Superhydrophobic PVDF and PVDF-HFP nanofibrous mats with antibacterial and anti-biofouling properties

Applied Surface Science 363 (2016) 363–371

Contents lists available at ScienceDirect

Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc

Superhydrophobic PVDF and PVDF-HFP nanofibrous mats with antibacterial and anti-biofouling properties M. Spasova a , N. Manolova a , N. Markova b , I. Rashkov a,∗ a b

Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St, bl. 103A, BG-1113 Sofia, Bulgaria Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev St, bl. 26, BG-1113 Sofia, Bulgaria

a r t i c l e

i n f o

Article history: Received 1 October 2015 Received in revised form 3 December 2015 Accepted 6 December 2015 Available online 12 December 2015 Keywords: Nanocomposites Electrospinning Superhydrophobicity PVDF PVDF-HFP Antibacterial activity

a b s t r a c t Superhydrophobic nanofibrous materials of poly(vinylidene fluoride) (PVDF) and poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) were prepared by one-pot electrospinning technique. The mats were decorated with ZnO nanoparticles with silanized surface and a model drug – 5-chloro-8hydroxyquinolinol (5Cl8HQ). The obtained hybrid nanofibrous materials were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), contact angle measurements, mechanical and microbiological tests. The results showed that the incorporation of ZnO nanoparticles into PVDF and PVDF-HFP nanofibers increased the hydrophobicity (contact angle 152◦ ), improved the thermal stability and imparted to the nanofibrous materials anti-adhesive and antimicrobial properties. The mats containing the model drug possessed antibacterial activity against Escherichia coli and Staphylococcus aureus. The results suggested that the obtained hybrid mats could find potential biomedical applications requiring antibacterial and anti-biofouling properties. © 2015 Elsevier B.V. All rights reserved.

1. Introduction In nature, biological micro/nanostructures have been developed as a result of millions of years of evolution and many plants and animals developed surfaces with specific properties. Recently, increased attention is paid on self-cleaning effect of the lotus leaf. The excellent water repellent properties of the lotus leaves known as lotus-leaf effect is due to the combination of a rough structure and the covering waxes on the leaves’ surface [1–3]. Inspired by nature, researchers have developed various methods for the preparation of superhyrdophobic surfaces such as phase separation [4], electrochemical deposition [5], chemical vapor deposition [6], crystallization control [7], photolithography [8], assembly [9], sol–gel methods [10] and solution-immersion methods [11]. Recently, electrospinning method was applied to obtain nanofibrous materials with controlled wettability [12–14]. Furthermore, considerable attention has been devoted to fluorine-containing polymers due to their unique characteristics, such as chemical, thermal and oxidative stability, hydrophobicity, excellent mechanical properties and biocompatibility. These

∗ Corresponding author. E-mail address: [email protected] (I. Rashkov). http://dx.doi.org/10.1016/j.apsusc.2015.12.049 0169-4332/© 2015 Elsevier B.V. All rights reserved.

characteristics were imparted by the specific properties of a fluorine atom [15]. Poly(vinylidene fluoride) (PVDF) is one of the most important fluorine-containing polymers with a wide range of medical applications (soft tissue repair and blood vessels prostheses) due to its good blood compatibility [16]. ZnO is a nontoxic, inexpensive material and has a photochemical and antibacterial activity. ZnO nanoparticles were successfully incorporated by electrospinning in poly (l-lactide) [17], poly(hydroxybutyrate-co-hydrohyvalerate) [18,19], alginate [20] and cellulose [21]. The aim of the present study is to increase the hydrophobicity and to impart anti-adhesive and antibacterial properties to PVDF and PVDF-HFP nanofibrous materials in order to use these new materials for potential biomedical application. To fulfil this aim, nanofibrous PVDF and PVDF-HFP mats decorated with ZnO nanoparticles and a model drug were fabricated by one-pot electrospinning. The morphology of the nanofibrous mats was observed by scanning electron microscopy (SEM), and the nanosized zinc oxide distribution – by transmission electron microscopy (TEM) and EDX mapping of zinc. The thermal degradation of the fibrous materials was evaluated by thermogravimetric analysis (TGA). The mechanical properties of the mats were determined by mechanical tests. The antibacterial activity of the materials against the pathogenic microorganism Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was tested.

364

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371

2. Materials and methods

crystallinity degree (c ) of the polymers in the fibrous materials was calculated using the following equation:

2.1. Materials c = Poly(vinylidene fluoride) (PVDF, Aldrich, France) with Mw = 180,000 g/mol and Ð = 2.53; poly(vinylidene fluoride-cohexafluoropropylene) (PVDF-HFP, Aldrich, USA) with Mw = 400,000 g/mol and Ð = 3.07 and commercial nanosized zinc oxide with silanized surface available under the trade mark Zano® 20 Plus of Umicore Zinc Chemicals – Belgium were used. The used Zn oxide is rod-like with diameter of 10–30 nm and length of 100 nm. N,N-dimethylformamide (DMF), acetone and 5-chloro-8quinolinol (5Cl8HQ) were of analytical grade of purity and were purchased from Sigma–Aldrich.

2.2. Preparation of nanofibrous PVDF and PVDF-HFP mats by electrospinning In the present study, PVDF, PVDF-HFP, PVDF/5Cl8HQ, PVDFHFP/5Cl8HQ, PVDF/ZnO and PVDF-HFP/ZnO nanofibrous mats were fabricated by one-pot electrospinning. The mats were prepared from the following polymer solutions and suspensions: (i) solutions of PVDF (25 wt%) and PVDF-HFP (20 wt%) in DMF/acetone (80/20 v/v); (ii) blend solutions of PVDF/5Cl8HQ (PVDF 25 wt%;) and PVDF-HFP/5Cl8HQ (PVDF-HFP 20 wt%) in DMF/acetone (80/20 v/v). 5Cl8HQ was 15 wt% of the polymer weight; and (iii) suspensions of PVDF/ZnO (PVDF 25 wt%;) and PVDF-HFP/ZnO (PVDF-HFP – 20 wt%) in DMF/acetone (80/20 v/v). ZnO was 30 wt% of the polymer weight. The suspensions were sonicated prior to be subjected to electrospinning (15 min in an ultrasonic bath Bandelin Sonorex, 160/640 W, 35 kHz). The spinning solutions and suspensions were loaded in a syringe equipped with a metal needle (gauge: 20GX1½ ) connected to the positively charged electrode of the high-voltage power supply (up to 30 kV). Electrospinning was conducted at a constant applied voltage of 25 kV and constant tip-to-collector distance of 20 cm using a grounded rotating aluminum collector (1000 rpm). The spinning suspensions were delivered at a constant rate of 0.5 mL/h enabled by the use of a pump Syringe Pump NE-300 (New Era Pump Systems, Inc.).

2.3. Characterization of the nanofibrous materials The rheological measurements of the used solutions and suspensions were performed using a Brookfield, DV – II+Pro, spindle CPE – 52, at 25 ◦ C. The morphology of the fibrous materials was evaluated by scanning electron microscopy (SEM). The samples (1 cm2 ) were vacuum-coated with gold and were observed with Jeol JSM-5510 (Jeol Ltd., Japan). The mean fiber diameter was estimated by Image J software [22], by measuring the diameters of at least 20 random fibers per sample from three different SEM micrographs for a total of 60 measurements and their morphology was assessed applying the criteria for overall evaluation of electrospun materials as described in details in [23]. TEM observations were carried out by JEM-2100 LaB6 (JEOL Co. Ltd.) operating at a voltage of 200 kV and equipped with EDS elemental analysis. In order to demonstrate that zinc oxide was incorporated into PVDF/ZnO and PVDF-HFP/ZnO fibers EDX mapping of zinc was applied. The thermal behavior of the obtained fibrous materials was evaluated by differential scanning calorimetry (DSC). The samples were heated in the temperature range from 0 to 200 ◦ C at heating rate of 10 ◦ C/min under nitrogen (TA Instruments, DSC Q2000, USA). The

H × 100%; Hm

where H is the melting enthalpy of the fibrous mats (J/g), Hm is the enthalpy when the PVDF is in a 100% crystalline state (104.7 J/g) [24]. The thermal stability of the fibrous materials was determined by thermogravimetry using TA Instruments TGA Q5000, USA in nitrogen. The heating was from r.t. to 800 ◦ C at a heating rate of 10 ◦ C/min. The water contact angles of the fibrous materials were measured using an Easy Drop DSA20E KRÜSS GmbH apparatus, Germany. Drops of distilled water with a volume of 10 ␮l were deposited on the surface of the test specimens (2 cm × 7 cm; cut in the direction of the collector rotation). The mean contact angle value was determined after averaging at least 10 measurements for each specimen. The tensile characteristics of the fibers were evaluated using a Zwick/Roell Z 2.5 apparatus (Germany), load cell 2 mV/V, type Xforce P, nominal force 2.5 kN, test Xpert II. The strain rate was 20 mm/min, the initial length between the clamps – 40 mm and the room temperature – 21 ◦ C. All samples were cut in the direction of collector rotation with dimensions of 20 mm × 60 mm and a thickness of ca. 100 ␮m. For the sake of statistical significance 10 specimens of each sample were tested, after which the average values of Young’s modulus, the ultimate stress and maximum deformation at break were determined. The antibacterial activity of the PVDF/ZnO and PVDF-HFP/ZnO fibrous materials against Gram-positive S. aureus 749 was evaluated by using the viable cell-counting method as described below. For comparison, the antibacterial activity of PVDF and PVDF-HFP against S. aureus was assessed as well. Upon appropriate dilution with sterilized 0.9% saline solution, a culture of about 105 cells/mL was prepared and used for antibacterial testing. The electrospun mats (0.1 g) were exposed to the bacteria (S. aureus) cell suspension (2 mL containing about 105 cells/mL). The growth of the pathogenic microorganism was estimated by illumination at  = 420 nm during the whole experiment. The distance between the lamp (10 W) and the surface of the Petri dishes was 10 cm. At specified time intervals (2, 4 and 24 h), aliquots of 50 ␮l were withdrawn from each suspension which had been in contact with the mats. Serial dilutions were made from the aliquots by deposition onto Petri dishes with nutrient agar (Sigma–Aldrich). The Petri dishes were incubated for 24 h at 37 ◦ C. The number of the survived bacteria was determined by counting the colony forming units (CFU) in triplicate for each experiment. Evaluation of the interaction between S. aureus and PVDF, PVDFHFP, PVDF/ZnO and PVDF-HFP mats was performed by direct SEM observation with Jeol JSM-5510 (Jeol Ltd., Japan) of S. aureus cells adhered to the surface of mats that had been in contact with a cell culture of S. aureus (ca. 105 cell/mL). Briefly, the mats were incubated in 2.0 mL of culture of S. aureus for 24 h. Then the samples were washed twice with phosphate buffered saline (PBS, pH 7.4) for removal of non-adhered bacteria. The adhered bacteria on the surface of the mats were fixed by immersion of the mats in 2.5 wt.% glutaraldehyde solution in PBS at 4 ◦ C for 5 h. Then the samples were washed carefully with PBS and freeze-dried. After 24-h contact of the mats with S. aureus cells and coating of the mats with gold, the bacterial morphology was observed with Jeol JSM-5510. The antibacterial activity of PVDF/5Cl8HQ and PVDFHFP/5Cl8HQ mats was monitored against S. aureus 749 and E. coli 3588 bacteria. In order to measure the inhibitory zones, in vitro studies were performed using Tryptone glucose extract agar (DIFCO Laboratories, Detroit, USA) solid medium. The surface of the solid agar was inoculated with a suspension of S. aureus and

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371

365

Fig. 1. SEM micrographs of: (A) PVDF (25 wt%) and (B) PVDF-HFP (20 wt%) nanofibrous materials; solvent DMF/acetone (80/20 v/v).

E. coli bacterial culture with a cell concentration of 1 × 105 cells/mL. In each Petri dish on the surface of the agar one disk (diameters of 17 mm and weight 2.0 mg) was placed. The Petri dishes were incubated for 24 h at 37 ◦ C and subsequently the zones of inhibition around the disks were measured. The average diameters of the zones of inhibition were determined using the ImageJ software based on 15 measurements in 15 different directions for each zone. 3. Results and discussion 3.1. Viscosity of the spinning solutions and suspensions It is well known that the viscosity of the spinning solutions has significant influence on the electrospinning process and the resultant fiber morphology [25]. The dynamic viscosity of the used in this study solutions and suspensions were measured. The viscosity of the PVDF (25 wt%) and PVDF-HFP (20 wt%) solutions in DMF/acetone was 585 and 125 cP, respectively. The incorporation of ZnO nanoparticles into the PVDF and PVDF-HFP solutions resulted in an increase of the viscosity. The measured viscosity for the PVDF/ZnO and PVDF-HFP/ZnO suspensions was 1050 cP and 1025 cP, respectively. The increase in suspension viscosity is most probably due to the strong interaction between the polar ZnO with large specific surface area and the polar polymer chains serves to constrain the mobility of PVDF and PVDF-HFP chains in the suspensions leading to a high viscosity. Our results are in agreement with the results obtained by Chae et al., where such an increase in the viscosity of PAN/ZnO suspensions has been demonstrated [26]. The incorporation of a low molecular weight drug into the PVDF and PVDF-HFP solutions did not alter the viscosity of the blend solutions in comparison to the PVDF and PVDF-HFP solutions (585 and 125 cP). The measured viscosities of the PVDF/5Cl8HQ and PVDFHFP/5Cl8HQ solutions were 590 and 180 cP, respectively. 3.2. Preparation of nanofibrous mats from PVDF, PVDF-HFP, PVDF/ZnO and PVDF-HFP/ZnO by electrospinning Nanofibrous mats of PVDF and PVDF-HFP were prepared by electrospinning of polymer solutions with concentration of 25 wt% for PVDF and 20 wt% for PVDF-HFP, respectively. The used polymer concentrations were selected after a series of preliminary experiments aimed to find the optimal conditions for electrospinning (polymer concentration, applied field strength). Based on the conducted preliminary experiments it was necessary to increase the polymer concentration to 25 wt% for the PVDF (dynamic viscosity – 585 cP) and to 20 wt% for the PVDF-HFP (dynamic viscosity – 125 cP) and to use tip-to-collector distance – 25 cm in order to obtain defect-free and dry fibers with uniform morphology that for use in the mechanical and biological tests.

Representative SEM images of the obtained PVDF and PVDF-HFP nanofibrous mats are shown in Fig. 1. The electrospinning of PVDF solutions (polymer concentration 25 wt%) under the selected conditions reproducibly resulted in obtaining nanofibers with mean fiber diameter of 146 ± 22 nm (Fig. 1A). The mean fiber diameter of the PVDF-HFP fibers was 138 ± 29 nm (Fig. 1B). It should be noted that the PVDF and PVDF-HFP fibers were with smooth surface. The electrospinning of PVDF/ZnO and PVDF-HFP/ZnO suspensions resulted in obtaining of hybrid fibrous PVDF and PVDF-HFP materials decorated with ZnO nanoparticles. The content of the nanosized ZnO filler was 30 wt% in respect to the polymer. The incorporation of ZnO nanoparticles into the PVDF and PVDF-HFP solutions resulted in preparation of fibers with larger diameters compared to the pristine PVDF and PVDF-HFP fibers. The increase in fiber diameter of the hybrid fibers was attributed to the significant increase in the viscosity of the PVDF/ZnO and PVDFHFP/ZnO suspensions, 1050 cР and 1025 cР, respectively. It is well known that the increase in solution viscosity resulted in an increase of the mean fiber diameter [27]. The mean fiber diameter of the hybrid PVDF/ZnO and PVDF-HFP fibers was 228 ± 50 nm and 147 ± 39 nm, respectively. It was observed that the incorporation of ZnO nanoparticles resulted in the preparation of fibers with rough surface which is most probably due to the formation of ZnO aggregates (Fig. 2A and C). TEM micrographs of the PVDF/ZnO and PVDF-HFP fibrous mats are presented in Fig. 2B and D. As seen from the TEM micrographs and EDX mapping of Zn (inset), the zinc oxide was distributed mainly in the fibers’ bulk; however some ZnO aggregates were formed. It may be assumed that one of the possible reasons for the presence of aggregates in the fibers is the solvent evaporation during the flight of the jet from the nozzle to the collector. 3.3. Preparation of nanofibrous mats from PVDF/5Cl8HQ and PVDF-HFP/5Cl8HQ In the present study an 8-hydroxyquinoline derivative was used as model drug: the 5-chloro-8-quinolinol (5Cl8HQ). The incorporation of 5Cl8HQ which possesses antibacterial and hydrophobic properties was expected to result in obtaining PVDF and PVDF-HFP fibrous mats with strong antibacterial activity. SEM micrographs of the obtained PVDF/5Cl8HQ and PVDF-HFP/5Cl8HQ fibers are presented in Fig. 3. The mean fiber diameter of PVDF/5Cl8HQ and PVDFHFP/5Cl8HQ fibers was 118 ± 20 nm and 107 ± 30 nm, respectively. The drug-containing fibers had smaller diameters as compared to the pristine fibers (146 ± 22 nm for PVDF and 138 ± 29 nm for PVDF-HFP fibers). The incorporation of a model drug led to a decrease of the mean diameter value of the fibers, which might be attributed to the ionogenic nature of the drug used. This decrease is in accordance with previous findings about fibrous materials

366

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371

Fig. 2. SEM (A and C) and TEM (B and D) micrographs and mapping of Zn (inset) of the fibrous mats of: PVDF/ZnO (A and B) and PVDF-HFP/ZnO (C and D).

Fig. 3. SEM micrographs of: (A) PVDF/5Cl8HQ and (B) PVDF-HFP/5Cl8HQ nanofibrous materials.

prepared by electrospinning of polymer solutions containing an ionogenic low molecular weight additive [28–30]. 3.4. Degree of crystallinity and thermal stability of the nanofibrous mats The processing conditions have a strong influence of the resulting structures of the composites. In the polymer–filler nanocomposites, the dispersed fillers’ particles in a polymer matrix always act as a heterogeneous nucleating agent for the spherulites. However, such nucleating effect of nanofillers is more pronounced at low loading levels, ca. 1–5 wt%. Above these levels, the fillers may hinder the movement of polymer chains, thus retarding the crystallization of spherulites. The restricted mobility of the chains imposed by higher filler content would not allow the growth of well developed lamellar crystals [31]. In the present study, mats of PVDF, PVDF/ZnO, PVDF/5Cl8HQ, PVDF-HFP, PVDF-HFP/ZnO and PVDF-HFP/5Cl8HQ were subjected to DSC analysis. Representative DSC thermograms are shown in

Table 1 Values of Tm , H and c in the PVDF, PVDF/ZnO, PVDF/5Cl8HQ, PVDF-HFP, PVDFHFP/ZnO and PVDF-HFP/5Cl8HQ mats. Fibrous mat

Tm , ◦ C

H, J/g

c , %

PVDF PVDF/ZnO PVDF/5Cl8HQ PVDF-HFP PVDF-HFP/ZnO PVDF-HFP/5Cl8HQ

168.9 168.9 166.7 142.7 142.1 140.5

57.4 40.8 56.3 30.2 22.8 30.3

54.8 38.9 53.7 28.8 21.7 28.9

Fig. 4. It was found that the melting temperature (Tm ), enthalpy and the degree of crystallinity were affected by the composition of the fibrous mats (Table 1). It was found that the pristine PVDF fibrous mat had the highest degree of crystallinity – 54.8%. The incorporation of ZnO nanoparticles in such concentration (30 wt%) resulted in decrease in the crystallinity and increase of the amorphous phase. As stated above, the addition of the filler (in high concentrations) hinders the

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371

367

Fig. 4. DSC thermograms (first heating run) of (A) PVDF, PVDF/ZnO and PVDF/5Cl8HQ and (B) PVDF-HFP, PVDF-HFP/ZnO and PVDF-HFP/5Cl8HQ.

Fig. 5. TGA thermograms of mats from: (A) PVDF, PVDF/ZnO and PVDF/5Cl8HQ and (B) PVDF-HFP, PVDF-HFP/ZnO and PVDF-HFP/5Cl8HQ.

movement of PVDF and PVDF-HFP chains and thus way hampers the polymer crystallization which resulted in decreased crystallinity of the hybrid materials. The results we have obtained are consistent with the literature data for decrease in the crystallinity degree of PCL in the presence of ZnO (concentrations higher than 1 wt%) [32]. The incorporation of the model drug resulted in slight decrease of the melting temperature, which is due to fact that the drug 5Cl8HQ melts at 125 ◦ C. The thermal stability and degradation of the fibrous materials was estimated by TGA analysis. The thermograms of PVDF, PVDF/ZnO, PVDF/5Cl8HQ, PVDF-HFP, PVDF-HFP/ZnO and PVDFHFP/5Cl8HQ are shown in Fig. 5. As seen, the pristine mats destructed thermally in one step and the thermal degradation temperatures for 5% and 50% weight loss were 450 ◦ C and 473 ◦ C for the PVDF mat and 439 ◦ C and 462 ◦ C for the PVDF-HFP mat, respectively. The incorporation of ZnO nanoparticles resulted in increase of the thermal stability of the obtained hybrid mats. The nanofibrous mats decorated with ZnO degrade at higher temperatures. This is due to the fact that ZnO particles have excellent thermal stability [33]. The incorporation of ZnO particles into the PVDF and PVDF-HFP mats increased their thermal stability and the thermal degradation temperatures for 5% and 50% weight loss were 462 and 497 ◦ C for the PVDF/ZnO mat, and 451 ◦ C and 492 ◦ C for the PVDF-HFP/ZnO mat, respectively. The presence of a small step at around 370 ◦ C in the TGA thermograms of the all mats containing ZnO particles is due to the thermal degradation of the organic phase in ZnO (the used ZnO is with silanized surface). PVDF/5Cl8HQ and PVDF-HFP/5Cl8HQ mats destructed thermally in two steps. The first stage was at ca. 142 ◦ C, and it is due to the

thermal degradation of the drug. In the second stage the thermal degradation temperature was ca. 430 ◦ C due to the polymer degradation. 3.5. Water contact angle of the nanofibrous mats In the present study development of superhydrophobic PVDF and PVDF-HFP fibrous mats decorated with ZnO nanoparticles and with an embedded model drug is achieved by combining biomimetic approaches and nanotechnology such as electrospinning. The relationship between the 3D micro- and nanoarchitecture of the mats and their hydrophobic behavior was found. The water contact angle values of the obtained nanofibrous materials and the images of the distilled water droplet (10 ␮l), deposited onto the surface of the mats are shown in Fig. 6. The average values of the water contact angle of PVDF and PVDF-HFP mats were 143 ± 2.3◦ and 141 ± 2.6◦ , respectively. The incorporation of ZnO particles led to hydrophobization of the mats and to the obtaining of fibrous PVDF and PVDF-HFP mats with superhydrophobic properties. It should be noted that the highest value of the water contact angle was determined for PVDF/ZnO mat −152◦ and the water droplet preserved its spherical shape after deposition. It is noteworthy that despite of the type of the polymer, the “decoration” with ZnO resulted in an increase of the water contact angle. This is due to the fabrication of hybrid fibrous mats with rough surface, resembling the lotus leave architecture, as well as because of the use of surface silanized ZnO nanoparticles that possessed hydrophobic properties. The obtained results demonstrate the influence of the topography of the surface of the fibrous materials on the water contact angle values.

368

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371

Fig. 6. Images of distilled water droplet (10 ␮l) onto mats from: (A) PVDF, (B) PVDF/5Cl8HQ, (C) PVDF/ZnO, (D) PVDF-HFP, (E) PVDF-HFP/5Cl8HQ and (F) PVDF-HFP/ZnO.

Fig. 7. Digital image of water droplets deposited onto the PVDF/ZnO mat. For better visualization the water droplets were coloured with reactive red dye. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Table 2 Mechanical properties (Et – Young’s modulus; εB – elongation at break;  B – tensile strength) of the nanofibrous mats. Sample

Et , MPa

PVDF PVDF/ZnO PVDF/5Cl8HQ PVDF-HFP PVDF-HFP/ZnO PVDF-HFP/5Cl8HQ

46.7 11.7 28.0 37.0 8.5 29.4

± ± ± ± ± ±

εB , % 4.4 2.2 1.6 4.5 1.9 1.6

32.7 32.5 38.2 57.9 34.8 46.5

 B , MPa ± ± ± ± ± ±

2.6 2.4 2.1 2.4 2.0 1.9

10.2 2.3 3.7 5.6 2.2 4.4

± ± ± ± ± ±

1.4 0.1 0.6 1.5 0.2 0.3

Digital image of water droplets deposited onto the PVDF/ZnO mat (water contact angle 152◦ ) were presented in Fig. 7. For the better visualization, the water was coloured with a Reactive Red dye. As seen, the droplets preserved their spherical shape and were not absorbed after their contact with the superhydrophobic mat. Moreover, after tilting of the mat, the droplets rolled away leaving the mat dry. 3.6. Mechanical behavior of the fibrous mats The mechanical properties of the fibrous materials obtained by electrospinning depend on the diameters and morphology of the fibers, their inter-connectivity, presence of defects, arrangement, incorporation of fillers, degree of crystallinity, etc. [34,35]. The results from the performed mechanical tests (typical tensile stress–strain curves) of the fibrous materials are presented in Fig. 8. In Table 2, the values of Young’s modulus, ultimate elongation at break and tensile strength of the mats are listed. The determination of the mechanical characteristics of the PVDF and PVDF-HFP mat revealed that the pristine fibres had the highest values of Young’s modulus, tensile strength and elongation at break. The greatest strength (10.2 MPa) was manifested by PVDF nanofibrous

mat which might be attributed to the highest degree of crystallinity, 54.8%. The values of Young’s modulus and the elongation at break for the PVDF mat was 46.7 MPa and 32.7%, respectively. The studies on the impact of the nanofillers (ZnO or 5Cl8HQ) on the mechanical behaviour of the mats revealed that the incorporation of the filler in such concentrations (30 wt% for ZnO and 15 wt% for 5Cl8HQ in respect to the polymer weight) retarded the mechanical properties of the hybrid mats. This is due to the fact that nanoparticles have high surface energy and are easy to aggregate, which leads to the poor dispersion of them in polymer matrix. The filler particles act as stress concentration centers which hinder the transfer of tension from the polymer matrix to the filler [32] and where cracks initiates on [31]. Furthermore, as mentioned earlier, the incorporation of the filler led to a drastic decrease in the crystallinity of the polymer. These results indicated that hybrid nanofibrous mats with lower crystallinity had weaker mechanical properties.

3.7. Microbiological tests Antibacterial activity of the nanofibrous PVDF, PVDF-HFP, PVDF/ZnO and PVDF-HFP/ZnO materials against S. aureus was tested in liquid medium after exposure for certain time intervals. The number of survived bacteria was subsequently assessed by plating and counting of CFU in solid medium. The log of the survival cells versus the exposure time for the nanofibrous mats is presented in Fig. 9. For comparison the growth of a control of the S. aureus was assessed as well. It was found that the control developed normally during the experiment. As seen from Fig. 9, significant decrease in the number of the viable cells was detected for the exposure time of 2 h for the mats decorated with ZnO. The hybrid PVDF/ZnO and PVDF-HFP/ZnO mats manifest antibacterial activity and for the contact of 24 h, a decrease of S. aureus titer by more than 3 log units was attained. In contrast, pristine PVDF and PVDF-HFP nanofibrous mats did not alter the bacterial growth. It is known that superhydrophobic surfaces can reduce the infections by reducing the S. aureus adherence, i.e. superhydrophobicity is one of the factors which influence the bacterial adhesion [36]. In order to evaluate the attachment of the bacterial cells to the surface of the fibrous materials, the PVDF, PVDF/ZnO, PVDF-HFP and PVDF-HFP/ZnO mats were incubated for 24 h in a culture medium containing S. aureus. Then, the cells were fixed with glutaraldehyde solution, washed with distilled water, and freeze-dried. SEM

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371

369

Fig. 8. Stress–strain curves of: (A) PVDF, PVDF/5Cl8HQ and PVDF/ZnO and (B) PVDF-HFP, PVDF-HFP/5Cl8HQ and PVDF-HFP/ZnO mats. The samples were cut in such a manner so that their length axis coincided with the direction of the collector rotation.

Fig. 9. Logarithmic plot of the viable bacteria cell number versus the exposure time Control – S. aureus; (in hours). Test has been carried out against S. aureus. PVDF; PVDF-HFP; PVDF/ZnO; PVDF-HFP/ZnO.

micrographs of the pristine PVDF and hybrid PVDF/ZnO mats that were in contact with S. aureus are shown in Fig. 10. It was found that a small number of S. aureus cells adhered to the pristine PVDF and PVDF-HFP mats. However, the cell morphology was not altered (remained round and with smooth surfaces). Moreover, a S. aureus cell which is dividing is shown in Fig. 10A. This indicates that PVDF and PVDF-HFP fibrous mats possess antiadhesive properties; however they do not alter the growth of pathogenic microorganisms. This result is in accordance with the microbiological test for the antimicrobial activity where it was

found that for the pristine PVDF and PVDF-HFP after 24 h contact with S. aureus, the number of the bacterial cells increased up to ca. 5.3 log units (Fig. 9). The small number of the adhered bacterial cells to the pristine fibrous mats can be explained with the high value of the contact angle of these mats, ca. 142◦ which imparts them anti-adhesive properties. In contrast, none adhered bacterial cells were detected onto the surface of the hybrid nanofibrous mats decorated with ZnO (Fig. 10B). This is due to the antibacterial properties of the incorporated ZnO and the preparation of superhybrophobic fibrous materials with a contact angle of 152◦ . This result is in agreement with the results obtained from the microbiological test where it was shown that the incorporation of ZnO into the PVDF and PVDF-HFP mats resulted in obtaining fibrous mats with antibacterial activity. The obtained results are in accordance with the results reported by the other authors that have been established that the increase of the value of the contact angle resulted in the decrease of the adhered S. aureus cells [37]. Moreover, it is known that ZnO possessed antibacterial properties [17,38,39]. The second approach that we have proposed in this study is the incorporation of a model drug into the PVDF and PVDF-HFP fibrous mats in order to impart to them antibacterial activity. 5Cl8HQ is a broad-spectrum antibacterial agent with minimum inhibitory concentration against S. aureus of 0.25 ␮g/mL [30,40]. The antibacterial activity of the PVDF/5Cl8HQ and PVDFHFP/5Cl8HQ mats was assessed by performance of microbiological tests against the S. aureus and E. coli microorganisms. The results obtained by determination of the zones of inhibition after a 24-h contact of the fibrous materials with the bacterial cells are shown in Fig. 11. For sake of comparison pristine PVDF and PVDF-HFP mats were also tested. A blank sample (without any fibrous material) is presented as well.

Fig. 10. SEM micrographs of (A) pristine PVDF mat and (B) hybrid PVDF-HFP/ZnO mat that were incubated in cell culture (S. aureus – 105 cells/mL) for 24 h at 37 ◦ C.

370

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371

Fig. 11. Digital images of the inhibitory zones against S. aureus (upper row) and E. coli (bottom), detected after a 24-h contact of the drug-(non)containing PVDF and PVDF-HFP fibrous materials with the bacterial cells. Left image – blank control.

As expected, pristine PVDF and PVDF-HFP mats did not exhibit any antibacterial activity. Well-distinguished zones of inhibition of the bacterial cells growth were detected for the drug-containing PVDF and PVDF-HFP mats. The diameters of the zones of inhibition of PVDF/5Cl8HQ and PVDF-HFP/5Cl8HQ mats were 4.4 cm and 5.5 cm against S. aureus and 3.5 cm and 4.9 cm against E. coli, respectively. The presence of inhibitory zones evidenced that the incorporated drug in the PVDF and PVDF-HFP mats imparted antibacterial activity to the mats. 4. Conclusion Novel hybrid PVDF and PVDF-HFP nanofibrous mats decorated with ZnO nanoparticles and a model drug were successfully prepared by one-pot electrospinning. It was found that the incorporation of ZnO particles resulted in the creation of superhydrophobic fibrous materials with contact angles of 152◦ . Moreover, the mats decorated with ZnO showed enhanced thermal stability and possessed anti-adhesive and antibacterial properties. The incorporation of the model drug – 5Cl8HQ into the PVDF and PVDFHFP fibrous materials imparted antibacterial properties against S. aureus and E. coli to them. The results revealed that the obtained superhybrophobic and antibacterial PVDF and PVDF-HFP nanofibrous materials can find potential biomedical applications. Acknowledgments MS thanks Prof. Ph. Dubois (University of Mons – UMONS, Belgium) for providing the possibility to perform mechanical analyses at the Laboratory of Polymeric and Composite Materials in the frame of the POLINNOVA project FP7-REGPOT-2012-2013-1, Grant 316086. References [1] A. Marmur, The lotus effect: superhydrophobicity and metastability, Langmuir 20 (2004) 3517–3519. [2] W. Barthlott, C. Neinhuis, Purity of the sacred lotus, or escape from contamination in biological surfaces, Planta 202 (1997) 1–8. [3] C. Neinhuis, W. Barthlott, Characterization and distribution of water-repellent, self-cleaning plant surfaces, Ann. Bot. 79 (1997) 667–677. [4] J.T. Han, X.R. Xu, K.W. Cho, Diverse access to artificial superhydrophobic surfaces using block copolymers, Langmuir 21 (2005) 6662–6665.

[5] M. Li, J. Zhai, H. Liu, Y. Song, L. Jiang, D. Zhu, Electrochemical deposition of conductive superhydrophobic zinc oxide thin films, J. Phys. Chem. B 107 (2003) 9954–9957. [6] B. Wang, J. Feng, Y. Zhao, T. Yu, Fabrication of novel superhydrophobic surfaces and water droplet bouncing behavior – part 1: stable ZnO–PDMS superhydrophobic surface with low hysteresis constructed using ZnO nanoparticles, J. Adhes. Sci. Technol. 24 (2010) 2693–2705. [7] T. Onda, S. Shibuichi, N. Satoh, K. Tsujii, Super-water-repellent fractal surfaces, Langmuir 12 (1996) 2125–2127. [8] D. Oner, T. McCarthy, Ultrahydrophobic surfaces. Effects of topography length scales on wettability, Langmuir 16 (2000) 7777–7782. [9] F. Shi, Z.Q. Wang, X. Zhang, Combining a layer-by-layer assembling technique with electrochemical deposition of gold aggregates to mimic the legs of water striders, Adv. Mater. 17 (2005) 1005–1009. [10] H. Shang, Y. Wang, S. Limmer, T. Chou, K. Takahashi, G.Z. Cao, Optically transparent superhydrophobic silica-based films, Thin Solid Films 472 (2005) 37–43. [11] S.T. Wang, L. Feng, L. Jiang, One-step solution-immersing process towards bionic superhydrophobic surfaces, Adv. Mater. 18 (2006) 767–770. [12] H. Wu, R. Zhang, Y. Sun, D. Lin, Z. Sun, W. Pan, P. Downs, Biomimetic nanofiber patterns with controlled wettability, Soft Matter 4 (2008) 2429–2433. [13] H.S. Lim, J.H. Baek, K. Park, H.S. Shin, J. Kim, J.H. Cho, Multifunctional hybrid fabrics with thermally stable superhydrophobicity, Adv. Mater. 22 (2010) 2138–2141. [14] D. Cho, H. Zhou, Y. Cho, D. Audus, Y.L. Joo, Structural properties and superhydrophobicity of electrospun polypropylene fibers from solution and melt, Polymer 51 (2010) 6005–6012. [15] G. Laroche, Y. Marois, R. Guidoin, M. King, L. Martin, T. How, Y. Douville, Polyvinylidene fluoride (PVDF) as a biomaterial: from polymeric raw material to monofilament vascular suture, J. Biomed. Mater. Res. 29 (1995) 1525–1536. [16] J.C. Lin, S.L. Tiong, C.Y. Chen, Surface characterization and platelet adhesion studies on fluorocarbons prepared by plasma-induced graft polymerization, J. Biomater. Sci. Polym. Ed. 11 (2000) 701–714. [17] D. Virovska, D. Paneva, N. Manolova, I. Rashkov, D. Karashanova, Electrospinning/electrospraying vs. electrospinning: a comparative study on the design of poly(l-lactide)/zinc oxide non-woven textile, Appl. Surf. Sci. 311 (2014) 842–850. [18] W. Yu, C.-H. Lan, S.-J. Wang, P.-F. Fang, Y.-M. Sun, Influence of zinc oxide nanoparticles on the crystallization behavior of electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanofibers, Polymer 51 (2010) 2403–2409. [19] R. Naphade, J. Jog, Electrospinning of PHBV/ZnO membranes: structure and properties, Fibers Polym. 13 (2012) 692–697. [20] K.T. Shalumon, K.H. Anulekha, S.V. Nair, K.P. Chennazhi, R. Jayakumar, Sodium alginate/poly(vinyl alcohol)/nano ZnO composite nanofibers for antibacterial wound dressings, Int. J. Biol. Macromol. 49 (2011) 247–254. [21] S. Ye, D. Zhang, H. Liu, J. Zhou, ZnO nanocrystallites/cellulose hybrid nanofibers fabricated by electrospinning and solvothermal techniques and their photocatalytic activity, J. Appl. Polym. Sci. 121 (2011) 1757–1764. [22] W.S. Rasband, ImageJ, U.S. National Institute of Health, Bethesda, MD, 1997–2006, http://rsb.info.nih.gov/ij/. [23] M. Spasova, R. Mincheva, D. Paneva, N. Manolova, I. Rashkov, Perspectives on: criteria for complex evaluation of the morphology and alignment of electrospun polymer nanofibers, J. Bioact. Compat. Polym. 21 (2006) 465–479.

M. Spasova et al. / Applied Surface Science 363 (2016) 363–371 [24] Y.D. Wang, M. Cakmak, Hierarchical structure gradients developed in injection-molded PVDF and PVDF–PMMA blends. I. Optical and thermal analysis, J. Appl. Polym. Sci. 68 (1998) 909–926. [25] Z.-M. Huang, Y.-Z. Zhang, M. Kotaki, S. Ramakrishna, A review on polymer nanofibers by electrospinning applications in nanocomposites, Compos. Sci. Technol. 63 (2003) 2223–2253. [26] D. Chae, B. Kim, Effects of interface affinity on the rheological properties of zinc oxide nanoparticle-suspended polymer solutions, Macromol. Res. 18 (2010) 772–776. [27] H. Fong, I. Chun, D. Reneker, Beaded nanofibers formed during electrospinning, Polymer 40 (1999) 4585–4592. [28] A. Toncheva, D. Paneva, N. Manolova, I. Rashkov, Electrospun poly(l-lactide) membranes containing a single drug or multiple drug system for antimicrobial wound dressings, Macromol. Res. 19 (2011) 1310–1319. [29] A. Toncheva, M. Spasova, D. Paneva, N. Manolova, I. Rashkov, Drug-loaded electrospun polylactide bundles, J. Bioact. Compat. Polym. 26 (2011) 161–172. [30] N. Stoyanova, D. Paneva, R. Mincheva, A. Toncheva, N. Manolova, Ph. Dubois, I. Rashkov, Poly(l-lactide) and poly(butylene succinate) immiscible blends: from electrospinning to biologically active materials, Mater. Sci. Eng. C 41 (2014) 119–126. [31] S. Tjong, Structural and mechanical properties of polymer nanocomposites, Mater. Sci. Eng. R 53 (2006) 73–197. [32] R. Augustine, H.N. Malik, D.K. Singhal, A. Mukherjee, D. Malakar, N. Kalarikkal, S. Thomas, Electrospun polycaprolactone/ZnO nanocomposite membranes as biomaterials with antibacterial and cell adhesion properties, J. Polym. Res. 21 (2014) 347–363.

371

[33] M. Murariu, A. Doumbia, L. Bonnaud, A.L. Dechief, Y. Paint, M. Ferreira, C. Campagne, E. Devaux, Ph. Dubois, High-performance polylactide/ZnO nanocomposites designed for films and fibers with special end-use properties, Biomacromolecules 12 (2011) 1762–1771. [34] S.-C. Wong, A. Baji, S. Leng, Effect of fiber diameter on tensile properties of electrospun poly(ε-caprolactone), Polymer 49 (2008) 4713–4722. [35] F. Chen, Y. Su, X. Mo, C. He, H. Wang, Y. Ikada, Biocompatibility, alignment degree and mechanical properties of an electrospun chitosan–P(LLA-CL) fibrous scaffold, J. Biomater. Sci. Polym. Ed. 20 (2009) 2117–2128. [36] P. Tang, W. Zhang, Y. Wang, B. Zhang, H. Wang, C. Lin, L. Zhang, Effect of superhydrophobic surface of titanium on Staphylococcus aureus adhesion, J. Nanomater. 2011 (2011) 1–8. [37] X. Zhang, L. Wang, E. Levanen, Superhydrophobic surfaces for the reduction of bacterial adhesion, RSC Adv. 3 (2013) 12003–120020. [38] D. Haciu, S. Saner, O. Türdo˘gru, U. Ünal, Study of antibacterial effects of a self-standing agar based film incorporated with ZnO, Front. Sci. 3 (2013) 96–101. [39] A. Sirelkhatim, S. Mahmud, A. Seeni, N. Kaus, L. Ann, S. Bakhori, H. Hasan, D. Mohamad, Review on zinc oxide nanoparticles: antibacterial activity and toxicity mechanism, Nano Micro Lett. 7 (2015) 219–242. [40] P. Hongmanee, K. Rukseree, B. Buabut, B. Somsri, P. Palittapongarnpim, In vitro activities of cloxyquin (5-chloroquinolin-8-ol) against Mycobacterium tuberculosis, Antimicrob. Agents Chemother. 51 (2007) 1105–1106.