pharmaceutics
Review
Electrospinning Proteins for Wound Healing Purposes:
Opportunities and Challenges
Alma Akhmetova
and Andrea Heinz *
LEO Foundation Center for Cutaneous Drug Delivery, Department of Pharmacy, University of Copenhagen,
2100 Copenhagen, Denmark; alma.akhmetova@sund.ku.dk
* Correspondence: andrea.heinz@sund.ku.dk
Abstract: With the growth of the aging population worldwide, chronic wounds represent an increasing burden to healthcare systems. Wound healing is complex and not only affected by the patient’s
physiological conditions, but also by bacterial infections and inflammation, which delay wound
closure and re-epithelialization. In recent years, there has been a growing interest for electrospun
polymeric wound dressings with fiber diameters in the nano- and micrometer range. Such wound
dressings display a number of properties, which support and accelerate wound healing. For instance,
they provide physical and mechanical protection, exhibit a high surface area, allow gas exchange,
are cytocompatible and biodegradable, resemble the structure of the native extracellular matrix,
and deliver antibacterial agents locally into the wound. This review paper gives an overview on
cytocompatible and biodegradable fibrous wound dressings obtained by electrospinning proteins
and peptides of animal and plant origin in recent years. Focus is placed on the requirements for
the fabrication of such drug delivery systems by electrospinning as well as their wound healing
properties and therapeutic potential. Moreover, the incorporation of antimicrobial agents into the
fibers or their attachment onto the fiber surface as well as their antimicrobial activity are discussed.
Keywords: antibacterial; antimicrobial; biomaterial; infection; microfibers; nanofibers; scaffold; tissue
engineering; wound dressing
Citation: Akhmetova, A.; Heinz, A.
Electrospinning Proteins for Wound
Healing Purposes: Opportunities and
1. Wound Healing and Electrospun Wound Dressings
Challenges. Pharmaceutics 2021, 13, 4.
The wound healing process is associated with four overlapping and well-orchestrated
stages: homeostasis, inflammation, proliferation and remodeling. Each stage involves a
cascade of events to ensure prevention of blood loss, elimination of bacterial contamination,
regeneration and formation of a new skin tissue, respectively. A variation from the norm
in this process results in a delay or prolongation of any of the healing stages, which in
turn leads to impaired healing [1]. The interruption in the healing process may occur
for a number of reasons connected to one’s lifestyle and health condition. For example,
smoking, malnutrition, obesity, low mobility, neuropathy, diabetes, vascular diseases and
skin disorders have been linked to the increasing chronicity of wounds, where healing has
not been achieved within 3–6 weeks [2–4].
Compromised wound healing represents a complex problem of multiple dependent
molecular and cellular processes that are closely intertwined. A slight dysregulation in
those processes leads to a development of a chronic non-healing condition, which requires
a combinational approach of diverse strategies to facilitate healing. Different polymeric
wound dressings have been created to supply favorable environment for wound healing,
to absorb exudate, allow vapor exchange across the scaffold, maintain moist conditions,
provide mechanical support and protect from further bacterial contamination. Such wound
dressings have also been employed to deliver active agents such as antibiotics, antiseptics,
anti-inflammatory drugs and biomolecules to direct the healing process to reach complete
healing [5,6] (Figure 1).
https://dx.doi.org/10.3390/pharma
ceutics13010004
Received: 18 November 2020
Accepted: 18 December 2020
Published: 22 December 2020
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license (https://creativecommons.org/
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Pharmaceutics 2021, 13, 4. https://dx.doi.org/10.3390/pharmaceutics13010004
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Pharmaceutics 2021, 13, 4
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Figure 1. Functions of protein-based nanofibrous mats with incorporated antimicrobial agents for wound healing. They allow fibroblast adhesion (often through cell-recognizing motifs the fiber carries), oxygen exchange and show bacteriostatic or
bactericidal activity.
The leading causes of non-healing chronic wounds are infection, pathological inflammation and formation of biofilms [2,6]. Therefore, wound care usually comprises of
debridement followed by antimicrobial treatment and application of wound dressings.
Debridement is required to clean the wound bed from exudate, necrotic tissue and bacterial load. Antimicrobial treatment prevents further bacterial growth and formation of
biofilms. Antimicrobial agents usually follow one or several strategies to attack bacterial cells, including disruption of the bacterial cell wall, interruption of nucleic acid and
protein synthesis, and dysregulation of metabolic pathways [7] (Figure 1). Antiseptics,
antibiotics or other biomolecules are either applied directly or incorporated into a wound
dressing [6]. In comparison to systemic administration of antimicrobial treatments, topical
application requires lower concentrations, displays fewer side effects and lowers the risk
of developing antibiotic resistance [6,8]. Topical application of antimicrobial agents such as
antibiotics often combines a rapid initial release to kill bacteria or inhibit bacterial growth
followed by a slower release to prevent further bacterial growth [9]. In order to prevent
development of microbial resistance to antibiotics, silver nanoparticles have been used
in certain materials for wound healing instead of antibiotics. However, recent studies
demonstrate that bacterial resistance also occurs against silver nanoparticles due to an
induction of nanoparticle aggregation as a result of the production of adhesive proteins by
the bacteria. This problem can be overcome by additional stabilization of the nanoparticles
by surfactants or polymers [10,11].
A variety of wound dressings facilitating wound healing are currently available on
the market and new advanced materials are being developed (e.g., films, hydrogels, foams,
hydrocolloids and nanoparticles). In particular, large research efforts have been directed to
fabricate nanofibers [5,12,13]. Unlike other types of biomaterials, nanofibers stand out due
to their unique structure and the tunability of their physical and mechanical properties.
Their versatility and the easy fabrication process facilitate obtaining materials with desired
Pharmaceutics 2021, 13, 4
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characteristics for the complex wound healing process. High surface area and homogenous
drug distribution makes nanofibers attractive as drug delivery systems with high drug
loading capacity and controlled release. Resemblance of nanofibers to collagen or elastin
fibers in the extracellular matrix (ECM) of healthy skin allows them to provide additional
support for fibroblasts and keratinocytes, which adhere to the fibers, migrate across the
wound bed and help regenerate and close the damaged tissue. Modifications of the surface
morphology of nanofibers and the porosity of the nanofibrous matrix further promote
adherence and migration of these cells [12] (Figure 1). However, even though electrospun
fibers are often have a high porosity, this property is dependent on the fiber diameter and is
difficult to control. This may also limit cell penetration into the scaffold in some cases [14].
A variety of methods to fabricate fibers have been developed over the years and mainly
include solution and melt electrospinning [15]. This review focuses on nanofibers created
from protein solutions using the solution electrospinning process. Electrospinning is based
on applying a high voltage to a polymer solution to transform a drop at the needle tip into
a cone shape in order to generate a jet. The ejected jet undergoes a number of instabilities,
during which the solvent from the solution is evaporated and dry fibers are collected on the
grounded or oppositely charged plate. The process is shown in Figure 2. The morphology,
diameter size and distribution of electrospun fibers can be adjusted and tuned according to
the solution (e.g., concentration, molecular weight, viscosity, conductivity, surface tension,
dielectric constant, evaporation rate and dipole moment) and process parameters (e.g.,
temperature, humidity, flow rate, voltage and working distance) [16]. For example, larger
fiber diameter is often associated with higher flow rate, higher applied voltage and lower
distance between the needle tip and the collector. However, there are exceptions to these
rules as for instance a higher voltage may lead to more solution deposition [15]. Therefore
both, the properties of the solution and the process parameters should be considered during
optimization of the electrospinning process [16].
Figure 2. Electrospinning process. A polymer solution is subjected to a high voltage output to create a polymer jet that
deposits as dry fibers on the collector.
The most widely used type of solution electrospinning is single-nozzle electrospinning
(also known as blend electrospinning), which itself has a few subcategories with some
variations including co-axial and emulsion electrospinning [15]. These techniques are commonly employed to incorporate drugs, including active biomolecules, and are summarized
in Figure 3. In blend electrospinning, the drug is mixed into the polymer solution—in this
Pharmaceutics 2021, 13, 4
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case the protein solution—directly. In contrast, in co-axial electrospinning two different
solutions are used, and the drug is incorporated either in the outer (shell) or inner (core)
solution [17]. Additionally, the drug can be incorporated into an emulsion to be electrospun,
where the final product is similar to that obtained by co-axial electrospinning due to the
lengthening of the emulsion within the jet, which creates a core-shell structure [18,19].
The electrospinning technique is chosen depending on the solubility of the polymer in a
particular solvent, as well as its stability during the electrospinning process and the desired
release kinetics of the electrospun nanofibers. During blend electrospinning, organic and
sometimes highly toxic solvents are commonly used and may affect structure, stability and
activity of the drug. Therefore, co-axial and emulsion electrospinning provide an alternative, where the drug can be dissolved in a more favorable solvent [20–23]. Nevertheless,
all of these techniques involve high voltage, which may potentially damage the therapeutic
agent [22,23]. In such a case, there is another method that is based on a functionalization of
the nanofiber surface after electrospinning by attachment of the drug (Figure 3). However,
a drawback of functionalization of the fibers as compared to other methods, where the drug
is incorporated into the fibers, is that the drug lacks a coating material, which normally
acts as a protective layer to provide longer shelf life [21].
Figure 3. Fabrication of protein-based electrospun fiber mats by different types of solution electrospinning, namely blend,
co-axial and emulsion electrospinning. The protein is first dissolved in a volatile solvent and starts unfolding, which is
a prerequisite for successful electrospinning of proteins. In blend electrospinning, the active agent is directly added to
the polymeric protein solution. In co-axial electrospinning, the active agent is either dissolved in the shell or the core
solution. In addition to the protein solution, a second natural or synthetic polymer is used in co-axial electrospinning.
In emulsion electrospinning, the drug is dissolved in the emulsion droplets (inner phase). In addition, the fibrous mat can
be functionalized by adding the active agent after electrospinning.
Pharmaceutics 2021, 13, 4
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2. Proteins as a Promising Starting Material for Electrospun Wound Dressings
The initial use of synthetic polymers in electrospinning has noticeably shifted towards
implementation of natural polymers such as proteins and carbohydrates [24]. In comparison to synthetic polymers such as polylactic acid (PLA) [25] and polyurethane (PU) [26,27],
natural polymers do not purely rely on the use of harsh and toxic organic solvents for
dissolution. Therefore, they provide an environmentally friendly alternative, which may
additionally offer better drug stability and activity as compared to pharmaceutical standard formulations, safer manufacturing and the possibility of an application on skin [28].
However, this comes at a cost of easy fabrication and reproducibility. Evaporation rate,
surface tension and conductivity of the employed solvent greatly affect electrospinnability
of the protein solution [15]. Moreover, electrospinning of proteins is more challenging
due to the intrinsic variations in complexity of their structures, molecular weight, surface
charge as well as ionic, hydrogen and disulfide bonds [29,30]. The electrospinnability of
proteins depends not only on their solubility in a specific solvent, but also on the degree of
protein unfolding in a particular solvent [29,31] and chain entanglement [31,32] (Figure 3).
The choice of the solvent further affects crystallinity, mechanical properties, fiber size and
morphology [29,31]. Therefore, the addition of synthetic polymers is often necessary to
electrospin the solution continuously and without artifacts [24,33].
Proteins demonstrate attractive features as antimicrobial delivery system due to their
natural origin, fast biodegradability and cytocompatibility [24,34]. Proteins used in electrospinning for wound healing applications are mainly obtained from two distinct sources:
plants and animals [13,33]. Their stability, activity and degradation depend on the protein
size, chemical structure, isolation and purification processes [5,35]. Different methods
for protein extraction and purification may affect the obtained raw material’s purity and
composition [5,36,37], which in turn impacts reproducibility of the electrospinning process
and properties of the final product [38].
Some of the main differences between plant- and animal-based proteins are their
availability and price. Plant proteins tend to be available in larger amounts and at a
lower cost [31,34,39,40]. As compared to synthetic polymers, proteins are in general
more challenging to electrospin due to their heterogeneity in structure and surface charge,
solvent-dependent protein unfolding and low viscosity, which lead to a non-continuous
electrospinning process and formation of beads [24,29]. Moreover, the final product may
lack stability in water, resulting in a loss of fiber structure [41,42]. To compensate for these
drawbacks, different strategies have been implemented that include the use of cross-linking
agents, toxic organic solvents and addition of synthetic polymers [33].
3. Electrospinning of Plant-Derived Proteins for Wound Healing Purposes
Plant proteins that have been used to prepare electrospun wound dressings alone or
in combination with other natural and/or synthetic polymers are summarized in Table 1.
These include zein protein, soy protein and pea protein.
Table 1. Electrospun plant-based proteins with antimicrobial activity.
Solvent
Antimicrobial
Agent
Uniaxial
Uniaxial
Co-axial
Water
NaOH
AA
CA
None
ATPPB
PU/CA
Uniaxial
DMF, MEK
Streptomycin
HA
PU
PCL, GA
Uniaxial
Uniaxial
Uniaxial
TFE, AA
DMF, THF
FA, AA
Salicylic acid
Ag NPs
GA
Protein
Co-Polymer
Pea
Soy
Zein
PVA, CA
PEO
None
Zein
Zein
Zein
Zein
Electrospinning
Type
Tested Bacterial Strain
Reference
E. coli, L. monocytogenes
S. aureus, P. aeruginosa
E. coli, S. aureus
E. coli, S. typhimurium,
V. vulnificus, S. aureus,
B. subtilis
S. aureus
E. coli, S. aureus
E. coli, S. aureus
[43]
[44]
[45]
[26]
[46]
[27]
[47]
Pharmaceutics 2021, 13, 4
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Table 1. Cont.
Electrospinning
Type
Solvent
Antimicrobial
Agent
Tested Bacterial Strain
Reference
Uniaxial,
multilayer
FA, AA
GA, C. officinalis
E. coli, S. aureus
[48]
PCL
Uniaxial
TFE, DCM
MRSA
[49]
Zein
Zein
Zein
Zein
None
None
None
None
Uniaxial
Uniaxial
Uniaxial
Co-axial
EtOH, water
EtOH, water
EtOH, water
EtOH, water
E. coli, S. aureus
E. coli, S. aureus
E. coli, Bacillus
E. coli
[50]
[51]
[52]
[53]
Zein
PEO
Co-axial
EtOH, water
E. coli, S. aureus
[54]
Zein
GT, PLA
Uniaxial
EtOH, water,
CHL
S. aureus, P. aeruginosa
[55]
Protein
Co-Polymer
Zein
PCL, GA
Zein
Tetracycline
hydrochloride
Ag NPs
Gentamicin
Ag NPs
OEO
Tetracycline
hydrochloride
Tetracycline
hydrochloride
Key: AA, acetic acid; ATPPB, allyltriphenylphosphonium bromide; CA, cinnamaldehyde; CHL, chloroform; DCM, dichloromethane; DMF,
N,N-dimethylformamide; EtOH, ethanol; FA, formic acid; GA, gum arabic; GT, gum tragacanth; HA, hyaluronic acid; MEK, methyl ethyl
ketone; MRSA, methicillin-resistant S. aureus; NaOH, sodium hydroxide; OEO, orange essential oil; PLA, polylactic acid; PU, polyurethane;
TFE, 2,2,2-trifluoroethanol; THF, tetrahydrofuran.
3.1. Zein Protein
Among the available plant-derived proteins, zein protein is extensively being used for
a variety of drug delivery systems such as films, gels, nanoparticles and nanofibers [56,57].
Its self-assembling nature and insolubility in water have made it interesting for application in
surface protection for food packaging, a variety of sensors and air filtration [58] as well as
vaccines and tissue engineering. Zein protein is extracted from corn seed and is categorized
into prolamines (α and δ) and glutelins (β and γ). It does not carry a nutritional value due
to the lack of the key amino acids lysine and tryptophan required for human diet. However,
the high amount of glutamic acid, proline, alanine and leucine in zein protein are responsible
for its hydrophobic nature [57]. Zein may carry genes that cause immunogenic reaction [59],
and studies on oral [60] and intramuscular administration [61] reveal controversial results.
Despite its wide use, the tertiary structure of zein still remains unknown and is not available
on the Protein Databank. Only a few studies have attempted to hypothesize on its structure, providing varying results from antiparallel helices forming a cylinder [62,63], triple
superhelices [64] to antiparallel helices arranged in hexagonal repeats [65].
For wound healing applications, zein protein has mostly been used either as film [66,67]
or nanofibrous scaffold [26,50]. One of the major advantages of electrospinning zein is
that the use of toxic solvents and cross-linkers can be avoided due to its sufficient solubility in aqueous ethanol and self-assembling nature, respectively [56,68,69]. However,
aqueous ethanol is not an ideal solvent for electrospinning zein protein [70]. Due to its
high evaporation rate, it leads to needle clogging, formation of ribbon-shaped fibers and
results in poor water stability of the fibers, which in turn leads to the loss of porous
structure of the fiber mat upon contact with water [41,42]. This can be overcome either
by cross-linking zein with UV light [71] or by co-axial electrospinning with ethanol [41]
or polyethylene oxide (PEO) as a shell solution [54]. The behavior of zein in aqueous
ethanol solution is described in more detail elsewhere [72]. There are various studies,
which focus on electrospinning zein to produce antimicrobial wound dressings (Table
1). Zein has been electrospun alone [45,50–53], together with synthetic polymers such as
polycaprolactone (PCL) [47,48], PEO [54], PU [27] and PLA [55] as well as with natural
polymers and substances including gum arabic [47,48], hyaluronic acid [46], cinnamaldehyde [26] and gum traganth [55]. Antimicrobial agents that have been incorporated into
zein-based fibers include a wide range of antibiotics [26,45,46,49,51,54,55], antibacterial
nanoparticles [27,50,52] as well as antimicrobial plant extracts [46,73].
It is worth mentioning that peptides produced from thermolysin-based hydrolysis
of zein protein are able to induce the production of angiotensin converting enzyme in-
Pharmaceutics 2021, 13, 4
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hibitor (ACEI) [74]. Inhibition of angiotensin receptors or conversion of angiotensin I to
angiotensin II by ACEI have shown to accelerate wound closure and prevent scar tissue
formation when ACEIs are administered topically [75] and orally [73], respectively. Further, more structurally organized collagen fibers resembling normal skin structure were
observed at the wound site due to the inhibition of tumor growth factor-β1 expression,
fibroblast proliferation and collagen production after ACEI administration [73,75].
3.2. Soy Protein
Unlike zein, soy proteins are widely researched not only for food packaging [76],
but also for their use in food products [77,78] due to their high availability and affordability [31]. However, in comparison to zein, soy protein has not been reported to be
electrospun on its own. Polymers such as polyvinyl alcohol (PVA) [30,79], PCL [80] and
PEO [44,76,81,82] are required to achieve a bead-free morphology and overcome brittleness
of the material (Table 1). Only a limited number of studies have, therefore, focused on
applying soy-based nanofibers for wound healing [44,81–83], even though soy protein
contains reactive amino acid residues such as arginine, glycine, aspartic acid and glutamine
that facilitate wound healing through cell attachment and proliferation [83–86]. Higher cell
proliferation in vitro has been demonstrated in electrospun soy protein in comparison to a
solvent cast film, which has been attributed to the porous nanofibrous matrix that allows
better nutrient access [83].
Soy protein isolates reach approximately 90% purity, and the presence of plant estrogens and isoflavones in the isolates demonstrates controversial biological impact [87,88].
On the one hand, isoflavones possess valuable antioxidant, antimicrobial and antiinflammatory properties [88]. In fact, the innate antimicrobial effect of soy protein against
E. coli and S. aureus has been shown (Table 1) [44]; however, nobody has attempted to
incorporate antimicrobial agents into soy protein-based nanofibers yet. On the other
hand, isoflavones have been shown to have carcinogenic and immunosuppressive effects.
The latter negatively affect the production of nitric oxide [88], leading to delayed wound
healing [89]. Moreover, soy protein isolates have been shown to contain two major allergens, glycinin and β-conglycinin [38,90]. However, to the best of our knowledge no study
has focused on these side effects associated with the development of soy protein-based
wound dressings. Therefore, more studies are required to better understand the influence
of soy protein biomaterials on wound healing.
3.3. Pea Protein
In comparison to other proteins, pea seed-derived proteins have only recently been
electrospun into nanofibers [29,43]. Pea seeds, unlike soybean, contain fewer proteinase
inhibitors and less phytic acid, resulting in lower allergic reaction in humans. Pea proteins
mostly consist of globulins up to 65% and albumin of around 25% [91]. Electrospinning
of pea protein remains a challenge due to the protein’s globular nature and absence of
molecular entanglement [43,92]. Despite the addition of PVA [43] or the use of a variety
of solvents [29], the fabricated fibers have been demonstrated to show heterogeneous
size distribution and the presence of artifacts. There is only one single study that has
explored incorporation of an antimicrobial agent (cinnamaldehyde) into pea protein-based
nanofibers, which showed a pronounced effect against E. coli and Listeria monocytogenes
(Table 1) [43]. However, even though cinnamaldehyde carries anti-inflammatory, antimicrobial, antifungal and anti-biofilm features, it is a well-known allergen that could cause
burning sensation [93,94]. Therefore, its application in wound healing should be considered
with caution.
Recent research using artificial intelligence and a deep learning approach predicted a
peptide within the pea protein genome with anti-aging properties that has a potential to be
used in wound healing. In particular, this peptide has been shown to facilitate proliferation
of keratinocytes and induce production of elastin and collagen from fibroblasts. The in vitro
Pharmaceutics 2021, 13, 4
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wound scratch assay demonstrated promising results of 40% decrease in wound area in
comparison to a control after 48 h of incubation [95].
4. Electrospinning Animal-Derived Proteins for Wound Healing Purposes
A lot of animal-derived proteins used in electrospinning are obtained from milk,
including casein, whey, lactoferrin and lysozyme, or connective tissue, such as collagen and
elastin. In comparison to plant-derived proteins that mostly require an incorporation of
antimicrobial agents, proteins obtained from milk possess innate antimicrobial properties
due to their iron-binding properties [96,97] and ability to disrupt bacterial cell walls [96,98].
Therefore, such proteins carry a dual function as a material basis with antimicrobial effect.
4.1. Casein
Casein has recently gained increasing attention as a biodegradable, cytocompatible,
self-assembling and highly available starting material for electrospinning. It is derived from
cow’s milk, comprises around 80% of the milk’s protein content and is potentially allergenic.
Approximately 55% of casein consists of polar amino acid groups, which allow the formation
of hydrogen bonds. This property facilitates the formation of films, but also leads to casein’s
poor electrospinnability, which is further compromised due to the low viscoelasticity of it in
solution [99–102]. Therefore, electrospinning fibers from casein is only possible upon addition
of synthetic polymers such as PEO [99] and PVA [103]. Moreover, the high hydrophilicity of
this protein leads to weak mechanical strength and water stability, which requires the use of
toxic cross-linking agents such as glutaraldehyde and silane [99,103]. Antimicrobial activity
has not been reported for casein, however, oligopeptides released during enzymatic digestion
of casein demonstrated inhibition of several bacterial strains [104]. No antimicrobial drugs
have been reported to be incorporated into casein-based nanofibers. However, casein-PEO
nanofibers with and without silver nanoparticles showed pronounced inhibition zones against
E. coli and S. aureus (Table 2) [105,106].
Table 2. Electrospun animal-based proteins with antimicrobial activity.
Protein
Co-Polymer
Electrospinning
Type
Solvent
Antimicrobial
Agent
Tested Bacterial
Strain
Casein
PEO
Uniaxial
Water
Ampicillin
α-lactoglobulin
PEO
Uniaxial
Water
Ampicillin
Lactoferrin
Gelatin
Uniaxial
FA, DMF
None
Lysozyme
CS, PVA
Uniaxial
AA, water
CS
Keratin
PVA, PEO
CS, PHBA,
gelatin
Uniaxial
Uniaxial,
multilayer
NaOH
Ag NPs
E. coli, S. aureus
E. coli,
P. aeruginosa,
B. thailandensis
E. coli, S. aureus
S. aureus, B.
subtilis, S. flexnery,
P. aeruginosa
E. coli, S. aureus
HFIP
Mupirocin
E. coli, S. aureus
[111]
Collagen
PLGA
Uniaxial,
multilayer
HFIP
E. coli, S. aureus
[112–114]
Collagen
PCL
Uniaxial
HFIP
E. coli
[115]
Collagen
PLA
Uniaxial
HFIP
Vancomycin
hydrochloride,
gentamicin
sulfate
Enterobacteria
phage T4
Levofloxacin
[25]
Collagen
-
Uniaxial
HFIP
Ag NPs
Collagen
CS
PCL (core),
PEO (shell)
Alginatedialde-hyde
Uniaxial
0.5 M AA
HFIP, glacial
AA
ZnO
E. coli, S. aureus
S. aureus,
P. aeruginosa
S. aureus, E. coli
[117]
Doxycycline
n.a.
[118]
Ciprofloxacin,
gentamicin
P. aeruginosa, S.
epidermidis
[119]
Keratin
Collagen
Gelatin
Co-axial
Uniaxial
AA(40% w/w)
Reference
[99]
[107]
[108]
[109]
[110]
[116]
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Table 2. Cont.
Protein
Co-Polymer
Electrospinning
Type
Solvent
Antimicrobial
Agent
Tested Bacterial
Strain
Reference
Gelatin
-
Uniaxial
TFE
Vancomycin,
caspofungin
MRSA, C. albicans
[120]
Gelatin
PMETAC
Uniaxial
FA, AA
PMETAC
Gelatin
PVA
Uniaxial
FA
Silk sericin
CS
TFA
Silk sericin
PLLA
Uniaxial
Uniaxial,
multilayer
Centella asiatica
extract
CS
S. aureus, E. coli,
MRSA,
A. baumannii
S. aureus, E. coli,
P. aeruginosa
E. coli, B. subtilis
TFA
Nitrafurazone
E. coli, B. subtilis
[124]
Silk sericin
CS, PVA
Uniaxial
Water
Cephalexin
hydrate
E. coli, B. subtilis
[125]
Silk fibroin
PCL
Uniaxial,
multilayer
HFIP
CS
S. aureus, E. coli
[126]
Silk fibroin
-
Uniaxial
FA
Oleuropein
[127]
Silk fibroin
Silk fibroin,
sulfated fibroin
CS
Uniaxial
HFIP, TFE
CS
PEI
Uniaxial
FA
PEI
Silk fibroin
-
Uniaxial
FA
Ag NPs
Silk fibroin
PVA
Uniaxial
Water
S. epidermidis,
E. coli
S. aureus, E. coli
S. aureus,
P. aeruginosa
S. aureus,
P. aeruginosa
S. aureus, S.
epidermidis, E. coli,
P. aeruginosa
Silk fibroin
PVA
Uniaxial
Water
Silk fibroin
Gelatin
Uniaxial
FA
Melaminemodified silk
fibroin
Silk fibroin
Silk fibroin
PCL
Uniaxial
HFIP
PEO
P(LLA-CL)
FA
HFIP
Silk fibroin
PCL, HA, PEO
Uniaxial
Uniaxial
Uniaxial,
multilayer
EGF,
ciprofloxacin
hydrochloride
LL-37
antimicrobial
peptide, EGF
Thyme essential
oil, doxycycline
monohydrate
Melaminemodified silk
fibroin
TiO2 NPs
Curcumin
FA, TFE, water
Thymol
Silk fibroin
CS, halloysite
nanotubes, PEO
Gelatin
Chlorhexidine
digluconate
Ceftazidime
Selenium NP
coating
[121]
[122]
[123]
[128]
[129]
[130]
[131]
S. epidermidis,
P. aeruginosa
[132]
S. aureus,
K. pneumoniae
[133]
S. aureus, E. coli
[134]
E. coli
S. aureus
S. aureus,
P. aeruginosa
[135]
[136]
S. aureus, E. coli
[138]
P. aeruginosa
[139]
S. aureus
[140]
[137]
Uniaxial
FA, AA, water
Uniaxial
FA
Uniaxial
FA
Uniaxial
HFIP, AA
Carboxymethyl
CS coating
S. aureus, E. coli
[141]
Silk fibroin
Uniaxial
HFIP, FA
Ag NP coating
S. aureus,
P. aeruginosa
[142]
Silk fibroin
Uniaxial
FA, water
Graphene oxide
coating
S. aureus, E. coli
[143]
Silk fibroin
Silk fibroin
Silk fibroin
Carboxymethyl CS
coating
Silk fibroin
PEO
Uniaxial
Water
Manuka honey
Silk fibroin
PEO
Uniaxial
Water
Cu2 O NPs
MRSA,
P. aeruginosa,
E. coli, S. aureus
S. aureus, E. coli
[144]
[145]
Key: AA, acetic acid; Ag NPs, silver nanoparticles; CS, chitosan; DMF, N,N-dimethylformamide; EGF, epidermal growth factor; FA, formic
acid; HA, hyaluronic acid; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; MRSA, methicillin-resistant S. aureus; NP, nanoparticle; PBS, phosphate
buffered saline; PCL, polycaprolactone; PEI, polyethylenimine; PMETAC, poly([2-(methacryloyloxy)ethyl] trimethylammoniumchloride);
PEO, polyethylene oxide; PHBA, poly(3-hydroxybutyric acid); PHBV, poly(3-hydroxybutyrate-co-3-hydroxyvalerate); PLLA, poly(L-lactic
acid); P(LLA-CL), poly(L-lactic acid-co-e-caprolactone); TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol.
Pharmaceutics 2021, 13, 4
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4.2. Whey
Whey is a globular protein found in milk and is obtained as a by-product of processing
dairy products such as casein and cheese. Whey proteins mostly consist of β-lactoglobulin
and α-lactoglobulin [36], and minor fractions include lactoferrin and lysozyme among
others. The source and manufacturing process greatly affect the composition and functionality of whey proteins, which can reach approximately 80% purity. Whey sub-fractions
such as lactoferrin pose manufacturing and purification challenges [146]. Whey proteins
have a high nutritional value and demonstrate antimicrobial, antioxidant, and anticancer
properties [36,147]. Additionally, whey proteins are often used for their foaming, gelling
and emulsifying properties [36]. Whey proteins have been electrospun with PEO [148] and
PCL [149], however, an incorporation of antimicrobial agents has not been reported yet.
4.2.1. Lactoglobulins
β-Lactoglobulin is a self-assembling protein [150], which potentially bears antimicrobial
properties due to its iron-binding ability. It has been electrospun into nanofibers using PEO,
but antimicrobial properties have not been investigated [151]. α-Lactoglobulin is also a selfassembling protein [152] and has been electrospun after addition of PEO. α-Lactoglobulin
fibers alone did not demonstrate antimicrobial properties, but an incorporation of ampicillin
showed time- and concentration-dependent inhibition of E. coli (Table 2) [107].
4.2.2. Lactoferrin
Lactoferrin is a globular glycoprotein with nutritional, antimicrobial, anti-inflammatory
and anti-oxidant properties [146]. Due to its iron-chelating property, it exerts a wide range
of bacteriostatic effects on a variety of pathogens [96,97], including Gram-positive and
Gram-negative bacteria, viruses and fungi [153,154]. Lactoferrin is also a part of preimmune defense system and can be found in different bodily fluids. Apart from its
bacteriostatic properties, lactoferrin also has antimicrobial and antibiofilm function due to
its ability to directly bind to bacterial membranes and initiate disruption [154]. More recent research shows that lactoferrin also possesses antioxidant and anticancer activities,
and is involved in modulation of metabolic system [155]. Similar to other proteins, lactoferrin has been electrospun into fibers after addition of other polymers such as PCL [156],
poly(lactic) acid (PLA) [153] and gelatin [108] (Table 2). A synthetic analog of lactoferrin
is available as the peptide GRRRRSVQWCA, known as hLF1-11, which possesses similar
bactericidal properties [157]. Nevertheless, there is no record of the peptide being used in
electrospinning yet. It has, however, already been functionalized on the titanium surface
demonstrating inhibition of bacterial growth and reduction in biofilm formation [158].
4.2.3. Lysozyme
Lysozyme is a 14 kDa enzyme with antimicrobial properties due to its ability to hydrolyze polysaccharides in bacterial cell wall [96,98]. It is abundantly present in the liver
and bodily fluids such as saliva, tears and milk. Lysozyme is well known for its antimicrobial activity and is widely researched and used in the food industry and biomedical
field. It can be obtained from both plants or animals [98], with lysozyme from hen egg
white mainly being used in electrospinning. Purification of lysozyme from eggs needs to be
carried out carefully because of impurities from other egg proteins that may contaminate
the final product and lead to allergic reactions [37]. Lysozyme has either been incorporated into fibers by blend [159,160], co-axial [23] and emulsion electrospinning [161] or
has been functionalized on the surface of the fibers [109,162] (Table 2). Lysozyme is not
electrospinnable on its own and has been electrospun with a variety of polymers such
as PLGA [23] and poly(vinylpyrrolidone) (PVP) [160]. The disruption of molecular conformation and subsequent decrease in activity of lysozyme has been shown during the
use of organic solvents such as chloroform and dimethyl formamide and application of
high voltage during electrospinning. When lysozyme was separated from organic solvents,
Pharmaceutics 2021, 13, 4
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and dissolved in aqueous solutions in the case of co-axial and emulsion electrospinning,
its activity was retained approximately above 80%, despite the applied electric field [23].
4.3. Keratin
Keratin proteins are found in epithelial tissues and can be easily extracted from hair,
wool and feathers [110]. They have an ability to self-assemble into fibrous structures [163]
and as compared to other epithelial components such as collagen and elastin, keratin is
more cytocompatible and non-immunogenic [164,165]. Keratin proteins are insoluble in
water, biodegradable, possess strong mechanical properties and contain cell-binding motifs
such as the arginine-glycine-aspartic acid (RGD) motif [33,166,167]. These advantages have
led to its use in the fabrication of materials for biomedical purposes. Electrospinning keratin
is challenging due to its high molecular weight and low viscoelasticity [33]. Therefore,
keratin has been electrospun into fibers after addition of other polymers such as PVA [110],
PEO [110,168] and chitosan (CS) [111]. Better cytocompatibility is achieved at higher keratin
concentrations in the fibers [169]; however, higher keratin concentrations make it more
challenging to electrospin and often result in beaded morphology [168,169]. As keratin
does not possess an intrinsic antimicrobial property, a few studies have incorporated
silver nanoparticles [110], antibiotics such as mupirocin [111] and the bactericidal agent
irgasan [168] into keratin-based fibers (Table 2).
4.4. Silk Fibroin and Sericin
Silk is a natural biopolymer and product of the secretion process of a number of arthropod lineages, including wasps (nests), silkworms (cocoons) and spiders (webs). It consists
of two major components: fibroin and sericin. Silk sericin (SS) is the sticky protein on the
outside of silk strands, makes up 15–35% of silk cocoons of the Bombyx mori silkworm and
is normally removed during extraction of the more versatile silk fibroin (SF) [170]. However, SS exhibits a number of useful properties such as cytocompatibility, biodegradability,
moisture absorption as well as antioxidant, antibacterial and UV resistance properties [171].
SS has further been shown to accelerate wound healing and collagen production [172].
SF is the main component of the natural silkworm thread obtained from the Bombyx
mori silkworm. It contains polypeptide chains with molecular weights between 200 kDa
and 350 kDa, which are composed of repetitive blocks of hydrophobic heavy chains and
hydrophilic light chains linked by disulfide bonds. In addition, a glycoprotein P25 is noncovalently linked to the heavy and light chains and is responsible for integrity of the structure.
Heavy SF forms anti-parallel β-sheets and highly organized crystalline domains due to
hydrogen bonding, van der Waals forces and hydrophobic interactions in repetitive domains
of the protein. These crystalline domains are cross-linked to an amorphous matrix formed
in the non-repetitive domains of the protein, which comprises random coils, β-turns and αhelices. Altogether, this generates a semi-crystalline fishnet structure, in which the crystalline
areas absorb pressure and distribute it throughout the entire fibroin network. SF has been
increasingly used as a biomaterial for tissue engineering in the last decade due to its availability,
low cost, cytocompatibility, bioactivity, biodegradability, thermostability, ideal mechanical
properties and low immunogenicity [33,34]. Its low weight (1.3 g cm−3 ) and high tensile
strength (~4.8 GPa) make it ideal for the production of electrospun fibers and its oxygen
and water vapor permeability allow its use for wound healing purposes. The mechanical
properties of SF fibers can also be altered through methanol treatment after electrospinning,
which increases β–sheet crystallinity and reduces water solubility. The degradation of SF
occurs via enzymatic surface erosion [170]. It is worth noting that SFs from other, so-called
non-mulberry silkworms Antheraea assama and Philosamia ricini possess RGD cell-binding
motifs that allow for interactions between the cells and the biomaterial.
To date, SS has only been used in a few electrospinning studies for wound healing purposes and has been blended with other synthetic polymers including PVA [125]
and poly(L-lactic acid) (PLLA) [124] and natural polymers such as CS [123,125] to improve the antimicrobial and mechanical properties of the fiber mats (Table 2). It is worth
Pharmaceutics 2021, 13, 4
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noting that SS-CS nanofibers without addition of an active pharmaceutical component
showed antimicrobial activity against both E. coli and B. subtilis with 100% growth inhibition at a nanofiber concentration of 0.4 mg mL−1 . Due to the positive surface electrical charge of the composite nanofibers, the antimicrobial activity of the fibers was
better against Gram-negative E. coli [123]. In contrast to SS, there are multiple studies
that have used SF as a starting material for electrospun wound dressings. SF has been
electrospun alone [127,130,140,142,143], in combination with synthetic polymers such as
PCL [126,134,137], polyethylenimine (PEI) [129], PVA [131,132], poly(L-lactic acid-co-ecaprolactone) [136], PEO [135,137,138,144,145], and in combination with natural polymers
such as gelatin [133] and CS as well as its derivatives [128,138,141] to tune the mechanical
and physicochemical properties of the fiber mats (Table 2). In many cases, the electrospun
SF-based fibers have been post-treated with ethanol, methanol, acetone or glutaraldehyde to increase β-sheet crystallinity or cross-link the material to improve its water stability [130,132,135,140,142,143]. Antimicrobial agents that have been incorporated into
SF-based fibers include a range antibiotics [131,133,138,139], antimicrobial peptides [132],
antimicrobial nanoparticles [130,135] as well as natural substances such as essential oils
and their components [127,131,137] and manuka honey [144]. Moreover, SF fibers have
been functionalized with antibacterial nanoparticle coatings [140,142,143]. In a complex
wound healing study that is worth mentioning, the authors have incorporated epidermal
growth factor (EGF) into SF-PVA composite fibers, which were coated with ciprofloxacin
hydrochloride to achieve enhanced wound healing. The fibers were tested in a rabbit
wound model, and accelerated wound healing, enhanced re-epithelialization, and lead to
highly vascularized granulation tissue and higher wound maturity in the case of fiber mats
from non-mulberry SFs as compared to Bombyx mori SF-based mats [131]. In a second study,
the authors have made similar observations when they incorporated the antimicrobial
peptide LL-37 and EGF into the SF-PVA fibers [132].
4.5. Collagen and Gelatin
Collagen is the most abundant protein in mammals and the main component of
the ECM in different organs and tissues including skin, bone, blood vessels, tendon and
ligaments. Collagen fibrils are composed of cross-linked tropocollagen units that comprise
three polypeptide chains that form a right-handed triple helix stabilized by interstrand
hydrogen bonding and intrastrand n→π interactions. Cross-linking between adjacent
tropocollagen units (length ≤ 300 µm) stabilizes the growing collagen fibrils (length ≤
1 cm). Collagen plays a vital role in maintaining the biological and structural integrity of the
ECM through its high tensile strength and mechanical resilience. Of the 29 collagen types
of collagen, only a few fibril-forming, in particular collagen I, are used in the production
of collagen-based biomaterials. Different inherent properties such as biodegradability,
weak antigenicity, controllable mechanical properties, interaction with different cell types
and formation of three-dimensional scaffolds, make collagen a relevant material for tissueengineering and clinical applications [173,174]. During electrospinning care needs to be
taken as denaturation of the collagen conformation may occur as a result of high voltage.
Collagen is often co-electrospun with other synthetic polymers such as PCL to increase the
stability of the fibers. In vivo, biodegradability of collagen fibers is achieved by endogenous
collagenases, such as matrix metalloproteinases.
Gelatin is a soluble protein obtained by partial alkaline, acid or enzymatic hydrolysis
of porcine, bovine or fish collagen. The manufacturing process has an impact on the
properties of gelatin such as molecular weight and isoelectric point, with type A gelatin
from acidic pre-treatment and type B gelatin from basic hydrolysis showing isoelectric
points of 8–9 and 4–5, respectively. Gelatin dissolves in hot water and forms gels on cooling.
The sol-gel transition occurs at temperatures between < 20 ◦ C and 30 ◦ C, and gelation
involves a partial restoration of the triple helices of collagen in the gelatin polymeric chains.
Gelatin is widely used in electrospinning due to its biodegradability, easy availability,
low antigenicity, versatile usability and low cost. Due to the presence of the RGD motif,
Pharmaceutics 2021, 13, 4
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gelatin—just like collagen—shows the ability for cell attachment through integrin-mediated
interactions. Due to its solubility, gelatin needs to be cross-linked to increase its stability
and the stability of electrospun fibers in aqueous solutions [175].
There are various studies that have used collagen and gelatin as a starting material
for electrospun wound dressings. Collagen has been electrospun alone [116], together
with synthetic polymers PCL [115,118], PEO [118] and PLA/PLGA [25,112–114] as well
as the natural polymer CS [117]. Gelatin has also been electrospun alone [120], in combination with synthetic polymers such as PVA [122] and poly([2-(methacryloyloxy)ethyl]
trimethylammoniumchloride) (PMETAC) [121] and together with the natural polymer alginate dialdehyde [119] (Table 2). In some cases, electrospun collagen-, and in particular,
gelatin-based fibers have to be cross-linked, for instance through exposure to ethanol [119] or
glutaraldehyde [116,121,122] to improve the material’s water stability. Antimicrobial agents
that have been incorporated into collagen- and gelatin-based fibers include a wide range
of antibiotics [25,112–114,118–120], antibacterial nanoparticles [116,117] as well as plant extracts [122]. Interestingly, in one study wound dressings were prepared from mixtures of
PCL and collagen in different ratios, and a virus (enterobacteria phage T4) was incorporated
into the fibers. Systems composed of 30% PCL and 70% collagen with the virus found to be
most promising as a hemostatic wound dressing with bacterial antibiotic function [115].
4.6. Elastin, Tropoelastin and Elastin-Like Recombinamers
Elastin, the core protein of elastic fibers, is an essential protein of the ECM of vertebrates, which imparts elasticity and resilience to organs and tissues such as blood vessels,
lung, skin, ligaments and cartilage. It is composed of extensively cross-linked units of its
precursor tropoelastin. Elastin shows unique properties such as a half-life of >70 years, extreme durability, reversible stretchability and resistance towards chemical and mechanical
influences. The Young’s modulus of elastic fibers ranges between 300 kPa and 600 kPa,
and they can be stretched up to 220% of their original length and undergo billions of cycles
of extension and recoil without failing [176]. Elastin is able to mediate cell interactions such
as dermal fibroblast attachment and spreading via interaction with integrins αVβ3 and
αVβ5 [177], which is useful for wound healing purposes. Elastin peptides, which occur as
a result of enzymatic elastin cleavage during aging, disease and physiological processes,
display a number of bioactive effects when released into the blood stream, including stimulation of proliferation, protease expression, vasodilatation, apoptosis as well as chemotaxis
of smooth muscle cells, endothelial cells and monocytes [178]. These may be used in a
targeted way in the context of wound healing.
Elastin is challenging to work with as a starting material for the fabrication of biomaterials by electrospinning due to its resistance and insolubility. However, elastin from
bovine and porcine tissues can be pre-treated by acidic or basic hydrolysis forming α- and
κ-elastin, respectively, which improves elastin’s solubility. Drawbacks of such preparations are their heterogeneity in composition, and they may lose their cellular signaling
ability, and require cross-linking by glutaraldehyde vapors to improve the water stability
of the fiber mats. In recent years, recombinant human tropoelastin has been increasingly
used for the fabrication of biomaterials for various tissue-engineering applications due to
ethical concerns associated with the use of elastin from animals. Moreover, elastin-like
peptides and artificial silk-elastin-like proteins (SELPs) are increasingly being used due
to the possibility to control the amino acid sequences, chain lengths as well as chemical,
mechanical and biological properties [179–181]. SELPs contain several repeats of silk- and
elastin-like blocks based on conserved amino acid motifs of the two native proteins SF and
elastin, and they combine the high tensile strength of SF with the elasticity and resiliency
of elastin. While the SF units self-assemble into packed antiparallel β-sheet structures of
great mechanical strength, the elastin-like building blocks display high flexibility [181].
Examples for electrospun elastin-based systems with potential applications in dermal tissue
replacement and wound healing are given in Table 3.
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Table 3. Electrospun elastin-based fiber mats for wound healing applications.
Protein
Co-Polymer
Electrospinning
Type
Solvent
Potential Applications
Reference
Elastin
ELR
Elastin
SELPs
Tropoelastin
Tropoelastin
SELPs
Collagen, PCL
Gelatin, CA
Collagen
Silk fibroin
Uniaxial
Uniaxial
Uniaxial
Uniaxial
Uniaxial
Uniaxial
Co-axial
HFIP
TFE
AA
Water, FA
HFIP
HFIP
Water
Skin grafting, dermal substitute for burn wounds
Wound dressings, skin tissue engineering
Skin injuries caused by trauma and diseases
Wound dressings, skin regeneration
Dermal tissue engineering
Dermal tissue engineering
Biomedical applications, drug delivery
[182]
[183]
[184]
[181]
[185]
[186]
[187]
Key: AA, acetic acid; CA, cellulose acetate; ELR, elastin-like recombinamer; FA, formic acid; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; PCL,
polycaprolactone; SELP, silk-elastin-like protein; TFE, 2,2,2-trifluoroethanol.
To the best of our knowledge, no antimicrobial agents have so far been incorporated
into such elastin-based fibers; however, focus was placed on stimulating the actual wound
healing process by providing scaffolds that mimic the physicochemical properties of native
skin ECM and support invasion, attachment and growth of cells and, hence, wound closure.
It has indeed been shown in vitro in cell culture [182–186] and in animal models [182] that
such scaffolds accelerate wound healing through increasing fibroblast invasion, attachment
and proliferation. In particular, composite fibrous mats containing elastin/tropoelastin and
collagen proved to be beneficial as the properties of both elastin (elasticity) and collagen
(tensile strength) are combined. In such formulations, elastin decreases the stiffness of the
scaffold, thus improving its mechanical properties [182,184,186].
5. Opportunities and Challenges of Using Proteins for Electrospun Drug Delivery
Systems for Wound Healing
As mentioned previously, there are a number of benefits that proteins display when
used as antimicrobial delivery systems. Most importantly, proteins show increased cytocompatibility and higher rate of biodegradation as compared to synthetic polymers [24,34].
Irrespective of their origin (plant or animal), proteins represent natural biomolecules that
are degraded by physiological mechanisms [55]. For example, degradation of zein in vivo
can be attributed to enzymatic and microbial activity as well as cell phagocytosis [188].
Another advantage of using proteins for antimicrobial drug delivery systems concerns their
cell interaction abilities. Proteins such as soy protein [83], keratin, collagen, gelatin and
elastin, for example, carry RGD or other cell-recognition motifs that facilitate recognition by
cells, cell adhesion to fibers and cell migration across the wound bed (Figure 1). Proteins
such as soy protein [88], lactoferrin [146] and lysozyme [98] further possess innate antimicrobial, anti-oxidant and anti-inflammatory activity that facilitate healing. The biological
properties and further beneficial features of proteins of plant and animal origin that are
used for electrospinning in the wound healing context are summarized in Table 4.
Using plant- and animal-derived proteins in electrospinning nanofibers not only comes
with benefits, but also with a few challenges. To begin with, the diversity in extraction
and purification methods has been shown to affect purity, composition and activity of the
proteins. This in turn may influence the reproducibility during electrospinning, which is
highly dependent on the homogeneity and surface charge of the material [5,36–38,57].
Unfortunately, there are currently no regulations in place to ensure homogeneity of structure and purity of protein-based materials [5]. Secondly, the use of organic solvents,
cross-linking agents and a high voltage that are required during electrospinning may
potentially inflict damage on the protein structure, which may result in loss of activity to
a certain degree [22,23]. Even though there are some hydrophilic proteins such as casein,
whey and SF (Table 2), using water as a solvent for electrospinning is a challenge in itself
due to its surface tension that often leads to non-continuous process and artifacts in fibers.
Moreover, aggregation and the low degree of protein unfolding in water further reduces
protein electrospinnability [29]. And even if electrospinning is successful, the final product
often requires cross-linking to ensure stability of fibers in water. For proteins with more
Pharmaceutics 2021, 13, 4
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amphiphilic nature such as zein, which can be dissolved in aqueous solvents, the final
product loses its fibrous structure upon contact with water and becomes more elastic [54].
Regarding the incorporation of antimicrobial agents into protein-based fibers, a high
diversity of antimicrobial agents can be integrated into fibers and elicit efficient antimicrobial response against Gram-negative and Gram-positive bacterial species (Tables 1 and 2).
Among plant-derived proteins, only three proteins (zein, soy and pea) have been reported
to be electrospun with antimicrobial agents. Zein is the most popular protein to electrospin with a wide variety of antimicrobial agents, mostly including antibiotics and silver
nanoparticles (Table 1). This may be attributed to its high availability as a manufacturing
by-product and the associated low price [57] (Table 4). Among animal-derived proteins, collagen/gelatin as well as SF are widely used in a wound healing context both alone (which
requires cross-linking) and in combination with synthetic polymers including PCL, PVA,
PEO, PL(G)A and CS (Table 2). Antimicrobial agents that have been incorporated into such
fibers include antibiotics, antimicrobial nanoparticles, antimicrobial peptides, antibacterial
plant extracts and even an antibacterial virus [115] (Table 2). In the case of some fibrous
mats made from SS or SF, instead of adding an antimicrobial agent, electrospinning with
antibacterial polymers such as CS proved to be enough to confer the electrospun wound
dressing with an antibacterial activity [123,126,128]. Overall, zein, gelatin and SF are the
only proteins that can be electrospun without toxic organic solvents. However, due to
their instability in water, all of them require either post-electrospinning cross-linking or the
addition of synthetic polymers during electrospinning.
Table 4. Biological properties and other relevant features of plant- and animal-derived proteins.
Protein
Zein
Soy
Pea
Casein
α-lactoglobulin
β-lactoglobulin
Lacto-ferrin
Lysozyme
Keratin
Collagen
Gelatin
Elastin
Silk sericin
Silk fibroin
Water
Soluble
Nutritional
Value
Industrial
ByProduct
X
X
X
X
Allergenic
X
X
X
Antimicrobial
X
Anti-oxidant
X
X
AntiInflammatory
Cell-Recognizing
Motifs
X
X
SelfAssembly
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
(X )
(X )
(X )
(X )
(X )
X
X
X
X
X
X
(X)
X
X
X
X
X
X
Note: X, present property; (X) weak manifestation of property; blank cell, not reported.
In summary, selecting proteins for electrospinning with antimicrobial agents depends
not only on the ease of electrospinnability and properties required to facilitate wound
healing, but also on the protein origin, availability, cost, manufacturing, purity, composition
and allergenicity. It is worth noting that proteins with low nutritional value and those which
occur as manufacturing by-products represent the most cost-effective and ecologically
friendly option for producing nanofibers. Developing wound dressings from naturallyderived materials by upcycling manufacturing by-products and products with low nutritive
value will help in establishing sustainable wound management approach with economically
and environmentally friendly alternative to synthetic polymers.
Pharmaceutics 2021, 13, 4
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Author Contributions: Conceptualization, A.A. and A.H.; investigation, A.A. and A.H.; writing
—original draft preparation, A.A. and A.H.; writing —review and editing, A.H. and A.A.; visualization, A.A. and A.H.; supervision, A.H.; project administration, A.H.; funding acquisition, A.H.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the LEO Foundation, grant 17063 (A.H.).
Conflicts of Interest: The authors declare no conflict of interest.
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