Highly Flexible Electrospun Hybrid (Polyurethane/Dextran/Pyocyanin) Membrane for Antibacterial Activity via Generation of Oxidative Stress.

A hybrid nanofibrous mat consisting of polyurethane, dextran, and 10 wt % of biopigment (i.e., pyocyanin) was facilely fabricated using a direct-conventional electrospinning method. The field emission scanning electron microscopy showed the bead-free fibers with a twisted morphology for the pyocyanin-loaded mat. The addition of pyocyanin enables the unprecedented approach to tailor the hydrophilicity of hybrid mat, as verified from the water contact measurement. Thermomechanical stabilities of electrospun mats were investigated in terms of thermogravimetric analysis, differential scanning calorimetry, and dynamic mechanical analysis. The bacterial inhibition test revealed that the antibacterial activity of electrospun mat containing pyocyanin was 98.54 and 90.2% toward Escherichia coli and Staphylococcus aureus, respectively. By the combined efforts of rapid release of pyocyanin and oxidative stress, the PU-dextran-pyocyanin (PUDP) electrospun mat significantly declined the viable cell number that disrupts the cell morphology. Hence, the proposed PUDP electrospun mat must meet the requirements of efficient antimicrobial material in various applications such as disinfectant wiping, food packaging, and textile industries.

Introduction

The development of disposable, cost-competitive, potential, and selective destabilization of bacteria is an urgent need in pharmaceuticals, filtrations, textiles, food packaging, and environmental monitoring, owing to their serious and often infections to the living environment. Despite various advancements in the medicinal chemistry domain, most of the natural compounds can only able to form folk medicines such as penicillin, carbapenems, and cephalosporins.[1−4] Although they are in current use after semi-synthetic modifications, still there remains a significant clinical concern. The continuous evolution of pathogenic bacterial resistance to existing semi-synthetic drugs in accordance with the evolutionary process of continuous drug developments demands an alternative emerging agent such as natural bioactive compounds pyocyanin (N-methyl-1-hydroxyphenazine). Pyocyanin is a pro-oxidant and redox-modulating secondary metabolite, which has superior antibacterial activity regulated by Pseudomonas aeruginosa.[5−7] Pyocyanin can readily diffuse inside the cell membrane of bacteria because of its low-molecular-weight (210 Da) zwitterion and subsequently can convert molecular O2 to superoxide O2× as a nonenzymatic oxidizing agent using NAD(P)H.[8,9] It was reported that about 90–95% of antimicrobial inhibitions of P. aeruginosa strains caused because of the generation of water-soluble secondary metabolite, that is, pyocyanin.[6] El-Zawawy et al. investigated about the combating efficiency of pyocyanin ointment against skin fungus, that is, Trichophyton rubrum.[10] In another one study, it was reported that substances such as pyocyanin secreted by P. aeruginosa showed antibiotic actions in vivo on Candida species which were grown on Sabroud’s Dextrose Agar.[11] The pyocyanin has been applied to fight against other pathogens to reduce crude oil degradation, whereas biosurfactants are used in textile industries and leather processing industry because of their antibacterial efficiency.[12] According to Saha et al., phenazine pigment pyocyanin can be exploiting as an antibacterial agent and food colorants in food and beverage industries.[13] There are reports on the antagonistic effect of P. aeruginosa against Staphylococcus aureus, Salmonella paratyphi, Escherichia coli, and Klebsiella pneumoniae by synthesizing pyocyanin.[6,13,14] The aforementioned reports certify that pyocyanin is a potential bioactive compound to destabilize the bacteria and can applicable in antibiotics, membrane filters, protective textiles, and food packaging. Different sorts of antibacterial carrier systems have been developed, by incorporation of antibacterial substances into a packet connected to the carrier, direct incorporation of antibacterial substances into carrier films/coating the antibacterial packages on the carrier matrix.

Polymer nanofibers hold considerable attention in diverse applications such as optical devices, filtrations, energy devices, and biomedical areas because of their exclusive features including ultrahigh specific surface area, flexibility, tailored morphology of fibers, tunable pore size, and drug-carrying competency. A number of techniques including pressurized gyration, phase separation, blowing, melt/solution blowing, and centrifugal spinning have been developed for the fabrication of polymer nanofibers. At present, electrospinning has emerged as a well-recognized engineering technique to fabricate seamless ultrafine fibers with tunable diameter size. In the electrospinning process, polymer solution/melt is driven electrostatically from the syringe and the discharged polymer solution jet is accelerated toward the grounded collector because of charge separation.

Further, the solvent evaporates, and the highly stretched polymer fiber deposits on a grounded collector.[15,16] By incorporation of a biocide in the electrospun nanofiber membrane, the unique result toward structural composition and excellent antibacterial activity can be achieved.[17] Kenaway et al. first reported about the incorporation of a biocide agent in nanofibers through electrospinning with controlled release.[18] Because numerous hydrophobic and hydrophilic biocides including chlorhexidine, triclosan, QACs, and PHMB have been applied to conventional fibers for various industrial and household disinfections as well as for antibacterial finishing,[19−21] these biocides are typically mixed in the polymer solutions prior to electrospinning and in consequence a burst release of those bioactive agents from the nanofibers inside any aqueous solutions. One previous study showed that the control release of ampicillin from electrospun poly (methyl methacrylate)–nylon 6 core/shell-based nanofibers was able to inhibit Listeria innocua growth effectively.[22] Kim et al. published that the sustained release of drug (Mefoxin) from electrospun poly(lactide-co-glycolide)-based scaffold nanofibers was effective to inhibit S. aureus growth up to 90%.[23]

Polyurethane (PU) is a cost-effective, biodegradable, chemical resistance, non-antigenic, and nonimmunogenic polymer. It is associated with intrinsic features such as elasticity, clarity, tunable mechanical properties, and self-healing effects.[24] The network of physical bond of PU is capable of rearrangement when it was subjected to different temperatures and different mechanical loads and while interaction with rigid heterogeneous domains.[25] Although, PU is suitable for an electrospun nanofiber membrane, it has low compatibility with biocide because of its extreme hydrophobic behavior.[26] If any hydrophilic polymer blends with PU, then, it enables the electrospun nanofiber membrane to encapsulate biocide. Dextran, produced by lactic acid bacteria, is a highly suitable polymer for producing the electrospun blend membrane because it is compatible with both hydrophobic PU and hydrophilic bioactive agents because of its amphiphilic behavior.[26] This unique solubility characteristic of dextran helps for directly blending with biodegradable polymers such as PU to prepare composite nanofibrous membranes through electrospinning after mixed solution in organic solvents. Because of its unique solubility characteristic, mechanical strength and swelling properties of dextran in water could thereby be modulated.[27] Although P. aeruginosa can cause cystic fibrosis by producing pyocyanin during infection, this study aims a trail to develop modified novel antibacterial PU–dextran (PUD) electrospun nanofibers by incorporating natural pyocyanin extracted from P. aeruginosa as a promising biomedical antibacterial active compound after considering its great bactericidal efficiencies against bacteria in order to apply in various fields such as biomedical, textile, and food packaging. Pyocyanin has been used as a barrier to prevent bacterial growth. The pyocyanin production by bacteria was determined by the LC–MS technology. Furthermore, the modified nanofibers were explored in order to assess an antibacterial efficiency against Gram-positive S. aureus and Gram-negative E. coli, and in vitro reactive oxygen species (ROS) generation in the presence of pyocyanin incorporated with nanofibrous scaffolds was also determined. The bacterial apoptotic death was analyzed by FACS. The scaffolds were further characterized by scanning electron, atomic force microscope (AFM), thermogravimetric analysis (TGA), dynamic mechanical analysis (DMA), and by Fourier transform infrared (FT-IR) technology.

Experimental Section

Materials

PU (medical grade Mw = 110 000) was received from Cardio Tech Intern., Japan. Dextran (from Leconostoc mesenteroides, average Mw = 8500–11 500) was purchased from Sigma-Aldrich. N,N-Dimethyl formamide (DMF) and methyl ethyl ketone/2-butanone (MEK) were procured from Samchun Chemicals, South Korea. The Luria–Bertani medium was obtained from BD Difco, United States. Standard pyocyanin and 2′,7′-dichlorofluorescin diacetate were supplied by Sigma-Aldrich. S. aureus (KACC 10768) and E. coli (KACC 11304) were collected from Korean Agricultural Culture Collection. P. aeruginosa (KCTC 1750) was obtained from Korean collection for type Cultures. FITC-Annexin V with a PI apoptosis detection kit was purchased from Bio-legend, San Diego, United States.

Pyocyanin Production

Bacto Peptone (20 g), MgCl2 (1.4 g), K2SO4 (10 g), glycerol (2%), and glucose (2%) were charged together into 1000 mL of DI water in a conical flask.[28,29] The solution was then transferred to an autoclave and sterilized at 121 °C for 15 min. Separately, P. aeruginosa PAO1 was grown in a Luria–Bertani medium at 37 °C for 12 h. The P. aeruginosa PAO1 suspension was inoculated into the above-prepared pyocyanin Broth medium (1:100 v/v) at 37 °C for 48 h. The obtained supernatants were collected, centrifuged (10 000 rpm for 10 min), and filter-sterilized at room temperature.

Pyocyanin Extraction and Purification

Pyocyanin was extracted according to the procedure reported in refs (30) and (31). The above-prepared filter-sterilized supernatant and chloroform (1:1 v/v) were mixed thoroughly by vortex in a test tube. The obtained pale blue suspension was collected and dried at room temperature for 72 h. The residue was then dissolved in 1 mL of acetonitrile and subjected to liquid chromatography–mass spectrometry (LC–MS). Pyocyanin was purified via a procedure reported in refs (10) and (13). The blue suspension (pyocyanin solution; ∼3 mL) was mixed into 1.5 mL of 0.2 M HCl in another test tube until attaining the pink color. The pink suspension was transferred to a fresh test tube, and the borate–NaOH buffer (0.4 M, pH—10) was added into this suspension until the color changes to blue. Afterward, the blue-colored pyocyanin was again re-extracted with chloroform, and this step was repeated twice to get a clear blue pure pyocyanin solution. Finally, the pyocyanin powder was recovered by completely evaporating the chloroform.

Electrospinning

The electrospun mats were prepared via an electrospinning method, in which the mixed solution of DMF/MEK (1:1) containing PU (80%) and dextran (20%) was stirred at room temperature for 72 h. Then, 10 wt % pyocyanin (with respect to polymer) was added with the above blend, and the mixture was stirred at room temperature until attaining a homogeneous solution. The mixture solution was loaded into the 5 mL syringe with a microtip. A conventional horizontal electrospinning setup, consisting of a high-voltage supplier, a capillary tube, a syringe pump with a stainless steel needle, and a metal drum collector, was used in this study. A voltage of 22 kV was created between the tip and collector, and the voltage has conquered the viscosity of polymeric solution thereby allows to form the interconnected fibers.

Characterizations

Morphological Characterizations

The surface morphology of electrospun mat specimens was captured by field emission scanning electron microscopy (FE-SEM) (SUPRA 40VP) comprising direct and cross detectors. The imaging was performed after gold sputtering. The surface roughness of mat specimens was investigated using an AFM (multimode-8 model), in a tapping mode. The shape deformation of bacteria (after inhibition) on PU, PUD, and PU–dextran–pyocyanin (PUDP) electrospun mats was observed using FE-SEM. Prior to analysis, the electrospun mats were immersed into the first fixation solution (2% paraformaldehyde + 2% glutaraldehyde in 0.05 M sodium cacodylate buffer) and second fixation solution (1% osmium tetroxide in 0.05 M sodium cacodylate buffer) for 12 h and 15 min, respectively. Subsequently, the samples were stained with 1% uranyl acetate in 0.05 M sodium cacodylate buffer. Dehydration was performed in a graded series of ethanol (30, 40, 50, 60, 70, 80, 90, and 100% each for 10 min), and the electrospun mats were dried in air at room temperature. Then, the mats were mounted on aluminum sample stubs and sputtered with gold.

Structural Characterizations

The functional groups in electrospun mat specimens were characterized by a FT-IR spectrometer (spectrum GX model) at the frequency range of 4000–400 cm–1, where the KBr pellet method was exploited for sample preparation. LC–MS (Agilent 6410B Triple Quadrupole) was used to confirm the production of pyocyanin form P. aeruginosa PAO1. An aliquot of 3 μL of the processed samples was injected into the high-performance liquid chromatography (1200 Series) equipped with a 2.6 μm C8 column (100 Å, 50 × 2.1 mm, Kinetex) maintained at 30 °C. Cone voltage, capillary voltage, and source offset were set to be 30 kV, 3 kV, and 30 V, respectively. The source temperature was maintained at 380 °C throughout the experiment. ESI was operated at the potential of +3000 V. Mobile phase A (water and 0.1% formic acid) and mobile phase B (0.1% formic acid and acetonitrile) were used for all experiments to separate the analyte at a flow rate of 0.5 mL/min over the time of 20 min. Nebulizer pressure, gas flow of desolvation, cone, fragmentor voltage, and collision voltage were set at 15 bar, 650 L/h, 150 L/h, 70 V, and 10 V, respectively. Data acquisition was performed with MassHunter Software (Version B.04.00).

Thermal and Mechanical Characterizations

The thermal behaviors of the specimens of electrospun mats were analyzed using the TA Instruments (Q-50 model), and the analysis was conducted from 30 to 800 °C at a heating rate increment of 5 °C/min under N2 gas atmosphere. Glass-transition temperatures of the electrospun mat specimens were investigated using differential scanning calorimetry (DSC) (Q20 thermal analysis system—TA instruments) at a heating rate of 10 °C/min under nitrogen atmosphere. The mechanical stabilities of the electrospun mat specimens were evaluated from the stress–strain curves obtained with a universal testing machine (LR5K plus 5 kN, Ametek Lloyd instruments Ltd) at a strain rate of 10 mm/min.

Measurements

Contact Angle

Wettability of electrospun mat samples was measured by static water contact angle using a contact angle goniometer (DSA10, Kruss GmbH analyser, Germany). The detailed procedure of the contact angle measurement can be found in the literature.[32]

In Vitro Drug Release

The % of pyocyanin in the electrospun mat was determined from the pyocyanin release in the buffer solution (0.15 mol L–1). A piece of PUDP electrospun sample with an area of 2 cm × 2 cm was soaked into 10 mL of buffer solution at 37 °C for 24 h. To measure pyocyanin release, 1 mL of above solution was exposed to UV-rays. By measuring the intensity of λmax of pyocyanin (i.e., 691 nm), the % of pyocyanin contained in the electrospun sample was determined.[33] The measurement was repeated three times for the electrospun mat using different specimens, and the average value was used for calculation. The % of pyocyanin release was calculated by using the following equationwhere C0 and C∞ refer to the concentration pyocyanin in the buffer solution before and after immersing an electrospun mat.

In Vitro Antibacterial Activity

Quantitative Analysis

Preactivated [subcultured in a fresh Luria–Bertani Broth medium at 37 °C under shaking condition (220 rpm) in an incubator for overnight] E. coli and S. aureus were inoculated separately in 5 mL of the LB liquid medium, incubated at 37 °C until the log growth phase was achieved (OD600 value ≈ 0.6). After incubation, the bacterial culture was centrifuged (8000 rpm for 10 min) and washed with the sterile phosphate-buffered solution (PBS). The cell pellets were suspended in the PBS solution to attain a final density of 106 cells/mL (standardized spectrophotometrically OD540 ≈ 1.0).[16] Followed by, the dynamic contact test was conducted to evaluate the antibacterial activity of the electrospun mat containing pyocyanin quantitatively.[20] A piece of electrospun fiber mat (2 cm × 2 cm) was soaked into 10 mL of bacterial suspension in a Erlenmeyer flask and kept under shaking condition (220 rpm) at 37 °C up to 12 h (Scheme ). To measure the viable cell count, the surface spread plate method was employed. Initially, 1 mL of suspension was taken from the bacterial sample at 2 h of interval over the period of 12 h and followed by decimal serial dilutions with PBS. From these dilutions, 0.05 mL was taken and spread onto sterile agar plates and incubated for 24 h at 37 °C. After incubation, the colonies were counted and expressed as mean colony-forming units per milliliter (CFU/mL) after multiplication with the dilution factor.

Scheme 1

Schematic Diagram of the Experimental Setup Used To Measure the Antibacterial Activity upon Exposure to the PUDP Mats

The antibacterial activity of electrospun mats were further assessed by calculating the percentage of bacterial inhibition. The required amount of the electrospun mat was immersed into the 5 mL E. coli or S. aureus liquid bacterial medium. To evaluate the antibacterial activity, the bacterial medium was exposed to UV-light. By measuring the optical density of the bacterial medium (i.e., 600 nm), the remaining quantity of bacteria was determined using a spectrophotometer. The optical density was measured at 2 h of intervals over a period of 12 h. The measurement was repeated three times for each electrospun mat using different samples, and the mean value was used for calculation. Following equation was used to calculate the % of bacterial inhibitionwhere IC and IS are the average optical density values of the control group and the experimental group, respectively.[23]

Qualitative Analysis of the Antimicrobial Activity

In the qualitative analysis, the antimicrobial activity was evaluated via a disk diffusion method.[34]E. coli or S. aureus (prior grown culture for overnight) was spread over the solid agar plates. Subsequently, electrospun mat samples (diameter 6 mm) were placed on the agar plates. The plates were then incubated at 37 °C for 24 h, and the zone of inhibition (ZOI) for each sample over the agar plate was visually examined. The experiment was repeated three times for each electrospun mat using different samples, and the reported value is the average of experiments.

ROS Detection

Accumulation of ROS was measured using an oxidation-sensitive fluorescent probe-2′,7′-dichlorofluorescein-diacetate (DCFH-DA).[35] Exponentially grown bacteria were harvested, washed with PBS buffer (pH—7.2), and resuspended in PBS buffer. Next, the bacterial cells were stained with 10 μM DCFH-DA for 30 min at 37 °C under dark condition. The cells were washed with copious amount of water until the removal of excess DCFH-DA. The stained bacterial cells were incubated with electrospun mats (2 cm × 2 cm) for 4 h. Finally, the samples were examined by a fluorescence microscope (LSM 510 META-Carl Zeiss, Jena, Germany) at an excitation wavelength of 488 nm and at an emission wavelength of 535 nm. The fluorescence intensity is directly proportional to the ROS level within the cell cytosol.[36]

Determination of Apoptosis

The dissipation of bacterial cells after treating with electrospun mats was examined with FITC-Annexin V with a PI apoptosis detection kit using a BD FACSCalibur flow cytometer (USA, BD). The electrospun mats (2 cm × 2 cm) were immersed into the bacterial solution containing the cells of 1 × 107 for 12 h. Subsequently, the mats were taken out, remaining bacterial solution centrifuged and washed with PBS buffer (pH—7.2) to remove bacterial media. The obtained cell pellet was resuspended in Annexin V binding buffer, and the staining process was carried out according to the protocol given in the kit. Finally, the stained cells were analyzed using a BD FACSCalibur flow cytometer, and data were acquired using FlowJo 10.0.7 software (Treestar Inc, Ashland, US).

Results and Discussion

Determination of Pyocyanin

The identification of the presence of pyocyanin in the culture filtrate of P. aeruginosa was performed via a sensitive analysis method by using LC–MS. The absorption peak of the culture filtrate showed the almost identical absorption peak of standard pyocyanin. Figure S1 (Supporting Information) displays the obtained chromatogram. The retention time of standard pyocyanin matches with the culture filtrate, and the full scan positive ion matches with the molecular mass of pyocyanin. Hence, it endorses the presence of pyocyanin in the supernatant.

Morphological Properties

The morphologies of the natural PU nanofibers, PUDP nanofibers, and hybrid PUDP nanofibers were analyzed by FE-SEM, and the micrographs are shown in Figure . The PU nanofibers exhibit a smooth surface and a flat weblike structure without beads or agglomerated with diameter 400–600 nm. The flat ribbon-like nanofibers have changed to ellipse shape spun homogeneous nanofibers after the addition of dextran. Compare to PU and PUD, the microstructure of PUDP nanofibers showed round fibers with a skin that sometimes twisted and wrinkled bend irregularly. An astute scrutiny in the morphology of PUDP nanofibers narrates well mixing of pyocyanin with nanofibers. It can be clearly observed that the PUDP nanofibers exhibited wrinkle and crumpled morphology where the aromatic and aliphatic chains of polymers and pyocyanin possibly clenched together. The attained crumpling was due to the Π–Π stacking and H-bonding between the polymer chains and pyocyanin, which enables the effectual dispersion of pyocyanin throughout the nanofiber mat. Scheme shows the possible interactions applied between the components in the hybrid nanofiber mat. Therefore, it demonstrates a good compatibility between pyocyanin, polymer, and solvent. The incorporation of pyocyanin seems to significantly alter the smooth fibrous morphology and diameter of nanofibers. The viscosity or conductivity of electrospinning solution influences the morphological structure and average size of the obtained fibers.[37] The slightly twisted structure of the PUDP nanofibers than that of pure PU nanofibers is presumably due to the increase of the solution viscosity or the solution conductivity which was caused by the introduction of pyocyanin and dextran as a foreign species in the electrospinning solution.[38] From Figure (inset), it can be observed that the water contact angle of PUDP was 38.8°, revealing the hydrophilic surface of the hybrid electrospun mat because of its hydrophilic functionalities. On the other side, the contact angle of bare PU is 125°. It is noteworthy that the hydrophilic character of PU enhanced after blending with dextran and pyocyanin. Collectively, the more hydrophilic surface of the PUDP electrospun mat facilitates the attachment of bacterial cells during antibacterial applications. The AFM images of electrospun mat specimens are displayed in Figure . The surface roughness that calculated for fibers was considerably increased after mixing with pyocyanin. Thus, pyocyanin had an influence on the increase in the surface roughness of the PUDP mats. It proves that successful incorporation of pyocyanin into the PUD matrix resulted in the interactions between the pyocyanin and PUD chain which may result in the enhanced surface roughness. Jaganathan et al. reported an increased surface roughness of the PU hybrid membrane after adding mustard oil, which is in agreement with this present study.[39] Furthermore, a rougher surface of the membrane is important for the antibacterial activity. The higher surface roughness is more advantageous for bacterial solution uptake and easy binding with bacteria.[40]

Figure 1

FE-SEM images of mats of (a) PU, (b) PUD, and (c) PUDP; insets of (a–c) corresponding water contact angle images. Red arrows refer to irregular twisted and wrinkled bend after incorporating pyocyanin.

Scheme 2

Possible Interactions Applied between the Components in the Hybrid Nanofiber Mat

Figure 2

2D AFM images of mats of (a) PU, (b) PUD, and (c) PUDP; 3D AFM images of mats of (d) PU, (e) PUD, and (f) PUDP; line profiles for mats of (g) PU, (h) PUD, and (i) PUDP.

Structural Properties

FT-IR spectra were recorded to investigate the intermolecular bond stretching in constructed electrospun mats, and Figure S2 portrays the obtained FT-IR spectra. The peak appearing at 3336 cm–1 in PU, PUD, and PUDP specifies the existence of −OH groups (attributed to hydrogen bonds) in electrospun mats. The peak appearing at 2960 cm–1 in PU, PUD, and PUDP specifies the existence of −OH groups (attributed to alcoholic groups) in electrospun mats. The characteristic peaks found at 1721 cm–1 are common for all of the three electrospun mats, which is attributed to the C=O stretching of PU and dextran. There is no new pyocyanin peak observed for PUDP because the functional groups of pyocyanin are also present in polymers.[10] The small peak shifts in hybrid electrospun mats with respect to pristine PU electrospun mat were observed, representing strong hydrogen-bonding interactions between the polar groups of (−OH, −O–, C=O) PU, dextran, and pyocyanin.

Thermal Stability

TGA thermographs of the fabricated electrospun mats are demonstrated in Figure S3. Pristine PU exhibited a single step weight drop with the onset temperature of 300 °C, owing to the crystallinity of PU, whereas the hybrid electrospun mats depicted two-step weight drops: (i) from 30 to 230 °C and (ii) from 240 to 450 °C. The former two is due to the desorption of free water molecules and detachment of hydrophilic functionalities. The second is because of disintegration of the aromatic backbones of the polymer. The thermal behavior of hybrid electrospun mats shifts toward the low temperature throughout the study, representing the manifestation of less thermal stability of the hybrid membrane, caused because of the hydrophilic functionalities of dextran and pyocyanin.

DSC thermograms were recorded in order to further scrutinize the thermal behaviors of electrospun mats. Figure S3 exhibits the all obtained endothermic inflexions (Tg) of electrospun mats. Compared with the pristine PU, slightly lower Tg is noticed for hybrid electrospun mats because of the sluggish reorganization of polymer backbones in hybrids caused by interfacial interactions such as H-bonding and Π–Π interactions. The Tg values yielded by PU, PUD, and PUDP mats are 174, 164, and 172 °C, respectively.

Thermomechanical Stability

Mechanical strength of the electrospun mats was investigated by DMA, and the results are presented in Figure S4. Figure S4a shows the modulus curves of electrospun mats. The maximum storage modulus of the bare PU membrane was 549 MPa, whereas the PUD and PUDP hybrid electrospun mats exhibited the maximum storage moduli of 558 and 221 MPa, respectively. The extended storage modulus of the PUD membrane was associated with the mutual interaction, such as hydrogen-bonding Π–Π interactions, between the backbone chains of PU and dextran. In the case of PUDP mat, the mat contains higher number of bound water molecules, which causes the rapid relaxation of hybrid mat during elongation. Therefore, the PUDP mat has a lower storage modulus compared to bare PU and PUD mats.

The pyocyanin release profiles from the PUDP hybrid mat are shown in Figure . The behavior of these electrospun mats showed an initial burst release of pyocyanin after immersing the PUDP sample into the PBS buffer with a pH of 7.4. Approximately, 60–80% of the pyocyanin was released in the burst phase within the first 24 h. The remaining pyocyanin was releasing at a gradual slower speed. The release profile of mixed bioactive agents was investigated by many researchers and reported to be a biphasic profile which follow initial burst release and further slower process.[18,20,23] Zheng et al. proposed that the rapid release of amoxicillin (AMX) may be because of the fact that the physical interaction between AMX and n-HA (e.g., hydrogen bonding) is not sufficiently strong.[38] Hence, in our study also, the burst release of pyocyanin in the starting phase is thought to be due to the same fact that the bonding interaction between pyocyanin and polymers (e.g., π–π interaction and H-bonding) is not strong enough. Moreover, the electrospun fiber contains high surface area with very small diameter which allocates more mass transfer through the short diffusion pathway.[20] Therefore, these factors could also induce the release of pyocyanin. However, it is of interest that the longer sustainable release of pyocyanin after 40 h could be a desirable feature for the antibacterial material.

Figure 3

In vitro pyocyanin release profile from the PUDP mat as a function of time.

Antibacterial Activity

Quantitative and Qualitative Analyses

The antibacterial activity was examined by immersing the PU, PUD, and PUDP membrane into the S. aureus or E. coli suspension. The count of viable cells was measured as a function of time via observation, and Figure a,b shows the corresponding results. It can be seen that there is no significant loss in the number of viable bacterial cells with pristine PU and PUD samples, whereas a significant decrease in the number of viable cells can be found with the PUDP sample, representing the antibacterial activity of pyocyanin. Further, the antibacterial activity of pristine PU, PUD, and PUDP samples was further assessed by calculating the percentage of bacterial inhibition after 12 h of time and represented in Figure c,d. The % of bacterial inhibition for the PUDP sample was found to be 98.54 and 90.2% for E. coli and S. aureus, respectively. The lower count of viable cells and higher % of bacterial inhibition with the hybrid electrospun mat sample are a clear manifestation of higher antibacterial activity of the hybrid electrospun mat containing pyocyanin. Because the deterioration of S. aureus is delayed by the thick peptidoglycan layer of its cell wall, the viable cell number of S. aureus increases in relation to the E. coli. The PUDP sample exhibited lower count of viable cells for E. coli and slightly higher count of viable cells for S. aureus. The antibacterial activity of prepared electrospun mat samples was further investigated by a disk diffusion method on the solid medium, as shown in Figure . Pristine PU and PUD nanofibers did not show activity, whereas the electrospun mat containing pyocyanin showed clear ZOI. The inhibition zone is slightly bigger for E. coli compared to the S. aureus plate, which is due to different cell membrane structures of both bacteria. The mean diameter of the inhibition zone for E. coli and S. aureus was around 22 ± 1.02 and 20.5 ± 1.17 mm, respectively. These results demonstrate that the pyocyanin-loaded electrospun fiber showed excellent antibacterial efficiency qualitatively against both S. aureus and E. coli. It is hypothesized that pyocyanin is a bioactive phenazine molecule which interacts with the bacterial cell membrane thereby interrupting the active metabolic transport process of bacteria.[41] Besides, the pyocyanin has high tendency to produce O2× and H2O2 via a nonenzymatic self-reduction and consequently destroy the cell membrane by oxidative stress.[6]Table lists the antibacterial activity results of various bioactive antimicrobial agent-based electrospun reported in the literature for comparison with the present study.

Figure 4

(a) Viable bacterial number of E. coli and (b) viable bacterial number of S. aureus on PU and hybrid mats; (c) growth inhibition of E. coli and (d) growth inhibition of S. aureus on PU and hybrid mats.

Figure 5

Growth inhibition of (a) E. coli and (b) S. aureus bacteria on an agar plate at the incubation time of 24 h.

Table 1

Comparison of Antibacterial Component-Based Hybrid Membranes

electrospun polymer	antibacterial agents	bacterial inhibition	ZOI (mm)	references
PU/D	pyocyanin	98.54%—E. coli	22—E. coli	present work
		90.2%—S. aureus	20.5—S. aureus
PU	quaternary ammonium moieties	99.9%—E. coli		(16)
		99.9%—S. aureus
PLGA/n-HA	amoxicillin	93.2%—S. aureus	14.4—S. aureus	(38)
PU/dextran	ciprofloxacin HCl		15—S. aureus	(32)
PAN	lavender oil		15—S. aureus	(47)
CDs	geraniol	100%—E. coli		(48)
		100%—S. aureus
PU	zein		12—E. coli	(49)
			8—S. aureus
PLLA	lidocaine and mupirocin		22—S. aureus	(50)
			26—S. aureus
PCL/PVP	herbal drugs		26—E. coli	(51)
			24—S. aureus
PVA	chitosan	90%—E. coli		(52)
		98%—S. aureus

Mechanism of Cell Death Induced by Physical Contact of PUDP Nanofibers with the Bacterial Surface

The morphological images of pristine PU, PUD, and PUDP electrospun mats were examined after antibacterial activity treatment for 12 h (Scheme ). Figure a–c shows all obtained images for S. aureus bacteria. Compared with pristine PU and PUD, the PUDP electrospun mat shows highly distributed S. aureus bacteria on its surface. The shape of S. aureus remained intact after 12 h contact with pristine PU and PUD mats. However, the shape of S. aureus was deformed (shrinkage and irregular shape of cells) significantly after 12 h contact with the PUDP fiber mat. In the case of pristine PU and PUD, S. aureus was distributed in between fibers rather than being located only on the surface. In contrast, S. aureus was entrapped only on the surface in the PUDP case, owing to the dense and defect-free morphology caused by the incorporation of pyocyanin. Figure d–f shows all obtained images for E. coli bacteria. It can be seen that numerous distinguishable E. coli cells were distributed over the surface of pristine PU, PUD, and PUDP fibers. The morphology of E. coli remains intact after 12 h contact with pristine PU and PUD fiber mats (Figure a,b), whereas the morphology of damaged cells of E. coli can be found after 12 h contact with the PUDP fiber mat surface, representing that pyocyanin penetration causes shrinkage and deformation of cells. In the case of E. coli, the number of disrupted cells is more, whereas relatively lower number of disrupted cells is observed for Gram-positive bacteria S. aureus. It is noteworthy to mention that the difference in the thickness of the peptidoglycan layer of Gram-positive and Gram-negative bacteria could be the reason for this variation. Altogether, the presence of pyocyanin enables the electrospun mat to be well suited for antibacterial applications. It was reported that the interaction of pyocyanin with the cell membrane causes the inhibition of bacterial growth.[42]

Figure 6

Morphological changes of S. aureus electrospun mats of (a) PU, (b) PUD, and (c) PUDP; morphological changes of E. coli on electrospun mats of (d) PU, (e) PUD, and (f) PUDP.

Detection of Intracellular Reactive Oxygen Radicals

Pyocyanin is a redox-active phenazine that can produce ROS after diffusing into the bacterial cell membrane from the electrospun mat. The intracellular ROS formation from PUDP-treated cells was evaluated by using the oxidation-sensitive fluorescent probe (DCFH-DA). DCFH-DA is a nonfluorescence intracellular ROS indicator which diffuses into cells and oxidizes to highly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF) using ROS.[9] It can be found that the PUDP-treated bacteria became DCF+ and exhibit high-fluorescence intensity, which is due to the intracellular ROS formation caused by pyocyanin (Figure ). In contrast, pristine PU- and PUD-treated cells have no fluorescent DCF+ because of the absence of pyocyanin. The fluorescence intensity and the number of DCF+ cells were found to be higher for E. coli with respect to S. aureus, which is owing to the rapid degradation of E. coli caused by more exposed surface for pyocyanin penetration. The fluorescence intensity depends on the amount of intracellular ROS production.[9] Upon obtained results, it is assumed that burst release of pyocyanin after immersing into the bacterial solution induces its diffusion to the cell membrane and stimulates oxidative stress via ROS.[6] This postulated hypothesis is summarized in Scheme .

Figure 7

In vitro ROS detection after adsorbing S. aureus on mats of (a) PU, (b) PUD, and (c) PUDP; in vitro ROS detection after adsorbing E. coli on mats of (d) PU, (e) PUD, and (f) PUDP.

Scheme 3

(i) Schematic Presentation of Pyocyanin Release from Mat and Get into the Cells; (ii) Proposed Mechanism of Antibacterial Activity of Electrospun Containing Pyocyanin through the Intracellular ROS Formation

Determination of Apoptosis

Oxidative stress of the electrospun mat is a crucial parameter which is necessary to be high to determine the effective apoptosis process. Apoptosis is a programmed cell death which can be induced through intracellular apoptotic effector molecules such as ROS. Thus, the apoptosis of bacterial cells was determined quantitatively by using Annexin V-FITC/PI-stain. Figure shows the corresponding results. The PUDP mat exhibited the late bacterial apoptosis of 89.6 and 83.42% toward E. coli and S. aureus, respectively. The % of early apoptosis was 0.32 for E. coli and 6.92 for S. aureus. Nevertheless, the significant apoptosis was not found for pristine PU and PUD mats toward both bacteria cells. The PUDP mat induces the apoptosis process via ROS provided by the bioactive compound (i.e., pyocyanin). Previously, it has been reported that apoptosis can be characterized in prokaryotic cells through (i) cell morphology, (ii) cell shrinkage, (iii) DNA fragmentation, and (iv) DNA condensation.[43] It can be seen that the obtained apoptosis results are consistent with other above-mentioned findings. Dwyer et al. reported that apoptosis induction takes place in response to ROS generation.[44] The contribution of pyocyanin for the generation of ROS in bacterial cells through oxidative stress has been well described by Rada et al.[45] Although various pyocyanin-based apoptosis were reported, a complete realization and detailed explanation on the electrospun mat-based apoptosis have rarely been investigated.[5,46] It is hypothesized that the vigorous release of pyocyanin from PUDP mats possibly induced the apoptosis in both bacteria. Therefore, PUDP mats can be used as a local treatment or disinfectant wiping purpose.

Figure 8

Detection of apoptosis by flow cytometric analysis of Annexin V-FITC/PI binding after (a–c) E. coli and (d,e) S. aureus contact with electrospun mats of (a,d) PU, (b,e) PUD, and (c,f) PUDP.

Conclusions

In the present study, a bioactive compound (i.e., pyocyanin) incorporated the hybrid membrane was successfully prepared through electrospinning, and their applications toward antimicrobial activity were investigated. A slightly twisted bead-free cylindrical fiber morphology was observed after incorporation of pyocyanin, whereas the pristine and dextran mixed fiber exhibited a flat ribbon-like structure. A more hydrophilic electrospun membrane was obtained after the addition of pyocyanin. The hybrid electrospun showed an initial burst release of 60–80% over the time of 20 h, which represent the effectual release profile of bioactive molecules. The PUDP mat showed the antimicrobial activity of 98.54 and 90.2% against E. coli and S. aureus, respectively, demonstrating the cytotoxic behavior of the hybrid membrane. The higher ZOI clearly depicted the bacterial efficiency of the hybrid electrospun mat. The morphology of bacteria after being adsorbed on PUDP mats was examined by FE-SEM, and shrinkage and irregular shape of cells was observed throughout the surface of mats. The apoptotic cell determination on the PUDP mat exhibited the late bacterial apoptosis of 89.6 and 83.42% toward E. coli and S. aureus, respectively. Altogether, the present results showed that pyocyanin-mixed electrospun mats can be applied as an antibacterial material in various applications such as household disinfectant product, textiles, and food industries.