Zwitterionic Polyhydroxybutyrate Electrospun Fibrous Membranes with

Feb 8, 2017 - We present a method for surface modification by thermal-evaporation self-assembling of poly(3-hydroxybutyrate) (PHB) fibrous membranes w...
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Zwitterionic Polyhydroxybutyrate Electrospun Fibrous Membranes with a Compromise of Bio-inert Control and Tissue-cell Growth Antoine Venault, Andre Subarja, and Yung Chang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04683 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Zwitterionic Polyhydroxybutyrate Electrospun Fibrous Membranes with a Compromise of Bio-inert Control and Tissue-cell Growth Antoine Venaulta*, Andre Subarja a, and Yung Changa* a

R&D Center for Membrane Technology and Department of Chemical Engineering, Chung

Yuan Christian University, Jhong-Li, Taoyuan 320, Taiwan. *

Corresponding

authors:

Antoine

Venault,

[email protected];

Yung

Chang,

[email protected]

ABSTRACT We present the surface modification by thermal-evaporation self-assembling of poly(3hydroxybutyrate) (PHB) fibrous membranes with a copolymer of hydrophobic octadecyl acrylate repeat units and hydrophilic zwitterionic 4-vinylpyridine blocks, zP(4VP-r-ODA), in view of controlling the biofoulants-fiber interactions. PHB is of interest as a material for bio-scaffolding, but suffers from its hydrophobicity, leading to unwanted interactions with proteins, blood cells or bacteria. The surface modification of electrospun PHB fibers addresses this issue, as the hydrophilicity of the membranes is improved, leading to important reduction of BSA (92%), lysozyme (73%) and fibrinogen (50%) adsorption. From a coating density of 0.78 mg/cm2, no bacteria interacted with the fibers, and from 1.13 mg/cm2, excellent hemocompatibility of

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membranes were measured from thrombocytes, erythrocytes, leukocytes and whole blood attachment tests. Additionally, HT-1080 fibroblasts were observed to develop in contact with the fibers after 3-7 days of incubation (cell density up to 329 ± 16 cells/mm2) suggesting that zP(4VP-r-ODA) provides an adequate humid environment to their growth. Providing an effective control of the surface chemistry and of the coating density, the association of PHB and zP(4VP-r-ODA) can promote the growth of fibroblasts, still maintaining resistance to unwanted biofoulants, and appears to be a promising composite material for tissue engineering.

KEYWORDS Polyhydroxybutyrate fibers; zwitterionic coating; bio-inert control; biofouling.

INTRODUCTION Tissue engineering is a multidisciplinary field that aims at developing scaffolds mimicking the bio-environment in order to promote cell ingrowth and tissue development, and eventually restore the functions of the injured tissue. Ideally, the scaffold should not only imitate the threedimensional organization of the extracellular matrix permitting the cells to spatially proliferate, but also have ideal physicochemical interfaces to enhance and guide the cell-cell interactions leading to the cell development favoring the birth of an healthy engineered tissue.1, 2 A brief survey of literature on recent advances in tissue engineering highlights that a wide variety of tissues can be repaired, or at least are the object of dedicated tissue studies, including soft and hard tissues such as liver,3,4 neurons,5,6 skin,7,8 vasculature9,10 or bone.11,12 Polymers, either selected for their biocompatibility, their biodegradability, their ability to promote hydration of the biomimetic extracellular matrix, their sturdiness,

or for their presence of an ideal

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combination of several of these major properties, are extensively used as scaffold for soft tissue engineering.13-16 Among them, poly(3-hydroxybutyric acid) (PHB), a thermoplastic biopolyester has been widely studied as a scaffolding polymer because it possesses a number of key properties favoring tissue ingrowth: it is stiff, biocompatible, degrades slowly in the body under the action of lipase and the biodegradation process does not leave behind harmful products.17-20 The one drawback of PHB is its hydrophobicity, as hydrophobic polymers exhibit a strong tendency to biofouling by proteins or cells. Biofouling on blood-contacted scaffolds is a serious issue: the hydrophobic-hydrophobic interactions established between the polymeric scaffold and the plasma proteins will irreversibly lead to a series of events resulting in the formation of blood clot.21,22 To prevent this, current research is oriented toward the hydrophilization of hydrophobic interfaces, which can be achieved by numerous surface/bulk modification processes.23-26 Indeed, one common criterion of nonfouling interfaces is their ability to retain water in their structure.27 The formation of a protective hydration layer permits to mitigate biofouling by preventing the proteins to approach the adsorption sites. Cells are also efficiently repelled because the proteins of their outer cell wall are less likely to establish interactions with the hydrophilic scaffold. There are two main classes of materials that enable to mitigate biofouling and that are used as surface/bulk modifiers of hydrophobic polymeric materials: poly(ethylene glycol) (PEG)derivatives28-30 and zwitterionic-derivatives.31-33 It is too early at this stage to definitely state on the most efficient materials, but PEG-derivatives may suffer from self-oxidation, which limits their long-term use.34 The zwitterionic or pseudo-zwitterionic moieties are somewhat more stable, and, provided a good control of surface charge distribution, lead to extremely efficient fouling resistance. They can be grown onto hydrophobic material by various surface modification methods, or more simply, coated onto the material. Coating, whether it is a dip-

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coating or a thermal evaporation coating should ideally lead to the homogeneous self-assembling of the zwitterionic polymers, which can likely be achieved if it is amphiphilic: the hydrophobic blocks act as anchoring sequences interacting with the hydrophobic surface to be modified, while the hydrophilic zwitterionic moieties fulfill their nonfouling functions. Recently, we presented a random copolymer made of poly(octadecyl acrylate) blocks and zwitterionic poly(4vinylpyridine) hydrophilic groups, named zP(4VP-r-ODA).35 There are many examples of carboxybetaine-based zwitterionic polymers allowing to reduce biofouling.31, 36, 37 Compared to them, z(P4VP-r-ODA) should be even more stable, through the introduction of the pyridine groups, and thus enhance the stability of the hydration layer. The association of the two polyesters, the long aliphatic polyester formed by the ODA group on one hand, and PHB on the other hand, should be readily achieved via a self-assembling method, given the similar nature of the chemical functional groups. zP(4VP-r-ODA) is likely to address the biofouling concern of PHB. Nevertheless, and despite our above-mentioned statement on the similarity of some functional groups forming the zwitterionic polymer and the biopolyester, the stability of the interactions have yet to be proven. Secondly, fouling mitigation of zwitterionic PHB fibers by proteins, bacteria or blood cells need to be supported by experimental analysis. Thirdly, the efficiency of the zwitterionic material to promote the cell growth by providing an adequate hydrated environment between the fibers has also to be demonstrated. It is essential to mention at this stage that human tissue cell interactions (e.g.: fibroblasts, osteoblasts,…) with the fibrous matrix is highly desirable while biofouling by blood plasma proteins, blood cells or bacteria has to be minimized as much as possible. Therefore, there is a compromise that needs to be found, associated to two factors: one chemical

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factor, related to the nature of the random copolymer and its ability to form a stable hydration layer, and the second linked to the surface porosity after self-assembling. The purpose of this work is to evaluate the feasibility of the combination of PHB material with zP(4VP-r-ODA) in the prevention of biofouling by proteins or blood cells without the inhibition of tissue cells growth. In other words, we want to know if zP(4VP-r-ODA)-coated PHB fibers can be used as a potential material for tissue engineering scaffold. In order to answer this core question, we have prepared a series of zP(4VP-r-ODA)-coated PHB fibers by thermalevaporation self-assembling method. After fully characterizing the physicochemical properties of the as-prepared scaffold, we moved onto the assessment of their low-biofouling properties using various static tests (protein adsorption, bacterial attachment, blood cells adhesion). At the end of this study, we lay the focus on the growth of HT-1080 fibroblasts in between the fibers, and discuss the cells development in the matrices presenting different coating densities. The important variables that should influence the tissue ingrowth in this last test are the coating density and the incubation time. We intend to shed light on the best combination, if any, that would potentially lead to an appropriate resistance to biofoulants such as bacteria or blood cells, still enabling the development of fibroblasts cells after an adequate incubation time.

EXPERIMENTAL METHODS

Materials. Poly(3-hydroxybutyrate) (PHB) powder was purchased from Sigma-Aldrich Co. Its chemical structure is available elsewhere and reminded in Figure S1.17, 18 Chloroform, purchased from Seechem Co. was used as a solvent for PHB without further purfication. N,NDimethlyformamide (DMF) 99.8% was bought from Tedia Company Inc. and used as a co-

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solvent as is without further purification. Phosphate buffered saline (PBS) powder used for the different analysis was bought from Sigma Chemical Co., and dissolved in deionized (DI) water. DI water used in the different analyses had a resistivity of 18 MΩ.cm and was obtained with a Millipore system. Bovine serum albumin (BSA, fraction V), lysozyme from chicken egg (LY) and human fibrinogen (FN) used in the protein adsorption tests were obtained from Sigma Chemical Co. Dulbecco’s Modified Eagle Medium used for HT1080 cells culture was bought from GIBCO BRL (Gaithersburg, MD, USA). Spinning of PHB Fibers. 5wt% of PHB powder was blended with chloroform (solvent) and DMF (co-solvent), and the solvent/co-solvent weight ratio was 90/10. The mixture was stirred at 320 rpm for 8 hours at 53°C. Once homogeneous, the solution was placed in a 10-mL-Terumo Japan glass syringe connected to a 22-gauge needle, positioned above a rotating collector drum (diameter: 10 cm, length: 50 cm). The distance between the needle and the collector working distance) was 20 cm. The voltage was set to 18.3 kV and the flow rate to 1 mL/hour. At the end of the electrospinning process (8 hours, 8 mL of spinning solution consumed), the fibrous sheet was placed in a vacuum dryer for 24 hours. Then, 13-mm-diameter samples were cut from the fibrous sheet, positioned in well-plates, and stored in 75% ethanol at room temperature. Eventually, samples were dried at 50°C before use. Zwitterionic Thermal-Evaporation Self-assembling of PHB Fibers. The synthesis and the structure of the zP(4VP-r-ODA) copolymer has been reported elsewhere.35 The 1H NMR spectra along with their description are reminded in Figure S1. This copolymer is insoluble in both water and PBS. Once prepared, it was used to modify the membranes by thermal-evaporation selfassembling. First, the zP(4VP-r-ODA) copolymer was dissolved in methanol at room temperature. The concentrations of the copolymer in the different coating baths were 50 mg/mL,

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100 mg/mL, 150 mg/mL and 250 mg/mL, corresponding to theoretical coating densities of 0.5 mg/cm2, 1 mg/cm2, 1.5 mg/cm2 and 2.5 mg/cm2, respectively. The required concentrations in the coating bath corresponding to the targeted theoretical coating densities were established after determining by trial and error the volume of solution needed to fully recover the surface of the PHB membrane samples. The value obtained (13.3 µL) was then multiplied by 2, because both surfaces would be coated. The amount of copolymer corresponding to the amount necessary for obtaining the targeted theoretical coating density was dissolved in the volume previously determined (for example, for a targeted coating density of 50 mg/cm2 on a 1.3-cm-diameter sample, 2 x 0.665 mg of copolymer had to be dissolved in 2 x 13.3 µL of methanol). The actual coating density will be discussed in the results and discussion section. Notice that it took about10 min to fully and homogenously dissolve the copolymer in its solvent, at room temperature. The coating solution were then either used directly to modify the membranes or stored at 4°C until used. For thermal-evaporation self-assembling, membrane disks (1.33 cm2) were placed in a 24well plate. Then, a 13.3-µL-volume was gently dropped at the center of the PHB fibrous membrane surface. The solvent quickly evaporated at room temperature (about 25°C). After 3 min, the fibers were turned up-side down in order to modify their second surface and a similar procedure was followed. Eventually, the self-assembled membranes were rinsed with DI water to remove the loosely attached zwitterionic polymer, and stored at 4°C until use. Physico-chemical Characterization of Zwitterionic PHB Fibers. The surface chemistry of self-assembled virgin PHB and zwitterionic PHB fibers was investigated by FT-IR and XPS. Regarding the first method, a Perkin-Elmer Spectrum One instrument was used in ATR mode, with zinc selenide (ZnSe) as the internal reflection element. Each spectrum reported in this study was obtained from 5 independent analyses, each corresponding to the average of 32 scans of the

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local surface of the sample, obtained with a 4 cm-1 resolution. FT-IR was also used to evaluate the stability of the thermal-evaporation self-assembling. For this the samples were immersed in DI water for four weeks, and their surface analyzed every week following the procedure abovedetailed. XPS was also used to evidence the efficient surface zwitterionization, through the detection of the N1s core level spectra. Details regarding the instrument and the method used are reported elsewhere.38 The physical structure of membranes was observed by scanning electron microscope. The instrument used was a Hitachi S-3000 SEM. Before observation, the PHB fibrous samples were sputter-coated with gold. The surfaces were analyzed under an accelerating voltage of 5-7 keV, depending on the samples. The porosity of the PHB fibrous membranes was assessed according to the same method as that used by Gu et al.39 Briefly, the dried scaffold (1.33 cm2) were weighed with a high precision micro-analytical balance (10-5 g, Mettler Toledo). Then, the samples were soaked in water for 24 hours. Afterwards, the superficial liquid was gently wiped out with tissue and the samples were immediately weighed. The following formula can then be used to calculate the porosity of the fibrous materials: ߝ=ఘ

ሺ௠మ ି௠భ ሻఘభ

(1)

భ ௠మ ାሺఘమ ିఘభ ሻ௠భ

Where, ɛ is the porosity, m2 is the weight of the membrane after soaking it in water, m1 is its initial dried weight, ρ1 is the density of PHB material (1.25 g/cm3 at room temperature), and ρ2 is the density of water. It is assumed that the overall density of our material is dominated by the density of PHB and that the coating does not significantly influences it. In addition, the values reported for the surface porosity correspond to the average of 6 independent measurements The actual coating density of the samples was evaluated by weight measurements. Samples before and after coating were all dried in a vacuum oven before measuring their weight. The samples (1.33 cm2) were weighed before, and after coating, using a 10-5 g microanalytical

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precision balance (Mettler Toledo). The difference of weight per unit surface area was taken as the coating density. Notice that both surfaces were accounted. In addition, the values reported for each sample correspond to the average of 6 independent measurements. The zeta potential of the PHB fibrous membranes was measured at different pH. A buffer solution (potassium chloride, 10-3 M) was prepared and its pH adjusted with either a hydrochloric acid solution (3 M) or a sodium hydroxide solution (3 M) depending on the pH at which the zeta potential would be measured. The samples were positioned on an adjustable cell of a SurPASSTM instrument (Anton Paar USA Ltd.), which was then infiltrated with the desired solution. The zeta potential of the sample could then be measured, and the data reported correspond to the average of 3 independent measurements. The hydration properties of the membranes were assessed by evaluating their hydration capacity and their water contact angle. Concerning the hydration property, the dried samples (1.33 cm2) were weighed (WD) with a precision analytical balance (Mettler Toledo) and immersed in DI water for 24 hrs. Thereafter, the superficial water was gently wiped out and the samples were weighed again (WW). The difference between Ww and WD per unit surface area was taken as the hydration capacity of the fibrous sample considered. Alike for the determination of the coating density, the two (top and bottom) surfaces were taken into account in the calculus. Also, the values reported correspond to the average of 3 independent tests. Regarding the measurement of the water contact angle, the technique and the instrument are the same as those used in our previous study. Therefore, one is invited to refer to that work for supplementary details.35 The water contact angle was measured at equilibrium, reached a few seconds after dropping the DI water droplet. Characterization of low-biofouling Properties of Zwitterionic PHB Fibers. The lowbiofouling properties of the membranes were assessed by evaluating their resistance to protein

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adsorption, using either bovine serum albumin (BSA), lysozyme (LY) or fibrinogen (FN), but also by incubating the membranes in bacterial solution of Escherichia coli. Starting with the protein adsorption tests, the method used with BSA and LY are similar. First, the series of membranes are immersed in ethanol and then in PBS, for 30 min and 2 hrs, respectively. These steps aim at swelling the samples and promote the diffusion of the proteins into the membranes, and so their adsorption. Then, the fibrous membranes were incubated with 1 mL of the protein solution considered (either BSA or LY), and the concentration of the protein solution was 1 mg/mL of PBS (pH 7.4). After a 2-hr-incubation time performed at ambient temperature (about 25°C), the absorbance of the incubation solution was determined with a UV-visible spectrophotometer at 280 nm. Note that the same wavelength was set in both protein adsorption tests. From the earlier determination of a calibration curve, the amount of protein adsorbed could be evaluated. Values reported in this work correspond to the average of 3 independent tests. The evaluation of the resistance to FN adsorption was done through an ELISA test. Once again, the method has been well documented elsewhere and one is invited to refer to our previous study for further details.36 Biofouling by bacterial species, Escherichia coli, was investigated according to the following experimental protocol. First, bacterial culture was prepared as follows. E. coli species were cultured at 37°C in a medium containing both peptone (5 mg/mL) and beef extract (3 mg/mL). All cultures were shaken at 100 rpm over the period of time of the culture, and until reaching the stationary phase. For E. coli, it corresponds to a cell concentration of 108 cells/mL. After preparing the culture of healthy bacteria, PHB fibrous membranes (surface area: 1.33 cm2) were positioned in a 24-well polystyrene culture plate and incubated at 37°C with 1 mL of bacterial suspension, for 3 hr. Afterwards, the bacterial suspension was removed from the wellplate and samples were thoroughly rinsed with PBS. Then, the bacteria adhering to the samples

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underwent a staining step, which was performed for 10 min with 0.2 mL of Live/Dead BacLightTM. Finally, the samples were washed again with PBS, and bacteria adhering at the surface and within the fibers were observed by confocal microscope (CLSM A1R, Nikon) at a 200X magnification. All images presented in the following section were taken at λex = 488 nm/λem = 520 nm. The quantitative data corresponding to these observations were obtained from the analysis of three independent images. Blood Compatibility of Zwitterionic PHB Fibers. In order to evaluate the hemocompatibility of the pristine and zwitterionic PHB fibers, four different adhesion tests were performed with either platelets concentrate, red blood cells concentrate, white blood cells concentrates or whole blood. Except for whole blood which was used as is, concentrates were prepared by centrifugation, from 250 mL of fresh blood obtained from a healthy Human volunteer, and according to a well-established procedure. In each case, 200 µL of solution (cell concentrate or whole blood) was used to perform the adhesion tests. The pristine and zwitterionic PHB fibers (1. 33 cm2) were equilibrated at 37°C with PBS (1 mL) for 24 hrs. Then, they were incubated with either one of the cells concentrate or the fresh blood, depending on the nature of the test performed. The incubation volume and incubation time were always 0.8 mL and 0.5 hr, respectively. At the end of the incubation, the cells concentrate or the fresh blood was removed and the fibrous membranes were thoroughly washed (5 times with at least 1 mL of PBS). Before observation, the cells adhering to the fibrous membranes were fixed with 0.8 mL of glutaraldehyde solution – prepared from a 25 wt% commercial glutaraldehyde solution (1 mL) in PBS (9 mL) – at 4°C and for 4 hrs, and eventually washed again with PBS. Notice that glutaraldehyde not only fixes the cells to the samples but also gives them autofluorescence. Then, the blood cells adhering to the surface of the membranes and within the fibrous

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membranes were observed by confocal microscope, according to the same settings as those used for bacterial attachment tests, and using the same instrument. The quantitative data corresponding to these observations were obtained from the analysis of three independent images, using the commercially available image processing and analysis ImageJ® software. HT1080 Adhesion and Development Onto Zwitterionic PHB Fibers. The adhesion of Human HT-1080 fibroblasts, possessing a green fluorescent protein, was studied according to the following method. First, the cells were grown at 37°C in a Dulbecco’s Modified Eagle Medium, supplemented with sodium pyruvate (1%), penicillin streptomycin (1%), nonessential amino acids (1%) and fetal bovine serum (10%). The medium was maintained in humid atmosphere with 5% carbon dioxide. Prior to the incubation, the fibrous membranes (1.33 cm2), positioned in a 24-well polystyrene culture plate were pre-treated with 1 mL of 75% ethanol at 25°C and for 1 hr, and then rinsed three times with PBS. Subsequently, they were incubated with 1 mL of HT1080 cells suspension at 37°C, under humid atmosphere containing 5% carbon dioxide, for either 1, 3 or 7 days. Finally, the samples were observed with a microscope (Nikon TS100) equipped with a 10X objective lens, and images taken at a x400 magnification. The quantitative data corresponding to these observations were obtained from the analysis by ImageJ® software of three independent images.

RESULTS AND DISCUSSION Physico-chemical Characterization of the Electrospun Fibers. The surface modification process chosen was thermal evaporation coating. A given volume of coating solution was deposited at the surface of membranes. This volume was calculated in order to obtain theoretical coating density of 0.5, 1, 2 or 2.5 mg/cm2. Experimental coating densities were measured by

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weight measurement and related results are presented in Figure 1. It is seen that all experimental coating densities are less than theoretical coating densities, which can be explained as follows. First, PHB fibrous membrane is porous, meaning that an appreciable, but difficult to quantify, amount of random copolymer must have penetrated in the deeper layers of the matrix and did not remain at the surface of the membranes, where coating density is evaluated. Second, it is assumed that all polymer brushes will interact with the fibrous material. Yet, local steric hindrance or misorientation of the brushes may result in the absence of interaction between some surface-modifier molecules and the PHB electrospun fibers. Last but not least, the actual coated surface area is unknown. Theoretical assessment of the coating density did not take into account the roughness nor the surface porosity of the fibrous membranes. Therefore, it is believed that the actual surface area was larger than that used in the preliminary calculation. These three effects, very hard to quantify, and not taken into account in similar studies on surface modification by coating, to the best of our knowledge, all contribute to a decreasing of the actual coating densities, as clearly observed on Figure 1. In addition, it is interesting to note that in our previous work in which poly(propylene) was the matrix polymer, an actual coating density of 1.1 was measured, corresponding to a theoretical value of 1 mg/cm2.35 In the present work making use of PHB, an actual coating density of 0.78 was measured, corresponding to the same theoretical value of 1 mg/cm2. This would suggest that the extent of interactions between z(P4VP-r-ODA) and PP are different from that between z(P4VP-r-ODA) and PHB. More precisely, stronger low energy interactions are created between PP and the hydrophobic moieties of the random copolymer (ODA block) than between PHB and ODA blocks. If one carefully analyzes the chemical structures at play, they will realize that the poly(ester) (PHB) is a little more polar than poly(ethylene). This change in polarity may partially have accounted for

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changes in the extent of interactions between the matrix polymer (PP or PHB) and the long backbone (17 CH2 groups) of ODA blocks. 2.0 2

Experimental coating density (mg/cm )

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1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

-0.5-

-1-

-1.5-

-2.52

Theoretical coating density (mg/cm )

Figure 1. Experimental vs. theoretical coating density. The coating of PHB fibers by z(P4VP-r-ODA) led to the decrease of the surface porosity, as qualitatively seen on the SEM images presented in Figure 2 and further quantified. This is explained by the quite high coating densities chosen. Even a coating density close to 0.5 mg/cm2 is enough to create some thin denser regions at the top surface of the membranes. However, the coating density is not high enough to create a homogeneous smooth dense top layer, as seen by the images at magnification 3K, even at the highest coating density (1.75 mg/cm2), which suggests that the scaffold remained porous, which is essential to the development of cells.

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Figure 2. SEM characterization of virgin and surface-modified PHB fibers. ε is the surface porosity. The numbers indicated in the IDs on top of the images correspond to the experimental coating densities. The top images were taken at magnification 1K while those of the bottom were taken at 3K.

As evidenced by the weight measurements performed to evaluate the coating density on the PHB fibers, the surface-modification was achieved. This was even better evidenced by the chemical characterization of the surfaces and in particular by the results of the FT-IR characterization presented in Figure 3 and Figure S2. The characteristic stretching bands of PHB polymer have been well documented elsewhere.40-42 Briefly, the prominent stretching band of the ester carbonyl function (νas(COO)) is seen at 1720 cm-1. Additionally, the stretching band observed at 1184 cm-1 corresponds to the asymmetric stretching variation of C-O-C ether group νas(COC), while that at 1099 cm-1 is assigned to the symmetric stretching variation of the same group νs(COC). Also, the intense band observed at 1283 cm-1 is attributed to the in-plane bending variation of the OH groups (δ(OH) non-bonded). Importantly, the stretching band observed at 1640 cm-1 corresponds to the quaternary ammonium salt held by z(P4VP-r-ODA),43, 44 and its intensity increases with surface modification. Results of FT-IR analysis were further supported by those of XPS analysis presented in Figure 4. Logically, no peak was found on the N1s spectrum of virgin PHB fibers. However, when coating PHB with zP(4VP-r-ODA), a new peak arose, at an approximate binding

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energy of 401.15 eV, attributed to the quaternized pyridine group.45, 46 The intensity of this peak tended to increase with the coating density. Therefore, the random copolymer successfully interacted with PHB fibers through hydrophobic-hydrophobic interactions between ODA moieties and PHB, and surface modification appeared to be fairly well controlled.

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zP(4VP-r-ODA)-1.75

zP(4VP-r-ODA)-1.13

Transmittance (%)

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zP(4VP-r-ODA)-0.78

zP(4VP-r-ODA)-0.33

Virgin PHB

4000

3500

3000

-1

Wavenumber (cm )

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Figure 3. FT-IR spectra of virgin and surface-modified PHB fibers. The numbers indicated in the IDs correspond to the experimental coating densities.

394

396

398

400

402

404

406

408

400

402

404

406

408

zP(4VP-r-ODA)-1.75

zP(4VP-r-ODA)-1.13

zP(4VP-r-ODA)-0.78

zP(4VP-r-ODA)-0.33

virgin PHB

394

396

398

Binding energy (eV) Figure 4. XPS analysis of virgin and surface-modified PHB fibers. The numbers indicated in the IDs correspond to the experimental coating densities.

Another evidence of the presence of copolymer is provided by the results of surface charge measurements presented in Figure 5. We performed zeta-potential measurements at different pH. Hydrolysis of PHB and PHB-derivatives may occur in a wide pH range,47,48 which could influence the results. However, it is lengthy and so not likely to have occurred during the present

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test. We initially assumed that coating the PHB matrix by a neutral copolymer would mask any potential surface charge, on condition that that the copolymer is pH-insensitive in the range tested In other words, a high copolymer density should lead to neutral PHB surfaces, provided that the copolymer interacts with PHB fibers. From the analysis presented in Figure 5, it is clear that the zeta potential of zP(4VP-r-ODA)-1.13 and zP(4VP-r-ODA)-1.75 is the closest to neutrality, suggesting that PHB fibers are well covered with zwitterionic moieties. However, for pristine PHB and low coating densities, the pH affects importantly the surface charge of membranes, especially at basic pH at which it is seen that virgin PHB, zP(4VP-r-ODA)-0.33 and zP(4VP-r-ODA)-0.78 membranes are significantly negatively charged. Virgin PHB zP(4VP-r-ODA)-0.33 zP(4VP-r-ODA)-0.78 zP(4VP-r-ODA)-1.13 zP(4VP-r-ODA)-1.75

15 10

Zeta potential (mV)

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

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5 0 -5 -10 -15 -20 -25 5

6

7

8

9

pH

Figure 5. Zeta potential of the virgin and surface-modified PHB fibers at different pH.

Effect of the Zwitterionic z(P4VP-r-ODA) Random Polymer on Low-biofouling Properties of PHB Electrospun Fibers. In order to apply the surface-modified PHB electrospun fibers as a potential material for biomedical application, interactions of fibers with diverse biofoulants such

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as proteins or bacteria must be minimized. To reach this goal, a hydrophilic protective layer should be formed around the fibers, as it is now well accepted that hydrophilicity is a prerequisite to nonfouling. The ability of the random copolymer to improve the surface hydrophilic properties of the PHB fibers was therefore evaluated, by measuring the water contact angle and the hydration capacity of the fibers. Results of this investigation are presented in Figure 6. Figure 6a shows an important decrease of water contact angle, from 131 ± 1° for virgin PHB membrane to 74 ± 2° for zP(4VP-r-ODA)-1.75 membrane, indicating a clear improvement of the surface hydrophilicity. Also, one should note that, although it is difficult to quantify it, another effect, besides the chemical change due to the presence of the zwitterionic copolymer, contributed to the reduction of the water contact angle: it is the reduction of the surface porosity after coating. Indeed, smooth interfaces are more readily wetted than rougher ones.49 Additionally, the ability of coated fibrous membranes to trap water within the hydrophilic moieties after immersion in a hydrophilic solvent is shown in Figure 6b: the hydration capacity of membranes can be multiplied by a 3.3 fold factor, suggesting that the zwitterionic zP(4VP-rODA) is likely to offer an efficient protection against biofouling. (a)

(b) 4.0 3.5

2

Hydration capacity (mg/cm )

120

Water contact angle (°)

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60

40

3.0 2.5 2.0 1.5 1.0

20 0.5 0

P gin Vi r

0.0

5 3 8 3 1.7 0.3 0.7 1.1 A)A)A)A)-OD -OD -OD -OD r r r r P P P P 4V 4V 4V 4V zP( zP( zP( zP(

HB

P gin Vi r

5 3 8 3 1.7 0.3 0.7 1.1 A)A)A)A)-OD -OD -OD -OD r r r r P P P P 4V 4V 4V 4V zP( zP( zP( zP(

HB

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Figure 6. Hydrophilic properties of virgin and surface-modified PHB fibers. (a) Water contact angle of membranes; (b) Hydration capacity of membranes. The numbers indicated in the IDs correspond to the experimental coating densities.

Biofouling involves different scales of interactions between the material and the biofoulants. Biofouling at the nanoscale occurs with proteins, while microscale is involved with larger biofoulants such as bacteria or cells. Ideally, both scales should be considered when studying the nonfouling properties of materials because proteins, cells or bacteria are commonly found in biological fluids. Also, biofouling by proteins can mediate that by bacteria or cells. Herein, we used several proteins, BSA, lysozyme and fibrinogen to study nanobiofouling. Adsorption tests were performed for a few hours, which is enough, considering that protein adsorption is a fast event, and occurs within seconds while the protein solution has been contacted with the interfaces. The results of this series of tests are presented in Figure 7. A coating density of 1.75 mg/cm2 can effectively reduce the BSA and lysozyme adsorption to 92% and 73% the limitation of virgin PHB membrane, while the adhesion of fibrinogen is reduced by half. Therefore, there is improvement of the resistance to protein adsorption.

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(a)

0.20

(b) 0.05

0.18 0.16 2

LY adsorbed (mg/cm )

2

BSA adsorbed (mg/cm )

0.14 0.12 0.10 0.08 0.06 0.04

0.04

0.03

0.02

0.01

0.02 0.00

gin Vir

B PH 4 zP(

0.3 A)-OD r VP

3 4 zP(

0.7 A)-OD r VP

0.00

5 3 1.7 1.1 A)A)-OD -OD r r P P 4V 4V zP( zP(

8

gin Vir

B PH 4 zP(

0.3 A)-OD r VP

3 4 zP(

0.7 A)-OD r VP

5 3 1.7 1.1 A)A)-OD -OD r r P P 4V 4V zP( zP(

8

(c) 120

Normalized FN adsorption (%)

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60

40

20

0

5 B 3 8 3 PH 1.7 0.3 0.7 1. 1 A) A) A) gin A)Vir -OD -OD -OD -OD r r r r P P P P 4V 4V 4V 4V zP( zP( zP( zP(

A SBM

Figure 7. Resistance to nanobiofouling by (a) BSA, (b) lysozyme and (c) fibrinogen of virgin and zwitterionic-coated PHB membranes. The numbers indicated in the IDs correspond to the experimental coating densities. SBMA stands for sulfobetaine methacrylate hydrogel

It is worth stressing on the fact that resistance to BSA was more readily obtained, followed by that to lysozyme. The least reduction to protein adsorption was obtained with fibrinogen. Fibrinogen presents the highest molecular weight, among the different proteins tested, which implies that it possesses numerous hydrophobic anchor blocks on its backbone that are likely to interact with the porous PHB matrix, despite its surface modification.50 In the past, we have investigated the formation of low-biofouling polyvinylidene fluoride membranes,51 and also

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noticed that resistance to BSA and lysozyme was more easily achieved than that to fibrinogen. We hypothesized back then that lower molecular weights could account for this difference. On the one hand, LY is the smallest protein among all proteins tested, which suggests that its diffusion in the pores of the PHB matrix should be facilitated, thus promoting the interactions with the fibers. On the other hand, one can reasonably consider that it possesses less hydrophobic domains than BSA or FN, because it is a smaller protein. In the present case, we assume that LY diffused well in the large pores of the virgin and coated fibers, ending up trapped in the membranes. Furthermore, not only the molecular weight of the protein is involved, but other factors such as the hydrophilic/hydrophobic balance of the protein or potential local electrostatic interactions between the protein and the matrix. At the pH of the measurements, the coated membranes are slightly positively charged, as supported by the zeta potential measurements. This slight charge bias from electroneutrality may be responsible for the adhesion via electrostatic interactions of BSA of FN. Also, despite a strong negative charge, fibrinogen has previously been shown to adhere to negatively charge surfaces, because its αC regions are locally positively charged.52 So, it is of major importance to achieve electrical neutrality of the PHB surface, but also surface homogeneity of the coating. Finally, we shall insist on the fact that all proteins (in particular LY, the smallest) may have penetrated in the deeper layers of the matrix during adsorption test, as matrices remained porous (even if the coating significantly decreased surface porosity). The deeper layers of the membranes are partially coated and surely not homogeneous (as we used a surface-modification process but we suspected that a non-negligible amount of zwitterionic molecules interacted with the PHB matrix inside the matrix). So, there are many potential interaction sites right underneath the surface, where proteins can interact. One may also have noted in Figure 7c that the highest coating density reached for z(P4VP-r-ODA)-

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1.75 sample still does not permit to obtain a resistance to FN as good as for pure SBMA hydrogel. A number of reasons can explain this difference among which the homogeneity of the material (pure zwitteironic material vs fibers coated with zwitterionic moieties) but also the different physical structures of the materials at play (SBMA hydrogel vs fibrous mat): there is clearly a supplementary physical penalty to fouling offered by porous materials as pores and rugosities provide physical anchoring sites that are not found in denser and smoother materials such as hydrogels. Eventually, resistance to BSA and lysozyme was quite high. Such levels of reduction are an indication of high resistance to fouling by bacteria where a larger scale is involved. Also, the adhesion of the very sticky fibrinogen protein was significantly reduced, and level obtained was believed to be good enough to considerably improve the blood cells compatibility of the asprepared membranes. The next step was therefore to study micro-biofouling by bacteria and cells. Escherichia coli is the most common bacteria employed when studying the nonfouling character of matrices used in water treatment, blood filtration or wound dressings. It might be due to the nature of its cell wall, and in particular to the thin peptidoglycan layer (2-6 nm) which makes it very deformable and so, able to stick and adhere to many surfaces.53 Therefore, resistance to Escherichia coli is a good indication to general resistance to micro-biofouling. Results of this test are presented in Figure 8 (quantitative analysis of the adhesion) and Figure S3 (confocal images showing the live bacteria adhering to the fibrous PHB). This analysis evidences that nonfouling by Escherichia coli species is achieved, as no bacteria can be seen adhering to z(P4VP-r-ODA)-1.75. The physical nature (highly porous, large pores) of the PHB virgin fibrous matrix is clearly favorable to physical entrapment of bacteria. No bacteria adhered onto z(P4VPr-ODA)-1.75 suggesting that the quality of surface modification was good enough to resist

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biofouling at a micro-scale, provided also that cell attachment will not be mediated by protein fouling. Thus, one should keep in mind that in the conditions of the test, there was no protein in the incubation medium, which must have partially influenced the very good final result. It is also important to mention again that coated membranes became denser and smoother with the coating density. Therefore, they were less likely to physically trapped bacteria like virgin PHB did. The surface modification of PHB by grafting from or grafting onto (coating) an antifouling polymer has very seldom been investigated. When done, the methods employed, geometry of the film, nature of the surface modifier or targeted application were significantly different, which makes difficult accurate comparisons of biofouling resistance results. For example, Woolnough et al. studied the control of biofouling and environmental degradation of PHB films and measure significant reduction of biofilm formation after 16 days of burial of the modified films, suggesting that their coating was efficient to reduce bacterial colonization.54 Furthermore, Handy et al. prepared a derivative of polyhydroxyalkanoate by chemical reaction, leading to zosteric acid-labeled ply[3-hydroxyalkanoates].55 These derivatives were found to significantly improve the resistance to biofilm formation, even though no clear details of the method employed for biofouling tests were provided. Finally, we shall stress on the work of Sin et al.,564 who selfassembled a PEGylated polymer (made of polystyrene and poly(ethylene glycol) methacrylate) and studied the resistance of the system to bacterial attachment using E. coli and S. epidermidis species. Their result indicate that they almost managed to prevent bacterial adhesion. In the present study, bacterial attachment was totally inhibited, which would suggest that the association of PHB and z(P4VP-r-ODA) leads to outstanding results in terms of the inhibition of bacteria colonization, and would compare with earliest reports.

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Number of bacteria adhering to the membranes (cells/mm )

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3500

3000

2500

2000

1500

1000

500

0

gin Vir

.75 .13 .33 .78 )-1 )-1 )-0 )-0 A A A A -OD -OD -OD -OD P-r P-r P-r P-r V V V V 4 4 4 4 zP( zP( zP( zP(

B PH

Figure 8. Effect of zwitterionic zP(4VP-r-ODA) on the resistance of PHB membranes to the adhesion of Escherichia coli. The numbers indicated in the IDs correspond to the experimental coating densities.

Effect of the Zwitterionic z(P4VP-r-ODA) Random Polymer on Hemocompatibility of PHB Electrospun Fibers. In previous section, we observed a decreasing of fibrinogen adsorption, a blood plasma proteins known to mediate platelet activation and clotting.57 This was a first indication of potential improvement of blood compatibility of the zwitterionic PHB fibers. Nonetheless, supplementary tests on the adhesion behavior of blood cells should be performed to reach an accurate conclusion on the hemocompatibility of the system. Here we performed red blood cells, white blood cells, platelet and whole blood adhesion tests and results are presented in Figure S4 (confocal images) and in Figure 9 (quantitative analysis).

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Confocal analysis clearly indicates that less blood cell adhere at the surface or are trapped within the fibers as the coating density increases. Several aspects should be emphasized at this point. First, the nonfouling behavior toward blood cells is independent on the nature of the blood cell. Hence, in all cases, very low attachment is seen for coating densities 0.33 mg/cm2. We should emphasize here that the adhesion of blood cells is controlled by the interactions between their cell wall proteins and the interfaces. As the nature of proteins varies with the type of cells, it shed lights on the excellent ability of the random copolymer to resist nonspecific protein adhesion, and also confirms results of nanobiofouling (Figure 6). Second, given the deformability of the cells and their ability to squeeze through capillaries with characteristic size smaller than their own diameter, results obtained for instance with red blood cells and for low coating densities are quite remarkable, given the high surface porosity and surface roughness of these matrices thus enabling physical trapping. For higher coating densities, we have seen in Figure 2 that the coating tended to importantly reduce the surface porosity, meaning that both a physical effect (smoother interfaces with smaller porosity) and a chemical effect (high surface content of zwitterionic copolymer0 contribute to the improvement of resistance to blood cells.

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Red blood cells White blood cells Platelets Cells from whole blood

2

adhering to the surface (cells/mm )

6000

Number of blood cells

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5000

4000

3000

2000

1000

0 P in ri g V

HB

( zP

4V

r-O P-

.33 -0 ) DA zP

r-O PV (4

DA

.7 )-0

8

( zP

4V

.13 -1 ) DA r-O P

( zP

P 4V

-1 A) D O -r-

.75

Figure 9. Quantitative analysis of the Effect of zwitterionic zP(4VP-r-ODA) on the resistance of PHB membranes to the adhesion of blood cells. The numbers indicated in the IDs correspond to the experimental coating densities.

Effect of the Zwitterionic zP(4VP-r-ODA) Random Polymer on Fibroblasts Adhesion and Proliferation. Coating of zwitterionic zP(4VP-r-ODA) onto PHB fibers was thus proven to positively influence the resistance to blood cells adhesion. We then moved onto the interaction and the development of fibroblasts in contact with the PHB fibers. The reason for running this test is the major role of fibroblasts in the growth of structural framework of animal tissues. Fibroblasts, the preponderant cells of connective tissues, play a key role in tissue growth, and

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their use as model cell is extensively reported in tissue-engineering applications of polymeric matrix.59-59 Results of these human cells incubation tests are displayed in Figures 10 and 11. In the case of tissue culture polystyrene (TCPS) control, cells seen after 1-day incubation actively proliferated, and eventually covered the scaffold surface almost completely (cells density of 650 ± 71 cells/mm2). This suggests that numerous hydrophobic-hydrophobic interactions were established between the hydrophobic polystyrene and the cell-walls of the fibroblasts. Even though the cell development was not important after the first day of culture on virgin PHB, active proliferation was also observed in the following days, with cells density found to be 590 ± 14 cells/mm2 and 1850 ± 71 cells/mm2 after 3 and 7 days of incubation, respectively. These results are in accordance with those of Sin et al., who also noted an important surface coverage of similar surfaces and attributed it to both their hydrophobicity and their cytocompatibility.564 The coated PHB fibers present very low cell density, owing to the nonfouling effect of the z(P4VP-r-ODA) copolymer such that very few cell were visible on the related fluorescent image of z(P4VP-r-ODA)-1.75 after the first day of culture (85 cells/mm2). In other words, in the case of the zwitterionic PHB fibers, there is no or few direct interactions between the cells and the matrix. This observation supports the results discussed in previous sections on the nonfouling ability of zP(4VP-r-ODA). However, when running fibroblasts adhesion tests for a longer time (up to 7 days), we did observe that cells actively proliferated on both control, virgin PHB and coated PHB fibers, as unveiled by the fluorescent images. The control TCPS and virgin PHB fibers logically exhibited a large cell density after 3 days (880 ± 28 cells/mm2 and 590 ± 14 cells/mm2, respectively) and 7 days (2050 ± 71 cells/mm2 and 1850 ± 71 cells/mm2, respectively) due to the establishment of numerous direct hydrophobic-hydrophobic interactions. But the cell proliferation also occurred

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onto coated PHB fibers, attributed to the adequate humid environment provided by the zwitterionic random polymer, thus favoring cell-cell communication and their further development. After 3 days of culture, a cell density as high as 329 ± 16 cells/mm2 was measured onto z(P4VP-r-ODA)-0.78 membrane. However, it was significantly lower for fibers exhibiting a higher coating density (170 ±14 cells/mm2 for z(P4VP-r-ODA)-1.13 membrane). For samples with high coating density (z(P4VP-r-ODA)-1.13 membrane and z(P4VP-r-ODA)-1.75 membrane), the low amount of cells can be attributed to both the nonfouling effect of the copolymer, that mitigates the cell-material interactions and adhesion to the fibers, but also to the lower surface porosity, preventing the embedding of cells and their subsequent division necessary to the growth of the tissue. But for low coating densities, cells can more readily interact with the fibers as the surface porosity is preserved. From all these results, it is believed that the zwitterionic PHB matrix can be an ideal matrix to promote tissue growth if the coating density is optimized. It has to be (1) high enough for preventing or mitigating the interactions with proteins, bacteria and blood cells that usually occur within seconds or minutes after contact, and for ensuring an adequate humid environment, and in the same time (2) low enough to preserve the matrix porosity and permit the trapping and development of the targeted fibroblasts cells. From these considerations and our observations, PHB fibers coated with z(P4VP-r-ODA) with an actual coating density of 0.78 mg/cm2, corresponding to an initial theoretical coating density of 1 mg/cm2 provides an ideal compromise between low-biofouling property (resistance to bacteria and blood cells) and skin cells growth (proliferation), and so, may be a suitable matrix for tissue engineering applications.

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Figure 10. Adhesion of fibroblasts onto virgin PHB and zP(4VP-r-ODA) coated PHB membranes. The numbers indicated in the IDs correspond to the experimental coating densities. The fluorescence is due to a green fluorescent protein (GFP). Day 1 Day 3 Day 7

2000

2

PHB fibers (cells/mm )

Density of HT1080 cells adhering to the

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1000

500

0 S co TCP

l ntro

HB .75 .78 .33 .13 in P A)-1 A)-0 A)-0 A)-1 Virg -OD -OD -OD -OD r r r r P P P P V V V V zP(4 zP(4 zP(4 zP(4

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Figure 11. Quantitative analysis of the adhesion of fibroblasts onto virgin PHB and zP(4VP-rODA) coated PHB membranes. The numbers indicated in the IDs correspond to the experimental coating densities.

CONCLUSIONS

In this work, we prepared poly(3-hydroxybutyrate) (PHB) fibrous membranes by electrospinning process, and surface-modified them with a zwitterionic copolymer, referred to as zP(4VP-rODA), by thermal-evaporation self-assembling method. The copolymer interacts with hydrophobic PHB through its hydrophobic octadecyl acrylate (ODA) repeat units, via hydrophobic-hydrophobic interactions, and the interactions were shown to be stable for at least 3 weeks. Once this novel material fully characterized, by carrying out numerous physico-chemical characterization tests (SEM, FT-IR and XPS tests; assessment of the coating density, porosity and zeta-potential), we laid the focus on the improvement of the surface hydrophilicity and on the resistance to protein (BSA, lysozyme and fibrinogen) adsorption due to the zwitterionic copolymer. From a copolymer density ranging from 0.78 mg/cm2 to 1.13 mg/cm2, the modified PHB fibers efficiently repelled proteins, but also bacteria (E. coli). Furthermore, these novel fibers were shown to be blood-inert, that is, they resisted to the adhesion of all types of blood cells, taken individually (single blood cells adhesion test), or mixed in whole blood. However, after incubating the fibers with HT-1080 fibroblasts cells, adhesion and growth importantly occurred on the fibers after 3 days. After 7 days, a cells density of 1610 cells/mm2 was measured onto the sample with a coating density of 0.78 mg/cm2. These results suggest that tuning the coating density enables to (1) efficiently repel common biofoulants, (2) provide a hydration layer

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essential to tissue cell development, and (3) maintain a porosity high enough to permit the interactions with fibroblasts and their growth. In other words, the zwitterionic zP(4VP-r-ODA)PHB fibers hold promise as a potential material for bio-scaffolding. Future essential in-depth investigations concern in particular the establishment of the biodegradability kinetic of the PHB/z(P4V-r-ODA) system, as well as the determination of a potential cytotoxic effect of the random copolymer.

AUTHOR INFORMATION

Corresponding Author * Antoine Venault: E-mail: [email protected]. Phone: 886-3-265-4113 *Yung Chang: E-mail: [email protected]. Phone: 886-3-265-4122. Fax: 886-3-265-4199 Author Contributions This manuscript was written through contributions of all authors. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

The authors would like to acknowledge the Ministry of Science and Technology of Taiwan (MOST 103-2221-E-033-078-MY3 and MOST 104-2221-E-033-066-MY3) for their financial support.

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ASSOCIATED CONTENT

Supporting Information Chemical structures and 1H NMR spectra, Stability tests, Bacterial attachment, Blood cells adhesion. Supplementary data associated with this article can be found in the online version.

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FIGURE CAPTIONS Figure 1. Experimental vs. theoretical coating density. Figure 2. SEM characterization of virgin and surface-modified PHB fibers. ε is the surface porosity. The numbers indicated in the IDs on top of the images correspond to the experimental coating densities. The top images were taken at magnification 1K while those of the bottom were taken at 3K.

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Figure 3. FT-IR spectra of virgin and surface-modified PHB fibers. The numbers indicated in the IDs correspond to the experimental coating densities.

Figure 4. XPS analysis of virgin and surface-modified PHB fibers. The numbers indicated in the IDs correspond to the experimental coating densities.

Figure 5. Zeta potential of the virgin and surface-modified PHB fibers at different pH. Figure 6. Hydrophilic properties of virgin and surface-modified PHB fibers. (a) Water contact angle of membranes; (b) Hydration capacity of membranes. The numbers indicated in the IDs correspond to the experimental coating densities. Figure 7. Resistance to nanobiofouling by (a) BSA, (b) lysozyme and (c) fibrinogen of virgin and zwitterioniccoated PHB membranes. The numbers indicated in the IDs correspond to the experimental coating densities. Figure 8. Effect of zwitterionic zP(4VP-r-ODA) on the resistance of PHB membranes to the adhesion of Escherichia coli. The numbers indicated in the IDs correspond to the experimental coating densities. Figure 9. Quantitative analysis of the Effect of zwitterionic zP(4VP-r-ODA) on the resistance of PHB membranes to the adhesion of blood cells. The numbers indicated in the IDs correspond to the experimental coating densities. Figure 10. Adhesion of fibroblasts onto virgin PHB and zP(4VP-r-ODA) coated PHB membranes. The numbers indicated in the IDs correspond to the experimental coating densities. The fluorescence is due to a green fluorescent protein (GFP).

Figure 11. Quantitative analysis of the adhesion of fibroblasts onto virgin PHB and zP(4VP-r-ODA) coated PHB membranes. The numbers indicated in the IDs correspond to the experimental coating densities.

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