174, the initial amount of phages in contact with each PEM matrix was about 5×106 (inoculum solution). After 18 hours incubation of the matrices in such a phagecontaining solution, no significant changes were observed between the amounts of infective phages in the inoculum and in the supernatant. However, significant variations of the amount of infective phages were evidenced in the PEM body compartments depending on the nature of the film considered. We observed indeed that PDADMAC-PAA is the matrix that contains the lowest amount of infective phages, and that PAH-HA is the matrix where this amount is largest (ca. 7×102 and 2×106 respectively). In addition, titration results reveals the quasiabsence (within experimental error) of infective phages in the last rinsing bath (between ca. 102 and 103) regardless the nature of the PEM, which indicates that practically all nonentrapped or PEM-adhered phages were already removed from the two previous rinsing steps. It further means that the rate of φx174 release from the PEM matrix should be significantly lower than the rate of phage accumulation inside the PEM volume. We further stress that the reduction in the amount of infective phages when transiting from the inoculum to the PEM films is in the 2-4 logs range for all tested matrices except for PAH-HA (for the latter the reduction is less than one log unit). Similar experiments were carried out with phage T4 (Figure 1b). Like φx174, the amount of infective T4 phages in the supernatant as compared to that in the inoculum was not significantly impacted by the presence of PEM matrices, except for PAH-HA system. Besides, titration of the last rinsing bath evidenced the significant presence therein of infective T4 phages (between c.a. 103 and 105) with an amount that is one to three orders of magnitude larger than that of infective φx174 depending on the PEM considered. In addition, the results evidence a significant 1 to 4 logs reduction of the amount of infective phages in the PEM matrices as compared to that in the inoculum solution.



In order to rationalize the reservoir capacity of infective phages for all tested PEM materials, we report in Figure 2a the entrapment ratio for each film defined by the ratio between the amount of infective phages in the PEM body after 18 hrs incubation and the amount of infective phages initially present in the inoculum solution. Concerning φx174, the best entrapment ratio was achieved for the PAH-HA system with ca. 30% of entrapped infective viruses, whereas this ratio remains systematically below 0.5% for all other PEM materials. Data thus suggest that φx174 entrapment performance of the PEM matrices is not directly related to the mechanical properties of the latter (see Table 3). Indeed, the softest PEM matrices with Young moduli lower than 20 kPa exhibit very low entrapment ratio (below 0.1%), a feature also found for stiffer PAH-PAA material with Young modulus of 6000 kPa. This result supports that φx174 entrapment is primarily mediated by the chemical nature of the polyelectrolytes constituting the 3D PEM reservoir and not by the stiffness or the hydration degree of the polyelectrolytic matrices (Table 3). For T4 phages, the best entrapment ratio is obtained for PAH-HA and PLL-HA matrices with ca. 5% to 10% of the infective phages present in the inoculum that are successfully entrapped. The poorest performance in terms of T4 infective phage entrapment is obtained for PDADMAC-PAA matrix (which also holds for φx174), with an entrapment ratio less than 0.01%. In addition, very similar T4 entrapment ratios were achieved for CHI-HA and PAH-PAA matrices despite their significantly different mechanical properties with Young moduli values of ca. 20 kPa and 6000 kPa, respectively. These results further indicate that absorption of infective phages by PEM films is not significantly driven by the defining mechanical PEM features (nor by their water content or swelling ratio) but rather by their very polyelectrolytic composition. At this stage of the analysis, it then becomes crucial to identify the process by which PEMs possibly mediate the infectivity of entrapped phages. Such identification necessarily asks for the prior measurement of the total amount of phages


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(including non-infective ones) PEMs may accommodate after 18 hrs incubation period. The next section is devoted to clarifying this issue.

3.2.

Do constituting PEM polyelectrolytes mediate phage infectivity? Before answering that question, we first evaluated the impact of the polyelectrolytes

constituting the PEMs on bacterial growth and metabolism. These measurements are required to detect any possible contribution of the polyelectrolytes in the outcome of the plaque bioassays performed on the originally phage-containing dissolved PEMs. This analysis is thus mandatory to differentiate a possible antimicrobial activity of the polyelectrolytes in solution (the situation met in plaque bioassays carried out on dissolved PEMs) from the here-targeted bacteriolytic activity of the phages (originally embedded in the PEMs). Accordingly, we determined the minimum inhibitory concentration (MIC100) of the different tested anionic and cationic polyelectrolytes toward E. coli C (Table 1 and Figure S1) targeted by φx174. We evidenced that the selected polyanions (HA and PAA) do not have any antimicrobial activity unlike the selected polycations (CHI, PLL, PAH and PDADMAC) for which we evaluated a MIC100 of about 0.05 g/L. This means that the presence of the polyanions in cell culture does not impact the outcome of the plaque bioassays (in terms of phage infectivity measurements) irrespective of the adopted polymer concentration. Figure S1 shows that the presence of tested polycations at concentration exceeding 0.05 mg/mL favors lysis of bacteria, a feature we detailed for another polycation, polyethylemine.54 Qualitatively, similar conclusions are obtained for polyelectrolyte MIC100 values pertaining to E. coli B targeted by T4 phages (i.e. absence and presence of cell growth inhibition for all tested polyanions and polycations, respectively). On a quantitative level, MIC100 of polycations are slightly lower against E. coli B as compared to those derived for E. coli C (see Table 1). Overall, the results highlight that polycations may interfere with measurements of the infectivity of PEM-entrapped φx174 and



T4 phages under conditions where polycation concentration exceed 0.01 mg/mL and 0.05 mg/mL, respectively. Considering that PEMs with final volumes of 4×10-4 mL to 9×10-4 mL (Table 1) were constructed with adopting polycation and polyanion concentrations of 1 mg/mL in solution and that PEMs were dissolved in 1 mL buffer solution (4 M), the polymer concentration relevant under plaque bioassays conditions are systematically much lower than the MIC100 values reported in Table 1. The results given in Figure 1 pertaining to the amounts of infective phage in PEMs thus solely reflect the bacteriolytic activity of originally entrapped viruses. To analyse the possible impact of the polyelectrolytes constituting the PEM materials on the infectivity of the phages entrapped therein, titration results obtained from plaque bioassays were compared to quantitative digital PCR measurements, the latter reflecting the total amount of genome copies (including those of infective and non-infective phages) in the PEM body volume. The results are collected in Figure 3a and Figure 3b for φx174. They show that the number of φx174 DNA copies detected by PCR in the inoculum suspension and in the PEM matrices (blue bars in Figure 3a) are one to three orders of magnitude larger than the number of infective phages observed according to plaque bioassay methodology (red bars in Figure 3a). Quantitatively, only 4% of the total amount of φx174 phages (Figure 3b) in the inoculum suspension are infective, showing that a small fraction of phages are naturally active and able to lyse host bacteria. The ratio (termed infectivity ratio) between the amount of infective φx174 phages entrapped in the PEM volume and the total amount of φx174 therein significantly depends on the type of matrix adopted: it is ca. 12% for PAH-HA, it drops to 7% for PAH-PAA and it remains below 0.5% for all other tested PEMs. It is remarkable that PAH-HA and PAH-PAA systems lead to a φx174 infectivity ratio that is ca. 2 to 3 times larger than that obtained for the inoculum compartment whereas other PEM systems basically promote a deactivation of the phages (10 to 40 times loss of infectivity).


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As far as T4 phages are concerned, infectivity ratios could be determined only in the inoculum solution (34% infectivity ratio) and in the PDADMAC-PAA system (24%) as polyelectrolytes (PAH, HA, PLL, CHI) were found to interfere with PCR kit-extraction reagents (Figure S3). These interferences led to a number of DNA copies detected by PCR that was systematically (and inconsistently) lower than the number of infective phages except for the PDADMAC-PAA matrix. Even partial, these results indicate that PDADMAC-PAA contributes to reduce T4 phages infectivity but to a lesser degree, it does for φx174. The above results make it transparent that the loss of- or gain in- phages infectivity after entrapment in PEM is strongly dependent on the PEM material and on the phage considered (Figure 3, Figure S3). Data suggest that the activity of φx174 phages is best preserved (and even enhanced) either in the soft PAH-HA and the rigid PAH-PAA system (Table 1 and Figure 3b). A possible explanation for this feature is that the slow dynamics of the constituting polyelectrolytic chains assembly (which tends to increase PEM stiffness55-56) reduces the number of collision events between the PEM constitutive components and the embedded phages, thereby maintaining phage infectivity at a level comparable to that in the inoculum solution. This element alone, however, is not sufficient to explain the lower infectivity ratio measured for PAH-PAA as compared to that of the softer PAH-HA. In turn, following previous work on the impact of phages aggregation on their very infectivity in aqueous media,57 we hypothesize that the physico-chemical microenvironment of the phages within the PEM body (salinity and local pH) strongly mediates their ability to form aggregates, thus modulating their infectivity.22 In order to explain changes in phage infectivity after entrapment, we may further argue that polycationic and/or polyanionic chains adsorb or attach to the viral capsids to an extent that depends on the affinity of the chemical polymer functions for the phage surface proteins. In turn, this induces a screening of the specific capsid receptors that are operational in cell recognition and infection processes.58



On the basis of the above anticipation, we attempted to identify the nature of the polyelectrolyte component of the PEMs that most significantly impacts phage infectivity. To do so, we restricted our analyses to plaque bioassays (which circumvents the inapplicability of PCR method to T4 phages, see discussion above) and measured the amount of infective phages in a solution containing a given anionic and cationic polyelectrolyte (listed in Table 1) at concentration 0 g/L (this corresponds to the inoculum solution with 109 PFU/mL total concentration of phages), 0.01, 0.10 and 1.00 g/L concentration. Results are reported in Figure 4 and are presented in terms of the ratio between detected amounts of infective phages in polymer-containing solution and in the inoculum solution. In the following, we term this ratio ‘Phage Activation Parameter (PAP in short)’: values lower (larger) than unity reflects polymer-mediated phage deactivation (activation, respectively). This ratio should not be confused with the infectivity ratio displayed in Figure 2 and defined after normalisation of the amount of entrapped infective phages by the total amount of phages in the PEM. For φx174, we observed that addition of polyanions (PAA and HA) leads to a slight increase of the PAP, whereas the addition of polycations (PLL, CHI, PDADMAC and PAH) significantly reduced PAP values (by a factor of ca. 2). Within experimental error, PAP is found to be independent of the polycation or polyanion concentration adopted. Qualitatively, similar conclusions hold for T4 phages (Figure 4b) except that the effects of the polycations (PLL, CHI, PDADMAC and PAH) and polyanions (PAA and HA) on PAP values are significantly more differentiated than for φx174. Indeed, polycations reduce T4 infectivity by ca. a factor 2 (PLL) to 10 (CHI) at 0.01g/L concentration. For each polycation considered, this reduction in phage infectivity is not a straightforward function of the polymer concentration. Unlike polycations, polyanions increase T4 phages infectivity by ca. a factor 2 with again no marked dependence on the polyelectrolyte concentration considered.


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The above data indicate that attractive electrostatic interactions between polycations and negatively charged capsids44 of the phages leads in fine to a loss of their infectivity whereas repulsive polymer-phage interactions tend to enhance the infectivity of the phages. These processes come at play for phages entrapped within PEM matrices. It is anticipated that these findings should be applicable for thinner films provided that two conditions are verified: (i) film thickness should exceed a critical value equal to several times the virus diameter, (ii) the molecular structure of the thinner films remains comparable to that of the thick films examined here. In order to decipher whether attractive polycation-phage interaction induces phage deactivation via screening of the specific capsid receptors or via the formation of polymer-phage hetero-aggregates, the stability of polyelectrolyte-phage suspension against aggregation was addressed by dynamic light scattering measurements detailed in the next section.

3.3.

Stability of polymer-phage suspensions against aggregation. To better fine the nature of the processes leading to enhancement or reduction of

phage infectivity, we performed dynamic light scattering (DLS) measurements on phage suspension prior to and after addition of polycations and polyanions at 0.1 g/L concentration, which corresponds to the concentration of polyelectrolytes chosen for PEM construction. DLS results collected in Table 4 are averages of 3 measurements, and size distribution of φx174 phage and T4 phage in the presence or absence of polyelectrolytes are detailed in Figures S4a and S4b, respectively. The size distribution of phage φx174 in the absence of polymers is bimodal. Each phage population exhibit diameters larger than 20-30 nm,44 the diameter range expected for isolated φx174 phage.59 This polydispersity in size reflects the presence of phage aggregates with diameters of about 140 and 550 nm. The size distribution obtained for T4 phages significantly differs from that of phage φx174. The distribution is indeed relatively



monodisperse with a mean diameter value of the order of 220 nm, which supports that phages T4 are present in the form of individual entities in the absence of polymers (the dimension of T4 phage was confirmed by AFM imaging as reported in Figure S5). After addition of the polyanion HA, DLS results show that a slight but significant reduction of phage aggregates size as φx174 size distribution is shifted to lower values (ca. 120-340 nm range). Measurements carried out for T4 phage also displays a shift of their size to smaller values (165 nm). Recalling that there is no aggregation for the T4 phage, this size reduction is probably connected to a contraction (triggered by the presence of the polyanion) of the phage tail. The possibility for T4 to contract its tail has been reported elsewhere 60 and it is further supported by AFM measurements provided in Figure S5. In the presence of PAH polycation, size distribution of phage φx174 becomes monomodal with a mean diameter value of 620 nm, thus indicating a significant phage aggregation induced by PAH. Regarding phage T4, after addition of PAH there was a rapid increase in turbidity of the suspensions, which renders impossible any size measurements (restricted to dilute systems). This is obviously the signature of a massive phage-polymer heteroaggregation. After a 20 minutes period necessary to let large aggregates sediment at the bottom of the cell, size measurements performed in the supernatant varied between 1435 nm and 3030 nm. The addition of PDADMAC polycation to φx174 phage suspension also leads to significant aggregation manifested by a marked turbidity of the solution. After 20 minutes, measurements indicated polymer-phages assembly in the supernatant up to 1780-3640 nm in diameter. In the case of the phage T4, PDADMAC addition did not lead to an increase in solution turbidity. The size distribution remains basically unchanged, reflecting here the absence of significant aggregation. The size distribution was further monomodal with a mean diameter value of 220 nm, i.e. the size obtained for the phage T4 in the absence of polymers.


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Altogether, putting aside the case of the PDADMAC-T4 system discussed below, all polycations favor phage aggregation. On the opposite, the tested polyanions tend to reduce the size of φx174 aggregates or reduce T4 size probably via triggering of tail contraction. These results are qualitatively in line with the increase or loss of phage infectivity upon addition of polyanions or polycations, respectively (see Figure 4). For PDADMAC-T4 system, it is likely that the reduction of T4 infectivity highlighted in Figure 4b results from screening of the T4 capsid receptors, which then become inoperational for bacterial recognition. It is expected that the stability of the polyelectrolyte-coated phages depends on the phage/polyelectrolyte concentration ratio. In particular, at sufficiently high polyelectrolyte concentrations, complete polymer coating at the phage surface would ensure phages stability against aggregation. However, in such a situation, phages should be inactive as a result of a full screening of their capsid surface receptors. The work detailed in the preceding sections identifies the intimate connection existing between polymer-phage interaction and extent of phage (de)activation in aqueous environment (see Figure 4 and Figure S4). In particular, it clearly evidences the paramount role played by electrostatics in governing phage-polymer hetero-aggregation and/or polymer adsorption on phages, and therewith phage deactivation. The results further evidenced that the macroscopic physico-chemical features of the PEM listed in Table 1 do not unambiguously explain the intricate dependence of phages infectivity ratio on the nature of the PEM film. Figure 4 and DLS-based analysis of polymer-phage suspension (Figure S4) rather suggests that an understanding of this dependence requires the analysis of the interactions locally experienced by the phages in the PEM film with polycationic and polyanionic constituents. In particular, the amount of mobile anionic and cationic chains in the whole PEM body -i.e. chains that are not engaged in polyelectrolyte film structure, appear as a critical parameter to



estimate the extent by which bacteriolytic activity of phages, as a result of their interactions with these available chains, may be reduced, fully inhibited or enhanced.

4.

Conclusions For the first time the suitability of different PEM films that differ in terms of

composition, nanomechanical and swelling properties, for hosting φx174 and T4 phages is addressed. A focus is given on the measurement of the infectivity ratio of the PEM films, a parameter that is of crucial importance for the evaluation of the bacteriolytic activity of the hybrid phage-PEM materials. It is found that the stiffness of the films, derived from spatiallyresolved AFM force measurements, and their water content (that impacts their swelling features) do not significantly control the film capacity to entrap the infective fraction of phages initially present in the outer inoculum solution. In this respect, the best PEM matrix among those tested is PAH-HA with which a 30% and 10% entrapment of φx174 and T4 infective phages is achieved, respectively. A major result of this study is the further demonstration that the activity of the phages, once entrapped in the PEM, changes as compared to that in the inoculum solution. Like the entrapment ratio (ratio between amounts of infective phages in the PEM and in the inoculum), the infectivity ratio (defined by the amount of entrapped infective phages normalized by the total amount of phages in the PEM) is not clearly rationalized by the macroscopic physicochemical film properties. For the sake of illustration, φx174 infectivity ratio data suggest that phages activity is enhanced in PAH-HA (ratio of 12%) and in PAH-PAA (7 %) as compared to situation in the inoculum (ratio of 4%), while it is basically suppressed for all other tested systems (PLL-HA, CHI-HA and PDADMAC-PAA, ratio below 0.5%). It is shown that this activation and deactivation of the phages stem from the repulsive or attractive electrostatic interactions they experience with anionic and cationic PEM components, respectively. These interactions indeed govern the


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extent by which the phage capsid receptors for cell recognition and infection are screened via the formation of either polymer-phage hetero-aggregates or polymer coating at the phage surface. Overall, the work opens a new route to optimize bacteriolytic activity of phageloaded PEM films as it identifies the necessity to fine tune, at the nanoscale, cationic polyelectrolyte-phage interactions rather than the macroscopic films properties for properly counteracting polymer-mediated phage aggregation and deactivation.

Acknowledgments The authors are indebted to the financial support of the French Agence Nationale de la Recherche (ANR) for MAGENTA project under the reference ANR-14-CE17-0005-01.

Supporting Information. The evaluation of the minimum inhibitory concentration of polyelectrolytes with respect to pathogen growth (Figure S1), representative AFM images of PEM films and distribution of films Young modulus (Figure S2), selectivity of PEM films in terms of T4 phages loading (Figure S3), details on phage size distribution in the presence of polyelectrolytes (Figure S4), AFM topographic images and height profiles of φx174 and T4 phages (Figure S5). The Supporting Information is available free of charge on the ACS Publications website.



References: (1) O'Sullivan, L.; Buttimer, C.; McAuliffe, O.; Bolton, D.; Coffey, A. Bacteriophage-Based Tools: Recent Advances and Novel Applications. F1000Research 2016, 5, 2782-2788. (2) Salmond, G. P. C.; Fineran, P. C. A Century of the Phage: Past, Present and Future. Nat. Rev. Microbiol. 2015, 13, 777-786. (3) Haq, I. U.; Chaudhry, W. N.; Akhtar, M. N.; Andleeb, S.; Qadri, I. Bacteriophages and Their Implications on Future Biotechnology: A Review. Virol. J. 2012, 9, 1-8. (4) Hayes, S.; Mahony, J.; Nauta, A.; van Sinderen, D. Metagenomic Approaches to Assess Bacteriophages in Various Environmental Niches. Viruses 2017, 9, 127. (5) Campbell, A. The Future of Bacteriophage Biology. Nat. Rev. Genet. 2003, 4, 471. (6) Vaks, L.; Benhar, I. Antibacterial Application of Engineered Bacteriophage Nanomedicines: Antibody-Targeted, Chloramphenicol Prodrug Loaded Bacteriophages for Inhibiting the Growth of Staphylococcus Aureus Bacteria. Methods Mol. Cell. Biol. 2011, 726, 187-206. (7) Sunderland, K. S.; Yang, M.; Mao, C. Phage-Enabled Nanomedicine: From Probes to Therapeutics in Precision Medicine. Angew. Chem., Int. Ed. 2017, 56, 1964-1992. (8) Petrenko, V. A.; Gillespie, J. W. Paradigm Shift in Bacteriophage-Mediated Delivery of Anticancer Drugs: From Targeted ‘Magic Bullets’ to Self-Navigated ‘Magic Missiles’. Expert Opin. Drug Delivery 2017, 14, 373-384. (9) Ryan, E. M.; Gorman, S. P.; Donnelly, R. F.; Gilmore, B. F. Recent Advances in Bacteriophage Therapy: How Delivery Routes, Formulation, Concentration and Timing Influence the Success of Phage Therapy. J. Pharm. Pharmacol. 2011, 63, 1253-1264. (10) Elbreki, M.; Ross, R. P.; Hill, C.; O'Mahony, J.; McAuliffe, O.; Coffey, A. Bacteriophages and Their Derivatives as Biotherapeutic Agents in Disease Prevention and Treatment. Journal of Viruses 2014, 2014, 1-20. (11) Henry, M.; Debarbieux, L. Tools from Viruses: Bacteriophage Successes and Beyond. Virology 2012, 434, 151-161. (12) Stanford, K.; McAllister, T. A.; Niu, Y. D.; Stephens, T. P.; Mazzocco, A.; Waddell, T. E.; Johnson, R. P. Oral Delivery Systems for Encapsulated Bacteriophages Targeted at Escherichia Coli O157:H7 in Feedlot Cattle. J. Food Prot. 2010, 73, 1304-1312. (13) Hussain, M. A.; Liu, H.; Wang, Q.; Zhong, F.; Guo, Q.; Balamurugan, S. Use of Encapsulated Bacteriophages to Enhance Farm to Fork Food Safety. Crit. Rev. Food Sci. Nutr. 2017, 57, 2801-2810. (14) Korehei, R.; Kadla, J. F. Encapsulation of T4 Bacteriophage in Electrospun Poly(Ethylene Oxide)/Cellulose Diacetate Fibers. Carbohydr. Polym. 2014, 100, 150-157.


Page 24 of 35

Page 25 of 35 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


(15) Ma, Y.; Pacan, J. C.; Wang, Q.; Xu, Y.; Huang, X.; Korenevsky, A.; Sabour, P. M. Microencapsulation of Bacteriophage Felix O1 into Chitosan-Alginate Microspheres for Oral Delivery. Appl. Environ. Microbiol. 2008, 74, 4799-4805. (16) Matinkhoo, S.; Lynch, K. H.; Dennis, J. J.; Finlay, W. H.; Vehring, R. Spray-Dried Respirable Powders Containing Bacteriophages for the Treatment of Pulmonary Infections. J. Pharm. Sci. 2011, 100, 5197-5205. (17) Choinska-Pulit, A.; Mitula, P.; Sliwka, P.; Laba, W.; Skaradzinska, A. Bacteriophage Encapsulation: Trends and Potential Applications. Trends Food Sci. Technol. 2015, 45, 212221. (18) Dini, C.; Islan, G. A.; de Urraza, P. J.; Castro, G. R. Novel Biopolymer Matrices for Microencapsulation of Phages: Enhanced Protection against Acidity and Protease Activity. Macromol. Biosci. 2012, 12, 1200-1208. (19) Dini, C.; Islan, G. A.; Castro, G. R. Characterization and Stability Analysis of Biopolymeric Matrices Designed for Phage-Controlled Release. Appl. Biochem. Biotechnol. 2014, 174, 2031-2047. (20) Bean, J. E.; Alves, D. R.; Laabei, M.; Esteban, P. P.; Thet, N. T.; Enright, M. C.; Jenkins, A. T. A. Triggered Release of Bacteriophage K from Agarose/Hyaluronan Hydrogel Matrixes by Staphylococcus Aureus Virulence Factors. Chem. Mater. 2014, 26, 7201-7208. (21) Vonasek, E.; Le, P.; Nitin, N. Encapsulation of Bacteriophages in Whey Protein Films for Extended Storage and Release. Food Hydrocolloids 2014, 37, 7-13. (22) Jończyk, E.; Kłak, M.; Międzybrodzki, R.; Górski, A. The Influence of External Factors on Bacteriophages - Review. Folia Microbiologica 2011, 56, 191-200. (23) Cooper, C. J.; Denyer, S. P.; Maillard, J. Y. Stability and Purity of a Bacteriophage Cocktail Preparation for Nebulizer Delivery. Lett. Appl. Microbiol. 2014, 58, 118-122. (24) Rode, T. M.; Axelsson, L.; Granum, P. E.; Heir, E.; Holck, A.; L'Abee-Lund, T. M. High Stability of Stx2 Phage in Food and under Food-Processing Conditions. Appl. Environ. Microbiol. 2011, 77, 5336-5341. (25) Rossi, P.; Aragno, M. Analysis of Bacteriophage Inactivation and Its Attenuation by Adsorption onto Colloidal Particles by Batch Agitation Techniques. Can. J. Microbiol. 1999, 45, 9-17. (26) Mora, P. T.; Young, B. G.; Rizvi, S. Inactivation of Bacteriophage with Polyanions, and Role of Amines in Activity. J. Biol. Chem. 1962, 237, 157-164. (27) Jiang, B.; Barnett, J. B.; Li, B. Advances in Polyelectrolyte Multilayer Nanofilms as Tunable Drug Delivery Systems. Nanotechnol., Sci. Appl. 2009, 2, 21-27. (28) Boudou, T.; Crouzier, T.; Ren, K.; Blin, G.; Picart, C. Multiple Functionalities of Polyelectrolyte Multilayer Films: New Biomedical Applications. Adv. Mater. 2010, 22, 441467.



(29) Seon, L.; Lavalle, P.; Schaaf, P.; Boulmedais, F. Polyelectrolyte Multi Layers: A Versatile Tool for Preparing Antimicrobial Coatings. Langmuir 2015, 31, 12856-12872. (30) Zhu, X.; Jańczewski, D.; Lee, S. S. C.; Teo, S. L.-M.; Vancso, G. J. Cross-Linked Polyelectrolyte Multilayers for Marine Antifouling Applications. ACS Appl. Mater. Interfaces 2013, 5, 5961-5968. (31) Wang, X.; Wang, Y.; Bi, S.; Wang, Y.; Chen, X.; Qiu, L.; Sun, J. Optically Transparent Antibacterial Films Capable of Healing Multiple Scratches. Adv. Funct. Mater. 2014, 24, 403411. (32) Chen, D.; Wu, M.; Li, B.; Ren, K.; Cheng, Z.; Ji, J.; Li, Y.; Sun, J. Layer-by-LayerAssembled Healable Antifouling Films. Adv. Mater. 2015, 27, 5882-5888. (33) Ladhari, N.; Hemmerle, J.; Haikel, Y.; Voegel, J.-C.; Ball, V. Polyelectrolyte Multilayer Films: A Sponge for Insulin? Bio-Med. Mater. Eng. 2010, 20, 217-225. (34) Saurer, E. M.; Jewell, C. M.; Roenneburg, D. A.; Bechler, S. L.; Torrealba, J. R.; Hacker, T. A.; Lynn, D. M. Polyelectrolyte Multilayers Promote Stent-Mediated Delivery of DNA to Vascular Tissue. Biomacromolecules 2013, 14, 1696-1704. (35) Wang, B.; Liu, H.; Sun, L.; Jin, Y.; Ding, X.; Li, L.; Ji, J.; Chen, H. Construction of High Drug Loading and Enzymatic Degradable Multilayer Films for Self-Defense Drug Release and Long-Term Biofilm Inhibition. Biomacromolecules 2018, 19, 85-93. (36) Vodouhe, C.; Le Guen, E.; Garza, J. M.; Francius, G.; Dejugnat, C.; Ogier, J.; Schaaf, P.; Voegel, J. C.; Lavalle, P. Control of Drug Accessibility on Functional Polyelectrolyte Multilayer Films. Biomaterials 2006, 27, 4149-4156. (37) Teng, W.; Wang, Q.; Chen, Y.; Huang, H. Controllably Local Gene Delivery Mediated by Polyelectrolyte Multilayer Films Assembled from Gene-Loaded Nanopolymersomes and Hyaluronic Acid. Int J Nanomedicine 2014, 9, 5013-5024. (38) Wang, B.; Xu, Q.; Ye, Z.; Liu, H.; Lin, Q.; Nan, K.; Li, Y.; Wang, Y.; Qi, L.; Chen, H. Copolymer Brushes with Temperature-Triggered, Reversibly Switchable Bactericidal and Antifouling Properties for Biomaterial Surfaces. ACS Appl. Mater. Interfaces 2016, 8, 2720727217. (39) Esteban, P. P.; Alves, D. R.; Enright, M. C.; Bean, J. E.; Gaudion, A.; Jenkins, A. T.; Young, A. E.; Arnot, T. C. Enhancement of the Antimicrobial Properties of Bacteriophage-K Via Stabilization Using Oil-in-Water Nano-Emulsions. Biotechnol. Prog. 2014, 30, 932-944. (40) Lobocka, M. B.; Glowacka, A.; Golec, P. Methods for Bacteriophage Preservation. Methods Mol. Biol. 2018, 1693, 219-230. (41) Leung, V.; Szewczyk, A.; Chau, J.; Hosseinidoust, Z.; Groves, L.; Hawsawi, H.; Anany, H.; Griffiths, M. W.; Ali, M. M.; Filipe, C. D. M. Long-Term Preservation of Bacteriophage Antimicrobials Using Sugar Glasses. ACS Biomater. Sci. Eng. 2017. (42) Pasco, E. V.; Shi, H.; Xagoraraki, I.; Hashsham, S. A.; Parent, K. N.; Bruening, M. L.; Tarabara, V. V. Polyelectrolyte Multilayers as Anti-Adhesive Membrane Coatings for Virus Concentration and Recovery. J. Membr. Sci. 2014, 469, 140-150.


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(43) Larsson, P. A.; Puttaswamaiah, S. G.; Ly, C.; Vanerek, A.; Hall, J. C.; Drolet, F. Filtration, Adsorption and Immunodetection of Virus Using Polyelectrolyte MultilayerModified Paper. Colloids Surf., B 2013, 101, 205-209. (44) Dika, C.; Duval, J. F. L.; Francius, G.; Perrin, A.; Gantzer, C. Isoelectric Point Is an Inadequate Descriptor of Ms2, Phi X 174 and Prd1 Phages Adhesion on Abiotic Surfaces. J. Colloid Interface Sci. 2015, 446, 327-334. (45) Lambert, R. J. W.; Pearson, J. Susceptibility Testing: Accurate and Reproducible Minimum Inhibitory Concentration (Mic) and Non-Inhibitory Concentration (Nic) Values. J. Appl. Bacteriol. 2000, 88, 784-790. (46) Dika, C.; Ly-Chatain, M. H.; Francius, G.; Duval, J. F. L.; Gantzer, C. Non-Dlvo Adhesion of F-Specific Rna Bacteriophages to Abiotic Surfaces: Importance of Surface Roughness, Hydrophobic and Electrostatic Interactions. Colloids Surf., A 2013, 435, 178-187. (47) Sneddon, I. N. The Relation between Load and Penetration in the Axisymmetric Boussinesq Problem for a Punch of Arbitrary Profile. Int. J. Eng. Sci. 1965, 3, 47-57. (48) Gavara, N.; Chadwick, R. S. Determination of the Elastic Moduli of Thin Samples and Adherent Cells Using Conical Afm Tips. Nat. Nanotechnol. 2012, 7, 733-736. (49) Polyakov, P.; Soussen, C.; Duan, J. B.; Duval, J. F. L.; Brie, D.; Francius, G. Automated Force Volume Image Processing for Biological Samples. PLoS One 2011, 6, e18887. (50) Lehaf, A. M.; Hariri, H. H.; Schlenoff, J. B. Homogeneity, Modulus and Viscoelasticity of Polyelectrolyte Multilayers by Nano-Indentation: Refining the Buildup Mechanism. Langmuir 2012, 28, 6348-6355. (51) Schneider, A.; Francius, G.; Obeid, R.; Schwinté, P.; Hemmerlé, J.; Frisch, B.; Schaaf, P.; Voegel, J.-C.; Senger, B.; Picart, C. Polyelectrolyte Multilayers with a Tunable Young's Modulus: Influence of Film Stiffness on Cell Adhesion. Langmuir 2006, 22, 1193-1200. (52) Tang, K.; Besseling, N. A. M. Formation of Polyelectrolyte Multilayers: Ionic Strengths and Growth Regimes. Soft Matter 2016, 12, 1032-1040. (53) Hübsch, E.; Ball, V.; Senger, B.; Decher, G.; Voegel, J.-C.; Schaaf, P. Controlling the Growth Regime of Polyelectrolyte Multilayer Films: Changing from Exponential to Linear Growth by Adjusting the Composition of Polyelectrolyte Mixtures. Langmuir 2004, 20, 19801985. (54) Krapf, M.-E. M.; Lartiges, B. S.; Merlin, C.; Francius, G.; Ghanbaja, J.; Duval, J. F. L. Polyethyleneimine-Mediated Flocculation of Shewanella Oneidensis Mr-1: Impacts of Cell Surface Appendage and Polymer Concentration. Water Res. 2012, 46, 1838-1846. (55) Nazaran, P.; Bosio, V.; Jaeger, W.; Anghel, D. F.; v. Klitzing, R. Lateral Mobility of Polyelectrolyte Chains in Multilayers. J. Phys. Chem. B 2007, 111, 8572-8581. (56) Volodkin, D.; von Klitzing, R.; Moehwald, H. Polyelectrolyte Multilayers: Towards Single Cell Studies. Polymers 2014, 6, 1502.



(57) Langlet, J.; Gaboriaud, F.; Gantzer, C. Effects of Ph on Plaque Forming Unit Counts and Aggregation of Ms2 Bacteriophage. J. Appl. Bacteriol. 2007, 103, 1632-1638. (58) Rowatt, E.; Williams, R. J. P. The Inhibition of Infectivity of Bacteriophage Φx174 by High-Valency Metal-Cations and Cyclic Polyamines. Biochem. J. 1987, 245, 641-647. (59) Bayer, M. E.; DeBlois, R. W. Diffusion Constant and Dimension of Bacteriophage Φx174 as Determined by Self-Beat Laser Light Spectroscopy and Electron Microscopy. J. Virol. 1974, 14, 975-980. (60) Kostyuchenko, V. A.; Chipman, P. R.; Leiman, P. G.; Arisaka, F.; Mesyanzhinov, V. V.; Rossmann, M. G. The Tail Structure of Bacteriophage T4 and Its Mechanism of Contraction. Nat. Struct. Mol. Biol. 2005, 12, 810-813.


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TABLES

Table 1. List of the polyelectrolytes used for PEM construction and details of their respective physicochemical and cell-growth inhibition properties.



Table 2. Sequences of the primers and probes used for T4 and φx174 genome quantification by PCR.

Table 3. Morphological and mechanical properties of the different PEM matrices adopted in this work.

Table 4. DLS size-measurements performed for ϕx174 and T4 phages in buffered solutions containing or not different polyelectrolytes at 0.1 g/L concentration.


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LIST OF FIGURES

Figure 1. Amounts of infective phages as measured in the different polyelectrolyte multilayer (PEM) films tested in this work. Infective φx174 (a) and T4 (b) phages were incubated with PEM films and then counted after 18h absorption (green bar). PEM films were rinsed three times and the last rinsing solution was titrated by plaque bioassay (orange bar). It is stressed that the supernatant was removed from the PEM films after 18h and infective phages therein were titrated (blue bar).



Figure 2. Representative infective (a) and total (b) phage entrapment ratios for the different polyelectrolytes multilayer films. Infective φx174 (red bar) and T4 (orange bar) phages were counted according to plaque bioassay method in both PEM films and inoculum solution. The values reported in the histogram (a) correspond to the ratios between the number of phages detected in the PEM films and the number of infective phages in the inoculum. * No accurate measurements could be obtained due to strong interferences between PEM components and PCR kit-extraction reagents for T4.


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Figure 3. Selectivity of PEM films in terms of infective φx174 phages loading. a) Infective (red) and total (blue) phages were counted in each PEM film according to plaque bioassay and qPCR methods, respectively. b) Infectivity ratio corresponding to the ratio between number of infective phages measured by plaque bioassays and number of DNA copies detected by qPCR.



Figure 4. Impact of cationic and anionic polyelectrolytes on phage infectivity. Is reported the Phage Activation Parameter (PAP) that corresponds to the ratio between amounts of infective phages detected by plaque bioassays in the polyelectrolyte solutions (concentration from 0.1 to 1.0 mg/mL) and the amount of infective phages detected in the absence of polymers. Data pertain to a) φx174 phages and b) T4 phages.


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PEM Reservoir

Polycation

Polyanion

ic n o y i t a ilit c ly ob o p m w ns o L ai ch

Hig h ch po ain lyc s m ati ob onic ilit y

Efficient antibacterial activity (phage stability against aggregation)

Antibacterial activity inhibition (phage-polyelectrolyte hetero-assemblies)

ACS Paragon Plus Environment

On the Infectivity of Bacteriophages in Polyelectrolyte Multilayer Films

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