poly (ethylene oxide) nanofibers

Accepted Manuscript Improvement of mechanical properties and antibacterial activity of crosslinked electrospun chitosan/poly (ethylene oxide) nanofibers Mirjana Grkovic, Dusica B. Stojanovic, Vladimir B. Pavlovic, Mirjana RajilicStojanovic, Milos Bjelovic, Petar S. Uskokovic PII:

S1359-8368(17)30973-3

DOI:

10.1016/j.compositesb.2017.03.024

Reference:

JCOMB 4958

To appear in:

Composites Part B

Received Date: 3 October 2016 Revised Date:

5 December 2016

Accepted Date: 17 February 2017

Please cite this article as: Grkovic M, Stojanovic DB, Pavlovic VB, Rajilic-Stojanovic M, Bjelovic M, Uskokovic PS, Improvement of mechanical properties and antibacterial activity of crosslinked electrospun chitosan/poly (ethylene oxide) nanofibers, Composites Part B (2017), doi: 10.1016/ j.compositesb.2017.03.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Improvement of mechanical properties and antibacterial activity of crosslinked electrospun chitosan/poly (ethylene oxide) nanofibers

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Mirjana Grkovica, Dusica B. Stojanovicb*, Vladimir B. Pavlovicc, Mirjana Rajilic-Stojanovicb, Milos Bjelovicd, Petar S. Uskokovicb

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a University of Belgrade, Innovation Centre, Faculty of Technology and Metallurgy, Karnegijeva 4, 11 000 Belgrade, Serbia

University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11 000 Belgrade, Serbia

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University of Belgrade, Faculty of Agriculture, Nemanjina 6, Zemun, 11080 Belgrade, Serbia

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University of Belgrade, Faculty of Medicine, Dr Subotica 8, 11 000 Belgrade, Serbia

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b

*Corresponding author: Dušica B. Stojanović, [email protected]; Tel: +381 11 3303754

Abstract

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In this study conditions for green crosslinking with citric acid of chitosan/PEO (polyethylene oxide) nanofibers were evaluated. The thermal in situ crosslinking enabled penetration of crosslinking agent into the matrix providing an improvement of antibacterial

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activity, thermal stability and mechanical properties of the prepared material. With an increase of temperature above 80°C antibacterial activity against Staphylococcus aureus

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and Escherichia coli, inversely decreased. Moreover crosslinking provided prolonged controlled drug release with outstanding increase of mechanical properties observed by nanoindentation measurements. Results of the investigation indicated crosslinking as an important parameter for producing material with multifunctional characteristics suitable for drug delivery and tissue engineering.

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Keywords: A Polymer-matrix composites (PMCs), Nano-structures, B Mechanical properties, Thermal properties

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1. Introduction

In order to fabricate material for biomedical applications, it is necessary to fulfill requirements such as to avoid inflammatory response, yield predicted mechanical

or excreted, and easy for processing [1].

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properties, ensure biodegradation and non-toxic degradation products that can be resorbed

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Chitosan is one of the most widely used biodegradable polymers. Biodegradability, biocompatibility and antimicrobial properties of chitosan qualify this material for biomedical applications, wound dressing and tissue engineering [2–4], and for food packaging [5, 6].

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Due to chitosan fast degradability some investigations are dedicated to crosslinking of the formed structures so the application in water based solutions could have sustained effect [7–9]. Crosslinking refers to establishment of links between polymer and crosslinking agent

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based on chemical and physical interactions. The most widely used chemical crosslinking agents are glutaraldehyde, sulfuric acid, genipin and oxidized glucose. The main problem

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with chemical crosslinking occurs in surface crosslinking, used in post treatment of obtained material, where limited penetrating crosslinking agent into material occurs. Dissolving chitosan in di-, tri- and multicarboxylic acids, used as both solvent and crosslinking agent, can provide improvement in some performances of material, although the material is not stable in aqueous solutions due to the physical crosslink [7, 8, 10, 11].

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Citric acid is commonly used as green or non-toxic crosslinking agent with favorable biocompatibility and antibacterial activity. As low cost and accessible material, citric acid is mainly used in food packing, as additive, and as cleaning agent. Crosslinking with citric

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acid has already been reported [12–14], even in low temperature conditions [15]. This crosslinking enables thermal stability and reduced dissolution with improved antibacterial activity and enhanced mechanical properties of prepared materials [11, 16, 17]. In addition

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to these characteristics citric acid is a promising constituent in systems for drug delivery [18] and [19].

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Biocompatibility and good mechanical properties of poly (ethylene oxide), (PEO), including high elongation and ability to orient when strained, stand out this material among other synthetic materials [20]. PEO can provide additional hydrogen bonding and improved interference of chitosan chains, which leads to an enhanced production of chitosan

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nanofibers [3]. Chitosan/PEO blend is well documented in various forms, such as films [21, 22], hydrogels [7, 23, 24], , nanoparticles [25, 26] and nanofibers [27–30], mainly for biomedical purposes.

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For several years, chitosan and its various blend systems were investigated for drug delivery [7, 23, 31], even chitosan/PEO system, which is of relevance in this study [32–34].

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Due to good solubility of PEO in aqueous systems, washing PEO from electrospun chitosan/PEO, increase the porosity of chitosan fibers [34]. One possibility for overcoming problems of prolonged drug delivery with nanofiber mats could be producing multilayer systems. This kind of system enables production of dual or multi drug release, specific prolonged release, or release with a delay [35, 36].

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Electrospinning is the most effective technique for fabricating fibrous mats with high performance, large surface area to volume ratio, and distinct porosity [37, 38]. Due to these advantages electrospinning method can be used for fabrication of biomedicals with desired

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properties. [35, 39, 40]. In the past few years the use of coaxial electrospinning opened new perspectives, through possibility of dual drug release, or combining two properties or functionalities of used materials to produce final product with outstanding multifunctional

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properties.

The aim of this study is to evaluate thermal crosslinking of obtained chitosan/PEO

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nanofibers with citric acid, as non-toxic crosslinking agent. The temperature of crosslinking was for the first time considered as an important parameter that influences properties of the obtained material especially their antibacterial activity. Morphology of the obtained nanofibers and the interaction between constituents were examined. Drug release from

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prepared matrix and antibacterial activity against representatives of Gram positive and Gram negative bacteria, Staphylococcus aureus and Escherichia coli were evaluated. Nanoindentation measurement was performed to evaluate mechanical properties of the

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materials.

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2. Materials and methods 2.1. Materials

In this study, we used low molecular weight chitosan (75 – 85% deacetylated) and PEO (Mw 900 kDa) purchased from Sigma Aldrich. Citric acid was supplied from Merck. The Gram-positive bacterium Staphylococcus aureus ATCC 25923 and Gram-negative bacterium Escherichia coli ATCC 25922 were used for antibacterial tests.

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2.2. Preparation of the solutions for electrospinning Five wt.% PEO and 8 wt.% chitosan solution were prepared separately by dissolving in deionized water and 10 wt.% aq. solution of citric acid, respectively. After overnight

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stirring the solutions were mixed in 80/20 (Chi/PEO) ratio at room temperature and stirred overnight. 0.1 wt.% Triton® X 100 (Merck) was added into the solution for lowering the surface tension, providing beadless nanofibers. Film was prepared by simple solvent

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casting method of prepared Chi/PEO solution. For control drug release model drug, Rhodamine B (RhB), was added in blend (0.1 wt.% based on the total weight of a

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polymer).

2.3. Electrospinning

Electrospinning of the solutions were performed in single nozzle and coaxial setup. The

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solutions were spinned immediately after they have been prepared. Electrospinning in single nozzle setup was carried out with flow rate of 0.5 ml/h, 15 cm distance from collectors and 30 kV.

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Coaxial electrospinning was performed using vertical setup containing a variable high DC voltage power supply and controlled syringe pumps. Chitosan and PEO solutions were

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poured in 20 ml plastic syringes placed in two separated pumps and the syringes were connected by PTFE (polytetrafluoroethylene) tubes to the inlets of the kit of modular coaxial needles with two layers (Linari Engineering, Pisa, Italy). The kit of modular coaxial needles consists of inner needle (i.d. 0.45 mm, o.d. 0.85 mm) concentrically placed in outer needle (i.d. 1.37 mm, o.d. 1.83 mm) which was grounded by an alligator clip. The produced fiber mats were collected on aluminum foil. Flow rates of both solutions were same, 0.5

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ml/h, and the electrospinning was performed with applied 30 kv voltage and 15 cm distance from ground collector. The coaxial setup is shown in Fig. 1.

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2.4. Thermal crosslinking

After electrospinning the nanofiber mats were dried in oven at 40 °C for 24 h, in order to evaporate residual water. Crosslinking was performed by heating obtained fibrous mats in

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oven at 60, 80, 100 and 145 °C (Chi/PEO 60, Chi/PEO 80, Chi/PEO 100 and Chi/PEO 145) for 15 minutes. Sample was pressed using N 840 D Hix Digital Press (Hix, Corp., USA) at

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a temperature of 25 ºC for 15 min under a pressure of 3 bar. The preparation and designations of the samples are presented in Table 1. All samples were kept in a desiccator. Scheme of crosslinking and proposed interactions between the constituents during the

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thermal crosslinking are presented in Fig. 2.

2.5. Morphology of electrospun nanofibers by scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

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Obtained nanofibers were observed by field emission scanning electron microscope (FESEM) (JSM 5800, Tescan Mira 3) and transmission electron microscopy (TEM) (JEM

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1400-Plus Jeol). In order to determine the mean fiber diameters and their distributions for each electrospun mats, 150 randomly selected fibers from each SEM image were measured using image analysis software Image Pro Plus.

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2.6. Fourier transform infrared spectroscopy (FTIR) The infrared spectra of the Fourier transform (FTIR) were recorded in the range 4000-400 cm-1, on BOMEM spectrophotometer (Hartmann & Braun, MB-series). All samples were

into a transparent plate by hydraulic press.

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2.7. Antibacterial activity test

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completely crushed with potassium bromide (KBr) and the resulting powder was pressed

The antibacterial properties of the material were evaluated by using a Gram-positive

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bacterium S. aureus and a Gram-negative E. coli indication strains and agar disk diffusion method. The samples were prepared in a form of tablet (10 mg) using 6 mm diameter press. Chi/PEO 40 was used as a control sample. The plates were prepared with tryptone soy agar (Torlak, Serbia) and inoculated with evenly spread fresh bacterial culture grown in tryptone

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soy broth. The total amount of the applied bacterial culture was ~1x107 CFU (cell forming units) and after application the plates were allowed to dry for ~15 minutes. The samples

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were placed on the agar plates and incubated at 37 ±1 °C for 24 h.

2.8. Model of drug controlled release

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Drug release was determined by soaking 50 mg of sample in 100 ml of phosphate buffer saline solution (pH = 7.4). The samples were incubated at 37 °C and the released RhB was measured at specific time intervals by measuring absorbance at 554 nm using UV-vis spectrophotometer (Shimadzu UV1700) by plotting against RhB calibration curve (10-8 – 10-5).

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2.9. Drug release kinetic studies The obtained data of model drug release were fitted with different kinetic models, including zero order, first order, Higuchi, Korsmeyer-Peppas and Weibull model. The regression

The equations of release kinetics are presented as followed [41]: −Zero order model: Qt =Q0 +K0t

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coefficients (r2) were used to evaluate the best fitting of obtained in vitro data to a model.

(1)

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where Qt is the amount of drug released in time t, Q0 is the initial amount of drug in the solution (most times Q0= 0), and K0 is the release constant.

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−First order model: logQt =logQ0 − K1t/2.303

(2)

where Qt is the amount of drug released at time t, Q0 is the initial amount of drug in the solution, and K1 is the first order release constant. −Higuchi model: Qt =KHt1/2

(3)

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where Qt is the amount of drug released at time t and KH is the release constant. −Korsmeyer–Peppas model: Qt/Q0=KKPtn

(4)

where Qt/Q0 is the fraction of drug released at time t, KKP is the release constant, and n is

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the release exponent, indicative of the drug mechanism. A detailed interpretation of the

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release exponent is shown in Table 2 [42].

−Weibull model: ln[ln(100/100-Qt)]=Kw lnt + Q0

(5)

where Qt is the amount of drug released at time t and Kw is the release constant. Q0 is the initial amount of drug in the solution.

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2.10. Differential scanning calorimetry analysis Thermal properties of the prepared samples were examined in a nitrogen atmosphere from room temperature to 110 °C at a heating rate of 10 °C/min using a differential scanning

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calorimeter (DSC, Q10 TA Instruments, USA). To determine the melting temperature (Tm), melting enthalpy (∆Hm) and the degree of crystallinity (χm), the samples were heated to 110 °C and kept for 10 min. Subsequently, they were reheated at a heating rate of 10 °C/min.

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Melting temperatures (Tm) were measured from the second cycle as the temperature at the top of the endothermic peak, Tm(max). The area under the endothermic peak determined the

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melting enthalpy, ∆Hm. The crystallinity degree of films and fibers (χm) was determined from DSC analysis based on the following Eq. (6):

χ m ,PEO =

∆H m ,PEO ∆H m ο, PEO ⋅ ω

(6)

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where ∆Hm,PEO is the melting enthalpy of PEO and ∆Hom,PEO is the melting enthalpy of 100% crystalline PEO (196.6 J/g) for the PEO molecular weight of 900 000 g/mol [43], ω

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is mass fraction of PEO.

2.11. Nanoindentation measurements

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The nanoindentation experiments on the prepared chitosan/PEO nanofibers were performed using a Triboscope T950 Nanomechanical Testing System (Hysitron, Minneapolis, MN) equipped with a Berkovich indenter type with in situ imaging mode. A peak load of 0.5 mN was applied for all samples with the load-hold-unload of 10-20-10 s for each segment. Nine indentation measurements were performed for each sample and the mean values and standard deviations are reported. Elastic recovery parameter (ERP) was determined by

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equation ERP = (hmax - hf) / hmax, where hmax is the maximum depth followed by the maximum indenter load in a sample and hf is non-recovered depth after unloading the

3. Results and discussion 3.1. Morphology of electrospun nanofibers

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indenter tip from the sample.

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FESEM images of electrospun nanofiber mats are presented in Fig. 3. Fig. 3a-c shows defect free nanofibers of Chi/PEO 40 with the mean diameter of 280 ± 80 nm. Heating the

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sample above melting point of citric acid (135 ºC) the sample became slightly yellowish which could be explained by dehydration and decarboxylation of the acid [16]. Crosslinking at 145 °C (Chi/PEO 145) showed uniformity in the mean diameter of nanofibers, with a slight decrease in the mean diameter (240 ± 25 nm) and no visual

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morphological changes (Fig. 3d-f), inspite of apparent change in the process of degradation of citric acid [5, 9]. TEM image with contrast showed detail morphology of the core-shell structure of nanofiber. As it is present in Fig. 3g-i the mean diameter of the core-shell

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indicates uniform structure and equal partition of core and shell, due to well tuned

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parameters of processing.

3.2. FTIR spectroscopy

FTIR analysis of the fibers indicates that the addition of the PEO in chitosan induced formation of double hydrogen bonds between the ether oxygen in the PEO and the hydroxyl groups on chitosan, and hydrogen on PEO with a quaternary ammonium group of chitosan [11]. Dissolving chitosan in citric acid and interaction between constituents was confirmed

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by occurrence of the peak of amide II group at 1560 cm-1, which indicated the formation of amide bonds between the carboxyl groups of citric acid and the amino group on the chitosan, and with a peak of an amide III group at 1310 cm-1. The increase in temperature

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during crosslinking caused the weakening of the peak at 1630 cm-1 (amide I) and strengthening the signal in the amide II and III groups. Ester bonding (C=O) between COOH groups of citric acid and OH groups of chitosan was indicated by presence of a peak

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at 1730 cm−1, which resulted shifting this peak towards higher wavenumbers indicating stronger interactions between these two constituents (Fig. 4). Shifting of these peaks

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compared to neat Chi/PEO spectra showed enhancement of ionic interactions between amino and carboxyl groups of all constituents.

3.3. Antibacterial disc diffusion test

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Antibacterial activity was used to highlight the influence of temperature on crosslinking of prepared nanofibers. The disc diffusion test was performed against the Gram positive and the Gram negative bacteria, E .coli and S. aureus, respectively. Formation of zone of

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inhibition is an indication of antimicrobial activity. Although the exact mechanism of the antimicrobial activity of chitosan and its derivates is not thoroughly investigated, it is

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assumed that the positively charged surface of chitosan destabilizes the structure of cell wall of bacteria, causing inhibition of normal bacteria metabolism [44]. Nanofibrous structure enabled better surface interaction and therefore intensified inhibition of bacteria [33]. The level of inhibition (indicated by a larger diameter of the inhibition zones) was higher against S. aureus when compared to E. coli for all samples, which is in correlation with results of others [45] (Fig. 5a,b). Due to the relatively smaller chitosan content in coreshell structure (50/50 polymer ratio) a smaller inhibition zone appeared compared to 11

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Chi/PEO nanofibers (80/20 ratio) prepared by single nozzle electrospinning setup (data not show). Crosslinking at 80 ºC induced a slight increase of antimicrobial activity, which could be explained by partial crosslinking of chitosan with citric acid and their synergistic

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interaction toward bacterial inhibition [12], [14] and [46]. Further increase of temperature provided better crosslinking causing decrease of amino groups (negatively charged centers) [8], [11] and [47], and eventually losing antibacterial activity of the obtained nanofibers

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(Fig. 5b,c). Crosslinking samples at 145 ºC induced loss of antibacterial activity apparent in

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disc diffusion test.

3.4. Control drug release and kinetic studies

Drug release is followed by several phenomena, such as diffusion of drug, water adsorption, swelling and erosion of matrix, which determine the kinetic release mechanism

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[48]. Based on the antibacterial activity test, the further investigation of the samples were devoted to sample with the highest antibacterial activity, Chi/PEO 80. In order to understand release mechanism, studies were performed on Chi/PEO film, Chi/PEO 40

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nanofibers, crosslinked Chi/PEO nanofibers (Chi/PEO 80) and compressed crosslinked Chi/PEO nanofibers (Chi/PEO PCNF). Figure 6 displays cumulative drug release vs. time

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of the tested samples. Chi/PEO film shows equilibrium within 5 hours [32]. Due to high surface area and porous morphology of compared to film, Chi/PEO 40 nanofibers show lower equilibrium point, within 4 hours. The effect of fast drug release is enhanced by quick dissolution of PEO in aqueous media. Recombination and new linkages between constituents via crosslinking with citric acid induce reduction of matrix solubility in aqueous solution and therefore elongation in drug release [19]. Sample of nanofiber Chi/PEO 80 revealed extend control release within 12 hours, without burst release, 12

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compared to both, film and nanofiber. Compression of crosslinked Chi/PEO 80 (Chi/PEO PCNF) nanofiber mats reveal prolonged control release up to 50 hours. Esterification of carboxylic functional group of citric acid and hydrogen groups on chitosan

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via crosslinking resulted with poor solubility in water, providing water stable nanofiber mats [11] and [14]. Crosslinking nanofiber mats indicated prolonged control, without burst release compared to film, with increasing control release from 5 hours up to 12 hours.

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Nevertheless, the crosslinking indicated possibility of prolonged control release, but the compression revealed further extent of release (up to 50 hours) due to better interface

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between constituents and increase of stiffness of the sample.

From obtained data of kinetic parameters in Table 3 the mechanism and the best fitting with model was evaluated. All mathematical models had reasonably good fit to the experimental

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data based on r2 value. Furthermore, comparison of r2 between zero and first order kinetics, showed better fitting with first order release kinetics, which indicated the dependence of concentration of loaded model drug. The diffusion of loaded drug was related to interaction

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between drug and matrix. Because of the weak non-covalent interactions of hydrophilic model drug and water soluble matrix, Chi/PEO film showed initial burst release and

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relative fast drug release in accordance with the previous report [49]. Changing the morphology into nanofibers, surface area and porosity increased causing faster diffusion of loaded drug, which was noted with the increase of kinetic constant. Crosslinking with citric acid had a multiplied influence; by reducing hydrophilicity of sample and entrapping the drug, the crosslinking provided slower diffusion, followed by lowering the kinetic constant. The effect of entrapping the drug is emphasized with compression, through increase of

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stiffness [50] providing slower and more controlled release. These observations are in correlation with n values of nanofiber samples (0.11-0.34), based on Kosmeyer-Peppas equation, which demonstrate that release mechanism follows Pseudo-Fickian diffusion.

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Good fitting of the obtained data with Weibull-model, also indicate that the release mechanism of model drug was presented as a combination of Fickian diffusion and erosion of the polymer matrix. As it is presented through modification of a process, from solvent

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be adjusted with desired kinetic mechanism.

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casting to electrospinning, or modification of matrix, like crosslinking, drug release could

3.5. DSC analysis and nanoindentation measurements

DSC analysis can be used as feasible a method for assessing the changes in polymer crystallinity [51–55], which are usually a consequence of reinforcement additon or blending

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with other polymers. DSC analysis revealed a decrease in enthalpy of fusion of nanofibers compared to film, indicating the change in crystallinity of the system caused by the process of electrospinning [51]. A large share of polymer, in polymer blend with PEO, limits

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nucleation of crystallization of PEO [22, 56, 57]. Both phenomena, the crosslinking chitosan with citric acid, via formation of peptide bonds, increases content of “free” PEO in

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matrix and the compression of nanofiber mats which induced increase of stiffness and better interaction between constituents cause the change in the thickness of crystalline lamella reflected in the change of Tm (Table 4). This change in morphology caused an enhancement of hardness and elastic modulus.

Similar to the reported results, our investigation revealed that crosslinking with citric acid has important part in increase of mechanical properties [11, 58]. The influence of 14

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crosslinking with glutaraldehyde (GA) on mechanical properties of nanofibrous Chi/PEO observed by nanoindentation was reported in work of Vondran et al. [30]. In reported work crosslinking of obtained sample was performed for 10 h with GA, with similar mechanical

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enhancement we observed in our study, with 15 min of citric acid crosslinking (data not shown). Albeit the fact that the nanoindentation method was applied to study mechanical properties of nanofibers [30, 59–61] the sensible morphology with low dimensions make

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the assessment of material mechanical properties difficult. Therefore, nanoindentation measurements were performed on compressed nanofiber mat samples and compared with

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non-compressed ones.

Figure 7a and 7b represent load-depth nanoindentation curves of both crosslinked samples, with and without compression. Elastic recovery parameter showed 63.75 % recovery of Chi/PEO 80 after unloading indenter tip and 48.46 % for Chi/PEO PCNF, which implied

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higher stiffness of the compressed sample and indicated higher elastic modulus and nanoindentation hardness [58] and [59]. Figure 7c and 7d present an outstanding increase of elastic modulus, from 35.6 ± 12 MPa to 2.15 ± 0.63 GPa, and from 6.5 ± 2.5 MPa to 144 ±

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62 MPa for hardness. This increase of mechanical properties was induced by several combined effects, such as chemical interaction via crosslinking, changing morphology of

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the sample and better interfaces between constituents due to compression. Thermal crosslinking with citric acid is a green and time saving option compared to crosslinking with GA that yields similar mechanical enhancement found in literature. Compared to film, electrospinning and crosslinking of the sample caused a change in crystallinity, with improved polymer orientation. Compression of crosslinked nanofiber

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mats, due to increase of stiffness provided coupled effect on improvement of mechanical properties. Within this study, we demonstrated possibility of producing biomaterial with benefit

control drug release and enhanced mechanical properties.

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4. Conclusions

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multifunctional characteristics, including enhanced antimicrobial activity, prolonged

Thermal in situ fast crosslinking with citric acid of Chi/PEO nanofibers provided a

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promising material with multifunctional characteristics suitable for drug delivery and tissue engineering. Crosslinking occurred as a result of ester, hydrogen and peptide bonds formation between constituents as observed in FTIR spectra.

The crosslinking temperature appeared to be an important parameter for inhibition of

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bacteria, which was demonstrated by a decrease of zone of inhibition with temperature increase above 80 ºC. Nevertheless, crosslinking nanofibers at 80 ºC indicated a slight increase of antibacterial activity, which derived from the synergistic effect of chitosan and

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citric acid. Crosslinking provided prolonged control without burst release, which could be extended with compression of the sample. The obtained data had the best fitting with

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Weibull model, indicating presence of both diffusion and erosion as kinetic profile mechanism. Nanoindentation of the compressed sample revealed enhanced mechanical properties due to increased stiffness, which occurred as a result of better interface of constituents and change of matrix crystallinity. Our investigation reveals a fast and green crosslinking alternative that can yield a material with improved mechanical properties, prolonged control drug release and enhanced antibacterial activity. Within the scope of this

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paper we demonstrated producing multifunctional material for drug delivery and tissue engineering.

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Acknowledgements

Financial support through the Ministry of Education, Science and Technological Development of the Republic of Serbia, Project III45019 and TR34011 are gratefully

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Figure captions Fig. 1. Photograph of coaxial electrospinning setup Fig. 2. Schematic view of proposed interactions occurring during the thermal crosslinking of Chi/PEO

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with citric acid

Fig. 3. FESEM images of nanofiber mats Chi/PEO 40 (a-c), Chi/PEO crosslinked at 145 ºC (d-f) and core-shell structure (g,h) with distribution of fiber diameters of polymer chitosan/PEO nanofiber; TEM

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image of core-shell Chi/PEO nanofiber (i)

Fig. 4. FTIR spectra of nanofibers, as followed: a) Chi/PEO 40, b) crosslinked Chi/PEO at 80 ºC, c)

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crosslinked Chi/PEO at 100 ºC, d) crosslinked Chi/PEO at 145 ºC

Fig. 5. Inhibition zone diameter of nanofiber mat Chi/PEO crosslinked at 80 and 100 °C against E. coli (a), decrease of inhibiton zone diameter of samples with increase of crosslinking temperature (b) and (c) against S. aureus (*as mentioned, the samples were prepared in tablets 6 mm in diameter) Fig. 6. Model drug release of different systems of prepared chitosan/PEO, (■) Chi/PEO film, (●)

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Chi/PEO 40, (▲) Chi/PEO 80 and (▼) Chi/PEO PCNF

Fig. 7. Representative load-depth curves for both Chi/PEO 80 (a) and Chi/PEO PCNF (b) showing higher stiffness for compressed sample; increase of reduced elastic modulus (c) and nanoindentation hardness

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(d) with compression of the crosslinked nanofiber mats

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Chi/PEO film

Chi/PEO CS

Chi/PEO 40

Chi/PEO 60

Temperature, ⁰C Compression, bar

40 -

40 -

40 -

60 -

Single nozzle setup

-

-

+

Coaxial setup Samples

-

+

-

Chi/PEO 80

Chi/PEO PCNF*

Chi/PEO 100

Temperature, ⁰C Compression, bar

80

80

100

-

3

-

Single nozzle setup

+ -

+ -

Coaxial setup

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Samples

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Table 1. Conditions of preparation and designations of samples

+ -

+ -

Chi/PEO 145 145 -

+ -

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*Sample was pressed using N 840 D Hix Digital Press (Hix, Corp., USA) at a temperature of 25 ºC for 15 min under a pressure of 3 bar.

Table 2. Interpretation of diffusional release mechanisms

Drug transport mechanism

n < 0.5 (0.45)

Pseudo-Fickian diffusion

n = 0.5 (0.45)

Diffusion mechanism

0.5 < n < 1 (0.89)

Non-Fickian diffusion (diffusion and erosion)

n=1

Case 2 transport (zero order release)

Super case 2 transport (erosion or relaxation)

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n > 1 (0.89)

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Release exponent (n)

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Table 3. Kinetic parameters for zero and first order model, Higuchi, Korsmeyer-Peppas and Weibull model for prepared drug loaded samples Chi/PEO film

Chi/PEO 40

Zero order kinetics

K0(%h-1)

26.78

28.33

Eq. (1)

r2

0.915

0.830

First order kinetics

K1(%h-1)

0.53

1.07

Eq. (2)

r2

0.924

0.973

KH(%h-1/2)

65.37

65.53

r2

0.821

0.936

n

0.54

KKP(%h-n)

Chi/PEO 80

Chi/PEO PCNF

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Sample / Model

1.90

0.875

0.829

0.31

0.16

0.977

0.923

32.63

14.83

0.953

0.921

0.11

0.34

0.18

22.49

52.20

24.12

14.60

r2

0.955

0.964

0.968

0.955

Kw(%h-1)

1.686

0.973

0.992

0.682

r2

0.982

0.974

0.978

0.966

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Higuchi Eq. (3)

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Korsmeyer-Peppas Eq. (4)

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Weibull Eq. (5)

Table 4. Result of DSC analysis for different Chi/PEO samples Melting enthalpy

Degree of crystallinity

Tm, ºC

∆Hm, J/g

χm , %

60

10.61

27

Chi/PEO 40

54

7.505

19

Chi/PEO 80

51

8.598

22

Chi/PEO PCNF

55

7.927

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Chi/PEO film

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Melting temperature Sample

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Fig. 1. Photograph of coaxial electrospinning setup

Fig.2. Schematic view of proposed interactions occurring during the thermal crosslinking of Chi/PEO with citric acid

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Fig.3. FESEM images of nanofiber mats Chi/PEO 40 (a-c), Chi/PEO crosslinked at 145 ºC (d-f) and coreshell structure (g,h) with distribution of fiber diameters of polymer chitosan/PEO nanofiber; TEM image of core-shell Chi/PEO nanofiber (i)

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Fig.4. FTIR spectra of nanofibers, as followed: a) Chi/PEO 40, b) crosslinked Chi/PEO at 80 ºC, c) crosslinked Chi/PEO at 100 ºC, d) crosslinked Chi/PEO at 145 ºC

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Fig. 5. Inhibition zone diameter of nanofiber mats Chi/PEO crosslinked at 80 and 100 °C against E. coli (a), decrease of inhibiton zone diameter of samples with increase of crosslinking temperature (b) and (c) against S. aureus (*as mentioned, the samples were prepared in tablets 6 mm in diameter)

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Fig.6. Model drug release of different systems of prepared chitosan/PEO, (■) Chi/PEO film, (●) Chi/PEO 40, (▲) Chi/PEO 80 and (▼) Chi/PEO PCNF

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Fig.7. Representative load-depth curves for both Chi/PEO 80 (a) and Chi/PEO PCNF (b) showing higher stiffness for compressed sample; increase of reduced elastic modulus (c) and nanoindentation hardness (d) with compression of the crosslinked nanofiber mats

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