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Coaxial Electrospinning for Encapsulation and Controlled Release of FragileWater-soluble Bioactive Agents

Hongliang Jiang, Liqun Wang, Kangjie Zhu

PII: S0168-3659(14)00243-0DOI: doi: 10.1016/j.jconrel.2014.04.025Reference: COREL 7135

To appear in: Journal of Controlled Release

Received date: 20 February 2014Revised date: 2 April 2014Accepted date: 10 April 2014

Please cite this article as: Hongliang Jiang, Liqun Wang, Kangjie Zhu, Coaxial Elec-trospinning for Encapsulation and Controlled Release of Fragile Water-soluble BioactiveAgents, Journal of Controlled Release (2014), doi: 10.1016/j.jconrel.2014.04.025

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Coaxial Electrospinning for Encapsulation and Controlled Release of

Fragile Water-soluble Bioactive Agents

Hongliang Jiang, Liqun Wang and Kangjie Zhu

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou

310027, China

Corresponding author:

Hongliang Jiang

Department of Polymer Science and Engineering

Zhejiang University

Hangzhou 310027, China

E-mail: hljiang@zju.edu.cn

Tel: 86-571-87952046

Fax: 86-571-87951773

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Abstract

Coaxial electrospinning is a robust technique for one-step encapsulation of fragile,

water-soluble bioactive agents, including growth factors, DNA and even living

organisms, into core-shell nanofibers. The coaxial electrospinning process eliminates

the damaging effects due to direct contact of the agents with organic solvents or harsh

conditions during emulsification. The shell layer serves as a barrier to prevent the

premature release of the water-soluble core contents. By varying the structure and

composition of the nanofibers, it is possible to precisely modulate the release of the

encapsulated agents. Promising work has been done with coaxially electrospun

non-woven mats integrated with bioactive agents for use in tissue engineering, in local

delivery and in wound healing etc. This paper reviews the origins of the coaxial

electrospinning method, its updated status and potential future developments for

controlled release of the class of fragile, water-soluble bioactive agents.

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1. Introduction

Electrospun non-woven mats have emerged as a novel drug delivery platform

within the last decade [1-3]. By simply applying an electric field between spinneret and

collector through a high voltage supply, solidified polymer fibers with sizes ranging

from tens of nanometers to several microns can be obtained without the need for any

post-treatment processing [4,5], such as solvent extraction which is used in

conventional spinning to eliminate solvents remaining in the fibers. This makes it

possible to incorporate bioactive agents into the fibers in one-step process with almost

no loss of the agents [6]. Such advantage for electrospun non-woven mat as a drug

delivery platform, coupled with the simplicity of electrospinning setup and their great

potential for tissue engineering [7,8], wound healing [9], postoperative anti-adhesion

[10], guided bone regeneration [11] and in vivo local delivery [3,12] etc., constitutes

the main reasons why electrospinning gained popularity in the field of controlled

release. Coaxial electrospinning is an innovative extension of electrospinning, which

uses two concentrically aligned capillaries to enforce the formation of the fibers with

a core-shell structure [13]. The primary motivation to make use of coaxial

electrospinning in controlled release is to circumvent the limitations of single-nozzle

electrospinning in the encapsulation of fragile, water-soluble bioactive agents which

play vital roles in regenerative medicine. Other advantages of coaxial electrospinning

over single-nozzle electrospinning include more sustained release of the encapsulated

agents as well as one-step co-encapsulation of multiple drugs with different solubility

characteristics.

The principle, technique details and biomedical applications of coaxial

electrospinning have been discussed at length in previous reviews [13,14]. Rather than

covering all aspects of coaxial electrospinning, we focus here on the historical

development of this important technique for encapsulation and controlled release of

fragile, water-soluble bioactive agents, including our own works and contributions

from other research groups, in order to give a clear image of the origins, current status

and potential development of this research area.

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2. Origin of coaxial electrospinning for controlled release of fragile

water-soluble bioactive agents

Just like microencapsulation of drugs into biodegradable microspheres through

emulsion-solvent evaporation [15], successful incorporation of bioactive agents into

electrospun fibers strongly relies on their solubility characteristics. For organo-soluble

drugs, direct electrospinning of drug/polymer mixed solution can facilely load the

drugs into the polymer fibers. It was until 2002 when Kenawy et al. initially studied

the incorporation and release behavior of drugs from electrospun non-woven mats

using tetracycline as a model organo-soluble bioactive agent [6]. The release rate of

tetracycline from the electrospun membranes was found to be always faster than that

from solvent-casting films, apparently because of higher surface area-to-volume ratio

of the electrospun membranes. Later studies further systematically investigated the

effects of several factors, including additives in the spinning fluid, drug lipophilicity

and polymer composition etc., on drug incorporation and release [16-17].

Although there were several attempts to incorporate water-soluble bioactive

agents into biodegradable polymer fibers by direct electrospinning of the suspension

of drug particles in polymer solution, serious burst release was frequently reported.

Kim et al. [18] prepared a suspension of fine hydrophilic antibiotics (Mefoxin)

particles in polymer solution by slowly adding the aqueous drug solution into

poly(lactide-co-glycolide) (PLGA)/DMF solution under vigorous stirring.

Electrospinning of the suspension generated Mefoxin-loaded non-woven PLGA mats.

Although the electrospinning process was found to have no effect on drug structure

and bioactivity, very serious burst release was observed. Similar method was used for

incorporating DNA into PLGA nanofibers [19]. More than 60% of the incorporated

DNA was released from the fibers within 2 hours. A strategy was later developed by

the same research group to inhibit the DNA burst release [20,21]. Individual DNA

molecules dissolved in an aqueous medium were first condensed into nanoparticles

with a water-miscible organic solvent. The nanoparticles were further encapsulated

into amphiphilic polylactide-b-polyethylene glycol (PLA-b-PEG) through a

self-assembly process. Electrospinning of the suspension of the encapsulated DNA

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nanoparticles in PLGA solution yielded DNA-laden non-woven mats displaying much

less DNA burst release.

In general, direct electrospinning of the suspension of water-soluble drug particles

in polymer solution requires a delicate mixing and always results in serious burst

release. In addition, the contact of fragile bioactive agents, such as growth factors and

DNA, with organic solvents has great possibility of denaturing the agents [22,23]. To

circumvent the limitations, Sanders et al. developed a method called “two-phase

electrospinning” (later termed as emulsion electrospinning) in 2003 for encapsulating

bovine serum albumin (BSA) into non-woven poly(ethylene-co-vinyl acetate) (EVA)

mats [24]. Water-in-oil (W/O) emulsion with aqueous BSA droplets suspended in

EVA/dichloromethane solution was first prepared and subjected to electrospinning to

produce BSA-loaded EVA mats. This method was later widely adopted for

encapsulation of proteins into biodegradable polymer fibers [25-29]. For example,

human α-nerve growth factor (NGF) was incorporated into electrospun poly(ethyl

ethylene phosphate) (PEEP) fibers by emulsion electrospinning [25]. NGF could be

released from the non-woven mats for more than 3 months. However, extremely low

loading efficiency of NGF in the fibers was found. Qi et al. [26] made a modification

of the above technique to incorporate BSA into electrospun PLLA fibers. BSA-loaded

alginate microspheres were first prepared by W/O emulsion method. After the alginate

microspheres were crosslinked with calcium chloride, PLLA was dissolved in the oil

phase of the emulsion (i.e., chloroform) to form a suspension of BSA-loaded alginate

microspheres in PLGA/chloroform solution. The suspension was subjected to

electrospinning to form fibrous membranes. Compared with the alginate microspheres,

the fibers significantly prolonged the BSA release duration. Xu et al. [30,31] later

found that electrospinning of W/O emulsion with aqueous PEO droplets suspended in

PLA-b-PEG/chloroform solution generated the nanofibers with distinctive core-shell

structure. PEO located in the core section of the nanofibers and PLA-b-PEG formed

the shell layer. It was speculated by the authors that stretching and evaporation of the

jets during electrospinning process induced de-emulsification of the emulsion,

resulting in the observed results.

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Emulsion electrospinning is reminiscent of water-in-oil-in-water (W/O/W)

emulsion-solvent evaporation method widely used for microencapsulation of proteins

into biodegradable polymers (Figure 1), where protein denaturation was always a big

concern because of the violent emulsification process and enrichment of proteins at

W/O interface [32]. As to emulsion electrospinning, such concern also exists. In

addition, the thermodynamically unstable nature of emulsion raises a challenge for

smooth electrospinning.

Figure 1 Illustration of W/O/W emulsion-solvent evaporation and emulsion electrospinning for encapsulation of

proteins, noting that the two techniques share common problems with protein stability.

3. Current status of coaxial electrospinning for controlled release of fragile

water-soluble bioactive agent

In 2003, Sun et al. reported the formation of core-shell fibers through coaxial

electrospinning where a spinneret composed of two coaxial capillaries was used

instead of single nozzle [33]. Two polymer fluids were independently fed through

inner/outer capillaries and formed a compound droplet at the exit of the spinneret.

When surface charges of the droplet induced by electrical field were high enough to

overcome the surface tension of the fluids, a compound jet was initiated, and then

substantially elongated and thinned because of the bending instability process.

Evaporation of the solvents contained in the jet resulted in the formation of the

nanofibers with core-shell structure. Although the report did not involve any bioactive

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agents to be incorporated into the core section of the fibers, the implication that there

was no mixing of core and shell fluids during electrospinning process did show the

promise of the technique for mild encapsulation of diverse bioactive agents. In a

subsequent study, Zhang et al. [34] attempted to apply the technique to biomedical

field. They successfully fabricated core-shell fibers with gelatin as a core material and

poly(ε-caprolactone) (PCL) forming the shell layer through coaxial electrospinning,

using trifluoroethanol (TFE) as a solvent for both PCL and gelatin. However, basic

methodology on how to apply coaxial electrospinning for controlled drug release was

not reported.

3.1. Basic methodology

With the goal of how to facilely incorporate fragile growth factors into electrospun

fibers, we demonstrated the feasibility of coaxial electrospinning for encapsulation

and controlled release of proteins [35]. The configuration of coaxial electrospinning

setup and compositions of inner/outer fluids are shown in Figure 2. In order to avoid

possible mixing of inner/outer fluids, PCL was dissolved in chloroform/DMF (7/3, v/v)

mixed solvent which was found to be immiscible with inner aqueous fluid. This

would prevent the formation of polymer clotting at the tip of compound spinneret.

DMF was used to slow down the drying of the compound jet and lower the interfacial

tension between the inner and outer fluids, thereby facilitating smooth electrospinning

[36]. In addition, poly(ethylene glycol) (PEG) was added into the core fluid to

stabilize the proteins [37]. By varying the feed rate of the inner fluid, core-shell

nanofibers with various shell thickness were prepared. The proteins could be

encapsulated into the fibers with entrapment efficiency close to 100%. Lysozyme was

used as a model enzyme for evaluating the effect of electrospinning process on protein

stability. No apparent loss of enzymatic activity was observed. It was also found that

as the membrane was soaked in release buffer, open pores emerged on fiber surface,

resulting from the dissolution of water-soluble core content located in the vicinity to

fiber surface [34,38]. Taking account of the very slow degradation nature of PCL and

compact internal structure of the PCL shell layer [39], it was speculated that the

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protein was released from the core-shell fibers mainly through the open pores, rather

than through bulk diffusion. Based on this assumption, PEG was further added into

outer PCL solution prior to coaxial electrospinning to act as a porogen of the shell

layer [40]. It was found that the protein release rate from core-shell fibers could be

manipulated over wide ranges by varying PEG content in core and shell layers.

Similar methodology was reported soon thereafter by other research groups [41-43].

The important role of PEG as a porogen in protein release was also verified by Liao et

al. [42] who found that in the absence of PEG in the shell layer the release of platelet

derived growth factor-bb (PDGF-bb) from PDGF-bb/PCL core/shell fibers was almost

negligible (Figure 3). PDGF-bb itself could not generate open pores throughout the

shell layer because of its very tiny amount in the core section. Owing to its

macromolecular nature, the diffusion of PDGF-bb through the bulk PCL shell was too

slow to take place within the evaluated period of time. The presence of PEG in the

shell layer could create porous structure of the PCL shell and facilitate the release of

the incorporated growth factor. The composition and electrospinning conditions

developed in all these contributions were generally adopted for encapsulation and

controlled release of proteins in the later studies [44-54].

Figure 2 Coaxial electrospinning for encapsulation of proteins into core-shell nanofibrous non-woven mats [35].

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Figure 3 Controlled release and bioactivity of encapsulated PDGF-bb (20 µg) from coaxial nanofibers. (A) The

effect of PEG as a porogen of the PCL shell layer on PDGF-bb release; (B) Bioactivity of PDGF-bb released into

the supernatant solution as determined by the enhanced proliferation rate of NIH 3T3 cells. Cell proliferation is

normalized to negative control (no PDGF-bb). D1, 7, 14 and D20 refers to the supernatant solutions collected at

day 1, 7, 14 and 20 [42].

3.2. Coaxial electrospinning for controlled release of proteins and DNA

The studies related to coaxial electrospinning for controlled protein release are

summarized in Table 1. In most cases, biodegradable crystalline polymers, such as

PCL and P(LLA-CL), were used as the shell materials. Amorphous PLGA was also

occasionally adopted. Unfortunately, the dimensional stability of PLGA-based coaxial

mats was not studied in the related works [44,50]. Significant shrinkage was reported

to occur for single-nozzle electrospun PLGA mats when they were incubated in

aqueous media at physiologic temperature, due to the relaxation of aligned PLGA

chains which has a glass transition temperature lower than 37 oC in aqueous media

[55]. Porogens, such as PEG, dextran, PVA and BSA, were commonly added into the

shell or core solutions prior to electrospinning. Either water-miscible solvents (TFE,

HFP) or water-immiscible mixed solvents were used for dissolving the shell materials.

The formation of core-shell fibers with the proteins encapsulated in core section was

reported in all the studies. The protein release could be modulated from several days

to months, depending on the compositions of the inner/outer fluids. It seemed that the

presence of PCL-b-PEG in PCL shell layer could promote the rapid release of the

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incorporated bFGF [51]. Nonetheless, modeling and prediction of protein release from

the core-shell fibers have yet not been systematically explored. The protein release

profile was affected by numerous factors including composition and concentration of

shell polymers, solvent of outer dope, type and concentration of the additives in the

core section, protein amount, electrospinning condition and even ambient humidity.

This makes it difficult to make a comparison of the results from different studies.

Several research groups compared controlled release capacity of core-shell fibers with

their counterparts of monolithic fibers prepared by single-nozzle electrospinning

[44-46,52]. In all the cases, the protein release from core-shell fibers exhibited much

more sustained release profiles because of the presence of shell barrier. Up to date,

coaxial electrospinning technique has been extended to numerous types of growth

factors for various biomedical applications. The combination of several growth factors

or a growth factor with other type of bioactive agent has also been attempted to

achieve synergistic effects [46,49,52,53]. Depending on the solubility characteristics

of the bioactive agents, they were either co-localized in core section or separately

resided in core and shell layers.

In vivo efficacy of coaxially electrospun fibers integrated with growth factors has

been evaluated in several studies for different application purposes [48,49,51,53,54].

Liao et al. [49] prepared a tissue engineered construct by culturing FVIII-producing

skeletal myoblasts on aligned electrospun fibers and found the constructs could

seamlessly integrate with the host tissue within one month upon subcutaneous

implantation into hemophilic mice. The construct was further covered with an

additional layer of coaxial fibers integrated with angiogenic or lymphangiogenic

growth factor which served to stimulate local lymphatic (VEGFC) or vascular

(VEGFA and PDGF-bb) systems to enhance the transport of FVIII from the implant

site to blood circulation. It was found that the engineered constructs that induced

angiogenesis resulted in sustained elevation of plasma FVIII and significantly reduced

blood coagulation time for at least 2-months. Liu et al. [48] observed similar nerve

regeneration ability among nerve autograft group and coaxially electrospun conduits

with NGF embedded in the core of core-shell fibers. Both groups all showed

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significantly greater efficacy than electrospun conduit blended with NGF. Choi et al.

[51] fabricated coaxially electrospun non-woven mats integrated with dual growth

factors, FGF and EGF, as wound dressings. In order to independently modulate the

release profiles of the two agents, FGF was loaded into the core of the core-shell

fibers, while EGF was chemically immobilized on the shell surface. It was found that

the bi-phasic release of bFGF and EGF greatly supported tissue recovery with the

similar phenotypes as the original keratinized tissue. Other related studies also

confirmed the role of sustained release of growth factors from core-shell fibers for the

corresponding applications [53,54].

In order to circumvent the limitations of liposome for controlled protein release,

Mickova et al. [56] encapsulated protein-loaded liposome into core-shell fibers

through coaxial electrospinning. PCL was used as the shell material and

liposome-containing PVA constituted the core content. It was found that in contrast

with single-nozzle electrospinning, coaxial electrospinning could preserve the

integrity of the liposome and protect the encapsulated enzyme. Only less than 10% of

enzymatic activity of horseradish peroxidase (HRP) was preserved after the enzyme

was subjected to single-nozzle electrospinning process, irrespective of whether the

enzyme was incorporated into the liposome or not prior to electrospinning. On the

contrary, coaxial electrospinning preserved 62% of the enzyme activity for the

liposome-incorporated HRP. Furthermore, the incorporation of growth factors into the

liposome could significantly prolong their release from the core-shell fibers when

compared with their naked counterparts.

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Table 1 List of the studies related to coaxial electrospinning for controlled release of proteins

Bioactive

agents

Core fluid Shell fluid

Release

duration

Application Ref.

BSA or lysozyme BSA (or lysozyme) and PEG

(or dextran) in water

PCL and PEG in CHCl3/DMF >30 days / [35,40]

BSA BSA and PEG in water PCL in TFE >30 days / [41]

PDGF-bb PDGF-bb in water PCL and PEG in

CH2Cl2/ethanol

>30 days / [42]

bFGF bFGF and BSA in Tris

buffer

PLGA in HFP >2 weeks Bone marrow stem

cells

[44]

PDGF-bb PDGF-bb, dextran and BSA

in water

P(LLA-CL) in CHCl3/DMF >28 days Vascular smooth

muscle cell

[47]

NGF NGF and BSA in PBS P(LLA-CL) in TFE / Nerve regeneration [48]

VEGFA, VEGFC,

PDGF-bb

VEGFA (or VEGFC) and

PDGF-bb in water

PU in CHCl3/ethanol 30 days Hemophilia [49]

VEGF VEGF, BSA, heparin and

dextran in water

PLGA in CHCl3/TFE >30 days Vascular tissue

engineering

[50]

bFGF, EGF bFGF and PVA in water PCL and PCL-PEG in

CHCl3/methanol

6 days Wound dressing [51]

BMP BMP and dextran in PBS P(LLA-CL) and collagen in

HFP

>22 days Bone tissue

engineering

[46]

EIF EIF and BSA in water P(LLA-CL) and gelatin in HFP >15 days Skin regeneration [52]

VEGF or PDGF VEGF (or PDGF) in chitosan

hydrogel

P(LLA-CL)-b-PEG in

CHCl3/TFE

~28 days Blood vessel [53]

BMP BMP and PEG in water PCL in CHCl3/DMF >30 days Guided bone

regeneration

[54]

Abbreviation: bFGF-basic fibroblast growth factor; HFP-hexafluoro-2-propanol; VEGF- vascular endothelial

growth factor; PU-polyurethane; PVA-poly(vinyl alcohol); EGF-epidermal growth factor; BMP-bone

morphogenetic protein; EIF-epidermal induction factors.

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Although the bioactivity of the growth factors encapsulated within core-shell

fibers was assessed in most studies, quantitative measurement was rarely performed

[42,56]. The coaxial electrospinning process, including high voltage applied, shearing

force imposed at the interface between core and shell fluids and rapid protein

dehydration, might still be harmful for the stability of fragile bioactive agents [57,58].

Some reports did show the loss of bioactivity of the enzymes subjected to coaxial

electrospinning [45,56]. To avoid possible protein denaturation induced by

electrospinning process, we developed a strategy to immobilize growth factors onto

fibrous non-woven mats by combining coaxial electrospinning with heparin

affinity-based growth factor delivery system [59,60]. This work was motivated by the

following two facts: 1) the nanofibrous structure of electrospun membranes is

beneficial for adsorption in terms of both adsorption capacity and speed [61], and 2)

heparin can specifically bind and stabilize numerous growth factors [62].The structure

and final composition of the non-woven mats are illustrated in Figure 4. Coaxial

electrospinning was used to fabricate the core-shell fibers with PCL as the core

material and crosslinked cationized gelatin (CG) forming the shell layer. The presence

of PCL in the fibers strengthened the fibers in aqueous media, while CG hydrogel

shell could accommodate sufficient amount of anionic heparin through electrostatic

adsorption [62]. Growth factors were finally immobilized onto the fibers through

specific binding to the adsorbed heparin. The immobilization could be performed by

simply spraying aqueous growth factor solution onto the heparinized membranes. It

was found VEGF could be strongly fixed in the membranes and steadily released in

its original active state for almost 15 days. Compared with one-step encapsulation

strategy, the post-immobilization method provides a milder, flexible way to load

fragile bioactive agents into electrospun membranes. The PCL/CG core/shell

non-woven mats also have the potential to immobilize DNA onto the fibers through

electrostatic interaction [63].

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Figure 4 Combination of coaxial electrospinning with heparin affinity-based drug delivery system for mild

immobilization and controlled release of growth factors [60].

Gene delivery from nanofibrous mats can also be achieved through coaxial

electrospinning. Saraf et al. [64] encapsulated plasmid DNA into core-shell fibers

where the shell was composed of PCL and poly(ethylenimine)-hyaluronic acid

(PEI-HA), while DNA-containing PEG constituted the core content. The gene vector,

PEI-HA, and DNA were separately located in the shell and core layer. Such

arrangement was reported to arise from the observation that co-incorporation of

PEI-HA/DNA complex into core section resulted in negligible release of the complex.

The observation was explained by the authors who speculated that interactions

between the core and sheath polymers limited incorporation of PEI-HA/DNA

complexes into the coaxial fibers. The released DNA was found to be able to transfect

the cells attached to the membranes. The transfection was believed to result from the

in situ formation of DNA/PEI-HA complexes in cell culture medium.

3.3. Coaxial electrospinning for encapsulation of living organisms

Yarin et al. predicted that shear stress at the interface of core and shell fluids was

of the order of 500 Pa during electrospinning process, which approaches the lower

level of mechanical stress lethal to many biologic agents including cells and virus

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(103-10

5 Pa) [38,57]. The prediction coincided with the practice of the studies about

using coaxial electrospinning for encapsulation of living organisms [65-72]. Such

practice represents the major strategic development of coaxial electrospinning for

biomedical applications. Up to date, living organisms including cells, bacteria, virus

and cell organelles have been successfully encapsulated into core-shell fibers through

coaxial electrospinning. In most cases, they could survive from the coaxial

electrospinning process. However, there were also reports indicating the harmful

effect of the process on the encapsulated species [69,71]. The release of the living

organisms from the fibers was achieved by addition of porogen such as PEG into the

shell layer.

3.3.1 Cells

Jayasinghe et al. [66,67] investigated the effect of coaxial electrospinning process

on the viability of cells, using liquid poly(dimethylsiloxane) (PDMS) as shell fluid.

Figure 5 shows the compound jet just below Taylor cone, indicating successful

encapsulation of the cells. No apparent loss of cell viability was found for two types

of cells, primary porcine vascular and rabbit aorta smooth muscle cells, through

long-term flow cytometry analysis.

Figure 5 Characteristic high-speed photographs of the compound jets developed during coaxial electrospinning,

indicating the successful encapsulation of the cells into PDMS stream [66].

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3.3.2. Bacteria

López-Rubio et al. [68] encapsulated bifidobacterial into core-shell fibers with

poly(vinyl alcohol) (PVA) as the shell material and bifidobacterial-containing milk

constituting the core content, and evaluated the protective effect of the core-shell

fibers on the bacteria under different storage conditions. It was found that the

encapsulation process had a beneficial effect on their storage stability instead of

damaging the bacterial.

Coaxial electrospinning was used by Klein et al. [69] to encapsulate three

different species of bacteria into core-shell fibers for use in bioremediation of

pollutants in water systems. In contrast with previous relevant studies [66-68] where

the cells were instantly de-encapsulated as the membranes contacted with aqueous

media so that cell viability could be accurately determined by plate counting, this

report used PCL as the shell material to retain the bacteria within the fibers for a

certain period of time. This raises practical applicability of such bio-system. PEG was

added in the shell layer as a porogen to facilitate transport of small molecules through

shell barrier. Efficacy of the bacteria-loaded system was assessed by analyzing

activity of cell membrane enzymes, while bacteria viability was indirectly evaluated

by cell respiration and their ability to synthesize proteins. It was found that only

partial bacterial survived from coaxial electrospinning process.

3.3.3. Virus

Liao et al. [65] encapsulated adenovirus encoding the gene for green fluorescent

protein into core-shell fibers. PEG was contained in PCL shell as a porogen so that the

virus could be released from the fibers to in situ transfect the cells attached on the

non-woven mats. Figure 6 depicts the evolution of porous structure of the PCL shell

containing different amount of PEG after the membranes were soaked in aqueous

media, confirming the role of PEG as a porogen to generate open pores in the PCL

shell. It was found that the released virus could locally transfect the cells seeded on

the non-woven mats. The attached cells expressed high level of green fluorescent

protein over a month, while the cells in scaffold supernatant showed only transient

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expression for a week.

Figure 6 Emergence of open pores on the surface of core-shell fibers with PEG contained in the PCL shell layer

[65], noting that the porosity increased with the PEG content and incubation time in aqueous media.

3.3.4. Cell organelles

Very recently, Buzgo et al. [70] incorporated platelet granules which could serve as

an efficient source of natural growth factors into coaxial fibers. The system was found

to preserve α-granule bioactivity and stimulate cell viability and chondrogenic

differentiation of mesenchymal stem cells, showing great potential for cartilage

engineering.

4. Challenges and future direction

Coaxial electrospinning has been extensively explored for encapsulation and

controlled release of growth factors. The limited number of growth factors approved

for clinical use restricts the commercialization of growth factor-loaded coaxial

non-woven mats [14,71]. Furthermore, combination of multiple growth factors in their

biologically determined ratio is always necessary to mimic their in vivo environment

where one growth factor only works as a part of growth factor network. In such

context, natural source of growth factors, such as platelet rich plasma (PRP) which

contains several different growth factors and other cytokines, has gained popularity in

academic and clinical settings over the past years [72]. The integration of PRP with

electrospun nanofibers has been achieved by either single nozzle electrospinning of

activated PRP [73,74] or encapsulation of platelet α-granules into coaxial fibers [70].

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The resultant non-woven mats could deliver multiple growth factors in a controlled

manner over a long period of time and promote proliferation of the attached cells,

showing great prospect for regenerative medicine.

Extension of coaxial spinneret to more complex configuration can further endow

the fibers with diverse structure and functionalities [75-79]. For instance,

triple-layered core-shell-corona fibers could be constructed by triaxial electrospinning

[76-79]. Very recently, this technique has been introduced to the field of controlled

release [79,80]. The basic thought was to use the intermediate shell layer for slowing

down the release of bioactive agents contained in the core section and use the outer

corona for improving the biocompatibility of non-woven mats. For this purpose,

biodegradable polymers such as PCL could serve as the intermediate material, while

hydroscopic polymers, e.g., collagen and gelatin, constitute the outmost layer. The

sustained release capacity of the triple-layered fibrous non-woven mats, as well as

their cell friendly characteristics, renders triaxial electrospinning a robust process for

creating ideal tissue engineering scaffolds. Nevertheless, there were only limited

proof-of-concept studies reported up to date, possibly because of the challenges in

smooth electrospinning. Compared with coaxial electrospinning, there are even more

coupled parameters governing triaxial electrospinning process and the properties of

resultant non-woven mats [59]. Alignment of three concentric capillaries is also a

tricky work. This will make it difficult to compare the results from different research

groups. Standardization of triaxial electrospinning setup, therefore, becomes urgent

for its widespread utilization. This is also true for coaxial electrospinning.

Standardization of coaxial/triaxial electrospinning setup and conditions relies on

comprehensive understanding of electrospinning physics, discovering of key

parameters governing electrospinning process and development of new techniques

[57,81].

Reznik et al. found that the extruding of core nozzle outside the shell counterpart

by around 0.5 of its radius facilitated formation of core-shell jets and nanofibers in

coaxial electrospinning [57]. Similar theoretical and experimental studies will help

rational designing of coaxial/triaxial spinneret.

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It is still not very clear how the solution pairs of inner/outer fluids affect

electrospinning process and fiber structure. Contradictory results on this issue can be

found in previous studies. Sun et al. [33] reported the formation of core-shell fibers

even when same polymer solutions were used as core and shell fluids, while Li et al.

[82] found the immiscibility of inner/outer solutions were essential for preventing

mixing of the two fluids during electrospinning process. Clarification of this question

is important because appropriate selection of solution pairs is critical not only for the

formation of the expected core-shell structure, but also for the stability of fragile

bioactive agents. The mixing or partial mixing of inner/outer dopes could cause direct

contacting of bioactive agents with organic solvents and the subsequent denaturation.

In addition, solidification process of the compound jet, especially the dehydration of

inner aqueous protein solution and effect of outer fluid and ambient conditions on

water evaporation, remained unexplored. Since proteins are subjected to

reversible/irreversible arrangement in secondary structure during the dehydration

process [85], elucidation of the aforementioned question can help formulation of an

aqueous protein solution with enhanced protein stability to be used as the core fluid of

coaxial electrospinning.

The development of new techniques can promote the standardization of coaxial

electrospinning setup. Srivastava et al. [84] designed and constructed a prototype

coaxial microfluidic device through soft lithography technique (Figure 7). Two layers

of non-intersecting, stacked microchannels arranged in a branching tree pattern are

created within the device to provide constant flow of core/shell fluids to each of eight

outlet spinnerets. The coaxial spinneret which consisted of concentric stainless steel

tubes were aligned and punched through the elastomer device. The advantages of the

microfluidic device include simple molding methodology for the elastomeric device

fabrication, flexible control over channel dimensions and geometry and parallel

coaxial electrospinning etc. In order to avoid the need for concentric spinneret,

hydrodynamic focusing method was adopted in a following study by the same group

to generate a coaxial stream of two immiscible fluids within a microfluidic device

(Figure 8) [85]. Electrospinnig of the coaxial stream resulted in the nanofibers with

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core-shell structure. Besides controlled release of bioactive agents, this technique

could be very useful for encapsulation of living organisms into core-shell fibers to

eliminate their possible agglomeration and evenly disperse the organisms in the fibers

by taking advantages of the sorting effect of hydrodynamic focusing which has been

successfully applied for flow cytometry [86].

Figure 7 (A) Schematic representation of the PDMS microfluidic device source using branching microchannel

architecture and coaxial spinnerets for parallel electrospinning of hollow and core/sheath type nanofibers. (B)

Optical microscopy image of the microfluidic channels illustrating the coaxial spinnerets and connection ports to

feed the sheath (PVP) and core (heavy mineral oil or pyrrole + PVP) solutions [86].

Figure 8 (a) Top–down schematic representation of the microchannel layout in the PDMS 2D flow-focusing device.

The sheath fluid is introduced through inlets A, C and D, while the core fluid is introduced through inlet B. (b)

Illustration of the 2D flow-focusing process in the microfluidic device. Core fluid enters the central microchannel

at the A–B junction, is initially focused in the vertical direction at the A–C junction, and subsequently focused

laterally into a single stream at the A–D junction prior to exiting the device [87].

5. Conclusion

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The introduction of the coaxial electrospinning to the field of controlled release

provides a powerful tool for the encapsulation of fragile, water-soluble bioactive

agents. This can include growth factors and DNA which play important roles in

regenerative medicine. The method also provides fine control over the release rate of

the agents.

Coaxial electrospinning furnishes another tool that can be used to integrate the

method of controlled-release into tissue engineering and other biomedical applications.

Although significant progress has been made over the last ten years, the true potential

of coaxial electrospinning has yet to be realized. Recent applications of coaxial

electrospinning for the encapsulation of cells, viruses, bacteria and platelet α-granules

show promise in biofabrication.

However, a fundamental understanding of the coaxial electrospinning process,

including the evolution of compound jets, solvent evaporation, and the discharge of

the resultant core-shell fibers, has yet to be achieved. This is critical for the

standardization of the setup and to achieve a stable operation of the process, which in

turn is necessary for the formation of defect-free core-shell nanofibers, and adequate

stabilization of the agents. The introduction of new techniques and equipment, such as

microfluidic devices, also show promise in the standardization of the technique.

Acknowledgements

This work was supported by National Science Foundation of China (21174125).

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Graphic abstract

Fragile, water-soluble bioactive agents, including proteins, growth factors, DNA and

even living organisms, could be encapsulated into core-shell fibers through coaxial

electrospinning technique.