Layer-by-Layer Assembled, Amphiphilic and Antibacterial Hybrid Electrospun Mat Made from Polypropylene and Chitosan Fibers

In this study, hybrid fiber mat (HFPP-CS) consisting of both chlorinated polypropylene and chitosan fibers (FPP-Cl and FCS) is obtained by assembling layer-by-layer for the first time using electrospinning process. Morphological, wettability, structural and thermal properties of HFPP-CS are investigated in detail by Scanning electron microscopy (SEM), water contact angle (WCA) measurement, fourier transform infrared spectroscopy, thermogravimetric analysis and differential scanning calorimetry analyses, respectively, comparing with FPP-Cl and FCS. Furthermore, the antibacterial activity of all samples was evaluated against to gram positive Staphylococcus aureus (S. aureus) and gram negative Escherichia coli (E. coli) bacteria. SEM analysis proves to HFPP-CS has a circular and smooth morphology and also comprises of microscale FPP-Cl and nanoscale FCS layers within the range of 2.1–2.7 μm and 175–475 nm, respectively. The incorporation of nano-FCS layer on micro-FPP-Cl layer leads to diminution in the hydrophobicity from WCA value of 137° ± 2 to 124° ± 2, but enhancement in the thermostability and glass transition temperature of the resulting fiber mat. The antibacterial activity results show that HFPP-CS has higher inhibition effect against to S. aureus and E. coli than FPP-Cl but lower than FCS. In general, it is anticipated that the prepared amphiphilic and antibacterial HFPP-CS can be employed as potential biomaterial for a variety of bioengineering applications. The novel hybrid fiber mat having natural and synthetic polymer layers has prepared by assembling layer-by-layer using electrospinning. The achieved amphiphilic hybrid fiber mat demonstrates higher thermal properties than that of each pristine fiber layer. The hybrid fiber mat has good antibacterial activity against to both gram positive and gram negative bacteria.


Introduction
Polyolefins (POs), in general consisting of only hydrogen and carbon atoms, are widely utilized in a variety of emerging commodity applications covering from plastics, elastomers to fibers, due to their advantages of superior thermal and environmental stability, easy processability, high recyclability, corrosion and chemical resistance [1,2]. In this context, they account for a very large part of the annual production capacity of the global plastic industry [3]. However, besides their many unique features, they represent some drawbacks that limit their usage, including low reactivity, poor adhesion, high photo oxidation sensitivity and incompatibility with other materials [4,5]. Thus, the modification of POs without sacrificing their service performance has long been a highly desirable subject of study for both industrial and academic researchers [6,7]. Halogenation process [8], copolymerization [9], grafting [10], plasma treatment [11], impregnation [12], blending [13] and nanoparticle introducing [14] are among the reasonable addressed ways in the literature to extend the range of their applications. The introduction of functional groups by halogenation enhances the adhesion, solvent resistance, and gas permeability but also provide reactive sides which can be used in several well-known chemical reactions of the olefin backbone [15,16]. Among the halogenated POs, chlorinated polypropylene (PP-Cl) has several unique features such as heat and light stability, and resistance to abrasion, age, acid and alkali [17] and has also been extensively evaluated in many research studies for different purposes [18][19][20][21][22].
Chitosan (CS), as a kind of polysaccharide biopolymer, has distinct properties such as biodegradability, nontoxicity, low immunogenicity, good biocompatibility, antibacterial activity and film forming capacity, making it an interesting compound for bio-applications [23]. The combination of these many special properties with those of the synthetic polymers that build up the backbone is widely required in biomaterial design and development. Therefore, researchers have proposed the combined use of synthetic and natural polymers in recent years. However, studies on chlorinated POs in order to combine their properties with natural polymers requires more challenging, harsh and restrictive operating conditions as well as additional reaction steps [24][25][26]. On the other hand, there was no any research paper in the literature on direct usage of chitosan with polypropylene due to the immiscibility problem. The chlorinated polypropylene can be used to overcome this drawback by taking advantage of its polarity enhancing chlorine functionality. Based on different studies in the literature, the idea that hybrid fiber mat possessing FPP-Cl and FCS segments can be used in many different biomedical applications including biosensors, tissue engineering and drug delivery has emerged. In this regard, the layer-by-layer (LBL) assembly technique is regarded as a low-cost, simple, reliable and effective technique for producing uniform films through sequential deposition of species via secondary or electrostatic interactions, or hydrogen bonding [27]. LBL assembly can be used as an effective one-step surface modification technique to alter the wettability of electrospun mats. Two alternative routes are possible so as to achieve amphiphilization of electrospun mats: (i) preparation of electrospinning solution by mixing the hydrophobic and hydrophilic or amphiphilic polymer and (ii) LBL deposition of these polymers on each other during the electrospinning.
Among the fiber production techniques [28], electrospinning providing the high surface area to volume ratio, is utilized to fabricate the fibers with well-controlled surface topography, morphology and size distribution at micro-or nano-scale range. This method, which can be applied to both natural and synthetic polymers and inorganic materials when appropriate conditions are provided, is a versatile for many applications particularly like sensor technology, textile, filtration, protective clothing, wound dressing, drug delivery, etc. [29][30][31][32][33][34]. It is clearly understood that the LBL deposition of different fibers made from different polymers by electrospinning is an effective process that will be able to utilize to produce composite multilayer scaffolds.
The preparation of CS based materials has received growing attention in extensive biomedical fields due to its inherent properties particularly including nontoxicity, antibacterial activity, biocompatibility and biodegradability [35]. Especially, it is well known that the antibacterial activity of chitosan is high against a wide variety of fungi, yeast, bacteria and viruses [36]. Meanwhile, the electrospinning of chitosan is hard to achieve due to the complicated behavior of its solution. In this regard, some polymers such as PLA [37], PVA [38] or PEO [39] were used to improve electrospinnability of chitosan. Furthermore, it was proven by the studies in the literature that the smooth and bead-free nanofibers of pristine CS were attained from its solution in TFA/DCM cosolvent system [40,41]. On the other hand, the electrospun microfibers of PP-Cl are achieved under ambient conditions in our laboratory for the first time in the literature [21]. However, to the best of our knowledge, there is no report on evaluating the LBL assembled PP-Cl and CS fibers based hybrid mat fabricated by electrospinning and its antibacterial properties. Based on the above information, hybrid fiber mat possessing FPP-Cl and FCS were developed by LBL technique using electrospinning. The concerned properties of resultant composite fibrous mat and its precursors were investigated accordingly in terms of morphology, wettability and thermal properties. The main focus was to find out to what extent PP-Cl / CS bicomponent fiber mats will exhibit antibacterial activity.

Instrumentation
The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of the samples were recorded by Perkin-Elmer Spectrum BX spectrometer over a range of wavenumber 4000-400 cm −1 with 4 cm −1 resolution and by averaging16 scans. Water wettability of surfaces of the specimens was determined on a Theta Optical Tensiometer device (CAM-200) and water droplet photographs were captured with a conventional digital camera. The other surface properties such as topography and morphology of the samples were examined by Scanning electron microscope (SEM, JEOL JSM 6510-LV, IET International Equipment Trading Ltd. Mundelein, Illinois USA) observations. The samples were coated with palladium using Polaron Sputter Coater SC7640 (Quorum Technologies, East Sussex, UK) to be examined by means of SEM. Thermal stabilities of the samples were determined by conducting thermogravimetric analysis (TGA, SeikoSII TG-DTA 6300 TG/DTA analyzer) under nitrogen atmosphere at the scanning range of 25-600 °C and heating rate of 10 °C/min. The glass transition temperatures of the samples were determined by differential scanning calorimetry (DSC, Mettler Toledo DSC analyzer) analysis at the scanning range of 25 to 100 °C and 25 to 400 °C by 10 °C/min heating rate using a nitrogen flow. The samples were weighed approximately to 3-5 mg before the both thermal analyses.

Fabrication of Electrospun Mat Made of Polypropylene and Chitosan Fibers (HFPP-CS)
The layer-by-layer (LBL) assembly technique was used to fabricate the amphiphilic HFPP-CS and its precursor fibers by means of electrospinning. Firstly, pure PP-Cl electrospinning solution (20%, w/w) was prepared in THF at room temperature under constant stirring for three hours until a homogeneous solution was achieved. Then, the microfibers of PP-Cl were fabricated by electrospinning by using parameters of tip-to-collector distance (TCD) of 17 cm, 15 kV applied voltage and 1.3 mL h −1 flow rate. On the other hand, pristine CS electrospinning solution (5%, w/w) prepared in TFA and DCM (7:3, v/v) solvent mixture at room temperature for overnight. The TCD, applied voltage and flow rate was kept at 10 cm, 15 kV and 0.1 mL h −1 , respectively to achieve the nanofibers of CS. The HFPP-CS composite mat was prepared by electrospinning from the pristine PP-Cl firstly, and then CS was electrospun onto the surface of as-spun PP-Cl fibers. In all cases, a typical laboratory scale NE-100 model electrospinning set up (Inovenso, Turkey) was utilized. The solutions were poured into a plastic syringe (5 mL) with a metal needle tip which was driven by a syringe pump (New Era Pump Systems Inc.) and a high voltage power supply (Elektrosis, PW1010, Turkey). Moreover, electrospinning durations were 1 h and 30 min for PP-Cl and CS, respectively. During the electrospinning processes, laboratory temperature was 23.1 °C, whereas relative humidity was 50%.

Water Contact Angle Measurements
The static water contact angle (WCA) of the achieved composite fiber mat and its pristine precursors coated on microscope glass slides was analyzed by placing 5 µL of ultra-pure water onto sample surfaces by a computer-controlled liquid dispensing system. The system consists of a syringe pump, a micro-syringe equipped with stainless steel needle. A conventional digital camera was utilized to capture the representative images of water droplet on the sample surfaces. The average of left and right contact angles was reported after eight measurements on different places of the specimens with ± SD by using Laplace-Young fitting algorithm.

In vitro Bactericidal Efficacy Test
The lowest antimicrobial agent concentration at which a microorganism is visibly inhibited from growth is described as the "Minimal Inhibitory Concentration (MIC)". Dilution methods are used to determine the MIC values of antimicrobial agents. Microdilution test; It is widely preferred in the MIC determination of compounds with potential antimicrobial activity due to advantages such as sensitivity, reproducibility, and ease of application. In this work, microdilution test was performed according to CLSI (Clinical and Laboratory Standards Institute) instructions.

Bacterial Suspension Preparation Procedure
Escherichia coli (ATCC 25,922) and S. aureus (ATCC 29,213) were inoculated into Muller Hinton Agar medium and incubated for 18-24 h. At the end of the incubation period, a single colony was selected from the plate and the density of the bacterial suspension homogenized by vortex agitation was adjusted by reading the 0.5 McFarland turbidity (1 × 10 8 CFU/mL) standard with physiological sterile saline solution (0.9% NaCl) using a densitometer (Densimat; Biome´rieux, Marcy l'Etoile, France). The bacterial suspension (1 × 10 8 CFU/mL) adjusted to 0.5 McFarland was diluted 1/10 to obtain 10 7 CFU/mL. 5 µL final bacteria suspension was pipetted into microplate wells for each cases. Schematic representation for preparation of bacterial suspensions was illustrated in Fig. 1.

Application of the Microdilution Test
96-well U-bottom sterile Tissue Culture Testplate microplate (TPP, Switzerland) was used for the test. 100 μL of cation-adjusted sterile BBL Müller Hinton Broth II (CAMHB, Becton Dickinson, USA) was pipetted under aseptic conditions into all wells of the microplate. The entire surface of microscope glass slides covered with electrospun fibers was scraped and 1 µg was taken. 1 µg of electrospun fibers were homogenized in 1 mL CAMHB and 100 μL was pipetted into the first well. Serial dilution was made by taking 100 μL from this first well and transferring it to the other well. Dilution was continued until well number 8 and 100 μL was ejected from this well. Wells numbered 9-10 were arranged as sterility control (SC) and well number 11 and 12 were labelled as E. coli and S. aureus negative bacteria growth control well (NC), respectively. From the standardized bacterial suspension of 10 7 CFU/mL, 5 µL was pipetted into all wells except sterility control wells numbered 9-10.

Results and Discussion
Advances in the modification of composite fibers produced by the LBL assembly technique in solutions and in combining the properties of natural and synthetic polymers gave us the idea for the production of PP-Cl and CS based fibrous mats for biomedical applications. In this purpose, firstly, the fabricated electrospun FPP-Cl, FCS and HFPP-CS were morphologically compared by using SEM analysis (Fig. 2). As illustrated in Fig. 2, pristine FPP-Cl and FCS exhibited nearly uniform fiber morphology and cylindrical fibers within the range of 2.1-2.7 μm and 175-475 nm, respectively. Moreover, the comparison between precursor micro and nanofibers and HFPP-CS fibers, it was worth noting that CS nanofibers were successfully adhered onto PP-Cl microfibers by LBL deposition (Blue arrows indicate the FPP-Cl, purple arrows show the FCS). The supporting information about this finding was the probable intermolecular electrostatic interactions between chlorine functionalities of PP-Cl and CS main chain [42].
By using the second LBL approach mentioned in introduction part, the first layer was hydrophobic PP-Cl microfibers, whereas the second layer was hydrophilic CS nanofibers. It has been well-known from the literature that the polymers having long saturated hydrocarbon chains like polypropylene (PP) has hydrophobic surface properties [43]. On the other hand, it was observed that chlorination of PP to obtain PP-Cl did not caused any significant alteration in hydrophobicity, may be due to insufficient chlorine functionality [44]. Sessile drop technique was used for investigating the resulting hydrophilic-hydrophobic transitions. After the assembly of a hydrophobic FPP-Cl and FCS possessing hydrophilic groups [45] by the LBL technique using electrospinning, a WCA value of 124° ± 2 determined for HFPP-CS which was between the WCA of both polymers proved that the HFPP-CS obtained was amphiphilic, as expected (Fig. 3).
The LBL assembling of electrospun FPP-Cl and FCS was structurally elucidated by observing specific group peaks by comparative FT-IR spectroscopy study, as shown in Fig. 4. First, it was found that the absorption observed around 2940 and 735 cm -1 corresponding to the asymmetric and symmetric stretching vibrations of C-H and stretching vibrations of C-Cl groups of electrospun FPP-Cl, respectively [16]. The specific signals, including the -OH stretching at 3450 cm −1 , C-H stretching at 2900 cm −1 , bending of amide I at 1670 cm −1 , amide II at 1520 cm −1 and amide III at 1420 cm −1 , C-O stretching vibrations at 1370 and 1030 cm −1 were clearly observed for electrospun FCS [42]. After the LBL assembling by electrospinning, the intensified -OH, amide I, II and III stretching vibrations of HFPP-CS compared to pristine FCS clearly confirmed the presence of the intermolecular electrostatic interactions between PP-Cl and CS main chains [42]. In addition, the other important finding was the preservation of all the major characteristic signals of both polymers in the HFPP-CS spectrum.
Evaluating the thermal features of electrospun mats consisting of different species is an important consideration, especially in the fields of biomedical applications. In this context, thermal stabilities and glass transition temperatures (T g s) of both precursor FPP-Cl and FCS as well as their LBL assembled mat were determined and comparatively studied by TGA and DSC analyses (Figs. 5 and 6). It is necessary to examine the thermal stability/structure relationship of chlorine-containing polymers, which are generally easy to thermally degrade [46]. TGA was conducted to present the decomposition pattern and hence the compositional differences of the specimens at the scanning range of 25-600 °C. Thermogravimetric curves of pristine and LBL assembled electrospun were illustrated in Fig. 5. As seen in this figure, the exhibited two thermal maximum decomposition stages (T 1 and T 2 ) recorded at 215 °C and 350 °C for FPP-Cl due to the dehydrochlorination and degradation of PP-Cl main chain, while the same number of decomposition stages represented at around 100 and 200-500 °C was determined for FCS resulting from evaporation of the adsorbed water and degradation of polymeric CS backbone, respectively, in accordance with previously published results [46,47]. TGA curves also illustrated that the maximum degradation temperatures slightly shifted to higher for electrospun HFPP-CS specimen after LBL assembling of the precursors (Table 1). This result may be another indication of the presence of electrostatic interactions between the FPP-Cl and FCS. It was also seen from the DSC curves that LBL assembling positively affected the T g of the resulting mat. As can be seen in Fig. 6, T g value of micro FPP-Cl was 45 °C, after the nano-FCS deposition on it, the T g value enhanced to higher temperature due to the restrictive effect of hard CS rings on segmental mobility achieved mat or its more oriented chains [48]. Meanwhile, the endothermic and exothermic peaks of FCS curve determined at approximately between 80-120 °C and 275-300 °C were corresponded to the evaporation of residual solvent and decomposition of chitosan backbone [49,50]. All achieved results from the thermal analyses were also summarized in Table 1.
The different nanotechnologies providing the large surface area have increasingly been utilized in order to develop the antibacterial materials especially for biomedical applications [51]. The electrospinning method facilitates the production of antibacterial fiber composites through the layered of neat polymeric fiber components from a solution or melt [52]. The antibacterial properties of HFPP-CS developed on the basis of these cases were assessed by determining MIC via broth dilution method (Fig. 7) [53]. After pipetting as described in application of the microdilution test section, the microplate was covered with adhesive foil to prevent drying and incubated at 37 °C for 16-20 h. The lowest concentration of fibers without visible turbidity, which prevents the growth of bacteria, was evaluated as the minimum inhibitory concentration. The photographs above Fig. 7 visually show the amount of turbidity after the samples were seeded with bacteria. Consequently, the amount of turbidity of the sample with the most antibacterial effect is the least. The 64-fold diluted FCS was effective in inhibiting the growth of S. aureus and E. coli, whereas no growth was observed in media including 16-fold diluted FPP-Cl and 8-fold diluted FPP-Cl for S. aureus and E. coli, respectively. In the case of  HFPP-CS, 32-fold dilution and 16-fold dilution were enough the inhibiting the growth of same bacteria. It was clearly seen from the Fig. 7 that HFPP-CS had a lower than FPP-Cl, but higher MIC than FCS against both bacteria. These findings were as anticipated when the results from the WCA and SEM analyses were considered [28,[54][55][56]. When the antibacterial activity of the mixture/blend of other chlorinated polymers with various natural or non-natural compounds/ polymers was considered, it was observed that the inhibition effect of HFPP-CS obtained from this study was not negligible [57].

Conclusions
Materials that are susceptible to bacterial invasion and infections compel researchers to develop various properties of these materials with innovative methods for many research areas. In the present research, a new dual-layer hybrid electrospun fiber mat possessing both synthetic and natural polymer with antibacterial activity was successfully fabricated. The lower layer included chlorinated polypropylene fibers surrounded by chitosan fibers as an external layer. SEM analysis proved that the lower FPP-Cl layer was thicker than FCS external layer. A significant effect of FCS deposition on the FPP-Cl on wettability, thermostability and T g of the hybrid fiber mat was observed. Ultimately, the nano-FCS coated hybrid fibers had a good antibacterial activity against both gram positive and gram negative bacteria. The MIC of HFPP-CS was lower than FPP-Cl but higher comparing to FCS, as expected. Therefore, it is anticipated that the findings of presented study will provide new contributions to researchers particularly working on biomedical applications. Authors Contribution GA: formal analysis, investigation, project administration, data curation, writing-original draft, methodology, conceptualization, software, supervision, writing-review and editing BA: formal analysis, methodology, investigation, writing-original draft. EA: formal analysis, antibacterial test, investigation.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.