A versatile and straightforward process to turn plastics into antibacterial materials

We demonstrate that antibacterial activity can be conferred to common plastic materials using amphiphilic cationic methacrylate-based block copolymers, specifically quaternized poly(butyl methacrylate)-b-poly( N,N -dimethyl- aminoethyl methacrylate) (PBMA- b -PDMAEMA) with 64 mol% of DMAEMA and M n close to 20,000 g mol -1 . With 0.5 - 2 wt% of these copolymers simply dispersed in the given matrix by extrusion, the modified materials prove to be active against E. coli , S. aureus , Listeria monocytogenes and enterohemorrhagic E. coli without toxicity against two cell lines, HaCaT and L929 fibroblasts, while keeping the mechanical properties of the materials intact. In addition, the study of the mechanism of action shows that the antibacterial materials target the bacterial membrane which is expected to avoid antibacterial resistance. Our protocol is a cost-effective solution to generate antibacterial materials for several applications, including packaging or medical devices, without modification of the production process.


Introduction
Infection remains the third leading cause of death in developed countries and the second worldwide. Indeed, the continuing decline of effectiveness of existing antibiotic options due to the ability of microbes to develop and disseminate mechanisms of resistance against multiple classes of traditional antimicrobials, raise international healthcare concerns as they are associated with increased morbidity and mortality and also higher hospital costs. 1-3 Antimicrobial surfaces and antimicrobial materials are thus becoming a very important class of products that could contribute to mitigate the problem. Among them, antimicrobial plastics have received considerable attention for numerous applications, including appliances, filters, packaging, nonwoven fabrics and textiles, and sanitary materials. 4 To confer antibacterial functionality to plastics, surface modification based on an array of methodologies have been exploited, such as surface casting, immersion, surface finishing, plasma treatment and deposition. 5,6 Although these methods afford plastic products with effective antibacterial properties, the processing methods are complex and are associated with high costs. Moreover, the antibacterial properties will be weakened if the antibacterial coating is damaged. To obtain antibacterial functions, blending is a simpler method. Many antibacterial compounds can be employed and include either inorganic species (such as copper nanoparticles, silver nanoparticles and silver-doped glass) or organic entities (for example quaternary ammonium salts, N-haloamines, natural biocide such as thymol or carvacrol and classic antibiotics). 4,7,8 However, the sometimes low antimicrobial efficiency and leaching of antimicrobial components along with the poor durability of their antimicrobial properties limit their application. 9 To prepare long-lasting or non-leaching antibacterial composites, polymer-based compounds were used as additives to impart antibacterial properties to pristine materials. [10][11][12][13] Such an approach is usually carried out either with a small library of known antibacterial polymers such as poly(hexamethylene guanidine hydrochloride) (PHMG), chitosan or poly(tert-butylamino methacrylate). 14,15 In these cases, because of the incompatibility of mixing the polymers with each other, a significant amount of additive has to be inserted into the matrix, potentially altering the material properties (mechanical properties, chemical and thermal resistance, etc.) of the plastics. More complex polymers that feature a backbone similar to the matrix and are decorated with antibacterial moieties or antibacterial polymers [18][19][20][21][22][23][24] have been developed to enhance the compatibilization of the additive to the plastics. Such approaches are efficient, yet specific macromolecular antibacterial architectures have to be designed for each matrix. Recently, we developed cationic amphiphilic poly(butyl methacrylate)-b-poly(N,N-dimethyl-aminoethyl methacrylate) (PBMA-b-PDMAEMA) diblock copolymers based on a methacrylate backbone that are potent antibacterial copolymers in solution as well as a promising additives to specific polymer matrices (polystyrene (PS) and poly(methyl methacrylate) (PMMA)). 16,17 N,N-dimethyl-aminoethyl methacrylate (DMAEMA) was chosen as hydrophilic monomer and butyl methacrylate (BMA) as hydrophobic monomer. A structure-activity analysis was subsequently performed, and we selected diblock copolymers with a molar composition of 64 mol% DMAEMA and obtained permanent cationic charges after quaternization of the amine groups by methyl iodide (MeI) to obtain the highest antibacterial activity (i.e., low MIC values) along with the lowest toxicity. In addition, a number-average molar mass (M n as measured by SEC/DMF) of close to 20,000 g mol -1 was targeted. Such large number average molecular weights, M n , compared to those typically employed in antibacterial copolymers, were expected to ensure a good dispersion of the copolymer in common industrial organic polymer matrices, while avoiding any leaching of the active copolymer and thus a loss of activity over time.
Herein, we demonstrate that quaternized PBMA-b-PDMAEMA copolymers can be efficiently used as an additive to confer antibacterial properties to four model industrial organic polymer matrices: Poly(ethylene terephthalate glycol) (PETG), Please do not adjust margins Please do not adjust margins polylactide (PLA) and both high density and low density polyethylene (HDPE and LDPE respectively). Such matrices were chosen since they have a large range of applications, especially for packaging, and are produced on a very large scale. The dispersion of the copolymer is performed by simply coextruding a low amount of the antibacterial copolymer (≤ 2 wt%) within the given matrix without impacting the innocuity of the material and its inherent mechanical properties (Figure 1). Such an approach constitutes a simple, efficient and versatile solution to limit bacterial infections.

Materials
Commercial organic matrices, i.e. PLA (Natureworks), PETG (Skygreen S2008), HDPE (Dow Chemical KT 10000) and LDPE (Total LA 0710). All the reagents and solvent were used without further purification. The synthesis of the B(A10%)Me copolymer is described in ESI following our previous study. Human normal skin cells HaCaT were obtained from Creative Bioarray (Shirley, NY 11967, USA) and mouse fibroblastic lung cells-L929 (C3H/An) were purchased from Sigma.

Elaboration of antibacterial organic materials
The extrusion step was done with a Thermo Scientific HAAKE MiniLab II mini-extruder. The pellets were dried at 90 °C under vacuum for 4h before extrusion. Once dry, 5 g of pellets are manually loaded into the extruder. For mechanical testing, tensile specimens (Type 5A) were prepared by injection moulding using a Thermo Scientific HAAKE Minijet II injector. The polymer melt at the exit of the extruder is collected in the nozzle heated to 200 °C of the injector and then injected with a pressure of 500 bar in a test specimen mold (Tensile Bar ISO527-2-5A) heated at 60 °C. Bioassays are done on circular shaped films of 3 cm diameter and 100 μm thickness. Upon exiting the extruder, the recovered rushes are cooled in the air then cut into small pellets. are finally shaped using a Specac hydraulic press equipped with heating platens and a thin film kit. The pellets are pressed between two aluminium sheets heated at 170 °C and under 2 tons of pressure for 10 s.

Antibacterial tests
The bactericidal activity of matrices was evaluated as previously described.1 Bacteria used in the study were either Gram positive (Staphylococcus aureus, ATCC CRM-6538P) or Gram negative (Escherichia coli, ATCC 8739) and were routinely grown on Lysogeny broth (LB) agar plates. The bactericidal activity of the different matrices was evaluated according to an adapted ISO 22196 procedure. Briefly, pieces of matrices (squares of 4 × 4 mm) were sterilely prepared from extruded films and were placed at the bottom of the wells of sterile polypropylene 96well microplates (Greiner BioOne). Overnight bacterial suspensions (3 ml of LB broth) of E. coli or S. aureus were prepared from single colonies isolated on LB agar plates. Tubes were incubated overnight at 37 °C under agitation (200 rpm). The next day, bacterial suspensions (Optical density OD > 1.0) were diluted 1/100 in 3 ml of fresh culture media and incubated at 37 °C, 200 rpm until bacteria reached log phase growth (OD around 0.6). Bacteria were then diluted in sterile phosphate buffer saline (PBS) to reach a cell density around 10 5 bacteria per ml. 10 µl of these suspensions (corresponding to ∼1000 bacteria) were added onto the surface of matrices. After 60 min incubation at 37 °C, bacteria were collected by adding 90 µl of sterile PBS into the wells containing the matrices and repeated up and down pipetting. Bacteria were then serially diluted (1 in 10 dilution) in sterile PBS before plating 10 µl of each bacterial dilution onto LB agar plates. After overnight incubation at 37 °C, plates were observed, and colonies were counted allowing the determination of the bacterial killing. Experiments were conducted in independent triplicate (n = 3). Similar protocol was used to evaluate the bactericidal activity of the matrices against food-born bacterial pathogens, using Listeria monocytogenes (ATCC 15313) and enterohemorrhagic E. coli EHEC (ATCC K88). Finally, the bactericidal activity of the matrices was evaluated using the brine of commercial mozzarella naturally containing mixed bacterial community. Commercial mozzarella was bought from food supermarket (Mozzarella Galbani, from Carrefour supermarket). The brine was collected sterilely and 10 µl of brine was used to evaluate bactericidal effect of the matrices as explained above.

Extractables and Leachables
To identify and quantify the extractables, we used a dissolution/precipitation process. 6 g of material were dissolved at reflux in 50 mL of hot toluene under stirring. After complete dissolution, the polymer was precipitated by slowly pouring 100 mL of methanol under stirring. Precipitated polymers were removed from the additive solution by filtration on a paper filter. The solution that contains the extractables was evaporated by rotavapor at 60°C under vacuum. To identify and quantify the leachables, 6 g of material were immersed in 15 mL of ethanol during 72h at 37°C in glass vials. The material was then removed and ethanol that contains the leachables for these conditions of storage was evaporated by rotavapor at 60°C under vacuum. Before injection in the HPLC system, the dry residue containing extractables or leachables was dissolved in a mixture of 0.5 mL of tetrahydrofuran (HPLC grade Prolabo) and 0.5 mL of acetonitrile (HPLC grade Prolabo) and the solution was filtered on a 0.2 μm filter. 20 L were injected on a Lichrospher 100 RP 18 (5 microns) using a 100% Acetonitrile phase; the apparatus was a HPLC Ultimate 3000 series (Dionex) equipped with an automatic injector, a gradient pump and an UV detector with a diode array (190-400 nm) Characterization and possible identification of the compounds was performed by using their retention time, their UV spectrum and by comparing them to those of references.

Antibacterial activity
Synthetic antimicrobial copolymers are inspired by antimicrobial peptides, i.e. synthetic amphiphilic cationic copolymers. 25 Since the pioneering work of DeGrado and colleagues, 11 studies covering synthetic antimicrobial copolymers experienced an exponential growth with more than 1,500 publications per year. These constructs have many advantages such as simple manufacturing on a large scale. Another advantage of these synthetic copolymers compared to peptides is that a large library of monomers can be used, as well as diverse architectures (generally random or block copolymers), lengths (M n between 1,000 to 80,000 g.mol -1 ) and compositions (various hydrophilic/hydrophobic balances). [26][27][28][29] This chemical variety allows identifying optimum antimicrobial systems that feature the best compromise between high antimicrobial activity and low toxicity. 25 Several families of polymers have been reported in the literature, including polyacrylate and polymethacrylate derivatives, poly(oxa)norbornene, polymaleimides, polyionenes and polycarbonates. 25,30 Herein we prepare an antibacterial copolymer constituted of a methacrylate backbone 16 due to the exothermic mixing enthalpy of PMMA derivatives with a wide range of commercially relevant plastics (e.g., poly(vinyl chloride) PVC, poly(styrene-co-acrylonitrile) PSAN, poly(vinylidene fluoride) PVDF, epoxy matrices, etc.), allowing for their facile incorporation into different organic matrices. 31 The diblock copolymer was synthesized via NMP technique 32 using acrylonitrile ACN as comonomer 33 to ensure good control of the polymerization reaction (PBMA-b-PDMAEMA, F DMAEMA = 0.64, M n = 18,400 g mol -1 ). ACN was used to lower the average main equilibrium constant, K, between the macroradical and macroalkoxyamine. This equilibrium is shifted towards active species in the case of methacrylate derivatives, impeding to obtain a good control/livingness of the polymerization. The addition of only 10% of some comonomers restored the correct equilibrium, enabling an living and controlled polymerization. 33 DMAEMA was selected as hydrophilic and BMA as hydrophobic monomer. The copolymer was quaternized using methyl iodide prior to use (Figure 1a). 16 To be consistent with our previous work, 16, 17 this copolymer will be denoted B(A10%)Me herein. Specifically, "B", "A" and "Me" refer respectively to block copolymer, acrylonitrile and methyl iodide. To prepare the antibacterial materials, pellets of commercial grades of various polymer matrices (LDPE, HDPE, PETG, PLA) and the antibacterial diblock copolymer B(A10%)Me are blended during the coextrusion using a micro-extruder that allows working on a gramscale ( Figure 1b). The experimental conditions of the extrusions are given in Supporting Information (SI -Table S1) and are very close to the recommendations of the matrix suppliers. The strips that were obtained directly from the micro-extruders were hot pressed to obtain films of 3 cm of diameter and 100 microns thickness (refer to the section I.4 of SI). Also, it was possible to directly employ -after micro-extrusion -a miniinjection moulding machine to prepare samples for mechanical testing (Tensile Bar ISO527-2-5A). We used a ratio of 0, 0.5 and 2wt% B(A10%)Me copolymer in the organic matrices to ensure that the antibacterial agent is only an additive, expecting that the mechanical properties of the matrices will not be altered.
The antibacterial activity of the prepared solid materials was subsequently investigated using S. aureus and E. coli as model Gram-positive and Gram-negative bacteria respectively, following an ISO-22196 adapted procedure. 16 Indeed, the size of the sample is supposed to be 5 × 5 cm in this procedure and thus requiring a high amount of additive and film to be prepared. To screen the various matrices and concentration of the additives more efficiently, the surface that is used in our study is decreased to 4 × 4 mm, enabling the use of 96-well plates. The tests was performed in triplicate and carried out using 1 h of time-exposure ( Figure 2). It has been reported that synthetic antibacterial copolymers are very efficient, displaying biocide action after a few minutes. 34 The results gathered in Figure 2 correspond to the percentage of bacteria that was killed compared to the negative control. In all cases, the pristine matrices do not show any antibacterial activity. The incorporation of 2 wt% of B(A10%)Me proved to be sufficient to confer a high antibacterial activity to commercial PETG, LDPE, HDPE and PLA matrices. The bacterial killing ranged from 88 % (for PETG) to 100 % (for HDPE, LDPE, PLA) on S. aureus and were of 100 % with all matrices for E. coli. At 0.5 wt% of incorporation of B(A10%)Me, bactericidal activities were lower for HDPE, LDPE and PETG matrices (bacterial killing ranging from 85 to 95 % for E. coli and from 74 to 90 % for S. aureus). Importantly, among the various matrices, only the additivated PLA matrix remains fully active with no viable bacteria (either S. aureus and E. coli) observed after one hour of contact with only 0.5 wt% of incorporation of B(A10%)Me copolymer. These tests confirm the antibacterial activity of these films, but more studies are required to investigate their antibiofilm behaviour as well as their effectiveness against aging.

Mechanism of action
The mechanism of action of such antibacterial methacrylatebased copolymers has been studied in the literature and is hypothesized to be mainly a membrane destabilization mechanism leading to the lysis of the bacterial membrane. 35 Thus we were interested in investigating the mechanism of action of the herein presented antibacterial copolymer embedded into a polymer matrix. A propidium iodide assay was used to establish if the bactericidal activity of the matrices is related to membranolytic effects. 36 Indeed, such compounds are known to enter cells and form fluorescent complex with DNA only after membrane damages and are therefore a good probe to monitor membrane integrity. [36][37][38] Incubation of E. coli as model bacteria and propidium iodide onto PLA -that displayed very good antibacterial properties -was monitored by fluorescence (the details of the test are described in ESI). Cetyl trimethylammonium bromide (CTAB) was used as a positive control (added in solution on a non additivated PLA surface), since this compound is known to lyse efficiently all the bacteria present in the medium and would represent the upper limit for the fluorescent value. The experiments performed using the additivated PLA present an increase in the fluorescent value with time, reaching the upper limit within error margin of the positive control (Figure 3a), confirming that the antibacterial activity is based on a permeabilization of the bacteria membrane. The comparison of the increase in fluorescence intensity between the positive control and the additivated PLA show that the process of permeabilization of the copolymer embedded into the matrix is slower than the liquid biocide reference, yet remains rapid (∼90 % permeabilization in less than 1 h) compared to the activity of leachable biocides that generally requires hours to be efficient. The other matrices (HDPE, LDPE and PETG) were also tested and showed similar behaviours within error margin (Figure 3b).

Cytotoxicity Study
Once the antibacterial activity had been demonstrated, we moved to assess the in vitro innocuity of such materials using human normal keratinocytes as model (HaCaT cells). Such kind of organic matrices could be utilized for applications in which the main contact is with skin. The cells were incubated for 48 h on the films and the cell viability was determined using the resazurin assay (The details of the tests are described in ESI). The results are presented in Figure 4a. The cell viability on all pristine polymer organic matrices were close to 100% compared to the positive control. Figure 4. a) Innocuity testing of the matrices using human normal keratinocytes (HaCaT cells) as model. HaCaT cells grown in 96-well plate were exposed to matrices for 48h before measurement of the cell viability using resazurin assay. CTAB (300 µM) was used as positive control of cell toxicity. Results correspond to means +/-S.D (n=3). b) Fibroblast viability referring to the MTT test results after 48h of contact between the cells and the material; the control (+) corresponds to phenol. Results correspond to means +/-S.D (n=6).
When 0.5 wt% of the additive was embedded into the matrices, the cell viability is identical to the pristine matrices, demonstrating that there is no impact on the additive at such low concentrations. When the amount of additive was increased from 0.5 to 2 wt%, the HaCaT cell viability (Figure 4a) is still close to 100%, showing that the additive does not impact the innocuity of all the materials. In order to assess if these results are dependent on the cell line, similar experiments were performed using L929 fibroblasts (Figure 4b) using the LDPE films. In that case, the cell viability is also not dependent on the concentration of the additive, showing that this approach confers an antibacterial activity without altering the cell viability for all the matrices. The main advantage of blending the antibacterial copolymer with the matrix is related to the non-leaching of the active antibacterial additive. The co-extrusion ensures a long-lasting efficiency compared to leaching approaches (either antibiotics/biocides or inorganic salts), avoiding the increase of antibiotic resistance due to the dispersion of antibiotics in the environment. Besides the action mechanism of antibacterial copolymer involving alteration of the bacterial membrane, the eventual leaching of such materials in the environment will not induce bacterial resistance. The non-leaching feature was assessed by using LDPE as model polymer and ethanol as a solvent. First, we determined the extractables, i.e., all the molecules that will be able to diffuse and migrate outside the polymer matrix and which will be extracted using aggressive solvent conditions. To identify and quantify the extractables, a dissolution/precipitation process was conducted using toluene at reflux. After complete dissolution, the matrix was precipitated by slowly pouring 100 mL of methanol under stirring. The filtrate was subsequently analysed by HPLC ( Figure  5a). A peak at 1.5-2 min was observed that is in good agreement with the B(A10%)Me copolymer (refer to Figure S3 in the SI). Unlike extractables that require the dissolution of the matrix, leachables refer to the compounds that are effectively leached inside a media for conditions that are generally milder and near the normal conditions of use. In our case, the films were immersed in 15 mL of ethanol during 72 h at 37 °C in glass vials. The material was subsequently removed and supernatant ethanol that contains the leachables (for these conditions of storage) was analysed (Figure 5c). Analysis of the leachables in ethanol by HPLC show similar chromatograms for the LDPE with and without antibacterial additive (Figure 5c): the main leachable in this case was the BHT, a phenolic antioxidant that was originally present in the pristine matrix. To confirm that the antibacterial copolymer and/or degradation side-products do not leach out and do not impact the biocompatibility of the matrix, the strips was also immersed in a culture medium for 48h at 37°C and this medium was then later used for incubating L929 fibroblasts. The results presented in Figure 5d showed no decrease of cell viability, invariant to the amount of antibacterial copolymer used as additive (Figure 5d). The absence of cytotoxicity of the additivated matrix is of prime importance to envision a biomedical application of the prepared antibacterial materials.

Mechanical properties
With applications ranging from biomaterials to packaging, the mechanical properties of the materials are of paramount importance. The modification of the materials to confer antibacterial activity should ideally not affect their mechanical properties. In our approach, the use of low amounts (0.5 -2 wt%) of antibacterial copolymer as additive is not expected to alter the bulk properties of the materials. For confirmation tensile tests were performed on PETG, PLA and HDPE samples to determine the Young Modulus. The samples of both pristine and additivated matrices (2 wt%) were prepared by injection moulding. We assessed the materials with the higher amount of antibacterial copolymer to determine the maximum modification of the mechanical properties. The comparison of these values (Figure 6a, details in ESI) demonstrates that the incorporation of copolymer in the matrix does not change the mechanical properties of the pristine material.
To confirm the conservation of the mechanical properties, dynamic mechanical analysis (DMA) tests were performed on PETG, PLA and HDPE samples to determine the viscoelastic properties in a range of temperatures.
The comparison of storage (E') and loss moduli (E") curves in function of temperature related in Figure 6b-d demonstrates that materials have the same viscoelastic properties on a large range of temperature. In conclusion, the addition of the copolymer into the polymer matrix does not change the mechanical properties of materials in a large range of temperatures and strain levels.

Surface characterization
According to the antibacterial studies, the use of a macromolecular antibacterial additive that is not leached out in the medium confer good activity by membrane permeabilization even with a small amount of copolymer. The localization of the copolymer in the materials is thus an intriguing question. Due to the blending in the micro-extruder, one would expect a homogeneous dispersion of the amphiphilic copolymer into the polymer matrix, but the partial or complete migration of the copolymer to the surface cannot be excluded. To answer this question, we performed Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) analyses on PETG, LDPE and PLA materials containing 2 wt% of copolymers (see ESI-section VI for details). Using ToF-SIMS, the specific mass response of the copolymer may be detected in the background of the matrix. Even if all the copolymer is migrated towards the surface of the materials, the low amount of additive is still Please do not adjust margins Please do not adjust margins difficult to detect. As shown in Figure S2, there is no increased intensity in the Tof-SIMS spectrum of the organic matrix with the copolymer at m/z values corresponding to peaks in the copolymer spectrum, for any of the three materials. These results showed that it was not possible to detect the antibacterial additive on the surface samples even using the sensitive ToF-SIMS analysis. These indirect results prove that whatever the matrix is, the copolymer does not migrate to the surface and likely stays well dispersed in the material. To obtain further insights, we focused on the matrix that differs the most from the copolymer in term of structure (LDPE with an apolar totally carbon-based backbone) to maximize the chance to detect the copolymer. The surface of the LDPE films was characterized by using Atomic Force Microscopy (AFM) in tapping mode and nanoscale Infrared Spectroscopy (nanoIR), allowing the acquisition of local Fourier Transform Infrared Spectroscopy (FTIR) spectra on the sample with a high lateral resolution (all the techniques are detailed in ESI). The pristine LDPE film has a heterogeneous and rough (Figure 7a) surface as shown by the local FTIR spectra (Figure 7b) and the hydrophobicity mapping (Figure 7i)). The non-homogeneous surface polarity and low contact angles observed for such an apolar polymer (Figure 7i) may be the result of a surface oxidation (C=O band at 1724 cm -1 on figure 7b) and/or of the presence of lubricant rich areas (see amide bands between 1700 and 1590 on Figure 7c. The addition of the antibacterial copolymer results in a slight increase in roughness (Figure 7h). The hydrophobicity is nevertheless not significantly modified and it is not obvious to attribute rich antibacterial copolymer areas on the film surface: oxidation band and amide lubricant bands (green arrow on Figure 7e)) are still observed, yet no other bands significantly appear, even if, for the film containing 2 wt% of copolymer, small bands around 1100 cm -1 (ether stretching band on figure 7g)) are locally detected and may be the result of the presence of the copolymer but cannot be ascribed with certainty to the additive. Figure 7. a) AFM height image (5 × 5 m) of the LDPE film surface without the antibacterial copolymer b) FTIR spectra corresponding to the red points on figure a; c) FTIR spectra corresponding to another area of the film surface without antibacterial copolymer d) AFM height image (10 × 10 m) of the LDPE film surface with 0.5% of antibacterial copolymer e) FTIR spectra corresponding to the red and green points on figure d; f) AFM height image (10 × 10 m ) of the LDPE film surface with 2 wt% of antibacterial copolymer g) FTIR spectra corresponding to the blue, red and green points on figure f; h) Root-mean-square roughness R q for the different LDPE films; i) Water contact angle mapping of the LDPE film surface for LDPE without antibacterial copolymer and containing 0.5 and 2 wt% of the copolymer. A 7 × 20 mm surface was investigated with 300 pL water droplets j) water mean contact angle for each surface (for the 168 droplets deposited onto the surface).

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Please do not adjust margins The combination of all these techniques showed that the surface is not sufficiently modified to detect the copolymer. This is a striking difference with the materials whose antibacterial properties are brought by various surface modification techniques. 39

Antibacterial packaging application
Finally, to demonstrate that our approach could be realistically envisioned as a solution to prevent bacterial infection in a real world scenario, we prepared antibacterial LDPE films containing 2 wt% of copolymer as additive and test them as packaging for mozzarella as already reported. 40 Figure 8. a-b) development of pathogen bacteria (Listeria monocytogenes (a) and enterohemorragic E. coli (b)) in contact to LDPE with 2 wt% of B(A10%)Me copolymer (noted +) compared to the pristine LDPE as reference (noted -) c-d) Survival of cheese bacteria from brine mozzarella after contact with LDPE with 2 wt% of B(A10%)Me copolymer (noted +) (c) compared to the he pristine LDPE as reference (noted -) (d).
The idea was to show whether such a plastic that is common to prepare films used in food packaging applications could be useful to (i) avoid the development of pathogen bacteria such as Listeria monocytogenes and enterohemorrhagic E. coli that are responsible every year of contaminations widely reported in the media 41, 42 and (ii) preserve the bacteria within the cheese that are necessary impart gustative properties. 43 Figure 8a-b clearly proves that both Listeria monocytogenes and enterohemorrhagic E. coli were completely killed after contact with the additive containing film. In the case of bacteria already present in cheese, we observe that a large fraction of them survive (Figure 8c-d) with nevertheless a lower amount than the pristine film. This result could be interesting to increase the shelf-life of the food and allows us to envision the possibility of using B(A10%)Me copolymer containing plastics as food packaging materials.

Conclusions
We demonstrate that simply adding an amphiphilic synthetic cationic diblock copolymer based on methacrylic monomers into common organic matrices (such as HDPE, LDPE, PLA and PETG) via an extrusion process confers antibacterial properties to the pristine materials after 1 h of exposure. The effect of concentration for such additives has been studied and we demonstrate that even 0.5 wt% is sufficient to impart good antibacterial efficiency (> 80 % of bacterial killing) for both E. coli and S. aureus. The increase to 2 wt% led to complete inhibition of both bacteria on HDPE, LDPE and PLA and more than (90 % of bacterial killing) on PETG. The mechanism of action was also investigated and the permeabilization of the bacteria membrane was monitored via the propidium iodide assay, suggesting that the killing of bacteria occurred by contact and membrane destabilization. This action mechanism is expected to limit the development of bacterial resistance. In addition, the study on HaCaT and L929 fibroblast cell lines reveals no toxicity of the materials. Further analysis of the materials demonstrated that the mechanical properties over a wide range of temperatures are not altered. The stable mechanical properties are of prime importance for the utilization of these materials in their specific applications. For a better understanding of the mode of action of such materials, a complete characterization of the surface has been carried out. ToF-SIMS, Nano-IR spectroscopy and AFM did not reveal any specific modification of the surface, suggesting that there is a homogeneous incorporation of the additive in the materials without a complete migration towards the surface. This is a critical advantage compared to antibacterial materials whose properties are given by a surface modification (antibacterial coatings). Lastly, to demonstrate its application in food packaging, a LDPE film containing 2 wt% of additive was shown to inhibit the growth of Listeria monocytogenes and enterohemorrhagic E. coli. At the same time, the additive containing film is slowing down the development of cheese bacteria from brine mozzarella, which could increase the shelflife of the food contained in the LDPE film.