143 (2022) 213178

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Biomaterials Advances
journal homepage: www.journals.elsevier.com/materials-science-and-engineering-c

Cytotoxicity evaluation of poly(ethylene) oxide nanofibre in MCF-7 breast

cancer cell line
Munirah Jamil a, *, Iskandar Shahrim Mustafa a, *, Naser Mahmoud Ahmed a, b,
Shahrul Bariyah Sahul Hamid c
a
School of Physics, Universiti Sains Malaysia, Gelugor 11800, Penang, Malaysia
b
Department of Medical Instrumentation Engineering, Dijlah University College, Baghdad, Iraq
c
Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas 13200, Penang, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: Biocompatible polymers have received significant interest from researchers for their potential in diagnostic
Poly(ethylene) oxide applications. This type of polymer can perform with an appropriate host response or carrier for a specific pur­
Nanofibre pose. The current study aims to fabricate and characterise poly(ethylene) oxide (PEO) nanofibres with different
MCF-7 cell lines
concentrations for cytotoxicity evaluation in human breast cancer cell lines (MCF-7) and to get an optimal PEO
Cytotoxicity
nanofibre concentration (permissible limit) as a suitable polymer matrix or carrier with potential use in diag­
MTS assay
Colony formation assay nostic applications. The fabrication of PEO nanofibres was done using electrospinning and was characterised by
structure and morphology, surface roughness, chemical bonding and release profiles. The functional effects of
PEO nanofibres were evaluated with MTS assay and colony formation assay in MCF-7 cells. The results showed
that viscosity plays a vital role in synthesising a polymer solution in electrospinning for producing beadless
nanofibrous mats ranging from 4.7 Pa⋅s to 77.7 Pa⋅s. As the PEO concentration increases, the nanofibre diameter
and thickness will increase, but the surface roughness will be decreased. The average fibre diameter for 5 wt%
PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 129 ± 70 nm, 185 ± 55 nm and 192 ± 53 nm, respectively. In
addition, the fibre thickness for 4 wt% PEO, 5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 269 ± 3
μm, 664 ± 4 μm, 758 ± 7 μm and 1329 ± 44 μm, respectively. Contrarily, the surface roughness for 4 wt% PEO,
5 wt% PEO, 6 wt% PEO and 7 wt% PEO nanofibres were 55.6 ± 9 nm, 42.8 ± 6 nm, 42.7 ± 7 nm and 36.6 ± 1
nm, respectively. PEO nanofibres showed the same burst release pattern and rate due to the same molecular
weight of PEO with a stable release rate profile after 15 min. It also demonstrates that the percentage of PEO
nanofibre release increased with the increasing PEO concentration due to the fibre diameter and thickness. The
findings showed that all PEO nanofibres formulations were non-toxic to MCF-7 cells. It is suggested that 5 wt%
PEO nanofibre exhibited non-cytotoxic characteristics by maintaining the cell viability from dose 0–1000 μg/ml
and did not induce the number of colonies. Therefore, 5 wt% PEO nanofibre is the optimal nanofibre concen­
tration and was suggested as a suitable base polymer matrix or carrier with potential use for diagnostic purposes.
The findings in this study have demonstrated the influence of cell growth and viability, including the effects of
PEO nanofibre formulations on cancer progress characteristics to achieve a permissible PEO nanofibre concen­
tration limit that can be a benchmark in medical applications, particularly diagnostic applications.

1. Introduction
Electrospinning has evolved as a versatile and cost-effective tech­
nique for producing functional nanofibres from various chemical ma­
terials. This method starts by applying a high voltage to a droplet of a
viscous polymer solution (Taylor's cone) from a spinneret to overcome
its surface tension and evaporation of the solvent while forming a
continuous fibrous mat on a metal collector. It is an effective and
adaptable method to produce fibres that act as a matrix to encapsulate
drugs or nanoparticles while performing as a carrier to the targeted site.
Nanofibre produced via electrospinning is a one-dimensional (1D)
nanostructure with a unique morphology. This technique has been well-

* Corresponding authors.
E-mail addresses: munirah_jamil@yahoo.com (M. Jamil), iskandarshah@usm.my (I.S. Mustafa), naser@usm.my (N.M. Ahmed), shahrulbariyah@usm.my
(S.B. Sahul Hamid).

https://doi.org/10.1016/j.bioadv.2022.213178
Received 15 July 2022; Received in revised form 17 October 2022; Accepted 28 October 2022
Available online 2 November 2022
2772-9508/© 2022 Elsevier B.V. All rights reserved.
M. Jamil et al.
Fig. 1. Setup of the electrospinning system.
received worldwide in fabricating fibres with nano to micro-sized di­
ameters. It has also been used in numerous applications due to its
excellent mechanical properties, simplicity with large-scale production
and unique properties [Alshaya et al. [3], Eren Boncu and Ozdemir [17],
Zhang et al. [62]].
Biocompatible polymers are the main factor of choice for diagnostic
and therapeutic purposes. These polymers can be natural and synthetic
that are non-toxic to induce a minor inflammatory response. In vitro
studies are essential in evaluating the cytotoxic level of materials
through half maximal inhibitory concentration (IC50) value. Another
aspect to focus on is the biodegradability of a polymer, as it is one of the
most promising strategies for a control-release system in drug delivery,
bone and dental implantation [Calzoni et al. [9]]. Synthetic polymers
such as polylactide acid (PLA), polyglycolic acid (PGA), poly­
caprolactone (PCL), polyethylene glycol (PEG), and polyglutamic acid
(PGLu) have been successfully employed as nanocarriers in previous
studies [Alven et al. [4], Ferrari et al. [19], Kopeček and Yang [32]]. On
the other hand, chitosan, hyaluronan, albumin, gelatin and collagen are
natural polymers actively used in nanomedicine in therapeutic appli­
cations [Chen et al. [10], Singh et al. [52]].
Poly(ethylene) oxide (PEO) is flexible, non-ionic, water-soluble, and
hydrophilic. As certified by the Food and Drug Administration (FDA),
PEO is biocompatible, biodegradable, bioinert, non-toxic, and exhibits
excellent mechanical properties. It has received tremendous attention in
various industrial applications, especially in the biomedical field, such
as bone tissue engineering [Hong [24], Singh et al. [53]], drug delivery
[Elsadek et al. [16], Guo et al. [22], Harish et al. [23], Li et al. [36]],
green composite [Wu et al. [58]], wound dressing [Khandaker et al.
[28], Khunová et al. [29], Yuan et al. [61]], and many more. It has
shown high versatility as a polymer carrier. PEO not only dissolve in the
water but also dissolve in different kinds of organic solvents. Water-
soluble polymers are commonly used as thickening agents to control
the rheology of aqueous fluids and exhibit an excellent stabilising agent,
emulsifier and lubricant [Ebagninin et al. [15]]. Polyethylene is recog­
nised in medical implantation and has been extensively applied in
fabricating high-porous scaffolds for facial and cranial reconstruction
[Paxton et al. [41]].
Cancer is a leading cause of death worldwide and is reported to cause
nearly 10 million deaths in 2020 [WHO [57]]. Breast cancer is the
second leading cause of cancer death in women after lung cancer,
contributing to 12.5 % of the total new cases diagnosed in 2020 [WCRF
[56]]. Therefore, early detection of breast cancer is necessary to prevent
death. The breast cancer cell line (MCF-7) was isolated from the breast
tissue of a 69 years old woman with metastatic adenocarcinoma in 1970.
The term MCF-7 is an acronym of Michigan Cancer Foundation-7 by
referring to the institute in Detroit where the cell line was established in
1973 [Lee et al. [34]].
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Biomaterials Advances 143 (2022) 213178
A recent study has been conducted on the cytotoxic effects of elec­
trospun PEO/rGO scaffold in epithelial colon cancer cells. According to
the study, PEO was chosen as a polymer matrix because of its ability to
reduce the cytotoxicity effects of carbon-based nanofillers and its high
electro-spinnability [Ivanoska-Dacikj et al. [26]]. The previous study
has demonstrated that the chitosan/PEO nanofibre was non-toxic and
induced the L929 fibroblastic cells in vitro [Rahimi et al. [46]]. A similar
study was performed using chitosan/PEO with fumed silica-cefazolin on
the wounded skin of the female Wistar rats and has been reported to heal
almost the entire wound after 10 days [Fazli and Shariatinia [18]]. In
addition, the encapsulation of cerium oxide and peppermint oil in PEO/
graphene oxide nanofibre revealed low cytotoxicity towards L929
fibroblast cells [Bharathi and Stalin [8]]. Therefore, the findings showed
that PEO nanofibre could be a suitable antibacterial wound dressing in
biomedical applications. In addition, a previous study on
tripolyphosphate-crosslinked chitosan/PEO nanofibre also proved a
controlled and sustained release of ranitidine hydrochloride that is
suitable for oral drug carriers and therapeutic response [Darbasizadeh
et al. [12]].
Even though PEO is considered a non-toxic polymer, the permissible
limit of PEO has not been investigated in detail for the influence of cell
growth and viability, as well as the effects of PEO concentrations on
cancer progress characteristics, which is crucial in diagnostic applica­
tions. In the current research, PEO nanofibres were synthesised and
fabricated with different concentrations using electrospinning for cyto­
toxicity evaluation in MCF-7 cells to get an optimal PEO nanofibre
concentration (permissible limit) as a suitable polymer matrix or carrier
with potential use for diagnostic purposes. The characterisation of the
PEO nanofibre was performed to evaluate the performance of PEO so­
lution viscosity, nanofibre diameter and thickness, surface roughness,
chemical bonding, and PEO nanofibre release stability. Meanwhile, the
functional effects of PEO nanofibres were evaluated with MTS assay and
colony formation assay in MCF-7 cells with a dose from 0 to 1000 μg/ml.
These assays were performed to investigate the influence of cell growth
and viability, including the effects of PEO nanofibre formulations on
cancer progress characteristics.
2. Experimental section
2.1. Chemicals and reagents
All chemicals and reagents used in this study were analytical grades
without further purification. Poly(ethylene) oxide (PEO) powder with
an average molecular weight of 900,000 (98 % hydrolysed), Dulbecco's
Modified Eagle's Medium (DMEM) and trypsin EDTA solution were
purchased from Sigma Aldrich. Stabilised penicillin-streptomycin mixed
solution (penicillin 10000 units/ml, streptomycin 10000 μg/ml) was
M. Jamil et al.
Fig. 2. MTS cell proliferation assay flowchart.
purchased from Nacalai Tesque. Fetal bovine serum (FBS) was pur­
chased from Tico Europe. CellTiter 96 AQueous One Solution Cell Pro­
liferation Assay was purchased from Promega, and MCF-7 cells (HTB-
22) were purchased from American Type Culture Collection (ATCC).
Sterile ultrapure water was used as a solvent for this experiment.
2.2. Preparation of PEO solution
PEO solutions were prepared with varying concentrations from 4 wt
% to 8 wt%. PEO powder was dissolved in sterile ultrapure water and
stirred for 24 h at room temperature (20∘C) with a magnetic stirrer
(Heidolph MR Hei-Mix D) [Bahlouli et al. [7], Huang and Kobayashi
[25]]. Finally, the PEO solution was sonicated using an ultrasonic
homogeniser (BioLogics Model 150 VT) with an output power of 50 W
for 15 min to attain a homogenous solution. The same PEO molecular
weight was used throughout the experiment. Therefore, it does not
impact the molecular weight but only causes the molecules to become
less mobile in a homogenous solution [Taghizadeh and Asadpour [54]].
2.3. Preparation of PEO nanofibre
In this study, PEO nanofibres were prepared using electrospinning.
The electrospinning system consisted of a spinneret (a 27-gauge stainless
steel needle), a syringe pump, a high-voltage power supply (15 kV), and
a grounded metal collector. Fig. 1 illustrates the electrospinning system
used in this research.
PEO solution has been loaded into a 5 ml Luer lock syringe attached
to a 27-G hypodermic needle. The solution's flow rate and targeted
volume were set at 1 ml/h and 5 ml, respectively. The inner diameter of
the syringe was set at 12.54 mm. The applied high voltage was set at 13
kV, and the flat vertical metal collector was kept constant at 18 cm. The
experiment was performed at room temperature (20 ∘C) and moderate
humidity (relative humidity 40–50 %). The resulting nanofibre was
collected on the collector covered with greaseproof paper. This tech­
nique was required to ensure that the nanofibre could be taken off
smoothly from a greaseproof paper without any damage.
3. Characterisation
The viscosity of PEO solutions was measured using a viscometer
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Biomaterials Advances 143 (2022) 213178
(VISCO BASIC Plus L) at room temperature (20 ∘C) before being
rendered into electrospun nanofibres. The electrospun PEO nanofibres'
morphology was characterised using Field Emission Scanning Electron
Microscope (FESEM) (Quanta FEG 650). The nanofibres were coated
with platinum using a sputter coater (Quorum Q150R S) to reduce the
charging effect and improve the image contrast before scanning. The
secondary electron (SE) technique was used to study the morphology
surface and measure the PEO nanofibre diameter. Then, a cross-
sectional technique was performed to measure the thickness of the
PEO nanofibre. The PEO nanofibre's thickness and diameter were
measured from the smallest to the largest sizes using ImageJ 1.53 s
software.
The nanofibre's surface roughness was determined using Atomic
Force Microscopy (AFM) (Bruker Dimension Edge). The average
roughness (Ra) was measured and calculated from five different regions
using NanoScope Analysis v1.20 software (Veeco Instruments). The
Fourier Transform Infrared Resonance (FTIR) analysis for the chemical
bonding was executed from 500 to 4000 cm− 1 wavenumbers (Perki­
nElmer Spectrum GX). PEO nanofibres release was performed to eval­
uate the stability of the nanofibre using UV–Vis spectroscopy (Shimadzu
UV-1800) with a wavelength from 200 nm to 800 nm. The cumulative
percentage of the PEO nanofibre release was calculated using the
formula:
PEO nanofibre release =
PEO absorbance at interval time
× 100% (1)
PEO final absorbance
All characterisations were measured in triplicates, and the data were
reported as mean ± standard deviation (SD).
3.1. Cell culture media
For the preparation of complete culture media, Dulbecco's Modified
Eagle's Medium (DMEM) and Fetal bovine serum (FBS) were used in this
study. According to previous reports, 1 % stabilised penicillin-
streptomycin mixed solution (Pen-strep) and 10 % FBS was added to
the DMEM [Franqui et al. [21], Lesniak et al. [35], Luis et al. [39]].
3.2. Cell culture
The cryovial containing MCF-7 cells was thawed by gentle agitation
M. Jamil et al.
Fig. 3. Colony formation assay flowchart.
in a water bath at 37 ∘C [ATCC [5]]. The cell suspension was transferred
into a 15 ml falcon tube and centrifuged at 125 ×g for 10 min. The su­
pernatant was discarded, and the cell pellet was resuspended with one
ml of complete media. Subsequently, the cells were then transferred into
a 25 cm3 (T25) flask. The flask was incubated at 37 ∘C, with 5 % CO2
humidity, and left overnight. Culture media was replaced with new
media until cells reached confluency.
3.3. MTS cell proliferation assay
The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium) (MTS) assay is a colourimetric method to
measure cellular metabolic activity to indicate viable cells, proliferation
and cytotoxicity. The MCF-7 cells treatment with PEO nanofibres (5 wt
%, 6 wt%, and 7 wt%) was performed after the cells achieved 80 %–90 %
confluence in the T25 culture flask. MCF-7 cells were seeded in tripli­
cates in a 96-well plate containing 10,000 cells per well and incubated
overnight at 37 ∘C with 5 % CO2 humidity. Each PEO nanofibre was
UV–sterilised for 15 min before the treatment. Next, the samples were
dissolved in the complete media. The next day, cells were treated with
PEO nanofibres with concentrations ranging between 0 and 1000 μg/ml.
The MTS assay was performed according to the manufacturer's protocol
after 24 h [Promega [44]]. A total of 20 μl of CellTiter 96 reagent was
pipetted into each well that contained the sample in 100 μl of culture
media and incubated at 37 ∘C with 5 % CO2 humidity for 2 h. After in­
cubation, the absorbance was recorded using a microplate reader (Bio-
Tek Instruments) at 490 nm. The flowchart of the MTS cell proliferation
assay procedure is simplified in Fig. 2.
3.4. Colony formation assay
The colony formation assay was performed to measure the effect of
PEO nanofibres on the growth characteristics of the MCF-7 cells. The
cells were treated with PEO nanofibres after achieving between 80 %–
90 % of confluence in the T25 culture flask. MCF-7 cells were seeded
triplicated in a 6-well plate at a density of 3000 cells per well and were
left overnight in an incubator at 37 ∘C with 5 % CO2 humidity to achieve
adherent cells.
The culture media in each well was discarded after 24 h. The cells
were treated with PEO nanofibres in 10 μg/ml doses. Each PEO
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Biomaterials Advances 143 (2022) 213178
nanofibre was UV-sterilised for 15 min before the treatment and dis­
solved in complete media to prepare a 10 μg/ml stock concentration.
The cells were treated with different PEO nanofibre formulations (5 wt
%, 6 wt%, and 7 wt%), then incubated at 37 ∘C with 5 % CO2 humidity
and the culture media was replaced with fresh media every 3-day in­
tervals. The flow chart of the colony formation assay procedure illus­
trates in Fig. 3.
The culture media from each well was discarded after 9 days of in­
cubation. The cell layer was rinsed with 2 ml of sterile PBS to remove
residues of the complete media. The next step was followed by adding 1
ml of 100 % methanol to each well, and the plate was swirled gently to
ensure the cell layer was covered with methanol. The cells were incu­
bated at room temperature (20∘C) for 20 min. After that, the methanol
solution was discarded from each well, and the cell layer was rinsed with
2 ml of distilled water to ensure no methanol residue was left on the
cells. Next, 1 ml of crystal violet solution was added to the wells and
incubated at room temperature for 15 min. The crystal violet solution
was prepared by adding 0.5 % crystal violet powder to 25 % of meth­
anol. The ultraviolet solution was discarded after 15 min, and the cells
were rinsed with 2 ml of distilled water thrice. The plates were inverted
on the tissue to dry overnight. The cells were visualised under a phase-
contrast microscope to capture the images. The percentage of the colony
was calculated using the formula:
Total colony after treatment
Percentage of the colony (%) = × 100% (2)
Total colony before treatment
3.5. Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics 27
(Chicago, IL, USA) software and expressed as mean ± standard devia­
tion (SD). Data were analysed and compared using a one-way analysis of
variance (one-way ANOVA), followed by Tukey HSD or Bonferroni's post
hoc. The comparison of the mean between the control and treatment
within the group was assessed with the PEO nanofibre concentrations'
paired t-test. A p-value < 0.05 was considered significantly different.
M. Jamil et al.
Fig. 4. The viscosity of the different concentrations of PEO solution.
4. Results and discussion
4.1. Viscosity test
The viscosity of as-prepared PEO solutions ranging from 4 wt% to 8
wt% is shown in Fig. 4. From Fig. 4, the viscosity increases with the
Fig. 5. Morphology of nanofibres under FESEM with a 50 k× magnification and nanofibre diameter distribution for (a) 4 wt% PEO nanofibre, (b) 5 wt% PEO
nanofibre, (c) 6 wt% PEO nanofibre, and (d) 7 wt% PEO nanofibre.
5
Biomaterials Advances 143 (2022) 213178
increment of PEO concentration. The viscosity for 4 wt% PEO is 0.8 Pa⋅s,
followed by 4.7 Pa⋅s for 5 wt% PEO, 10.3 Pa⋅s for 6 wt% PEO, 31.5 Pa⋅s
for 7 wt% PEO and 77.7 Pa⋅s for 8 wt% PEO. It can be seen that higher
concentration resulted in higher viscosity. It is due to the larger mole­
cules exerting friction and interacting with one another in the polymer
solution to become more concentrated. The results supported the theory
that solution concentration correlated with the viscosity of the polymer
[Ebagninin et al. [15], Filip and Peer [20], Hong [24], Jung and Hu
[27]]. A previous study also demonstrated that polymer concentration
increased the viscosity, thus causing the molecules in the solution to
become less mobile, and the velocity gradients around the collapsing
bubbles became smaller [Taghizadeh and Asadpour [54]].
4.2. Structure and morphology
The morphology of the PEO nanofibres was observed using FESEM,
as shown in Fig. 5. The selection of 100 nanofibre diameters was
measured using ImageJ, and the data was presented as nanofibre
distribution.
Nanofibre is a continuous string-like spider web structure. Due to the
bead-like structures, the nanofibre diameter for 4 wt% PEO was unable
to be measured. A continuous polymer fibre is difficult to be achieved if
the concentration is too low. Thus, polymer beads are obtained. This
phenomenon happens due to the surface tension of the solution
becoming the dominant factor. As a result, no fibre formation will be
formed. In addition, the viscosity is low compared to the surface tension
of the solution at the lowest concentration. Consequently, the beads are
created as Gibb's surface free-energy minimises [Cohades and Michaud
M. Jamil et al. Biomaterials Advances 143 (2022) 213178

Fig. 5. (continued).

Fig. 6. The thickness of the PEO nanofibres under FESEM with 100× magnification for (a) 4 wt% PEO nanofibre, (b) 5 wt% PEO nanofibre, (c) 6 wt% PEO nanofibre,
and (d) 7 wt% PEO nanofibre.

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M. Jamil et al. Biomaterials Advances 143 (2022) 213178

Meanwhile, the average nanofibre diameter for 5 wt% PEO nanofibre

Fig. 7. The graph for the average thickness of PEO nanofibres. Asterisk (***)
indicates significance p < 0.000.
[11], Deitzel et al. [13], Schneider et al. [50]]. The finding reveals that
the viscosity of the PEO solution (0.8 Pa⋅s) will not produce a good
nanofibre mat.
was 129 ± 70 nm. The average nanofibre diameters for 6 wt% PEO and
7 wt% PEO nanofibres were 185 ± 55 nm and 192 ± 53 nm, respec­
tively. In general, even using the same needle size, a higher PEO con­
centration will produce a larger nanofibre diameter, and the diameter
tends to decrease with decreasing PEO concentration.
Eventually, 8 wt% PEO was impossible to be fabricated into nano­
fibre due to the viscosity level. Moreover, the needle size selection (27-
G) is unsuitable for the 8 wt% PEO concentration. When the viscosity is
too high, the electrostatic force on the solution is not strong enough to
initiate a jet from the spinneret, resulting in the formation of droplets. At
this point, the applied electric field needed to initiate a jet would be on
the same order of magnitude as the electric breakdown voltage of the
polymer. Therefore, an appropriate polymer concentration is critical to
electrospinning [Hong [24]]. This study proved that sizeable helix-
shaped fibres formed if the viscosity was reduced (but maintained
relatively high) by decreasing the solution concentration, resulting in
smooth, continuous fibres [Schneider et al. [50], Wang et al. [55], Yang
et al. [60]].
Fig. 6 illustrates the thickness of the PEO nanofibres. The nanofibre
thickness was measured using ImageJ to select three areas of nanofibre
thickness. The nanofibre's cross-section area is thicker at the centre than
the edge due to the stretching of the fibre surface and the deformation of
soft fibres while preparing the cross-section sample. In terms of the
nanofibre thickness, it is noted that 4 wt% PEO nanofibre was less thick

Fig. 8. 3D view images of surface roughness and profile of PEO nanofibres: (a) 4 wt% PEO nanofibre, (b) 5 wt% PEO nanofibre, (c) 6 wt% PEO nanofibre, and (d) 7
wt% PEO nanofibre.

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M. Jamil et al. Biomaterials Advances 143 (2022) 213178

Table 1
Wavenumbers and assignments of FTIR bands exhibited by PEO nanofibres.
Where ν = stretch, δ = scissor/deformation, ω= wag, ρ = rock, τ = twist.
The subscripts as = asymmetric vibrational mode.
Frequency (cm− 1) Assignments

2870 ν (CH2)
2229 (C ≡ O)
2169 (C ≡ O)
1748 (C––O)
1639 (C––O)
1464 δ (CH2)
1354 ω (CH2)
1283 τ (CH2)
967 ρas (CH2) or τ (CH2) or ω (CH2)
852 ν (C–O) or τ (CH2)

roughness for 5 wt% PEO and 6 wt% PEO nanofibres were almost
similar, which were 42.8 ± 6 nm and 42.7 ± 7 nm, respectively.
From Fig. 9, the result also interpreted that the largest nanofibre
diameter has the lowest surface roughness and vice versa. Consequently,
the smaller the nanofibre diameter, the higher the nanofibrous mem­
brane surface area and the rugged surface of the nanofibres [Moghadam
Fig. 9. The average surface roughness (Ra) of the PEO nanofibres. Asterisk (**)
et al. [40]]. Surface roughness is necessary for the adherence and growth
indicates significance p < 0.01–0.001.
of cell lines. A higher surface roughness will lead to a high hydrophobic
surface that causes lower cell attachment [Dubey et al. [14], Rabiatul
et al. [45], Razali et al. [47]]. Thus, it is a crucial parameter affecting cell
behaviour and should be considered when designing a scaffold for
diagnostic applications.

4.4. Chemical bonding

Fig. 10 depicts the FTIR spectra of chemical bonding and functional

groups in PEO nanofibres, ranging from 500 cm− 1 to 4000 cm− 1.
The FTIR spectra of PEO nanofibres showed a sharp peak at 2870
cm− 1, corresponding to the CH2 stretching mode. This band was
confirmed in previous studies [Aydogdu et al. [6], Rengifo et al. [48],
Rezaei et al. [49]]. It is worth mentioning that peaks were detected at
2229 cm− 1, and 2169 cm− 1 indicated that the triple bonding (C ≡ C)
appeared in PEO nanofibres. Double carbonyl bonding (C = C) was
confirmed when the peaks existed at 1748 cm− 1 and 1639 cm− 1
[Aydogdu et al. [6]]. Peaks observed at 1464 cm− 1, 1354 cm− 1 and
1283 cm− 1 were attributed to the CH2 scissoring, CH2 wagging, and CH2
twisting modes, respectively [Abdelrazek et al. [1], Aydogdu et al. [6],

Fig. 10. FTIR spectra of PEO nanofibres.

compared to others, which was 269 ± 3 μm. Meanwhile, the thickness of

5 wt% PEO and 6 wt% PEO nanofibres were 664 ± 4 μm and 758 ± 7
μm, respectively. Among the PEO nanofibres, 7 wt% PEO nanofibre was
the thickest, 1329 ± 44 μm. Statistical results showed significant dif­
ferences between all PEO nanofibres, where p = 0.000 (Fig. 7). This
finding concluded that the PEO concentration also influences the
thickness of the nanofibre.

4.3. Surface roughness

AFM was used to reveal the 3D surface roughness of the PEO nano­
fibres with a scan size of 10 μm in Fig. 8. Five different regions in the
nanofibre were selected and measured with an area of 2 μm × 2 μm.
The surface roughness of the nanofibre is directly proportional to the
PEO concentration. The average surface roughness of 7 wt% PEO
nanofibre (36.6 ± 1 nm) is significantly different from 4 wt% PEO
nanofibre (55.6 ± 9 nm), where p = 0.009. Meanwhile, the surface Fig. 11. PEO nanofibres release profiles.

8
M. Jamil et al.
Fig. 12. Cell culture growth inside the T25 flask with 100× magnification for (a) thawed cells and (b) after three days, the cells have become stabilised and attached
to the flask surface. (c) The cells have reached 80–90 % confluency after seven days with 100× magnification. (d) The MCF-7 cells with 200× magnification with
80–90 % confluency.
Kianfar et al. [30], Wang et al. [55]]. A peak at 967 cm− 1 was assigned to
asymmetric CH2 rocking mode. A study by Aydogdu et al. [6] confirmed
this band, although Latonen et al. [33] assigned this band to the CH2
twisting vibration mode and Rengifo et al. [48] assigned this band to the
CH2 wagging mode. The band at 852 cm− 1 is ascribed to the C–O
stretching mode [Abdelrazek et al. [1], Aydogdu et al. [6], Latonen et al.
[33], Li et al. [37]] but Rengifo et al. [48] ascribed this band to the CH2
twisting vibration mode. A study presented by Wang et al. [55] showed
that PEO chains in the crystalline state adopt a 7/2 helix structure, the
internal rotations of the succession bonds (O–CH2–CH2–O) con­
structing a trans, and gauche conformation. The band assignments for
each peak are presented in Table 1.
4.5. PEO nanofibre release profiles
Fig. 11 shows release profiles of PEO nanofibres (4 wt%, 5 wt%, 6 wt
% and 7 wt%) for 120 min. Upon the placement of the PEO nanofibre in
the PBS (pH 7.5), an initial large bolus of PEO is released before the
release rate reaches a stable profile. All the PEO nanofibres showed the
same burst release pattern and reached a stable release after 15 min. The
study showed that the percentage of the PEO nanofibre release increased
with the increasing PEO concentration. Furthermore, the percentage of
the nanofibre release is also associated with the fibre diameter and
thickness. When the fibre diameter becomes larger, the percentage of
the nanofibre release will be higher. Simultaneously, thicker nanofibres
will have a higher percentage of the nanofibre release rate. The PEO
nanofibres showed the same burst release rate due to the same molecular
weight of the PEO used [Filip and Peer [20], Kim et al. [31], Shojaee
et al. [51], Xu et al. [59]]. In addition, a previous study proved that a
biodegradable polymer demonstrated a high burst release rate [Abid
et al. [2]]. PEO is a biodegradable polymer with a glass transition
temperature (Tg) of approximately − 50∘C. It promises extensive chain
flexibility and is easily dissolved in organic solvent materials [Polaskova
9
Biomaterials Advances 143 (2022) 213178
et al. [42]]. Meanwhile, 4 wt% PEO nanofibre has a lower burst release
at minute 5 than others due to its morphology (beading formation).
4.6. Cell culture
The MCF-7 cells were grown in DMEM media supplemented with
antibiotics and FBS. It was maintained for two passages preceding
treatment with PEO nanofibres (Fig. 12).
4.7. Cell proliferation assay
Based on the nanofibre morphology characterisation results, three
samples of PEO nanofibre concentrations (5 wt%, 6 wt%, and 7 wt%)
were selected to proceed with the cytotoxicity test of MCF-7 cell lines.
The PEO nanofibres' influence on cell growth and viability was exam­
ined for 24 h in different dose concentrations (0–1000 μg/ml) of PEO
nanofibre solutions on MCF-7 cells. Dose-response curves with different
PEO concentrations, which are 5 wt% PEO, 6 wt% PEO and 7 wt% PEO
nanofibres, are provided (Fig. 13).
As shown in Fig. 13a, the cells were treated with <100 μg/ml for all
three nanofibre concentrations. There are three different patterns of cell
viability for each concentration of the PEO nanofibre. The viability of
the MCF-7 cells was maintained with 5 wt% PEO nanofibre. However,
the MCF-7 cells were able to proliferate in 6 wt% PEO nanofibre. In
contrast, proliferation was reduced with 7 wt% PEO nanofibre. The
following experiment treated cells with >100 μg/ml. The viability of
MCF-7 cells was still maintained even with a high dose concentration for
the 5 wt% PEO nanofibre formulation (Fig. 13b). Similarly, the MCF-7
cells exhibited proliferation with a 6 wt% PEO nanofibre formulation.
On the other hand, the viability of the MCF-7 cells was lower with the 7
wt% PEO nanofibre formulation at 100 μg/ml compared with 1000 μg/
ml.
Findings suggest that the results interpret that all PEO nanofibre
M. Jamil et al. Biomaterials Advances 143 (2022) 213178

Fig. 13. Dose-response curve with different poly(ethylene) oxide (PEO) concentrations in the MCF-7 cells: (a) Lower dose concentration from 10 to 100 μg/ml and
(b) Higher dose concentration from 100 to 1000 μg/ml. All experiments were performed in triplicates (n = 3). The asterisk (*) indicates significance p < 0.05–0.01,
(**) indicates significance p < 0.01–0.001 and (***) indicates significance p < 0.000.

formulations were non-toxic to the MCF-7 cells. Despite reduced cell

Table 2
growth with 7 wt% PEO nanofibre formulation, the viability of the MCF-
Total number of colonies after treatment with 10 μg/ml of PEO nanofibre
7 cells did not reach the 50 % inhibition of cell viability. Hence, the IC50
formulations.
values for all the PEO nanofibres were not achieved. It indicates the
suitability of the nanofibre formulation for diagnostic purposes. Treatment The average number of Percentage of a colony
colonies (%)
As a carrier in nanofibre, PEO should be non-toxic to the cells. Recent
studies showed that poly(ethylene) glycol (PEG) induces cell viability Untreated cells 23 ± 2 –
5 wt% PEO 25 ± 1 108
dependent on the PEG molecular weight and concentration. It is sug­
nanofibre
gested that this polymer can be a therapeutic agent [Postic and Shear­ 6 wt% PEO 37 ± 2 160
down [43]]. On the other hand, a previous study also reported that PEG nanofibre
derivatives are almost non-toxic to the human cervical cancer cells 7 wt% PEO 10 ± 1 43.5
(HeLa) and cell line of fibroblast-derived from mice (L929) but moder­ nanofibre

ately toxic at high concentrations [Liu et al. [38]]. In the present study,
the 5 wt% PEO nanofibre formulation was chosen as a base polymer or
carrier due to its non-cytotoxic characteristics in the MCF-7 cells.
The statistical analysis was performed using one-way ANOVA. The
results indicate a significant difference between all PEO nanofibres,
where p = 0.000 (p < 0.05). Paired t-test within doses was performed in
each PEO nanofibre normalised to the negative control (MCF-7 cells).
There was no significant difference within doses for 5 wt% PEO nano­
fibre in lower dose concentrations. Meanwhile, there were significant
differences between each dose for 6 wt% PEO and 7 wt% PEO nanofibres
for lower dose concentrations. Compared to the higher dose concen­
trations, there were significant differences between 700 μg/ml and 800
μg/ml for 5 wt% PEO nanofibre, where p = 0.006 and p = 0.009,
respectively. There are also significant differences between 500 μg/ml,
900 μg/ml, and 1000 μg/ml for 6 wt% PEO nanofibre, where p = 0.002,
p = 0.016 and p = 0.039, respectively. Additionally, there were signif­
icant differences between 200 μg/ml, 400 μg/ml, 700 μg/ml, and 900
μg/ml for 7 wt% PEO nanofibre, where p = 0.005, p = 0.026, p = 0.001
and p = 0.034, respectively.
4.8. Colony formation assay
A colony formation assay was performed to determine the effects of
PEO nanofibre formulation on cancer progress characteristics. It is
assessed by the number of colonies formed. In this experiment, 10 μg/ml
of each PEO nanofibre formulation was chosen as a safe dose to proceed
with the colony assay experiment. A total number of colonies was
counted after 9 days of incubation with a 10 μg/ml dose from different
PEO nanofibre concentrations (Table 2).
The results indicated that the 5 wt% PEO nanofibre gave a slightly
similar number of colonies formed in 9 days of incubation, which was 25
compared to the negative control (p < 0.05). The highest number of
colonies noted after 9 days of incubation was 37 with 6 wt% PEO
nanofibre formulation, followed by 10 colonies with 7 wt% PEO nano­
fibre formulation. It proves that the 5 wt% PEO nanofibre is the optimal
concentration for use in diagnosis as it does not induce the number of
colonies compared to the 6 wt% PEO nanofibre formulation. However,
the number of colonies was significantly reduced with the 7 wt%

10
M. Jamil et al.
Fig. 14. A paired t-test (between nanofibre samples) normalised to negative control MCF-7 cells. All experiments were performed in triplicates (n = 3). Asterisk (*)
indicates significance p < 0.05–0.01 and (**) indicates significance p < 0.01–0.001.
nanofibre formulation.
There were significant differences between all nanofibres when
normalised to the negative control (MCF-7 cells), as shown in Fig. 14.
The significant difference for 5 wt% PEO nanofibre was p = 0.020, fol­
lowed by p = 0.021 for 6 wt% PEO nanofibre and p = 0.006 for 7 wt%
PEO nanofibre.
5. Conclusion
In summary, PEO nanofibres using the electrospinning technique
were successfully synthesised and fabricated. The polymer concentra­
tion was correlated to the solution viscosity is vital in producing bead­
less nanofibrous mat. As the PEO concentration increased, the nanofibre
diameter and thickness also increased. Conversely, the surface rough­
ness of the nanofibre was higher when the PEO concentration was lower.
PEO nanofibres showed the same burst release pattern and rate due to
the same molecular weight of PEO with a stable release rate profile after
15 min. It also demonstrated that the percentage of PEO nanofibre
release increased with the increasing PEO concentration due to the fibre
diameter and thickness. PEO nanofibres are considered non-toxic to the
MCF-7 cells based on the cell proliferation assay results, and the values
of IC50 were not achieved. Additionally, the IC50 value is not required for
diagnostic purposes. In conclusion, our findings suggest that the 5 wt%
PEO nanofibre is the optimal nanofibre concentration as it exhibited
non-cytotoxic characteristics by maintaining the cell viability and did
not induce the number of colonies, therefore was suggested as a suitable
base polymer matrix or carrier with potential use for diagnostic pur­
poses. The findings in this study have demonstrated the influence of cell
growth and viability, including the effects of PEO nanofibre formula­
tions on cancer progress characteristics to achieve a permissible PEO
nanofibre concentration limit that can be a benchmark in medical ap­
plications, particularly diagnostic applications.
CRediT authorship contribution statement
Munirah Jamil: conception of the study, data curation, formal
analysis, methodology, investigation, and writing (review and editing).
Iskandar Shahrim Mustafa: conception of the study, investigation,
funding acquisition, project administration, investigation, and supervi­
sion. Naser Mahmoud Ahmed: electrospinning acquisition, investiga­
tion, and supervision. Shahrul Bariyah Sahul Hamid: conception of cell
culture study, formal analysis, investigation, cell culture administration,
and supervision. All authors have approved the final version of this
manuscript.
Declaration of competing interest
The authors declare no competing interests.
11
Biomaterials Advances 143 (2022) 213178
Data availability
Data will be made available on request.
Acknowledgements
The authors are grateful for the research facilities provided by Uni­
versiti Sains Malaysia (USM). The Ministry of Higher Education
Malaysia funded this research under the Fundamental Research Grant
Scheme with project code FRGS/1/2019/STG07/USM/02/19 and
project ID 17443 (203/ PFIZIK/6711769). The authors would like to
thank the School of Physics, USM, who sponsored the tuition fees and
ZAWAIN USM for financially supporting the author's study for two se­
mesters (Semester I and II Session 2019/2020). The authors also express
their gratitude to the School of Physics (Mdm. Aznorhaida Ramli, Mr.
Mohd Rizal Mohamad Rodin, Mr. Hazhar Hassan, Mdm. Ee Bee Choo,
Mr. Yushamdan Yusof, Mdm. Mahfuzah Mohamad Fuad, Mr. Abdul
Jamil Yusuf, and Thair Hussien Khazaalah), and Advanced Medical and
Dental Institute (Auni Fatin Abdul Hamid, Nur Wahida Zulkifli, and Siti
Aishah Abu Bakar) for their valuable advice and assistance in this
research.
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