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Non-woven Carboxylated Agarose-based Fiber Meshes with Antimicrobial Properties Aurelien Forget, Neha Arya, Rotsiniaina Randriantsilefisoa, Florian Miessmer, Marion Buck, Vincent Ahmadi, Daniel Jonas, Anton Blencowe, and V. Prasad Shastri Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01401 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 9, 2016

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Non-woven Carboxylated Agarose-based Fiber Meshes with Antimicrobial Properties Aurelien Forget,1,2 Neha Arya,2,3 Rotsiniaina Randriantsilefisoa,2 Florian Miessmer,2 Marion Buck,4 Vincent Ahmadi,2 Daniel Jonas,4 Anton Blencowe,5 and V. Prasad Shastri,2,3,6,* 1

Future Industries Institute, University of South Australia, Mawson Lakes 5095, SA, Australia;

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Institute for Macromolecular Chemistry, University of Freiburg, 79104, Freiburg, Germany;

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Helmholtz Virtual Institute on Multifunctional Biomaterials for Medicine, Kantstr 55,14513

Teltow, Germany; 4Department of Environmental Health Science, Universitätklinikum Freiburg, Freiburg 79106, Germany; 5School of Pharmacy and Medical Science, University of South Australia, Mawson Lakes 5095, SA, Australia; 6BIOSS–Centre for Biological Signaling Studies, University of Freiburg, 79104, Freiburg, Germany.

KEYWORDS: agarose, wound dressing, ionic liquid, electrospinning, antimicrobial

Abstract

Hydrogel forming polysaccharides, such as the seaweed derived agarose, are well suited for wound dressing applications as they have excellent cell and soft tissue compatibility. For wound dressings, fibrous structure is desirable as the high surface area can favor adsorption of wound exudate and promote drug delivery. Although electrospinning offers a straightforward means to produce non-woven fibrous polymeric structures, processing agarose and its derivatives into

ACS Paragon Plus Environment

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fibers through electrospinning is challenging as it has limited solubility in solvents other than water. In this study we describe the processing of carboxylated agarose (CA) fibers with antibacterial properties by electrospinning from a solution of the ionic liquid (IL) 1-butyl-3methylimidazolium chloride ([Bmim]+Cl-) possessing antimicrobial properties. The extent of carboxylation was found to impact fiber diameter, mesh elastic modulus, fiber swelling, and the loading and release of IL. IL-bearing CA fibers inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa, bacteria’s commonly found in wound exudate. In sum non-woven CA fibers processed from IL are promising as biomaterials for wound dressing applications.

Introduction Naturally occurring polysaccharides (PS) have shown increased applications in regenerative medicine due to physicochemical similarities with mammalian PS. Agarose, a marine algae derived PS, has been extensively used in cell encapsulation, topical and injectable applications.1– 3

We have recently shown that the key properties of hydrogels composed of α-helical PS such as

agarose and κ-carrageenan, including gelation temperature, material stiffness and topography can be altered by evolving new secondary structures through backbone modification4 and blending modified PS with native PS.5 These findings therefore present new opportunities to extend their processibility. Electrospinning (ES) is commonly used for the processing of polymers into fibers6–8 via ‘jetting’ of polymer solutions, preferably in a volatile organic solvent, under the influence of an electric field. Since PS are water soluble and their viscosity is difficult to control, their processing into fibers through electrospinning is challenging.9 In the case of agarose, it has been only been electrospun into fibers as a blend with chitosan.10 Since solvent is an important parameter in ES, approaches were solvent-PS interactions may be leveraged to promote ES have


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been explored and ionic liquids (ILs) have emerged as an alternative to volatile organics.11 ILs are organic salts that exist in a liquid state below 100°C due to weak coordination between ions; and have been investigated in fiber extrusion and ES of diverse synthetic and natural polymers including cellulose.12–14 Their unique physicochemical properties offer control over the viscosity of PS solutions, a solution variable critical for optimizing ES conditions.15 Another consequence of the IL chemical structure, is that the imidazolium cation can function like a surfactant and is capable of selectively disrupting prokaryotic cell membranes while displaying reduced toxicity towards eukaryotic cells.16–20 Herein, we describe the processing of carboxylated agarose (CA)4 (Figure 1) into non-woven meshes that possess antibacterial properties by extrusion of a highly viscous solution of CA in 1butyl-3-methylimidazolium chloride ([Bmim]+Cl-) (Figure 1) under the influence of an electric field into a non-solvent. This wet electrospinning technique yielded micron-sized fiber meshes of CAs that incorporated [Bmim]+Cl- and showed bioactivity against both gram-positive and gramnegative infectious bacteria commonly found in human wound. Thus, this work represents a step toward exploiting the antimicrobial properties of imidazolium cations in wound dressings.

Figure 1. Chemical structures of native and carboxylated agarose (CA), and the ionic liquid (IL), 1-butyl-3-methylimidazolium chloride ([Bmim]+Cl-) CA with three different degrees of carboxylation (28% (CA28), 60% (CA60) and 93 % (CA93)) were prepared as previously described.4 The CA derivatives were dissolved in the IL, 1butyl-3-methylimidazolium chloride ([Bmim]+Cl-). Maximal solubilization of CA in the IL was


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found to be at 7.5 w/v% and this solution was used for processing into fibers (Supporting Information Table S1).21,22

Materials and Methods Materials: Native agarose was obtained from Merck (Germany), 99% (2,2,6,6tetramethylpiperidin-1-yl)oxyl (TEMPO), 99% NaBr, 15% v/v NaOCl solutions, 1-butyl-3methylimidazolium chloride ([Bmim]+ Cl-), 99.99% NaBH4, BioXtra ≥ 99.5% NaCl, Phosphate buffer saline (PBS) and BioXtra ≥ 99.0%KBr was purchased from Sigma Aldrich (Germany) and used as received. Ethanol (technical grade) was used as received. Deionized water was obtained from a laboratory ion exchanger. Synthesis of carboxylated agarose: Native agarose (1 g) was transferred into a 3-necked round bottom flask equipped with a mechanical stirrer and pH-meter, and dissolved in water at a concentration of 1 % w/v by heating to 90 °C. The flask was cooled down to 0 °C, using an ice bath, under vigorous mechanical stirring in order to prevent gelation of the agarose, and the reactor was charged with TEMPO (20.6 mg, 0.16 mmol), NaBr (0.1 g, 0.9 mmol) and NaOCl (2.5 ml, 15 % solution). As the reaction occurs, the solution becomes acidic. The pH of the solution was maintained at 10.8 by dropwise addition of NaOH (0.1M) throughout the duration of the reaction. The degree of carboxylation was back calculated by using the volumes of NaOH (0.1 M) solution added during the reaction. The reaction was quenched by the addition of NaBH4 (0.1 g), following which the solution was acidified to pH 8 (0.1 M HCl) and stirred for 1 h. The modified agarose was then precipitated by the sequential addition of NaCl (12 g, 0.2 mol) and ethanol (500 ml). The product was collected by vacuum filtration using a fritted glass funnel and then washed using ethanol (500 mL). The ethanol, catalyst and salts were removed by extensive


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dialysis against water for 2 days with replacement of the water every 12 h. The modified agarose was then freeze-dried (Beta 2-8 LD; Christ, Germany) overnight to yield a white solid. The degree of carboxylation was verified as described earlier4 by the appearance of peaks associated with aliphatic carboxylic acid groups via FTIR (KBr) (νc=o: 1750 cm-1) and NMR (13C: 180 ppm). Preparation of CA-IL Fibers: Carboxylated agarose (CA) was dissolved in 1-butyl-3methylimidazolium chloride ([Bmim]+Cl-) (Sigma Aldrich, Germany) at a concentration of 7.5 % w/v under stirring at 90 °C for 30 min. The solutions were kept at 60 °C (above the sol-gel transition) for further processing. For wet electrospinning, an upward setup was used (Supporting Information Figure S2): a 2 ml glass syringe (Hamilton, USA) was loaded with CA-IL solution and positioned on a syringe pump (Cole Palmer, USA). The syringe pump was held upwards above a custom-made plastic collector containing the non-solvent (ethanol). A screw was passed through the plastic material and the head of the screw was adjusted to the plastic dish bottom. An aluminum foil was positioned on top of the screw head and the plastic dish was filled up with ethanol. A 30 kV current generator (Heinzinger, Germany) was wired to the 0.8 mm blunt syringe needle (Braun, Germany), and another current generator was wired to the screw positioned under the plastic dish. A positive potential was applied to the needle (+7.5 kV) and a negative potential was applied to the collector plate (-3 kV). The CA-IL solution was extruded at a speed of 125 µl / min with a current of 0.001 mA. After processing, the fiber meshes were removed from the collecting plate and allowed to dry at room temperature for 24 h prior to further characterization. Thermogravimetric Analysis (TGA): Thermal degradation of the CA derivatives, CA-IL Fibers and IL was assessed by TGA with a STA 409 from Netzsch (Germany). Mass loss of the


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sample was measured as a function of the temperature from 50 to 550 °C under oxidative conditions. Mechanical properties: For each formulation of CA-IL fiber meshes, 3 samples were cut to equal sizes with identical weights and mounted into an EZ-LX mechanical tester (Shimadzu, Japan). The samples were positioned in test grips and tensile measurements were performed with a 50 N load cell (Shimadzu, Japan). The samples were elongated at a rate of 1 mm/min and the elastic modulus was directly measured from the resulting curves. Measurement of fiber diameters: Fibers were fixed on a carbon tape and SEM images were obtained using a Quanta 250 FEG (FEI, USA) environmental scanning electron microscope (20 kV at 100 Pa). Images shown are representative of different areas of several batches of a given composition. Images of three different samples per formulation were randomly taken and for each formulation > 100 fibers were measured. Fibers stability: Fiber meshes (100 mg) were placed in a 100 µm nylon mesh cell strainer (BD Falcon, USA). The lid of the cell strainer was cut off and the nylon mesh was positioned in a 6 well plate. PBS (2 ml) was added to each well, and the solution was changed every day after the fibers and cell strainer were weighted. The measurements were normalized to the weight of the dry fibers. Fourier transform infrared (FTIR): The fiber mesh was directly measured on a FTIR Vector 22 (Bruker, Germany) equipped with an Attenuated Total Reflectance Golden Gate (Specac, United Kingdom). The spectra were recorded in absorbance mode using the Opus Software (Bruker, Germany) provided by the manufacturer. Ionic liquid and ethanol release: A similar set up as for the fiber stability study was used; fiber meshes (100 mg) were loaded in a 40 µm nylon mesh (cell strainer) positioned in a 6 well


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plate and 2 ml of PBS were added to the well. At regular intervals the whole solution of PBS was removed and kept in a separate vial for characterization. The amount of 1-butyl-3methylimidazolium released in the PBS solution was measured by UV-vis spectroscopy on a Nanodrop 2000C (Thermo Fisher Scientific, USA). The quantity of IL was then calculated by using a calibration curve giving the relation between absorption and concentration. Using the same solution, the amount of ethanol released by the fibers was measured using a 7820A gas chromatography system (Agilent, USA). Antimicrobial properties: Staphylococcus aureus (S. auerus, ATCC 29213) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) were obtained from ATCC (Wesel, Germany). The minimum inhibition concentration (MIC) was determined using the Mueller Hinton (MH) broth methodology. Briefly, IL (500 mg/ml in PBS) was serially diluted in a 50 µl volume of MH broth in a 96 well plate starting from a concentration of 250 mg/ml up to 0.977 mg/ml. Ceftazidime (GlaxoSmitthKline, UK) was used as the antibiotic standard and was plated at a concentration range of 128 µg/ml to 0.5 µg/ml in a separate plate. Following this, bacteria were introduced in a 50 µl volume into each well. Overnight culture of the bacterial strains on blood agar was used to inoculate MH broth to reach an absorbance of 0.07 and 0.11 for S. auerus and P. aeruginosa, respectively. The inoculum was then diluted 1:100 before being introduced into each well. The negative controls contained only 100 µl MH broth while 100 µl MH broth containing bacteria served as a positive control. Plates were incubated for 24 h at 36 °C and the MIC was determined as the concentration of IL in the well that did not allow any bacterial colony formation. MIC was determined by three independent experiments with duplicates run at each time. CFU/ml for each bacterial inoculum was calculated following spread plating 10 µl of 1:100 dilution of the bacterial inoculum (used for MIC studies) on blood agar plates. The end


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concentration of the bacterial inoculum was maintained in the range of 0.5 x 105 to 0.7 x 106 CFU/ml. Zone of Inhibition (ZOI): CA-IL fiber meshes were punched into discs of 9 mm diameter. As mentioned in the MIC study, S. aureus and P. aeroginosa at an absorbance of 0.07 and 0.11 respectively in MH broth were prepared from an overnight culture on blood agar plate. They were then spread end-to-end on MH agar plates using a cotton swab in three different directions. Following this, the CA-IL disks were positioned on the agar plates and incubated for 24 h at 36 °C. A similar set up was used for the control discs. The ZOI was measured around the disc where no bacterial growth was observed. The ZOI was represented as the normalized log weight of each disc.

Results and Discussion IL enables ES of carboxylated agarose and influence fiber properties: In order to assess the fiber characteristics when obtained through extrusion alone, CA-IL solutions were extruded through a needle under gravity into an ethanol bath (a non-solvent for CA) using a conventional vertical setup. However, this approach only afforded a viscous hydrogel mass in all CA compositions with no distinct fiber-like structures (Supporting Information Figure S1). Therefore, a voltage bias was applied between the extrusion needle and the collector plate (Supporting Information Figure S2), to investigate if the presence of an electric filed could facilitate thinning of the polymer solution during extrusion. This resulted in a vigorous ejection of the CA-IL solution through the syringe and elongation of the extruded filament along the applied electric field, leading to efficient fiber formation on the aluminum foil target. Since it is well known that the viscosity, polymer concentration, solution conductivity, extrusion speed and


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voltage influence the quality of electrospun fibers,23 these parameters were kept constant (Supporting Information Table S1) to allow the effect of degree of carboxylation on fiber formation to be interpreted. For each of the CA-IL formulations, fiber meshes were prepared and characterized using environmental scanning electron microscopy (ESEM) (Figure 2). Analysis of the fiber diameter distribution from ESEM images revealed a trend of decreasing mean fiber diameter with increasing carboxylation as well as a narrower size distribution of 183±153, 117±58 and 63±54 µm for CA28-IL, CA60-IL and CA93-IL, respectively (Figure 2), which were statistically significant (CA28-CA60 p = 4.3E-5; CA28-CA93 p = 4.3E-16; CA60-CA93 p = 1.9E-19). This decrease in fiber diameter maybe attributed partially to a decrease in shear modulus with increasing carboxylation as shown previously,4 which could translate into a lower shear force to induce jet thinning.

Figure 2. SEM images (top) and fiber diameter distribution (bottom) for electrospun (a) CA28IL, (b) CA60-IL and (c) CA93-IL fiber meshes; scale bars 1 mm and 100 µm (insets). ES CA fibers show low swelling and good elastic modulus: Polymer fiber meshes are particularly valuable for biomedical applications such as wound dressings. For heavily exuding wounds, the dressings need to absorb the lesion secretions and maintain their structural


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integrity.24 In order to assess the water absorption capacity of CA-IL fibers, the weight variation of the fiber meshes was monitored in vitro under physiological conditions. After 1 day in contact with phosphate buffered saline (PBS), the fiber meshes gained up to 35 % of their dry mass for CA60-IL, 23 % for CA28-IL and 21 % for CA93-IL (Figure 3a). After 3 days, the wet weight of the fibers reached equilibrium and resulted in a gain of 10-20 % of their dry mass and remained unchanged for 5 days. These results demonstrate that the CA-IL fiber meshes are stable over prolonged periods, which provides a major advantage over other degradable PS fiber meshes used in wound dressings such as alginate in which the dissociation of calcium ion (Ca2+) crosslinks leads to loss of fiber integrity and the local release of calcium ions.25 Since any topical wound dressing will experience mechanical deformation, fiber integrity is an important consideration. In this regard the elastic modulus (E) of a material can be a good determinant of this metric. Tensile testing of the CA-IL fiber meshes yielded average E values of 46, 122 and 255 kPa for CA28-IL, CA60-IL and CA93-IL, respectively (Figure 3b). Surprisingly, fibers made of CA93 displayed the highest elastic modulus compare to CA60 and CA28. This trend is in contrast to the shear modulus behavior of CAs gels, where hydrogels made of CA93 showed the lowest modulus.3 The inverse trends in the shear and elastic moduli may be attributed in part to the differences in the molecular characteristics of polymer networks formed in presence of IL versus the association of helices and β-sheet structures in the CA by themselves and is the subject of ongoing investigations.


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Figure 3. (a) Mass variation of dry CA-IL fibers over 5 days in contact with PBS. (b) Elastic modulus of the CA-IL fibers. CA fibers spun from IL solutions possess antimicrobial properties: Fibrous meshes possessing antimicrobial properties are highly desirable for wound care applications. The application of imidazolium based IL as antimicrobial agents has been demonstrated previously.18,26–29 While the 1-butyl-3-methylimidazolium cation exhibits cytotoxicity toward prokaryotic cells it shows relatively low toxicity towards eukaryotic cells.30,31 Therefore, the IL [Bmim]+Cl- used in this study could serve as a model system to test the premise that PS fibers with antimicrobial properties may be realized by processing from an IL solution. TGA of the CA-IL fibers was performed to assess the amount of IL encapsulated within the fibers and the results were compared to pure CA (Figure 4a and b, respectively). The amount of IL loaded in each CA-IL composition was estimated by calculating the percentage of weight loss attributed to pure CA. With IL% = 100-(100(CA%) / IL-CA%), we obtained an IL weight percentage of 45±3, 38±2 and 17±2 % for CA28-IL, CA60-IL and CA93-IL, respectively. Considering that the IL is composed of an imidazolium cation it was surprising that encapsulation showed an inverse correlation with carboxylation. This may be attributed to solubility differences of IL in the CA fibers and interactions between the imidazolium cation and CA backbone. It was previously shown that solubilization of agarose in different IL is impacted by the length of side chain of the imidazolium cation as well by the size of the anion.32 Additionally, it has been suggested that xanthan gum, a carboxyl bearing polysaccharide can interact with the imidazolium cation in water33 and that the xanthan gum and imidazole cation can form complexes impacting the mechanical properties of the resulting hydrogels.34 However, in our system, the CA is dissolved in pure [Bmim]+Cl- without addition of water. Since no marked differences was observed in the


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FTIR spectra across the three formulations (SI, Figure S3), the most probable interaction modality between the IL and the CA could be via the hydroxyl groups in the sugar moieties and the imidazolium, as also reported for cellulose and imidazolium.35,36 Therefore, the observed differences in encapsulation efficiency may be attributed to a combination of reduced molecular interactions between the IL and CA fiber with increasing carboxylation and swelling of the fibers during formation leading to leaching out of the IL during gelation of the fibers. The release of the 1-butyl-3-methylimidazolium of the IL from the CA-IL fiber meshes in PBS was measured via UV spectroscopy and it was found that the majority of the [Bmim]+ was released over a 30 min period upon exposure to PBS, which was followed by a constant release that was sustained over 4 days of the experiment (Figure 4c). Consistent with the IL loading, the release of [Bmim]+ increased in the order CA28-IL > CA60-IL > CA93-IL. Thus demonstrating the fact that degree of carboxylation impacts IL encapsulation and its release offers the possibility to tune the release behavior of IL by co-spinning different formulations.


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Figure 4. TGA weight loss thermograms (left) and 1st derivative curves (right) for (a) the CA-IL fiber meshes and IL, and (b) CA derivatives and IL. (c) Release profile of the 1-butyl-3methylimidazolium of the IL from CA-IL fiber meshes as monitored over 4 days via UV spectroscopy, standard deviations are marked in black. The antibacterial activity of the CA-IL fibers was assessed using an agar diffusion assay by positioning pre-cut circular CA-IL meshes on agar plates swabbed with either gram-positive or gram-negative bacteria: Staphylococcus aureus and Pseudomonas aeruginosa, respectively, which are typically found in infected wounds.37 The zone of inhibition (ZOI) was measured after 24 h of exposure and normalized to the log weight (Figure 5). Interestingly, the ZOI was smaller for S. aureus compared to P. aeruginosa, although the inhibition was complete in the former and only partial in the latter. This difference could be attributed to strain heterogeneity in P.


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aeruginosa and loss of porins.38 Additionally, there was no difference in ZOI/log weight across the different fiber formulations for P. aeruginosa despite the differences in IL loadings and release profiles. However, CA28-IL demonstrated higher S. aureus inhibition as compared to CA60-IL and CA93-IL (Figure 5a), which correlates to the in vitro IL release data (Figure 4c).

Figure 5. ZOI normalized to log weight for the CA-IL fiber meshes with (a) S. aureus (ATCC 29213) and (b) P. aeruginosa (ATCC 27853) (n = 12); *** represents P < 0.005 and * represents P < 0.05. To confirm that the antibacterial properties are due to the 1-butyl-3-methylimidazolium of the IL, control samples were prepared by incubating the CA-IL fiber meshes in PBS for a period of 5 days in order to remove the IL, and then they were placed in agar plates swabbed with S. aureus and P. aeruginosa. After 24 h of incubation, no ZOI was observed, thereby demonstrating that the CA fibers alone have no antibacterial activity (Supporting Information Figure S4). Additionally, as the fibers were collected in an ethanol bath after extrusion, one could envisage that traces of solvent may be present and possibly contribute to the observed antibacterial effect. To rule this out, the amount of ethanol released from the fibers in vitro was measured by gas chromatography, and found to be < 0.004 µl/ml, which is well below the concentration needed to exhibit a bacteriostatic effect (Supporting Information Figure S5).39

Conclusions


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In summary, by processing a high viscosity solution of carboxylated agarose in an IL with antimicrobial properties using electric field assisted extrusion, micron-sized non-woven fiber meshes with good mechanical properties that exhibit antibacterial properties against grampositive and gram-negative strains of wound bacteria has been achieved. This simple approach offers a novel paradigm for the development and production of antimicrobial non-woven fiber meshes for wound-care dressings.

Supporting Information. Provided in the supporting information is the viscosity of the polymer solutions utilized in this study, photographs of extruded CA in the absence of the electric field, scheme of the electrospinning setup, table summarizing the viscosity and conductivity of the CAIL solutions, table summarizing the minimum inhibitory concentration of the IL for the bacteria strains utilized. Pictures of the agar diffusion assay of carboxylated agarose fibers alone and loaded with IL and the amount of ethanol released from the carboxylated agarose fibers after electrospinning. Author Information Corresponding Author *Email: [email protected] Author Contributions AF and VPS conceived the study. AF, NA, DJ, AB and VPS designed the experiments, RR and FM carried out synthesis of carboxylated agarose and electrospinning, NA, MB, and DL carried out the bacterial inhibition studies, VA carried out SEM analysis, AF, NA, DJ, AB and VPS


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analyzed the data and wrote the manuscript. All authors have given approval to the final version of the manuscript. Acknowledgment This work was supported by the excellence initiative of the German Federal and State Governments Grant EXC 294 (Centre for Biological Signaling Studies) and the University of Freiburg. Abbreviations PS, polysaccharide; ES, electrospinning; PBS, phosphate buffer saline; TGA, thermogravimetric analysis; CA, carboxylated agarose; IL, ionic liquid; SEM, scanning electronic microscope; MIC, minimum inhibition concentration; ZOI, zone of inhibition

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