Investigation of antimicrobial activity and cytotoxicity of synthesized surfactant-modified carbon nanotubes/polyurethane electrospun nanofibers

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Nano-Structures & Nano-Objects

journal homepage:www.elsevier.com/locate/nanoso

Investigation of antimicrobial activity and cytotoxicity of synthesized surfactant-modified carbon nanotubes/polyurethane electrospun nanofibers

Mansab Ali Saleemi

^a^,¹

, Phelim Voon Chen Yong

^a^,¹

, Eng Hwa Wong

^b^,^∗^,¹

aSchool of Biosciences, Faculty of Health and Medical Sciences, Taylor’s University Lakeside Campus, 47500 Subang Jaya, Selangor Darul Ehsan, Malaysia

bSchool of Medicine, Faculty of Health and Medical Sciences, Taylor’s University Lakeside Campus, 47500 Subang Jaya, Selangor Darul Ehsan, Malaysia

a r t i c l e i n f o

Article history:

Received 17 August 2020

Received in revised form 17 October 2020 Accepted 21 October 2020

Keywords:

Carbon nanotubes Thermoplastic polyurethane Electrospun nanofibers Antimicrobial activity Cytotoxicity

a b s t r a c t

Carbon nanotubes (CNTs) are attractive materials because of their excellent physicochemical properties, electrical and thermal conductivity, mechanical strength, and chemical durability. Therefore, CNTs can be used in a wide range of various biological and pharmaceutical sectors. The impact of CNTs- fiber composites on the growth of microbial cells needs to be fully explored. In this work, we have investigated the antimicrobial role of thermoplastic polyurethane (TPU) nanofibers containing various concentrations of surfactant-modified CNTs, such as double-wall (DW) and multi-wall (MW) CNTs against different representative Gram-positive and Gram-negative bacterial and the fungal strains. Be- sides, the cytotoxic effects of synthesized nanofibers were also studied on the human adenocarcinomic lung epithelial cell line (A549). Various concentrations of surfactant-modified CNTs were prepared and then mixed with 10 % solution of polymer (TPU) in N, N-Dimethylformamide (DMF) solvent by using a magnetic stirrer. The prepared solution was passed through the electrospinning apparatus to obtain electrospun nanofibers using a highly stable dispersion. Fourier transform infrared spectroscopy (FTIR) results exhibited that polyurethane polymer was covalently attached to the sidewalls of functionalized CNTs. Further, the morphology of synthesized nanofibers and interaction between pathogens and TPU/f-CNTs fibers were studied by using a field-emission scanning electron microscope (FESEM), transmission electron microscope (TEM) and fluorescence microscopy. In conclusion, the highest rate of microbial growth inhibition was recorded when using the surfactant-modified CNTs concentration of 100 —g/mL with 10 % TPU solution. The antimicrobial activity and cytotoxicity of TPU/f-CNTs nanofibers were both dependent on the treatment time and concentrations. The antimicrobial findings demonstrated the excellent microbicidal and prolonged microbial growth inhibition properties of electrospun nanofibers which propose their applicability as sustained antimicrobial biomaterials.

1. Introduction

At present, nanotechnology is a rapidly growing research field that considerably contributes to the improvement of active biomaterials. It is necessary to keep the progress of development at the present pace in order to excel in this field and innovative nanoparticle with unique and novel features are also required.

Among several carbon nanomaterials, carbon nanotubes (CNTs) have attracted growing attention by several researchers to work since their discovery by Iijima in 1991 [1] because of their remarkable physicochemical properties and are likely to be applied

∗ Corresponding author.

E-mail address: [email protected](E.H. Wong).

1 All the authors have contributed equally to the preparation of manuscript draft.

in a large number of emerging and existing applications, such as composite materials science [2], electronics [3], water purification [4], energy storage [5], medical field, and biotechnology [6,7].

The ability of carbon atom can bond itself to form a structure with an extremely low dimension. Carbon can form diverse allotropes based on the formation of either sp²or sp³bonds between carbon atoms made up of graphite and have been built in cylindrical tubes with several millimeters in length and nanometer-scale in diameter. The arrangement of these atoms at the nanoscale level is particularly substantial where each carbon allotrope displays unique properties. In the field of nanotechnology, researchers have concentrated on their prevalence rate because of the association with the living tissue and their fate in organisms [8] as well as significance for the environment [9].

In addition, CNTs have triggered a great interest by the researchers because of their immense potential as an antibacterial

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agent. Many researches have been conducted to investigate the antimicrobial activity of CNTs in medical instruments [10–17].

Previous literature shows that various approaches have been applied to increase the antimicrobial properties of biomedical apparatus, including bactericidal nanoparticles [18–20], antimicrobial peptides [21], polyethylene glycol [22,23], and bactericidal agents-cationic polymers [24]. The study conducted by Seoktae et al. (2007) showed that a highly purified form of single-wall carbon nanotubes (SWCNTs) demonstrated significant antimicrobial activity [25]. The CNTs’ antibacterial mechanisms involve [26,27], (1) physical interactions between CNTs and pathogens induced damage to cell membranes; (2) disruption of the microbial cell membrane; (3) electronic structure-based microbial cells oxida- tion. In addition, when CNTs are applied as an antimicrobial agent in medical devices, there are two main barriers that would restrict the CNTs application. Firstly, the toxicity of CNTs on human cells, although it has great potential to inhibit microbial growth. Yang et al. (2010) demonstrated the highest toxicity of single-wall carbon nanotubes due to their longer length [28].

Moreover, certain parameters such as length, diameter, electronic structure, and dispersion state of CNTs were also influ- enced upon their toxicity [14,15,28]. Secondly, CNTs are hardly dispersed into medical devices due to their self-reaggregation state. Thus, it is required to propose the optimum strategies for increasing the antimicrobial properties of CNTs while re- ducing their aggregation state and toxicity to the human cell.

Sayes et al. (2006) demonstrated that surface modification of SWCNTs had a great impact on the cytotoxicity of carbon nanotubes that increased the degree of sidewall functionalization and decreased the toxicity of SWCNTs [29]. However, functionalization of CNTs is a crucial step towards increasing the antimicrobial properties of carbon nanotubes. Yuan et al. (2008) synthesized a nanohybrid consisting of silver nanoparticles embedded in the dendritic poly(amidoamine) dendrimer functionalized MWCNTs to study the antibacterial properties against Gram- positive and Gram-negative pathogens [30]. Hao et al. (2016) synthesized nanocomposites comprising MWCNTs-grafted car- boxymethylated chitosan contained remarkable stability and solubility in DMSO and other aqueous solution [31].

Electrospinning (ES) is an efficient and sophisticated technique for the synthesis of CNTs/polymer composite fibers with nanoscale diameter. The reasons for the selection of this technique include the simplicity and efficiency of the process, rela- tively low startup costs, and ability to handle various factors, such as composition, fiber diameter and orientation. The structures of electrospun nanofibers owing to their higher porosity, high surface area and similarity with the human extra cellular matrix (ECM) have shown various advantages over other techniques [32].

The mechanical properties of CNTs/polymer nanofibers could be increased by stretching and making composites. Some previous studies reported the synthesis of electrospun nanofibers, which showed weekly alignment and randomly oriented nonwoven structures leading to low molecular orientation, resulted in low mechanical properties [33,34]. Therefore, it is necessary to synthesis aligned nanocomposite fibers in order to apply in a wide range of biomedical applications. This study shows that electrospinning apparatus used for the synthesis off-CNTs/polymer nanofibers with different parameters could increase the diameter distribution, decrease the nanofiber diameter, and improve the electrospun nanofiber alignment. Thus, for thef-CNTs-reinforced TPU nanofibers, high alignment, uniform dispersion, and strong interfacial adhesion were significant factors in order to improve the physical properties of composite nanofibers.

In this work, we have studied the antimicrobial role of thermoplastic polyurethane (TPU) nanofibers containing different concentrations of surfactant-modified CNTs, such as double-wall

Fig. 1. Schematic structure of polyurethane.

(DW) and multi-wall (MW) CNTs against different representative bacterial and the fungal strains. According to our understanding, no work has been reported on the antimicrobial activities off-DWCNTs/polyurethane electrospun nanofibers. We have also observed that preparedf-CNTs/polyurethane nanofibers contained broad-spectrum antimicrobial effects. Besides, very limited preliminary studies were conducted in order to check the antimicrobial activity off-CNTs/polyurethane electrospun nanofiber against Klebsiella pneumoniae. The thermoplastic polyurethane polymer was used due to its remarkable abrasion resistance, transparency, resistance to microorganism, excellent low- temperature flexibility, toughness, and hydrolytic stability. More- over, transmission electron microscopy (TEM), field-emission scanning electron microscopy (FESEM) and fluorescence microscopy were applied to investigate the morphology of synthesized electrospun fibers and to study the interactions between pathogens andf-CNTs/polyurethane nanofibers. The cytotoxicity study of prepared electrospun nanofibers was also conducted using human lung epithelial cell line (A549). Conse- quently, this study showed that carbon nanotubes/polyurethane nanofibers contained the antimicrobial activity against selected pathogens and also significantly repressed the viability of cell (A549) based on dose and treatment time. The results con- firm that antimicrobial activity of TPU/f-MWCNTs nanofibers is greater than TPU/f-DWCNTs nanofibers. The surfactant-modified MWCNTs electrospun nanofibers may be a good option as a microbial growth inhibiting agent. Therefore, the synthesized electrospun nanofibers can be used in a wide range of biomedical sectors.

2. Experimental procedure 2.1. Materials

Carbon nanotubes (CNTs) were supplied by the NE Scientific Enterprise, Malaysia with length of 10–20µm, the outer diameter of 2–4 nm, and 90% of purity for DWCNTs, while the length of ∼100 µm, outer diameter of 10–15 nm, and >^{95% purity} for MWCNTs. Thermoplastic polyurethane (TPU) was received from the IDL Scientific Co, Ltd., Malaysia, and its structure has shown in Fig. 1. The N, N-Dimethylformamide (DMF) solvent, and sodium dodecylbenzene sulfonate (SDBS) were obtained from Sigma-Aldrich Co., Ltd.

2.2. Purification and functionalization of CNTs

As previously used the purification method [35], the pristine form of CNTs was heated in a furnace at 450^◦C and then treated with 6 M HCl in order to eradicate the metallic catalyst (e.g. Ni, Cu, etc.) produced during the synthesis of carbon nanotubes.

The acidic solution was passed through the membrane filtration assembly and filtered nanotubes were treated with the solution of sodium hydroxide (NaOH) and again heated at 100 ^◦C temperature to remove the residues of aluminum oxides [36]. Later, suspensions of nanotubes were quickly passed through the membrane filtration assembly and the filtered nanomaterials washed

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Fig. 2. Schematic of the electrospinning machine for synthesizing the electrospun nanofibers.

with distilled water to obtain pH neutral. The purified CNTs were dried in the oven at 60^◦C. After drying, carbon nanotubes (CNTs) were treated with the suspension of 0.05 wt% sodium dodecylbenzene sulfonate (SDBS) solution. To obtain the SDBS adsorbed surface of CNTs as previously reported [35], the suspensions of nanotubes were passed through the ultrasonication process for 30 min. The centrifuge machine was used to obtain the functionalized CNTs suspension at the bottom of the centrifuge tube. The filter paper (0.⁴⁵µm) was used for the filtration of functionalized CNTs and washed the filtered nanotubes with distilled water to obtain pH neutral. Lastly, suspensions of nanotubes were dried in the oven [37].

2.3. Synthesis of f-CNTs/TPU nanofibers using electrospinning appa- ratus

The polymer thermoplastic polyurethane (TPU) was dissolved in the DMF solvent at a concentration of 10 wt %. After drying, functionalized CNTs (DWCNTs and MWCNTs) were also sepa- rately added and efficiently dispersed in a prepared solution of TPU in DMF solvent with different concentrations. The magnetic stirrer was used to obtain the homogeneous solution of f-CNTs/TPU at 500 rpm for 24 h. Then the prepared solution of nanotubes/polymer was loaded into a syringe to initiate the electrospinning process, as shown in Fig. 2. While the flow rate was adjusted to 1 mL/h and the applied voltage power supply was 10 kV. The distance between the collector surface and metallic needle was maintained at 15 cm. The electrospinning process was conducted for one hour and the nanofiber was collected on aluminum foil. The square weight of synthesized one electrospun nanofiber layer was estimated to be approximately 900 mg/m² (rotational electrode).

2.4. Surface characterization

Fourier transform infrared spectroscopy (FTIR) was used to investigate that the polymer (TPU) covalently attached to the sidewalls of CNTs. The surface morphology of pristine and functionalized CNTs was observed by using transmission electron microscopy (TEM, Hitachi Limited, Tokyo, Japan). Field-emission scanning electron microscopy (FESEM) was used to observe the morphology of synthesized electrospun nanofibers. The average diameters of electrospun nanofibers were measured from SEM images using Digimizer image analysis software. SEM images were captured by using field-emission scanning electron microscope (FESEM, Hitachi Limited, Tokyo, Japan) with an applied voltage of 20 kV. A small section of nanofibers was taken from the as-prepared electrospun nanofibers and observed under the scanning electron microscope.

2.5. Antimicrobial property 2.5.1. Preparation of microbial cells

In this work, different microbial strains, such asStaphylococ- cus aureus(ATCC 25923),Pseudomonas aeruginosa(ATCC 15692), Klebsiella pneumoniae(ATCC 43816), andCandida albicans(ATCC 10231) were included to investigate the antimicrobial activity of synthesized electrospun nanofibers. For the bacterial strains, cells were grown in Luria–Bertani (LB, Oxoid) broth, and yeast peptone dextrose (YPD, Oxoid) broth was used to grow yeast cells in a shaking incubator. The microbial growth media was passed through the centrifugation process at 6000 rpm, as previously reported [35]. The microbial cells were washed with 0.9% of NaCl for the removal of residual macromolecules and growth medium.

Again, the cells were resuspended in NaCl (0.9%) for further use.

2.5.2. Treatment of microbial cells by surfactant-modified CNTs/TPU nanofibers

Before microbial cells treated with electrospun nanofibers, serial dilution (10-fold) of cells was prepared using 0.9% of NaCl in order to obtain the concentrations (∼10⁷to 10⁸CFU/mL). The suspension of microbial cells (150µL) was added into the centrifuge tubes with a small section off-CNTs/polyurethane electrospun nanofibers (9 mm) prepared at different concentrations of functionalized carbon nanotubes (DWCNTs and MWCNTs). While the suspension of microbial cells (150µL) was introduced into the another centrifuge tubes with 20µL of deionized (DI) water as a control sample. The centrifuge tubes were placed on the mixer to spin for one hour.

2.5.3. Measurement of optical density growth curve

The sample mixtures were subjected to the tubes after one hour of treatment and placed into the LB and YPD medium. In a shaking incubator, bacterial cells were grown at 37^◦C and yeast cells cultivated at 28^◦C. After incubation, the reading was taken using enzyme-linked immunosorbent assay (ELIZA) microplate reader (BioTek, USA) to calculate the optical density (OD) at a wavelength of 600 nm.

2.5.4. Viable cell study by surface plating method

To determine the viable cell number, microbial culture was treated with different concentrations of carbon nanotubes used for the synthesis of electrospun nanofibers. A conventional surface plating method was used to observe the reduction in viable cells number. The electrospun nanofibers treated microbial cells and control samples were serially diluted (10-fold) with NaCl (0.9%) solution. The concentration (0.1 mL) was taken from an appropriate cell dilution placed onto the agar plate. Different growth media were used, such as mannitol salt agar (MSA) for Staphylococcus aureus, cetrimide and MacConkey agar forPseu- domonas aeruginosa and Klebsiella pneumoniae, and yeast peptone dextrose agar (YPD) for Candida albicans. The number of viable cells was enumerated by using surface plating method. The colonies were counted for Gram-positive and Gram-negative bacterial strains after 24 h at 37^◦C, while the cells were enumerated after 48 h at 28^◦C for fungal strain. Hence, the reduction in the number of viable cells was confirmed as colony-forming units per milliliter (CFU/mL) [38].

2.5.5. Disc diffusion method

The antimicrobial activity of pure polyurethane (PU) andf- CNTs/PU electrospun nanofibers at various surfactant-modified CNTs (DWCNTs and MWCNTs) ratios was evaluated using disc diffusion method. In this approach, Mueller hinton (MH) and sabouraud dextrose (SD) agar was used to investigate the zone of inhibition produced by the selected pathogens. The agar was

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poured into the petri dish and allowed the plates to solidify for 5–

10 mins. The concentration of microbial cells (10⁷to 10⁸CFU/mL) was taken and spread on the surface of solidified agar plates [39].

Then, the small sections of f-CNTs/polyurethane electrospun nanofibers (9 mm) prepared at different concentrations of f- CNTs were placed on the microbial culture plates. Petri dishes were sealed and incubated for 24 h at 37 ^◦C. For fungal strain, the incubation time was 48 h at 28 ^◦C. Zone of inhibition was observed around the small sections of electrospun nanofibers and measured in mm radii along with the control samples. The control samples were TPU nanofibers and a disc containing 50µ^{L of DMF} solvent.

2.6. Fluorescence microscopy

The fluorescence microscopy (Nikon ECLIPSE Ni, Japan) was used to take the images by using a FITC-HYQ (fluorescein isothiocyanate) filter cube (460–500 nm excitation and 510–560 nm emission filter). Initially, microbial cells were treated with the synthesized electrospun nanofibers as follows: a small section of nanofibers (9 mm) was taken from the as-prepared electrospun nanofibers and then mixed with the microbial cell concentrations (∼10⁷to 10⁸CFU/mL) in deionized (DI) water and kept spinning for 30 min. A drop of 1µL from the sample mixtures was taken and dropped onto the microscopic glass slide. The glass cover slips were used to cover the samples and fluorescence images were captured immediately.

2.7. Cells cultivation

The human adenocarcinomic lung epithelial cell line (A549) was used to study the cytotoxicity of prepared electrospun nanofibers. The A549 cells were grown in Dulbecco’s modified ea- gle medium (DMEM) supplemented with penicillin/streptomycin (1%) as well as 10% of fetal bovine serum (FBS). The cells (A549) were incubated and kept at 37^◦C with CO₂(5%) in a humidified atmosphere in this work and routinely checked the growth of cells under the microscope. The cells were cultivated in the 25 cm²cell culture flask and trypsinized to harvest the cells after obtaining confluence (∼80%) for further MTT assay.

2.8. Cytotoxicity test (MTT assay)

MTT assay was applied to assess the cytotoxicity of synthesized electrospun nanofibers on human adenocarcinomic lung epithelial cell line (A549). Briefly, the cells were grown in the 96-wells microplates at a density of (1.2×10⁶cells/mL) and then placed at 37^◦C incubator with CO₂(5%) to allow the cells attach- ment. Then the medium was discarded and washed the cells with phosphate-buffered saline (PBS) and added the fresh medium with small sections (9 mm) of nanofibers at different concentration (0, 20µ^{g/mL, 40}µ^{g/mL, 60}µ^{g/mL, 80}µ^{g/mL, 100}µ^{g/mL) of} surfactant-modified CNTs used to prepare electrospun nanofibers with polyurethane. After 24 h, 10 µL of MTT (5 mg/mL) was subjected to each treated well and the microplates were further incubated for 4 h. After incubation, all the remaining supernatants were discarded and replaced with 10% of dimethyl sulfoxide (DMSO) to dissolve the resulting crystals of formazan. Lastly, the absorbance reading was taken by the enzyme-linked immunosorbent assay (ELIZA) microplate reader (BioTek, USA) to calculate the cell viability percentage at 570 nm using the formula: Cell viability (%)=mean OD₅₇₀ of treated cells/mean OD₅₇₀ of control cells) × 100. The cell viability percentages were applied in order to determine the IC50 value that is the concentration of surfactant-modified CNTs (DWCNTs and MWCNTs) used to prepare electrospun nanofibers to inhibit the growth of cells compared to control cultures (untreated).

Fig. 3. FTIR spectra of polyurethane (PU), puref-DWCNTs, pure f-MWCNTs, polyurethane/f-double-wall CNTs (PU/f-DWCNTs), and polyurethane/f-multi- wall CNTs (PU/f-MWCNTs).

2.9. Statistical analysts

Each experiment was conducted in triplicate (n=3) and the values were measured as the mean±SD. The statistical study was carried out using a single factor of variance (one-way ANOVA) and the p-values<0.05 were considered significant.

3. Results and discussion 3.1. FTIR spectroscopy

ATR-FTIR spectroscopy was used to analyze the intermolecular interactions between polymer (TPU) and surfactant-modified carbon nanotubes (CNTs), such as DWCNTs and MWCNTs from the prepared homogeneous mixtures. The FTIR apparatus assists us to investigate the interaction of amide carbonyl group of PUA on the CNTs surface. Previous study on infrared (IR) demonstrates that hydrogen bonding is arrested in the PUA because of functional group proximity of amide carbonyl and intermolecular interaction with the CNTs in the PU matrix [40]. The ATR-FTIR spectra of TPU, pure f-CNTs and TPU/f-CNTs are shown in Fig. 3. The peaks appeared within the range of 2700–3000 cm⁻¹and 3200–

3470 cm⁻¹ could be ascribed to the N–H and C–H stretching vibrations, respectively [41]. The HTPB-based polyurethane shifts the amide C==O absorption at a low frequency because it contains olefinic conjugation in the backbone chain. The peak occurs at 1645 cm⁻¹may be attributed to the stretching vibration of amide carbonyl. While a sharp peak appears at 1050 cm⁻¹ is identified as absorption for C==C stretch. The peak occurs at 1645 cm⁻¹can be attributed to urethane carbonyl of nanocomposites with low intensity than neat PU due to pi-pi interactions of polyurethane with carbon nanotubes in the homogeneous mixtures. The other peaks within the range of 1149–1400 cm⁻¹are recognized as N–

H bending amides vibration. However, vibrational bending peaks and N–H broad shoulder become sharper and also increase their intensity [42]. Thus, it was confirmed the interfacial interaction between surfactant-modified CNTs and PU in the homogeneous mixtures. Consequently, modified carbon nanotubes (CNTs) embedded in the polyurethane electrospun fibers were obtained after running the electrospinning apparatus.

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Fig. 4. TEM images of pristine DWCNTs (A), SDBS-modified DWCNTs (B), pristine MWCNTs (C), and SDBS-modified MWCNTs.

3.2. Surface characterization

Fig. 4 indicates transmission electron microscope (TEM) images of pristine CNTs and surfactant-modified CNTs. The pristine CNTs (DWCNTs and MWCNTs) are tightly packed with each other, as shown in (Fig. 4A, C), while CNTs after functionalized with a surfactant (SDBS) become considerably untied and strongly dispersed the CNTs without causing any structural damage (Fig. 4B, D). Thus, non-covalently dispersed CNTs plays a significant part in increasing the dispersion power of CNTs.

However, the synthesized electrospun nanofibers using electrospinning apparatus were analyzed by field-emission scanning electron microscope (FESEM microscopy). FESEM images were clearly shown inFig. 5. The images showed the weblike structure of f-DWCNTs with a diameter of 2–4 nm, as shown in Fig. 5b and f-MWCNTs with diameter of 10–15 nm were observed in synthesized electrospun nanofibers with PU, as shown inFig. 5c than neat polyurethane nanofibers without CNTs (Fig. 5a). The neat PU nanofiber diameter was found between 156.81 and 621.27 nm, while the average electrospun nanofiber diameter was 329.59 nm. The produced electrospun nanofibers were fine enough without any beads formation. The fibers produced by electrospinning apparatus were found similar according to the previous study conducted by Emad Abdoluosefi et al. (2017) [43].

3.3. Antimicrobial activity of electrospun nanofibers

The antibacterial and antifungal activity of TPU/f-CNTs electrospun nanofibers was assessed after treatment with pathogens at OD600 nm. The optical density curves of treated pathogen were compared with various concentrations of TPU/f-MWCNTs and TPU/f-DWCNTs nanofibers, as displayed inFigs. 6and7. Previous reports showed that pristine carbon nanotubes did not cause any microbial cell damage due to the poor solubility in aqueous solution and large diameter of nanotubes than functionalized ones [44,45]. Therefore, dispersion of f-CNTs in the composites plays a very important role to increase their antimicrobial activity. The less antimicrobial effectiveness of non-modified CNTs

in composites that could be ascribed to their functional and structural properties. Highly dispersed CNTs provide greater interaction with the cell and probably thus, the rate of microbial cell lysis is high [44]. Besides, enhanced antimicrobial activity of electrospun nanofibers may be attributed to (1) a large surface area for interaction with microbial cell surface, (2) a small diameter of nanotubes that enables the partial nanotubes penetration into the cell membrane, and (3) unique electronic and chemical properties that convey higher chemical reactivity. Thus, an appropriate dispersion of CNTs could play a significant role in their antimicrobial activity.

The surfactant solution of sodium dodecylbenzene sulfonate (SDBS) was selected for the dispersion of CNTs, as previously reported [35]. Sodium dodecylbenzene sulfonate is an anionic surfactant that consists of benzene ring on the head and hydrocarbon chain towards the tail. The benzene ring adsorbs more strongly on the sidewall of CNTs due to intense pi-pi interaction. The efficiency of adsorption and dispersing power of the surfactant depends on its tail length. Longer tail shows high spatial volume and more steric hindrance. Thus, it provides a more electrostatic repulsion among the individual nanotubes and assists to disperse the CNTs. In this study, the functionalization of CNTs was an ini- tial step before synthesizing the TPU/CNTs electrospun nanofibers by using electrospinning apparatus. The optical density growth curves for these selected bacterial strains and fungal strain are reported inFigs. 6and7. The antimicrobial activity of electrospun nanofibers increases with rising the concentration off-DWCNTs andf-MWCNTs. When the concentration of functionalized MWC- NTs for the synthesis of electrospun nanofibers is increased to 100 µg/mL, microbial cell growth is not apparently observed within 24 h and 48 h, respectively (Fig. 6). This indicates that a high concentration of functionalized MWCNTs can almost kill the cells in the samples. In contrary, the antimicrobial activity of functionalized DWCNTs for the synthesis of electrospun nanofibers demonstrated less and slow inhibition of microbial cell growth at 100 µ^{g/mL of} ^f-DWCNTs (Fig. 7). These findings confirmed

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Fig. 5. FESEM images of (A) polyurethane (PU) electrospun nanofibers, (B) polyurethane/double-wall carbon nanotubes (PU/f-DWCNTs) nanofibers, and (C) polyurethane/multi-wall carbon nanotubes (PU/f-MWCNTs) nanofibers. The arrow indicates weblike structure off-CNTs embedded in the PU nanofibers.

Fig. 6. The optical density curves obtained whenStaphylococcus aureus(A),Pseudomonas aeruginosa(B),Klebsiella pneumoniae(C), andCandida albicans(D) at a concentration of∼10⁷to 10⁸CFU/mL treated with various concentrations of TPU/f-MWCNTs electrospun nanofibers and then treated microbial cultures grown in LB and YPD broth in the incubator. The suspension of cells in deionized water (DI) was used as a control. PU/CNF indicates polyurethane/CNTs electrospun nanofibers.

that electrospun nanofibers produced by functionalized MWC- NTs exhibited microbicidal activity, while functionalized DWCNTs nanofibers showed bacteriostatic and fungistatic activity.

Moreover, the prepared electrospun nanofibers with a minimum concentration of surfactant-modified CNTs (20 µ^{g/mL) in} the synthesized electrospun nanofibers demonstrated a maximum value for the growth of microbial cells. At low concentration of functionalized CNTs, the time required to reach the exponential log phase growth cycle is significantly different. The growth time of microbial cells is delayed by approximately 4 h and 8 h corresponding to the f-MWCNTs electrospun nanofibers treatment with concentration of 60 µ^{g/mL, 80} µg/mL and 100 µ^g/mL, respectively. A longer delay growth time indicates less surviving the cells and thus stronger antimicrobial activity. A compari- son of Figs. 6 and 7 indicates that the delay in the growth time of cells by TPU/f-DWCNTs electrospun nanofibers is shorter than the TPU/f-MWCNTs nanofiber under the same concentrations. This proposes that antimicrobial activity of TPU/f-MWCNTs nanofibers is greater as compared to other ones. The thermoplastic polyurethane electrospun nanofibers were also allowed the microbial cells to grow with minimum inhibition of cells. How- ever, functionalized double-wall and multi-wall CNTs with 80 and 100µg/mL concentrations in the prepared electrospun nanofibers were demonstrated a significant microbial growth inhibition.

In this work, we have observed that Gram-negative bacteria (Pseudomonas aeruginosa and Klebsiella pneumoniae) are highly susceptible towards electrospun nanofibers prepared at high concentrations of surfactant-modified CNTs, while fungal strain (Can- dida albicans) showed less inhibition compared to other pathogens. In contrary, a significant microbial growth inhibition was also observed for Gram-positive bacteria (Staphylococcus aureus). The results displayed that both types of TPU/f-MWCNTs and TPU/f-DWCNTs electrospun nanofibers exhibited the antimicrobial activity against selected pathogens. As far as we know, no report have been published on the antimicrobial activity of TPU/f-DWCNTs electrospun nanofibers. The earlier reports [46–

48] demonstrated the antibacterial property of single-wall and multi-wall CNTs/TPU electrospun nanofibers, but still lack of reports on the double-wall/TPU nanofibers. This work indicates that electrospun nanofibers synthesized by TPU/f-DWCNTs have the tendency to prevent microbial growth and cause cell membrane destruction. Thus, the observed antimicrobial activity of TPU/f-MWCNTs fibers was high compared to electrospun fibers produced by TPU/f-DWCNTs, which may be because of multiple graphene sheets of MWCNTs [49].

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Fig. 7. The optical density curves obtained whenStaphylococcus aureus(A),Pseudomonas aeruginosa(B),Klebsiella pneumoniae(C), andCandida albicans(D) at a concentration of∼10⁷to 10⁸CFU/mL treated with different concentrations of TPU/f-DWCNTs electrospun nanofibers and then treated microbial cultures cultivated in LB and YPD broth in the incubator. The suspension of cells in deionized water (DI) was used as a control. PU/CNF indicates polyurethane/CNTs electrospun nanofibers.

3.4. Viability study

The microbial cell reduction was observed after treatment with various concentrations of both types of surfactant-modified CNTs used in the synthesis of nanofibers using electrospinning apparatus, as shown in Fig. 8A, B. The antibacterial activity of TPU/f-CNTs nanofibers depends on the treatment time and concentration [50]. After 24 h, the viability of cells after treatment with different concentrations of f-MWCNTs/TPU nanofibers reduced by 35, 49, 64, 75, and 83 percent, respectively, against Staphylococcus aureus. Similarly, the viability of cells reduced with increasing concentrations of f-DWCNT/TPU nanofibers against Staphylococcus aureus. The reduction in viable cell numbers was observed as the dose of both types of surfactant-modified CNTs increased in the synthesized electrospun nanofibers. In case of Pseudomonas aeruginosa, the viability of cells reduced by 45, 59, 71, 86, and 95 percent by rising the concentration of f- MWCNTs/TPU nanofibers, but the reduction in viable cells observed less after treatment of cells with f-DWCNTs/TPU nanofibers. The findings of this study verify from the earlier reports that double-wall CNTs demonstrate less antimicrobial activity than multi-wall CNTs [51,52].

In contrast, the TPU/f-MWCNTs and TPU/f-DWCNTs electrospun nanofibers treatment with different concentrations showed the reduction in viable cells by 39, 53, 69, 81, and 89 percent and 25, 35, 44, 66, and 73 percent against Klebsiella pneumo- niae, respectively. While, it was observed the viability of fungal strain (Candida albicans) decreased by 30, 44, 60, 72, and 80 percent and 15, 27, 35, 58, and 63 percent after treatment withf-MWCNTs/TPU andf-DWCNTs/TPU electrospun nanofibers for 48 h. However, the polyurethane nanofibers (without CNTs) showed very less reduction in the viable cells of selected microbial strains. The reduction in microbial cells after nanofibers treatment exhibited the antimicrobial activity of synthesized TPU/f- CNTs electrospun fibers. Besides, viable cell reduction demonstrates the delayed observations in the exponential log phase of selected isolates treated with electrospun nanofibers, which con- firms the antimicrobial activity of TPU/f-MWCNTs nanofibers is

more than TPU/f-DWCNTs nanofibers. Thus, surfactant-modified MWCNTs electrospun nanofibers may be a good option as a microbial growth inhibiting agent. These modified nanofibers can be applied in a broad range of applications in the antimicrobial field.

However, the effect of treatment time on inhibiting the microbial cell growth treated with synthesized electrospun nanofibers was also observed. The reduction in microbial cells number after treatment with 100 µ^g/mL concentration of f-CNTs/TPU nanofibers for various treatment time was shown in Fig. 8C, D. The study showed that f-MWCNTs/TPU and f-DWCNTs/TPU electrospun nanofibers exhibit a similar pattern of microbial cell reduction with respect to treatment time, indicating the microbial cell reduction shows a positive association with treatment time.

Thus, a significant decline in the microbial cells number was observed as the treatment time increased. Consequently, the treatment time effect on the reduction in viable cell was more clear for f-MWCNTs/TPU electrospun nanofibers. However, the impact of f-CNTs/TPU electrospun nanofibers on the growth of microbial cells needs to be fully explored. For this purpose, we discuss the antimicrobial property and mechanisms of synthesized nanofibers against selected pathogens and also explain the cytotoxic results of these electrospun nanofibers using the human lung epithelial cell line (A549).

3.5. Diameter of inhibition zone for electrospun nanofibers The antimicrobial effectiveness of surfactant-modified CNTs/

TPU electrospun nanofibers was evaluated against different selected microbial strains, such as Staphylococcus aureus, Pseu- domonas aeruginosa,Klebsiella pneumoniae, and Candida albicans using the disc diffusion method.Fig. 9(a)demonstrates the images of antimicrobial activity of tested electrospun nanofibers prepared using different functionalized MWCNTs ratios.Table 1 exhibits the diameter inhibition zone of surfactant-modified MWCNTs/TPU electrospun nanofibers containing different concentrations of functionalized MWCNTs (20µ^{g/mL, 40}µ^{g/mL, 60} µ^{g/mL, 80} µg/mL and 100 µg/mL) to assess the microbicidal

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Fig. 8. The antimicrobial activity of prepared TPU/f-CNTs nanofibers depends on the dose and treatment time. The microbial cells (∼10⁷to 10⁸CFU/mL) incubated with different concentration (A, B) and treatment time at 100µg/mL (C, D) off-CNTs/TPU nanofibers for 24 h for Gram-negative and Gram-positive bacteria, while 48 h for fungal strain. The viability of cells was studied by colony counting method and calculated as a percentage with regards to untreated microbial cells incubated with distilled water (DW). The treatment of microbial cells with distilled water used as a control.

effect of functionalized MWCNTs that integrated with TPU to form the nanofibers. The results show that inhibition zone diameter for f-MWCNTs/TPU nanofibers is about 14 mm, 21 mm, 18 mm and 13 mm for the bacterial and fungi strains, respectively after ap- plying concentration (100µg/mL) of modified MWCNTs based on the weight of TPU. Besides, TPU nanofibers demonstrated no zone of inhibition against Gram negative bacteria, such asPseudomonas aeruginosa, andKlebsiella pneumoniae, while it showed less inhibition zone againstStaphylococcus aureusandCandida albicans. It is noticed that antimicrobial activity of TPU nanocomposites fibers has improved with the surfactant-modified CNTs.

Similarly, the zone of inhibition of functionalized DWCNTs/

TPU nanofibers is increased with increasing the concentrations of functionalized DWCNTs as shown in Fig. 9(b)and Table 2. The results show that inhibition zone diameter for f-DWCNTs/TPU nanofibers is about 12 mm, 16 mm, 15 mm and 10 mm against selected pathogens. Thus, the fabricatedf-CNTs/TPU electrospun nanofibers exhibited good antimicrobial activity against different selected Gram positive, Gram negative and fungal strains. While the antimicrobial efficiency of functionalized MWCNTs is higher than functionalized DWCNTs.

Moreover, it is projected that inhibitory effect of non- covalently functionalized CNTs against pathogens is not com- pletely understood. Previous studies showed that cellular protein was deactivated, and DNA lost its ability to replicate after CNTs interaction with the microbial cells [39,53]. In this work, it is hypothesized that both types of functionalized CNTs release from the synthesized TPU nanocomposite fibers, which may strongly inhibit the growth of microorganisms. Overall, the prepared mul- tipurpose electrospun nanofibers may be good choice as a microbial growth inhibiting agent and have potential to be applied in various biomedical fields.

3.6. Microbial cells aggregation with electrospun nanofibers To comprehend the antimicrobial mechanisms of TPU/f-CNTs electrospun nanofibers with 80 µ^g/mL concentration of surfactant-modified CNTs, the fluorescence microscopy was used to investigate the interaction of synthesized nanofibers with microbial cells. It was observed that the production of cell aggregates with both types of TPU/f-MWCNTs and TPU/f-DWCNTs nanofibers in a similar pattern. Fig. 10 shows the production of cell aggregates with electrospun nanofibers in deionized water. The uniformly distributedStaphylococcus aureuswithout any

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Fig. 9(a). Antimicrobial activity of surfactant-modified MWCNTs/TPU electrospun nanofibers againstStaphylococcus aureus(a),Pseudomonas aeruginosa(b),Klebsiella pneumoniae(c), andCandida albicans(d). The discs containing 50µL of DMF solvent used as a control.

Table 1

Diameter of inhibition zone (mm) for the synthesizedf-MWCNTs/TPU electrospun nanofibers.

Strains Surfactant-modified MWCNTs/TPU electrospun nanofiber with different concentrations off-MWCNTs

20µ^g/mL ⁴⁰µ^g/mL ⁶⁰µ^g/mL ⁸⁰µ^g/mL ¹⁰⁰µ^g/mL TPU-nanofiber

Diameter of inhibition zone (mm) Staphylococcus

aureus

8 10 11 12 14 7

Pseudomonas aeruginosa

– 10 16 18 21 –

Klebsiella pneumoniae

– 8 11 15 18 –

Candida albicans 5 7 9 11 13 6

Table 2

Diameter of inhibition zone (mm) for the synthesizedf-DWCNTs/TPU electrospun nanofibers.

Strains Surfactant-modified DWCNTs/TPU electrospun nanofiber with different concentrations off-DWCNTs

20µ^g/mL ⁴⁰µ^g/mL ⁶⁰µ^g/mL ⁸⁰µ^g/mL ¹⁰⁰µ^g/mL TPU-nanofiber

Diameter of inhibition zone (mm) Staphylococcus

aureus

6 8 9 10 12 7

Pseudomonas aeruginosa

– 5 10 14 16 –

Klebsiella pneumoniae

– 6 9 13 15 –

Candida albicans – 2 7 9 10 6

treatment with nanofibers can be seen inFig. 10A. While the fluorescence images of cell aggregates after mixing with electrospun nanofibers are obvious inFig. 10B, C. Interestingly, a small clump of cells was observed after the cells treated with TPU/f-DWCNTs fibers than TPU/f-MWCNTs fibers that showed more aggregation of cells. All the cells were treated with electrospun nanofibers for 30 min in the shaking incubator [54]. The treatment ofPseu- domonas aeruginosa(Fig. 10D) with these nanofibers showed that the cells aggregated at the center with TPU/f-MWCNTs fibers (Fig. 10F), but less aggregation of microbial cells was observed

after treatment with TPU/f-DWCNTs fibers (Fig. 10E). A similar pattern was observed when Klebsiella pneumoniae treated with the synthesized electrospun nanofibers, as shown in (Fig. 10G–

I). The fluorescence images showed that microbial cells were started to form the small aggregates followed by the addition of TPU/f-MWCNTs nanofibers into the cells suspension in DI water and rarely free cells were observed. However, the prepared TPU/f-DWCNTs nanofibers were also acted in a similar pattern to produce cell aggregates in DI water.Fig. 10L shows the formation of clumps byCandida albicanswith electrospun nanofibers,

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Fig. 9(b). Antimicrobial activity of surfactant-modified DWCNTs/TPU electrospun nanofibers againstStaphylococcus aureus(a),Pseudomonas aeruginosa(b),Klebsiella pneumoniae(c), andCandida albicans(d). The discs containing 50µL of DMF solvent used as a control.

while Fig. 10K demonstrates that the cells are still randomly distributed after treatment and show less cells aggregation. Thus, it was observed the increasing size of cell aggregates during cell–

nanofibers aggregation in all cases, but changed with the size for different types of CNTs.

The detailed antimicrobial mechanisms of cell aggregation formation with electrospun nanofibers are yet to be fully investigated. Both the synthesized TPU/f-MWCNTs and TPU/f-DWCNTs electrospun nanofibers have the capacity to produce cell aggregates in the suspensions, while the antimicrobial activity of f-MWCNTs nanocomposite fibers is more compared to other ones. There may be certain factors associated with the formation of cells–CNTs nanocomposite fibers aggregation, such as van der Waals forces between cell surfaces and molecules of CNTs may be the main factor that induces the formation of aggregation between cells and CNTs nanofibers [49]. However, the less strong interactions between cells and f-DWCNTs nanocomposite fibers within the aggregates may be due to the weakly effective van der Waals forces. In addition, strong contacts between cells and MWCNTs are more possibly to disrupt the cell walls, accounting for the higher antimicrobial property of MWCNTs, while the weak interactions of cells with DWCNTs do not cause severe cell wall damage. Another study reported that long length of MWCNTs may attributed to their higher antimicrobial activity [38]. The larger surface area of MWCNTs may facilitate greater interaction with the cell membrane of the pathogen than DWCNTs. Kang et al. (2008) verified the cell membrane damage by the efflux of higher amount of RNA and DNA of cells (E. coli) treated with SWCNTs at 37^◦C compared to control samples [14]. Thus, there are very limited reports available to understand the significant difference in antimicrobial activity of DWCNTs and MWCNTs;

further studies are required to provide a comprehensive detailed mechanistic explanation of this difference.

3.7. Cytotoxic effects of electrospun nanofibers

It was observed that the synthesized electrospun nanofibers repressed the viability of human lung epithelial cells (A549) in a dose and treatment time dependent manner. The investigated half-maximal inhibitory concentrations (IC50) of TPU/f- MWCNTs and TPU/f-DWCNTs electrospun nanofibers were 80 and 60µg/mL after 48 h, respectively (Fig. 11A, B).

We studied the cytotoxic effects of electrospun nanofibers on the viability of A549 cells. The values of IC50 were 80 µ^g/mL of TPU/f-MWCNTs nanofibers (Fig. 11A) compared to TPU/f- DWCNTs nanofibers (Fig. 11B), which was 60µg/mL after 48 h of incubation with A549 cells. The results indicate that the cell viability was maximum after 24 h of incubation, which gradually decreased after 48 h. Thus, the viability of cells was minimum with a 100µg/mL concentration of both types (TPU/f-MWCNTs and TPU/f-DWCNTs) of electrospun nanofibers. It indicates a direct association between treated time and dose off-CNTs in the synthesis of electrospun nanofibers to induce cytotoxicity. These findings are based on the previous work conducted by Ursini et al.

(2012) that CNTs could be toxic for the human lung epithelial cells (A549) and also revealed that the viability of cell decreases as the concentration of carbon nanotubes increases [55]. Another study conducted by Di Giorgio et al. (2011) reported the genotoxic and cytotoxic effects of CNTs (SWCNTs and MWCNTs) on the mouse macrophage cell (RAW 264.7) [56]. They observed that MWCNTs with concentration (200–400 mg/mL) induced dose-dependent cytotoxicity after exposure to rat glioma cells (C6 cells). Hence, it has also been proposed that large sizes of CNTs may be less cytotoxic than smaller ones [57]. According to the current study, it can be deduced that electrospun nanofibers produced by TPU/f- MWCNTs are more suitable for biomedical applications because of less cytotoxicity compared to the other ones. Previous studies have reported that cytotoxicity of nanoparticles is mainly affected by the size, purity, and shape of nanomaterials [49,58,59]. Some

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Fig. 10. Fluorescence images of (A–C)Staphylococcus aureus, (D–F)Pseudomonas aeruginosa, (G–I)Klebsiella pneumoniae, and (K–L)Candida albicans. The images were taken using a FITC-HYQ (fluorescein isothiocyanate) filter.

Fig. 11. The viability of human lung epithelial cells (A549) by TPU/f-CNTs fibers. The cells were treated with different concentrations of (A) TPU/f-MWCNTs fibers and (B) TPU/f-DWCNTs fibers for 24 and 48 h. The results were expressed as the mean±SD and p-values<0.05 were considered significant compared to control samples.

earlier data reported that CNTs can produce more toxic effects on the lung because this organ is the most susceptible to the CNTs-induced toxicity compared to other organs [60–63].

4. Conclusion

In this work, the antimicrobial activity and cytotoxicity of synthesized electrospun nanofibers have studied. For antimicrobial activity, different bacterial and fungal strains have selected

and human lung epithelial cells (A549) used for cytotoxicity investigation. The results show that TPU/f-MWCNTs and TPU/f- DWCNTs electrospun nanofibers have the potential to inhibit the growth of microbial cells. The reduction in the microbial cells viability and A549 cell line depends on the treatment time and dose of surfactant-modified CNTs in the synthesis of electrospun nanofibers. The fluorescence images show the formation of cell aggregates after treatment with electrospun nanofibers that may be due to the van der Waals forces between cell surfaces and molecules of CNTs. Moreover, the morphology of prepared electrospun nanofibers was evaluated by field-emission

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scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Fourier transform infrared spectroscopy (FTIR) findings exhibited that polyurethane polymer was covalently attached to thef-CNT’s sidewalls. The number of viable cell count, diameter of inhibition zone and optical density growth curve values confirmed the rate of microbial growth inhibition after treatment with nanofibers. However, cytotoxicity results indicate that cell viability was maximum after 24 h of incubation, which gradually decreased after 48 h. Thus, it indicates a direct association between treatment time and dose of surfactant-modified CNTs in the synthesis of electrospun nanofibers to induce cytotoxicity. This study may pave the way for developingf-CNTs-fiber composites which offer various opportunities to apply in a wide range of applications in the antimicrobial field.

CRediT authorship contribution statement

Mansab Ali Saleemi:Designed, Conceived, and conducted the experimental work, Analyzed and interpreted the results, Pre- pared the figures, Reviewed, and approved the final draft.Phelim Voon Chen Yong:Edited the draft, Reviewed, and approved the final manuscript. Eng Hwa Wong: Designed, Conceived, Inter- preted the results, Contributed to the materials/reagents analysis tools, Reviewed, and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing finan- cial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was supported by the Faculty of Health and Medi- cal Sciences, and Faculty of Innovation and Technology, Taylor’s University Lakeside Campus, Malaysia.

Funding

This project was financially supported by Taylor’s University Flagship Research, Malaysia Grant with project code:

TUFR/2017/001/05.

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