1.1.

Nanofibers

Nanofibers are fibers with diameters less than 1000 nm [1]. Varying in length from tens

of nanometers to a few microns, fiber features create surface topographies that affect various

applications used in the nano- and biotechnology fields. Nanofibers tailored from natural

and synthetic polymers have gained much interest because they are easy to synthesize and

the structural, functional, and compositional properties of these nanofibers are tunable [2-5].

They can be produced by interfacial polymerization, electrospinning (ES), and electrostatic

spinning. Carbon nanofibers are graphitized fibers synthesized under catalytic conditions.

Among the possible techniques used to prepare nanofibers, such as phase separation,

template synthesis, self-assembly, and drawing, ES is one of the most efficient, simple, and

versatile methods owing to its relatively simple and cost-effective setup [6-8].

In the ES process, polymer nanofibers are produced by applying a strong electric field

between a grounded target and the polymer solution. The polymer solution is fed with the

use of a syringe pump through a metallic needle (spinneret) at a constant and controllable

rate. The collector plate acts as the counter electrode on which the fibers are collected as a

non-woven mesh or membrane. The process conditions and properties of the polymer

solution influence the diameter of polymer nanofibers, which range from 10 to 1000 nm.

The key advantage of producing polymer nanofibers with extremely small diameters is their

large surface-to-mass ratio, high porosity, and superior mechanical performance [4,9].

Moreover, the functionality of the polymer nanofibers can be affected by the polymer

molecules located at the surface of the polymer nanofibers, thus enabling customization of

the nanofiber properties by tailoring the nanofiber surface compositions and morphologies.

This ability to customize has led to diverse applications of nanofibers in wound healing,

biosensors, drug delivery systems, medical implants, tissue engineering, dental materials
[10-12], filtration membranes, military protective clothing, and other industrial applications

[7,13].

Spinning polymer blends to create composite nanofibers allows for further tunability of

nanofibers that can fulfill specific industrial requirements in terms of their material

properties, thereby increasing their potential applications. Most recently, investigations have

targeted the inclusion of other nanoscale structures within the nanofibers to produce

structures with added functionality. For example, silver nanoparticles have been included in

synthetic polymer nanofibers to produce a highly antimicrobial material [14]. In another

study, emulsion droplets were successfully included in nanofibers. Self-assembled structures

such as liposomes, micelles, and micro-emulsions have gained much attention in recent

years [15-17]. They have been used as carrier systems for the delivery of antimicrobial

agents, drugs, flavors, dyes, antioxidants, enzymes, and other functional compounds [18-

20]. A combination of nanofibers and self-assembled structures, such as micelles, can thus

create a novel delivery system with superior properties and many potential applications.

Owing to the advantageous features of nanofibers and magnetic nanoparticles (MNPs),

many researchers have incorporated MNPs into biodegradable nanofibers to produce

paramagnetic nanofiber scaffolds. To prepare these composite nanofibers, one of the most

commonly used techniques is the mixing of dry inorganic powder with a polymeric solution

followed by ES, although the nanocomposites formed are not stable and tended to

agglomerate. To overcome this problem, various surface treatments have been used

including salinization, polymer coating, and grafting. In addition to this type of surface

coating, dispersion of Fe3O4 nanoparticles in the nanofibers has been performed in both

water and organic solvents, as well as sodium citrate and oleic acid, although the complete

dispersion of Fe3O4 nanoparticles was not achieved due to these incompatible interfaces
[21].

1.2. Hybrid Nanofiber System

The addition of inorganic components to the polymer system facilitates the preparation

of nanofibers with specific functionalities. A recent patent described an approach for

preparing hybrid nanofibers by adding nanoparticles into the polymers [22]. In this patent,

nanoparticle dispersion was easily carried out, and a porous structure was also created using

salt dissolution. Antibacterial nanofibers utilizing Ag as an antibacterial agent have been

prepared using ES [23]. To prepare the hybrid nanofibers by ES, the chemical precursor of

silver NPs, AgNO3, was mixed with cellulose acetate (CA) or polyacrylonitrile (PAN)

solution. The ES process was then followed by photo-reduction to form the silver NPs

within the nanofibers formed. Chemical precursors of other metals were also used to obtain

other types of hybrid nanofibers. For example, the chemical precursor of Pd was used to

make poly(acrylonitrile-co-acrylic acid) (PAN-co-PAA)/Pd by ES [24]. In a separate study,

gold NPs (AuNPs) were directly mixed with polymers prior to the ES process and

subsequently electrospun (E-spun) to obtain hybrid nanofibers of poly(vinyl pyrrolidone)

(PVP)/Au [25]. Other than with these metals, nanoparticles have also been prepared with

functional metal oxides [26]. Table 1 summarizes the different inorganic components used

for the preparation of hybrid nanofibers.

Table 1. Hybrid nanofibers and their applications.

Hybrid Precursor and Polymer/Dopant Potential Referenc

Nanofiber Application e
CO3O4 1. Cobalt acetate; 2. PVA/ H2O Biomarker [16]
Fe3O4 1. Iron (II) chloride; 2. Graft copolymer,Drug carrier [19]

poly(ethylene oxide) (PEO) or PVA

CeO2 1. Cerium nitrate; 2. PVA/H2O Catalyst [27]
SiO2 1. Tetraethylorthosilicate; 2. HCl Drug carrier [28]
Ca10(PO4)6(O 1. Ca(NO3)2; 2. P(C2H5O)3 Artificial bone [29]

H, F)2
Ta2O5 1. Tantalum isopropyl oxide; 2. PVAC/DMF Implant [30]

(or acetic acid)

Yang et al. reported a new method for preparing aligned fibrous arrays of composite

magnetic nanofibers by ES [31]. As shown in Figure 1, nanofibrous arrays using polylactic

acid (PLA) fibers can be applied in scaffolds without any structural changes. Also, the fiber

morphologies remain intact after loading the MNPs. Moreover, their functionality can be

controlled by adding selected types of NPs.

Recent studies have demonstrated the possibility of obtaining composite nanofibers by

ES of ceramics and biopolymers. Hydroxyapatite (HA), a major component of bone, is a

widely used bioceramic. Hybrid E-spun nanofibers containing HA as a bone regeneration

implant material revealed high mechanical strength and good biocompatibility [28].

Observations from a scanning electron microscope (SEM) image revealed that the

incorporation of HA did not change the required morphology and had a final structure

consisting of smooth and interconnected nanofibers with high volume. A nanofibrous

PLA/HA composite prepared by ES had good mechanical strength with fibers on the

nanometer scale. This composite is promising as a temporary substrate for bone tissue

regeneration. Inorganic compound-loaded nanofibers have been prepared using a

combination of ES and the sol-gel process using common precursors such as SiO2, TiO2,

and AhO3 [33].

Figure 1. Characterization of the super-paramagnetic nanofibrous scaffolds. (a) The scaffold

pellet with diameter of ~5 mm; (b): Schematic representation of the scaffold pellet

implanted into the lumbar transverse defect of a white rabbit lumbar vertebral segment L5;

(c) SEM image of the scaffold showing randomly tangled nanofibers with diameters ranging

from 300 to 1000 nm; and (d) The TEM image of a single fiber. Reprinted with permission

from [32].

7 8 9

Polymer nanofibers loaded with Au, Ag, Pt, or Pd nanoparticles can be produced by ES

with the addition of metal salt solutions as precursors. The diameters of the nanoparticles

were in the range of 5 to 15 nm. These nanofibers have also been reported to have highly

effective catalytic properties.

2. Methods to Prepare MNPs (Magnetic Nanoparticles)

MNPs are prepared via basic inorganic chemistry methods. Specifically, MNPs are

prepared with magnetite, maghemite or iron alloys as the core magnetic material. MNPs can

be prepared either by a single-step or a multi-step procedure, each of which has its

advantages and disadvantages. There is no universal technique available for MNP synthesis.
Commonly used methods for MNP synthesis will be briefly discussed in the following

section.

2.1. Precipitation

One simple chemical method available for the preparation of MNPs is the precipitation

method. It was developed to use aqueous solutions of iron (II or III) ions. Precipitation of

MNPs can be accomplished using one of two methods: wet precipitation or co-precipitation.

The wet precipitation method was developed first for MNPs preparation [34]. In the co-

precipitation method, used for the preparation of iron oxide particles (Fe3O4), two

stoichiometric solutions containing Fe 2+ and Fe3+ ions are mixed with a base [35]. This co-

precipitation method results in large nanoparticle sizes that are dependent on the pH of the

solution. To synthesize MNPs successfully, the oxidation of the iron (II) precursor should be

avoided because it leads to the conversion of Fe3O4 (magnetite) to Fe2O3 (maghemite),

which might impair advantageous properties of Fe3O4 (magnetite) in its application as a

contrast agent in magnetic resonance imaging (MRI). It has been shown that in the spinel

structure of magnetite, cationic vacancies are in the octahedral positions, which result in a

lower net spontaneous magnetization [36]. In particular, Basti et al. found that Fe3O4

magnetite provided stronger proton relaxivities in MRI than Fe2O3 maghemite [37]. As the

process involves a large quantity of water, however, it is very difficult to scale-up the

process [38]. One widely used method to effectively prevent oxidation is by bubbling N2,

which leads to a reduction of the particle sizes. However, it is not easy to perform both

precipitation and the addition of protective coating materials to the magnetic particles

because maintaining the pH is laborious.

2.2. Reverse Micelle Formation

Micelle formation is a classic process in surfactant chemistry [39]. Normal micelles are
usually synthesized in aqueous medium, whereas reverse micelles are formed in a mixture

of a non-polar solvent and water. To produce iron oxide-based magnetic particles, the

inorganic precursor of iron (III) chloride dissolved in aqueous medium is slowly added to

the oily medium, followed by the addition of pH regulators [30,40-43]. The advantage of the

reverse micelle method is to obtain organic-coated MNPs with controlled particle size. Also,

it is possible to obtain inorganic-coated MNPs using reverse micelles [29,44-48]. The

disadvantages of this method are that the remaining monomer hinders the coating of MNPs,

it is difficult to scale-up the process due to the use of large amounts of organic solvent

required, and it is not easy to prepare particles with a range of 20 to 500 nm because particle

sizes depend on the size of the micelles [49-54].

2.3. Thermal Decomposition

Thermal decomposition is a popular method used in industry to synthesize MNPs

because it does not use organic solvents [55]. The use of this method has led to advances in

the preparation of metallic nanocrystals and semiconductors. Ferric and ferrous fatty acid

complexes are widely used precursors of iron oxide super-paramagnetic particles because

they are cheap, less toxic, and easier to scale-up for mass production [56]; However, the

disadvantage of this method is that it is very difficult to control the particle size.

2.4. Liquid Phase Reduction

Strong reducing agents, such as NaBH4 and LiAlH4, are used to prepare MNPs through

the reduction of magnetic or non-magnetic metal oxides to magnetic metal oxides. NaBH4

is the most commonly used reductant because it is soluble in both methanol and water [57-

60]. The advantages of hybrid MNPs produced by this method are that they are very active,

even under mild conditions, and can penetrate the polymer coating; however, they are

difficult to handle due to their sensitivity to moisture.

 3. Preparation of MNP-Functionalized Nanofibers

The development of MNP-functionalized nanofibers has attracted interest due to their

potential use in scientific and industrial applications. The most widely used method for the

preparation of MNP-loaded nanofibers is ES.

The first documented use of electro-hydrodynamics to modify the shape of a liquid

meniscus under the influence of an electric field was reported by William Gilbert in the late

16th century [61]. In particular, he noticed that, when a suitably electrically charged piece of

amber was brought near a droplet of water, it formed a cone shape and small water droplets

were ejected from the tip of the cone. This was, in fact, the first recorded observation of

electrospraying. The process of electrospinning (ES), developed by Anton Formhals in the

thirties and forties of the last century [62-64] can be viewed as a special case of

electrospraying. Larsen et al. [65] was the first to combine electrospinning with sol-gel

methods to design nanofibers from inorganic oxides and hybrid materials. ES is a very

simple method to produce nanofibers of a diameter range of 3 nm to 10 p,m [66,67]. The

technique mainly relies on the electrostatic repulsion of surface charges on the charged

polymer solution and other variables, such as the solution flow rate, solution concentration,

applied voltage magnitude, and the distance between the needle and the collector [68,69]. As

shown in Figure 2, the ES setup involves a high-voltage direct current (DC) supply, a

syringe pump, and a grounded collector. The basic ES technique involves the application of

voltage on the polymeric droplet, thereby charging the droplet, followed by Taylor cone

formation of a charged fiber jet, and accumulation on the grounded collector. Synthetic or

natural polymers are widely used in making electrospun nanofibers [70], as well as the

combination of synthetic and natural polymers, such as alginate/chitosan composite fibers

[71]. Luong-Van et al. [72] prepared MNP-loaded poly(s-caprolactone) (PCL) and MNP-
functionalized poly(lactic-co-glycolide) (PLGA) nanofibers using ES. The size and

morphology of magnetically-functionalized E-spun nanofibers can be controlled by

changing the polymer concentration. Singh et al. reported the development of magnetic

nanofibrous scaffolds composed of PCL and MNPs for bone regeneration [73]. In this study

different weight ratio % of MNPs were distributed in PCL solutions up to 5 to 20 wt % and

subsequently E-spun into nonwoven nanofibrous webs. It was demonstrated that the fiber

diameter was dependent on the MNPs added which in turn changes the electrical

conductivity and viscosity of the solution. Other studies [74-77] have also used synthetic

polymers like poly(L-lactic acid) (PLLA) and polyurethane to make electrospun nanofibers.

Furthermore, Fan et al. [78] prepared PAN/Fe2O3 nano-composite fibers by suspending the

Fe2O3 nanoparticles in PAN/DMF solution, followed by ES. The addition of MNPs to the

precursor solution for the preparation of magnetic nanofibers can be beneficial because the

spatial distribution of MNPs can be confined within the range of 200-500 nm in a one-

dimensional structure (without stacking of the nanoparticles in the radial direction) [79];

however, size deformation of the nanofibers may occur due to the change of viscosity

resulting from the addition of the MNPs to the precursor solution. Multi-functional

polystyrene-based nanofibers with embedded MNPs have also been obtained in a single-step

ES process [80].

Power supply

Figure 2. Schematic representation of the ES setup showing arrangement for syringe,

precursor solution, needle, liquid jet, collector, and a power supply.

4. Biomedical Applications of MNP-Functionalized Nanofibers

Nanofibers functionalized with MNPs have gained attention because of their potential

applications (Table 2), including the use in sensors, tissue regeneration scaffolds, and drug

delivery systems [81,82]. Many kinds of magnetic nanofibers have been synthesized,

although they have not been applied in many practical applications. In the following section,

several applications of nanofibers loaded with MNPs are discussed.

Table 2. Applications of nanofibers loaded with MNPs.

Source ofKind of MNPs Technolog Application In Vitro and in Referenc

Nanofiber y Vivo e
Chitosan /Fe3O4 ES Bone MG63 human[83]

poly(vinyl regeneration osteoblast-like

alcohol) (PVA) cells

Hydroxyapatite Super- ES Bone tissueWhite rabbit[32]

(HA) paramagnetic formation andmodel of lumbar

nanoparticles and Fe2O3 remodeling in

poly(lactic acid) rabbit defects transverse defects
nanoparticles
(PLA)
Poly(s- MNPs ES Bone Osteoblastic cells[73]

caprolactone) regeneration and

(PCL) subcutaneously
implanted in rats
Hydroxyapatite MNPs Immersion Bone repair ROS 17/2.8 and[84]

(HA) of MNPs MC3T3-E1 cells

into HA

scaffold
Magnetic poly(L-Fe3O4 ES Enhanced effectsMC3T3-E1 [85]

lactic acid) on cell

(PLLA) attachment and

proliferation

Table 2. Cont.

Source ofKind of MNPs Technology Application In Vitro andReferenc

Nanofiber in Vivo e
Poly(D,L-lactic Superpara- ES Cell proliferationOsteoblast [86]

acid) (PDLLA) magnetic iron and induction ofcells

oxide the cell orientation

nanoparticles

(SPIONs)
Chitosan E-CHS-Fe3O4 ES Hyperthermia HFL1and [87]

treatment of tumorcaco-2 cells

cells
Cross-linked Fe3O4 ES byReduction ofTumor cells [88]

chitosan iminodiacetic tumor cell

acid (IDA) proliferation

Polystyrene (PS)Magnetic NP-ES withIsolation andLymph nodes

and nanofibers (NF) surface- activation

poly(styrene-co- embedded Tof primary CD3+harvested [89]

maleic anhydride) cell receptorT lymphocytes from

(PSMA) ligand C57BL/6

mice
Indomethacin, T1
Porous Up-conversion ES magnetic MC 3T3-E1[90]

hydroxyapatite of luminescent resonance imagingcells

composite and MNPs (MRI) contrast

agents, and

luminescent

nanoparticles
Hydroxyapatite Doped with ES In situ monitoringHuman [91]

nanocrystals gadolinium of bone tissuemesenchymal

within PCL (Gd) regeneration bystem cells

MR
Amphiphilic Macrocyclic GdP-sheet MRI Tibialis [92]

peptide (III) amino acid anterior

sequence muscle of a

murine model
4.1. Scaffold for Bone Regeneration

Bone regeneration by tissue engineering is very important for bone defects resulting

from tumor resection, trauma, and skeletal abnormalities. Even though many kinds of

scaffolds used for tissue engineering for bone repair have been investigated [93,94], the

development of a successful scaffolding material is needed to satisfy clinical requirements.

Using the ES technique, Meng et al. prepared a novel nanofibrous scaffold composed of

maghemite super-paramagnetic MNPs, hydroxyapatite, and PLA [32]. The nanofibrous

pellet was implanted into the lumbar transverse defect of a white rabbit. The rabbits were

raised in rabbit cages fixed with permanent magnets to provide a static magnetic field after

surgery. The observed enhancement of tissue regeneration in the lumbar defect when the

magnetic field was applied pointed to a novel strategy to improve bone tissue regeneration
based on MNPs-functionalized nanofibers.

The resulting nanofiber webs became more hydrophilic with improved mechanical

properties due to the addition of MNPs into the PCL scaffold. The addition of MNPs

resulted in magnetic properties of the nanofibrous scaffolds, typical for weak ferromagnetic

or super-paramagnetic materials, as well as increased hydrophilicity, accelerated scaffold

degradation, and apatite-forming ability. When osteoblast cells were cultured on the MNP-

loaded nanofibers versus on pure PCL nanofibers, initial cell adhesion and penetration

increased. Furthermore, rats implanted with MNP-loaded PCL nanofibers showed

significantly better bone regeneration with minimal adverse reactions. Singh et al. [73]

incorporated MNPs in PCL and studied adhesion, spreading, and penetration of

mesenchymal stem cells (MSCs) (Figure 3), which revealed enhanced cell penetration depth

with higher content of MNPs.

Another study [85] reported MNP-functionalized PLLA using trifluoroethanol (TFE) as a

cosolvent. The composite nanofibers formed by ES showed paramagnetic properties with

minimum cytotoxicity and enhanced cell attachment.

Hydroxyapatite (HA), used extensively for bone regeneration, has also been applied as a

scaffold. In one study [84], MNPs were loaded into the pores of HA to make a magnetic

biomimetic scaffold for bone repair. This magnetic HA scaffold had good cell adhesion,

differentiation, and proliferation ability.

Figure 3. MC3T3-E1 cell adhesion and penetration tests on the nanofiber scaffolds. (a)

Initial cell adhesion level on the nanofiber scaffolds during culture for up to 16 h, presented

as % initial seeding. Significantly higher levels of cell adhesion noticed on the PCL-MNP

scaffolds vs. PCL (*p < 0.05); (b) Cell adhesion morphology taken from confocal

microscopy of immunofluorescent stained cells at 2 h and 4 h of culture; nuclei in blue, F-

actin in red and FAK in green; (c-e) Cell penetration assay through the nanofiber scaffolds;

exemplar image showing that z-stacking of immunofluorescence-stained cells (F-actin in

green and nuclei in blue) were unfolded on xz- and yz-planes to reveal 2D constructed

images (c), which were then combined to complete construction of depth profile of cells on

2D plane view (d, compared images of PCL and 10MNP samples at a culture period of 3, 6

or 9 days), and the quantification of depth profile (e, shown average positions of cell

penetration depth), showing significant improvement in cell penetration within the

nanofibers incorporating MNPs. Reprinted with permission from [73].

4.2. Cancer Therapy

 Hyperthermia-based cancer therapy using MNPs has recently gained wide interest;

However, the application of free MNPs has several limitations due to low solubility, poor

cancer targeting, and leakage of MNPs from the tumor location. Therefore, current strategy

involves MNP-loaded electrospun nanofibers for localized hyperthermia-based tumor

treatment.

In a recent study, 50-nm iron oxide nanoparticles (IONPs) were loaded into polystyrene

(PS) electrospun nanofibers to allow for repeated heating by applying an alternating

magnetic field (AMF) with minimum IONP leakage. IONP-loaded PS nanofibers were

made by spontaneous ES of IONPs after dispersing of IONPs in a PS solution containing a

mixture of tetrahydrofuran and dimethylformamide (1:3 volume ratio) to form uniform

nanoscale electrospun fibers. Most of the human SKOV-3 ovarian cancer cells attached to

IONP-loaded PS nanofiber mats through applying an AMF were dead as a result of the

cancer hyperthermia effect [88].

In another study [89], electrospun MNP-loaded chitosan nanofibers were prepared by

two different methods: a) direct adsorption of MNPs into nanofibers by immersion of MNPs

into a chitosan solution, and b) direct immersion of chitosan in an Fe 2+/Fe3+ solution and co-

precipitation of MNPs by ammonium hydroxide. However, there were not many differences

in the morphologies and in vitro hyperthermic effects in Caco-2 cells between the nanofibers

made by the two different methods; although, cross-linking of the MNP-loaded chitosan

nanofibers with gluteraldehyde during ES was performed. Similarly, in another study [90],

cross-linked chitosan nanofibers using iminodiacetic acid (IDA) were prepared using the co-

precipitation method to increase the loading of the MNPs into the nanofibers. In addition,

Ganesh et al. loaded MNPs into thermoplastic poly(ethylene terephthalate) (PET)

nanofibers [91]. MNP-loaded nanofibers have potential to be used as cancer hyperthermia

therapy through the application of an AMF.

4.3. Tissue Engineering

Tissue engineering is the substitution of any human organ or tissue with artificial

functional materials using a combination of biology, medicine, and engineering. Recently,

Preslar et al. manipulated target cells in a scaffold using physical means, such as magnetic

force [92]. Nanofibers loaded with MNPs used as the scaffold play an important role in

wound healing [93], because the MNPs in the nanofiber scaffold assemble the tissue and aid

tissue formation by magnetic force. Using this technique, the magnetic force-based tissue

engineering and mechano-transduction methods can control cellular signaling, artificial

blood vessel development, and bone tissue formation [85,94]. In addition, this method is an

effective way to mimic signal transduction in vivo and convert extracellular mechanical

stress to intracellular chemical cues [94]. Furthermore, MNP-loaded nanofiber scaffolds

have been applied in cell sheet engineering for skin tissue formation through the creation of

multi-layered keratinocytes by magnetic force [95].

5. Conclusions and Future Prospects

In this review, magnetically functionalized E-spun nanofibers have been discussed from

the standpoint of biomedical applications, including scaffolds, regenerative medicine, and

cancer therapy. These fibers combine inherent characteristics of standard E-spun nanofibers,

such as high porosity, high

surface-area-to-volume ratio, flexibility, and ease of fiber production, with unique and

advantageous magnetic properties.

In the future, magnetic E-spun nanofibers will likely be used as a functionalized scaffold

in tissue engineering because MNP-loaded nanofiber scaffolds have in vitro osteogenesis

and tissue compatibility and the ability to regenerate bone in vivo due to their increased
hydrophilicity, accelerated degradation, ability to form apatites, and enhanced mechanical

properties by magnetic force, suggesting that they can be used as a new class of bone

regenerative materials. In addition, electrospun nanofiber mats with a conformal coating on

the surface of the mats may be used as an ideal wound-dressing material because they are

antibacterial, nontoxic, non-antigenic, permeable for gaseous exchange, resistant to shearing

forces (due to their elasticity), and are highly water-absorbent. However, the use of highly

engineered electrospun nanofiber mats tailored for wound healing in the clinic requires

further study. Furthermore, nanofiber systems may be used in the future as an alternative

approach to gastro-retentive drug delivery systems for enhanced bioavailability and the

controlled release of drugs because they provide prolonged contact time with the gastric

mucosa, controlled release of the drug, and good stability.