Fabrication and investigation of silica nanofibers via electrospinning

Accepted Manuscript Fabrication and electrospinning

investigation

of

silica

nanofibers

via

Mehran Shahhosseininia, Saeed Bazgir, Morteza Daliri Joupari PII: DOI: Reference:

S0928-4931(17)33635-4 doi:10.1016/j.msec.2018.05.068 MSC 8626

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

8 September 2017 6 May 2018 21 May 2018

Please cite this article as: Mehran Shahhosseininia, Saeed Bazgir, Morteza Daliri Joupari , Fabrication and investigation of silica nanofibers via electrospinning. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi:10.1016/j.msec.2018.05.068

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ACCEPTED MANUSCRIPT Fabrication and Investigation of Silica Nanofibers via Electrospinning

Mehran Shahhosseininia1, Saeed Bazgir*2, Morteza Daliri Joupari3 1

Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran

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Department of Animal and Marine Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

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Department of Polymer Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran (E-mail: [email protected]; Tel/fax: +98-21-4486 5328)

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Abstract

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Electrospinning is a versatile and cost-effective method for fabricating nanofibers of different materials suitable for various applications. In this work, silica nanofibers have produced

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using the electrospinning method followed by the heat treatment. To fabricate silica nanofibers,

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polyvinylpyrrolidone (PVP), tetraethyl orthosilicate (TEOS) and Butanol were used to prepare the dope solutions. The optimized concentration for polymer in the dope solutions was then

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measured at 0.1 g/ml. The electrospinning process was conducted under the optimum

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circumstances of voltage, injection flow, tip to collector distance, ambient temperature (25°C) and the humidity of 47%. Having conducted the thermal analysis (TG/DTA), electrospun fibers

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were exposed to thermal analysis in three different temperatures of 500, 700, and 1000 °C for 5 hours. Following this, the morphology and the diameter of the fibers, as well as the chemical composition and the crystallinity of each sample were analyzed using scanning electron microscopy (SEM), fourier transform infrared spectrometer (FT-IR), and x-ray diffractometry (XRD), respectively. The noteworthy conditions of 700 °C and 5 hours of heat treatment (i.e., calcination) have provided satisfactory results in terms of silica nanofibers morphology and

ACCEPTED MANUSCRIPT fibers; diameter, i.e., 110 and 600 nm. For cytotoxicity assay, murine fibroblast cells L929 were cultured on a mat of as-spun silica nanofibers. After 24 h and 48 h cultivation time, samples showed no evidence of cytotoxicity effect, which will be a promising result.

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Keywords: Electrospinning; Silica nanofibers; Morphology; Cytotoxicity effect.

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1. Introduction

In the last decades, electrospinning has attracted much more attentions for fabricating

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nanofibers due to its capability to generate ultrafine fibers within micrometer up to nanometer

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diameter range [1, 2]. Electrospinning is a simple and affordable method which relies on electrostatic forces to fabricate nanofibers from viscoelastic solutions or melt. Important features

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of electrospun fibers include high surface to volume/weight ratio, three-dimensional

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interconnected structure and fine porosities on the fibers’ surface [3, 4]. Inorganic materials, especially silica, have been considered as one of the most promising options to fabricate

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nanofibers due to their astonishing characteristics. Due to a number of unique properties, such as

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low thermal conductivity, chemically inert and non-toxicity, not only for tissue engineering purposes, but also for biosensors and drug delivery applications, silica nanofibers have attracted

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considerable attenetion during the last decades. Moreover, the osteoblastic cells were found to successfully adhere to bio-active glass (silicate compounds) and undergo the cells proliferation [5-9].

However, silica nanofibers cannot be produced by electrospining directly, therefore, the aim of the study is prepration of a gel sample with proper rheology properties for electrospining process [10,11]. A number of researches have used different polymer–solvent systems to fabricate silica nanofibers, for instance. The Polyvinylbutyral (PVB)-Ethanol (solvent) system

ACCEPTED MANUSCRIPT [12-16]. In some researches, after the fabrication of silica nanofibers by sol-gel process, several studies have been done on the surface of silicate compounds. Surprisingly, protein/DNA binding and adult Stem cell culture have been investigated on silica nanoparticles which have had appropirate results. However, in this work, the qualitative cell cytotoxicity of silica nanofibers

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for fibroblast cell culture will be examined [21-26].

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In this research, polyvinylpyrrolidone (PVP) used as the carrier for Silanol groups and

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butanol is the alcoholic solvent during the electrospinning process. PVP is a nontoxic, odorless, and enviromental-friendly polymer, which has widely been used as a functional material in

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various industries. There is no doubt that PVP is an ideal organic component for the producing of

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organic/inorganic composite nanofibers due to significant compablity with inorganic materials [18,19,22]. In this work, Calcination reaction was exclusively employed to prepare silica

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nanofibers which alongside with usage of the tetraethyl orthosilicate (TEOS) as a silica

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precursor. Moreover, morphology and crystallographic changes as well as chemical components have been investigated by various calcination temperatures. Eventually, murine fibroblast cells

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were cultured on silica nanofibrous scaffold, which distinguishes this research in the published

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literature; this is because the same investigation has not been done before on silica fibrous mat.

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2. Materials and Methods 2.1. Preparation of electrospinning solution Three major substances including TEOS (tetraethyl orthosilicate; Purity: 98%; Sigma Aldrich), PVP (polyvinylpyrrolidone; PVPK25, MW=1300000; Purity: 98%; Rahavard Tamin Pharmaceutical Co, Iran) and butanol (Solubility: 77 gr/lit; Purity: 99.9%; Merck) were used for preparing the dope solution. Dope solution with concentration of 0.1 gr (PVP)/mL (TEOS+butanol) was prepared: initialy, 14 ml of butanol and 24 ml of TEOS were mixed and

ACCEPTED MANUSCRIPT well-stirred at 80 °C for 30 min. Then, 4 gr of PVP was added to this mixture and the mixing was then continued at 120 °C for 90 min. The resultant solution was then kept for 24 h under ambient condtions for realxing the polymer chains. Viscosity and conductivity of solution were evaluated for obtaining the righ rheology characteristic for electroespining process.

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2.2. Preparation of Silica/PVP nanofibers

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An injection syringe (50 mL) equipped with a stainless steel neddle (18 guage) was filled up by the dope solution. It is worth quoting that due to high viscosity of the dope solution it was

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impossible to use 20 and 21 guage needdles.

The plastic syringe was attached to the syringe pump (TOP Syringe Pump TOP-5300

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,Tokyo ,Japan). Afterward, the stainless steel needle was connected to the electrode at a distance

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of 19 cm away from the counterelectrode. A stainless steel rotating drum (with the diameter of 8

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cm) was connected to the counterelectrode and was employed as the collector. A high voltage DC generator (ES60 ,Gamma Hight Voltage Res ,USA) was applied to supply 16 kV for

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electrospinning at ambient conditions (temperature of 25°C and humidity of 47%). The

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electrospun fibers were then dried at room temperature for 24 h to allow the residual solvent removal. The as-spun fibers were then characterized for their morphological features and

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chemical structure using XRD, SEM and STA methods. 2.3. Cytotoxicity assay In order to analyze the qualitative cell cytotoxicity of silica nanofibers, murine fibroblast cells (L929) were used. Silica fibers were treated at a temperature of 700 °C for 5 hours. The fibrous mat was cut into 1×1 cm2 samples and sterilized by the UV Irradiation for 1.5 h, and was then placed on 6-well tissue culture plates. The cell colonies were suspended in the RPMI-1640

ACCEPTED MANUSCRIPT culture media containing 75 µg/ml streptomycin and 75 IU/ml penicillin supplemented with 10% fetal bovine serum. About 5 ml of cell suspension with a concentration of 1×105 cells/ml was added to each well, containing silica fibrous mats and a control well without any sample. Plates were incubated, at least for 48 h at 37 o C in an atmosphere of 5% CO2 and 85% humidity. Plates

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were then checked at 24 h and 48 h.

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After incubation period, plates were analyzed using the inverted microscope and

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morphological feature of cells was evaluated. For cellular adherence on silica mats, samples were removed and rinsed twice with PBS to remove non-adherent cells. Cells were then fixed

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with 2.5% glutaraldehyde at 4 °C for 3.5 h. afterward; the samples were dehydrated through the

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various grades of ethanol solutions and dried in air overnight. Dried samples were then sputtered

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with gold for SEM observation of cells morphology on the nanofibers surface.

3.1.Microstructure

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3. Results and Discussion

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3.1.1. Structure of PVP and Silica/PVP fibers

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Figure 1 shows the SEM images of PVP fibers and silica/PVP fibers. As could be observed, additon of silica components to fibers decreased electrospinnability of fiber because of

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Silnol and Silatrane groups , which have been formed by TEOS and PVP and the solvent(butanol). As a result, silica/PVP fibers have larger fiber diameter compared to PVP asspun fibers [19]. In the silica/PVP nanofibrous mat, polymer palyed the binder-carrier role and caused the formation of fibers.

ACCEPTED MANUSCRIPT 3.1.2.Structure of calcined Silica/PVP at 500,700 and 1000˚C Figure 2-b demonstrates that the diameter of silica/PVP fibers is decreased after heat treatment at 500 ˚C. This is because of polymer decomposition and PVP removal from the fibers’ network. Moreover, some nodes are formed on the fibers’ structure after decomposition of

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polymer and formation of Si-O-Si bonds. This is due to the trapped Silanol groups in the fibers. That is why the EDS analysis (see Figure 3) showed high amouts of Si and O atoms in the nodes

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area. As could be observed in Figure 2-c, at 700 ˚C silica fibers demonstrated desired structrue,

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i.e., Si-O-Si bonds were shrunk, because of which it preserved the fibrous structure. Calcined fibers at 1000 ˚C (see Figure 2-d) do not show desirable arrangement compered with calcined

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fibers at 700 ˚C.

Figure 4 shows that Si-O-Si bonds were shrunk more at 1000 ˚C and several fibers have

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been incorporated. Therefore, fibers’ dimension has increased compared to 700 oC, so there were

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some ruptures in the structure. Undoubtedly, rupture in structures is caused by remelting and it

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3.2.Rheology

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has might been because of nanostructured fibers and subsequently extra calcination rate or time.

The viscosity and the conductivity of the dope solution with the ideal concentration of

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TEOS/PVP/butanol were measured [11]. The brookfield viscosity (BF35, AMTEK,USA) and the conductivity (C912, Metrohm, Switzerland) of desirable electrospinning solution were measured at 640 centipoise and 0.67 at 25 ˚C and humidity 40%, respectively. 3.3 Phase and Thermal analysis Thermal decomposition behavior of nanofibers was evaluated using the TG/DTA analysis. As could be observed in Figure 5, samples weight decreases in three zones: about 5%

ACCEPTED MANUSCRIPT weight loss from 40 to 60 ˚C could be attributed to desorption of water and solvent. Second weight loss of 4% can be related to the water and solvent’s release from the framework of silica/PVP fibers. For the third zone, the 91% weight loss between 347 to 460 ˚C were related to decomposition of PVP. It is worth quoting that at 347 ˚C PVP burns out [21,22].

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The endothermic and exothermic peaks in DTA (see Figure 5) were identified by the

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XRD and FT-IR analyzes for three cancination temperatures of 500, 700 and 1000 ˚C. As could

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be observed in Figure 6, FT-IR graph of the sample containes PVP and silica/PVP fibers (Figure 6-b) illustrated the Si-O-Si bonds at 844 and 1103 Cm‾¹, which don’t appear in the FT-IR graph

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of the PVP samples (Figure 6-a). Moreover, the Exothermic peak at 347 ˚C (see Figure 5)

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indicated that PVP content has burned, which was confirmed by removing some fuctional groups of silica/PVP fibers after heated at 500 ˚C (see Figure 7-a) including C=C and C-N bonds.

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Additionally, the C=O and C-H bonds were not removed completely at 500˚C as they have been

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trapped in the silica/PVP fibers` network. All these peaks and reference bonds can be seen in Figure 6 and Figure 7.

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Another exothermic peak at 870 ˚C (see Figure 5) indicated that amorphous SiO2

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particulates were crystallized. Therefore, the FT-IR pattern for silica fibers after heat treatment at 1000 ˚C shows deeper Silanol groups (Si-O-Si) compared to those heat treated at 500 and 700 ˚C

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(see Figure 7).

The XRD pattern for the calcinated samples at 500, 700 and 1000 ˚C (see Figure 8) indicated the glassy characteristics of silica fibers. Crystalization peak did not show itself in the XRD pattern at 1000 ˚C for SiO2, rather it stems from low calcination time (about 5 hours) and consequently low kinetic rate of molecules to elstablish “Quartz” or “Cristobalite” or “Tridymite” crystalline phase structures.

ACCEPTED MANUSCRIPT 3.4. Cytotoxicity of the Silica nanofibers Light microscopy images showed cells with elongated and flattened structure on the fibers’ surface with compact population, especially after 48 hours incubation. Wells with samples also showed similar morphology as the control group indicating the material is not

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cytotoxic. Figure 9 shows images of cultured cell and cells in boundaries with samples, respectively.

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Images from scanning electron microscopy observation revealed the growth and

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attachment of L929 fibroblast cells in the control and silica fiber samples which exhibit pattern of silica fibers and also proper flattening with discrete Filopodia and desirable growth of

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fibroblast cell on the nanofibrous structure (see Figure 10).

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One of the highlighted advantages of proposed fibrous mats to prepare scaffolds is indeed

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the easy setup as it can be heated up to 250 ˚C in the oven (for sterilization), with no significant

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4. Conclusion

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change occurring in the chemical structure and compound of the sample.

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In this study, polymer has been used as the carrier-binder in the production of silica nanofibers. The applied methods were electrospinning followed by calcination which made it unique. SEM images demonstrated that silica/PVP fibers size has increased in comparison to pure PVP fibers due to existence of Si-O-Si bonds. In addition, the results from SEM illustrated that most appropriate morphology of silica nanofibers are those calcinated at 700 ˚C. Moreover, the FT-IR and EDAX tests indicated the presence of Silanol groups (i.e., Si-O-Si bonds). The amorphous structure in the heat-treated samples in XRD results demonstrated that 5 hours have

ACCEPTED MANUSCRIPT not been sufficient for crystallization of the fibers. Based on the derived results, bio-inert silica nanofibers could be used as scaffold in tissue engineering, taking into account on cell proliferation and attachment. No cytotoxicity was observed and firm cellular attachment could be detected. Quantitative and qualitative attachment of cells should be undertaken as part of another

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study. The most suited fibers in terms of crystallinity and structure, depending on the temperature

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and heat treatment time, are to be derived calcination temperature and heating time, on which

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further research should be done.

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Acknowledgements

This research was supported by the Nano Polymer Research Laboratory (NPRL) of

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Islamic Azad University (Science and Research Branch), Tehran, IRAN.

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ACCEPTED MANUSCRIPT Figure Captions Figure 1- SEM images of the PVP (a and c) and TEOS/PVP (b and d) electrospun fiber samples. Figure 2- SEM images of electrospun fibers: (a) TEOS/PVP, (b) TEOS/PVP calcined at 500 °C, (c) TEOS/PVP calcined at 700 °C, and (d) TEOS/PVP calcined at 1000 °C. Figure 3- EDAX spectra of TEOS/PVP electrospun fibers calcined at 500 °C.

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Figure 5- TG/DTA curves of TEOS/PVP electrospun fibers.

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Figure 4- SEM Images of TEOS/PVP electrospun fibers: (a and c) calcined at 700 °C, (b and d) calcined at 1000 °C.

Figure 6- Fourier-trasformed infrared spectoscopy of: (a) PVP electrospun nanofibers, (b) TEOS/PVP electrospun fibers.

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Figure 7- Fourier-trasformed infrared spectoscopy of TEOS/PVP electrospun fibers: heat treated at 500 °C, (b) heat treated at 700 °C, (c) heat treated at 1000 °C.

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Figure 8- X-ray diffraction pattern of electrospun fibers: (a) TEOS/PVP non calcined, (b) calcined at 500 °C, (c) calcined at 700 °C, (d) calcined at 1000 °C.

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Figure 9- Optical Microscopy images of fibroblast cell culture: (a and b) control sample after 24 and 48 hours; (c and d) silica fibers after 24 and 48 hours, repectively.

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Figure 10- SEM Images of: (a) fibroblast cell culture of control sample after 48 hours; (b) silica fibers scaffold before fibroblast cell culture; (c and d) fibroblast cell culture on silica fibers after 48 hours.

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Figure 1- SEM images of the PVP (a and c) and TEOS/PVP (b and d) electrospun fiber samples.

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Figure 2- SEM images of electrospun fibers: (a) TEOS/PVP, (b) TEOS/PVP calcinated at 500°C, (c) TEOS/PVP calcinated at 700 °C, and (d) TEOS/PVP calcinated at 1000 °C.

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Figure 3- EDAX spectra of TEOS/PVP electrospun fibers calcinated at 500 °C.

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Figure 4- SEM Images of TEOS/PVP electrospun fibers: (a and c) calcinated at 700 °C, (b and d) calcinated at 1000 °C.

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Figure 5- TG/DTA curves of TEOS/PVP electrospun fibers.

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Figure 6- Fourier-trasformed infrared spectoscopy of: (a) PVP electrospun nanofibers, (b)

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TEOS/PVP electrospun fibers.

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Figure 7- Fourier-trasformed infrared spectoscopy of TEOS/PVP electrospun fibers: heat treated

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at 500 °C, (b) heat treated at 700 °C, (c) heat treated at 1000 °C.

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Figure 8- X-ray diffraction pattern of electrospun fibers: (a) TEOS/PVP non calcined, (b)

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calcined at 500 °C, (c) calcined at 700 °C, (d) calcined at 1000 °C.

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Figure 9- Optical Microscopy images of fibroblast cell culture: (a and b) control sample after 24

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Figure 10- SEM Images of: (a) fibroblast cell culture of control sample after 48 hours; (b) silica fibers scaffold before fibroblast cell culture; (c and d) fibroblast cell culture on silica fibers after 48 hours.

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Highlights Preparation and characterization of Silica- Composite Nanofibers via electrospinning Unique properties of Silica fibers based on calcination temperature Investigation of fibroblast cell culture for Cytotoxicity assay Heat treatment (calcination) whit no changes in Crystallinity structure