IJBIOMAC D 21 03969 - Reviewer

International Journal of Biological Macromolecules
Design, optimization and in vitro characterization of novel biomimetic and bioactive

chitosan/polyethylene oxide nanofibers as wound dressings
--Manuscript Draft--
Manuscript Number:
Article Type: Research Paper
Section/Category: Carbohydrates, Natural Polyacids and Lignins
Keywords: chitosan, nanofibers, biocompatibility, biological effects, Wound dressings
Abstract: Although chitosan based nanofibers (CS-NFs) are excellent artificial extracellular
matrices (ECMs), due to the resemblance of CS with the glycosaminoglycans of the
natural ECMs, the poor electrospinnability and mechanical properties of CS are
responsible for important limitations in respect with its biomedical applications. In order
to improve its physico-chemical properties, new bioactive and biomimetic CS based
nanofibers, having incorporated different active components (ACs), with beneficial
effects for healing, were formulated. SEM morphology revealed well-alignment,
unidirectional arrays, with small diameters, no beads and smooth surfaces
for CS/PEO-ACs NFs. The most suitable for wound healing applications seems to be
CS-PEO@P_C which showed improved hemolysis index (2.92±0.16%), is non-toxic
(cell viability degree more than 97%) and has also significant radical scavenging effect
(DPPH inhibition more than 65%). In addition, CS-PEO@P_C presents increased
antimicrobial effects, especially for Staphylococcus aureus, which is an important
feature, knowing that wound infection leads to delaying the healing process.
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Response to Reviewers
Dear Sir,
Thank you for your time and recommendations. All changes in the manuscript were highlighted (in red
color). The answers to recommendation of Editor are following:
Nr. Editor recommendations Author’s answers
1. The word "novel" is The word “novel” was eliminated from the title
unacceptable in the title since The title: Design, optimization and in vitro characterization of
only novel investigations are novel biomimetic and bioactive chitosan/polyethylene oxide
accepted by Carbohydrate nanofibers as wound dressings
Polymers. was changed as:
Design, preparation and in vitro characterization of biomimetic and
bioactive chitosan/polyethylene oxide based nanofibers as wound
dressings
2. 2.1. There is a vast literature of All recommended articles were included in Introduction and
electrospun chitosan/PEO fiber Discussion sections and were proper reviewed, highlighting the
that you do not acknowledge in progress of our work, as follow:
the introduction or the 1. Introduction
discussion (see example of Biomedical research focused on wound care management is
some of this literature below). constantly seeking new options with respect to promoting the
This literature must be healing process (Halstead et al., 2015) and minimizing therapy
adequately reviewed with costs (Hamdan et al., 2017). Normally, wound healing involves
discussion of how your four phases, haemostasis, inflammation, formation of granulation
investigation provides tissue and remodeling (Fazil & Nikhat, 2020). Various factors
advancement over the prior increase the risk of wound closure delay (Stejskalova & Almquist,
endeavors. 2017). Among these, systemic factors such as diabetes, smoking,
Bhattarai, N., et al., malnutrition and the age of the patient, are considered major risk
Electrospun chitosan-based factors (Das et al., 2020). Impairment in the inflammatory phase,
nanofibers and their cellular due to leakage of superoxide, hydrogen peroxide or hydrochloric
compatibility. Biomaterials, acid from neutrophil cells to the wound site, contributes to a delay
2005. 26(31): p. 6176-6184. in healing. In addition, malfunction of glucose metabolism at this
Desai, K., et al., stage leads to cytotoxicity and normal wound healing becoming
Morphological and Surface prolonged (Oh et al., 2021). Moreover, there are local factors, such
Properties of Electrospun as moisture, ischemia and/or necrosis of the tissue and oedema,
Chitosan Nanofibers. which are involved in healing (Shah & Amini-Nik, 2017).
Biomacromolecules, 2008. Although several wound dressings materials have already been
9(3): p. 1000-1006. developed, a proper wound healing is far from being easily solved.
Doshi, J. and D.H. Reneker, It is known that natural extracellular matrices (ECMs) are
Electrospinning process and composed of two types of macromolecules: proteoglicans and
applications of electrospun fibrous proteins with diameter ranging 50 nm to 150 nm (Bhattarai
fibers. Journal of et al., 2005). Studies have also shown that cells attach and
Electrostatics, 1995. 35(2): p. proliferate well in micro and nanostructured materials (Varnaite-
151-160. Žuravliova et al., 2019).
Duan, B., et al., Novel wound dressing such as electrospun nanofibers (NFs) bring
Electrospinning of chitosan the benefit of enhancing tissue formation (Wang et al., 2018). NFs
solutions in acetic acid with are artificial ECMs (Boateng & Cantazano, 2015) and show two
poly(ethylene oxide). Journal key properties: a high surface/volume ratio and high porosity -
1
of Biomaterials Science, properties which are responsible to promote cellular adhesion,
Polymer Edition, 2004. 15(6): proliferation and differentiation (Barbosa et al., 2017; Farkhani et
p. 797-811 al., 2014; Jang et al., 2009). The design of NFs offers a wide range
Fuh, Y.K., S.Z. Chen, and Z.Y. of prospects with respect to polymer and active components (ACs)
He, Direct-write, highly selection (Weng & Xie, 2015). Blends of natural and synthetic
aligned chitosan-poly(ethylene polymers are suitable to overcome low spinnability of pure natural
oxide) nanofiber patterns for polymers (Hassiba et al., 2017).
cell morphology and spreading Chitosan (CS) is a linear polycation, nontoxic and biodegradable
control. Nanoscale Research polysaccharide - a derivative of chitin, which is extracted from the
Lettera, 2013. 8(97). exo-skeleton of the shell of crustaceae or fungi (Azuma et al.,
García-López, E., D. Olvera- 2014; Desai et al., 2008). It is composed of beta-(1-4)-linked D-
Trejo, and L.F. Velásquez- glucosamine and N-acetyl-D-glucosamine units and the D-
García, 3D printed multiplexed glucosamine content is dependent on the degree of deacetylation of
electrospinning sources for CS (Iacob et al., 2018; Desai et al., 2008).
large-scale production of CS acts in wound healing, tissue restoration and, in addition
aligned nanofiber mats with displays antibacterial (Wei et al., 2020; Kenawyet et al., 2019; Fuh
small diameter spread. et al., 2013), antioxidant (Anraku et al., 2018), hemostatic and
Nanotechnology, 2017. 28(42): immunomodulatory properties (Klossner et al., 2008). During the
p. 425302 depolymerization process, the N-acetyl-D-glucosamine initiates
Grkovic, M., et al., fibroblast proliferation and promotes hyaluronic acid synthesis at
Improvement of mechanical the wound site (Schuerer et al., 2018). The molar mass of CS
properties and antibacterial directs its antimicrobial mechanism. Indeed, a low-molar mass CS
activity of crosslinked (LMW-CS) can enter the microbial/fungal cell and attach itself to
electrospun chitosan/poly the DNA and suppress protein synthesis. A high molar mass CS
(ethylene oxide) nanofibers. (HMW-CS) triggers cell leakage by covering the cell wall (Garcia
Composites Part B: et al., 2019).
Engineering, 2017. 121: p. 58- Chitosan based nanofibers (CS-NFs) have already been described
67 as artificial ECMs due to the resemblance of CS and the
Klossner, R.R., et al., glycosaminoglycans of the natural ECMs (Gadkari et al., 2019;
Correlation of Chitosan’s Huang et al., 2019).
Rheological Properties and Its The electrospinnability of CS is limited, mainly because of its
Ability to Electrospin. polycationic nature, specific inter- and intra-molecular interactions
Biomacromolecules, 2008. and high viscosity of polymeric solution, which prevents the free
9(10): p. 2947-2953. movement of polymeric chain segments exposed to the electrical
Kriegel, C., et al., field, leading to jet break up during the electrospinning (Aduba
Electrospinning of chitosan– &Yang, 2017; Desai et al., 2008; Klossner et al., 2008). Because
poly(ethylene oxide) blend CS has poor electrospinning properties by itself, CS-copolymer
nanofibers in the presence of nanofibers are preffered CS-NFs (Aduba &Yang, 2017).
micellar surfactant solutions. In order to increase the spinnality of CS, different parameters were
Polymer, 2009. 50(1): p. 189- have been studied such as: polymer concentration, polymer
200. molecular weight, deacetylation degree, type and concentration of
Matsumoto, H., et al., the solvents (trifluoroacetic acid, acetic acid) and the use the
Characterization of chitosan crosslinking agents (poly(ethylene oxide) (PEO), poly(vinyl)
nanofiber fabric by alcohol (PVA)) (Klossner et al., 2008).
electrospray deposition: PEO is an amphiphilic, water-soluble and biocompatible, spinnable
Electrokinetic and adsorption polymer (Doshi & Reneker, 1995) which is often used in the
2
behavior. Journal of Colloid electrospinning process of CS (Abid et al., 2019; Ahire et al., 2017;
and Interface Science, 2007. Kuntzler et al., 2018), expecially the ultrahigh molecular weight
310(2): p. 678-681. PEO (Yuan et al., 2012). PEO solutions have already been
Mengistu Lemma, S., F. electrospun by themselves (Doshi & Reneker, 1995) and with the
Bossard, and M. Rinaudo, help of a 3-D printer that generated aligned PEO nanofibers
Nanofibers by Electrospinning (Garcia-Lopez et al., 2017).
in the Presence of To obtain better fibrous structure at high CS-PEO ratio, which is
Poly(ethylene oxide). Int. J. often required for tissues engineering applications, different
Mol. Sci., 2016. 17: p. 1790. surfactants (anionic, cationic, nonionic) and solvents (acetic acid,
Naseri, N., et al., Porous citric acid, succinic acid) were studied to increase the spinnality
electrospun nanocomposite rate and to obtain highly aligned CS-NFs (Bhattarai et al., 2005).
mats based on chitosan– Previous studies developed small diameter CS-PEO NFs, with the
cellulose nanocrystals for help of Triton X-100 (Bhattarai et al., 2005), citric acid (Grkovic et
wound dressing: effect of al., 2017), succinic acid (Stie et al., 2019), micellar surfactants
surface characteristics of (Kriegel et al., 2009, Ziani et al., 2011), genipin (Naseri et al.,
nanocrystals. Cellulose, 2015. 2015) or dimethyl sulfoxide (DMSO) (Fuh et al., 2013, Zhang et
22: p. 521-534. al., 2008).
Pakravan, M., M.-C. Heuzey, When designing the electrospinning experiment, parameters vary
and A. Ajji, A fundamental for every device used and must be adjusted accordingly (Stie et al.,
study of chitosan/PEO 2019, Varnaitė-Žuravliova et al., 2019). In case of CS-PEO NFs,
electrospinning. Polymer, their fabrication may occur for example at low flow rates (0.1
2011. 52(21): p. 4813-4824 mL/h) (Duan et al., 2004), by using a device that sprays the
Stie, M.B., et al., Acids polymer solution through a multicapillary (Matsumoto et al., 2007)
‘generally recognized as safe’ or at moderate temperature (40°C-70°C) (Pakravan et al., 2011).
affect morphology and Morphological characterization proved that CS-PEO nanofibers
biocompatibility of electrospun differ when the solution is produced by blending the two solutions
chitosan/polyethylene oxide of separately dissolved CS and PEO or by dissolving PEO into the
nanofibers. Carbohydrate CS solution (Lemma et al., 2016).
Polymers, 2019. 215: p. 253- The association of CS with different ACs is of benefit due to CS’s
262. properties as potential biological action enhancer (Smith et al.,
Varnaitė-Žuravliova, S., et al., 2004, Rosenthal et al., 2012)
Electrospinning of Chitosan Honey is a natural derivative with trophic and antibacterial effects,
Biopolymer and Polyethylene reactive oxygen species (ROS) scavenging activity and increased
Oxide Blends. AUTEX osmotic gradient (Sarkaret al., 2018). It also reduces inflammation,
Research Journal, 2019. absorbs excessive exudate therefore avoiding maceration, promotes
Yuan, H., et al., Stable jet angiogenesis, granulation tissue production, wound contraction and
electrospinning for easy epithelialization (Ruckriemen et al., 2017). Honey consists of water
fabrication of aligned ultrafine (20%), fructose (40%), glucose (30%), sucrose (5%), and other
fibers. Journal of Materials substances (minerals, vitamins, amino acids and enzymes) (Zekry
Chemistry, 2012. 22(37): p. et al., 2020). Manuka honey’s antibacterial effect is especially due
19634-19638. to its most important constituent, being methylglyoxal (Yang et al.,
Zhang, Y., et al., Electrospun 2017).
biomimetic nanocomposite Propolis is a resin made by bees with important biological effects
nanofibers of based on its chemical composition which can vary depending on
hydroxyapatite/chitosan for the local flora, climate condition, weather and the presence of other
bone tissue engineering. components, such as wax or pollen. The main chemical compounds
3
Biomaterials, 2008. 29(32): p. of biological interest are flavonoids, phenolic acids and their esther
4314-4322. derivatives. Propolis is a valuable candidate for treating wounds,
Zhang, Y.Z., et al., Chitosan given its antioxidant, anti-inflammatory, antimicrobial and
Nanofibers from an Easily antifungal properties. Data from literature describe the benefits of
Electrospinnable UHMWPEO- propolis-incorporating NFs in treating different chronic wounds
Doped Chitosan Solution such as leg ulcers (Liu et al., 2019; Patil et al., 2015).
System. Biomacromolecules, Calendula officinalis is used as infusion, tincture or ointment on
2008. 9(1): p. 136-141. skin inflammations, wounds, superficial burns, leg ulcers and insect
Ziani, K., et al., Effect of bites (Agatonovic-Kutrin et al., 2015). Its curative properties are
nonionic surfactant and acidity due to the presence of flavonoids and saponins (Nicolaus et al.,
on chitosan nanofibers with 2017; Rad et al., 2019).
different molecular weights. Insulin is a peptide hormone and growth factor which reduces
Carbohydrate Polymers, 2011. blood glucose levels and promotes healing of damaged skin (Oryan
83(2): p. 470-476. et al., 2018). If applied on wounded skin, insulin leads to faster re-
epithelialization and contraction of the wound by promoting the
production of granulation tissue (Emanuelli et al., 2016).
L-arginine is a basic alpha-amino acid which acts as a precursor of
nitric oxide (NO) (Debats et al., 2009), a signal molecule involved
in collagen synthesis, epithelialization and formation of granulation
tissue, which are essential processes for wound healing (Bennacef-
Heffar et al., 2017). In addition, NO regulates the immune
responses and angiogenesis (Iacob et al., 2018). At the wound site,
a low concentration of L-arginine is present due to the high
prevalence of arginase, an enzyme which hydrolyzes L-arginine to
urea and L-ornithine (Rieger, 2016).
In this work bioactive and biomimetic NFs were prepared via
conventional electrospinning of a blend containing CS (medium
molecular) and PEO (high molecular weight) in ratio of 2:1 in
acetic acid 50%, in which were incorporated Manuka honey,
propolis, Calendula officinalis, insulin and L-arginine as ACs with
beneficial effect for wound healing. The successful incorporation of
the ACs into the CS-based nanoscaffolds was proved by IR spectral
analysis. Well-alignment, unidirectional arrays, with small
diameters, no beads and smooth surfaces NFs were obtained, which
can work as artificial ECMs and so be proper for tissue engineering
applications. The CS/PEO-ACs NFs showed a good
biodegradability, increased hemocompatibility and reduced
cytotoxicity degree. In addition, good antioxidant and antimicrobial
effects were noted for developed NFs which make them suitable,
especially, for chronic wounds, being known the role of oxidative
stress and infection risk in delaying healing.
2. Materials and methods

2.3. Electrospinning process of CS-PEO-ACs solutions
The nanofibrous scaffolds were fabricated via conventional
electrospinning technique (Doshi & Reneker, 1995; Varnaite-
4
Žuravliova et al., 2019), using Nanospinner Inovenso device
equipped with an infusion pump, generator and a plate collector as
a negative electrode. A syringe of 5 mL with 23-gauge needles
served as positive electrode. The parameters were constant during
the experiments, with a tip-to-collector distance of 25 cm, an
infusion rate of 0.2 to 0.6 mL/h and a voltage below 22 kV (Table
1). The counter electrode of the setup was a grounded collector.
3. Results and Discussion
3.1. Characterization of the CS-PEO-ACs solutions
…………………..
The viscosity of the polymeric solutions depends on the
intermolecular interactions occurring between the polymeric
chains, thereby influencing the electrospinnability of the solution
and the morphology of the resulting NFs. It is directly related to
concentration, molecular weight, and the structure of the polymer
as well as solvent type (Ziani et al., 2011). During electrospinning,
the elongation of the jet takes place at very high shear rates.
………………………………
3.2. Characterization of the CS-PEO-NFs

3.2.1. SEM morphology and fiber diameter
The diameter and orientation of NFs in fibrous scaffolds is one of
the most important features for tissue engineering applications,
because the fiber orientation influences the cell growth and related
functions (Varnaite-Žuravliova et al., 2019). Well-alignment,
unidirectional NFs arrays, with small diameters, no beads and
smooth surfaces were observed for the developed NFs, probably
due of the physical characteristics of the used ACs. It was noted
that the average diameter increased with the addition of ACs into
the CS-PEO solutions (Fig. 1).
…………………………………..
3.6. In vitro cytotoxicity assay

The cell viability (%) recorded at all tested concentrations (12.5
g/mL, 25 g/mL, 50 g/mL, 100 g/mL) exceed 90% (Fig. 4),
which means that CS-PEO-NFs are noncytotoxic, in agreement
with ISO 10993-5 (Standardization, 2009). At 100 g/mL, the
lowest cell viability value was recorded for CS_PEO@M_L
(88±2.30). Our results are in agreement with previouly published
data on CS NFs including honey (Zekry et al., 2020).
………………………….
3.7. In vitro antioxidant assays

……………………
Our results were even better than other studies that reported the
DPPH scavenging activity of propolis/cellulose
5
acetate/polycaprolactone nanofibrous mats (Khoshnevisan et al.,
2019) and polyamide-6/propolis electrospun fibers (Razavizadeh &
Niazmand, 2020).
……………………….
3.2.8. Antimicrobial assay

Agar disk diffusion method is often used as a means of determining
the antimicrobian effects of NFs (Grkocic et al., 2017). Most of
reported studies use only two bacterial strains, Staphylococcus
aureus and Escherichia coli, which mean a limited view of
antibacterial effects (Tian et al., 2016, Ullah et al., 2020). Our study
included also Pseudomonas aeruginosa, a multi-drug resistant
Gram negative bacteria, associated with severe nosocomial
infections, and Candida albicans, a fungal strain. The antimicrobial
effects of the tested CS-PEO-NFs, expressed as diameters of the
inhibition area (mm) are presented in Table 4.
…………….
It was noted that the samples showed good antibacterial effects, in
most cases with inhibition diameter higher than 24 mm, much
better than other studies have reported (Tian et al., 2016, Tang et
al., 2019, Eskandarinia et al., 2020). For example the NFs including
honey, developed by Tang et al. (2019), displayed a diameter of
inhibition area of up 11.3 mm for Escherichia coli and up to 13.6
mm for Staphylococcus aureus. In our study the sample including
Manuka honey (CS_PEO@M_L, CS_PEO@M_L_P) display
improved inhibition diameter for both bacterial strains (24 mm/25
mm for Staphylococcus aureus and 25 mm/20 mm for Escherichia
coli).
……………………
2.2. Your active compounds The term "encapsulated" was changed with "incorporated"
are not "encapsulated" into the
PEO/chitosan nanofiber but
rather they are "incorporated".
2.3. PEO/chitosan nanofibers More evidencs of novelty and the significance of the selected
have been used previously to compounds and of the our work were included:
deliver active compounds, ………………
relative to this prior work you Honey is a natural derivative with trophic and antibacterial effects,
need to provide evidence of reactive oxygen species (ROS) scavenging activity and increased
novelty and significance of the osmotic gradient (Sarkaret al., 2018). It also reduces inflammation,
compounds you have selected absorbs excessive exudate therefore avoiding maceration, promotes
angiogenesis, granulation tissue production, wound contraction and
epithelialization (Ruckriemen et al., 2017). Honey consists of water
(20%), fructose (40%), glucose (30%), sucrose (5%), and other
substances (minerals, vitamins, amino acids and enzymes) (Zekry
et al., 2020). Manuka honey’s antibacterial effect is especially due
6
to its most important constituent, being methylglyoxal (Yang et al.,
2017).
Propolis is a resin made by bees with important biological effects
based on its chemical composition which can vary depending on
the local flora, climate condition, weather and the presence of other
components, such as wax or pollen. The main chemical compounds
of biological interest are flavonoids, phenolic acids and their esther
derivatives. Propolis is a valuable candidate for treating wounds,
given its antioxidant, anti-inflammatory, antimicrobial and
antifungal properties. Data from literature describe the benefits of
propolis-incorporating NFs in treating different chronic wounds
such as leg ulcers (Liu et al., 2019; Patil et al., 2015).
Calendula officinalis is used as infusion, tincture or ointment on
skin inflammations, wounds, superficial burns, leg ulcers and insect
bites (Agatonovic-Kutrin et al., 2015). Its curative properties are
due to the presence of flavonoids and saponins (Nicolaus et al.,
2017; Rad et al., 2019).
Insulin is a peptide hormone and growth factor which reduces
blood glucose levels and promotes healing of damaged skin (Oryan
et al., 2018). If applied on wounded skin, insulin leads to faster re-
epithelialization and contraction of the wound by promoting the
production of granulation tissue (Emanuelli et al., 2016).
L-arginine is a basic alpha-amino acid which acts as a precursor of
nitric oxide (NO) (Debats et al., 2009), a signal molecule involved
in collagen synthesis, epithelialization and formation of granulation
tissue, which are essential processes for wound healing (Bennacef-
Heffar et al., 2017). In addition, NO regulates the immune
responses and angiogenesis (Iacob et al., 2018). At the wound site,
a low concentration of L-arginine is present due to the high
prevalence of arginase, an enzyme which hydrolyzes L-arginine to
urea and L-ornithine (Rieger, 2016).
In this work bioactive and biomimetic NFs were prepared via
conventional electrospinning of a blend containing CS (medium
molecular) and PEO (high molecular weight) in ratio of 2:1 in
acetic acid 50%, in which were incorporated Manuka honey,
propolis, Calendula officinalis, insulin and L-arginine as ACs with
beneficial effect for wound healing. The successful incorporation of
the ACs into the CS-based nanoscaffolds was proved by IR spectral
analysis. Well-alignment, unidirectional arrays, with small
diameters, no beads and smooth surfaces NFs were obtained, which
can work as artificial ECMs and so be proper for tissue engineering
applications. The CS/PEO-ACs NFs showed a good
biodegradability, increased hemocompatibility and reduced
cytotoxicity degree. In addition, good antioxidant and antimicrobial
effects were noted for developed NFs which make them suitable,
especially, for chronic wounds, being known the role of oxidative
7
stress and infection risk in delaying healing.
2.4. Statistical significance The statistical significance was included all comparisons.
should be included in any
comparisons.
2.5. For acceptance by The comparationa between our results and other studies were
Carbohydrate Polymers, this performed, as follow:
evidence of significance and
novelty would require direct 3. Results and Discussion
comparisons between the 3.1. Characterization of the CS-PEO-ACs solutions
results obtained in your …………………..
investigations and those of The viscosity of the polymeric solutions depends on the
prior studies in terms of intermolecular interactions occurring between the polymeric
"activity", whether chains, thereby influencing the electrospinnability of the solution
antibacterial or other type of and the morphology of the resulting NFs. It is directly related to
activity (antioxidant etc.), as concentration, molecular weight, and the structure of the polymer
well as safety as well as solvent type (Ziani et al., 2011). During electrospinning,
(biocompatibility). the elongation of the jet takes place at very high shear rates.
………………………………
3.2. Characterization of the CS-PEO-NFs

3.2.1. SEM morphology and fiber diameter
The diameter and orientation of NFs in fibrous scaffolds is one of
the most important features for tissue engineering applications,
because the fiber orientation influences the cell growth and related
functions (Varnaite-Žuravliova et al., 2019). Well-alignment,
unidirectional NFs arrays, with small diameters, no beads and
smooth surfaces were observed for the developed NFs, probably
due of the physical characteristics of the used ACs. It was noted
that the average diameter increased with the addition of ACs into
the CS-PEO solutions (Fig. 1).
…………………………………..
3.6. In vitro cytotoxicity assay

The cell viability (%) recorded at all tested concentrations (12.5
g/mL, 25 g/mL, 50 g/mL, 100 g/mL) exceed 90% (Fig. 4),
which means that CS-PEO-NFs are noncytotoxic, in agreement
with ISO 10993-5 (Standardization, 2009). At 100 g/mL, the
lowest cell viability value was recorded for CS_PEO@M_L
(88±2.30). Our results are in agreement with previouly published
data on CS NFs including honey (Zekry et al., 2020).
………………………….
3.7. In vitro antioxidant assays

……………………
Our results were even better than other studies that reported the
8
DPPH scavenging activity of propolis/cellulose
acetate/polycaprolactone nanofibrous mats (Khoshnevisan et al.,
2019) and polyamide-6/propolis electrospun fibers (Razavizadeh &
Niazmand, 2020).
……………………….
3.2.8. Antimicrobial assay

Agar disk diffusion method is often used as a means of determining
the antimicrobian effects of NFs (Grkocic et al., 2017). Most of
reported studies use only two bacterial strains, Staphylococcus
aureus and Escherichia coli, which mean a limited view of
antibacterial effects (Tian et al., 2016, Ullah et al., 2020). Our study
included also Pseudomonas aeruginosa, a multi-drug resistant
Gram negative bacteria, associated with severe nosocomial
infections, and Candida albicans, a fungal strain. The antimicrobial
effects of the tested CS-PEO-NFs, expressed as diameters of the
inhibition area (mm) are presented in Table 4.
…………….
It was noted that the samples showed good antibacterial effects, in
most cases with inhibition diameter higher than 24 mm, much
better than other studies have reported (Tian et al., 2016, Tang et
al., 2019, Eskandarinia et al., 2020). For example the NFs including
honey, developed by Tang et al. (2019), displayed a diameter of
inhibition area of up 11.3 mm for Escherichia coli and up to 13.6
mm for Staphylococcus aureus. In our study the sample including
Manuka honey (CS_PEO@M_L, CS_PEO@M_L_P) display
improved inhibition diameter for both bacterial strains (24 mm/25
mm for Staphylococcus aureus and 25 mm/20 mm for Escherichia
coli).
……………………
3. Please justify why your Based on complex composition of many of the active components
antimicrobial testing did not (ACs) used in our study, such as Manuka honey, propolis,
include minimum inhibitory Calendula officinalis, which have different content of flavonoids,
concentration (MIC) and phenolic acids, carbonyl and esther derivatives, is not possible to
AATCC Test Method 100 express the minimum inhibitory concentration, for which an unitary
"assessment of Antimicrobial compound is necesarry. This was the reason we choose a
Finishes on Textile Materials". qualitative method, based on diamater of inhibtion area. In addition
Zone of inhibition testing has this method was reported by similar studies:
limited usefulness since it 1. Tian, J., Tu, H., Shi, X., Wang, X., Deng, H., Li, B., & Du, Y.
depends on active agent (2016). Antimicrobial application of nanofibrous mats self-
release which diminishes over assembled with chitosan and epigallocatechin gallate. Colloids and
time. Surfaces B: Biointerfaces, 145, 643–652.
https://doi.org/10.1016/j.colsurfb.2016.05.008
2. Tang,Y., Lan, X., Liang, C., Zhong, Z., Xie, R., Zhou, Y., Miao,
X., Wang, H., Wang, W. (2019). Honey loaded alginate/PVA
9
nanofibrous membranes as potential bioactive wound dressing.
Carbohydrate Polymers, 219, 113-129.
https://doi.org/10.1016/j.carbpol.2019.05.004
3. Grkovic, M., Stojanovic, D. B., Pavlovic, V. B., Rajilic-
Stojanovic, M., Bjelovic, M., & Uskokovic, P. S. (2017).
Improvement of mechanical properties and antibacterial activity of
crosslinked electrospun chitosan/poly (ethylene oxide) nanofibers.
Composites Part B: Engineering, 121, 58–67.
https://doi.org/10.1016/j.compositesb.2017.03.024
4. Ullah, A., Ullah, S., Khan, M. Q., Hashmi, M., Nam, P. D., Kato,
Y., Tamada, Y., & Kim, I. S. (2020). Manuka honey incorporated
cellulose acetate nanofibrous mats: Fabrication and in vitro
evaluation as a potential wound dressing. International Journal of
Biological Macromolecules, 155, 479–489.
https://doi.org/10.1016/j.ijbiomac.2020.03.237
10
Response to Reviewers
Dear Editor,
We are very grateful all Reviewers for them evaluation of the quality of our manuscript and them constructive suggestions.
All changes in the manuscript were highlighted (in red color) and the detailed responses are following:
Reviewers’ comments Response to reviewers

Reviewer 1
The abstract should report the main The Abstract was improved including more relevant data, as follow:
results (numbers) and not a description Abstract
of the performed measurements. Although chitosan based nanofibers (CS-NFs) are excellent artificial extracellular matrices (ECMs),
due to the resemblance of CS with the glycosaminoglycans of the natural ECMs, the poor
electrospinnability and mechanical properties of CS are responsible for important limitations in respect
with its biomedical applications. In order to improve its physico-chemical properties, new bioactive
and biomimetic CS based nanofibers, having incorporated different active components (ACs), with
important beneficial effects for healing, were formulated. Manuka honey (trophic and antimicrobial
effects), propolis (antimicrobial effects), Calendula officinalis infusion (antioxidant effect,
reepithelization stimulating agent), insulin (trophic effect) and L-arginine (angiogenetic effect) were
selected as ACs. SEM morphology analysis revealed well-alignment, unidirectional arrays, with small
diameters, no beads and smooth surfaces for developed CS/PEO-ACs NFs. The developed NFs
showed good biodegradability (NFs mats lost up to 60% of their initial weight in PBS), increased
hemocompatibility (hemolytic index less than 4%) and a reduced cytotoxicity degree (cell viability
degree more than 90%). In addition, significant antioxidant and antimicrobial effects were noted for
developed NFs which make them suitable, especially for chronic wounds, being known the role of
oxidative stress and infection risk in delaying healing. The most suitable for wound healing
applications seems to be CS-PEO@P_C which showed improved hemolysis index (2.92±0.16%), is
non-toxic (cell viability degree more than 97%) and has also significant radical scavenging effect
(DPPH inhibition more than 65%). In addition, CS-PEO@P_C presents increased antimicrobial
effects, especially for Staphylococcus aureus, which is an important feature, knowing that wound
infection leads to delaying the healing process. It can be concluded that the developed CS/PEO-ACs
nanofibers are very promising biomaterials for wound care, especially CS-PEO@P_C.
1
Figure 1 should show SEM images at The Fig. 1 was corrected. SEM images at low magnification (1 μm) and at the same magnification,
Page
the same magnification. Moreover, a were included:
wide field SEM images at low
magnification should be presented to
understand the general distribution and
uniformity of the nanofibers.
CS_PEO@I_P
CS_PEO@M_L
2
Page
CS_PEO@P_C CS_PEO@P
Page
3
CS_PEO@M_L_P
CS_PEO
It is stated that "The IR spectra analysis The sentence The IR spectra analysis proved the successful incorporation of the ACs into the CS-based
proved the successful encapsulation". NFs.
Is it proved? The FTIR proved the was rephrased as:
existence of the ACs in the structure of The presence of ACs into the nanofibrous scaffold was proved by IR spectroscopy
the nanofibers but It cannot prove their
4
encapsulation. Encapsulation should
Page
involve a bi-component
electrospinning using a coaxial
configuration. Please rephrase.
Despite the results can show and The sentence was rephrased as follow:
increase in the swelling ability stating Both results, in PBS and PECF, support the capacity of the tested NFs to uptake physiological fluids,
that the tested NFs absorb exudates and which is useful in terms of wound surface interactions, to initiate the delivery of ACs incorporated into
can be useful for wound management polymer matrix and so to promote the biological effects.
is quite speculative since the of
electrospun nanofibers are very thin
films. Very useful for surface
interaction and to promote mass
transfer or antimicrobial effects but not
for exudate adsorption. Please
rephrase.
An antioxidant assay control only with A DPPH antioxidant assay control with ACs and CS-PEO-ACs polymeric solutions was performed
the ACs extracts should be performed and consequently the results and discussion sections were improved as follow:
to prove that the flavonoid content of
Calendula officinalis is responsible for
the observed effect.
Fig. 6. The DPPH antiradical scavenging effect of (a) CS-PEO-ACs polymeric solutions (1:
CS_PEO@I_P, 2: CS_PEO@M_L, 3: CS_PEO@P, 4: CS_PEO@P_C, 5: CS_PEO@M_L_P, 6:
CS_PEO).*p˂0.05, **p˂0.01, when compared to CS_PEO and (b) ACs solution (1: M, 2: L, 3: P, 4:
C).
......................
5
Increased radical scavenging effects were also noted for propolis and Calendulla officinalis infusion,
Page
both as CS-PEO-ACs polymeric (Fig. 6a) and ACs (Fig. 6b) solutions.
The inhibition zones test is not the Thank you for your suggestion.
more appropriate in this context since it We have chosen this method, according to references from literature, in order to compare our results
is not clear which is the contribution of with other ones to prove the improved effects of the developed formulations (please see the table). We
the leaching effect of ACs and CS. The plan to apply also the suggested method because we consider that it will be very useful to compare the
standard shake flask method (ASTM- results.
E2149-01) should be performed. Then, Diameter of inhibition area Refs.
the antibacterial effect should be Polymeric (mm)
reported in log reduction to understand nanofibers/Active
if a bactericidal or bacteriostatic compounds S. E. P. C.
activity is present. aureus coli aeruginosa albicans
Poly (E- 7.6 7.6 ND ND Motealleh et al., J Biomed Mater Res B, 2013,
caprolactone)- 102(5), https://doi.org/10.1002/jbm.b.33078
Polystyren (PCL-
PS/Chamomile
Poly -2- 17 18 ND ND Rammalingam et al., J Biomed Mater Res
Hydroxyethyl A,2014, 103(1), https://doi.org/
Methacrylate 10.1002/jbm.a.35138
(pHEMA)/curcumin
Polyacrylonitrile 12 15 ND ND Fayemy et al., ACS Omega, 2018, 3(5),
(PAN)/Moringa leaf https://doi.org/10.1021/acsomega.7b01981
extract
bacterial 24 17 17 ND Marquele-Oliveira et al., Int J Biol.
celullose/propolis Macromol, 2019, 136,
membrane (BC/PP) https://doi.org/10.1016/j.ijbiomac.2019.05.135
sulphonated 18 28 ND 17 Ekambaram & Dharmalingam,Mat Sci Eng C,
6
Page
polyether ether 2020, 115, https://doi.org/10.1016/j.msec.2020
ketone
(SPEEK)/Aloe vera
CS_PEO/insulin- 26 24 25 25 our study
propolis
CS_PEO/L- 24 25 25 27 our study
arginine-Manuka
Honey
Reviewer 2
- use the word "concentration" The word concentration was replaced with content in whole manuscript
correctly, it is reserved for sample
composition given in mol/L, what is
not in your cases. Therefore use
amount or content of polymers or ACs
or percentage. Corrections are needed
through whole text
- Calendula officinalis is a plant, it is Calendula officinalis was replaced with Calendula officinalis infusion in whole manuscript
not incorporated in nanofibers,
therefore, use through the whole text
Calendula officinalis infusion
- L. 87 CS has polycationic nature and Lines 88-91 were omitted and the corresponding sentences were rephrased:
inter- and intramolecular repulsive Because CS has poor electrospinning properties, mainly because of its polycationic nature, specific
interaction. To improve the inter- and intra-molecular interactions and high viscosity (Aduba &Yang, 2017), blending CS with
spinnability PEO is added to shade other polymers such as poly(ethylene oxide) (PEO), poly(vinyl)alcohol (PVA) or polycaprolactone
them. Lines 88-91 omit (PCL) was useful (Aduba &Yang, 2017).
- L. 99 Sentence "PEO ,,, " omit The sentence PEO solutions have already been electrospun by themselves (Doshi &Reneker, 1995)
and with the help of a 3-D printer that generated aligned PEO nanofibers (Garcia-Lopez et al., 2017),
has been ommited
- L-104-108 "Previous studies ..." omit, The sentence Previous studies developed small diameter CS-PEO NFs, with the help of Triton X-100
7
it is not the subject of this manuscript (Bhattarai et al., 2005), citric acid (Grkovic et al., 2017), succinic acid (Stie et al., 2019), micellar
Page
surfactants (Kriegel et al., 2009, Ziani et al., 2011), genipin (Naseri et al., 2015) or dimethyl sulfoxide
(DMSO) (Fuh et al., 2013, Zhang et al., 2008), has been omitted
- L.218 - Replace "an infusion rate" infusion rate was replace with flow rate
with a solution or dispersion, to
prevent confusion with Calendula
officinalis infusion,
- L. 257 - Absorbtion? water (PBS/PECF) absorbtion capacity was replaced with water (PBS/PECF) uptake capacity
-L. 306-3o9 How were cells incubated? The details about sample preparation were included:
They grew in the present of nanofibers A sample of 100 μg CS-PEO-NFs was incubated with 1 mL alpha-MEM medium, for 72 h at 37 °C,
or what means extract, according to after which the serially dilutions were made to obtain different concentrations (50 µg/mL, 25 µg /mL,
what 12,5 um/mL ....Actuality, the 12.5 µg /mL).
supernatant solution was tested. ……………….
The cells were then incubated for 72 h with 100 μl of previously prepared CS-PEO-NFs samples (12.5
µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL) or alpha-MEM medium (used also as control)
- L. 317 - Sample was immersed in The sentence A sample of 60 mg of the CS-PEO-NFs was immersed in 5 mL of ethanol, after which a
ethanol and mixed ???' for ??? time set volume of the ethanolic extract was used for the antioxidant assay was rephrase as
and then supernatant solution were A sample of 60 mg of the CS-PEO-NFs was mixed with 5 mL ethanol for 1 h, and then the ethanolic
withdrawn. Extract is not usually used supernatant was separate. A set volume of this solution was used for the antioxidant assays.
in such cases. Correct also in L. 339 L 339 was also corrected
- L. 401 - Omit the sentence The sentence Usually, electrospinnable polymeric solutions display conductivities between 0.05 and
"Usually,.." 30 mS. was omitted
- L. 419 - Replace concentration with The sentence It is directly related to concentration, molecular weight, and the structure of the polymer
polymer content , anyhow is this as well as solvent type (Ziani et al., 2011). was omitted
sentence here redundant
- L. L. 493 - 503 - 493 rewrite the The paragraph The lowest values belong to the samples which contain propolis (CS_PEO@P) which
results ... the best biocompatibility has a γSLvalue of 2.08±0.68mN/m. Low values were obtained also for the samples containing propolis
show empty Cs-PEO and samples with and Calendula officinalis infusion (CS_PEO@P_C, γSLvalue of 3.83±0.39 mN/m) and the samples
propolis. Delete the rest of the text, containing Manuka honey (CS_PEO@M_L, γSL value of 2.33±0.34 mN/m; CS_PEO@M_L_P γSL
everthing is seen from the Table 3. values of 2.88±0.54 mN/m).
was rephrased as:
8
The lowest γSL values were recorded for CS_PEO@P (2.08±0.68), CS_PEO@M_L (2.33±0.34) and
Page
CS_PEO@M_L_P (2.88 ± 0.54), which means these polymeric solutions have the best
biocompatibility.
- Improve the readability of the words The readability of the words in the Figures was improved
in the Figures
- Table 4 - I find it hard to trust that We have chosen the aplied antimicrobial method, according to references from literature, in order to
tested natural active compounds are compare our results with other ones to prove the improved effects of the developed formulations
almost equally effective as CIP or (please see the table).
VRC. Please carefully justify the
results! Diameter of inhibition area Refs.
Polymeric (mm)
nanofibers/Active
compounds S. E. P. C.
aureus coli aeruginosa albicans
Poly (E- 7.6 7.6 ND ND Motealleh et al.,J Biomed Mater Res B, 2013,
caprolactone)- 102(5), https://doi.org/10.1002/jbm.b.33078
Polystyren (PCL-
PS/Chamomile
Poly -2- 17 18 ND ND Rammalingam et al., J Biomed Mater Res
Hydroxyethyl A,2014, 103(1), https://doi.org/
Methacrylate 10.1002/jbm.a.35138
(pHEMA)/curcumin
Polyacrylonitrile 12 15 ND ND Fayemy et al.,ACS Omega, 2018, 3(5),
(PAN)/Moringa leaf https://doi.org/10.1021/acsomega.7b01981
extract
bacterial 24 17 17 ND Marquele-Oliveira et al., Int J Biol.
celullose/propolis Macromol, 2019, 136,
membrane (BC/PP) https://doi.org/10.1016/j.ijbiomac.2019.05.135
9
Page
sulphonated 18 28 ND 17 Ekambaram & Dharmalingam,Mat Sci Eng C,
polyether ether 2020, 115, https://doi.org/10.1016/j.msec.2020
ketone
(SPEEK)/Aloe vera
CS_PEO/insulin- 26 24 25 25 our study
propolis
CS_PEO/L- 24 25 25 27 our study
arginine-Manuka
Honey
- The number of cited paper is huge After revision according to the reviewers comments the number of references was shortened.
(95) for research paper.
L.94 -Please omit "crosslinking agents" The sentence was rephrased as follow:
from the sentence ".... and the use of
crosslinking agents (poly(ethylen In order to increase the electrospinnability of CS, different parameters were have been studied such as:
oxide) (PEO), poly (vinyl ...." polymer content, polymer molecular weight, deacetylation degree, type of solvents (trifluoroacetic
acid, acetic acid), ratio polymer/solvent and ratio CS/PEO or CS/PVA (Klossner et al., 2008).
General advice to the authors - include Some sentences were ommited:
only the important text and the Conductivity:
important results in the article, which The conductivity is temperature dependent; therefore, measurements were carried out at the same
meaningfully express and confirm the temperature (23.4°C).
content according to the title. Skip Viscosity:
everything else. During electrospinning, the elongation of the jet takes place at very high shear rates.
For better manuscript, the conductivity, The viscosity of the polymeric solutions with ascending shear rates is divided between a first region
surface free energy and in vitro (solution at rest), a second shear-thinning region (the non-Newtonian behavior of the solution during
antioxidant testing would be much electrospinning) and a third Newtonian region (plateau) seen at very high shear rates, where the
shorter. alignment of polymeric chains is at its maximum.
Reviewer 3
10
In this manuscript, the authors describe The rationale design of the bioactive and biomimetic CS-NFs for wound management started from
Page
the preparation and characterization of having a polymeric matrix, based on CS, that not only protects the wound from external damaging
chitosan/polyethylene oxide agents, yet act through biological effects, CS having antibacterial, antioxidant, hemostatic and
nanofibers, designed for wound healing immunomodulatory properties. The active components chosen to be incorporated into polymer
applications. Authors have chosen scaffold were taken into consideration for their benefits in speeding the healing process. Therefore,
several compounds, with any trophic agents (manuka honey and insulin), antimicrobial agents (propolis, manuka honey), an
relationship to each other, to angiogenetic compound (L-arginine) and an antioxidant/reepithelization stimulating agent (Calendula
investigate the properties that they officinalis infusion) were selected for this study.
confers to a PEO-Ch based polymer
matrix. They should explain and The correponding paragraph was reformulated as follow:
motivate this choice, that risk of In this work, bioactive and biomimetic NFs were prepared via conventional electrospinning of a blend
sounding random. Moreover, it is containing CS (medium molecular weight) and PEO (high molecular weight) in ratio of 2:1 in acetic
particularly interesting to investigate acid 50%, in which were incorporated different active components (ACs) with beneficial effect for
the biological effects of different wound healing. Manuka honey (trophic and antimicrobial effects), propolis (antimicrobial effects),
(natural) components on biological Calendula officinalis infusion (antioxidant effect, reepithelization stimulating agent), insulin (trophic
properties of a material. But in some effect) and L-arginine (angiogenetic effect) were selected as ACs.
cases, it can be equally interesting to
understand the effect that the polymer In our preliminary experiments we investigated different ratio between CS, PEO and solvent in order
matrix has to the embedded additive. In to obtain stable NFs. Only CS:PEO in ratio of 2:1 in acetic acid 50% was succesful. In the table bellow
this case, the investigation has been we present the results of these experiments:
carried out on a unique polymer Needle-
Acetic
formulation of Ch-PEO (1:0,5 %), but Chitosan PEO Feed rate Collector Voltage Observations
acid
for a more complete and in-depth distance
evaluation it might be very interesting 3g/100 mL 1g/100 mL 1 mL/h 25 cm 24 kV 50 % Dripping, large
to evaluate the same properties with diameter fibers
different polymer composition. with beads
2g/100 mL 1g/100 mL 0.9 mL/h 25 cm 22 kV 50% Beads
formation
1g/100 mL 1g/100 mL 0.8mL/h 25 cm 20 kV 70% Dripping
1.5g/100 mL 1g/100 mL 0.7mL/h 25 cm 20 kV 10% Dripping
Highlights should be improved (i.e. The highlights were improved as follow:
 Novel CS/PEO-ACs biomimetic and bioactive nanofibers were designed and successful
11
good biodegradability rate is a too
general statement). prepared via electrospinning
Page
 Manuka honey, propolis, Calendula off., insulin and L-arginine, with beneficial effects for
wound healing were chosed as ACs
 CS/PEO-ACs nanofibers showed increased hemocompatibility (hemolytic index less than 4%),
reduced cytotoxicity degree (cell viability more than 90%), as well as significant antioxidant
and antimicrobial effects
 CS-PEO@P_C is the most promising healing formulation
Page 1, line 29, the sentence has to be The Abstract was improved including more relevant data, as follow:
rewritten ("but difficult electrospinning Although chitosan based nanofibers (CS-NFs) are excellent artificial extracellular matrices (ECMs),
and poor mechanical properties are due to the resemblance of CS with the glycosaminoglycans of the natural ECMs, the poor
important disadvantages"). electrospinnability and mechanical properties of CS are responsible for important limitations in respect
line 32, the acronym ACs has not yet with its biomedical applications. In order to improve its physico-chemical properties, new bioactive
been defined. and biomimetic CS based nanofibers, having incorporated different active components (ACs), with
important beneficial effects for healing, were formulated. Manuka honey (trophic and antimicrobial
effects), propolis (antimicrobial effects), Calendula officinalis infusion (antioxidant effect,
reepithelization stimulating agent), insulin (trophic effect) and L-arginine (angiogenetic effect) were
selected as ACs. SEM morphology analysis revealed well-alignment, unidirectional arrays, with small
diameters, no beads and smooth surfaces for developed CS/PEO-ACs NFs. The developed NFs
showed good biodegradability (NFs mats lost up to 60% of their initial weight in PBS), increased
hemocompatibility (hemolytic index less than 4%) and a reduced cytotoxicity degree (cell viability
degree more than 90%). In addition, significant antioxidant and antimicrobial effects were noted for
developed NFs which make them suitable, especially for chronic wounds, being known the role of
oxidative stress and infection risk in delaying healing. The most suitable for wound healing
applications seems to be CS-PEO@P_C which showed improved hemolysis index (2.92±0.16%), is
non-toxic (cell viability degree more than 97%) and has also significant radical scavenging effect
(DPPH inhibition more than 65%). In addition, CS-PEO@P_C presents increased antimicrobial
effects, especially for Staphylococcus aureus, which is an important feature, knowing that wound
infection leads to delaying the healing process. It can be concluded that the developed CS/PEO-ACs
nanofibers are very promising biomaterials for wound care, especially CS-PEO@P_C.
Page 2, line 56, the statement "normal The sentence In addition, malfunction of glucose metabolism at this stagenleads to cytotoxicity and
wound healing becoming prolonged" normal wound healing becoming prolonged (Oh et al., 2021).
12
has to be rewritten; was rephrased:
In addition, insulin deficiency in diabetes affects the carbohydrate, protein and lipid metabolism and is
Page
responsible for impaired cellular function and tissue synthesis in wound healing (Oh et al., 2021).
Page 2, line 77, please correct the Low/high molar mass was replaced with low/high molecular weight
typing mistake with "high molar mass";
Table 1, the column "polymers" is The table was improved according to the suggestions:
unnecessary, as the ratio Ch/PEO is the
same for all the samples. Same thing CS-PEO-NFs/ Flow-rate
No. ACs
for the "solvent" column, since the CS-PEO-ACs mL/h
same solvent is used for all samples 1 CS_PEO@I_P I: 46 IU/mL, P: 7.5% (wt/v) 0.2
and the details of the only preparation 2 CS_PEO@M_L M: 7.5% (wt/v), L: 7.5% (wt/v) 0.5
that was different are reported in the 3 CS_PEO@P P: 7.5% (wt/v) 0.2
text below the table. The unit of 4 CS_PEO@P_C P: 7.5% wt/v 0.2
measurement can be put in the title of 5 CS_PEO@M_L_P MH: 7.5% (wt/v), L: 7.5% (wt/v), 0.5
column, P: 7.5% (wt/v)
to avoid repeating them next to each 6 CS_PEO - 0.6
value. The whole table is not easy to
read.
Why the authors choose to mix some The active components chosen to be incorporated were taken into consideration for their benefits in
components and not others? The speeding the healing process. Therefore, trophic agents (manuka honey and insulin), antimicrobial
rationale is not clear. agents (propolis, manuka honey), an angiogenetic compound (L-arginine) and an
antioxidant/reepithelization stimulating agent (Calendula officinalis infusion) were selected for this
study.
The correponding paragraph was reformulated as follow:

In this work, bioactive and biomimetic NFs were prepared via conventional electrospinning of a blend
containing CS (medium molecular weight) and PEO (high molecular weight) in ratio of 2:1 in acetic
acid 50%, in which were incorporated different active components (ACs) with beneficial effect for
wound healing. Manuka honey (trophic and antimicrobial effects), propolis (antimicrobial effects),
Calendula officinalis infusion (antioxidant effect, reepithelization stimulating agent), insulin (trophic
effect) and L-arginine (angiogenetic effect) were selected as ACs.
Page 5, line 158, why the solutions The solutions were electrospun immediately after being homogenous, and no later than 48 h.
were electrospun 48h later? Were they The sentence was reformulated as follow:
13
held under stirring all along? This is The solutions were electrospun immediately after becoming homogenous, and no later than 48 h, to
Page
not clear. prevent any damage, such as polymer degradation or aging of the solution.
Page 6, line 216, the sentence has to be In this study we proposed to present the design, preparation and in biological evaluation of developed
re-written. The first result obtained CS-NFs using in vitro assays. As next research we plan to study these nanomaterials in wound model
from the swelling studies is about the to rats and a kinetic study will be also performed to have complete data for biological effects.
absorption ability of material, not about Indeed the kinetic study is very important to understand the behavior of the developed NFs, and we
the drug release. Moreover, in this will do it next.
regard, I consider that the study of the
release kinetics of bioactive The sentence was rephrased as follow:
components is absolutely necessary in Both results, in PBS and PECF, support the capacity of the tested NFs to uptake physiological fluids,
this kind of investigations to better which is useful in terms of wound surface interactions, to initiate the delivery of ACs incorporated into
understand the behavior and the polymer matrix and so to promote the biological effects.
potentialities of prepared materials.
Page 7, line 234, please correct with The correction was made
"quantified".
Page 7, line 241, by analogy, it could The correction was made
be better to indicate with the same code
the weight of material in the dry state
in both swelling and degradation
studies.
Page 10, line 363, the conductivity The values of CS-PEO is presented in the table - 2.28±0.03
value of CS_PEO is not reported in the
table.
Page 11, line 387, please correct with The correction was made
"shown".
It would be particularly interesting to Discussion about interactions between different ACs and polymeric matrix were added, as follow:
exploit the FT-IR analysis to assess the
interaction between the different active In reference with the IR spectrum of CS_PEO, the CS_PEO_ACs NFs presented an intense and large
components and the polymer matrix, absorption band at ̴ 3300 cm-1 and at ̴ 2875 cm-1, due to the interactions between –OH, -NH2 and -
rather than use this technique just to CH2- groups of CS and PEO respectively and the functional groups of ACs, especially -OH , -NH-
demonstrate the presence of a and -NH-CO-. Also, based on hydrogen interactions, a shift of -CH stretching frequencies band from
14
component which is certainly present the ACs-polymer matrix spectrum to shorter wavelengths was observed, when compared with the CS-
in the material thus obtained. By the PEO. A shift of the characteristic absorption bands of insulin (at ̴1650 cm-1 and at ̴ 1540 cm-1) and L-
Page
way, the figure has to be improved. the arginine (at ̴ 1632 cm-1) were also noted in reference with pure insulin (at ̴ 1679 cm-1 and ̴ 1576 cm-
legend behind the curves is not legible 1
) and L-arginine (at ̴ 1632 cm-1) (Liu et al. 2019), also based on interactions between the functional
and it is not easy to identify the groups of CS, PEO and ACs.
described peaks.
The Fig. 2 was improved in order legend to be more legible:
With regard to the swelling plots, in The sweeling results were presented as classic line and symbol curve as follow:
my opinion results could be more
immediately readable with a classic
line+symbol curve, instead of column
chart.
15
Page
Page
16
Sincerely yours,
Prof. Dr. Lenuta Profire

University of Medicine and Pharmacy „Grigore T. Popa” of Iasi
16 University Street
700115, Iasi, Romania
e-mail: lenuta.profire@umfiasi.ro
17
Page
Highlights (for review)
Design, optimization and in vitro characterization of novel biomimetic and

bioactive chitosan/polyethylene oxide nanofibers as wound dressings
Oana Maria Ionescua,, Andreea-Teodora Iacoba,, Arn Mignonb, Sandra Van Vlierberghec,
Mihaela Baicand, Maricel Danue,f, Constanța Ibănescue,f, Natalia Simionescuf,g, Lenuța
Profirea,*
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and
Pharmacy of Iași, 16 University Street, Iasi, Romania
b
Smart Polymeric Biomaterials, Surface and Interface Engineered Materials, Campus Group T, KU Leuven,
Andreas Vesaliusstraat 13, 3000 Leuven, Belgium
c
Polymer Chemistry and Biomaterials Group, Center of Macromolecular Chemistry, Department of Organic and
Macromolecular Chemistry, Ghent University, Krijgslaan 281, S4-bis, 9000 Ghent, Belgium
d
Department of Pharmaceutical Physics, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and
e
Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and Environmental
Protection, “Gheorghe Asachi” Technical University of Iaşi, Mangeron Avenue 73, 700050 Iaşi, Romania
f
“Petru Poni” Institute of Macromolecular Chemistry, Centre of Advanced Research in Bionanoconjugates and
Biopolymers, 41A Grigore Ghica Voda Alley, 700487, Iasi, Romania
g
“Prof. Dr. Nicolae Oblu” Emergency Clinical Hospital, 2 Ateneului Street, 700309, Iasi, Romania
HIGHLIGHTS
 Novel CS/PEO-ACs biomimetic and bioactive nanofibers were designed and
successful prepared via electrospinning
 Manuka honey, propolis, Calendula off., insulin and L-arginine were chosed as ACs in
order to improved the biological effects of CS/PEO nanoscafold
 The successful encapsulation of ACs into CS/PEO nanoscafold was proved by IR-
spectroscopy
 CS/PEO-ACs nanofibers showed good swelling degree, good biodegradable rate,
increased hemocompatibility, reduced cytotoxicity degree, as well as antioxidant and
antimicrobial effects
 CS/PEO-ACs nanofibers are very promising biomaterials for wound care, expecially
CS-PEO@P_C.

bioactive chitosan/polyethylene oxide based nanofibers as wound dressings
Profirea,*
a
b
c
d
e
f
Biopolymers,41A Grigore Ghica Voda Alley, 700487, Iasi, Romania
g
* Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa”
University of Medicine and Pharmacy of Iași, 16 University Street, Iasi, Romania,
E-mail address: lenuta.profire@umfiasi.ro (L.P.).

Contributed equally to this work: Oana Maria Ionescu (O.M.I) and Andreea-Teodora Iacob (A.T.I.)
Keywords: Chitosan Nanofibers, Biocompatibility, Biological effects, Wound dressings
HIGHLIGHTS
 Manuka honey, propolis, Calendula off., insulin and L-arginine, with beneficial effects
for wound healing were chosed as ACs
 CS/PEO-ACs nanofibers showed increased hemocompatibility (hemolytic index less
than 4%), reduced cytotoxicity degree (cell viability more than 90%), as well as
significant antioxidant and antimicrobial effects

bioactive chitosan/polyethylene oxide based nanofibers as wound dressings
Profirea,*
a
b
c
d
e
f
Biopolymers,41A Grigore Ghica Voda Alley, 700487, Iasi, Romania
g
* Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa”
University of Medicine and Pharmacy of Iași, 16 University Street, Iasi, Romania,
E-mail address: lenuta.profire@umfiasi.ro (L.P.).

Contributed equally to this work: Oana Maria Ionescu (O.M.I) and Andreea-Teodora Iacob (A.T.I.)
Keywords: Chitosan Nanofibers, Biocompatibility, Biological effects, Wound dressings
HIGHLIGHTS
 Manuka honey, propolis, Calendula off., insulin and L-arginine, with beneficial effects
for wound healing were chosed as ACs
 CS/PEO-ACs nanofibers showed increased hemocompatibility (hemolytic index less
than 4%), reduced cytotoxicity degree (cell viability more than 90%), as well as
significant antioxidant and antimicrobial effects
Abstract
1 Design, preparation and in vitro characterization of biomimetic and bioactive

2 chitosan/polyethylene oxide based nanofibers as wound dressings
3
4 Oana Maria Ionescua,, Andreea-Teodora Iacoba,, Arn Mignonb, Sandra Van Vlierberghec,
5 Mihaela Baicand, Maricel Danue,f, Constanța Ibănescue,f, Natalia Simionescuf,g, Lenuța Profirea,*
6
7 a
8 Pharmacy of Iași, 16 University Street, Iasi, Romania
9 b
10 Andreas Vesaliusstraat 13, 3000 Leuven, Belgium
11 c
12 Macromolecular Chemistry, Ghent University, Krijgslaan 281, S4-bis, 9000 Ghent, Belgium
13 d
14 Pharmacy of Iași, 16 University Street, Iasi, Romania
15 e
Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and Environmental Protection,
16 “Gheorghe Asachi” Technical University of Iaşi, Mangeron Avenue 73, 700050 Iaşi, Romania
17 f
18 Biopolymers,41A Grigore Ghica Voda Alley, 700487, Iasi, Romania
19 g
20
21 * Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa”
22 University of Medicine and Pharmacy of Iași, 16 University Street, Iasi, Romania,
23 E-mail address: lenuta.profire@umfiasi.ro (L.P.).

24 Contributed equally to this work: Oana Maria Ionescu (O.M.I) and Andreea-Teodora Iacob (A.T.I.)
25
26 Keywords: Chitosan Nanofibers, Biocompatibility, Biological effects, Wound dressings
27
28 Abstract
29 Although chitosan based nanofibers (CS-NFs) are excellent artificial extracellular matrices
30 (ECMs), due to the resemblance of CS with the glycosaminoglycans of the natural ECMs, the
31 poor electrospinnability and mechanical properties of CS are responsible for important
32 limitations in respect with its biomedical applications. In order to improve its physico-chemical
33 properties, new bioactive and biomimetic CS based nanofibers, having incorporated different
34 active components (ACs), with important beneficial effects for healing, were formulated.
35 Manuka honey (trophic and antimicrobial effects), propolis (antimicrobial effects), Calendula
36 officinalis infusion (antioxidant effect, reepithelization stimulating agent), insulin (trophic effect)
37 and L-arginine (angiogenetic effect) were selected as ACs. SEM morphology analysis revealed
38 well-alignment, unidirectional arrays, with small diameters, no beads and smooth surfaces for
39 developed CS/PEO-ACs NFs. The developed NFs showed good biodegradability (NFs mats lost
40 up to 60% of their initial weight in PBS), increased hemocompatibility (hemolytic index less
41 than 4%) and a reduced cytotoxicity degree (cell viability degree more than 90%). In addition,
42 significant antioxidant and antimicrobial effects were noted for developed NFs which make them
43 suitable, especially for chronic wounds, being known the role of oxidative stress and infection
44 risk in delaying healing. The most suitable for wound healing applications seems to be CS-
45 PEO@P_C which showed improved hemolysis index (2.92±0.16%), is non-toxic (cell viability
1
46 degree more than 97%) and has also significant radical scavenging effect (DPPH inhibition more
47 than 65%). In addition, CS-PEO@P_C presents increased antimicrobial effects, especially for
48 Staphylococcus aureus, which is an important feature, knowing that wound infection leads to
49 delaying the healing process. It can be concluded that the developed CS/PEO-ACs nanofibers are
50 very promising biomaterials for wound care, especially CS-PEO@P_C.
2
Manuscript File (Word Document ONLY) Click here to view linked References
1
2
3
4
5 1 Design, preparation and in vitro characterization of biomimetic and bioactive
6 2 chitosan/polyethylene oxide based nanofibers as wound dressings
7
8 3
9 4 Oana Maria Ionescua,, Andreea-Teodora Iacoba,, Arn Mignonb, Sandra Van Vlierberghec,
10 5 Mihaela Baicand, Maricel Danue,f, Constanța Ibănescue,f, Natalia Simionescuf,g, Lenuța Profirea,*
11
12 6
13 7 a
14 8 Pharmacy of Iași, 16 University Street, Iasi, Romania
15 9 b
16 10 Andreas Vesaliusstraat 13, 3000 Leuven, Belgium
17 11 c
18
12 Macromolecular Chemistry, Ghent University, Krijgslaan 281, S4-bis, 9000 Ghent, Belgium
19
20 13 d
21 14 Pharmacy of Iași, 16 University Street, Iasi, Romania
22 15 e
Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and Environmental Protection,
23 16 “Gheorghe Asachi” Technical University of Iaşi, Mangeron Avenue 73, 700050 Iaşi, Romania
24 17 f
25 18 Biopolymers,41A Grigore Ghica Voda Alley, 700487, Iasi, Romania
26 19 g
27
28
20
29 21 * Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa”
30 22 University of Medicine and Pharmacy of Iași, 16 University Street, Iasi, Romania,
31 23 E-mail address: lenuta.profire@umfiasi.ro (L.P.).

32 24 Contributed equally to this work: Oana Maria Ionescu (O.M.I) and Andreea-Teodora Iacob (A.T.I.)
33 25
34 26 Keywords: Chitosan Nanofibers, Biocompatibility, Biological effects, Wound dressings
35 27
36
37 28 Abstract
38 29 Although chitosan based nanofibers (CS-NFs) are excellent artificial extracellular matrices
39 30 (ECMs), due to the resemblance of CS with the glycosaminoglycans of the natural ECMs, the
40
41 31 poor electrospinnability and mechanical properties of CS are responsible for important
42 32 limitations in respect with its biomedical applications. In order to improve its physico-chemical
43 33 properties, new bioactive and biomimetic CS based nanofibers, having incorporated different
44
45 34 active components (ACs), with important beneficial effects for healing, were formulated.
46 35 Manuka honey (trophic and antimicrobial effects), propolis (antimicrobial effects), Calendula
47 36 officinalis infusion (antioxidant effect, reepithelization stimulating agent), insulin (trophic effect)
48
49 37 and L-arginine (angiogenetic effect) were selected as ACs. SEM morphology analysis revealed
50 38 well-alignment, unidirectional arrays, with small diameters, no beads and smooth surfaces for
51 39 developed CS/PEO-ACs NFs. The developed NFs showed good biodegradability (NFs mats lost
52
40 up to 60% of their initial weight in PBS), increased hemocompatibility (hemolytic index less
53
54 41 than 4%) and a reduced cytotoxicity degree (cell viability degree more than 90%). In addition,
55 42 significant antioxidant and antimicrobial effects were noted for developed NFs which make them
56
43 suitable, especially for chronic wounds, being known the role of oxidative stress and infection
57
58 44 risk in delaying healing. The most suitable for wound healing applications seems to be CS-
59 45 PEO@P_C which showed improved hemolysis index (2.92±0.16%), is non-toxic (cell viability
60
61
62
1
63
64
65
1
2
3
4 46 degree more than 97%) and has also significant radical scavenging effect (DPPH inhibition more
5
6 47 than 65%). In addition, CS-PEO@P_C presents increased antimicrobial effects, especially for
7 48 Staphylococcus aureus, which is an important feature, knowing that wound infection leads to
8 49 delaying the healing process. It can be concluded that the developed CS/PEO-ACs nanofibers are
9
10 50 very promising biomaterials for wound care, especially CS-PEO@P_C.
11 51
12
13
52 1. Introduction
14 53 Biomedical research focused on wound care management is constantly seeking new
15 54 options with respect to promoting the healing process [1] and minimizing therapy costs [2].
16
55 Normally, wound healing involves four phases, haemostasis, inflammation, formation of
17
18 56 granulation tissue and remodeling [3]. Various factors increase the risk of wound closure delay
19 57 [4]. Among these, systemic factors such as diabetes, smoking, malnutrition and the age of the
20 58 patient, are considered major risk factors [5]. Impairment in the inflammatory phase, due to
21
22 59 leakage of superoxide, hydrogen peroxide or hydrochloric acid from neutrophil cells to the
23 60 wound site, contributes to a delay in healing. In addition, insulin deficiency in diabetes affects
24 61 the carbohydrate, protein and lipid metabolism and is responsible for impaired cellular function
25
26 62 and tissue synthesis in wound healing [6]. Moreover, there are local factors, such as moisture,
27 63 ischemia and/or necrosis of the tissue and oedema, which are involved in healing [7].
28 64 Although several wound dressings materials have already been developed, a proper
29
30 65 wound healing is far from being easily solved. It is known that natural extracellular matrices
31 66 (ECMs) are composed of two types of macromolecules: proteoglicans and fibrous proteins with
32 67 diameter ranging 50 nm to 150 nm [8]. Studies have also shown that cells attach and proliferate
33
34 68 well in micro and nanostructured materials [9].
35 69 Novel wound dressing such as electrospun nanofibers (NFs) bring the benefit of
36 70 enhancing tissue formation [10]. NFs are artificial ECMs [11] and show two key properties: a
37
71 high surface/volume ratio and high porosity - properties which are responsible to promote
38
39 72 cellular adhesion, proliferation and differentiation. The design of NFs offers a wide range of
40 73 prospects with respect to polymer and active components (ACs) selection [12]. Blends of natural
41
74 and synthetic polymers are suitable to overcome low spinnability of pure natural polymers [13].
42
43 75 Chitosan (CS) is a linear polycation, nontoxic and biodegradable polysaccharide - a
44 76 derivative of chitin, which is extracted from the exo-skeleton of the shell of crustaceae or fungi
45 77 [14,15]. It is composed of beta-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine units
46
47 78 and the D-glucosamine content is dependent on the degree of deacetylation of CS [15,16].
48 79 CS acts in wound healing, tissue restoration and, in addition displays antibacterial [17–
49 80 19], antioxidant [20], hemostatic and immunomodulatory properties [21]. During the
50
51 81 depolymerization process, the N-acetyl-D-glucosamine initiates fibroblast proliferation and
52 82 promotes hyaluronic acid synthesis at the wound site [22]. The molecular weight of CS directs its
53 83 antimicrobial mechanism. Indeed, low-molecular weight CS (LMW-CS) can enter to
54
55 84 microbial/fungal cell and attach itself to the DNA and suppress protein synthesis while high
56 85 molecular weight CS (HMW-CS) triggers cell leakage by covering the cell wall [23].
57 86 Chitosan based nanofibers (CS-NFs) have already been described as artificial ECMs due
58
59 87 to the resemblance of CS and the glycosaminoglycans of the natural ECMs [24,25].
60
61
62
2
63
64
65
1
2
3
4 88 Because CS has poor electrospinning properties, mainly because of its polycationic
5
6 89 nature, specific inter- and intra-molecular interactions and high viscosity [26], blending CS with
7 90 other polymers such as poly(ethylene oxide) (PEO), poly(vinyl)alcohol (PVA) or
8 91 polycaprolactone (PCL) was useful [27].
9
10 92 In order to increase the electrospinnability of CS, different parameters were have been
11 93 studied such as: polymer content, polymer molecular weight, deacetylation degree, type of
12 94 solvents (trifluoroacetic acid, acetic acid), ratio polymer/solvent and ratio CS/PEO or CS/PVA
13
14 95 [21].
15 96 PEO is an amphiphilic, water-soluble and biocompatible, spinnable polymer [28] which
16 97 is often used in the electrospinning process of CS [29–31], expecially the ultrahigh molecular
17
18 98 weight PEO [32].
19 99 To obtain better fibrous structure at high CS-PEO ratio, which is often required for
20 100 tissues engineering applications, different surfactants (anionic, cationic, nonionic) and solvents
21
22 101 (acetic acid, citric acid, succinic acid) were studied to increase the spinnability rate and to obtain
23 102 highly aligned CS-NFs [8].
24 103 When designing the electrospinning experiment, parameters vary for every device used
25
104 and must be adjusted accordingly [9,33]. In case of CS-PEO NFs, their fabrication may occur for
26
27 105 example at low flow rates (0.1 mL/h) [34], by using a device that sprays the polymer solution
28 106 through a multicapillary [35] or at moderate temperature (40°C-70°C) [36]. Morphological
29
30
107 characterization proved that CS-PEO nanofibers differ when the solution is produced by
31 108 blending the two solutions of separately dissolved CS and PEO or by dissolving PEO into the CS
32 109 solution [37].
33
110 Honey is a natural derivative with trophic and antibacterial effects, reactive oxygen
34
35 111 species (ROS) scavenging activity and increased osmotic gradient [38]. It also reduces
36 112 inflammation, absorbs excessive exudate therefore avoiding maceration, promotes angiogenesis,
37 113 granulation tissue production, wound contraction and epithelialization [39]. Honey consists of
38
39 114 water (20%), fructose (40%), glucose (30%), sucrose (5%), and other substances (minerals,
40 115 vitamins, amino acids and enzymes) [40]. Manuka honey’s antibacterial effect is especially due
41
116 to its most important constituent, being methylglyoxal [41].
42
43 117 Propolis is a resin made by bees with important biological effects based on its chemical
44 118 composition which can vary depending on the local flora, climate condition, weather and the
45 119 presence of other components, such as wax or pollen. The main chemical compounds of
46
47 120 biological interest are flavonoids, phenolic acids and their esther derivatives. Propolis is a
48 121 valuable candidate for treating wounds, given its antioxidant, anti-inflammatory, antimicrobial
49 122 and antifungal properties. Data from literature describe the benefits of propolis-incorporating
50
51 123 NFs in treating different chronic wounds such as leg ulcers [42,43].
52 124 Calendula officinalis is used as infusion, tincture or ointment on skin inflammations,
53 125 wounds, superficial burns, leg ulcers and insect bites [44]. Its curative properties are due to the
54
55 126 presence of flavonoids and saponins [45,46].
56 127 Insulin is a peptide hormone and growth factor which reduces blood glucose levels and
57 128 promotes healing of damaged skin [47]. If applied on wounded skin, insulin leads to faster re-
58
59
129 epithelialization and contraction of the wound by promoting the production of granulation tissue
60 130 [48].
61
62
3
63
64
65
1
2
3
4 131 L-arginine is a basic alpha-amino acid which acts as a precursor of nitric oxide (NO)
5
6 132 [49], a signal molecule involved in collagen synthesis, epithelialization and formation of
7 133 granulation tissue, which are essential processes for wound healing [50]. In addition, NO
8 134 regulates the immune responses and angiogenesis [16]. At the wound site, a low content of L-
9
10 135 arginine is present due to the high prevalence of arginase, an enzyme which hydrolyzes L-
11 136 arginine to urea and L-ornithine [51].
12 137 In this work, bioactive and biomimetic NFs were prepared via conventional
13
14 138 electrospinning of a blend containing CS (medium molecular weight) and PEO (high molecular
15 139 weight) in ratio of 2:1 in acetic acid 50%, in which were incorporated different active
16 140 components (ACs) with beneficial effect for wound healing. Manuka honey (trophic and
17
18
141 antimicrobial effects), propolis (antimicrobial effects), Calendula officinalis infusion
19 142 (antioxidant effect, reepithelization stimulating agent), insulin (trophic effect) and L-arginine
20 143 (angiogenetic effect) were selected as ACs. The successful incorporation of the ACs into the CS-
21
144 based nanoscaffolds was proved by IR spectral analysis. Well-alignment, unidirectional arrays,
22
23 145 with small diameters, no beads and smooth surfaces NFs were obtained, which can work as
24 146 artificial ECMs and so be proper for tissue engineering applications. The CS/PEO-ACs NFs
25 147 showed a good biodegradability, increased hemocompatibility and reduced cytotoxicity degree.
26
27 148 In addition, good antioxidant and antimicrobial effects were noted for developed NFs which
28 149 make them suitable, especially, for chronic wounds, being known the role of oxidative stress and
29 150 infection risk in delaying healing.
30
31 151
32 152 2. Materials and methods
33
34 153 2.1. Materials
35
36 154 Chitosan (CS) (medium molar mass), poly(ethylene oxide) 1 million g/mol, glacial acetic
37 155 acid (≥99.7%), L-arginine∙HCl, α-bromo-naphtalene (97%), DPPH (2,2-Diphenyl-1-
38
39 156 picrylhydrazyl), ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), sodium chloride,
40 157 disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Sigma
41 158 Aldrich. Calendula officinalis (Fares, Romania), insulin (NovoRapid Penfill 100 U/mL,
42
43
159 NovoNordisk, Dennmark), propolis (Apiland, Romania), Manuka honey 550+ (Manuka Health
44 160 New Zealand Ltd),double distilled water, formamide (Calbiochem).Gram positive
45 161 (Staphylococcus aureus ATCC 25923), Gram negative (Escherichia coli ATCC 25922,
46
162 Pseudomonas aeruginosa ATCC 27853) bacterial strains and pathogenic yeasts (Candida
47
48 163 albicans ATCC 10231) were provided by Department of Microbiology, “Grigore T. Popa”
49 164 University of Medicine and Pharmacy, Iasi, Romania. Normal dermal fibroblasts (NHDF,
50 165 PromoCell, Heidelberg, Germany), alpha-MEM medium (Lonza, Basel, Switzerland), fetal
51
52 166 bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA USA) 1% Penicillin-
53 167 Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland).
54
55
56
168 2.2. Preparation of CS-PEO-ACs solutions
57 169 CS-PEO solutions were prepared by dissolving CS (1%, wt/v) and PEO (0.5%, wt/v) in
58
59 170 acetic acid 50% (v/v), after which different ACs such as Manuka honey (M), propolis (P),
60
61
62
4
63
64
65
1
2
3
4 171 Calendula officinalis infusion (C), insulin (I), L-arginine∙HCl (L), were added (Table 1). The
5
6 172 solutions were blended on a tabletop shaker until the composition was homogenous.
7 173
8 174 Table 1
9
10 175 The composition of CS-NFs/CS-PEO-ACs and optimal flow-rateused for electrospinning.
11 176
12 CS-PEO-NFs/ Flow-rate
13 No. ACs
14 CS-PEO-ACs mL/h
15 1 CS_PEO@I_P I: 46 IU/mL, P: 7.5% (wt/v) 0.2
16 2 CS_PEO@M_L M: 7.5% (wt/v), L: 7.5% (wt/v) 0.5
17
3 CS_PEO@P P: 7.5% (wt/v) 0.2
18
19 4 CS_PEO@P_C P: 7.5% wt/v 0.2
20 5 CS_PEO@M_L_P MH: 7.5% (wt/v), L: 7.5% (wt/v), 0.5
21 P: 7.5% (wt/v)
22
23 6 CS_PEO - 0.6
24 177 CS-PEO-NFs = chitosan-PEO based nanofibers, CS-PEO-ACs = chitosan-PEO based polymeric
25 178 solution with different active components, I = insulin, P = propolis, M = Manuka honey, L = L-
26
27 179 arginine∙HCl, C = Calendula officinalis infusion.
28 180
29 181 The CS_PEO@P_C solution was prepared similarly, with slight modifications. Firstly,
30
31 182 the Calendula officinalis infusion was prepared, according to the European Union herbal
32 183 monograph on Calendula officinalis L., flos [45], from 1.5 g of herba and 150 mL water (70°C).
33 184 After filtration, the resulting solution was kept at room temperature and used in the first 24 h in a
34
35 185 1:1 ratio with glacial acetic acid (v/v), as a solvent for CS, PEO and propolis.
36
37 186 2.3. Characterization of the CS-PEO-ACs solutions
38
39 187 The CS-PEO-ACs solutions were characterized rheologically and had their conductivity
40 188 measured before being spun. The solutions were electrospun immediately after becoming
41
42
189 homogenous, and no later than 48 h, to prevent any damage, such as polymer degradation or
43 190 aging of the solution. A slightly modified protocol was used with regards to the CS-PEO
44 191 solutions containing M, which were spun after one-week-resting time, according to literature
45
192 data [52].
46
47 193 The viscosity of CS-PEO-ACs solutions was measured with a Physica MCR rheometer
48 194 from Anton Paar (Austria) with a 50 mm upper plate mounted in a parallel-plate system. The
49 195 flow curves were determined with shear-rates ranging from 0.01 to 500 s-1. Before each
50
51 196 measurement, the solutions were equilibrated at 25℃. The rheological behaviour was studied
52 197 using the Ostwald model to display the pseudoplastic behaviour and the Carreau-Yassuda model
53 198 to display the zero shear viscosity of the CS-PEO-ACs solutions. The apparent viscosity of the
54
55 199 solutions was determined at 100 s-1. The conductivity of the CS-PEO-ACs solutions was
56 200 determined using the Oakton PC510 Conductometer. For each solution a volume of 30 mL was
57 201 used and the recorded values are shown in mS. The measurements were performed in triplicate
58
59 202 and the values were expressed as mean ± standard deviation (SD).
60
61
62
5
63
64
65
1
2
3
4 203 2.3. Electrospinning process of CS-PEO-ACs solutions
5
6
204
7 205 The nanofibrous scaffolds were fabricated via conventional electrospinning technique [9,28],
8 206 using Nanospinner Inovenso device equipped with a pump, generator and a plate collector as a
9 207 negative electrode. A syringe of 5 mL with 23-gauge needles served as positive electrode. The
10
11 208 parameters were constant during the experiments, with a tip-to-collector distance of 25 cm, a
12 209 flow rate of 0.2 to 0.6 mL/h and a voltage below 22 kV (Table 1). The counter electrode of the
13 210 setup was a grounded collector.
14
15
16 211 2.4. Characterization of the CS-PEO-NFs
17
18 212 2.4.1. SEM morphology and fiber diameter
19
20 213 Fiber morphology and diameter were carried out using the scanning electron microscopy
21 214 (SEM). Measurements were performed with a Inspect S electron microscope with acceleration
22
23
215 voltages in high vacuum, using secondary electron beam with energies of 30keV. For
24 216 diminishing the electrical charging, the samples were capped with a thin gold film. The fiber
25 217 distribution was analysed using the Phenom FiberMetric software by measuring at least 150
26
218 fibers per image and three images per sample. Data are shown as average diameter ± standard
27
28 219 deviation (SD).
29
30 220 2.4.2. Fourier transform infrared spectroscopy (FTIR)
31
32 221 The IR spectra of the developed CS-PEO-NFs were recorded using the IR ABB MB300
33 222 Spectrophotometer. Each spectrum was collected in the wave number range 4000-600cm−1 at a
34
35 223 resolution of 4 cm−1.
36
37 224 2.4.3. Surface free energy and biocompatibility
38
39 225 In order to quantify the biocompatibility features of developed CS-PEO-NFs, the surface
40 226 free energy (SFE) was determined using the acid-base approach of van Oss and Good. To attain
41
227 the constituents of the SFE, the contact angles values at equilibrium between the electrospun
42
43 228 nanomat surface and pure liquids were measured, using the sessile drop method and the CAM-
44 229 PLUS Micro goniometer instrument from KSV-Finland. Three pure liquids including double-
45 230 distilled water, formamide and α-bromo-naphthalene, were used. The polymeric solutions were
46
47 231 directly spun onto a glass surface (blade) and dried. The measurements were recorded within 10s
48 232 after 1 μL of the pure liquid was placed on the NFs surface. At least 10 measurements were
49 233 carried out on different areas of the surface and the average contact angle value was calculated.
50
51 234 The Young–Laplace equation divides the SFE into dispersive interactions (Lifshitz–van der
52 235 Waals: γsLW) and polar acid–base interactions (Lewis: γsAB), which are also subdivided into
53 236 electron donor γs- (Lewis base) and electron acceptor γs+ (Lewis acid) [16]:
54
55 (1+cosθ) γLTOT=2(√γsLW γLLW+√γs+ γL- +√γs- γL+) (1)
56
57 237 where: θ is the contact angle, γLTOT is the liquid total surface tension, and γLLWand γsLW are the
58 238 apolar Lifshitz–van der Waals components of the liquid and the solid, respectively, whereas γ AB,
59
60
61
62
6
63
64
65
1
2
3
4 239 γs+ γL- andγs- γL+ are the Lewis acid–base components of either the solid or the liquid phase as
5
6 240 indicated by the subscripts.
7 241 Based on γAB and γLW values, the γSL parameter, indicative for the biocompatibility range, was
8 242 calculated, using the following formula [53]:
9
10 γSL=(7.14-√γAB)2+(4.67-√γLW)2 (2)
11
12 243 2.4.4. Swelling degree assay
13
14 244 The swelling degree (SD) measure the water (PBS/PECF) uptake capacity, which could
15 245 be translates into physiological exudate uptake capacity. According to literature data [29], the
16
246 CS-PEO-NFs were cut into 1x1 cm2 pieces. The samples were firstly weighed in dry state and
17
18 247 were immersed in phosphate buffered solution, pH 7.4 (PBS) and in pseudo extracellular fluid
19 248 solution (PECF), respectively, at 37°C. The PECF solution was prepared by dissolving NaHCO 3
20 249 (2.5 g), NaCl (0.68g), KCl (0.22 g) and NaH2PO4 (0.35 g) in 100 mL distilled water (adjusting
21
22 250 the pH to 8.5) [54].
23 251 The CS-PEO-NFs samples were finely blotted in order to remove the superficial layer of
24 252 PBS/PECF and were weighed on a four-digit balance. The SD (%) was calculated according to
25
26 253 the formula [54]:
27
28 SD (%) =(Wt-W1/W1) x100 (3)
29
30
254 where: Wt is the weight of the mats in the hydrated state (at set time interval), W1 is the weight of
31 255 the mats in the dry state. All experiments were performed in triplicate and the results are
32 256 expressed as mean ± standard deviation (SD).
33
34
35
257 2.4.5. In vitro degradation assay
36 258 The protocol used for in vitro degradation, quatified as remaining weight (RW), was
37
38 259 similar to the SD assay. The difference is that the degradation assay has been conducted for a
39 260 longer time frame (4 weeks), and therefore the values are depicted as two independent variables.
40 261 In brief, CS-PEO-NFs were cut into 1x1 cm2 pieces, weighed in dry state and immersed in PBS,
41
42 262 pH 7.4 and PECF solution, respectively, at 37°C. After blotting the samples were weighed on a
43 263 four-digit balance. The RW (%) was calculated according to the following formula [54]:
44
45 RW (%) = (Wf-W1/W1) x 100 (4)
46
47 264 where: Wf is the final weight of the mats in the hydrated state (after four weeks), W1 is the
48 265 weight of the mats in the dry state. All experiments were performed in triplicate and the results
49 266 are expressed as mean ± standard deviation (SD).
50
51
52 267 2.4.6. In vitro hemocompatibility assay
53 268 The capacity of the CS-PEO-NFs to generate hemolysis, as a parameter to predict their
54
55 269 hemocompatibility, was evaluated according to a protocol from literature [55]. Briefly, a volume
56 270 of whole blood from healthy volunteers was diluted in normal saline (0.9% NaCl) in a 4:5 ratio
57 271 (v/v). The CS-PEO-NFs were cut into 1x1 cm2 pieces and each sample was immersed in 5 mL of
58 272 normal saline and was incubated for 30 min at 37°C. Afterwards, 0.1 mL of the previously
59
273 diluted blood was added followed by further incubation for 2 h at 37°C. The samples were then
60
61
62
7
63
64
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1
2
3
4 274 centrifuged at 1500 rpm for 10 min and the supernatant was removed. A negative control
5
6 275 (containing 0.1 mL of diluted blood in 5 mL 0.9% NaCl) and a positive control (containing 0.1
7 276 mL of diluted blood in 5 mL double distilled water), were also used. The absorbance was
8 277 measured at λ=545 nm. The hemolysis (%) was calculated according to the following formula
9 278 [55]:
10
11 Hemolysis (%) = (As-Anc)/(Apc-Anc) x 100 (5)
12
13 279 where: As is the absorbance of the sample; Anc is the absorbance of the negative control; Apc is
14 280 the absorbance of the positive control. The experiments were performed in triplicates and the
15
281 results are expressed as mean ± standard deviation (SD).
16
17
18 282 2.4.7. In vitro cytotoxicity (MTS) assay
19
20 283 The cytotoxicity degree of the CS-PEO-NFs was assessed by an MTS assay using the
21 284 CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI USA),
22 285 according to the manufacturer’s instructions and through an indirect contact procedure adapted
23
24 286 from ISO 10993-5 [56]. A sample of 100 μg CS-PEO-NFs was incubated with 1 mL alpha-MEM
25 287 medium, for 72 h at 37 °C, after which the serially dilutions were made to obtain different
26 288 concentrations (50 µg/mL, 25 µg /mL, 12.5 µg /mL). The normal dermal fibroblasts cells, grown
27
28 289 in alpha-MEM medium supplemented with 10% fetal bovine serum and 1% Penicillin-
29 290 Streptomycin-Amphotericin B mixture, were seeded at a density of 0.5×105 cells/mL into 96-
30 291 well tissue culture-treated plates and allowed to adhere for 24 h. The cells were then incubated
31
32
292 for 72 h with 100 μl of previously prepared CS-PEO-NFs samples (12.5 µg/mL, 25 µg/mL, 50
33 293 µg/mL, 100 µg/mL) or alpha-MEM medium (used also as control). MTS reagent (tetrazolium
34 294 inner salt) (20 µL) was added 3 h prior to absorbance readings at 490 nm on a FLUOstar®
35
295 Omega microplate reader (BMG LABTECH, Ortenberg, Germany). The cell viability was
36
37 296 expressed as percentage (%) of control cells’ viability. All experiments were performed in
38 297 triplicate and the results are expressed as mean ± standard deviation (SD).
39
40
298 2.4.8. In vitro antioxidant assays
41
42 299 The radical scavenging activity of CS-PEO-NFs toward 1,1-diphenyl-2-picrylhydrazyl
43
44 300 (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was measured. The
45 301 total antioxidant capacity and ferric reducing antioxidant power were also evaluated. A sample of
46 302 60 mg of the CS-PEO-NFs was mixed with 5 mL ethanol for 1 h, and then the ethanolic
47
48 303 supernatant was separated. A set volume of this solution was used for the antioxidant assays. The
49 304 measurements were performed using an UV–VIS spectrophotometer GBC Cintral. All
50 305 experiments were performed in triplicate and the results are expressed as mean ± standard
51
52
306 deviation (SD).
53
54 307 2.4.8.1. DPPH radical scavenging assay
55
56 308 This assay is one of most used method to evaluate the radical scavenging effect and is
57 309 based on the reduction of DPPH, which is violet in ethanol solution but yellow in the presence of
58 310 a proton donating agent [57]. A volume of 0.25 mL of ethanolic supernatant (CS-PEO-NFs)
59
60 311 previously obtained was mixed with 1 mL of ethanolic DPPH solution (100 μM). The resulting
61
62
8
63
64
65
1
2
3
4 312 mixture was kept in the dark at room temperature and the absorbance was measured at λ=517
5
6 313 nm, after 30 min and 60 min respectively, against a blank (ethanol instead of the set volume of
7 314 CS-PEO-NFs ethanolic extract). Similar experiments were performed for CS-PEO-ACs
8 315 polymeric and ACs solutions in an equivalent content to CS-PEO-NFs.
9
10 316 The DPPH radical-inhibiting capacity (inhibition/scavenging activity) (%) was calculated
11 317 according to the formula [58]:
12
13 Inhibition (Scavenging activity) %= [(ADPPH-As)/ADPPH]x100 (6)
14
15 318 where: ADPPH is the absorbance of the DPPH solution; As is the absorbance of the CS-PEO-NFs
16 319 sample.
17
18 320 2.4.8.2. ABTS•+radical scavenging assay
19
20 321 An aqueous solution of ABTS (7 mM) with ammonium persulfate (2.45 mM) was
21
22
322 prepared. The solution was kept in the dark for 16 h to initiate the formation of the ABTS•+ and
23 323 then diluted with ethanol to obtain an absorbance of 0.700±0.02 at 734 nm, as described in
24 324 literature [59]. In the presence of hydrogen donating agents, the blue chromophore ABTS•+ is
25
325 reduced and as result the absorbance decreases [60]. A sample of 1 mL ABTS ethanolic solution
26
27 326 was added to 0.25 mL of CS-PEO-NFs ethanolic supernatant and after 6 min the absorbance of
28 327 each sample was measured at λ=734 nm, against a blank (ethanol instead of the set volume of
29 328 CS-PEO-NFs ethanolic extract).
30
31 329 The ABTS•+radical-inhibiting capacity (scavenging activity) (%) was calculated
32 330 according to the following formula [59,60]:
33
34 Inhibition (Scavenging activity) %=[(AABTS–As)/AABTS]x100 (7)
35
36 331 where: AABTSis absorbance of the ABTS•+solution; Asis absorbance of the CS-PEO-NFs sample.
37
38 332 2.4.8.3. Phosphomolybdenum reducing antioxidant power (PRAP) assay
39
40 333 The total antioxidant capacity of CS-PEO-NFs was evaluated using the phospho-
41 334 molybdenum assay according to the protocol described in literature [61] with slight
42
43
335 modifications. The method is based on spectrophotometric quantification of green colored
44 336 phosphomolibdenum complex, formed after the reduction of Mo(VI) to Mo(V), in the presence
45 337 of electron donating agents in acidic solution [62]. A volume of 2 mL of reagent solution (0.6 M
46
338 sulfuric acid, 28 mM disodium hydrogen phosphate and 4 mM ammonium molybdate) was
47
48 339 added over 0.2 mL of CS-PEO-NFs ethanolic supernatant. The samples were then incubated at
49 340 95°C for 90 min in an oven and after cooling (to room temperature), the absorbance was
50
341 measured at λ=695 nm against a blank (ethanol instead of the set volume of CS-PEO-NFs
51
52 342 ethanolic supernatant). An increase in optical density implies a superior antioxidant effect.
53
54 343 2.4.8.4. Ferric reducing antioxidant power (FRAP) assay
55
56 344 The ferric reducing antioxidant power of CS-PEO-NFs was quantified by a method
57 345 described in literature [61]. The method is based on the reduction of ferricyanide into blue
58
59 346 ferrocyanide in the presence of electron donating agents, which can be spectrophotometrically
60 347 quantified. A volume of 0.5 mL of 0.2 M phosphate buffer, pH 6.6, was added to 0.5 mL of CS-
61
62
9
63
64
65
1
2
3
4 348 PEO-NFs ethanolic supernatant. The reaction was then initiated by the addition of 0.5 mL of
5
6 349 potassium ferricyanide 1% (wt/v), after which the samples were incubated at 50 °C for 20 min.
7 350 The completion of the reaction took place by addition of 0.5 mL trichloroacetic acid 10% (wt/v).
8 351 1 mL of the resulting solution of each sample was diluted with 1 mL double distilled water and
9
10 352 finally 0.2 mL of ferric chloride 0.1% (wt/v) was added. The mixture was left at room
11 353 temperature for 10 min and then the absorbance was measured at λ=700 nm against a blank
12 354 (ethanol instead of the set volume of CS-PEO-NFs ethanolic supernatant). An increase in optical
13
14 355 density implies a superior antioxidant effect.
15
16 356 2.4.9. Antimicrobial assay
17
18 357 Antibacterial and antifungal activity, measured as the diameter of the inhibition area,
19 358 were evaluated by the agar disc diffusion method (Performance Standards for Antimicrobial
20
359 Susceptibility Testing [63]) using Gram positive (Staphylococcus aureus ATCC 25923) and
21
22 360 Gram negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853)
23 361 bacterial strains and Candida albicans ATCC 10231 as pathogenic yeasts. The bacterial and
24 362 fungal strains suspensions (106 CFU/mL in NaCl 0.9%) were inoculated on the surface of the
25
26 363 culture medium (Mueller Hinton for bacterial strains and Sabouraud for fungal strain) spread at a
27 364 volume of 25 mL/sterile Petri plate. Sterile stainless steel cylinders (5 mm internal diameter and
28 365 10 mm height) were applied on the agar surface in Petri plates and 200 µL of CS-PEO-ACs
29
30 366 tested was added into cylinders. Commercial discs containing Ciprofloxacin (5 µg/disc) and
31 367 Voriconazole (1 µg/disc) were used as controls. The plates were incubated at 37°C for 24 h
32 368 (antibacterial activity assessment) and at 35°C for 48 h (antifungal activity assessment). After
33
34 369 incubation, the diameters of the inhibition area (mm) including disc size were measured.
35
36 370 2.5. Data analysis
37
38 371 Experiments were performed in triplicate and the statistical significance was calculated
39 372 with t-Test and analysis of variance (ANOVA). A p value ˂ 0.05 was considered statistically
40 373 significant.
41
42 374
43
44
45 375 3. Results and Discussion
46
47 376 3.1. Characterization of the CS-PEO-ACs solutions
48
49
377 A certain conductivity of the polymeric solutions is needed for proper electrospinning, it
50 378 being considered the elementary force when it elongates towards the collector. If such a solution
51 379 shows a high conductivity, then a lower polymeric content can be used for electrospinning. This
52
380 will in turn leads to smaller diameters of NFs and aids with reducing bead formation. The data
53
54 381 referring to the conductivity of the CS-PEO-ACs solutions are presented in Table 2.
55 382 In our experiment the lowest conductivity (2.28±0.03 mS) was recorded for CS_PEO.
56
383 Any addition of ACs to the CS_PEO solution was associated with a conductivity increase. The
57
58 384 highest conductivity was noted for the polymeric solutions containing L-arginine in combination
59 385 with Manuka honey (CS_PEO@M_L, 10.07±0.05). A high conductivity was also noted for the
60
61
62
10
63
64
65
1
2
3
4 386 polymeric solution containing L-arginine, Manuka honey and propolis (CS_PEO@M_L_P,
5
6 387 8.72±0.07mS). This could be explained by the presence of the basic amino-acid, L-arginine,
7 388 which increases the number of -NH3+ groups in the polymeric solution [64]. For the polymeric
8 389 solution containing propolis (CS_PEO@P, 2.35±0.04 mS), propolis-Calendula officinalis
9
10 390 infusion (CS_PEO@P_C, 2.32±0.05 mS) and propolis-insulin (CS_PEO@I_P, 2.40±0.06 mS)
11 391 the conductivity was lower than those with L-arginine.
12 392
13
14 393 Table 2
15 394 The conductivity and rheological parameters of the CS-PEO-ACs solutions.
16 395
17
18 Carreau- Apparent
Ostwald I Model
19 Conductivity Yassuda Model viscosity, at
No. CS-PEO-ACs
20 value (mS) Flow behaviour Consistency Zero shear 100 s-1 (ηa,100,
21 index, a (K) coefficient, b (n) viscosity, η0 (Pa∙s) Pa s)
22 1 CS_PEO@I_P 2.40±0.06* 0.47 0.83 0.36 0.22
23
24 2 CS_PEO@M_L 10.07±0.05 0.63 0.83 1.42 0.31
25 3 CS_PEO@P 2.35±0.04 0.52 0.81 0.37 0.23
26 4 CS_PEO@P_C 2.32±0.05 0.50 0.80 0.51 0.21
27 5 CS_PEO@M_L_P 8.72±0.07* 0.11 0.92 1.58 0.08
28 6 CS_PEO 2.28±0.03 0.03 0.97 2.10 0.03
29 7 PEO 2.40±0.06 5.31 0.56 1.90 0.79
30 396 *where p˂0.05 when compared to CS_PEO
31
32 397 The viscosity of the polymeric solutions depends on the intermolecular interactions
33
398 occurring between the polymeric chains, thereby influencing the electrospinnability of the
34
35 399 solution and the morphology of the resulting NFs.
36 400 The rheological characteristics of CS-PEO-ACs polymeric solution, based on Carreau-
37
401 Yassuda model, are show in Table 2. For the CS precursor solution a high zero shear viscosity
38
39 402 (2.10 Pa∙s) was recorded, due to the strong intermolecular bonds between the polymer chains. A
40 403 reduction of the zero shear viscosity can be seen once the ACs are blended into CS-PEO
41 404 solution. The addition of Manuka honey to the CS-based polymeric solution was partly
42
43 405 responsible for the higher zero shear viscosity (CS_PEO@M_L, 1.42 Pa∙s; CS_PEO@M_L_P,
44 406 1.58 Pa∙s) when compared to other formula containing insulin, propolis and Calendula officinalis
45 407 infusion (CS_PEO@I_P; 0.36 Pa∙s; CS_PEO@P, 0.37 Pa∙s; CS_PEO@P_C, 0.51 Pa∙s).
46
47 408 Similar to other literature data, the flow behavior index (K) for all CS-based polymeric
48 409 solutions, is smaller than 1, following a pseudo plastic behaviour. This behavior may be due to
49 410 the strong intramolecular hydrogen bonds occurring between the CS chains which prevent
50
51 411 disentanglements at higher shear rates [65]. K values of the neat CS solution (0.03) significantly
52 412 increased after the addition of PEO and ACs respectively. In the latter case, the K values ranging
53 413 between 0.11 (CS_PEO@M_L_P) and 0.63 (CS_PEO@M_L). If polymers are not sufficiently
54
55
414 entangled, droplets may be deposited on the collector instead of fibers. Although the
56 415 CS_PEO@M_L_P showed the lowest K value, the high conductivity (8.72±0.07) noted for this
57 416 polymeric solution, may have counteracted the lower chitosan chain entanglements.
58
59
60
417 3.2. Characterization of the CS-PEO-NFs
61
62
11
63
64
65
1
2
3
4 418 3.2.1. SEM morphology and fiber diameter
5
6 419 The diameter and orientation of NFs in fibrous scaffolds are some of the most important
7
8
420 features for tissue engineering applications, because the fiber orientation influences the cell
9 421 growth and related functions [9]. Well-alignment, unidirectional NFs arrays, with small
10 422 diameters, no beads and smooth surfaces were observed for the developed NFs, probably due of
11
423 the physical characteristics of the used ACs. It was noted that the average diameter increased
12
13 424 with the addition of ACs into the CS-PEO solutions (Fig. 1). The highest diameter was noted for
14 425 CS_PEO@M_L_P) (200.97±5.91 nm) and CS_PEO@M_L (188.81±6.65 nm) while for the
15 426 others CS-PEO-NFs the diameter was similar to CS-PEO (163.54±16.29 nm). This could be
16
17 427 explained by the presence of Manuka honey which increases the viscosity of the corresponding
18 428 spinning solutions [66]. Therefore less stretching occurs which leads to a larger diameter [52].
19 429 Furthermore, the nanofibers without Manuka honey (CS_PEO@P, CS_PEO@P_C,
20
21 430 CS_PEO@P_I) had a smoother morphology and a homogeneous appearance, while those with
22 431 Manuka honey (CS_PEO@M_L, CS_PEO@M_L_P) showed a more non-uniform morphology
23 432 with a wider diameter distribution (Fig. 1).
24
25 433
26
27
28
29
30
CS_PEO@I_P
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
CS_PEO@M_L
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
12
63
64
65
1
2
3
4
5
6
7
8
9
CS_PEO@P
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CS_PEO@P_C
24
25
26
27
28
29
30
31
32
33
34
35
36
37
CS_PEO@M_L_P
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
13
63
64
65
1
2
3
4
5
6
7
8
9
10
CS_PEO
11
12
13
14
15
16
17
18
19
20
21 434 Fig. 1. SEM micrographs and diameter/average diameter ± SD (nm) of CS-PEO-NFs (for all
22 435 samples p ˂0.05 when compared to CS_PEO).
23
24 436 3.2.2. Fourier transform infrared spectroscopy (FTIR)
25
26 437 The presence of ACs into the nanofibrous scaffold was proved by IR spectroscopy. As noted in
27 438 Fig. 2, CS displays a characteristic band at 1560 cm-1 assigned to the N-H groups (bending
28
29 439 vibration) [67] and a specific band at 3357 cm-1assigned to intermolecular hydrogen bonding and
30 440 N-H and OH stretching vibrations. PEO displays characteristic bands at 2875 cm-1 due to the -
31 441 CH2- groups, at ̴ 1095 cm-1 due to the C-O-C groups (anti-symmetric stretching) and at ̴ 840 cm-1
32
33
442 due to the bending C-O-C groups. The addition of PEO explains the vibration band of OH/NH2
34 443 at 1543cm-1. Manuka honey displays its characteristic bands at ̴ 2910 cm-1 (C-H), ̴ 1650 cm-1
35 444 (C=O) and ̴ 1054 cm-1 (C-O) [41].
36
37
445 The specific absorption bands for propolis were noticed at 3336 cm-1 (aromatic OH
38 446 group) and at ̴ 1617 cm-1, ̴ 1496 cm-1 and ̴ 1450 cm-1 (C=C stretches of aromatic rings) [68]. L-
39 447 arginine displayed a characteristic -C=N stretching vibration of the guanidine group at ̴ 1632 cm-
40 448 1
[42]. Two absorption bands, at ̴1650 cm-1 and at ̴ 1540 cm-1, are characteristic of two amide
41
42 449 groups belonging to insulin. The insulin monomer contains ionizable groups resulting from the
43 450 six aminoacid residues able to carry a positive charge. Also, ten amino acid residues are
44 451 available to attain a negative charge [69]. Calendula officinalis infusion displays peaks which
45
46 452 correspond to alkyl and C-H groups (̴ 2940 cm-1), flavonoids ( ̴1050 cm-1) and ether groups
47 453 at ̴1030 cm-1, which is in agreements with literature data [46].
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
14
63
64
65
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22 454
23
24 455 Fig. 2. FT-IR spectra of CS_PEO_NFs.
25 456
26 457 In reference with the IR spectrum of CS_PEO, the CS_PEO_ACs NFs presented an
27
28 458 intense and large absorption band at ̴ 3300 cm-1 and at ̴ 2875 cm-1, due to the interactions
29 459 between –OH, -NH2 and -CH2- groups of CS and PEO respectively and the functional groups of
30 460 ACs, especially -OH , -NH- and -NH-CO-. Also, based on hydrogen interactions, a shift of -CH
31
32 461 stretching frequencies band from the ACs-polymer matrix spectrum to shorter wavelengths was
33 462 observed, when compared with the CS-PEO. A shift of the characteristic absorption bands of
34 463 insulin (at ̴1650 cm-1 and at ̴ 1540 cm-1) and L-arginine (at ̴ 1632 cm-1) were also noted in
35
36
464 reference with pure insulin (at ̴ 1679 cm-1 and ̴ 1576 cm-1) and L-arginine (at ̴ 1632 cm-1) [70],
37 465 also based on interactions between the functional groups of CS, PEO and ACs.
38
39 466 3.2.3. Surface freeenergy and biocompatibility
40
41
467 Surface free energy can predict the materials´ biological properties, such as cell
42 468 attachment, which is often used to measure the materials’ biocompatibility. A strong correlation
43 469 between the total surface energy and the cell attachment has been demonstrated It state that a
44 470 surface free energy value lower than 4, is correlated with a reduced cell attachment [71]. The
45 471 values recorded for the tested samples were close to this value (Table 3) which confirm their
46
47 472 biocompatibility.
48 473 The lowest γSL values were recorded for CS_PEO@P (2.08±0.68), CS_PEO@M_L
49 474 (2.33±0.34) and CS_PEO@M_L_P (2.88 ± 0.54), which means these polymeric solutions have
50
475 the best biocompatibility.
51
52
476
53
54 477
55 478
56 479
57 480
58
59 481
60 482
61
62
15
63
64
65
1
2
3
4 483 Table 3
5
6 484 The contact angle and γSL parameter values recorded for CS-PEO-ACs solutions.
7 485
8 Contact angle value (°)
9 Sample double-distilled formamide α-bromo- γSL (mN/m)
10
11
water naphtalene
12 CS_PEO@I_P 53.13 ± 0.15* 25.84 ± 0.85* 66.42 ± 0.63* 5.94 ± 0.75*
13
14 CS_PEO@M_L 43.94 ± 0.27* 49.45 ± 0.45* 33.91 ± 0.35 2.33 ± 0.34*
15
16 CS_PEO@P 104.47 ± 0.34* 99.78 ± 0.87* 57.31 ± 0.96* 2.08 ± 0.68
17
18 CS_PEO@P_C 52.41 ± 0.67* 35.94 ± 0.44* 33.91 ± 0.19 3.83 ± 0.39
19
20 CS_PEO@M_L_P 30.68 ± 0.38* 31.78 ± 0.72* 57.99 ± 0.66* 2.88 ± 0.54
21
22 CS_PEO 16.26 ± 0.77 8.10 ± 0.95 35.94 ± 0.79 3.73 ± 0.87
23
24 486 *where p˂0.05 when compared to CS_PEO
25
26 487 3.2.4. Swelling degree and remaining weight
27
28 488 The swelling degree and remaining weight values recorded for the CS-PEO-NFs are
29 489 presented in Fig. 3. It was noted that the swelling capacity of the tested samples in PBS solution
30
31 490 increased during the experiment; after 24 h the SD (%) was ranging between 74±0.34
32 491 (CS_PEO@P_C) and 181±0.46 (CS_PEO@I_P) (Fig. 3a). Except for CS_PEO@P_C, the SD
33 492 values recorded for the tested samples were higher than for CS-PEO (118±0.39). The lowest
34
35 493 value recorded for CS_PEO@P_C is possibly due to the saponin content of Calendula officinalis
36 494 infusion.
37
38 495
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
16
63
64
65
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
496 Fig. 3. Swelling degree (%) (a: PBS, b: PECF;forall samples, p˂0.05 when compared to CS_PEO),
22 497 remaining weight (%) (c) (PBS: CS_PEO@I_P, CS_PEO@M_L, CS_PEO@P, p ˂0.05; PECF:
23 498 CS_PEO@M_L, CS_PEO@P_C, CS_PEO@M_L_P, p ˂0.05 when compared to CS_PEO) and hemolysys
24
25
499 values (d) of CS-PEO-NFs(CS_PEO@P_C, CS_PEO@M_L_P, p ˂0.05 when compared to CS_PEO).
26
500 The values recorded in PECF were higher than in PBS (Fig. 3b). The absorption capacity
27
28 501 increased during the first 2 h, within values ranging between 284±0.90 (CS_PEO@I_P) and
29 502 509±0.24 (CS_PEO). At the end of the experiment the values recorded for the swelling capacity
30
503 ranged between 280±0.56 (CS_PEO@I_P) and 374±0.87 (CS_PEO). Both results, in PBS and
31
32 504 PECF, support the capacity of the tested NFs to uptake physiological fluids, which is useful in
33 505 terms of wound surface interactions, to initiate the delivery of ACs incorporated into polymer
34 506 matrix and so to promote the biological effects. CS-NFs mimic the extracellular matrix and the
35
36 507 tested samples display good biodegradable properties (Fig. 3c). The degradation process was
37 508 more pronounced in PBS than in PECF. After four weeks, the NFs mats lost up to 60% of their
38 509 initial weight in PBS.
39
40
41 510 3.5. In vitro hemocompatibility assay
42
43 511 Hemocompatibility of blood-contacting biomaterials is one of the most important criteria
44 512 for their successful application as wound dressing [72]. According to ISO 10993-4 [73], the
45 513 materials whose hemolytic index is less than 5% are considered safe and hemocompatible. It was
46
514 reported that CS has a low hemolytic index [18,74] and PEO is biocompatible, hemocompatible
47
48 515 and non-toxic [75]. In our experiment, all samples, including CS_PEO, showed hemolysis values
49 516 less than 4, which resembles a good hemocompatibility (Fig. 3d). The most biocompatible NFs
50
517 seems to be CS-NFs loaded with propolis and Calendula officinalis infusion (CS_PEO@P_C),
51
52 518 with an index of 2.92±0.16%. Good hemocompatibility was shown also for CS-NFs loaded with
53 519 propolis (CS_PEO@P) and propolis and insulin (CS_PEO@I_P), for which the hemolysis index
54 520 was 3.20±0.09% and 3.33±0.14%, respectively. The Manuka honey-encapsulating NFs displayed
55
56 521 a hemolysis of 3.78±0.08% (CS_PEO@M_L) and 3.40±0.04 respectively (CS_PEO@M_L_P).
57 522 The data suggest that all samples meet the hemocompatibility criteria of biomaterials and may be
58 523 used without causing any acute hemolysis.
59
60
61
62
17
63
64
65
1
2
3
4 524 3.6. In vitro cytotoxicity assay
5
6 525 The cell viability (%) recorded at all tested contents (12.5 g/mL, 25 g/mL, 50 g/mL,
7
8
526 100 g/mL) exceed 90% (Fig. 4), which means that CS-PEO-NFs are noncytotoxic, in
9 527 agreement with ISO 10993-5 [56]. At 100 g/mL, the lowest cell viability value was recorded
10 528 for CS_PEO@M_L (88±2.30). Our results are in agreement with previously published data on
11 529 CS-NFs including honey [40].
12
530
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28 531
29 532
30
31 533 Fig. 4. Cell viability (%) of CS-PEO-NFs tested in presence of normal human dermal fibroblasts,
32 534 at different contents (CS_PEO@M_L_P, p ˂0.05when compared to CS_PEO).
33
34 535 3.7. In vitro antioxidant assays
35
36 536 An imbalance in the reactive oxygen species (ROS) production and biochemical
37 537 antioxidant mechanism may result in the onset of oxidative stress. ROS present in the wound site
38
39 538 may delay the normal rate of wound healing. An enhanced antioxidant activity [76] suggests the
40 539 ability to control the overproduction of ROS and promote wound repair [77].
41 540 The analysis of the data presented in Fig. 5 revealed that the best antioxidant effect was
42
43
541 obtained for CS_PEO@P_C in all four assays. For this sample type the value of scavenging
44 542 ability for the DPPH radical was 60.79±2.08% (after 60 min of incubation) and 65.46±2.54%
45 543 (after 30 min of incubation), respectively (Fig. 5a). This effect could be explained mainly
46
544 through the high flavonoid content of Calendula officinalis infusion, because the sample which
47
48 545 contains only propolis (CS_PEO@P) didn’t present a noticeable effect. A correlation between
49 546 phenolic compounds, mainly flavonoids, and antioxidant activity, was also reported earlier
50 547 [44,78]. Moreover, it was also noted that the effect of CS_PEO@P_C was significantly better (p
51
52 548 ˂0.5) when compared to CS_PEO, used as negative control. Increased radical scavenging effects
53 549 were also noted for propolis and Calendulla officinalis infusion, both as CS-PEO-ACs polymeric
54 550 (Fig. 6a) and ACs (Fig. 6b) solutions.
55
56 551 Our results were even better than other studies that reported the DPPH scavenging
57 552 activity of propolis/cellulose acetate/polycaprolactone nanofibrous mats [67] and polyamide-
58 553 6/propolis electrospun fibers [79].
59
60
61
62
18
63
64
65
1
2
3
4 554 The high radical scavenging ability of the CS_PEO@P_C is strongly supported also by
5
6 555 the data of the ABTS assay. The ABTS inhibition recorded for CS_PEO@P_C was
7 556 96.83±0.18%, being substantially higher than for CS_PEO@P (15.59±0.49%) and for CS_PEO
8 557 (3.69±0.56%) (Fig. 5b). CS_PEO@P_C showed important antioxidant effects measured as
9
10 558 PRAP and FRAP capacity (Fig. 5c, d), with increased absorbance values, i.e. 0.96±0.11 (PRAP)
11 559 and 0.85±0.07 (FRAP), respectively. Moreover, the values recorded for CS_PEO were 0.20±0.04
12 560 (PRAP) and 0.07±0.02 (FRAP).
13
14 561 For PRAP and FRAP assays it is important to note that all samples are more active than
15 562 CS-PEO. In addition, the PRAP assay showed an appreciable effect also for samples containing
16 563 Manuka honey, CS_PEO@M_L (0.89±0.06), CS_PEO@M_L_P (0.69±0.03) and propolis,
17
18
564 CS_PEO@P (0.61±0.13). In the FRAP assay, a noticeable effect was observed only for samples
19 565 containing Manuka honey, CS_PEO@M_L (0.36±0.06), CS_PEO@M_L_P (0.54±0.05). The
20 566 data are in good agreement with other studies which proved the antioxidant effect of Manuka
21
567 honey [80]and propolis [81,82] via PRAP and FRAP assays, based on their phenolic compounds
22
23 568 content.
24 569
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52 570 Fig. 5. The antioxidant effects of CS-PEO-NFs: antiradical scavenging effect (a: DPPH radical
53 571 scavengin effect; b: ABTS radical scavenging effect); c: PRAP capacity; d: FRAP capacity.1:
54
55 572 CS_PEO@I_P, 2: CS_PEO@M_L, 3: CS_PEO@P, 4: CS_PEO@P_C, 5: CS_PEO@M_L_P, 6:
56 573 CS_PEO.*p˂0.05, **p˂0.01, when compared to CS_PEO.
57
58
59
60
61
62
19
63
64
65
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16 574 Fig. 6. The DPPH antiradical scavenging effect of (a) CS-PEO-ACs polymeric solutions (1:
17 575 CS_PEO@I_P, 2: CS_PEO@M_L, 3: CS_PEO@P, 4: CS_PEO@P_C, 5: CS_PEO@M_L_P, 6:
18 576 CS_PEO).*p˂0.05, **p˂0.01, when compared to CS_PEO and (b) ACs solution (1: M, 2: L, 3:
19
20 577 P, 4: C).
21
22 578 3.2.8. Antimicrobial assay
23
24 579 Agar disk diffusion method is often used as a means of determining the antimicrobian
25 580 effects of NFs [83]. Most of reported studies use only two bacterial strains, Staphylococcus
26
581 aureus and Escherichia coli, which mean a limited view of antibacterial effects [84,85]. Our
27
28 582 study included also Pseudomonas aeruginosa, a multi-drug resistant Gram negative bacteria,
29 583 associated with severe nosocomial infections, and Candida albicans, a fungal strain. The
30 584 antimicrobial effects of the tested CS-PEO-NFs, expressed as diameters of the inhibition area
31
32 585 (mm) are presented in Table 4.
33 586
34 587 Table 4
35
588 Antimicrobial effects of the CS-PEO-NFs tested on bacterial and fungal strains.
36
37 589
38 Diameter of inhibition area (mm)
39 Sample S. aureus E. coli P. aeruginosa C. albicans
40 ATCC 25923 ATCC 25922 ATCC 27853 ATCC 10231
41
42 CS_PEO@I_P 26 24 25 25
43 CS_PEO@M_L 24 25 25 27
44 CS_PEO@P 25 0 25 27
45 CS_PEO@P_C 26 17 24 24
46
47
CS_PEO@M_L_P 25 20 26 25
48 CS_PEO 24 20 25 26
49 CIP (5µg/ disc) 26 28 30 nt
50 VRC (1µg/disc) nt nt nt 27
51
590 CIP = Ciprofloxacin, VRC = Voriconazole, nt = untested
52
53 591
54 592 It was noted that the samples showed good antibacterial effects, in most cases with
55 593 inhibition diameter higher than 24 mm, much better than other studies have reported [66,84,86].
56
57 594 For example the NFs including honey, developed by Tang et al. [66], displayed a diameter of
58 595 inhibition area of up 11.3 mm for Escherichia coli and up to 13.6 mm for Staphylococcus
59 596 aureus. In our study the sample including Manuka honey (CS_PEO@M_L, CS_PEO@M_L_P)
60
61
62
20
63
64
65
1
2
3
4 597 display improved inhibition diameter for both bacterial strains (24 mm/25 mm for
5
6 598 Staphylococcus aureus and 25 mm/20 mm for Escherichia coli).
7 599 The most active were CS_PEO@I_P and CS_PEO@P_C, with their effect being
8 600 identical to Ciprofloxacin, used as control. Concerning the antifungal assessment, the effect of
9
10 601 CS_PEO@M_L and CS_PEO@P was similar to Voriconazole. Considering the infection risk of
11 602 wounds, the dressing materials which possess antibacterial activity are of high interest given
12 603 their beneficial effects towards wound healing.
13
14 604
15
16 605 4. Conclusion
17
18 606 New chitosan/polyethylene oxide biomimetic and bioactive nanofibers were prepared and
19 607 characterized in terms of physico-chemical and structural features including, morphology,
20
21 608 swelling degree, surface free energy parameters, biodegradation, biocompatibility, cytotoxicity
22 609 and were tested for their antioxidant and antimicrobial effects. The developed CS/PEO-ACs
23 610 nanofibers showed good swelling degrees, in both PBS and PECF medium, which supports their
24
611 capacity to absorb wound exudate. The CS/PEO-ACs nanofibers showed a good
25
26 612 biodegradability, increased hemocompatibility and reduced cytotoxicity degree. The most suited
27 613 sample type for wound healing applications seems to be CS-PEO@P_C which, besides showing
28
614 improved physiological liquids uptake and being biocompatible, biodegradable and non-toxic,
29
30 615 showed also increased antioxidant effects in terms of radical scavenging ability (DPPH, ABTS)
31 616 and antioxidant capacity (PRAP and FRAP). In addition, CS-PEO@P_C presents increased
32 617 antimicrobial effects, especially for Staphylococcus aureus and, even similar to Ciprofloxacin,
33
34 618 which is the most relevant parameter, because it is known that wound infection, leads to a delay
35 619 of the healing process. It can be concluded that the developed CS/PEO-ACs nanofibers are very
36 620 promising biomaterials for wound care, especially CS-PEO@P_C.
37
38 621
39 622 Acknowledgements
40 623 This work was financially supported by UEFISCDI grants PN-III-P1-1.1-MC-2017-2462, PN-
41
42 624 III-P1-1.1-MC-2018-2451, PN-III-P4-ID-PCE-2020-2687, COST-STSM-Request-CA16122-
43 625 45799, COST-CA19140, AUF-IFA 2019-2020 (contract no. 28/2019), University of Medicine
44 626 and Pharmacy “Grigore T. Popa” of Iasi (contract no. 27496/20.12.2018) and by the European
45
46
627 Social Fund for Regional Development, Competitiveness Operational Programme Axis 1,
47 628 InoMatPol, ID P_36_570(contract no. 142/10.10.2016).S. Van Vlierberghe would like to
48 629 acknowledge Ghent University for providing a BOF grant from the Special Research Fund. Also
49
630 authors thank to Department of Science and Engineering of Oxide Materials and Nanomaterials,
50
51 631 Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest for
52 632 acquisition of SEM images of CS/PEO_ACs NFs.
53
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International Journal of Biological Macromolecules

Design, optimization and in vitro characterization of novel biomimetic and bioactive

Article Type: Research Paper

Section/Category: Carbohydrates, Natural Polyacids and Lignins

Keywords: chitosan, nanofibers, biocompatibility, biological effects, Wound dressings

2. Materials and methods

3.2. Characterization of the CS-PEO-NFs

3.6. In vitro cytotoxicity assay

3.7. In vitro antioxidant assays

3.2.8. Antimicrobial assay

3.2. Characterization of the CS-PEO-NFs

3.6. In vitro cytotoxicity assay

3.7. In vitro antioxidant assays

3.2.8. Antimicrobial assay

Reviewers’ comments Response to reviewers

sulphonated 18 28 ND 17 Ekambaram & Dharmalingam,Mat Sci Eng C,

The correponding paragraph was reformulated as follow:

Prof. Dr. Lenuta Profire

Design, optimization and in vitro characterization of novel biomimetic and

Design, preparation and in vitro characterization of biomimetic and

Keywords: Chitosan Nanofibers, Biocompatibility, Biological effects, Wound dressings

Design, preparation and in vitro characterization of biomimetic and

Keywords: Chitosan Nanofibers, Biocompatibility, Biological effects, Wound dressings

1 Design, preparation and in vitro characterization of biomimetic and bioactive

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