Chapter 1

1.0 INTRODUCTION

Polymeric nanofibrous mats are important class of nanomaterials and have wide
applications in nanotechnology field. The diameter of nanofibers is less than 100nm.
In contrast to other fibers fabrication techniques, electrospinning technique is the most
applicable, easy processing, cost effective and extensively used technique for the
fabrication of fibers or membranes with fiber diameter in the range of micro to
nanometer scale (Abdelhady, Honsy & Kurakula, 2015). A high voltage is used to
fabricate nanofibers with high surface to volume ratio and reduced pore size in
electrospinning technique. Through electric source negative and positive polarity is
created. Fibers can be fabricated by using polymer solution and melt of polymer in
electrospinning technique (Chen, Lee & Liu, 2019).

1.1 HISTORY

The electrospinning process is gaining extraordinary importance from last two

decades due to many advantages. These advantages include low-cost production,
chemical versatility, fabrication of materials having good porosity, fibers with nano to
micron level, enhanced electrical, optical and mechanical properties. Fibers with new
and existing characteristics can be fabricated by managing configuration of
membranes and varying the electrospinning parameters (Persano et al.,2017). The
electrospinning process was first reported by William Gilbert in 17 th century. He
described that water drop on dry surface was deformed to conical shape under the
influence of static electricity (Wu et al., 2020).

The electrospun fibers were firstly made by the efforts of Cooley and Morton in
1900 (Wu et al., 2020). Jhon Zeleny published his work in 1914 related to the drop
behavior at the tip of capillary (Hong et al., 2011). Lot of work was performed by
early scientists to develop electrospinning experimental process. In 1969 scientist
Geoffrey Ingram Taylor made theoretical studies and determined that conical shape of
droplet was formed in the presence of electricity which is known as Taylor cone
(Asmatulu & Khan, 2011; Razzaq & Hofman, 2012). Since, 1995 publications related
to electrospinning technique and its advantages increased exponentially.

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1.2 COMPONENTS OF MACHINE
Following are the main parts of electrospinning machine;

a. The syringe and needle

b. The syringe pump

c. High voltage power supply

d. The collector plate

The first step to start the electrospinning procedure, the syringe is filled with melt
polymer or its solution. Then the syringe is fitted on a pump which is movable. Its
function is to control the rate of feeding of polymeric solution. Electrical power
supply produces electric field between the collector plate and metallic needle and
provided high voltage for the fabrication of nanofibers. The collector plate consists of
a surface made up of metals like Cu, on which solid fibers are deposited. The collector
is in the form of rotating drum or a static surface. The collector plate can be enfolded
with the help of a foil called Aluminum foil. When specified value of voltage in the
range of 1 - 30kV is applied through power supply, solution gets charged and ejected
from the tip of nozzle and on the collector plate fibers are deposited (Ding et al.,
2020). Through electro spinning process a variety of fibers can be fabricated by using
different polymers containing different properties.

Figure 1.1 Electrospinning Machine

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1.3 ELECTROSPINNING MECHANISM

In spite of the fact that the process of electrospinning is simple but the fiber
production mechanism is little bit complex. To start process of electrospinning, the
syringe is filled up with prepared solution of polymer and then fixed in a pump.
Between the collector plate and spinneret, an electric field is created by the application
of high voltage. At specified value of feed rate or flow rate solution is pumped out
from syringe to needle and produce a solution droplet at the tip of spinneret. When
electric field is not applied, due to equilibrium between mutual repulsive and
gravitational forces the droplet sustained its spherical shape. The surface tension at the
drop surface is responsible for the presence of these forces. The needle tip is
connected with positive electrode and collector is connected with negative electrode.
On the application of high voltage, positive charges are induced on the surface of
solution in needle. The mutual repulsion of induced charges results in the production
of an opposite force to control the surface tension. After which the drop shape appears
in distorted shape (Reneker & Yarin, 2008). The shape of droplet changes at spinneret
and changed into a shape of cone, known as Taylor cone. On further increase in
applied voltage, the charged solution jet ejected towards collector plate. During the
flight of solution jet from spinneret to collector plate, the solvent evaporate and solid
fibers are collected (Haider & Kang, 2018).

Figure 1.2 Mechanism of Electrospinning

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1.4 ELECTROSPINNING PARAMETERS

The factors that can affect the process of electrospinning are classified as (a)
solution (b) environmental and (c) process parameters.
The process parameters include TCD distance, diameter of needle, flow rate and
applied voltage. The solution parameters are solvent viscosity, conductivity of
solution, and their concentration. All these parameters can be changed according to
required conditions. Thus, smooth and beadless nanofibers can be fabricated by
controlling these parameters in a certain optimal range (Rodoplu & Mutlu, 2012).
Feng et al. studied the effect of these parameters for polydioxanone nanofibers. He
observed that diameter of nanofibers decreases by increasing voltage, injection rate
and increases by increasing concentration of the solution (Wang et al., 2019).

1.4.1 Solution Parameters

1.4.2 Molecular Weight

The nanofibers structure is affected by molecular weight of polymer. The
electrospun nanofibers could only be prepared when polymers having certain value of
molecular weight. The solution of low molecular weight polymer results in beaded
nanofibers during the process of electrospinning. Through an increase in molecular
weight of the desired polymer, bead formation can be minimized (Wang et al., 2019).

1.4.3 Polymer Solution Concentration

The electrospun fibers are produced when charged solution jet is ejected from
the nozzle tip. The charged jet show stretching or bending while reaching at collector
plate. This stretching ability of charged solution jet is influenced by varying the
concentration of solution of polymer. If the polymeric solution with low concentration
is electrospun, the applied voltage can easily overcome the surface tension at droplet
surface. The entangled polymeric chain then breaks into its small fragments (Haider &
Kang, 2018). These fragments are collected on collector plate in the form of beads.
Thus, increases the concentration leads to the formation of uniform fibers. After
reaching critical value the further increase in concentration results in very viscous
solution that cause the droplet to dry at the needle tip and blocks it. Therefore, the
critical value of concentration is essential for beadless or uniform fibers production.

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1.4.4 Viscosity
Viscosity of solution is the parameter that is directly related with
concentration. The solution viscosity increases by increasing its concentration. At
high concentration molecular chains of polymer get entangled more strongly in
solution and viscosity of solution is also increased (Wang et al., 2019). With low
viscosity, beaded or defective fibers are formed at collector surface while very high
viscosity cause inability to the flow of solution. An optimum viscosity results in
formation of smooth nanofibers.

1.4.5 Surface Tension

Surface tension of solution drops is another factor that influences the structure
of nanofibers. During electrospinning process when the repulsive forces due to electric
field reduce the surface tension of drop, charged jet is ejected. To fabricate uniform
and smooth nanofibers, surface tension can be decreased by enhancing the
concentration of the polymer solution. The surface tension decreases with increase in
concentration due to breaking of hydrogen bonding in solution. The surface tension
parameter can also be controlled by varying solvents and with the addition of
surfactants to the solution (Amariei et al., 2017). The electrospinning of solution with
less surface tension can be done by applying low electric field.

1.4.6 Solution Conductivity

The electrospinning process can be processed with polymers having
conductive property. Due to conductivity, electric charges can move from electrode
surface to solution droplet. The conductivity shows the availability of ionic species in
polymeric solution and controls the formation of continuous nanofibers. The ions
availability in polymeric solution is affected with increase in solvent or polymer
concentration (Amarie et al.,2017).The diameter of fibers can be controlled by
conductivity of solution. If ions are small sized and have high mobility, they tend to
produce fibers with small diameter as it can reduce defects present in nanofibers. The
polymer solutions without conductivity cannot be electrospun. Their conductivity can
be improved by adding inorganic salts such as sodium chloride or ionic organic
compounds (Hu et al., 2019).

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1.4.7 Solvents Property
The solvent property is an important parameter that will influence the
electrospinning process. These properties include their volatility, boiling point and
solubility of polymer in solvent. The structure or morphology of fibers is affected by
volatility of solvents. The solvents with optimum volatility have good evaporation rate
which allows solvent to evaporate easily during their flight from needle to collector. If
volatility is too low, then drying of fibers is difficult due to high boiling point and low
evaporation rate. If solvent volatility is too high, spinning solution will dry at needle
tip due the evaporation of solvent. The solubility of polymer in solvent affects surface
tension and viscosity of solution (Hu et al., 2019). Thus, for electrospinning process
solvent must have suitable volatility, vapor pressure and solubility to polymers.

1.4.8 Process Parameters

1.4.9 Applied Voltage

In the electrospinning process, applied voltage is very important factor that
remarkably influence the diameter of nanofibers. When voltage is applied an electric
field is generated that induces electric charge in polymeric solution via metallic
needle. This induction of charges will cause the spherical drop of solution to deform
and result in the formation of Taylor cone. A high voltage is required to overcome the
repulsive forces present between these electric charges. At a critical value of applied
voltage, charged solution jet is ejected from Taylor cone. This critical value is
different for different polymers. At high voltage fibers with smaller fiber diameter
could produce (Amariei et al., 2017). On the other hand, high voltage beyond its
optimum range beaded nanofibers will produce on collector plate as the Taylor cone
size reduce and the velocity of jet is also increased for similar flow rate (Aman et al.,
2020).Thus, high voltage affects the stretching of ejected solution jet and greatly
influence the fibers morphology.

1.4.10 Solution Flow Rate

Flow rate at which polymeric solution pumped out is also an important factor
that influences the surface morphology of nanofibers. The amount of polymeric
solution filled in syringe can be determined from this flow rate. Mostly low flow rate
is applied as it is effective for evaporation of solvent from fibers. If the flow rate is too
low the spinning solution is unable to eject from needle tip as fiber jet and shape of

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Taylor cone cannot be sustained (Amariei et al., 2017). When the flow rate is too high
diameter of ejected jet is increased and evaporation of solvent is also incomplete
before coming to collector plate.

1.4.10.1 Needle tip to collector distance

The distance between Tip to Collector Distance plate is another important

parameter that greatly influence the morphology of nanofibers. The solvent
evaporation and deposition time of solution on collector depends on distance between
the collector plate and needle tip. If the distance is too low or too high beaded
electrospun fibers can produce. The strength of electric field is also reduced if distance
is high that increases the jet velocity and fibers with large diameter will be obtained.
Therefore, optimized distance is required to obtain the fibers with reduce diameter
(Aman et al., 2020).

1.4.11 Environmental Parameters

1.4.12 Humidity and Temperature

Ambient or environmental factors also influence the morphology of
electrospun fibers. These factors include humidity and temperature. Humidity affects
the solvent volatility as high humidity cause decrease in evaporation rate. It also
influences the surface morphology of fibers as porous fibers are formed when
hydrophobic polymers are dissolved in organic solvents and humidity is high (Sun et
al., 2019). Temperature is another factor that cause two opposing effects on
morphology of spinnable solution. An increase in temperature cause increase in
conductivity of solution by rising the movement of molecules in solution. The
evaporation rate of solvent also increases with increase in temperature. In addition, the
viscosity of polymeric solution can also decrease by increasing temperature. So, fibers
diameter can be changed by varying these parameters in electrospinning process. The
effect of these parameters on morphology of fibers is summarized in a table (Wang &
Hu, 2020; Haider & Kang, 2018).

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Table 1.1 Effects of Electrospinning Parameters on Diameter of Nanofibers

Parameters Effect on diameter & morphology

of electrospun fibers
Solution Molecular mass & Less beaded, uniform, and smooth fibers
Parameters within optimum range
Concentration\viscosity

Surface tension Instability of jet and beaded fibers

Conductivity Decrease in diameter with wide distribution

Process Applied voltage Initially, then at hi4h voltage defective

Parameters fibers are produced

Feed rate Low flow rate is better, too high flow rate
results in beaded fibers

Tip-collector distance Too low or too high distance produce

beaded fibers

Ambient Humidity Formation of pores on fibers surface,

Parameters porosity

Temperature Fiber diameter decreases

1.5 APPLICATIONS OF ELECTROSPUN FIBERS

Electrospun scaffolds produced from different natural or synthetic polymers, ceramics
and carbon have high surface/volume ratio, porosity and improved mechanical
characteristics. These enhanced properties have made electrospun nanofibers ideal for
various applications (Shi et al., 2015).

● In cosmetics, food packaging, environmental sensors.

● For biomedical applications including ultrafiltration, tissue engineering, cancer

treatment, wound dressing and delivery of drugs.

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● High alignment and flexibility of electrospun nanofibers make them essential for

production of electronic devices as transistors and diodes.

● In addition, energy storage or energy harvesting devices such as solar cells,

supercapacitor, and fuel cells can be fabricated by using electrospinning fibers.

Thus, electrospinning process is getting more importance at industrial level due to its
low-cost production and environmentally friendly nature (Xue et al., 2019). The
fabrication of electrospun scaffolds is becoming important in biomedical field as in
tissue engineering, wound dressings, and delivery of drug. The chemicals selection for
fabricating electrospun fibers must be done carefully (Arik et al., 2019). The materials
with good antimicrobial property, biocompatibility and biodegradability are also
important. Numerous bio/natural or synthetic polymers with antimicrobial agents are
greatly used for medical purpose due to having excellent biodegradability and
biocompatibility (Song et al.,2017).In our studies cost effective and environment
friendly Montmorillonite reinforced crosslinked PVA scaffolds loaded with plant
extract are fabricated for medical purpose.

Energy
storage

Drug
delivery Filteration

Electrospun
Fibers Tissue
Cosmetics Engineer-
ing

Bio-
Sensors
medical

Figure 1.3 Applications of Electrospinning

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1.6 POLYVINYL ALCOHOL (PVA)
Polyvinyl alcohol is a polymer with semi-crystalline and hydrophilic nature. The
structure of polyvinyl alcohol consists of hydroxyl groups (OH). Its synthesis involves
the polymerization of vinyl acetate to produce polyvinyl acetate firstly, then it sets
hydrolyzed to form polyvinyl alcohol. The physical properties of PVA such as,
solubility depends on the degree of hydrolysis and amount of acetate groups.

1.6.1 Structure of PVA

PVA polymer is hydrophilic in nature and soluble in polar solvents such as
water, ethanol, N-methyl pyrrolidone and dimethyl sulfoxide. The most important
solvent for PVA is water as hydrogen bonding is present in aqueous solution. Many
properties like pH, refractive index, viscosity, surface tension and solubility of PVA
depends on the extent of hydrolysis, polymerization, and temperature. It has been
observed that high degree of hydrolysis decreases the solubility of PVA in water. PVA
has emulsifying and adhesive properties. PVA films have been used for various
pharmaceutical and medical applications (Song et al., 2017).

Figure 1.4 Structure of PVA

The fabrications of PVA fibers have attracted widespread interest in medicinal

field due to its nontoxic nature, water solubility, biodegradability, chemical resistance
and biocompatibility. PVA is beneficial for biomedical purpose due to its bio-
adhesive, elastic and non-carcinogenic nature (Kadajji & Betageri, 2011). It helps
natural tissues to replicate and easily acceptable into body. Thus, it is used for
transdermal patches, contact lenses, artificial organs and drug delivery system
(Ardekani et al., 2019). The electro-spinnability of poly-vinyl alcohol can be

10
improved by using its blended solution with other synthetic polymers due to easy
modification and good biocompatibility (Soud et al., 2020).

1.6.2 Applications of PVA

● In food industry for packaging of food product.

● In textile industry for finishing purpose and sizing agent.

● For fabrication of paper products.

● In biomedical applications such as wound dressings, transdermal patches, tissue

adhesion barriers and drug delivery purpose.

● In adhesive mixtures, glues and paints PVA is used as thickening agent.

1.7 CROSSLINKING
The solubility of PVA in water enables its nanofibers to dissolve in water which
cause restriction to use these fibers for various applications. Many efforts have been
made to increase water resistance of PVA nanofibers. The simplest approach to
decrease their water solubility is to use blended solution with hydrophobic polymers.
An alternative approach is to use cross linkers (Sa’adon, Abd Razak, & Fakhruddin,
2019; Soud et al., 2020).
. The hydrophilicity of polymer can be reduced by its physical and chemical
crosslinking to form hydrogels. Physical crosslinking involves ionic-electrostatic
interaction and hydrogen bonding, while chemical crosslinking cause formation of
covalent bonds among chains of polymer (Hu et al., 2019). Crosslinking is a process
that involves the coupling of polymer chains with each other by chemical reaction
under broad range of conditions. Crosslinking occurs by many chemical reactions as:

● Condensation

● Ring closure

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 ● Addition

Crosslinking process cause remarkable changes in properties of polymeric fibers

(Nataraj & Reddy, 2020). The crosslinked hydrogels have certain characteristics:

● Enhanced solvent resistance

● Improved tensile strength, rigidity and stiffness

● Improved porous structure and swelling characteristics

1.7.1 Crosslinking of PVA

To improve water stability of electrospun PVA fibers, crosslinking is done by
using various crosslinkers like glutaraldehyde, citric acid, malic anhydride, glyoxal,
formaldehyde and bis(β-hydroxyethyl) sulfone (Sa’adon, Abd Razak & Fakhruddin,
2019).Other properties like morphology of PVA fibers, thermal stability and
mechanical properties will also be affected. In our research work PVA is crosslinked
with glutaraldehyde. It is a homodifunctionalized crosslinker of 5-carbons chain with
aldehyde group at both ends.

Figure 1.5 Structure of Glutaraldehyde

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Figure 1.6 Crosslinking of PVA

1.8 MONTMORILLONITE (MMT)

MMT is a natural inorganic material or hydrated aluminosilicate clay mineral. It
has layered crystal structure of 2:1, in which octahedral sheets of aluminum
sandwiched between two tetrahedral layers of silicon (Quinn et al., 2018). This crystal
structure is produced when one octahedral sheet for either aluminum or magnesium
hydroxide is bonded with two tetrahedral sheets. The weak Van der Waal forces are
present between layered structures of Montmorillonite. Due to these weak Van der
Waal forces polymeric molecules can be easily incorporated between layers.
Montmorillonite has a large surface area with following properties:

● Good swelling ability

● Good absorption capacity

● Cation exchange capability

● Drug carrying ability

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Figure 1.7 Structure of Montmorillonite
The Figure 1.7 shows the structure of Montmorillonite (Hu et al., 2019). The
hydrophilic nature of MMT reduces its dispersion only polymers with hydrophilicity.
The hydrated cations on its surface can be replaced by cationic surfactant to form
surface organophilic (Chen, Garcia & Zimmerman, 2020; Jiraskova et al., 2018). The
addition of MMT as nanofiller to polymeric solution increases its mechanical and
thermal properties.

1.9 GREEN SYNTHESIS

Green methods for synthesis and production of polymer matrix composite or
fibers have also been explored because such materials are environment friendly.
Bioactive plant extract due to its biocompatibility, can be used in the form of blend or
additives with polymer to produce composite. Plants that possess antioxidant,
antiproliferative and antibacterial properties have been utilized for medical treatment
(Maina, Wanyika & Gacanja, 2016).

1.9.1 Plants as Source of Medicines

Plants have been used for basic requirements by human beings since ages.
Medicinal plants show dominant role in healthcare system. In developing countries,
the use of herbal medicines has long history (Bahreini, Heydari & Namdari, 2017).
Medicinal plants show remarkable improvements in industries such as pharmaceutics,
cosmetics and drug discovery. The 40 percent of the drugs in the western world
are derived from plants that have been used for centuries. The World Health
Organization (WHO) promotes the addition of herbal drugs in national health care
programs due to their accessibility at low price. As the used of herbal drugs is safer as
compared to modern synthetic drugs. These herbal drugs include Lantana camara,
Moringa oleifera, Berberis lycium, Ginkgo biloba, Syzygium aromaticum etc.

1.9.2 Moringa oleifera (MO)

● It is commonly known as Drumstick tree or Sohanjana.

● Moringa oleifera is rapidly growing, transitive plant that is 10–12 m high.

● Moringa oleifera belongs to the Moringaceae family.

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 ● It is cultivated all over the tropical areas and is found in Bangladesh, India,

Afghanistan and Pakistan.

● Moringa oleifera had been cultivated for its super nutritious seeds, flowers and

edible leaves, fruit and roots.

● It can be used for various pharmaceutical applications such as anti-

inflammatory, antibacterial drugs, antiulcer, cosmetic oils, hepato-protective

effect and reduction of cholesterol.

● The leaves of the plant have widely been used because they are rich in bioactive

components such as vitamins, amino acids carotenoids, polyphenols, phenolic

acids, flavonoids, alkaloids, isothiocyanates and tannins (Barikloo & Ahmadi,
2021).

● The leaves of Moringa Oleifera are widely used for medicinal purposes as it has

antioxidant and antimicrobial properties which make it beneficial for the

treatment of various diseases.

Figure 1.8 Fresh Leaves of Moringa oleifera

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1.10 WOUND HEALING PROCESS
A wound is explained as an injury either to the organs, tissues or the skin due to
some microbial, thermal, physical and chemical disorder. Skin wounds are divided
into two types: (a) acute wounds are due to traumas, surgical procedures, irradiations,
and superficial burns and (ii) chronic wounds are due to specific diseases including
diabetic, pressure ulcers, and venous leg ulcers. Furthermore, homeostasis and release
of inflammation mediators are also observed on wound. There is also a high risk of
wound infection. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa
are bacterial organisms that cause wound infections within 48hrs when exposed
(Maina, Wanyika & Gacanja, 2016). Thus, the wound healing is biochemical process
of repairing the injured tissue to its normal state. Wound healing process consists of
four main steps including hemostasis, inflammation, proliferation and maturation.

In recent times, Dry gauze dressings are commonly used wound dressings as it
is easily available and inexpensive. However, several disadvantages of these dressings
are observed such as a high absorbent capacity, leading to increase in bacterial growth
and wound dehydration. Therefore, various complex dressings have been developed
with semipermeable properties. For example, hydrogels and hydrocolloids with
hydrophilic properties are good absorption of exudate and gas permeability. They also
have biological properties such as antimicrobial activity (Sun et al., 2019). Now,
electrospinning technique is also gaining interest for the fabrication of nanofibers in
wound healing applications.

1.11 ELECTROSPUN NANOFIBERS FOR WOUND HEALING

The high surface to volume ratio, highly porous structure, good mechanical
properties, high drug loading and controlled drug release capacity of electrospun
nanofibers make them suitable for biomedical applications (Fayemi et al., 2018).

The nanoscale structure and the topography of fibers support proliferation, adhesion
and differentiation of cells. The high surface area of nanofibers makes good
interaction with cell which is favorable for cell adhesion. The porosity and good pore
connectivity of nanofibers is also beneficial for transport of nutrients and gases during
healing process. The small size of pores is effective in blocking of bacterial invasion,
thus facilitates healing of wound. The electrospinning of polymers by encapsulating
them with bioactive molecules provides them variety of functions in tissue

16
engineering. The high surface area of nanofibers increases the capacity of drug
loading to accelerate the healing of wound (Sun et al., 2019).

Chapter 2

2.0 LITERATURE REVIEW

During the past few years, demand for the fabrication of polymeric nanofibers
has been increased due to their variety of applications which are including delivery of
drugs, tissue engineering, cosmetics, food packaging and devices for energy storage
(Ali et al., 2019). Due to which researchers have made many developments for the
fabrication of nanofibers such as electrospinning techniques and non-electrospinning
techniques (Dar, Shahnawaz & Qazi, 2017; Vergara-Jimenez et al., 2017). Non-
electrospinning techniques are self-assembly, phase drawing method, spinneret-based
tunable engineered parameter (STEP) technique, freeze-drying method, phase
separation, nanofibers interfacial polymerization and template synthesis (Pant, Park &
Park, 2019). Electrospinning techniques include blending, coaxial and emulsion
electro spinning (Alghoraibi & Alomari, 2018).

Table 2.1 Non- Electrospinning Techniques

Techniques Principle

Interfacial It is a step growth polymerization. It occurs at or near the

Polymerization interface of two different phases by dissolving two or more
monomers. The produced polymer precipitates out and can be

17
 drawn as film/fibers if it has good mechanical strength.

Phase Drawing It is important process of spinning and used to increase the

orientation of polymer molecule. Filaments of desired strength
and hardness are produced. Crystallinity zone of fibers are also
increased.

Spinneret-Based Through this technique 3D structure of fibers are produced. It

Tunable allows precise control on fiber diameters and further allows
Engineered deposition of fiber arrays in aligned configurations. Fibers with
Parameters diameter nano to micro level can be obtained.
(STEP)

Phase Separation In this technique phases will differentiate due to their

incompatible properties. The process involves four steps of
polymer dissolution, gelation, extraction and freeze drying. The
mechanical characteristics of nanofibers can be changed by
adjusting concentration of desired polymer.

Template In this synthesis, nanofibers of metals, polymers,

Synthesis semiconductors, or ceramics can be synthesized by the
implement of template or mold with various cylindrical pores
having thickness of 5–50 mm.

Self-Assembly The fabrication method through which molecules tends to

arrange themselves into structures in the presence of non-
covalent forces including electrostatic reactions and hydrogen
bonding. It is a complex technique with difficult control of
dimensions of fibers.

Table 2.2 Electrospinning Techniques

Technique Principle

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Coaxial In coaxial electrospinning spinneret is consists of an inner and outer
needle. It can produce fibers from variety of pairs of solutions,
hollow, functional fibers and core–sheath.

Multi-Needle The electrospinning process in which productivity is increased by

using number of needles is known as multi-needle electro-spinning.
For continuous electrospinning, a strong electric field is needed in
multi-needle electrospinning.

Centrifugal In centrifugal technique, the forces for the stretching of the solution
drop into fibers are both electrostatic force and the centrifugal force.
So, less voltage is required to overcome the surface tension of the
solution.

Blend In blend electrospinning, drug is loaded in polymeric solution prior

to electrospinning process.

Emulsion The emulsion electrospinning is based on two or multiple phases,

which are not miscible during the electrospinning process. It is
based on single nozzle formation of core-shell structure.

There is a growing interest for the fabrication of nanofibers loaded with plant
extract, essential oil and active ingredients by using electrospinning technique.
Amongst extraction methods, soaking or maceration process is the simplest one to
prepare extract of plants (Gugulothu et al., 2019).

Yang et.al. (2018) produced the polyvinyl alcohol based electrospun nanofibers
containing Coptis chinensis. 5, 10, and 15 % of water extract of Coptis chinensis was
added into 10 wt % of PVA solution. The morphological changes through SEM,
antimicrobial and antifungal properties of PVA with different concentrations of
extract were studied. The antibacterial activity of nanofibers against Staphylococcus
aureus and Staphylococcus epidermidis, and the antifungal activity against
Penicillium pinophilum and Aureobasidium pullulans were investigated. Antioxidant,

19
anti-inflammatory and cytotoxicity was also evaluated. These properties enhance their
availability to use nanofibers for medicinal purpose.

Tridax procumbens L. (T. procumbens) is a flowering plant mostly available in

America. It is rich in elements such as sodium, potassium, and calcium. The leaf
extract of T. procumbens, flowering plant heals wounds and has been used on the
injured part of the body.

Ganesan & Pradeepa. (2017) fabricated the PVA nanofibers with pore diameter of
2.26 μm. The nanofibers were loaded with methanolic T. procumbens leaf extract. The
nanofibers showed an inhibition zone up to 45 mm, 36 mm against S. aureus and E.
coli respectively indicating that the nanofibers possess antibacterial activity.

Ali et.al. (2019) prepared polyvinyl alcohol (PVA)-Azadirachta indica (neem)-

chitosan blended nanofibrous (PNCNM) by bi-layered technique through
electrospinning under optimum parameters. Azadirachta indica (neem), containing
many bioactive constituents such as assteroids, sesquiterpene lactones sugars,
triterpinoids, tannins, alkaloids, reducing sugars, phenolic compounds and flavonoids.
It has antimalarial, antifungal, antiulcer, antibacterial, antiviral, antioxidant, anti-
inflammatory activities. It has been extensively used for medicinal purpose. The
tensile behavior, bonding behavior and antibacterial activity against Staphylococcus
aureus (S.aureus) had been investigated. The study showed that the PVA/chitosan
nanofibers containing neem extract have a promising potential to use nanofibers as
wound dressings.

Han et.al. (2019) fabricated lutein-loaded pva and sodium alginate nanofibers and
investigated its release. lutein is an antioxidant present in fruits, flowers and
vegetables. polyvinyl alcohol and sodium alginate (sa) loaded with extract were
produced. electrospun pva/sa nanofibers were cross-linked by using mixture of
glutaraldehyde and saturated boric acid at room temperature to improve water
resistance capability of nanofibrous mats. After crosslinking the release of lutein from
the nanofibers was measured by an UV spectrophotometer.

Gugulothu et.al. (2019) investigated the fabrication of electrospun nanofibers by using

blend solution of polyvinyl alcohol, honey and Curcumin longa extract. The fibers
showed effective antibacterial activity against Staphylococcus aureus bacteria. The

20
characterization results reveals that nanofibers have great potential to be used as
wound dressing materials.

2.1 AIMS AND OBJECTIVES

● To fabricate Polymeric-MO leaf extract-based nanofibers by electrospinning

method.

● To fabricate biodegradable and biocompatible plant extract-based nanofibers.

● To study antimicrobial activity and wound healing ability of fabricated

nanofibers.

Chapter 3

3 Materials
3.0 MATERIALS AND METHODS
3.1 REAGENTS
Following reagents were used for the synthesis of plant extract loaded PVA
based electrospun nanofibers and their pharmacological testing.

3.1.1 Polyvinyl Alcohol (PVA)

PVA is a synthetic polymer with Mw = 72000 g/mol. The degree of hydrolysis
of PVA is 85.89 %. It is soluble in water and commercially available in the form of
granules. It was obtained from Sigma Aldrich.

3.1.2 Glutaraldehyde (GA)

It is a colorless oily liquid with pungent odor. It is used as a crosslinker in
preparation of nanofibers. It is obtained from Sigma Aldrich as 50 % PS solution.

3.1.3 Montmorillonite (MMT)

MMT (K-catalyst) is commercially available nano-clay having pH 3-4. It is
present in the form of greyish white fine powder. It was obtained from Sigma Aldrich
with surface area of 250m2/g.

21
3.1.4 Moringa oleifera Leaves
Moringa oleifera (MO) plant leaves were obtained from Benessere Health
Company, Multan.

3.1.5 Deionized Water

Deionized water is obtained from commercial sources.

3.2 METHODOLOGY

3.2.1 Extract Preparation

3.2.1.1 Collection

Fresh leaves of 500 Moringa oleifera plant were obtained from Benessere
Health Company, Multan in September 2020 and the sample plant were identified by
Dr Ghulam Murtaza at COMSATS University Lahore campus.

3.2.1.2 Preparation of plant sample

The fresh plant leaves were washed to remove dust particles, dried for 30 days
in shaded area at room temperature. The dried leaves were ground into fine powder by
using electrical grinder.

3.2.1.3 Maceration of dried plant leaves

The powdered leaves were macerated or soaked into water for about 10 days.
The stirring with glass rod and shaking was done at least 4 to 5 times daily. The
maceration of dried leaves was carried out at room temperature.

3.2.1.4 Crude extract filtration

The active ingredients were dissolved in solvent and come out from plant cells
into solvent by the process of diffusion. Extract was filtered through filter paper.
Filtrate was evaporated, dried and stored in a dry place.

22
 Figure 3.1 Flow Chart of Plant Extraction

3.2.2 Solution Preparation for Electrospinning

3.2.2.1 Preparation of PVA solution

To start electrospinning process 16wt% solution of PVA polymer was

prepared in deionized water. PVA powder (3.2g) was added by stirring into deionized
water to make 20ml solution. Then the mixture was placed on a hot plate for 4-5 hrs.
Stirring was done with the help of magnetic stirrer at a rotation speed of 250-300 rpm.
The temperature of hot plate was maintained at 60-80oC to get homogenous solution.

3.2.2.2 Addition of crosslinker

After preparation of homogenized polymer solution reduce the temperature to

40oC. Add 1-2 drops of glutaraldehyde with continuous stirring for 2hrs to complete
the crosslinking reaction between PVA and glutaraldehyde.

3.2.2.3 Addition of MMT nanoclay

Mont morillonite 1% nanoclay 0.032g was mixed separately in desired amount

of deionized water. This mixture was then added into crosslinked polymeric mixture
with vigorous stirring and heating.

3.2.2.4 Addition of leaf extract

The prepared nanocomposite solution was cooled down to room temperature

measured quantity of leaf extract has dissolved into appropriate amount of deionized
water. At the end, dissolve solution of leaf extract was added into polymeric mixture.
After complete dissolution, the resultant solution was ready to electrospun.

23
Figure 3.2 Prepared Solutions for Electrospinning

Four formulations were prepared by changing the concentrations of plant

extract. The quantities of PVA, glutaraldehyde and MMT nanoclay were kept
constant.

Table 3.1 Table of Formulations for Polymeric nanofibers

Sr. Formulatio Concentrat PVA Glutaraldehyde MMT

No n Code ion of Leaf
(wt %) (drops) (wt %)
Extract
(grams)

PNF-1 16 2 1
1. 0

PNF-2 0.1 (3 %) 16 2 1
2.

24
 3. PNF-3 0.2 (6 %) 16 2 1

PNF-4 0.4 (12 %) 16 2 1

4.

PNF = Polymeric nanofibers

3.2.3 Electrospinning of Polymeric Solution

The completely dissolved homogenous solution was subjected to
electrospinning process by using electrospinning machine of model FLUIDNA TEK
LE-10. A syringe of 5 ml with needle diameter of 11.99 mm was filled with polymeric
solution. The process parameters to fabricate nanofibers have been adjusted once at
optimum value and kept constant. When high voltage was applied, charged solution
jet was ejected from spinneret in the form of Tylor cone. The charged solution jet was
moved towards collector and deposited on the metallic collector plate in the form of
solid fibers. The collector plate was covered with aluminum foil from which fibers
were collected as a membrane and stored. All the four formulations were successfully
electrospun at same parameters.

25
 Figure 3.3 MMT Reinforced Crosslinked PVA Nanofibers

Table 3.2 Optimum Process Parameters for Montmorillonite

Sr. Parameters Optimum Values

No

1. Applied Voltage 15 KV

2. Needle tip to Collector distance 18 cm

3. Feed rate 0.5 ml hr-1

Plant Extraction Solution Preparation Electrospinning

Collection, Drying, Preparation of PVA Electrospinning of

Grinding solution
26 homogenized solution
Figure 3.4 Optimum Process Parameters

Figure 3.5 Optimum Process

3.3 Characterization Techniques

The electrospun crosslinked nanofibers loaded with leaf extract were subjected
to following characterization techniques.

3.3.1 Fourier Transformed Infrared Spectroscopy (FTIR)

The FTIR spectroscopy analysis was made by using “FT/IR-6600 type A”
Fourier Transform Spectrophotometer. The spectrum was scanned from 4700 cm -1 to

27
500 cm-1. It is studied to deal with both qualitative and quantitative analysis of organic
as well as inorganic materials.

3.3.2 Scanning Electron Microscopy (SEM)

Jeol, JSM 6400F was used to identify the surface morphology of nanofibers.

3.4 ANTIMICROBIAL ASSAYS

Crude extract of Moringa oleifera and electrospun nanofibers were tested against
microbes to find their antimicrobial activities. To determine antimicrobial activities of
crude extract and different fabrications following assays were used.

● Antibacterial assay

● Antifungal assay

3.4.1 Antibacterial Activity

Antibacterial activity of crude extract and extract loaded nanofibers were
determined by agar diffusion test against different bacterial strains.

3.4.1.1 Materials required

● Plant extract and fabricated samples (4mg/ml DMSO)

● Petri dishes

● DMSO

● Incubator

● Nutrient agar

● Standard antibiotics

● Sterile normal saline solution

28
 ● Micropipette

● Laminar flow hood

● Autoclave

● Filter paper discs

3.4.1.2 Microorganisms

To estimate the antibacterial activity, following strains which were gram positive
were used.

● Pseudomonas aeruginosa

● Staphylococcus aureus

These strains of microorganisms were preserved on nutrient agar media at 40oC.

3.4.1.3 Preparation of media

Media for antibacterial activity was prepared by adding 28 g of nutrient agar

into 1000 ml of distilled water. pH meter was used to adjust the pH at 7.4.

3.4.1.4 Sterilization of media

All glassware and media were sterilized in autoclave for 20 min at 121oC.

3.4.1.5 Procedure

Antibacterial activity was determined by utilizing agar diffusion method

(Balouiri, Sadiki & Ibnsouda, 2016). For the preparation of nutrient agar media, 28g
of nutrient agar was dissolved in 1litre of distilled water. The media was sterilized by
autoclaving at 121oC. Later, this media was permitted to cool down at room
temperature and 25ml was placed in sterilized petri plates and left it for solidification.
The inoculated bacterial strains were prepared in test tubes. 10µl of suspended
bacterial strains were transferred to nutrient agar containing plates. Approximately 5µl

29
of each test sample was assembled onto autoclaved 5mm paper disc. All the discs
were placed on particular position in the petri plates and plates were placed in an
incubator for 24hrs at 37oC. After 24hrs, the antibacterial activity of test samples was
observed.

3.4.2 Antifungal Activity

The plant extract and nanofibers were evaluated for antifungal activity by
using Disc Diffusion Method.

3.4.2.1 Materials required

● Extract and nanofibers sample (4 mg/ml DMSO)

● DMSO

● Fungal strains

● Petri plates

● Standard drug

● Incubator

● Subouraud Dextrose Agar

● Laminar Flow hood

3.4.2.2 Preparation of media

Media for antibacterial activity was prepared by adding 68g of Subouraud

Dextrose agar into 1000ml of distilled water. The pH meter was used to adjust the pH
at 7.4.

3.4.2.3 Sterilization of media

All glassware and media were sterilized in autoclave for 20 mins at 121oC.

30
3.4.2.4 Procedure

After removing media from the autoclave, about 20 ml of media was filled
effectively on petri plates and was allowed to harden. When it solidifies, the naturally
arrange contagious suspension into the media of each petri plates. At that point 5µL of
every tested samples solution was stacked on 5mm discs were set on the assigned spot
on the plate. The plates were named appropriately. The plates were then incubated for
a time of 37-48 hours at 28ºC. After 48hrs the distance across of zone of inhibition
measured, whether tested samples have any antifungal activity or not. Experiment was
performed in triplicates (Njunda et al., 2012).

3.4.3 Enzyme Inhibition

The leaf extract of Moringa oleifera was analyzed on monoamine oxidase A
and B isozymes (MAO A and MAO B) and their activity was measured as per
previously reported protocol. Freshly enzyme was prepared 15-20 min before, at cool
room temperature. The activity of MAO A and MAO B were blocked irreversibly by
using Clorgyline (60 nM) or Deprenyl (300 nM). 96 well plate was used to perform
this assay. The assay volume was 200 μL having 140 μL buffer, 10 μL test sample
(1mg/mL, 100% DMSO) followed by adding enzyme 10 μL (26 μg of protein for
MAO A and 5.0 μg for MAO B). The mixture was incubated for 15 min and 20 min
for MAO B and MAO A respectively, 20 μL of substrate and 20 μL of freshly
prepared Amplex red was added in the mixture. The final concentration of clorgyline
and Deprenyl was 0.1 mM used to determine non MAO A and MAO B activity. The
change in the fluorescence was determined by using fluorescence plate reader (BMG
Labtech GmbH, orten berg Germany). All experiments were repeated twice in
triplicate, and percentage (%age) Inhibition was calculated.

% Inhibition = 100-(Df/ NC)*100

Df = After read minus pre read.

NC = difference in Negative control (DMSO)

MAO = Monoamine oxidase

31
3.4.3.1 Cholinesterase inhibition studies

To evaluate the potency of samples (Extracts) to inhibit BuChE

(Butyrylcholine esterase) all samples were subjected to a slightly modified method of
Ellman's test. The reaction of released thiocholine to give a colored product with a
chromogenic reagent 5,5-dithio-bis(2-nitrobenzoic) acid (DTNB) is the basis of the
spectrophotometric method. At a concentration of 2.5 units/mL, the enzyme solutions
were prepared. The assay volume was 100μL having 60μL buffer 10μL test compound
(1mg/mL, 10% DMSO) followed by adding enzyme 10μL (0.04 U/well). The
mixture was incubated for 10min, 10μL of substrate (0.5mM) and 10μL of DTNB
(0.5mM) was added in the mixture. After 20 min, the production of the yellow anion
was recorded at 405nm. The percentage inhibition rate was calculated by the
following equation:

% Inhibition = 100-(Df/ NC)*100

Df = After read minus pre read.

NC = Negative control (DMSO)

Chapter 4

4.0 RESULTS AND DISCUSSIONS

4.1 INSTRUMENTAL CHARACTERIZATION

32
4.1.1 Scanning Electron Micrographs

Surface Morphology of nanofibers loaded with leaf extract of moringaoleifera

and without extract is studied to investigate the changes in surface properties of
fabricated nanofibers. SEM micrographs of nanofibers containing 0%, 6%, and 12%
of leaf extract are shown in Figure 4.1, Figure 4.2 and 4.3, respectively.

Figure 4.1 SEM Images of PNF-1 Nanofibers without Plant Extract

33
Figure 4.2 SEM Images of Nanofibers Containing 0.2 g of Plant Extract

34
Figure 4.3 SEM Images of PNF-4 Nanofibers Containing 0.4g of Plant Extract

Figure 4.1 illustrates the surface morphology of MMT reinforced crosslinked

PVA nanofibers. The SEM images showed that the fabricated nanofibers have
uniform, smooth and beadless surface. The fibers are randomly oriented and possess

35
high degree of porosity. The average diameter of nanofibers is approximately 300-330
nm. The SEM images of crosslinked PVA containing 0.2 g and 0.4 g of leaf extract of
moringaoleifera were shown in Figure 4.2 and Figure 4.3, respectively. The SEM
micrographs showed morphology of nanofibers with even distribution of leaf extract.
The results observed from micrographs showed that the morphology of fabricated
nanofibers were slightly changed by increasing the concentration of leaf extract. The
nanofibers with high concentration of extract (PNF-4) have uniform, randomly
oriented, beadless and smooth surface. But diameter of nanofibers is slightly increased
with increase in extract concentration due to increase in viscosity of solution. The
increase in viscosity reduces the mobilization of extract and leading to thicker
nanofibers (Fayemi et al., 2018). Beaded nanofibers are obtained at high concentration
of leaf extract. The PVA nanofibers with 0.2g and 0.4g of leaf extract show the
diameter in the range of 350-370 nm and 390-410 nm respectively.

4.1.2 Thermogravimetric Analysis

Different stages of degradation were studied by Thermogravimetric analysis of
crosslinked PVA: clay NF in the absence and presence of leaf extract.
Thermogravimetric curves represent the 1st derivative of TGA curves. Figure 4.4
shows the TGA thermogram of first formulation, PNF-1 with zero concentration of
extract.

Figure 4.4 TGA thermogram of PNF-1 Nanofibers without Extract

36
 The mass loss occurred in three distinct stages. In 1 st stage 5.1% mass loss
occurred between (40- 70oC) which is due to the evaporation of moisture present in
nanofibers and loss of air present in pores. At second stage 57% mass is lost in the
temperature range of 280-390oC. In this stage mass quickly decreases with maximum
loss. This is due to the loss of hydroxyl groups present in polyvinyl alcohol and
breakage of bonds in carbon chain. At the last stage 17% mass loss occurred due to
loss of hydroxyl groups of MMT in the temperature range of 400-470oC.

Figure 4.5 shows the TGA thermogram of formulation PNF-3 with 6% concentration
of leaf extract containing same concentration of PVA and nanoclay montmorillonite.

Figure 4.5 TGA Thermogram of PNF-3 Nanofibers Containing 0.2g of Leaf

Extract

The initial 6.7 % mass loss observed after 40 oC may be due to the evaporation
of moisture from the film. The 5.1 % mass loss from (120-270 oC) may be due to the
loss of highly volatile components present in extract. The maximum mass loss (56 %)
after 300 oC may be due to the elimination of amorphous part of PVA and other
volatile components in extract. The 12 % mass loss at 440 oC is due to the degradation
of hydroxyl groups present in montmorillonite..

37
Figure 4.6 TGA Thermogram of PNF-4 Nanofibers Containing 0.4 g of Leaf
Extract

Figure 4.6 shows the thermogram of sample PNF-4 with high concentration of
leaf extract (12% of PVA). The initial 7.4% mass loss in the temperature range of 37-
71oC is appeared due to the loss of moisture and gases present in porous structure of
fabricated nanofibers. The mass losses are like that of PNF-3 sample. The gradual
slope appears after 300oC with the maximum loss of 46.9% is observed due to the
degradation of PVA and volatile components of extract. The 14.7% mass loss from
367-465oC is due to degradation of hydroxyl groups in montmorillonite and some
residue which is left.

Thermal analysis was done to investigate the thermal stability of fabricated

nanofibers. Figure 4.4 showed the thermogram representation of MMT reinforced
PVA nanofibers in the absence of leaf extract and the first derivative of the TGA
thermogram (DTG). Three typical weight loss regions can be observed in TGA
thermogram. The initial weight loss in the range of 40-70 oC may be appeared due to
loss of moisture present in nanofibers. The mass loss from 280-390 oC may be due to
degradation of carbon chain present in PVA and loss of hydroxyl groups. The weight
loss from 400-470oC may be due to decomposition of -OH groups present in MMT.
Figure 4.5 shows similar behavior. The mass loss between 120-268oC may be due to

38
decomposition of volatile components present in leaf extract. The mass loss in the
range of 371-465oC may be due to loss of hydroxyl groups in MMT and some residue
(Thamer et al., 2021).

Figure 4.6 showed thermal behavior of nanofibers containing high concentration of

leaf extract. The mass loss between 110-260 oC was greater as compared with PNF-3
sample due to presence of large number of volatile components. The mass loss on
other stages was slightly decreases which showed the stability of nanofibers. The
results showed that thermal stability of fabricated nanofibers increases due to
interaction between hydroxyl groups present in PVA and leaf extract of Moringa
oleifera.

4.2 FOURIER TRANSFORM SPECTROSCOPY

Figure 4.7 illustrates the FT-IR results of montmorillonite reinforced crosslinked
PVA nanofibers. Black peaks represent the sample PNF-1 in which the concentration
of leaf extract was 0%. The red peaks represent the sample PNF-4 in which high
concentration (12%) of leaf extract was present.

39
Figure 4.7 FT-IR Spectra of PNF Loaded and Unloaded with Plant Extract

The FT-IR spectra showed that PVA have various absorbance peaks at
different frequencies and similar peaks were observed for nanofibers containing leaf
extract. The peaks for sample PNF-4 shows some degree of broadness due to the
presence of extract.

Figure 4.7 showed the FTIR spectra of PVA. The spectra show the O-H
stretching at 3313 cm-1, C=O at 1733 cm-1 , O-H bending at 1432 cm-1, 1365 cm-1 and
1320 cm-1, the stretching C-H at 2930 cm -1, and the bending C-H at 941 cm -1, 835 cm-
1
, and 610 cm-1 and C-O stretching at 1254 cm -1 and 1088 cm-1. There is no appearance
of new peak in the spectra of PNF containing leaf extract which showed the
interaction takes place between PVA and extract (Hikmawati, Rohmadanik & Putra,
2018). In FT-IR spectra of nanofibers loaded with root extract the broadening of peaks
observed due to the hydrogen bond between PVA and compounds present in Moring
oleifera, indicating that hydroxyl and carboxylic acid groups are present in extract.

40
4.2.1 X-ray Diffraction Spectroscopy
X-ray diffraction spectroscopy was made to determine the crystalline and
amorphous nature of electrospun polymeric nanofibers by varying the concentration of
plant extract. The XRD diffractogram of nanofibers shown in Figure 4.8.

Figure 4.8 XRD Pattern of Electrospun Nanofibers

XRD studies showed few peaks for fabricated nanofibers show four peaks. In
sample PNF-1 four different peaks appeared at 2θ= 12.57o, 20.30o, 30.98o, 41.85o. The
second sample PNF-3 showed almost same XRD pattern as sample PNF-1. The peaks
appeared at 2θ= 12.85o, 20.50o, 30.70o, 41.85o. However, one of the peak intensities
decrease in PNF-4 sample and it showed three peak intensities at 2θ= 12.71 o, 30.86o,
41.25o.

41
 Table 4.1 Comparison of d-spacing and Intensity of Curves Against 2θ When
Extract Concentration Varies

Sr. no Fabrication Code 2θ Intensity d-spacing

1 PNF-1 12.57 185.01 7.0

2 20.30 215.28 4.3

3 30.98 173.58 2.8

4 41.85 126.16 2.1

1 PNF-3 12.85 441.16 6.8

2 20.50 446.87 4.3

3 30.70 462.12 2.9

4 41.99 382.31 2.1

1 PNF-4 12.71 668.94 6.9

2 30.86 682.06 2.8

3 41.25 628.93 2.1

42
Table 4.2 Calculation of Lattice Parameters of PNF-1 Sample

Sr no 2θ Sin 2 θ Sin 2
θ x Sin 2
θ x Sin 2 θ x h 2 + k 2 + l2 hkl a
1 2 3 (Ao)
1 12.57 0.011 1.000 2.000 3.000 3 111 12.1
8
2 20.30 0.031 2.638 5.277 7.834 8 202 12.3
5
3 30.98 0.071 5.991 11.982 17.973 18 033 12.2
3
4 41.85 0.127 10.714 21.428 32.140 32 440 12.1
9

Figure 4.9 XRD Pattern of PNF-1

43
 Table 4.3 Calculation of Lattice Parameters of PNF-3

Sr 2θ Sin 2 θ Sin 2 θ x Sin 2

θ x Sin 2 θ x h 2 + k 2 + l2 Hkl a (Ao)
n 1 2 3
o

1 12.85 0.012 1.000 2.000 3.000 3 111 11.91

2 20.50 0.031 2.533 5.066 7.599 8 202 12.23

3 30.70 0.070 5.600 11.200 16.800 17 322 11.99

4 41.85 0.127 10.629 21.258 31.887 32 440 12.19

44
 Figure 4.10 XRD Pattern of PNF-3

Table 4.4 Calculation of Lattice Parameters of PNF-4

Sr 2θ Sin 2 θ Sin 2
θ x Sin 2
θ x Sin 2
θ x h 2 + k 2 + l2 Hkl a (Ao)
n 1 2 3
o

1 12.71 0.012 1.000 2.000 3.000 3 111 11.91

3 30.86 0.070 5.795 11.590 17.385 17 322 11.99

4 41.25 0.124 10.163 20.326 30.489 30 521 11.94

45
Lattice parameter = 11.9A° represents that the Bravais lattice is Face-Centered Cubic.

Figure 4.11 XRD Pattern of PNF-4

Lattice parameter “a” was calculated through following formula:

a = λ /2 x Sin 2 θ √ h2 +k2+ l2

Volume of cell was calculated by using following relation:

V = a3

V = (11.9) 3

V = 1685.15 (A°)3

Different material parameters like grain size, dislocation line density and strain were
calculated using different relations. Grain sizes of crystalline particles were
determined using the following formula:

D = 0.9 λ / β Cos θ

Where D = Grain size of particles

46
λ = wavelength of Cu source 1.54 cm-1

β = full wavelength at half maxima (FWHM)

θ = angle of diffraction (Bragg’s angle)

4.3 Thickness of Fibers

The thickness of fabricated nanofibers was measured by using Thickness Gauge.
Table 4.2 shows the results of thickness measurement.

Table 4.5 Thickness Measurement in Micrometer

Sr no. Thickness in micrometer

1. PNF-1 PNF-4

2. 23μm 24μm

Figure 4.8 shows that fabricated nanofibers consist of four different peaks. Three
different peaks were observed at 2θ=20.30o, 30.70o, 41.85o showing semi-crystalline
behavior of PVA fibers. The pure PVA nanofibers showed three characteristics peaks
at 20.4o, 30 o and 40o (Abdel Bary et al., 2018;. Hong et al., 2018). The peak of PVA
was slightly shifted to 2θ= 19.54o in electrospun nanofibers containing leaf extract of
moringa oleifera. The X-ray diffractogram of fabricated nanofibers containing
different concentrations shows that relatively broad peaks are appeared as compared
to PNF-1.

In PNF-4 (figure 4.11) the peak at 2θ= 20.50o was slightly disappeared, showing less
crystalline behavior of fabricated nanofibers. The electrospinning procedure causes
hindrance in the production of crystalline structures in fabricated nanofibers. The
interaction between PVA and leaf extract of Moringa oleifera results in the
disappearance of some peaks (Kebede, Dube & Nindi, 2018) The diffractogram
showed two peaks for MMT at 2θ= 12.85o, 19.54o. However, the loading of leaf
extract did not cause significant effect on the amorphous behavior of fabricated Nano
fibers (Kim et al., 2018).

47
 Table 4.1 showed the comparison of d-spacing value and intensity of curves
against 2θ when extract concentration varies. The lattice parameters at different angles
were also calculated as mentioned in Table 4.2, Table 4.3 and Table 4.4 for fabricated
nanofibers. The volume of the cell was 1685.15 (A°) 3 and grain size can also be
calculated by using different relations.

4.4 BIOLOGICAL ACTIVITIES

4.4.1 Antimicrobial Results

4.4.1.1 Antibacterial activity

Table 4.6 Zone of Inhibition (mm) of Extract Loaded and Non-loaded Nanofibers

Sr. Formulation Pseudomonas Staphylococcus

no
Code Aeruginosa Aureus

1 PNF-1 0 0

2 PNF-2 9±0.94mm 7±0.94mm

48
3 PNF-3 12±0.94mm 8±0.47mm

4 PNF-4 13±0.82mm 10±0.94mm

5 Reference 22±0.94mm 25±0.82mm

Norfloxacin

Antibacterial properties of electrospun scaffolds were evaluated through agar

diffusion method. The antibacterial efficacy of nanofibers with and without extract
was inspected against two bacterial strains i.e., Pseudomonas aeruginosa and
Staphylococcus aureus. A bar graph between %age inhibition of bacterial growth
against different sample scaffolds. PNF-2, PNF-3 and PNF-4 were active against
gram-positive and gram-negative bacterial strains. However, PNF-4 fraction has
shown highest zone of inhibition among all fractions and fraction without extract is
not active against these strains with the reference of Norfloxacin.

Figure 4.12 Antibacterial Results of Nanofibers

49
Figure 4.13 A Histogram Representation of Antibacterial Sensitivity of
Nanofibers

Antibacterial properties of fabricated nanofibers loaded with leaf extract of

Moringa oleifera were evaluated against Gram negative bacteria (Pseudomonas
aeruginosa) and Gram positive bacteria (Staphylococcus aureus). The MMT
reinforced PVA nanofibers containing 3%, 6% and 12% of leaf extract were evaluated
for their antibacterial properties. Norfloxacin was used as reference against which the
activity of nanofibers was observed. Figure 4.13 and Table 4.6 illustrates that the
fabricated nanofibers had concentration dependent properties against both Gram
negative and Gram positive bacteria.

The sample PNF-4 containing high concentration of leaf extract (12 %)

showed high antibacterial activity. The PNF-4 had shown higher antibacterial property
against Staphylococcus aureus with zone of inhibition of 10±0.94 mm and against
Pseudomonas aeruginosa with zone of inhibition of 13±0.82 mm. The sample PNF-1
did not showed any activity against bacteria due to the absence of leaf extract. The

50
results shows that the presence of leaf extract of Moringa oleifera transmitted the
antibacterial activity to the nanofibers.

The bioactive compounds present in leaf extract of Moringa oleifera cause cell
lysis and disruption of cell wall. The bioactive components also inhibit DNA
replication, formation of biofilms and stimulate production of reactive oxygen species.
The hydroxyl group present in phenolic compounds and flavonoids showed toxic
behavior to microorganism by hydroxylation of bacterial cell wall. The aromatic
groups containing compounds like quinones also made irreversible complex with
amino acids and polypeptides in cell wall. The hydroxylation and complexation
results in cell wall disruption. Alkaloids inhibit cell respiration and enzymes involved
in DNA replication and protein synthesis (Mickymaray, 2019).

4.4.1.2 Antifungal activity

Antifungal properties of electrospun scaffolds loaded with extract and without

extract were evaluated through disc diffusion method. Antifungal activity was
investigated against two fungal strains i.e., Candida albicans and Candida
parapsilosis. PNF-1, PNF-2, PNF-3 and PNF-4 showed zero activity against both
fungal strains with respect to Amphotericin as reference.

Table 4.7 Antifungal Zone of Inhibition (mm) of Extract Loaded and Non-loaded
Nanofibers

Sr. Formulation Candida albicans Candida parapsilosis

no
Code

1 PNF-1 0 0

2 PNF-2 0 0

51
3 PNF-3 0 0

4 PNF-4 0 0

5 Reference 18±0.47mm 22±0.082mm

Amphotericin

Figure 4.14 Antifungal Activity of Nanofibers

The antifungal properties of PNF’s unloaded and loaded with leaf extract of
Moringa oleifera were investigated against Candida albicans and Candida
parapsilosis. The PVA nanofibers with 3%, 6% and 12% of Moringa oleifera were
evaluated for their antifungal properties. From results it was observed that nanofibers
did not possess any antifungal property as shown in Table 3.7

4.4.2 Enzymes Inhibition Studies

Table 4.8 The Inhibition Potencies of Extract and Extract Loaded Nanofibers

52
 Sr Formulation MAO A MAOB Butyrylcholine
no %age inhibition
Code

1 PNF-2 49 13 17

2 PNF-3 50 28 21

3 PNF-4 53 34 41

4 PNF-Extract 87 57 64

5 Positive control 82 79 84

The inhibition potencies of pure plant extract and electrospun nanofibers

loaded with extract were evaluated against enzymes such as monoamine oxidase
(MAO), Butyrylcholine esterase (BuChE). A bar graph between % bacterial inhibition
against different sample scaffolds. PNF-2, PNF-3, PNF-4 and PNF-E have different %
inhibition with reference to PNF-F which is a positive control. PNF-4 fraction has
shown highest enzyme inhibition among other fractions.

Figure 4.15 A Histogram Representation of Inhibition Potencies of Nanofibers

53
 Enzymes are biologically active substances or proteins that catalyze all
metabolic reactions that occurred in human body. They possess active sites on which
substrate can attach.

E +S ⇌ ES → E +P

Many metabolic imbalances were observed due to activity of enzymes, so

enzymes inhibition is required. Inhibitors are chemical compounds that can bind on
active sites and stop the binding of substrate with enzymes active site and cause
hinderance for enzymes to catalyze the chemical reaction.

E +S +I ⇌ EI + S

BuChE, MAO A and B enzymes have been involved in many neuropsychiatric

disorders, such as Parkinson’s disease, Alzheimer’s disease, and depression. These
enzymes are important binding proteases or flavin (FAD) present in the outer
mitochondrial membrane. MAO are responsible for oxidation of monoamines such as
dopamine, adrenaline, norepinephrine, serotonin. BuChE cause loss of cholinergic
neurons by degradation of acetylcholine into acetate and thiocholine (Ardekani et al.,
2019).An imbalance of neurotransmitter degradation leads to neurological disorders.
Therefore, there is a need to inhibit these enzymes by using alternative inhibitors to
reduce neuropsychiatric disorders. Many medicinal plants are an important source for
the treatment of neurological disorders. Moringa oleifera is a natural plant that contain
phenolic compounds, alkaloids and flavonoids which are responsible for inhibition of
enzymes. Flavonoid compounds have antidepressant effects due to their
neuroprotective property. Flavonoids such as apigenin, quercetin, luteolin,
kaempferol, naringenin and galangin can inhibit MAO enzymes (Yang et al., 2018).

The enzyme inhibitory potential of leaf extract of Moringa oleifera and

fabricated nanofibers with different concentrations of leaf extract were evaluated
against MAO A, MAO B and BuChE as shown in Table 4.8. The results shows that
the inhibition potential of PNF against all enzymes increases with increase in leaf
extract concentration as shown in Figure 4.15. The bioactive components present in
leaf extract of Moringa oleifera may be responsible of inhibition of these enzymes
(Sa’adon, Abd Razak & Fakhruddin, 2019).

54
4.5 CONTACT ANGLE MEASUREMENTS
The wettability of MMT reinforced crosslinked PVA nanofibers is investigated
through contact angle analyzer. The wetting ability of samples PNF-1 with 0% of leaf
extract and PNF-4 containing 12% of extract is examined by using drop method.
Figure 4.16 shows contact angle images of sample without extract at instant time
and after 10 sec

a. At instant time b. After 10 sec

Figure 4.17 Contact Angle of Sample PNF-1

a. At Instant time b. After 10 sec

c. After 20sec

Figure 4.18 Contact Angle of Sample PNF-4

55
 d. After 30sec e. After 1 min
The wettability of fabricated nanofibers were examined by measuring contact
angle of nanofibers. Contact angle is the inverse measure of wettability. The materials
with less value of angle θ < 90oC, have high degree of hydrophilicity. Figure 3.16
showed less contact angle 40oC with high degree of hydrophilicity. Figure 3.17
showed that hydrophilicity of sample PNF-4 is increased till 50 oC due to interaction
between hydroxyl present in PVA chain and extract. In literature hydrophilicity is
increased by increasing concentration of plant extract. But in our research work it was
not justified. The hydrophilicity may be increased due to presence of some
hydrophobic tails in bioactive components.

56
 Chapter 5

5.0 CONCLUSION

MMT reinforced, crosslinked nanofibers have been fabricated with successful

loading of different concentrations of leaf extract of Moringa oleifera by using
electrospinning machine. Fabricated nanofibers possess good biocompatible and
biodegradable properties. SEM images indicated the porous, smooth and uniform
morphology with no presence of beads. Antibacterial results revealed that extract
loaded nanofibers can inactive the Gram-positive bacteria (Staphylococcus aureus)
and the Gram-negative bacteria (Pseudomonas aeruginosa) and the activity of
nanofibers was increased with increase in concentration of leaf extract. The enzyme
inhibition results reflected the concentration-dependent properties as increase in the
concentration of Moringa oleifera extract the inhibition potential of nanofibers was
also increased. The contact angle measurements have shown good hydrophilicity of
fabricated nanofibers which was suitable of wound healing application.

57
 Chapter 6

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