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Designing biodegradable PHA-based 3D scaffolds with antibiofilm properties for wound dressings: optimization of the micro/nanostructure Aracelys Maria Marcano, Naila Bou Haidar, Stéphane Marais, Jean Marc Valleton, and Anthony C Duncan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00552 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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ACS Biomaterials Science & Engineering

Designing biodegradable PHA-based 3D scaffolds with antibiofilm properties for wound dressings: optimization of the micro/nano structure Aracelys Marcano*, Naila Bou Haidar, Stéphane Marais, Jean-Marc Valleton, Anthony C. Duncan.

Normandie Univ, UNIROUEN, INSA Rouen, CNRS, PBS, 76000 Rouen, France

*Corresponding Author Tel.: +33-2-35-14-66-94; Fax: +33-2-35-14-67-04; E-mail: [email protected]

Abstract: One major factor inhibiting natural wound-healing processes is infection through bacterial biofilms, particularly in the case of chronic wounds. In this study, the micro/nanostructure of a wound dressing was optimized in order to obtain a more efficient antibiofilm protein-release profile for biofilm inhibition and/or detachment. A 3D substrate was developed with asymmetric polyhydroxyalkanoate (PHA) membranes to entrap Dispersin B (DB), the antibiofilm protein. The membranes were prepared using wet induced phase separation (WIPS). By modulating the concentration and the molecular weight of the porogen polymer, polyvinylpyrrolidone (PVP), asymmetric membranes with controlled porosity were obtained. PVP was added at 10, 30 and 50% w/w, relative to the total polymer concentration. The physical and kinetic properties of the quaternary non-solvent/solvent/PHA/PVP systems were studied and correlated with the membrane structures obtained. The results show that at high molecular weight (Mw= 360 KDa) and high PVP content (above 30%), pore size decreased and the membrane became extremely brittle with serious loss of physical integrity. This brittle effect was not observed for low molecular weight PVP (Mw= 40 KDa) at comparable contents. Whatever the molecular weight, porogen content up to 30% increased membrane surface porosity and consequently protein uptake. Above 30% porogen content, the pore size and the physical integrity/mechanical robustness both decreased. The PHA membranes were loaded with DB and their antibiofilm activity was evaluated against Staphylococcus epidermidis biofilms. When the bacterial biofilms

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were exposed to the DB-loaded PHA membrane, up to 33 % of the S. epidermidis biofilm formation was inhibited, while 26% of the biofilm already formed was destroyed. These promising results validate our approach based on the development of bioactive-protein-loaded asymmetric membranes for antibiofilm strategies in situations where traditional antibiotic therapies are ineffective.

Keywords: biomaterials, asymmetric PHA membranes, porogen, wet induced phase separation, drug delivery, Dispersin B, biofilm inhibition and/or detachment, chronic-wound dressing.

1. INTRODUCTION Bacterial biofilms have a major impact on public health, on the environment and on industrial activities. They are responsible for the development in living tissues of chronic infections such as mastitis, otitis, pneumonia, urinary tract infections, and osteomyelitis, amongst others. Within a mature biofilm, antibiotic resistance can increase 100- to 1000- fold compared to that for free planktonic bacteria1. Thus, once a biofilm has formed, bacterial eradication becomes either difficult or impossible. There are four possible approaches to prevent, reduce or treat biofilm-based infections: 1) preventing bacterial fixation, 2) disrupting the biofilm to allow the penetration of topical antimicrobial agents, 3) dispersing bacteria into their planktonic form, thus rendering them sensitive once again to conventional antibiotics, 4) interfering in quorum sensing2. Wound healing is a very complex process affected by many factors, such as the host, injury severity, medical care parameters, and so on. Bacterial biofilm infection is a major cause of wound-healing failure. This highlights the need for a suitable wound dressing that can protect wounds from external aggressions, such as bacteria and fungi, while concomitantly eliminating wound exudation3. However, while most conventional wound dressings address one or both of these issues, they are mainly antibiotic-based. This means they aim at killing planktonic bacteria, but do not address the issue of the increased resistance acquired by bacterial biofilms. Our substrate not only meets basic wound-dressing requirements, but also provides an efficient antibiofilm microenvironment over the wound4. Indeed, bacterial biofilms are composed of an extracellular matrix synthesized by the bacteria themselves, and in which they are entrapped7,8. This matrix, constituted predominantly of


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polysaccharides, allows bacteria to adhere in large amounts to a surface, ensures favorable growth environments, and protects bacterial cells from external agents, including antibiotics. Staphylococcus epidermidis is one of the bacteria playing a major role in the development of skin infections9, especially in patients whose immune system is compromised. The S. epidermidis matrix is composed of several extracellular substances, such as proteins, extracellular teichoic acids, extracellular DNA and polysaccharides9. Poly β-1,6-linked Nacetylglucosamine (PNAG), a polysaccharide secreted by S. epidermidis, is responsible for cell-to-cell adhesion9, making bacteria highly resistant to antibiotics and host defense mechanisms. For this reason, it is essential to destroy or weaken (disrupt) the biofilm matrix in order to render the embedded bacteria vulnerable to conventional antibiotics. Dispersin B, produced by the Actinobacillus actinomycetemcomitans pathogen11,12, has shown strong disrupting/destroying abilities on many bacterial biofilm matrices containing PNAG5,12, thus rendering the entrapped bacteria sensitive to antibiotics again5,12,12. Wound dressings loaded with bioactive components (e.g., vitamins, antibiotics, growth factors or minerals) for release at the wound site have been used in the past14. However, these are mainly antibacterial, not antibiofilm, dressings

15,16

. In addition, there is currently a strong interest in

replacing plastic petrochemical derivatives with sustainable materials17. Biodegradable polymers, with raw materials that are renewable and end-of-life controlled, are interesting alternatives to non-biodegradable petroleum-based materials. Moreover, biopolymers are the subject of great research interest for their properties of non-toxicity, resorption by the host body when necessary, and/or biomimicking abilities 17,18,19,19,21. Polyhydroxyalkanoates (PHAs) are biopolymers, produced naturally from microorganisms, that have properties similar to those of many synthetic thermoplastics derived from the petroleum industry15,21. In addition, unlike most of these thermoplastics, PHAs have the advantage of being durable, biodegradable and biocompatible. This is important in a growing context of sustainable development imperatives. To be effective, wound dressings must provide protection against external bacterial contamination and maintain proper environmental conditions for the wound (optimal hydration levels, possible gas exchanges). For instance, the structure of the wound-dressing material should allow water vapor and oxygen transmission rates to be controlled23,24.


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Furthermore, in the case of synthetic systems used for drug-release, it is essential to have a large number of surface sites to anchor the active component. One way to meet all the above criteria is through the use of asymmetric membranes (AMs) with a controlled micro/nanostructure. These AMs consist of a very thin dense layer (the skin) lying on a thicker porous underlayer. Thanks to its simplicity, wet induced phase separation has become the most frequently used technique for the preparation of AMs. When a thin layer of polymer solution is immersed in a non-solvent for the polymer, the polymer is solidified and an AM is formed. Three main elements are involved in this process: the polymer, which will form the membrane, the solvent, which has dissolved the polymer, and the non-solvent, which will precipitate the polymer, but is miscible with the solvent. Phase inversion creates both a polymer-poor and a polymer-rich phase. After liquid-liquid demixing, the polymer-rich phase solidifies and constitutes the AM skin. The polymer-poor phase is removed by successive washing and leaves room for the underlying porous membrane structure. Studies have shown that the structure of the AMs is highly dependent on the thermodynamic and kinetic parameters of the non-solvent/solvent/polymer system25. In preliminary work26 we developed a PHA-based wound-dressing membrane with double micro/nanoporosity capable of destroying S. epidermidis biofilms by continuous, progressive release of Dispersin B, the bioactive antibiofilm protein. However, we were not able to show significant inhibition effects. This was probably due to inadequate kinetic protein-release profiles that were unable to create an inhibiting antibiofilm microenvironment quickly enough. A “cocktail effect”, involving antibiotic/antibiofilm agents, that combines a broad spectrum activity with a high rate of biofilm matrix destruction is an interesting approach to be developed for the treatment of biofilm-infected wounds5,5. The main purpose of this study was to optimize the micro/nanostructure of a wound dressing in order to obtain release profiles that would be more efficient for biofilm inhibition and detachment. To that effect, PVP was used the porogen in order to increase membrane porosity and initial protein uptake. DB, used as the active agent against a PNAG biofilm matrix, was loaded into the AM samples. The antibiofilm activity of the drug-loaded PHA/PVP membranes was evaluated on a model of the S. epidermidis biofilm.


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2. MATERIALS AND METHODS 2.1. Materials The poly(3-hydroxybutyrate-co-4-hydroxybutyrate) called P(3HB-co-4HB), a statistically random copolymer containing 13 % 4HB27, was used as the mechanical support in this study. It was supplied under powder form by Tianjin GreenBioScience Ltd. (China). The weight-averaged molecular weight determined by gel permeation chromatography was estimated at 3.1·105 g.mol1

. The semi-crystalline P(3HB-co-4HB) matrix (crystallinity degree, XC = 32%) is in the rubbery

state at 37°C, since its glassy transient temperature Tg is -6 °C. The polyvinylpyrrolidones (PVP) K30 and K90 (Mw = 40 and 360 KDa, respectively) were obtained from Sigma-Aldrich®. The solvent N-methyl-2-pyrrolidone (NMP) was supplied by Merck & Co. Inc. (Rahway, NJ). The Dispersin B (DB) was provided as a lyophilized powder by Kane Biotech, Inc. (Canada). The 1 mg/ml flame-sealed glass ampoules of bovine serum albumin (BSA) in 0.15 M NaCl with 0.05% sodium azide and the QuantiproTM BCA Assay kit were obtained from Sigma-Aldrich®. Ultrapure water (Milli-Q) was used throughout the study.

2.2. Bacterial strains, media and growth conditions In this study Staphylococcus epidermidis (ATCC 35984 strain) was used as the bacterial strain and was pre-cultured in 25 ml of BHI nutrient medium (Brain Heart Infusion, from BD Biociences) at 37°C under agitation (140 rpm) overnight as described previously26.

2.3. Preparation of asymmetric PHA membranes Asymmetric PHA membranes were prepared using the wet induced phase separation (WIPS) method described previously26,28,29,30. The N-methyl-2-pyrrolidone (NMP) was used as the solvent for P(3HB-co-4HB) and PVP polymers; pure water was used for the coagulation bath (non-solvent for P(3HB-co-4HB)). PHA membranes were prepared at a polymer concentration of 17% w/w. The porogen agent (PVP) was added at various concentrations (10, 30 and 50% w/w), relative to the total polymer concentration. The polymer powder was dissolved in NMP under stirring at 75°C until the total amount was dissolved. After removal of any gas bubbles, the polymer solution was cast onto a glass plate in a 140µm-thick layer. Polymer-solution casting thickness was controlled using a frame with a calibrated thickness placed on the glass substrate. The coating was immediately quenched in an aqueous coagulation bath (water) and maintained


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at a constant room temperature of 21°C for at least 1 hour. After coagulation, the plate was then removed from the bath and rinsed 3 times with pure water in order to remove any residual solvent and porogen agent. The membrane was then carefully peeled off the glass substrate and dried at room temperature for a day. Films of thickness 75-126 µm were finally obtained. The resulting asymmetric structure consisted of a thin dense layer at the non-solvent side and a porous layer at the glass side, and was thereafter functionalized and loaded with the DB protein, as described below.

2.4. Optical analysis of the asymmetric membranes Optical microscopy in reflection mode using a Leica DMLM device was carried out to in order to investigate the effects of both the content and the molecular weight of the porogen on PHA membrane morphology. Moreover, the latter was observed by scanning electron microscopy (SEM) Neoscope JCM 6000. The cross-sections of membranes were prepared by fracturing the samples in liquid nitrogen. The cross-sections and the two surfaces were fixed on a carbon tape and subsequently cold-coated using a JFC 1300 Autofine coater (metallization step).

2.5. Porosity study Two different methods were used in order to measure the PHA membrane porosity. The first, a gravimetric method, allowed the void percentage (free volume at the macroscopic scale) of the polymer membrane to be determined by dividing the total volume of the membrane pores by that of the whole membrane. The samples (diameter Φ = 2.2 cm) were dried, weighed, then placed in pure water for 3 hours at room temperature of 21°C until the wet mass reached its maximum (equilibrium state). It was then assumed that all the membrane pores were completely filled with water. Once the equilibrium was established, the excess surface water was removed with a hydrophobic parchment paper (purchased from Alfapac, France) and the wet membrane was weighed on a sensitive electronic balance. It was assumed that all the membrane pores were completely filled with water. The assay was conducted in triplicate. The second method was based on image analysis of the optical micrographs of the membrane surfaces using Image J 1.51p software. This approach was based on the calculation of pixel area and value level statistics. Pores were identified as dark pixel clusters exceeding a minimum area size. The pore size and density at the PHA membrane surface was thus estimated.


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2.6. Water contact angle measurements The water contact angles of a dense PHA material (presenting no porosity) were measured using the sessile-drop method (Multiskop system, Optrel, Germany) at room temperature (21 ± 2°C). At equilibrium, water contact angles θwater were measured with CAM image software. The tests were carried out on dense PHA membranes obtained by solvent evaporation. This consisted in dissolving 100 mg of PHA powder (purchased from Tianjin GreenBioScience Ltd., China) in 10 ml of chloroform. The mixture was stirred at room temperature until complete dissolution was achieved. After removal of gas bubbles, the polymer solution was transferred onto a glass petri dish (8.5 cm diameter). After solvent evaporation for 24h, at 21 ± 2°C and atmospheric pressure, the ﬁlm was stored in a desiccator over P2O5.

2.7. Mechanical properties Uniaxial tensile strength was measured using an Instron 5543 testing machine with a 500 N sensor at crosshead speeds of 5 mm/min. The prepared dumbbell-shaped membranes were 30 mm long × 4 mm wide × 0.20 mm thick. The tests were performed at room temperature (23 ± 2°C) with relative humidity (29 ± 1 % RH). At least ten specimens were tested for each sample type. Averages and standard deviations are reported.

2.8. Surface treatment and PHA membrane loading The P(3HB-co-4HB) membranes were functionalized via hydrolyzation by leaving the porous surface in contact with a 1 M NaOH solution for 1 hour as described previously26. They were then rinsed several times in ultra-pure water (Milli-Q) to remove excess NaOH and dried. The membranes were subsequently immersed in phosphate-buffered saline (PBS 15 mM, pH 7.4) for at least 24 h, in order to reach swelling equilibrium. Discs of 1cm in diameter were then cut out and immersed in 1 ml of the protein solution for 24 h at 37 °C.

Protein adsorption was carried out with BSA and DB proteins. Due to the high cost of DB, BSA which has a similar molecular weight and isoelectric point, was used initially as the model protein. BSA solution was prepared by simple dilution of 1 mg / ml with PBS buffer 15 mM (pH 7.4) down to a protein concentration of 40µg / ml. For the antibiofilm evaluation, DB solutions at 20 and 50 µg / ml were prepared. DB was first dissolved in PBS (50 mM, pH 5.8) under stirring


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at a concentration of 1 mg/ml, then diluted down to concentrations of 20 and 50 µg / ml in PBS (0.15 M, pH 7.4). The pH of the buffer solutions was adjusted using a pH-meter (The Metrohm 632 pH meter, electrode XC 161, 12019-F10, calibration of pH 4 and 7) and solutions of 0.1 M HCl and 1 M NaOH. The amount of protein adsorbed onto the membrane was evaluated using a colorimetric BCA assay (QuantiPro ™ BCA Assay Kit, SIGMA-ALDRICH). Protein concentration was determined by measuring optical density at 562 nm using a UV-Visible spectrophotometer (Varian Cary 100 Bio model) and comparing it to the curve for the standard absorbance versus known protein concentration. Bulk protein solution concentration was measured before the adsorption step (Si) and after this step (Sf). The difference (Sf - Si) yielded the amount of protein adsorbed onto and absorbed into the polymer membrane. This is reported as an amount of adsorbed protein per square centimeter of membrane surface.

2.9. Release rate of BSA from PHA membranes BSA-loaded 1 cm membrane discs (adsorption conditions: BSA 40 µg/ml for 24 h at 37 °C) were immersed in 1 ml of the wash solution (PBS buffer, 15 mM, pH 7.4) at 37°C under stirring at a constant speed (35 rpm), and monitored over 48 h. The amount of protein released during the release period was determined via the BCA assay as described previously.

2.10. Evaluation of the antibiofilm properties of the modified PHA membrane Experiments were performed aseptically on a 24-well polystyrene microtiter plate, each well being inoculated with the same bacterial solution diluted to a concentration of 1 × 107 CFU/ml in BHI medium. The amount of biofilm biomass in preventive (inhibition tests) and curative (detachment tests) modes, was quantified as described previously26,30. Inhibition studies: aliquots of 1 ml of the bacterial solution were transferred to the wells, then 1.5 cm diameter membrane discs, with and without DB (+DB and -DB), were placed carefully, porous side down, on the floor of the wells. The plates were then incubated for 24 h at 37 °C. After incubation, the PHA surfaces and the culture medium were removed and the wells were washed once in pure water and then stained for 20 minutes with 1 ml of 1% w/v crystal violet aqueous solution, and rinsed again three times in pure water. The amount of biomass was


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quantified by redissolving the crystal violet (adsorbed onto the bacterial biofilm) with 30% v/v acetic acid for 20 min. The supernatant was withdrawn and its absorbance was measured at 595 nm. Detachment studies: the biofilms were first grown alone, without the membrane, for 24 hours at 37 °C in microtiter plates similar to those described previously. After incubation, the bacterial supernatant solution was removed and replaced with 1 ml of BHI fresh solution. Finally, membranes with and without DB (+DB and -DB) were added to their respective wells. After 24h at 37 °C, the amount of biomass was quantified as described above.

2.11. Evaluation of the antibiofilm activity of Dispersin B Biofilms were assayed by crystal violet staining, as described above, in the absence of membrane. Control wells were treated with either the medium (BHI) or the appropriate buffer (PBS). For inhibition studies, DB with two different concentrations of 20 µg/ml and 50 µg/ml was added before the biofilm formed, and the plate was incubated for 24h. For detachment studies, DB was added on a biofilm formed over 24h. The amount of biofilm biomass was quantified by measuring the optical densities (OD) of the wells using a Victor3 Perkin Elmer microplate reader set to 595 nm. The assay was conducted at least twice, each time in four wells.

3. RESULTS AND DISCUSSION 3.1. Asymmetric membrane preparation by phase inversion: effects of the concentration and the molecular weight of the porogen agent The effects of the concentration and the molecular weight of the porogen agent are shown in Figure 1. Micro-structure and membrane morphology were dependent on the concentration and on the molecular weight of the porogen agent. An increase in the pore number and porosity was observed with increasing porogen concentration. It has been demonstrated that macroporous structures form more easily when the polymer concentration is low. Indeed, the diffusion of solvent and non-solvent is facilitated when the viscosity of the polymer solution decreases28. Here, faster demixing took place, which accelerates the growth of the porous structures, generating larger pores. However, a decrease of the pore size was observed in the presence of PVP (K30 as well as K90) above 30% w/w concentration. With a polysulfone membrane, Dal-


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Cin et al.32 also showed that the average pore size may depend on the PVP concentration; increasing the PVP concentration resulted in a decrease in pore size. In our system, the addition of PVP up to 30% in the PHA-NMP solution decreased viscosity, resulting in higher exchange rates between the solvent and the non-solvent. The demixing process30 was thus accelerated and this led to the formation of a macroporous structure with greater pore size. Above 30% of PVP the trend was reversed. This may be attributed to high PVP content rendering the PVP-PHA polymer demixing process more difficult, thus considerably slowing down the overall phase separation process. It is this delayed phase separation process that could lead to smaller pore size. With respect to the effect of the molecular weight, one would expect a molecular weight increase to result in higher pore size. This was not the case (Figure 1). While Ratieuville33 showed that the increase in PVP molecular weight (from 10 kDa 360 kDa) led to a higher average pore size in a polyimide membrane prepared via the VIPS approach (vapor induced phase separation), Boom et al.34 showed, as we also found in this study, that the addition of a PVP above a certain molecular weight resulted in the reduction of the pore size. The reduction in size in the work of Boom et al. was such that they observed only micropores, whereas in our work macropores still existed. They showed that low molecular weight PVP (MW= 40 KDa) added to a polyethersulfone polymer (Victrex5200P) could readily diffuse from the solvent-poor to the solvent-rich phase, thus producing a macroporous structure. In contrast, in the case of high molecular weight PVP (MW= 160 and 360 KDa), both polymers, polyethersulfone and PVP, behaved as a single “body” thus leading to a completely different morphology. The occurrence of the solvent-rich phase may have hindered phase separation by limiting the access of the solvent to the non-solvent, confining the porogen macromolecular chains, thus leading to a more “porous” structure. However, longer PVP macromolecular chains (higher PVP molecular weight), also lower both mobility and the viscosity of the medium that further delay the phase separation process, probably explaining the change in morphology evidenced by the presence of smaller pores. In the present study, it is worth noting that when the porogen agent was added, the dense layer became porous, but without attaining the same porosity level as the porous side. It is clear that the formation of the porous structure inside an AM is a complex phenomenon dependent on various formulation parameters (e.g. temperature, pressure, polymer structure,


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concentration and molecular weight of the porogen, solvent/non-solvent ratio). A key parameter is the time taken for the porogen to migrate, which will in turn be linked to the viscosity of the medium, as well as to the affinity of the porogen with polymer and solvent.

Figure 1. Optical images of asymmetric membranes prepared with different concentrations of PVP. K30: Mw = 40KDa and K90: Mw= 360 KDa. Preparation conditions: non-solvent is water; casting solution thickness of 140 µm; coagulation bath at room temperature (21 °C).

The treatment of the optical images by Image J software can also enlighten us on the percentage of free space within the films. It is an indirect and incomplete approach, since it yields 2D information only from the visualized surface. Despite this limitation, the trends observed are consistent with previous observations by Dal-Cin et al.32 and Boom et al.34. In the case of the K90 membrane, increasing the PVP concentration above 10% resulted in decreased pore size (Figure 2-b). This has been attributed to the molecular entanglement brought about by the high molecular weight of the PVP K90 (MW = 360 KDa), which caused the phase separation to slow down, hence leading to smaller pores.


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In the case of K30 this behavior is less marked. The surface pore number, pore size and pore density increased with increasing porogen concentration up to 30% PVP (Figure 2-a and 2-b). However, a decrease in the mechanical resistance of the PHA membranes was observed when the number of pores increased. In certain cases, the membranes became brittle. This phenomenon was more pronounced for PVP K90-based membranes at high PVP concentrations, where a loss in mechanical properties was observed. This loss may be attributed to a stress-cracking process of the membrane35.

Figure 2. a) Surface porosity and pore density and b) Surface pore diameter of PHA asymmetric membranes prepared without porogen (orange), with 10, 30 and 50% PVP for low (K30) and high (K90) molecular weight (orange = 100% PHA). Results are shown as mean and standard deviation (n = 10).

According to the porosity study, the results obtained via the weighing method appeared to be much higher than those obtained via the Image J software. This is probably because the gravimetric 3-D approach takes into account the entire thickness of the membrane, in contrast to the 2-D Image J software technique. Nevertheless, both approaches indicated the same trend: increasing pore density with increased PVP porogen concentration up to 30% PVP (Table 1). No additional effect was observed above 30% PVP. Levine et al.36 used a similar gravimetric method in order to determine the cross-linked fraction of PHA scaffolds by extracting the non-crosslinked fraction (using chloroform as


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solvent) and measuring the mass of the material before and after extraction. Our goal was different. Our objective was to measure the free volume (void space) within our porous material, using a liquid that is a non-solvent for the polymer (i.e. water) and measuring the wet and dry mass difference. It is worth noting here that no significant difference in the dry mass of the membrane was observed before and after the hydration step, suggesting little or no leaching phenomena.

Table 1. Void ratio in the PHA membranes prepared with different K30 concentrations. Results are shown as mean and standard deviation (n = 10). PVP (%) 0 10 30 50

Weighing method (%) 53.2 69.4 92.4 90.7

Image J software (%) 6.4 ± 3.1 8.2 ± 0.5 29.1 ± 4.4 23.2 ± 3.7

Protein adsorption is a complex phenomenon involving various initial interactions - ionic, electrostatic, hydrogen, hydrophobic, and van der Waals interactions - between the protein and the surface of the material37. NaOH treatment (in relatively mild conditions) was designed to promote the emergence of carboxylic-charged groups by partial hydrolysis of some of the ester groups in the PHA backbone. The objective was to increase protein sorption capacity and to favor reversible protein adsorption by promoting predominantly electrostatic interactions between the proteins and the PHA surface (for instance between negatively-charged surface carboxylic groups and positivelycharged lysine moieties on the protein) and by increasing the hydrophilic character of the PHA surface. Indeed, water contact angle (θwater) measurements showed an increase in the hydrophilic character of the dense membrane surface after hydrolysis (Table 2). BSA uptake assays (performed in similar conditions as described above) on porous asymmetric membranes showed that the NaOH treatment allowed for a significant increase in the protein sorption capacity, confirming the benefit of the surface treatment (Table 2).


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Table 2. Effect of the NaOH surface treatment of PHA membranes on water contact angles and BSA sorption capacity. Untreated membrane Hydrolyzed membrane θwater (°) BSA ads (µg/cm2)

88 ± 2 2.2 ± 0.5

75 ± 3 5.1 ± 0.1

Protein uptake profiles are presented in Figure 3. At low PVP concentrations, a similar trend was observed for both K30 and K90 membranes. Increasing PVP porogen content up to 30 % resulted in higher pore density and protein sorption levels. Hence, at low PVP concentrations, protein uptake correlated with increased pore density. This was probably due to a greater internal specific surface area. The internal micro/nanostructure of the membrane is a key parameter in the interpretation of the adsorption results. Indeed, for identical surface chemistry, higher pore density will probably result in higher specific surface area, leading to more surface adsorption sites available to the proteins. However, at higher PVP contents, a different trend was observed. For K30 membranes, those at 50% PVP exhibited higher pore density, lower pore size, and lower BSA uptake than those at 30% PVP (Figures 2 and 3). For 50% PVP, the K90 membranes showed similar pore density to K30 membranes, but lower pore size and lower BSA protein uptake. At 50% PVP content, for both K30 and K90 membranes, the lower sorption levels observed may be due, to a large extent, to the protein-repellent effect of residual PVP. Indeed, protein adsorption inhibition by PVP is a well-known effect39. Thus, at high porogen content, residual PVP may significantly inhibit protein sorption. The partial loss of the physical integrity of the 50 % PVP membranes combined with the drastic loss of their mechanical properties may contribute to explaining the lower sorption levels, due to fewer adsorption sites.

In addition to its optimal specific surface (Figures 3, Table 1), the 30% PVP K30 membrane exhibited the best mechanical resistance (even after hydrolysis).


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Figure 3. Protein uptake in the loaded membranes as a function of PVP porogen content (orange = 100% PHA). Results are shown as mean and standard deviation (n = 3).

SEM micrographs of the membranes, shown in Figure 4, for various concentrations of PVP (0 up to 50 %) are in agreement with the optical data (Figure 1). Addition of PVP increased the pore size and density, particularly on the porous side of the PVP K30 membrane, where pinholes of 20-30µm can be clearly observed (Figure 4b). Thus, our data suggest the following general trend: higher PVP concentrations (up to 30 % PVP) resulted in higher pore density, thus leading to higher sorption rates, thanks to a greater specific surface.


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Figure 4. SEM micrographs of the (a) dense side (b) porous side and (c) the cross-section of the asymmetric membranes. Preparation conditions: 0, 10, 30 and 50 % w/w PVP for K30 and K90; the thickness of the casting solution was 140 µm for a coagulation bath at room temperature (23°C).

Tensile tests were carried out on the PHA membranes prepared with 0 and 30% PVP. Young’s elastic modulus of the membranes decreased with added PVP. A similar trend was observed for stress and strain at break (Table 3). The decrease may result from the fact that the presence of the porogen agent increased membrane porosity and heterogeneity. It is not surprising that a porous material is more fragile than when it is in its dense state. In addition pore formation may result in easier micro-crack propagation.


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Table 3. Properties of PHA membranes with and without porogen agent. Results are shown as mean and standard deviation (n= 10). PVP (%)

Parameter

0 5.4 ± 0.8 95.0 ± 12.5 12.0 ± 1.5

Young’s modulus (MPa) Stress break (KPa) Deformation at break (%)

30 1.5 ± 0.3 67.5 ± 14.5 5.0 ± 1.5

BSA-release profiles are presented in Figure 5. An initial fast release was observed, since approximately 50% of the initially-loaded BSA was released in barely 3 hours. A plateau was reached within 24h for all membranes. It is thus safe to consider that most of the loaded protein was released within two days. This double-release profile may be advantageous in our chronicwound-dressing context, since it will allow for the rapid creation of an antibiofilm environment that will be sustained over longer periods.

The 30 % PVP K30 membrane was chosen as the best antibiofilm membrane due to its optimal mechanical and protein-loading/releasing properties. The bioactivity results for this membrane are presented below.


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Figure 5. Protein release from the loaded membranes as a function of PVP porogen content. Results are shown as mean and standard deviation (n = 3).

3.2. Antibiofilm activity results Antibiofilm activity results for the 30% PVP K30 membrane are shown in Figure 6. BSA was used as a negative control protein (no antibiofilm activity was observed). For the inhibition tests, Figure 6 shows nearly 20% inhibition for the S. epidermidis biofilm with growth medium supplemented with DB, compared to no inhibition for the unsupplemented medium. For the detachment tests, a significant 60% of the S. epidermidis already formed (ATCC 35984) was detached by the growth medium supplemented with DB, compared to no detachment with the unsupplemented medium. These results are consistent with those of previous studies,


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demonstrating that DB was able to either inhibit or detach biofilms produced by various strains of S. epidermidis5,40. S. epidermis biofilm formation was significantly inhibited in the presence the DB-loadedPHA membrane. Compared to the unloaded membrane, increases of 24% and 33% inhibition were observed for the membrane loaded with the 20µg/ml and 50µg/ml DB solutions, respectively. Thus, increasing the concentration of the DB solution increased the final inhibition rate of the membrane. Violet crystal tests suggested that the DB-loaded membranes were more effective for biofilm inhibition, with a reduced effect on detachment for lower DB concentrations. Increasing the latter up to 50µg/ml DB showed an effect on both inhibition and detachment (since 26% biofilm detachment was achieved).

Figure 6. Inhibition and detachment of S. epidermidis biofilms by free solution DB and DBloaded membranes. Results are shown as mean and standard deviation (n = 8).


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Conclusion In the present study, using a wet induced phase separation (WIPS) approach, we were able to develop asymmetric membranes with controlled porosity by modulating both the concentration and the molecular weight of a PVP porogen added to the PHA/solvent solution. We have shown that porogen content increased membrane porosity and consequently protein uptake and release. However, a compromise had to be made between the loss of physical integrity/mechanical robustness and optimal release rates of the membrane. In that respect, the 30% PVP K30 PHA membrane exhibited optimal combined properties. This membrane, loaded with the biofilm-destructuring protein (DB), showed significant antibiofilm activity in both inhibition and detachment studies. These promising results validate our approach of using asymmetric membranes in antibiofilm strategies in situations where traditional antibiotic therapies are ineffective. We anticipate that this micro/nanostructured polymer, loaded with several antibiofilm proteins aimed at different targets of the biofilm matrix and combined with one or several conventional antibiotics – the cocktail effect – will be effective where conventional antibiotherapies fail. The present work provides the basis for reaching such a goal in the near future.

Acknowledgements: We thank Kane Biotech Inc. for generously supplying us with the Dispersin B protein. The authors also thank the GRR CRUNCh SESA (Normandy Region) and the European Regional Development Fund (ERDF) for funding this research (ORGA-PATHOP project).

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For Table of Contents Use Only


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Figure 1. Optical images of asymmetric membranes prepared with different concentrations of PVP. K30: Mw = 40KDa and K90: Mw= 360 KDa. Preparation conditions: non-solvent is water; casting solution thickness of 140 µm; coagulation bath at room temperature (21 °C). 155x101mm (150 x 150 DPI)


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Figure 2. a) Surface porosity and pore density and b) Surface pore diameter of PHA asymmetric membranes prepared without porogen (orange), with 10, 30 and 50% PVP for low (K30) and high (K90) molecular weight (orange = 100% PHA). Results are shown as mean and standard deviation (n = 10). 253x96mm (150 x 150 DPI)



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Figure 3. Protein uptake in the loaded membranes as a function of PVP porogen content (orange = 100% PHA). Results are shown as mean and standard deviation (n = 3). 127x84mm (150 x 150 DPI)


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Figure 4. SEM micrographs of the (a) dense side (b) porous side and (c) the cross-section of the asymmetric membranes. Preparation conditions: 0, 10, 30 and 50 % w/w PVP for K30 and K90; the thickness of the casting solution was 140 µm for a coagulation bath at room temperature (23°C). 164x131mm (150 x 150 DPI)



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Figure 5. Protein release from the loaded membranes as a function of PVP porogen content. Results are shown as mean and standard deviation (n = 3). 100x145mm (150 x 150 DPI)


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Figure 6. Inhibition and detachment of S. epidermidis biofilms by free solution DB and DB-loaded membranes. Results are shown as mean and standard deviation (n = 8). 130x92mm (150 x 150 DPI)


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