Evaluation of electrospun poly (vinyl alcohol)-based nanofiber mats incorporated with Zataria multiflora essential oil as potential wound dressing

Accepted Manuscript Evaluation of electrospun poly (vinyl alcohol)-based nanofiber mats incorporated with Zataria multiflora essential oil as potential wound dressing

Niloofar Torabi Ardekani, Mohammad Khorram, Kamiar Zomorodian, Somayeh Yazdanpanah, Hamed Veisi, Hojat Veisi PII: DOI: Reference:

S0141-8130(18)34879-7 https://doi.org/10.1016/j.ijbiomac.2018.12.085 BIOMAC 11244

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

12 September 2018 4 December 2018 8 December 2018

Please cite this article as: Niloofar Torabi Ardekani, Mohammad Khorram, Kamiar Zomorodian, Somayeh Yazdanpanah, Hamed Veisi, Hojat Veisi , Evaluation of electrospun poly (vinyl alcohol)-based nanofiber mats incorporated with Zataria multiflora essential oil as potential wound dressing. Biomac (2018), https://doi.org/10.1016/ j.ijbiomac.2018.12.085

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ACCEPTED MANUSCRIPT Evaluation of electrospun poly (vinyl alcohol)-based nanofiber mats incorporated with Zataria multiflora essential oil as potential wound dressing

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Niloofar Torabi Ardekani1, Mohammad Khorram1*, Kamiar Zomorodian2, Somayeh Yazdanpanah,3 Hamed Veisi, 4 Hojat Veisi4

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School of Chemical and Petroleum Engineering, Shiraz University, Shiraz, Iran.

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Basic Sciences in Infectious Diseases Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

3 4

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Department of Chemistry, Payame Noor university, Tehran, Iran.

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Departments of Medical Mycology and Parasitology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

*Corresponding Author:

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Mohammad Khorram, School of Chemical and Petroleum Engineering, Shiraz University, Shiraz Iran. Tel: +98 71 36133748; Fax: +98 71 36473748. Postal Code: 7193616511. E-mail Address: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT Infections, especially those caused by multi-drug resistant pathogens, result in serious problems in wound healing process. In this study, Zataria multiflora (ZM) essential oil, as a strong natural antimicrobial agent, is incorporated into chitosan-based nanofiber mats to fabricate a novel

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wound dressing. Different amounts of ZM essential oil (0, 2, 5 and 10% (v/v)) were incorporated

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into chitosan/poly(vinyl alcohol)/gelatin (CS/PVA/Gel) solutions and then were successfully

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electrospun into beadless and uniform fibers with 95±14, 154±27, 187±40 and 218±58 nm in diameters, respectively. The produced nanofiber mats (CS/PVA/Gel/ZM) were chemically

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crosslinked by glutaraldehyde vapor. The chemical compositions of ZM essential oil and

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nanofiber mats were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FTIR), respectively. The antimicrobial activity of the

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CS/PVA/Gel/ZM nanofiber mats was determined by the AATCC100 method. The nanofiber mat

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loaded with 10% of ZM essential oil completely inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans after 24h of incubation. Swelling investigations

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showed that the produced nanofibers have a substantial ability to take up water, in the range of

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400-900 %. Mechanical properties of the nanofiber mats were studied by tensile testing. Furthermore, they were found to be non-toxic by biocompatibility assays on mouse fibroblast

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(L929) cells. The obtained results have demonstrated that CS/PVA/Gel nanofiber mats, loaded with ZM essential oil, are promising alternatives to conventional wound dressings. Keywords: Electrospinning, Chitosan, Zataria multiflora essential oil, Antimicrobial activity, Cytotoxicity

ACCEPTED MANUSCRIPT 1. Introduction Human skin, the first defensive barrier against the external environment and the largest organ of the body, protects the body against microbial intrusion and excessive water loss [1, 2]. Patients who suffer from burn injuries and surgeries are particularly susceptible to infections, especially

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as they are prone to systemic immunosuppression. Up to 75% of the fatalities in burn patients are

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related to an infection [1]. In addition, the incidence of infections caused by drug-resistant

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pathogens is increasing as one of the most serious threats to human health [3, 4]. Therefore, the

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necessity of alternative treatments has encouraged researchers more than ever. Since ancient time, herbal medicines have been used to improve the wound healing process [5].

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Zataria multiflora (ZM) with the common Persian name of “Avishan-e-Shirazi” (Avishan is the

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Persian name of thyme and Shiraz is the name of a city in Iran), is a thyme-like plant that belongs to the lamiaceae family that grows extensively in central and southern Iran, Pakistan and

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Afghanistan. ZM is reported to have aromatic and medicinal properties. The leaves of this

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species are commonly used in food processing as an aromatic spice. Moreover, in traditional medicine, ZM has been used to treat various diseases such as cramps, muscle pains, nausea,

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indigestion, diarrhea and infectious diseases [6]. Modern pharmacological studies show that ZM

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has an extensive range of biological properties such as antibacterial, antifungal [6, 7], antiinflammatory, antinociceptive and spasmolytic effects [6]. Plant-derived essential oils have long been utilized to overcome bacterial infections. Antimicrobial properties and mechanism of action of ZM essential oil have reported in detail previously [8]. It has been shown that the antimicrobial activity of the ZM essential oil might be due to its high content of phenolic compounds such as carvacrol and thymol [6, 7]. These monoterpens exhibit their antimicrobial activities through disruption of the cytoplasmic membrane, which leads to in enhancing cell

ACCEPTED MANUSCRIPT membrane permeability and leakage of ions and ATP [8, 9]. Similarly, it has been reported the main sites of action of the essential oil on fungal cells is the plasma membrane and cell wall [10]. Additionally, the proposed mechanism of antifungal activity of thymol, as the main component of the essential oil, is inhibition of telomerase activity [11]. Currently, some pharmaceutical

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forms of ZM, such as vaginal creams, oral drops, syrups and soft capsules are available in

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antimicrobial agent, it has not been used in wound dressing so far.

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market[6]. Despite the availability of different pharmaceutical forms of the ZM as an

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Wound dressings for skin injuries are used to stop bleeding, protect wound against infections, absorb excess exudates and maintain wound hydration to accelerate the healing process [2, 12].

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Recently, electrospinning, a powerful and simple method of producing continuous nanofiber

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mats [13], has attracted a lot of attention as a technique to manufacture new generation of wound dressings. This technology can make nanofiber mats which are composed of different kinds of

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natural or synthetic (or their combinations [14]) polymers with suitable biological characteristics

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[2]. Electrospun nanofiber mats have unique properties such as high surface area to volume ratio, high porosity with variable pore size distribution, oxygen-permeability, and morphological

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similarity to the natural extracellular matrix (ECM) which make them promising materials for

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wound dressings [15, 16]. Therapeutic agents (such as natural remedies [17], growth factors, anti-inflammatory drugs, antimicrobial agents, and antioxidants) can be incorporated into the electrospun nanofiber mats which makes them great platforms for local delivery [2, 14]. In order to produce a desired scaffold for specific application, undoubtedly polymer blending has a great influence [5]. Nowadays, chitosan (CS), as a natural polymer, has been used frequently in tissue engineering and wound healing due to its intrinsic antimicrobial, hemostatic, non-toxic, healing-stimulant, biodegradable and biocompatible properties [18]. There are numerous reports

ACCEPTED MANUSCRIPT on chitosan-based electrospun nanofiber mats [12, 15, 17]. However, it has been shown that electrospinning of CS, due to the poor solubility in its pure form, is difficult [19, 20]. In order to assist CS fiber formation, researchers have used poly(vinyl alcohol) (PVA)[21] as a biodegradable, biocompatible, non-toxic and water soluble synthetic polymer [22]. In addition,

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gelatin (Gel), which is generally recognized as safe (GRAS) by the US Food and Drug

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Administration (FDA) [23], is another biodegradable, biocompatible, non-toxic [24], and water

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soluble biopolymer which is derived from partially hydrolyzed negative collagen [25], commonly used in biomedical applications [21]. In last decades, plant derived substances have

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attracted attention as antimicrobial agents [26]; however, only few studies are available on the

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incorporation of essential oils into electrospun nanofiber mats as antimicrobial dressing. For example, in a study by Rieger et al., chitosan/poly (ethylene oxide) electrospun nanofiber mat

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loaded with Cinnamaldehyde essential oil demonstrated very good antimicrobial activity against

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P.aeruginosa [27]. In another study, cellulose-based electrospun nanofiber mats encapsulating three different essential oils (Cinnamon, Lemongrass and Peppermint) were able to inhibit the

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growth of Escherichia coli [28]. Similarly, cellulose acetate electrospun nanofiber mats

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incorporated with Rosemary and Oregano essential oils were evaluated against S.aureus, Escherichia coli and C.albicans [26]. In this study, ZM essential oil was incorporated into the

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CS/PVA/Gel nanofiber mats via electrospinning process and its physicochemical, antimicrobial and cytotoxic properties were investigated. 2. Experimental section 2.1. Materials Chitosan (Mw=600-800 kDa) was purchased from Acros-Organic Co., (USA). PVA (Mw=72 kDa), edible bovine gelatin, dimethyl sulfoxide (DMSO), acetic acid (glacial) (AA),

ACCEPTED MANUSCRIPT glutaraldehyde (GA, 25% aqueous solution) and sabouroud dextrose media were obtained from Merck Co., (Germany). Roswell Park Memorial Institute (RPMI-1640) was obtained from Sigma Co., (St.Louis, MO, USA). Tryptone soya broth was obtained from Oxid Co., (UK) and muller hinton media was purchased from Himedia (Mumbai, India). Mouse fibroblast cells (L929, Cell

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bank number C161) were obtained from National Cell Bank of Iran, Pasteur Institute, Tehran,

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Iran. ZM essential oil was a gift from the lab of Dr. M.J. Saharkhiz, Professor of Medicinal and

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Aromatic Plants Production & Processing, at Shiraz University, Shiraz, Iran. All other analytical

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grade solvents and reagents were available. 2.2. Solution preparation

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The 6% (w/v) PVA solution was prepared by dissolving PVA in distilled water at 80℃, followed

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by stirring for 4h. The 3% (w/v) CS solution was prepared by dissolving CS and Gel in acetic

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acid solution at a weight ratio of 3:1. Then, CS-Gel solution was mixed with PVA solution in final concentration of acetic acid solution (30% v/v) and was stirred for 24h. Various amounts of

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the ZM essential oil (0, 2, 5 and 10% (v/v)) were added to polymeric solution and mixed for

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additional 24h.

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2.3. Electrospinning

The as-prepared polymer solution with different amounts of ZM essential oil (0, 2, 5 and 10% (v/v)) was loaded into a 5ml syringe equipped with stainless steel needle (27 gauge). High voltage of 21 kV (Fnm High Voltage Power Supply-OC Series, Iran) was applied, positive electrode of high voltage was connected to the capillary needle and negative electrode was connected to rotating collector (600 rpm) which was wrapped with aluminum foil. Injection of electrospinning solution was performed using a syringe pump with a constant feed rate of 0.2

ACCEPTED MANUSCRIPT ml/h. The distance between the needle tip to collector, kept at 15 cm. Glutaraldehyde vapor was used as crosslinking agent [30] to enhance stability and mechanical properties of the prepared nanofiber mats. All samples were crosslinked by glutaraldehyde vapor for 20, 30, 60, 120 and 180 min and then heated in a vaccum oven at 40℃ for 24h [14].

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2.4. Characterization

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2.4.1. Essential oil analysis by Gas Chromatography-Mass Spectrometry (GC-MS) The GC-MS analysis of the EO was carried out on a Thermoquest-Finnigan instrument equipped

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with a DB-5 column (60 m × 0.25 mm, 0.25 𝜇𝑚 film thickness). Same as previous reports [29-

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31], the GC conditions were set to have increasing temperature from 60 ℃ to 250 ℃ at a rate of 4 ℃/min and eventually held for 10 min; the temperature of the transfer line and injector were

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250℃. The selected carrier gas was helium at a flow rate of 1.1 ml/min. The sample injection, in

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the split mode was with the rate of 1/50. The quadrupole mass spectrometer was scanned over the 35-600 amu with an ionization current of 150 mA and an ionization voltage of 70 eV. By

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using a Thermoquest-Finnigan instrument equipped with a DB-5 column (60 m × 0.25 mm, 0.25

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μm film thickness), the GC-flame ionization detector (FID) analysis of the EO was carried out. The selected carrier gas was nitrogen at the constant flow of 1.1 ml/min; the split ratio was same

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as that for GC-MS. The temperature of oven was increased from 60℃ to 250 ℃ at 4℃/min rate and held for 10 min. The temperatures of injector and detector (FID) were held at 250℃ and 280℃, respectively. Without using the correction factors, the semi-quantitative data analysis was gained from FID area percentages. Calculation of retention indices (RI) was done by using retention times of normal alkanes (C6 -C24 ) which were injected after essential oil at the same

ACCEPTED MANUSCRIPT chromatography conditions. Identification of the compounds were made by comparing the retention indices and mass spectra with those reported in Wiley library [7, 32]. 2.4.2. SEM analysis

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The morphology and diameter of the electrospun nanofibers were studied under scanning

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electron microscopy (SEM, TESCAN Vega3, Czech Republic). Before the SEM observations,

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nanofibers were cut into 1cm × 1cm squares for sputter coating with gold (Dsr1, Nanostructured Coating Co., Iran). ). In order to calculate the mean value of nanofibers diameters, 50 different

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nanofibers from SEM images were selected randomly and their diameters were measured by

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using ImageJ 1.44p software (National Institute of Health, USA). 2.4.3. Fourier-Transform Infrared Spectroscopy (FTIR)

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FTIR spectroscopy (Spectrum RXI, Perkin Elmer, USA) was used to characterize the presence of

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particular chemical groups in the materials and the produced nanofiber mats. FTIR spectra were

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recorded in the range of 400-4000 cm−1 by means of KBr pellets at a controlled ambient condition.

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2.4.4. Antimicrobial evaluation of ZM essential oil

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To determine the minimum inhibitory concentration (MIC) values of the essential oil, the broth microdilution method was used as recommended by CLSI, with some modifications [33, 34]. Briefly explaining, for determination of antibacterial activities against S.aureus and P.aeruginosa, serial dilutions of the essential oil (0.031-16 μl/ml and 0.125-64μl/ml, respectively) were prepared in 96-well microtiter plates using Muller-Hinton broth media and to determine the antifungal activity against C.albicans, serial dilutions of the essential oil (0.03116μl/ml) were prepared in RPMI-1640 media buffered with MOPS. Stock inoculum of bacteria

ACCEPTED MANUSCRIPT and yeast strains were suspended in media and the turbidity of the cells was adjusted to 0.5 McFarland scale at 530 nm wavelength using a spectrophotometric method to yield stock suspension of 1-5 × 106 cell/ml for yeast and 1-1.5 × 108 cell/ml for bacteria. Working suspensions were prepared by making 1/100 and 1/1000 dilutions of the stock suspensions with

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appropriate media for bacteria and yeast, respectively. One hundred microliters of the working

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inoculum were added to the wells of microtiter plates, which were incubated at 37℃ for 24h

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(bacteria) or at 30℃ for 24-48h (yeast). As a sterility control (blank), 0.2 ml of un-inoculated

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medium free of essential oil was added to the first column. Besides, medium with inoculum but without essential oil was added to the last column of microtiter plates as growth control. In order

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to evaluate the growth in each well, it was compared with the growth in the control well. MICs were defined as the lowest concentration of essential oil which prevents any viable growth and

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determined visually. Each experiment was performed in duplicate. For determination of

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minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), 10

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μl of media from optically clear wells, with microorganism showing no visible growth, were further cultured on Muller-Hinton agar and Sabouroud dextrose agar, respectively. MFC and

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MBSs were defined as the lowest concentration of the essential oil that yields 98% mortality

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effects as compared to the initial inoculum [7, 32]. 2.4.5. Antimicrobial evaluation of electrospun nanofiber mat The antimicrobial activity of produced electrospun nanofibers were evaluated quantitatively by standard test method of AATCC 100. Standard species of S.aureus (ATCC 25923), P.aeruginosa (ATCC 27853), and C.albicans (ATCC 10261) were used in this study. Circular swatches (2.4 ± 0.1 cm in diameter, ≈3 mg) were cut from electrospun nanofibers and sterilized under UV light for 20 min. The tested microorganisms were grown in nutrient broth at 37℃ for 24h, and the

ACCEPTED MANUSCRIPT cells were separated by centrifugation (10000rpm, 6 min). According to AATCC100document, test yeasts and bacteria strains were suspended in appropriate media and the cell densities were adjusted to 0.5 McFarland standards at 630 nm wavelength using a spectrophotometric method (this yields stock suspension of 1-5 × 106 cells/ml for yeasts and 1-1.5 × 108 cells/ml for

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bacteria). The sterilized swatches were then inoculated with 0.1 ml microbial suspension of each

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concentration and incubated for 24h. After that contact period, 10 ml neutralizing solution (PBS,

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pH = 7.4) was added to the falcon tubes containing the inoculated treated swatches. After 1min

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shaking, 10 µl of the solution was cultured on nutrient agar plates and incubated for 24h at 37℃. The reduction of microbes were calculated by the following equation: 𝐶−𝐴 𝐶

× 100

(1)

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%R=

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Where %R is percent of microbial reduction, A is the number of microorganisms recovered from

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inoculated nanofibers after 24h and C is the number of microorganisms recovered from the

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inoculated untreated control (at “0” contact time).

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2.4.6. Swelling measurement of the electrospun mats In order to determine water uptake ability of the as-prepared electrospun nanofiber mat (with and

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without the ZM essential oil), the swelling tests were performed in phosphate buffer saline (PBS, pH = 7.4) at 37 ℃ for 1h and 24 hours. A preweighed amount of dry electrospun nanofibers was immersed in PBS at 37℃. The samples were removed from PBS at specified times and excess surface water was blotted out using a filter paper before weighing. The percent of swelling degree was calculated according to equation (2): 𝑀− 𝑀𝑖

Degree of swelling (%) = [

𝑀i

]×100

(2)

ACCEPTED MANUSCRIPT Where M is the mass of swollen sample after immersion in the PBS for 1h and 24h which was dried by a filter paper and 𝑀𝑖 is the initial dry mass of sample [17]. 2.4.7. Tensile properties of the nanofibers

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The mechanical properties of the produced electrospun nanofiber mats (with (10% v/v) and

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without the ZM essential oil) were measured by using a single column testing machine

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(Brookfield CT3 texture analyzer, UK) according to the ASTM standard test method D882-02 (ASTM, 2002). The nanofiber samples (20 to 40 micrometer in thicknesses) were cut into

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approximately 50 mm × 10 mm (length×width). A 4.5 kg load cell was applied to the samples,

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under a crosshead speed of 0.1mm/s. The Young modulus (MPa) of the samples were calculated by the slope of the initial linear portion of the stress-strain curve and averaged among the

𝐿𝑚𝑎𝑥

× 100 (3)

𝐹𝑚𝑎𝑥 𝐴

(4)

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UTS =

𝐿0

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Eb =

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equations (3) and (4), respectively.

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samples. Elongation at break (Eb) and ultimate tensile strength (UTS) (MPa) were calculated by

Where Lmax is the extension at the moment of rupture (m), L0 is the initial length of the

(𝑚2 )[35].

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specimen (m), Fmax is the maximum load (N) and A is the cross-sectional area of the specimen

2.4.8. Cytotoxicity The cytotoxicity of nanofiber mats was evaluated in accordance with ISO 10993-5 standard test method (indirect contact)[36] using mouse fibroblast (L929) cells. First, cells were cultured in RPMI medium containing 10% (v/v) fetal bovine serum (FBS- Serum, Germany), 100 U/ml

ACCEPTED MANUSCRIPT penicillin, and 100 µg⁄ml streptomycin and incubated in humidified air containing 5% CO2 at 37℃. The nanofiber mats were sterilized with UV radiation for 1h and then were extracted via immersion in culture media for 24h at 37℃ (based on ISO 10993-12, [37]). Pure culture medium, kept under similar conditions, was applied as a negative control. L929 cells separately

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were seeded in 96-well culture plates (with 100 µl culture media) at a density of 1×104 cells/well

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and incubated for 24h to allow cell attachment. Then, the medium in each well was replaced with

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100 µl of the above mentioned extract containing 10% FBS and cells were incubated for another

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24h. In the next step, 100µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/ml [Sigma, USA]) was replaced with extraction medium in each well,

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and incubated for 4h. Finally, isopropanol (Sigma, USA) was used to solubilize the formed formazan crystals. The optical density was measured at 570 nm using a microplate reader (Stat

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Fax 2100, USA). The results were standardized with respect to control sample [38]. The

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percentage of viability was calculated for each concentrations of ZM essential oil extraction and

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average values are presented for five independent experiments as mean ± SD.

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3. Results and discussion

3.1. Essential oil analysis by GC-MS

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The chemical composition of the ZM essential oil is presented in Table 1. In total, 29 compounds representing 99.97 % of the oil were identified by GC-MS. Similar to previous studies [7, 39], the main constituent of the essential oil was thymol (52.8%) followed by cymen(o-) (13.89%), and carvacrol (5.96%). While others reported carvacrol or linalool as the main component of the essential oil [7, 40]. These variations maybe due to geographical location or ecotype of the plants where collected.

ACCEPTED MANUSCRIPT Table 1: Chemical compositions (%) of the ZM essential oil 𝑅𝑇𝑎

Area (%)

Thujene (α-)

6.80

0.06

Pinene (α-)

7.03

3.14

Camphene

7.38

0.18

Octanone (3-)

7.89

1.17

Pinene (β-)

8.12

Octanol(3-)

8.31

Myrcene

8.40

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Compounds

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0.26

Phellandrene (α-)

0.10

9.28

0.75

9.39

13.89

9.65

1.22

10.62

3.48

Sabinene hydrate (cis-)

10.75

0.16

Linalool

11.85

1.64

Borneol

14.30

0.43

Terpinen-4-ol

14.81

0.73

Terpineol (α-)

15.24

0.87

Thymol, methyl ether

16.99

4.30

Carvacrol, methyl ether

17.40

0.89

Thymol

19.32

52.80

Carvacrol

19.58

5.97

Thymol acetate

21.47

1.85

Carvacrol acetate

22.19

0.14

Cineole

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Terpinene (γ-)

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Cymene (o-)

0.67

8.88

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Terpinene (α-)

0.14

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2.78

Aromadendrene

25.77

0.46

Humulene

26.25

0.13

Viridiflorene

27.85

0.32

Spathulenol

30.49

0.61

Caryophyllene oxide

30.70

0.83

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Caryophyllene (E-)

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Main components of the ZM essential oil are indicated by bold letters, a: Retention time

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3.2. CS/PVA/Ge/ZM nanofiber mat physical characteristics

The CS/PVA/Gel (3:6:1) solutions containing different amounts of the ZM essential oil (0, 2, 5

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and 10% (v/v) to polymer solution) were prepared and electrospun successfully. When the

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amount of the ZM essential oil was greater than 10% (v/v), the polymer solution degraded and lost its continuous form, so it could not be electrospun. The SEM images and diameter

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distributions of the CS/PVA/Gel/ZM nanofiber mats are displayed in Figure 1. The SEM images

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illustrated that all of the nanofiber mats were composed of beadless, uniform and continuous fibers. Uniform nanofibers of the CS/PVA/Gel/ZM (0, 2, 5, and 10%), before crosslinking (fig.1

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a, b, c, and d), with average fiber diameters (n = 50) in range of 95 ±14, 154 ± 27, 187 ± 40, and

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218 ± 58 nm, were respectively achieved. This result demonstrated that the ZM essential oil was well incorporated within the fibers and as the amount of the essential oil increased, the average diameter slightly increased, too.

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Fig. 1. SEM image and diameter distribution of CS/PVA/Gel nanofiber mats incorporated with 0, 2, 5, and 10% of ZM essential oil (a, b, c, and d) before crosslinking and (e, f, g, and h) after crosslinking.

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As mentioned, all the samples were crosslinked for 20, 30, 60, 120, and 180 min by glutaraldehyde vapor and then heated in a vaccum oven at 40℃ for 24h [17]. After 20 min

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exposure time, the color of the nanofiber mats changed from very light yellow to light brown.

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The color changing is the result of reactions between the glutaraldehyde and the nanofiber mats composed of chitosan [41] and gelatin [42]. So, among different exposure time, 20 min was

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selected as appropriate exposure time for properties evaluations. As shown in Figure 1 (e, f, g, and h), after crosslinking, the average diameters of the CS/PVA/Gel/ZM (0, 2, 5 and 10%) nanofiber mats were determined to be 126 ± 21, 178 ± 30, 200 ± 41, and 231 ± 32 nm, respectively. Based on the independent sample t-test, the difference of mean of nanofibers’ diameter between crosslinked and non-cross linked groups were not statistically significant (pvalue= 0.57) and it seems that the density of fibers has increased because of inter-fiber linkages after crosslinking.

ACCEPTED MANUSCRIPT 3.3. FTIR spectroscopy The FTIR spectra of CS, PVA, and Gel powder, ZM essential oil and CS/PVA/Gel nanofiber mat with and without the ZM essential oil shown in Figure 2. The CS powder exhibits a peak at 3435cm−1 which is attributed to N‒H and O‒H stretching vibration and inter molecular

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hydrogen bonding of chitosan backbone [16, 43]; the amino characteristic peak is at

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1601cm−1 [44], and the peaks at 1324 cm−1 and 1261 cm−1 are assigned to C‒H and C‒O

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bonds, respectively. PVA powder shows FTIR peaks at 3405, 2942, 1441, and 1095 cm−1 which

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are related to O‒H, C‒H, CHOH and C‒O groups, respectively [16]. The gelatin powder bands appeared in 3306, 1668, 1535, and 1238 cm−1 , which represent amide A (N-H), amide Ι (C═O),

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amide ΙΙ (N‒H and C‒H), and amide ΙΙΙ (C‒N stretch plus N‒H in phase bending), respectively

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[45].

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The FTIR spectra of the ZM essential oil shows peak at 3429 cm−1 which corresponds to a phenolic group [15]. The observed peaks at 2962 cm−1 and 2871 cm−1 are related to asymmetric

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and symmetric methyl C‒H stretching, respectively. In addition, the C═C‒C ring-related

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vibration peaks can be seen near 1600 to 1400cm−1. Furthermore, the peak at 1258 cm−1is assigned to aromatic ether, aryl‒O stretching [46, 47]. In addition there is an obvious peak at

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around 810cm−1 which is characteristic of thymol [48].

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Fig. 2. FTIR spectra of (a) PVA powder, (b) CS powder, (c) Gel powder, (d) ZM essential oil, (e) nanofiber mat without ZM essential oil, and (f) nanofiber mat incorporated with ZM essential oil.

The FTIR spectra of CS/PVA/Gel nanofiber mat demonstrates peaks at 3365, 2940, 1651, 1554, and 1260 cm−1 which indicate the presence of O‒H and N‒H, C‒H, C═O (Amide Ι), N‒H (AmideΙΙΙ ) bands, respectively [16, 49]. Therefore, the produced nanofiber mat is composed of all CS, PVA and Gel compounds. On the other hand, the FTIR spectra of CS/PVA/Gel nanofiber

ACCEPTED MANUSCRIPT mat incorporated with the ZM essential oil (10% (v/v)) was evaluated to specify whether the ZM essential oil remains in the nanofiber mat structure after the electrospinning process. Based on the obtained results, it can be seen that beside the peaks at 3364, 2939, and 1258 cm−1 which manifest the presence of CS, PVA, and Gel in the nanofiber mat, four new peaks at 1595, 1418,

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1374, and 810 cm−1appeared which are attributed to aromatic rings [47], symmetric methyl

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groups [46], and thymol [48], confirms the incorporation of ZM essential oil into the nanofiber

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mat.

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3.4. Antimicrobial evaluation of ZM essential oil

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The essential oil of ZM exhibited remarkable antimicrobial activity against yeast and Grampositive and Gram-negative bacteria, as shown in Table 2. Similar to previous studies [7, 50] the

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ZM essential oil inhibited the growth of the tested bacteria at concentrations ranging from 2-4

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µl/ml. Moreover, it showed bactericidal activity against tested bacteria at concentrations similar to their corresponded MICs. In addition, it had fungistatic and fungicidal activities at

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concentrations of 0.062µl/ml and 0.5µl/ml, respectively, which is comparable to the findings of

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previous investigations [7, 29].

Table 2: Antimicrobial activities (MIC, MBC, and MFC) of the ZM essential oil according to broth microdilution method

MBC or MFC (μl/ml)

0.062

0.5

P.aeruginosa

2

2

S.aureus

4

4

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MIC (μl/ml)

Microorganism C.albicans

MIC: Minimum Inhibitory Concentration; MBC: Minimum Bactericidal Concentration; MFC: Minimum Fungicidal Concentration

ACCEPTED MANUSCRIPT 3.5. Antimicrobial evaluation of electrospun nanofiber mats The antimicrobial activities of the nanofiber mats against the tested microorganisms are shown in Figure 3. The CFU reduction rate (R%) of the bare nanofiber mats against S.aureus and P.aeruginosa were 30.34% and 77.59%, respectively. As indicated in this figure, the

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CS/PVA/Gel nanofiber mat (without ZM essential oil, 0%) revealed no antifungal activity against C.albicans, while considerable reduction of bacterial colonies observed. It has been

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shown previously that effective inhibitory concentration of Chitosan for C.albicans is about 1

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mg/ml [51]. The final concentration of chitosan in our mats was 0.001 mg/ml that is much less than the mentioned effective concentration. As it has been previously reported that chitosan has

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antibacterial activity [18, 52], it could be assumed that chitosan is the active antimicrobial

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component of CS/PVA/Gel. Although all researchers generally reported that chitosan has antibacterial activities, its effect on Gram-negative and Gram-positive bacteria is different [53,

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54]. Some studies revealed that chitosan was more effective against Gram-negative bacteria [55,

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56] which is similar to our results, whereas others found that it exhibits a better antibacterial activity against Gram-positive bacteria [57-59]. As the hydrophilicity of Gram-negative bacteria

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is remarkably higher than that of Gram-positive ones, so more amount of chitosan, as a

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polycationic polymer, might be adsorbed to these Gram-negative bacteria with high electronegative charge [54, 55].

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80 60

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40 20

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Reduction(%) of colonies

100

0%

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0

2%

5%

10%

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ZM essential oil (%)

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Fig. 3. The antimicrobial activities of CS/PVA/Gel/ZM (0, 2, 5 and 10%) nanofiber mats in term of reduction (%) of colonies (CFU), against C.albicans, S.aureus, and P.aeruginosa.

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As shown in the Figure 4, incorporation of ZM essential oil into the CS/PVA/Gel nanofiber mats

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enhanced the reduction rate of tested microorganisms. Complete elimination (100%) of the C.albicans colonies treated with CS/PAVA/Gel/ZM (2%) in comparison with other tested

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bacteria might be related to the lower MIC of ZM essential oil against C.albicans. Moreover,

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CS/PVA/Gel/ZM reduced the number of S.aureus and P.aeruginosa colonies in a dose

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dependent manner, which is concordant with the results of the antimicrobial susceptibility test. 3.6. Swelling degree of the nanofiber mats The mean swelling degree of CS/PVA/Gel nanofiber mats immersed in PBS for 1 and 24 hours were 530.94±37.97 and 800.28±47.23, respectively; and their difference was statistically significant (p-value < 0.05).As shown in this figure 4, swelling was decreased with increasing amount of the ZM essential oil in a dose dependent manner. One-way analysis of variance (ANOVA) indicates that mean swelling degree between different concentrations of

ACCEPTED MANUSCRIPT the essential oil after 1h and 24h immersing in PBS was statically significant (p-value < 0.05). Post-hoc analysis by LSD method revealed that these differences were due to the difference of mean swelling degrees between all concentrations of essential oil both after 1h and 24h, except concentration of 0% with 5% and 10%, and 2% with 10% in the mats immersed for 1 hour.

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1000

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800

24h

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700

1h

600

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500 400 300 200

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Swelling degree(%)

900

100

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0 0%

2%

5%

10%

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ZM essential oil (% v/v to polymer solution)

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Fig. 4. Swelling degree (%) of CS/PVA/Gel nanofiber mats with different amounts of ZM essential oil (0, 2, 5, and 10% (v/v)). The data are demonstrated as mean ± SD from three independent experiments.

According to these results, the produced nanofiber mats demonstrated a high degree of swelling,

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this result may be attributed to the hydrophilic nature of chitosan [15], PVA [60] and gelatin [61]

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which enhance the water uptake ability [17, 62]. The obtained swelling degree ranges of the nanofiber mats in this study were similar to swelling results of Archana et al. investigations [63]. In addition, the decreasing pattern in swelling degree of the nanofiber mats with increasing the amounts of the ZM essential oil is may be due to the hydrophobic nature of the essential oil.

ACCEPTED MANUSCRIPT 3.7. Tensile properties of the nanofibrous mats The results of the mechanical properties of both as-spun and cross-linked nanofiber mats (with and without the ZM essential oil) are presented in Table 3. When ZM essential oil was not used in the nanofiber mats, the UTS and the elongation at break decreased significantly after

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crosslinking (p-value≤ 0.05). These patterns might be due to the increase in nanofiber’s brittleness after crosslinking [64]. The same patterns were observed by Chen et al. (2008), in the

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mechanical properties of the electrospun collagen/chitosan/polyethylene oxide nanofiber [65].

0% (as-spun)a

169.32 ± 13.22

14.25 ± 0.17

61.97 ± 28.19

26.23 ± 3.47

4.805 ± 0.715

102.81 ± 18.37

15.67 ± 1.55

a,b and a,c

-

a,b

6.38 ± 0.045

c

Statistics*

*: p-value < 0.05

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10%(cross-linked)

3.325 ± 0.005 d

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0% (cross-linked) 10% (as-spun)

127.14 ± 2.72

Elongation at break (Eb)(%) 17.28 ± 0.2

8.05 ± 0.1 b

Young's modulus (MPa)

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Ultimate tensile strength (UTS)(MPa)

Sample

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Table 3. Mechanical properties of the ZM essential oil (0 and 10 % v/v) CS/PVA/Gel as-spun and cross-linked electrospun nanofiber mats. Each value represents the mean ± SD.

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Not enough mechanical data have been reported on chitosan nanofibers incorporated with the

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essential oils, but different researchers have focused on the effects of the essential oil on the mechanical properties of the chitosan films [35, 66].

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After addition of 10% ZM essential oil of mats, only UTS in as-spun mats significantly decreased (p-value≤ 0.05). According to the obtained results in our investigation, the UTS and Young's modulus values were decreased and the elongation at break (%) values were increased in the presence of the ZM essential oil compared with the nanofiber mats without essential oil, but only the reduction of UTS is statistically significant (p-value≤ 0.05). Hosseini et al. (2009) believed that the addition of essential oil results in the reduction of UTS. This might be due to the increase in moisture content, which leads to the disorder of film network [66]. Moreover, in

ACCEPTED MANUSCRIPT the presence of ZM essential oil, it seems that crosslinking was not affected the tensile properties of nanofiber mat, significantly (p-value> 0.05). 3.8. Cytotoxicity of the nanofiber mats

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The cytotoxicity of CS/PVA/Gel nanofiber mat alone and loaded with ZM essential oil (2, 5, and

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10% (v/v)) was investigated using non-cytotoxicity test (ISO 10993-5) on L929 cells. As

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illustrated in Figure 5, cell viability of L929 in the presence of CS/PVA/Gel nanofiber mat extraction has confirmed the non-cytotoxicity of obtained neat (without ZM essential oil) fibers.

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Besides, nanofiber extraction at all amounts of ZM essential oil (2, 5, and 10% (v/v)) were nontoxic to the cells, represented by cell viability in the range of 91-106%. The results reveal that

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these electrospun nanofiber mats in each obtained amount of ZM essential oil are non-toxic to

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L929 cells. No significant difference in cell viability was found between control cells and those

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110 100 90 80 70 60 50 40 30 20 10 0

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Cell viability (%)

treated with 10% essential oil embedded mats (p-value >0.05).

Control

0%

2%

5%

10%

Fig. 5. The percentage of cell viability in L929 cells at varying concentration of ZM essential oil (0, 2, 5, and 10 %) incorporated into CS/PVA/Gel nanofiber mats: Each value represents the mean ± standard deviation of five independent determinations.

ACCEPTED MANUSCRIPT 4. Conclusions In this study, the CS/PVA/Ge nanofiber mats incorporated with ZM essential oil were successfully electrospun. The nanofiber mats found to be safe against the tested cell line and exhibited suitable swelling and mechanical properties. Moreover, GC-MS results showed that

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thymol is the main constituent of the ZM essential oil and therefore might contribute to the

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antimicrobial activities of the produced nanofiber mats against C.albicans, P.aeruginosa and, S.aureus. According to results, the obtained electrospun nanofiber mats with their biocompatible,

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antimicrobial and mechanical properties can be used as wound dressings in surgery and burn

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wounds. Funding:

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The study was partially supported by Shiraz University of Medical sciences [grant number 1396-

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01-43-14735].

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References:

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Graphical abstract

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Highlights

• Zataria multiflora (ZM) essential oil was incorporated into PVA-based electrospun nanofiber mats. • ZM essential oil enhanced the antimicrobial activity of fabricated nanofiber mats dramatically.

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• Nanofibers demonstrated a strong swelling capability.

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• The produced nanofiber mats exhibited no cytotoxicity for L929 cells and showed suitable biocompatibility.