Antibacterial bi-layered polyvinyl alcohol (PVA)-chitosan blend nanofibrous mat loaded with Azadirachta indica (neem) extract

International Journal of Biological Macromolecules 138 (2019) 13–20

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Antibacterial bi-layered polyvinyl alcohol (PVA)-chitosan blend nanofibrous mat loaded with Azadirachta indica (neem) extract Ayub Ali a,⁎, Md. Abdus Shahid a, Md. Delwar Hossain a, Md. Nurul Islam b a b

Department of Textile Engineering, DUET, Gazipur 1707, Dhaka, Bangladesh Department of Chemistry, DUET, Gazipur 1707, Dhaka, Bangladesh

a r t i c l e

i n f o

Article history: Received 8 May 2019 Received in revised form 1 July 2019 Accepted 2 July 2019 Available online 03 July 2019 Keywords: Electrospinning Chitosan-neem nanofiber Bi-layered technique

a b s t r a c t The present study suggests the formation of polyvinyl alcohol (PVA)-Azadirachta indica (neem)-chitosan blend nanofibrous mat (PNCNM) by bi-layered technique under optimum processing conditions. The antibacterial activity against Staphylococcus aureus (S. aureus) bacteria, morphology, bonding behavior, thermal stability, tensile behavior and moisture management properties of the developed sample had been investigated. The scanning electron microscopy (SEM) images revealed the homogeneous and smooth fibers produced having average diameter of 213.52 nm (nm) with the minimum and maximum diameter of 152 nm and 298 nm respectively. Besides, it showed 91% porosity which is indicative of porous structure. The presence of PVA, neem constituents and chitosan was established by Fourier Transform Infrared Spectroscopy (FTIR) indicating the formation of hydrogen bonding among them. The addition of neem extracts led to enhanced thermal stability and moisture management properties. In addition, the developed mat showed a tensile strength of 18.78 N corresponding to the elongation value of 4.98 mm. Besides, the incorporation of neem extract into the nanofiber mat exhibited a significant synergistic antibacterial activity against bacterial cells through the formation of inhibition zone. Thus, the newly developed nanofibrous mat could turn out to be a suitable material for the wound dressing purpose. © 2019 Elsevier B.V. All rights reserved.

1. Introduction The emergence of electrospun nanofibers from natural and modified natural polymers as novel materials in biomedical applications including wound dressing, tissue engineering and drug delivery is a contemporary research field both in academic and industrial sectors because of their biocompatibility, biodegradation, low toxicity, high porosity, light weight, excellent pore interconnectivity and most prominently large surface area [1–4]. Electrospinning technique being incredibly straightforward, simple and cheaper is usually used to produce these fibers due to its highly versatility, cost effectiveness, production of continuous nanofibers, ease of surface functionalization and industrial application as well as unique capability to generate fibers from several micrometers to nanometers under applied electrical forces [3,5–13]. The large surface to volume ratio offers a high active contact surface of nanofibrous mat and their porous structure favors cell adhesion, proliferation and differentiation [3,4,14–17]. In addition, it attains uniform adherence on wet wound surface without any fluid accumulation and thus, can meet the requirements such as higher gas permeation and protection of wound from infection and dehydration [18]. Usually, metal nanoparticles are used to impart ⁎ Corresponding author. E-mail addresses: [email protected] (A. Ali), [email protected] (M.A. Shahid), [email protected] (M.D. Hossain).

https://doi.org/10.1016/j.ijbiomac.2019.07.015 0141-8130/© 2019 Elsevier B.V. All rights reserved.

antibacterial properties to the nanofibers [19,20] but the detrimental effect of these nanoparticles and synthetic antibiotics on the environment, human health, and/or bacteria resistance issues has triggered the use of natural antimicrobial compounds because they are expected to be nontoxic, environmentally sustainable, and less prone to create resistant bacteria. Consequently, electrospun nanofibrous mats incorporating natural remedial plant extract showed potentiality to be used invarious biomedical applications especially for wound dressings due to the inherent medicinal properties [18,21–24]. For instance, nanofibers from polyacrylonitrile-moringa extract was fabricated and characterized for antimicrobial and wound healing applications [25]. Majd et al. [26] developed chitosan/PVA nano fiber to be used as wound dressing for streptozotocin-induced diabetic rats and found significant acceleration in diabetes wound healing on the rats. Besides, nanofibrous mat from the mixer of PVA-aloe vera, PVA-henna, PEO-henna, polycarbonate/leaf extract, wool keratin/polycaprolactone, starch, collagen, PCL/GT nanofiber loaded with curcumin and PCL-natural extract were also investigated by several studies [7,27–33]. Azadirachta indica, prominently called as neem, containing more than 140 bioactive constituents with azadirachtin as main constituents (Fig. 1) has been well known in the Indian subcontinent as one of the most versatile medicinal plants exhibiting a wide spectrum of biological activities such as antimalarial, antiulcer, antibacterial, antifundal, antiviral, antioxidant, anti-inflammatory, antimutagenic and anticarcinogenic [34,35]. It

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A. Ali et al. / International Journal of Biological Macromolecules 138 (2019) 13–20

O O O

CO2 Me OH

O

O

H OAc CO2Me

O OH

OH Fig. 1. Chemical structure of azadirachtin [35].

has been extensively used traditionally for the treatment of inflammation, infections, fever, skin diseases and dental disorders [36–38]. The presence of different bioactives such as assteroids, sugars, triterpinoids, alkaloids, reducing sugars, tannins, flavonoids, sesquiterpene lactones, and phenolic compounds [39,40] are mainly responsible for its inherent antibacterial properties [41–46]. However, the natural form of these herbs offers a limited acceptability in wound dressing purpose which has been overcome by adopting new approaches including electrospinning, electrospraying, solution blow spinning and film casting to use plant extracts in the form of suitable products to serve as wound dressing materials [22,47]. On the other hand, chitosan, a deacetylated derivative of chitin, the second most abundant polysaccharide after cellulose, is a natural polysaccharide present in the exoskeletons of crustaceans, insects, and some fungi. It consists of a linear chain of β (1 → 4) linked N-acetyl Dglucosamine monomer units and the presence of amino moieties displays its unique polycationic, chelating, and film-forming properties. The protonated amino groups exert an antimicrobial function due to electrostatic interactions between the polycationic structure of chitosan and anionic cell wall of microorganisms. Chitosan shows a wide spectrum of antimicrobial activities against both gram-positive and gram-negative bacteria [4]. Polycationic chitosan could interact with anionic groups on the cell surface thereby causing an increase in membrane permeability which in turn leads to disruption and leakage of cellular proteins. This electrostatic interaction facilitates the higher activity of chitosan against gramnegative (E. coli) than gram-positive bacteria (S. aureus) which can be overcome by adding another antibacterial plant extract [48,49]. Moreover, chitosan is nontoxic towards mammalian cells and biodegradable [50–53]. Fabrication of chitosan nanofibrous mat with or without different natural and manmade chemicals like henna, PVA, PEO, sericine, silk fibroin, lysozyme-CLEA for different purposes such as wound dressing and antibacterial materials have been studied by several researchers [4,54–61]. Besides, application of functionalized chitosan by O-amine for antibacterial and antioxidative activity [62], artificial skin from chitosan composites [63], composite membranes from chitosan-hyaluronan mixer loaded with mitoQ for wound healing [64], polymeric foam dressings of modified chitosan blends for topical wound delivery of chloramphenicol [65], chitosan associated chlorhexidine in gel form for healing wounds applications [66], wound dressing films from the mixer of chitosan/copaiba oleoresin for would dressing [67], formation and evaluation of glycerol plasticized chitosan/PVA blends for burn wounds [68], wound dressing based on electrospun PVA/chitosan/starch hybrid mixer [69], hydrogels of chitosan-alkali lignin as potential wound healing materials [70], development of polyelectrolyte nanocomplexes based on chitosan derivatives for wound healing applications [71], application of chitosan aerogel particles loaded with vancomycin for chronic wound applications [72] are some prominent studies conducted by different researchers in recent years. But the formation of nanomat using chitosan incorporated with neem herb for wound dressing purpose has not yet been studied thoroughly by researchers. The extreme brittleness of pure chitosan polymer and its collection difficulty prevent it from using

alone. This has been avoided in this study employing bi-layered technique which paves the way for using chitosan alongside neem to be transformed into something capable of being used conveniently. In this study, PVA/chitosan blend nanofibrous mat loaded with neem extract were fabricated by bi-layer technique to enhance antimicrobial efficacy and biocompatibility of nanofibrous mats for wound dressing purpose. In the resulting developed mat PVA is used as a carrier polymer because of its nontoxicity, biocompatibility, good fiber forming ability and wide applications in the biomedical field [73] whereas chitosan and neem have played the role of co-polymer and biocide respectively. The synergistic effect of chitosan and neem to impart antibacterial property was studied by disc diffusion method. The bonding behavior and the thermal stability were evaluated by ATR/FTIR spectroscopy and thermal analysis (TGA and DSC). The morphology of nanomat and its moisture management properties were investigated by SEM and moisture management test (MMT) respectively. 2. Materials and methods 2.1. Materials The leaves of Azadirachta indica were collected from local area of Gazipur city, Dhaka, Bangladesh. Chitosan (medium MW, degree of deacetylation: 75–85%, 200–800 cps) and cationizer (ForCat BCD) were purchased from Sigma-Aldrich Co, USA and Fortune Top Pte Ltd., Taiwan respectively. Polyvinyl alcohol (PVA) with molecular weight (MW) of 1,15,000, DP of 1700–1800, Viscosity: 26–32 cps, 99% hydrolyzed granules was sourced from loba chemical (India). Reagent grade acetic acid (99.7%) and absolute methanol with purity of 99% were purchased from Merck, Germany. All the chemicals were of analytical reagent grade and used without further purification. 2.2. Preparation of neem leaves extract The fresh leaves were collected and oven-dried at a temperature of 45 °C. The dried leaves were then ground into a fine powder using a mixer grinder. Then 30 g of dried leaves powder was suspended in 150 mL of absolute methanol and kept for 24 h. The process is repeated for three times with fresh methanol every time. The extract was filtered through nylon mesh and later through a Whatman No. 1 filter paper under suction. The resulting solution was then evaporated to remove the solvent at 40 °C. Then the remaining solvent was removed and stored at 4 °C for further use [74]. 2.3. Preparation of electrospinning solutions Chitosan solution was prepared at 3 wt% concentrations in 50 wt% aqueous acetic acid solution. The solution was made at room temperature using a laboratory magnetic stirrer for 24 h maintaining the pH of solution at 4. Cationizer (as a surfactant) (1.5 mL) was added to chitosan solution to reduce surface tension and then again stirred for 1 h. The final neem loaded solutions was made by adding neem extract [75] to the blend solution. This solution was stirred for 5 h to ensure complete dissolution and resulted in a homogeneous mixture. Besides, the PVA solution was prepared by dissolving 10 g PVA into 100 mL of distilled water. Then solution was stirred at 80 °C for 3 h using magnetic stirrer to get a homogenous and crystal-clear solution. 2.4. Parameters optimization of the electrospinning process In this experiment, electrospinning set-up consisted of a high voltage supply (−20 kV and +50 V), a syringe pump (TL-F6, Tong Li Tech, China), a rotary drum collector (diameter-158 mm, length-500 mm, 500 rpm), a syringe (30 mL), a heater (0.5 kW) and 5 needles (20 Guage). All the components were used as a single electrospinning machine (model: TL-ProBM, company: Tong Li Tech, origin: China). Since, natural polymer like

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chitosan is difficult to collect after electrospinning because of its brittleness that's why a new approach has been followed in this study which includes the formation of background nanomat by PVA solution and then chitosan/neem mixer was inserted onto that in nanofibrous form. At first, about 20 mL PVA solution was placed into the pump and connected to needles by a pipe. After that 50 mL chitosan/neem solution was electrospun on it. The best spinning condition was achieved at −12.3 kV, +23 kV, 0.45 kW with a flow rate of 3 mL per hour with ambient condition 65% relative humidity and 27 °C respectively. Nanofibrous mat was collected on aluminum foil dried overnight and conditioned appropriately for characterization. The process sequence of producing nanofibrous mat from the mixer of PVA-chitosan and neem is shown in the Fig. 2. 2.5. Characterizations 2.5.1. Tensile property The tensile property of the nanofibrous mat was evaluated using a universal tensile testing machine (Testometric, M250-3CT, India) equipped with a precise load cell capacity of 300 N. Testing was conducted according to ASTM D5034 test method with the nanomat thickness of 0.4 mm. Test speed of 300 mm/min was used to perform the test under ambient conditions. Since, the PVA nanonat is so weak that it is not possible to determine its tensile property under the aforementioned load and that's why only PNCNM was tested. 2.5.2. Attenuated Total Reflectance (ATR)/Fourier transform infrared spectroscopy (FTIR) The chemical structure of neem powder and PVA nanofibrous mats with and without neem-chitosan was characterized using spectroscopy (IRPrestige21, Shimadzu Corporation, Japan). The spectra of the samples were recorded in 698–4000 cm−1 range with 4 cm−1 resolution. 2.5.3. Morphology The orientation of nanofibers along with their diameter was measured by scanning electron microscope (SEM) (SU 1510, Hitachi, Japan) at a magnification of 15 k times. Besides, the porosity of dry nanofibrous mat was determined by weighting the samples and calculating their volume from the thickness and area measurements. Porosity of the fibrous mat was estimated from Eq. (1).  ρ Porosity ¼ 1− Þ  100 ð1Þ ρ0

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electrospun mat was determined by measuring the mass to volume ratio of the sample, which was chopped into 5.9 cm × 5.9 cm dimension with a thickness of 0.4 mm and then has weighed using a precise balance. The sample thickness was measured at several points by digital thickness meter and the measured values were averaged. 2.5.4. Thermal properties Thermal behavior i.e. thermogravimetric analysis (TGA) of the developed mats was evaluated by a thermal analyzer (SDT 650, Discovery, USA) within the temperature range of 50-300 °C at a constant heating rate of 5 °C per minute. Approximately 7.5 mg nanofibrous mat was taken as sample and subjected to heat to evaluate its characteristics. 2.5.5. Measurement of moisture management Moisture management property of the developed mats was evaluated by a moisture management tester (M290, SDL Atlas, UK) according to the method of AATCC 195-2009. Wetting time, absorption rate, maximum wetted radius and spreading speed of inner and outer surface including accumulative one-way transport capacity (R) and overall moisture management capacity (OMMC) were evaluated following the above mentioned standard to categorize the mat due to its interaction with liquid. 2.5.6. Antibacterial assay Antibacterial activity of the electrospun nanofibrous mat was studied using disc diffusion method against Staphylococcus aureus (S. aureus) bacteria (as gram-negative like E. coli is less and grampositive like S. aureus is more responsible for wound infection) and the zone of inhibition was measured. A qualitative disk diffusion test using 0.5 × 106 colony forming units (CFUs)/mL of the S. aureus was cultured on TSA plates while PVA nanomat was used as control. The sample for the study was prepared by pelletizing the nanofibrous mat of 13 mm diameter disc on the agar plate and placed in the incubator overnight at 37 °C. Next, the zone of inhibition formed by sample was measured. 3. Results and discussion 3.1. Tensile behavior

where ρ is the apparent density of the electrospun mat and ρ0 is the density of the bulk polymer. The bulk densities of PVA, chitosan and neem extract were 1.19, 1.33 and 0.58 g/cm3 respectively. The density of the

Since the wound dressing materials being wrapped on the wound area are likely to endure pulling forces in order to adhere the mat smoothly and effectively to skin, their mechanical properties especially tensile strength plays a crucial role. To investigate the amount of pulling forces it can sustain up to the break, tensile test was conducted and plotted in the stress-strain curve as given in the Fig. 3. Maximum tensile strength of the nanomat was

Fig. 2. Schematic representation for the development of PNCNM.

Fig. 3. Force versus elongation behavior during tensile test.

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2856 cm−1 (C\\H stretching) and 1720 cm−1 (C_O stretching). Besides the attributable peaks have been found at 3433 cm−1 (O\\H stretching), 2927 cm−1 (C\\H stretching) and 1095 cm−1 (C\\O\\C stretching) in case of PVA polymer and PVA nanofiber. This is in accordance with the signature peaks of PVA and neem as reported in previous studies [76,77]. On the other hand, the strong characteristic peak observed at 1558 cm −1 is attributed to the amine band in chitosan in PNCNM sample [78,79]. Besides, the peaks at 2879 cm −1 (C\\H stretching) and 1728 cm−1 (C_O stretching) in the same sample indicated the presence of neem constituents while PVA peaks is identified at 1026 cm−1. The broadening in the band after 3000 cm−1 to 3400 cm−1 with a very large number of individual molecules indicates the formation of hydrogen bond in the developed sample.

3.3. Impact of porous structure Fig. 4. ATR-FTIR spectra of neem, PVA polymer, PVA nanofiber and PNCNM respectively.

found 18.78 N corresponding to the elongation value of 4.98 mm. This value of strength at break is good enough to be used for wound dressing purpose (as not so much pulling force is applied here). 3.2. Fourier transforms infrared spectroscopy (FTIR) Infrared spectroscopy confirmed the presence of functional group of PVA, neem constituents, chitosan in the developed sample as shown in the Fig. 4. The functional group region is identified in the range of 4000–1450 cm −1 whereas the finger print region corresponds to the region of 1450–500 cm−1. The characteristic peaks of neem powder in FTIR spectra have been identified at wave number

In order to enhance the effectiveness of wound dressing materials, the diffusion and permeability of oxygen from air to the skin is provided through tiny pores present in it. The existence of such tiny pores does not only allow air ventilation but also restricts the penetration of bacteria in the wound area [80,81]. Fig. 5 shows the images of inner surface, outer surface and SEM view which clearly shows the porous structure. As per the Eq. (1), the mat shows 91% porosity which is advantageous for adherence and proliferation of cells. This higher porous nature may be due to the mesho structure of the nanofibers which on overlapping led to this effect. The distribution of fiber diameter along with their respective frequency has also been shown in the Fig. 5. Twenty five samples of fibers were measured in various portion of the image and an average diameter of 213.52 nm was found. The minimum and the maximum fiber diameters were observed at 152 and 298 nm respectively.

Fig. 5. (a) Top view (b) inside view (c) SEM image at 15 k magnification and (d) fiber diameter (nm) distribution with respective frequency.

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above 250 °C where the initial degradation of PVA nanofiber starts at around 215 °C. Although the melting point of PVA nanofiber is 190 °C or 227 °C due to its higher amorphous region [82,83], the addition of neem-chitosan mixer shows enhanced initial degradation at a higher temperature of 230 °C. This shows that PNCNM is much more stable towards thermal decomposition when compared with PVA nanofiber. The increased stability may be due to the interactions of neem and chitosan with PVA which retard the mobility of polymer chains, reduce segmental motion and as a result a higher energy is required to move the polymer chains. Up to 225 °C, the developed mat shows thermal stability though there was some weight loss happened due to the removal of moisture without any polymer degradation. 3.5. Moisture management properties

Fig. 6. Curve showing the weight loss with respect to temperature.

3.4. TGA analysis The thermal behavior of PVA-neem-chitosan system nanomat and PVA alone in the form of TGA curve is shown in Fig. 6. The TG curve shows a linear degradation behavior with an initiation of degradation

Despite having the biological action, the nanofibrous mat must also need to offer a high comfort at dermal level of human skin. Hence, it is necessary to evaluate the capacity to transfer liquids (water or perspiration) from the skin to the environment. The results of moisture management properties of the sample produced from the mixer of PVA-neemchitosan solution have been presented in the Fig. 7(a).The wetting time (sec) of top and bottom surface of the developed mat lies in the range of 3–5 indicting its fast wetting nature according to AATCC standard. Given the behavior of absorption rate, inner surface i.e. neem-chitosan layer

Fig. 7. (a) Quantitative grading of moisture behavior of PNCNM, (b) PVA nanofiber and (c) water and time location diagram.

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shows a better grade (4) than outer surface (PVA layer) corresponding to the value of 61.84% which lies in the range of 50–100. This fast absorbing nature of inner surface may be due to the less adhesive nature of neem and chitosan that helps absorb the liquid of wound area very quickly which is an additional advantage for wound healing. However, the lower absorption rate of outer surface may be due to adhesive nature of PVA polymer which restricts the easiness of moisture adherence on it. Maximum wetted radius was observed 5 mm for both surfaces as shown in Fig. 7(b).The value of liquid/ moisture spreading speed for inner surface is slightly higher than outer but both surfaces are to be graded as 1 and hence exhibit a poor liquid spreading nature. This nature of the developed mat can prevent it from being wetted very quickly due to the absorption of liquids from wound area. However, the value of one-way transport capacity and overall moisture management capacity (OMMC) has been calculated using all parameters that indicate that the mat is

poor in moisture transport property and thus should be considered as water repellent. 3.6. Antibacterial activity Besides biocompatibility, thermal stability, mechanical and moisture management properties, the antibacterial activity of the nanofibrous mat plays a vital role in wound dressing purpose. Therefore, the antibacterial property of the prepared PNCNM against S. aureus is discussed in this section. The antibacterial activity of PVA nanomat and PNCNM nanofibrous mat is presented in Fig. 8 that shows the formation of inhibition zones. The formation of zone with a value of 14.5 mm was found only for PNCNM whereas no zone was formed in case of PVA nanomat. Usually, the thicker cell wall of S. aureus makes it resistant to chitosan and the presence of polycationic group in chitosan molecules facilitates the disruption of gram-negative bacteria [76]. But the addition of neem

Fig. 8. (a) Formation of zone of inhibition of PNCNM, (a1) PVA nanofiber (b) mechanism of the antibacterial effect of chitosan S. aureus [93].

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extract has enhanced the formation of inhibition zone against S. aureus which indicates their combined effect. This may be due to leaching of antibacterial bioactive constituents from the nanomat which is incorporated by the addition of neem extract. This finding is consistent with previous research on the antibacterial characteristics of neem reported by several researchers [84–90]. Additionally, the mesho structures of the developed nanomat can prevent the penetration of any bacteria because of the presence of tiny pores and thus can nullify the surrounding infections effectively [91,92] Although the mechanism of killing S. aureus bacteria by chitosan have been illustrated by researchers as shown in the Fig. 8(b), further studies are essential to understand the exact mode of action of bioactive compounds of neem extract and the mechanism of growth inhibition in S. aureus. 4. Conclusion The new way of developing PVA-neem-chitosan nanofibrous mat (PNCNM) through electro- spinning technique under optimum processing conditions has been introduced that offers bacterial resistance against S. aureus bacteria. The morphological structure has demonstrated the formation of a smooth nanofiber with a particular diameter having characteristic peaks of individual chemical has been confirmed by FTIR spectra. Regarding thermal behavior, the developed mat has shown enhanced thermal stability in comparison with PVA nanofiber alone due to reduction in chain movement at an elevated temperature. Besides, the moisture management properties indicate the fast absorbing nature of the inner surface which may help quick absorption of wound liquids. PVA nanomat incorporated with neem extract and chitosan also leads to the formation of inhibition zone against S. aureus bacteria in liquid medium. Since, this study deals only with the formation and characterization of PNCNM, the biocompatibility test was not conducted. This might be done during the application stage of the developed mat in the subsequent studies. The overall findings of this study suggest the formation and potential application of PVA-neemnanofibrous mat (PNCNM) as wound dressing material having bacterial resistance and moisture properties with porous structure. Therefore, the present study shows that PNCNM holds a great promise to be used as a biodegradable, bio-based and antibacterial wound dressing material. Acknowledgements The authors are grateful to the Department of Textile Engineering, DUET for providing lab facility. Although no funding was available for this study, a special gratitude to the Department of Biochemistry of Dhaka University for providing antibacterial test facilities for this research. The Institute of Energy Engineering (IEE), DUET provided an untiring support to conduct the thermal analysis which is worth mentioning gratefully. References [1] M.M.R. Khan, M. Tsukada, X. Zhang, H. Morikawa, Preparation and characterization of electrospun nanofibers based on silk sericin powders, J. Mater. Sci. 48 (10) (2013) 3731–3736. [2] T.T.T. Nguyen, B. Tae, J.S. Park, Synthesis and characterization of nanofiber webs of chitosan/poly (vinyl alcohol) blends incorporated with silver nanoparticles, J. Mater. Sci. 46 (20) (2011) 6528–6537. [3] V. Guarino, L. Ambrosio, Electrofluidodynamic Technologies (EFDTs) for Biomaterials and Medical Devices: Principles and Advances, Woodhead Publishing, 2018. [4] R. Zhao, X. Li, B. Sun, Y. Zhang, D. Zhang, Z. Tang, X. Chen, C. Wang, Electrospun chitosan/sericin composite nanofibers with antibacterial property as potential wound dressings, Int. J. Biol. Macromol. 68 (2014) 92–97. [5] Y.-F. Goh, I. Shakir, R. Hussain, Electrospun fibers for tissue engineering, drug delivery, and wound dressing, J. Mater. Sci. 48 (8) (2013) 3027–3054. [6] J. He, Y. Qin, S. Cui, Y. Gao, S. Wang, Structure and properties of novel electrospun tussah silk fibroin/poly (lactic acid) composite nanofibers, J. Mater. Sci. 46 (9) (2011) 2938–2946.

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